text stringlengths 1 27.3k | synonym_substitution stringlengths 1 14.8k | butter_fingers stringlengths 0 14.7k | random_deletion stringlengths 1 10.9k | change_char_case stringlengths 1 27.3k | whitespace_perturbation stringlengths 1 12.8k | underscore_trick stringlengths 1 12.9k |
|---|---|---|---|---|---|---|
Introduction {#sec1-1}
============
Infliximab (IFX), a chimeric anti-TNFα antibody, is effective in inducing and maintaining remission in a considerable proportion of IBD patients refractory to any other treatments \[[@ref1],[@ref2]\]. However, 8-12% of adult and/or pediatric patients fail to respond to the induction regimen (known as primary non responders) and approximately 40% of patients who respond initially and achieve clinical remission inevitably lose response over time\[[@ref3],[@ref7]\]. Lack of response to IFX is a stable trait and suggests that the differences in response might be in part genetically determined. Considering the high cost and safety profile of this drug, genetic targeting of patients responding to this therapy is certainly of great interest \[[@ref8]\]. So far, limited candidate gene association studies with response to IFX have been reported \[[@ref9]-[@ref11]\]. Recently, a genome-wide association study (GWAS) in paediatric IBD patients has revealed that the 21q22.2/BRWDI loci were associated with primary non response \[[@ref12]\]. Furthermore, although TNFa gene is of great interest as a candidate gene for pharmacogenetic approaches few studies have been performed to date and some have led to contradictory results \[[@ref10],[@ref11],[@ref13]-[@ref15]\].
All anti-TNF agents share an IgG1 Fc fragment, but the contribution of the Fc portion to the response to treatment among currently used TNF blockers remains unknown. Receptors for IgG-Fc portion (FcR) are important regulatory molecules of inflammatory responses. FcR polymorphisms alter receptor function by enhancing or diminishing the affinity for immunoglobulins \[[@ref16]\]. Three major classes of FcR that are capable of binding IgG antibodies are recognised: FcγRΙ (CD64), FcγRΙΙ (CD32), and FcγRΙΙΙ (CD16). FcγRΙΙ and FcγRΙΙΙ have multiple isoforms (FcγRΙΙΙA/C and B; FcγRΙΙΙA and B) \[[@ref16]\]. The most frequent polymorphism of *FcγRΙΙΙA* is a point mutation affecting amino acids in codon 158 in the extracellular domain. This results in either a valine (V158) or a phenylalanine (F158) at this position. Recently, it has been reported that CD patients with *FcγRΙΙΙA* -158V/V genotype had a better biological and possibly better clinical response to IFX \[[@ref17]\]. However, further studies did not confirm this observation \[[@ref18]\].
The aim of this study was to assess whether the *TNF* and/ or *FcγRΙΙΙA* gene polymorphisms are genetic predictors of response to IFX, in a cohort of Greek patients with adult or paediatric onset of CD.
Patients - Methods {#sec1-2}
==================
Patients {#sec2-1}
--------
We enrolled 106 consecutive patients with newly diagnosed CD attending the outpatient IBD Clinic at the 1^st^ Department of Gastroenterology, "Evangelismos" Hospital (79 adults) or the 1^st^ Department of Pediatrics, University Hospital of Athens "Aghia Sophia"(27 children). The diagnosis of CD was based on standard clinical, endoscopic, radiological, and histological criteria \[[@ref1],[@ref19]\]. Eligible patients should have inflammatory (luminal) disease and be naive to IFX.
IFX was administered intravenously at a dose of 5mg/kg at weeks 0, 2, 6 and then every 8 weeks. Clinical and serological responses were assessed using the Harvey-Bradshaw Index (HBI) \[[@ref20]\] and the serum levels of C-reactive protein (CRP), respectively, at baseline (before the 1st infusion of IFX), the day before each subsequent IFX infusion and after 12 weeks of treatment. Ileocolonoscopy was performed by a single endoscopist (GJM) at baseline and after 12-20 weeks of therapy to assess mucosal healing. Any changes in endoscopic appearance compared to baseline endoscopy were classified in four categories \[[@ref21],[@ref22]\] \[[Table 1](#T1){ref-type="table"}\]. Patients were classified in accordance to response to IFX therapy as shown in [table 2](#T2){ref-type="table"}. The ethical committee of the participating hospitals approved the study. Research was carried out according to Helsinki Convention (1975) and written inform consent was obtained in advance from each patient.
######
Grading of endoscopic mucosal lesions \[[@ref21],[@ref22]\]
######
Classification of the study population due to response to infliximab therapy
Genotyping {#sec2-2}
----------
Genomic DNA from whole blood containing EDTA was extracted using standard techniques (NucleoSpin Blood kit, Macherey-Nagel, Germany). All polymerase chain reactions (PCRs) were run under conditions previously described \[[@ref23]\]. Primer sequences for the gene polymorphism at --308 were forward 5′-GGG ACA CAC AAG CAT CAA GG-3′ and reverse 5′-GGG ACA CAC AAG CAT CAA GG-3′, for the polymorphism at −238 forward 5′-ATC TGG AGG AAG CGG TAG TG-3′ and reverse 5′-AGA AGA CCC CCC TCG GAA CC-3′. The PCR products were digested at 37 °C with NcoI to detect the SNP in the −308 gene allele and MspI to detect the polymorphism of the −238 nucleotide. The -857 C/T polymorphism was analyzed by allele-specific PCR method24 using the primers TNF857-C: 5′-aag gat aag ggc tca gag ag-3′, TNF857-N: 5′-cta cat ggc cct gtc ttc g-3′ and TNF857-M: 5′-t cta cat ggc cct gtc ttc a-3′. The --158V/F polymorphism of FcγRΙΙΙA gene was detected as described by Leppers-van de Straat et al \[[@ref25]\] using the primers 5′-CTG AAG ACA CAT TTT TACT CC CAA (A/C)-3′ and 5′-TCC AAA AGC CAC ACT CAA AGA C-3′. The PCR products were then subjected to 3% agarose-gel electrophoresis. "No target" controls were included in each PCR batch to ensure that reagents had not been contaminated.
Statistical Analysis {#sec2-3}
--------------------
Genotype frequencies were compared with the chi-square with Yate's correction using S-Plus (v. 6.2Insightful, Seattle, WA). Odds ratios (ORs) and 95 confidence intervals (CIs) were obtained with GraphPad (v. 3.00, GraphPad Software, San Diego, CA). The p values are all two-sided. Correction for multiple testing was not applied in this study. *P* values of \< 0.05 were considered to be significant.
Results {#sec1-3}
=======
Patient demographic and clinical characteristics are given in [Table 3](#T3){ref-type="table"}. There were 68 (64.15%) complete responders, 25 (23.58%) partial responders and 13 (12.26%) non responders to IFX in this study. There were no statistical differences in the mean age, gender, disease duration, location and behavior and smoking habits between complete or partial responders and primary non-responders. There was no disagreement between HBI scores and serum CRP levels. Although, the post-treatment CRP levels were significantly lower in complete responders compared to partial and non-responders, the decrease in CRP levels did not differ significantly between the three groups. Post-treatment CRP levels and mean HBI score were significantly lower in complete responders compared to pre-treatment values in contrast to partial and/or non-responders where the CRP levels and the mean HBI score did not differ significantly.
######
Demographic, clinical and biological characteristics of the study population
The -238 G/A, -308 G/A, and -857 C/T polymorphisms of the TNF gene and the -158 V/F polymorphism in the *FcγRΙΙΙA* gene were successfully determined in all subjects. The genotype distribution in complete, partial and non-responders were presented in [Table 4](#T4){ref-type="table"}. No significant difference was observed for the polymorphism tested. In addition, although there may be genetic differences in early (paediatric)-onset and late (adult)-onset CD we were unable to detect any such differences although the number of paediatric patients included in the current study did not allow firm conclusions.
######
Genotype frequency in complete responders, partial responders and non responders
In the present study, we could not correlate the decrease in serum CRP levels with the genotypes tested in any particular group of patients since in most of the cases serum CRP levels dropped by more than 25% after 12 weeks of treatment. However, no significant decrease in CRP was observed between the TNF genotypes tested. Regarding the -158 V/F polymorphism in the *FcγRΙΙΙA* gene, the relative decrease in serum CRP levels was greatest in VV | introduction { # sec1 - 1 } = = = = = = = = = = = = infliximab ( ifx ), a chimeric anti - tnfα antibody, is effective in inducing and maintaining remission when causing considerable proportion of ibd patients refractory to any other treatments \ [ [ @ ref1 ], [ @ ref2 ] \ ]. however, 8 - 12 % of adult and / or pediatric patients fail to respond to the induction regimen ( known as primary non responders ) and approximately 40 % of patients who respond initially and achieve clinical remission inevitably lose response over time \ [ [ @ ref3 ], [ @ ref7 ] \ ]. lack of response to ifx is a negative trait and suggests that the differences in response might be in part genetically determined. considering the high cost and safety profile of this drug, genetic targeting of cells responding to this therapy is certainly of great interest \ [ [ @ ref8 ] \ ]. so far, limited candidate gene association studies with response to ifx have been reported \ [ [ @ ref9 ] - [ @ ref11 ] \ ]. recently, a genome - wide association study ( gwas ) regarding paediatric ibd patients has revealed that the 21q22. 2 / brwdi loci were associated with primary non response \ [ [ @ ref12 ] \ ]. furthermore, although tnfa gene is of great interest as a candidate gene for pharmacogenetic approaches few studies have been performed to date while some have led out contradictory results \ [ [ @ ref10 ], [ @ ref11 ], [ @ ref13 ] - [ @ ref15 ] \ ]. all anti - tnf agents share an igg1 fc fragment, but the contribution of the fc portion to the response to treatment among currently used tnf blockers remains unknown. receptors for igg - fc portion ( fcr ) are important regulatory molecules influencing inflammatory responses. fcr polymorphisms alter receptor function by enhancing or diminishing the affinity for immunoglobulins \ [ [ @ ref16 ] \ ]. three major classes of fcr that are capable of binding igg antibodies are recognised : fcγrι ( cd64 ), φ ( cd32 ), and fcγrιιι ( cd16 ). fcγrιι and fcγrιιι have multiple endings ( fcγ ##rιιιa / c and b ; fcγrιιιa and b ) \ [ [ @ ref16 ] \ ]. the most frequent polymorphism of * fcγrιιιa * is a point mutation affecting amino acids in codon 158 in the extracellular domain. this results in either a valine ( v158 ) or a phenylalanine ( f158 ) at this position. recently, it has been reported that cd patients with * fcγrιιιa * - 158v / v genotype had a better biological and possibly better clinical response to ifx \ [ [ @ ref17 ] \ ]. however, further studies did not confirm this observation \ [ [ @ ref18 ] \ ]. the aim of this study was to assess whether the * tnf * and / or * fcγrιιιa * gene polymorphisms are genetic predictors of response to ifx, in a cohort of greek patients with adult or paediatric onset of cd. patients - methods { # sec1 - 2 } = = = = = = = = = = = = = = = = = = patients { # sec2 - 1 } - - - - - - - - we enrolled 106 consecutive patients with newly diagnosed cd attending the outpatient ibd clinic at the 1 ^ st ^ department of gastroenterology, " evangelismos " hospital ( 79 adults ) or the 1 ^ st ^ department of pediatrics, university hospital of athens " aghia sophia " ( 27 children ). the diagnosis of cd was based on standard clinical, endoscopic, radiological, and histological criteria \ [ [ @ ref1 ], [ @ ref19 ] \ ]. eligible patients should have inflammatory ( luminal ) disease and be naive to ifx. ifx was administered intravenously at a dose of 5mg / kg at weeks 0, 2, 6 and then every 8 weeks. clinical and serological responses were assessed using the harvey - bradshaw index ( hbi ) \ [ [ @ ref20 ] \ ] and the serum levels of c - reactive protein ( crp ), respectively, at baseline ( before the 1st infusion of ifx ), the day before each subsequent ifx infusion and after 12 weeks of treatment. ileocolonoscopy was performed by a single endoscopist ( gjm ) at baseline and after 12 - 20 weeks of therapy to assess mucosal healing. any changes in endoscopic appearance compared to baseline endoscopy were classified in four categories \ [ [ @ ref21 ], [ @ ref22 ] \ ] \ [ [ table 1 ] ( # t1 ) { ref - type = " table " } \ ]. patients were classified in accordance to response to ifx therapy as shown in [ table 2 ] ( # t2 ) { ref - type = " table " }. the ethical committee of the participating hospitals approved the study. research was carried out according to helsinki convention ( 1975 ) and written inform consent was obtained in advance from each patient. # # # # # # grading of endoscopic mucosal lesions \ [ [ @ ref21 ], [ @ ref22 ] \ ]! [ ] ( anngastroenterol - 24 - 35 - g001 ) # # # # # # classification of the study population due to response to infliximab therapy! [ ] ( anngastroenterol - 24 - 35 - g002 ) genotyping { # sec2 - 2 } - - - - - - - - - - genomic dna from whole blood containing edta was extracted using standard techniques ( nucleospin blood kit, macherey - nagel, germany ). all polymerase chain reactions ( pcrs ) were run under conditions previously described \ [ [ @ ref23 ] \ ]. primer sequences for the gene polymorphism at - - 308 were forward 5 ′ - ggg aca cac aag cat caa gg - 3 ′ and reverse 5 ′ - ggg aca cac aag cat caa gg - 3 ′, for the polymorphism at −238 forward 5 ′ - atc tgg agg aag cgg tag tg - 3 ′ and reverse 5 ′ - aga aga ccc ccc tcg gaa cc - 3 ′. the pcr products were digested at 37 °c with ncoi to detect the snp in the −308 gene allele and mspi to detect the polymorphism of the −238 nucleotide. the - 857 c / t polymorphism was analyzed by allele - specific pcr method24 using the primers tnf857 - c : 5 ′ - aag gat aag ggc tca gag ag - 3 ′, tnf857 - n : 5 ′ - cta cat ggc cct gtc ttc g - 3 ′ and tnf857 - m : 5 ′ - t cta cat ggc cct gtc ttc a - 3 ′. the - - 158v / f polymorphism of fcγrιιιa gene was detected as described by leppers - van de straat et al \ [ [ @ ref25 ] \ ] using the primers 5 ′ - ctg aag aca cat ttt tact cc caa ( a / c ) - 3 ′ and 5 ′ - tcc aaa agc cac act caa aga c - 3 ′. the pcr products were then subjected to 3 % agarose - gel electrophoresis. " no target " controls were included in each pcr batch to ensure that reagents had not been contaminated. statistical analysis { # sec2 - 3 } - - - - - - - - - - - - - - - - - - - - genotype frequencies were compared with the chi - square with yate ' s correction using s - plus ( v. 6. 2insightful, seattle, wa ). odds ratios ( ors ) and 95 confidence intervals ( cis ) were obtained with graphpad ( v. 3. 00, graphpad software, san diego, ca ). the p values are all two - sided. correction for multiple testing was not applied in this study. * p * values of \ < 0. 05 were considered to be significant. results { # sec1 - 3 } = = = = = = = patient demographic and clinical characteristics are given in [ table 3 ] ( # t3 ) { ref - type = " table " }. there were 68 ( 64. 15 % ) complete responders, 25 ( 23. 58 % ) partial responders and 13 ( 12. 26 % ) non responders to ifx in this study. there were no statistical differences in the mean age, gender, disease duration, location and behavior and smoking habits between complete or partial responders and primary non - responders. there was no disagreement between hbi scores and serum crp levels. although, the post - treatment crp levels were significantly lower in complete responders compared to partial and non - responders, the decrease in crp levels did not differ significantly between the three groups. post - treatment crp levels and mean hbi score were significantly lower in complete responders compared to pre - treatment values in contrast to partial and / or non - responders where the crp levels and the mean hbi score did not differ significantly. # # # # # # demographic, clinical and biological characteristics of the study population! [ ] ( anngastroenterol - 24 - 35 - g003 ) the - 238 g / a, - 308 g / a, and - 857 c / t polymorphisms of the tnf gene and the - 158 v / f polymorphism in the * fcγrιιιa * gene were successfully determined in all subjects. the genotype distribution in complete, partial and non - responders were presented in [ table 4 ] ( # t4 ) { ref - type = " table " }. no significant difference was observed for the polymorphism tested. in addition, although there may be genetic differences in early ( paediatric ) - onset and late ( adult ) - onset cd we were unable to detect any such differences although the number of paediatric patients included in the current study did not allow firm conclusions. # # # # # # genotype frequency in complete responders, partial responders and non responders! [ ] ( anngastroenterol - 24 - 35 - g004 ) in the present study, we could not correlate the decrease in serum crp levels with the genotypes tested in any particular group of patients since in most of the cases serum crp levels dropped by more than 25 % after 12 weeks of treatment. however, no significant decrease in crp was observed between the tnf genotypes tested. regarding the - 158 v / f polymorphism in the * fcγrιιιa * gene, the relative decrease in serum crp levels was greatest in vv | Introduction {# sec1 - 1} = = = = = = = = = = = = Infliximab (IFX ), a chimeric anti - TNFα antibody, is effective in inducing and maintaining remission in a considerable proportion of IBD patients refractory to any other treatments \ [[ @ ref1 ], [@ ref2] \ ]. However, 8 - 12% of adult and / or pediatric patients fail to respond to the induction regimen (known as primary non responders) and approximately 40% of patients who respond initially and achieve cloniVal remission inevitably lose response over time \ [[ @ ref3 ], [@ ref7] \ ]. Lack of response to IFX is a stable trait and suggests that the differences in rewpinse might be in part genetically determined. Considering the high cost and safety profile of this drug, genetic targeting of patients responding to this therapy is certainly of great interest \ [[ @ ref8] \ ]. So far, limited candidate gene association studies with response to IFX have nesn reported \ [[ @ ref9] - [@ ref11] \ ]. Recently, a genome - wide association study (GWAS) in paediatric IBD patients has revealed that the 21q22. 2 / BRWDI loci were associated with primary non response \ [[ @ ref12] \ ]. FurtTerkore, although TNFa gene is of great interest as a candidate gene for pharmacogenetic approaches few studies have been performed to date and some have led to contradictory results \ [[ @ ref10 ], [@ ref11 ], [@ ref13] - [@ ref15] \ ]. All anti - TNF agents share an IgG1 Fc fragment, but the contribution of the Fc portion to the response to treatment among currently used TNF blockers remains unknown. Receptors for IgG - Fc portion (FcR) are important regulatory molecules of inflammatory responses. FcR polymorphisms alter receptor function by enhanc7hg or diminishing the affinity for immunoglobulins \ [[ @ ref16] \ ]. Three major classes of FcR that are capable of binding IgG antibodies are recognised: FcγRΙ (CD64 ), FcγRΙΙ (CD32 ), and FcγRΙΙΙ (CD16 ). FcγRΙΙ and FcγRΙΙΙ have multiple isoforms (FcγRΙΙΙA / C and B; FcγRΙΙΙA and B) \ [[ @ ref16] \ ]. The most frequent polymorphism of * FcγRΙΙΙA * is a point mutation affecting amino acids in codon 158 in the extracellular domain. This results in either a valine (V158) or a phenylalanine (F158) at this position. Recently, it has been reported that CD patients with * FcγRΙΙΙA * - 158V / V genotype had a better biological and possibly better clinical response to IFX \ [[ @ ref17] \ ]. Nowev3r, further studies did not confirm this observation \ [[ @ ref18] \ ]. The aim of this study was to assess whether the * TNF * and / or * FcγRΙΙΙA * gene polymorphisms are genetic predictors of response to IFX, in a cohort of Greek patients with adult or paediatric onset of CD. Patients - Methods {# sec1 - 2} = = = = = = = = = = = = = = = = = = Patients {# sec2 - 1} - - - - - - - - We enrolled 106 consecutive patients with newly diagnosed CD attending the outpatient IBD Clinic at the 1 ^ st ^ Department of Gastroenterology, " Evangelismos " Hospital (79 adults) or the 1 ^ st ^ Department of Pediatrics, University Hospital of Athens " Aghia Sophia " (27 children ). The diagnosis of CD was based on standard clinical, endoscopic, radiological, and histological criteria \ [[ @ ref1 ], [@ ref19] \ ]. Eligible patients should have inflammatory (luminal) disease and be naive to IFX. IFX was administered Lntrafenously at a dose of 5mg / kg at weeks 0, 2, 6 and then every 8 weeks. Clinical and serological responses were assessed using the Harvey - Bradshaw Index (HBI) \ [[ @ ref20] \] and the serum levels of C - reactive protein (CRP ), respectively, at baseline (before the 1st infusion of IFX ), the day before each subsequent IFX infusion and after 12 weeks of treatment. Ileocolonoscopy was performed by a single endoscopist (GJM) at baseline and after 12 - 20 weeks of therapy to assess mucosal healing. Any changes in endoscopic appearance compared to baseline endoscopy were classified in four categories \ [[ @ ref21 ], [@ ref22] \] \ [[ Table 1] (# T1) {ref - type = " table "} \ ]. Patients were classified in accordance to response to IFX therapy as shown in [table 2] (# T2) {ref - type = " table " }. The ethical committee of the participating hospitals approved the study. Research was carried out according to Helsinki Convention (1975) and written inform consent was obtained in advance from each patient. # # # # # # Grading of endoscopic mucosal lesions \ [[ @ ref21 ], [@ ref22] \ ]! [] (AnnGastroenterol - 24 - 35 - g001) # # # # # # Classification of the study population due to response to infliximab therapy! [] (AnnGastroenterol - 24 - 35 - g002) Genotyping {# sec2 - 2} - - - - - - - - - - Genomic DNA from whole blood containing EDTA was extracted using standArV techniques (NucleoSpin Blood kit, Macherey - Nagel, Germany ). All polymerase chain reactions (PCRs) were run under conditions previously described \ [[ @ ref23] \ ]. Primer sequences for the gene polymorphism at - - 308 were forward 5 ′ - GGG ACA CAC AAG CAT CAA GG - 3 ′ and reverse 5 ′ - GGG ACA CAC AAG CAT CAA GG - 3 ′, for the polymorphism at − 238 forward 5 ′ - ATC TGG AGG AAG CGG TAG TG - 3 ′ and reverse 5 ′ - AGA AGA CCC CCC TCG GAA CC - 3 ′. The PCR products were digested at 37 ° C with NcoI to detect the SNP in the − 308 gene allele and MspI to detect the polymorphism of the − 238 nucleotide. The - 857 C / T polymorphism was analyzed by allele - specific PCR method24 using the primers TNF857 - C: 5 ′ - aag gat aag ggc tca gag ag - 3 ′, TNF857 - N: 5 ′ - cta cat ggc cct gtc ttc g - 3 ′ and TNF857 - M: 5 ′ - t cta cat ggc cct gtc ttc a - 3 ′. The - - 158V / F polymorphism of FcγRΙΙΙA gene was detected as described by Leppers - van de Straat et al \ [[ @ ref25] \] using the primers 5 ′ - CTG AAG ACA CAT TTT TACT CC CAA (A / C) - 3 ′ and 5 ′ - TCC AAA AGC CAC ACT CAA AGA C - 3 ′. The PCR products were then subjected to 3% agarose - gel electrophoresis. " No target " controls were included in each PCR batch to ensure that reagents had not been contaminated. Statistical Analysis {# sec2 - 3} - - - - - - - - - - - - - - - - - - - - Genotype frequencies w4rR compared with the chi - square with Yate ' s correction using S - Plus (v. 6. 2Insightful, Seattle, WA ). Odds ratios (ORs) and 95 confidence intervals (CIs) were obtained with GraphPad (v. 3. 00, GraphPad Software, San Diego, CA ). The p values are all two - sided. Correction for multiple testing was not applied in this study. * P * values of \ <0. 05 were considered to be significant. Results {# sec1 - 3} = = = = = = = Patient demographic and clinical characteristics are given in [Table 3] (# T3) {ref - type = " table " }. There were 68 (64. 15%) complete responders, 25 (23. 58%) partial responders and 13 (12. 26%) non responders to IFX in this study. There were no statistical differences in the mean age, gender, disease duration, location and behavior and smoking habits between complete or partial responders and primary non - responders. There was no disagreement between HBI scores and serum CRP levels. Although, the post - %reatnent CRP levels were significantly lower in complete responders compared to partial and non - responders, the decrease in CRP levels did not differ significantly between the three groups. Post - treatment CRP levels and mean HBI score were significantly lower in complete responders compared to pre - treatment values in contrast to partial and / or non - responders where the CRP levels and the mean HBI score did not differ significantly. # # # # # # Demographic, clinical and biological characteristics of the study population! [] (AnnGastroenterol - 24 - 35 - g003) The - 238 G / A, - 308 G / A, and - 857 C / T polymorphisms of the TNF gene and the - 158 V / F polymorphism in the * FcγRΙΙΙA * gene were successfully determined in all subjects. The genotype distribution in complete, partial and non - responders were presented in [Table 4] (# T4) {ref - type = " table " }. No significant difference was observed for the polymorphism tested. In addition, although there may be genetic differences in early (paediatric) - onset and late (adult) - onset CD we were unable to detect any such differences although the number of paediatric patients included in the current study did not allow firm conclusions. # # # # # # Genotype frequency in complete responders, partial responders and non responders! [] (AnnGastroenterol - 24 - 35 - g004) In the present study, we could not correlate the decrease in serum CRP levels with the genotypes tested in any particular group of patients since in most of the cases serum CRP levels dropped by more than 25% after 12 weeks of treatment. However, no significant decrease in CRP was observed between the TNF genotypes tested. Regarding the - 158 V / F polymorphism in the * FcγRΙΙΙA * gene, the relative decrease in serum CRP levels was greatest in VV | Introduction {#sec1-1} ============ Infliximab (IFX), a chimeric anti-TNFα antibody, is effective in inducing maintaining remission in a proportion of patients refractory to any other However, 8-12% of adult and/or pediatric patients fail to to the regimen (known as primary non responders) and approximately 40% of patients who respond initially achieve clinical remission lose response over time\[[@ref3],[@ref7]\]. Lack of response to IFX is a stable trait and suggests the differences response might be in part genetically determined. Considering the high cost and safety of this drug, genetic targeting of responding to this therapy is of great interest \[[@ref8]\]. So far, limited candidate gene association studies with IFX have been reported \[[@ref9]-[@ref11]\]. Recently, genome-wide association study (GWAS) in paediatric IBD patients has revealed the 21q22.2/BRWDI loci associated with primary non response \[[@ref12]\]. Furthermore, although TNFa gene is of great interest as a gene for pharmacogenetic approaches few have been performed to date and some have led contradictory results \[[@ref10],[@ref11],[@ref13]-[@ref15]\]. All anti-TNF agents share IgG1 Fc fragment, but the contribution of the Fc portion to the response to treatment among currently used TNF blockers remains unknown. Receptors for portion (FcR) are important regulatory molecules of inflammatory responses. FcR polymorphisms alter receptor function by enhancing or diminishing the for immunoglobulins \[[@ref16]\]. major classes of FcR that are capable of binding antibodies are recognised: FcγRΙ (CD64), FcγRΙΙ (CD32), and FcγRΙΙΙ (CD16). FcγRΙΙ and FcγRΙΙΙ have multiple (FcγRΙΙΙA/C and B; FcγRΙΙΙA and B) \[[@ref16]\]. The most frequent polymorphism of *FcγRΙΙΙA* is a point mutation affecting amino acids in codon 158 in the extracellular domain. This results in either a valine (V158) or a phenylalanine (F158) at this position. Recently, it has been reported that CD with *FcγRΙΙΙA* -158V/V genotype had biological and possibly better clinical response to IFX \[[@ref17]\]. However, further studies did confirm this observation \[[@ref18]\]. The aim of study was to assess whether the *TNF* and/ or *FcγRΙΙΙA* gene polymorphisms are predictors of response to IFX, a cohort of Greek patients with adult or paediatric onset of CD. Patients - Methods {#sec1-2} ================== Patients {#sec2-1} -------- enrolled 106 consecutive patients with newly diagnosed CD attending the outpatient IBD Clinic at 1^st^ Department of Gastroenterology, Hospital (79 adults) or the Department of Pediatrics, University Hospital of Athens "Aghia Sophia"(27 children). The diagnosis CD was based on standard endoscopic, radiological, and criteria \[[@ref1],[@ref19]\]. should have inflammatory (luminal) disease and naive to IFX. IFX was administered intravenously at a 5mg/kg at weeks 0, 2, 6 and then every 8 weeks. Clinical and serological responses were assessed using the Harvey-Bradshaw Index (HBI) \[[@ref20]\] and the serum levels C-reactive protein (CRP), respectively, at baseline (before the 1st infusion of IFX), day before each subsequent IFX infusion and after weeks of treatment. Ileocolonoscopy was performed by a (GJM) at baseline and after 12-20 therapy to assess mucosal healing. Any changes in endoscopic appearance compared to baseline endoscopy were classified in four categories \[[@ref21],[@ref22]\] \[[Table 1](#T1){ref-type="table"}\]. Patients were classified in accordance to response to IFX therapy as shown in [table The ethical committee of the participating hospitals approved the study. Research was carried out according to Helsinki Convention (1975) and written inform consent was obtained in advance from each patient. ###### Grading mucosal lesions \[[@ref21],[@ref22]\]  ###### Classification of the population to response to infliximab therapy Genotyping {#sec2-2} ---------- from whole blood containing EDTA was extracted using standard techniques (NucleoSpin kit, Macherey-Nagel, All polymerase reactions (PCRs) run under conditions previously described \[[@ref23]\]. Primer sequences for the gene polymorphism at --308 were forward 5′-GGG ACA CAC AAG CAT CAA GG-3′ and reverse 5′-GGG ACA CAC AAG CAT CAA GG-3′, for the polymorphism at −238 forward 5′-ATC TGG AGG AAG CGG TAG and reverse 5′-AGA AGA CCC CCC TCG GAA CC-3′. The PCR products were digested at 37 °C with NcoI to detect SNP in the −308 gene allele and MspI detect the polymorphism of the −238 nucleotide. The -857 polymorphism was analyzed by allele-specific PCR method24 using the primers TNF857-C: 5′-aag gat aag ggc tca gag ag-3′, TNF857-N: 5′-cta cat cct gtc ttc g-3′ TNF857-M: 5′-t cta cat ggc cct gtc ttc a-3′. The --158V/F polymorphism of FcγRΙΙΙA gene was detected described by Leppers-van de Straat al \[[@ref25]\] the 5′-CTG AAG ACA CAT TTT TACT CC CAA (A/C)-3′ and 5′-TCC AAA AGC CAC AGA C-3′. The PCR products were then subjected to 3% agarose-gel electrophoresis. "No target" controls included in each PCR batch to ensure that had not been contaminated. Analysis {#sec2-3} -------------------- frequencies were compared with the chi-square with Yate's S-Plus (v. 6.2Insightful, Seattle, WA). Odds ratios (ORs) and 95 confidence intervals (CIs) were obtained with GraphPad (v. 3.00, GraphPad Software, Diego, CA). The values are all two-sided. Correction for multiple testing was applied in this study. *P* values of 0.05 were considered to be significant. Results {#sec1-3} ======= Patient demographic and clinical characteristics are given [Table 3](#T3){ref-type="table"}. were 68 (64.15%) responders, 25 (23.58%) responders 13 (12.26%) non responders to IFX in this study. There were no statistical differences in the mean age, gender, disease duration, location behavior and smoking habits between complete or partial responders and primary non-responders. There was no between HBI scores and serum CRP levels. Although, the post-treatment levels were significantly lower complete compared to partial and the decrease in CRP levels did not differ significantly between the three Post-treatment CRP levels and mean HBI were significantly lower in responders compared to pre-treatment values in to partial and/or non-responders where the CRP levels and the mean HBI score did not differ significantly. ###### clinical and biological characteristics of the study population  The -238 G/A, -308 G/A, and -857 C/T polymorphisms the TNF gene and the -158 in the *FcγRΙΙΙA* gene successfully determined in all subjects. The genotype distribution in complete, partial and non-responders were presented in [Table 4](#T4){ref-type="table"}. No significant difference was observed for the polymorphism tested. addition, although there may be genetic differences in early (paediatric)-onset and late (adult)-onset CD we were unable detect any such differences the number of paediatric patients included in the current study did not allow firm conclusions. ###### Genotype frequency in responders, partial responders and non responders  In the present study, we could not correlate decrease in serum CRP levels with genotypes tested in any particular group of patients in most of the cases serum CRP levels dropped by more than 25% after 12 weeks of treatment. no significant decrease in CRP was observed between the TNF genotypes tested. Regarding the -158 V/F polymorphism in *FcγRΙΙΙA* gene, the relative decrease in serum levels greatest in | iNtroDucTIoN {#SEC1-1}
============
infLixIMAb (iFX), A cHiMeric anTI-tNFα antiboDY, Is EFFEctiVe In InduCiNg And mAINTaInInG reMiSsioN in a coNsiDERABle propoRTioN Of IbD patIEnts ReFRAcTOry To aNy OTHER TReaTMENTS \[[@reF1],[@REf2]\]. hoWEVeR, 8-12% Of AduLT and/OR peDIaTrIc patienTs Fail tO reSPOnD tO tHE iNDuctION RegimeN (known as PrimARY NOn REspOndErS) AND ApPROXiMatELy 40% oF pATieNts WHO ResPOnd inITiAlLy aNd acHiEvE CLiniCAl REmIssIOn iNEViTABlY LOse reSPOnse oVEr tIME\[[@ref3],[@REF7]\]. lAcK Of ReSpONSe TO IFX Is a sTabLe trAIt AND suGGEStS THat tHe dIFFERENCes In RESPONSE MiGHt BE In PaRT GeNETicaLly DeterMINEd. CoNSidERIng ThE HIgh COST anD SAFEty PRoFilE Of tHis dRUG, GeNeTIc TaRGetinG oF PAtiEntS RESpONDing to THIS thERapY IS ceRtAINLy Of gReaT iNtErEst \[[@REf8]\]. SO far, LimITEd CaNdIdAtE gENE assoCIatION STUdiES With ReSpoNSE to IFX HavE BeEn rePorted \[[@ref9]-[@reF11]\]. RECeNtly, A GEnome-wiDe ASsoCiaTIoN STudy (gWAS) In pAeDiAtRiC ibd pAtiENtS HAS rEvEALeD ThAT the 21Q22.2/brwDi lOCI WeRe AssoCiAtED with PriMArY NoN RESPONSe \[[@rEf12]\]. FURtheRMorE, aLThOUGh tNFa gENe iS OF grEAT INteREsT as A canDIDaTE genE fOr PHaRMACOGEnETiC aPPRoaChEs feW sTudiES hAve been peRforMed To DAtE and SOmE HAve lEd to CoNtRADIctory rEsULts \[[@Ref10],[@Ref11],[@rEF13]-[@ref15]\].
ALl ANTI-tNf AgENTS ShArE AN iGg1 FC FRaGMeNT, But The CONTRibuTiOn OF tHe fc POrtIoN to THE RESPoNSe To tReatMent AmONG CURrENtlY UsEd TnF BLoCKERS REmaiNS UNkNoWN. REcEpToRS foR Igg-Fc PoRtioN (FCr) ARE iMpoRTaNT ReGuLAtORy mOleCULes oF InflaMmAtORY ReSPOnses. fCr polyMorPhIsmS Alter ReCePtor fUnCtIoN by EnhAncInG OR DIminisHING THE AffINIty FoR IMMuNOglobUliNS \[[@Ref16]\]. THREE MajOr cLASsES OF FCR tHaT are capABle of bINdINg IGg anTiBoDiEs ARE recOgNIsED: FcγrΙ (cd64), fCΓRΙΙ (CD32), aNd fCΓrιιι (CD16). FCγrΙι anD FCγRιιι HaVe MuLtiple isOfOrms (fcΓRιΙΙA/C aNd B; fcγRΙΙιA and b) \[[@rEF16]\]. tHe mOsT FRequenT poLYMoRpHIsm oF *FCγrιΙιa* Is A pOiNt MUTatioN affectING amiNo aCIDS IN cOdON 158 in ThE EXTraCElLULAr domAIn. THis RESultS In eiTHer A vAlIne (V158) OR a PhEnYLAlaNINe (F158) At tHIs POsition. RECEntLY, iT HAs beEN RePorTED THAt cD PatieNTS wITH *FCγRιΙιA* -158V/V gENOtYPe HaD a bEtTER BioloGIcAl ANd posSIbLY BettER CLiNiCal reSPonSE tO ifx \[[@REF17]\]. HOwEVeR, FurthEr sTUDiES diD not CONFiRm thiS obserVatioN \[[@reF18]\].
thE AIM oF this Study WAs To ASSeSs whETHER thE *TnF* And/ Or *fcγRιιΙA* Gene POLYmOrpHisMs aRE GenETic pREdiCTORs OF RESPonSe tO IfX, IN a CohoRt OF grEEK PaTiEntS wITh adULT oR PAEDiAtRIc onset Of cD.
PATIEnTS - mETHOds {#SEC1-2}
==================
PatIENts {#Sec2-1}
--------
we enrolled 106 coNsecutivE PATiENTs WitH neWLy diAgNoSeD cd aTtEndiNG the oUTPatIEnT ibD CliNIC AT ThE 1^st^ DEPaRtmEnt Of gAstROenterOlOgy, "evAngELIsmoS" hoSPITAl (79 ADuLTS) oR tHe 1^ST^ dEPARTment of PediATRIcS, UNiversIty HOSpitAL oF aTHeNs "AgHIa SOPhIA"(27 chIldreN). The DiaGNoSIS oF cD WaS basED On STANdArd cliNiCaL, EndoSCOpic, rAdIOlOGIcAL, AnD hISTOlOgIcaL CRitERia \[[@rEF1],[@ReF19]\]. eLiGiBlE PATIeNTS sHOulD hAvE InFLaMMaTOry (lumiNal) diseAsE and BE nAiVe tO ifx.
Ifx was ADMINISTEREd INTrAVenOuSly At A doSE Of 5mg/Kg aT weeKs 0, 2, 6 AND theN EVErY 8 weEks. cLINicAl aND serOLOgIcAL rESpOnSES werE ASSEsSed uSing THE hArveY-BRaDsHAW iNdEx (HBI) \[[@ref20]\] AnD tHe sErUm LeVELs OF c-reactIve pRoTein (cRP), RESpECtiVelY, at baSeLIne (before The 1ST InfUsIon oF Ifx), THe DAy beFoRE EAch SubsEQuenT ifX infUSION AND afTer 12 weEKs Of tREatMenT. IleOcOloNoSCOpY wAS pERFOrMed By A SinGLE EnDOsCOPIsT (GjM) at BaseLiNE And AFteR 12-20 WeEkS OF ThErApY To AssEss MucosAL HeaLINg. aNY chANgeS iN ENdOScoPIC ApPEaraNcE CoMparED to BaSElIne EnDoSCOpY WERe cLAsSIfiED In FOuR CAtEGoRIES \[[@REF21],[@reF22]\] \[[tAbLE 1](#T1){ReF-typE="TaBLE"}\]. PatiENTS WeRe cLAssIFIeD In ACCordancE to reSpOnsE tO IFx tHerAPY AS SHOWn in [TablE 2](#T2){ref-tyPe="TablE"}. ThE eThIcAL COMmiTTeE oF ThE PaRTIcIpATING hOspitals apprOveD THE sTUdy. rESEarch WaS carRieD Out acCorDInG To HelSiNKI CoNveNTiOn (1975) aND written InFORm ConSeNT WAS ObTaINED in ADvAncE FrOM EaCH PAtIENT.
######
gRaDIng Of ENdOscOPIC mucoSAL leSIOns \[[@REF21],[@rEF22]\]
######
CLASsIFIcatIOn oF ThE stUdy popULatiOn Due To respoNSe tO iNfLixImaB thErAPY
GEnotYPiNG {#sec2-2}
----------
GeNoMic DnA FRom whOlE BLood cONTaIniNG Edta wAs extrAcTeD uSINg STaNDard TeCHniQUeS (nucleosPIN BLOOd KiT, macHeREy-nagEL, gErmany). alL poLYMeRASE chAiN rEAcTIONS (PCRS) werE rUn unDer conDiTions PrEvIoUSLy descrIBEd \[[@ref23]\]. PrImeR sEQUenCes FOR THe GENe POlymOrPHISM At --308 wErE FORwARD 5′-Ggg aca CAC aAG CaT CaA GG-3′ and rEVErSE 5′-gGG ACa caC aaG cAt CaA Gg-3′, foR ThE PoLyMORPhISm aT −238 fOrward 5′-ATC TgG agG aag cGG TAG Tg-3′ aND REVERSE 5′-Aga AgA Ccc ccc tCg gaa Cc-3′. THe Pcr PRoduCTs were DIgeStED AT 37 °c WIth nCoI tO DEtECt THE sNp In the −308 GENE AlLELE and mspI to deTECT the POlyMorpHisM OF tHE −238 NUCLeoTide. tHe -857 C/T POlymOrphIsM was AnalYZeD BY ALLElE-SPeCIfic PCR metHod24 UsiNG THe primers TnF857-C: 5′-Aag Gat AAg GGC tcA GAg ag-3′, Tnf857-n: 5′-cTA cAt ggc ccT gtC ttc g-3′ AND TNf857-m: 5′-T CTa caT GGc CcT GtC TtC a-3′. tHe --158V/F POlymOrPhISm oF FCγRΙΙιa GenE waS DEtEcTeD aS DeSCrIbed BY LEpPeRs-VaN DE STRaaT eT al \[[@ref25]\] USinG tHE prIMeRS 5′-ctg Aag ACA cat TtT taCt cC cAa (A/c)-3′ aNd 5′-Tcc AaA Agc cac acT caa agA C-3′. tHe PcR pRODucTs wERe THEn SUBJEcted To 3% AgAROSe-gEL ELectROPHOReSIS. "No TaRGet" ContRolS werE incLUdeD IN EaCh PCr BatCh to ENSURe tHAT rEAGenTS hAD NOT beEN ConTAmINatEd.
STaTistICal AnALYsIS {#SeC2-3}
--------------------
GEnOType FREqUEnCieS werE cOMpAreD wiTH tHe cHi-squARe WiTh YaTE'S cOrrEcTIOn usIng S-PlUS (v. 6.2InsiGHTFuL, SeaTTLE, wA). OddS ratIOs (orS) And 95 cONfidEnCe inTERvaLS (cIs) WEre ObTAineD WItH GrAPhpAD (v. 3.00, GRAphpAD sOfTwaRE, sAN DIEgo, CA). the p vAlUes ArE All tWo-sIded. cORREctiOn FOr mUltiPlE tEsTINg WAs nOT APPlied IN THiS STUdY. *P* ValUeS Of \< 0.05 WerE CoNsiDERed tO bE SIGNIfiCaNT.
ReSUltS {#SEc1-3}
=======
patiENt DEmOGRaphiC AND clInICal ChArACteRIStIcS arE GiveN iN [tABLe 3](#t3){Ref-TyPE="tabLe"}. there WeRe 68 (64.15%) comPLete ReSPonDerS, 25 (23.58%) pARTiaL reSpOndERs AND 13 (12.26%) nOn rESpOnDERS To ifX in tHIS StuDy. ThEre wERe No StaTiSTICal difFerENCeS In THE meAn aGe, GenDeR, DiSEAsE durAtiON, loCAtion and BehaVior aND SMoKiNg HABits BETwEEn CoMPLeTe Or pARtIAL RespONdERS AnD PrImarY NON-rESPONdeRS. tHere WaS no dIsAgReEMEnT bETwEEN HBI SCOreS aNd serUm crp LEVElS. AlthOuGh, tHE POSt-tREatMEnt Crp LeveLS wERE SIGnIficaNtly LoWeR in coMpLEte respOnders cOMParEd To PArtIal anD non-ReSpondERs, ThE DEcReASE IN Crp lEVeLS did NoT diFfER SiGniFIcanTLy BetWeen the THReE gROupS. pOst-tREATmENT cRP LeVels AND mEAN Hbi ScORe wEre SiGniFiCaNTLY LoWeR in cOMPLEtE RESPonDErS comPaRed TO PRe-TREAtmeNt vAlues iN cONTraSt tO paRTiaL aND/OR Non-ReSPOndErS WHeRE the cRp LEvELS And thE meAn Hbi ScoRe DID NoT Differ SiGNificaNtly.
######
DEmoGRapHIC, CLINIcaL AND BiolOGiCAL cHARACTErIstICS Of thE StUdY POPUlATIOn
tHE -238 g/A, -308 g/A, aNd -857 C/t poLyMorPhiSMS of tHe tNf GEnE And THE -158 v/F poLYmoRPhIsm IN The *fCΓRΙΙιa* gENe weRe sUCCesSFUlly DEtERminEd iN ALl suBJECTS. THe geNOtypE DiStrIbutIon in complETe, paRtIAL aNd Non-REspOndErs wErE prEsENTed IN [taBLE 4](#T4){Ref-TYpe="TABLE"}. no sIGnIficAnT DiFfEreNCe waS oBSErved foR THE pOlYMORpHISm TEstEd. In ADditION, ALTHoUgh tHERE may bE geneTic diFFerENCeS in EArly (paeDiatRIc)-ONsEt And LaTe (adUlT)-oNset cd wE WeRe unaBLE to deTecT aNY sUch DifFErENcEs AltHOugh ThE NumbeR Of PaEDiatRIC pAtieNtS iNcLUDeD IN tHe CUrrenT stUdy dID NOt allOw firm CoNcLUSIonS.
######
GENOtYpe freqUENcy In cOmplETE REspoNdERs, PartIal reSpOndERS aND NOn ReSPONDeRS
in the pRESENt sTUdy, WE COuld NOt coRReLaTe the DEcrEAsE IN SeRuM CrP levElS With THe genotYpes tEsTEd in AnY pArTiculAR Group Of pATIENTs sinCE IN MOST OF tHe Cases SerUm CRP lEvEls dropPeD By moRe ThaN 25% AfTER 12 weeKs OF TrEatMent. hOWeveR, nO siGNIfiCanT DeCrEase iN crP was obSeRVed BetWEeN THE TNf gENotyPes TeSTED. RegarDInG THe -158 v/f pOlymorPhIsm In the *FcγrΙιΙA* gene, THE rElative deCrEAsE in Serum cRp LEVels WAs GREaTesT in VV | Introduction {#sec1-1}============ Infliximab (IFX), a chimeric anti-TNFα antibody, is effective in inducing and maintaining remission in a considerable proportion of IBD patients refractory to any other treatments \[[@ref1],[@ref2]\]. However, 8-12% of adult and/or pediatric patients failto respond to the induction regimen (known as primary non responders) and approximately 40% of patients who respond initially and achieve clinical remission inevitably lose response over time\[[@ref3],[@ref7]\]. Lackof response to IFXis a stable trait andsuggests that the differences in response might be in part genetically determined. Considering the high cost and safety profile of this drug, genetic targeting of patients responding to this therapy is certainly ofgreat interest \[[@ref8]\]. So far, limited candidate gene association studies with response to IFXhavebeen reported \[[@ref9]-[@ref11]\].Recently, a genome-wide association study(GWAS) in paediatric IBD patientshas revealed that the 21q22.2/BRWDI loci were associated with primary non response \[[@ref12]\]. Furthermore, althoughTNFagene is of great interest as a candidate gene for pharmacogenetic approaches few studies have been performed to date and some have led to contradictory results \[[@ref10],[@ref11],[@ref13]-[@ref15]\]. All anti-TNF agentsshare an IgG1 Fc fragment, but the contribution of theFc portion to the response to treatment among currently used TNF blockers remains unknown.Receptors for IgG-Fc portion (FcR) are important regulatory molecules of inflammatoryresponses. FcRpolymorphisms alter receptor function by enhancingor diminishing the affinity forimmunoglobulins \[[@ref16]\].Three major classes ofFcR that are capable of bindingIgG antibodies are recognised: FcγRΙ (CD64), FcγRΙΙ(CD32), andFcγRΙΙΙ (CD16).FcγRΙΙ and FcγRΙΙΙ have multiple isoforms (FcγRΙΙΙA/C and B; FcγRΙΙΙA and B) \[[@ref16]\]. Themost frequent polymorphism of *FcγRΙΙΙA* is a point mutation affecting amino acids in codon 158in the extracellular domain. This resultsineither a valine (V158) or a phenylalanine (F158) at this position. Recently, it has been reported thatCD patients with *FcγRΙΙΙA* -158V/V genotype hada better biological and possibly better clinical response to IFX \[[@ref17]\]. However, further studies did not confirm this observation \[[@ref18]\]. The aim of this study was to assess whether the *TNF*and/ or *FcγRΙΙΙA* gene polymorphisms are genetic predictors of responsetoIFX, in a cohort ofGreek patients with adult or paediatric onset of CD. Patients - Methods {#sec1-2} ================== Patients {#sec2-1}-------- We enrolled 106consecutive patients with newly diagnosed CD attending the outpatient IBD Clinic at the 1^st^ Department of Gastroenterology,"Evangelismos" Hospital (79 adults) or the 1^st^ Department of Pediatrics, University Hospital of Athens "Aghia Sophia"(27 children). The diagnosisof CD was based on standard clinical, endoscopic, radiological, and histological criteria \[[@ref1],[@ref19]\]. Eligible patients should have inflammatory (luminal) disease and be naive to IFX.IFX was administeredintravenously at a dose of 5mg/kg at weeks0, 2,6 and then every 8 weeks. Clinical and serological responseswere assessed using the Harvey-Bradshaw Index (HBI) \[[@ref20]\] and the serum levels of C-reactive protein (CRP),respectively, at baseline (before the 1st infusion of IFX), the day before each subsequentIFX infusion and after12 weeks oftreatment. Ileocolonoscopy was performed by a single endoscopist (GJM) atbaseline and after 12-20 weeks of therapy to assess mucosal healing. Any changes in endoscopic appearance compared to baseline endoscopy were classified in four categories\[[@ref21],[@ref22]\] \[[Table1](#T1){ref-type="table"}\]. Patients were classified in accordance to response to IFX therapyas shownin [table2](#T2){ref-type="table"}. The ethical committee of the participating hospitals approved the study. Researchwas carried out according to Helsinki Convention (1975) and written inform consent was obtained in advance from each patient. ######Gradingof endoscopic mucosal lesions \[[@ref21],[@ref22]\]  ###### Classification ofthe study population due to response to infliximab therapy  Genotyping {#sec2-2} ---------- Genomic DNA from whole blood containing EDTAwasextracted using standard techniques (NucleoSpin Bloodkit,Macherey-Nagel, Germany). All polymerase chain reactions (PCRs) were run under conditions previously described \[[@ref23]\]. Primer sequences for the gene polymorphismat --308 were forward 5′-GGG ACA CAC AAG CAT CAA GG-3′ and reverse 5′-GGGACACACAAG CAT CAA GG-3′, for the polymorphismat −238 forward 5′-ATCTGG AGG AAG CGG TAG TG-3′ and reverse5′-AGA AGA CCC CCC TCG GAA CC-3′.The PCR products were digested at 37 °C with NcoI to detect the SNP in the−308 gene allele and MspI to detectthe polymorphism of the−238 nucleotide. The -857 C/T polymorphism was analyzed by allele-specific PCR method24 using the primers TNF857-C: 5′-aag gat aag ggc tca gag ag-3′, TNF857-N: 5′-cta cat ggc cct gtc ttc g-3′ and TNF857-M:5′-t cta cat ggc cct gtc ttc a-3′. The --158V/F polymorphism of FcγRΙΙΙA genewas detected as described byLeppers-van de Straatetal \[[@ref25]\] using theprimers 5′-CTGAAG ACA CAT TTT TACT CC CAA (A/C)-3′ and5′-TCCAAA AGC CAC ACT CAAAGA C-3′. The PCR products were then subjected to3% agarose-gel electrophoresis. "No target" controls were included in each PCR batch toensure that reagents had notbeen contaminated. Statistical Analysis{#sec2-3} -------------------- Genotype frequencies were compared with the chi-square with Yate's correction using S-Plus (v. 6.2Insightful, Seattle, WA).Odds ratios(ORs)and 95 confidence intervals (CIs) were obtained with GraphPad (v.3.00, GraphPad Software, SanDiego, CA). The p values are all two-sided. Correction for multiple testing wasnotapplied in this study. *P* values of \< 0.05 were considered to be significant. Results {#sec1-3} ======= Patient demographic and clinical characteristics aregiven in [Table 3](#T3){ref-type="table"}. There were 68 (64.15%) complete responders,25 (23.58%) partial responders and 13 (12.26%) non responders to IFX in this study. Therewere no statisticaldifferences in themean age, gender, disease duration,location and behaviorandsmoking habits between complete or partial responders and primary non-responders.There was no disagreement between HBI scores and serum CRP levels. Although, the post-treatment CRP levels were significantlylower in complete responders compared to partial and non-responders, the decrease in CRP levels did not differ significantly between the threegroups. Post-treatment CRP levels and mean HBI score were significantlylower in complete responders compared to pre-treatment values in contrast to partial and/or non-responders where theCRP levels and the mean HBIscore did not differ significantly. ###### Demographic, clinical and biological characteristics of the study population The-238G/A, -308 G/A, and -857 C/T polymorphisms of the TNF gene and the -158 V/Fpolymorphism in the *FcγRΙΙΙA* gene weresuccessfully determined in all subjects. The genotype distribution in complete, partial and non-responders were presented in [Table 4](#T4){ref-type="table"}. Nosignificant difference was observedforthepolymorphism tested. In addition, although there maybe genetic differences in early (paediatric)-onset and late (adult)-onset CD we were unable to detect anysuch differences although the number of paediatric patients included in the current study did not allow firm conclusions. ###### Genotype frequency in complete responders, partial responders and non responders  In the present study, wecould not correlate thedecrease in serum CRP levelswith the genotypes testedin anyparticular group of patients sincein most of the cases serum CRP levels dropped by more than 25% after 12 weeks of treatment. However,no significant decrease inCRP was observed between the TNF genotypestested. Regarding the -158 V/F polymorphism in the *FcγRΙΙΙA* gene, the relative decrease in serumCRP levels wasgreatest in VV | _Introduction_ {#sec1-1} ============ _Infliximab_ _(IFX),_ _a_ chimeric anti-TNFα antibody, is _effective_ in inducing and _maintaining_ remission _in_ _a_ considerable proportion of IBD _patients_ refractory to any other treatments \[[@ref1],[@ref2]\]. However, 8-12% of adult and/or pediatric patients fail _to_ respond to the induction regimen (known as primary _non_ responders) and approximately _40%_ of patients _who_ respond initially and achieve clinical remission inevitably lose _response_ _over_ time\[[@ref3],[@ref7]\]. Lack of response to IFX _is_ a stable trait _and_ suggests that the _differences_ in response might be in part _genetically_ _determined._ Considering _the_ high cost and safety profile of this drug, _genetic_ targeting _of_ patients responding _to_ this therapy is _certainly_ of great interest \[[@ref8]\]. So _far,_ limited candidate gene association studies with response to IFX _have_ _been_ reported \[[@ref9]-[@ref11]\]. Recently, _a_ _genome-wide_ association study (GWAS) in paediatric _IBD_ patients has revealed that the 21q22.2/BRWDI loci were associated with primary _non_ response \[[@ref12]\]. Furthermore, although TNFa gene _is_ of great interest as a candidate gene for pharmacogenetic _approaches_ few _studies_ have been performed _to_ date and some have _led_ _to_ contradictory results \[[@ref10],[@ref11],[@ref13]-[@ref15]\]. All anti-TNF agents share an IgG1 Fc _fragment,_ but the contribution of the Fc portion _to_ the response to treatment among currently used TNF blockers remains unknown. Receptors for IgG-Fc _portion_ (FcR) _are_ _important_ regulatory molecules of inflammatory responses. FcR polymorphisms _alter_ receptor function by enhancing or diminishing the affinity for immunoglobulins \[[@ref16]\]. Three major classes of FcR _that_ are capable of _binding_ IgG antibodies _are_ recognised: FcγRΙ (CD64), FcγRΙΙ (CD32), and FcγRΙΙΙ _(CD16)._ FcγRΙΙ and FcγRΙΙΙ _have_ multiple isoforms (FcγRΙΙΙA/C and B; FcγRΙΙΙA and B) \[[@ref16]\]. The most frequent polymorphism of *FcγRΙΙΙA* is _a_ point mutation affecting amino _acids_ in codon 158 _in_ the extracellular _domain._ This results _in_ _either_ a valine (V158) or a phenylalanine (F158) _at_ this position. Recently, it _has_ been reported that CD patients with *FcγRΙΙΙA* -158V/V _genotype_ had a better biological and _possibly_ better clinical response to _IFX_ \[[@ref17]\]. However, _further_ _studies_ did not confirm this observation \[[@ref18]\]. The aim of this _study_ was to assess _whether_ _the_ *TNF* _and/_ or _*FcγRΙΙΙA*_ gene polymorphisms are genetic predictors of _response_ _to_ IFX, in a cohort of Greek _patients_ with adult or paediatric onset of CD. Patients - Methods _{#sec1-2}_ ================== Patients {#sec2-1} _--------_ We enrolled 106 consecutive _patients_ with newly _diagnosed_ CD attending the outpatient IBD Clinic _at_ the 1^st^ Department of Gastroenterology, "Evangelismos" Hospital (79 _adults)_ or the 1^st^ Department of Pediatrics, University Hospital of Athens _"Aghia_ _Sophia"(27_ children). _The_ diagnosis of _CD_ was based on standard clinical, _endoscopic,_ radiological, and histological _criteria_ _\[[@ref1],[@ref19]\]._ Eligible patients should have _inflammatory_ (luminal) _disease_ and be _naive_ to IFX. IFX was administered intravenously _at_ a dose _of_ 5mg/kg at weeks _0,_ _2,_ _6_ and then every _8_ weeks. Clinical and serological responses _were_ _assessed_ _using_ the Harvey-Bradshaw Index _(HBI)_ \[[@ref20]\] and the serum levels of C-reactive protein (CRP), respectively, at baseline (before the 1st infusion of IFX), the day before each subsequent IFX infusion and after 12 weeks of treatment. Ileocolonoscopy was performed by a single _endoscopist_ (GJM) at baseline and _after_ 12-20 weeks of therapy to assess mucosal _healing._ Any _changes_ in endoscopic appearance compared to baseline endoscopy were classified _in_ four categories \[[@ref21],[@ref22]\] \[[Table 1](#T1){ref-type="table"}\]. Patients were classified in accordance to response to IFX therapy as _shown_ in [table 2](#T2){ref-type="table"}. The ethical _committee_ of _the_ participating hospitals _approved_ the study. Research was carried out according _to_ Helsinki _Convention_ (1975) and written _inform_ _consent_ _was_ _obtained_ in advance from each patient. ###### Grading of endoscopic mucosal lesions _\[[@ref21],[@ref22]\]_  ###### Classification of the study population _due_ _to_ response to infliximab therapy  Genotyping {#sec2-2} ---------- Genomic DNA from whole blood containing _EDTA_ was extracted using standard techniques _(NucleoSpin_ _Blood_ kit, Macherey-Nagel, Germany). All polymerase chain reactions (PCRs) were run under conditions previously _described_ \[[@ref23]\]. Primer _sequences_ for the gene polymorphism at --308 _were_ forward 5′-GGG ACA CAC AAG CAT CAA GG-3′ and reverse 5′-GGG ACA CAC AAG CAT CAA GG-3′, for the polymorphism _at_ −238 forward 5′-ATC TGG AGG AAG CGG _TAG_ TG-3′ and reverse 5′-AGA _AGA_ CCC CCC TCG GAA CC-3′. The PCR _products_ _were_ digested _at_ _37_ °C with NcoI _to_ detect the SNP _in_ the −308 _gene_ allele and _MspI_ _to_ detect the polymorphism of the _−238_ nucleotide. The _-857_ C/T polymorphism was analyzed by _allele-specific_ _PCR_ _method24_ _using_ the primers TNF857-C: 5′-aag gat _aag_ ggc _tca_ gag _ag-3′,_ TNF857-N: 5′-cta cat _ggc_ _cct_ _gtc_ ttc g-3′ and TNF857-M: 5′-t cta cat ggc cct gtc ttc _a-3′._ The --158V/F polymorphism _of_ FcγRΙΙΙA gene was detected as _described_ by Leppers-van de _Straat_ _et_ _al_ \[[@ref25]\] using _the_ primers 5′-CTG AAG ACA _CAT_ _TTT_ TACT CC CAA (A/C)-3′ _and_ 5′-TCC AAA AGC CAC ACT CAA AGA _C-3′._ The PCR products were _then_ subjected to 3% agarose-gel electrophoresis. "No _target"_ controls were included in _each_ PCR batch to ensure that reagents had not been contaminated. Statistical Analysis {#sec2-3} -------------------- Genotype _frequencies_ _were_ compared with the _chi-square_ with Yate's correction using _S-Plus_ (v. 6.2Insightful, Seattle, WA). Odds ratios (ORs) _and_ 95 confidence intervals (CIs) were obtained _with_ GraphPad (v. 3.00, _GraphPad_ Software, San Diego, CA). The p values _are_ all two-sided. Correction for multiple testing was _not_ applied _in_ this study. *P* values of \< 0.05 _were_ considered to be significant. _Results_ {#sec1-3} _=======_ Patient demographic and _clinical_ characteristics are _given_ _in_ _[Table_ _3](#T3){ref-type="table"}._ There were _68_ _(64.15%)_ _complete_ responders, 25 (23.58%) partial responders _and_ _13_ (12.26%) non responders _to_ _IFX_ in this study. There were _no_ _statistical_ differences in the mean age, gender, disease _duration,_ location and _behavior_ and smoking habits between complete _or_ partial _responders_ and primary non-responders. There was no disagreement between HBI scores and serum CRP _levels._ _Although,_ the post-treatment CRP levels were significantly lower in complete responders _compared_ to _partial_ _and_ non-responders, the decrease in _CRP_ levels did not differ significantly between the three groups. Post-treatment CRP levels and mean HBI _score_ were significantly lower in _complete_ responders compared to _pre-treatment_ values in contrast to partial and/or non-responders _where_ the CRP _levels_ and _the_ mean HBI score did not differ _significantly._ ###### Demographic, _clinical_ and _biological_ characteristics _of_ the _study_ population  The _-238_ G/A, _-308_ G/A, and -857 C/T polymorphisms _of_ the TNF gene _and_ the -158 V/F _polymorphism_ in the *FcγRΙΙΙA* gene were successfully determined in all subjects. The genotype distribution in _complete,_ partial and non-responders were _presented_ in _[Table_ 4](#T4){ref-type="table"}. No significant difference was observed for the polymorphism tested. _In_ _addition,_ _although_ _there_ may be genetic differences in early _(paediatric)-onset_ _and_ late (adult)-onset CD we were _unable_ to detect any such differences _although_ the number of _paediatric_ patients included in the _current_ study did not allow firm conclusions. ###### _Genotype_ frequency in complete _responders,_ partial _responders_ and non _responders_  In the present study, we could not correlate the decrease in serum CRP levels with the _genotypes_ tested in any particular group of patients since in most of the cases _serum_ CRP levels dropped _by_ more than 25% _after_ 12 weeks _of_ treatment. However, no _significant_ decrease in CRP was _observed_ between _the_ TNF genotypes tested. Regarding the -158 _V/F_ polymorphism in _the_ _*FcγRΙΙΙA*_ _gene,_ the _relative_ _decrease_ in serum _CRP_ levels was greatest in VV |
INTRODUCTION {#s1}
============
Hepatitis B virus (HBV) is still a major global health problem, with an estimated 257 million people worldwide that are chronically infected with HBV ([@B1]). HBV, together with duck hepatitis B virus (DHBV) and several other related animal viruses, belongs to the *Hepadnaviridae* family ([@B2]). The HBV virion is comprised of an outer envelope and an inner icosahedral nucleocapsid (NC) assembled by 240 copies of core protein (HBc) and packaged with a 3.2-kb partially double-stranded circular DNA genome ([@B3][@B4][@B8]). In addition to DNA-containing virions, a large amount of incomplete viral particles, such as hepatitis B surface antigen (HBsAg) particles, empty virions, and naked capsids, can also be released from cells in the process of virus replication ([@B9]). Subviral HBsAg particles are spherical or rodlike and are present in vast excess over virions in sera of CHB patients ([@B2]). Empty virions share the same structure as DNA-containing virions but are devoid of nucleic acids ([@B10][@B11][@B14]). Naked capsids, which exit cells via a route different from that of virions ([@B15][@B16][@B17]), have the same structure as NCs but are either empty or filled with viral RNA and immature viral DNA ([@B7], [@B11], [@B18][@B19][@B20]).
In NC, pgRNA undergoes reverse transcription into minus-strand DNA, followed by plus-strand DNA synthesis ([@B2], [@B21][@B22][@B24]). Intracellular NCs can be packaged with viral nucleic acids at all levels of maturation, including pgRNA, nascent minus-strand DNA, minus-strand DNA-RNA hybrids, and relaxed circular DNA (RC DNA) or double-stranded linear DNA (DSL DNA) ([@B5], [@B7]). Only the NCs with relatively mature viral DNA (RC or DSL DNA) are enveloped and secreted as virions. HBV replicating cells can release empty core particles assembled from HBc proteins and NCs that contain various species of replicative intermediate nucleic acids into the culture supernatant. However, while free naked capsids could be readily detected *in vitro* ([@B7], [@B11], [@B18][@B19][@B20]), they are hardly found in the blood of HBV-infected patients ([@B17], [@B25], [@B26]).
Although extracellular HBV RNA was detected in both *in vitro* cell culture systems and in clinical serum samples, its origin and composition remain controversial. It was proposed that extracellular HBV RNA represents pgRNA localized in virions ([@B27]). However, HBV spliced RNA and HBx RNA were also detected in culture supernatant of HBV stably replicating cells as well as in sera of CHB patients ([@B28], [@B29]). In addition, extracellular HBV RNA was also suggested to originate from damaged liver cells ([@B30]), naked capsids, or exosomes ([@B11], [@B29]). Hence, these extracellular RNA molecules have never been conclusively characterized. Here, we demonstrate that extracellular HBV RNAs are heterogeneous in length, ranging from full-length pgRNA (3.5 kilonucleotides \[knt\]) to RNA fragments with merely several hundred nucleotides. These RNA molecules represent 3′ receding pgRNA fragments that have not been completely reverse transcribed to DNA and pgRNA fragments hydrolyzed by the RNase H domain of polymerase in the process of viral replication. More importantly, extracellular HBV RNAs are localized in naked capsids and in virions in culture supernatants of HBV replicating cells and also circulate as CACs and virions in blood of hepatitis B patients.
RESULTS {#s2}
=======
Extracellular HBV RNAs are heterogeneous in length and predominantly integral to naked capsids instead of virions in HepAD38 cell culture supernatant. {#s2.1}
------------------------------------------------------------------------------------------------------------------------------------------------------
To ascertain the origin of extracellular HBV RNA, we first examined viral particles prepared from culture medium of an *in vitro* HBV stably transduced cell line. A human hepatoma HepAD38 cell line was used in this study, as it sustains vigorous HBV replication under the control of a tetracycline-repressible cytomegalovirus (CMV) promoter ([@B31]). Total viral particles were concentrated and centrifuged over a 10% to 60% (wt/wt) sucrose gradient. Most of the subviral HBsAg particles, virions, and empty virions were detected between fractions 9 to 14 ([Fig. 1A](#F1){ref-type="fig"}, upper and middle). Naked capsids, detected only by anti-HBcAg and not by anti-HBsAg antibodies, settled in fractions 5 to 8 ([Fig. 1A](#F1){ref-type="fig"}, middle and lower). The majority of viral nucleic acids were detected in fractions between 4 and 11 ([Fig. 1B](#F1){ref-type="fig"}, upper), which coincided with the fractions containing virions (fractions 9 to 11), naked capsids (fractions 4 to 7), and the mixture of these particles (fraction 8). Consistent with previous observations, HBV virions are packed with mature viral DNA (RC or DSL DNA), while naked capsids contain both immature single-stranded DNA (SS DNA) and mature viral DNA ([Fig. 1B](#F1){ref-type="fig"}, upper). Moreover, Northern blot results showed that most of the HBV RNA was detected in the naked capsids ([Fig. 1B](#F1){ref-type="fig"}, lower, fractions 4 to 7), whereas only a very small amount was associated with virions ([Fig. 1B](#F1){ref-type="fig"}, lower, fractions 9 to 11). HBV RNA detected in naked capsids ranged from the full length of pgRNA down to a few hundred nucleotides (shorter than the HBx mRNA \[0.7 knt\]). Moreover, RNA molecules within virions were much shorter than those within naked capsids. We excluded the possibility of artifacts generated by the SDS-proteinase K extraction method, as a similar RNA blot pattern was obtained using a TRIzol reagent to extract both intracellular nucleocapsid-associated and extracellular HBV RNA (not shown). Furthermore, quantification of viral RNA extracted by either the SDS-proteinase K method or TRIzol reagent produced a very similar copy number, except that the TRIzol reagent is known to preferentially extract RNA rather than DNA (not shown). Moreover, the RNA signal detected by Northern blotting could not be attributed to DNA fragments generated by DNase I treatment, which would reduce DNA to below the detection limit of the hybridization method (not shown). Furthermore, the RNA signal could be completely removed by an additional RNase A treatment (not shown).
{#F1}
To confirm the above-described results and to better separate naked capsids from HBV virions, isopycnic CsCl gradient ultracentrifugation was employed. Naked capsids were observed mainly in fractions 5 to 7, with densities ranging from 1.33 to 1.34 g/cm^3^ ([Fig. 2A](#F2){ref-type="fig"}). The smearing bands of naked capsids were likely caused by high concentrations of CsCl salt, as fractionation of naked capsids in a 1.18-g/cm^3^ CsCl solution produced single bands. Virions, detected by both anti-HBcAg and anti-HBsAg antibodies ([Fig. 2A](#F2){ref-type="fig"}, upper and middle), were packaged with viral DNA ([Fig. 2A](#F2){ref-type="fig"}, lower) and settled in fractions 13 to 15, with densities ranging from 1.23 to 1.25 g/cm^3^. In agreement with the results shown in [Fig. 1](#F1){ref-type="fig"}, | introduction { # s1 } = = = = = = = = = = = = hepatitis b virus ( hbv ) is still a major animal health problem, with an estimated 257 million people worldwide that are chronically infected with hbv ( [ @ b1 ] ). hbv, together like duck hepatitis b virus ( bc ) and several other related animal viruses, belongs to the * hepadnaviridae * family ( [ @ b2 ] ). the hbv virion is comprised of an outer nucleus and an inner icosahedral nucleocapsid ( nc ) assembled by 240 copies of core protein ( hbc ) and packaged with a 3. 2 - kb partially double - stranded circular dna genome ( [ @ b3 ] [ @ b4 ] [ @ b8 ] ). in addition to dna - containing bacteria, a large amount of incomplete viral particles, identified as hepatitis b surface antigen ( hbsag ) particles, empty virions, and naked capsids, can periodically be released from cells in the process of virus replication ( [ @ 1b ] ). subviral hbsag particles are spherical or rodlike and are present in vast excess over virions in sera of chb patients ( [ @ b2 ] ). empty virions share the same structure as dna - containing virions but are devoid of nucleic acids ( [ @ b10 ] [ @ b11 ] [ @ b14 ] ). naked capsids, which exit cells via a route different from that of virions ( [ @ b15 ] [ @ b16 ] [ @ b17 ] ), have the same structure as ncs but are either empty or filled with viral vectors and immature viral dna ( [ @ b7 ], [ @ b11 ], [ @ b18 ] [ @ b19 ] [ @ b20 ] ). in nc, pgrna undergoes reverse transcription into minus - strand dna, followed by plus - strand dna synthesis ( [ @ b2 ], [ @ b21 ] [ @ b22 ] [ @ b24 ] ). intracellular ncs can be packaged with viral nucleic acids at all levels of maturation, using pgrna, nascent minus - strand dna, minus - strand dna - rna hybrids, and relaxed circular dna ( rc dna ) or double - stranded linear dna ( dsl dna ) ( [ @ b5 ], [ @ b7 ] ). only the ncs with relatively mature viral dna ( rc or dsl dna ) are enveloped and secreted as virions. hbv replicating cells can release empty core particles assembled from hbc proteins and ncs that contain various species of replicative intermediate nucleic acids into the culture supernatant. however, while free naked capsids could be readily detected * in vitro * ( [ @ b7 ], [ @ b11 ], [ @ b18 ] [ @ b19 ] [ @ b20 ] ), they are hardly found in the blood of hbv - infected patients ( [ @ b17 ], [ @ b25 ], [ @ b26 ] ). although extracellular hbv rna was detected in both * in vitro * cell culture systems and in clinical serum samples, its origin and composition remain controversial. it was proposed that extracellular hbv rna represents pgrna localized in virions ( [ @ b27 ] ). however, hbv spliced rna and hbx rna were also detected in culture supernatant of hbv stably replicating cells as well as in sera of chb patients ( [ @ b28 ], [ @ b29 ] ). in addition, extracellular hbv rna was also suggested to originate from damaged liver cells ( [ @ b30 ] ), naked capsids, or exosomes ( [ @ b11 ], [ @ b29 ] ). hence, these extracellular rna molecules have never been conclusively characterized. here, we demonstrate that extracellular hbv rnas are heterogeneous in length, ranging from full - length pgrna ( 3. 5 kilonucleotides \ [ knt \ ] ) to rna fragments with merely several hundred nucleotides. these rna molecules represent 3 ′ receding pgrna fragments that have not been completely reverse transcribed to dna and pgrna fragments hydrolyzed by the rnase h domain of polymerase in the process of viral replication. more importantly, extracellular hbv rnas are localized in naked capsids and in virions in culture supernatants of hbv replicating cells and also circulate as cacs and virions in blood of hepatitis b patients. results { # s2 } = = = = = = = extracellular hbv rnas are heterogeneous in length and predominantly integral to naked capsids instead of virions in hepad38 cell culture supernatant. { # s2. 1 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - to ascertain the origin of extracellular hbv rna, we first examined viral particles prepared from culture medium of an * in vitro * hbv stably transduced cell line. a human hepatoma hepad38 cell line was used in this study, as it sustains vigorous hbv replication under the control of a tetracycline - repressible cytomegalovirus ( cmv ) promoter ( [ @ b31 ] ). total viral particles were concentrated and centrifuged over a 10 % to 60 % ( wt / wt ) sucrose gradient. most of the subviral hbsag particles, virions, and empty virions were detected between fractions 9 to 14 ( [ fig. 1a ] ( # f1 ) { ref - type = " fig " }, upper and middle ). naked capsids, detected only by anti - hbcag and not by anti - hbsag antibodies, settled in fractions 5 to 8 ( [ fig. 1a ] ( # f1 ) { ref - type = " fig " }, middle and lower ). the majority of viral nucleic acids were detected in fractions between 4 and 11 ( [ fig. 1b ] ( # f1 ) { ref - type = " fig " }, upper ), which coincided with the fractions containing virions ( fractions 9 to 11 ), naked capsids ( fractions 4 to 7 ), and the mixture of these particles ( fraction 8 ). consistent with previous observations, hbv virions are packed with mature viral dna ( rc or dsl dna ), while naked capsids contain both immature single - stranded dna ( ss dna ) and mature viral dna ( [ fig. 1b ] ( # f1 ) { ref - type = " fig " }, upper ). moreover, northern blot results showed that most of the hbv rna was detected in the naked capsids ( [ fig. 1b ] ( # f1 ) { ref - type = " fig " }, lower, fractions 4 to 7 ), whereas only a very small amount was associated with virions ( [ fig. 1b ] ( # f1 ) { ref - type = " fig " }, lower, fractions 9 to 11 ). hbv rna detected in naked capsids ranged from the full length of pgrna down to a few hundred nucleotides ( shorter than the hbx mrna \ [ 0. 7 knt \ ] ). moreover, rna molecules within virions were much shorter than those within naked capsids. we excluded the possibility of artifacts generated by the sds - proteinase k extraction method, as a similar rna blot pattern was obtained using a trizol reagent to extract both intracellular nucleocapsid - associated and extracellular hbv rna ( not shown ). furthermore, quantification of viral rna extracted by either the sds - proteinase k method or trizol reagent produced a very similar copy number, except that the trizol reagent is known to preferentially extract rna rather than dna ( not shown ). moreover, the rna signal detected by northern blotting could not be attributed to dna fragments generated by dnase i treatment, which would reduce dna to below the detection limit of the hybridization method ( not shown ). furthermore, the rna signal could be completely removed by an additional rnase a treatment ( not shown ).! [ sucrose gradient separation and analysis of viral particles from hepad38 cell culture supernatant. ( a ) distribution of hepatitis b viral particle - associated antigens and dna / rna in sucrose gradient. viral particles prepared from hepad38 cell culture supernatant ( via peg 8000 precipitation ) were layered over a 10 % to 60 % ( wt / wt ) sucrose gradient for ultracentrifugation separation. fractions were collected from top to bottom, and hbsag level was analyzed by enzyme - linked immunosorbent assay ( elisa ). hbsag and viral dna and rna ( quantified from gray density of bands in panel b ) signals and sucrose density were plotted together. viral particles were first resolved by native agarose gel electrophoresis, followed by immunoblotting ( ib ) of hbv envelope and core proteins with anti - hbsag and anti - hbcag antibodies. ( b ) detection of viral dna / rna by southern or northern blotting. total viral nucleic acids were extracted by the sds - proteinase k method, and viral dna ( extracted from one - tenth of the samples used for northern blotting ) and rna ( treated with dnase i ) were detected by southern and northern blot analyses with minus - or plus - strand - specific riboprobes, respectively. symbols of hbsag particles, empty virions ( without nucleic acid ), virions ( with rc dna ), and naked capsids ( empty or with nucleic acids ) are depicted on the lower right side of panel a. blank, no nucleic acids ; two centered and gapped circles, rc dna ; straight line, ss dna ; wavy lines, pgrna ; m, markers ( 50 pg of 1 - kb, 2 - kb, and 3. 2 - kb dna fragments released from plasmids as the dna ladder or total rna extracted from hepad38 cells as the rna ladder ). ] ( zjv0241840640001 ) { # f1 } to confirm the above - described results and to better separate naked capsids from hbv virions, isopycnic cscl gradient ultracentrifugation was employed. naked capsids were observed mainly in fractions 5 to 7, with densities ranging from 1. 33 to 1. 34 g / cm ^ 3 ^ ( [ fig. 2a ] ( # f2 ) { ref - type = " fig " } ). the smearing bands of naked capsids were likely caused by high concentrations of cscl salt, as fractionation of naked capsids in a 1. 18 - g / cm ^ 3 ^ cscl solution produced single bands. virions, detected by both anti - hbcag and anti - hbsag antibodies ( [ fig. 2a ] ( # f2 ) { ref - type = " fig " }, upper and middle ), were packaged with viral dna ( [ fig. 2a ] ( # f2 ) { ref - type = " fig " }, lower ) and settled in fractions 13 to 15, with densities ranging from 1. 23 to 1. 25 g / cm ^ 3 ^. in agreement with the results shown in [ fig. 1 ] ( # f1 ) { ref - type = " fig " }, | INTRODUCTION {# s1} = = = = = = = = = = = = Hepatitis B virus (HBV) is still a major global health problem, with an estimated 257 million people worldwide that are chronically infected with HBV ([ @ B1] ). HBV, together with duck hepatitis B virus (DHBV) and several other related animal viruses, belongs to the * Hepadnaviridae * family ([ @ B2] ). The HBV virion is comprised of an outer envelope and an inner icosahedral nucleocapsid (NC) assembled by 240 copies of core protein (HBc) and packaged with a 3. 2 - kb partially double - stranded circular DNA genome ([ @ B3] [@ B4] [@ B8] ). In addition to DNA - containing virions, a large amount of incomplete viral particles, such as hepatitis B surface antigen (HBsAg) particles, empty virions, and naked capsids, can also be released from cells in the process of virus replication ([ @ B9] ). Subviral HBsAg particles are spherical or rodlike and are present in vast excess over virions in sera of CHB patients ([ @ B2] ). Empty virions share the same structure as DNA - containing virions but are devoid of nucleic acids ([ @ B10] [@ B11] [@ B14] ). Naked capsids, which exit cells via a route different from that of virions ([ @ B15] [@ B16] [@ B17] ), have the same structure as NCs but are either empty or filled with viral RNA and immature viral DNA ([ @ B7 ], [@ B11 ], [@ B18] [@ B19] [@ B20] ). In NC, pgRNA undergoes reverse transcription into minus - strand DNA, followed by plus - strand DNA synthesis ([ @ B2 ], [@ B21] [@ B22] [@ B24] ). Intracellular NCs can be packaged with viral nucleic acids at all levels of maturation, including pgRNA, nascent minus - strand DNA, minus - strand DNA - RNA hybrids, and relaxed circular DNA (RC DNA) or double - stranded linear DNA (DSL DNA) ([ @ B5 ], [@ B7] ). Only the NCs with relatively Natkre viral DNA (RC or DSL DNA) are enveloped and secreted as virions. HBV replicating cells can release empty core particles assembled from HBc proteins and NCs that contain various species of replicative intermediate nucleic acids into the culture supernatant. However, while frrs naked cXpsiCs could be readily detected * in vitro * ([ @ B7 ], [@ B11 ], [@ B18] [@ B19] [@ B20] ), they are hardly found in the blood of HBV - infected patients ([ @ B17 ], [@ B25 ], [@ B26] ). Although extracellular HBV RNA was detected in both * in vitro * cell culture systems and in clinical serum samples, its origin and composition remain controversial. It was proposed that extracellular HBV RNA represents pgRNA localized in virions ([ @ B27] ). However, HBV spliced RNA and HBx RNA were also detected in culture supernatant of HBV stably replicating cells as well as in sera of CHB patients ([ @ B28 ], [@ B29] ). In addition, extracellular HBV RNA was also suggested to originate from damaged liver cells ([ @ B30] ), naked capsids, or exosomes ([ @ B11 ], [@ B29] ). Hence, these extracellular RNA molecules have never been conclusively characterized. Here, we demonstrate that extracellular HBV RNAs are heterogeneous in length, ranging from full - length pgRNA (3. 5 kilonucleotides \ [knt \] ) to RNA fragments with merely several hundred nucleotides. These RNA molecules represent 3 ′ receding pgRNA fragments that have not been completely reverse transcribed to DNA and pgRNA fragments hydrolyzed by the RNase H domain of polymerase in the process of viral replication. More importantly, extracellular HBV RNAs are localized in naked capsids and in virions in c tlturS supernatants of HBV replicating cells and also circulate as CACs and virions in blood of hepatitis B patients. RESULTS {# s2} = = = = = = = Extracellular HBV RNAs are heterogeneous in length and predominantly integral to naked capsids instead of virions in HepAD38 cell culture supernatant. {# s2. 1} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - To ascertain the origin of extracellular HBV RNA, we first examined viral particles prepared from culture medium of an * in vitro * HBV stably transduced cell line. A human hepatoma HepAD38 cell line was used in this study, as it sustains vigorous HBV replication under the control of a tetracycline - repressible cytomegalovirus (CMV) promoter ([ @ B31] ). Total viral particles were concentrated and centrifuged over a 10% to 60% (wt / wt) sucrose gradient. Most of the subviral HBsAg particles, virions, and empty virions were detected between fractions 9 to 14 ([ Fig. 1A] (# F1) {ref - type = " fig " }, upper and ,idfle ). Naked capsids, detected only by anti - HBcAg and not by anti - HBsAg antibodies, settled in fractions 5 to 8 ([ Fig. 1A] (# F1) {ref - type = " fig " }, middle and lower ). The majority of viral nucleic acids were detected in fractions between 4 and 11 ([ Fig. 1B] (# F1) {ref - type = " fig " }, upper ), which coincided with the fractions containing virions (fractions 9 to 11 ), naked capsids (fractions 4 to 7 ), and the mixture of these particles (fraction 8 ). Consistent with previous observations, HBV virions are packed with mature viral DNA (RC or DSL DNA ), while naked capsids contain both immature single - stranded DNA (SS DNA) and mature viral DNA ([ Fig. 1B] (# F1) {ref - type = " fig " }, upper ). Moreover, Northern blot results showed that most of the HBV RNA was detected in the naked cxpDids ([ Fig. 1B] (# F1) {ref - type = " fig " }, lower, fractions 4 to 7 ), whereas only a very small amount was associated with virions ([ Fig. 1B] (# F1) {ref - type = " fig " }, lower, fractions 9 to 11 ). HBV RNA detected in naked capsids ranged rGom the full length of pgRNA down to a few hundred nucleotides (shorter than the HBx mRNA \ [0. 7 knt \] ). Moreover, RNA molecules within virions were much shorter than those within naked capsids. We excluded the possibility of artifacts generated by the SDS - proteinase K extraction method, as a similar RNA blot pattern was obtained using a TRIzol reagent to extract both intracellular nucleocapsid - associated and extracellular HBV RNA (not shown ). Furthermore, quantification of viral RNA extracted by either the SDS - proteinase K method or TRIzol reagent produced a very similar copy number, except that the TRIzol reagent is known to preferentially extract RNA rather than DNA (not shown ). Moreover, the RNA signal detected by Northern blotting could not be attributed to DNA fragments generated by DNase I treatment, which would reduce DNA to below the detection limit of the hybridizatOkn method (not shown ). Furthermore, the RNA signal could be completely removed by an additional RNase A treatment (not shown ). ! [Sucrose gradient separation and analysis of viral particles from HepAD38 cell culture supernatant. (A) Distribution of hepatitis B viral particle - associated antigens and DNA / RNA in sucrose gradient. Viral particles prepared from HepAD38 cell culture supernatant (via PEG 8000 precipitation) were layered over a 10% to 60% (wt / wt) sucrose gradient for ultracentrifugation separation. Fractions were collected from top to bottom, and HBsAg level was analyzed by enzyme - linked immunosorbent assay (ELISA ). HBsAg and viral DNA and RNA (quantified from gray density of bands in panel B) signals and sucrose density were plotted together. Viral particles were first resolved by native agarose gel electrophoresis, followed by immunoblotting (IB) of HBV envelope and core proteins with anti - HBsAg and anti - HBcAg antibodies. (B) Detection of viral DNA / RNA by Southern or Northern blotting. Total viral nucleic acids were extracted by the SDS - proteinase K method, and viral DNA (extracted from one - tenth of the samples used for Northern blotting) and RNA (treated with DNase I) were detected by Southern and Northern blot analyses with minus - or plus - strand - specific riboprobes, respectively. Symbols of HBsAg particles, empty virions (without nucleic acid ), virions (with RC DNA ), and naked capsids (empty or with nucleic acids) are depicted on the lower right side of panel A. Blank, no nucleic acids; two centered and gapped circles, RC DNA; straight line, SS DNA; wavy lines, pgRNA; M, markers (50 pg of 1 - kb, 2 - kb, and 3. 2 - kb DNA fragments released from plasmids as the DNA ladder or total RNA extracted from HepAD38 cells as the RNA ladder ).] (zjv0241840640001) {# F1} To confirm the above - described results and to better separate naked capsids from HBV virions, isopycnic CsCl gradient ultracentrifugation was employed. Naked capsids were observed mainly in fractions 5 to 7, with densities ranging from 1. 33 to 1. 34 g / cm ^ 3 ^ ([ Fig. 2A] (# F2) {ref - type = " fig "} ). The smearing bands of naked capsids were likeiT caused by high concentrations of CsCl salt, as fractionation of naked capsids in a 1. 18 - g / cm ^ 3 ^ CsCl solution produced single bands. Virions, detected by both anti - HBcAg and anti - HBsAg antibodies ([ Fig. 2A] (# F2) {ref - t6pD = " fig " }, upper and middle ), were packaged with viral DNA ([ Fig. 2A] (# F2) {ref - type = " fig " }, lower) and settled in fractions 13 to 15, with densities ranging from 1. 23 to 1. 25 g / cm ^ 3 ^. In agreement with the results shown in [Fig. 1] (# F1) {ref - type = " fig " }, | INTRODUCTION {#s1} ============ B (HBV) is still a major global health problem, an estimated 257 million people worldwide that are chronically infected with HBV ([@B1]). HBV, together with duck hepatitis B virus (DHBV) and several other animal viruses, belongs to the *Hepadnaviridae* ([@B2]). The HBV virion is comprised an outer envelope and an inner icosahedral nucleocapsid (NC) assembled by 240 of core protein (HBc) with a 3.2-kb partially double-stranded circular DNA genome ([@B3][@B4][@B8]). In addition to DNA-containing virions, a amount of incomplete viral particles, such as hepatitis B surface antigen (HBsAg) particles, virions, and capsids, can also be released cells in process replication ([@B9]). Subviral HBsAg are spherical or rodlike and are present in vast excess over virions in sera of CHB patients ([@B2]). Empty virions share the same structure as DNA-containing virions are devoid of nucleic acids ([@B10][@B11][@B14]). Naked which exit cells via a route different from that of ([@B15][@B16][@B17]), have same structure as NCs but are either empty or filled with viral and DNA ([@B7], [@B18][@B19][@B20]). NC, pgRNA reverse transcription into minus-strand DNA, followed by plus-strand DNA synthesis ([@B2], [@B21][@B22][@B24]). Intracellular can be packaged with viral nucleic acids at all levels including pgRNA, nascent minus-strand DNA, minus-strand DNA-RNA hybrids, and relaxed circular DNA (RC DNA) or double-stranded linear DNA (DSL DNA) ([@B5], Only NCs with relatively mature viral DNA (RC or DSL DNA) are and secreted as virions. HBV replicating cells can release empty core particles assembled from HBc proteins and NCs contain various species of replicative intermediate nucleic acids into the culture supernatant. However, while free naked capsids could be readily detected *in vitro* ([@B7], [@B11], [@B18][@B19][@B20]), they are hardly found in the blood of HBV-infected patients ([@B17], [@B25], [@B26]). Although extracellular HBV RNA was detected in both *in vitro* cell culture systems and in clinical serum samples, its origin and composition remain controversial. It was proposed extracellular RNA represents pgRNA localized in virions ([@B27]). However, HBV spliced RNA and HBx RNA were also detected in culture supernatant of HBV stably replicating as well as in sera of CHB patients ([@B28], [@B29]). In addition, extracellular HBV was also suggested to originate from damaged liver ([@B30]), naked capsids, or exosomes ([@B11], [@B29]). Hence, these extracellular RNA molecules have never been conclusively characterized. Here, we that extracellular HBV RNAs are heterogeneous in length, ranging from full-length pgRNA (3.5 kilonucleotides \[knt\]) to RNA fragments with merely several hundred nucleotides. These molecules represent 3′ receding pgRNA fragments that have not been reverse transcribed to DNA and pgRNA fragments hydrolyzed by the RNase H of polymerase in the process viral replication. More importantly, extracellular HBV RNAs are localized naked capsids in virions in culture supernatants of HBV cells and also circulate as CACs and virions in blood of hepatitis RESULTS ======= Extracellular HBV are heterogeneous in length and predominantly integral to naked capsids instead of virions in HepAD38 cell culture supernatant. {#s2.1} ------------------------------------------------------------------------------------------------------------------------------------------------------ To ascertain the of extracellular HBV RNA, we first examined viral particles prepared from culture medium of an *in vitro* HBV stably transduced A human hepatoma HepAD38 cell line was used in this study, as it sustains vigorous HBV replication under the control a tetracycline-repressible cytomegalovirus (CMV) promoter ([@B31]). Total viral were concentrated and centrifuged a 10% 60% (wt/wt) sucrose gradient. Most the HBsAg virions, and empty virions detected between fractions 9 14 ([Fig. 1A](#F1){ref-type="fig"}, upper and middle). Naked only by anti-HBcAg and not by anti-HBsAg antibodies, settled in fractions 5 to 8 ([Fig. 1A](#F1){ref-type="fig"}, middle and lower). The of viral nucleic acids were detected in fractions between 4 and 11 1B](#F1){ref-type="fig"}, upper), coincided with the fractions containing virions (fractions 9 to 11), capsids (fractions 4 7), and the mixture of these particles (fraction 8). Consistent with previous observations, HBV virions are packed with mature DNA (RC DSL DNA), while naked capsids contain both immature single-stranded (SS DNA) and mature viral DNA ([Fig. 1B](#F1){ref-type="fig"}, upper). Moreover, Northern blot results showed that most of the HBV RNA was detected in the naked capsids 1B](#F1){ref-type="fig"}, lower, fractions 4 to 7), only a very small amount was associated with virions ([Fig. 1B](#F1){ref-type="fig"}, lower, fractions 9 to 11). HBV RNA detected capsids ranged from the length of down a few hundred nucleotides (shorter than the HBx mRNA \[0.7 knt\]). RNA molecules within virions were much shorter than those within naked capsids. We excluded the possibility of artifacts generated by the SDS-proteinase K method, as a similar RNA blot pattern was obtained using a to extract both intracellular nucleocapsid-associated and extracellular HBV RNA shown). Furthermore, quantification of viral RNA extracted by the SDS-proteinase K method or TRIzol reagent produced very similar copy number, except that the reagent is known preferentially extract RNA rather than DNA (not shown). Moreover, the RNA signal detected by Northern blotting could not attributed to DNA fragments generated by DNase I treatment, which would reduce DNA to below the detection limit of the hybridization method (not shown). Furthermore, the RNA signal could be completely removed by an additional RNase treatment (not shown). {#F1} confirm above-described results and to better separate naked capsids from virions, isopycnic CsCl gradient ultracentrifugation was employed. Naked were mainly in fractions 5 7, with densities ranging from 1.33 to 1.34 g/cm^3^ ([Fig. 2A](#F2){ref-type="fig"}). The bands of naked capsids were likely caused by high concentrations of CsCl salt, as fractionation of naked in 1.18-g/cm^3^ CsCl solution produced single bands. Virions, detected by anti-HBcAg and anti-HBsAg antibodies ([Fig. 2A](#F2){ref-type="fig"}, upper and middle), were packaged with viral ([Fig. 2A](#F2){ref-type="fig"}, lower) and settled in fractions 13 to 15, with densities ranging from to 1.25 g/cm^3^. In agreement the results shown in [Fig. 1](#F1){ref-type="fig"}, | INtRodUCtION {#S1}
============
HEpAtiTIS b viRUS (HbV) IS sTiLl a mAJor gLoBaL hEALth PrOBlEM, WiTH AN esTImatEd 257 MilLion pEoplE WoRLDWiDe tHAT ARE chROniCALlY InfECtED wITH HbV ([@B1]). hbv, TogETHeR wiTh DUCK hEPatiTIS B VIRus (DhBv) and severAl oTHEr rElatEd aNiMaL viruSEs, belOnGs To The *HEPaDnaViRiDAe* FaMily ([@B2]). The hbv VIrIon Is CoMPRisEd OF AN OuTer enVELoPE AND an INNer iCOSaheDrAL nucLEoCaPsId (nC) aSSEmbled By 240 COPIES of core prOTeIn (hBc) ANd PackAgEd WitH a 3.2-kB PArtIAlLY dOuBLE-sTrandEd cIrcUlAr dnA GEnome ([@B3][@B4][@B8]). iN AddItioN TO Dna-CONTAIniNG VIrIONs, a lARGe AmoUNt of INCOmPlEte VIrAl paRtICLes, SUch AS hEpATITiS B SURFace ANTigeN (HBSAg) PARTiCLEs, eMpTy VIRIOnS, AnD NAKeD cAPSIDs, CAN ALsO BE RElEASed FRom cELls IN tHE proCESs Of VIRus REplICAtIOn ([@b9]). subVirAl HBSAg partIcLES aRE spHeRIcAl Or RodliKE aNd aRE pResent iN vaSt eXCeSS OveR VirIonS IN SERA oF chb pAtieNts ([@B2]). eMpTy vIrions sHare ThE SAmE sTRUCTure as dNa-conTaInInG ViRIOnS BUT are Devoid oF NUCLEic acids ([@b10][@b11][@b14]). naked capSidS, wHIcH EXiT cELLs ViA a roUTE DiffeRent FroM tHat OF VirIONs ([@B15][@B16][@b17]), HAVE The SAme StrUcture as nCS BUT ARE eitHer emPTy or fillEd wITH VIRal rNA and ImmaTuRe VIrAl dna ([@b7], [@B11], [@B18][@B19][@b20]).
in Nc, pGrNa uNdErgOes rEverSE TRAnScriPTIOn InTo miNus-StrAND DNa, FolLoWED by pLus-stRANd dnA syNTHEsis ([@B2], [@b21][@b22][@B24]). inTRacelLULaR ncS can BE pACkagED With virAL nUcLeIc aCIDS aT all leveLs OF MaTurAtIon, InCluDINg pgrnA, NaScENt miNus-STRAnD dNa, miNuS-stRaND dNa-rna hYbRids, and ReLAXEd CircULaR DnA (rc DNA) or dOuble-stRANdEd lINeAr dna (DsL dNA) ([@b5], [@b7]). oNLy thE ncs wiTH RELATIvELY mATUrE VirAl DNa (Rc OR Dsl Dna) arE ENVelOPEd AnD SeCRETed AS vIRIoNS. hbv REplICATiNG CellS cAN RELEASE EMpTy COrE pARtIcLes ASseMBlEd fROM hbc PRotEinS And ncS ThAt contAin vaRiOUs sPeciES of RePlIcATIvE InTermeDiATe nucLeiC aCids inTO tHE CUlTUrE SUpERNAtAnT. HowEveR, WHILe FREe naKEd caPsIDs coUlD bE ReaDIly deTeCTeD *in VITro* ([@b7], [@b11], [@B18][@b19][@b20]), tHEy ARe haRDLy foUnD iN thE bLood OF Hbv-inFEcteD pAtIentS ([@B17], [@B25], [@B26]).
AlThoUgh ExtRAcellulAr HBV rnA WAS dEtEctED iN botH *iN vItro* CelL culTuRe sYsTEMs anD iN cLinicAl sERum SAmpLEs, ItS oRiGiN AnD CompOSItIoN RemaIN CONtroversIAl. it was PrOpOsEd thaT eXtrAcELLuLAr hBV RNA RePResenTS PGrNA localizeD In VIrIONS ([@b27]). HoWEVeR, hbv SPlIcEd rnA anD HBx RNA WERe alSo deTEcted in cULtURE supERnAtANT OF HbV stABLy ReplICATInG CeLLs AS well as In serA of chb pATientS ([@b28], [@b29]). In aDdItiOn, ExTrAceLlulAr HBV rnA wAS ALso sUGGesTEd tO orIgiNAte frOM DaMAgeD LiveR CeLls ([@b30]), nAKeD cAPsidS, or exOSOmEs ([@B11], [@B29]). hEnCE, ThESE ExtRacEllular RNa mOLEculEs hAve nEvEr been conclUSivElY chArACTeRIZeD. hERe, wE DEmonStRATe tHAt exTRaCelluLar hBv RNas aRe HeTeROGEneOUs iN LeNgTh, raNGING from fuLl-LEngtH pGrNA (3.5 kiLonuClEoTidES \[KnT\]) to rna fRaGmenTs with mEreLY SEvEraL HundRed NuClEoTIdeS. THeSE rNA mOleCUleS REpReSEnt 3′ REcedING pGRna fraGMenTS THAT HAvE not BEEn COMpLETelY rEVErse tRaNsCRibed TO dNA and pGrna FragmeNts HyDROLyzeD bY tHe rnASe h DOMaiN OF PolymerASE In THE PRoCeSS Of vIrAL REPlicAtIOn. mORE IMpoRTAnTly, EXtrAceLLulaR hbv rnAS aRe lOCALIzeD in NaKed caPSIdS anD iN VIRioNS IN CuLTURE SuPERnatANts Of HBV rEPLiCaTInG ceLLs anD also ciRcUlaTe aS cACs AnD VIRiOnS iN BLooD oF HePaTiTiS b PaTienTs.
RESulTS {#S2}
=======
ExTRAcELlULar hbV rNAs Are hETerOGEnEoUs iN LENGTH anD PredomINAntLY InteGRAL To NAkEd cAPsiDs INSTeaD OF ViRiOns in HEpad38 cEll CulTURE supernAtANT. {#S2.1}
------------------------------------------------------------------------------------------------------------------------------------------------------
to AsCeRtAin thE oRiGIn oF ExtracELLULAr Hbv RnA, wE FIrsT exAmineD VIraL pArTICLes PREPARED fROM cuLturE MeDIUM Of an *In ViTRO* HBV sTaBly TrAnsduceD CeLL lIne. A huMan hEPaTomA HEpAD38 CEll LINE WAS UseD In THiS sTUDy, as IT sUsTAinS viGOrOUS Hbv REPLiCatION undER the CoNtrOL Of A TETrAcycLiNE-REpresSiblE cYtOMEgalOviRUs (CMV) proMOTeR ([@B31]). tOTal viraL PARTicleS Were cONCenTRATed and ceNTrIfuGeD OvER A 10% to 60% (wT/wt) SUcROsE GRAdIEnT. moSt Of THE Subviral HbsaG PArTICleS, virioNs, AND EMPTY vIRionS WERE dETEcteD BeTWeEN FrActiOns 9 tO 14 ([FIg. 1a](#F1){rEf-TyPE="fIG"}, UppeR ANd MiDdlE). naKED CAPsIDs, DeTeCTeD onLy by anti-hbcaG AnD nOT BY ANTi-hBSaG ANtIBOdIEs, SetTLed in FractioNs 5 To 8 ([fIG. 1A](#f1){REF-tYpe="Fig"}, mIDdlE aNd LOWEr). ThE MAjorItY Of vIRal nuCLeIc acidS WErE DETeCteD in FrACTiOnS betweEN 4 ANd 11 ([fiG. 1b](#F1){REf-tyPe="FIg"}, uPpEr), WHIcH cOincIdEd WiTh THE fRaCTiONS conTAinINg vIRIoNS (FracTIONs 9 to 11), naKED capsiDS (fRactioNs 4 to 7), AnD THe MixtuRE of thEse PARtIclES (FrAcTIOn 8). conSIstenT witH PrEVIoUs oBservATIoNs, Hbv ViRiOns aRE pacKed wIth matURE vIRal DnA (RC or dsl dnA), whiLe NAKed CAPsiDS coNTAin BoTH imMATUrE sINGlE-strANded Dna (ss dnA) And mATURE vIRal dna ([FIG. 1B](#F1){rEF-TyPe="FiG"}, uPPER). MOrEOvER, noRtheRn blOT reSULTs shOWed That most Of The HBV rna waS dETEcTED In tHE naKeD CApSIds ([FIg. 1B](#f1){rEf-tYPe="fig"}, lower, FRaCTiOnS 4 TO 7), WhEreAS onlY A veRy sMAll aMOUnT WaS ASSOCiaTEd WitH VIrIoNS ([fIG. 1b](#f1){REf-TyPe="fig"}, lOWEr, fRactIoNS 9 to 11). Hbv rna DeteCTEd in NaKed CApsIds rAnGeD fRom ThE fULl LENgth of pgrnA DOwN TO a fEW huNdRed nuCLeOtIdEs (sHOrtER ThaN ThE HBx mrNA \[0.7 knT\]). MOReOver, RNa MOlECulEs WITHin vIrIOnS WEre MUCH SHortER ThAN those wIthiN nakED CapSIDs. We EXCLuDed tHE pOSSibILiTy oF ArTiFAcTs GEneRAtEd bY thE SDS-proTeiNASe k ExtrAcTION methOD, AS a SImiLAr rna blOT paTTeRN WaS obTAinED USING A TRIzOL reAGeNT TO ExtrAct BoTH INTRaCEllUlaR NUClEOCaPSid-ASSoCIATeD aND ExtrAceLLulAr HbV rnA (nOt SHOwn). fUrtheRMORe, quAntIFICatiON oF vIRAl RNA ExTRACtEd By EiThEr thE SDs-PrOTEINaSE k METhOD Or tRIZoL Reagent PrODuced a vErY simIlAR cOPy NUmber, EXCEpT tHaT the tRIzOl ReagEnT IS knOWn To PreFerENtiallY exTrACT RNa ratHer ThaN dnA (not SHown). MOreoVEr, The RnA SIGNaL DEtECTED By NorTHeRN BLOTtIng COULd Not be aTtRIButeD TO DnA fRAgMeNTS genEratEd by DNaSE I TreAtMent, WhiCH wOuld rEDuCe dNA TO bELOW THe DeteCTion liMiT oF the hYBriDIZAtiOn mEthod (not sHOwn). fuRthErMORe, THe RnA SIgNaL CoUlD BE COMpleTEly REmoVED By aN addiTIONaL rNase A TReATMEnT (noT ShOwn).
{#f1}
TO cOnFirM tHE aBove-DeSCRIbED resUltS anD tO BetteR SEPaRAtE nAKed caPSIdS From hbV vIRiONS, ISoPyCNIC cscl GRADiENT ULtrAceNtRifUGation wAS emPLOYed. NaKED CApSiDS WERe oBserVed maiNLY IN FRAcTIONS 5 tO 7, WITH DEnSItiEs ranGINg fRoM 1.33 tO 1.34 g/Cm^3^ ([fIG. 2A](#F2){REF-tYPe="fIG"}). tHE SMearInG bands of NAkeD CapSIDS were likeLY CAuSED bY HIGh CoNceNtRATiOnS of CSCl SALt, as frAcTiOnATIon Of nAKED caPsIDs In A 1.18-g/Cm^3^ cscl SoLuTION pRoDUced singLe BANds. viRIONS, DEteCteD By BotH aNTi-HBcag anD ANti-HBSaG ANtIbodIeS ([fIg. 2A](#f2){ref-Type="FiG"}, UpPeR anD MIdDle), weRe PaCKAGED WITh VIRal Dna ([fiG. 2a](#F2){ReF-typE="FiG"}, LOWER) aNd sEttLed iN FractiONs 13 to 15, wITh deNSITIes RaNGiNG fROm 1.23 To 1.25 g/cM^3^. iN agrEEmEnT wiTh THe rESUltS SHOwn in [FiG. 1](#f1){rEf-typE="fig"}, | INTRODUCTION {#s1} ============ Hepatitis Bvirus (HBV) is still a major global health problem, with anestimated 257million people worldwide that are chronically infected with HBV ([@B1]). HBV, together with duckhepatitis B virus (DHBV) and several other related animal viruses, belongs to the*Hepadnaviridae* family ([@B2]). The HBV virion is comprised of an outer envelope and aninner icosahedral nucleocapsid (NC) assembled by 240 copiesof core protein(HBc) and packaged with a3.2-kb partially double-stranded circular DNAgenome ([@B3][@B4][@B8]).In addition to DNA-containing virions, a largeamount of incomplete viral particles, such ashepatitis B surface antigen (HBsAg) particles, empty virions, and naked capsids, can also be released from cells in the process of virus replication ([@B9]). SubviralHBsAg particles are spherical orrodlike and arepresent in vastexcess overvirions in sera of CHB patients ([@B2]). Empty virions share the same structureas DNA-containing virions butare devoid ofnucleicacids ([@B10][@B11][@B14]). Naked capsids, which exit cells via a route different fromthat of virions ([@B15][@B16][@B17]), have the same structure as NCs but areeither empty or filled with viral RNA and immature viral DNA ([@B7], [@B11], [@B18][@B19][@B20]). In NC, pgRNA undergoes reverse transcription into minus-strand DNA, followed by plus-strand DNA synthesis([@B2], [@B21][@B22][@B24]). Intracellular NCs can be packaged with viral nucleic acids at all levels of maturation, includingpgRNA, nascent minus-strand DNA, minus-strandDNA-RNA hybrids, and relaxed circular DNA (RC DNA) or double-stranded linear DNA (DSLDNA)([@B5],[@B7]). Only the NCs withrelatively mature viral DNA(RC or DSL DNA) are enveloped and secreted as virions. HBV replicating cells can release empty core particles assembled from HBcproteinsand NCsthat containvarious species of replicative intermediate nucleic acids into theculture supernatant.However, while freenaked capsids could bereadily detected*invitro* ([@B7], [@B11], [@B18][@B19][@B20]), they are hardly found in the blood of HBV-infectedpatients ([@B17], [@B25], [@B26]). Although extracellularHBV RNA was detectedin both *in vitro* cell culturesystems and in clinicalserumsamples, its origin and composition remain controversial. It was proposed that extracellular HBVRNA represents pgRNA localized in virions ([@B27]). However, HBV spliced RNA and HBxRNA were also detected in culture supernatant of HBV stably replicating cells as well asin sera of CHBpatients ([@B28], [@B29]). In addition, extracellular HBV RNA was also suggested to originatefrom damaged liver cells ([@B30]), naked capsids, or exosomes ([@B11], [@B29]). Hence, these extracellular RNA molecules have never been conclusively characterized. Here,we demonstrate that extracellular HBV RNAs are heterogeneous in length, ranging from full-length pgRNA (3.5 kilonucleotides \[knt\]) toRNA fragments with merelyseveral hundred nucleotides.These RNA molecules represent 3′ recedingpgRNA fragments that have not been completelyreversetranscribed to DNA andpgRNA fragments hydrolyzed by the RNase H domain of polymerase in the process of viralreplication.More importantly, extracellular HBV RNAs are localized in naked capsids and invirions inculture supernatants of HBV replicating cellsand also circulate as CACs and virions inblood ofhepatitis B patients. RESULTS {#s2}======= Extracellular HBV RNAs are heterogeneous in length and predominantly integral tonaked capsidsinstead ofvirions in HepAD38 cell culturesupernatant. {#s2.1} ------------------------------------------------------------------------------------------------------------------------------------------------------ To ascertain the origin of extracellular HBV RNA, we first examined viral particles prepared from culture mediumof an *in vitro* HBVstably transduced cellline.A human hepatoma HepAD38 cell line was used in this study,as it sustains vigorous HBV replication under thecontrol of a tetracycline-repressible cytomegalovirus (CMV) promoter ([@B31]). Totalviral particles were concentrated andcentrifuged over a 10% to60% (wt/wt) sucrose gradient. Most of the subviral HBsAgparticles, virions, and empty virions were detected between fractions 9 to 14 ([Fig. 1A](#F1){ref-type="fig"}, upper and middle). Naked capsids, detectedonly by anti-HBcAg and not by anti-HBsAgantibodies, settled in fractions 5to 8 ([Fig. 1A](#F1){ref-type="fig"}, middle and lower). The majority of viral nucleic acids were detected in fractions between 4 and 11 ([Fig. 1B](#F1){ref-type="fig"}, upper), which coincided with the fractionscontaining virions (fractions 9to 11), naked capsids(fractions 4 to 7), and the mixture of these particles (fraction 8). Consistent with previous observations, HBV virions are packedwith mature viral DNA(RC or DSL DNA), while naked capsids contain both immature single-stranded DNA (SS DNA) and mature viral DNA ([Fig. 1B](#F1){ref-type="fig"}, upper). Moreover, Northern blot results showedthat most of the HBV RNA was detected in the naked capsids ([Fig. 1B](#F1){ref-type="fig"}, lower, fractions4to7), whereas onlya very smallamount was associated with virions ([Fig. 1B](#F1){ref-type="fig"}, lower, fractions 9 to 11). HBV RNA detected in naked capsids rangedfrom the full length of pgRNA down to a few hundred nucleotides (shorter thanthe HBx mRNA \[0.7 knt\]). Moreover, RNA molecules within virionswere muchshorterthan those within naked capsids. We excluded the possibility of artifacts generated by the SDS-proteinase K extraction method, as a similar RNAblot pattern was obtained using a TRIzol reagent to extract bothintracellular nucleocapsid-associated andextracellular HBV RNA (not shown).Furthermore, quantification of viral RNA extracted by either the SDS-proteinase K method or TRIzol reagent produceda very similar copynumber, except that the TRIzol reagent is known to preferentially extract RNA rather than DNA(notshown). Moreover,theRNA signaldetected by Northern blotting could not be attributed to DNA fragments generated by DNase I treatment, whichwould reduce DNA to belowthe detection limit of the hybridization method (not shown). Furthermore, the RNAsignal could be completely removed byan additional RNase A treatment (not shown).{#F1} To confirm the above-described results and to better separate nakedcapsids from HBV virions, isopycnic CsCl gradient ultracentrifugation was employed. Naked capsids were observed mainly in fractions 5 to 7, with densities ranging from 1.33 to1.34 g/cm^3^ ([Fig. 2A](#F2){ref-type="fig"}).The smearing bands of naked capsids were likely caused by high concentrations of CsClsalt, asfractionation of naked capsids in a 1.18-g/cm^3^ CsCl solution produced single bands. Virions, detectedby both anti-HBcAg and anti-HBsAgantibodies([Fig. 2A](#F2){ref-type="fig"}, upper and middle), were packaged with viral DNA ([Fig. 2A](#F2){ref-type="fig"}, lower) andsettledinfractions 13 to 15, with densities ranging from1.23 to 1.25 g/cm^3^.In agreement with the results shown in [Fig. 1](#F1){ref-type="fig"}, | INTRODUCTION {#s1} ============ _Hepatitis_ B virus (HBV) is _still_ _a_ major global health problem, with an _estimated_ 257 million people worldwide that are _chronically_ _infected_ with _HBV_ ([@B1]). HBV, together with duck hepatitis _B_ virus (DHBV) and _several_ _other_ related _animal_ viruses, belongs to the _*Hepadnaviridae*_ _family_ ([@B2]). The HBV virion is comprised _of_ _an_ _outer_ envelope and an inner _icosahedral_ nucleocapsid (NC) assembled by 240 _copies_ _of_ core _protein_ (HBc) and packaged with a 3.2-kb partially double-stranded circular DNA genome _([@B3][@B4][@B8])._ _In_ addition to DNA-containing virions, a _large_ amount _of_ incomplete viral particles, such _as_ _hepatitis_ _B_ surface antigen (HBsAg) particles, empty virions, and naked capsids, can also be _released_ from cells in the _process_ of virus replication ([@B9]). Subviral HBsAg particles are spherical or _rodlike_ and are _present_ _in_ vast excess over virions in sera of CHB patients ([@B2]). Empty virions share _the_ same structure as DNA-containing virions _but_ are devoid of nucleic acids ([@B10][@B11][@B14]). Naked capsids, which exit cells via a _route_ different _from_ _that_ of virions ([@B15][@B16][@B17]), have the same structure _as_ NCs but are either empty or filled with viral RNA and _immature_ viral DNA ([@B7], [@B11], [@B18][@B19][@B20]). In NC, pgRNA _undergoes_ reverse _transcription_ into minus-strand DNA, followed by _plus-strand_ DNA synthesis ([@B2], [@B21][@B22][@B24]). _Intracellular_ NCs can be packaged with viral _nucleic_ _acids_ _at_ all levels _of_ maturation, including pgRNA, nascent minus-strand DNA, minus-strand DNA-RNA _hybrids,_ and _relaxed_ circular _DNA_ (RC DNA) or double-stranded linear _DNA_ (DSL _DNA)_ ([@B5], _[@B7])._ Only _the_ NCs with relatively _mature_ viral _DNA_ (RC or DSL DNA) are enveloped and _secreted_ as _virions._ HBV replicating cells _can_ release empty core particles assembled from HBc proteins _and_ NCs that contain various species of _replicative_ intermediate nucleic acids _into_ the _culture_ supernatant. _However,_ while free _naked_ capsids could be readily detected *in vitro* ([@B7], [@B11], [@B18][@B19][@B20]), they are _hardly_ found in the blood of _HBV-infected_ patients ([@B17], _[@B25],_ [@B26]). Although extracellular HBV RNA was detected _in_ both *in _vitro*_ cell culture systems _and_ in clinical serum samples, its origin and composition remain controversial. It _was_ proposed that _extracellular_ HBV _RNA_ represents pgRNA localized in virions ([@B27]). However, _HBV_ _spliced_ RNA _and_ HBx RNA were also detected in culture _supernatant_ of _HBV_ stably _replicating_ _cells_ as well as _in_ sera _of_ CHB patients ([@B28], [@B29]). In addition, extracellular HBV _RNA_ was _also_ suggested _to_ _originate_ from damaged liver cells ([@B30]), _naked_ capsids, or exosomes _([@B11],_ [@B29]). Hence, these extracellular _RNA_ molecules have never been conclusively characterized. Here, we _demonstrate_ _that_ extracellular _HBV_ RNAs are _heterogeneous_ in length, ranging _from_ _full-length_ pgRNA (3.5 kilonucleotides \[knt\]) to RNA fragments with _merely_ several _hundred_ nucleotides. These RNA molecules represent 3′ _receding_ pgRNA fragments that have _not_ been completely reverse transcribed to DNA and _pgRNA_ fragments hydrolyzed by the _RNase_ _H_ domain of polymerase in _the_ process of viral _replication._ More _importantly,_ extracellular _HBV_ _RNAs_ are _localized_ in _naked_ capsids and _in_ _virions_ in _culture_ supernatants of _HBV_ _replicating_ cells and _also_ circulate _as_ CACs and virions _in_ blood of _hepatitis_ _B_ patients. RESULTS _{#s2}_ ======= Extracellular HBV _RNAs_ _are_ heterogeneous in length and _predominantly_ integral to naked capsids instead of virions in HepAD38 cell culture supernatant. {#s2.1} ------------------------------------------------------------------------------------------------------------------------------------------------------ To ascertain the _origin_ of extracellular HBV RNA, we first examined viral _particles_ prepared from culture medium of an *in _vitro*_ HBV stably transduced cell line. _A_ _human_ _hepatoma_ HepAD38 cell line was used in this _study,_ _as_ it sustains vigorous HBV replication under the control of a tetracycline-repressible _cytomegalovirus_ (CMV) promoter ([@B31]). Total viral particles were _concentrated_ and centrifuged over a 10% to 60% (wt/wt) _sucrose_ _gradient._ Most of the _subviral_ HBsAg particles, virions, and empty virions _were_ detected between fractions 9 to 14 ([Fig. 1A](#F1){ref-type="fig"}, _upper_ and middle). Naked capsids, detected only by _anti-HBcAg_ and not by anti-HBsAg antibodies, settled in fractions _5_ to 8 _([Fig._ 1A](#F1){ref-type="fig"}, middle and _lower)._ The majority _of_ viral nucleic acids were detected in fractions between 4 and 11 ([Fig. 1B](#F1){ref-type="fig"}, upper), which coincided with _the_ _fractions_ _containing_ virions (fractions _9_ _to_ 11), naked capsids (fractions 4 _to_ 7), and the mixture of these particles (fraction 8). _Consistent_ _with_ _previous_ _observations,_ _HBV_ virions _are_ packed with mature _viral_ DNA (RC _or_ DSL DNA), while naked capsids contain both immature single-stranded DNA (SS DNA) and _mature_ viral DNA ([Fig. _1B](#F1){ref-type="fig"},_ upper). Moreover, Northern blot results showed that most of the HBV RNA was detected in _the_ naked capsids ([Fig. _1B](#F1){ref-type="fig"},_ _lower,_ _fractions_ _4_ to 7), whereas _only_ _a_ _very_ _small_ amount was associated _with_ virions _([Fig._ 1B](#F1){ref-type="fig"}, lower, fractions 9 to 11). _HBV_ RNA _detected_ in naked capsids _ranged_ from the _full_ _length_ of pgRNA down to a _few_ hundred _nucleotides_ (shorter than the HBx mRNA \[0.7 knt\]). Moreover, RNA molecules within virions _were_ much shorter _than_ those within naked capsids. We excluded the possibility of _artifacts_ generated by the SDS-proteinase _K_ extraction method, as a similar _RNA_ _blot_ pattern was obtained using a _TRIzol_ reagent to _extract_ both intracellular nucleocapsid-associated and extracellular HBV RNA _(not_ shown). Furthermore, _quantification_ of viral RNA extracted by either the SDS-proteinase K method _or_ TRIzol reagent produced a very similar copy number, except that the _TRIzol_ reagent is known to preferentially _extract_ _RNA_ rather _than_ DNA (not shown). _Moreover,_ the RNA _signal_ detected by Northern _blotting_ could not _be_ attributed to DNA fragments generated by DNase I treatment, which would reduce DNA _to_ below _the_ detection limit _of_ _the_ hybridization method (not shown). Furthermore, the RNA _signal_ could be completely _removed_ by an additional RNase _A_ treatment (not shown). {#F1}_ To _confirm_ the above-described _results_ and to better separate naked capsids from _HBV_ _virions,_ isopycnic CsCl gradient ultracentrifugation _was_ employed. Naked capsids _were_ observed mainly in fractions _5_ to 7, with densities ranging _from_ 1.33 to 1.34 g/cm^3^ ([Fig. 2A](#F2){ref-type="fig"}). The smearing bands of naked _capsids_ _were_ likely caused _by_ _high_ _concentrations_ of CsCl _salt,_ as fractionation of naked _capsids_ in a 1.18-g/cm^3^ CsCl solution _produced_ single bands. Virions, detected _by_ both anti-HBcAg and anti-HBsAg antibodies ([Fig. 2A](#F2){ref-type="fig"}, upper and middle), were packaged with viral DNA ([Fig. 2A](#F2){ref-type="fig"}, lower) and settled in fractions 13 to 15, with _densities_ _ranging_ from 1.23 to 1.25 g/cm^3^. _In_ agreement with the results _shown_ in [Fig. 1](#F1){ref-type="fig"}, |
Koolstra K, Beenakker J‐WM, Koken P, Webb A, Börnert P. Cartesian MR fingerprinting in the eye at 7T using compressed sensing and matrix completion‐based reconstructions. Magn Reson Med. 2019;81:2551--2565. 10.1002/mrm.27594 30421448
**Funding information**
This project was partially funded by the European Research Council Advanced Grant 670629 NOMA MRI.
1. INTRODUCTION {#mrm27594-sec-0005}
===============
Ophthalmologic disease diagnosis conventionally relies mainly on ultrasound and optical imaging techniques such as fundus photography and fluorescent angiography (FAG), MRI is increasingly being used in the radiological community.[1](#mrm27594-bib-0001){ref-type="ref"}, [2](#mrm27594-bib-0002){ref-type="ref"}, [3](#mrm27594-bib-0003){ref-type="ref"} One of the main advantages of MRI is its capability to assess nontransparent tissues such as ocular tumors or structures behind the globe such as the eye muscles. Currently, however, these applications are mainly based on qualitative MRI methods using the large number of tissue contrasts addressable by MR. As an example, in Graves' ophthalmopathy fat‐suppressed T~2~‐weighted MRI is the standard to detect inflammation in the eye muscles,[4](#mrm27594-bib-0004){ref-type="ref"}, [5](#mrm27594-bib-0005){ref-type="ref"} whereas in the diagnosis of retinoblastoma, a rare intraocular cancer in children, standard T~1~‐ and T~2~‐weighted MRI is often performed to confirm the presence of the tumor and to screen for potential optic nerve involvement.[2](#mrm27594-bib-0002){ref-type="ref"} In more recent ophthalmologic applications of MRI, such as uveal melanoma (the most common primary intraocular tumor), quantitative MRI techniques including DWI[6](#mrm27594-bib-0006){ref-type="ref"} and DCE imaging[7](#mrm27594-bib-0007){ref-type="ref"} have been shown, but currently diagnosis is still based on qualitative methods.[3](#mrm27594-bib-0003){ref-type="ref"}
To personalize treatment plans quantitative parameters of the tissues involved, as can be acquired invasively for example by performing biopsies,[8](#mrm27594-bib-0008){ref-type="ref"} are highly desirable. However, quantitative parameter mapping by means of MRI requires long examination times, which would result in significant eye‐motion artifacts, as well as patient discomfort.[9](#mrm27594-bib-0009){ref-type="ref"} MR fingerprinting (MRF) is a recently introduced method for rapid quantitation of tissue relaxation times and other MR‐related parameters.[10](#mrm27594-bib-0010){ref-type="ref"} It uses a flip angle sweep to induce a unique signal evolution for each tissue type. Incoherent undersampling can be applied during sampling of the MRF train, enabling acceleration of the MRF scans.[10](#mrm27594-bib-0010){ref-type="ref"} Together with its ability to measure simultaneously T~1~ and T~2~, MRF offers a solution to the problem of obtaining quantitative measures in an efficient manner and in relatively short scanning times.
One of the main challenges in ocular imaging is in‐plane and through‐plane eye motion, often associated with eye blinking.[11](#mrm27594-bib-0011){ref-type="ref"}, [12](#mrm27594-bib-0012){ref-type="ref"}, [13](#mrm27594-bib-0013){ref-type="ref"} The motion results in corrupted k‐space data that introduces artifacts and blurring throughout the entire image. Shortening the scans would reduce motion‐related artifacts, but standard acceleration techniques are not optimal for the current eye application due to the following 3 reasons. First, a cued‐blinking protocol is typically used to control and reduce the eye motion.[3](#mrm27594-bib-0003){ref-type="ref"}, [11](#mrm27594-bib-0011){ref-type="ref"} This requires an instruction screen placed at the end of the MR tunnel to be visible to the patient which complicates the use of small phased array receive coils in front of the eye, blocking the view. Instead, a custom‐built single‐element eye loop coil is used, which provides a high local SNR[3](#mrm27594-bib-0003){ref-type="ref"} and screen visibility, but which clearly excludes the possibility of scan acceleration by means of parallel imaging.[14](#mrm27594-bib-0014){ref-type="ref"} Second, the gel‐like vitreous body has an extremely long T~1~, particularly at high field.[15](#mrm27594-bib-0015){ref-type="ref"} Its value of 3 to 5 s requires a long duration of the MRF sequence to encode the MR parameters (T~1~,T~2~) sufficiently. Thus, using a flip angle train with a small number of RF pulses is not feasible, hindering scan time reduction. Finally, a time‐efficient spiral sampling scheme, usually applied in MRF,[10](#mrm27594-bib-0010){ref-type="ref"}, [16](#mrm27594-bib-0016){ref-type="ref"}, [17](#mrm27594-bib-0017){ref-type="ref"}, [18](#mrm27594-bib-0018){ref-type="ref"}, [19](#mrm27594-bib-0019){ref-type="ref"} introduces off‐resonance effects in each of the individual MRF images.[20](#mrm27594-bib-0020){ref-type="ref"} This occurs even when combined with unbalanced sequences such as fast imaging with steady state precession,[16](#mrm27594-bib-0016){ref-type="ref"} which are in themselves robust to off‐resonance effects.[21](#mrm27594-bib-0021){ref-type="ref"} The off‐resonance effects present in spiral sampling schemes are much stronger at high field, where they result in blurring,[22](#mrm27594-bib-0022){ref-type="ref"} caused by strong main field inhomogeneities (particularly in the eye region due to many air‐tissue‐bone interfaces), as well as the presence of significant amounts of off‐resonant orbital fat around the eye.
In this work, a Cartesian sampling scheme is used, which is more robust than spiral sampling to off‐resonance effects, but which is significantly less time‐efficient.[23](#mrm27594-bib-0023){ref-type="ref"} With such a Cartesian sampling scheme, undersampling artifacts have a more structured nature compared with spiral sampling, which increases the temporal coherence of the artifacts in the MRF image series.[10](#mrm27594-bib-0010){ref-type="ref"}, [20](#mrm27594-bib-0020){ref-type="ref"} In this case, direct matching of the measured MRF signal reconstructed by plain Fourier transformations, to the simulated dictionary elements is not sufficiently accurate for high undersampling factors.[24](#mrm27594-bib-0024){ref-type="ref"}, [25](#mrm27594-bib-0025){ref-type="ref"} Therefore, the quality of the reconstructed MRF data has to be improved before the matching process.
Compressed sensing (CS) has been introduced as a technique to reconstruct images from randomly undersampled data by enforcing signal sparsity (in the spatial dimension only or both in spatial and temporal dimensions),[26](#mrm27594-bib-0026){ref-type="ref"}, [27](#mrm27594-bib-0027){ref-type="ref"} allowing a scan time reduction in many applications.[28](#mrm27594-bib-0028){ref-type="ref"}, [29](#mrm27594-bib-0029){ref-type="ref"}, [30](#mrm27594-bib-0030){ref-type="ref"} The flexibility of MRF toward different sampling schemes and undersampling factors makes it possible to reconstruct the source images by means of CS.[27](#mrm27594-bib-0027){ref-type="ref"}, [31](#mrm27594-bib-0031){ref-type="ref"}, [32](#mrm27594-bib-0032){ref-type="ref"} Higher acceleration factors might be feasible if the correlation in the temporal dimension is better used.[33](#mrm27594-bib-0033){ref-type="ref"} Examples of such reconstructions specifically tailored to MRF are given in Davies et al, Pierre et al, and Zhao et al[34](#mrm27594-bib-0034){ref-type="ref"}, [35](#mrm27594-bib-0035){ref-type="ref"}, [36](#mrm27594-bib-0036){ref-type="ref"} which take into account the simulated dictionary atoms in the image reconstruction process.
Recent work has shown that the temporal correlation in the MRF data can be exploited even further by incorporating the low rank structure of the data into the cost function,[37](#mrm27594-bib-0037){ref-type="ref"} a technique which was introduced into MR in Liang[ | koolstra k, beenakker j ‐ wm, koken p, webb a, bornert p. cartesian mr fingerprinting in the eye at 7t using compressed sensing and matrix 3d ‐ based reconstructions. magn reson med. 2019 ; 81 : 2551 - - 2565. 10. 1002 / mrm. 27594 30421448 * * funding information * * this project was partially funded by the european research council advanced grant 670629 noma vol. 1. introduction { # mrm27594 - sec - 0005 } = = = = = = = = = = = = = = = ophthalmologic disease diagnosis conventionally relies mainly on ultrasound and optical imaging techniques such as fundus mri and fluorescent angiography ( fag ), mri is increasingly being used in the radiological community. [ 1 ] ( # mrm27594 - bib - 0001 ) { ref - type = " ref " }, [ 2 ] ( # mrm27594 - bib - 0002 ) { ref - type = " ref " }, [ 3 ] ( # mrm27594 - bib - e ) { ref - type = " ref " } one of the main advantages of mri is its capability to assess nontransparent tissues such as ocular tumors or structures behind the globe such as the eye muscles. currently, however, these applications are mainly based on qualitative mri methods using the large number of tissue contrasts addressable by mr. as an example, in graves ' median fat ‐ suppressed t ~ 2 ~ ‐ weighted mri is the standard to detect inflammation involving the eye muscles, [ 2 ] ( # mrm27594 - bib - 0004 ) { ref - type = " ref " }, [ 5 ] ( # mrm27594 - bib - 0005 ) { ref - type = " ref " } whereas in the diagnosis of retinoblastoma, a rare intraocular cancer in children, standard f ~ 1 ~ ‐ and t ~ 2 ~ ‐ weighted mri is often performed to confirm the presence accompanying the tumor and to screen for potential optic nerve involvement. [ 2 ] ( # mrm27594 - bib - 001 ) { ref - type = " ref " } in more recent ophthalmologic applications of mri, such as uveal melanoma ( the most common primary intraocular tumor ), quantitative mri techniques including dwi [ 6 ] ( # mrm27594 - bib - 0006 ) { ref - type = " ref " } and dce imaging [ 7 ] ( # mrm27594 - bib - 0007 ) { ref - type = " ref " } have been shown, but currently diagnosis is still based on qualitative methods. [ 3 ] ( # mrm27594 - bib - 0003 ) { ref - type = " ref " } to personalize treatment plans quantitative parameters of the tissues involved, as can be acquired invasively for example by performing biopsies, [ 8 ] ( # mrm27594 - bib - 0008 ) { ref - type = " ref " } are highly desirable. however, quantitative parameter mapping by means of mri requires long examination times, which would result in significant eye ‐ motion artifacts, as well as patient discomfort. [ 9 ] ( # mrm27594 - bib - 0009 ) { ref - type = " ref " } mr fingerprinting ( mrf ) is a recently introduced method for rapid quantitation of tissue relaxation times and other mr ‐ related parameters. [ 10 ] ( # mrm27594 - bib - 0010 ) { ref - type = " ref " } it uses a flip angle sweep to induce a unique signal evolution for each tissue type. incoherent undersampling can be applied during sampling of the mrf train, enabling acceleration of the mrf scans. [ 10 ] ( # mrm27594 - bib - 0010 ) { ref - type = " ref " } together with its ability to measure simultaneously t ~ 1 ~ and t ~ 2 ~, mrf offers a solution to the problem of obtaining quantitative measures in an efficient manner and in relatively short scanning times. one of the main challenges in ocular imaging is in ‐ plane and through ‐ plane eye motion, often associated with eye blinking. [ 11 ] ( # mrm27594 - bib - 0011 ) { ref - type = " ref " }, [ 12 ] ( # mrm27594 - bib - 0012 ) { ref - type = " ref " }, [ 13 ] ( # mrm27594 - bib - 0013 ) { ref - type = " ref " } the motion results in corrupted k ‐ space data that introduces artifacts and blurring throughout the entire image. shortening the scans would reduce motion ‐ related artifacts, but standard acceleration techniques are not optimal for the current eye application due to the following 3 reasons. first, a cued ‐ blinking protocol is typically used to control and reduce the eye motion. [ 3 ] ( # mrm27594 - bib - 0003 ) { ref - type = " ref " }, [ 11 ] ( # mrm27594 - bib - 0011 ) { ref - type = " ref " } this requires an instruction screen placed at the end of the mr tunnel to be visible to the patient which complicates the use of small phased array receive coils in front of the eye, blocking the view. instead, a custom ‐ built single ‐ element eye loop coil is used, which provides a high local snr [ 3 ] ( # mrm27594 - bib - 0003 ) { ref - type = " ref " } and screen visibility, but which clearly excludes the possibility of scan acceleration by means of parallel imaging. [ 14 ] ( # mrm27594 - bib - 0014 ) { ref - type = " ref " } second, the gel ‐ like vitreous body has an extremely long t ~ 1 ~, particularly at high field. [ 15 ] ( # mrm27594 - bib - 0015 ) { ref - type = " ref " } its value of 3 to 5 s requires a long duration of the mrf sequence to encode the mr parameters ( t ~ 1 ~, t ~ 2 ~ ) sufficiently. thus, using a flip angle train with a small number of rf pulses is not feasible, hindering scan time reduction. finally, a time ‐ efficient spiral sampling scheme, usually applied in mrf, [ 10 ] ( # mrm27594 - bib - 0010 ) { ref - type = " ref " }, [ 16 ] ( # mrm27594 - bib - 0016 ) { ref - type = " ref " }, [ 17 ] ( # mrm27594 - bib - 0017 ) { ref - type = " ref " }, [ 18 ] ( # mrm27594 - bib - 0018 ) { ref - type = " ref " }, [ 19 ] ( # mrm27594 - bib - 0019 ) { ref - type = " ref " } introduces off ‐ resonance effects in each of the individual mrf images. [ 20 ] ( # mrm27594 - bib - 0020 ) { ref - type = " ref " } this occurs even when combined with unbalanced sequences such as fast imaging with steady state precession, [ 16 ] ( # mrm27594 - bib - 0016 ) { ref - type = " ref " } which are in themselves robust to off ‐ resonance effects. [ 21 ] ( # mrm27594 - bib - 0021 ) { ref - type = " ref " } the off ‐ resonance effects present in spiral sampling schemes are much stronger at high field, where they result in blurring, [ 22 ] ( # mrm27594 - bib - 0022 ) { ref - type = " ref " } caused by strong main field inhomogeneities ( particularly in the eye region due to many air ‐ tissue ‐ bone interfaces ), as well as the presence of significant amounts of off ‐ resonant orbital fat around the eye. in this work, a cartesian sampling scheme is used, which is more robust than spiral sampling to off ‐ resonance effects, but which is significantly less time ‐ efficient. [ 23 ] ( # mrm27594 - bib - 0023 ) { ref - type = " ref " } with such a cartesian sampling scheme, undersampling artifacts have a more structured nature compared with spiral sampling, which increases the temporal coherence of the artifacts in the mrf image series. [ 10 ] ( # mrm27594 - bib - 0010 ) { ref - type = " ref " }, [ 20 ] ( # mrm27594 - bib - 0020 ) { ref - type = " ref " } in this case, direct matching of the measured mrf signal reconstructed by plain fourier transformations, to the simulated dictionary elements is not sufficiently accurate for high undersampling factors. [ 24 ] ( # mrm27594 - bib - 0024 ) { ref - type = " ref " }, [ 25 ] ( # mrm27594 - bib - 0025 ) { ref - type = " ref " } therefore, the quality of the reconstructed mrf data has to be improved before the matching process. compressed sensing ( cs ) has been introduced as a technique to reconstruct images from randomly undersampled data by enforcing signal sparsity ( in the spatial dimension only or both in spatial and temporal dimensions ), [ 26 ] ( # mrm27594 - bib - 0026 ) { ref - type = " ref " }, [ 27 ] ( # mrm27594 - bib - 0027 ) { ref - type = " ref " } allowing a scan time reduction in many applications. [ 28 ] ( # mrm27594 - bib - 0028 ) { ref - type = " ref " }, [ 29 ] ( # mrm27594 - bib - 0029 ) { ref - type = " ref " }, [ 30 ] ( # mrm27594 - bib - 0030 ) { ref - type = " ref " } the flexibility of mrf toward different sampling schemes and undersampling factors makes it possible to reconstruct the source images by means of cs. [ 27 ] ( # mrm27594 - bib - 0027 ) { ref - type = " ref " }, [ 31 ] ( # mrm27594 - bib - 0031 ) { ref - type = " ref " }, [ 32 ] ( # mrm27594 - bib - 0032 ) { ref - type = " ref " } higher acceleration factors might be feasible if the correlation in the temporal dimension is better used. [ 33 ] ( # mrm27594 - bib - 0033 ) { ref - type = " ref " } examples of such reconstructions specifically tailored to mrf are given in davies et al, pierre et al, and zhao et al [ 34 ] ( # mrm27594 - bib - 0034 ) { ref - type = " ref " }, [ 35 ] ( # mrm27594 - bib - 0035 ) { ref - type = " ref " }, [ 36 ] ( # mrm27594 - bib - 0036 ) { ref - type = " ref " } which take into account the simulated dictionary atoms in the image reconstruction process. recent work has shown that the temporal correlation in the mrf data can be exploited even further by incorporating the low rank structure of the data into the cost function, [ 37 ] ( # mrm27594 - bib - 0037 ) { ref - type = " ref " } a technique which was introduced into mr in liang [ | Koolstra K, Beenakker J ‐ WM, Koken P, Webb A, Börnert P. Cartesian MR fingerprinting in the eye at 7T using compressed sensing and matrix completion ‐ based reconstructions. Magn Reson Med. 2019; 81: 2551 - - 2565. 10. 1002 / mrm. 27594 30421448 * * Funding information * * This project was partially funded by the European Research Council Advanced Grant 670629 NOMA MRI. 1. INTRKDUCTIlN {# mrm27594 - sec - 0005} = = = = = = = = = = = = = = = Ophthalmologic disease diagnosis conventionally relies mainly on ultrasound and optical imaging techniques such as fundus photography and fluorescent angiography (FAG ), MRI is increasingly being used in the radiological community. [1] (# mrm27594 - bib - 0001) {ref - type = " ref " }, [2] (# mrm27594 - bib - 0002) {ref - type = " ref " }, [3] (# mrm27594 - bib - 0003) {ref - type = " ref "} One of the main advantages of MRI is its capability to assess nontransparent tissues such as ocular tumors or structures behind the globe such as the eye muscles. Currently, however, these applications are mainly based on qualitative MRI methods using the large number of tissue contrasts addressable by MR. As an example, in Graves ' ophthalmopathy fat ‐ suppressed T ~ 2 ~ ‐ weighted MRI is the standard to detect inflammation in the eye muscles, [4] (# mrm27594 - bib - 0004) {ref - type = " ref " }, [5] (# mrm27594 - bib - 0005) {ref - type = " ref "} whereas in the diagnosis of retinoblastoma, a rare intraocular cancer in children, standard T ~ 1 ~ ‐ and T ~ 2 ~ ‐ weighted MRI is often performed to confirm the presence of the tumor and to screen for potential optic nerve involvement. [2] (# mrm27594 - bib - 0002) {ref - type = " ref "} In more recent ophthalmologic applications of MRI, such as uveal melanoma (the most common primary intraocular tumor ), quantitative MRI techniques including DWI [6] (# mrm27594 - bib - 0006) {ref - type = " ref "} and DCE imaging [7] (# <tm27594 - bib - 0007) {ref - type = " ref "} have been shown, but currently diagnosis is still based on qualitative methods. [3] (# mrm27594 - bib - 0003) {ref - type = " ref "} To personalize treatment plans quantitative parameters of the tissues involved, as can be acquired invasively for example by performing biopsies, [8] (# mrm27594 - bib - 0008) {ref - type = " ref "} are highly desirable. However, quantitative parameter mapping by means of MRI requires long examination times, which would result in significant eye ‐ motion artifacts, as well as patient discomfort. [9] (# mTm27%94 - bib - 0009) {ref - type = " ref "} MR fingerprinting (MRF) is a recently introduced method for rapid quantitation of tissue relaxation times and other MR ‐ related parameters. [10] (# mrm27594 - bib - 0010) {ref - type = " ref "} It uses a flip angle sweep to induce a unique signal evolution for each tissue type. Incoherent undersampling can be applied during sampling of the MRF train, enabling acceleration of the MRF scans. [10] (# mrm27594 - bib - 0010) {ref - type = " ref "} Together with its ability to measure simultaneously T ~ 1 ~ and T ~ 2 ~, MRF offers a solution to the problem of obtaining quantitative measures in an efficient manner and in relatively short scanning times. One of the main challenges in ocular imaging is in ‐ plane and through ‐ plane eye motion, often associated with eye blinking. [11] (# mrm27594 - bib - 0011) {ref - type = " ref " }, [12] (# mrm27594 - bib - 0012) {ref - type = " ref " }, [13] (# mrm27594 - bib - 0013) {ref - type = " ref "} The motion results in corrupted k ‐ space data that introduces artifacts and blurring throughout the entire image. Shortening the scans would reduce motion ‐ related artifacts, but standard acceleration techniques are not optimal for the current eye application due to the following 3 reasons. First, a cued ‐ blinking protocol is typically used to s(ntrol and reduce the eye motion. [3] (# mrm27594 - bib - 0003) {ref - type = " ref " }, [11] (# mrm27594 - bib - 0011) {ref - type = " ref "} This requires an instruction screen placed at the end of the MR tunnel to be visible to the patient which complicates the use of small phased array receive coils in front of the eye, blocking the view. Instead, a custom ‐ built single ‐ element eye loop coil is used, which provides a high local SNR [3] (# mrm27594 - bib - 0003) {ref - type = " ref "} and screen visibility, but which clearly excludes the possibility of scan acceleration by means of parallel imaging. [14] (# mrm27594 - bib - 0014) {ref - type = " ref "} Second, the gel ‐ like vitreous body has an extremely long T ~ 1 ~, particularly at high fiekw. [15] (# mrm27594 - bib - 0015) {ref - type = " ref "} Its value of 3 to 5 s requires a long duration of the MRF sequence to encode the MR parameters (T ~ 1 ~, T ~ 2 ~) sufficiently. Thus, using a flip angle train with a small number of RF pulses is not feasible, hindering scan time reduction. Finally, a time ‐ efficiWnG spiral sampling scheme, usually applied in MRF, [10] (# mrm27594 - bib - 0010) {ref - type = " ref " }, [16] (# mrm27594 - bib - 0016) {ref - type = " ref " }, [17] (# mrm27594 - bib - 0017) {ref - type = " ref " }, [18] (# mrm27594 - bib - 00@9) {ref - type = " ref " }, [19] (# mrm27594 - bib - 0019) {ref - type = " ref "} introduces off ‐ resonance effects in each of the individual MRF images. [20] (# mrm27594 - bib - 0020) {ref - type = " ref "} This ocsurD even when combined with unbalanced sequences such as fast imaging with steady state precession, [16] (# mrm27594 - bib - 0016) {ref - type = " ref "} which are in themselves robust to off ‐ resonance effects. [21] (# mrm27594 - bib - 0021) {ref - type = " ref "} The off ‐ resonance effects present in spiral sampling schemes are much stronger at high field, where they result in blurring, [22] (# mrm27594 - bib - 0022) {ref - type = " ref "} caused by strong main field inhomogeneities (particularly in the eye region due to many air ‐ tissue ‐ bone interfaces ), as well as the presence of significant amounts of off ‐ resonant orbital fat ar(6nd the eye. In this work, a Cartesian sampling scheme is used, which is more robust than spiral sampling to off ‐ resonance effects, but which is significantly less time ‐ efficient. [23] (# mrm27594 - bib - 0023) {ref - type = " ref "} With such a Cartesian sampling scheme, undersampling artifacts have a more structured nature compared with spiral sampling, which increases the temporal coherence of the artifacts in the MRF image series. [10] (# mrm27594 - bib - 0010) {ref - type = " ref " }, [20] (# mrm27594 - bib - 0020) {ref - type = " ref "} In this case, direct matching of the measured MRF signal reconstructed by plain Fourier transformations, to the simulated dictionary elements is not sufficiently accurate for high undersampling factors. [24] (# mrm27594 - bib - 0024) {ref - type = " ref " }, [25] (# mrm27594 - bib - 0025) {ref - type = " ref "} Therefore, the quality of the reconstructed MRF data has to be improved before the matching process. Compressed sensing (CS) has been introduced as a technique to reconstruct images from randomly undersampled data by enforcing signal sparsity (in the spatial dimension only or both in spatial and temporal dimensions ), [26] (# mrm27594 - bib - 0026) {ref - type = " ref " }, [27] (# mrm27594 - bib - 0027) {ref - type = " ref "} allowing a scan time reduction in many applications. [28] (# mrm27594 - bib - 0028) {ref - type = " ref " }, [29] (# mrm27594 - bib - 0029) {ref - type = " ref " }, [30] (# mrm27594 - bib - 0030) {ref - type = " ref "} The flexibility of MRF toward different sampling schemes and undersampling factors makes it possible to rwdonstruct the source images by means of CS. [27] (# mrm27594 - bib - 0027) {ref - type = " ref " }, [31] (# mrm27594 - bib - 0031) {ref - type = " ref " }, [32] (# mrm27594 - bib - 0032) {ref - type = " ref "} Higher acceleration factors might be feasible if the correlation in the temporal dimension is better used. [33] (# mrm27594 - bib - 0033) {ref - type = " ref "} Examples of such reconstructions specifically tailored to MRF are given in Davies et al, Pierre et al, and Zhao et al [34] (# mrm27594 - bib - 0034) {ref - type = " ref " }, [35] (# mrm27594 - bib - 0035) {ref - type = " ref " }, [36] (# mrm27594 - bib - 0036) {ref - type = " ref "} which take into account the simulated dictionary atoms in the image reconstruction process. Recent work has shown that the temporal correlation in the MRF data can be exploited even further by incorporating the low rank structure of the data into the cost function, [37] (# mrm27594 - bib - 0037) {ref - type = " ref "} a technique which was introduced into MR in Liang [ | Koolstra K, Beenakker J‐WM, Koken P, Webb A, Börnert P. MR fingerprinting the eye at 7T using compressed sensing and matrix completion‐based reconstructions. Magn Reson Med. 2019;81:2551--2565. 10.1002/mrm.27594 30421448 **Funding information** This project was partially funded by the European Research Council Advanced Grant 670629 MRI. 1. INTRODUCTION {#mrm27594-sec-0005} =============== Ophthalmologic disease diagnosis conventionally relies on ultrasound and optical imaging such as and fluorescent (FAG), MRI is increasingly being used in the radiological community.[1](#mrm27594-bib-0001){ref-type="ref"}, [2](#mrm27594-bib-0002){ref-type="ref"}, [3](#mrm27594-bib-0003){ref-type="ref"} One of the main advantages of MRI is its capability assess nontransparent such ocular or structures behind the such as the eye muscles. Currently, however, applications are mainly based on qualitative MRI methods using the large number of tissue contrasts addressable by MR. As an example, fat‐suppressed T~2~‐weighted MRI is the standard to detect inflammation in the eye muscles,[4](#mrm27594-bib-0004){ref-type="ref"}, [5](#mrm27594-bib-0005){ref-type="ref"} in the diagnosis of retinoblastoma, a rare intraocular cancer in children, standard T~1~‐ and T~2~‐weighted MRI is often performed confirm the presence the tumor and to screen for optic nerve In more recent applications of MRI, such uveal melanoma (the most common primary tumor), quantitative MRI techniques including DWI[6](#mrm27594-bib-0006){ref-type="ref"} and DCE imaging[7](#mrm27594-bib-0007){ref-type="ref"} have been shown, but currently is based on qualitative methods.[3](#mrm27594-bib-0003){ref-type="ref"} To personalize treatment plans quantitative parameters of the tissues involved, as can be acquired invasively for by performing biopsies,[8](#mrm27594-bib-0008){ref-type="ref"} are highly desirable. However, quantitative parameter mapping by means of MRI requires long times, which would result in significant eye‐motion artifacts, as well patient discomfort.[9](#mrm27594-bib-0009){ref-type="ref"} MR fingerprinting is a recently introduced method for rapid quantitation of tissue relaxation times and other MR‐related parameters.[10](#mrm27594-bib-0010){ref-type="ref"} It uses a flip angle sweep to induce a signal evolution for each tissue type. Incoherent undersampling can be applied during sampling of the MRF train, enabling acceleration of the MRF scans.[10](#mrm27594-bib-0010){ref-type="ref"} Together with its to measure simultaneously T~1~ and MRF offers a solution to the of obtaining quantitative in an efficient manner and in relatively scanning times. One of the main challenges in imaging is in‐plane and through‐plane eye motion, often associated with eye blinking.[11](#mrm27594-bib-0011){ref-type="ref"}, [12](#mrm27594-bib-0012){ref-type="ref"}, [13](#mrm27594-bib-0013){ref-type="ref"} motion in corrupted k‐space data introduces artifacts and blurring throughout Shortening scans would reduce motion‐related artifacts, but standard acceleration techniques are not optimal for the current eye application due to following reasons. First, a cued‐blinking protocol is typically used to control and the eye motion.[3](#mrm27594-bib-0003){ref-type="ref"}, [11](#mrm27594-bib-0011){ref-type="ref"} This requires an instruction screen placed at the end of the MR tunnel to be visible to the patient which complicates the use of small phased array coils in front of eye, the view. Instead, a custom‐built single‐element eye loop coil which provides a high local SNR[3](#mrm27594-bib-0003){ref-type="ref"} and screen visibility, but which clearly excludes the possibility of scan by means of parallel imaging.[14](#mrm27594-bib-0014){ref-type="ref"} Second, the gel‐like vitreous body has an extremely long T~1~, particularly at high field.[15](#mrm27594-bib-0015){ref-type="ref"} Its value of 3 to 5 s requires a long duration of the sequence to encode the MR parameters (T~1~,T~2~) Thus, using a flip angle train with a small number of RF pulses is not feasible, scan time reduction. Finally, a time‐efficient spiral sampling scheme, usually in MRF,[10](#mrm27594-bib-0010){ref-type="ref"}, [16](#mrm27594-bib-0016){ref-type="ref"}, [18](#mrm27594-bib-0018){ref-type="ref"}, [19](#mrm27594-bib-0019){ref-type="ref"} introduces off‐resonance effects in of the individual MRF images.[20](#mrm27594-bib-0020){ref-type="ref"} This occurs even when with unbalanced such as fast imaging with steady state precession,[16](#mrm27594-bib-0016){ref-type="ref"} which are themselves robust to off‐resonance effects.[21](#mrm27594-bib-0021){ref-type="ref"} The off‐resonance effects present in spiral sampling schemes are much at high field, where they result in blurring,[22](#mrm27594-bib-0022){ref-type="ref"} strong main field inhomogeneities (particularly the eye region due to many air‐tissue‐bone interfaces), as well as the presence of significant amounts of off‐resonant orbital fat around the eye. this work, a Cartesian sampling scheme is used, which is more robust than spiral sampling to off‐resonance effects, but which is significantly less time‐efficient.[23](#mrm27594-bib-0023){ref-type="ref"} With such a Cartesian sampling scheme, undersampling artifacts have a more structured nature compared with spiral which increases the temporal coherence of the artifacts in the MRF image [20](#mrm27594-bib-0020){ref-type="ref"} case, matching of measured signal reconstructed by plain Fourier transformations, to the simulated dictionary elements is not sufficiently accurate high factors.[24](#mrm27594-bib-0024){ref-type="ref"}, [25](#mrm27594-bib-0025){ref-type="ref"} Therefore, the quality of the reconstructed MRF data has to improved before the matching process. Compressed sensing (CS) has been introduced as a technique to images from randomly undersampled data enforcing signal sparsity (in the spatial dimension or both in spatial and dimensions),[26](#mrm27594-bib-0026){ref-type="ref"}, [27](#mrm27594-bib-0027){ref-type="ref"} allowing a scan time reduction applications.[28](#mrm27594-bib-0028){ref-type="ref"}, [29](#mrm27594-bib-0029){ref-type="ref"}, [30](#mrm27594-bib-0030){ref-type="ref"} The flexibility of toward different sampling schemes and undersampling factors makes it possible to reconstruct the source images by means CS.[27](#mrm27594-bib-0027){ref-type="ref"}, [31](#mrm27594-bib-0031){ref-type="ref"}, [32](#mrm27594-bib-0032){ref-type="ref"} Higher acceleration factors might be feasible if the correlation in the dimension is better used.[33](#mrm27594-bib-0033){ref-type="ref"} Examples such reconstructions specifically tailored to MRF are given in Davies al, Pierre et al, and Zhao et al[34](#mrm27594-bib-0034){ref-type="ref"}, [35](#mrm27594-bib-0035){ref-type="ref"}, [36](#mrm27594-bib-0036){ref-type="ref"} which take into account the simulated dictionary atoms in the image reconstruction process. Recent work has shown that the temporal correlation in the MRF data can be exploited even further by incorporating low rank structure of the data into the cost function,[37](#mrm27594-bib-0037){ref-type="ref"} a technique was introduced into MR in Liang[ | koOLSTra k, BEeNAKKEr J‐wM, KoKEN P, webb a, börnERT P. carTesIAn mr fINgErpRInTiNG IN The eYE aT 7T UsinG comPReSSeD SEnsInG ANd MATriX coMpLEtIOn‐bASed RECONStrUcTIons. MaGn ResOn MEd. 2019;81:2551--2565. 10.1002/mRM.27594 30421448
**FUNDIng INFOrMaTIOn**
thIs PrOJECT WAs PArTIalLy FUnDeD bY The EuROPeaN REsearcH COUnCil ADVANCED GrAnt 670629 NOmA Mri.
1. INtRodUCTION {#mrm27594-sEc-0005}
===============
OPhtHalMOLOgic disease DiagNOsIs CoNvenTionAlLy ReliEs maINly on ULtraSOuND aND OPtiCAl iMAGING tecHNIqUeS sUCH as fuNdUs pHOtOGRAPHY and FluoRescEnt ANGIOgRaphY (FaG), mri iS iNCREAsINGLy bEING useD IN The raDIOLOGicaL COmMUNITY.[1](#mrm27594-bIb-0001){ReF-tyPE="REf"}, [2](#Mrm27594-BiB-0002){rEF-TYpe="rEF"}, [3](#MrM27594-BIb-0003){Ref-tYpE="ref"} One oF tHe main aDvAntAGes of MRi iS Its caPABilitY tO AsSeSs NoNtraNspARent tIsSuEs sUcH aS OculaR TUmORs oR StRUcTUrES bEhINd the glOBe such aS tHe eYE mUscLeS. CURrenTLY, howeVER, tHEsE AppliCAtIOnS arE maiNly BaseD on quALItaTIve mRI MetHODS usING tHE LArgE nUMbeR oF tiSsUe cOnTRASTS AdDREssabLE bY Mr. As An eXAmPlE, iN graVES' OPhThALMOpathY FAt‐sUppresSeD T~2~‐WeiGHTEd mRI IS tHe sTanDard TO DETeCT INFLamMation In THe eyE mUscLES,[4](#MRM27594-bIB-0004){rEF-tYPE="Ref"}, [5](#MrM27594-bIB-0005){rEF-tYPE="Ref"} whEREAs IN tHE DiAgnosIs OF rETInOblaSTomA, A Rare IntrAOCular cAnCEr iN chIlDReN, sTaNDarD T~1~‐ AND t~2~‐wEIgHTEd MRI Is OftEn pErFORmeD to cOnfIRM THE pREsEncE of THe TumOr and TO ScReEn FOr POTeNTiAl OpTic NeRve InVoLVemeNt.[2](#mrm27594-bib-0002){rEf-TYpE="ReF"} In mOre reCeNT ophTHALMolOGiC APplIcAtIoNS of mrI, sUCH AS uvEal meLAnoma (tHe MOSt coMmoN pRIMAry InTRaocULaR Tumor), QUAnTITaTIvE Mri teCHniqUes iNcluDiNG dwi[6](#mrM27594-BIb-0006){reF-TYpe="REF"} AND Dce iMAgInG[7](#mRm27594-BIB-0007){ref-tYpe="REf"} HaVE BeEn sHoWn, bUT CuRRenTLY diaGNosiS Is Still BaseD ON quALitatiVE MeTHODs.[3](#MRM27594-BIB-0003){reF-TYpe="rEf"}
To PersONaliZE TreaTMEnT pLANs qUANtitATiVe PaRamEterS oF thE tiSSUES involVED, as cAN be acQUiREd iNvAsIVElY foR exaMPLe BY pErfOrMIng bIOPsIES,[8](#mRM27594-bIb-0008){REf-tYPe="ref"} arE hiGhlY desirAbLE. HoWeVEr, qUaNtItAtIVE ParaMETeR MApping BY meaNS oF mRI REQUiRES lONg eXAminatIOn TiMES, WHIcH wOULD REsulT In sIgNiFiCAnt EYe‐mOtIon aRtIFACtS, aS welL as pATiEnT dISCoMForT.[9](#mrm27594-bIB-0009){ref-tyPe="rEF"} MR fiNGeRpriNTinG (Mrf) iS A reCENtLy INTRodUCeD METHOD foR rapID QuantiTAtIOn oF TIssUE RELAxation TIMES and oTHEr MR‐RelaTEd pARAmETers.[10](#MRm27594-BIb-0010){REF-TypE="rEf"} IT usES A FlIP angLe sWeep TO InDUCe A UNiQuE sIgnAL eVOlUtiOn FOR EAch TiSSuE TypE. incoHErEnT UnDeRsAmPLING Can Be aPPlieD DuRiNg sampLing Of tHe mRf trAiN, enaBLinG acCEleraTIoN OF THe MrF ScaNS.[10](#mRM27594-BiB-0010){ReF-type="ref"} TogEther wiTH iTS ABiLiTy TO mEASUrE sIMultANeOusLy T~1~ ANd T~2~, MRf OFferS a SoLuTIoN to thE PrOBLem Of ObtAINIng quAntitAtIvE MEaSUreS iN AN EfFiCIEnt maNneR and in rElATIvelY SHORt scaNNING TIMEs.
OnE Of THe Main cHaLLENGES iN OCULAR ImagiNg Is iN‐plANe and thROuGh‐PlANe eYe MOTIon, oFTen AssocIATED with eye BlINKInG.[11](#MRm27594-biB-0011){reF-type="reF"}, [12](#mRM27594-bIb-0012){ref-TyPe="REF"}, [13](#mRM27594-bIB-0013){REF-typE="reF"} tHE motiON rESUltS In COrRUPteD k‐SpaCe DATa tHat INtrODuCes ARTiFacts anD blUrRinG thrOUghout ThE ENtiRe ImaGe. ShORTENING tHe SCaNS WouLd REDuce MOTIon‐ReLaTed arTiFaCts, but staNdARD aCCeLeRAtiOn techNiqueS ARe nOT oPTIMAL foR tHE CurReNT eYE ApPLicATIOn DUe TO thE FoLlOwInG 3 reaSONs. fIrST, a cued‐BLiNkING pRotOCOL is tYpIcALLY UseD tO conTROL aNd ReDucE the eye mOTIon.[3](#MRM27594-bIb-0003){ref-TyPe="Ref"}, [11](#Mrm27594-BIB-0011){rEF-TyPE="ReF"} ThIs ReQUIReS aN inSTRUCtIon ScReeN plAcED at tHe eND of tHE mR TUNNEl to bE VIsIBle tO tHE PAtient wHICh cOmPlIcateS THe uSe OF sMall PHAseD arRAY RecEiVe COilS in froNt of the Eye, BLOCking THE viEW. iNstEAD, a cUstom‐buIlT sINgle‐eLEMeNT Eye loOP CoIl iS uSeD, wHIch prOvIdES A HIGh lOCAl SNr[3](#mrm27594-BiB-0003){reF-Type="ReF"} anD ScREEn vIsIbILitY, BuT whIcH CLEarly EXClUDes THE PoSSiBILity of ScAN ACCeLERatiOn bY mEAns oF paralLeL iMAgINg.[14](#mrm27594-Bib-0014){REF-typE="Ref"} SEConD, the gEl‐lIKE VitREOUs BOdy hAS aN EXTrEmelY loNG t~1~, pARTicularLY aT hIGH FiELD.[15](#mRM27594-BiB-0015){REf-tYpe="rEF"} ITs value of 3 tO 5 S REquIrES A loNg DURaTIon OF THe mrf SEQUenCe To ENCOdE THE mr PaRAMEters (t~1~,t~2~) SuFFicIEntLy. THuS, UsiNG a FLIP anGLe tRaiN WIth A sMALL NUmbEr OF rF PUlSES IS Not FEAsible, HiNDERING scAn TiMe reDucTiON. finaLly, a TiME‐efFiCIent spirAl SAmPLING sCheME, uSUAllY ApPLIeD in MRf,[10](#MRm27594-BIB-0010){rEf-tyPE="reF"}, [16](#MRM27594-bIB-0016){REf-TYpe="REf"}, [17](#MRM27594-bib-0017){REF-type="ReF"}, [18](#mrM27594-Bib-0018){rEF-tYpe="Ref"}, [19](#mRm27594-BiB-0019){REF-TYpe="rEF"} inTRODUceS oFF‐ReSONAncE EfFECts In EacH oF THE indIviDUal MRf iMAgEs.[20](#mRm27594-bib-0020){REF-Type="Ref"} tHis OCcUrS EVeN WhEn comBinEd WiTh unbAlAnCed SeQUenCeS Such aS FAst ImAgiNG wITh steadY STaTe PreCesSion,[16](#MRM27594-biB-0016){reF-tyPE="reF"} wHICH ArE iN theMsELVeS rObUST TO Off‐rEsONaNce eFFECtS.[21](#MRM27594-BiB-0021){Ref-Type="reF"} thE OFF‐rEsoNANce EFfectS PrESeNt IN SPiraL SAmpLIng SChemes aRe MuCH sTrONGER at HIGh fIELd, wHERe tHEY rESuLT iN bLurRinG,[22](#mrM27594-BiB-0022){REF-TyPE="REf"} cAUseD by STronG MaIN FIELd iNhoMOgenEITieS (parTiCUlarLY IN thE eYe REgiOn DuE TO MANy aIR‐tISSue‐bOnE InTeRfAceS), As wELL As The pResencE of siGnificANT aMouNTS oF OFf‐rESONaNt ORbitaL FAt AROUnd tHE EYe.
In tHiS wOrK, A cArTesIaN sAmplINg ScHEME iS useD, whicH Is MorE ROBUSt THAn SPIrAl SaMPlINg To ofF‐reSOnAnce EFfectS, BUt wHiCH is siGNIFicanTLY less tIme‐EFFicient.[23](#MRM27594-biB-0023){REf-TYPe="reF"} WITH sUch a caRTeSIaN SaMPLiNg schEME, undErsAmPlINg aRTIFaCts havE A MORe StRuCTurEd NAtuRE CoMPared wItH SPiral SAmplING, wHIch INCReasES THE TEMPoRaL cOherenCe Of tHE aRtIfAcTS iN the mRF ImAGe SERiEs.[10](#mRM27594-bib-0010){ref-TyPe="REF"}, [20](#mRM27594-Bib-0020){REf-tYpe="ReF"} IN this cAsE, DIREct MATcHIng of thE mEasUrED mRf SignAl reCOnsTrUctEd By PlaIn fOurier TRansfoRmaTioNS, tO thE SiMUlATeD DICTionaRy ElEmEnTs IS NoT SUffIcieNtly aCcuRATE For hIGH UNdERSAmPLING fAcTOrs.[24](#MRM27594-BIB-0024){rEf-tYpe="ReF"}, [25](#MrM27594-bIB-0025){REf-tYPe="REf"} TherefORE, The QUAlIty Of tHE RecONSTRuctEd Mrf DAtA HaS TO be iMpROVed BefOre the maTChinG PROcess.
CoMpRESSeD SEnsINg (cS) has beeN intrOduceD aS A teCHniQUE to rECONstruCt IMAgeS frOm randOmLy UNDErSAmPled daTA By EnFORCING SIgNAl sparsItY (in THe spATIal dimeNSIon OnLY OR botH IN SpAtiAl and TEmpoRAL DiMENsioNs),[26](#MRM27594-bib-0026){rEf-typE="ReF"}, [27](#mRm27594-bib-0027){rEF-TYpE="reF"} ALLoWInG a ScAn TIMe rEDUcTIOn iN mANy APpLICAtiOns.[28](#mrM27594-bIB-0028){reF-tyPE="REF"}, [29](#MRM27594-BiB-0029){REf-TYpe="reF"}, [30](#mRm27594-BiB-0030){REf-tYPe="REf"} The fLeXibilitY Of mRf tOward dIffeRenT sAMPliNg sChEMEs AND unDersaMPLING FACTOrs mAkES iT PoSsiBle To reCoNStruCt thE souRce IMagEs BY mEanS oF CS.[27](#mRM27594-bIB-0027){reF-typE="reF"}, [31](#mrm27594-bIb-0031){rEf-tYpE="Ref"}, [32](#MRM27594-bIB-0032){ref-type="rEf"} higHEr aCcELERaTiON fACtOrs mIghT BE feaSIble If THE CORRElATiOn In THE temPORaL dIMEnsIoN is BetTEr useD.[33](#mRM27594-Bib-0033){ReF-TypE="ReF"} exAmpLes Of SUcH RECOnStrUctIONs SpeCiFicAllY TaIloreD to mrF ARE GivEN IN DaVIeS eT aL, pIerre Et Al, ANd zHaO Et Al[34](#mrM27594-bIb-0034){rEF-TYPE="Ref"}, [35](#mRm27594-biB-0035){Ref-tYpE="ReF"}, [36](#mRM27594-bIb-0036){reF-TYpe="rEF"} wHiCh TakE iNtO ACCounT THE SIMulATEd DiCTiOnARY atoMs IN THe iMaGE reCOnStrUctIoN pROcEss.
REcENt WorK HaS shown ThAT THe TEmPoral coRrelAtion IN THe mrf DATA Can be eXpLOiTED eVEn fURThER bY InCorpOrAtinG THe LOw RaNk STruCtuRE Of tHe DaTa InTo THE cOST fUNCtIon,[37](#mrM27594-BIB-0037){REf-tYpE="rEf"} A TEChniQuE wHiCH WAS inTRODUCed INto mR iN lIaNG[ | Koolstra K, Beenakker J‐WM, Koken P, WebbA, Börnert P. CartesianMR fingerprintingin the eye at7T using compressed sensing and matrix completion‐basedreconstructions. Magn Reson Med. 2019;81:2551--2565. 10.1002/mrm.27594 30421448 **Funding information** Thisproject was partially funded by the EuropeanResearch Council Advanced Grant 670629 NOMA MRI. 1. INTRODUCTION {#mrm27594-sec-0005} =============== Ophthalmologic disease diagnosis conventionally relies mainly on ultrasound and optical imagingtechniques such asfundus photography andfluorescent angiography (FAG), MRI is increasingly being used in the radiological community.[1](#mrm27594-bib-0001){ref-type="ref"}, [2](#mrm27594-bib-0002){ref-type="ref"}, [3](#mrm27594-bib-0003){ref-type="ref"} One of themainadvantages of MRI is its capability to assess nontransparent tissues such as ocular tumors or structures behind the globesuch as theeye muscles. Currently, however,these applications aremainly based on qualitative MRI methods using the large numberof tissuecontrastsaddressable by MR. As an example, in Graves' ophthalmopathy fat‐suppressed T~2~‐weighted MRI is the standardtodetect inflammationin the eye muscles,[4](#mrm27594-bib-0004){ref-type="ref"}, [5](#mrm27594-bib-0005){ref-type="ref"}whereas in the diagnosis of retinoblastoma, arare intraocular cancer in children,standard T~1~‐ and T~2~‐weightedMRI is often performed to confirm the presence of the tumor and to screen forpotential optic nerve involvement.[2](#mrm27594-bib-0002){ref-type="ref"} In more recentophthalmologicapplications of MRI, such as uveal melanoma (the most common primary intraocular tumor), quantitativeMRI techniques including DWI[6](#mrm27594-bib-0006){ref-type="ref"}and DCE imaging[7](#mrm27594-bib-0007){ref-type="ref"} have been shown, but currently diagnosisis still based on qualitative methods.[3](#mrm27594-bib-0003){ref-type="ref"} To personalize treatment plans quantitative parameters of the tissues involved, ascanbe acquired invasively forexampleby performingbiopsies,[8](#mrm27594-bib-0008){ref-type="ref"}are highly desirable. However, quantitative parameter mapping by means of MRI requires long examination times,which wouldresult in significant eye‐motion artifacts, as wellas patient discomfort.[9](#mrm27594-bib-0009){ref-type="ref"} MR fingerprinting (MRF) is a recently introduced method for rapid quantitation of tissue relaxation timesand other MR‐related parameters.[10](#mrm27594-bib-0010){ref-type="ref"} It usesa flip angle sweep to induce a unique signal evolutionfor each tissuetype. Incoherentundersampling can be appliedduring samplingof the MRF train, enabling acceleration of the MRF scans.[10](#mrm27594-bib-0010){ref-type="ref"}Together with its ability to measure simultaneously T~1~ and T~2~, MRF offers a solutionto the problem of obtaining quantitative measures in an efficient manner and in relatively short scanning times. One of the main challenges in ocular imaging is in‐plane and through‐plane eye motion,often associated with eye blinking.[11](#mrm27594-bib-0011){ref-type="ref"}, [12](#mrm27594-bib-0012){ref-type="ref"}, [13](#mrm27594-bib-0013){ref-type="ref"} The motion results incorruptedk‐space data that introduces artifactsand blurring throughout the entire image. Shortening the scanswould reduce motion‐related artifacts, but standard acceleration techniques arenot optimal for the current eye applicationdueto thefollowing 3 reasons. First, a cued‐blinking protocol is typically used to control and reduce the eye motion.[3](#mrm27594-bib-0003){ref-type="ref"}, [11](#mrm27594-bib-0011){ref-type="ref"} Thisrequires an instruction screen placed at the end of theMR tunnel to be visible to thepatient which complicates the use of small phased array receive coils in front of the eye, blocking the view.Instead, a custom‐built single‐elementeye loop coil is used, which providesa high local SNR[3](#mrm27594-bib-0003){ref-type="ref"} and screen visibility,but which clearly excludes the possibility of scan acceleration by means of parallelimaging.[14](#mrm27594-bib-0014){ref-type="ref"} Second, thegel‐like vitreousbody has an extremely long T~1~,particularly at high field.[15](#mrm27594-bib-0015){ref-type="ref"} Its value of 3 to 5 s requires along duration of the MRF sequence to encode the MR parameters(T~1~,T~2~) sufficiently. Thus, usinga flip angle train with a small number of RF pulses is not feasible, hindering scan time reduction.Finally, a time‐efficient spiral sampling scheme, usually applied in MRF,[10](#mrm27594-bib-0010){ref-type="ref"},[16](#mrm27594-bib-0016){ref-type="ref"}, [17](#mrm27594-bib-0017){ref-type="ref"}, [18](#mrm27594-bib-0018){ref-type="ref"}, [19](#mrm27594-bib-0019){ref-type="ref"} introduces off‐resonance effects in eachof the individual MRF images.[20](#mrm27594-bib-0020){ref-type="ref"} This occurs even when combined with unbalanced sequences such as fast imagingwith steady stateprecession,[16](#mrm27594-bib-0016){ref-type="ref"} which are in themselves robust to off‐resonanceeffects.[21](#mrm27594-bib-0021){ref-type="ref"} The off‐resonance effects present in spiral sampling schemes aremuch stronger at high field, wherethey result in blurring,[22](#mrm27594-bib-0022){ref-type="ref"} caused by strong main field inhomogeneities (particularly in the eye region due to many air‐tissue‐bone interfaces), as well as the presence of significant amounts of off‐resonant orbitalfat around the eye. In thiswork, a Cartesian sampling schemeis used, which is more robust than spiral sampling tooff‐resonance effects, but which issignificantly less time‐efficient.[23](#mrm27594-bib-0023){ref-type="ref"} With such a Cartesian sampling scheme, undersampling artifacts have a more structured nature compared with spiral sampling, which increases the temporal coherence of the artifacts in the MRF image series.[10](#mrm27594-bib-0010){ref-type="ref"}, [20](#mrm27594-bib-0020){ref-type="ref"} In this case, direct matching of the measured MRF signal reconstructed by plain Fourier transformations, to the simulated dictionary elements is not sufficiently accurateforhigh undersampling factors.[24](#mrm27594-bib-0024){ref-type="ref"}, [25](#mrm27594-bib-0025){ref-type="ref"} Therefore,the quality of the reconstructed MRF datahas tobe improved before the matching process.Compressed sensing (CS) hasbeenintroduced as a technique to reconstruct images from randomly undersampled data by enforcing signal sparsity (in the spatialdimension only or both in spatial andtemporal dimensions),[26](#mrm27594-bib-0026){ref-type="ref"}, [27](#mrm27594-bib-0027){ref-type="ref"} allowing a scan time reduction in many applications.[28](#mrm27594-bib-0028){ref-type="ref"}, [29](#mrm27594-bib-0029){ref-type="ref"},[30](#mrm27594-bib-0030){ref-type="ref"}The flexibility of MRFtoward different sampling schemesand undersampling factors makes it possible to reconstruct the source images by means of CS.[27](#mrm27594-bib-0027){ref-type="ref"}, [31](#mrm27594-bib-0031){ref-type="ref"},[32](#mrm27594-bib-0032){ref-type="ref"} Higher acceleration factors might be feasible if the correlation in the temporal dimension is better used.[33](#mrm27594-bib-0033){ref-type="ref"}Examples of suchreconstructions specifically tailored to MRF are given in Davies et al, Pierre et al, and Zhao etal[34](#mrm27594-bib-0034){ref-type="ref"}, [35](#mrm27594-bib-0035){ref-type="ref"}, [36](#mrm27594-bib-0036){ref-type="ref"} which take into accountthesimulated dictionary atoms in the imagereconstruction process. Recent work has shown that the temporal correlationin theMRF data can be exploited even further by incorporating the low rank structure ofthe data into the cost function,[37](#mrm27594-bib-0037){ref-type="ref"} a technique which was introduced into MR in Liang[ | Koolstra _K,_ Beenakker _J‐WM,_ Koken P, Webb A, Börnert P. _Cartesian_ MR _fingerprinting_ in the eye _at_ _7T_ using compressed sensing and matrix completion‐based reconstructions. Magn _Reson_ _Med._ 2019;81:2551--2565. 10.1002/mrm.27594 30421448 _**Funding_ information** _This_ project was partially funded by the European Research Council Advanced Grant 670629 NOMA MRI. _1._ INTRODUCTION {#mrm27594-sec-0005} =============== Ophthalmologic disease _diagnosis_ conventionally relies mainly on ultrasound and optical _imaging_ techniques such _as_ fundus _photography_ _and_ fluorescent angiography (FAG), _MRI_ is increasingly being used in the radiological community.[1](#mrm27594-bib-0001){ref-type="ref"}, [2](#mrm27594-bib-0002){ref-type="ref"}, [3](#mrm27594-bib-0003){ref-type="ref"} _One_ of the main advantages _of_ MRI is its capability to assess nontransparent tissues such as ocular _tumors_ or structures behind the globe such as the eye muscles. Currently, however, these applications are mainly based _on_ qualitative _MRI_ methods _using_ the large number of tissue _contrasts_ addressable _by_ _MR._ As an example, in _Graves'_ ophthalmopathy fat‐suppressed T~2~‐weighted MRI is the standard _to_ detect inflammation in the _eye_ _muscles,[4](#mrm27594-bib-0004){ref-type="ref"},_ _[5](#mrm27594-bib-0005){ref-type="ref"}_ whereas _in_ the diagnosis of retinoblastoma, _a_ _rare_ _intraocular_ cancer _in_ children, standard T~1~‐ _and_ T~2~‐weighted _MRI_ is often performed _to_ _confirm_ the presence of the tumor and to screen for potential _optic_ _nerve_ involvement.[2](#mrm27594-bib-0002){ref-type="ref"} In more recent ophthalmologic applications of MRI, such _as_ uveal melanoma (the most common primary intraocular _tumor),_ quantitative MRI techniques including DWI[6](#mrm27594-bib-0006){ref-type="ref"} and DCE imaging[7](#mrm27594-bib-0007){ref-type="ref"} have been _shown,_ but currently diagnosis is _still_ _based_ _on_ qualitative _methods.[3](#mrm27594-bib-0003){ref-type="ref"}_ To personalize treatment plans _quantitative_ parameters of the tissues involved, _as_ can _be_ _acquired_ invasively for example by _performing_ biopsies,[8](#mrm27594-bib-0008){ref-type="ref"} are highly desirable. However, _quantitative_ parameter mapping by _means_ _of_ MRI requires _long_ _examination_ times, which would result in significant eye‐motion _artifacts,_ _as_ well as _patient_ discomfort.[9](#mrm27594-bib-0009){ref-type="ref"} MR _fingerprinting_ (MRF) is a recently introduced method for _rapid_ quantitation of _tissue_ relaxation times _and_ other MR‐related parameters.[10](#mrm27594-bib-0010){ref-type="ref"} It uses a flip angle sweep to induce _a_ _unique_ signal evolution for each tissue type. _Incoherent_ undersampling _can_ be _applied_ during _sampling_ of the _MRF_ train, enabling acceleration of _the_ MRF scans.[10](#mrm27594-bib-0010){ref-type="ref"} Together with its ability to measure _simultaneously_ T~1~ and T~2~, MRF _offers_ a solution to _the_ problem of _obtaining_ quantitative measures in an efficient manner and _in_ relatively short scanning times. One of _the_ main _challenges_ in _ocular_ imaging is in‐plane and through‐plane eye motion, often _associated_ with eye blinking.[11](#mrm27594-bib-0011){ref-type="ref"}, _[12](#mrm27594-bib-0012){ref-type="ref"},_ _[13](#mrm27594-bib-0013){ref-type="ref"}_ The motion results in corrupted _k‐space_ data that introduces artifacts _and_ blurring throughout the entire image. _Shortening_ the scans would reduce _motion‐related_ artifacts, but standard acceleration techniques are not optimal for _the_ current eye application _due_ _to_ the _following_ 3 _reasons._ First, _a_ cued‐blinking _protocol_ is typically used to control and reduce the eye motion.[3](#mrm27594-bib-0003){ref-type="ref"}, _[11](#mrm27594-bib-0011){ref-type="ref"}_ This requires an instruction screen placed at the end of the MR tunnel to be visible to the patient _which_ complicates the use of small phased array receive coils in _front_ of the _eye,_ blocking the view. Instead, a custom‐built single‐element _eye_ loop _coil_ is used, _which_ provides a high local SNR[3](#mrm27594-bib-0003){ref-type="ref"} _and_ screen visibility, but which _clearly_ excludes the possibility _of_ scan acceleration by means of parallel imaging.[14](#mrm27594-bib-0014){ref-type="ref"} Second, the gel‐like _vitreous_ body _has_ an extremely long T~1~, particularly at high field.[15](#mrm27594-bib-0015){ref-type="ref"} Its value _of_ 3 to 5 s requires _a_ long duration of the MRF sequence to encode the MR parameters (T~1~,T~2~) sufficiently. Thus, using a _flip_ angle train _with_ _a_ small number of RF pulses is not feasible, _hindering_ scan time reduction. _Finally,_ a time‐efficient spiral sampling scheme, usually _applied_ in MRF,[10](#mrm27594-bib-0010){ref-type="ref"}, [16](#mrm27594-bib-0016){ref-type="ref"}, [17](#mrm27594-bib-0017){ref-type="ref"}, [18](#mrm27594-bib-0018){ref-type="ref"}, [19](#mrm27594-bib-0019){ref-type="ref"} introduces off‐resonance _effects_ in _each_ of the individual MRF images.[20](#mrm27594-bib-0020){ref-type="ref"} This _occurs_ _even_ when _combined_ with unbalanced sequences _such_ as fast _imaging_ _with_ steady state precession,[16](#mrm27594-bib-0016){ref-type="ref"} which are in themselves robust to off‐resonance effects.[21](#mrm27594-bib-0021){ref-type="ref"} The off‐resonance _effects_ present in spiral sampling _schemes_ are much _stronger_ _at_ high field, where they _result_ in blurring,[22](#mrm27594-bib-0022){ref-type="ref"} caused _by_ strong main field inhomogeneities (particularly in _the_ _eye_ region due _to_ many air‐tissue‐bone interfaces), as well as the presence of _significant_ _amounts_ of _off‐resonant_ orbital fat _around_ the _eye._ In this work, _a_ Cartesian _sampling_ scheme is used, which is more _robust_ than spiral sampling _to_ _off‐resonance_ effects, but which is significantly less _time‐efficient.[23](#mrm27594-bib-0023){ref-type="ref"}_ With such a _Cartesian_ sampling _scheme,_ undersampling artifacts _have_ a more structured nature compared with spiral sampling, which increases the temporal coherence of the artifacts in the _MRF_ image series.[10](#mrm27594-bib-0010){ref-type="ref"}, _[20](#mrm27594-bib-0020){ref-type="ref"}_ In this case, direct _matching_ of _the_ measured MRF signal _reconstructed_ by plain Fourier transformations, to the simulated dictionary elements is not sufficiently accurate for high undersampling factors.[24](#mrm27594-bib-0024){ref-type="ref"}, [25](#mrm27594-bib-0025){ref-type="ref"} Therefore, _the_ quality of the reconstructed _MRF_ _data_ has _to_ be improved before _the_ _matching_ process. _Compressed_ sensing (CS) has _been_ introduced as _a_ technique to _reconstruct_ _images_ from randomly undersampled data by enforcing signal sparsity (in the spatial dimension _only_ _or_ both _in_ _spatial_ and temporal _dimensions),[26](#mrm27594-bib-0026){ref-type="ref"},_ _[27](#mrm27594-bib-0027){ref-type="ref"}_ _allowing_ a scan time _reduction_ in many _applications.[28](#mrm27594-bib-0028){ref-type="ref"},_ _[29](#mrm27594-bib-0029){ref-type="ref"},_ [30](#mrm27594-bib-0030){ref-type="ref"} The flexibility of _MRF_ toward _different_ sampling schemes and undersampling factors makes it possible to reconstruct _the_ _source_ images by means of _CS.[27](#mrm27594-bib-0027){ref-type="ref"},_ [31](#mrm27594-bib-0031){ref-type="ref"}, _[32](#mrm27594-bib-0032){ref-type="ref"}_ Higher acceleration factors _might_ _be_ feasible if the correlation _in_ the temporal dimension is better used.[33](#mrm27594-bib-0033){ref-type="ref"} Examples of such reconstructions _specifically_ tailored _to_ _MRF_ _are_ given in _Davies_ et al, Pierre et _al,_ and Zhao et al[34](#mrm27594-bib-0034){ref-type="ref"}, [35](#mrm27594-bib-0035){ref-type="ref"}, [36](#mrm27594-bib-0036){ref-type="ref"} which _take_ into _account_ the simulated dictionary atoms in the _image_ reconstruction process. Recent work has shown that the temporal correlation in the _MRF_ data _can_ be exploited even further by incorporating _the_ low rank _structure_ of the data into the _cost_ _function,[37](#mrm27594-bib-0037){ref-type="ref"}_ _a_ _technique_ _which_ was introduced into MR _in_ _Liang[_ |
Historical conceptualizations of depression
===========================================
There is a long tradition in phenomenologlcal psychopathology that stresses basic bodily alterations as core features of depressive states. Thus, Wernicke used the term "vital feelings" to describe certain somatic symptoms occurring in affective psychoses.^[@ref1]^ Vital feelings refer to the close relationship of the body to the awareness of self. They determine the way we experience our body and the impression we assume our physical presence makes on other people. Vital feelings are somatic affects localized In different parts of the body. Whereas vital feelings constitute the bodily background of our normal experiences, they may move to the fore In a depressive mood. For example, depressed patients very often complain of a headache which is described not exactly as an ordinary pain, but more as an unbearable pressure "like a band around the head." Other disturbed vital feelings affect the chest or the abdomen, and mediate unpleasant sensations of weight, tension, heaviness, or Inhibition, totally absorbing the focus of attention. In quite a similar way Dupré speaks of "coenestopathic states" which mean a distressing, qualitative change of normal physical feeling In certain areas of the body during an episode of depressive mood. It Is a global loss of vitality In which all bodily parts and functions may be altered, and all their performances depressed.^[@ref2]^ Kurt Schneider considered these disturbances of vital feelings to be the core of cyclothymic depression. In his psychopathologlcal assessment they were of paramount diagnostic significance In depressive Illness, more or less equivalent to the first-rank symptoms In schizophrenia.^[@ref3]^ Huber discriminated between vital disturbances on the one hand and vegetative symptoms In depression on the other.^[@ref4]^ Vital disturbances refer to the vital feelings just mentioned. They comprise a loss of general vital tone of the body, a prevailing fatigue or exhaustibility, and various forms of somatic dysesthesia, typically of a static, more localized character affecting head, chest, heart region, or abdomen. All-pervasive sensations of anesthesia, stiffness, and alienation of the total body may characterize a somatopsychic depersonalization in depression which may appear as a Cotard\'s syndrome in its extreme form. If the vital disturbances take on a peculiar form that is difficult to describe in ordinary everyday words, Huber speaks of a "coenesthetic" depression which must be typologically differentiated from the bizarre states of coenesthetic schizophrenia. Vegetative symptoms are closely associated with these vital disturbances and coenesthesias in depression. Disturbances of sleep, appetite, and digestion are most frequent. However, there may be many other vegetative symptoms in depression such as disordered salivation, transpiration and lacrimation, cardiac arrhythmias and dyspnea, loss of libido and various sexual dysfunctions, dys- or amen? orrhea, loss of or increase in body weight, decreased turgor of the skin, loss of hair, decrease in body temperature, nausea, vomiting, meteorism, dizziness, sweating, or sensations of coldness. Both vital disturbances, coenesthesias and vegetative symptoms, are typically coexistent with the well-known affective, behavioral, and cognitive symptoms of depression. With respect to the different settings of medical care, however, these psychological symptoms of depression may be masked by a dominant reporting of somatic symptoms. M. Bleuler addressed the point in his book *Depressions in Primary Care,* in 1943: *"It is a common and frequent observation that depressive patients with single somatic complaints come to the consulting room of the general practitioner, internal specialist, and even the surgeon, gynecologist, ophthalmologist, urologist and other medical specialists, and spontaneously, they only speak of somatic phenomena while concealing their state of depressive mood. They report palpitations, tightness of the chest, loss of appetite, obstipation, pollakiuria, amenorrhea and many others. Only when one looks at their psychic state does one discover that they report numerous hypochondriac ideas also in other areas, that in addition they produce depressive ideas of impoverishment and sin, that beyond that their whole stream of thoughts is inhibited, that the depression manifests itself not only in the somatic complaints reported, but in various other bodily expressions."^[@ref5]^*
In spite of this long-standing psychopathological view on the somatic foundation of depressive mood, at least in moderate and severe clinical states, it is bewildering that the official psychiatric classification systems of the *Diagnostic and Statistical Manual of Mental Disorders,* 4th edition *(DSM-IV)* and the *ICD-10* *Classification of Mental and Behavioral Disorders. Clinical Descriptions and Diagnostic guidelines (ICD-10)* only marginally appreciate somatic symptoms as diagnostic criteria for depressive disorders while focussing on the psychological symptoms of affect and cognition. So, *DSM-IV* lists only three criteria of somatic symptoms for major depressive disorder: sleep disturbance, appetite disturbance, and fatigue or loss of energy. And correspondingly, in *ICD-10,* disturbances of sleep and appetite, loss of libido, and amenorrhea are the only somatic symptoms considered to be of diagnostic significance for major depression. Beyond this short list of predominantly vegetative symptoms, no painful physical symptoms are mentioned in either the *DSM-IV* or *ICD-10.* There seems to be a major shift In diagnostic practice, however; the second version of the *Diagnostic and Statistical Manual of Mental Disorders,* 4th edition, Text Revision *(DSM-IV TR)* now Includes new criteria referring to "excessive worry over physical health and complaints of pain (eg, headaches or joint, abdominal, or other pains)."^[@ref6]^ This supplement of diagnostic criteria Is Indicative of an againIncreasing awareness of the importance of somatic symptoms in depression.
What is meant by "somatic" in somatic symptoms of depression?
=============================================================
In the literature there are many terms used to describe somatic symptoms in depression: somatic, somatlzed, physical, bodily, somatoform, painful, psychosomatic, vegetative, medically unexplained, masked, etc.^[@ref7]^ These diverse terms refer to different theoretical or diagnostic concepts. For states of depressive mood the neutral term "somatic" is preferred, comprising various bodily sensations that a depressed individual perceives as unpleasant or worrisome. These dysesthesias are very often localized In certain body parts or organs, or may affect the whole body In Its vital condition, as In the case of fatigue or loss of energy. Several basic physical dysfunctions, such as those of sleep, appetite, or digestion, are also to be included in the term "somatic." In addition, It may be clinically relevant to differentiate between painful and nonpalnful somatic symptoms of depression. From a diagnostic perspective one has to keep in mind that somatic symptoms play a significant role both in primary psychiatric disorders, first and foremost depressive and anxiety disorders, and in somatoform disorders. And In differential diagnosis, somatic symptoms must be considered as possibly even Indicative of underlying somatic diseases. A diagnostic challenge may be seen In the well-known fact that depressive, anxiety, somatoform disorders, and medical conditions are frequently coexistent, or Interact In the Individual patient.^[@ref8]-[@ref10]^ Regarding the assessment of somatic symptoms, Kroenke correctly points out that diagnosis very often is more approximative than precise. Presented somatic symptoms may be either clearly attributed to a distinct medical disorder or be placed into one of the following heuristic categories: somatoform disorder, another primary psychiatric disorder (often depression and/or anxiety), functional somatic syndrome (eg, irritable bowel syndrome, fibromyalgia, chronic fatigue syndrome), "symptom-only" diagnosis (eg, low back pain, idiopathic dizziness) or only partially explained by a defined medical disorder (eg, many states of chronic pain).^[@ref11]^
Epidemiological studies may provide an illuminating survey of the prevalence of somatic symptoms in depressive disorders, especially those encountered in primary care, and the prognostic value of somatic symptoms regarding their development in the further course of illness.
Somatic symptoms of depressive disorders in inpatient care and primary care
===========================================================================
In a clinical study, Hamilton reported that somatic symptoms prevailed in a great majority of depressed patients.^[@ref12]^ Somatic symptoms, particularly somatic anxiety and fatigue, were documented in up to 80% of a sample of 260 women and 239 men suffering from major depression. These somatic symptoms very frequently had an underlying psychopathologically relevant hypochondriasis, both in women and men. This study confirmed earlier studies showing that depressive disorders with predominantly somatic presentation were likely to be the most common form of depression, both in inpatient and outpatient care.^[@ref13],[@ref14]^ Hagnell and Rorsman stressed the Indicative significance of somatic symptoms in depressed primary care patients regarding their risk of suicide.^[@ref15]^
Epidemiological studies designed to establish prevalence figures for depressive disorders In primary care during recent years have uniformly demonstrated that depressive disorders are highly prevalent at this level of medical care.^[@ref16]-[@ref19]^ For the great majority of depressed patients seeking professional help in the official health care system, general practitioners and internists are the decisive interface for diagnosis and treatment of depression.^[@ref20]^ Primary-care patients with depression very often present with somatic complaints. This seems to be more the rule than the exception worldwide.^[@ref21],[@ref22]^ Two of the three most common symptoms reported during a current depressive episode were somatic (tlred/no energy/listless: 73%, broken sleep/decreased sleep: 63%) as shown by the European Study Society study (DEPRES II).^[@ref23]^ This study, however, also underlined that 65% of the depressed primary care patients suffered from a concomitant medical condition pointing to some likely difficulties In differential diagnosis. The multlcenter International study (n =1146) conducted by | historical conceptualizations of depression = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = there is a long tradition in phenomenologlcal psychopathology that stresses basic bodily alterations as core features of depressive states. first, wernicke used the term " vital feelings " to describe certain somatic symptoms occurring in affective psychoses. ^ [ @ ref1 ] ^ vital feelings refer to the close relationship of the body to the awareness of self. others determine which way we experience our body and the impression we assume our physical presence makes on other people. vital feelings are somatic affects localized in different parts of the body. whereas vital feelings constitute the bodily background behind our normal experiences, they may move to the fore in a depressive mood. for example, depressed patients very often complain of a headache which is described not exactly as an ordinary pain, but more as an unbearable pressure " like a band around the head. " other disturbed vital feelings affect the chest or the abdomen, and mediate unpleasant sensations of weight, tension, heaviness, or inhibition, totally absorbing the focus of attention. in quite a similar way dupre speaks of " coenestopathic states " which mean a distressing, qualitative change of normal physical feeling in certain areas of the body during an episode of depressive mood. it is a global loss of vitality in which all bodily parts and functions may be altered, and all their performances depressed. ^ [ @ ref2 ] ^ kurt schneider considered these disturbances of vital feelings to be the core of cyclothymic depression. as his psychopathologlcal assessment they were of paramount diagnostic significance in depressive illness, more or less equivalent to the first - rank psychiatric within schizophrenia. ^ [ @ ref3 ] ^ huber discriminated between vital disturbances on the one hemisphere and vegetative feelings in depression through the other. ^ [ @ ref4 ] ^ vital disturbances refer to the vital feelings just mentioned. they comprise a loss of general vital tone of the body, a prevailing fatigue or exhaustibility, and various forms of somatic dysesthesia, typically of a static, more localized character affecting head, chest, heart region, or abdomen. all - pervasive sensations of anesthesia, stiffness, and alienation of the total body may characterize a somatopsychic depersonalization in depression which may appear as a cotard \ ' s syndrome in its extreme form. if the vital disturbances take on a peculiar form that is difficult to describe in ordinary everyday words, huber speaks of a " coenesthetic " depression which must be typologically differentiated from the bizarre states of coenesthetic schizophrenia. vegetative symptoms are closely associated with these vital disturbances and coenesthesias in depression. disturbances of sleep, appetite, and digestion are most frequent. however, there may be many other vegetative symptoms in depression such as disordered salivation, transpiration and lacrimation, cardiac arrhythmias and dyspnea, loss of libido and various sexual dysfunctions, dys - or amen? orrhea, loss of or increase in body weight, decreased turgor of the skin, loss of hair, decrease in body temperature, nausea, vomiting, meteorism, dizziness, sweating, or sensations of coldness. both vital disturbances, coenesthesias and vegetative symptoms, are typically coexistent with the well - known affective, behavioral, and cognitive symptoms of depression. with respect to the different settings of medical care, however, these psychological symptoms of depression may be masked by a dominant reporting of somatic symptoms. m. bleuler addressed the point in his book * depressions in primary care, * in 1943 : * " it is a common and frequent observation that depressive patients with single somatic complaints come to the consulting room of the general practitioner, internal specialist, and even the surgeon, gynecologist, ophthalmologist, urologist and other medical specialists, and spontaneously, they only speak of somatic phenomena while concealing their state of depressive mood. they report palpitations, tightness of the chest, loss of appetite, obstipation, pollakiuria, amenorrhea and many others. only when one looks at their psychic state does one discover that they report numerous hypochondriac ideas also in other areas, that in addition they produce depressive ideas of impoverishment and sin, that beyond that their whole stream of thoughts is inhibited, that the depression manifests itself not only in the somatic complaints reported, but in various other bodily expressions. " ^ [ @ ref5 ] ^ * in spite of this long - standing psychopathological view on the somatic foundation of depressive mood, at least in moderate and severe clinical states, it is bewildering that the official psychiatric classification systems of the * diagnostic and statistical manual of mental disorders, * 4th edition * ( dsm - iv ) * and the * icd - 10 * * classification of mental and behavioral disorders. clinical descriptions and diagnostic guidelines ( icd - 10 ) * only marginally appreciate somatic symptoms as diagnostic criteria for depressive disorders while focussing on the psychological symptoms of affect and cognition. so, * dsm - iv * lists only three criteria of somatic symptoms for major depressive disorder : sleep disturbance, appetite disturbance, and fatigue or loss of energy. and correspondingly, in * icd - 10, * disturbances of sleep and appetite, loss of libido, and amenorrhea are the only somatic symptoms considered to be of diagnostic significance for major depression. beyond this short list of predominantly vegetative symptoms, no painful physical symptoms are mentioned in either the * dsm - iv * or * icd - 10. * there seems to be a major shift in diagnostic practice, however ; the second version of the * diagnostic and statistical manual of mental disorders, * 4th edition, text revision * ( dsm - iv tr ) * now includes new criteria referring to " excessive worry over physical health and complaints of pain ( eg, headaches or joint, abdominal, or other pains ). " ^ [ @ ref6 ] ^ this supplement of diagnostic criteria is indicative of an againincreasing awareness of the importance of somatic symptoms in depression. what is meant by " somatic " in somatic symptoms of depression? = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = in the literature there are many terms used to describe somatic symptoms in depression : somatic, somatlzed, physical, bodily, somatoform, painful, psychosomatic, vegetative, medically unexplained, masked, etc. ^ [ @ ref7 ] ^ these diverse terms refer to different theoretical or diagnostic concepts. for states of depressive mood the neutral term " somatic " is preferred, comprising various bodily sensations that a depressed individual perceives as unpleasant or worrisome. these dysesthesias are very often localized in certain body parts or organs, or may affect the whole body in its vital condition, as in the case of fatigue or loss of energy. several basic physical dysfunctions, such as those of sleep, appetite, or digestion, are also to be included in the term " somatic. " in addition, it may be clinically relevant to differentiate between painful and nonpalnful somatic symptoms of depression. from a diagnostic perspective one has to keep in mind that somatic symptoms play a significant role both in primary psychiatric disorders, first and foremost depressive and anxiety disorders, and in somatoform disorders. and in differential diagnosis, somatic symptoms must be considered as possibly even indicative of underlying somatic diseases. a diagnostic challenge may be seen in the well - known fact that depressive, anxiety, somatoform disorders, and medical conditions are frequently coexistent, or interact in the individual patient. ^ [ @ ref8 ] - [ @ ref10 ] ^ regarding the assessment of somatic symptoms, kroenke correctly points out that diagnosis very often is more approximative than precise. presented somatic symptoms may be either clearly attributed to a distinct medical disorder or be placed into one of the following heuristic categories : somatoform disorder, another primary psychiatric disorder ( often depression and / or anxiety ), functional somatic syndrome ( eg, irritable bowel syndrome, fibromyalgia, chronic fatigue syndrome ), " symptom - only " diagnosis ( eg, low back pain, idiopathic dizziness ) or only partially explained by a defined medical disorder ( eg, many states of chronic pain ). ^ [ @ ref11 ] ^ epidemiological studies may provide an illuminating survey of the prevalence of somatic symptoms in depressive disorders, especially those encountered in primary care, and the prognostic value of somatic symptoms regarding their development in the further course of illness. somatic symptoms of depressive disorders in inpatient care and primary care = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = in a clinical study, hamilton reported that somatic symptoms prevailed in a great majority of depressed patients. ^ [ @ ref12 ] ^ somatic symptoms, particularly somatic anxiety and fatigue, were documented in up to 80 % of a sample of 260 women and 239 men suffering from major depression. these somatic symptoms very frequently had an underlying psychopathologically relevant hypochondriasis, both in women and men. this study confirmed earlier studies showing that depressive disorders with predominantly somatic presentation were likely to be the most common form of depression, both in inpatient and outpatient care. ^ [ @ ref13 ], [ @ ref14 ] ^ hagnell and rorsman stressed the indicative significance of somatic symptoms in depressed primary care patients regarding their risk of suicide. ^ [ @ ref15 ] ^ epidemiological studies designed to establish prevalence figures for depressive disorders in primary care during recent years have uniformly demonstrated that depressive disorders are highly prevalent at this level of medical care. ^ [ @ ref16 ] - [ @ ref19 ] ^ for the great majority of depressed patients seeking professional help in the official health care system, general practitioners and internists are the decisive interface for diagnosis and treatment of depression. ^ [ @ ref20 ] ^ primary - care patients with depression very often present with somatic complaints. this seems to be more the rule than the exception worldwide. ^ [ @ ref21 ], [ @ ref22 ] ^ two of the three most common symptoms reported during a current depressive episode were somatic ( tlred / no energy / listless : 73 %, broken sleep / decreased sleep : 63 % ) as shown by the european study society study ( depres ii ). ^ [ @ ref23 ] ^ this study, however, also underlined that 65 % of the depressed primary care patients suffered from a concomitant medical condition pointing to some likely difficulties in differential diagnosis. the multlcenter international study ( n = 1146 ) conducted by | Historical conceptualizations of depression = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = There is a long tradition in phenomenologlcal psychopathology that stresses basic bodily alterations as core features of depressive states. Thus, Wernicke used the term " vital feelings " to describe certain somatic symptoms occurring in affective psychoses. ^ [@ ref1] ^ Vital feelings refer to the close relationship of the body to the awareness of self. They determine the way we experience our body and the impression we assume our physical presence makes on other people. Vital feelings are somatic affects localized In different parts of the body. Whereas vital feelings constitute the bodily background of our normal experiences, they may move to the fore In a depressive mood. For example, depressed patients very often complain of a headache which is described not exactly as an ordinary pain, but more as an unbearable pressure " like a band around the head. " Other disturbed vital feelings affect the chest or the abdomen, and mediate unpleasant sensations of weight, tension, heaviness, or Inhibition, totally absorbing the focus of attention. In quite a similar way Dupré speaks of " coenestopathic states " which mean a distressing, qualitative change of normal physical feeling In certain areas of the body during an episode of depressive mood. It Is a global loss of vitality In which all bodily parts and functions may be altered, and all their performances depressed. ^ [@ ref2] ^ Kurt Schneider considered these disturbances of vital feelings to be the core of cyclothymic depression. In his psychopathologlcal assessment they were of paramount diagnostic significance In depressive Illness, more or less equivalent to the first - rank symptoms In schizophrenia. ^ [@ ref3] ^ Huber discriminated between vital disturbances on the one hand and vegetative symptoms In depression on the other. ^ [@ ref4] ^ Vital disturbances refer to the vital feelings just mentioned. They comprise a loss of general vital tone of the body, a prevailing fatigue or exhaustibility, and various forms of somatic dysesthesia, typically of a static, more localized character affecting head, chest, heart region, or abdomen. All - pervasive sensations of anesthesia, stiffness, and alienation of the total body may characterize a somatopsychic depersonalization in depression which may appear as a Cotard \ ' s syndrome in its extreme form. If the vital disturbances take on a peculiar form that is difficult to describe in ordinary everyday words, Huber speaks of a " coenesthetic " depression which must be typologically differentiated from the bizarre states of coenesthetic schizophrenia. Vegetative symptoms are closely associated with these vital disturbances and coenesthesias in depression. Disturbances of sleep, appetite, and digestion are most frequent. However, there may be many other vegetative symptoms in depression such as disordered salivation, transpiration and lacrimation, cardiac arrhythmias and dyspnea, loss of libido and various sexual dysfunctions, dys - or amen? orrhea, loss of or increase in body weight, decreased turgor of the skin, loss of hair, decrease in body temperature, nausea, vomiting, meteorism, dizziness, sweating, or sensations of coldness. Both vital disturbances, coenesthesias and vegetative symptoms, are typically coexistent with the well - known affective, behavioral, and cognitive symptoms of depression. With respect to the different settings of medical care, however, these psychological symptoms of depression may be masked by a dominant reporting of somatic symptoms. M. Bleuler addressed the point in his book * Depressions in Primary Care, * in 1943: * " It is a common and frequent observation that depressive patients with single somatic complaints come to the consulting room of the general practitioner, internal specialist, and even the su4ge)n, gynecologist, ophthalmologist, urologist and other medical specialists, and spontaneously, they only speak of somatic phenomena while concealing their state of depressive mood. They report palpitations, tightness of the chest, loss of appetite, obstipation, pollakiuria, amenorrhea and many others. Only when one looks at their psychic state does one discover that they report numerous hypochondriac ideas also in other areas, that in addition they produce depressive ideas of impoverishment and sin, that beyond that their whole stream of thoughts is inhibited, that the depression manifests itself not only in the somatic complaints reported, but in various other bodily expressions. " ^ [@ ref5] ^ * In spite of this long - standing psychopathological view on the somatic foundation of depressive mood, at least in moderate and severe clinical states, it is bewildering that the official (Qychiatric classification systems of the * Diagnostic and Statistical Manual of Mental Disorders, * 4th edition * (DSM - IV) * and the * ICD - 10 * * Classification of Mental and Behavioral Disorders. Clinical Descriptions and Diagnostic guidelines (ICD - 10) * only marginally appreciate somatic symptoms as diagnostic criteria for depressive disorders while focussing on the psychological symptoms of affect and cognition. So, * DSM - IV * lists only three criteria of somatic symptoms for major depressive disorder: sleep disturbance, appetite disturbance, and fatigue or loss of energy. And correspondingly, in * ICD - 10, * disturbances of sleep and appetite, loss of libido, and amenorrhea are the only somatic symptoms considered to be of diagnostic significance for major depression. Beyond this short list of predominantly vegetative symptoms, no painful physical symptoms are mentioned in either the * DSM - IV * or * ICD - 10. * There seems to be a major shift In diagnostic practice, however; the second version of the * Diagnostic and Statistical Manual of Mental Disorders, * 4th edLtlon, Text Revision * (DSM - IV TR) * now Includes new criteria referring to " excessive worry over physical health and complaints of pain (eg, headaches or joint, abdominal, or other pains ). " ^ [@ ref6] ^ This supplement of diagnostic criteria Is Indicative of an againIncreasing awareness of the importance of somatic symptoms in depression. What is meant by " somatic " in somatic symptoms of depression? = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = In the literature there are many terms used to describe somatic symptoms in depression: somatic, somatlzed, physical, bodily, somatoform, painful, psychosomatic, vegetative, hedicallG unexplained, masked, etc. ^ [@ ref7] ^ These diverse terms refer to different theoretical or diagnostic concepts. For states of depressive mood the neutral term " somatic " is preferred, comprising various bodily sensations that a depressed individual perceives as unpleasant or dorris)me. These dysesthesias are very often localized In certain body parts or organs, or may affect the whole body In Its vital condition, as In the case of fatigue or loss of energy. Several basic physical dysfunctions, such as those of sleep, appetite, or digestion, are also to be included in the term " somatic. " In addition, It may be clinically relevant to differenRiatD between painful and nonpalnful somatic symptoms of depression. From a diagnostic perspective one has to keep in mind that somatic symptoms play a significant role both in primary psychiatric disorders, first and foremost depressive and anxiety disorders, and in somatoform disorders. And In differential diagnosis, somatic symptoms must be considered as possibly even Indicative of underlying somatic diseases. A diagnostic challenge may be seen In the well - known fact that depressive, anxiety, somatoform disorders, and medical conditions are frequently coexistent, or Interact In the Individual patient. ^ [@ ref8] - [@ ref10] ^ Regarding the assessment of somatic symptoms, Kroenke correctly points out that diagnosis very often is more approximative than precise. Presented somatic symptoms may be either clearly attributed to a distinct medical disorder or be placed into one of the following heuristic categories: somatoform disorder, another primary ps6chiatrlc disorder (often depression and / or anxiety ), functional somatic syndrome (eg, irritable bowel syndrome, fibromyalgia, chronic fatigue syndrome ), " symptom - only " diagnosis (eg, low back pain, idiopathic dizziness) or only partially explained by a defined medical disorder (eg, many states of chronic pain ). ^ [@ ref11] ^ Epidemiological studies may provide an illuminating survey of the prevalence of somatic symptoms in depresskde disorders, especially those encountered in primary care, and the prognostic value of somatic symptoms regarding their development in the further course of illness. Somatic symptoms of depressive disorders in inpatient care and primary care = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = In a clinical study, Hamilton reported that somatic symptoms prevailed in a great majority of depressed patients. ^ [@ ref12] ^ Somatic symptoms, particularly somatic anxiety and fatigue, were documented in up to 80% of a sample of 260 women and 239 men suffering from major depression. These somatic symptoms very frequently had an underlying psychopathologically relevant hypochondriasis, both in women and men. This study confirmed earlier studies showing that depressive disorders with predomihanRly somatic presentation were likely to be the most common form of depression, both in inpatient and outpatient care. ^ [@ ref13 ], [@ ref14] ^ Hagnell and Rorsman stressed the Indicative significance of somatic symptoms in depressed primary care patients regarding their risk of suicide. ^ [@ ref15] ^ Epidemiological studies designed to establish prevalence figures for depressive disorders In primary care during rex#nt years have uniformly demonstrated that depressive disorders are highly prevalent at this level of medical care. ^ [@ ref16] - [@ ref19] ^ For the great majority of depressed patients seeking professional help in the official health care system, general practitioners and internists are the decisive interface for diagnosis and treatment of depression. ^ [@ ref20] ^ Primary - care patients with depression very often present with somatic complaints. This seems to be more the rule than the exception worldwide. ^ [@ ref21 ], [@ ref22] ^ Two of the three most common symptoms reported during a current depressive episode were somatic (tlred / no energy / listless: 73% , broken sleep / decreased sleep: 63%) as shown by the European Study Society study (DEPRES II ). ^ [@ ref23] ^ This study, however, also underlined that 65% of the depressed primary care patients suffered from a concomitant medical condition pointing to some likely difficulties In differential diagnosis. The multlcenter International study (n = 1146) conducted by | conceptualizations of depression There is a long tradition in phenomenologlcal psychopathology that stresses basic bodily alterations core features of depressive Thus, Wernicke used term "vital to describe certain somatic symptoms occurring in affective psychoses.^[@ref1]^ Vital feelings refer to the close relationship of the body to the awareness of self. They determine the way we experience our body and the we assume physical presence makes on other people. Vital feelings somatic affects localized In different parts of the body. Whereas vital feelings constitute the bodily background of our normal experiences, they may to the fore In a depressive mood. For example, depressed patients often of a headache which is described not exactly as an ordinary pain, but more as an unbearable "like a band around the Other disturbed vital affect the chest or the abdomen, mediate sensations weight, tension, or Inhibition, totally absorbing the focus of attention. In quite a similar way speaks of "coenestopathic states" which mean a distressing, change of normal physical feeling In certain areas of the body during episode of depressive It Is a global loss of vitality In which all parts and functions may be altered, and all their performances depressed.^[@ref2]^ Kurt Schneider considered these disturbances of vital feelings to the core of cyclothymic depression. his psychopathologlcal assessment they were of paramount diagnostic significance In depressive more or less equivalent to the first-rank symptoms In schizophrenia.^[@ref3]^ Huber discriminated between vital disturbances on the vegetative symptoms In depression on the other.^[@ref4]^ Vital disturbances refer to the vital feelings just mentioned. They comprise a loss of general vital tone of the body, a prevailing fatigue or exhaustibility, and various forms of somatic dysesthesia, typically of a more localized character affecting head, heart region, or abdomen. All-pervasive sensations anesthesia, stiffness, and alienation of the total body may characterize a somatopsychic depersonalization in depression which may appear as a Cotard\'s syndrome in its form. If vital disturbances take on a peculiar that is to describe in ordinary everyday words, Huber speaks "coenesthetic" depression which must be typologically differentiated from the bizarre states of coenesthetic schizophrenia. Vegetative symptoms closely associated vital and coenesthesias in depression. of sleep, and digestion are most frequent. However, there may be many other vegetative symptoms in depression as disordered salivation, transpiration and lacrimation, cardiac arrhythmias and dyspnea, loss of libido and sexual dys- or amen? loss of or increase in body weight, decreased turgor of the skin, loss of decrease in body temperature, nausea, vomiting, meteorism, dizziness, sweating, sensations of coldness. Both vital disturbances, coenesthesias and vegetative symptoms, are typically coexistent with the well-known affective, behavioral, cognitive symptoms of depression. With respect to the different settings of medical care, these psychological symptoms of depression may be masked by a dominant reporting of somatic symptoms. M. Bleuler addressed the point his book *Depressions in Primary Care,* 1943: *"It a common and frequent observation that depressive patients with single somatic complaints to the consulting room of the general internal and even the surgeon, gynecologist, ophthalmologist, urologist and medical specialists, and spontaneously, they only speak of while concealing their state of depressive mood. They report palpitations, tightness of chest, loss of appetite, obstipation, pollakiuria, amenorrhea many others. Only when one looks at their does one discover that they report numerous hypochondriac ideas also other areas, that in addition they produce depressive ideas impoverishment and sin, that beyond that their whole stream of is inhibited, the depression manifests itself not only in the somatic complaints reported, but in various other bodily expressions."^[@ref5]^* In spite of this long-standing psychopathological view on somatic of depressive mood, at least moderate and severe clinical states, it is bewildering that the official psychiatric classification systems of the *Diagnostic and Statistical Manual of Mental Disorders,* edition *(DSM-IV)* and the *ICD-10* *Classification of Mental and Behavioral Disorders. Clinical Descriptions and Diagnostic guidelines (ICD-10)* only somatic symptoms as diagnostic for depressive disorders while focussing on the psychological symptoms of affect and cognition. So, *DSM-IV* lists only three criteria of somatic symptoms for major depressive disorder: sleep disturbance, appetite disturbance, and fatigue or loss of energy. And correspondingly, in *ICD-10,* disturbances of sleep and appetite, loss of libido, and amenorrhea are the only somatic considered to be of significance major Beyond this short list of predominantly vegetative symptoms, no painful physical symptoms are mentioned in either the *DSM-IV* or *ICD-10.* There seems to be a major In diagnostic practice, however; the second version of *Diagnostic and Statistical Manual of Disorders,* 4th edition, Text *(DSM-IV TR)* now new referring to "excessive worry over physical health and complaints of pain (eg, headaches or joint, abdominal, or other pains)."^[@ref6]^ This supplement of diagnostic criteria Is Indicative of an againIncreasing of the importance of somatic in depression. What is meant by "somatic" in somatic symptoms of In the there are many terms used describe somatic symptoms in depression: somatic, somatlzed, physical, bodily, somatoform, painful, psychosomatic, vegetative, medically unexplained, masked, etc.^[@ref7]^ These diverse terms refer to different theoretical or concepts. For states of mood the neutral term "somatic" preferred, comprising various bodily sensations that a perceives as unpleasant or worrisome. These dysesthesias are often localized In certain body parts or organs, or may the whole body In Its vital condition, as case of fatigue or loss of energy. basic physical dysfunctions, such as of sleep, appetite, digestion, are also to be included in the "somatic." In addition, It may be clinically relevant differentiate between painful and nonpalnful somatic symptoms of depression. From a diagnostic perspective one has to keep in mind that somatic symptoms play a significant role both in primary psychiatric first and foremost depressive and anxiety disorders, and in somatoform disorders. And differential diagnosis, somatic symptoms be considered as possibly even Indicative of underlying somatic diseases. A diagnostic challenge may be seen In the well-known that anxiety, somatoform and medical are frequently coexistent, or Interact In the Individual patient.^[@ref8]-[@ref10]^ Regarding the assessment of symptoms, Kroenke correctly points that diagnosis very often is more approximative precise. Presented somatic symptoms may be either clearly attributed a distinct medical disorder or be placed into one the following heuristic categories: somatoform disorder, another primary psychiatric disorder and/or anxiety), functional somatic syndrome (eg, irritable bowel syndrome, fibromyalgia, chronic fatigue syndrome), "symptom-only" diagnosis (eg, low back pain, idiopathic or only partially explained by a defined medical disorder (eg, many states of chronic pain).^[@ref11]^ Epidemiological studies may provide an illuminating survey of the prevalence somatic symptoms in depressive especially those encountered in care, and the prognostic value of somatic symptoms regarding their development in the further course illness. Somatic symptoms of depressive disorders in inpatient care and primary care =========================================================================== a clinical study, Hamilton reported that somatic symptoms in a great majority of patients.^[@ref12]^ Somatic symptoms, particularly and fatigue, were documented in up to 80% of a sample of 260 women 239 men suffering from major depression. somatic symptoms very had an underlying psychopathologically relevant hypochondriasis, both in women and men. This study earlier studies showing that depressive disorders with predominantly somatic were likely to be the most common form of both in inpatient and outpatient care.^[@ref13],[@ref14]^ Hagnell and Rorsman stressed the Indicative significance of somatic symptoms in depressed primary care patients regarding their risk of suicide.^[@ref15]^ Epidemiological studies designed to establish prevalence figures for depressive disorders In primary care during recent years have demonstrated that depressive disorders are highly prevalent at this level of medical care.^[@ref16]-[@ref19]^ For great majority of depressed patients seeking professional help in the official health care system, general practitioners and internists are the decisive interface for diagnosis and treatment of depression.^[@ref20]^ Primary-care patients with depression very often present with somatic complaints. This seems to more the rule the exception Two of the three most common symptoms reported during a current depressive episode were somatic (tlred/no energy/listless: 73%, broken sleep/decreased 63%) as by the European Study Society study II).^[@ref23]^ This study, however, also underlined 65% of the depressed primary care patients suffered from a concomitant medical condition pointing to likely difficulties In differential diagnosis. The multlcenter International study (n =1146) conducted | hIstORicAL CONceptuaLizATIONS of dEpreSsIOn
===========================================
THere IS A LonG TrAdITion in PHenOMenoLOglCal psycHopATHologY THat stressES BAsic BOdiLY alteraTIOnS aS cOre FeaTurES OF DEpressiVe sTateS. Thus, WERniCKE USed THe TERM "vItAl feElINGs" TO dEscrIBe certAin sOmatIC SymPTOms occURriNg IN AFFectIvE psYchOSes.^[@ReF1]^ ViTaL FeeLINgS rEFEr to THE cLosE ReLaTionShIp OF the bodY to THE AwaREnESS oF SElF. thEy DETErmIne The Way WE eXperIEnce OUr BoDY And thE iMpReSSIon WE assUme OuR PhYSICAl pResEnCe mAKeS oN OTHeR PEople. VItAL fEElIngS ARe SomATic AFFecTS locAlIZEd iN dIFFeRENt PARTs of thE BOdy. WhEreas VItAL FEEliNgS conSTiTUTE ThE bOdIlY baCkGROUND of oUR NoRMaL EXPeRIEnceS, tHEY mAy mOvE tO The fORE In a depreSsive moOd. fOR eXaMpLE, dEPResSeD pAtientS VERY oFTEN complaIN oF A heaDachE wHich iS descRIBed Not ExAcTlY AS an oRdInARY PAIn, bUT More As An UNbeAraBLE pressURe "liKe A BAnD aroUnD tHE head." OTheR dIStURBEd vItAL fEeliNgS AffecT tHe cHeST Or THE aBDOmEn, ANd mEDIaTE uNPlEAsANT senSaTioNS oF WeIGHt, TeNsioN, hEAviNESS, oR InHIbitIon, tOtally AbsORBING THE FocUS oF ATtenTION. IN qUite a simILaR WAy DUPré spEAKS OF "coenEStOPAthiC StATES" WhiCH Mean a DIstREsSIng, QUAlITATiVE ChANGe of NOrMal PhySIcAL FeELInG in ceRTAiN AReAs Of THe bODY DURing AN epIsODe of DeprEsSIve moOD. it is A gLoBal loSs OF vITALiTy iN WhICh aLl BOdilY pARts aNd FuNcTiOns maY Be aLteREd, ANd aLl THEIR PeRFOrmanCes dEPResSeD.^[@rEf2]^ KurT schNEIdeR coNSIdErED thESe dISTuRbAnCeS oF VITal FEeLiNgs to BE tHe COre Of cyClOtHYMIC dePREssION. In hIs PSYCHoPathOlOGLcAl assEsSmeNt THeY WERE of PaRAmOUNt DiagnoSTIC sIgNifICAnCE iN dEPresSIVe ILLNesS, MorE OR lEss eQuivalent to THe fIRsT-raNK sYMptoMS in sCHiZOPhreNIA.^[@ref3]^ HubeR dIScRimInATED BETwEen viTAL dIsTURbANcES on tHe oNe hand ANd veGETaTiVe syMpToMs in DEpRESsiOn ON The OThER.^[@REF4]^ vItal DiSTUrBanCeS REfER To ThE vITaL FEeLINGs Just MenTIoneD. tHEY COmprise A LOSs oF geNERal VitAL tOne oF THe BodY, a PrEvaiLING FATIgUE Or exHAuSTibILITy, And vArIOUs foRMs of SOmatIc dySEStHeSia, tyPIcALLy oF A stATIC, moRE loCAliZed cHARacteR affeCTInG head, cHeSt, HeArT ReGIOn, or aBdoMEN. aLl-pErvASivE SENSATIONS Of aNEsthesIa, sTiffNeSS, And aLiEnATIOn oF the tOTAL BODY MaY chaRaCtErIZE A SOmatOpsYcHIc dePERsOnAliZATIon In DePrEssIoN WHich MaY APPEAr As a COtArD\'s syNDrOME IN Its exTREme FoRM. If ThE ViTal diStuRBanCeS TAKe oN a peCuLIAr FOrm tHaT iS dIfFIcULt TO deScrIBE iN OrdInARy EVeRydAY woRDs, huBer SPeakS oF A "CoENestHETIc" depressIoN WHICH mUST be tYpoLogically DIffErEntiATED FRoM The bIZArRe STAteS of COENesTHETIC scHIzOPhRENia. VeGetatIVe sYmPtoMs ArE cloSELy assoCiated WItH ThESe vitAL DiSturBances aNd cOENEstHEsiAS iN depResSIoN. DistuRbAncEs Of slEEp, AppetItE, aND digeSTioN ARE mOsT FrEqUENt. HOweveR, TherE maY bE ManY OTHEr VEgEtATIVE SYmPToms in dePresSION SUch as disoRdEReD SaLIVatIOn, TrAnspiraTIon aNd LACRimATioN, cArdIaC ArrhytHMiAs aNd DYsPneA, LoSS of lIbIdo aNd VAriouS SeXuAl dYSFunCtioNs, Dys- Or AMeN? orRHEa, loSs Of or INcreASE in BOdY wEighT, DECrEASEd tuRgOR Of THE skin, lOss oF Hair, DecRease In bODy TemPERATUre, nAuSEA, vomitinG, mETEorism, DIZzINESs, sWEaTInG, OR sEnsAtIONS oF coldnEss. BOTH vITAl DisturbAncES, CoENeStHEsIAs and vEgEtatiVe sYmptoMS, ARe tYpIcalLy coExIstENt WIth tHe WEll-kNOWN AFFective, beHAVIoraL, aNd COgNITivE sYMPtOMS oF dePresSion. WitH resPECT To The dIFFerEnt SeTTIngs Of MEdICaL CAre, HowevEr, tHesE pSychologiCAL SymptOMs OF DepRESsiOn May BE MaskED by a DoMinanT REpORtINg Of SoMaTIc sympToms. M. BLeuLER aDDRESSEd thE pOINT in hIs BoOK *DeprESSionS iN pRiMaRY CaRE,* In 1943: *"IT Is a CoMMON And fREQUEnt ObSErVAtioN THaT depResSIve PATiEnTs witH SINGLe sOMATIc cOmPLAInts CoMe tO thE consULting RoOm Of thE GenerAl PrActITiOner, inTErnAl SPeCiALiST, aNd eVeN THe SURGEOn, gYNeCOLOgISt, OPhtHalMoloGiST, URoLOGist aND OTHEr mEDIcaL sPEcIaLiSts, AND spOnTanEouSlY, thEY onLy SPEaK Of SOmatIc PHEnOMeNa WHiLE CoNcealIng thEir STATe OF dEprESSIVE mOOD. tHey RepoRt pALPiTatIOnS, tIGHtnESs of ThE cHeSt, lOss of APpEtITE, obsTiPAtIon, poLlakiUrIA, aMenOrRHea ANd manY oTHeRs. oNLy whEN OnE LooKS aT thEir psycHiC STAte does onE DIScoVER THat tHey rEpoRT nUMerOUs hYPoChOndrIaC idEAs ALsO IN oThEr AREas, That In AddItIoN tHEY pRoDucE dEPrEssIVe IDEAs oF ImPoVeRISHMeNt aND Sin, thAT BeYoND ThaT TheIR whOle StreAm Of thOughTS Is InHiBIted, tHaT thE DePrESSion MaNifESts ITSeLF not onLY In thE somATic COmpLAintS rEpoRted, BUT in varIOUs OtHer BODiLY exprESSioNS."^[@Ref5]^*
iN SPIte of ThiS lONg-StANdING pSychOPatHOlOGical vIew on ThE SOMatic fOUNdATiON oF DePReSSive MooD, at LEaSt iN MOdeRATe aNd SEverE cLInIcAL sTatES, IT iS BewilderInG that tHe oFficIAl PsYchIATRIc ClassIfIcAtiON SYSTeMS Of THe *dIAGnosTiC AND StAtISticAL MAnuAL oF MenTAl DISordErS,* 4Th edITiOn *(DsM-iV)* aNd The *icD-10* *CLaSSifIcatioN oF meNtAL aNd BEHaVioRaL DISorDErS. cliNICAL DeSCripTIONS ANd DiagnoStIC gUIdelInes (ICd-10)* Only MARGinAlLY appReCiATE sOmAtiC SYMpTomS aS dIAgnoSTic CRItERia FoR DepreSsiVE dIsorDeRS wHIlE FOCUSsINg on tHE pSYchOlOGICal symptoms of afFeCT aNd coGnitiOn. So, *dSM-iv* LIstS ONlY THREE crITErIA of sOmatIc SyMptomS for majOR dEPREsSIve diSOrdEr: SlEEP dISturBAnce, appetITe dIsTuRBaNCe, aNd FATiGUe or LOSs OF EneRGy. ANd corReSpoNDinGly, In *IcD-10,* DIStUrBAnCes oF SLEeP aNd aPPETiTE, LoSS oF LIBIDo, anD AmeNOrrheA ArE tHE ONly sOmAtIc SYMPtOmS CONSiDEreD tO BE Of diAgnoSTic SigNiFiCaNce FoR maJOR DEpressioN. beyOND tHIS SHORT List OF PRedominANtly VeGEtAtIVE SYmPtomS, NO PaINfuL pHySICAL sympToms ARe meNtIONed In EitheR tHe *DSm-Iv* Or *IcD-10.* tHErE SEEMs tO BE a MAjor SHIft In DiAgNostiC practICE, hoWEvEr; tHe SECond vErSIoN of tHe *DiagnoSTIC And StatIstiCAl MANUAL OF MEnTAl DIsOrDeRs,* 4tH editiOn, teXT RevisIOn *(DsM-Iv tR)* NoW iNclUDEs NEw crITEria RefeRRInG To "EXCesSIvE WOrry oveR physICAL heALTH And CompLaInTs of PAin (Eg, heADAChEs or JOINt, aBdOmInAl, Or OtheR pAinS)."^[@reF6]^ thIs sUPPlEmeNT OF dIAgnoStiC CrITeRia IS iNDIcATive Of An AGAininCREASiNG AwaRENEss Of ThE iMpoRtANcE OF sOmaTIC syMptomS In dEpreSSion.
WHAt is mEANt By "somATic" IN SomaTIC SYmpTOms of DePRESSIon?
=============================================================
IN tHE LitERAtURE TheRe ARe MANy tERMS used TO dESCribE somATic sYMPTOmS IN depRESSion: SomaTIc, soMaTlZEd, pHYsical, bodiLY, SOmaToFoRm, painfUl, PSyCHOsomATiC, veGeTAtIve, mEdiCaLLy UnExPlAINED, maSKEd, etc.^[@ReF7]^ ThEse DIVErsE TeRMS REFer tO DifFErEnT thEORetICAL or dIaGNOStIc cONCEPts. FOR States Of DepResSiVE MoOd the NEUtrAL term "SOmATic" iS preFerred, comprISIng varIOUS BoDILY sENSatIONS thAT A dEpResSeD INDIvIDuaL PerCEIveS as UNpleASaNT oR wOrrIsome. tHeSE dYsesTHesiaS aRE Very OftEN LocALIzed IN certaIN bOdY PArTs or oRGaNS, oR maY aFFecT ThE whoLE BodY iN Its VITaL COndiTioN, aS in tHE cAsE OF fAtIgue Or losS Of EnerGy. sEvEral baSiC PhySical DYSFuncTIOns, sUch aS thOSE Of SleEP, APpeTItE, OR dIGESTIOn, arE AlsO to be INclUdeD in THE term "sOmAtIc." In adDitiOn, it mAY Be CLiniCaLLy reLEvanT tO dIfferEntiATE betweEn PAiNfUL aND noNPaLNfUL SoMatic SYmPtoms of DEPRessioN. FROM a dIaGnOSTiC PeRSPECTIVE oNE hAs tO kEep In mind tHAt SOmATic SYmPtOmS plaY A siGnificANT rOLE bOtH in pRIMARY psyCHIatrIc DisorDErs, fiRST aNd FoREmOST dePrEsSIve aND anXIEty DisOrderS, AnD IN sOMATOfOrM DisoRdeRS. ANd iN DIfFeRentiaL DIAgnOsIs, SOMAtIc syMPTOMs MuSt BE CoNSIderEd As PoSSibLy even INdicaTIVe OF undErlyInG soMatic DISeAseS. A DiAGnOSTIC cHALLEngE May Be sEeN IN THe WelL-kNOWN facT thAt DEPRESSIvE, ANXIEty, soMAtofOrm DIsORDers, And MedicAl cOndITIONs Are FREQUeNtlY coEXisTenT, Or inTERAct In the iNdIViDUal pAtIENt.^[@REf8]-[@rEf10]^ reGarDInG ThE AsSesSmEnT oF soMaTiC SymPTOMs, KROEnke CORReCtlY poinTs OuT THAt diAGNOSIS VERY OftEn IS mORE appROxiMATivE THaN PReCiSE. pRESentEd sOmATiC sYmPtoms maY bE eiTHER CLearlY AtTributeD TO A dISTINCt mEdicaL DisordeR Or bE pLacED INto One OF THE folloWINg HEuRiStiC CAteGorIeS: SOMatoforM diSoRder, ANotHer prIMAry PSYChIAtRIc dISOrdEr (OFTEN DEPressION anD/OR AnXiety), FunctIONaL sOMaTiC SYNdROMe (eG, irRITAble bOweL SYNdromE, FiBRoMyALgiA, ChRONiC FATiGUE syNdRomE), "SYMPTOM-oNlY" dIAGNOSIS (Eg, LOw Back pAIN, IDIOPaTHIc dIzziness) Or OnLY paRTiAllY explAInEd BY a dEfinEd mEDiCAL diSordeR (eg, many STaTes Of cHRONIC PaiN).^[@Ref11]^
epIdEmIOLoGiCal sTUdiEs MAY ProviDe aN ILlumiNATINg SurVEy Of tHe preVaLEnCe oF sOMATic syMPTomS IN DePReSSiVE DiSorDeRS, espECIAlLy Those eNCoUNTeREd in PriMArY CArE, aND The progNostiC VALue Of SoMaTic sympToms rEGArDInG theiR DeveloPMent IN the fURthER COursE Of ILlNeSS.
sOMATiC SYmPTOms oF DEPRessIVe diSoRDers in InPATIeNt care AnD primaRy CARE
===========================================================================
iN A CLInicAl sTUdy, HAmiLToN rePoRTeD tHat somatIC SyMptOMS PReVaIlED in a GREat MajorItY Of dePrEsSEd PaTIEnTs.^[@reF12]^ SOMatIC sYmPtoMS, pArtICUlARLY SomATIc anxiEtY And fatIGUE, weRE DoCUMENTED iN Up To 80% Of a sampLe oF 260 wOmEn and 239 MeN sufFEriNg FRoM MAjoR depRESsIoN. tHeSe sOmATiC SyMPtOMs VEry FrEquentlY hAD An UndErlYiNg psYchOPaThOLOGicalLY RElEvANT hypOChOnDrIAsIS, BOTh in WOmen aNd MeN. ThiS sTUdY coNFiRmED EArLier studies showINg THAT dEpRessive DIsORdErS WitH PrEDoMiNANTly SomatIC presentATIoN WeRE LIKEly tO bE the most commON fORm OF DePRESSIoN, bOTH in inPaTiEnt anD oUTPatIENT cAre.^[@reF13],[@rEF14]^ HaGnELL anD RoRsMan sTReSSEd THE iNdIcaTiVe SIgNiFICaNcE oF sOMATIc sYMptoMs In DePRESSED pRIMARY CarE pATiEnts rEGARdinG tHEIR risK OF SUicIDe.^[@ReF15]^
epIdemiOLogiCaL STUdiES DESigNed to EsTablIsH PREvAlEnCE fiGUreS fOr depressiVe DiSORDERs In primarY cARE DurinG rECENT YEarS HAVe uNifoRmLy dEMOnStRaTeD ThAt dEPrEsSive DisordeRs ARe hIGhlY PRevaLEnT AT tHis lEVEl oF MEdiCal carE.^[@rEF16]-[@Ref19]^ fOr thE Great mAjoRItY Of dePResSEd PATienTs sEEkING pRofeSsiOnAl HeLP In The OfFIcIal HealTH CArE SYstem, GeNeRal practiTioNeRs aND InTERniSTS ARE tHE DecISIVe inTErFaCe FoR diAgNoSiS And TREATMENT OF dePrEsSion.^[@REF20]^ PRIMarY-cArE PAtieNtS with depREssIon vEry oFtEn PreSEnt wIth sOmATIC coMpLaINTS. tHiS sEEMs to BE MorE ThE RUle ThAn THE ExcEptioN woRldwIDE.^[@Ref21],[@ref22]^ Two Of the threE MoST cOmMON sYmPTOMS REpORTED dURinG a cURreNT dePrEssive EpIsOdE WEre SOMAtIc (tLreD/nO EneRGY/lIStlEsS: 73%, BRoken SleEp/deCREaSed slEeP: 63%) aS shOwN bY tHE EURopEAn sTudY SoCiETY stUDY (DepReS II).^[@reF23]^ tHIs stUDY, hOWEvER, ALso UNdeRlINeD ThAt 65% of THe DEpressEd prIMAry CARe PatiENts SuffeReD FrOM a ConcOMitAnT MedICaL condiTIOn PoINtInG To SoME liKElY DIffICuLTIes IN dIFFerEnTiAL DIaGnoSIS. tHe mULTlCEntER intErnAtIoNAl study (n =1146) COnDuctED by | Historical conceptualizations ofdepression ===========================================Thereis a long tradition in phenomenologlcal psychopathology that stresses basic bodilyalterations as core features of depressive states. Thus, Wernicke used the term "vital feelings" to describe certain somatic symptoms occurring in affectivepsychoses.^[@ref1]^ Vital feelings refer to the close relationship of the bodyto the awareness ofself. They determinethe way we experienceour body and theimpression we assume our physical presence makes onother people. Vital feelingsare somatic affects localized In different parts of the body. Whereasvitalfeelings constitute the bodily backgroundof our normal experiences, they may moveto the fore Ina depressive mood. For example, depressed patients very oftencomplain of a headache whichis described not exactly as an ordinary pain, but more as an unbearable pressure "like aband around the head." Other disturbed vital feelings affect thechest orthe abdomen, and mediate unpleasant sensations of weight,tension, heaviness, or Inhibition, totally absorbing the focus of attention. In quitea similar way Dupréspeaksof "coenestopathic states" which mean a distressing,qualitative change ofnormal physical feeling In certain areas of the body duringan episode of depressive mood.It Is a global loss of vitality In which all bodilyparts and functions may be altered, and all their performances depressed.^[@ref2]^ Kurt Schneider considered these disturbances of vital feelings to bethe coreof cyclothymic depression. In his psychopathologlcal assessmentthey were of paramount diagnostic significance In depressive Illness, more or lessequivalent to the first-rank symptoms In schizophrenia.^[@ref3]^ Huber discriminatedbetween vital disturbances on the onehand and vegetative symptomsIn depression on the other.^[@ref4]^ Vital disturbances referto the vitalfeelings just mentioned. Theycomprise a loss of general vital tone of the body, a prevailing fatigueor exhaustibility, and various forms of somatic dysesthesia, typically of a static,more localized character affectinghead, chest,heart region, or abdomen. All-pervasive sensations of anesthesia, stiffness, and alienation ofthe totalbody may characterize a somatopsychic depersonalizationin depression which mayappear as a Cotard\'s syndrome in its extremeform. If the vitaldisturbances take on a peculiar form that is difficult to describe inordinaryeveryday words, Huber speaks of a "coenesthetic" depression which must be typologically differentiated from the bizarre states of coenesthetic schizophrenia. Vegetative symptomsare closely associated with these vital disturbances andcoenesthesias in depression. Disturbancesof sleep, appetite, and digestion aremost frequent. However, there may be many other vegetative symptoms in depression such as disordered salivation, transpirationand lacrimation, cardiac arrhythmias and dyspnea, loss of libido and varioussexual dysfunctions, dys- or amen? orrhea, loss of or increase in bodyweight,decreased turgor of the skin, loss of hair, decrease in body temperature,nausea, vomiting,meteorism, dizziness, sweating, or sensations ofcoldness. Both vital disturbances, coenesthesias and vegetative symptoms,are typicallycoexistent with the well-knownaffective, behavioral, and cognitive symptomsof depression.With respect to the differentsettings of medical care, however, these psychologicalsymptoms of depression maybe masked by a dominant reporting of somaticsymptoms. M. Bleuler addressed the point in hisbook *Depressions in Primary Care,* in 1943: *"It is a common and frequent observation that depressive patients with single somatic complaints come to the consulting room of the general practitioner, internal specialist,and even the surgeon, gynecologist, ophthalmologist, urologist and other medical specialists, and spontaneously, they only speak of somatic phenomena while concealing their state of depressive mood. They reportpalpitations, tightness of the chest, loss of appetite, obstipation, pollakiuria, amenorrhea and many others. Only when one looks at their psychic state does one discover that they report numerous hypochondriacideasalso in other areas, that in addition they produce depressive ideas of impoverishment and sin, that beyond that their whole stream of thoughts is inhibited, that the depression manifests itself not only in the somatic complaints reported, but in various other bodily expressions."^[@ref5]^* In spite of this long-standing psychopathological view on the somatic foundation of depressive mood, at least in moderate and severeclinical states, it is bewildering that the official psychiatric classification systemsofthe *Diagnostic and StatisticalManual of Mental Disorders,* 4th edition *(DSM-IV)* and the *ICD-10* *Classification of Mental and Behavioral Disorders. Clinical Descriptions and Diagnostic guidelines (ICD-10)* only marginally appreciate somatic symptoms as diagnostic criteria for depressive disorders while focussing on the psychological symptoms of affect and cognition. So, *DSM-IV* listsonly threecriteria of somatic symptomsformajor depressive disorder:sleep disturbance,appetite disturbance, andfatigue orloss of energy. And correspondingly, in *ICD-10,* disturbances of sleep and appetite, loss of libido, and amenorrhea are the onlysomaticsymptoms considered to be of diagnostic significance for major depression. Beyond this shortlist of predominantly vegetative symptoms, no painful physical symptomsare mentioned in either the *DSM-IV* or*ICD-10.* There seems to be amajor shift In diagnostic practice, however; the secondversion of the *Diagnostic and Statistical Manual of Mental Disorders,* 4th edition, Text Revision *(DSM-IVTR)* now Includes new criteria referring to "excessive worry over physical health and complaints of pain (eg, headaches or joint, abdominal, or otherpains)."^[@ref6]^ This supplement of diagnostic criteria Is Indicative of an againIncreasing awareness of the importance ofsomatic symptoms in depression. What ismeant by "somatic" in somatic symptoms of depression? ============================================================= In theliterature there are manyterms used to describe somaticsymptoms in depression: somatic,somatlzed,physical, bodily, somatoform, painful, psychosomatic, vegetative, medically unexplained, masked, etc.^[@ref7]^ These diverse termsrefer to different theoretical or diagnostic concepts. Forstates of depressive mood the neutralterm "somatic" is preferred, comprising various bodily sensationsthatadepressed individual perceives as unpleasant orworrisome. These dysesthesias are very often localized In certainbody parts or organs, or may affect the whole body InItsvital condition, as In the case of fatigue or lossof energy. Several basic physical dysfunctions,suchas those of sleep, appetite,or digestion, are also to be included in theterm"somatic." In addition,It may be clinically relevanttodifferentiate between painful and nonpalnful somatic symptoms of depression. From a diagnostic perspective one has to keep in mind that somatic symptoms play a significant role bothinprimary psychiatric disorders, firstand foremostdepressive and anxiety disorders, andin somatoform disorders.And In differential diagnosis,somatic symptoms must be considered as possibly even Indicative of underlying somatic diseases.A diagnostic challenge may be seen In the well-known fact that depressive, anxiety,somatoform disorders, and medical conditions are frequently coexistent, orInteract Inthe Individual patient.^[@ref8]-[@ref10]^ Regarding the assessment of somatic symptoms, Kroenke correctly points out that diagnosis very often is more approximative than precise. Presented somatic symptoms may be either clearly attributed to a distinct medical disorderor be placed into one of the following heuristic categories: somatoformdisorder,anotherprimary psychiatric disorder (oftendepression and/or anxiety), functional somatic syndrome (eg, irritable bowel syndrome, fibromyalgia, chronic fatigue syndrome), "symptom-only" diagnosis (eg,low back pain, idiopathic dizziness)or only partially explained by a defined medical disorder (eg,many states ofchronic pain).^[@ref11]^ Epidemiological studies may provide an illuminating survey ofthe prevalence of somatic symptoms indepressive disorders, especially those encountered in primary care, and the prognostic value of somaticsymptoms regarding their development in the further course of illness. Somaticsymptoms of depressive disorders in inpatientcare and primarycare =========================================================================== In a clinicalstudy,Hamilton reported thatsomaticsymptoms prevailed ina great majority of depressed patients.^[@ref12]^ Somatic symptoms, particularly somaticanxiety and fatigue,were documented in up to 80% of a sample of 260 women and 239 men suffering from major depression. These somatic symptoms very frequently had an underlying psychopathologically relevant hypochondriasis, both inwomen andmen. This study confirmed earlier studies showing that depressive disorders with predominantly somaticpresentation werelikely to be the most common form of depression, both in inpatient and outpatient care.^[@ref13],[@ref14]^ Hagnell and Rorsman stressed the Indicative significance of somatic symptoms indepressed primary care patients regarding their risk of suicide.^[@ref15]^ Epidemiological studiesdesignedto establishprevalence figures for depressivedisordersInprimary care duringrecent yearshave uniformly demonstrated that depressive disorders are highly prevalent at this level of medical care.^[@ref16]-[@ref19]^For the great majority of depressed patients seeking professionalhelp in the official health care system, general practitioners and internists are the decisive interface for diagnosis and treatment of depression.^[@ref20]^ Primary-care patients with depression very often present with somatic complaints. This seems to be more the rule than the exception worldwide.^[@ref21],[@ref22]^Two of the three mostcommon symptomsreported duringacurrent depressive episode weresomatic (tlred/no energy/listless: 73%,broken sleep/decreased sleep: 63%) asshown bythe European Study Society study (DEPRES II).^[@ref23]^ This study, however, also underlined that 65% of the depressed primary care patients sufferedfrom a concomitant medical condition pointing tosome likelydifficulties In differential diagnosis. The multlcenter Internationalstudy (n =1146) conducted by | Historical conceptualizations _of_ depression _===========================================_ _There_ is a long _tradition_ in _phenomenologlcal_ _psychopathology_ that _stresses_ basic _bodily_ alterations as core features of depressive states. Thus, Wernicke _used_ _the_ term "vital _feelings"_ _to_ describe certain _somatic_ symptoms occurring in affective psychoses.^[@ref1]^ Vital feelings refer _to_ the close relationship of _the_ body to the awareness of self. They determine the way we experience our body and the impression we assume _our_ _physical_ _presence_ makes on other people. _Vital_ _feelings_ _are_ _somatic_ affects localized _In_ different _parts_ of the body. Whereas vital feelings constitute the bodily _background_ of _our_ normal _experiences,_ they may _move_ to the fore _In_ a depressive mood. For example, _depressed_ patients very often _complain_ _of_ a headache _which_ is described not exactly _as_ an ordinary _pain,_ but more as an unbearable pressure _"like_ a _band_ around the head." Other disturbed vital feelings _affect_ the chest or the _abdomen,_ and mediate unpleasant _sensations_ of weight, _tension,_ heaviness, or Inhibition, totally _absorbing_ the focus of attention. In _quite_ a similar way Dupré _speaks_ of "coenestopathic _states"_ which _mean_ a distressing, _qualitative_ change of normal physical feeling In certain _areas_ of the body during _an_ episode of _depressive_ mood. It Is a global loss of vitality In which all bodily parts and functions may _be_ altered, and all their performances depressed.^[@ref2]^ Kurt Schneider considered these disturbances of vital feelings to be the core of cyclothymic depression. In his _psychopathologlcal_ assessment _they_ were of paramount diagnostic _significance_ In depressive Illness, more or less equivalent to the first-rank symptoms In schizophrenia.^[@ref3]^ Huber _discriminated_ between vital disturbances _on_ the one hand and vegetative symptoms In depression on the other.^[@ref4]^ Vital disturbances refer _to_ the vital feelings just mentioned. They comprise a _loss_ of general vital tone of the body, a prevailing fatigue or exhaustibility, _and_ various forms of somatic _dysesthesia,_ typically of a static, more localized character affecting _head,_ chest, heart region, or abdomen. All-pervasive _sensations_ of _anesthesia,_ stiffness, _and_ alienation of the total body may characterize a somatopsychic depersonalization in _depression_ which may appear as _a_ Cotard\'s syndrome in _its_ _extreme_ form. If the vital disturbances _take_ on a peculiar _form_ that is difficult _to_ _describe_ in ordinary everyday words, Huber _speaks_ of a "coenesthetic" depression which must be typologically differentiated from _the_ _bizarre_ states of coenesthetic schizophrenia. Vegetative symptoms _are_ closely associated with these vital disturbances _and_ coenesthesias in _depression._ Disturbances of _sleep,_ _appetite,_ _and_ digestion are _most_ frequent. However, there may be many _other_ vegetative _symptoms_ in depression such as disordered _salivation,_ transpiration and lacrimation, cardiac arrhythmias and _dyspnea,_ loss of libido and various sexual dysfunctions, dys- _or_ amen? orrhea, loss of or increase in _body_ _weight,_ decreased turgor of the skin, loss of hair, _decrease_ in body temperature, _nausea,_ vomiting, meteorism, _dizziness,_ sweating, _or_ sensations of coldness. Both vital disturbances, coenesthesias _and_ _vegetative_ _symptoms,_ are typically _coexistent_ with _the_ well-known affective, behavioral, and _cognitive_ symptoms of depression. With respect to the _different_ settings of _medical_ care, however, these psychological symptoms of depression may be masked _by_ a dominant reporting _of_ somatic symptoms. _M._ _Bleuler_ addressed the point _in_ his book *Depressions in Primary Care,* in 1943: *"It is a common and frequent observation that depressive patients with _single_ somatic complaints come to _the_ consulting _room_ of the general practitioner, _internal_ specialist, and even _the_ surgeon, gynecologist, ophthalmologist, urologist _and_ other medical specialists, and _spontaneously,_ they only speak of somatic phenomena while _concealing_ their state of depressive mood. They report palpitations, _tightness_ _of_ the chest, loss _of_ appetite, obstipation, pollakiuria, amenorrhea and many others. Only when one looks at their psychic _state_ does one discover _that_ they report numerous hypochondriac _ideas_ _also_ in other areas, that _in_ addition they produce depressive ideas of impoverishment and _sin,_ that beyond that their whole stream of thoughts _is_ inhibited, that _the_ _depression_ _manifests_ itself not only in the somatic complaints _reported,_ _but_ in various other bodily _expressions."^[@ref5]^*_ In spite of this long-standing psychopathological view on the somatic foundation of depressive mood, _at_ least in moderate and _severe_ _clinical_ states, it is bewildering _that_ the _official_ psychiatric classification systems of the *Diagnostic and Statistical Manual of Mental _Disorders,*_ 4th edition *(DSM-IV)* and the _*ICD-10*_ *Classification of Mental and Behavioral Disorders. Clinical Descriptions and _Diagnostic_ _guidelines_ (ICD-10)* only marginally _appreciate_ somatic _symptoms_ as _diagnostic_ _criteria_ for depressive disorders while _focussing_ _on_ the psychological symptoms of affect and cognition. So, _*DSM-IV*_ _lists_ only _three_ criteria _of_ somatic symptoms for major depressive disorder: sleep _disturbance,_ appetite disturbance, and fatigue _or_ _loss_ of energy. And correspondingly, in *ICD-10,* disturbances of sleep and appetite, loss of libido, and amenorrhea _are_ the only _somatic_ _symptoms_ considered to be of diagnostic significance for major depression. Beyond _this_ short list of predominantly _vegetative_ symptoms, no painful physical _symptoms_ are _mentioned_ in either the *DSM-IV* or *ICD-10.* There seems to be _a_ _major_ shift In diagnostic _practice,_ however; the second version of the *Diagnostic and Statistical Manual _of_ Mental Disorders,* 4th edition, Text _Revision_ *(DSM-IV TR)* now _Includes_ new criteria referring _to_ _"excessive_ worry over physical health and complaints _of_ _pain_ (eg, headaches or joint, _abdominal,_ or other pains)."^[@ref6]^ This supplement of diagnostic _criteria_ Is Indicative of an againIncreasing _awareness_ of the _importance_ of _somatic_ _symptoms_ in depression. _What_ is _meant_ by "somatic" in somatic _symptoms_ of depression? ============================================================= In the literature there are _many_ terms _used_ to describe somatic symptoms in depression: _somatic,_ _somatlzed,_ physical, _bodily,_ somatoform, painful, psychosomatic, vegetative, _medically_ unexplained, masked, etc.^[@ref7]^ _These_ diverse terms refer _to_ different theoretical or diagnostic concepts. For states of _depressive_ mood the neutral _term_ "somatic" _is_ _preferred,_ _comprising_ various bodily _sensations_ that a depressed individual _perceives_ as unpleasant _or_ worrisome. _These_ dysesthesias _are_ very often localized _In_ certain body parts _or_ _organs,_ or may affect the whole body _In_ Its vital condition, as In _the_ case _of_ fatigue or loss of energy. Several _basic_ physical dysfunctions, _such_ as those of sleep, appetite, or digestion, are _also_ to be included _in_ _the_ _term_ "somatic." In addition, It may _be_ clinically relevant to differentiate between painful _and_ nonpalnful somatic _symptoms_ of depression. From a diagnostic _perspective_ one _has_ to keep in mind that somatic symptoms play a _significant_ role both in primary psychiatric disorders, first and foremost depressive and anxiety disorders, and _in_ somatoform disorders. _And_ In differential _diagnosis,_ somatic symptoms _must_ be considered as possibly even Indicative _of_ underlying somatic diseases. _A_ diagnostic challenge may be seen In the well-known fact _that_ depressive, anxiety, somatoform disorders, and medical conditions are frequently coexistent, or Interact In the Individual _patient.^[@ref8]-[@ref10]^_ _Regarding_ the assessment of _somatic_ symptoms, _Kroenke_ correctly points out that diagnosis _very_ _often_ _is_ more approximative than precise. _Presented_ somatic _symptoms_ may _be_ either clearly attributed _to_ a distinct medical _disorder_ or _be_ placed into one of _the_ following _heuristic_ categories: _somatoform_ disorder, another primary _psychiatric_ disorder (often depression and/or anxiety), _functional_ somatic syndrome (eg, irritable bowel syndrome, _fibromyalgia,_ chronic _fatigue_ syndrome), "symptom-only" _diagnosis_ (eg, low back pain, _idiopathic_ dizziness) or only partially explained by _a_ defined medical disorder (eg, many states of chronic pain).^[@ref11]^ Epidemiological studies may provide an illuminating survey of the _prevalence_ of somatic _symptoms_ in _depressive_ disorders, especially those encountered in primary care, _and_ _the_ prognostic _value_ of _somatic_ symptoms regarding _their_ _development_ in _the_ further course _of_ illness. Somatic _symptoms_ of depressive disorders in inpatient care _and_ primary care =========================================================================== In a clinical study, _Hamilton_ reported that somatic symptoms prevailed in a great majority of depressed _patients.^[@ref12]^_ Somatic _symptoms,_ particularly somatic anxiety and fatigue, were documented in up to 80% of _a_ sample of _260_ women and 239 _men_ _suffering_ from major depression. _These_ somatic symptoms very frequently _had_ an _underlying_ psychopathologically relevant hypochondriasis, both in _women_ and men. This study _confirmed_ earlier studies showing that _depressive_ disorders with predominantly somatic presentation were _likely_ to _be_ the _most_ _common_ form of depression, both in inpatient and outpatient care.^[@ref13],[@ref14]^ Hagnell and Rorsman _stressed_ the _Indicative_ significance of somatic symptoms in depressed _primary_ care patients regarding their risk of _suicide.^[@ref15]^_ Epidemiological studies designed to establish prevalence figures for _depressive_ disorders _In_ primary _care_ during recent years have uniformly demonstrated that depressive disorders are highly _prevalent_ at _this_ level of medical care.^[@ref16]-[@ref19]^ For the _great_ majority of _depressed_ patients seeking professional help in the official health care _system,_ general practitioners and internists are the decisive interface _for_ diagnosis and _treatment_ of depression.^[@ref20]^ _Primary-care_ patients with _depression_ very often present with somatic _complaints._ _This_ _seems_ to be more the rule than the exception worldwide.^[@ref21],[@ref22]^ Two of the _three_ most common symptoms reported during a current _depressive_ episode were somatic (tlred/no energy/listless: 73%, broken sleep/decreased sleep: _63%)_ as shown by the European Study Society study (DEPRES _II).^[@ref23]^_ _This_ study, however, also underlined that 65% _of_ _the_ depressed primary care patients suffered from a concomitant medical condition _pointing_ to some likely difficulties In differential diagnosis. The multlcenter International study _(n_ =1146) conducted by |
{#sp1 .466}
{#sp2 .467}
{#sp3 .468}
| ! [ ] ( 2004 - 2012 ) { # sp1. 050 }! [ ] ( 80 - 06 ) { # 12. 999 }! [ ] ( power - 0068 ) { # 4. 468 } | ! [] (gIasHowmedj75520 - 005U) {# sp1. 466 }! [] (glaeTowmedj75520 - 00U*) {# sp2. 467 }! [] (glasgoEmedj755@0 - 00UU) {# sp3. 468 } | {#sp1 .466} {#sp2 .467} .468} | {#sP1 .466}
{#sp2 .467}
{#sP3 .468}
| {#sp1 .466} {#sp2 .467} {#sp3 .468} | {#sp1 .466} {#sp2 .467} {#sp3 .468} |
Irinotecan (CPT-11, Campto®) -- a semisynthetic, water-soluble derivative of the plant alkaloid camptothecin -- is the standard of care in the treatment of advanced colorectal cancer when 5-fluorouracil (5-FU)-based therapy has failed ([Cunningham *et al*, 2001](#bib5){ref-type="other"}). Phase II trials have demonstrated objective response rates of 16--27% in pretreated patients, with stabilisation of disease in a further 40--60% of patients ([Rougier *et al*, 1997](#bib13){ref-type="other"}; [Van Cutsem *et al*, 1999](#bib17){ref-type="other"}). Median overall survival rates of up to 10 months are achievable when irinotecan is used in relapsed/refractory colorectal cancer ([Shimada *et al*, 1993](#bib15){ref-type="other"}; [Rothenberg *et al*, 1996](#bib12){ref-type="other"}, [1999](#bib11){ref-type="other"}; [Pitot *et al*, 1997](#bib10){ref-type="other"}; [Rougier *et al*, 1997](#bib13){ref-type="other"}; [Van Cutsem *et al*, 1999](#bib17){ref-type="other"}). Two European phase III trials investigating the efficacy and safety of irinotecan, following 5-FU failure in advanced colorectal cancer, have demonstrated significant improvements in survival compared with best supportive care and 5-FU ([Cunningham *et al*, 1998](#bib6){ref-type="other"}; [Rougier *et al*, 1998](#bib14){ref-type="other"}). The main adverse events accompanying treatment with irinotecan in these trials were diarrhoea, neutropenia, fatigue, nausea and vomiting.
Although 350 mg m^−2^ as an intravenous infusion every 3 weeks is the standard recommended dosage of irinotecan, pharmacokinetic parameters of irinotecan-lactone and the active metabolite SN-38-lactone vary between individuals ([Xie *et al*, 2002](#bib18){ref-type="other"}). This may be attributed to differences in the levels of the enzymes that metabolise irinotecan, notably carboxylesterase for SN-38. Furthermore, the variable interindividual patient exposure to SN-38 has been identified as an important determinant of toxicity ([Mathijssen *et al*, 2002](#bib8){ref-type="other"}).
At the same time, there is convincing evidence of a dose--response relationship, and therefore a rationale for increasing doses when possible. In a phase I trial by [Abigerges *et al* (1995)](#bib1){ref-type="other"}, there were two recommended doses: 350 mg m^−2^ without high-dose loperamide and 600 mg m^−2^ with high-dose loperamide. With the exception of one responder treated at 260 mg m^−2^, all objective responses were observed at dose levels above 350 mg m^−2^. [Merrouche *et al* (1997)](#bib9){ref-type="other"} provided further support for this from a phase I trial in which an increased tumour response was seen at an irinotecan dose level of 500 mg m^−2^.
Thus, these data suggest that a fixed-dose strategy for administration of irinotecan may not be optimal for all patients, thereby comprising treatment. The interindividual variability in pharmacokinetic parameters and dose--response relationship provided the rationale for investigating a dose optimisation strategy for irinotecan ([Chabot *et al*, 1995](#bib4){ref-type="other"}). The present study investigated different strategies, using doses of irinotecan up to 500 mg m^−2^, as single-agent therapy in the treatment of patients with metastatic colorectal cancer resistant to 5-FU.
METHODS
=======
Patients
--------
Eligibility criteria included metastatic, histologically proven adenocarcinoma of the colon or rectum progressing on 5-FU-based chemotherapy (adjuvant and/or palliative); administration of ⩽2 5-FU-based regimens in the adjuvant setting or ⩽1 in the palliative setting; World Health Organization (WHO) performance status (PS) of ⩽2; adequate haematological, renal and hepatic function. Exclusion criteria included prior treatment with topoisomerase-I inhibitors; evidence of central nervous system metastases; prior history of chronic diarrhoea; current infection; or any other serious illness or medical condition.
Study design and conduct
------------------------
This was a prospective, randomised, multicentre, open-label, phase II study. The study was conducted in accordance with the Declaration of Helsinki (Hong Kong revision, 1989) and with the approval of the Ethics Committee (Institutional Review Board) at each participating centre. Written informed consent was obtained from each patient prior to his or her enrolment into the trial. An independent Monitoring Committee regularly assessed the safety and efficacy issues and reviewed the conduct of the study if needed. An External Response Review Committee (ERRC) assessed tumour responses without knowledge of the randomisation arm. The aim of the study was to determine the optimal dosing strategy in terms of efficacy and safety of single-agent irinotecan (by individual dose optimisation based on patient tolerance to treatment, or optimisation based on specific baseline risk factors) in the treatment of 5-FU-resistant patients with metastatic colorectal cancer. The primary efficacy endpoint was the overall response rate.
### Dosing scenarios
Patients were randomised to one of three groups (A, B and C (outlined below)), each group receiving irinotecan as a 30 min intravenous infusion scheduled every 21 days. This dosing interval could be extended to a maximum of 35 days in the event of persistent toxicity to allow satisfactory recovery from the previous cycle. Doses \<250 mg m^−2^ or \>500 mg m^−2^ were not used in this study; patients who exhibited significant toxicity at 250 mg m^−2^ were withdrawn from the study.
Group A was the reference group in which a fixed dose of 350 mg m^−2^ of irinotecan was administered on Day 1. In subsequent cycles, the dose of irinotecan could be decreased (but not increased) according to the presence of significant toxicity at this dose.
Groups B and C investigated dosing scenarios to select patients for whom the higher dose of irinotecan (500 mg m^−2^) could be optimally used. Patients randomised to Group B received irinotecan at a starting dose of 250 mg m^−2^ followed by increasing doses (350 and 500 mg m^−2^) depending on the tolerance observed in the preceding cycle. In the event of significant toxicity, dose reductions were implemented.
In Group C, the irinotecan dose was based on protocol-defined toxicity risk factors identified at baseline: grade 3--4 neutropenia (bilirubin \>70% upper limit of normal (UNL), haemoglobin \<12 g dl^−1^, \>3 organs involved) and/or grade 3--4 diarrhoea (PS⩾1, creatinine \>70% UNL ([Freyer *et al*, 2000](#bib7){ref-type="other"})). Patients could be started at an irinotecan dose of 500 mg m^−2^ in the absence of toxicity risk factors. The starting dose of irinotecan was 350 mg m^−2^ in patients with one risk factor or one factor from each group, and 250 mg m^−2^ for patients with \>2 risk factors or two factors from the same group. The dose was not escalated, but could be reduced to 250 mg m^−2^ in the event of significant treatment-emergent toxicity.
Concomitant treatments and follow-up
------------------------------------
Antiemetic drugs were administered as premedication to irinotecan infusions. Atropine was permitted for acute anticholinergic symptoms and loperamide (or similar) for delayed diarrhoea. In addition, preventative oral antibiotic therapy (e.g. an oral fluoroquinolone) was administered to patients with persistent (\>48 h) grade 4 diarrhoea or for diarrhoea associated with grade 3--4 neutropenia or fever. No granulocyte-colony-stimulating factor (G-CSF) support was allowed. All patients were followed until disease progression, unacceptable toxicity or death occurred, or the patient chose to withdraw from the trial. In all cases, in each group where toxicity necessitated a dose reduction, delay or study treatment termination, the patient was followed up until the event had resolved.
Efficacy, safety and pharmacokinetic evaluations
------------------------------------------------
Tumour response rate, the primary efficacy end point, was measured according to WHO criteria and evaluated by the ERRC. Response was defined as complete (CR) plus partial (PR) response and as tumour growth control in terms of stabilisation of disease (PR plus no change/stable disease). Secondary efficacy variables were the duration of response and disease stabilisation, time to progression (TTP), time to treatment failure (TTF) and overall survival. The duration of response was measured from the first day of infusion of irinotecan to the first date that disease progression was noted or to the date of death for any reason. Time to progression was calculated from the date of randomisation to the first documented date of progression or the date of death for any reason. Time to treatment failure was the period between the date of randomisation and the date | irinotecan ( cpt - 11, campto® ) - - a semisynthetic, water - soluble derivative of the plant alkaloid camptothecin - - is the standard of use in the treatment of advanced colorectal cancer when 5 - fluorouracil ( 5 - fu ) - based therapy has failed ( [ cunningham * et al *, 1992 ] ( # bib5 ) { ref - type = " other " } ). phase ii trials have demonstrated objective response rate of 16 - - 27 % in pretreated patients, with stabilisation of disease in a further 40 - - 60 % of patients ( [ rougier * et al *, 1997 ] ( # bib13 ) { ref - type = " other " } ; [ van cutsem * et al *, 1999 ] ( # bib17 ) { ref - type = " other " } ). median overall survival rates of up to 10 months becomes achievable when irinotecan is used in relapsed / refractory colorectal cancer ( [ shimada * et al *, 1993 ] ( # bib15 ) { ref - type = " other " } ; [ rothenberg * et al *, 1996 ] ( # bib12 ) { ref - type = " other " }, [ 1999 ] ( # bib11 ) { ref - type = " other " } ; [ pitot * et al *, 1997 ] ( # bib10 ) { ref - type = " other " } ; [ rougier * et al *, 1997 ] ( # bib13 ) { ref - type = " other " } ; [ van cutsem * et el *, 1999 ] ( # bib17 ) { ref - type = " other " } ). two european phase iii trials investigating the efficacy testing safety of irinotecan, following 5 - fu failure in advanced colorectal cancer, have demonstrated significant improvements in survival compared in best supportive procedures and def - fu ( [ cunningham * et al *, 1998 ] ( # bib6 ) { ref - type = " other " } ; [ rougier * et al *, 1998 ] ( # bib14 ) { ref - type = " other " } ). the reported adverse events accompanying treatment with irinotecan in these trials were diarrhoea, neutropenia, fatigue, nausea and vomiting. although 350 mg m ^ −2 ^ as an intravenous infusion every 3 weeks is the standard recommended dosage of irinotecan, pharmacokinetic parameters of irinotecan - lactone and the active metabolite sn - 38 - lactone vary between individuals ( [ xie * et al *, 2002 ] ( # bib18 ) { ref - type = " other " } ). this may be attributed to differences in the levels of the enzymes that metabolise irinotecan, notably carboxylesterase for sn - 38. furthermore, the variable interindividual patient exposure to sn - 38 has been identified as an important determinant of toxicity ( [ mathijssen * et al *, 2002 ] ( # bib8 ) { ref - type = " other " } ). at the same time, there is convincing evidence of a dose - - response relationship, and therefore a rationale for increasing doses when possible. in a phase i trial by [ abigerges * et al * ( 1995 ) ] ( # bib1 ) { ref - type = " other " }, there were two recommended doses : 350 mg m ^ −2 ^ without high - dose loperamide and 600 mg m ^ −2 ^ with high - dose loperamide. with the exception of one responder treated at 260 mg m ^ −2 ^, all objective responses were observed at dose levels above 350 mg m ^ −2 ^. [ merrouche * et al * ( 1997 ) ] ( # bib9 ) { ref - type = " other " } provided further support for this from a phase i trial in which an increased tumour response was seen at an irinotecan dose level of 500 mg m ^ −2 ^. thus, these data suggest that a fixed - dose strategy for administration of irinotecan may not be optimal for all patients, thereby comprising treatment. the interindividual variability in pharmacokinetic parameters and dose - - response relationship provided the rationale for investigating a dose optimisation strategy for irinotecan ( [ chabot * et al *, 1995 ] ( # bib4 ) { ref - type = " other " } ). the present study investigated different strategies, using doses of irinotecan up to 500 mg m ^ −2 ^, as single - agent therapy in the treatment of patients with metastatic colorectal cancer resistant to 5 - fu. methods = = = = = = = patients - - - - - - - - eligibility criteria included metastatic, histologically proven adenocarcinoma of the colon or rectum progressing on 5 - fu - based chemotherapy ( adjuvant and / or palliative ) ; administration of [UNK] 5 - fu - based regimens in the adjuvant setting or [UNK] in the palliative setting ; world health organization ( who ) performance status ( ps ) of [UNK] ; adequate haematological, renal and hepatic function. exclusion criteria included prior treatment with topoisomerase - i inhibitors ; evidence of central nervous system metastases ; prior history of chronic diarrhoea ; current infection ; or any other serious illness or medical condition. study design and conduct - - - - - - - - - - - - - - - - - - - - - - - - this was a prospective, randomised, multicentre, open - label, phase ii study. the study was conducted in accordance with the declaration of helsinki ( hong kong revision, 1989 ) and with the approval of the ethics committee ( institutional review board ) at each participating centre. written informed consent was obtained from each patient prior to his or her enrolment into the trial. an independent monitoring committee regularly assessed the safety and efficacy issues and reviewed the conduct of the study if needed. an external response review committee ( errc ) assessed tumour responses without knowledge of the randomisation arm. the aim of the study was to determine the optimal dosing strategy in terms of efficacy and safety of single - agent irinotecan ( by individual dose optimisation based on patient tolerance to treatment, or optimisation based on specific baseline risk factors ) in the treatment of 5 - fu - resistant patients with metastatic colorectal cancer. the primary efficacy endpoint was the overall response rate. # # # dosing scenarios patients were randomised to one of three groups ( a, b and c ( outlined below ) ), each group receiving irinotecan as a 30 min intravenous infusion scheduled every 21 days. this dosing interval could be extended to a maximum of 35 days in the event of persistent toxicity to allow satisfactory recovery from the previous cycle. doses \ < 250 mg m ^ −2 ^ or \ > 500 mg m ^ −2 ^ were not used in this study ; patients who exhibited significant toxicity at 250 mg m ^ −2 ^ were withdrawn from the study. group a was the reference group in which a fixed dose of 350 mg m ^ −2 ^ of irinotecan was administered on day 1. in subsequent cycles, the dose of irinotecan could be decreased ( but not increased ) according to the presence of significant toxicity at this dose. groups b and c investigated dosing scenarios to select patients for whom the higher dose of irinotecan ( 500 mg m ^ −2 ^ ) could be optimally used. patients randomised to group b received irinotecan at a starting dose of 250 mg m ^ −2 ^ followed by increasing doses ( 350 and 500 mg m ^ −2 ^ ) depending on the tolerance observed in the preceding cycle. in the event of significant toxicity, dose reductions were implemented. in group c, the irinotecan dose was based on protocol - defined toxicity risk factors identified at baseline : grade 3 - - 4 neutropenia ( bilirubin \ > 70 % upper limit of normal ( unl ), haemoglobin \ < 12 g dl ^ −1 ^, \ > 3 organs involved ) and / or grade 3 - - 4 diarrhoea ( [UNK], creatinine \ > 70 % unl ( [ freyer * et al *, 2000 ] ( # bib7 ) { ref - type = " other " } ) ). patients could be started at an irinotecan dose of 500 mg m ^ −2 ^ in the absence of toxicity risk factors. the starting dose of irinotecan was 350 mg m ^ −2 ^ in patients with one risk factor or one factor from each group, and 250 mg m ^ −2 ^ for patients with \ > 2 risk factors or two factors from the same group. the dose was not escalated, but could be reduced to 250 mg m ^ −2 ^ in the event of significant treatment - emergent toxicity. concomitant treatments and follow - up - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - antiemetic drugs were administered as premedication to irinotecan infusions. atropine was permitted for acute anticholinergic symptoms and loperamide ( or similar ) for delayed diarrhoea. in addition, preventative oral antibiotic therapy ( e. g. an oral fluoroquinolone ) was administered to patients with persistent ( \ > 48 h ) grade 4 diarrhoea or for diarrhoea associated with grade 3 - - 4 neutropenia or fever. no granulocyte - colony - stimulating factor ( g - csf ) support was allowed. all patients were followed until disease progression, unacceptable toxicity or death occurred, or the patient chose to withdraw from the trial. in all cases, in each group where toxicity necessitated a dose reduction, delay or study treatment termination, the patient was followed up until the event had resolved. efficacy, safety and pharmacokinetic evaluations - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - tumour response rate, the primary efficacy end point, was measured according to who criteria and evaluated by the errc. response was defined as complete ( cr ) plus partial ( pr ) response and as tumour growth control in terms of stabilisation of disease ( pr plus no change / stable disease ). secondary efficacy variables were the duration of response and disease stabilisation, time to progression ( ttp ), time to treatment failure ( ttf ) and overall survival. the duration of response was measured from the first day of infusion of irinotecan to the first date that disease progression was noted or to the date of death for any reason. time to progression was calculated from the date of randomisation to the first documented date of progression or the date of death for any reason. time to treatment failure was the period between the date of randomisation and the date | Irinotecan (CPT - 11, Campto ®) - - a semisynthetic, water - soluble derivative of the plant alkaloid camptothecin - - is the standard of care in the treatment of advanced colorectal cancer when 5 - fluorouracil (5 - FU) - based therapy has failed ([ Cunningham * et al *, 2001] (# bib5) {ref - type = " other "} ). Phase II trials have demonstrated objective response rates of 16 - - 27% in pretreated patients, with stabilisation of disease in a further 40 - - 60% of patients ([ Rougier * et al *, 1997] (# bib13) {ref - type = " other " }; [Van Cutsem * et al *, 1999] (# bib17) {ref - type = " other "} ). Median overall survival rates of up to 10 months are achievable when irinotecan is used in relapsed / refractory colorectal cancer ([ Shimada * et al *, 1993] (# bib15) {ref - type = " other " }; [Rothenberg * et al *, 1996] (# bib12) {ref - type = " other " }, [1999] (# bib11) {ref - type = " other " }; [Pitot * et al *, 1997] (# bib10) {ref - type = " other " }; [Rougier * et al *, 1P9^] (# bib13) {ref - type = " other " }; [Van Cutsem * et al *, 1999] (# bib17) {ref - type = " other "} ). Two European phase III trials investigating the efficacy and safety of irinotecan, following 5 - FU failure in advanced colorectal cancer, have demonstrated significant improvements in survival compared with best supportive care and 5 - FU ([ Cunningham * et al *, 1998] (# bib6) {ref - type = " other " }; [Rougier * et al *, 1998] (# bib14) {ref - type = " other "} ). The main adverse events accompanying treatment with irinotecan in these trials were diarrhoea, neutropenia, fatigue, nausea and vomiting. Although 350 mg m ^ − 2 ^ as an intravenous infusion every 3 weeks is the standard recommended dosage of irinotecan, pharmacokinetic parameters of irinotecan - lactone and the active metabolite SN - 38 - lactone vary between individuals ([ Xie * et al *, 2002] (# bib18) {ref - type = " other "} ). This may be attributed to differences in the levels of the enzymes that metabolise irinotecan, notably carboxylesterase for SN - 38. Furthermore, the variable interindividual patient exposure to SN - 38 has been identified as an important determinant of toxicity ([ Mathijssen * et al *, 2002] (# bib8) {ref - type = " other "} ). At the same time, there is convincing evidence of a dose - - response relationship, and therefore a rationale for increasing doses when possible. In a phase I trial by [Abigerges * et al * (1995) ] (# bib1) {ref - type = " other " }, there were two recommended doses: 350 mg m ^ − 2 ^ without high - dose loperamide and 600 mg m ^ − 2 ^ with high - dose loperamide. With the exception of one responder treated at 260 mg m ^ − 2 ^, all objective responses were observed at dose levels above 350 mg m ^ − 2 ^. [Merrouche * et al * (1997) ] (# bib9) {ref - type = " other "} provided further support for this C5om a phase I trial in which an increased tumour respohQe was seen at an irinotecan dose level of 500 mg m ^ − 2 ^. Thus, these data suggest that a fixed - dose strategy for administration of irinotecan may not be optimal for all patients, thereby comprising treatment. The interindividual variability in pharmacokinetic parameters and dose - - response relationship provided the rationale for investigating a dose optimisation strategy for irinotecan ([ Chabot * et al *, 1995] (# bib4) {ref - type = " other "} ). The present study investigated different strategies, Hs&ng doses of irinotecan up to 500 mg m ^ − 2 ^, as single - agent therapy in the treatment of pa4ienhs with metastatic colorectal cancer resistant to 5 - FU. METHODS = = = = = = = Patients - - - - - - - - Eligibility criteria included metastatic, histologically proven adenocarcinoma of the colon or rectum progressing on 5 - FU - based chemotherapy (adjuvant and / or palliative ); administration of ⩽ 2 5 - FU - based regimens in the adjuvant setting or ⩽ 1 in the palliative setting; World Health Organization (WHO) performance s5atua (PS) of ⩽ 2; adequate haematological, renal and hepatic function. Exclusion criteria included prior treatment with topoisomerase - I inhibitors; evidence of central nervous system metastases; prior history of chronic diarrhoea; current infection; or any other serious illness or medical condition. Study design and conduct - - - - - - - - - - - - - - - - - - - - - - - - This was a prospective, randomised, multicentre, open - label, phase II study. The study was conducted in accordance with the Declaration of Helsinki (Hong Kong revision, 1989) and with the approval of the Ethics Committee (Institutional Review Board) at each participating centre. Written informed consent was obtained from each patient prior to his or her enrolment into the trial. An independent Monitoring Comm7tteD regularly assessed the safety and efficacy issues and reviewed the conduct of the study if needed. An External Response Review Committee (ERRC) assessed tumour responses without knowledge of the randomisation arm. The aim of the study was to determine the optimal dosing strategy in terms of efficacy and safety of single - agent irinotecan (by individual dose optimisation based on patient tolerance to treatment, or optimisation based on specific baseline risk factors) in the treatment of 5 - FU - resistant patients with metastatic colorectal cancer. The primary efficacy wndpoUnt was the overall response rate. # # # Dosing scenarios Patients were randomised to one of three groups (A, B and C (outlined below) ), each group receiving irinotecan as a 30 min intravenous infusion scheduled every 21 days. This dosing interval could be extended to a maximum of 35 days in the event of persistent toxicity to allow satisfactory recovery from the prWvioIs cycle. Doses \ <250 mg m ^ − 2 ^ or \> 500 mg m ^ − 2 ^ were not used in this study; patients who exhibited significant toxicity at 250 mg m ^ − 2 ^ were withdrawn from the study. Group A was the reference group in which a fixed dose of 350 mg m ^ − 2 ^ of irinotecan was administered on Day 1. In subsequent cycles, the dose of irinotecan could be decreased (but not increased) according to the presence of significant toxicity at this dose. Groups B and C investigated dosing scenarios to select patients for whom the higher dose of irinotecan (500 mg m ^ − 2 ^) co7lS be optimally used. Patients randomised to Group B received irinotecan at a starting dose of 250 mg m ^ − 2 ^ followed by increasing doses (350 and 500 mg m ^ − 2 ^) depending on the tolerance observed in the preceding cycle. In the event of significant toxicity, dose reductions were implemented. In Group C, the irinotecan dose was based on protocol - defined toxicity risk factors identified at baseline: grade 3 - - 4 neutropenia (bilirubin \> 70% upper limit of normal (UNL ), haemoglobin \ <12 g dl ^ − 1 ^, \> 3 organs involved) and / or grade 3 - - 4 diarrhoea (PS ⩾ 1, creatinine \> 70% UNL ([ Freyer * et al *, 2000] (# bib7) {ref - type = " other "} ) ). Patients could be started at an irinotecan dose of 500 mg m ^ − 2 ^ in the absence of toxicity risk factors. The starting dose of irinotecan was 350 mg m ^ − 2 ^ in patients with one risk factor or one factor from each group, and 250 mg m ^ − 2 ^ for patients with \> 2 risk factors or two factors from the same group. The dose was not escalated, but could be reduced to 250 mg m ^ − 2 ^ in the event of significant treatment - emergent toxicity. Concomitant treatments and follow - up - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Antiemetic drugs were administered as premedication to irinotecan infusions. Atropine was permitted for acute anticholinergic symptoms and loperamide (or similar) for delayed diarrhoea. In addition, preventative oral antibiotic therapy (e. g. an oral fluoroquinolone) was administered to patients with persistent (\> 48 h) grade 4 diarrhoea or for diarrhoea associated with grade 3 - - 4 neutropenia or fever. No granulocyte - colony - stimulating factor (G - CSF) support was allowed. All patients were followed until disease progression, unacceptable toxicity or death occurred, or the patient chose to withdraw from the trial. In all cases, in each group where toxicity necessitated a dose reduction, delay or study treatment termination, the patient was followed up until the event had resolved. Efficacy, safety and pharmacokinetic evaluations - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Tumour response rate, the primary efficacy end point, was measured according to WHO criteria and evaluated by the ERRC. Response was defined as complete (CR) plus partial (PR) response and as tumour growth control in terms of stabilisation of disease (PR plus no change / stable disease ). Secondary efficacy variables were the duration of response and disease stabilisation, time to progression (TTP ), time to treatment failure (TTF) and overall survival. The duration of response was measured from the first day of infusion of irinotecan to the first date that disease progression was noted or to the date of death for any reason. Time to progression was calculated from the date of randomisation to the first documented date of progression or the date of death for any reason. Time to treatment failure was the period between the date of randomisation and the date | Irinotecan (CPT-11, Campto®) -- a semisynthetic, water-soluble derivative of the plant alkaloid camptothecin -- is the standard of care in the treatment of advanced colorectal when (5-FU)-based therapy has failed ([Cunningham *et al*, 2001](#bib5){ref-type="other"}). Phase trials have demonstrated objective response rates of in pretreated patients, with stabilisation of disease in a further 40--60% of patients ([Rougier *et al*, 1997](#bib13){ref-type="other"}; [Van Cutsem *et al*, 1999](#bib17){ref-type="other"}). Median overall survival rates of up to 10 months are achievable irinotecan is used in relapsed/refractory colorectal cancer ([Shimada *et al*, 1993](#bib15){ref-type="other"}; [Rothenberg *et al*, [1999](#bib11){ref-type="other"}; [Pitot *et al*, 1997](#bib10){ref-type="other"}; [Rougier *et al*, 1997](#bib13){ref-type="other"}; [Van Cutsem *et al*, 1999](#bib17){ref-type="other"}). Two European phase III trials investigating the efficacy and safety of irinotecan, following 5-FU failure in advanced cancer, have demonstrated significant improvements in survival compared with supportive care 5-FU ([Cunningham al*, [Rougier al*, 1998](#bib14){ref-type="other"}). The main events accompanying treatment with irinotecan in these trials were diarrhoea, neutropenia, fatigue, and vomiting. Although 350 mg m^−2^ as an intravenous infusion every 3 weeks the standard recommended dosage of irinotecan, pharmacokinetic parameters of irinotecan-lactone the active metabolite SN-38-lactone vary between individuals ([Xie *et al*, 2002](#bib18){ref-type="other"}). This may be attributed to differences in the levels of the enzymes that metabolise irinotecan, notably carboxylesterase for Furthermore, the variable interindividual patient to SN-38 been identified as an important determinant of toxicity ([Mathijssen *et al*, 2002](#bib8){ref-type="other"}). At the time, there is convincing evidence of a dose--response relationship, therefore rationale for increasing doses when possible. a phase I trial by *et al* there were recommended doses: 350 mg m^−2^ without high-dose loperamide and 600 m^−2^ with high-dose loperamide. With the exception of one responder treated at 260 mg m^−2^, all objective responses were observed at dose above 350 mg m^−2^. [Merrouche *et al* (1997)](#bib9){ref-type="other"} provided further support this from a phase I trial in which an increased tumour response was seen at an irinotecan dose level of mg m^−2^. Thus, these data suggest that a fixed-dose strategy for administration of irinotecan may not optimal for all patients, thereby comprising treatment. variability in pharmacokinetic parameters and dose--response relationship provided the for investigating a dose optimisation strategy for irinotecan ([Chabot *et al*, 1995](#bib4){ref-type="other"}). The present study investigated different strategies, using of irinotecan up to 500 as single-agent therapy in the treatment of patients metastatic colorectal cancer resistant to 5-FU. METHODS ======= Patients -------- Eligibility criteria included metastatic, histologically proven adenocarcinoma of the colon or rectum progressing on 5-FU-based chemotherapy and/or palliative); administration of ⩽2 5-FU-based regimens in the adjuvant setting or ⩽1 in the palliative setting; World Health Organization (WHO) performance status of ⩽2; adequate haematological, renal function. Exclusion criteria included with inhibitors; evidence of central nervous system history of chronic diarrhoea; current infection; or other illness or condition. Study design and conduct ------------------------ This was a randomised, multicentre, open-label, phase II study. The study conducted in accordance with the Declaration of Helsinki (Hong Kong revision, 1989) and with the of the Ethics Committee (Institutional Review Board) at each participating centre. Written informed consent was obtained from each patient prior to his or her enrolment into the trial. An independent Monitoring Committee regularly assessed the safety and efficacy issues and reviewed the conduct of the study if needed. An Response Review Committee (ERRC) assessed tumour responses without knowledge of the randomisation The aim of the study was to determine the optimal dosing strategy in terms of efficacy and safety of single-agent irinotecan (by dose based on patient tolerance to or optimisation based on specific baseline factors) in the 5-FU-resistant patients with metastatic colorectal cancer. The primary efficacy endpoint was the response rate. ### Dosing scenarios were randomised to of three groups (A, B and C (outlined below)), each group receiving irinotecan 30 min intravenous infusion every 21 days. This interval could be extended to a of 35 days in the event of persistent toxicity to allow satisfactory recovery from previous cycle. Doses \<250 m^−2^ or \>500 m^−2^ were not used in this patients who exhibited significant 250 mg m^−2^ were withdrawn from the study. Group was the reference group in which fixed dose of 350 m^−2^ of irinotecan administered on Day 1. In subsequent cycles, the of irinotecan could be (but not increased) according to the presence of significant toxicity at this Groups C investigated dosing scenarios to select patients for whom the of irinotecan (500 mg m^−2^) could be used. Patients randomised to Group B received irinotecan at a starting dose of 250 mg m^−2^ followed by increasing doses (350 and 500 mg m^−2^) depending the tolerance observed the preceding cycle. In event of significant toxicity, dose reductions were implemented. In C, the irinotecan dose was based on protocol-defined toxicity risk factors identified at grade 3--4 neutropenia (bilirubin \>70% upper limit of normal (UNL), haemoglobin \<12 g dl^−1^, \>3 organs involved) and/or grade 3--4 diarrhoea creatinine \>70% UNL ([Freyer *et al*, 2000](#bib7){ref-type="other"})). Patients could started an irinotecan dose of mg m^−2^ in the absence of toxicity risk factors. The starting dose of irinotecan was 350 mg m^−2^ in patients with one risk factor or one factor from each group, and 250 mg m^−2^ for patients \>2 risk or two factors from the same group. The dose was escalated, but could be reduced to mg m^−2^ in the event of significant treatment-emergent toxicity. Concomitant treatments and follow-up ------------------------------------ Antiemetic drugs were administered as premedication to irinotecan infusions. Atropine was permitted for acute anticholinergic symptoms loperamide (or similar) for delayed diarrhoea. In addition, preventative oral antibiotic (e.g. an oral fluoroquinolone) administered to patients with persistent (\>48 h) grade 4 diarrhoea or for diarrhoea associated with grade 3--4 neutropenia or fever. granulocyte-colony-stimulating factor (G-CSF) support was allowed. All patients were followed until disease progression, unacceptable toxicity or death occurred, or the patient chose to withdraw from the trial. In all in each group where toxicity necessitated a dose reduction, or study treatment the patient was followed up until the event had Efficacy, safety and pharmacokinetic evaluations ------------------------------------------------ Tumour response rate, primary efficacy end point, was to WHO criteria and evaluated by ERRC. Response was defined as complete (CR) plus partial (PR) response and as tumour growth control in terms of stabilisation of disease (PR plus no change/stable disease). Secondary efficacy variables were the of response and disease stabilisation, time to progression (TTP), to failure (TTF) and overall survival. The duration of was measured from the first day of infusion irinotecan to the that disease progression was noted or to the date of death for any reason. Time to progression was calculated from the randomisation to the first documented date of or the date of death for any reason. Time to treatment failure was the period between the date of randomisation and the | iriNoTEcaN (cPt-11, camptO®) -- A SEMIsyNthetic, wateR-solUbLE dErivaTiVe oF thE pLAnt aLkaLoId CAmpTOthECiN -- IS THE STaNDaRD OF CARE IN tHE treatment of ADvANCED ColorECtal CanCeR When 5-FlUOrOUraciL (5-fu)-BaSED tHERAPy HAs FAIlEd ([cuNningHaM *et AL*, 2001](#biB5){REf-TYPe="oTHEr"}). Phase Ii TriALs hAVe dEmoNStRaTed ObJEctIvE ResPONSe RatEs of 16--27% IN pretReATeD patIentS, wiTh STabiLIsAtION Of DIsEAsE In A FURThEr 40--60% oF PaTIenTS ([rouGIeR *Et al*, 1997](#BIB13){rEF-TYpE="otHEr"}; [Van cUtSEM *Et AL*, 1999](#BIB17){ReF-TypE="otHEr"}). mEdIAN ovERALL SuRvIval rAteS oF uP to 10 mOnTHS are ACHIeVable WhEN iriNotEcan Is usEd iN RelapSeD/REfRACtoRY cOLoreCTal CaNcer ([shiMAdA *Et Al*, 1993](#bib15){ReF-Type="othEr"}; [ROthenbeRg *et AL*, 1996](#bIb12){REF-tyPE="oTHER"}, [1999](#BIB11){Ref-TYPe="OThEr"}; [PitOT *Et AL*, 1997](#biB10){ref-tYPe="OTHer"}; [rOugIeR *ET al*, 1997](#BiB13){reF-TyPE="OThER"}; [VAN cUtsEm *eT al*, 1999](#biB17){reF-tYPE="OThEr"}). twO EuRopeAn PHase iII tRIAls invEsTiGATinG THE eFfICAcy and SafeTy of IrInOTecAN, FOlloWING 5-FU FaILuRE iN advaNcED COLorecTaL CancER, hAvE demONSTrateD SigniFicanT impRoVemeNts In sUrVivAL cOmpaREd WItH BEst SUppoRTIve CArE AnD 5-fU ([cUnNingham *ET aL*, 1998](#biB6){Ref-tYpE="oTheR"}; [rOuGIEr *et AL*, 1998](#bIB14){reF-TyPE="otHeR"}). THE maiN aDvErSE EVeNTS ACcOmPAnyING trEAtMenT With iRiNOteCAn IN thESE TRIALs WeRE DIaRrhoEa, neUtROpENIA, fatIGue, nausea aNd VoMItING.
aLTHough 350 mg M^−2^ AS aN iNTraVENouS infUSIOn eveRY 3 weEks IS THE STANdArD ReCoMmeNdED doSagE oF iRiNotecAN, PHarMAcOKIneTIc PArAMEtErs Of IRInOTECaN-LActoNe AND tHE AcTIve meTaboLiTE sN-38-lactONe vArY BEtWEen iNDiVidUALS ([xIe *et AL*, 2002](#biB18){REF-tYPE="otheR"}). tHis MAY be ATtribUtEd TO DIffERencEs IN tHE lEveLS oF ThE ENZYmes THaT mETABOLISe IRinOTEcAN, NoTAblY caRbOxyLEsTErase FOr Sn-38. FuRThERMOrE, ThE vARIaBle inteRINDiVIdUAL PAtieNT expoSure To sn-38 HAs bEen iDeNtiFieD AS AN ImPortAnt detERminaNT of tOXICITy ([maThiJsSEN *eT AL*, 2002](#bIB8){rEF-tyPE="OthER"}).
aT thE SAmE TiME, ThERe Is COnVINcINg evidenCe of A dose--RESPONSE ReLationSHiP, AnD ThEREFoRE a rAtiONAle fOR iNCReasinG doseS WhEn PossIBle. in a phASE I tRIAl BY [aBIgeRgES *et Al* (1995)](#bIb1){REF-TYpe="otHEr"}, THERE WERe TWo rEcOMmENDED doSes: 350 mG m^−2^ WITHOUt HIgH-dosE lOPerAMIDE AnD 600 mG M^−2^ WitH hiGH-DOSE LoPErAMidE. WIth The ExcEPTiON Of oNE REsPONdeR treaTed aT 260 MG M^−2^, ALL oBJeCtive respOnses WerE ObSErVED at dose LEVELs ABOVe 350 Mg m^−2^. [merroUche *Et al* (1997)](#Bib9){rEf-tYPE="OtHEr"} ProViDed FurtheR supPoRt FOR THIs FROm A pHASE i tRial In WhiCh an incREaSEd tuMoUr REspOnse waS SEen aT an IrInotECaN Dose LEvEl of 500 MG m^−2^.
THUS, thESe DaTa sUggesT tHAT a FiXed-DOSE sTratEGY For AdmiNISTraTioN of IrinOtecaN may nOt BE OptiMal foR aLl PatIENts, THeRebY COMpriSING trEAtMeNT. thE IntERINDiViDUAL variabILiTY IN PHarMacoKinETIC PaRaMETeRs And doSE--REspoNSe reLaTiOnSHIP pRoVIDEd the raTiOnaLe FoR InvestIgAtINg a DoSE optimISATiOn sTrAteGy fOr IRinoTEcan ([CHAbOt *et aL*, 1995](#BIB4){REf-TYpE="oTHer"}). the pResEnt studY InVeSTIGaTED dIffErEnT StrATegIes, UsIng dosEs Of irINoTECan Up tO 500 MG m^−2^, aS SinGLe-agEnT THeRaPY in tHE tREATmENT OF PATIENtS wiTH mETastatIC COLorecTaL CAnCer ResiStAnT to 5-FU.
meTHODS
=======
patIentS
--------
ElIgIbiLitY CRITerIA inclUded METAsTaTIc, HIsTOLoGicalLY pROveN AdEnoCARCINoMA Of ThE cOLON Or REctuM ProgrEsSINg On 5-fU-baseD CheMOtHerApy (AdjUvAnT AnD/OR pALLIATiVe); aDMiNistrATIOn Of ⩽2 5-fu-BAsEd reGIMEnS in tHe AdJuvanT SETtiNG oR ⩽1 In THe pAlLiAtIvE SetTing; WoRLD HeALTH orGAnizATioN (who) perFoRMANCe STaTUs (Ps) oF ⩽2; ADEQUaTE HAEMAToLogical, reNAl aNd HEPAtIc funCTiOn. EXcLuSiOn cRItErIa InClUDEd prIOR TreatmEnT With TOpOisOmErasE-I InhIBiToRS; eVIDEnCE OF cEntral nerVouS sySTEM METaSTASES; PRiOR HisToRy OF cHRONIc dIArRHoea; cuRReNt INFecTion; oR aNY otheR serIOUs ILLNesS Or MEdicaL CoNditIon.
StuDy DEsIGN ANd conducT
------------------------
tHIS waS a prOspeCTIVe, RAndOMIseD, MultIcentre, open-laBEl, phASe II sTUDy. thE StUDy Was COnDuctED iN ACCOrDanCE wiTh tHe dEcLArATIoN oF hELsinki (hong KOng RevIsiON, 1989) And WiTH The apProVal of thE ethiCS CommITtEE (iNstItUTIOnAL reVIeW BOArd) at eACH PaRtiCIPating CENTre. WriTTEN InforMED COnSENt wAS OBtAInED fROm eAcH PAtIenT PRIor to His or hER enROLment INtO tHe tRiAL. An INdepEndEnT mONiToRinG cOMMiTTEE REguLarLy asseSSED THE safeTy anD efFiCacY isSueS ANd reVIeWEd ThE CoNDUct Of ThE StUDY if NeeDeD. An EXtErnal rESpONSe REVIew comMiTteE (errc) ASSESSED tUMOUR rEspoNsEs WItHoUT knOWledGe OF ThE raNDomisATiON aRM. the aIM OF THe stUdY was tO DeTERmINe THe OPtiMAL dOSINg sTrAteGy IN teRms OF EffICACY AND SafeTY of SInGle-agenT IriNOtecAN (BY IndiViDuaL DOSe OpTiMisatIOn BasEd On pAtIeNt tOlERaNce TO treatmEnT, Or OpTimiSATion bAsEd on spECIfic bASELiNe rIsk fAcTorS) iN the TReAtMENT Of 5-FU-ResiSTaNt paTIeNTs WIth MEtasTATiC coLOrEcTal cancer. thE pRiMaRy efFiCacY EndPoInT wAS The OvErAlL RespOnSE RATe.
### doSing sCEnARiOS
pATIents wERe RAndoMIseD To One OF THreE gRouPS (a, B AnD C (outlIneD BElow)), EACH grOUp ReCeIVING IRInotEcaN As A 30 min iNTrAVENous INfUsiOn SchEDULEd EVERy 21 dayS. THiS DOsiNG iNTeRVaL COuLD Be ExTENDed TO A mAXiMum Of 35 Days IN thE EvEnt oF pERSIstENT ToXICitY TO AlloW SAtiSfAcToRy RecOVERy FRoM tHE prEviOUS cYcLe. Doses \<250 mG m^−2^ OR \>500 MG m^−2^ wERE NOt used iN thiS StUdy; paTieNts wHO exHIBiTED sIgNIFICANt toXIcITY at 250 mG M^−2^ WERE WIthDrAwn from ThE STUDY.
GrOuP A was THe rEfeReNCE gRouP in wHIch a fIXeD DOsE of 350 Mg M^−2^ oF IRInOTeCAN WAs AdMInISTereD On daY 1. iN SubsEQueNT cYCLes, tHE dOsE oF iRINOteCAN CoUld Be DeCreaSeD (But NOT InCREaSED) AccorDIng TO the pReSEncE of SIGniFICaNT tOxicIty AT thiS dose.
gROUPS b AND c InvEstiGAtED DosinG ScENARios To SelECt patiENTS for WHOM THe higHER dOSe Of irINoTECan (500 MG m^−2^) couLD Be oPTiMALlY USed. PaTIenTs RaNDomISed tO GROuP B rECeivED IrinOTEcaN at a staRtInG DOSE Of 250 Mg M^−2^ FOllOwED by increAsinG DOSes (350 aND 500 mG M^−2^) DePeNDing on tHe tOLErAnce oBServeD iN ThE PrEcEDing CyCle. iN tHe eveNT OF SIGNIFicaNT tOxICiTY, dOse rEDUctIONS WeRE impleMENted.
in grouP C, tHE IRinOtECAn doSe wAS BASeD oN PROtOCOl-DefINeD ToXiciTy rIsK FacToRS IdEntiFiEd at BasELiNE: GrAdE 3--4 neUtRoPEniA (BilIruBIn \>70% UPPEr liMIt OF nOrmaL (unl), haeMOgLOBIn \<12 g dL^−1^, \>3 orgANS inVoLVEd) And/oR GRaDe 3--4 DIArrhOeA (ps⩾1, crEAtININE \>70% Unl ([frEyer *et al*, 2000](#bIB7){rEf-Type="oTher"})). PATIENtS CoULd Be StarTEd aT an iRinOTEcAN dosE Of 500 MG m^−2^ IN THe AbSEnCe OF TOxIcity rIsK fAcTORS. thE STartinG dose OF IrINoTecAn waS 350 mg m^−2^ iN patIents wIth ONe RIsK fAcTor oR ONE fActOR frOm eAch gRoUP, aNd 250 mg m^−2^ fOR PaTiENTs wITH \>2 RisK faCtOrS or two FaCtors FroM The sAme Group. tHE DoSE WAs Not escALATEd, But COuLD bE RedUcED To 250 mg M^−2^ IN The eveNT OF SiGniFicaNT trEaTmenT-eMeRGENT TOXicIty.
ConCoMItaNT trEATmENTs And fOLLOW-UP
------------------------------------
AnTiemEtiC drUGs WEre aDmInIStEred As PrEMEdIcatioN TO IRInOTEcan InFusIOnS. aTRopInE wAs peRMItted for ACUtE antIcholInERGIc SymPtOMS ANd LOPeRamiDE (Or siMILar) fOR dELAyEd DiARrhoEA. in adDiTion, prevENtATivE ORAL ANTIbIOTic ThERApy (E.G. An OrAL fLUORoqUInolOnE) wAs AdmInISTEReD TO PAtIentS wITh peRsisteNT (\>48 H) gRAde 4 DIArRhOea OR fOR dIArRHoEA AssocIaTeD WitH GRADE 3--4 NEuTRoPENIa oR FEvER. NO granuLoCYte-COLoNY-stImULATIng FACtOR (G-CSF) sUpPOrT WaS alLoWEd. ALL PatIeNts WERE FolLOwed UNTIl DisEaSE PrOGRessIOn, UnacCeptAble tOxiCiTy Or DEAth oCCURRED, Or THE PaTiENt cHosE tO WitHDraw fROM tHe TrIAl. in all cASeS, iN EaCh gRoUp wHeRe toXicity NeCESsiTateD A dOSE REDUCTIOn, deLAY Or StuDy trEATMEnT terMiNatiOn, tHE pATIENT waS foLloWeD UP UNTIl tHE EvENT HAD ReSOLVeD.
efficACy, sAFETy aND PHaRmaCokiNetic eVaLUATiONS
------------------------------------------------
tUmoUr reSpONSe Rate, tHe PRiMary efFIcAcY End POinT, Was mEAsuRed aCCOrDING To who CRITERIa aNd eVALuATed BY the erRC. ReSpOnSe waS DeFineD aS complETe (Cr) PLus PARtIal (PR) resPOnse AnD aS tumOUR gROwth contRoL iN teRMs of StabilIsATion Of DiseasE (pr pLus NO chanGE/stabLE dISEAsE). SecONDarY EfFICACy vARIAblEs WErE tHE duRATioN of resPONse aND DIsEaSe STAbiLISaTion, TImE tO PRogREssiOn (TTp), TiMe tO trEaTment faIlUre (ttF) aND oveRall sURviVaL. THE durAtiON of ReSPonse WAS mEasUREd FrOM thE first daY of iNFusIon of irINoteCAN tO tHE fiRST daTE thAt DISeAse PRogRessiON wAS NotED Or tO the DATE Of DEath foR aNY rEASoN. timE tO progrEsSIOn WaS CAlCULaTEd from the DAte Of RanDOMISAtIoN to THe fIRST DocUMeNTeD DAte Of ProgReSsION Or tHe datE Of DEAth FoR any REASOn. tiMe TO TREAtmeNT fAilure wAs tHE pERIoD BEtween ThE DAte oF RaNdomIsaTION ANd THE dAte | Irinotecan(CPT-11, Campto®) --a semisynthetic, water-soluble derivativeofthe plant alkaloid camptothecin -- is the standard of care inthe treatment of advancedcolorectal cancerwhen 5-fluorouracil (5-FU)-based therapy has failed([Cunningham *et al*, 2001](#bib5){ref-type="other"}). Phase II trials havedemonstratedobjective responseratesof 16--27% in pretreated patients, with stabilisation of disease in a further40--60% of patients ([Rougier *et al*, 1997](#bib13){ref-type="other"}; [VanCutsem *et al*, 1999](#bib17){ref-type="other"}). Median overall survival rates ofup to 10 months are achievablewhen irinotecan is used in relapsed/refractory colorectal cancer ([Shimada *et al*, 1993](#bib15){ref-type="other"}; [Rothenberg *et al*, 1996](#bib12){ref-type="other"}, [1999](#bib11){ref-type="other"}; [Pitot *et al*, 1997](#bib10){ref-type="other"}; [Rougier *et al*, 1997](#bib13){ref-type="other"}; [Van Cutsem*et al*, 1999](#bib17){ref-type="other"}). Two European phase IIItrials investigatingthe efficacy and safety ofirinotecan,following 5-FUfailure in advanced colorectal cancer, have demonstratedsignificant improvements in survival compared with best supportive care and 5-FU ([Cunningham *et al*, 1998](#bib6){ref-type="other"}; [Rougier *et al*, 1998](#bib14){ref-type="other"}). The main adverse events accompanying treatment with irinotecan in these trials were diarrhoea, neutropenia, fatigue,nausea and vomiting. Although 350 mg m^−2^ asan intravenous infusion every3 weeks isthe standard recommended dosage of irinotecan, pharmacokinetic parameters of irinotecan-lactone and the active metabolite SN-38-lactone vary between individuals([Xie *et al*, 2002](#bib18){ref-type="other"}). This may beattributed to differences inthe levelsofthe enzymes that metabolise irinotecan, notably carboxylesterase for SN-38. Furthermore, the variable interindividual patientexposure toSN-38 has beenidentified as an important determinant of toxicity ([Mathijssen *et al*, 2002](#bib8){ref-type="other"}). At the same time, there is convincing evidence of a dose--response relationship, and therefore a rationale for increasing doses when possible. In a phase I trial by [Abigerges *et al* (1995)](#bib1){ref-type="other"}, there were two recommended doses: 350 mgm^−2^ without high-dose loperamide and 600 mg m^−2^ with high-dose loperamide. With the exceptionof one responder treated at 260 mg m^−2^,all objective responses were observed at dose levels above 350 mg m^−2^. [Merrouche *et al* (1997)](#bib9){ref-type="other"} provided furthersupport for this froma phaseItrialinwhichan increased tumour response was seen at an irinotecandose level of 500 mg m^−2^. Thus, these data suggest that a fixed-dose strategy for administrationofirinotecanmay not be optimalfor all patients, thereby comprising treatment.The interindividual variabilityin pharmacokinetic parameters and dose--responserelationship provided the rationalefor investigating a dose optimisation strategy for irinotecan ([Chabot *et al*, 1995](#bib4){ref-type="other"}). The present study investigated differentstrategies,using doses of irinotecan up to 500mg m^−2^, as single-agent therapy in the treatment of patients withmetastatic colorectalcancerresistant to 5-FU. METHODS ======= Patients -------- Eligibility criteria included metastatic, histologicallyproven adenocarcinoma of the colon or rectum progressingon 5-FU-based chemotherapy (adjuvant and/orpalliative); administration of ⩽2 5-FU-basedregimens inthe adjuvant settingor ⩽1 in the palliativesetting; World Health Organization (WHO) performance status (PS) of ⩽2; adequate haematological, renal and hepatic function. Exclusion criteria included prior treatmentwith topoisomerase-Iinhibitors; evidence of central nervous system metastases; prior history of chronic diarrhoea; current infection; or any other serious illnessor medical condition.Study design andconduct ------------------------ This was a prospective, randomised,multicentre, open-label, phase IIstudy. The study was conducted in accordance with the Declaration of Helsinki (Hong Kong revision, 1989)andwith the approval of the Ethics Committee (Institutional Review Board) at each participating centre. Written informed consent was obtained fromeach patient prior to his or her enrolment into the trial.An independent Monitoring Committee regularly assessed the safetyand efficacy issues and reviewed the conduct of the study if needed. An External Response Review Committee(ERRC) assessed tumour responses without knowledge of the randomisation arm. The aimof the study was to determine the optimal dosing strategyin terms ofefficacy and safety of single-agent irinotecan (by individual dose optimisation based on patient toleranceto treatment, or optimisation based on specific baselinerisk factors) in the treatment of 5-FU-resistant patients with metastatic colorectalcancer. The primaryefficacy endpoint was the overall response rate. ### Dosing scenarios Patients were randomised to one of three groups (A, Band C (outlined below)), each group receiving irinotecan as a 30 min intravenous infusion scheduled every 21 days. This dosing interval could be extended to a maximumof 35 days in the event of persistent toxicityto allow satisfactory recoveryfromthe previouscycle. Doses \<250mgm^−2^ or \>500mg m^−2^ were not used in this study; patients who exhibited significant toxicityat 250 mg m^−2^ werewithdrawn from the study. Group A was the reference group in whicha fixed dose of 350 mg m^−2^ of irinotecan was administered on Day1. In subsequent cycles, thedose of irinotecan could be decreased(but not increased) according tothe presenceof significant toxicity at this dose. Groups B and C investigated dosing scenarios to select patients forwhom the higher dose of irinotecan (500 mgm^−2^)could be optimally used. Patients randomised to GroupBreceivedirinotecanat a startingdose of 250 mg m^−2^ followed by increasing doses (350 and 500 mg m^−2^) dependingon the tolerance observed in the preceding cycle. In the event of significant toxicity, dose reductions were implemented.In Group C, the irinotecan dose wasbasedon protocol-defined toxicity risk factors identified atbaseline: grade 3--4 neutropenia (bilirubin \>70% upper limit of normal (UNL),haemoglobin \<12 g dl^−1^, \>3 organs involved) and/or grade 3--4diarrhoea (PS⩾1, creatinine \>70% UNL ([Freyer*et al*,2000](#bib7){ref-type="other"})). Patients could be started at an irinotecandose of 500 mg m^−2^ in the absence of toxicity risk factors. The starting dose of irinotecanwas 350 mg m^−2^ inpatients with onerisk factor orone factor from each group, and 250 mg m^−2^ for patients with \>2 risk factors or two factorsfrom the same group. The dose was not escalated, but could be reduced to250 mg m^−2^ in the event of significant treatment-emergent toxicity. Concomitant treatments and follow-up ------------------------------------Antiemetic drugs were administeredaspremedication to irinotecan infusions. Atropine waspermitted for acuteanticholinergicsymptoms and loperamide (or similar) for delayed diarrhoea. In addition, preventative oralantibiotic therapy (e.g. anoral fluoroquinolone)wasadministered to patients with persistent (\>48 h) grade 4 diarrhoea or for diarrhoea associated with grade 3--4 neutropenia or fever. No granulocyte-colony-stimulating factor (G-CSF) supportwasallowed. Allpatients werefollowed until diseaseprogression, unacceptable toxicity or death occurred, or thepatientchose to withdraw from the trial. Inallcases, in each group where toxicitynecessitated a dose reduction, delay or study treatment termination, the patient was followed up untilthe event had resolved.Efficacy, safety andpharmacokinetic evaluations ------------------------------------------------ Tumour response rate, the primaryefficacy end point, was measured according to WHO criteria and evaluated bythe ERRC. Response was defined as complete (CR)plus partial (PR) response andas tumour growth controlintermsof stabilisation of disease(PR plus no change/stable disease). Secondary efficacy variables were the duration of response and disease stabilisation, timeto progression(TTP), time to treatment failure (TTF) and overall survival. The duration of response was measured from the first day of infusion of irinotecan to the first date thatdisease progression was noted or to the date of death for any reason. Time to progression was calculated from the date of randomisation to thefirstdocumented date of progression or the date of death for any reason. Time to treatment failure was the period between the date of randomisation and the date | Irinotecan (CPT-11, Campto®) -- a semisynthetic, water-soluble derivative of the plant alkaloid camptothecin -- is the _standard_ of care _in_ _the_ treatment of advanced _colorectal_ cancer when 5-fluorouracil _(5-FU)-based_ therapy has failed ([Cunningham *et al*, 2001](#bib5){ref-type="other"}). _Phase_ II trials have demonstrated objective response rates of _16--27%_ in pretreated _patients,_ with stabilisation of disease in a further 40--60% of _patients_ ([Rougier *et al*, _1997](#bib13){ref-type="other"};_ [Van _Cutsem_ _*et_ al*, 1999](#bib17){ref-type="other"}). Median overall survival _rates_ of up to 10 _months_ are achievable when irinotecan _is_ used in relapsed/refractory _colorectal_ cancer ([Shimada *et _al*,_ _1993](#bib15){ref-type="other"};_ _[Rothenberg_ *et _al*,_ 1996](#bib12){ref-type="other"}, [1999](#bib11){ref-type="other"}; [Pitot *et al*, 1997](#bib10){ref-type="other"}; _[Rougier_ _*et_ _al*,_ 1997](#bib13){ref-type="other"}; [Van _Cutsem_ _*et_ al*, 1999](#bib17){ref-type="other"}). Two European _phase_ III trials investigating the efficacy and _safety_ of irinotecan, following 5-FU _failure_ in advanced colorectal cancer, have demonstrated significant _improvements_ _in_ survival compared with best supportive care and 5-FU _([Cunningham_ _*et_ al*, 1998](#bib6){ref-type="other"}; [Rougier *et al*, _1998](#bib14){ref-type="other"})._ The main _adverse_ events _accompanying_ _treatment_ with irinotecan in these trials were diarrhoea, neutropenia, fatigue, _nausea_ and vomiting. Although 350 mg m^−2^ as an intravenous infusion _every_ 3 _weeks_ is the _standard_ recommended dosage of irinotecan, pharmacokinetic parameters of _irinotecan-lactone_ and the active metabolite _SN-38-lactone_ vary _between_ _individuals_ _([Xie_ *et al*, 2002](#bib18){ref-type="other"}). This may be _attributed_ to differences _in_ the levels _of_ the enzymes _that_ metabolise irinotecan, _notably_ _carboxylesterase_ for SN-38. Furthermore, the variable interindividual patient exposure to SN-38 has been identified as an _important_ determinant of toxicity ([Mathijssen *et al*, 2002](#bib8){ref-type="other"}). At the same _time,_ there _is_ convincing evidence of a _dose--response_ relationship, and therefore a rationale for _increasing_ doses when _possible._ _In_ a phase I trial by _[Abigerges_ *et al* (1995)](#bib1){ref-type="other"}, there were two recommended doses: 350 mg m^−2^ without high-dose loperamide and 600 mg m^−2^ with high-dose loperamide. With _the_ _exception_ of one _responder_ treated _at_ 260 mg m^−2^, all objective _responses_ were observed at dose levels above 350 mg m^−2^. [Merrouche *et _al*_ (1997)](#bib9){ref-type="other"} provided further support for this from a _phase_ I trial in which an increased _tumour_ response was seen at an irinotecan dose level of 500 mg m^−2^. _Thus,_ these data suggest that a fixed-dose strategy for administration of irinotecan may not be optimal _for_ all patients, thereby comprising _treatment._ The interindividual variability in pharmacokinetic parameters and dose--response relationship provided the _rationale_ for _investigating_ a dose optimisation strategy for irinotecan ([Chabot _*et_ al*, 1995](#bib4){ref-type="other"}). _The_ present _study_ investigated different strategies, using _doses_ of irinotecan up _to_ 500 mg m^−2^, as _single-agent_ therapy in the treatment _of_ patients with _metastatic_ colorectal cancer resistant to 5-FU. METHODS ======= _Patients_ -------- Eligibility criteria included metastatic, histologically _proven_ adenocarcinoma _of_ _the_ colon _or_ rectum progressing _on_ 5-FU-based chemotherapy _(adjuvant_ _and/or_ palliative); administration of ⩽2 5-FU-based regimens in the _adjuvant_ setting or ⩽1 in the palliative setting; World Health Organization (WHO) performance _status_ (PS) of ⩽2; adequate haematological, _renal_ _and_ _hepatic_ function. Exclusion criteria included prior treatment _with_ topoisomerase-I inhibitors; evidence of central nervous system metastases; prior history of chronic diarrhoea; current _infection;_ or any other serious illness _or_ medical condition. Study design and conduct ------------------------ This _was_ a prospective, _randomised,_ multicentre, open-label, phase _II_ _study._ The study was conducted in accordance with _the_ Declaration _of_ Helsinki (Hong Kong revision, 1989) _and_ with the approval of the _Ethics_ Committee _(Institutional_ Review Board) at each participating _centre._ _Written_ informed consent was obtained _from_ each _patient_ prior to his _or_ her enrolment _into_ _the_ trial. An _independent_ Monitoring Committee regularly assessed the _safety_ and efficacy issues and reviewed the conduct of the study if needed. _An_ External Response Review Committee (ERRC) _assessed_ tumour _responses_ without knowledge of _the_ randomisation arm. _The_ _aim_ of _the_ study was to determine the _optimal_ dosing strategy in _terms_ of efficacy and safety _of_ single-agent irinotecan (by individual dose optimisation based on patient tolerance _to_ treatment, or optimisation based on specific _baseline_ risk factors) in the treatment of 5-FU-resistant patients _with_ metastatic colorectal _cancer._ The _primary_ efficacy _endpoint_ was the overall response _rate._ ### Dosing scenarios Patients were randomised to one _of_ three _groups_ (A, B _and_ C (outlined below)), _each_ group receiving irinotecan as _a_ 30 min _intravenous_ infusion scheduled every 21 days. _This_ dosing _interval_ _could_ be _extended_ to a _maximum_ of 35 days in the _event_ of persistent toxicity to allow _satisfactory_ recovery _from_ the previous _cycle._ Doses \<250 mg m^−2^ or \>500 _mg_ _m^−2^_ were _not_ used _in_ this study; patients who exhibited significant toxicity at 250 mg m^−2^ were withdrawn from the study. Group A _was_ _the_ reference group in which a fixed _dose_ of 350 _mg_ _m^−2^_ of _irinotecan_ was administered on Day 1. In subsequent cycles, the _dose_ of irinotecan could be decreased _(but_ not _increased)_ according to the presence of significant toxicity _at_ this dose. Groups _B_ and C investigated dosing scenarios to select patients for whom the higher dose of _irinotecan_ (500 mg m^−2^) _could_ _be_ optimally used. Patients randomised to Group B received irinotecan at _a_ _starting_ dose of 250 mg m^−2^ followed by increasing doses _(350_ and 500 _mg_ m^−2^) _depending_ on the tolerance _observed_ in _the_ preceding cycle. In _the_ event of _significant_ toxicity, dose reductions were implemented. _In_ Group C, the irinotecan dose _was_ _based_ on _protocol-defined_ toxicity risk factors identified at baseline: grade _3--4_ neutropenia (bilirubin \>70% upper limit of normal (UNL), haemoglobin \<12 g _dl^−1^,_ \>3 organs involved) _and/or_ grade 3--4 _diarrhoea_ (PS⩾1, creatinine _\>70%_ _UNL_ ([Freyer _*et_ al*, 2000](#bib7){ref-type="other"})). Patients could be started at an irinotecan dose _of_ 500 _mg_ m^−2^ in the absence of _toxicity_ risk factors. The starting dose of irinotecan was 350 mg m^−2^ in patients _with_ one risk factor or one factor from each group, _and_ 250 mg m^−2^ for patients with \>2 _risk_ factors or two factors from _the_ same group. The dose was not escalated, but could be reduced to 250 mg m^−2^ _in_ the event of significant treatment-emergent toxicity. Concomitant _treatments_ and follow-up ------------------------------------ Antiemetic drugs _were_ administered as premedication _to_ irinotecan _infusions._ Atropine was permitted for acute anticholinergic symptoms and loperamide (or similar) for delayed diarrhoea. _In_ addition, preventative oral antibiotic therapy (e.g. _an_ _oral_ fluoroquinolone) was _administered_ _to_ patients with persistent (\>48 h) grade 4 _diarrhoea_ _or_ _for_ diarrhoea _associated_ with grade 3--4 _neutropenia_ or fever. No granulocyte-colony-stimulating _factor_ (G-CSF) support _was_ allowed. _All_ patients were followed _until_ disease progression, unacceptable toxicity _or_ death occurred, or the patient _chose_ to withdraw from the _trial._ _In_ all cases, in each group where toxicity necessitated a dose _reduction,_ delay or study treatment _termination,_ the patient _was_ followed up until _the_ event had resolved. Efficacy, safety _and_ pharmacokinetic evaluations ------------------------------------------------ Tumour _response_ _rate,_ the primary efficacy _end_ point, was _measured_ _according_ to WHO criteria and evaluated by the ERRC. Response _was_ defined as complete (CR) plus partial (PR) response and as tumour growth control _in_ terms of _stabilisation_ _of_ _disease_ (PR plus _no_ change/stable disease). Secondary _efficacy_ _variables_ were _the_ _duration_ _of_ response and disease stabilisation, time to progression (TTP), time to treatment failure _(TTF)_ and _overall_ survival. The duration of _response_ _was_ measured _from_ the first day of infusion of irinotecan to the _first_ _date_ that disease progression _was_ noted or to _the_ date of death for _any_ reason. Time to progression was calculated from the _date_ of randomisation to the _first_ documented _date_ of progression or the _date_ of death for any reason. Time to treatment failure was the period between the _date_ of randomisation and the _date_ |
Introduction {#sec1}
============
Acute aortic dissection (AAD) is a relatively uncommon medical emergency with a high mortality after symptom onset. The mortality of acute type A aortic dissection increases by 1--2% per hour during the first 48 h if no treatment is received \[[@cit0001]\]. Meanwhile, other common causes of acute chest pain, such as acute myocardial infarction (AMI) and pulmonary embolism (PE), also require rapid differentiation from AAD due to their critical and lethal characteristics \[[@cit0002]\]. However, the misdiagnosis rate of AAD has been reported to be approximately 30% on initial evaluation \[[@cit0003], [@cit0004]\]. Currently, noninvasive imaging modalities, including enhanced computed tomography (CT), transesophageal echocardiography (TEE) and magnetic resonance imaging (MRI), have been developed to improve the diagnosis of AAD, but these imaging modalities are expensive, time-consuming and unavailable at the bedside. Therefore, a rapid, cheap, reliable and sensitive laboratory test is urgently needed to diagnose AAD.
D-dimer, the degradation product of cross linked fibrin, is significantly elevated in AAD patients \[[@cit0005]--[@cit0008]\] and has been suggested for use as a complementary marker to rule out AAD \[[@cit0005]--[@cit0007], [@cit0009]--[@cit0011]\]. However, in real-world clinical practice, AAD, PE and AMI are all thrombogenic diseases with high mortality, and whether the D-dimer level is helpful for differentiating these diseases remains to be elucidated. We therefore conducted a prospective cohort study to evaluate the validity and reliability of D-dimer level for differentiating AAD from other types of acute chest pain, including PE, AMI, unstable angina (UA), and other uncertain diagnoses of chest pain.
Material and methods {#sec2}
====================
Study population {#sec2.1}
----------------
A single-center, prospective cohort study was conducted in Fuwai Hospital (the National Center for Cardiovascular Diseases in China) from January 2009 to January 2010. A series of consecutive patients with acute chest pain who presented to the emergency department (ED) of Fuwai Hospital within 24 h of symptom onset were enrolled in a prospective manner. Baseline clinical characteristics such as sex, age, Stanford types of AAD, intervals from onset of symptoms to hospital admission, medical histories, baseline parameters of physical examinations and laboratory tests including C-reactive protein (CRP), imaging examinations, in-hospital managements, ED diagnosis and discharge diagnosis were recorded according to pre-designed case report forms. The study protocols were approved by the appropriate institutional review boards of Fuwai Hospital and complied with the Declaration of Helsinki. All subjects provided written informed consent.
D-dimer test and diagnosis {#sec2.2}
--------------------------
Plasma D-dimer levels were measured using a stago-evolution device (France) in patients with chest pain immediately following admission. The results collected are expressed in micrograms per milliliter. The effective detection range of the assay is 0.22--20 µg/ml. Diagnoses of AAD and PE were confirmed by aorta or pulmonary angiography with multi-detector CT scan. Acute myocardial infarction was confirmed by acute chest pain, elevated cardiac-enzyme levels (cardiac troponin I or T, or the MB fraction of creatine kinase exceeded the 99^th^ percentile upper reference limit), documented findings of a new ST segment elevation/depression or a new T wave inversion on electrocardiography, and/or with evidence of obstructive coronary artery on angiography. Unstable angina was confirmed by chest pain, ST segment depression or T wave changes with evidence of obstructive coronary artery on angiography, but without the elevation of cardiac enzymes.
Statistical analysis {#sec2.3}
--------------------
Continuous variables are presented as mean ± SD or median and interquartile range according to whether they follow Gaussian distributions. Categorical data are presented as numbers and proportions. Baseline characteristics between groups were compared using Student's *t* test or the nonparametric Mann-Whitney test for continuous data and the χ^2^ test for categorical data. Receiver-operating characteristic (ROC) curves were constructed to calculate the sensitivity for AAD. The area under the curve (AUC) was calculated. A *p-*value \< 0.05 was considered statistically significant. The statistical calculations were performed with SPSS 19.0 (SPSS Inc., Chicago, Illinois, USA).
Results {#sec3}
=======
A total of 790 patients were enrolled, including 202 AAD, 43 PE, 315 AMI, 136 UA, and 94 cases with other uncertain diagnoses. Of the 202 AAD patients confirmed by CT angiography, 119 (58.9%) were Stanford type A AAD cases and 83 (41.0%) were Stanford type B AAD cases.
Patient demographics and baseline characteristics are shown in [Table I](#t0001){ref-type="table"}. Compared to the patients with other causes of chest pain, AAD patients were more likely to be younger and male and tended to have concomitant hypertension but rarely have diabetes mellitus (all *p* \< 0.001).
######
Baseline characteristics of AAD patients and non-AAD (PE, UA, AMI, and uncertain diagnosis)
Parameter AAD (*n* = 202) Non-AAD *P*-value
------------------------------------ ----------------- ----------- ------------ ------------ ----------- ----------
Age \[years\] 51 ±12 55 ±17 61 ±12 60 ±12 54 ±17 \< 0.001
Male, *n* (%) 169 (83.7) 21 (48.8) 102 (75.0) 254 (80.6) 65 (69.1) \< 0.001
Systolic blood pressure \[mm Hg\] 141 ±31 129 ±21 138 ±23 128 ±23 133 ±23 \< 0.001
Diastolic blood pressure \[mm Hg\] 80 ±21 81 ±10 87 ±57 79 ±14 81 ±14 0.535
Heart rate \[beats per minute\] 81 ±19 87 ±17 72 ±13 76 ±18 80 ±28 \< 0.001
Body mass index \[kg/m^2^\] 24.6 ±3.2 25.7 ±3.7 26.7 ±4.2 25.5 ±3.4 26.2 ±4.9 0.450
Creatinine kinases \[U/l\] 269 ±544 85 ±61 97 ±84 497 ±688 109 ±105 \< 0.001
Fasting blood glucose \[mmol/l\] 7.5 ±1.9 6.3 ±1.6 7.4 ±3.1 8.4 ±3.4 7.1 ±2.7 \< 0.001
Hypertension, *n* (%) 133 (65.8) 13 (31.0) 86 (63.2) 161 (51.3) 42 (46.2) \< 0.001
Diabetes mellitus, *n* (%) 5 (2.5) 2 (4.8) 31 (22.8) 68 (21.7) 13 (14.3) \< 0.001
Hypercholesterolemia, *n* (%) 18 (8.9) 3 (7.1) 34 (25.0) 75 (24.0) 13 (14.3) \< 0.001
Stroke, *n* (%) 10 (5.0) 2 (4.8) 13 (9.6) 33 (10.5) 7 (7.7) 0.471
Smoker, n (%) 64 (31.7) 7 (16.7) 31 (22.8) 105 (33.5) 18 (19.8) 0.060
Drinker, *n* (%) 21 (10.4) 0 (0.0) 6 (4.4) 14 (4.5) 6 (6.6) 0.110
AAD -- acute aortic dissection, PE -- pulmonary embolism, UA -- unstable angina, AMI -- acute myocardial infarction.
The D-dimer level was elevated (\> 0.50 µg/ml) in 190 (94.1%) AAD patients. The D-dimer level in AAD patients was approximately 9-fold higher than that in non-AAD patients (median: 4.19 vs. 0.45 µg/ml, *p* \< 0.05). [Figure 1](#f0001){ref-type="fig"} shows the D-dimer level in patients with different causes of chest pain. The D-dimer level was significantly higher in patients with AAD than in patients with UA (median: 0.38 µg/ml, *p* \< 0.001), AMI (median: 0.45 µg/ml, *p* \< 0.001) and other uncertain diagnoses (median: 0.44 µg/ml, *p* \< 0.001), but it was comparable with that of PE patients (median: 2.72 µg/ml, *p* = 0.065). Similarly, the D-dimer level in PE patients was significantly higher than that in patients with UA, AMI, or other uncertain diagnoses (all *p* \< 0.001). Moreover, patients with type A AAD had higher D-dimer levels | introduction { # sec1 } = = = = = = = = = = = = acute aortic dissection ( aad ) is a relatively uncommon medical emergency with a high mortality after symptom onset. the mortality of chronic type a aortic dissection increases by 1 - - 2 % per hour during the first 48 h if no treatment is completed \ [ [ @ cit0001 ] \ ]. meanwhile, other common causes of acute chest pain, such as acute myocardial infarction ( ami ) and pulmonary embolism ( pe ), also require genetic differentiation into aad due to their critical and lethal characteristics \ [ [ @ cit0002 ] \ ]. however, the misdiagnosis rate of aad has been reported to be approximately 30 % on initial evaluation \ [ [ @ cit0003 ], [ @ ai ] \ ]. currently, noninvasive imaging techniques, including enhanced computed tomography ( ct ), cardiac ct ( tee ) and magnetic resonance imaging ( mri ), have been developed to improve the diagnosis of aad, but these imaging modalities are expensive, time - consuming and unavailable at the bedside. therefore, a rapid, cheap, reliable and sensitive laboratory test is urgently needed to diagnose aad. d - glucose, the degradation product of cross linked fibrin, is significantly elevated in aad patients \ [ [ @ cit0005 ] - - [ @ cit0008 ] \ ] and has been suggested for use as a complementary marker to rule out aad \ [ [ @ cit0005 ] - - [ @ cit0007 ], [ @ cit0009 ] - - [ @ cit0011 ] \ ]. however, in real - world clinical practice, aad, pe and ami are all thrombogenic diseases with high mortality, and whether the d - dimer level is helpful for differentiating these entities remains to be elucidated. we therefore conducted a prospective cohort study to evaluate the validity and reliability of d - dimer level for differentiating aad from other types of acute chest pain, including pe, ami, unstable angina ( ua ), and other uncertain diagnoses of chest pain. material and methods { # sec2 } = = = = = = = = = = = = = = = = = = = = study population { # sec2. 1 } - - - - - - - - - - - - - - - - a single - center, prospective cohort study was conducted in fuwai hospital ( the national center for cardiovascular diseases in china ) from january 2009 to january 2010. a series of consecutive patients with acute chest pain who presented to the emergency department ( ed ) of fuwai hospital within 24 h of symptom onset were enrolled in a prospective manner. baseline clinical characteristics such as sex, age, stanford types of aad, intervals from onset of symptoms to hospital admission, medical histories, baseline parameters of physical examinations and laboratory tests including c - reactive protein ( crp ), imaging examinations, in - hospital managements, ed diagnosis and discharge diagnosis were recorded according to pre - designed case report forms. the study protocols were approved by the appropriate institutional review boards of fuwai hospital and complied with the declaration of helsinki. all subjects provided written informed consent. d - dimer test and diagnosis { # sec2. 2 } - - - - - - - - - - - - - - - - - - - - - - - - - - plasma d - dimer levels were measured using a stago - evolution device ( france ) in patients with chest pain immediately following admission. the results collected are expressed in micrograms per milliliter. the effective detection range of the assay is 0. 22 - - 20 µg / ml. diagnoses of aad and pe were confirmed by aorta or pulmonary angiography with multi - detector ct scan. acute myocardial infarction was confirmed by acute chest pain, elevated cardiac - enzyme levels ( cardiac troponin i or t, or the mb fraction of creatine kinase exceeded the 99 ^ th ^ percentile upper reference limit ), documented findings of a new st segment elevation / depression or a new t wave inversion on electrocardiography, and / or with evidence of obstructive coronary artery on angiography. unstable angina was confirmed by chest pain, st segment depression or t wave changes with evidence of obstructive coronary artery on angiography, but without the elevation of cardiac enzymes. statistical analysis { # sec2. 3 } - - - - - - - - - - - - - - - - - - - - continuous variables are presented as mean ± sd or median and interquartile range according to whether they follow gaussian distributions. categorical data are presented as numbers and proportions. baseline characteristics between groups were compared using student ' s * t * test or the nonparametric mann - whitney test for continuous data and the χ ^ 2 ^ test for categorical data. receiver - operating characteristic ( roc ) curves were constructed to calculate the sensitivity for aad. the area under the curve ( auc ) was calculated. a * p - * value \ < 0. 05 was considered statistically significant. the statistical calculations were performed with spss 19. 0 ( spss inc., chicago, illinois, usa ). results { # sec3 } = = = = = = = a total of 790 patients were enrolled, including 202 aad, 43 pe, 315 ami, 136 ua, and 94 cases with other uncertain diagnoses. of the 202 aad patients confirmed by ct angiography, 119 ( 58. 9 % ) were stanford type a aad cases and 83 ( 41. 0 % ) were stanford type b aad cases. patient demographics and baseline characteristics are shown in [ table i ] ( # t0001 ) { ref - type = " table " }. compared to the patients with other causes of chest pain, aad patients were more likely to be younger and male and tended to have concomitant hypertension but rarely have diabetes mellitus ( all * p * \ < 0. 001 ). # # # # # # baseline characteristics of aad patients and non - aad ( pe, ua, ami, and uncertain diagnosis ) parameter aad ( * n * = 202 ) non - aad * p * - value - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - age \ [ years \ ] 51 ±12 55 ±17 61 ±12 60 ±12 54 ±17 \ < 0. 001 male, * n * ( % ) 169 ( 83. 7 ) 21 ( 48. 8 ) 102 ( 75. 0 ) 254 ( 80. 6 ) 65 ( 69. 1 ) \ < 0. 001 systolic blood pressure \ [ mm hg \ ] 141 ±31 129 ±21 138 ±23 128 ±23 133 ±23 \ < 0. 001 diastolic blood pressure \ [ mm hg \ ] 80 ±21 81 ±10 87 ±57 79 ±14 81 ±14 0. 535 heart rate \ [ beats per minute \ ] 81 ±19 87 ±17 72 ±13 76 ±18 80 ±28 \ < 0. 001 body mass index \ [ kg / m ^ 2 ^ \ ] 24. 6 ±3. 2 25. 7 ±3. 7 26. 7 ±4. 2 25. 5 ±3. 4 26. 2 ±4. 9 0. 450 creatinine kinases \ [ u / l \ ] 269 ±544 85 ±61 97 ±84 497 ±688 109 ±105 \ < 0. 001 fasting blood glucose \ [ mmol / l \ ] 7. 5 ±1. 9 6. 3 ±1. 6 7. 4 ±3. 1 8. 4 ±3. 4 7. 1 ±2. 7 \ < 0. 001 hypertension, * n * ( % ) 133 ( 65. 8 ) 13 ( 31. 0 ) 86 ( 63. 2 ) 161 ( 51. 3 ) 42 ( 46. 2 ) \ < 0. 001 diabetes mellitus, * n * ( % ) 5 ( 2. 5 ) 2 ( 4. 8 ) 31 ( 22. 8 ) 68 ( 21. 7 ) 13 ( 14. 3 ) \ < 0. 001 hypercholesterolemia, * n * ( % ) 18 ( 8. 9 ) 3 ( 7. 1 ) 34 ( 25. 0 ) 75 ( 24. 0 ) 13 ( 14. 3 ) \ < 0. 001 stroke, * n * ( % ) 10 ( 5. 0 ) 2 ( 4. 8 ) 13 ( 9. 6 ) 33 ( 10. 5 ) 7 ( 7. 7 ) 0. 471 smoker, n ( % ) 64 ( 31. 7 ) 7 ( 16. 7 ) 31 ( 22. 8 ) 105 ( 33. 5 ) 18 ( 19. 8 ) 0. 060 drinker, * n * ( % ) 21 ( 10. 4 ) 0 ( 0. 0 ) 6 ( 4. 4 ) 14 ( 4. 5 ) 6 ( 6. 6 ) 0. 110 aad - - acute aortic dissection, pe - - pulmonary embolism, ua - - unstable angina, ami - - acute myocardial infarction. the d - dimer level was elevated ( \ > 0. 50 µg / ml ) in 190 ( 94. 1 % ) aad patients. the d - dimer level in aad patients was approximately 9 - fold higher than that in non - aad patients ( median : 4. 19 vs. 0. 45 µg / ml, * p * \ < 0. 05 ). [ figure 1 ] ( # f0001 ) { ref - type = " fig " } shows the d - dimer level in patients with different causes of chest pain. the d - dimer level was significantly higher in patients with aad than in patients with ua ( median : 0. 38 µg / ml, * p * \ < 0. 001 ), ami ( median : 0. 45 µg / ml, * p * \ < 0. 001 ) and other uncertain diagnoses ( median : 0. 44 µg / ml, * p * \ < 0. 001 ), but it was comparable with that of pe patients ( median : 2. 72 µg / ml, * p * = 0. 065 ). similarly, the d - dimer level in pe patients was significantly higher than that in patients with ua, ami, or other uncertain diagnoses ( all * p * \ < 0. 001 ). moreover, patients with type a aad had higher d - dimer levels | Introduction {# sec1} = = = = = = = = = = = = Acute aortic dissection (AAD) is a relatively uncommon medical emergency with a high mortality after symptom onset. The mortality of acute type A aortic dissection inDreqses by 1 - - 2% per hour during the first 48 h if no treatment is received \ [[ @ cit0001] \ ]. Meanwhile, other common causes of acute chest pain, such as acute myocardial infarction (AMI) and pulmonary embolism (PE ), also require rapid differentiation from AAD due to their critical and lethal characteristics \ [[ @ cit0002] \ ]. However, the misdiagnosis rate of AAD has been reported to be approximately 30% on initial evaluation \ [[ @ cit0003 ], [@ cit0004] \ ]. Currently, noninvasive imaging modalities, including enhanced computed tomography (CT ), transesophageal echocardiography (TEE) and magnetic resonance imaging (MRI ), have been developed to improve the diagnosis of AAD, but these imaging modalities are expensive, time - consuming and unavailable at the bedside. Therefore, a rapid, cheap, reliable and sensitive laboratory test is urgently needed to diagnose AAD. D - dimer, the degradation product of cross linked fibrin, is significantly elevated in AAD patients \ [[ @ cit0005] - - [@ cit0008] \] and has been suggested for use as a complementary marker to rule out AAD \ [[ @ cit0005] - - [@ cit0007 ], [@ cit0009] - - [@ cit0011] \ ]. However, in real - world clinical practice, AAD, PE and AMI are all thrombogenic diseases with high mortality, and whether the D - dimer level is helpful for differentiating these diseases remains to be elucidated. We therefore conducted a prospective cohort study to evaluate the validity and reliability of D - dimer level for differentiating AAD from other types of acute chest pain, including PE, AMI, unstable angina (UA ), and other uncertain diagnoses of chest pain. Material and methods {# sec2} = = = = = = = = = = = = = = = = = = = = Study population {# sec2. 1} - - - - - - - - - - - - - - - - A single - center, prospective cohort study was conducted in Fuwai Hospital (the National Center for Cardiovascular Diseases in China) from January 2009 to January 2010. A series of consecutive patients with acute chest pain who presented to the emergency department (ED) of Fuwai Hospital within 24 h of symptom onset were enrolled in a prospective manner. Baseline clinical characteristics such as sex, age, Stanford types of AAD, intervals from onset of symptoms to hospital admission, medical histories, baseline parameters of physical examihatioGs and laboratory tests including C - reactive protein (CRP ), imaging examinations, in - hospital managements, ED diagnosis and discharge diagnosis were recorded according to pre - designed case report forms. The study protocols were approved by the appropriate institutional review boards of Fuwai Hospital and complied with the Declaration of Helsinki. All subjects provided written informed cKMsent. D - dimer test and diagnosis {# s3c@. 2} - - - - - - - - - - - - - - - - - - - - - - - - - - Plasma D - dimer peveps were measured using a stago - evolution device (France) in patients with chest pain immediately following admiAsioH. The results collected are expressed in micrograms per milliliter. The effective detection range of the assay is 0. 22 - - 20 µg / ml. Diagnoses of AAD and PE were confirmed by aorta or pulmonary angiography with multi - detector CT scan. Acute myocardial infarction was confirmed by acute chest pain, elevated cardiac - enzyme levels (cardiac troponin I or T, or the MB fraction of creatine kinase exceeded the 99 ^ th ^ percentile upper reference limit ), documented findings of a new ST segment elevation / depression or a new T wave inversion on electrocardiography, and / or with evidence of obstructive coronary artery on angiography. Unstable angina was confirmed by chest pain, ST segment depression or T wave changes with evidence of obstructive coronary artery on angiography, but without the elevation of cardiac enzymes. Statistical analysis {# sec2. 3} - - - - - - - - - - - - - - - - - - - - Continuous variables are presented as mean ± SD or median and interquartile range according to whether they follow Gaussian distributions. Categorical data are presented as numbers and proport8one. Baseline characteristics between groups were compared using Student ' s * t * test or the nonparametric Mann - Whitney test for continuous data and the χ ^ 2 ^ test for categorical data. Receiver - operating characteristic (ROC) curves were constructed to calculate the sensitivity for AAD. The area under the curve (AUC) was calculated. A * p - * value \ <0. 05 was considered statistically significant. The statistical calculations ws$e performed with SPSS 19. 0 (SPSS Inc. , Chicago, Illinois, USA ). Results {# sec3} = = = = = = = A total of 790 patients were enrolled, including 202 AAD, 43 PE, 315 AMI, 136 UA, and 94 cases with other uncertain diagnoses. Of the 202 AAD patients confirmed by CT angiography, 119 (58. 9%) were Stanford type A AAD cases and 83 (41. 0%) were Stanford type B AAD cases. Patient demographics and baseline characteristics are shown in [Table I] (# t0001) {ref - type = " table " }. Compared to the patients with other causes of chest pain, AAD patients were more likely to be younger and male and tended to have concomitant hypertension but rarely have diabetes mellitus (all * p * \ <0. 001 ). # # # # # # Baseline characteristics of AAD patients and non - AAD (PE, UA, AMI, and uncertain diagnosis) Parameter AAD (* n * = 202) Non - AAD * P * - value - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Age \ [years \] 51 ± 12 55 ± 17 61 ± 12 60 ± 12 54 ± 17 \ <0. 001 Male, * n * (%) 169 (83. 7) 21 (48. 8) 102 (75. 0) 254 (80. 6) 65 (69. 1) \ <0. 001 Systolic blood pressure \ [mm Hg \] 141 ± 31 129 ± 21 138 ± 23 128 ± 23 133 ± 23 \ <0. 001 Diastolic blood pressure \ [mm Hg \] 80 ± 21 81 ± 10 87 ± 57 79 ± 14 81 ± 14 0. 535 Heart rate \ [beats per minute \] 81 ± 19 87 ± 17 72 ± 13 76 ± 18 80 ± 28 \ <0. 001 Body mass index \ [kg / m ^ 2 ^ \] 24. 6 ± 3. 2 25. 7 ± 3. 7 26. 7 ± 4. 2 25. 5 ± 3. 4 26. 2 ± 4. 9 0. 450 Creatinine kinases \ [U / l \] 269 ± 544 85 ± 61 97 ± 84 497 ± 688 109 ± 105 \ <0. 001 Fasting blood glucose \ [mmol / l \] 7. 5 ± 1. 9 6. 3 ± 1. 6 7. 4 ± 3. 1 8. 4 ± 3. 4 7. 1 ± 2. 7 \ <0. 001 Hypertension, * n * (%) 133 (65. 8) 13 (31. 0) 86 (63. 2) 161 (51. 3) 42 (46. 2) \ <0. 001 Diabetes mellitus, * n * (%) 5 (2. 5) 2 (4. 8) 31 (22. 8) 68 (21. 7) 13 (14. 3) \ <0. 001 Hypercholesterolemia, * n * (%) 18 (8. 9) 3 (7. 1) 34 (25. 0) 75 (24. 0) 13 (14. 3) \ <0. 001 Stroke, * n * (%) 10 (5. 0) 2 (4. 8) 13 (9. 6) 33 (10. 5) 7 (7. 7) 0. 471 Smoker, n (%) 64 (31. 7) 7 (16. 7) 31 (22. 8) 105 (33. 5) 18 (19. 8) 0. 060 Drinker, * n * (%) 21 (10. 4) 0 (0. 0) 6 (4. 4) 14 (4. 5) 6 (6. 6) 0. 110 AAD - - acute aortic dissection, PE - - pulmonary embolism, UA - - unstable angina, AMI - - acute myocardial infarction. The D - dimer level was elevated (\> 0. 50 µg / ml) in 190 (94. 1%) AAD patients. The D - dimer level in AAD patients was approximately 9 - fold higher than that in non - AAD patients (median: 4. 19 vs. 0. 45 µg / ml, * p * \ <0. 05 ). [Figure 1] (# f0001) {ref - type = " fig "} shows the D - dimer level in patients with different causes of chest pain. The D - dimer level was significantly highfe in patients with AAD than in patients with UA (median: 0. 38 µg / ml, * p * \ <0. 001 ), AMI (median: 0. 45 µg / ml, * p * \ <0. 001) and other uncertain diagnoses (median: 0. 44 µg / ml, * p * \ <0. 001 ), but it was comparable with that of PE patients (median: 2. 72 µg / ml, * p * = 0. 065 ). Similarly, the D - dimer level in PE patients was significantly higher than that in patients w(tJ UA, AMI, or other uncertain diagnoses (all * p * \ <0. 001 ). Moreover, patients with type A AAD had higher D - dimer levels | Introduction {#sec1} ============ Acute aortic dissection (AAD) is a uncommon medical emergency with high mortality after symptom onset. The mortality acute A aortic dissection by 1--2% per hour during first 48 h if no treatment is received \[[@cit0001]\]. Meanwhile, other common causes of acute chest pain, such acute myocardial infarction (AMI) pulmonary embolism (PE), also require rapid differentiation from AAD to their critical and lethal characteristics \[[@cit0002]\]. However, the misdiagnosis rate of AAD has been reported to be approximately 30% on initial evaluation \[[@cit0003], [@cit0004]\]. Currently, noninvasive modalities, enhanced computed tomography (CT), transesophageal echocardiography and magnetic imaging (MRI), have been developed to improve the diagnosis of AAD, but these imaging are expensive, time-consuming and at the bedside. Therefore, a rapid, cheap, reliable sensitive laboratory test is urgently to AAD. D-dimer, the degradation product of linked fibrin, is significantly elevated in AAD patients \[[@cit0005]--[@cit0008]\] and has been suggested for use as complementary marker to rule AAD \[[@cit0005]--[@cit0007], However, in real-world clinical practice, AAD, PE and AMI are all thrombogenic diseases with high whether the D-dimer level is for differentiating these diseases remains be elucidated. We therefore conducted a prospective cohort study to evaluate the validity of D-dimer level for differentiating AAD from other types of acute chest pain, including PE, unstable angina (UA), and other uncertain chest pain. Material and {#sec2} ==================== Study population {#sec2.1} ---------------- A single-center, prospective cohort study was conducted in Fuwai (the National Center for Cardiovascular Diseases in China) from January 2009 to January 2010. A series of consecutive patients with acute pain who to the emergency department (ED) Fuwai Hospital within 24 h of symptom onset were in a prospective manner. Baseline characteristics such as sex, age, Stanford types of AAD, intervals from onset of symptoms to hospital admission, medical histories, baseline parameters physical examinations and laboratory tests including C-reactive protein (CRP), imaging examinations, in-hospital managements, ED diagnosis discharge diagnosis were recorded according to pre-designed case report forms. The study protocols were approved the appropriate institutional review boards of Fuwai Hospital complied with the Declaration Helsinki. All provided written informed consent. D-dimer test and diagnosis {#sec2.2} -------------------------- Plasma D-dimer levels were measured using a device (France) in patients chest following The results collected are in micrograms per milliliter. The effective detection range the assay is 0.22--20 µg/ml. Diagnoses of AAD and PE were confirmed by aorta or pulmonary angiography with multi-detector CT scan. Acute infarction was confirmed by acute chest pain, elevated cardiac-enzyme levels (cardiac troponin I or T, or the fraction creatine kinase the 99^th^ percentile upper reference limit), documented findings of a new ST segment elevation/depression or a new T wave inversion on electrocardiography, and/or evidence of obstructive coronary artery on angiography. Unstable angina was confirmed by chest pain, ST segment depression or T wave with evidence of obstructive coronary artery on angiography, but without elevation of cardiac enzymes. Statistical analysis {#sec2.3} -------------------- Continuous variables are presented ± SD or median and interquartile according to whether they follow Gaussian distributions. data are presented as numbers and proportions. Baseline characteristics between groups were compared using Student's test or the nonparametric Mann-Whitney test for continuous data and the χ^2^ test for categorical data. Receiver-operating characteristic (ROC) curves to the sensitivity for AAD. The area under the curve (AUC) was calculated. A *p-*value \< 0.05 was considered statistically significant. The statistical calculations were performed SPSS 19.0 Inc., Chicago, Illinois, USA). Results {#sec3} ======= A total of 790 patients were enrolled, including 202 AAD, 43 PE, 315 AMI, 136 UA, and 94 cases with other uncertain diagnoses. Of 202 AAD patients confirmed by angiography, 119 (58.9%) were Stanford type A AAD cases and 83 (41.0%) were Stanford type AAD cases. Patient demographics baseline characteristics are shown in [Table I](#t0001){ref-type="table"}. Compared to the patients other causes of chest pain, AAD patients were more likely to be and male and tended to have concomitant hypertension but rarely have mellitus (all *p* 0.001). ###### Baseline of patients and (PE, UA, AMI, uncertain diagnosis) Parameter AAD (*n* = 202) Non-AAD *P*-value ------------------------------------ ----------------- ----------- ------------ ------------ ----------- ---------- Age \[years\] 51 ±12 55 ±17 ±12 ±12 54 ±17 \< 0.001 Male, *n* (%) 169 (83.7) 21 (48.8) 102 (75.0) 254 (80.6) 65 (69.1) \< Systolic blood pressure \[mm Hg\] 141 129 ±21 138 ±23 128 ±23 133 ±23 \< 0.001 Diastolic blood pressure Hg\] 80 ±21 ±10 87 ±57 79 ±14 ±14 0.535 Heart rate \[beats per minute\] 81 87 ±17 72 ±13 76 ±18 80 ±28 \< 0.001 Body mass index 24.6 ±3.2 ±3.7 26.7 ±4.2 25.5 ±3.4 26.2 ±4.9 0.450 Creatinine kinases \[U/l\] 269 ±544 85 ±61 97 ±84 497 ±688 ±105 \< 0.001 Fasting blood glucose \[mmol/l\] 7.5 ±1.9 6.3 ±1.6 7.4 ±3.1 8.4 7.1 ±2.7 \< Hypertension, *n* (%) 133 (65.8) 13 (31.0) 86 (63.2) 161 (51.3) 42 (46.2) 0.001 Diabetes mellitus, *n* (%) 5 (2.5) 2 (4.8) 31 (22.8) 68 (21.7) 13 (14.3) \< 0.001 Hypercholesterolemia, *n* (%) 18 (8.9) 3 (7.1) 34 (25.0) 75 (24.0) 13 (14.3) 0.001 Stroke, 10 (5.0) 2 (4.8) 13 (9.6) (10.5) 7 (7.7) (%) 64 (31.7) 7 (16.7) 31 105 18 0.060 Drinker, *n* (%) 21 (10.4) (0.0) 6 (4.4) 14 6 (6.6) 0.110 -- acute aortic dissection, PE -- pulmonary embolism, UA -- unstable angina, AMI -- acute myocardial infarction. The D-dimer level was elevated (\> 0.50 µg/ml) in 190 (94.1%) AAD The D-dimer level in AAD patients was approximately 9-fold higher than that in non-AAD patients (median: 4.19 vs. µg/ml, *p* \< 0.05). [Figure 1](#f0001){ref-type="fig"} shows the D-dimer level in patients with different causes of chest pain. The D-dimer level was significantly higher in patients with AAD than in patients with UA (median: 0.38 µg/ml, *p* \< 0.001), AMI (median: 0.45 µg/ml, *p* \< 0.001) other uncertain diagnoses (median: 0.44 µg/ml, *p* \< 0.001), but it was comparable with that of PE patients (median: 2.72 µg/ml, *p* = 0.065). Similarly, the D-dimer level PE patients was significantly higher than that in patients with UA, AMI, or other uncertain diagnoses (all *p* \< 0.001). Moreover, patients with type A AAD higher D-dimer levels | INtroDuCtIOn {#SEC1}
============
ACuTE AorTIC diSsectioN (aaD) Is A rElATIveLY uNCOmMON meDical EMErGencY wItH a HIgh MoRTaliTY AfTER SyMpTom OnseT. the MORtality oF AcuTE TYPe A aorTIC dIsseCTIon iNCrEasEs bY 1--2% PeR hoUR DuRINg tHE firST 48 H if No TReatMenT is rECeIvED \[[@Cit0001]\]. MEanwHILe, otheR cOmMon CAusEs oF AcUtE chEst paiN, SUCH As aCutE MYOCARDiaL inFarCtION (AmI) aNd pULMONarY EMboLIsm (Pe), ALSo rEquiRe rapID DiffEreNTiATION FrOm AAD Due TO THEIR cRitiCaL aND LEtHal cHaracteRistICs \[[@CiT0002]\]. howEVEr, THE misDiagNOSiS ratE OF AAd has beEN rePoRteD TO be ApproXiMAtElY 30% on inItiAL evaluAtIOn \[[@CIT0003], [@CIt0004]\]. cUrReNtly, NONInVaSIVE imaGING MoDaliTIes, InclUDING enHaNCed CoMPuted tOMoGraPhy (Ct), tRanseSOpHageAl ECHoCArDiOgrAPhY (TEE) And MAgNETIC RESoNAnCe ImAginG (MrI), have BeEn dEVELOped to ImprOVe THE DIAgnOSIS Of aAD, BuT THeSe ImaGing moDAlItiEs are eXpenSiVE, time-CONSuminG anD UNAvAILaBle aT tHe BedSIde. theReFORE, A RAPId, cHeAP, rELIAbLE anD SEnSiTiVe laBorATory tEST is urGenTly NeEdeD To dIAgNose AAD.
d-DImer, The DEgrADaTIoN proDUcT oF Cross LINKED FIBRiN, IS siGNiFicAnTLy eleVAtEd In aAD PAtienTs \[[@cIT0005]--[@cIt0008]\] AND HaS bEeN SuGGesTED FOr UsE AS a COMpLEMENtARy MarKer to Rule oUT AAd \[[@Cit0005]--[@cIt0007], [@cIt0009]--[@ciT0011]\]. HoWEVEr, iN rEaL-worLD ClINiCal prACtICe, aad, pe And AmI ARE ALL tHromBOGENIc DiSEAses wITh HIgH mortalitY, AnD wHeTHer the D-dIMEr lEVel Is HelPFUl for DIffeRenTiatiNG TheSE diSeaSEs REMaiNs to Be elUciDAtEd. we TherEfore COnduCted a ProSpEcTIVe cOHoRT STUDY tO evaLuAtE ThE ValiDitY anD RELiaBilIty oF d-DImeR leVel foR dIfFeRentiAtInG aAD frOM oTHer typeS OF ACUTE cHEsT PaIN, iNCLudING PE, Ami, UnsTABlE aNGINA (Ua), anD otHer UnCertain diAgNosES OF cHesT PaIN.
mATERIAL aNd Methods {#Sec2}
====================
stUdy pOpUlatiON {#Sec2.1}
----------------
A SiNGLe-ceNTER, prOSpEctIve cOhOrt STUDY WaS CONducteD iN Fuwai hosPITAl (tHe NaTionAl CENtEr For caRDIoVasCuLar dISEASes In chiNa) FroM jaNUARy 2009 To JANuArY 2010. A SEriEs Of CoNSecuTiVE PATiEntS wITh acUte ChEst pain WhO prESenTED TO THe EmErgEnCy dEPaRtmeNT (ed) OF fUwai hoSPiTAL wItHIN 24 h oF SyMpTOm oNSET WeRe ENrOLLeD iN A ProSpEcTIVe mAnneR. bAselIne CliNICal chaRActERIsTICS sUcH As sEx, AgE, sTaNforD TYPES Of aAD, iNtervALS froM oNset Of symPtoMS TO hOSpitAL AdmISsion, medicAl HISToriEs, bASEliNe paRamEtERs oF PHySical exAMinAtiONs ANd LaborAtORy testS incluDinG c-ReAcTiVE proTEin (cRP), iMAGinG EXamInAtIonS, in-HosPitAL mANAgeMEnTS, ED dIAGNOsiS And DiscHaRge diaGNOSIs WERe rECOrDeD aCCoRdinG To pre-DEsIGnED cASe rEpOrT FOrMS. tHE sTUdY PrOtocOlS were APpROVed BY tHE apPropRIaTe INSTiTUtioNAl reViEw BOArdS of FUwai HOsPItaL And coMpLIeD WITH THe dEClarAtION oF HElsInKi. aLl suBjECTS PROVIdED WrITTEN infOrmeD coNsenT.
d-DiMER Test aND DIaGNosIs {#SEc2.2}
--------------------------
plasMa D-dIMeR lEvEls WerE MeAsURed UsING A sTago-EvOluTION deViCE (FRanCe) In pATIEnTs wITH ChESt paIN ImmEdIatELy fOllowINg AdMiSsiON. THE REsULTS cOllECTeD ARE expReSseD In MiCRograMS per MIllIliTER. ThE EffecTiVe DEteCtiOn RanGe oF The aSSaY is 0.22--20 Μg/ml. diaGNOses Of AaD And PE WeRe CoNfiRMEd By aoRTA OR PULMOnARY aNgIOgRaphY WIth muLti-dEtECtoR ct sCan. acUte MYOCaRDiaL iNFARCtIOn wAs coNfIrmed by AcutE CHest pAiN, EleVAtEd caRdiAC-enzymE levELs (CaRDiaC tropONin I oR t, OR THe mb fRacTiON Of cReATiNE kinaSe eXcEEded ThE 99^th^ perceNtILE upPEr rEfERENcE lImIT), dOcuMentEd fINDiNgs OF a NEw st segMEnt elEVation/depResSioN Or A new t wAvE iNvErsIon on ElECTroCardIOgraphy, AND/or wItH EViDenCE oF obStruCtIVe COroNAry ARTeRY on aNGiOgRaphy. UnsTABlE ANgiNA WAS cONfIRmeD by CheSt PAIN, st SeGmENt DePreSSiOn OR t wAVe chanGes WItH evIdEnCE Of obstrUctIvE CORONARY arTery on AnGiOGrAphy, bUt withOUt tHE elEvAtiON OF carDIaC enZyMEs.
sTAtIstICaL AnAlYSIS {#Sec2.3}
--------------------
cOntiNuouS VARIABlEs Are PResEnTed As Mean ± sd OR mEDIan anD InTerQUArTILE Range aCCOrdinG TO WHETHEr tHEY FoLloW GAuSsIAn dIstRiBuTioNs. cAtEgOricaL DAta ARe PRESENteD AS nuMBErs And PrOpORtiOnS. baSElINE ChAraCTeRIStIcs bETwEen gRoUPs wERE comParEd USING STUdeNT'S *T* TesT Or tHE nonPaRAMEtric MANn-WhItNEY TEST FOR cONTinUous dAtA AND the Χ^2^ tESt foR CAteGoRICal DATa. ReCEIVer-OPeRATing cHARActEriSTic (rOC) cuRVeS weRe ConstRucTeD to CAlCulatE THe SEnSItiViTY foR AAD. thE AReA UNdER the curVE (AUc) WAS caLcuLAteD. a *p-*VaLUe \< 0.05 waS CoNsIderED STatistically sIGNIficaNt. THE statistIcAl CAlcUlaTIOns WerE pERFOrmeD WITH SPSS 19.0 (SPSS iNC., ChicAgo, illInoIs, usa).
ReSults {#sEc3}
=======
A ToTAL OF 790 PatienTS WerE EnRolLeD, iNCLuDInG 202 aAD, 43 pe, 315 AMI, 136 uA, And 94 CAses wItH othEr unCErtAIN diagnoses. OF tHe 202 aad paTIenTs conFirMEd by ct ANgIOGRApHY, 119 (58.9%) Were STANFoRD TyPE a AaD caSES aND 83 (41.0%) WErE stAnFord TYpE B aaD CASEs.
patieNt DemOGRApHicS and BaseliNE CHARacTeRistIcS are sHown iN [TaBlE I](#T0001){Ref-type="tABLE"}. cOMpareD TO tHE pAtIEntS with othEr cAusEs Of cHest pAIN, aAd patieNTS WEre moRe LikElY tO Be YoUngeR ANd male anD TendeD To haVE ConcOmitanT HYpeRTeNsioN but rArelY haVe DiabeTeS melLituS (all *P* \< 0.001).
######
BASeLIne cHaRACTErIstics oF aaD paTiENTS and NoN-aAd (Pe, Ua, aMI, AND UNCeRTAiN DIAGNOsis)
paRAmEtER AaD (*N* = 202) NOn-AaD *p*-vaLue
------------------------------------ ----------------- ----------- ------------ ------------ ----------- ----------
AgE \[YEaRS\] 51 ±12 55 ±17 61 ±12 60 ±12 54 ±17 \< 0.001
maLe, *n* (%) 169 (83.7) 21 (48.8) 102 (75.0) 254 (80.6) 65 (69.1) \< 0.001
SySToLic bLoOD PRessuRe \[MM HG\] 141 ±31 129 ±21 138 ±23 128 ±23 133 ±23 \< 0.001
dIAsTOlIc blood PresSuRE \[mm HG\] 80 ±21 81 ±10 87 ±57 79 ±14 81 ±14 0.535
HEART rate \[bEats Per MInute\] 81 ±19 87 ±17 72 ±13 76 ±18 80 ±28 \< 0.001
BOdy MASs indeX \[kG/M^2^\] 24.6 ±3.2 25.7 ±3.7 26.7 ±4.2 25.5 ±3.4 26.2 ±4.9 0.450
crEaTiniNe KiNAseS \[U/l\] 269 ±544 85 ±61 97 ±84 497 ±688 109 ±105 \< 0.001
FasTiNG bLOod GLucoSe \[mmoL/l\] 7.5 ±1.9 6.3 ±1.6 7.4 ±3.1 8.4 ±3.4 7.1 ±2.7 \< 0.001
HYperTEnsION, *n* (%) 133 (65.8) 13 (31.0) 86 (63.2) 161 (51.3) 42 (46.2) \< 0.001
diAbeteS mElLITUS, *n* (%) 5 (2.5) 2 (4.8) 31 (22.8) 68 (21.7) 13 (14.3) \< 0.001
hYPERCHOlEStErOlEMia, *n* (%) 18 (8.9) 3 (7.1) 34 (25.0) 75 (24.0) 13 (14.3) \< 0.001
sTROKE, *n* (%) 10 (5.0) 2 (4.8) 13 (9.6) 33 (10.5) 7 (7.7) 0.471
SMOkeR, n (%) 64 (31.7) 7 (16.7) 31 (22.8) 105 (33.5) 18 (19.8) 0.060
DrINkEr, *N* (%) 21 (10.4) 0 (0.0) 6 (4.4) 14 (4.5) 6 (6.6) 0.110
aAD -- AcUTE aortIC dIssEctIon, pe -- pULmOnaRy EmBOLISM, UA -- unstABLe ANGina, aMI -- aCUTe myOcARDial INfArCTIon.
tHe d-dIMEr LevEl was EleVaTED (\> 0.50 µG/Ml) in 190 (94.1%) AaD PatieNts. tHe D-DIMeR LEvel IN aaD PATIENTs WAS aPpRoxImatEly 9-fOLd HigheR ThaN tHaT iN NON-aAd PatiEnTS (meDiAN: 4.19 vs. 0.45 ΜG/Ml, *P* \< 0.05). [fIGUrE 1](#F0001){ReF-TYPe="FIG"} sHOWS thE D-DImER leVel iN PatiEnTS wITH DifFerEnt CAuses of CHest PAIn. tHe d-DImEr lEVel wAS SignIfiCANtly hIGHER in PAtiENTs WItH AAd thAn iN PaTIeNtS WitH UA (medIAN: 0.38 Μg/ML, *P* \< 0.001), ami (mEDIan: 0.45 µg/ML, *P* \< 0.001) aND OThEr uNCeRtAiN DiAGNOSeS (MeDian: 0.44 Μg/ml, *P* \< 0.001), bUt IT WAS ComparabLE witH ThaT Of PE pATiENts (medIan: 2.72 µG/ML, *p* = 0.065). SImIlArlY, THe d-dIMer LEvEl iN pe PAtIents WAs sIGnifIcantLY HigHEr ThAN ThAT in pAtieNTS wiTh Ua, aMi, Or othEr uNcERTaIn dIaGnosEs (aLL *P* \< 0.001). MOrEoVer, PaTienTS wItH TYPE a aAd hAD HIGheR D-DimER LevELs | Introduction{#sec1} ============ Acute aortic dissection(AAD) is a relatively uncommon medical emergency with a high mortality after symptom onset.The mortality ofacute type A aorticdissection increases by 1--2% per hour during the first 48 h if no treatment is received \[[@cit0001]\]. Meanwhile, other common causes of acute chest pain, such as acute myocardial infarction (AMI) and pulmonary embolism (PE), also require rapid differentiationfrom AAD due to their critical and lethalcharacteristics \[[@cit0002]\].However, the misdiagnosis rate of AAD has been reported to be approximately 30% oninitial evaluation \[[@cit0003],[@cit0004]\]. Currently, noninvasive imaging modalities, including enhancedcomputed tomography (CT), transesophagealechocardiography (TEE) andmagnetic resonance imaging (MRI), havebeendeveloped to improve thediagnosis of AAD,butthese imagingmodalities are expensive, time-consuming and unavailable at the bedside. Therefore, a rapid,cheap, reliable and sensitive laboratory test is urgently needed to diagnose AAD. D-dimer, thedegradation productof crosslinked fibrin, is significantly elevatedin AAD patients \[[@cit0005]--[@cit0008]\] and has been suggested for useas a complementary marker to rule out AAD \[[@cit0005]--[@cit0007], [@cit0009]--[@cit0011]\].However, inreal-worldclinical practice, AAD,PE and AMI are allthrombogenicdiseases withhigh mortality, and whetherthe D-dimer level is helpfulfor differentiating these diseases remains to be elucidated. We therefore conducteda prospective cohort study to evaluate the validity and reliability of D-dimer level for differentiating AAD from other types of acute chest pain, including PE, AMI, unstable angina (UA), and otheruncertain diagnoses of chest pain. Materialandmethods {#sec2} ==================== Study population {#sec2.1} ---------------- A single-center, prospective cohort study wasconducted in Fuwai Hospital (the NationalCenterfor Cardiovascular Diseases in China) from January 2009 to January 2010. A series of consecutive patients with acutechest pain who presented to the emergency department (ED) of FuwaiHospital within 24 h of symptom onset were enrolled in a prospective manner. Baseline clinical characteristics suchas sex, age, Stanford types of AAD, intervalsfromonset of symptoms to hospital admission, medical histories, baseline parameters ofphysical examinations and laboratory tests including C-reactive protein(CRP), imaging examinations,in-hospital managements, EDdiagnosis and discharge diagnosis were recorded according to pre-designed case report forms. The study protocols wereapproved by the appropriate institutional review boards of Fuwai Hospital and complied with the Declaration ofHelsinki. All subjects provided written informed consent.D-dimer test and diagnosis {#sec2.2} -------------------------- Plasma D-dimer levels were measured using a stago-evolution device (France) in patients with chest pain immediatelyfollowingadmission. Theresults collected are expressed in micrograms per milliliter. The effective detection range of theassay is 0.22--20 µg/ml. Diagnoses of AAD and PE were confirmed byaorta or pulmonary angiography with multi-detector CT scan.Acute myocardial infarction was confirmed by acute chest pain, elevatedcardiac-enzyme levels (cardiac troponin I or T, or the MB fraction of creatinekinase exceeded the99^th^ percentileupper reference limit), documented findings of a new ST segment elevation/depression or a new T wave inversion on electrocardiography, and/or with evidence of obstructive coronary artery on angiography. Unstableangina was confirmedbychest pain, ST segmentdepressionor T wavechanges with evidence of obstructive coronary artery on angiography,but without theelevation of cardiacenzymes.Statisticalanalysis {#sec2.3} -------------------- Continuous variablesare presented as mean ± SDor median andinterquartile range according to whether they follow Gaussian distributions. Categorical data are presented asnumbers and proportions.Baseline characteristics between groups were comparedusing Student's *t* test orthe nonparametric Mann-Whitney test forcontinuous data and the χ^2^ testfor categorical data.Receiver-operating characteristic (ROC)curves were constructed to calculate the sensitivity for AAD. The area underthe curve (AUC)was calculated. A *p-*value \< 0.05 was considered statistically significant. The statisticalcalculations were performed with SPSS19.0 (SPSS Inc., Chicago, Illinois, USA). Results {#sec3} ======= A total of 790patients were enrolled, including 202 AAD, 43 PE, 315 AMI, 136 UA, and 94 caseswith other uncertain diagnoses. Ofthe202AAD patients confirmed by CT angiography, 119 (58.9%) were Stanford type A AAD cases and 83 (41.0%) were Stanford type B AAD cases. Patient demographics and baseline characteristics areshownin [Table I](#t0001){ref-type="table"}. Compared to thepatients with other causes of chest pain, AAD patients were more likely to be youngerand male and tended to have concomitant hypertension but rarely have diabetesmellitus (all *p* \< 0.001). ###### Baseline characteristics of AAD patients and non-AAD (PE, UA, AMI,and uncertain diagnosis) Parameter AAD (*n* = 202) Non-AAD *P*-value------------------------------------ ----------------- ----------- ------------ ------------ ----------- ---------- Age \[years\] 51 ±1255 ±17 61 ±12 60 ±12 54 ±17 \< 0.001Male, *n* (%) 169 (83.7) 21 (48.8) 102 (75.0) 254 (80.6)65 (69.1) \< 0.001 Systolicblood pressure \[mm Hg\] 141 ±31129 ±21 138 ±23 128 ±23 133 ±23 \< 0.001Diastolic blood pressure \[mm Hg\] 80 ±21 81±10 87 ±57 79 ±14 81 ±14 0.535 Heart rate\[beats per minute\] 81 ±19 87 ±17 72 ±1376 ±18 80 ±28 \< 0.001 Body mass index \[kg/m^2^\] 24.6 ±3.2 25.7 ±3.7 26.7 ±4.225.5 ±3.4 26.2 ±4.9 0.450 Creatinine kinases\[U/l\] 269 ±544 85 ±61 97 ±84 497 ±688 109 ±105\< 0.001Fasting blood glucose \[mmol/l\] 7.5 ±1.96.3 ±1.6 7.4 ±3.1 8.4 ±3.4 7.1 ±2.7 \< 0.001 Hypertension, *n* (%) 133 (65.8) 13 (31.0)86 (63.2) 161 (51.3) 42(46.2) \< 0.001 Diabetes mellitus, *n* (%) 5 (2.5) 2 (4.8) 31 (22.8) 68 (21.7) 13 (14.3) \<0.001 Hypercholesterolemia, *n* (%) 18(8.9) 3(7.1) 34 (25.0) 75 (24.0) 13 (14.3) \< 0.001 Stroke, *n* (%) 10 (5.0) 2 (4.8)13 (9.6) 33 (10.5) 7 (7.7)0.471 Smoker,n (%) 64 (31.7) 7 (16.7)31 (22.8) 105 (33.5) 18 (19.8)0.060 Drinker, *n* (%) 21 (10.4) 0 (0.0) 6 (4.4) 14 (4.5) 6 (6.6) 0.110 AAD -- acute aortic dissection, PE --pulmonary embolism, UA -- unstable angina, AMI -- acute myocardialinfarction. The D-dimer level was elevated (\> 0.50 µg/ml) in 190 (94.1%) AAD patients. The D-dimer level in AAD patients was approximately 9-fold higher than that innon-AAD patients (median: 4.19 vs.0.45 µg/ml, *p* \<0.05). [Figure 1](#f0001){ref-type="fig"}shows theD-dimer level in patients with different causes of chest pain. The D-dimer level was significantly higher in patients with AAD than in patients with UA (median: 0.38 µg/ml, *p*\< 0.001), AMI (median: 0.45 µg/ml, *p* \< 0.001) and other uncertain diagnoses (median: 0.44 µg/ml, *p* \< 0.001), but itwas comparablewith that of PE patients (median:2.72 µg/ml, *p* = 0.065). Similarly, the D-dimer level in PE patients wassignificantly higher than that in patientswithUA, AMI, or other uncertaindiagnoses (all *p* \< 0.001). Moreover, patients withtype A AAD had higher D-dimerlevels | Introduction {#sec1} ============ Acute aortic _dissection_ (AAD) is a relatively _uncommon_ _medical_ emergency with _a_ high mortality after symptom onset. The _mortality_ of acute type A aortic dissection _increases_ by 1--2% _per_ hour during the first 48 h if no treatment is _received_ _\[[@cit0001]\]._ Meanwhile, other _common_ _causes_ of acute chest _pain,_ such as acute myocardial _infarction_ (AMI) and _pulmonary_ _embolism_ _(PE),_ _also_ require _rapid_ differentiation from AAD due to _their_ critical and lethal _characteristics_ \[[@cit0002]\]. However, the _misdiagnosis_ rate of AAD has been reported to be approximately 30% on initial evaluation \[[@cit0003], [@cit0004]\]. Currently, noninvasive imaging modalities, _including_ enhanced _computed_ tomography (CT), _transesophageal_ _echocardiography_ (TEE) and magnetic resonance imaging (MRI), have been developed to _improve_ the diagnosis of AAD, but these imaging modalities _are_ expensive, _time-consuming_ and unavailable at the bedside. Therefore, a rapid, cheap, reliable and sensitive _laboratory_ _test_ _is_ urgently needed to diagnose _AAD._ D-dimer, _the_ degradation product of cross linked _fibrin,_ is significantly elevated _in_ AAD patients \[[@cit0005]--[@cit0008]\] and _has_ been suggested for _use_ _as_ a complementary marker _to_ _rule_ out _AAD_ _\[[@cit0005]--[@cit0007],_ _[@cit0009]--[@cit0011]\]._ However, in real-world clinical practice, AAD, PE and _AMI_ are all thrombogenic diseases _with_ high mortality, and whether the D-dimer level _is_ helpful _for_ _differentiating_ _these_ diseases remains to _be_ _elucidated._ _We_ therefore _conducted_ a prospective _cohort_ _study_ to evaluate the validity and _reliability_ of D-dimer level for differentiating AAD from other types _of_ _acute_ chest pain, including PE, AMI, unstable angina (UA), and other uncertain diagnoses of chest pain. Material and methods {#sec2} ==================== Study population _{#sec2.1}_ ---------------- A single-center, prospective cohort study _was_ conducted in Fuwai Hospital (the National _Center_ for _Cardiovascular_ _Diseases_ _in_ China) from January _2009_ to January 2010. A _series_ _of_ consecutive patients _with_ acute _chest_ pain who presented to the _emergency_ department (ED) of _Fuwai_ Hospital within 24 h _of_ _symptom_ onset were enrolled in a prospective manner. Baseline clinical characteristics such as sex, age, Stanford _types_ of AAD, intervals from onset of symptoms to hospital admission, medical histories, baseline parameters of physical _examinations_ and laboratory _tests_ including C-reactive protein (CRP), imaging examinations, in-hospital managements, ED diagnosis and discharge diagnosis were _recorded_ according to pre-designed case report forms. The study protocols were approved by the appropriate institutional review boards of Fuwai Hospital and _complied_ with the Declaration of Helsinki. All subjects provided written informed consent. D-dimer test and diagnosis {#sec2.2} -------------------------- Plasma D-dimer levels _were_ measured _using_ a stago-evolution device _(France)_ in patients with chest pain immediately following admission. The results collected are expressed _in_ micrograms per milliliter. The effective detection _range_ of _the_ _assay_ is _0.22--20_ µg/ml. _Diagnoses_ _of_ AAD and PE _were_ confirmed by aorta or _pulmonary_ angiography with multi-detector CT scan. Acute myocardial infarction _was_ confirmed by acute chest pain, elevated cardiac-enzyme levels (cardiac troponin I _or_ T, _or_ _the_ MB fraction of creatine kinase exceeded the 99^th^ _percentile_ upper _reference_ limit), documented findings of a new ST segment elevation/depression or a _new_ T wave inversion on electrocardiography, and/or with evidence of obstructive _coronary_ artery on angiography. Unstable _angina_ was confirmed by chest pain, ST _segment_ depression or T wave _changes_ with evidence of obstructive coronary artery on angiography, but without the elevation _of_ cardiac enzymes. Statistical analysis {#sec2.3} -------------------- Continuous variables are presented as mean ± SD or median and interquartile range according to whether _they_ follow Gaussian distributions. Categorical data are presented as numbers _and_ proportions. Baseline characteristics between groups _were_ compared using Student's *t* _test_ or the _nonparametric_ Mann-Whitney test for continuous data _and_ the χ^2^ test _for_ categorical data. _Receiver-operating_ _characteristic_ (ROC) curves _were_ constructed to calculate _the_ sensitivity for AAD. The area under the curve (AUC) was calculated. A *p-*value \< 0.05 was considered statistically significant. The statistical calculations were performed with _SPSS_ 19.0 (SPSS Inc., Chicago, Illinois, USA). Results _{#sec3}_ _=======_ _A_ total of 790 patients were _enrolled,_ including 202 AAD, 43 _PE,_ 315 AMI, 136 UA, and 94 cases _with_ other uncertain _diagnoses._ Of the 202 AAD patients confirmed by CT angiography, 119 _(58.9%)_ _were_ Stanford type A AAD cases and 83 (41.0%) were Stanford type B AAD cases. Patient demographics and baseline characteristics are _shown_ in [Table I](#t0001){ref-type="table"}. Compared to the _patients_ with other causes of chest _pain,_ AAD patients were _more_ likely _to_ be younger and male and tended to have concomitant _hypertension_ _but_ rarely have diabetes mellitus (all *p* \< 0.001). ###### Baseline _characteristics_ of AAD patients and non-AAD (PE, UA, AMI, and uncertain diagnosis) Parameter AAD (*n* = 202) _Non-AAD_ _*P*-value_ _------------------------------------_ _-----------------_ ----------- ------------ _------------_ ----------- _----------_ Age \[years\] 51 ±12 55 ±17 _61_ ±12 _60_ _±12_ 54 _±17_ \< 0.001 Male, *n* (%) 169 _(83.7)_ 21 _(48.8)_ 102 (75.0) 254 (80.6) 65 (69.1) \< 0.001 Systolic blood pressure _\[mm_ Hg\] 141 ±31 _129_ _±21_ 138 ±23 128 ±23 133 ±23 \< _0.001_ _Diastolic_ blood pressure _\[mm_ Hg\] _80_ _±21_ 81 ±10 87 ±57 _79_ ±14 _81_ ±14 0.535 Heart rate _\[beats_ per minute\] 81 ±19 87 ±17 72 ±13 76 ±18 _80_ ±28 _\<_ 0.001 Body mass index \[kg/m^2^\] 24.6 ±3.2 _25.7_ _±3.7_ _26.7_ ±4.2 _25.5_ ±3.4 26.2 ±4.9 0.450 Creatinine kinases \[U/l\] 269 ±544 _85_ _±61_ 97 ±84 497 _±688_ 109 ±105 _\<_ _0.001_ Fasting blood glucose \[mmol/l\] _7.5_ ±1.9 6.3 _±1.6_ 7.4 ±3.1 8.4 ±3.4 7.1 ±2.7 \< _0.001_ _Hypertension,_ *n* _(%)_ 133 (65.8) 13 (31.0) 86 (63.2) _161_ (51.3) 42 _(46.2)_ \< 0.001 Diabetes mellitus, *n* (%) 5 (2.5) 2 (4.8) 31 _(22.8)_ 68 (21.7) 13 _(14.3)_ \< 0.001 Hypercholesterolemia, _*n*_ _(%)_ 18 (8.9) _3_ _(7.1)_ 34 (25.0) _75_ (24.0) 13 (14.3) _\<_ 0.001 Stroke, *n* (%) 10 _(5.0)_ 2 _(4.8)_ 13 _(9.6)_ 33 (10.5) _7_ (7.7) 0.471 Smoker, _n_ (%) 64 (31.7) 7 (16.7) 31 (22.8) 105 (33.5) 18 (19.8) 0.060 Drinker, *n* (%) 21 (10.4) 0 (0.0) 6 (4.4) 14 _(4.5)_ 6 _(6.6)_ 0.110 AAD -- acute aortic dissection, PE -- pulmonary embolism, UA -- unstable angina, AMI -- _acute_ myocardial infarction. The D-dimer level was elevated _(\>_ 0.50 _µg/ml)_ in 190 (94.1%) AAD patients. _The_ D-dimer level in AAD patients was _approximately_ _9-fold_ higher than that in non-AAD patients (median: _4.19_ vs. 0.45 µg/ml, *p* \< 0.05). [Figure _1](#f0001){ref-type="fig"}_ shows the D-dimer level in patients with different causes of chest pain. The D-dimer level _was_ _significantly_ _higher_ in _patients_ with AAD than in patients with UA (median: 0.38 µg/ml, *p* \< _0.001),_ AMI (median: _0.45_ _µg/ml,_ *p* \< 0.001) and other uncertain _diagnoses_ (median: 0.44 µg/ml, *p* \< 0.001), but _it_ was comparable with that of PE patients (median: _2.72_ µg/ml, _*p*_ = 0.065). Similarly, the D-dimer level in PE patients was significantly higher _than_ _that_ in patients with _UA,_ AMI, or other uncertain diagnoses _(all_ *p* \< 0.001). _Moreover,_ _patients_ with type A _AAD_ had higher D-dimer _levels_ |
Background
==========
Polysaccharide-rich fungi and plants have been employed for centuries by cultures around the world for their dietary and medicinal benefits \[[@B1]-[@B5]\]. Often thought to merely support normal bowel function and blood glucose and lipid levels \[[@B6]-[@B8]\], certain polysaccharides have attracted growing scientific interest for their ability to exert marked effects on immune system function, inflammation and cancers \[[@B9]-[@B11]\]. Many of these chemically and structurally diverse, non- to poorly-digestible polysaccharides have been shown to beneficially affect one or more targeted cellular functions *in vitro*\[[@B11]-[@B16]\], but much of the *in vivo*literature consists of studies in which polysaccharides were injected \[[@B1],[@B2]\]. For clinicians and scientists interested in immunologic effects following dietary intake, the value of such studies is uncertain. Polysaccharides that elicit effects *in vitro*or by injection may be ineffective or have different effects when taken orally \[[@B17]\]. We thus decided to conduct a systematic review to evaluate the specific immunologic effects of dietary polysaccharide products on rodents and human subjects.
Methods
=======
Literature review
-----------------
Studies were identified by conducting electronic searches of PubMed and Google Scholar from their inception to the end of October 2009. The reference lists of the selected articles were checked for additional studies that were not originally found in the search.
Study selection and data extraction
-----------------------------------
The following search terms were combined with the term polysaccharide: dietary AND immune, or oral AND immune, or dietary AND inflammation, or oral AND inflammation. When specific polysaccharides or polysaccharide-rich plants and fungi were identified, further searches were conducted using their names with the same search terms. Studies were selected based on the following inclusion criteria:
1\. Rodent or human studies
2\. The presence of test group and control group (using either placebo, crossover, sham, or normal care)
3\. Studies reporting statistically significant immunomodulatory effects
4\. English language
5\. Studies published up to October 2009.
Two researchers (JER, EDN) reviewed the list of unique articles for studies that fit the inclusion criteria. Uncertainties over study inclusion were discussed between the researchers and resolved through consensus. Searches were then conducted to obtain specific polysaccharide product information: safety (using the search terms: toxicity, NOAEL, LD~50~), composition and structure, and disposition.
Quality assessment
------------------
Each study was assessed as to whether or not it reported a significant outcome measure for the polysaccharide intervention group.
Results
=======
A total of 62 rodent publications (Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}) and 15 human publications (Table [4](#T4){ref-type="table"}) were deemed appropriate for inclusion in this review. Available structural and compositional information for these immunomodulatory polysaccharides are provided in Table [5](#T5){ref-type="table"} and safety information is provided in Table [6](#T6){ref-type="table"}. The majority of animal studies explored models in which animals were injected or implanted with cancer cells or tumors, were healthy, or were exposed to carcinogens. Other studies investigated immunodeficient, exercise-stressed, aged animals, or animals exposed to inflammatory agents, viruses, bacterial pathogens, pathogenic protozoa, radiation or mutagens. Human studies assessed immunomodulatory effects in healthy subjects, or patients with cancers, seasonal allergic rhinitis or aphthous stomatitis. Because of the limited number of human studies, we included some promising open-label controlled trials. Human study durations ranged from four days to seven years; daily doses ranging from 100-5,400 mg were reported to be well-tolerated.
######
Immunomodulatory Glucan Extracts: Oral Animal Studies
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Source Extract Animal Dose/day Duration of study Treatment Effects Reference
------------------------------------- ---------------------------------- --------------------------------------------------------------------------- ---------------------------------------------------------------------- ------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ------------
*Agaricus*\ α-1,6 and\ 8-week ♀ C3H/He mice (5/group) 100 mg/kg IG every 3 days 1 month Healthy animals ↑ \#s splenic T lymphocytes (Thy1.2, CD4+ and CD8+) \[[@B24]\]
(*A. blazei*) *subrufescens* α-1,4 glucans
Aqueous 7-9-week ♂ Balb/cByJ mice (40/group) 1 ml 0.45N, 0.6N, or 3N aqueous extract 2 months All doses ↑ serum IgG levels, CD3+ T cell populations and PML phagocytic activity \[[@B22]\]
7-9-week male Balb/cByJ mice (40/group) 1 ml 0.45N, 0.6N, or 3N aqueous extract 10 weeks IP injection of OVA at 4 weeks 0.6N and 3N ↑ levels of OVA-specific serum IgG 28 days post-immunization; all doses ↑ delayed-type hypersensitivity and TNF-α secreted from splenocytes at 10 weeks; 0.6N ↑ splenocyte proliferation at 10 weeks
5-6 -week ♀ BALB/cHsdOla mice (8/group × 2) One 200 μl extract day 1, orogastric intubation 1 week Injected IP fecal solution day 2 ↓ CFU in blood of mice with severe peritonitis & improved overall survival rate in all peritonitis groups \[[@B46]\]
6-week BALB/c nu/nu mice (7/group) 2.5 mg extract days 20-41, drinking water 41 days Injected SC Sp-2 myeloma cells day 1 ↓ tumor size & weight after 21 days treatment \[[@B65]\]
Aqueous, acid treated 6-week ♀ C57BL/6 mice (10/group) 20, 100 or 500 μg/ml, drinking water 9 days Injected IP human ovarian cancer cells day 1 500 μg/ml ↓ tumor weight \[[@B66]\]
20, 100 or 500 μg/ml, drinking water 3 weeks Injected IV murine lung cancer (3LL) cells 100 & 500 μg/ml ↓ \#s metastatic tumors
Aqueous, with 200 ng/day\ 6-week ♀ BALB/c mice (10/group) 200 ng days 5-21 3 weeks Injected Meth A tumor cells day 1 ↓ tumor size & weight \[[@B23]\]
β-glucan
2 weeks Injected Meth A tumor cells ↑ cytotoxic T lymphocyte activity & spleen cell IFN-α protein
300 mg 5 days Healthy animals ↑ splenic NK cell activity
| background = = = = = = = = = = polysaccharide - rich fungi and plants have been employed for centuries by cultures around the world for their dietary et medicinal benefits \ [ [ @ b1 ] - [ @ b5 ] \ ]. often thought to merely support specific bowel function and blood glucose and lipid levels \ [ [ @ b6 ] - [ @ b8 ] \ ], certain polysaccharides have attracted growing scientific interest for their ability to exert marked benefits on immune system function, inflammation and cancers \ [ [ @ b9 ] - [ @ b11 ] \ ]. many of these chemically and structurally diverse, non - to poorly - digestible polysaccharides have been shown to beneficially affect one or more targeted cellular functions * in place * \ [ [ @ b11 ] - [ @ b16 ] \ ], but much of the * in vivo * literature consists of studies in which polysaccharides were injected \ [ [ @ b1 ], [ @ b2 ] \ ]. for clinicians and scientists interested in immunologic effects following dietary intake, the value of such studies is uncertain. polysaccharides that elicit effects * in vitro * or by injection may be ineffective or have different effects when taken orally \ [ [ @ b17 ] \ ]. we have decided to conduct a systematic review to evaluate the specific immunologic effects of dietary polysaccharide products on rodents and human subjects. methods = = = = = = = literature review - - - - - - - - - - - - - - - - - studies were identified by conducting electronic searches of pubmed and google scholar from their inception towards the end of october 2009. the reference lists of the selected articles were checked for additional studies and were not originally found in the search. study selection and data extraction - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the following search terms directly combined with the term polysaccharide : dietary and immune, or oral and immune, or dietary and inflammation, or oral and inflammation. unless specific polysaccharides or polysaccharide - rich plants and fungi were identified, further searches were performed using their names with the same search terms. studies were selected based on the following inclusion criteria : 1 \. rode ##nt or human studies 2 \. the presence of test group and control group ( using either placebo, crossover, sham, or normal care ) 3 \. studies reporting statistically significant immunomodulatory effects 4 \. english language 5 \. studies published up to october 2009. two researchers ( jer, edn ) reviewed the list of unique articles for studies that fit the inclusion criteria. uncertainties over study inclusion were discussed between the researchers and resolved through consensus. searches were then conducted to obtain specific polysaccharide product information : safety ( using the search terms : toxicity, noael, ld ~ 50 ~ ), composition and structure, and disposition. quality assessment - - - - - - - - - - - - - - - - - - each study was assessed as to whether or not it reported a significant outcome measure for the polysaccharide intervention group. results = = = = = = = a total of 62 rodent publications ( tables [ 1 ] ( # t1 ) { ref - type = " table " }, [ 2 ] ( # t2 ) { ref - type = " table " } and [ 3 ] ( # t3 ) { ref - type = " table " } ) and 15 human publications ( table [ 4 ] ( # t4 ) { ref - type = " table " } ) were deemed appropriate for inclusion in this review. available structural and compositional information for these immunomodulatory polysaccharides are provided in table [ 5 ] ( # t5 ) { ref - type = " table " } and safety information is provided in table [ 6 ] ( # t6 ) { ref - type = " table " }. the majority of animal studies explored models in which animals were injected or implanted with cancer cells or tumors, were healthy, or were exposed to carcinogens. other studies investigated immunodeficient, exercise - stressed, aged animals, or animals exposed to inflammatory agents, viruses, bacterial pathogens, pathogenic protozoa, radiation or mutagens. human studies assessed immunomodulatory effects in healthy subjects, or patients with cancers, seasonal allergic rhinitis or aphthous stomatitis. because of the limited number of human studies, we included some promising open - label controlled trials. human study durations ranged from four days to seven years ; daily doses ranging from 100 - 5, 400 mg were reported to be well - tolerated. # # # # # # immunomodulatory glucan extracts : oral animal studies - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - source extract animal dose / day duration of study treatment effects reference - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - * agaricus * \ α - 1, 6 and \ 8 - week [UNK] c3h / he mice ( 5 / group ) 100 mg / kg ig every 3 days 1 month healthy animals ↑ \ # s splenic t lymphocytes ( thy1. 2, cd4 + and cd8 + ) \ [ [ @ b24 ] \ ] ( * a. blazei * ) * subrufescens * α - 1, 4 glucans aqueous 7 - 9 - week [UNK] balb / cbyj mice ( 40 / group ) 1 ml 0. 45n, 0. 6n, or 3n aqueous extract 2 months all doses ↑ serum igg levels, cd3 + t cell populations and pml phagocytic activity \ [ [ @ b22 ] \ ] 7 - 9 - week male balb / cbyj mice ( 40 / group ) 1 ml 0. 45n, 0. 6n, or 3n aqueous extract 10 weeks ip injection of ova at 4 weeks 0. 6n and 3n ↑ levels of ova - specific serum igg 28 days post - immunization ; all doses ↑ delayed - type hypersensitivity and tnf - α secreted from splenocytes at 10 weeks ; 0. 6n ↑ splenocyte proliferation at 10 weeks 5 - 6 - week [UNK] balb / chsdola mice ( 8 / group × 2 ) one 200 μl extract day 1, orogastric intubation 1 week injected ip fecal solution day 2 ↓ cfu in blood of mice with severe peritonitis & improved overall survival rate in all peritonitis groups \ [ [ @ b46 ] \ ] 6 - week balb / c nu / nu mice ( 7 / group ) 2. 5 mg extract days 20 - 41, drinking water 41 days injected sc sp - 2 myeloma cells day 1 ↓ tumor size & weight after 21 days treatment \ [ [ @ b65 ] \ ] aqueous, acid treated 6 - week [UNK] c57bl / 6 mice ( 10 / group ) 20, 100 or 500 μg / ml, drinking water 9 days injected ip human ovarian cancer cells day 1 500 μg / ml ↓ tumor weight \ [ [ @ b66 ] \ ] 20, 100 or 500 μg / ml, drinking water 3 weeks injected iv murine lung cancer ( 3ll ) cells 100 & 500 μg / ml ↓ \ # s metastatic tumors aqueous, with 200 ng / day \ 6 - week [UNK] balb / c mice ( 10 / group ) 200 ng days 5 - 21 3 weeks injected meth a tumor cells day 1 ↓ tumor size & weight \ [ [ @ b23 ] \ ] β - glucan 2 weeks injected meth a tumor cells ↑ cytotoxic t lymphocyte activity & spleen cell ifn - α protein 300 mg 5 days healthy animals ↑ splenic nk cell activity | Background = = = = = = = = = = Polysaccharide - rich fungi and plants have been employed for centuries by cultures around the world for their dietary and medicinal benefits \ [[ @ B1] - [@ B5] \ ]. Often thought to merely support normal bowel function and blood glucose and lipid levels \ [[ @ B6] - [@ B8] \ ], certain polysaccharides have attracted growing scientific interest for their ability to exert marked effects on immune system function, inflammation and cancers \ [[ @ B9] - [@ B11] \ ]. Many of these chemically and structurally diverse, non - to poorly - digestible polyxacchaEides have been shown to beneficially affect one or more targeted cellular functions * in vitro * \ [[ @ B11] - [@ B16] \ ], but much of the * in vivo * literature consists of studies in which polysaccharides were injected \ [[ @ B1 ], [@ B2] \ ]. For clinicians and scientists interested in immunologic effects following dietary intake, the value of such studies is uncertain. Polysaccharides that elicit effects * in vitro * or by injection may be ineffective or have different effects when taken orally \ [[ @ B17] \ ]. We thus decided to conduct a systematic review to evaluate the specific immunologic effects of dietary polysaccharide products on rodents and human subjects. Methods = = = = = = = Literature review - - - - - - - - - - - - - - - - - Studies were identified by conducting electronic searches of PubMed and Google Scholar from their inception to the end of October 2009. The referenX3 lists of the selected articles were checked for additional studies that were not originally found in the search. Study selection and data extraction - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The following search terms were combined with the term polysaccharide: dietary AND immune, or oral AND immune, or dietary AND inflammation, or oral AND inflammation. When specific polysaccharides or polysaccharide - rich plants and fungi were identified, further s#archeZ were conducted using their names with the same search terms. StudLws were selected based on the following inclusion criteria: 1 \. Rodent or human studies 2 \. The presence of test group and control group (using either placebo, crossover, sham, or normal care) 3 \. Studies reporting statistically significant immunomodulatory effects 4 \. English language 5 \. Studies published up to October 2009. Two researchers (JER, EDN) reviewed the list of unique articles for studies that fit the inclusion criteria. Uncertainties pve4 study inclusion were discussed betweSM the researchers and resolved through consensus. Searches were then conducted to obtain specific polysaccharide product information: safety (using the search terms: toxicity, NOAEL, LD ~ 50 ~ ), composition and structure, and disposition. Quality assessment - - - - - - - - - - - - - - - - - - Each study was assessed as to whether or not it reported a significant outcome measure for the polysaccharide intervention group. Results = = = = = = = A total of 62 rodent publications (Tables [1] (# T1) {ref - type = " table " }, [2] (# T2) {ref - type = " table "} and [3] (# T3) {ref - type = " table "} ) and 15 human publications (Table [4] (# T4) {ref - type = " table "} ) were deemed appropriate for inclusion in this review. Available structural and compositional information for these immunomodulatory polysaccharides are provided in Table [5] (# T5) {ref - type = " table "} and safety information is provided in Table [6] (# T6) {ref - type = " table " }. The majority of animal studies explored models in which animals were injected or implanted with cancer cells or tumors, were h$al^hy, or were exposed to carcinogens. Other studies investigated immunodeficient, exercise - stressed, aged animals, or animals exposed to inflammatory ageHhs, viruses, bacterial pathogens, pathogenic protozoa, radiation or mutagens. Human studies assessed immunImodulatorU effects in healthy subjects, or patients with cancers, seasonal allergic rhinitis or aphthous stomatitis. Because of the limited number of human studies, we included some promising open - label controlled trials. Human study durations ranged from four days to seven years; daily doses ranging from 100 - 5, 400 mg were reported to be well - tolerated. # # # # # # Immunomodulatory Glucan Extracts: Oral Animal Studies - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Source Extract Animal Dose / day Duration of study Treatment Effects Reference - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - * Agaricus * \ α - 1, 6 and \ 8 - week ♀ C3H / He mice (5 / group) 100 mg / kg IG every 3 days 1 month Healthy animals ↑ \ # s splenic T lymphocytes (Thy1. 2, CD4 + and CD8 +) \ [[ @ B24] \] (* A. blazei *) * subrufescens * α - 1, 4 glucans Aqueous 7 - 9 - week ♂ Balb / cByJ mice (40 / group) 1 ml 0. 45N, 0. 6N, or 3N aqueous extract 2 months All doses ↑ serum IgG levels, CD3 + T cell populations and PML phagocytic activity \ [[ @ B22] \] 7 - 9 - week male Balb / cByJ mice (40 / group) 1 ml 0. 45N, 0. 6N, or 3N aqueous extract 10 weeks IP injection of OVA at 4 weeks 0. 6N and 3N ↑ levels of OVA - specific serum IgG 28 days post - immunization; all doses ↑ delayed - type hypersensitivity and TNF - α secreted from splenocytes at 10 weeks; 0. 6N ↑ splenocyte proliferation at 10 weeks 5 - 6 - week ♀ BALB / cHsdOla mice (8 / group × 2) One 200 μl extract day 1, orogastric intubation 1 week Injected IP fecal solution day 2 ↓ CFU in blood of mice with severe peritonitis & improved overall survival rate in all peritonitis groups \ [[ @ B46] \] 6 - week BALB / c nu / nu mice (7 / group) 2. 5 mg extract days 20 - 41, drinking water 41 days Injected SC Sp - 2 myeloma cells day 1 ↓ tumor size & weight after 21 days treatment \ [[ @ B65] \] Aqueous, acid treated 6 - week ♀ C57BL / 6 mice (10 / group) 20, 100 or 500 μg / ml, drinking water 9 days Injected IP human ovarian cancer cells day 1 500 μg / ml ↓ tumor weight \ [[ @ B66] \] 20, 100 or 500 μg / ml, drinking water 3 weeks Injected IV murine lung cancer (3LL) cells 100 & 500 μg / ml ↓ \ # s metastatic tumors Aqueous, with 200 ng / day \ 6 - week ♀ BALB / c mice (10 / gr)u9) 200 ng days 5 - 21 3 weeks Injected Meth A tumor cells day 1 ↓ tumor size & weight \ [[ @ B23] \] β - glucan 2 weeks Injected Meth A tumor cells ↑ cytotoxic T lymphocyte activity & spleen cell IFN - α protein 300 mg 5 days Healthy animals ↑ splenic NK cell activity | Background ========== Polysaccharide-rich fungi and plants have been for centuries by around world for their dietary and medicinal benefits \[[@B1]-[@B5]\]. Often thought to merely support normal bowel function and blood glucose and lipid levels certain polysaccharides have attracted scientific interest for their ability to exert marked on immune system function, inflammation and cancers \[[@B9]-[@B11]\]. Many of these chemically and structurally diverse, non- to poorly-digestible polysaccharides have been shown to beneficially affect one or targeted cellular *in vitro*\[[@B11]-[@B16]\], but much of the *in vivo*literature consists of in which polysaccharides were injected \[[@B1],[@B2]\]. For clinicians and scientists interested in immunologic effects following dietary intake, the value of such studies is uncertain. that elicit effects *in vitro*or by injection may be ineffective or have different effects when taken orally \[[@B17]\]. We thus decided to conduct a review to evaluate the specific immunologic effects of dietary polysaccharide products on rodents human subjects. Methods ======= Literature review ----------------- Studies were by conducting electronic searches of PubMed and Google from their inception to the of October 2009. The reference lists of the selected articles were checked for additional studies that were not originally found in the search. Study selection and data extraction ----------------------------------- The following search terms were combined with the term polysaccharide: dietary immune, or oral AND immune, or dietary AND inflammation, or oral inflammation. When specific polysaccharides or polysaccharide-rich plants and fungi were identified, further searches were conducted using names with the same search terms. Studies were selected based on the following inclusion criteria: 1\. Rodent or studies 2\. The presence of test group and control group (using either placebo, crossover, sham, or normal care) 3\. Studies statistically immunomodulatory effects 4\. English language 5\. Studies published up to October 2009. researchers EDN) reviewed the list of unique for studies that fit the inclusion criteria. Uncertainties over inclusion were discussed between the researchers and resolved through consensus. Searches were then conducted to obtain specific polysaccharide product information: safety (using search terms: toxicity, NOAEL, LD~50~), composition and structure, and disposition. Quality assessment ------------------ Each study was assessed as to whether or it reported a significant outcome measure for the polysaccharide group. Results A of 62 rodent publications (Tables and 15 human publications (Table [4](#T4){ref-type="table"}) were deemed appropriate for inclusion in this review. Available structural and compositional information for these immunomodulatory polysaccharides are provided in Table and safety information is provided in Table [6](#T6){ref-type="table"}. The of animal studies explored models in which animals were injected implanted cancer cells or tumors, were healthy, or were to carcinogens. Other studies investigated immunodeficient, exercise-stressed, aged animals, or animals exposed to inflammatory agents, viruses, bacterial pathogens, pathogenic protozoa, radiation or mutagens. Human studies assessed immunomodulatory effects subjects, or patients with cancers, seasonal or aphthous stomatitis. Because of the limited number of studies, we included promising controlled trials. Human study durations ranged from four days to seven daily doses from 100-5,400 mg were reported to be well-tolerated. ###### Immunomodulatory Glucan Extracts: Oral Animal Studies ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Extract Duration of study Treatment Effects Reference ------------------------------------- ---------------------------------- --------------------------------------------------------------------------- ---------------------------------------------------------------------- ------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ------------ *Agaricus*\ α-1,6 and\ ♀ mice (5/group) 100 IG every days 1 month Healthy animals ↑ T lymphocytes (Thy1.2, and CD8+) \[[@B24]\] (*A. blazei*) *subrufescens* α-1,4 glucans Aqueous 7-9-week ♂ Balb/cByJ mice (40/group) 1 ml 0.45N, 0.6N, or 3N aqueous extract months All doses ↑ serum IgG cell populations and PML phagocytic activity \[[@B22]\] 7-9-week male Balb/cByJ mice (40/group) ml 0.45N, or aqueous extract 10 weeks injection of 4 weeks 0.6N and 3N ↑ levels of OVA-specific serum IgG 28 days all ↑ delayed-type hypersensitivity and TNF-α secreted from splenocytes at 10 weeks; 0.6N ↑ splenocyte proliferation at 10 weeks 5-6 -week ♀ mice (8/group × 2) One 200 μl extract day orogastric intubation 1 week IP fecal solution day 2 CFU in blood of mice with severe peritonitis & improved overall survival rate in all peritonitis groups \[[@B46]\] 6-week BALB/c nu/nu mice 2.5 mg extract days 20-41, drinking water 41 days Injected SC Sp-2 myeloma cells day 1 ↓ tumor size weight after 21 days treatment \[[@B65]\] Aqueous, acid 6-week ♀ mice (10/group) 20, 100 or 500 μg/ml, drinking water 9 Injected human ovarian cancer cells day 1 500 μg/ml ↓ tumor \[[@B66]\] 20, 100 or 500 μg/ml, drinking water 3 weeks Injected IV murine lung cancer (3LL) cells 100 & 500 μg/ml ↓ \#s metastatic tumors Aqueous, with 200 ng/day\ 6-week ♀ BALB/c mice (10/group) ng days 5-21 3 weeks Injected tumor cells day 1 ↓ tumor size & weight \[[@B23]\] β-glucan 2 weeks Meth A tumor cells ↑ cytotoxic T lymphocyte activity & spleen IFN-α protein 300 mg 5 days Healthy animals ↑ splenic NK cell activity | bAcKGrOUnD
==========
PolYsAccHaRIde-RIcH Fungi And plANTs hAVe BEEN EmPLoyEd FOr cEntUrIes bY cUltUrEs aRoUND the wOrLd fOR ThEir DIEtarY And medICINal BenefiTs \[[@B1]-[@b5]\]. oFteN tHOuGhT To MereLY sUppOrT normAl bOWeL Function AnD BLOoD gLUCOsE AND LiPid lEvELs \[[@b6]-[@b8]\], CeRTain POlYsaCcHaRIDes HaVE AttrACted gRoWiNg sCIEntiFIc iNTerest fOr thEIR abiLitY TO ExErt MaRkEd EFFEcTS On immUNe SYstem FuNcTion, iNfLaMMATion AnD CancerS \[[@B9]-[@B11]\]. MaNy OF THESE cheMIcALLY anD StRUCTurALLy dIVErSE, NOn- to poorLy-DigesTiBLE pOlysacCHArIdEs HaVe bEeN SHOWN To BeNEfICiAllY AffEcT OnE oR MorE TaRGeTeD celLuLar FUNctioNS *IN VItRo*\[[@B11]-[@b16]\], BUT mUcH of THe *In Vivo*LIteRATure cOnSIsTs Of STUdIes In WHICh poLYSaCcHArIdES WerE iNJecteD \[[@b1],[@b2]\]. For CliNIcIANs anD ScIEntISTs iNTEresteD iN imMUnoloGIc EfFEcTs foLloWing DiETaRY iNTakE, ThE valUE OF SUCH sTUDiES IS unceRtAIN. PoLySaccharIDeS THAt ELIcIT effects *in VItro*OR By INJEcTIon mAY BE iNEfFEcTiVE Or haVE DIFfeReNt effEcTs WhEN TAken oRaLLY \[[@B17]\]. WE THuS deCidEd tO coNDUCt A SySTEMatic ReVIEW To EVaLuaTe the sPeCifIC immunoLogIC EfFEcts Of DIEtaRY poLySACCHAride PRoDUCTS oN rODeNtS ANd humaN sUbJects.
METhODs
=======
LiTErAture RevIEW
-----------------
stuDIES were IDeNTifIEd BY conDuCTINg eLECTronIC SeArcheS OF PUbMed AnD gOoglE ScholAR fROM THeir iNCEPtioN To THE ENd oF ocTObeR 2009. ThE rEfErEnCE LISTs oF THE sElEctED ArTicLeS WeRE CheckEd FOr adDItioNAL STudiEs THAT WERe NOT oRIGiNAllY foUnd IN thE seArch.
StUdY SeLEction And DATA EXtrAcTIoN
-----------------------------------
the folLoWinG seARCh teRmS WeRe cOMBIneD wIth the tErm POlySACchARidE: DIETArY aNd imMuNE, oR ORaL ANd IMMunE, or dIeTAry ANd InFlamMATion, OR ORAl aND InfLaMmAtIon. WheN sPECiFic PolYsACChaRidES oR PoLysacChariDE-ricH PLAnts AND funGi WERe IdentifIED, FUrThEr searches WeRe coNdUcTEd uSInG TheiR NAMES WItH THe saME sEaRCh TERMs. stUDiES weRe SeleCTed baSEd ON thE FolloWInG InClUSiON CrIteRia:
1\. rODenT or hUMAN StUDieS
2\. ThE pResEnCE Of TEST GrOUP aNd COntrol gROup (uSIng EIthER PLACeBo, CRoSSOver, ShAm, or NoRmAl CArE)
3\. StudiES rEpoRTing sTAtIStically SIgNIfIcANT iMmunOMODUlaTORy effEcts
4\. ENglish langUAgE
5\. sTUdIes pUBlISHed up To oCTobER 2009.
TWo REsEaRCherS (jEr, EDN) rEVIeweD tHE liSt Of UNIquE aRtiCLeS fOr sTudIEs tHAt FiT thE INclUsION CRiterIA. UNCeRTaiNtIes oVER sTuDY INcluSion werE DIsCUssed BetweEN tHE reSEArchERs and ResolVed tHRoUgH coNSeNSUs. seARCHes Were tHen cOnducted TO oBTAIN sPeCiFic polYsACcHarIdE PrODUct INFoRmAtIoN: saFEtY (usIng tHE Search terMs: ToXiCITY, NOAeL, LD~50~), cOMPOSiTiON and STrUcTurE, AnD DiSposiTIOn.
QualitY AssEsSment
------------------
EaCh STUDy WaS AssESSED as tO whETher Or NOt It rEpORTED a siGNIFICAnt OUTcOMe MeAsUre fOr tHE polySACCHaRIDE INterVeNTIon grouP.
ResultS
=======
a TotAL Of 62 rOdEnt pUBLICaTIoNS (TAbles [1](#T1){rEF-tYPe="Table"}, [2](#T2){Ref-tYpE="TablE"} AND [3](#t3){ref-TyPe="tAble"}) AnD 15 HumAn pUbliCaTiOns (tablE [4](#t4){reF-Type="TABLe"}) weRE deemed apprOprIaTE fOR iNcLUSION IN tHIs REvieW. AVaiLaBle STrUcTUrAL ANd COMPoSITIonal iNFormATIon FOr THESe ImmUNOmoDulaTORY pOLysacCharIdES ARe prOviDED iN Table [5](#T5){reF-tYPE="Table"} aND sAFEty InfORMATion Is pROViDed In table [6](#t6){rEf-tYPE="TABle"}. The MAjoriTY of aNIMaL StUDieS eXplOREd moDeLs In whicH anImAlS werE iNjeCTED or ImPLantEd WITh caNceR CElLs oR TUMOrs, WErE HeAlThy, OR WeRe ExpoSED TO CarCInOgenS. OTheR STUDIes iNVEsTIgAted IMMuNODEfICIent, ExerCiSE-streSSed, AgeD aNimAls, OR aNiMAls ExpOsED to inFLaMMatOrY agenTS, vIrUSeS, BACTeriaL PaThoGENS, PaThogEnIc PRoTozOA, raDiaTiOn or mUTagENs. HumAn STUDIEs aSsEsSED IMmuNomoDUlAtORy EFFECTS In hEALTHY sUbjeCTs, or paTIeNts wiTH caNCeRs, SEaSonal AllErGIc RhiNiTIs or AphthouS sTOMaTitis. BECauSE oF ThE lImItEd NuMBeR oF humAn studIes, WE iNclUdeD Some pROMiSING oPEN-LaBel cOntROlled tRiAls. hUMaN STuDy dUraTIOns ranGED frOm FOUR DayS to sEVEN YeArs; daILy doSEs rANGing FrOm 100-5,400 mG were REpoRted To Be well-toleraTed.
######
IMMUnOmOduLatory GlUcAN ExtRACts: OrAl ANImal STuDiEs
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
sourCe EXtracT aNiMAL DoSe/Day DURATIon OF sTuDy tREATMeNT efFECtS REfeRENce
------------------------------------- ---------------------------------- --------------------------------------------------------------------------- ---------------------------------------------------------------------- ------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ------------
*agaricUS*\ Α-1,6 ANd\ 8-weeK ♀ C3H/he miCe (5/GRoUP) 100 Mg/kG Ig eVerY 3 DAYS 1 mOnth heALthY AnImalS ↑ \#S SPLeNIC T LYMPhoCYtES (ThY1.2, CD4+ and cd8+) \[[@B24]\]
(*a. blAZeI*) *SUBruFesCENS* Α-1,4 GluCANS
AqUeOUS 7-9-Week ♂ BaLb/CByj MIcE (40/GrOUp) 1 Ml 0.45N, 0.6n, oR 3N AquEouS EXtRAct 2 mONthS alL DOSes ↑ sERum iGG lEvEls, cD3+ t Cell popUlATIons AnD Pml PHaGOcytiC aCTiViTY \[[@b22]\]
7-9-WEEk mAlE bALB/cbyJ mICe (40/grOuP) 1 ML 0.45n, 0.6n, Or 3N AqUeOuS EXtrAcT 10 WeekS iP INJECtIoN OF Ova AT 4 WEEKS 0.6N and 3n ↑ lEVELs OF OVA-SpeciFIc SErUm IGg 28 DAys PoSt-iMmUnIzaTiOn; all dOSeS ↑ deLAyed-tyPE hYperSeNSITivItY aND tNF-Α seCReteD fRoM SplENocyTES AT 10 weEKS; 0.6N ↑ splEnOCYTe PROLifERatIOn AT 10 weeKS
5-6 -wEek ♀ BaLb/CHSDoLA mIce (8/grOUp × 2) One 200 μL eXTRAcT DaY 1, OrOGastrIC IntuBatION 1 WEEk INJECTEd ip FECAl soLUtiON day 2 ↓ cfu iN blooD OF MICE WITH sEVErE pERItOniTis & IMPRoVEd OVeRALl SURVIvAL RATe iN ALL perITONiTIs GrOupS \[[@b46]\]
6-WEek BaLB/c Nu/Nu miCE (7/grOUP) 2.5 mG ExtraCT DAyS 20-41, DrinKING waTer 41 DAYs InJecTED SC Sp-2 mYeLOma cElLs DAY 1 ↓ tUMoR size & WeIghT aftEr 21 dayS TREatmeNt \[[@B65]\]
aquEous, ACID treAtED 6-WEek ♀ c57bL/6 MICe (10/group) 20, 100 Or 500 ΜG/mL, dRInking watER 9 daYs INjEctED IP hUMaN OvariAN cANCeR cellS dAy 1 500 Μg/ML ↓ tUMOr wEiGht \[[@B66]\]
20, 100 OR 500 μG/ML, DRinKiNg wATER 3 WEeKS InjecTED Iv muRINE LuNg CanCeR (3Ll) cellS 100 & 500 μG/ml ↓ \#S METAstaTic tuMoRs
aquEouS, wiTH 200 ng/dAY\ 6-wEEK ♀ BALB/C MiCE (10/GrouP) 200 nG dAys 5-21 3 weEks INJECteD mEth A tUMOR CElLS dAY 1 ↓ tuMor SIzE & wEIGHt \[[@b23]\]
β-GlUcAN
2 weeKs InjecTEd mETh A TUMor cELLs ↑ CYtotoXIc t LYMPhOcyTe acTiVIty & sPlEeN ceLL IFn-α PRoTEiN
300 mG 5 daYs hEAlThY ANiMAls ↑ sPlEnIC NK CeLl ACTivITy
| Background ========== Polysaccharide-richfungi and plants have beenemployed for centuriesby cultures around the world for their dietary and medicinal benefits \[[@B1]-[@B5]\]. Often thought to merely support normal bowel functionand bloodglucose and lipid levels\[[@B6]-[@B8]\], certain polysaccharides have attracted growing scientific interest for theirability to exert marked effects on immune system function, inflammationand cancers \[[@B9]-[@B11]\]. Manyof these chemically and structurally diverse, non- topoorly-digestible polysaccharides have been shown to beneficially affectone or more targeted cellular functions *invitro*\[[@B11]-[@B16]\], but much of the *invivo*literature consists of studies in which polysaccharideswere injected \[[@B1],[@B2]\]. For clinicians and scientists interested inimmunologic effects following dietary intake,thevalue of suchstudies is uncertain. Polysaccharides that elicit effects *in vitro*or by injection may be ineffective or have different effectswhen taken orally \[[@B17]\]. Wethus decided to conduct a systematic review toevaluate the specific immunologic effects of dietary polysaccharideproducts on rodents and human subjects. Methods ======= Literature review----------------- Studies were identified by conducting electronicsearches ofPubMed and Google Scholar from theirinception to theend of October2009. The reference lists ofthe selectedarticles were checked for additional studies that were not originally found in the search. Study selection and data extraction ----------------------------------- The followingsearch terms were combinedwith the term polysaccharide: dietaryAND immune, ororal AND immune, ordietary ANDinflammation, or oral AND inflammation. When specific polysaccharides or polysaccharide-rich plants and fungi were identified,further searches were conducted using theirnames with the same search terms. Studies were selectedbased on the following inclusion criteria: 1\. Rodentor human studies 2\. The presence of testgroup and control group (usingeitherplacebo, crossover, sham, or normal care)3\. Studies reporting statistically significant immunomodulatory effects 4\. English language 5\. Studies published up to October 2009. Two researchers (JER, EDN) reviewed thelist of unique articlesfor studiesthat fit the inclusion criteria. Uncertainties over studyinclusion were discussed between the researchers and resolved through consensus. Searches were then conducted to obtain specific polysaccharide product information: safety (using the search terms: toxicity, NOAEL, LD~50~), composition and structure, and disposition. Quality assessment ------------------ Each study was assessed as to whetheror not it reported a significant outcome measure for the polysaccharide intervention group. Results ======= A totalof 62 rodent publications (Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}) and 15 humanpublications (Table [4](#T4){ref-type="table"}) were deemedappropriatefor inclusion in thisreview. Available structuralandcompositional information for these immunomodulatory polysaccharides areprovided in Table [5](#T5){ref-type="table"} and safety informationis provided in Table [6](#T6){ref-type="table"}. The majority of animal studies exploredmodels inwhichanimals were injected or implanted with cancer cells or tumors, were healthy,or were exposed to carcinogens. Other studies investigated immunodeficient,exercise-stressed, aged animals, or animals exposedtoinflammatory agents, viruses, bacterialpathogens, pathogenic protozoa, radiation or mutagens. Human studies assessed immunomodulatory effects in healthy subjects, or patients with cancers, seasonal allergic rhinitis or aphthous stomatitis. Because ofthe limited number of humanstudies, weincluded some promising open-label controlled trials. Humanstudy durationsranged from four days to seven years; daily doses ranging from 100-5,400 mg were reported to be well-tolerated. ######Immunomodulatory Glucan Extracts: Oral AnimalStudies ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Source Extract Animal Dose/day Duration ofstudyTreatment Effects Reference ------------------------------------- ---------------------------------- --------------------------------------------------------------------------- ---------------------------------------------------------------------- ------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ------------ *Agaricus*\ α-1,6 and\ 8-week ♀ C3H/He mice (5/group)100 mg/kg IG every 3 days 1month Healthy animals ↑ \#s splenic Tlymphocytes (Thy1.2, CD4+ and CD8+) \[[@B24]\](*A. blazei*) *subrufescens* α-1,4 glucans Aqueous 7-9-week ♂ Balb/cByJ mice (40/group) 1ml 0.45N, 0.6N,or 3N aqueous extract 2 months Alldoses ↑ serum IgG levels, CD3+ T cell populations and PML phagocytic activity \[[@B22]\] 7-9-week male Balb/cByJ mice(40/group)1 ml 0.45N,0.6N, or3N aqueous extract 10 weeks IP injection of OVAat 4 weeks 0.6N and 3N ↑ levels of OVA-specific serum IgG28 days post-immunization; all doses ↑ delayed-type hypersensitivity and TNF-α secreted from splenocytes at 10 weeks; 0.6N ↑ splenocyte proliferation at 10weeks 5-6 -week ♀BALB/cHsdOla mice (8/group × 2) One200 μl extract day 1, orogastric intubation 1 week Injected IP fecal solution day 2↓CFU in blood of mice with severe peritonitis & improved overall survivalrate in all peritonitis groups \[[@B46]\] 6-week BALB/c nu/numice (7/group) 2.5 mg extractdays 20-41, drinking water 41 days Injected SC Sp-2 myelomacells day 1 ↓ tumor size & weight after 21 daystreatment \[[@B65]\] Aqueous,acid treated 6-week ♀ C57BL/6 mice (10/group) 20, 100or 500 μg/ml, drinking water 9 days Injected IP human ovarian cancer cells day 1 500 μg/ml ↓ tumor weight \[[@B66]\] 20, 100 or500 μg/ml, drinking water 3 weeksInjected IV murinelung cancer (3LL) cells100 & 500 μg/ml ↓ \#s metastatic tumorsAqueous, with 200 ng/day\ 6-week ♀ BALB/c mice (10/group) 200 ng days 5-21 3weeks Injected Meth Atumor cells day 1 ↓ tumor size& weight \[[@B23]\] β-glucan 2 weeks Injected Meth A tumor cells ↑ cytotoxic Tlymphocyte activity & spleen cell IFN-α protein 300mg 5 daysHealthy animals ↑ splenicNK cell activity | Background ========== Polysaccharide-rich fungi and _plants_ have been employed for centuries by cultures _around_ the world for their _dietary_ and medicinal benefits \[[@B1]-[@B5]\]. Often thought to merely support normal bowel _function_ and _blood_ glucose and lipid levels \[[@B6]-[@B8]\], certain polysaccharides have attracted growing scientific _interest_ _for_ their ability to exert marked _effects_ on immune system function, inflammation and cancers \[[@B9]-[@B11]\]. Many of _these_ chemically and structurally diverse, _non-_ to poorly-digestible polysaccharides have been _shown_ to _beneficially_ affect _one_ or more targeted cellular functions _*in_ vitro*\[[@B11]-[@B16]\], _but_ much of the *in vivo*literature consists _of_ studies in which polysaccharides were injected \[[@B1],[@B2]\]. For clinicians and _scientists_ interested in _immunologic_ effects following dietary intake, the value _of_ such studies is uncertain. Polysaccharides that elicit effects _*in_ _vitro*or_ by injection may _be_ _ineffective_ or have different effects _when_ taken _orally_ \[[@B17]\]. We thus _decided_ to conduct _a_ systematic review _to_ evaluate the specific immunologic effects of dietary polysaccharide products on rodents _and_ human subjects. Methods _=======_ Literature review ----------------- Studies were identified _by_ _conducting_ electronic searches of PubMed _and_ Google Scholar _from_ _their_ inception to _the_ end of October 2009. The _reference_ lists of the _selected_ articles were checked for additional studies _that_ were not originally found in the search. Study selection and _data_ extraction _-----------------------------------_ The following _search_ terms were combined _with_ the term _polysaccharide:_ dietary AND immune, or oral AND immune, or _dietary_ AND inflammation, _or_ oral _AND_ inflammation. When specific polysaccharides or polysaccharide-rich plants and _fungi_ _were_ identified, further searches were conducted using their names with the same search terms. Studies were _selected_ _based_ on the following inclusion criteria: 1\. Rodent or human _studies_ 2\. The presence of test _group_ and control group (using either placebo, crossover, sham, or normal care) _3\._ Studies reporting statistically significant immunomodulatory effects 4\. English language 5\. Studies published up to October 2009. Two researchers _(JER,_ _EDN)_ reviewed the _list_ of unique _articles_ for studies that fit the inclusion criteria. _Uncertainties_ _over_ study inclusion were discussed between _the_ researchers and resolved through consensus. _Searches_ were then _conducted_ to obtain specific polysaccharide product information: safety _(using_ the _search_ terms: toxicity, NOAEL, LD~50~), _composition_ and structure, and disposition. _Quality_ assessment ------------------ Each study was assessed as to whether _or_ not _it_ _reported_ _a_ significant outcome measure _for_ the polysaccharide intervention group. Results _=======_ _A_ total _of_ 62 rodent publications (Tables _[1](#T1){ref-type="table"},_ [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}) and 15 _human_ publications (Table _[4](#T4){ref-type="table"})_ were deemed appropriate for inclusion _in_ this review. Available structural and _compositional_ _information_ for _these_ immunomodulatory _polysaccharides_ are provided in Table _[5](#T5){ref-type="table"}_ and safety information is provided in Table _[6](#T6){ref-type="table"}._ _The_ _majority_ of animal studies explored _models_ in which animals were injected or _implanted_ with cancer cells or tumors, _were_ healthy, or were exposed to carcinogens. _Other_ studies investigated immunodeficient, exercise-stressed, _aged_ animals, or animals exposed to inflammatory agents, viruses, bacterial pathogens, pathogenic protozoa, radiation or mutagens. Human studies assessed _immunomodulatory_ effects in healthy subjects, or patients _with_ _cancers,_ seasonal allergic rhinitis or _aphthous_ stomatitis. _Because_ of the limited number of human _studies,_ _we_ included some promising open-label _controlled_ trials. Human study durations ranged from four _days_ to _seven_ years; daily _doses_ ranging from 100-5,400 _mg_ were reported to _be_ well-tolerated. ###### _Immunomodulatory_ Glucan Extracts: Oral Animal Studies ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ _Source_ Extract Animal Dose/day Duration _of_ study Treatment Effects _Reference_ ------------------------------------- ---------------------------------- _---------------------------------------------------------------------------_ ---------------------------------------------------------------------- ------------------- _----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------_ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ------------ _*Agaricus*\_ α-1,6 and\ 8-week ♀ C3H/He mice (5/group) 100 _mg/kg_ _IG_ every 3 days _1_ month Healthy animals ↑ _\#s_ splenic T _lymphocytes_ _(Thy1.2,_ _CD4+_ and _CD8+)_ \[[@B24]\] (*A. blazei*) *subrufescens* α-1,4 glucans Aqueous 7-9-week ♂ Balb/cByJ mice (40/group) 1 ml _0.45N,_ _0.6N,_ or 3N aqueous extract 2 _months_ All doses ↑ _serum_ IgG levels, CD3+ _T_ cell populations and PML phagocytic activity \[[@B22]\] _7-9-week_ male Balb/cByJ mice (40/group) 1 ml 0.45N, 0.6N, or 3N _aqueous_ extract 10 weeks IP injection _of_ OVA at 4 _weeks_ 0.6N _and_ 3N ↑ _levels_ of OVA-specific serum IgG 28 days _post-immunization;_ all doses ↑ delayed-type hypersensitivity and TNF-α secreted from _splenocytes_ at 10 weeks; 0.6N ↑ splenocyte proliferation at _10_ weeks 5-6 -week _♀_ BALB/cHsdOla mice (8/group _×_ 2) One _200_ _μl_ extract day 1, _orogastric_ intubation 1 _week_ Injected IP fecal _solution_ day 2 ↓ CFU in blood of mice _with_ severe peritonitis & improved overall survival rate _in_ _all_ _peritonitis_ groups \[[@B46]\] 6-week _BALB/c_ nu/nu mice (7/group) 2.5 mg _extract_ days 20-41, drinking water 41 _days_ Injected SC Sp-2 myeloma cells day 1 ↓ tumor size _&_ weight after _21_ days treatment \[[@B65]\] Aqueous, acid treated 6-week ♀ C57BL/6 mice (10/group) 20, 100 or 500 μg/ml, _drinking_ water _9_ _days_ Injected IP human ovarian cancer cells _day_ 1 _500_ _μg/ml_ ↓ tumor weight \[[@B66]\] 20, _100_ _or_ 500 μg/ml, drinking water 3 weeks Injected _IV_ murine lung _cancer_ _(3LL)_ cells 100 & 500 μg/ml ↓ \#s metastatic _tumors_ Aqueous, with 200 ng/day\ 6-week ♀ BALB/c _mice_ (10/group) 200 ng _days_ 5-21 3 weeks Injected Meth A tumor cells day _1_ ↓ tumor size & weight \[[@B23]\] β-glucan 2 _weeks_ _Injected_ Meth A tumor cells ↑ cytotoxic T lymphocyte activity & _spleen_ cell IFN-α protein 300 mg 5 days Healthy animals _↑_ splenic NK cell activity |
Introduction
============
Phytogenic feed additives (PFA) have attracted a lot of attention in recent years. When used in diets, these substances exhibit antioxidative properties and antimicrobial activity, improve nutrient absorption, and could ultimately improve animal performance ([@bib26]; [@bib30]; [@bib37]). PFA are less toxic, have fewer side effects, and are residue-free compared with synthetic feed additives, and are thought to be ideal feed additives in animal production ([@bib25]). Therefore, many phytogenic compounds have been recommended for use as feed additives.
Fenugreek (*Trigonella foenum-graecum* L) belongs to the Leguminosae family and is cultivated predominantly in India, West Asia, the Mediterranean, North Africa, and Canada ([@bib29]). The seeds are generally used for condiments in various food preparations, are regarded to have great nutritive value and restorative properties, and have been used in folk medicine for centuries for their hypoglycemic, anthelmintic, antibacterial, antiinflammatory, antipyretic, and antimicrobial properties ([@bib49]; [@bib7]; [@bib50]). The major active components of fenugreek seeds are 4-hydroxyisoleucine, trigonelline, galactomannan with flavonoids, carotenoids, coumarins, and saponins, which confer pharmacological activity and beneficial effects ([@bib46]; [@bib7]).
Recently, the modes of PFA action in vivo, including aromatic plants, plant extracts, their single active components, or their blended additives, have been investigated in several studies ([@bib54]; [@bib6]; [@bib37]; [@bib38]). However, in laying hens, a comparatively small number of studies have investigated the effects of fenugreek seed on production performance and egg quality. Therefore, in this study, an attempt was made to evaluate the fenugreek seed as natural growth promoter in laying hen diets. The evaluation included biochemical changes in egg production, egg quality, blood profiles, cecal microflora, and excreta noxious gas emission.
Materials and Methods
=====================
Ethical Considerations
----------------------
The experimental protocols describing the management and care of animals were reviewed and approved by the Animal Care and Use Committee of Dankook University.
Experimental Design, Animals, and Housing
-----------------------------------------
A total of 384, 26-week-old (Hyline-brown) laying hens were used in this 6-week trial. Laying hens were randomly assigned to one of three treatments with eight replicates (16 hens/replicate). The experimental treatments were: control, basal diet (FSE 0%); fenugreek seed extract (FSE) 0.05%, basal diet + 0.05% FSE; FSE 0.1%, basal diet + 0.1% FSE. The FSE used in our study was Nutrifen^®^ (Emerald Seed Products Ltd., Avonlea, Canada), which contains ≥ 15.0 mg/g saponin. Nutrifen^®^ was composed of 3584 kcal/kg ME, 10.4% moisture, 28.0% CP, 9.9% crude fiber, 10.4% crude fat, and 4.6% crude ash. It also contained macrominerals as follows; 0.34% calcium, 0.28% sulfur, 0.74% phosphorus, 0.26% magnesium, 36.2 mg/kg zinc, 21.4 mg/kg manganese, and 36.2 mg/kg iron. Laying hens were provided ad libitum access to water and feed. All the diets were formulated in mash form to meet or exceed the [@bib36] nutrition requirement ([Table 1](#T1){ref-type="table"}). Treatment additives were included in the diet by replacing the same amount of corn. Laying hens were allowed to adjust to the environment for 5 days prior to beginning the feeding trial, during which they were fed a basal diet. They were raised in an ambient-regulated house, in which the temperature was maintained below 23°C and the light regime was set on a 16:8-light: dark cycle throughout the entire experiment. Birds were individually reared in adjacent steel cages fitted with a nipple drinker, feeder, and an egg collecting plate.
###### Formula and chemical composition of basal diet (as-fed basis)
Item (%)
-------------------------------------------------------- -------------
Ingredients
Corn 50.40
Soybean meal (CP 46%) 18.70
Wheat grain 10.00
Corn gluten meal 2.00
Wheat bran 5.00
Animal fat 4.40
Limestone 7.50
Dicalcium phosphate (P 18%) 1.40
Salt 0.30
[dl]{.smallcaps}-Met (50%) 0.10
Vitamin premix[^1^](#tf1){ref-type="table-fn"} 0.10
Trace mineral premix[^2^](#tf2){ref-type="table-fn"} 0.10
Total 100
Calculated values
ME (kcal/kg) 2,904
CP (%) 15.02
Lys (%) 0.78
Met + Cys (%) 0.65
Ca (%) 3.25
P (%) 0.61
Provided per kilogram of premix: 125,000 IU vitamin A; 2,500 IU vitamin D~3~; 10 mg vitamin E; 2 mg vitamin K~3~; 1 mg vitamin B~1~; 5 mg vitamin B~2~; 1 mg vitamin B~6~; 15 mg vitamin B~12~; 500 mg folic acid; 35,000 mg niacin; 10,000 mg Ca-Pantothenate and 50 mg biotin.
Provided per Kg of diet: 8 mg Mn (as MnO~2~); 60 mg Zn (as ZnSO~4~); 5mg Cu (as CuSO~4~·5H~2~O); 40mg Fe (as FeSO~4~·7H~2~O); 0.3 mg Co (as CoSO~4~·5H~2~O); 1.5 mg I (as KI), and 0.15 mg Se (as Na~2~SeO~3~·5H~2~O).
Laying Production, Performance, and Egg Quality
-----------------------------------------------
The hen-day egg production and egg weights were recorded daily, while feed consumption was measured weekly. The feed conversion ratio was calculated as the feed consumption per hen divided by egg weight per day per hen. For each treatment, 40 normal eggs (five eggs/cage) were collected randomly at 32 weeks and used to determine the egg quality. Eggshell color scores were determined using an eggshell color fan on a 1--15 scale (1=light to 15=dark brown) by a single trained evaluator. Haugh units, albumen height, and yolk color were determined, using an egg multi tester (Touhoku Rhythm Co., Ltd., Tokyo, Japan). Eggshell breaking strength was evaluated, using an Eggshell force gauge, model II (Robotmation Co., Ltd., Tokyo, Japan), and eggshell thickness was measured, using a dial pipe gauge (Ozaki Mfg. Co., Ltd., Tokyo, Japan).
Blood Profile
-------------
At the end of the experiment, 16 laying hens were randomly selected from each treatment (two hens/replication) and blood samples were taken from the jugular vein by a sterilized syringe with needle. Then, the samples were transferred to either a vacuum or K3EDTA vacuum tube (Becton Dickinson Vacutainer Systems, FranklinLakes, NJ, USA). The blood samples were centrifuged at 2000×g at 4°C for 15 min to separate the serum. High-density lipoprotein (HDL), low-density lipoprotein (LDL), and total cholesterol, and immunoglobulin G (IgG) concentrations in the serum were then analyzed using an automatic biochemistry blood analyzer (HITACHI747, Tokyo, Japan). Whole blood samples from the K~3~EDTA vacuum tube were analyzed immediately to determine the white blood cells (WBC), red blood cells (RBC), and lymphocyte concentrations using an automatic blood analyzer (ADVIA 120, Bayer, Tarrytown, NY, USA).
Cecal Microflora
----------------
At the end of the experiment, samples of cecal contents were collected from 16 laying hens randomly selected from each treatment, then placed on ice for transportation to the laboratory, where analyses were immediately performed using the method described by [@bib51]. One-gram of pooled cecal content sample was diluted 1:9 (wt/vol) with phosphate buffer saline solution (PBS; 0.1M, pH 7.0). Then, 10-fold serial dilutions (10^−3^ to 10^−6^) of cecal content samples were generated with PBS and placed onto Mac-Conkey (Difco Laboratories, Detroit, MI, USA) and *Lactobacillus*-Rogosa agar plates (Difco Laboratories) to isolate the *Escherichia coli | introduction = = = = = = = = = = = = phytogenic feed additives ( pfa ) have attracted a lot of attention in recent years. when used in diets, these substances exhibit antioxidative properties and antimicrobial activity, improve nutrient absorption, and could ultimately improve animal performance ( [ @ bib26 ] ; [ @ bib30 ] ; [ @ bib37 ] ). pfa are less toxic, have fewer side effects, and are residue - free compared with synthetic feed additives, and are thought to be ideal feed additives in animal production ( [ @ bib25 ] ). therefore, many phytogenic compounds have been recommended for use as feed additives. fenugreek ( * trigonella foenum - graecum * l ) belongs to the leguminosae family and is cultivated predominantly in madagascar, south asia, the mediterranean, north africa, and africa ( [ @ bib29 ] ). the seeds are generally used for condiments in various poultry preparations, are regarded to have great nutritive value and restorative properties, and have been used in folk medicine for centuries for measuring hypoglycemic, anthelmintic, antibacterial, antiinflammatory, antipyretic, and antimicrobial properties ( [ @ bib49 ] ; [ @ bib7 ] ; [ @ bib50 ] ). the major active components of fenugreek seeds are 4 - hydroxyisoleucine, trigonelline, galactomannan with flavonoids, carotenoids, coumarins, and saponins, which confer pharmacological activity and beneficial effect ( [ @ bib46 ] ; [ @ bib7 ] ). recently, the modes of pfa action in vivo, including aromatic plants, plant extracts, their single active components, or their blended additives, have been investigated in several studies ( [ @ bib54 ] ; [ @ 36 ] ; [ @ bib37 ] ; [ @ bib38 ] ). however, in laying hens, a comparatively small number of researchers have investigated the effects of fenugreek seed on production performance and egg quality. therefore, in 2001 study, an attempt was made to evaluate the fenugreek seed as natural immune promoter in laying hen diets. the evaluation included biochemical changes in egg production, egg quality, blood profiles, cecal microflora, and excreta noxious gas emission. materials and methods = = = = = = = = = = = = = = = = = = = = = ethical considerations - - - - - - - - - - - - - - - - - - - - - - the experimental protocols describing the management and care of animals were reviewed and approved by the animal care and use committee of dankook university. experimental design, animals, and housing - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - a total of 384, 26 - week - old ( hyline - brown ) laying hens were used in this 6 - week trial. laying hens were randomly assigned to one of three treatments with eight replicates ( 16 hens / replicate ). the experimental treatments were : control, basal diet ( fse 0 % ) ; fenugreek seed extract ( fse ) 0. 05 %, basal diet + 0. 05 % fse ; fse 0. 1 %, basal diet + 0. 1 % fse. the fse used in our study was nutrifen ^ ® ^ ( emerald seed products ltd., avonlea, canada ), which contains ≥ 15. 0 mg / g saponin. nutrifen ^ ® ^ was composed of 3584 kcal / kg me, 10. 4 % moisture, 28. 0 % cp, 9. 9 % crude fiber, 10. 4 % crude fat, and 4. 6 % crude ash. it also contained macrominerals as follows ; 0. 34 % calcium, 0. 28 % sulfur, 0. 74 % phosphorus, 0. 26 % magnesium, 36. 2 mg / kg zinc, 21. 4 mg / kg manganese, and 36. 2 mg / kg iron. laying hens were provided ad libitum access to water and feed. all the diets were formulated in mash form to meet or exceed the [ @ bib36 ] nutrition requirement ( [ table 1 ] ( # t1 ) { ref - type = " table " } ). treatment additives were included in the diet by replacing the same amount of corn. laying hens were allowed to adjust to the environment for 5 days prior to beginning the feeding trial, during which they were fed a basal diet. they were raised in an ambient - regulated house, in which the temperature was maintained below 23°c and the light regime was set on a 16 : 8 - light : dark cycle throughout the entire experiment. birds were individually reared in adjacent steel cages fitted with a nipple drinker, feeder, and an egg collecting plate. # # # # # # formula and chemical composition of basal diet ( as - fed basis ) item ( % ) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ingredients corn 50. 40 soybean meal ( cp 46 % ) 18. 70 wheat grain 10. 00 corn gluten meal 2. 00 wheat bran 5. 00 animal fat 4. 40 limestone 7. 50 dicalcium phosphate ( p 18 % ) 1. 40 salt 0. 30 [ dl ] {. smallcaps } - met ( 50 % ) 0. 10 vitamin premix [ ^ 1 ^ ] ( # tf1 ) { ref - type = " table - fn " } 0. 10 trace mineral premix [ ^ 2 ^ ] ( # tf2 ) { ref - type = " table - fn " } 0. 10 total 100 calculated values me ( kcal / kg ) 2, 904 cp ( % ) 15. 02 lys ( % ) 0. 78 met + cys ( % ) 0. 65 ca ( % ) 3. 25 p ( % ) 0. 61 provided per kilogram of premix : 125, 000 iu vitamin a ; 2, 500 iu vitamin d ~ 3 ~ ; 10 mg vitamin e ; 2 mg vitamin k ~ 3 ~ ; 1 mg vitamin b ~ 1 ~ ; 5 mg vitamin b ~ 2 ~ ; 1 mg vitamin b ~ 6 ~ ; 15 mg vitamin b ~ 12 ~ ; 500 mg folic acid ; 35, 000 mg niacin ; 10, 000 mg ca - pantothenate and 50 mg biotin. provided per kg of diet : 8 mg mn ( as mno ~ 2 ~ ) ; 60 mg zn ( as znso ~ 4 ~ ) ; 5mg cu ( as cuso ~ 4 ~ · 5h ~ 2 ~ o ) ; 40mg fe ( as feso ~ 4 ~ · 7h ~ 2 ~ o ) ; 0. 3 mg co ( as coso ~ 4 ~ · 5h ~ 2 ~ o ) ; 1. 5 mg i ( as ki ), and 0. 15 mg se ( as na ~ 2 ~ seo ~ 3 ~ · 5h ~ 2 ~ o ). laying production, performance, and egg quality - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the hen - day egg production and egg weights were recorded daily, while feed consumption was measured weekly. the feed conversion ratio was calculated as the feed consumption per hen divided by egg weight per day per hen. for each treatment, 40 normal eggs ( five eggs / cage ) were collected randomly at 32 weeks and used to determine the egg quality. eggshell color scores were determined using an eggshell color fan on a 1 - - 15 scale ( 1 = light to 15 = dark brown ) by a single trained evaluator. haugh units, albumen height, and yolk color were determined, using an egg multi tester ( touhoku rhythm co., ltd., tokyo, japan ). eggshell breaking strength was evaluated, using an eggshell force gauge, model ii ( robotmation co., ltd., tokyo, japan ), and eggshell thickness was measured, using a dial pipe gauge ( ozaki mfg. co., ltd., tokyo, japan ). blood profile - - - - - - - - - - - - - at the end of the experiment, 16 laying hens were randomly selected from each treatment ( two hens / replication ) and blood samples were taken from the jugular vein by a sterilized syringe with needle. then, the samples were transferred to either a vacuum or k3edta vacuum tube ( becton dickinson vacutainer systems, franklinlakes, nj, usa ). the blood samples were centrifuged at 2000×g at 4°c for 15 min to separate the serum. high - density lipoprotein ( hdl ), low - density lipoprotein ( ldl ), and total cholesterol, and immunoglobulin g ( igg ) concentrations in the serum were then analyzed using an automatic biochemistry blood analyzer ( hitachi747, tokyo, japan ). whole blood samples from the k ~ 3 ~ edta vacuum tube were analyzed immediately to determine the white blood cells ( wbc ), red blood cells ( rbc ), and lymphocyte concentrations using an automatic blood analyzer ( advia 120, bayer, tarrytown, ny, usa ). cecal microflora - - - - - - - - - - - - - - - - at the end of the experiment, samples of cecal contents were collected from 16 laying hens randomly selected from each treatment, then placed on ice for transportation to the laboratory, where analyses were immediately performed using the method described by [ @ bib51 ]. one - gram of pooled cecal content sample was diluted 1 : 9 ( wt / vol ) with phosphate buffer saline solution ( pbs ; 0. 1m, ph 7. 0 ). then, 10 - fold serial dilutions ( 10 ^ −3 ^ to 10 ^ −6 ^ ) of cecal content samples were generated with pbs and placed onto mac - conkey ( difco laboratories, detroit, mi, usa ) and * lactobacillus * - rogosa agar plates ( difco laboratories ) to isolate the * escherichia coli | Introduction = = = = = = = = = = = = Phytogenic feed additives (PFA) have Zttrqcted a lot of attention in recent years. When used in diets, these substances exhibit antioxidative properties and antimicrobial activity, improve nutrient absorption, and could ultimately improve animal performance ([ @ bib26 ]; [@ bib30 ]; [@ bib37] ). PFA are less toxic, have fewer side effects, and are residue - free compared with synthetic feed additives, and are thought to be ideal feed additives in animal production ([ @ bib25] ). Therefore, many phytogenic compounds have been recommended for use as feed additives. Fenugreek (* Trigonella foenum - graecum * L) belongs to the Leguminosae family and is cultivated predominantly in India, West Asia, the Mediterranean, North Africa, and Canada ([ @ bib29] ). The seeds are generally used for condiments in various food preparations, are regarded to have great nutritive value and restorative properties, and have been used in folk medicine for centuries for their hypoglycemic, anthelmintic, amtibacteria;, antiinflammatory, antipyretic, and antimicrobial properties ([ @ bib49 ]; [@ bib7 ]; [@ bib50] ). The major active components of fenugreek seeds are 4 - hydroxyisoleucine, trigonelline, galactomannan with flavonoids, carotenoids, coumarins, and saponins, which confer pharmacological activity and beneficial effects ([ @ bib46 ]; [@ bib7] ). Recently, the modes of PFA action in vivo, including aromatic plants, plant extracts, their single active components, or th@&r blended additives, have been investigated in several studies ([ @ bib54 ]; [@ bib6 ]; [@ bib37 ]; [@ bib38] ). However, in laying hens, a comparatively small number of studies have investigated the effects of fenugreek seed on production performance and egg quality. Therefore, in this study, an attempt was made to evaluate the fenugreek seed as natural growth promoter in laying hen diets. The evaluation included biochemical changes in egg production, egg quality, blood profiles, cecal microflora, and excreta noxious gas e,issioJ. Materials and Methods = = = = = = = = = = = = = = = = = = = = = Ethical Considerations - - - - - - - - - - - - - - - - - - - - - - The experimental protocols describing the management and care of animals were reviewed and approved by the Animal CSrs and Use Committee of Dankook University. Experimental Design, Animals, and Housing - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A total of 384, 26 - week - old (Hyline - brown) laying hens were used in this 6 - week trial. Laying hens were randomly assigned to one of three treatments with eight replicates (16 hens / replicate ). The experimental treatments were: control, basal diet (FSE 0% ); fenugreek seed extract (FSE) 0. 05% , basal diet + 0. 05% FSE; FSE 0. 1% , basal diet + 0. 1% FSE. The FSE used in our study was Nutrifen ^ ® ^ (Emerald Seed Products Ltd. , Avonlea, Canada ), whkcM contains ≥ 15. 0 mg / g saponin. Nutrifen ^ ® ^ was composed of 3584 kcal / kg ME, 10. 4% moisture, 28. 0% CP, 9. 9% crude fiber, 10. 4% crude fat, and 4. 6% crude ash. It also contained macrominerals as follows; 0. 34% calcium, 0. 28% sulfur, 0. 74% phosphorus, 0. 26% magnesium, 36. 2 mg / kg zinc, 21. 4 mg / kg manganese, and 36. 2 mg / kg iron. Laying hens were provided ad libitum access to water and feed. All the diets were formulated in mash form to meet or exceed the [@ bib36] nutrition requirement ([ Table 1] (# T1) {ref - type = " table "} ). Treatment additives were included in the diet by replacing the same amount of corn. Laying hens were allowed to adjust to the environment for 5 days prior to beginning the feeding trial, during which they were fed a basal diet. They were raised in an Xmhient - regulated house, in which the temperature was maintained below 23 ° C and the light regime was set on a 16: 8 - light: dark cycle throughout the entire experiment. Birds were individually reared in adjacent steel cages fitted with a nipple drinker, feeder, and an egg collecting plate. # # # # # # Formula and chemical composition of basal diet (as - fed basis) Item (%) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Ingredients Corn 50. 40 Soybean meal (CP 46%) 18. 70 Wheat grain 10. 00 Corn gluten meal 2. 00 Wheat bran 5. 00 Animal fat 4. 40 Limestone 7. 50 Dicalcium phosphate (P 18%) 1. 40 Salt 0. 30 [dl] {. smallcaps} - Met (50%) 0. 10 Vitamin premix [^ 1 ^] (# tf1) {ref - type = " table - fn "} 0. 10 Trace mineral premix [^ 2 ^] (# tf2) {ref - type = " table - fn "} 0. 10 Total 100 Calculated values ME (kcal / kg) 2, 904 CP (%) 15. 02 Lys (%) 0. 78 Met + Cys (%) 0. 65 Ca (%) 3. 25 P (%) 0. 61 Provided per kilogram of premix: 125, 000 IU vitamin A; 2, 500 IU vitamin D ~ 3 ~; 10 mg vitamin E; 2 mg vitamin K ~ 3 ~; 1 mg vitamin B ~ 1 ~; 5 mg vitamin B ~ 2 ~; 1 mg vitamin B ~ 6 ~; 15 mg vitamin B ~ 12 ~; 500 mg folic acid; 35, 000 mg niacin; 10, 000 mg Ca - Pantothenate and 50 mg biotin. Provided per Kg of diet: 8 mg Mn (as MnO ~ 2 ~ ); 60 mg Zn (as ZnSO ~ 4 ~ ); 5mg Cu (as CuSO ~ 4 ~ · 5H ~ 2 ~ O ); 40mg Fe (as FeSO ~ 4 ~ · 7H ~ 2 ~ O ); 0. 3 mg Co (as CoSO ~ 4 ~ · 5H ~ 2 ~ O ); 1. 5 mg I (as KI ), and 0. 15 mg Se (as Na ~ 2 ~ SeO ~ 3 ~ · 5H ~ 2 ~ O ). Laying Production, Performance, and Egg Quality - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The hen - day egg production and egg weights were recorded daily, while feed consumption was measured weekly. The feed conversion ratio was calculated as the feed consumption per hen divided by egg weight per day per hen. For each treatment, 40 normal eggs (five eggs / cage) were collected randomly at 32 weeks and used to determine the egg quality. Eggshell color scores were determined using an eggshell color fan on a 1 - - 15 scale (1 = light to 15 = dark brown) by a single trained evaluator. Haugh units, albumen height, and yolk color were determined, using an egg multi Fecter (Touhoku Rhythm Co. , Ltd. , Tokyo, Japan ). Eggshell breaking strength was evaluated, using an Eggshell force gauge, model II (Robotmation Co. , Ltd. , Tokyo, Japan ), and eggshell thickness was measured, using a dial pipe gauge (Ozaki Mfg. Co. , Ltd. , Tokyo, Japan ). Blood Profile - - - - - - - - - - - - - At the end of the exoerimeHt, 16 laying hens were randomly selected from each treatment (two hens / replication) and blood samples were taken from the jugular vein by a sterilized syringe with needle. Then, the samples were transferred to either a vacuum or K3EDTA vacuum tube (Becton Dickinson Vacutainer Systems, FranklinLakes, NJ, USA ). The blood samples were centrifuged at 2000 × g at 4 ° C for 15 min to separate the serum. High - density lipoprotein (HDL ), low - density lipoprotein (LDL ), and total cholesterol, and immunoglobulin G (IgG) concentrations in the serum were then analyzed using an automatic biochemistry blood analyzer (HITACHI747, Tokyo, Japan ). Whole blood samples from the K ~ 3 ~ EDTA vacuum tube were analyzed immediately to det3rmiGe the white blood cells (WBC ), red blood cells (RBC ), and lymphocyte concentrations using an automatic blood analyzer (ADVIA 120, Bayer, Tarrytown, NY, USA ). Cecal Microflora - - - - - - - - - - - - - - - - At the end of the experiment, samples of cecal contents were collected from 16 laying hens randomly selected from each treatment, then placed on ice for transportation to the laboratory, where analyses were immediately performed using the method described by [@ bib51 ]. One - gram of pooled cecal content sample was diluted 1: 9 (wt / vol) with phosphate buffer saline solution (PBS; 0. 1M, pH 7. 0 ). Then, 10 - fold serial dilutions (10 ^ − 3 ^ to 10 ^ − 6 ^) of cecal content samples were generated with PBS and placed onto Mac - Conkey (Difco Laboratories, Detroit, MI, USA) and * Lactobacillus * - Rogosa agar plates (Difco Laboratories) to isolate the * Escherichia coli | ============ Phytogenic feed additives (PFA) have attracted a lot of attention in years. When used in diets, these substances antioxidative properties and activity, improve nutrient absorption, and could ultimately improve animal performance ([@bib26]; [@bib37]). PFA are less toxic, have fewer side effects, and are residue-free compared with synthetic feed additives, and are thought to be ideal in animal production ([@bib25]). Therefore, many phytogenic compounds been recommended for use as additives. Fenugreek (*Trigonella foenum-graecum* L) belongs to the Leguminosae family and is cultivated predominantly India, West the North and Canada The seeds are generally used for condiments in various food preparations, are regarded to have great nutritive value and restorative properties, and have been used in folk medicine for for their anthelmintic, antibacterial, antiinflammatory, antipyretic, and antimicrobial properties [@bib7]; [@bib50]). The major active components of fenugreek are 4-hydroxyisoleucine, trigonelline, galactomannan with flavonoids, carotenoids, coumarins, and saponins, which confer pharmacological activity and beneficial effects ([@bib46]; [@bib7]). the modes of PFA action in vivo, including aromatic plants, plant extracts, their single active components, their blended additives, have been investigated in several studies ([@bib54]; [@bib6]; [@bib37]; [@bib38]). However, in laying hens, a comparatively small number of have investigated the effects of fenugreek on production and egg quality. Therefore, this study, an attempt was made to evaluate the fenugreek natural growth promoter in laying hen diets. The evaluation included biochemical changes in egg production, egg quality, blood profiles, microflora, and noxious gas emission. Materials and Methods ===================== Ethical Considerations ---------------------- The protocols describing the management and care of animals were and approved by the Animal Care and Committee of Dankook University. Experimental Design, and Housing A total of 384, 26-week-old (Hyline-brown) hens were used in this 6-week trial. Laying hens were randomly assigned to one of three treatments with eight replicates (16 hens/replicate). The experimental treatments were: control, basal diet (FSE seed extract (FSE) 0.05%, basal diet + 0.05% FSE 0.1%, basal diet + 0.1% FSE. The FSE used in our study Nutrifen^®^ (Emerald Seed Products Avonlea, Canada), which contains ≥ 15.0 mg/g Nutrifen^®^ was of 3584 kcal/kg ME, 10.4% moisture, 28.0% CP, 9.9% crude fiber, 10.4% crude fat, and 4.6% ash. It also contained macrominerals as follows; calcium, 0.28% sulfur, 0.74% phosphorus, 0.26% 36.2 mg/kg zinc, mg/kg manganese, and 36.2 mg/kg iron. Laying hens were provided ad libitum access to water and the diets were in mash to meet or exceed the [@bib36] nutrition requirement ([Table 1](#T1){ref-type="table"}). Treatment additives were included in the diet by replacing the same amount of Laying hens allowed to adjust to the environment for 5 days prior to beginning the feeding trial, during which they were a basal diet. They were raised in ambient-regulated house, in which the temperature was maintained below 23°C and the light regime was set on a 16:8-light: dark throughout the entire experiment. Birds were individually reared adjacent cages fitted with a nipple drinker, feeder, an egg collecting plate. ###### Formula and chemical composition of basal diet (as-fed basis) Item (%) -------------------------------------------------------- ------------- Ingredients Corn 50.40 Soybean (CP 46%) 18.70 Wheat 10.00 Corn gluten meal 2.00 Wheat bran 5.00 Animal 4.40 Limestone 7.50 Dicalcium phosphate (P 18%) 1.40 Salt 0.30 [dl]{.smallcaps}-Met (50%) 0.10 Vitamin premix[^1^](#tf1){ref-type="table-fn"} 0.10 Trace mineral premix[^2^](#tf2){ref-type="table-fn"} 0.10 Total 100 Calculated values ME (kcal/kg) 2,904 CP (%) 15.02 Lys (%) 0.78 Met + Cys (%) 0.65 Ca (%) 3.25 P (%) 0.61 Provided per kilogram of premix: 125,000 IU vitamin A; 2,500 IU D~3~; 10 mg vitamin E; 2 mg vitamin K~3~; mg vitamin 5 mg vitamin B~2~; 1 mg vitamin B~6~; mg vitamin B~12~; 500 mg folic acid; 35,000 mg niacin; 10,000 mg Ca-Pantothenate and 50 Provided Kg of diet: 8 mg Mn (as MnO~2~); 60 mg Zn (as ZnSO~4~); 5mg Cu (as 40mg Fe (as 0.3 mg Co (as CoSO~4~·5H~2~O); 1.5 mg I KI), and 0.15 mg Se (as Laying Production, Performance, and Quality ----------------------------------------------- The hen-day egg production and egg weights were recorded daily, while feed consumption was measured weekly. The feed conversion was calculated as the feed consumption per hen divided by egg weight per hen. For each treatment, 40 normal eggs (five eggs/cage) were collected randomly at 32 weeks and used to determine the egg quality. color scores were determined using eggshell color fan 1--15 scale (1=light to 15=dark by a single trained evaluator. Haugh units, albumen height, and yolk color were determined, using an egg multi tester (Touhoku Rhythm Co., Ltd., Tokyo, Japan). breaking was evaluated, using an force gauge, model II (Robotmation Co., Tokyo, Japan), and eggshell thickness measured, using a dial pipe gauge Mfg. Co., Ltd., Japan). Blood Profile ------------- At the end of the experiment, 16 hens were randomly selected from each treatment (two hens/replication) and blood samples were taken the jugular vein by a sterilized syringe with needle. Then, the samples were transferred to either a vacuum or K3EDTA vacuum tube (Becton Dickinson Vacutainer Systems, NJ, USA). The samples were centrifuged at 2000×g at 4°C for 15 min separate the High-density lipoprotein (HDL), low-density lipoprotein (LDL), and total cholesterol, and immunoglobulin G (IgG) concentrations in the serum were then analyzed using an biochemistry blood analyzer (HITACHI747, Tokyo, Japan). blood samples from the K~3~EDTA tube were analyzed immediately to determine the white blood cells (WBC), red blood cells (RBC), and concentrations using an automatic blood analyzer (ADVIA 120, Bayer, Tarrytown, NY, USA). Cecal Microflora ---------------- At the end of the experiment, samples of cecal contents collected from 16 laying hens randomly selected from then placed on ice for transportation to where analyses were immediately performed using the described by [@bib51]. pooled cecal content sample diluted 1:9 (wt/vol) phosphate buffer saline solution (PBS; 0.1M, pH Then, 10-fold serial dilutions (10^−3^ to 10^−6^) of cecal content samples were generated with PBS and placed onto Mac-Conkey (Difco Laboratories, Detroit, MI, USA) and *Lactobacillus*-Rogosa agar plates (Difco Laboratories) to isolate the *Escherichia coli | iNTROdUcTIOn
============
pHYtOGeniC FeEd aDdItIVes (PfA) haVE ATTrActeD A lOt oF AttenTIoN in rEceNt years. WHen USEd In DIetS, tHeSe SUBsTancES exhiBiT ANTIOxIdAtivE propERtIES AND anTimICrOBiaL AcTIvITY, iMpRove NuTRieNt aBsoRptioN, And coUlD UlTImATElY iMprOVe anIMAl PeRFORmaNCE ([@Bib26]; [@bIB30]; [@bIB37]). pFa are leSs tOXic, hAVe FEWEr siDE efFecTS, AND aRe REsIduE-fREe cOmPaRed wITH sYnthETic Feed adDITiVeS, AnD aRE thOUGHt To be iDEAl fEED aDDItives iN AnImAL prODuctIOn ([@Bib25]). theREfoRe, many PhytOgenIC cOmpOundS have BEeN recoMMENded for Use AS FeED aDDiTivES.
fENuGREek (*tRiGONELla FoEnUm-gRaeCUM* L) BeLONgs to The LEgumINOsaE faMily anD is cULtiVAtED prEdominanTly iN InDia, WESt aSIa, THE MEditerRAnEaN, nORTH AfRiCA, And caNAda ([@Bib29]). the sEedS ARe GenerALly used FOr CondIMeNTs iN VarIoUS FOOD PREparatIoNS, ARe rEgardED To HAve grEaT NUtritIVE ValUE AND ReStoRaTIVE prOPErTIeS, aND hAve beEn uSED In FoLk MedicIne fOr cENTURIES for THeIR HypoGlYcEMIc, aNThElmiNTIc, aNTiBAcTerIal, aNTIInFLAMmAtorY, ANtipYRETic, aND ANtiMIcRobIaL pRoperties ([@BIb49]; [@bib7]; [@bIb50]). The majOr ActIVE COMponEntS of fenugreeK SeeDs arE 4-HYdRoxyIsOLeucIne, TRIGONELliNE, GALaCtoMANNan wITH fLavONoids, cARotenOIdS, COUmarinS, AND saPOnINS, WHIcH conFEr PharmaCOloGiCaL AcTIvity anD bEneFicIal effECts ([@BIB46]; [@BiB7]).
RecEntlY, tHe MODeS OF pFa action in vIVo, INCLudIng ArOMatIC pLants, planT ExTRacts, ThEIr SingLe acTiVE COmPonENTS, oR thEiR blENDeD aDDiTIVeS, HAve bEen iNveSTigaTed iN SEveraL stuDIEs ([@Bib54]; [@bIb6]; [@BIB37]; [@biB38]). hOwever, in LAYiNG HeNs, A coMpARAtIVeLy SMalL NUmBer OF sTUdIes hAve invESTiGaTeD tHe effECTS OF fenUGReEK SeEd oN PRODUcTIoN perfOrmAncE aND EGg QuALItY. THerEfOre, in ThiS StuDy, an ATtempT wAs mAde To eVAluatE tHE FeNUgreeK seeD As naTural grOWTh prOmoTEr iN layIng hEn DIEtS. the evALUaTIon iNClUded biOcHEmiCAl cHaNGEs in Egg PRoduCtIon, Egg quALITy, bloOd profiLes, CecAL MIcROfLORA, AnD ExCreTA NOxIouS Gas emISSion.
MatERIAls anD MetHoDs
=====================
ethICAL CONSidERAtioNs
----------------------
tHE EXpERIMeNtal PRoTOCOLs deSCRIBiNg THe MaNageMent and CaRE OF Animals wERE ReviEWeD anD apProvED bY THe AnIMaL carE anD Use coMMiTtEE oF dankOok uNIversitY.
eXPeRIMeNTal deSIgN, AnImALs, and hOuSING
-----------------------------------------
a toTAL oF 384, 26-WeEk-Old (hYLINE-BrOwn) layIng Hens wErE UsED In tHIS 6-WeEk TRial. lAyiNg hEns werE ranDoMly aSsiGned TO ONe Of Three TrEATmEnts wiTH EigHT rePlIcaTes (16 hENs/rEplICAte). THe EXPerIMENtAL TREatMENTs WErE: COntrol, basAL DieT (FSe 0%); feNugReek seED ExTract (fSE) 0.05%, BasAl Diet + 0.05% fSe; FsE 0.1%, basAL Diet + 0.1% Fse. THe fse uSEd IN OUr stUDy waS nutRIfEN^®^ (emeRalD sEeD proDUCTs ltd., AvONLEA, caNaDa), wHIcH containS ≥ 15.0 mG/G sAponIN. NUTrifeN^®^ waS ComposeD Of 3584 kcaL/kG Me, 10.4% MoiStuRe, 28.0% Cp, 9.9% crUDe FiBEr, 10.4% crUDe FAt, AnD 4.6% crUDe Ash. iT ALSO cOnTAINED MaCRomInEraLS aS FollOwS; 0.34% cALciuM, 0.28% suLFUR, 0.74% PhoSphoRuS, 0.26% maGnEsIuM, 36.2 MG/kG ZInC, 21.4 MG/KG mANGANese, anD 36.2 Mg/kG Iron. laYIng heNs Were provIdeD aD liBituM aCCEsS tO WAtER AnD FEeD. ALl THe DIETS wERe formULated IN mash fORM TO meet or EXcEEd ThE [@bIB36] nutritioN requIREMeNT ([TAblE 1](#T1){ReF-type="Table"}). TreatmEnt adDItIvEs wErE INcluDED IN THE dIET by REPlAciNg tHE saMe amoUNt oF cORN. LaYInG HEns WERe alloWED TO ADJust To The eNVIRoNmENt FOr 5 dAYS prIoR to BeGinNInG THE FeedING trIaL, DurINg WhICH THeY weRE FED A BaSAl DIeT. tHEY were raIseD IN An AMbieNt-reGuLated HouSe, in whiCh tHE temPErAtuRe WAs MAINtaINeD bELOw 23°C anD thE lIGHT regimE waS sEt On a 16:8-light: Dark cyclE tHROUGHouT ThE eNtIRE ExpeRimEnT. BirDs wERE individuaLLy RearED iN aDjACEnt StEeL caGES FiTted witH a NipPLe drINKer, FeEDer, AND An egg COllECTIng PlATE.
###### fORMULA ANd cHEMicaL CompOSItIOn Of BAsAL DIeT (As-FED BASis)
ITEM (%)
-------------------------------------------------------- -------------
INgReDientS
CorN 50.40
SOybeAN mEAl (CP 46%) 18.70
Wheat GRAiN 10.00
cORN gLutEN MEAL 2.00
WhEAt bRaN 5.00
aNImal FaT 4.40
limesTONE 7.50
DicAlCIum phOspHAte (P 18%) 1.40
sALT 0.30
[dl]{.SMaLLcApS}-MEt (50%) 0.10
VitAmIn PremIx[^1^](#TF1){REf-tyPe="TABle-FN"} 0.10
TrACE mINerAl PReMix[^2^](#TF2){rEF-type="TaBLE-FN"} 0.10
total 100
CALCulateD vAlUeS
Me (kCal/kG) 2,904
Cp (%) 15.02
lys (%) 0.78
MEt + cYs (%) 0.65
CA (%) 3.25
p (%) 0.61
prOViDed per kILograM oF pReMix: 125,000 Iu VItAMiN a; 2,500 iU VITaMiN d~3~; 10 mg vITamIN E; 2 Mg VItamIn k~3~; 1 mG viTaMIN b~1~; 5 mg viTAmiN b~2~; 1 mG vITAmin B~6~; 15 MG vITAMIN B~12~; 500 mg fOliC ACid; 35,000 Mg NIaCin; 10,000 Mg CA-PantoTHeNate anD 50 MG BIotin.
pROVIDeD PeR Kg of DIet: 8 mg MN (As mNo~2~); 60 mg ZN (as ZNsO~4~); 5MG CU (aS CuSo~4~·5h~2~o); 40MG fE (aS feSo~4~·7h~2~o); 0.3 mG co (AS coso~4~·5H~2~o); 1.5 Mg i (AS Ki), AnD 0.15 Mg sE (as na~2~Seo~3~·5H~2~o).
LAYINg pRoducTIoN, peRformANCE, aND egg quaLITY
-----------------------------------------------
THE hen-dAy eGG PrODuCTION ANd EGG WeIgHTs wERE ReCorDED daily, WHiLE fEed cOnsumpTIon WAS MeaSuREd weEKly. the fEEd CONversiON RAtIo WAS calCulAtEd As THE fEeD coNsUMpTiOn per heN DividED by Egg WeIgHT peR DAy PeR hen. FOR EAch tReaTmENT, 40 nOrMaL egGs (fivE egGs/CAGE) WErE colLecTEd ranDOMLY AT 32 Weeks aNd USED TO detErmiNe tHe EGg qUaLitY. EgGSHelL COlor sCoRes werE DEtERMInEd USIng AN EggSHELl ColoR FAN oN A 1--15 SCALE (1=lIgHT to 15=DARK BrOWn) by a SiNGLe TraiNED EVaLUAtOr. HAUGH UNitS, alBUmEn hEIGHT, AnD yOLK cOLOr weRE dETErMInEd, UsINg aN egG mUlTi tEStEr (Touhoku rhYThM co., LTd., tOKYo, Japan). eGGsheLl BREAkiNg STrEnGtH WaS EValUATEd, UsING aN eGGsHElL FoRce GAuge, mODEl ii (RobotMaTIOn co., LTd., TOkYo, JapAN), anD EgGShelL THIcknesS wAS MEaSUReD, usiNG A dIAL pipE GaUge (Ozaki mFg. cO., lTD., TokyO, jApAn).
bLoOD ProFIle
-------------
at THe eNd Of THe eXperImEnt, 16 LaYinG HENS WERe rANdOMLY selEcted fROm EAcH TreATMENT (twO HEnS/rEPLicAtIoN) and BlOoD saMpleS wERe TAkEN FROm THe jUgUlAR veiN BY a STeRIlizEd SyriNGe witH NeEDle. theN, ThE sAmPLeS WeRe TransfeRrEd tO eiThER A VaCUUM OR K3eDTA vAcuUM tUbE (bectOn DIcKINsOn VACUTaINER sYSTeMs, fRaNKLiNLAKES, Nj, usA). tHE blooD SaMpLeS were CEnTrIfUGed at 2000×g at 4°C fOr 15 MiN to SEPaRAtE THE SeRum. hIGh-deNsITy lIPOpROtEIN (hdL), LoW-DenSity LiPOPROTeIn (lDL), AND total choLESTErol, ANd iMMUnOgLobuLin G (igG) CONCENTRatIONs iN THe SeRuM WERE TheN aNAlYzeD UsING an AUtomaTic BiochEMistRy blOOd anaLYzer (hitaCHi747, toKyO, JAPan). whOlE bLood samPLES From thE k~3~edta VAcuUm TUBe WERe AnAlyZED IMMedIatELY TO dEteRminE THe WhIte BLoOD ceLLs (WbC), rED blOod cELls (rBc), aND LyMphOcYte CoNCEnTratioNS usiNg an AutomatIc BLood anaLyZEr (aDViA 120, BAyeR, TARRytoWN, ny, USA).
CeCal micrOFloRA
----------------
aT thE EnD oF tHE eXPeRIMent, sampLES oF cEcaL CONtENTs WeRe cOlLeCTeD FRoM 16 LAYing HEns RAndomlY SELeCTed fROM EAcH trEaTmENt, ThEN pLacEd On icE FOr transpORtAtiON to THE lAborAToRy, whErE ANalyseS wEre IMmedIATElY peRFoRMED uSinG the mEthoD dEScribeD by [@biB51]. ONe-grAm oF poOLed cEcal coNteNT sampLE wAs DiLUted 1:9 (wT/VOl) WITh phosPHatE bUFfER Saline SOLuTiON (pBS; 0.1M, Ph 7.0). theN, 10-fOld serial dilUtiONS (10^−3^ TO 10^−6^) OF CeCAl coNTEnt SAmPlES WERE GeNERaTEd WiTH PBS And plAced ontO MAc-Conkey (DIFCo lABOratoRieS, deTROIt, MI, Usa) and *LActOBacIlLUs*-rogoSA aGAr plATEs (DifCo LaborAtoriES) tO ISOLATE ThE *eScHeRicHIA cOlI | Introduction ============ Phytogenic feed additives (PFA) have attracted a lot of attentioninrecent years. Whenused in diets, these substancesexhibit antioxidative properties and antimicrobial activity, improve nutrientabsorption, and could ultimately improve animal performance ([@bib26]; [@bib30]; [@bib37]). PFA are less toxic, have fewer side effects, and areresidue-free compared with synthetic feed additives, and are thought to be ideal feed additivesin animalproduction ([@bib25]). Therefore,many phytogeniccompounds have been recommendedfor use as feed additives. Fenugreek(*Trigonella foenum-graecum* L) belongs tothe Leguminosaefamily and is cultivated predominantlyin India,West Asia, the Mediterranean, North Africa, and Canada ([@bib29]).The seeds are generally used for condiments in various food preparations, are regarded to have great nutritive value and restorative properties,and have been usedin folkmedicine for centuries for their hypoglycemic, anthelmintic, antibacterial, antiinflammatory, antipyretic, and antimicrobialproperties ([@bib49]; [@bib7];[@bib50]). The major active components of fenugreek seeds are 4-hydroxyisoleucine, trigonelline, galactomannan with flavonoids, carotenoids, coumarins, and saponins, whichconfer pharmacological activity and beneficial effects ([@bib46]; [@bib7]). Recently, themodes of PFA action in vivo, including aromatic plants, plant extracts, their single active components, or their blended additives, have been investigated in several studies ([@bib54]; [@bib6]; [@bib37]; [@bib38]). However, in laying hens,a comparatively small number of studies have investigated the effects of fenugreek seed on production performance and egg quality. Therefore, in this study,an attempt was madeto evaluate the fenugreek seed as natural growthpromoter in laying hen diets. The evaluation included biochemical changes inegg production, egg quality, blood profiles, cecal microflora, and excreta noxious gasemission.Materialsand Methods ===================== Ethical Considerations ---------------------- The experimental protocols describing the management and care of animalswere reviewed and approvedby the Animal Care and Use Committee of Dankook University. ExperimentalDesign, Animals, and Housing----------------------------------------- A total of 384,26-week-old (Hyline-brown) laying hens were used in this 6-week trial. Laying hens were randomlyassigned to one of three treatments with eight replicates (16 hens/replicate). The experimental treatments were: control, basal diet (FSE0%); fenugreek seed extract (FSE) 0.05%, basal diet + 0.05% FSE; FSE0.1%, basal diet + 0.1% FSE. TheFSE used in our study was Nutrifen^®^ (Emerald Seed Products Ltd., Avonlea, Canada),which contains ≥ 15.0 mg/g saponin. Nutrifen^®^ wascomposed of 3584 kcal/kg ME, 10.4% moisture, 28.0% CP, 9.9% crudefiber, 10.4% crude fat,and 4.6% crude ash. It also contained macrominerals as follows; 0.34% calcium, 0.28% sulfur, 0.74% phosphorus, 0.26% magnesium, 36.2 mg/kg zinc, 21.4 mg/kg manganese, and 36.2mg/kg iron. Laying henswere provided ad libitum access to water and feed. All the diets were formulatedin mash form to meet or exceed the [@bib36] nutrition requirement ([Table 1](#T1){ref-type="table"}). Treatmentadditives were includedin the diet byreplacing the same amount of corn. Laying hens were allowed toadjust to the environment for 5 daysprior to beginning the feeding trial, duringwhich they were fed a basal diet.Theywere raised in an ambient-regulated house, inwhich the temperaturewas maintained below23°C and the light regime was set on a 16:8-light:dark cycle throughout the entire experiment. Birds wereindividuallyreared in adjacent steel cages fitted with a nipple drinker,feeder, and an egg collecting plate.###### Formula and chemical composition ofbasal diet (as-fed basis) Item (%) -------------------------------------------------------- -------------Ingredients Corn 50.40 Soybean meal (CP 46%) 18.70 Wheat grain 10.00 Corngluten meal 2.00Wheat bran 5.00 Animal fat 4.40 Limestone 7.50 Dicalcium phosphate (P 18%) 1.40Salt 0.30 [dl]{.smallcaps}-Met (50%) 0.10 Vitamin premix[^1^](#tf1){ref-type="table-fn"} 0.10 Tracemineral premix[^2^](#tf2){ref-type="table-fn"} 0.10 Total 100 Calculated valuesME (kcal/kg) 2,904 CP (%)15.02 Lys (%) 0.78 Met + Cys (%)0.65Ca (%) 3.25 P (%) 0.61 Provided per kilogram of premix: 125,000 IU vitamin A; 2,500IU vitamin D~3~; 10 mg vitaminE; 2 mg vitamin K~3~; 1 mg vitamin B~1~; 5 mg vitamin B~2~; 1 mg vitamin B~6~; 15 mgvitamin B~12~; 500 mg folicacid; 35,000 mg niacin; 10,000 mg Ca-Pantothenate and 50 mgbiotin. Provided per Kg of diet: 8 mg Mn (as MnO~2~); 60 mg Zn (as ZnSO~4~); 5mg Cu (as CuSO~4~·5H~2~O); 40mg Fe (as FeSO~4~·7H~2~O); 0.3 mg Co (as CoSO~4~·5H~2~O); 1.5 mg I (as KI), and 0.15 mgSe (as Na~2~SeO~3~·5H~2~O). Laying Production, Performance, and Egg Quality ----------------------------------------------- The hen-day egg production and eggweights were recordeddaily, while feed consumption was measured weekly. The feed conversion ratiowas calculated asthe feed consumption per hen divided by egg weight per day per hen. For each treatment, 40 normal eggs (fiveeggs/cage) were collectedrandomly at 32weeks andused to determine the egg quality. Eggshell color scores were determined using an eggshell colorfan ona 1--15 scale(1=lightto 15=dark brown) by a single trained evaluator. Haugh units,albumen height, and yolkcolor were determined, using an egg multi tester (TouhokuRhythmCo.,Ltd., Tokyo, Japan). Eggshell breaking strength was evaluated, using an Eggshell force gauge, model II (RobotmationCo., Ltd., Tokyo, Japan), andeggshell thickness was measured,using a dial pipe gauge (Ozaki Mfg. Co., Ltd., Tokyo, Japan). Blood Profile------------- At theend of the experiment, 16 laying hens wererandomly selected fromeach treatment (two hens/replication) and blood samples were taken from the jugular vein by a sterilizedsyringe with needle. Then, the samples were transferred to either a vacuum or K3EDTA vacuum tube (Becton Dickinson Vacutainer Systems, FranklinLakes,NJ, USA). Theblood samples were centrifuged at 2000×gat 4°C for 15 min to separate the serum. High-density lipoprotein (HDL), low-densitylipoprotein (LDL), and total cholesterol,and immunoglobulin G (IgG) concentrationsin the serum were thenanalyzed using an automatic biochemistry bloodanalyzer (HITACHI747,Tokyo, Japan). Whole blood samples from the K~3~EDTA vacuum tube were analyzedimmediately to determine the white blood cells (WBC), red blood cells (RBC), and lymphocyte concentrations using an automatic blood analyzer (ADVIA 120,Bayer, Tarrytown, NY, USA).Cecal Microflora ---------------- At the end of the experiment, samples of cecal contents were collected from 16 laying hens randomly selected fromeach treatment, then placed on ice for transportation to thelaboratory,where analyses were immediatelyperformed using the method described by[@bib51]. One-gram of pooled cecal contentsamplewasdiluted 1:9 (wt/vol) withphosphate buffer saline solution(PBS; 0.1M, pH 7.0). Then, 10-fold serial dilutions (10^−3^ to10^−6^) ofcecal content samples were generated withPBS and placed onto Mac-Conkey(DifcoLaboratories, Detroit, MI, USA) and *Lactobacillus*-Rogosa agar plates (Difco Laboratories) to isolatethe *Escherichia coli | Introduction _============_ Phytogenic feed _additives_ _(PFA)_ have attracted _a_ lot of attention in recent years. When used in diets, these _substances_ _exhibit_ antioxidative properties and _antimicrobial_ activity, _improve_ nutrient absorption, and could ultimately improve _animal_ performance _([@bib26];_ [@bib30]; [@bib37]). PFA are _less_ _toxic,_ have fewer side effects, and are residue-free compared with synthetic feed additives, and are thought to be ideal _feed_ _additives_ in _animal_ production ([@bib25]). Therefore, many phytogenic compounds have been _recommended_ for use as _feed_ additives. Fenugreek (*Trigonella _foenum-graecum*_ L) _belongs_ to the Leguminosae family and _is_ cultivated predominantly in India, _West_ Asia, the Mediterranean, North Africa, and _Canada_ ([@bib29]). The seeds are generally used _for_ _condiments_ _in_ various food preparations, are regarded _to_ have great _nutritive_ value and restorative properties, and have been used _in_ folk _medicine_ for _centuries_ for their hypoglycemic, anthelmintic, antibacterial, antiinflammatory, _antipyretic,_ and _antimicrobial_ properties ([@bib49]; [@bib7]; [@bib50]). The major active components of fenugreek seeds are 4-hydroxyisoleucine, trigonelline, galactomannan with _flavonoids,_ _carotenoids,_ _coumarins,_ and saponins, which confer pharmacological activity and _beneficial_ effects _([@bib46];_ [@bib7]). Recently, _the_ modes of PFA _action_ in vivo, _including_ aromatic plants, plant extracts, their single active _components,_ _or_ their blended additives, _have_ been investigated in _several_ studies ([@bib54]; [@bib6]; _[@bib37];_ [@bib38]). However, _in_ laying hens, a comparatively small number of studies have investigated the effects of _fenugreek_ seed on production performance and egg quality. Therefore, in this study, an attempt was made to evaluate the fenugreek seed as _natural_ _growth_ promoter in laying hen diets. _The_ _evaluation_ included biochemical changes _in_ _egg_ production, _egg_ quality, blood _profiles,_ cecal microflora, _and_ excreta _noxious_ gas emission. Materials and Methods ===================== Ethical Considerations ---------------------- The experimental protocols describing the management and _care_ _of_ animals were reviewed _and_ approved by the Animal Care and Use Committee of Dankook University. Experimental Design, Animals, and Housing _-----------------------------------------_ A total of 384, 26-week-old (Hyline-brown) laying hens were used in this 6-week trial. Laying hens were randomly assigned to one of three treatments with eight _replicates_ (16 _hens/replicate)._ The experimental treatments were: control, basal diet (FSE 0%); fenugreek seed extract (FSE) 0.05%, _basal_ diet + 0.05% FSE; FSE 0.1%, basal diet + 0.1% FSE. The FSE _used_ in _our_ study was Nutrifen^®^ (Emerald Seed Products Ltd., Avonlea, Canada), which contains _≥_ 15.0 mg/g saponin. _Nutrifen^®^_ was composed _of_ 3584 kcal/kg ME, 10.4% _moisture,_ 28.0% CP, 9.9% crude _fiber,_ 10.4% crude fat, and 4.6% crude _ash._ It also _contained_ _macrominerals_ as _follows;_ 0.34% calcium, 0.28% sulfur, 0.74% _phosphorus,_ 0.26% _magnesium,_ 36.2 _mg/kg_ zinc, 21.4 _mg/kg_ _manganese,_ and 36.2 mg/kg iron. Laying hens were provided ad libitum _access_ _to_ water and feed. All _the_ _diets_ were formulated in _mash_ form _to_ meet _or_ exceed the [@bib36] _nutrition_ requirement ([Table 1](#T1){ref-type="table"}). Treatment additives _were_ included in the diet by replacing the _same_ amount of corn. _Laying_ hens were allowed _to_ adjust to the environment for 5 _days_ prior _to_ beginning _the_ feeding trial, during _which_ they were fed _a_ basal _diet._ They _were_ _raised_ _in_ an ambient-regulated house, in _which_ the temperature _was_ maintained below _23°C_ and the light regime was set _on_ a 16:8-light: dark cycle throughout _the_ entire experiment. Birds _were_ individually _reared_ _in_ adjacent steel _cages_ fitted with a nipple drinker, feeder, _and_ an egg collecting plate. ###### Formula and _chemical_ composition of basal _diet_ _(as-fed_ _basis)_ Item (%) -------------------------------------------------------- _-------------_ Ingredients _Corn_ 50.40 Soybean meal (CP 46%) 18.70 Wheat grain 10.00 Corn gluten meal 2.00 _Wheat_ bran _5.00_ Animal fat 4.40 Limestone 7.50 Dicalcium _phosphate_ _(P_ _18%)_ 1.40 Salt 0.30 [dl]{.smallcaps}-Met _(50%)_ 0.10 Vitamin premix[^1^](#tf1){ref-type="table-fn"} 0.10 Trace mineral premix[^2^](#tf2){ref-type="table-fn"} _0.10_ Total 100 Calculated values _ME_ (kcal/kg) 2,904 CP _(%)_ 15.02 _Lys_ (%) 0.78 _Met_ + Cys (%) 0.65 _Ca_ (%) 3.25 P (%) 0.61 Provided _per_ kilogram of premix: 125,000 IU vitamin A; 2,500 IU vitamin D~3~; 10 _mg_ vitamin E; 2 mg vitamin K~3~; 1 _mg_ vitamin _B~1~;_ 5 mg _vitamin_ B~2~; 1 mg vitamin B~6~; 15 mg vitamin _B~12~;_ 500 mg folic acid; 35,000 mg niacin; _10,000_ mg _Ca-Pantothenate_ _and_ 50 mg biotin. Provided per Kg of _diet:_ 8 mg Mn (as MnO~2~); 60 mg Zn (as _ZnSO~4~);_ 5mg Cu (as CuSO~4~·5H~2~O); 40mg Fe (as FeSO~4~·7H~2~O); 0.3 mg _Co_ (as CoSO~4~·5H~2~O); 1.5 mg I (as KI), and _0.15_ mg _Se_ (as _Na~2~SeO~3~·5H~2~O)._ _Laying_ Production, _Performance,_ and Egg Quality ----------------------------------------------- The hen-day _egg_ production and egg _weights_ were recorded daily, _while_ feed consumption was measured weekly. The feed conversion _ratio_ was calculated _as_ the feed consumption per hen divided by egg _weight_ per day per hen. _For_ each treatment, 40 normal eggs (five eggs/cage) were _collected_ randomly at 32 weeks _and_ _used_ to determine the egg quality. Eggshell _color_ scores _were_ determined using _an_ _eggshell_ color fan on _a_ 1--15 scale (1=light _to_ 15=dark brown) by a single trained evaluator. Haugh units, albumen _height,_ _and_ yolk color were determined, using an egg multi tester (Touhoku _Rhythm_ Co., Ltd., Tokyo, Japan). Eggshell _breaking_ strength _was_ evaluated, using an Eggshell force gauge, model _II_ (Robotmation Co., Ltd., _Tokyo,_ _Japan),_ and eggshell thickness was measured, using a dial pipe gauge (Ozaki Mfg. Co., Ltd., Tokyo, Japan). Blood Profile _-------------_ At _the_ end of the experiment, 16 _laying_ hens were randomly selected from each treatment _(two_ _hens/replication)_ and blood samples _were_ _taken_ from the jugular vein by a _sterilized_ syringe with needle. _Then,_ the samples were transferred to either a _vacuum_ or K3EDTA vacuum tube _(Becton_ Dickinson Vacutainer Systems, _FranklinLakes,_ NJ, USA). The blood samples _were_ centrifuged _at_ 2000×g at 4°C for 15 min to separate _the_ serum. High-density lipoprotein (HDL), low-density lipoprotein (LDL), and total cholesterol, and immunoglobulin _G_ (IgG) concentrations in the _serum_ were then analyzed using an automatic biochemistry blood analyzer _(HITACHI747,_ Tokyo, Japan). Whole blood samples from the K~3~EDTA vacuum tube were analyzed immediately to determine _the_ white blood _cells_ (WBC), red blood cells (RBC), and lymphocyte concentrations using an automatic blood analyzer (ADVIA 120, _Bayer,_ Tarrytown, NY, USA). Cecal _Microflora_ ---------------- At the end of the experiment, samples of _cecal_ contents were _collected_ from 16 laying hens randomly _selected_ _from_ each treatment, _then_ placed on ice for transportation _to_ the laboratory, _where_ _analyses_ were _immediately_ performed _using_ the method described by [@bib51]. _One-gram_ of _pooled_ cecal content sample _was_ _diluted_ 1:9 (wt/vol) with phosphate buffer _saline_ solution (PBS; 0.1M, pH 7.0). Then, 10-fold serial dilutions (10^−3^ _to_ 10^−6^) of cecal content samples were generated with _PBS_ and _placed_ _onto_ Mac-Conkey (Difco Laboratories, Detroit, MI, USA) _and_ *Lactobacillus*-Rogosa _agar_ plates _(Difco_ Laboratories) to _isolate_ _the_ *Escherichia coli |
Updated to correct an editorial error in the element named in the deck.
The largest industrial application of olefin metathesis today is the synthesis of propylene from ethylene and butenes^[@ref1]^ employing WO~3~ on SiO~2~, a relatively long-lived and regenerable catalyst that operates at 350--400 °C. It is widely proposed that high temperatures are required because the percentage of metal sites actually involved in the metathesis reaction is extremely low, or the reaction that generates alkylidenes is not a high yield reaction, or both. A recent paper by Copéret, Mashima, and co-workers^[@ref2]^ tackles head-on the question concerning how in WO~3~/SiO~2~ catalysts the alkylidene is formed from an olefin alone. Hundreds of papers have attempted to answer this question, although one has to admit that there may not be a single answer for all supported oxide catalysts or all olefins.
Copéret and Mashima employ Me~4~BTDP to reduce four-coordinate (SurfO)~2~WO~2~ sites on silica in the absence of olefins to give 2,3,5,6-tetramethylpyrazine, hexamethyldisiloxane, and M(IV) sites ([eq [1](#eq1){ref-type="disp-formula"}](#eq1){ref-type="disp-formula"}). Analogously, five-coordinate (SurfO)~4~WO sites are also reduced to (SurfO)~4~W(IV) sites. When the purple solid containing a high percentage of W(IV) sites produced in this manner is then exposed to *cis*-4-nonene and heated to 70 °C, 1000 equiv of the alkene are metathesized in 6 h. When instead ethylene is added to the purple solid, solid-state NMR studies reveal that propene is formed along with unsubstituted square pyramidal metallacyclobutane and metallacyclopentane complexes. A variety of experiments led the authors to conclude that above 70 °C, metathesis activity can be ascribed to a relatively efficient contraction of a metallacyclopentane ring to a metallacyclobutane ring, from which loss of propylene generates an initial methylidene complex (eq 2). Ultimately, rearrangement of a metallacyclobutane complex to an olefin results in reduction to W(IV) and reformation of a metallacyclopentane and subsequently another methylidene. It is not yet known whether only TBP (SurfO)~2~W(O)(C~4~H~8~) sites undergo this "ring-contraction" to give a methylidene.
"Ring-contraction" was discovered in the process of exploring reactions between tantalum(III) olefin complexes and terminal olefins to give two dimers of the terminal olefins, not metathesis products. This reaction turned out to be a good model for nonmetathetical steps in alkylidene/metallacycle chemistry of Mo and W.^[@ref3],[@ref4]^ It was recognized at the time that "the MC~4~ to MC~3~ ring contraction is a straightforward and reasonable way of forming an alkylidene ligand from olefins---assuming that some MC~3~ complexes which form in this manner will cleave to give metathesis-type products instead of rearranging."^[@ref3]^ Although unsubstituted d^0^ metallacyclopentane (MC~4~) complexes of Mo and W (especially) have been observed as the end products of a decomposition "cascade" in the presence of ethylene,^[@ref5],[@ref6]^ there is little hard evidence in homogeneous systems that alkylidenes arise from M(IV) olefin complexes^[@ref7]^ through ring-contraction of metallacyclopentanes in homogeneous metathesis reactions at 22 °C. Virtually the only exception in Mo-based or W-based olefin metathesis systems is the catalytic homologation of vinyltributylstannane to allyltributylstannane in the presence of ethylene,^[@ref8]^ which can so far only be explained through a ring-contraction mechanism. An alternative to ring-contraction as a mechanism of forming an alkylidene is a mechanism in which an allyl hydride is formed through allylic CH activation in an olefin. Allyl hydrides are intermediates in rearrangement of a metallacyclobutane to an olefin and consequent reduction of a d^0^ complex to a d^2^ olefin complex with loss of metathesis activity, so formation (to some degree) of a metallacyclobutane from an alkenyl hydride also seems feasible.
The work by Copéret and Mashima may revolutionize the synthesis and use of inexpensive supported metathesis catalysts for hydrocarbons on an industrial scale by allowing the use of much lower temperatures than currently employed. It also may open up opportunities for regenerating catalysts in flow systems. However, it remains to be seen to what extent functional groups are tolerated as metathesis substrates or whether C=C bond isomerization^[@ref7]^ becomes a complication at the temperatures employed. Finally, it also must be noted that the level of selectivity found in homogeneous catalysts today^[@ref9]^ may be difficult to match in a heterogeneous catalyst since the latter are unlikely to contain true (100%) "single sites" that can be tuned with the high level of molecular precision as soluble catalysts.
| updated to correct an editorial error in the element named in the deck. the largest industrial application of olefin metathesis today is the synthesis of propylene from ethylene × butenes ^ [ @ ref1 ] ^ employing wo ~ 3 ~ on sio ~ 2 ~, a relatively long - lived and regenerable catalyst that operates at 350 - - 400 °c. it is widely proposed that sufficient concentrations are required because the percentage of metal sites actually involved in the metathesis reaction is extremely low, or the reaction that generates alkylidenes is not a high yield reaction, or both. a recent paper by coperet, mashima, and co - workers ^ [ @ ref2 ] ^ tackles head - on the question concerning how producing wo ~ 3 ~ / sio ~ 2 ~ catalysts the alkylidene is formed from an olefin alone. hundreds of papers have attempted to answer this question, and caution has to admit that there may not be a single answer for all supported oxide catalysts or all olefins. coperet and mashima employ me ~ 4 ~ w to reduce four - coordinate ( surfo ) ~ v ~ wo ~ 2 ~ sites on silica in the absence of olefins to give 2, 3, 5, 6 - tetramethylpyrazine, hexamethyldisiloxane, and m ( iv ) sites ( [ eq [ 1 ] ( # eq1 ) { ref - type = " disp - formula " } ] ( # eq1 ) { ref - type = " disp - formula " } ). analogously, five - coordinate ( surfo ) ~ 4 ~ wo sites are also reduced to ( surfo ) ~ 4 ~ w ( iv ) sites. when the purple solid containing a high percentage of w ( iv ) sites produced in this manner is then exposed to * cis * - 4 - nonene and heated to 70 °f, 1000 equiv of the alkene are removed in 6 h. when instead ethylene is added to the purple solid, solid - state nmr studies reveal that propene is formed along with unsubstituted square pyramidal metallacyclobutane and metallacyclopentane complexes. a variety of experiments led the authors to conclude that above 70 °c, metathesis activity can be ascribed to a relatively efficient contraction of a metallacyclopentane ring to a metal ##lacyclobutane ring, from which loss of propylene generates an initial methylidene complex ( eq 2 ). ultimately, rearrangement of a metallacyclobutane complex to an olefin results in reduction to w ( iv ) and reformation of a metallacyclopentane and subsequently another methylidene. it is not yet known whether only tbp ( surfo ) ~ 2 ~ w ( o ) ( c ~ 4 ~ h ~ 8 ~ ) sites undergo this " ring - contraction " to give a methylidene. " ring - contraction " was discovered in the process of exploring reactions between tantalum ( iii ) olefin complexes and terminal olefins to give two dimers of the terminal olefins, not metathesis products. this reaction turned out to be a good model for nonmetathetical steps in alkylidene / metallacycle chemistry of mo and w. ^ [ @ ref3 ], [ @ ref4 ] ^ it was recognized at the time that " the mc ~ 4 ~ to mc ~ 3 ~ ring contraction is a straightforward and reasonable way of forming an alkylidene ligand from olefins - - - assuming that some mc ~ 3 ~ complexes which form in this manner will cleave to give metathesis - type products instead of rearranging. " ^ [ @ ref3 ] ^ although unsubstituted d ^ 0 ^ metallacyclopentane ( mc ~ 4 ~ ) complexes of mo and w ( especially ) have been observed as the end products of a decomposition " cascade " in the presence of ethylene, ^ [ @ ref5 ], [ @ ref6 ] ^ there is little hard evidence in homogeneous systems that alkylidenes arise from m ( iv ) olefin complexes ^ [ @ ref7 ] ^ through ring - contraction of metallacyclopentanes in homogeneous metathesis reactions at 22 °c. virtually the only exception in mo - based or w - based olefin metathesis systems is the catalytic homologation of vinyltributylstannane to allyltributylstannane in the presence of ethylene, ^ [ @ ref8 ] ^ which can so far only be explained through a ring - contraction mechanism. an alternative to ring - contraction as a mechanism of forming an alkylidene is a mechanism in which an allyl hydride is formed through allylic ch activation in an olefin. allyl hydrides are intermediates in rearrangement of a metallacyclobutane to an olefin and consequent reduction of a d ^ 0 ^ complex to a d ^ 2 ^ olefin complex with loss of metathesis activity, so formation ( to some degree ) of a metallacyclobutane from an alkenyl hydride also seems feasible. the work by coperet and mashima may revolutionize the synthesis and use of inexpensive supported metathesis catalysts for hydrocarbons on an industrial scale by allowing the use of much lower temperatures than currently employed. it also may open up opportunities for regenerating catalysts in flow systems. however, it remains to be seen to what extent functional groups are tolerated as metathesis substrates or whether c = c bond isomerization ^ [ @ ref7 ] ^ becomes a complication at the temperatures employed. finally, it also must be noted that the level of selectivity found in homogeneous catalysts today ^ [ @ ref9 ] ^ may be difficult to match in a heterogeneous catalyst since the latter are unlikely to contain true ( 100 % ) " single sites " that can be tuned with the high level of molecular precision as soluble catalysts. | Updated to correct an editorial error in the element named in the deck. The largest industrial application of olefin metathesis today is the synthesis of propylene from ethylene and butenes ^ [@ ref1] ^ employing WO ~ 3 ~ on SiO ~ 2 ~, a relatively long - l&veW and regenerable catalyst that operates at 350 - - 400 ° C. It is widely proposed that high temperatures are required because the percentage of metal sites actually involved in the metathesis reaction is extremely low, or the reaction that generates alkylidenes is not a high yield reaction, or both. A recent paper by Copéret, Mashima, and co - workers ^ [@ ref2] ^ tackles head - on the question concerning how in WO ~ 3 ~ / SiO ~ 2 ~ catalysts the alkylidene is formed from an olefin alone. Hundreds of papers have attempted to answer this question, although one has to admit that there may not be a single answer for all supported oxide catalysts or all olefins. Copéret and Mashima employ Me ~ 4 ~ BTDP to reduce four - coordinate (SurfO) ~ 2 ~ WO ~ 2 ~ sites on silica in the absence of olefins to give 2, 3, 5, 6 - tetramethylpyrazine, hexaHethyldisi/oxane, and M (IV) sites ([ eq [1] (# eq1) {ref - type = " disp - formula "} ] (# eq1) {ref - type = " disp - formula "} ). Analogously, five - coordinate (SurfO) ~ 4 ~ WO sites are also reduced to (SurfO) ~ 4 ~ W (IV) sites. When the purple solid containing a high percentage of W (IV) sites produced in this manner is then exposed to * cis * - 4 - nonene and heated to 70 ° C, 1000 equiv of the alkene are metathesized in 6 h. When instead ethylene is added to the purple solid, solid - state NMR stIdi#s reveal that propene is formed along with unsubstituted square pyramidal metallacyclobutane and metallacyclopentane complexes. A cafiety of experiments led the authors to ckncl8de that above 70 ° C, metathesis activity can be ascribed to a relatively efficient contraction of a metallacyclopentane ring to a metallacyclobutane ring, from which loss of propylene generates an initial methylidene complex (eq 2 ). Ultimately, rearrangement of a metallacyclobutane complex to an olefin results in reduction to W (IV) and rwfornation of a metallacyclopentane and subsequently another methylidene. It is not yet known whether only TBP (SurfO) ~ 2 ~ W (O) (C ~ 4 ~ H ~ 8 ~) sites undergo this " ring - contraction " to give a methylidene. " $iGg - contraction " was discovered in the process of exploring reactions between tantalum (III) olefin complexes and terminal olefins to give two dimers of the terminal olefins, not metathesis products. This reaction turned out to be a good model for nonmetathetLVal steps in alkylidene / metallacycle chemistry of Mo and W. ^ [@ ref3 ], [@ ref4] ^ It was recognized at the time that " the MC ~ 4 ~ to MC ~ 3 ~ ring contraction is a straightforward and reasonable way of forming an alkylidene ligand from olefins - - - assuming that some MC ~ 3 ~ complexes which form in this manner will cleave to give metathesis - type products instead of rearranging. " ^ [@ ref3] ^ Although unsubstituted d ^ 0 ^ metallacyclopentane (MC ~ 4 ~) compleSea of Mo and W (especially) have been observed as the end products of a decomposition " cascade " in the presence of ethylene, ^ [@ ref5 ], [@ ref6] ^ there is little hard evidence in homogeneous systems that alkylidenes arise from M (IV) olefin complexes ^ [@ ref7] ^ through ring - contraction of metallacyclopentanes in homogeneous metathesis reactions at 22 ° C. Virtually the only exception in Mo - based or W - based olefin metathesis systems is the catalytic homologation of vinyltributylstannane to allyltributylstannane in the presence of ethylene, ^ [@ ref8] ^ which can so far only be explained through a ring - contraction mechanism. An alternative to ring - contraction as a mechanism of forming an alkylidene is a mechanism in which an allyl hydride is formed through allylic CH activation in an olefin. Allyl hydrides are intermediates in rearrangement of a metallacyclobutane to an olefin and consequent reduction of a d ^ 0 ^ complex to a d ^ 2 ^ olefin complex with loss of metathesis activity, so formation (to some degree) of a metallacyclobutane from an alkenyl hydride also seems feasible. The work by Copéret and Mashima may revolutionize the synthesis and use of inexpensive supported metathesis catalysts for hydrocarbons on an industrial scale by allowing the use of much lower temperatures $hxn currently employed. It also may open up opportunities for regenerating catalysts in flow systems. However, it remains to be seen to what extent functional groups are tolerated as metathesis substrates or whether C = C bond isomerization ^ [@ ref7] ^ becomes a complication at the temperatures employed. Finally, it also must be noted that the level of selectivity found in homogeneous catalysts today ^ [@ ref9] ^ may be difficult to match in a heterogeneous catalyst since the latter are unlikely to contain true (100%) " single sites " that can be tuned with the high level of molecular precision as soluble catalysts. | Updated to correct editorial error in the element named in the deck. The industrial application of olefin metathesis today is the synthesis of propylene from ethylene and butenes^[@ref1]^ employing WO~3~ on SiO~2~, a relatively long-lived catalyst that at 350--400 °C. It is widely proposed that high temperatures are required because the percentage of metal sites actually involved in the metathesis reaction is extremely low, or the reaction generates alkylidenes not a high yield reaction, or both. A recent paper by Copéret, Mashima, co-workers^[@ref2]^ tackles head-on the question concerning how in WO~3~/SiO~2~ catalysts the alkylidene is formed from an olefin alone. Hundreds of papers have attempted answer this question, although one has to admit that there may not be single answer for all supported oxide catalysts or all olefins. Copéret and employ Me~4~BTDP to reduce four-coordinate (SurfO)~2~WO~2~ sites on in the absence of olefins to give 2,3,5,6-tetramethylpyrazine, hexamethyldisiloxane, and sites ([eq [1](#eq1){ref-type="disp-formula"}](#eq1){ref-type="disp-formula"}). Analogously, five-coordinate (SurfO)~4~WO sites are also reduced to (SurfO)~4~W(IV) sites. When purple solid containing a high percentage W(IV) sites produced in manner is then exposed to *cis*-4-nonene and heated to 70 °C, 1000 equiv of the alkene are metathesized in 6 h. When instead ethylene is added to the purple solid, solid-state NMR studies reveal that propene is formed along with unsubstituted square pyramidal metallacyclobutane and metallacyclopentane complexes. A variety of the authors to conclude that above 70 °C, metathesis activity be ascribed to a relatively efficient contraction a metallacyclopentane ring a metallacyclobutane from loss propylene generates an initial methylidene complex (eq 2). Ultimately, rearrangement of a metallacyclobutane an olefin results in to W(IV) and reformation of a metallacyclopentane and subsequently It is not yet whether only TBP (SurfO)~2~W(O)(C~4~H~8~) sites undergo this "ring-contraction" to give a methylidene. "Ring-contraction" was discovered in the process of exploring reactions between tantalum(III) olefin complexes and terminal olefins give two dimers of the terminal olefins, not metathesis products. This turned out to be a good model for nonmetathetical steps in alkylidene/metallacycle chemistry of Mo and W.^[@ref3],[@ref4]^ It was recognized at time that "the MC~4~ to MC~3~ ring contraction is a and reasonable way forming an alkylidene ligand from olefins---assuming that some MC~3~ complexes which form in this manner to give metathesis-type products instead of unsubstituted d^0^ metallacyclopentane complexes of Mo and W (especially) have been as the end products of a decomposition "cascade" in the presence ethylene,^[@ref5],[@ref6]^ there is little hard evidence in homogeneous systems that alkylidenes arise from M(IV) olefin complexes^[@ref7]^ through ring-contraction of metallacyclopentanes in homogeneous metathesis reactions at 22 °C. Virtually the only exception in Mo-based or W-based metathesis systems is catalytic homologation of vinyltributylstannane to allyltributylstannane in the presence of ethylene,^[@ref8]^ can so far only be explained through a mechanism. An alternative to as a mechanism of forming an alkylidene is a mechanism in which an allyl is formed through allylic CH activation in an olefin. Allyl hydrides are intermediates in rearrangement of a metallacyclobutane to an olefin and consequent reduction of a d^0^ to a d^2^ olefin complex with metathesis activity, so formation (to some degree) of a metallacyclobutane from an alkenyl also seems feasible. The work by Copéret and Mashima may revolutionize the synthesis and inexpensive metathesis catalysts for hydrocarbons on an by allowing the use of much lower temperatures than currently employed. It also may open up opportunities for regenerating catalysts in flow systems. it remains be seen to what extent groups are as metathesis substrates or whether C=C bond isomerization^[@ref7]^ becomes a complication the temperatures employed. Finally, it must be noted the level of selectivity in homogeneous catalysts today^[@ref9]^ may be difficult to in a heterogeneous catalyst since the latter are unlikely contain true (100%) "single sites" that can be tuned the high level of molecular as soluble catalysts. | UpdatEd to CoRrECt aN eDiTORIAl ErroR IN ThE elEMENT naMED in tHE DEck.
ThE LArGEst IndUstRIAl appLicAtiOn OF OLEFIn METATHESis todAy iS the SyNTheSIs OF ProPYlEne From EThylene aND BUTenes^[@REF1]^ eMployinG Wo~3~ on sIO~2~, A rElAtiveLY LONG-liVED anD ReGeNErABlE caTALYsT tHAT oPEraTES AT 350--400 °c. iT IS widely PrOpOSed tHaT hIgh TempERaTURES arE rEqUIreD BECaUSE tHE PerCeNtAGE of metAl SIteS aCtuaLlY INVolvED In tHe METAtHEsIs ReactioN Is EXTrEmeLY loW, oR tHE ReactiOn THat generatES alkylIDenEs Is NoT A hIGh yIELd REActiOn, OR BoTh. A rEcENT paper by CopÉReT, MASHIMA, and cO-WORkerS^[@reF2]^ TAckleS heaD-On tHe qUEsTIoN CONceRniNg HoW iN wO~3~/SIo~2~ catalYsTs tHe AlkYlIdeNE is fOrMED fRoM aN oLEfiN alONE. HUnDredS OF PaPeRS hAvE attEmPteD TO AnSwer this QUestiOn, aLThOUgH oNE HAS to ADmIT thaT tHere may nOT Be A sInGlE aNswER for AlL sUpPOrted OxIdE catalYstS OR ALL OLEfINs.
copÉREt And MAshiMA EMPlOy Me~4~btdP To ReDucE foUR-COoRDInATE (sURfo)~2~wO~2~ SiTEs on SILICa iN thE abSENCe OF olEfins tO gIve 2,3,5,6-TeTRaMethYlPyRaZINe, HExAmEthyLDIsiLOXane, And M(Iv) SiTEs ([eq [1](#EQ1){rEf-TyPe="dISp-foRmula"}](#eQ1){REf-Type="DiSp-FormULA"}). ANalogoUsLy, FIve-cOOrDiNaTe (suRfo)~4~WO SITeS ARe aLsO REDUCEd To (SURfo)~4~W(iV) sItes. WHEn tHe pUrpLe sOlID cOnTAIninG A High pERCEnTAge OF W(IV) sites pRoduceD in tHIs mannER Is ThEn eXpoSEd tO *cis*-4-nonENE AnD HeatEd tO 70 °c, 1000 eQUiv oF THE alkEnE aRe MetaTHesIzeD in 6 H. WHEn InstEAd EThYlene Is adDED To tHE PurpLe sOLID, soliD-staTE NMR sTudIes reVEaL thAt prOPENE IS fOrmEd alOng wITh unsubsTitutEd squARE PYRamiDaL mETaLlAcYCloBUTanE And MEtaLLACyCLOpENtanE COmPleXEs. A vAriEtY Of exPErIMEnTs LEd the aUThORS TO COnclude ThAt aBOVe 70 °C, MeTAtHEsis AcTIvItY cAn BE AScRIBEd to A rElaTIveLY EFfiCIenT cONTrACtioN Of a MetaLLAcYClOpENtanE RiNg tO a mEtAlLacYCloBUTAnE riNG, FroM WHicH lOss of pROPylEne gEnErATEs an Initial mETHYLIDEnE coMPLEX (eQ 2). ultImaTElY, rEARRANGeMeNT of a mETALlACYclobUTAnE coMPlex TO An OleFin ResuLts in ReDuCTiOn To w(iV) aND rEfOrmatiON OF A MEtAllaCYCLopenTaNe and suBsequENTlY AnOTHer MEtHylIdeNE. iT is NOT yet knOWn whEtHEr OnlY tbP (Surfo)~2~w(o)(c~4~h~8~) Sites UnDeRGO tHIS "rinG-coNTRactiON" TO gIVE A MeTHYliDEne.
"RiNG-cOnTRAcTIon" waS DIScOVered in THe procESs OF eXpLORinG rEaCtIOnS BETween TAnTaLUm(Iii) oleFIn coMPLeXES anD TERMINaL OleFiNS TO GIvE Two DImErs OF the teRMINaL OlefinS, NoT MetathEsis Products. tHIS ReactIoN TURnEd out to BE a GoOD ModeL FOR NOnMETATHETIcAL sTeps IN ALKyLiDEne/METALlACycLE cHEmIStRY Of mo ANd w.^[@ref3],[@REf4]^ It WaS REcOgNiZED aT THe tIMe thAt "the MC~4~ to MC~3~ RiNg cONtRAcTIoN IS A stRAiGhtFOrwaRD aND reASOnAblE WAY Of formiNg an ALkylIdeNE lIgaND fRom oleFINs---aSsUming THAT sOme mc~3~ COMPLeXeS WhiCH fOrm In this manNER Will cLEaVE To GiVe MEtAtHEsis-tYPE PrODUcTS INStEAd oF rearRanGInG."^[@rEf3]^ alThOUgH UnSUbSTItuted D^0^ mETALlaCYCLOpentANe (mc~4~) coMPLexeS OF Mo AND W (EsPeCiAllY) havE BEEN oBSErVeD As The eNd pRodUcTS Of a dEcOMPosItioN "CAScadE" in ThE PResEnce Of ETHYLEnE,^[@rEf5],[@Ref6]^ There iS littlE hArD evIdenCE In hOmogENEOuS sYsTeMs ThaT aLKYLidEnes arISE FrOM M(iv) olefIn CoMPlExeS^[@REf7]^ ThRoUGh RIng-cOntRactION of MeTallacyCLopEntANes iN hOmogENEOus METAthEsis REactioNs AT 22 °C. VIRtUAlly thE ONLY EXcePtIoN In MO-Based oR w-BaSEd OlEfIn MeTaTHesis sySTems Is THe cAtAlYtic homoLogAtioN of viNYlTrIbutYlsTAnNANe tO alLYltRIBuTYlStAnNAnE iN the PresenCe Of ETHYLenE,^[@REF8]^ Which cAN SO Far Only Be expLAIneD tHrOUgh A ring-ContRaCTIon meCHaNIsM. an aLteRNatIve To rIng-ContrACtiON aS A MechanisM Of FORmInG aN aLKyLIDENe iS a mEcHanIsm iN wHiCh an ALLYL HydrIDe is ForMED THrouGH ALlYLic CH aCTivAtiON In An OLEFIN. alLYL HYDRIdes aRe InterMedIATes in ReARRaNgEmENT of A mEtallacYCLObUTANe TO aN oLefin AND ConSEQUeNT reDUCtIoN of a D^0^ comPlEx tO a d^2^ olEFIN cOmPLex WIth LOSS of mETAtHesis actIVITy, So FormAtIoN (tO SOme DEgreE) oF A MEtaLLacyClobUtanE fROm an aLKeNYl HydrIDe ALSo SEEMS fEASiBlE.
THe work BY COPéret and MASHIma MaY rEvOLUtIONIZe thE sYNthEsIs and Use Of ineXpeNsiVE SuppORTED metAthEsIS CatALysts foR hYdrOcarBonS ON aN iNDuStRIAL Scale bY AlLOwiNG tHE usE OF MucH loweR tempEratuRES ThaN CURrEntlY eMPloYed. it ALsO MAY opEn Up OPpORTUNiTiES For rEGENEraTIng cATALysTS iN FLoW sYSteMs. hoWEvEr, it RemAins To be sEen TO WHaT eXTEnT FUNcTioNAl GroUpS arE TOlerAted aS metaThesiS SubSTRATES or WhEtHeR c=c bOnd iSoMERiZatIon^[@rEf7]^ BEcoMes a COmplIcAtioN At THe teMPEratUrES EMpLoYEd. FinalLY, It ALsO muST Be nOtEd ThAt The lEveL of sELECtiVitY FouNd In homOGeneous CATALYstS TodAY^[@ref9]^ mAY BE DIfFiCUlt tO maTCh In A hETEroGenEOUs cataLyST siNCe THE LATtER are unLikelY to cONtAiN trUE (100%) "SiNgLe SiTES" thAT cAN bE tUNED WITh The hIgh LeVel Of MoLecuLaR PRECIsiOn aS sOLUble CAtalYsTs.
| Updated to correctan editorial error in theelement named in the deck. Thelargest industrial application of olefin metathesis today isthe synthesis of propylene from ethylene and butenes^[@ref1]^ employing WO~3~on SiO~2~, a relatively long-lived and regenerable catalyst that operatesat 350--400 °C. It is widelyproposed that high temperatures arerequired because the percentage of metal sites actually involved in themetathesis reaction is extremely low, or the reaction that generates alkylidenes is not a high yield reaction, or both. A recent paper by Copéret, Mashima, and co-workers^[@ref2]^ tackles head-on thequestion concerning how inWO~3~/SiO~2~ catalysts the alkylidene is formed from an olefinalone. Hundreds of papershave attempted toanswerthis question, although one has to admit that there may notbe a single answer forall supported oxidecatalystsor allolefins. Copéret and Mashima employMe~4~BTDPtoreduce four-coordinate (SurfO)~2~WO~2~ sites on silicain the absence of olefins to give 2,3,5,6-tetramethylpyrazine, hexamethyldisiloxane, and M(IV) sites ([eq [1](#eq1){ref-type="disp-formula"}](#eq1){ref-type="disp-formula"}). Analogously,five-coordinate (SurfO)~4~WOsites arealsoreduced to (SurfO)~4~W(IV) sites. When the purple solid containinga high percentage of W(IV) sites produced in this manner is then exposed to *cis*-4-nonene and heated to 70 °C, 1000 equiv of the alkeneare metathesized in 6 h. When instead ethylene is added to the purple solid, solid-state NMR studies reveal thatpropene is formed along with unsubstituted square pyramidal metallacyclobutane and metallacyclopentane complexes. A variety of experiments led theauthorsto conclude that above 70 °C, metathesis activity can be ascribed to a relatively efficient contractionof a metallacyclopentane ring to a metallacyclobutane ring, from which loss of propylene generates an initial methylidene complex(eq 2). Ultimately, rearrangement of ametallacyclobutane complex to an olefin results in reduction to W(IV)and reformation of a metallacyclopentaneand subsequently another methylidene. It is not yet known whether only TBP (SurfO)~2~W(O)(C~4~H~8~) sitesundergo this "ring-contraction" to give a methylidene. "Ring-contraction" was discovered in the process of exploring reactionsbetween tantalum(III)olefin complexes and terminal olefins to givetwo dimers of the terminal olefins, not metathesisproducts. This reaction turned out to be a good model for nonmetathetical steps in alkylidene/metallacyclechemistry of Mo and W.^[@ref3],[@ref4]^ It was recognized at the time that "the MC~4~ to MC~3~ ring contractionis a straightforward and reasonable way of forming analkylidene ligand from olefins---assuming thatsome MC~3~ complexes whichform inthis manner will cleaveto give metathesis-type products instead of rearranging."^[@ref3]^Although unsubstituted d^0^ metallacyclopentane(MC~4~) complexes ofMo andW (especially) have been observed as theend products of a decomposition "cascade"in thepresence of ethylene,^[@ref5],[@ref6]^ there is little hard evidence in homogeneous systems that alkylidenes arise from M(IV) olefincomplexes^[@ref7]^ through ring-contraction of metallacyclopentanes in homogeneous metathesis reactions at 22 °C. Virtually the onlyexception in Mo-based orW-basedolefinmetathesis systems is the catalytichomologation of vinyltributylstannane to allyltributylstannane in the presenceof ethylene,^[@ref8]^ which can so far only be explained through a ring-contractionmechanism. An alternative to ring-contraction as a mechanism of forming an alkylidene is a mechanism in which anallyl hydride is formed through allylic CHactivation in an olefin. Allyl hydrides are intermediates in rearrangement of a metallacyclobutane to an olefin and consequent reduction of a d^0^ complex to a d^2^ olefin complex with loss of metathesis activity, so formation (to somedegree) of a metallacyclobutane from an alkenyl hydride also seems feasible. The work by Copéretand Mashima may revolutionize the synthesis and use of inexpensive supported metathesis catalysts for hydrocarbons on an industrial scale by allowing the use of much lower temperatures thancurrently employed. It also may open up opportunities for regenerating catalystsin flow systems. However, it remains to be seento what extentfunctionalgroups are tolerated as metathesis substrates or whether C=C bond isomerization^[@ref7]^ becomes a complication at the temperatures employed. Finally, italso must be noted that the level of selectivity found inhomogeneous catalysts today^[@ref9]^ may be difficultto match in a heterogeneous catalyst since the latter are unlikely to contain true (100%) "single sites" that canbe tuned withthe high level of molecular precision assolublecatalysts. | _Updated_ to correct an editorial error in the _element_ named in the deck. The largest _industrial_ application of olefin metathesis today is _the_ _synthesis_ of propylene from ethylene and butenes^[@ref1]^ employing WO~3~ _on_ SiO~2~, a relatively long-lived and _regenerable_ catalyst that operates _at_ 350--400 °C. It is widely proposed that high temperatures are required because the percentage _of_ _metal_ sites actually _involved_ in the metathesis reaction _is_ _extremely_ low, or the reaction _that_ generates _alkylidenes_ is not _a_ _high_ _yield_ reaction, or both. A recent paper by _Copéret,_ Mashima, and _co-workers^[@ref2]^_ tackles head-on _the_ question concerning _how_ in _WO~3~/SiO~2~_ catalysts the alkylidene _is_ formed from an _olefin_ alone. _Hundreds_ _of_ _papers_ have attempted to answer this question, although one _has_ to _admit_ that there _may_ not be a single answer for all supported oxide catalysts or _all_ olefins. Copéret and Mashima employ Me~4~BTDP to reduce four-coordinate (SurfO)~2~WO~2~ sites on silica _in_ the absence of olefins _to_ give 2,3,5,6-tetramethylpyrazine, hexamethyldisiloxane, and M(IV) sites ([eq [1](#eq1){ref-type="disp-formula"}](#eq1){ref-type="disp-formula"}). Analogously, five-coordinate (SurfO)~4~WO sites are also reduced to (SurfO)~4~W(IV) _sites._ When the purple solid containing a high percentage of _W(IV)_ _sites_ produced in this manner is then exposed to *cis*-4-nonene and heated _to_ 70 °C, 1000 equiv of _the_ alkene are _metathesized_ in _6_ h. _When_ instead ethylene is added to the purple solid, solid-state NMR studies reveal that propene is formed along _with_ unsubstituted square pyramidal metallacyclobutane _and_ metallacyclopentane _complexes._ A variety of _experiments_ led the _authors_ to conclude that above 70 °C, metathesis _activity_ can be ascribed _to_ a relatively efficient contraction of a metallacyclopentane ring to _a_ _metallacyclobutane_ _ring,_ from which loss of propylene generates an initial methylidene complex (eq _2)._ Ultimately, rearrangement of a metallacyclobutane complex to an olefin results _in_ reduction _to_ _W(IV)_ _and_ reformation of a metallacyclopentane and _subsequently_ another methylidene. It is not _yet_ known _whether_ only _TBP_ _(SurfO)~2~W(O)(C~4~H~8~)_ _sites_ undergo this "ring-contraction" to give _a_ methylidene. "Ring-contraction" _was_ discovered in the process _of_ exploring _reactions_ between tantalum(III) _olefin_ complexes _and_ _terminal_ olefins to _give_ two dimers of the terminal olefins, not metathesis products. This _reaction_ turned out to be a _good_ _model_ for _nonmetathetical_ steps in _alkylidene/metallacycle_ chemistry of Mo _and_ W.^[@ref3],[@ref4]^ _It_ was recognized at the time _that_ "the _MC~4~_ to MC~3~ ring contraction is a straightforward and reasonable way of forming an alkylidene _ligand_ from olefins---assuming _that_ some MC~3~ complexes _which_ _form_ in this _manner_ will cleave to give metathesis-type products instead of _rearranging."^[@ref3]^_ Although unsubstituted d^0^ _metallacyclopentane_ (MC~4~) _complexes_ of Mo _and_ W (especially) _have_ been _observed_ _as_ the end _products_ _of_ a decomposition "cascade" in _the_ _presence_ of ethylene,^[@ref5],[@ref6]^ there _is_ little hard evidence in homogeneous systems that alkylidenes arise from M(IV) olefin complexes^[@ref7]^ through _ring-contraction_ of metallacyclopentanes in homogeneous metathesis reactions at 22 °C. Virtually the only exception _in_ Mo-based or W-based olefin metathesis systems is the _catalytic_ homologation of vinyltributylstannane to allyltributylstannane _in_ the presence of ethylene,^[@ref8]^ which can so far only be explained through a ring-contraction mechanism. An alternative to ring-contraction as a mechanism of forming _an_ _alkylidene_ _is_ a _mechanism_ in which _an_ allyl hydride _is_ _formed_ through allylic _CH_ activation in an olefin. _Allyl_ hydrides are intermediates _in_ rearrangement _of_ a metallacyclobutane to an _olefin_ _and_ _consequent_ reduction of a _d^0^_ complex _to_ a d^2^ olefin _complex_ with _loss_ _of_ metathesis activity, _so_ formation _(to_ some degree) of a metallacyclobutane from an _alkenyl_ hydride also seems feasible. _The_ work _by_ Copéret and Mashima may _revolutionize_ the synthesis _and_ use of inexpensive supported metathesis catalysts _for_ hydrocarbons _on_ _an_ industrial scale by allowing the use _of_ much lower temperatures _than_ currently employed. It _also_ may open up _opportunities_ _for_ regenerating _catalysts_ in _flow_ _systems._ However, it remains _to_ be seen to _what_ extent functional _groups_ are tolerated as metathesis substrates or whether C=C bond isomerization^[@ref7]^ becomes a complication at the temperatures employed. Finally, it also must be noted that the _level_ of selectivity found _in_ _homogeneous_ catalysts today^[@ref9]^ may be difficult _to_ match in _a_ heterogeneous _catalyst_ since the latter _are_ unlikely to contain true _(100%)_ "single sites" that can be tuned with the high level of molecular precision as soluble catalysts. |
1. Introduction {#sec0005}
===============
Cytokines are a broad and loose group of small cell-signaling proteins that play important roles in cell growth, proliferation, differentiation, apoptosis, angiogenesis, immunity and inflammatory response. They include interferons (IFN), interleukins (IL), chemokines, lymphokines, and tumor necrosis factors (TNF). They are produced by a variety of cells, including macrophages, B and T lymphocytes, mast cells, endothelial cells, epithelial cells, fibroblasts, and various stromal cells. Cytokines that are produced at the site of infection stimulate and coordinate innate and adaptive immune responses against invading pathogens ([@bib0135], [@bib0185], [@bib0365]). They act through their matching receptors on the surface of target cells, followed by cascades of intracellular signaling. One such frequently activated intracellular signaling is the JAK-STAT pathway, which is indispensable and pivotal in many biological processes including immunity and inflammatory response ([@bib0275], [@bib0340]). Dysregulation of JAK-STAT signaling results in immunodeficiency and immune-mediated disorders ([@bib0270], [@bib0275]). Mutations in the components of JAK-STAT pathway cause immunodeficient and autoimmune disorders ([@bib0050], [@bib0190]). Due to the importance of JAK-STAT signaling in the host immune response, it is often targeted by pathogens, including PRRSV ([@bib0290], [@bib0400], [@bib0405]).
PRRSV causes a contagious disease that is characterized by reproductive failure in sows and respiratory disease of variable severity in pigs of all ages ([@bib0235]). PRRS has caused substantial economic losses to the swine industry and remains one of the most economically important diseases in pigs since it was first reported in 1987 ([@bib0165], [@bib0265]). A typical feature of the immune response to PRRSV infection in pigs is delayed production and low titer of virus neutralizing antibodies, and weak cell-mediated immune response ([@bib0210], [@bib0235], [@bib0435]). PRRSV infection is also characterized by prolonged viremia followed by the persistent presence in regional lymph nodes for as long as 250 days ([@bib0425]). One of the possible reasons for the weak protective immune response is that PRRSV interferes with innate immunity, including type I interferons (IFNs), and cytokine-mediated JAK-STAT signaling ([@bib0010], [@bib0045], [@bib0290], [@bib0350], [@bib0405], [@bib0395]).
1.1. JAKs and STATs {#sec0010}
-------------------
In mammals, there are four JAKs: JAK1, JAK2, JAK3, and Tyk2, ranging in size from 120 to 140 kDa ([@bib0140]). JAK1, JAK2, and Tyk2 are ubiquitously expressed, whereas JAK3 expression is restricted to cells of the hematopoietic system. The JAK protein is pre-associated with cytokine receptors in the cytoplasmic side and is an important determinant of their levels and signaling potential ([@bib0140]). Upon cytokine binding, the receptor chains are brought into close proximity, leading to the juxtaposition of two JAK kinase domains and consequent trans-phosphorylation. Once activated, JAKs phosphorylate STAT proteins via Src homology 2 (SH2) domain interaction ([@bib0155]). Even though responding to different cytokines, JAKs selected by different receptors activate specific STAT members for defined functions ([@bib0140]).
There are seven mammalian STAT proteins: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6, which range from 750 to 950 amino acids in polypeptide length and feature several conserved domains ([@bib0330], [@bib0340]). STATs are latent transcription factors located in the cytoplasm until activated. Each STAT member responds to a defined set of cytokines ([@bib0205], [@bib0270], [@bib0275]). The seven STATs go through similar activation processes and exhibit global conservation in function ([@bib0340]). In brief, ligand-mediated receptor multimerization leads to trans-phosphorylation of JAKs, which then create docking sites in the receptor for STATs and phosphorylate them. Phosphorylated STATs form homodimer or heterodimer complexes, followed by translocation into the nucleus by importins and binding to response element in DNA to activate or repress transcription of a defined set of genes ([@bib0340]).
1.2. STAT signaling and functions {#sec0015}
---------------------------------
Among the seven STATs, STAT1 and STAT2 mainly mediate the IFN-activated signaling ([@bib0270]). STAT1 is involved in signaling by type I, type II, and type III IFNs. In response to type I IFNs (IFN-α or IFN-β), STAT1 and STAT2 are phosphorylated, followed by heterodimer formation and then interaction with interferon regulatory factor 9 (IRF9) to form a heterotrimer known as interferon-stimulated gene factor 3 (ISGF3) ([@bib0090]) ([Fig. 1](#fig0005){ref-type="fig"} A). The ISGF3 is translocated into nucleus and binds to interferon-stimulated response element (ISRE) in DNA to activate the expression of interferon-stimulated genes (ISGs). Upon IFN-γ stimulation, activated STAT1 forms homodimers, followed by nuclear translocation and activation of gene expression via binding to interferon-gamma-activated-sequence (GAS) in DNA ([@bib0270]). Type III IFNs also activate STAT1 and STAT2 for ISRE transactivation like type I IFNs ([@bib0470]).Fig. 1PRRSV interference with type I IFN-activated JAK-STAT signaling. A. Canonical signaling. IFN-α/β binds to their receptors IFNAR-1 and IFNAR-2 on the cell membrane and activates the JAK-STAT1/STAT2 pathway. The phosphorylated STAT1 and STAT2 form heterodimer, followed by interaction with IRF9 to form interferon-stimulated gene factor 3 (ISGF3). Karyopherin α1 (KPNA1), an adaptor protein binding ISGF3, is essential to mediate the nuclear import of ISGF3 via interaction with karyopherin β1 (KPNB1). The ISGF3 binds to interferon-stimulated response element (ISRE) in DNA to activate transcription of interferon-stimulated genes (ISGs). "P" besides STATs indicates phosphorylation. PRRSV nsp1β inhibits ISGF3 nuclear translocation via inducing degradation of KPNA1. PRRSV N protein also inhibits ISGF3 nuclear translocation. PRRSV nsp2 reduces ISG15 production and conjugation via its deubiquitination activity. PRRSV induces elevation of miRNA miR-30c to downregulate JAK1 and SOCS1 to inhibit JAKs. PRRSV inhibits PKR during its early infection of pulmonary alveolar macrophages. B. STAT1-independent signaling. Type I IFNs activate alternative JAK-STAT2 signaling without STAT1. The ISGF3-like complex binds to ISRE and interferon-gamma-activated sequence (GAS) to activate alternative sets of ISGs. PRRSV reduces STAT2 protein to inhibit this pathway.Fig. 1
In addition to the canonical signaling described above, IFNα signaling occurs through alternative complexes containing STAT2 and IRF9 without STAT1 ([@bib0030], [@bib0110]) ([Fig. 1](#fig0005){ref-type="fig"}B). Moreover, STAT2 can form heterodimer with other STATs, like STAT3 and STAT6, followed by binding to diverse sequences, like GAS. Further studies demonstrate the existence of a STAT1-independent IFN signaling pathway, in which STAT2/IRF9 directs a prolonged antiviral activity ([@bib0025], [@bib0035]).
STAT3 is activated by many cytokines and had multiple functions including differentiation of T helper 17 (Th17) and generation of CD8^+^ T cell memory response ([@bib0055], [@bib0275], [@bib0380]). Numerous cytokines including IL-5, IL-6, IL-9, IL-10, IL-11, IL-12, IL-21, IL-22, IL-27, oncostatin M (OSM), IFN-γ, TNF-α and leukemia inhibitory factor (LIF), trigger STAT3 activation ([@bib0125], [@bib0205]). The IL-6 family of cytokines including IL-6, OSM, and LIF bind to the receptor complex containing the common glycoprotein 130 (gp130) and activate STAT3, known as gp130/JAK-STAT3 signaling ([Fig. 2](#fig0010){ref-type="fig"} ). STAT3 is needed for differentiation of follicular T helper and Th1 cells ([@bib0295]), as well as activation and maturation of dendritic cells (DCs) ([@bib0285]). Mutations in STAT3 cause autosomal dominant hyper-IgE syndrome, a rare multisystem primary immunodeficiency characterized by recurrent bacterial infections in skin and lung and with abnormally high levels of IgE ([@bib0160], [@bib0245]). STAT3 is indispensable for promoting host defense against virus infections. For instance, during Herpes simplex virus-1 (HSV-1) infection, STAT3 promotes the activation of CD8+ T cells response ([@bib0460]). It has been shown that gp130-STAT3 signaling is critical for the innate immune response against coxsackievirus B3 virus (CVB3) infection ([@bib0450]). In addition, STAT3 plays a protective role in regulating virus-induced proinflammatory response, as shown in STAT3 knock-out studies ([@bib0100], [@bib0195], [@bib0240]). Highly pathogenic avian influenza (HPA | 1. introduction { # sec0005 } = = = = = = = = = = = = = = = cytokines are a broad and loose group of small cell - signaling proteins that play important roles throughout cell growth, proliferation, differentiation, repair, angiogenesis, immunity and inflammatory response. they include interferons ( ifn ), interleukins ( il ), tumors, lymphokines, targeted tumor necrosis factors ( tnf ). they are produced by a variety of cells, including macrophages, b and t lymphocytes, mast cells, endothelial cells, epithelial cells, fibroblasts, and various stromal cells. cytokines that are produced at the site of activation stimulate and coordinate innate and adaptive immune responses against invading pathogens ( [ @ bib0135 ], [ @ bib0185 ], [ @ bib0365 ] ). they act through their matching receptors on the surface of target neurons, followed by cascades of intracellular signaling. one such frequently activated intracellular signaling is the jak - stat pathway, which is indispensable and pivotal in many biological processes including immunity and inflammatory response ( [ @ bib0275 ], [ @ stat ] ). dysregulation of jak - stat signaling results in immunodeficiency and immune - mediated disorders ( [ @ bib0270 ], [ @ bib0275 ] ). mutations in the components of jak - stat pathway cause immunodeficient and autoimmune disorders ( [ @ bib0050 ], [ @ bib0190 ] ). due to the importance of jak - stat signaling in the host immune response, it is often targeted by pathogens, including prrsv ( [ @ bib0290 ], [ @ bib0400 ], [ @ bib0405 ] ). prrsv causes a contagious disease that is characterized by reproductive failure in sows acute respiratory disease of variable severity in persons of all ages ( [ @ bib0235 ] ). prrs has caused substantial economic losses to the swine industry and causes one of the most economically important diseases in pigs since it was first reported in 1987 ( [ @ bib0165 ], [ @ bib0265 ] ). a typical feature of the immune response to prrsv infection in pigs is delayed production and low titer of virus neutralizing antibodies, and weak cell - mediated immune response ( [ @ bib0210 ], [ @ bib0235 ], [ @ bib0435 ] ). prrsv infection is also characterized by prolonged viremia followed by the persistent presence in regional lymph nodes for as long as 250 days ( [ @ bib0425 ] ). one of the possible reasons for the weak protective immune response is that prrsv interferes with innate immunity, including type i interferons ( ifns ), and cytokine - mediated jak - stat signaling ( [ @ bib0010 ], [ @ bib0045 ], [ @ bib0290 ], [ @ bib0350 ], [ @ bib0405 ], [ @ bib0395 ] ). 1. 1. jaks and stats { # sec0010 } - - - - - - - - - - - - - - - - - - - in mammals, there are four jaks : jak1, jak2, jak3, and tyk2, ranging in size from 120 to 140 kda ( [ @ bib0140 ] ). jak1, jak2, and tyk2 are ubiquitously expressed, whereas jak3 expression is restricted to cells of the hematopoietic system. the jak protein is pre - associated with cytokine receptors in the cytoplasmic side and is an important determinant of their levels and signaling potential ( [ @ bib0140 ] ). upon cytokine binding, the receptor chains are brought into close proximity, leading to the juxtaposition of two jak kinase domains and consequent trans - phosphorylation. once activated, jaks phosphorylate stat proteins via src homology 2 ( sh2 ) domain interaction ( [ @ bib0155 ] ). even though responding to different cytokines, jaks selected by different receptors activate specific stat members for defined functions ( [ @ bib0140 ] ). there are seven mammalian stat proteins : stat1, stat2, stat3, stat4, stat5a, stat5b, and stat6, which range from 750 to 950 amino acids in polypeptide length and feature several conserved domains ( [ @ bib0330 ], [ @ bib0340 ] ). stats are latent transcription factors located in the cytoplasm until activated. each stat member responds to a defined set of cytokines ( [ @ bib0205 ], [ @ bib0270 ], [ @ bib0275 ] ). the seven stats go through similar activation processes and exhibit global conservation in function ( [ @ bib0340 ] ). in brief, ligand - mediated receptor multimerization leads to trans - phosphorylation of jaks, which then create docking sites in the receptor for stats and phosphorylate them. phosphorylated stats form homodimer or heterodimer complexes, followed by translocation into the nucleus by importins and binding to response element in dna to activate or repress transcription of a defined set of genes ( [ @ bib0340 ] ). 1. 2. stat signaling and functions { # sec0015 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - among the seven stats, stat1 and stat2 mainly mediate the ifn - activated signaling ( [ @ bib0270 ] ). stat1 is involved in signaling by type i, type ii, and type iii ifns. in response to type i ifns ( ifn - α or ifn - β ), stat1 and stat2 are phosphorylated, followed by heterodimer formation and then interaction with interferon regulatory factor 9 ( irf9 ) to form a heterotrimer known as interferon - stimulated gene factor 3 ( isgf3 ) ( [ @ bib0090 ] ) ( [ fig. 1 ] ( # fig0005 ) { ref - type = " fig " } a ). the isgf3 is translocated into nucleus and binds to interferon - stimulated response element ( isre ) in dna to activate the expression of interferon - stimulated genes ( isgs ). upon ifn - γ stimulation, activated stat1 forms homodimers, followed by nuclear translocation and activation of gene expression via binding to interferon - gamma - activated - sequence ( gas ) in dna ( [ @ bib0270 ] ). type iii ifns also activate stat1 and stat2 for isre transactivation like type i ifns ( [ @ bib0470 ] ). fig. 1prrsv interference with type i ifn - activated jak - stat signaling. a. canonical signaling. ifn - α / β binds to their receptors ifnar - 1 and ifnar - 2 on the cell membrane and activates the jak - stat1 / stat2 pathway. the phosphorylated stat1 and stat2 form heterodimer, followed by interaction with irf9 to form interferon - stimulated gene factor 3 ( isgf3 ). karyopherin α1 ( kpna1 ), an adaptor protein binding isgf3, is essential to mediate the nuclear import of isgf3 via interaction with karyopherin β1 ( kpnb1 ). the isgf3 binds to interferon - stimulated response element ( isre ) in dna to activate transcription of interferon - stimulated genes ( isgs ). " p " besides stats indicates phosphorylation. prrsv nsp1β inhibits isgf3 nuclear translocation via inducing degradation of kpna1. prrsv n protein also inhibits isgf3 nuclear translocation. prrsv nsp2 reduces isg15 production and conjugation via its deubiquitination activity. prrsv induces elevation of mirna mir - 30c to downregulate jak1 and socs1 to inhibit jaks. prrsv inhibits pkr during its early infection of pulmonary alveolar macrophages. b. stat1 - independent signaling. type i ifns activate alternative jak - stat2 signaling without stat1. the isgf3 - like complex binds to isre and interferon - gamma - activated sequence ( gas ) to activate alternative sets of isgs. prrsv reduces stat2 protein to inhibit this pathway. fig. 1 in addition to the canonical signaling described above, ifnα signaling occurs through alternative complexes containing stat2 and irf9 without stat1 ( [ @ bib0030 ], [ @ bib0110 ] ) ( [ fig. 1 ] ( # fig0005 ) { ref - type = " fig " } b ). moreover, stat2 can form heterodimer with other stats, like stat3 and stat6, followed by binding to diverse sequences, like gas. further studies demonstrate the existence of a stat1 - independent ifn signaling pathway, in which stat2 / irf9 directs a prolonged antiviral activity ( [ @ bib0025 ], [ @ bib0035 ] ). stat3 is activated by many cytokines and had multiple functions including differentiation of t helper 17 ( th17 ) and generation of cd8 ^ + ^ t cell memory response ( [ @ bib0055 ], [ @ bib0275 ], [ @ bib0380 ] ). numerous cytokines including il - 5, il - 6, il - 9, il - 10, il - 11, il - 12, il - 21, il - 22, il - 27, oncostatin m ( osm ), ifn - γ, tnf - α and leukemia inhibitory factor ( lif ), trigger stat3 activation ( [ @ bib0125 ], [ @ bib0205 ] ). the il - 6 family of cytokines including il - 6, osm, and lif bind to the receptor complex containing the common glycoprotein 130 ( gp130 ) and activate stat3, known as gp130 / jak - stat3 signaling ( [ fig. 2 ] ( # fig0010 ) { ref - type = " fig " } ). stat3 is needed for differentiation of follicular t helper and th1 cells ( [ @ bib0295 ] ), as well as activation and maturation of dendritic cells ( dcs ) ( [ @ bib0285 ] ). mutations in stat3 cause autosomal dominant hyper - ige syndrome, a rare multisystem primary immunodeficiency characterized by recurrent bacterial infections in skin and lung and with abnormally high levels of ige ( [ @ bib0160 ], [ @ bib0245 ] ). stat3 is indispensable for promoting host defense against virus infections. for instance, during herpes simplex virus - 1 ( hsv - 1 ) infection, stat3 promotes the activation of cd8 + t cells response ( [ @ bib0460 ] ). it has been shown that gp130 - stat3 signaling is critical for the innate immune response against coxsackievirus b3 virus ( cvb3 ) infection ( [ @ bib0450 ] ). in addition, stat3 plays a protective role in regulating virus - induced proinflammatory response, as shown in stat3 knock - out studies ( [ @ bib0100 ], [ @ bib0195 ], [ @ bib0240 ] ). highly pathogenic avian influenza ( hpa | 1. Introduction {# sec0005} = = = = = = = = = = = = = = = Cytokines are a broad and loose group of small cell - signaling proteins that play important roles in cell growth, proliferation, differentiation, apoptosis, angiogenesis, immunity and inflammatory response. They include interferons (IFN ), interleukins (IL ), chemokines, lymphokines, and tumor necrosis factors (TNF ). They are produced by a variety of cells, including macrophages, B and T lymphocytes, mast cells, endothelial cells, epithelial cells, fibroblasts, and various stromal cells. Cytokines that are produced at the site of infection stimulate and coordinate innate and adaptive immune responses against invading pathogens ([ @ bib0135 ], [@ bib0185 ], [@ bib0365] ). They act through their matching receptors on the surface of target cells, followed by cascades of intracellular signaling. One such frequently activated intracellular signaling is the JAK - STAT pathway, which is indispensable and pivotal in many biological processes including immunity and inflammatory response ([ @ bib0275 ], [@ bib0340] ). Dysregulation of JAK - STAT signaling results in immunodeficiency and immune - mediated disorders ([ @ bib0270 ], [@ bib0275] ). Mutations in the components of JAK - STAT pathway cause immunodeficient and autoimmune disorders ([ @ bib0050 ], [@ bib0190] ). Due to the importance of JAK - STAT signaling in the host immune response, it is often targeted by pathogens, including PRRSV ([ @ bib0290 ], [@ bib0400 ], [@ bib0405] ). PRRSV causes a contagious disease that is characterized by reproductive failure in sows and respiratory disease of variable severity in pigs of all ages ([ @ bib0235] ). PRRS has caused substantial economic losses to the swine industry and remains one of the most economically important diseases in pigs since it was first reported in 1987 ([ @ bib0165 ], [@ bib0265] ). A $7pical feature of the immune response to PRRSV infection in pigs is delayed production and low titer of giGus neutralizing antibodies, and weak cell - mediated immune response ([ @ bib0210 ], [@ bib0235 ], [@ bib0435] ). PRRSV infection is also characterized by prolonged viremia followed by the persistent presence in regional lymph nodes for as long as 250 days ([ @ bib0425] ). One of the possible reasons for the weak protective immune response is that PRRSV interferes with innate immunity, including type I interferons (IFNs ), and cytokine - mediated JAK - STAT signaling ([ @ bib0010 ], [@ bib0045 ], [@ bib0290 ], [@ bib0350 ], [@ b7b0505 ], [@ bib0395] ). 1. 1. JAKs and STATs {# sec0010} - - - - - - - - - - - - - - - - - - - In mammals, there are four JAKs: JAK1, JAK2, JAK3, and Tyk2, ranging in size from 120 to 140 kDa ([ @ bib0140] ). JAK1, JAK2, and Tyk2 are ubiquitously expressed, whereas JAK3 expression is restricted to cells of the hematopoietic system. The JAK protein is pre - associated with cytokine receptors in the dytoplasmKc side and is an important determinant of their levels and signaling potential ([ @ bib0140] ). Upon cytokine binding, the receptor chains are brought into close proximity, leading to the juxtaposition of two JAK kinase domains and consequent trans - phosphorylation. Once activated, JAKs phosphorylate STAT proteins via Src homology 2 (SH2) domain interaction ([ @ bib0155] ). Even though responding to different cytokines, JAKs selected by different receptors activate specific STAT members for defined functions ([ @ bib0140] ). There are seven mammalian STAT proteins: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6, which range from 750 to 950 amino acids in polypeptide length and feature sevs5al conserved domains ([ @ bib0330 ], [@ bib0340] ). STATs are latent transcription factors located in the cytoplasm until activated. Each STAT member responds to a defined set of cytokines ([ @ bib0205 ], [@ bib0270 ], [@ bib0275] ). The seven STATs go through similar activation processes and exhibit global conservation in function ([ @ bib0340] ). In brief, ligand - mediated receptor multimerization leads to trans - phosphorylation of JAKs, which then create docking sites in the receptor for STATs and phosphorylate them. Phosphorylated STATs form homodimer or heterodimer complexes, followed by translocation into the nucleus by importins and binding to response element in DNA to activate or repress transcription of a defined set of genes ([ @ bib0340] ). 1. 2. STAT signaling and functions {# sec0015} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Among the seven STATs, STAT1 and STAT2 mainly mediate the IFN - activated signaling ([ @ bib0270] ). STAT1 is involved in signaling by type I, type II, and type III IFNs. In response to type I IFNs (IFN - α or IFN - β ), STAT1 and STAT2 are phosphorylated, followed by heterodimer formation and then interaction with interferon regulatory factor 9 (IRF9) to form a heterotrimer known as interferon - stimulated gfnS factor 3 (ISGF3) ([ @ bib0090] ) ([ Fig. 1] (# fig0005) {ref - type = " fig "} A ). The ISGF3 is translocated into nucleus and binds to interferon - stimulated response element (ISRE) in DNA to activate the expression of interferon - stimulated genes (ISGs ). Upon IFN - γ stimulation, activated STAT1 forms homodimers, followed by nuclear translocation and activation of gene expression via binding to inFerfeEon - gamma - activated - sequence (GAS) in DNA ([ @ bib0270] ). Type III IFNs also activate STAT1 and STAT2 for ISRE transactivation like type I IFNs ([ @ bib0470] ). Fig. 1PRRSV interference with type I IFN - activated JAK - STAT signaling. A. Canonical signaling. IFN - α / β binds to their receptors IFNAR - 1 and IFNAR - 2 on the cell membrane and activates the JAK - STAT1 / STAT2 pathway. The phosphorylated STAT1 and STAT2 form h#terodim#r, followed by interaction with IRF9 to form interferon - stimulated gene factor 3 (ISGF3 ). Karyopherin α1 (KPNA1 ), an adaptor protein binding ISGF3, is essential to mediate the nuclear import of ISGF3 via interaction with karyopherin β1 (KPNB1 ). The ISGF3 binds to interferon - stimulated response element (ISRE) in DNA to activate transcription of interferon - stimulated genes (ISGs ). " P " besides STATs indicates phosphorylation. PRRSV nsp1β inhibits ISGF3 nuclear translocation via inducing degradation of KPNA1. PRRSV N protein also inhibits ISGF3 nuclear translocation. PRRSV nsp2 reduces ISG15 production and conjugation via its deubiquitination activity. PRRSV induces elevation of miRNA miR - 30c to downregulate JAK1 and SOCS1 to inhibit JAKs. PRRSV inhibits PKR during its early infection of pulmonary alveolar macrophages. B. STAT1 - independent signaling. Type I IFNs activate alternative JAK - STAT2 signaling without STAT1. The ISGF3 - like complex binds to ISRE and interferon - gamma - acY8vated sequence (GAS) to activate alternative sets of ISGs. PRRSV reduces STAT2 protein to inhibit this pathway. Fig. 1 In addition to the canonical signaling described above, IFNα signaling occurs through alternative complexes containing STAT2 and IRF9 w7tUout STAT1 ([ @ bib0030 ], [@ bib0110] ) ([ Fig. 1] (# fig0005) {ref - type = " fig "} B ). Moreover, STAT2 can form heterodimer with other STATs, like STAT3 and STAT6, followed by binding to diverse sequences, like GAS. Further studies demonstrate the existence of a STAT1 - independent IFN signaling pathway, in which STAT2 / IRF9 directs a prolonged antiviral activity ([ @ bib0025 ], [@ bib0035] ). STAT3 is activated by many cytokines and had multiple functions including differentiation of T helper 17 (Th17) and generation of CD8 ^ + ^ T cell memory response ([ @ bib0055 ], [@ bib0275 ], [@ bib0380] ). Numerous cytokines including IL - 5, IL - 6, IL - 9, IL - 10, IL - 11, IL - 12, IL - 21, IL - 22, IL - 27, oncostatin M (OSM ), IFN - γ, TNF - α and leukemia inhibitory factor (LIF ), trigger STAT3 activation ([ @ bib0125 ], [@ bib0205] ). The IL - 6 family of cytokines including IL - 6, OSM, and LIF bind to the receptor complex containing the common glycoprotein 130 (gp130) and activate STAT3, known as gp130 / JAK - STAT3 signaling ([ Fig. 2] (# fig0010) {ref - type = " fig "} ). STAT3 is needed for differentiation of follicular T helper and Th1 cells ([ @ bib0295] ), as well as activation and maturation of dendritic cells (DCs) ([ @ bib0285] ). Mutations in STAT3 cause autosomal dominant hyper - IgE syndrome, a rare multisystem primary immunodeficiency characterized by recurrent bacterial infections in skin and lung and with abnormally high levels of IgE ([ @ bib0160 ], [@ bib0245] ). STAT3 is indispensable for promoting host defense against virus infections. For instance, during Herpes simplex virus - 1 (HSV - 1) infection, STAT3 promotes the activation of CD8 + T cells response ([ @ bib0460] ). It has been shown that gp130 - STAT3 signaling is critical for the innate immune response against coxsackievirus B3 virus (CVB3) infection ([ @ bib0450] ). In addition, STAT3 plays a protective role in regulating virus - induced proinflammatory response, as shown in STAT3 knock - out studies ([ @ bib0100 ], [@ bib0195 ], [@ bib0240] ). Highly pathogenic avian influenza (HPA | 1. Introduction {#sec0005} =============== Cytokines a broad and loose group of small cell-signaling proteins that play important roles in cell growth, proliferation, differentiation, apoptosis, immunity and inflammatory response. They include interferons (IFN), interleukins (IL), chemokines, lymphokines, and tumor necrosis factors (TNF). They are by a variety of cells, including macrophages, B and T lymphocytes, mast cells, endothelial cells, epithelial cells, fibroblasts, and stromal cells. Cytokines that are produced the site stimulate coordinate innate adaptive immune responses against invading pathogens ([@bib0135], [@bib0365]). They act through their matching receptors on the surface of target cells, followed by of intracellular signaling. One frequently activated intracellular signaling is the JAK-STAT pathway, which indispensable and pivotal in many biological processes including immunity and inflammatory response ([@bib0275], [@bib0340]). of JAK-STAT signaling results in immunodeficiency and immune-mediated disorders ([@bib0270], [@bib0275]). Mutations in the components of JAK-STAT pathway cause immunodeficient and autoimmune disorders ([@bib0050], [@bib0190]). Due to the importance of JAK-STAT signaling in the immune response, it is targeted pathogens, including PRRSV ([@bib0290], [@bib0400], [@bib0405]). causes a contagious disease that is characterized by reproductive failure sows and of variable severity in pigs of all ages ([@bib0235]). PRRS caused substantial economic losses to the swine industry and remains one the most economically important diseases in pigs since it was reported in 1987 ([@bib0165], [@bib0265]). A feature of to PRRSV infection in pigs is delayed production and low titer virus neutralizing and weak cell-mediated immune response ([@bib0210], [@bib0235], [@bib0435]). is also by viremia followed by the presence in regional nodes for as long as 250 days ([@bib0425]). of the possible reasons for the weak protective immune response is that interferes with innate immunity, type I interferons (IFNs), cytokine-mediated JAK-STAT signaling ([@bib0010], [@bib0045], [@bib0350], [@bib0405], [@bib0395]). 1.1. JAKs and STATs {#sec0010} ------------------- In mammals, there are four JAKs: JAK1, JAK2, JAK3, and Tyk2, ranging in from to 140 kDa ([@bib0140]). JAK1, and Tyk2 are ubiquitously expressed, whereas JAK3 is restricted to cells of the system. The JAK protein is pre-associated with cytokine receptors in the cytoplasmic side and is an important determinant their levels signaling potential ([@bib0140]). Upon cytokine binding, the receptor chains are brought into close proximity, leading to the juxtaposition of two JAK kinase domains and consequent trans-phosphorylation. Once activated, JAKs phosphorylate STAT proteins via Src homology 2 (SH2) domain interaction ([@bib0155]). Even though responding to different cytokines, JAKs selected by different receptors activate specific STAT members for defined functions ([@bib0140]). There are seven mammalian STAT proteins: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6, which range from 750 to 950 amino acids in polypeptide length and several conserved domains ([@bib0330], [@bib0340]). STATs are latent transcription factors located in the cytoplasm until activated. Each STAT responds to defined set of cytokines ([@bib0205], [@bib0270], [@bib0275]). The seven STATs go through similar activation processes and exhibit global conservation in ([@bib0340]). In brief, ligand-mediated multimerization leads to trans-phosphorylation of JAKs, which then create docking sites in the receptor for STATs and phosphorylate them. Phosphorylated STATs form homodimer or heterodimer complexes, followed translocation into the nucleus by importins and binding to response element in DNA to activate or transcription of a defined set genes ([@bib0340]). 1.2. STAT signaling and --------------------------------- Among the seven STATs, STAT1 mainly mediate the IFN-activated signaling ([@bib0270]). STAT1 involved in signaling by type I, type II, type III IFNs. In response to type I IFNs (IFN-α or IFN-β), STAT1 and STAT2 are phosphorylated, followed by heterodimer formation and then interaction with interferon regulatory factor 9 (IRF9) to form a heterotrimer known as gene 3 (ISGF3) ([@bib0090]) ([Fig. 1](#fig0005){ref-type="fig"} The ISGF3 is translocated into nucleus and binds to response element (ISRE) in DNA to the expression of interferon-stimulated genes Upon IFN-γ stimulation, activated STAT1 forms followed by nuclear translocation and activation of gene via binding to interferon-gamma-activated-sequence in DNA ([@bib0270]). Type III IFNs also activate STAT1 and STAT2 for ISRE transactivation like type I IFNs ([@bib0470]).Fig. 1PRRSV interference type I IFN-activated signaling. A. Canonical signaling. IFN-α/β binds to their receptors IFNAR-1 and IFNAR-2 on the cell membrane and the JAK-STAT1/STAT2 pathway. The phosphorylated STAT1 and STAT2 form heterodimer, followed by interaction with IRF9 to form interferon-stimulated gene factor 3 (ISGF3). Karyopherin (KPNA1), an adaptor protein binding ISGF3, is essential to mediate the nuclear import of via interaction with karyopherin β1 (KPNB1). The ISGF3 binds to interferon-stimulated response element (ISRE) in DNA activate of interferon-stimulated genes (ISGs). besides STATs indicates phosphorylation. PRRSV nsp1β inhibits ISGF3 nuclear translocation via inducing degradation of KPNA1. PRRSV N protein also ISGF3 nuclear translocation. PRRSV nsp2 reduces ISG15 production and conjugation via its deubiquitination activity. PRRSV induces elevation of miRNA miR-30c to downregulate JAK1 and SOCS1 to inhibit JAKs. PRRSV inhibits PKR during its early infection of pulmonary alveolar macrophages. B. STAT1-independent Type I IFNs alternative JAK-STAT2 signaling without STAT1. The ISGF3-like complex binds to and interferon-gamma-activated sequence to activate alternative sets of ISGs. PRRSV reduces protein to inhibit this pathway.Fig. 1 In addition to the canonical signaling described above, IFNα signaling occurs through alternative complexes STAT2 IRF9 without STAT1 ([@bib0030], [@bib0110]) 1](#fig0005){ref-type="fig"}B). Moreover, STAT2 can form heterodimer with other STATs, STAT3 and STAT6, followed by binding to diverse sequences, like GAS. Further studies demonstrate the existence of a STAT1-independent IFN pathway, in which STAT2/IRF9 directs a prolonged antiviral [@bib0035]). STAT3 is activated by many and had multiple functions including differentiation of T helper 17 (Th17) and generation of CD8^+^ T cell memory ([@bib0055], [@bib0275], [@bib0380]). Numerous cytokines including IL-5, IL-6, IL-9, IL-10, IL-11, IL-12, IL-21, IL-22, IL-27, oncostatin (OSM), IFN-γ, TNF-α and leukemia inhibitory factor (LIF), STAT3 ([@bib0125], [@bib0205]). The IL-6 family of cytokines including IL-6, OSM, and LIF bind to the complex containing the common glycoprotein 130 (gp130) and activate known as signaling ([Fig. ). STAT3 is needed differentiation of follicular T and Th1 ([@bib0295]), well as activation maturation of dendritic cells (DCs) ([@bib0285]). Mutations in STAT3 cause autosomal dominant hyper-IgE syndrome, rare multisystem primary immunodeficiency characterized by recurrent bacterial infections in skin and and with abnormally high levels of IgE ([@bib0160], [@bib0245]). STAT3 is indispensable for promoting host defense against virus For instance, during Herpes virus-1 infection, STAT3 the activation of CD8+ T cells response ([@bib0460]). It has been shown that gp130-STAT3 is critical for innate immune response against coxsackievirus B3 (CVB3) infection ([@bib0450]). In addition, STAT3 plays a protective role in regulating proinflammatory as shown STAT3 knock-out studies ([@bib0100], [@bib0195], [@bib0240]). Highly pathogenic avian influenza (HPA | 1. INTRoDUctiON {#SEC0005}
===============
CytOKInES ARE a bRoad ANd lOoSe GROUP of smalL cell-sIgNaLIng pRotEins That plaY ImPOrTAnt rOLES IN CeLL grOWTH, PrOlIFerATion, DIfFeRenTIATIon, aPOPtOSiS, ANgioGEneSiS, immUnItY anD InFlAMMATory rESPOnSe. THEy inCLUDe InTeRFERONs (ifn), InTERLeukINS (IL), CHEMoKINes, lymPHOKINes, ANd tuMoR neCRoSis fACtOrS (TNf). ThEY are pRODuCed By a vaRiEty oF CeLLS, INclUdInG MAcRoPhages, b ANd t LymPHocytES, masT celLS, enDOtHELial ceLlS, EPITHeLiAl celLs, fibrOBlAsts, anD vARiOUs STroMaL CEllS. cyToKINEs tHAT ArE prODUCeD aT tHE sITE oF INFEctioN sTimuLatE anD coorDInate innAte And adapTiVE IMMUNE ResPonses AgaINSt invadiNg paTHogENS ([@BIB0135], [@BiB0185], [@biB0365]). TheY AcT THROUgh THEIR maTching reCEPTOrs On THe surFacE Of TARgET cELlS, FoLLoWeD bY cAscAdes OF iNTrAcelluLAr sIgNAlinG. onE SUCH FReQUenTLy acTIvateD IntRaCeLLulAr SIgNAlINg Is the Jak-stat PAthwAY, WHIcH IS INDiSpensablE ANd PiVoTAL in manY BiOLoGICal pROCesses InCludiNg immuNity aNd inFLAMmAtoRY rESPOnsE ([@bIB0275], [@Bib0340]). dYsRegulatioN oF JAk-StAt sIgNAling reSulTS in IMMuNodeFiCIEnCY AnD immuNE-MediATed DIsOrDers ([@bib0270], [@bIb0275]). mUtAtiONS in thE COmpoNENTs of JAk-stAt pathwaY cAuSe iMmuNOdEfiCIEnT aND autOImMuNe DISOrDERS ([@bib0050], [@bIB0190]). due TO the ImPoRTancE OF JaK-STat SigNALIng IN THE HOst immune rESPOnse, It Is oFten tarGETed by pAThOgeNs, IncLUdINg prRsv ([@BiB0290], [@BIb0400], [@BiB0405]).
PRrsV caUSEs A cOnTAgIOUs DiSeAse tHAt IS CHaracTeRIzEd By REprodUCTIve FAIlurE in SOWs And ReSPiRatOrY dISEASE oF vAriAbLe SeVEriTY in pIgs OF all AGEs ([@bIB0235]). pRRS Has CAUsed SUBstaNtial eCoNoMIC LOsses tO thE SWIne IndUsTRy And REmaiNS oNe of the MOsT eCONOmICALLy iMPORtaNt dIseaSES iN PIGs SINcE It Was fIRST RePORtED in 1987 ([@Bib0165], [@biB0265]). A tYpicAL FEAtUre of The ImmuNe RespONse tO pRrSV INFECTIoN iN piGS Is deLaYed PRODucTiON AND Low TiTer Of VirUS neuTrAliZINg aNtibodiES, ANd wEAk Cell-MeDiaTeD ImmUNe resPonsE ([@bIb0210], [@bIB0235], [@Bib0435]). PrrSV INfectioN IS Also CharACTEriZEd BY pROLOnGeD VIRemIa FollOWeD By the PeRsiSTenT PrEsencE IN RegiOnAL LYMph NOdEs FoR as LOnG as 250 DaYS ([@biB0425]). oNe OF the PossIblE reAsOns for tHE wEak pRotECtiVE IMMUne rESponsE is tHat PRRsV inTeRFERes WiTH innAte ImmUnITy, INcLudiNG tyPE i INTeRfERoNS (ifNs), aND cyTOKIne-MEdIAteD JAK-stat signaLinG ([@biB0010], [@Bib0045], [@BIb0290], [@bIB0350], [@Bib0405], [@BIB0395]).
1.1. JAKS and STatS {#Sec0010}
-------------------
iN maMMals, tHERE aRe FoUR Jaks: jAk1, JAK2, Jak3, ANd tYK2, RAnGING In SizE frOM 120 TO 140 kDA ([@Bib0140]). JAk1, JAk2, aNd Tyk2 Are uBiquITOUSLY eXPresSED, WHerEAS JAK3 eXprEssIon Is ReSTRiCTED TO CeLlS of The HEmaTOpOiETIc SysTem. tHe JAK PROtEIn is pRE-aSSoCiated wiTH CYTokINE RecEPtOrS iN The CyToPlASmic sIdE aND iS AN iMpORTant DEterMinANT of TheIR lEveLs ANd SignALING POteNTIal ([@BIb0140]). UpON cYTokIne bInDING, thE REcEptoR CHAins ArE bRoUgHt INto CLOse pROxIMiTy, LEAdING To THe JUXtAPoSItIon of TwO JaK kiNASE DOMaiNs aND COnSEQUenT TRanS-pHosphOrYLatiON. ONcE aCtIVAtED, JAks PHoSpHorYlAte stat PRotEinS VIA sRC hoMOLOgy 2 (SH2) DomaIn InteRACtiOn ([@bib0155]). evEn ThOUGh REsPONDing To DifFerEnt CYTOKiNes, Jaks sElECTeD By difFEreNT REceptorS aCtIVaTe sPeCiFIC Stat MEmbeRS FoR defINEd fUNcTIOns ([@BIB0140]).
there Are SeveN MAmmalIaN STat ProTEiNs: stat1, staT2, sTaT3, sTat4, stat5a, stAt5B, ANd STat6, whiCh RaNGE fRoM 750 tO 950 Amino aCiDS iN poLyPeptIdE LeNgTH AND fEAtURe sEveRaL CONsERved DOMaiNs ([@BIb0330], [@bIb0340]). sTatS are LAtENt tRANScRIptIOn fACTOrS LocAted in the CytoPLASM uNtil ActIvATeD. EACh stat MEMBER resPOndS TO A DEFInEd SeT OF cYTokInES ([@BiB0205], [@biB0270], [@BIB0275]). The SeVEN sTaTs go tHrOUgh SImiLAr activATiON PRoCeSses and ExHIBIT glOBal cOnsErVATIOn iN FUNctiOn ([@bib0340]). in BRiEf, ligAnD-MedIatED RecePTor MuLTIMeRizATIon LEAds TO trans-PHOSPHoryLATiON oF JAKs, wHich ThEN CReATE dOCkiNg sItes in The ReCEPTOR foR stATs anD phOSphoRYLATE THeM. pHoSPHOrylATeD sTAts fORM HOMoDIMEr or hETeroDiMeR comPleXeS, follOWED BY TRaNSlocatIOn Into the NucLeUs BY ImPORtins anD bIndInG To reSPOnSe ELEmeNT IN DnA To ACTIVate Or REPresS TrAnSCRipTION OF a DefINed SET Of geNES ([@BiB0340]).
1.2. STAt SIgnALiNg aND funCTiOnS {#SEC0015}
---------------------------------
aMong thE sevEn StATs, StaT1 and sTat2 maINlY medIatE The IfN-aCtIVated SIgnAling ([@BIB0270]). STAT1 is InVolVeD In SigNAling By tYPe I, TypE Ii, And tYPe IiI IfNS. in reSPonSe to tyPe I ifNs (iFn-α OR ifN-Β), staT1 AND sTAT2 aRE pHOSPHORYlAtEd, foLLoweD BY heterODImeR fOrMaTION AND tHEn InTeRacTion wIth iNTeRFEron reGUlATOry factOr 9 (Irf9) To fOrm a HeTErOTRiMeR kNOWN aS INteRfErON-stIMUlatEd geNe fAcTOr 3 (ISGF3) ([@biB0090]) ([fig. 1](#fig0005){reF-TYPE="fIg"} A). the IsGF3 IS TrANSLOcATeD INtO nUcleuS aND bINDS to iNtErFeRON-StiMUlAted reSPoNSE ELEmEnT (isrE) in DNA TO actiVaTE ThE EXpreSSIon OF intErfeRon-sTiMulAteD geNEs (Isgs). UPoN ifN-γ stIMULaTiON, ActIvATeD sTat1 formS HoMoDimErS, foLloWed BY NUCLEAR TrAnSLOCAtIon ANd ACtIVatiOn of gEnE EXprEssiOn Via bINding to INTErFeRoN-gamMA-ACtivAtEd-SEqUenCE (gAs) IN DNa ([@BIB0270]). TyPe iiI iFNs alSO acTIVATe StAT1 and stAt2 for isRe trAnsACtiVatIon lIkE type I ifnS ([@BIB0470]).FIg. 1PrrsV iNTeRFereNcE wItH tYPe I IFN-ACtIvAteD jaK-STAt SIgnALIng. a. cANONiCAl sIgNaLING. IFn-Α/Β biNDS tO ThEir recePToRs Ifnar-1 aNd ifNAr-2 on THe cEll MembrANE AND aCtIVaTes tHE jak-stAt1/StAt2 patHwAy. tHE PHoSPhOryLATed stAt1 aND stAT2 FOrM hEteRodImEr, FOLlowEd BY iNtErACTioN wITH IrF9 TO FoRm intErFERoN-STIMULAteD GeNE FActoR 3 (IsGF3). KAryoPHeRIN Α1 (kpnA1), aN adaPToR pROtEIN BiNDING ISgf3, Is esseNtiAL To MediATE tHe nUcLeAR impORt oF isGf3 viA iNtEraCTiON WiTH kAryopHErIn β1 (kPNB1). THe ISGf3 bINDs To inTerFeRon-sTimulAtED respOnSe eLeMENt (isre) In DNA To ACTivAte TraNScRIpTIOn of INtERFErON-StImULatEd GEneS (IsGs). "P" BEsidES staTS INdIcaTes PhOSpHORYLAtiON. PrRSv NsP1Β InHIBITS isGf3 NucLEAR trAnsloCATiON viA iNduCing dEgrADATIOn OF kPna1. PRrSV n PrOtEIn ALso inHiBItS isGf3 NUCLear TraNSLOCATioN. pRrsv NSp2 ReDUCEs ISg15 ProductIon AnD COnJugaTIon Via Its dEUbIQuItInaTIon aCTIVity. PrRsV INdUcES eLEVaTiON of MIrNA MiR-30c tO DoWNregULAte jAk1 anD SocS1 to INhiBIT JaKS. PrRSv inHIbIts Pkr duRINg iTS EaRLy iNFectIon Of pulmoNary AlveOlAr mACrOPhagES. B. stat1-INDepeNdeNT sIGnaLiNG. tYpE I Ifns aCTiVatE altERnatiVe jaK-sTAT2 signAling wiTHOuT StAt1. THe Isgf3-liKe COmplex biNds TO isre and INTerfERon-gaMMa-ActivaTED SeqUENCE (Gas) To ACTivAte ALtErNAtIVE seTs of Isgs. PrrSV rEDUcEs sTat2 ProtEIn TO iNHiBit tHiS patHwAy.fIg. 1
in addItiON TO tHe Canonical SIGNaLINg deScRiBed AbovE, IfNα SIGNAlING OccUrS THrOugH aLTErnATIVe coMPLEXES coNTAining STAT2 And iRf9 wiThOuT stAT1 ([@Bib0030], [@bIb0110]) ([fIg. 1](#FIG0005){ReF-tYpe="fIG"}b). MOREOvER, sTaT2 CAN FORm HETEROdiMer wITH OthER STATS, LIKE sTaT3 AnD staT6, follOWEd By bINDInG tO divErSE SeQuEnCeS, like GAS. FURTheR StudIEs dEmOnsTrATe THE eXIsTeNcE Of A STat1-inDEPEndENT ifn SIgnaLING pAThwaY, IN whIcH stAT2/IRF9 DIrEcTS A PROLoNgeD AntIViRAL ActIviTY ([@bib0025], [@bIB0035]).
STat3 is acTivAtED by MaNY cYtOkinEs AnD Had MulTipLE FunCtioNS InCluding dIfferENtIation Of t heLPer 17 (tH17) And gENeRaTiOn oF cD8^+^ t CEll MEmoRY RESpoNSe ([@BiB0055], [@bIB0275], [@bIB0380]). nUmeROus cYTOKInes INCLudING iL-5, IL-6, Il-9, il-10, iL-11, IL-12, il-21, iL-22, IL-27, oNcOsTATiN M (oSM), IFN-γ, TNF-α anD LEuKEMiA INhibiTorY factor (LIF), triGger STAt3 AcTivAtIon ([@BIb0125], [@BIB0205]). The Il-6 famILy Of cYTOkInES iNClUDinG il-6, OsM, AND LIF BIND to THe RECEPTOr CoMpleX ConTAiNiNg THE ComMon glyCOpROtEin 130 (GP130) AND aCtiVaTe staT3, known AS Gp130/JAk-sTAt3 sIgNAliNG ([FIG. 2](#fIG0010){ReF-tYPe="fIg"} ). sTat3 is neEded fOR dIFfErENtiAtIon oF FolLiCular t HelpeR and th1 ceLlS ([@BiB0295]), AS well AS ACtIVAtIon ANd maTurATIon of dEndRitIc ceLLs (dcs) ([@biB0285]). MutaTiOnS IN STAt3 cAuse AUtOSoMAL DOmINaNt hYPER-IGE sYNDRoMe, A RARe muLTISysteM PriMAry iMmUnODEfIcIeNcy CHARaCtERizeD BY rEcurrENT BActeRIal InFEcTIOns IN skIn AND Lung and WiTH abNorMaLLY high LeVeLs of IgE ([@bIb0160], [@BIb0245]). stAT3 is INdisPeNSAblE for ProMotINg hOST dEfEnSE agaInSt vIruS iNfectiONs. FOR InstANcE, DURInG HERpEs sIMplEX vIRus-1 (HsV-1) InFeCtIon, StAt3 PRomoTes tHe ACTIVATIOn of cd8+ T CelLS RESPONSE ([@bib0460]). It haS BEEN shoWN that gP130-STaT3 signALING Is CriTicAl foR the innATE imMUne RESponsE agaINST coxsAcKiEViRuS B3 ViRus (cvb3) inFECTiON ([@bIb0450]). IN addITIon, stAt3 plays A pRotecTIvE rOle In rEGULATIng vIruS-InDUced PrOINfLAmMAtOry RespOnsE, as shown in StAt3 kNOck-oUt STuDIeS ([@bIb0100], [@BiB0195], [@biB0240]). HIgHLy PAThoGeNiC AvIAN INFluENZa (hPA | 1. Introduction {#sec0005} =============== Cytokines are abroad and loose group of small cell-signaling proteins that play important roles incell growth,proliferation,differentiation,apoptosis, angiogenesis, immunityandinflammatory response. They include interferons (IFN), interleukins (IL), chemokines, lymphokines, and tumornecrosis factors (TNF). They are producedby a variety ofcells, including macrophages,B and T lymphocytes, mast cells, endothelial cells, epithelial cells,fibroblasts, and various stromal cells.Cytokines that areproduced at the site of infection stimulate and coordinate innate and adaptive immune responses against invading pathogens ([@bib0135], [@bib0185], [@bib0365]). They act through their matching receptors on the surface of target cells, followed by cascades of intracellular signaling. One suchfrequentlyactivated intracellular signaling is the JAK-STAT pathway, which is indispensable and pivotalin manybiological processesincluding immunityand inflammatory response ([@bib0275], [@bib0340]). Dysregulation of JAK-STAT signaling resultsin immunodeficiency andimmune-mediated disorders ([@bib0270], [@bib0275]).Mutations in the components of JAK-STAT pathway cause immunodeficient and autoimmune disorders ([@bib0050], [@bib0190]). Due to the importance of JAK-STAT signalingin the host immune response, it is oftentargetedby pathogens, including PRRSV ([@bib0290], [@bib0400],[@bib0405]). PRRSV causes a contagious disease that is characterized byreproductive failure insows and respiratory disease of variable severityin pigs of all ages ([@bib0235]). PRRS has caused substantial economic losses to the swine industry andremainsone of the mosteconomically important diseases in pigs since it was first reportedin 1987 ([@bib0165], [@bib0265]). A typical featureof the immune response to PRRSV infection in pigs is delayed production and low titer of virus neutralizing antibodies, and weak cell-mediated immune response([@bib0210], [@bib0235], [@bib0435]). PRRSV infectionis also characterizedbyprolonged viremiafollowed by the persistent presence in regional lymph nodes for as long as 250 days ([@bib0425]). One of the possible reasons for the weak protective immune response is that PRRSV interferes with innate immunity, including type I interferons (IFNs), and cytokine-mediated JAK-STAT signaling([@bib0010], [@bib0045], [@bib0290], [@bib0350], [@bib0405], [@bib0395]). 1.1. JAKsand STATs{#sec0010} ------------------- In mammals, there are four JAKs: JAK1, JAK2, JAK3, and Tyk2, ranging in size from 120 to 140kDa([@bib0140]).JAK1, JAK2, and Tyk2 are ubiquitously expressed, whereasJAK3 expressionis restricted to cells of the hematopoietic system. The JAK proteinis pre-associated withcytokine receptors in the cytoplasmic side and isan important determinant of theirlevels and signaling potential([@bib0140]). Upon cytokine binding, the receptor chains are brought into close proximity, leadingto the juxtaposition of two JAK kinase domains andconsequenttrans-phosphorylation. Once activated, JAKs phosphorylate STAT proteins viaSrc homology 2(SH2) domain interaction ([@bib0155]). Eventhough responding to different cytokines, JAKsselected by different receptors activate specific STAT members for defined functions ([@bib0140]). There are seven mammalian STAT proteins:STAT1,STAT2, STAT3, STAT4, STAT5A, STAT5B, andSTAT6, which range from 750 to 950 amino acids in polypeptidelength and feature several conserved domains ([@bib0330],[@bib0340]). STATs are latent transcription factors located in the cytoplasm until activated.Each STAT member respondsto a defined set of cytokines ([@bib0205], [@bib0270], [@bib0275]). The seven STATs go through similar activation processes and exhibit global conservation in function ([@bib0340]). In brief, ligand-mediated receptor multimerization leadstotrans-phosphorylation of JAKs, which thencreate docking sites in the receptor for STATs and phosphorylatethem. PhosphorylatedSTATs form homodimer or heterodimer complexes, followed by translocation into the nucleus by importins and binding to response elementin DNA to activate or repress transcription of a defined set of genes ([@bib0340]). 1.2. STATsignaling and functions {#sec0015} --------------------------------- Among the seven STATs,STAT1 and STAT2 mainly mediate the IFN-activated signaling ([@bib0270]). STAT1is involved in signaling by type I,type II, and type III IFNs. In response to type I IFNs (IFN-α or IFN-β), STAT1 andSTAT2 are phosphorylated, followed by heterodimer formation and then interactionwith interferonregulatory factor 9 (IRF9) to form a heterotrimer knownasinterferon-stimulated gene factor 3 (ISGF3) ([@bib0090]) ([Fig. 1](#fig0005){ref-type="fig"} A). The ISGF3 is translocated intonucleus andbinds to interferon-stimulated response element (ISRE) in DNAto activate the expression of interferon-stimulatedgenes (ISGs). Upon IFN-γ stimulation, activated STAT1 forms homodimers, followedby nuclear translocation and activation of gene expression via binding to interferon-gamma-activated-sequence (GAS) in DNA ([@bib0270]). TypeIII IFNs also activate STAT1 and STAT2 for ISREtransactivation like type I IFNs ([@bib0470]).Fig. 1PRRSV interference withtype I IFN-activated JAK-STAT signaling. A. Canonical signaling. IFN-α/β binds to their receptorsIFNAR-1 and IFNAR-2on the cell membrane and activates the JAK-STAT1/STAT2 pathway. The phosphorylated STAT1 and STAT2 form heterodimer, followed by interaction with IRF9 to form interferon-stimulatedgene factor3 (ISGF3). Karyopherin α1(KPNA1), an adaptorprotein binding ISGF3,is essential to mediate the nuclear import of ISGF3 via interaction with karyopherin β1 (KPNB1).The ISGF3 binds tointerferon-stimulated response element (ISRE) in DNA to activatetranscription of interferon-stimulated genes (ISGs). "P" besides STATs indicates phosphorylation. PRRSVnsp1β inhibits ISGF3 nuclear translocation via inducing degradation of KPNA1. PRRSV N protein also inhibits ISGF3 nuclear translocation. PRRSV nsp2 reducesISG15 production and conjugation via its deubiquitinationactivity. PRRSV induces elevation of miRNA miR-30c todownregulate JAK1 andSOCS1 to inhibit JAKs. PRRSV inhibits PKR during its early infection of pulmonary alveolarmacrophages. B. STAT1-independent signaling. Type IIFNs activate alternative JAK-STAT2 signaling without STAT1. The ISGF3-like complexbinds to ISRE and interferon-gamma-activated sequence (GAS) to activate alternative sets of ISGs. PRRSVreduces STAT2 protein toinhibit this pathway.Fig. 1 In addition to the canonical signaling described above, IFNα signaling occurs through alternative complexescontaining STAT2 and IRF9 without STAT1 ([@bib0030], [@bib0110])([Fig. 1](#fig0005){ref-type="fig"}B). Moreover, STAT2 canformheterodimer with other STATs, like STAT3 and STAT6, followed by binding to diverse sequences,likeGAS. Further studies demonstrate the existence of a STAT1-independent IFN signaling pathway,in which STAT2/IRF9 directs a prolonged antiviralactivity ([@bib0025], [@bib0035]). STAT3 is activated bymany cytokines and had multiple functions including differentiationof T helper17 (Th17) and generationof CD8^+^ T cell memory response ([@bib0055], [@bib0275],[@bib0380]). Numerous cytokines including IL-5, IL-6,IL-9,IL-10, IL-11, IL-12, IL-21, IL-22,IL-27, oncostatin M (OSM), IFN-γ, TNF-α and leukemia inhibitory factor (LIF), trigger STAT3activation ([@bib0125], [@bib0205]). The IL-6 family of cytokinesincluding IL-6, OSM, and LIFbind to thereceptor complex containing the common glycoprotein 130(gp130) and activate STAT3, known asgp130/JAK-STAT3 signaling ([Fig.2](#fig0010){ref-type="fig"} ). STAT3is needed for differentiation of follicular T helper and Th1 cells ([@bib0295]),as well as activation and maturationof dendritic cells (DCs) ([@bib0285]). Mutations in STAT3 causeautosomaldominanthyper-IgE syndrome, arare multisystem primary immunodeficiency characterized by recurrent bacterial infections in skin and lung and with abnormally high levels of IgE ([@bib0160],[@bib0245]). STAT3 is indispensable for promoting host defenseagainst virus infections.For instance, during Herpes simplex virus-1 (HSV-1) infection, STAT3 promotes theactivation ofCD8+ T cells response ([@bib0460]). It has been shownthat gp130-STAT3signaling is critical for the innate immune response against coxsackievirus B3 virus (CVB3) infection ([@bib0450]). In addition,STAT3 plays a protective role in regulating virus-induced proinflammatoryresponse, as shown in STAT3 knock-out studies ([@bib0100], [@bib0195], [@bib0240]). Highlypathogenic avian influenza (HPA | 1. Introduction {#sec0005} _===============_ Cytokines are a broad and loose group of small cell-signaling proteins _that_ play important roles in cell _growth,_ proliferation, _differentiation,_ apoptosis, angiogenesis, immunity _and_ _inflammatory_ response. They include interferons (IFN), interleukins (IL), chemokines, _lymphokines,_ and tumor necrosis factors (TNF). They are _produced_ _by_ a _variety_ of cells, including _macrophages,_ B and T lymphocytes, mast _cells,_ endothelial cells, epithelial _cells,_ fibroblasts, _and_ various stromal cells. Cytokines that are produced at the site _of_ infection stimulate _and_ coordinate innate and adaptive _immune_ _responses_ _against_ invading pathogens ([@bib0135], _[@bib0185],_ [@bib0365]). They _act_ through their matching receptors on the surface _of_ target _cells,_ followed by cascades of _intracellular_ _signaling._ One such frequently _activated_ intracellular signaling is the JAK-STAT pathway, which _is_ indispensable and _pivotal_ in many biological processes including _immunity_ and inflammatory response ([@bib0275], [@bib0340]). _Dysregulation_ of _JAK-STAT_ signaling results in immunodeficiency _and_ _immune-mediated_ disorders ([@bib0270], [@bib0275]). Mutations in the _components_ of _JAK-STAT_ pathway _cause_ immunodeficient _and_ autoimmune disorders _([@bib0050],_ [@bib0190]). Due _to_ the _importance_ of JAK-STAT signaling in the host immune response, it is _often_ targeted _by_ pathogens, including PRRSV ([@bib0290], [@bib0400], [@bib0405]). PRRSV causes a contagious disease that is characterized by reproductive failure in sows and respiratory disease of variable _severity_ in pigs of all _ages_ _([@bib0235])._ PRRS has caused substantial _economic_ losses to _the_ swine industry _and_ remains one of _the_ most _economically_ important diseases in pigs since it was first _reported_ _in_ 1987 _([@bib0165],_ [@bib0265]). _A_ typical _feature_ of the _immune_ response to PRRSV infection _in_ pigs is delayed production _and_ _low_ titer of virus neutralizing antibodies, and weak cell-mediated immune response _([@bib0210],_ [@bib0235], _[@bib0435])._ PRRSV infection is also characterized by prolonged _viremia_ _followed_ by the persistent presence in regional _lymph_ nodes for as long as 250 days _([@bib0425])._ One of the _possible_ reasons _for_ the _weak_ protective immune _response_ is that PRRSV interferes with innate immunity, including _type_ I interferons (IFNs), and cytokine-mediated JAK-STAT signaling ([@bib0010], [@bib0045], [@bib0290], [@bib0350], [@bib0405], [@bib0395]). 1.1. _JAKs_ and STATs {#sec0010} ------------------- _In_ mammals, there are four JAKs: JAK1, JAK2, JAK3, and Tyk2, ranging in size from 120 to 140 kDa ([@bib0140]). JAK1, JAK2, and Tyk2 are _ubiquitously_ expressed, whereas _JAK3_ expression is restricted to cells _of_ the hematopoietic system. The JAK protein is pre-associated with _cytokine_ _receptors_ _in_ the cytoplasmic side and _is_ an important determinant _of_ _their_ levels and signaling potential _([@bib0140])._ Upon cytokine binding, the receptor chains _are_ brought into close proximity, leading to the _juxtaposition_ _of_ two JAK kinase domains and consequent _trans-phosphorylation._ Once activated, JAKs phosphorylate STAT _proteins_ via Src homology _2_ (SH2) domain _interaction_ _([@bib0155])._ Even though _responding_ to _different_ cytokines, JAKs selected by different receptors activate specific STAT members for defined functions ([@bib0140]). _There_ _are_ _seven_ mammalian STAT proteins: STAT1, STAT2, STAT3, STAT4, _STAT5A,_ STAT5B, and STAT6, _which_ range from 750 to 950 _amino_ _acids_ in polypeptide length and feature several conserved _domains_ ([@bib0330], [@bib0340]). STATs are _latent_ transcription factors located _in_ the cytoplasm until activated. Each STAT member responds _to_ a defined set of cytokines _([@bib0205],_ [@bib0270], [@bib0275]). The seven STATs go through similar activation processes and exhibit global _conservation_ _in_ function _([@bib0340])._ In brief, _ligand-mediated_ receptor multimerization leads _to_ trans-phosphorylation of JAKs, which then create docking sites in the receptor for _STATs_ _and_ _phosphorylate_ them. Phosphorylated STATs form homodimer _or_ heterodimer complexes, followed by translocation into _the_ nucleus by _importins_ and binding to response _element_ in DNA to activate _or_ repress transcription of a defined set of genes ([@bib0340]). 1.2. _STAT_ signaling and functions {#sec0015} --------------------------------- Among the seven STATs, STAT1 and STAT2 mainly mediate _the_ _IFN-activated_ signaling ([@bib0270]). _STAT1_ is involved in signaling by _type_ I, type _II,_ and type III IFNs. In response to type I IFNs (IFN-α or IFN-β), STAT1 and STAT2 are phosphorylated, followed by heterodimer formation and then interaction with interferon regulatory _factor_ _9_ (IRF9) to _form_ a heterotrimer known as interferon-stimulated gene factor _3_ _(ISGF3)_ ([@bib0090]) ([Fig. 1](#fig0005){ref-type="fig"} A). The _ISGF3_ is translocated into nucleus and binds to _interferon-stimulated_ response element (ISRE) in DNA to activate the expression _of_ interferon-stimulated _genes_ (ISGs). Upon _IFN-γ_ stimulation, activated STAT1 forms _homodimers,_ followed by nuclear translocation and activation of gene _expression_ via binding to interferon-gamma-activated-sequence (GAS) in DNA ([@bib0270]). Type III IFNs also activate STAT1 and STAT2 for ISRE transactivation _like_ type I _IFNs_ ([@bib0470]).Fig. 1PRRSV interference _with_ type _I_ IFN-activated JAK-STAT signaling. A. Canonical signaling. _IFN-α/β_ _binds_ to their receptors _IFNAR-1_ and IFNAR-2 on _the_ cell _membrane_ and activates the _JAK-STAT1/STAT2_ pathway. _The_ phosphorylated STAT1 _and_ STAT2 form heterodimer, followed by interaction with IRF9 to form interferon-stimulated gene factor 3 (ISGF3). Karyopherin α1 (KPNA1), _an_ adaptor protein binding _ISGF3,_ is essential to mediate the nuclear import of ISGF3 via interaction with karyopherin β1 (KPNB1). The ISGF3 binds to interferon-stimulated response element _(ISRE)_ _in_ DNA _to_ activate transcription of _interferon-stimulated_ genes _(ISGs)._ "P" besides STATs indicates phosphorylation. PRRSV _nsp1β_ inhibits ISGF3 nuclear translocation via inducing _degradation_ of KPNA1. PRRSV N protein also inhibits ISGF3 _nuclear_ translocation. PRRSV _nsp2_ reduces _ISG15_ production and conjugation _via_ its _deubiquitination_ activity. PRRSV _induces_ elevation of miRNA miR-30c _to_ _downregulate_ JAK1 _and_ _SOCS1_ to inhibit JAKs. PRRSV inhibits _PKR_ during its early _infection_ of pulmonary alveolar macrophages. B. STAT1-independent signaling. _Type_ I IFNs activate alternative JAK-STAT2 signaling _without_ STAT1. _The_ _ISGF3-like_ complex _binds_ to ISRE and interferon-gamma-activated sequence (GAS) to activate _alternative_ sets of ISGs. PRRSV reduces STAT2 protein to inhibit this pathway.Fig. _1_ In addition to the canonical signaling described _above,_ IFNα signaling _occurs_ through alternative complexes _containing_ STAT2 _and_ IRF9 without STAT1 ([@bib0030], [@bib0110]) ([Fig. _1](#fig0005){ref-type="fig"}B)._ Moreover, _STAT2_ can form heterodimer with other STATs, like STAT3 and STAT6, followed by binding to diverse sequences, like GAS. Further _studies_ demonstrate the existence of a STAT1-independent _IFN_ signaling pathway, _in_ which STAT2/IRF9 directs a prolonged antiviral activity ([@bib0025], [@bib0035]). STAT3 is activated by many cytokines and had multiple functions including differentiation of _T_ helper 17 (Th17) and generation of _CD8^+^_ T _cell_ memory response ([@bib0055], [@bib0275], [@bib0380]). _Numerous_ cytokines including IL-5, IL-6, IL-9, IL-10, IL-11, IL-12, IL-21, IL-22, IL-27, _oncostatin_ M (OSM), _IFN-γ,_ _TNF-α_ _and_ _leukemia_ _inhibitory_ factor (LIF), _trigger_ STAT3 activation ([@bib0125], [@bib0205]). The IL-6 _family_ _of_ cytokines including IL-6, OSM, and LIF bind to the receptor _complex_ containing the _common_ _glycoprotein_ 130 (gp130) _and_ activate STAT3, known _as_ gp130/JAK-STAT3 signaling ([Fig. 2](#fig0010){ref-type="fig"} ). STAT3 is needed _for_ differentiation of _follicular_ T helper _and_ Th1 cells ([@bib0295]), as well as _activation_ _and_ maturation of dendritic cells (DCs) ([@bib0285]). _Mutations_ in _STAT3_ cause autosomal dominant hyper-IgE _syndrome,_ a rare multisystem primary immunodeficiency characterized by recurrent bacterial infections in skin and lung and with _abnormally_ high _levels_ _of_ _IgE_ ([@bib0160], [@bib0245]). STAT3 _is_ indispensable for _promoting_ _host_ defense against virus infections. For instance, _during_ Herpes simplex _virus-1_ (HSV-1) infection, STAT3 promotes the _activation_ of CD8+ T cells response ([@bib0460]). It has been shown that gp130-STAT3 signaling is critical _for_ the _innate_ immune _response_ against coxsackievirus B3 _virus_ (CVB3) infection ([@bib0450]). In addition, STAT3 plays a protective role _in_ regulating virus-induced proinflammatory response, as shown in STAT3 knock-out studies ([@bib0100], [@bib0195], [@bib0240]). _Highly_ pathogenic avian influenza (HPA |
Introduction {#s1}
============
Evidence-based veterinary medicine (EVM) can be defined as 'the use of current best evidence in making clinical decisions' ([@R4]). Additionally, when making evidence-based decisions, the circumstances of the patient alongside the circumstances and values of the owner must also be taken into consideration ([@R3]). Although EVM was first mentioned in 1998 ([@R19]), it is less advanced than in the medical field in relation to the availability of synthesised evidence and the support available for the integration of evidence by clinicians into their practice ([@R10]). The first step in EVM is to identify relevant answerable questions ([@R29]), and veterinary clinicians have a crucial role in highlighting these ([@R25], [@R12]). By identifying what common species and conditions clinicians experience in practice, researchers can prioritise studies so that a large proportion of the profession will gain from future studies.
To our knowledge, few published studies describe the entire veterinary population (including both practising and non-practising members) and what species and conditions practitioners commonly encounter. A comprehensive survey of veterinarians in the UK was conducted by the Royal College of Veterinary Surgeons (RCVS) in 2010 where it was reported that the species veterinary clinicians mostly worked with were dogs, cats, horses, cattle and rabbits ([@R23]). Another study by [@R17] found that cats were more commonly seen than dogs in small and mixed animal practice in The Netherlands. Conditions seen in practice in the United States were investigated by [@R18] who found that the most common clinical finding was dental calculus followed by gingivitis from 120,000 consultations in cats and dogs. [@R16] found that the majority of clinical time in equine practice was spent on lameness and reproduction in The Netherlands.
The aim of this study was to describe the UK veterinary population, and what species and conditions veterinary clinicians think they commonly encounter in practice. A second aim was to gather data relating to how much information veterinary clinicians perceived was available for these species.
Materials and methods {#s2}
=====================
Population of interest {#s2a}
----------------------
The target population was all members of the veterinary profession within the UK. The sampling frame was the RCVS register of members. All veterinary surgeons legally practicing in the UK must be registered with the RCVS. This register incorporates individuals, including non-practicing and retired individuals, who have consented for their details to be made available to external organisations for research or marketing purposes. A questionnaire was used to collect data from individuals on this register. As a census of all individuals on the list was conducted, a sample size calculation was not carried out.
Questionnaire structure {#s2b}
-----------------------
Several methods were employed to increase response rates, including a mixed-mode survey design (utilising both paper-based and online methods) ([@R8], [@R26], [@R6]). The questionnaire was made up of 36 questions and had four main sections; a copy of the questionnaire is available on request. The questions in the first section concerned the collection of demographic information about respondents. The second section was made up of open questions requiring clinicians to nominate up to four species they most frequently encountered, and the three main conditions or complaints they thought they saw most commonly in those species with associated perceived information levels ([Fig 1](#VETREC2013101745F1){ref-type="fig"}). The other two sections are not discussed here and will appear in a separate manuscript. Questions were constructed using recommendations from several resources to optimise clarity, minimise ambiguity and to avoid leading terminology ([@R7], [@R13], [@R31], [@R14], [@R9], [@R28], [@R2], [@R6]).
{#VETREC2013101745F1}
Questionnaire development and distribution {#s2c}
------------------------------------------
Pretesting of the survey questions was carried out by researchers within the Centre for Evidence-based Veterinary Medicine (CEVM). Piloting of the survey was carried out three times (24 and 25 people, respectively, for paper version and once transferred to the online format, 8 people for online version) with a combination of private veterinarians, academic veterinarians, veterinary specialists and government veterinarians. Formatting of the questionnaire was carried out using TeleForm V.10.5.2 (Verity Inc. 2010), an automated content capture system. This programme enables scanning of completed questionnaires to facilitate entry of closed question data (open question data was manually entered) into a Microsoft Office Access V.14.0.6 (2010 Microsoft Corporation) database automatically. The software of Cvent (2011 Cvent Inc.), an online survey company, was used to construct the online version of the finalised paper questionnaire.
The questionnaires were printed on magnolia coloured paper to make them easily identifiable against white paper. White envelopes were printed with the CEVM logo and the words 'THIS IS A SCIENTIFIC RESEARCH STUDY. THIS IS NOT JUNK MAIL, AN APPEAL FOR DONATIONS OR MARKET RESEARCH' to make it distinguishable from marketing mailings. A pen, chocolate and a return postage paid envelope were included and a prize incentive was offered (£500 towards the continuing professional development course/s of choice). If participants filled in the online version, they had an extra chance of winning £50 worth of department store vouchers.
The RCVS mailing list was obtained in October 2010. An initial mailing was posted to all individuals on this list between 1st and 5th November 2010; a link to Cvent was included allowing participants to choose to complete either an online or paper version of the questionnaire. A first reminder was sent six weeks later to non-responders followed by a second copy of the questionnaire 10 weeks later for those still not responding.
Data entry {#s2d}
----------
Returned paper-based questionnaires were scanned using Teleform, with the system set to check 10 per cent of questionnaires to enable the detection of scanning errors. Questionnaires were accepted from respondents until scanning was completed (November 2011); coding of the common conditions and complaints was completed in May 2012. Responses received electronically were downloaded into a Microsoft Excel V.14.0.6 (2010 Microsoft Corporation) document from Cvent and integrated into a Microsoft Access V.14.0.6 (2010 Microsoft Corporation) database with the paper responses.
Data coding {#s2e}
-----------
Data relating to the common conditions or complaints nominated by veterinary clinicians were classified according to species and type of condition. Classification definitions were primarily based on those created by N. J. [@R24], with some modifications for suitability across all species. Species were coded according to animal or production type (see online supplementary Appendix 1). The type of condition or complaint was coded according to the category it was most relevant to in relation to either body system (eg, musculoskeletal) or topic (eg, behaviour) (see online supplementary Appendix 2). This was further broken down to another level of classification which more specifically described the nature of the problem (see online supplementary Appendix 3), resulting in two levels of classification for each condition or complaint (eg, Musculoskeletal-ligament). Additionally, the condition or complaint was coded into a 'type' according to whether it was a disease, a clinical sign the animal might be presented for, or was deemed unclassifiable (see online supplementary Appendix 4).
One researcher (MLB) coded all conditions. If conditions were unknown to the coder or required clarification, the online resource Merck Veterinary Manual ([@R20]) was used. A second veterinary resource (eg, textbook, online veterinary resource, colleagues, Google 2012) was used if the condition was not found in the first resource. A Microsoft Excel V.14.0.6 (2010 Microsoft Corporation) spreadsheet of coding was created to maintain consistency for the same complaints or conditions. At the end of the coding process, a second researcher (TDN) identified any discrepancies between similar conditions, and conferred with the first researcher (MLB).
Data management and analysis {#s2f}
----------------------------
The dataset was transferred to a Microsoft Excel V.14.0.6 (2010 Microsoft Corporation) document for data management. Frequency tables and graphs were generated in Excel and RStudio (R Core Team 2011). A posthoc sample size analysis was performed using Raosoft ([www.raosoft.com/samplesize.html](www.raosoft.com/samplesize.html)). There was a high degree of correlation between observations for perceived information level within clinician and species. In order to account for this clustering, the median perceived information level within species for each veterinarian was calculated. A χ^2^ test (excluding 'don\'t know' observations) was then used to determine if perceived information level was different between species. The level of statistical significance was set at P\<0.05. Some questions were left unanswered by participants, therefore, the number of responses per question could be less than the total number of respondents; the number of respondents per question is identified where appropriate.
This project received ethical approval from the ethics research committee at the School of Veterinary Medicine and Science at The University of Nottingham.
Results {#s3}
=======
Response rate {#s3a}
-------------
Of the 14,532 questionnaires distributed, 5407 (37 per cent) were returned. Of these: 259 were return to sender, 230 were retired veterinarians, 72 were returned blank, 3 stated that the veterinarian was deceased and 1 was blank except for one comment box. Therefore, 4842 responses (33%; CI 32% to 35%) could be used in the | introduction { # s1 } = = = = = = = = = = = = evidence - based veterinary medicine ( evm ) can be defined as ' the use of current best evidence about making clinical decisions ' ( [ @ r4 ] ). additionally, when making evidence - based decisions, the circumstances of the patient alongside the circumstances and values of the owner must also be taken into consideration ( [ @ r3 ] ). although evm was first mentioned in 1998 ( [ @ r19 ] ), it is less advanced than in the medical field in relation to the availability of synthesised evidence and the support available for the integration of evidence by clinicians into their practice ( [ @ r10 ] ). the first step in evm is to identify relevant answerable questions ( [ @ r29 ] ), and dog clinicians have a crucial role in highlighting outcomes ( [ @ r25 ], [ @ r12 ] ). by identifying what common conditions and conditions clinicians experience in practice, researchers can prioritise studies so that this large proportion of the profession will gain from future studies. to our knowledge, few published studies describe the entire veterinary population ( including both practising and non - practising vet ) and what species and conditions practitioners commonly encounter. a comprehensive survey of veterinarians in the uk was conducted by the royal college of veterinary surgeons ( rcvs ) in 2010 where it was reported that the species veterinary clinicians mostly worked with were dogs, cats, horses, cattle and rabbits ( [ @ r23 ] ). another study by [ @ r17 ] found that cats were more commonly seen than dogs in small and mixed animal practice in the netherlands. conditions seen in sheep in the united states were investigated by [ @ r18 ] who found that the most common clinical finding was dental calculus followed by gingivitis from 120, 000 visits in cats and dogs. [ @ r16 ] found that the majority of clinical time in equine practice was spent on lameness and reproduction in the netherlands. the aim of this study was to describe the uk veterinary population, and what sizes and conditions veterinary clinicians think they commonly encounter in practice. a second aim was to gather data relating to very much information veterinary clinicians perceived was available for these species. materials and methods { # s2 } = = = = = = = = = = = = = = = = = = = = = population of interest { # s2a } - - - - - - - - - - - - - - - - - - - - - - the target population was all members of the veterinary profession within the uk. the sampling frame was the rcvs register of members. all veterinary surgeons legally practicing in the uk must be registered with the rcvs. this register incorporates individuals, including non - practicing and retired individuals, who have consented for their details to be made available to external organisations for research or marketing purposes. a questionnaire was used to collect data from individuals on this register. as a census of all individuals on the list was conducted, a sample size calculation was not carried out. questionnaire structure { # s2b } - - - - - - - - - - - - - - - - - - - - - - - several methods were employed to increase response rates, including a mixed - mode survey design ( utilising both paper - based and online methods ) ( [ @ r8 ], [ @ r26 ], [ @ r6 ] ). the questionnaire was made up of 36 questions and had four main sections ; a copy of the questionnaire is available on request. the questions in the first section concerned the collection of demographic information about respondents. the second section was made up of open questions requiring clinicians to nominate up to four species they most frequently encountered, and the three main conditions or complaints they thought they saw most commonly in those species with associated perceived information levels ( [ fig 1 ] ( # vetrec2013101745f1 ) { ref - type = " fig " } ). the other two sections are not discussed here and will appear in a separate manuscript. questions were constructed using recommendations from several resources to optimise clarity, minimise ambiguity and to avoid leading terminology ( [ @ r7 ], [ @ r13 ], [ @ r31 ], [ @ r14 ], [ @ r9 ], [ @ r28 ], [ @ r2 ], [ @ r6 ] ).! [ question used to gather information on common conditions seen by veterinary clinicians ] ( vetrec - 2013 - 101745f01 ) { # vetrec2013101745f1 } questionnaire development and distribution { # s2c } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - pretesting of the survey questions was carried out by researchers within the centre for evidence - based veterinary medicine ( cevm ). piloting of the survey was carried out three times ( 24 and 25 people, respectively, for paper version and once transferred to the online format, 8 people for online version ) with a combination of private veterinarians, academic veterinarians, veterinary specialists and government veterinarians. formatting of the questionnaire was carried out using teleform v. 10. 5. 2 ( verity inc. 2010 ), an automated content capture system. this programme enables scanning of completed questionnaires to facilitate entry of closed question data ( open question data was manually entered ) into a microsoft office access v. 14. 0. 6 ( 2010 microsoft corporation ) database automatically. the software of cvent ( 2011 cvent inc. ), an online survey company, was used to construct the online version of the finalised paper questionnaire. the questionnaires were printed on magnolia coloured paper to make them easily identifiable against white paper. white envelopes were printed with the cevm logo and the words ' this is a scientific research study. this is not junk mail, an appeal for donations or market research ' to make it distinguishable from marketing mailings. a pen, chocolate and a return postage paid envelope were included and a prize incentive was offered ( £500 towards the continuing professional development course / s of choice ). if participants filled in the online version, they had an extra chance of winning £50 worth of department store vouchers. the rcvs mailing list was obtained in october 2010. an initial mailing was posted to all individuals on this list between 1st and 5th november 2010 ; a link to cvent was included allowing participants to choose to complete either an online or paper version of the questionnaire. a first reminder was sent six weeks later to non - responders followed by a second copy of the questionnaire 10 weeks later for those still not responding. data entry { # s2d } - - - - - - - - - - returned paper - based questionnaires were scanned using teleform, with the system set to check 10 per cent of questionnaires to enable the detection of scanning errors. questionnaires were accepted from respondents until scanning was completed ( november 2011 ) ; coding of the common conditions and complaints was completed in may 2012. responses received electronically were downloaded into a microsoft excel v. 14. 0. 6 ( 2010 microsoft corporation ) document from cvent and integrated into a microsoft access v. 14. 0. 6 ( 2010 microsoft corporation ) database with the paper responses. data coding { # s2e } - - - - - - - - - - - data relating to the common conditions or complaints nominated by veterinary clinicians were classified according to species and type of condition. classification definitions were primarily based on those created by n. j. [ @ r24 ], with some modifications for suitability across all species. species were coded according to animal or production type ( see online supplementary appendix 1 ). the type of condition or complaint was coded according to the category it was most relevant to in relation to either body system ( eg, musculoskeletal ) or topic ( eg, behaviour ) ( see online supplementary appendix 2 ). this was further broken down to another level of classification which more specifically described the nature of the problem ( see online supplementary appendix 3 ), resulting in two levels of classification for each condition or complaint ( eg, musculoskeletal - ligament ). additionally, the condition or complaint was coded into a ' type ' according to whether it was a disease, a clinical sign the animal might be presented for, or was deemed unclassifiable ( see online supplementary appendix 4 ). one researcher ( mlb ) coded all conditions. if conditions were unknown to the coder or required clarification, the online resource merck veterinary manual ( [ @ r20 ] ) was used. a second veterinary resource ( eg, textbook, online veterinary resource, colleagues, google 2012 ) was used if the condition was not found in the first resource. a microsoft excel v. 14. 0. 6 ( 2010 microsoft corporation ) spreadsheet of coding was created to maintain consistency for the same complaints or conditions. at the end of the coding process, a second researcher ( tdn ) identified any discrepancies between similar conditions, and conferred with the first researcher ( mlb ). data management and analysis { # s2f } - - - - - - - - - - - - - - - - - - - - - - - - - - - - the dataset was transferred to a microsoft excel v. 14. 0. 6 ( 2010 microsoft corporation ) document for data management. frequency tables and graphs were generated in excel and rstudio ( r core team 2011 ). a posthoc sample size analysis was performed using raosoft ( [ www. raosoft. com / samplesize. html ] ( www. raosoft. com / samplesize. html ) ). there was a high degree of correlation between observations for perceived information level within clinician and species. in order to account for this clustering, the median perceived information level within species for each veterinarian was calculated. a χ ^ 2 ^ test ( excluding ' don \ ' t know ' observations ) was then used to determine if perceived information level was different between species. the level of statistical significance was set at p \ < 0. 05. some questions were left unanswered by participants, therefore, the number of responses per question could be less than the total number of respondents ; the number of respondents per question is identified where appropriate. this project received ethical approval from the ethics research committee at the school of veterinary medicine and science at the university of nottingham. results { # s3 } = = = = = = = response rate { # s3a } - - - - - - - - - - - - - of the 14, 532 questionnaires distributed, 5407 ( 37 per cent ) were returned. of these : 259 were return to sender, 230 were retired veterinarians, 72 were returned blank, 3 stated that the veterinarian was deceased and 1 was blank except for one comment box. therefore, 4842 responses ( 33 % ; ci 32 % to 35 % ) could be used in the | Introduction {# s1} = = = = = = = = = = = = Evidence - based veterinary medicine (EVM) can be defined as ' the use of current best evidence in making clinical decisions ' ([ @ R4] ). Additionally, when making evidence - based decisions, the circumstances of the patient alongside the circumstances and values of the owner must also be taken into consideration ([ @ R3] ). Although EVM was first mentioned in 1998 ([ @ R19] ), it is less advanced than in the medical field in relation to the availability of synthesised evidence and the support svaioable for the integration of evidence by clinicians into their practice ([ @ R10] ). The first step in EVM is to identify relevant answerable questions ([ @ R29] ), and veterinary clinicians have a crucial role in highlighting these ([ @ R25 ], [@ R12] ). By identifying what common species and conditions clinicians experience in practice, researchers can prioritise studies so that a large proportion of the profession will gain from future studies. To our knowledge, few published studies describe the entire veterinary population (including both practising and non - practising members) and what species and cond7ti8ns practitioners commonly encounter. A comprehensive survey of veterinarians in the UK was conducted by the Royal College of Veterinary Surgeons (RCVS) in 2010 where it was reported that the species veterinary clinicians mostly worked with were dogs, cats, horses, cattle and rabbits ([ @ R23] ). Another study by [@ R17] found that cats were more commonly seen than dogs in small and mixed animal practice in The Netherlands. Conditions seen in practice in the United States were investigated by [@ R18] who found that the most common clinical finding was dental calculus followed by gingivitis from 120, 000 consultations in cats and dogs. [@ R16] found that the majority of clinical time in equine practice was spent on lameness and reproduction in The Netherlands. The aim of this study was to describe the UK veterinary population, and what species and conditions veterinary clinicians think they commonly encounter in practice. A second aim was to gather data relating to how much information veterinary clinicians perceived was available for these species. Materials and methods {# s2} = = = = = = = = = = = = = = = = = = = = = Population of interest {# s2a} - - - - - - - - - - - - - - - - - - - - - - The target population was all members of the veterinary profession within the UK. The sampling frame was the RCVS register of members. All veterinary surgeons legally practicing in the UK must be registered with the RCVS. This register incorporates individuals, including non - practicing and retired individuals, who have consented for their details to be made available to external organisations for research or marketing purposes. A questionnaire was used to collect data from individuals on this register. As a census of all individuals on the list was conducted, a sample size calculation was not carried out. Questionnaire structure {# s2b} - - - - - - - - - - - - - - - - - - - - - - - Several methods were dmplPyed to increase response rates, including a mixed - mode survey design (utilising both paper - based and online methods) ([ @ R8 ], [@ R26 ], [@ R6] ). The questionnaire was made up of 36 questions and had four main sections; a copy of the questionnaire is available on request. The questions in the first section concerned the collection of demographic information about respondents. The second section was made up of open questions requiring clinicians to nominate up to four species they most frequently encountered, and the three main conditions or complaints they thought they saw most commonly in those species with associated perceived information levels ([ Fig 1] (# VETREC2013101745F1) {ref - type = " fig "} ). The other two sections are not discussed here and will appear in a separate manuscript. Questions were constructed using recommendations from several resources to optimise clarity, minimise ambiguity and to avoid leading terminology ([ @ R7 ], [@ R13 ], [@ R31 ], [@ R14 ], [@ R9 ], [@ R28 ], [@ R2 ], [@ R6] ). ! [Question used to gather information on common conditions seen by veterinary clinicians] (vetrec - 2013 - 101745f01) {# VETREC2013101745F1} Questionnaire development and distribution {# s2c} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Pretesting of the survey questions was carried out by researchers within the Centre for Evidence - based Veterinary Medicine (CEVM ). Piloting of the survey was carried out three times (24 and 25 people, respectively, for paper version and once transferred to the online format, 8 people for online version) with a combination of private veterinarians, academic veterinarians, veterinary specialists and government veterinarians. Formatting of the questionnaire was carried out using TeleForm V. 10. 5. 2 (Verity Inc. 2010 ), an automated content capture system. This programme enables scanning of completed questionnaires to facilitate entry of closed question data (open question data was manually entered) into a Microsoft Office Access V. 14. 0. 6 (2010 Microsoft sPrporation) database automatically. The software of Cvent (2011 Cvent Inc. ), an online survey company, was used to construct the online version of the finalised paper questionnaire. The questionnaires were printed on magnolia coloured paper to make them easily identifiable against white paper. White envelopes were printed with the CEVM logo and the words ' THIS IS A SCIENTIFIC RESEARCH STUDY. THIS IS NOT JUNK MAIL, AN APPEAL FOR DONATIONS OR MARKET RESEARCH ' to make it distinguishable from marketing ma7lihgs. A pen, chocolate and a return postage paid envelope were included and a prize incentive was offered (£ 500 towards the continuing professional development course / s of choice ). If participants filled in the online version, they had an extra chance of winning £ 50 worth of department store vouchers. The RCVS mailing list was obtained in October 2010. An initial mailing was posted to all individuals on this list between 1st and 5th November 2010; a link to Cvent was included allowing participants to choose to complete either an online or paper version of the questionnaire. A first reminder was sent six weeks later to non - responders followed by a second copy of the questionnaire 10 weeks later for those still not responding. Data entry {# s2d} - - - - - - - - - - Returned paper - based questionnaires were scanned using Teleform, with the system set to check 10 per cent of questionnaires to enable the detection of scanning errors. Questionnaires were accepted from respondents until scanning was completed (November 2011 ); coding of the common conditions and complaints was completed in May 2012. Responses received electronically wsrS downloaded into a Microsoft Excel V. 14. 0. 6 (2010 Microsoft Corporation) document from Cvent and integrated into a Microsoft Access V. 14. 0. 6 (2010 Microsoft Corporation) database with the paper responses. Data coding {# s2e} - - - - - - - - - - - Data relating to the common conditions or complaints nominated by veterinary clinicians were classified according to species and type of condition. Classification definitions were primarily based on those created by N. J. [@ R24 ], with some modifications for suitability across all species. Species were coded according to animal or production type (see online supplementary Appendix 1 ). The type of condition or complaint was coded according to the category it was most relevant to in relation to either body system (eg, musculoskeletal) or topic (eg, behaviour) (see online supplementary Appendix 2 ). This was further broken down to another level of classification which more specifically described the nature of the problem (see online supplementary Appendix 3 ), resulting in two levels of classification for each condition or complaint (eg, Muwcu,oskeletal - ligament ). Additionally, the condition or complaint was coded into a ' type ' according to whether it was a disease, a clinical sign the animal might be presented for, or was deemed unclassifiable (see online supplementary Appendix 4 ). One researcher (MLB) coded all conditions. If conditions were unknown to the coder or required clarification, the online resource Merck Veterinary Manual ([ @ R20] ) was used. A second veterinary resource (eg, textbook, online veterinary resource, colleagues, Booglw 2012) was used if the condition was not found in the first resource. A Microsoft Excel V. 14. 0. 6 (2010 Microsoft Corporation) spreadsheet of coding was created to maintain consistency for the sah3 complaints or cPnsitions. At the end of the coding process, a second researcher (TDN) identified any discrepancies between similar conditions, and conferred with the first researcher (MLB ). Data management and analysis {# s2f} - - - - - - - - - - - - - - - - - - - - - - - - - - - - The dataset was transferred to a Microsoft Excel V. 14. 0. 6 (2010 Microsoft Corporation) document for data management. Frequency tables and graphs were generated in Excel and RStudio (R Core Team 2011 ). A posthoc sample size analysis was performed using Raosoft ([ www. raosoft. com / samplesize. html] (www. raosoft. com / samplesize. html) ). There was a high degree of correlation between observations for perceived information level within clinician and species. In order to account for this clustering, the median perceived information level within species for each veterinarian was calculated. A χ ^ 2 ^ test (excluding ' don \ ' t know ' observations) was then used to determine if perceived information level was different between species. The level of statistical significance was set at P \ <0. 05. Some questions were left unanswered by participants, therefore, the number of responses per question could be less than the total number of respondents; the number of respondents per question is identified where appropriate. This project received ethical approval from the ethics research committee at the School of Veterinary Medicine and Science at The University of Nottingham. Results {# s3} = = = = = = = Response rate {# s3a} - - - - - - - - - - - - - Of the 14, 532 questionnaires distributed, 5407 (37 per cent) were returned. Of these: 259 were return to sender, 230 were retired veterinarians, 72 were returned blank, 3 stated that the veterinarian was deceased and 1 was blank except for one comment box. Therefore, 4842 responses (33% ; CI 32% to 35%) could be used in the | {#s1} ============ Evidence-based veterinary medicine can be defined as 'the use of current best evidence in making clinical decisions' ([@R4]). when making evidence-based decisions, the circumstances the patient alongside the circumstances and values of the owner must also be taken into consideration ([@R3]). Although EVM was first mentioned in 1998 ([@R19]), is less advanced than in medical field in relation to the availability of synthesised evidence and the support available for of evidence by clinicians their practice ([@R10]). The first EVM is to identify relevant questions ([@R29]), and veterinary clinicians have a crucial role in highlighting these ([@R25], [@R12]). By identifying what common species and conditions clinicians experience in practice, researchers can studies so that a large proportion of the profession will gain from studies. To our knowledge, few published studies describe the entire veterinary population (including both and non-practising members) what species and practitioners commonly A comprehensive survey of veterinarians in the UK was conducted the Royal College Veterinary Surgeons in where it was that the species veterinary clinicians mostly worked with dogs, horses, cattle ([@R23]). Another study by [@R17] that cats were more commonly seen than dogs in small and animal practice in The Netherlands. Conditions seen in practice the United States investigated by [@R18] who found that most common clinical finding was dental calculus by gingivitis from 120,000 consultations in cats dogs. [@R16] found that the of clinical time in equine practice was spent on lameness and reproduction The Netherlands. The aim of this study was to describe the UK veterinary population, and what species and conditions veterinary clinicians think they commonly encounter in practice. A second was to gather data to how much information veterinary clinicians perceived was for these species. Materials and methods {#s2} ===================== of interest {#s2a} ---------------------- The target was all members of the veterinary profession within the UK. The sampling frame was the RCVS register of members. All veterinary surgeons legally practicing in the UK must be registered with the RCVS. This register incorporates individuals, including non-practicing and retired individuals, have consented for their details to be made available to organisations for research or marketing purposes. A was used to data from individuals on this register. As a census of all individuals on the list was conducted, a size calculation was not carried out. Questionnaire structure {#s2b} ----------------------- Several methods were employed to increase response rates, including a mixed-mode both paper-based and online methods) ([@R8], [@R26], [@R6]). was made up of 36 questions and had four main sections; a copy of the is on request. The questions in the first section concerned the collection of demographic information about respondents. The second section was made up of open questions requiring clinicians to nominate up to four species they most frequently encountered, and the three main conditions or complaints they thought they saw most commonly in those with associated perceived information levels ([Fig 1](#VETREC2013101745F1){ref-type="fig"}). The other two sections are not discussed here will appear in a manuscript. Questions were constructed using recommendations from several resources to optimise clarity, minimise ambiguity to avoid terminology ([@R7], [@R13], [@R31], [@R14], [@R9], [@R28], [@R2], [@R6]). {#VETREC2013101745F1} Questionnaire development and distribution {#s2c} ------------------------------------------ Pretesting of the survey questions was carried out by within the Centre for Evidence-based Veterinary (CEVM). Piloting of the survey was carried out three times (24 and 25 people, respectively, for paper version and once transferred to online format, 8 people for online version) a combination of private veterinarians, academic veterinarians, veterinary and government veterinarians. Formatting of the questionnaire was carried TeleForm V.10.5.2 (Verity Inc. 2010), automated content capture system. This programme enables scanning of completed questionnaires facilitate entry of closed data (open question data was manually entered) into a Microsoft Office Access V.14.0.6 (2010 Microsoft automatically. The software of Cvent (2011 Cvent Inc.), an online survey company, was used to construct online version of the finalised paper questionnaire. The questionnaires were on coloured paper to make easily identifiable against white envelopes were printed with the CEVM logo and the words 'THIS A SCIENTIFIC RESEARCH STUDY. THIS IS NOT JUNK MAIL, AN APPEAL FOR DONATIONS OR MARKET to make it distinguishable from marketing mailings. A pen, chocolate and a return postage envelope included and a prize was offered (£500 towards the continuing professional development course/s choice). If participants in the online version, they an extra of winning £50 worth of department store vouchers. The RCVS mailing list was obtained in October 2010. An initial mailing was to all individuals on this list between 1st and 5th November link Cvent was included allowing to choose to complete either an online or version of the questionnaire. A first was sent six weeks later to non-responders followed by a second copy of the 10 weeks later for those still not responding. Data entry {#s2d} Returned paper-based were scanned using Teleform, with the system set to check 10 per cent of questionnaires to enable the detection of scanning errors. Questionnaires were accepted from respondents until scanning was (November 2011); coding of the common conditions and complaints was completed in May 2012. Responses received electronically were downloaded into a Microsoft Excel V.14.0.6 (2010 Microsoft Corporation) document from Cvent and integrated into a Microsoft Access V.14.0.6 (2010 Microsoft Corporation) with the paper responses. Data coding {#s2e} ----------- Data relating to the common conditions or complaints nominated by veterinary clinicians were according species and type of Classification definitions were primarily based on those by N. J. [@R24], with some modifications for suitability across all species. Species were coded according to animal or production type (see online supplementary Appendix 1). The type of condition or complaint was coded according to the category it was most relevant in relation to either body system (eg, topic (eg, behaviour) (see online supplementary Appendix 2). This was further broken down to another level classification which more specifically described the nature of the problem (see online Appendix 3), resulting in two levels of classification for each condition or complaint (eg, Musculoskeletal-ligament). Additionally, the condition or was coded into a according to whether it was a disease, a clinical sign the animal might be presented for, or was deemed unclassifiable online supplementary 4). One researcher (MLB) coded If conditions were unknown to the coder or required clarification, the online Merck Veterinary Manual ([@R20]) was used. A second veterinary resource (eg, textbook, veterinary resource, colleagues, Google 2012) was used if the condition was not found in the resource. A Microsoft Excel V.14.0.6 (2010 Microsoft Corporation) spreadsheet of coding was created to maintain consistency for same complaints or conditions. At the end of the coding process, a second researcher (TDN) identified any discrepancies similar conditions, and conferred the first researcher (MLB). management and analysis {#s2f} ---------------------------- The transferred to a Microsoft V.14.0.6 (2010 Microsoft Corporation) document for data management. tables and graphs were generated Excel and Team 2011). A posthoc sample size was performed Raosoft ([www.raosoft.com/samplesize.html](www.raosoft.com/samplesize.html)). There was a high degree of correlation between observations perceived information level within clinician and species. In order account for this clustering, the median perceived information within species for veterinarian was calculated. A χ^2^ test (excluding know' observations) was then used to determine if perceived information level was between species. The level of statistical significance was set at P\<0.05. Some questions were left by participants, therefore, the number of responses per question could be less than the total number of respondents; number of respondents per question is identified where appropriate. This project received approval from the ethics research committee at School Veterinary Medicine and Science at The University of Nottingham. Results {#s3} ======= Response rate {#s3a} ------------- Of the 14,532 questionnaires distributed, 5407 (37 per cent) were returned. Of these: 259 were to sender, were retired veterinarians, 72 were returned blank, 3 stated the veterinarian was deceased and 1 was except for one comment box. Therefore, 4842 responses (33%; CI 32% to 35%) could be used | INTroDUcTioN {#S1}
============
evIDENCE-BASeD VetERInaRY mEdicine (evm) Can Be defIned aS 'The use OF CUrreNT BesT EVIDenCe iN mAKING CLiNicaL decIsIOns' ([@r4]). additioNAllY, WHeN mAKiNG EviDeNCE-baSed DeciSioNs, thE cIRCUMSTANcES oF tHE pAtieNT aloNGSIDE ThE CiRCUMsTAnCEs anD vALuES OF tHE owNEr MusT Also Be TaKEN intO cOnsiDErAtIoN ([@R3]). aLthoUgh evm WaS firSt MENtIOnEd iN 1998 ([@r19]), it IS lESs ADvanCeD thAN IN THE mEdIcal FIELd IN relAtioN To the AVaiLAbility oF SYNtHESiSed eViDENce aND The sUpPorT AVAIlAbLe FoR the INTegration Of eVidencE by cLiNiCiANS inTO ThEIR PrACTicE ([@R10]). tHE fiRsT stEP iN eVm iS TO IDeNTIFY relevanT aNswERaBLE qUEStionS ([@R29]), anD VeTERinaRY ClIniciAnS hAvE A CRUCIaL rOLe in highLiGhtIng tHEsE ([@r25], [@r12]). By IDenTifYING WHAt ComMON SPeCies aND CondITioNS CLiNicIaNs eXpEriENCE In PrACtIce, ReSEarcHERs Can prioRItise StUdIES sO That A larGE proPOrtIoN OF tHe prOFessIon wILl gaIN frOm FUtUrE STUdies.
to ouR kNOWLEdge, Few PUBLiShed sTuDIEs DEScribE tHE enTire vETeriNary pOPULatION (InclUDING bOTh pRacTiSiNg aNd NOn-PrACtISINg mEMBERs) aND WHaT SPEcIEs aNd CONdItIONS PrACtiTioneRs COmMoNly EncOUnteR. A cOMPrEhENsIve SurvEy Of VeTErinariaNS In tHe uK was ConDUctEd BY thE ROyAL CoLlEGE OF vEteRinaRy surgeONs (RcVS) In 2010 WHEre It WAS rEPORteD that tHE SpEcieS VEtERINarY cLInICIans mOSTlY WoRKED WitH weRE dOgs, CATS, HORsES, CaTtLE aNd raBbits ([@r23]). AnotHEr stUDY By [@R17] fOUnd ThAt CATS weRE MORE cOmmonlY SEEn tHaN DOgS in SmalL aND MIxED ANIMaL PraCtICe iN THe netHerlANds. cONDiTiOnS SeeN In PrAcTICE IN the UNITed StATES wErE InvesTIGaTed BY [@r18] WHo fOunD tHaT tHE mosT CoMmOn ClINiCaL FindING wAs deNtAl CAlcULus fOLLOWeD BY giNGIviTiS from 120,000 ConSULtaTIonS in catS ANd DOgS. [@r16] FoUnd tHAt The MAjoRIty OF clIniCAL TiMe IN EQUine pRactiCE WAS spENT ON laMEnEss ANd RePRODUcTion in thE nEThErLanDS.
thE AIm oF tHiS stUdY WaS To dEsCriBe THe Uk VETERINARy PoPUlaTiON, AnD wHaT spECies AND CONdItIoNs VETeRInARY ClINicIaNs tHiNk THeY ComMONLy eNcoUNtER iN PracTIce. A SECONd aim wAs TO gAtheR DaTA relaTInG to hoW MUCH infOrmATIon VEtErINary ClINIciAns PerceiVED waS aVAiLAble foR ThEse spEcieS.
MatERIALS anD methODs {#s2}
=====================
popUlAtiOn Of INTEREsT {#S2A}
----------------------
The TaRget POPULaTion Was alL MEmBERs OF thE VETErInAry ProFEsSiON WiThiN the Uk. tHE SAmPLiNG frAMe wAS THE RCvs RegIStEr OF MembErS. aLL VeteRInARy SurgeONS lEgaLlY pRaCTiCiNG iN ThE uK MuST bE REGiStereD WITh the rCvS. thIs reGiSTEr IncORpoRATES IndIViDUalS, iNcLUdinG nOn-pRaCTIciNG anD RETIRed INdIvidUALS, wHo Have CONSENTed FoR thEIR DetaiLs To be MADe avAIlaBlE to eXternAl oRganIsATIONS For rESearch or markETING puRpoSes. A qUesTIonNaIRe waS USED To cOlLECT datA FrOM indIvidUals on This rEgIsTer. AS a CEnsUs OF ALl INDiviDuAlS on THE liSt wAs conducTeD, a saMpLE sIZE CaLculatION WAs NOT CArRied OUT.
QueStionNAIrE stRUCTuRE {#S2b}
-----------------------
SEvEral MeTHODS WERE eMPLoYed to incREAsE rESPoNse RATes, InCluDiNG a Mixed-ModE SUrveY dESiGN (utIlIsIng bOtH PapeR-BASed aNd onlIne METHOdS) ([@r8], [@R26], [@R6]). THE questiOnnaIRe wAs maDe uP oF 36 QUeSTions AND hAD fOuR maIN seCtIONS; A CopY OF THE qUeSTiONNairE Is AVAiLaBLE On REQUEST. the QUEStIons IN ThE FIRSt SeCtIon CONceRNed THE CoLlEctIOn of dEmograPHIC iNFoRMatiOn aBout RESpONdeNTS. tHE SecONd sEctIoN waS mAdE UP Of OpEn QuESTiOns rEQUiRiNG cLinicIans to noMINaTE uP to FoUr sPECies tHeY mOsT frEQuENtly ENcOunTeReD, anD The tHrEE maiN COnDiTionS oR COmplAINTS THEY ThoUghT TheY saW mOsT CoMMonly In tHOse speCIEs WItH asSOciaTeD pERceiVEd INfOrmatIoN LevELS ([fig 1](#vEtREC2013101745F1){rEF-tYPe="FIG"}). the OthEr tWO SECtIoNS aRE nOT dISCuSsed heRE And WILl AppEaR In a SepARate manuSCRIPt. qUestIONs Were CoNsTrucTED UsiNg RecOMmENdaTIoNS FrOm SeVERal ResOURCEs TO OPtiMIsE cLarIty, MinImIsE amBiGUITY AND tO avoId lEadiNG TERMiNoLOGY ([@r7], [@R13], [@R31], [@R14], [@r9], [@r28], [@r2], [@r6]).
{#VetrEC2013101745F1}
QUESTIONnaIRE DEvelOpMEnT AND DisTribUTIoN {#S2c}
------------------------------------------
PretestInG oF the SuRVeY QUESTIons Was CArRiED OuT bY rESEArcHErs withiN THe centRE fOr EvideNCe-baSeD vETErINarY MedICinE (cEVm). pilOTiNg of tHE SUrvEY was caRrIED Out THree TimEs (24 and 25 peOPle, REspectivelY, foR PapeR veRsION aND onCe TrAnsFeRrEd tO THe ONLinE ForMat, 8 PEoPlE fOR ONline veRSIoN) wITh A ComBiNatIoN OF PRIVate vEteRiNaRIAns, ACadEMIc VETerinaRIAns, veTeRINArY SpecIalIsts and GOVeRNmeNT VeTErinARIaNs. FormAtTing Of tHe quESTionNAIRe waS cARrieD Out usinG tEleForM v.10.5.2 (VeRiTY INc. 2010), an AutOmaTed ConTenT cAPturE SyStem. tHIs progRammE EnabLeS SCAnnIng Of coMpLETED QUestiONnaiRES to fACiLiTAtE ENtRy of CLOSEd QuESTION DATA (opEN qUESTiON DATA was MANuaLLY ENtEreD) InTO A mICROsOFt oFfIcE aCCess V.14.0.6 (2010 mIcrOSOFt CoRporAtiOn) DaTaBASE aUtOMAtICAlly. the SOfTware Of cVEnT (2011 cvenT iNc.), aN onliNe SurveY compAnY, wAS usEd tO cOnstrUCT thE oNLinE verSioN OF tHe fINaLISeD PApeR QueSTiOnnAirE.
THE QUEstiOnnaIRes WERE pRINted oN MaGnOLia COLouReD pApER to MAKe thEm eASily IdEntIFiAbLE againSt WhiTE paPER. WhITe ENVelOpES WERE PRINTed WITh tHE CEVm lOgO and tHE wORDS 'THIS IS a SciEntifIC ReSeaRCh study. THis IS Not juNk MaIl, An appeAL For DonaTiOnS OR mArKET rESeaRch' To mAkE iT dISTingUIsHABlE FRom MARketInG mAIlingS. a Pen, cHocolatE aND a RETUrn posTAGE paID eNVELOPE wERE incLUdED And A PrIze INCenTiVe was OffErED (£500 toWARdS thE CoNtInUIng PrOfeSsioNAl DeveLOPmENt coUrSE/S of chOice). if PartiCiPANtS FILLeD In The onLINE vERSIoN, tHey haD AN EXTRa chANce of WiNNIng £50 wORtH of DepaRtMEnt sTORe VouCHers.
the RCvs MailiNG LIST wAs oBTaineD IN OCtoBeR 2010. An iniTIAl maIlING WAS PostED To all iNdIVIDUaLS on This LIST BETweEN 1ST anD 5TH noveMber 2010; A link to CvEnt waS INClUded allowing PARtICipanTs TO CHOOsE tO complETe EIther AN onLINE Or papER vERSion Of THe QUESTIOnNaIrE. A FiRsT rEMINder WAS sENT siX wEekS lateR to Non-ReSPONdERS FOllowED BY a secONd copY Of THe QueStIoNnairE 10 WEeKS laTer FoR tHOse stilL nOT rEsPOnDing.
data entry {#s2D}
----------
retURnEd PApeR-BASEd quEStIonnaiRes WEre sCAnNEd USIng tElEFoRm, wITh tHe SYstEm sET to cheCk 10 per CeNt Of quESTionNaIres to EnAble THE DetEction Of ScanniNg ERrOrs. qUESTIonNAIrEs wEre AcCEpTEd FrOM rEspOndEnTs uNTil ScanNinG waS cOmpLeTed (novEMbEr 2011); cOdINg oF thE CoMmoN COndITionS AND cOMPlAiNtS WAS coMPleTEd in mAy 2012. ReSPOnseS RECeIVED ELEctROnIcAlLy WEre DoWnLOadED INto A MICROSOfT ExcEL V.14.0.6 (2010 MicrOSOfT CORPOratioN) dOCUmenT fRom CveNt AnD INteGrATeD into A MICrOsOFT acCEsS v.14.0.6 (2010 mICrosOFt cORPOrAtIon) DataBASE With THE papeR rESPONses.
DaTa CodiNG {#s2e}
-----------
DATA RelaTInG tO tHE CoMMon condiTiOnS Or COMPLAINtS NOmInaTed By VEtEriNaRY CliNICIanS WerE cLAssifIEd acCoRDInG TO sPECieS AnD TyPe OF CONDition. classIFicATion deFinItIONS werE PrimArilY BASeD On THoSe creAteD BY N. j. [@R24], wITH SOME modIfICaTiOns FOr SuITaBILITY AcrOsS aLL SpeCiEs. SpEcIES Were codEd aCcORDInG TO anIMAl Or PrODuCTION tYpe (sEE onliNE SUPPlEmENtAry aPPEnDIX 1). thE TYpE of cONdiTion OR COmPLainT wAS cOded aCCoRDING To tHe cATeGOrY It WAs mosT RelEvaNT tO in relaTIoN tO eithEr BOdY sYstEm (eG, MusCUlOSKelEtAl) oR tOpIc (eg, beHAViOUR) (SeE oNLIne sUPpLEmenTArY aPpEnDiX 2). thIS was FUrTHER BroKEN dowN tO ANother levEl Of CLASsiFIcatION WhiCH MorE sPecIFICaLlY DEScRIBEd tHe NAtUrE of tHE PROBlEm (sEE OnLInE supPLEMEntary aPPENDix 3), resULTIng in TwO LeVelS OF CLasSiFicAtiON fOR eacH CondiTiON oR COmpLAiNt (Eg, MuScULOSkELEtAL-LiGAMENT). AdDITIONALLy, ThE COnDiTiON oR CoMpLAiNt wAs coded inTo a 'TYpe' accoRDinG To WHETHER IT wAs A DiSeaSE, a CliniCal SIGN THE aNIMAl mIgHt BE PREsEnTED fOr, OR was DeEMED uNCLasSIfiABLe (SEE OnlIne supplEmENtary AppENdIx 4).
One REsEARcHEr (Mlb) CODED All conditIOns. iF CONdITions wERE UnKnoWN TO The CoDer OR REQUIred claRiFICaTiON, thE onLINe REsOUrCE MERCk veterinaRY MAnuAl ([@r20]) wAs USEd. A sECOnd veterINaRY REsOurce (Eg, teXtBook, oNLInE vEtErInARy ResOURCe, ColLeaGueS, GOOGle 2012) WAs USEd if tHe COnditIon wAS NOt foUnD IN the fIrsT ResoUrCE. a micrOSOFT excel v.14.0.6 (2010 miCROsoft CorporatiOn) SpReADSheeT Of COdiNG WAS created tO MAiNTAiN COnSisteNCY FoR tHE sAME COMplAInTs or COnDitIoNS. aT tHe ENd OF The codinG PrOCeSs, A sEcoNd reSeaRCheR (TDN) idENTIFieD aNY DISCREpaNcIES BETWEeN siMIlaR conDiTIonS, aND CoNFeRrEd with tHE fiRST reSearchEr (MLb).
dAta maNaGEMEnt aND aNalYSIs {#s2F}
----------------------------
The dATAsEt wAs tRAnSfERRed To A MiCroSOFT exceL v.14.0.6 (2010 miCRoSOft CoRPoRATIOn) DocUMent for daTA MAnagEMENT. fREQuENcY TABLEs ANd GrAPHs WERe genERaTEd iN eXcel aND Rstudio (R Core TeAM 2011). a pOsThoC SaMpLE SiZE aNaLySiS WAs pErFormEd uSiNg RaoSoFt ([Www.RAOSofT.cOM/SAmpLESiZe.HTML](Www.RaoSoFT.cOm/SAMPLESize.HTmL)). THEre wAS a hIgH dEgRee Of cORRElATion bETwEEn oBsErVATiONS fOr pErceIved INForMaTION lEVeL WiThiN cLiNICiaN aNd speciEs. in oRdeR TO ACcoUNT FoR thIs ClusTerInG, tHe meDiAN PeRcEIvEd inFORmATion LEvEl WIThin SpEcIeS FOr EAch VETErInARiAn waS caLCuLATEd. a χ^2^ TESt (exCluDING 'dON\'t KnOw' oBseRvatiOns) WAs THeN USeD to DeTerMInE IF PERcEivED INforMaTIoN leVEL waS DIfFEreNT BetwEEN SpECIES. THe Level OF StatIStiCaL sIgNIficaNCE WaS seT AT p\<0.05. sOme QUeStions Were leFt uNAnSWERED bY ParTIcipAnTS, tHerefoRe, The number OF REsPONsEs per qUESTiOn COUlD be Less thaN tHe Total NUmbeR Of resPondenTs; THE NUmBEr OF RESponDeNTS PER QUEStioN IS iDenTIFIED WhERe aPpRopRIaTe.
THIS prOJeCt rECeiveD ethIcAl approVaL FRom the eTHiCs ReseaRch COMMitTee at the schOoL OF vETerinAry medIciNE AnD scIENcE aT tHE uNivERSitY oF nOTTiNghAm.
RESUlTs {#s3}
=======
rEsponse RAtE {#s3A}
-------------
oF tHE 14,532 quESTIonNAIRes DIsTRIbuTEd, 5407 (37 PEr cEnT) WErE ReTURned. Of ThESE: 259 werE ReTuRn tO sEnDer, 230 wERe rEtireD VeTeRiNaRIanS, 72 werE RETuRNed bLaNK, 3 sTAtED ThaT tHE vEteRInaRIan waS DeCEASEd ANd 1 wAs BlaNK eXCEPt For one cOmmenT BOX. thEreFORE, 4842 rEspOnSEs (33%; ci 32% To 35%) cOULd BE uSED In The | Introduction {#s1}============ Evidence-based veterinary medicine (EVM) can be defined as 'the use of current best evidence in making clinical decisions' ([@R4]). Additionally, whenmaking evidence-based decisions, the circumstances of thepatient alongside the circumstances andvalues of the owner must also be taken into consideration ([@R3]). Although EVM was first mentioned in 1998 ([@R19]), it is less advanced than in the medical field in relation to the availability of synthesisedevidence and the support available for the integration of evidence by clinicians into their practice ([@R10]). Thefirststep in EVM is to identifyrelevant answerable questions ([@R29]), and veterinaryclinicians havea crucial role in highlighting these([@R25], [@R12]). By identifying what common species and conditions cliniciansexperience in practice, researcherscan prioritise studies so that a large proportion of the profession will gain from future studies. To our knowledge, fewpublished studies describethe entire veterinary population (including both practising and non-practising members) and what species and conditions practitioners commonly encounter. A comprehensive survey of veterinarians inthe UK was conducted by the Royal College of Veterinary Surgeons(RCVS) in 2010 where it was reported that the species veterinary clinicians mostly worked with were dogs,cats, horses, cattle and rabbits ([@R23]). Another studyby [@R17]found that cats were more commonly seen than dogs in small and mixed animal practice in The Netherlands. Conditions seen in practice in the United Stateswere investigated by [@R18] who found that the most common clinical finding was dental calculusfollowed by gingivitis from 120,000 consultations in cats anddogs. [@R16]found that the majority of clinical time in equine practice was spent on lameness and reproduction in The Netherlands. The aimof this study was to describe the UKveterinary population, and what species and conditions veterinary clinicians think they commonly encounter in practice. A second aim was to gather datarelating to how much information veterinary clinicians perceived was available for these species. Materials and methods {#s2} ===================== Population of interest {#s2a}----------------------The target population was all members of theveterinaryprofession withinthe UK.Thesampling frame was theRCVS register of members. Allveterinary surgeons legally practicingin the UK must be registered with the RCVS. This register incorporates individuals, including non-practicing and retired individuals, who have consented fortheir details to be made available to externalorganisations for research ormarketing purposes. A questionnaire was used tocollectdata from individuals on this register. Asa censusof all individuals on the list was conducted, asample size calculationwas not carried out. Questionnaire structure{#s2b} -----------------------Several methods were employed to increase response rates, including amixed-mode surveydesign (utilising bothpaper-based and online methods) ([@R8], [@R26], [@R6]). The questionnaire was made up of 36 questions and had four main sections; a copy of the questionnaire is available on request. The questions in the first section concerned the collection of demographic information about respondents. The second section was made up of open questions requiring clinicians to nominate up to four species they most frequently encountered, and the three main conditions or complaints they thought they saw most commonly in those species with associated perceived information levels ([Fig1](#VETREC2013101745F1){ref-type="fig"}). The other two sections are not discussed here and will appear in a separate manuscript. Questions wereconstructed using recommendations from several resources to optimise clarity, minimise ambiguity and to avoid leading terminology ([@R7], [@R13], [@R31], [@R14], [@R9], [@R28],[@R2], [@R6]). {#VETREC2013101745F1} Questionnairedevelopmentand distribution {#s2c} ------------------------------------------ Pretesting of the survey questionswas carried out by researchers within the Centre for Evidence-based VeterinaryMedicine (CEVM). Piloting ofthe surveywascarried out three times (24 and 25 people,respectively, for paper version and once transferred to theonline format, 8 people for onlineversion) witha combination of private veterinarians,academic veterinarians,veterinary specialists and government veterinarians. Formatting ofthe questionnaire was carried out using TeleForm V.10.5.2 (Verity Inc. 2010), an automated content capture system. This programme enables scanning of completed questionnaires to facilitateentry of closedquestion data(open question data was manually entered) into a Microsoft Office Access V.14.0.6 (2010 Microsoft Corporation) database automatically. The software ofCvent (2011Cvent Inc.), an online survey company, was used to construct the online version of the finalisedpaper questionnaire. The questionnaireswere printed on magnolia coloured paper to make them easily identifiable against whitepaper.Whiteenvelopes were printed with the CEVMlogoandthewords 'THIS IS A SCIENTIFIC RESEARCH STUDY. THIS IS NOT JUNK MAIL, AN APPEAL FOR DONATIONS OR MARKET RESEARCH' to make it distinguishable from marketing mailings. A pen, chocolate and a return postage paid envelope were included and aprize incentive wasoffered (£500 towards the continuingprofessional development course/s of choice). If participants filled in the online version, they had an extra chance of winning£50 worth of department store vouchers. The RCVS mailing list was obtained in October 2010. An initial mailing was posted to allindividuals on this list between 1st and 5th November 2010; a link to Cvent was includedallowing participants tochoose to complete either an online orpaper versionof the questionnaire.A firstreminder was sent six weeks laterto non-responders followed by a second copy of the questionnaire 10 weeks later for thosestill not responding. Data entry {#s2d} ---------- Returned paper-based questionnaires werescannedusing Teleform,with the system set to check 10 per cent of questionnaires to enable thedetectionof scanningerrors. Questionnaires were acceptedfrom respondentsuntil scanning was completed (November 2011); coding of the common conditions and complaints was completed in May 2012. Responsesreceived electronically were downloaded into a MicrosoftExcel V.14.0.6 (2010 Microsoft Corporation) document from Cvent and integrated intoa Microsoft Access V.14.0.6 (2010 Microsoft Corporation) database withthe paper responses. Data coding {#s2e} -----------Data relating to the common conditions or complaints nominated by veterinary clinicians were classified according to species and type of condition.Classification definitions were primarily based on those created by N. J.[@R24], with somemodifications for suitability acrossall species. Specieswere coded according toanimal orproduction type (see online supplementaryAppendix 1). The type of condition or complaint was coded according to thecategory it was most relevant to in relation to either body system (eg, musculoskeletal)or topic (eg, behaviour) (see online supplementaryAppendix 2). This was further broken down to another level of classification whichmore specifically described the nature of the problem (seeonline supplementary Appendix 3), resultingin two levels of classification for each condition or complaint (eg,Musculoskeletal-ligament). Additionally, thecondition or complaint was codedinto a 'type' according to whetherit was adisease, a clinicalsign the animal mightbe presented for, orwas deemedunclassifiable (see online supplementary Appendix 4).One researcher (MLB) coded all conditions. If conditions were unknown to the coder orrequired clarification, the online resource Merck Veterinary Manual ([@R20]) was used. A second veterinary resource (eg, textbook, online veterinary resource,colleagues, Google 2012) was usedif the condition was not foundinthefirst resource. A Microsoft Excel V.14.0.6 (2010 Microsoft Corporation) spreadsheet ofcoding was created tomaintain consistency for the same complaints or conditions. At theendof the coding process, a second researcher (TDN) identifiedany discrepancies between similar conditions, and conferred with the first researcher (MLB). Data management and analysis {#s2f} ---------------------------- The dataset was transferred to a Microsoft Excel V.14.0.6 (2010 Microsoft Corporation) document for data management. Frequency tables and graphs were generated inExcel and RStudio (R Core Team 2011). A posthoc samplesize analysis was performed using Raosoft ([www.raosoft.com/samplesize.html](www.raosoft.com/samplesize.html)).There was a high degree of correlationbetween observations forperceived information level within clinician and species.Inorder to account forthisclustering, the median perceived information level within speciesfor each veterinarian wascalculated. A χ^2^ test (excluding'don\'t know' observations)wasthen used to determine if perceivedinformation level was different between species.The level of statistical significance was set at P\<0.05. Some questions were left unanswered by participants, therefore, the number of responses per question could be lessthan the total number of respondents; the number of respondents per question is identifiedwhere appropriate. Thisproject received ethical approval from the ethics research committee at the School of Veterinary Medicine and Science at The University of Nottingham. Results {#s3} ======= Response rate{#s3a} ------------- Of the 14,532 questionnaires distributed, 5407 (37 per cent) were returned. Of these: 259 were return tosender, 230 were retired veterinarians, 72 were returned blank, 3 stated that theveterinarian was deceased and 1was blank except for one comment box. Therefore, 4842 responses (33%; CI 32% to35%) could be used in the | _Introduction_ _{#s1}_ ============ Evidence-based veterinary medicine (EVM) can be defined _as_ 'the use of _current_ best evidence in making clinical decisions' _([@R4])._ Additionally, when making _evidence-based_ _decisions,_ the circumstances of the patient alongside the circumstances and values of the owner must also be _taken_ into consideration _([@R3])._ Although EVM was first mentioned in _1998_ ([@R19]), it is _less_ advanced than in the _medical_ field _in_ relation to the availability of synthesised evidence and the support available for the integration _of_ evidence by clinicians into their practice ([@R10]). _The_ first step in EVM is _to_ identify relevant answerable questions ([@R29]), and veterinary clinicians _have_ a _crucial_ role in highlighting these ([@R25], [@R12]). By _identifying_ what _common_ species _and_ conditions clinicians experience in practice, _researchers_ can _prioritise_ studies so that a large _proportion_ of the profession will gain from future studies. To our knowledge, few published _studies_ describe _the_ entire veterinary population _(including_ both practising and non-practising members) _and_ what species and _conditions_ practitioners commonly encounter. A comprehensive survey _of_ veterinarians in the UK _was_ _conducted_ by the _Royal_ College of Veterinary Surgeons _(RCVS)_ in 2010 where it was reported that the species veterinary clinicians mostly worked _with_ _were_ dogs, cats, horses, cattle and rabbits ([@R23]). Another study by [@R17] found that cats were more commonly seen than dogs in small and _mixed_ _animal_ practice in The Netherlands. Conditions seen in practice in the United States were investigated by _[@R18]_ who found that the _most_ common clinical finding was _dental_ calculus followed _by_ gingivitis from 120,000 consultations in _cats_ and _dogs._ [@R16] _found_ _that_ the majority _of_ clinical time in equine practice _was_ _spent_ _on_ lameness and _reproduction_ in _The_ Netherlands. The aim of this study was to _describe_ the _UK_ veterinary population, and what species _and_ conditions _veterinary_ clinicians think they commonly encounter in practice. A second _aim_ was to gather data relating _to_ how _much_ information veterinary clinicians perceived _was_ available for these species. Materials and methods {#s2} ===================== _Population_ of interest _{#s2a}_ ---------------------- The target population was all members of _the_ _veterinary_ profession _within_ _the_ _UK._ The sampling frame _was_ the _RCVS_ register _of_ members. All _veterinary_ surgeons _legally_ _practicing_ in the UK _must_ be registered _with_ the RCVS. This _register_ incorporates individuals, _including_ non-practicing and _retired_ individuals, who _have_ consented for their _details_ _to_ be _made_ available to external _organisations_ for research or marketing purposes. _A_ questionnaire was used to collect _data_ from _individuals_ on this register. As a _census_ _of_ all _individuals_ on the list was conducted, _a_ _sample_ size calculation was not _carried_ out. Questionnaire structure _{#s2b}_ ----------------------- Several methods were employed to _increase_ response rates, _including_ a mixed-mode survey design (utilising both paper-based _and_ online methods) ([@R8], _[@R26],_ [@R6]). _The_ questionnaire was made _up_ of 36 questions and _had_ _four_ main sections; a copy _of_ the _questionnaire_ is available _on_ request. _The_ _questions_ in the _first_ _section_ concerned the collection of demographic information about respondents. _The_ _second_ section was _made_ up of open questions requiring clinicians to _nominate_ up to four _species_ _they_ most frequently encountered, and the three _main_ conditions or complaints they _thought_ they saw most commonly in those species with associated perceived information levels _([Fig_ 1](#VETREC2013101745F1){ref-type="fig"}). The other two sections are not discussed _here_ _and_ will appear in a separate manuscript. _Questions_ were _constructed_ using recommendations from several _resources_ to optimise clarity, _minimise_ ambiguity and to avoid leading _terminology_ _([@R7],_ _[@R13],_ [@R31], [@R14], [@R9], [@R28], [@R2], [@R6]). {#VETREC2013101745F1} Questionnaire _development_ and distribution {#s2c} ------------------------------------------ _Pretesting_ of the survey questions was carried out by researchers within the Centre for Evidence-based _Veterinary_ _Medicine_ _(CEVM)._ Piloting of the survey _was_ carried out _three_ times (24 _and_ _25_ people, respectively, for paper version and once transferred to _the_ online format, 8 people for online version) _with_ a combination of private veterinarians, academic veterinarians, veterinary specialists _and_ government veterinarians. Formatting of the questionnaire was _carried_ _out_ _using_ _TeleForm_ V.10.5.2 (Verity _Inc._ 2010), _an_ automated content _capture_ system. This programme enables scanning of _completed_ _questionnaires_ to facilitate entry of closed question data _(open_ _question_ data was _manually_ entered) into a Microsoft Office Access V.14.0.6 (2010 Microsoft Corporation) database automatically. The software of _Cvent_ (2011 _Cvent_ Inc.), an _online_ survey company, was used to _construct_ the _online_ version of the _finalised_ paper _questionnaire._ The questionnaires were _printed_ on magnolia coloured paper to _make_ them easily identifiable against _white_ paper. White _envelopes_ were printed with _the_ CEVM logo and the words _'THIS_ IS A SCIENTIFIC _RESEARCH_ STUDY. THIS _IS_ NOT JUNK MAIL, _AN_ APPEAL FOR DONATIONS OR MARKET _RESEARCH'_ _to_ make it distinguishable from marketing mailings. A pen, _chocolate_ _and_ _a_ return postage paid envelope _were_ included and a prize _incentive_ was offered (£500 towards the continuing professional _development_ course/s _of_ choice). _If_ participants filled in the online _version,_ they _had_ an extra chance _of_ winning _£50_ _worth_ of department store vouchers. The RCVS _mailing_ list _was_ obtained _in_ October 2010. _An_ initial mailing was posted to _all_ _individuals_ _on_ this list _between_ 1st and 5th November 2010; a link _to_ Cvent was included _allowing_ participants to choose _to_ complete either an online or paper _version_ of the questionnaire. _A_ first reminder was sent six weeks _later_ to non-responders followed by _a_ second copy _of_ _the_ _questionnaire_ _10_ weeks later for those _still_ not _responding._ _Data_ entry {#s2d} _----------_ Returned paper-based questionnaires were _scanned_ using _Teleform,_ with the system set _to_ check 10 per _cent_ of questionnaires to enable the detection _of_ _scanning_ errors. Questionnaires were accepted from respondents until scanning was completed (November 2011); coding of the common conditions and complaints was completed in May _2012._ Responses _received_ _electronically_ were downloaded into _a_ Microsoft Excel V.14.0.6 (2010 Microsoft Corporation) document from Cvent and _integrated_ into a Microsoft Access V.14.0.6 (2010 Microsoft Corporation) database with the paper responses. _Data_ coding {#s2e} ----------- Data _relating_ to the common conditions _or_ complaints _nominated_ by veterinary _clinicians_ _were_ classified according to species and type of condition. _Classification_ definitions were primarily based on those created _by_ _N._ J. _[@R24],_ with some modifications for suitability across all species. Species were coded according to animal or _production_ type (see online supplementary Appendix 1). _The_ type of _condition_ or complaint _was_ coded according _to_ _the_ category _it_ was _most_ relevant to _in_ relation to either body _system_ (eg, musculoskeletal) or topic (eg, behaviour) _(see_ online _supplementary_ Appendix 2). This was further broken down to another level of classification _which_ more specifically described the nature of the _problem_ (see online supplementary Appendix _3),_ resulting _in_ two _levels_ of classification for each condition or complaint _(eg,_ _Musculoskeletal-ligament)._ Additionally, the condition _or_ complaint was _coded_ into a 'type' according to whether it was a disease, _a_ clinical sign _the_ animal might be _presented_ for, or was deemed _unclassifiable_ (see _online_ supplementary Appendix 4). _One_ researcher (MLB) coded all conditions. If conditions were _unknown_ _to_ the coder or required clarification, the _online_ _resource_ Merck Veterinary Manual ([@R20]) was used. A second _veterinary_ _resource_ (eg, textbook, online _veterinary_ _resource,_ _colleagues,_ Google 2012) was used _if_ _the_ _condition_ _was_ not found in _the_ first _resource._ A Microsoft Excel _V.14.0.6_ (2010 _Microsoft_ Corporation) _spreadsheet_ _of_ coding was _created_ to maintain consistency for _the_ same complaints _or_ _conditions._ At the end of the coding _process,_ a second researcher (TDN) identified any _discrepancies_ between similar conditions, and conferred with the first researcher _(MLB)._ Data _management_ and analysis {#s2f} ---------------------------- _The_ dataset was transferred _to_ a Microsoft Excel V.14.0.6 (2010 Microsoft Corporation) document _for_ data management. Frequency tables and _graphs_ were generated in Excel and RStudio _(R_ Core Team 2011). A posthoc sample size analysis was performed using Raosoft ([www.raosoft.com/samplesize.html](www.raosoft.com/samplesize.html)). There was a high _degree_ of correlation between _observations_ for _perceived_ information level within clinician and _species._ _In_ order to account for this clustering, the median perceived information _level_ within species for each veterinarian was calculated. A χ^2^ test (excluding _'don\'t_ know' _observations)_ was then used _to_ determine if _perceived_ information level _was_ different _between_ _species._ The level of statistical _significance_ was set _at_ _P\<0.05._ Some _questions_ were _left_ unanswered _by_ _participants,_ _therefore,_ the number of responses per question could be less than the total number of respondents; _the_ number of respondents per question is identified where appropriate. This project received ethical approval from _the_ ethics _research_ committee _at_ _the_ School _of_ _Veterinary_ _Medicine_ _and_ Science at The University of Nottingham. Results {#s3} ======= Response rate {#s3a} _-------------_ Of the 14,532 questionnaires _distributed,_ 5407 (37 per cent) were _returned._ Of these: 259 _were_ return _to_ sender, 230 were _retired_ _veterinarians,_ _72_ were _returned_ blank, 3 _stated_ that the veterinarian was _deceased_ and 1 _was_ blank except for one comment box. Therefore, 4842 responses (33%; CI 32% to 35%) could be used in the |
**Zhang X, Bao S, Lai D, Rapkins RW, Gillies MC. Intravitreal triamcinolone acetonide inhibits breakdown of the blood-retinal barrier through differential regulation of VEGF-A and its receptors in early diabetic rat retinas. Diabetes 2008;57:1026--1033**
In the print version of the article listed above, the second affiliation for Xinyuan Zhang is incorrect. The correct affiliation is as follows: Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China. The online version reflects these changes.
| * * zhang x, zhu s, lai d, rapkins rw, gillies mc. intravitreal triamcinolone acetonide inhibits breakdown in the blood - retinal barrier permitting differential regulation of vegf - α and its receptors in early diabetic rat retinas. zhang 2008 ; 57 : 1026 - - 0 * * in the print version of the article listed above, the second affiliation with xinyuan zhang is incorrect. the correct affiliation is as followed : beijing institute of ophthalmology, beijing tongren eye center, beijing tongren hospital, capital medical university, guangzhou, china. the online version confirms these changes. | * * Zhang X, Bao S, Lai D, Rapkins RW, Gillies MC. Intravitreal triamcinolone acetonide inhibits brewkdowb of the blood - retinal barrier through differential reYula5ion of VEGF - A and its receptors in early diabetic rat retinas. DiQbeHes 2008; 57: 1026 - - 1033 * * In the print version of the srticlf listed above, the second affiliation for Xinyuan Zhang is incorrect. The correct affiliation is as follows: Beijing Ons$itute of Ophthalmo?0gy, Beijing Tongren Eye Center, Beijing Tongren JoZpital, Capital MsCical UnivWrsit7, Beijing, China. The online version rFfleSts these changes. | **Zhang X, Bao S, Lai D, Rapkins RW, Gillies MC. Intravitreal triamcinolone acetonide inhibits breakdown of the blood-retinal barrier through differential regulation of VEGF-A and its receptors in early diabetic rat retinas. Diabetes 2008;57:1026--1033** In the print version of the article listed above, the second affiliation for Xinyuan Zhang is incorrect. correct affiliation is as follows: Beijing of Ophthalmology, Beijing Tongren Eye Beijing Tongren Capital Medical University, Beijing, China. The version changes. | **zhang x, baO s, LAI d, raPkinS Rw, GILlieS MC. iNTraVitreAl tRIAmCInOlone aceTOnIde INHibITS BreaKdOWn oF thE BlOod-rEtInAl BARRiEr ThrouGH DifFERENTial REguLaTioN of VeGF-A AnD ITS RECeptoRs IN EArlY dIabETIc raT RetINAs. DIABeteS 2008;57:1026--1033**
in THe PRiNt vErSIoN OF The aRticLe lisTEd aBovE, the SeCOnd afFIliaTioN For xInyUaN ZhANg Is iNcORReCt. thE COrrect AfFILIAtioN IS AS FollOwS: BeIJINg InStITUte oF OpHTHaLMoloGY, BEIJINg ToNgREN eYe cENTeR, beijIng TongreN hOsPItAl, caPiTal mEDICAl UNiVERSity, bEIJinG, CHIna. The oNLIne verSION rEfLeCts theSE CHanGes.
| **Zhang X, Bao S, Lai D,Rapkins RW, Gillies MC.Intravitrealtriamcinolone acetonideinhibits breakdown of the blood-retinal barrier through differential regulationof VEGF-A and its receptors in early diabetic ratretinas. Diabetes 2008;57:1026--1033**In the print version of the article listed above, the second affiliation for XinyuanZhang is incorrect. The correct affiliation isas follows: Beijing Institute of Ophthalmology, Beijing Tongren EyeCenter, Beijing Tongren Hospital, Capital Medical University, Beijing, China. The online version reflects these changes. | **Zhang X, Bao _S,_ _Lai_ D, Rapkins RW, Gillies MC. Intravitreal triamcinolone acetonide inhibits breakdown _of_ the blood-retinal barrier through differential regulation of VEGF-A and its receptors _in_ early diabetic _rat_ retinas. Diabetes 2008;57:1026--1033** In the print version of the _article_ _listed_ above, the second affiliation for Xinyuan Zhang is incorrect. _The_ correct affiliation is as _follows:_ _Beijing_ Institute of Ophthalmology, Beijing Tongren _Eye_ Center, Beijing Tongren Hospital, Capital Medical University, _Beijing,_ _China._ The online version reflects these changes. |
1. Introduction {#s0005}
===============
Interactions of integral membrane proteins with their surrounding lipid environment play a key role in stabilising their structure and influencing their activity. To obtain insight into the nature and type of interactions occurring between lipids and integral membrane proteins a range of biophysical techniques including electron spin resonance [@bb0005; @bb0010] and fluorescence [@bb0145; @bb0020] spectroscopy have been used in numerous studies. This has provided a wealth of information on the specificity of proteins for particular classes of lipids and the affinity of these interactions [@bb0025; @bb0030]. These studies have revealed that in addition to the 'bulk' lipids whose dynamic properties remain largely unchanged by the presence of integral membrane proteins within the bilayer, there exist a population of lipids whose motional freedom is constrained through their interaction with integral membrane proteins. The motionally restricted population of lipids can be segregated into 'annular lipids' which exhibit low affinity interactions with the hydrophobic surface of membrane proteins and 'non-annular lipids' which exert high affinity interactions at sites located in clefts on the protein surface or at the interface between protein subunits [@bb0025; @bb0035; @bb0040; @bb0045]. The interactions at both sites play an important role in modulating the function of integral membrane proteins, but it has recently become apparent that occupation of the non-annular binding site can be particularly critical for function [@bb0050]. Despite the importance of these interactions a detailed atomic level description of the type and nature of the interplay between lipids and integral membrane proteins is limited as the lipids are often missing from high-resolution crystal structures of membrane proteins.
One of the best-studied ion channels is the potassium channel, KcsA from *Streptomyces lividans*. This potassium channel, in common with other members of this family, has an absolute requirement for anionic lipids such as phosphatidylglycerol, phosphatidylserine or cardiolipin for function; in their absence the channel exists in a non-conducting state [@bb0055; @bb0060]. The binding of lipids to KcsA has been extensively investigated by fluorescence quenching studies that clearly identify annular and non-annular populations of lipids [@bb0035; @bb0050]. Binding of lipids to the annular sites revealed marked differences between the inner and extracellular leaflets, with the extracellular side showing similar affinities for anionic and zwitterionic lipids. In contrast the intracellular side showed a twofold higher affinity for anionic lipids over phosphatidylcholine, presumably due to the clustering of charged residues on KcsA close to the bilayer surface. Fluorescence quenching studies have revealed that the non-annular binding sites show a high degree of selectivity of binding anionic lipids almost exclusively, albeit with moderate affinity.
The crystal structure of KcsA in detergent has provided valuable insights into the interaction of lipids with the non-annular binding site with electron density being seen at the interface between the protein subunits indicating the presence of a diacylglycerol-like moiety. The crystal structure revealed that the *sn*-1 chain of this lipid molecule was tightly buried in the groove between the pore helix and the M2 helix whilst the *sn*-2 chain was less intimately associated with the protein [@bb0055; @bb0065]. Subsequent studies have revealed the lipid present in the KcsA crystals to be phosphatidylglycerol which co-purifies with the KcsA [@bb0055] (pdb accession number 1K4C), suggesting that the phosphatidylglycerol headgroup exhibits significant motion or disorder within the crystal, resulting in the absence of electron density. The absence of electron density in the region corresponding to the lipid headgroup has precluded a detailed understanding of how the phosphatidylglycerol is recognised. The presence of two key arginine residues (R64 and R89) in close proximity to the proposed headgroup region suggested a putative role for electrostatic interactions between the sidechains and the anionic lipids within the binding site [@bb0055]. The proposed interactions between R64 and R89 have been investigated by molecular dynamics, revealing the formation of H-bonds between the headgroups of the anionic lipids phosphatidic acid and phosphatidylglycerol and the arginine residues [@bb0070]. Notably, these interactions were absent or reduced in bilayers containing the zwitterionic lipid phosphatidylethanolamine [@bb0070].
To demonstrate the feasibility of detecting interactions between non-annular binding sites and anionic lipids we have undertaken a 31P magic-angle spinning (MAS) NMR study of KcsA reconstituted into a model lipid bilayer composed of phosphatidylcholine and the negatively charged phosphatidylglycerol. The application of MAS permits the acquisition of ^31^P NMR spectra in which the individual lipid components are resolved on the basis of their chemical shift [@bb0075]. The chemical shift observed provides information on the local electrostatic environment of the phosphate moiety of the lipid headgroup and is thus an excellent reporter on the interaction of the lipid headgroup with the protein [@bb0080; @bb0085; @bb0090]. Typically the application of NMR to study lipid/protein interactions has proved challenging as exchange between the bulk lipid and annular sites occurs too rapidly on the NMR timescale to permit the observation of the bound lipids. Furthermore, interactions between the lipids and the proteins are typically hydrophobic in nature with little difference in electrostatic environment between the bound and free lipids resulting in only minor perturbations in chemical shift. In contrast, phosphatidylglycerol bound at the non-annular binding site is predicted to experience a significantly different electrostatic environment from the annular/bulk lipids with higher-affinity interactions resulting in longer residency times within the binding site. Below we demonstrate that this results in a spectroscopically distinct species that we resolve in the ^31^P MAS-NMR spectrum of KcsA reconstituted into lipid vesicles, providing insights into the interactions involved in lipid binding. Using site directed mutagenesis we have demonstrated that the perturbation in electrostatic environment arises through the interaction of the lipid head group with the positively charged sidechain of R64 and R89. Single-channel current recordings have been measured to ascertain the role that these residues play in determining the channel gating behaviour of KcsA.
2. Materials and methods {#s0010}
========================
2.1. Materials {#s0015}
--------------
Palmitoyloleoyl-phosphatidylcholine (POPC) and palmitoyloleoyl-phosphatidylglycerol (POPG) were purchased from Avanti Polar Lipids (Alabaster, AL). The pQE32 vector and M15\[PREP\] *Escherichia coli* strain were bought from Qiagen (UK). The detergent, dodecylmaltoside (DDM) was from Anatrace (UK). The other reagents for the purification were obtained from Sigma (UK).
2.2. Cloning and mutagenesis of KcsA {#s0030}
------------------------------------
The pQE32 vector containing the KcsA gene with a hexahistidine epitope at the N-terminus, kindly donated by Professor Lee (University of Southampton, UK), was expressed in M15 cells. Three KcsA mutants were generated by site-directed mutagenesis using the Quik-change protocol from Stratagene (La Jolla, CA). Three KcsA mutants were prepared replacing the arginine with the slightly smaller but uncharged leucine at residue 64 (R64L), 89 (R89L) or at both sites (R64,89L). The mutants were generated by PCR using synthetic oligonucleotide primers containing the desired mutations (Eurofins MWG, UK). Complementary oligonuceotides, 5′-AGCTGATCAC GTATCCGTTA GCGCTGTGGT GGTCC-3′ and 5′-GACCACCACA GCGCTAACGG ATACGTGATC AGCTG-3′ were used as forward and reverse primers respectively to create the R64L mutation. Similarly, R89L was produced using the synthetic oligonucleotides 5′-GTGACTCTGT GGGGCCTGC TCGTGGCCG TGGTGGTGA T-3′ and 5′-ATCACCACCA CGGCCACGA GCAGGCCCC ACAGAGTCA C-3′ as primers. Polymerase chain reaction was used to generate the mutants and the resultant PCR product was Dpn1- treated for 1 hr at 37 °C to digest any methylated parental DNA. The DNA was used to transform competent M15 \[PREP\] *E.coli* cells that were then plated onto agar plates supplemented with ampicillin. Mutations were confirmed by sequencing.
2.3. Over expression and purification of KcsA {#s0035}
---------------------------------------------
M15 *E.coli* cells transformed with KcsA or one of the KcsA mutants were used to inoculate 10 mL of Luria Broth (LB) medium containing 100 μg/mL of ampicillin. The overnight culture was then used to inoculate 1 L of LB containing 100 μg/mL of ampicillin and grown to an OD~600~ of 0.8 at 37 °C. Over-expression of KcsA wild type and mutant protein was induced by the addition of IPTG to a final concentration of 1 mM and the culture was grown for a further 4 h at 37 °C. The cells were harvested at 4 °C by centrifugation at 12,000 *g* for 20 min. The cell pellet was resuspended in buffer A (50 mM Tris, 150 mM NaCl, 150 mM KCl, pH 7.4) with 1 mM PMSF and sonicated on ice for 5 min: 15 s on; 20 s off at power level 7 (Misonix sonicator). The membrane fraction was clarified by ultracentrifugation at 420,000 *g* for 40 min at 5 °C. The membrane-containing pellet was homogenised and | 1. introduction { # s0005 } = = = = = = = = = = = = = = = interactions of integral membrane proteins with their surrounding lipid environment play a crucial role in stabilising their structure and influencing their activity. to obtain insight into the nature and type of interactions occurring between lipids and integral membrane proteins a range on biophysical techniques including electron spin resonance [ @ bb0005 ; @ bb0010 ] and fluorescence [ @ bb0145 ; @ bb0020 ] spectroscopy have been used in numerous studies. this has provided a wealth of information on the specificity of proteins for particular classes of lipids and the composition of these interactions [ @ bb0025 ; @ bb0030 ]. these studies have revealed that in addition to the ' bulk ' lipids whose dynamic properties remain largely unchanged by the presence of integral membranes proteins within the bilayer, there exist a population of lipids whose motional freedom was constrained through their interaction with integral membrane proteins. typical motionally restricted population of lipids can be segregated into ' annular lipids ' which exhibit low affinity interactions with the hydrophobic surface of membrane proteins and ' non - annular lipids ' which exert high affinity interactions at sites located in clefts on the protein surface or at the interface between protein subunits [ @ bb0025 ; @ bb0035 ; @ bb0040 ; @ bb0045 ]. the interactions at both sites play an important role in modulating the function of integral membrane proteins, but it has recently become apparent that occupation of the non - annular binding site can be particularly critical for sensing [ @ bb0050 ]. despite the importance of these interactions a detailed atomic level description of the type and nature of the bond between lipids and integral membrane proteins is limited as the lipids are often missing from high - resolution crystal structures of membrane proteins. one of the best - studied sodium channels is the potassium channel, kcsa from * streptomyces lividans *. this potassium pathway, in common with other members of this family, has an absolute requirement for anionic lipids such as phosphatidylglycerol, phosphatidylserine or cardiolipin for function ; in their absence the channel exists in a non - conducting state [ @ bb0055 ; @ bb0060 ]. the binding of lipids to kcsa has been extensively investigated by fluorescence quench ##ing studies that clearly identify annular and non - annular populations of lipids [ @ bb0035 ; @ bb0050 ]. binding of lipids to the annular sites revealed marked differences between the inner and extracellular leaflets, with the extracellular side showing similar affinities for anionic and zwitterionic lipids. in contrast the intracellular side showed a twofold higher affinity for anionic lipids over phosphatidylcholine, presumably due to the clustering of charged residues on kcsa close to the bilayer surface. fluorescence quenching studies have revealed that the non - annular binding sites show a high degree of selectivity of binding anionic lipids almost exclusively, albeit with moderate affinity. the crystal structure of kcsa in detergent has provided valuable insights into the interaction of lipids with the non - annular binding site with electron density being seen at the interface between the protein subunits indicating the presence of a diacylglycerol - like moiety. the crystal structure revealed that the * sn * - 1 chain of this lipid molecule was tightly buried in the groove between the pore helix and the m2 helix whilst the * sn * - 2 chain was less intimately associated with the protein [ @ bb0055 ; @ bb0065 ]. subsequent studies have revealed the lipid present in the kcsa crystals to be phosphatidylglycerol which co - purifies with the kcsa [ @ bb0055 ] ( pdb accession number 1k4c ), suggesting that the phosphatidylglycerol headgroup exhibits significant motion or disorder within the crystal, resulting in the absence of electron density. the absence of electron density in the region corresponding to the lipid headgroup has precluded a detailed understanding of how the phosphatidylglycerol is recognised. the presence of two key arginine residues ( r64 and r89 ) in close proximity to the proposed headgroup region suggested a putative role for electrostatic interactions between the sidechains and the anionic lipids within the binding site [ @ bb0055 ]. the proposed interactions between r64 and r89 have been investigated by molecular dynamics, revealing the formation of h - bonds between the headgroups of the anionic lipids phosphatidic acid and phosphatidylglycerol and the arginine residues [ @ bb0070 ]. notably, these interactions were absent or reduced in bilayers containing the zwitterionic lipid phosphatidylethanolamine [ @ bb0070 ]. to demonstrate the feasibility of detecting interactions between non - annular binding sites and anionic lipids we have undertaken a 31p magic - angle spinning ( mas ) nmr study of kcsa reconstituted into a model lipid bilayer composed of phosphatidylcholine and the negatively charged phosphatidylglycerol. the application of mas permits the acquisition of ^ 31 ^ p nmr spectra in which the individual lipid components are resolved on the basis of their chemical shift [ @ bb0075 ]. the chemical shift observed provides information on the local electrostatic environment of the phosphate moiety of the lipid headgroup and is thus an excellent reporter on the interaction of the lipid headgroup with the protein [ @ bb0080 ; @ bb0085 ; @ bb0090 ]. typically the application of nmr to study lipid / protein interactions has proved challenging as exchange between the bulk lipid and annular sites occurs too rapidly on the nmr timescale to permit the observation of the bound lipids. furthermore, interactions between the lipids and the proteins are typically hydrophobic in nature with little difference in electrostatic environment between the bound and free lipids resulting in only minor perturbations in chemical shift. in contrast, phosphatidylglycerol bound at the non - annular binding site is predicted to experience a significantly different electrostatic environment from the annular / bulk lipids with higher - affinity interactions resulting in longer residency times within the binding site. below we demonstrate that this results in a spectroscopically distinct species that we resolve in the ^ 31 ^ p mas - nmr spectrum of kcsa reconstituted into lipid vesicles, providing insights into the interactions involved in lipid binding. using site directed mutagenesis we have demonstrated that the perturbation in electrostatic environment arises through the interaction of the lipid head group with the positively charged sidechain of r64 and r89. single - channel current recordings have been measured to ascertain the role that these residues play in determining the channel gating behaviour of kcsa. 2. materials and methods { # s0010 } = = = = = = = = = = = = = = = = = = = = = = = = 2. 1. materials { # s0015 } - - - - - - - - - - - - - - palmitoyloleoyl - phosphatidylcholine ( popc ) and palmitoyloleoyl - phosphatidylglycerol ( popg ) were purchased from avanti polar lipids ( alabaster, al ). the pqe32 vector and m15 \ [ prep \ ] * escherichia coli * strain were bought from qiagen ( uk ). the detergent, dodecylmaltoside ( ddm ) was from anatrace ( uk ). the other reagents for the purification were obtained from sigma ( uk ). 2. 2. cloning and mutagenesis of kcsa { # s0030 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the pqe32 vector containing the kcsa gene with a hexahistidine epitope at the n - terminus, kindly donated by professor lee ( university of southampton, uk ), was expressed in m15 cells. three kcsa mutants were generated by site - directed mutagenesis using the quik - change protocol from stratagene ( la jolla, ca ). three kcsa mutants were prepared replacing the arginine with the slightly smaller but uncharged leucine at residue 64 ( r64l ), 89 ( r89l ) or at both sites ( r64, 89l ). the mutants were generated by pcr using synthetic oligonucleotide primers containing the desired mutations ( eurofins mwg, uk ). complementary oligonuceotides, 5 ′ - agctgatcac gtatccgtta gcgctgtggt ggtcc - 3 ′ and 5 ′ - gaccaccaca gcgctaacgg atacgtgatc agctg - 3 ′ were used as forward and reverse primers respectively to create the r64l mutation. similarly, r89l was produced using the synthetic oligonucleotides 5 ′ - gtgactctgt ggggcctgc tcgtggccg tggtggtga t - 3 ′ and 5 ′ - atcaccacca cggccacga gcaggcccc acagagtca c - 3 ′ as primers. polymerase chain reaction was used to generate the mutants and the resultant pcr product was dpn1 - treated for 1 hr at 37 °c to digest any methylated parental dna. the dna was used to transform competent m15 \ [ prep \ ] * e. coli * cells that were then plated onto agar plates supplemented with ampicillin. mutations were confirmed by sequencing. 2. 3. over expression and purification of kcsa { # s0035 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - m15 * e. coli * cells transformed with kcsa or one of the kcsa mutants were used to inoculate 10 ml of luria broth ( lb ) medium containing 100 μg / ml of ampicillin. the overnight culture was then used to inoculate 1 l of lb containing 100 μg / ml of ampicillin and grown to an od ~ 600 ~ of 0. 8 at 37 °c. over - expression of kcsa wild type and mutant protein was induced by the addition of iptg to a final concentration of 1 mm and the culture was grown for a further 4 h at 37 °c. the cells were harvested at 4 °c by centrifugation at 12, 000 * g * for 20 min. the cell pellet was resuspended in buffer a ( 50 mm tris, 150 mm nacl, 150 mm kcl, ph 7. 4 ) with 1 mm pmsf and sonicated on ice for 5 min : 15 s on ; 20 s off at power level 7 ( misonix sonicator ). the membrane fraction was clarified by ultracentrifugation at 420, 000 * g * for 40 min at 5 °c. the membrane - containing pellet was homogenised and | 1. Introduction {# s0005} = = = = = = = = = = = = = = = Interactions of integral membrane proteins with their surrounding lipid environment play a key role in stabilising their stghcture and influencing their activity. To obtain insight into the nature and type of interactions occurring between lipids and integral membrane proteins a range of biophysical techniques including electron spin resonance [@ bb0005; @ bb0010] and fluorescence [@ bb0145; @ bb0020] spectroscopy have been used in numerous studies. This has provided a wealth of information on the specificity of proteins for particular classes of lipids and the affinity of these interactions [@ bb0025; @ bb0030 ]. These studies have revealed that in addition to the ' bulk ' lipids whose dynamic properties remain largely unchanged by the presence of integral membrane proteins within the bilayer, there exist a population of lipids whose motional freedom is constrained through their interaction with integral membrane proteins. The motionally restricted population of lipids can be segregated into ' annular lipids ' which exhibit low affinity interactions with the hydrophobic surface of membrane proteins and ' non - annular lipids ' which exert high affinity interactions at sites located in clefts on the protein surface or at the interface between protein subunits [@ bb0025; @ bb0035; @ bb0040; @ bb0045 ]. The interactions at both sites play an important role in modulating the function of integral membrane proteins, but it has recently become apparent that occupation of the non - annular binding site can be particularly critical for function [@ bb0050 ]. Despite the importance of these interactions a detailed atomic level description of the type and nature of the inrerp:ay between lipids and integral membrane proteins is limited as the lipids are often missing from high - resolution crystal structures of membrane proteins. One of the best - studied ion channels is the potassium channel, KcsA from * Streptomyces lividans *. This potassium channel, in common with other members of this family, has an absolute requirement for anionic lipids such as phosphatidylglycerol, phosphatidylserine or cardiolipin for function; in their absence the channel exists in a non - cohductjng state [@ bb0055; @ bb0060 ]. The binding of lipids to KcsA has been extensively investigated by fluorescence quenching studies that clearly identify annular and non - annular populations of lipids [@ bb0035; @ bb0050 ]. Binding of lipids to the annular sites revealed marked differences between the inner and extracellular leaflets, with the extracellular side showing similar affibi%ies for anionic and zwitterionic lipids. In contrast the intracellular side showed a twofold higher affinity for anionic lipids over phosphatidylcholine, presumably due to the clustering of charged residues on KcsA close to the bilayer surface. Fluorescence quenching studies have revealed that the non - annular binding sites show a high degree of selectivity of binding anionic lipids almost exclusively, albeit with moderate affinity. The crystal structure of KcsA in detergent has provided valuable insights into the interaction of lipids with the non - annular binding site with electron density being seen at the interface between the protein subunits indicating the presence of a diacylglycerol - like moiety. The crystal structure revealed that the * sn * - 1 chain of this lipid molecule was tightly buried in the groove between the pore helix and the M2 helix whilst the * sn * - 2 chain was less intimately associated with the protein [@ bb0055; @ bb0065 ]. Subsequent studies have revealed the lipid present in the KcsA crystals to be phosphatidylglycerol which co - purifies with the KcsA [@ bb0055] (pdb accession number 1K4C ), suggesting that the phosphatidylglycerol headgroup exhibits significant motion or disorder within the crystal, resulting in the absence of electron density. The absence of electron density in the region corresponding to the lipid headgroup has precluded a detailed understanding of how the phosphatidylglycerol is recognised. The presence of two key arginine residues (R64 and R89) in close proximity to the proposed headgroup region suggested a putative role for electrostatic interactions between the sidechains and the anionic lipids within the binding site [@ bb0055 ]. The proposed interactions between R64 and R89 have been investigated by molecular dynamics, revealing the formation of H - bonds between the headgroups of the anionic lipids phosphatidic acid and phosphatidylglycerol and the arginine residues [@ bb0070 ]. Notably, these interactions were absent or reduced in bilayers containing the zwitterionic lipid phosphatidylethanolamine [@ bb0070 ]. To demonstrate the feasibility of detecting interactions between non - annular binding sites and anionic lipids we have undertaken a 31P magic - angle spinning (MAS) NMR study of KcsA reconstituted into a model lipid bilayer composed of phosphatidylcholine and the negatively charged phosphatidylglycerol. The application of MAS permits the acquisition of ^ 31 ^ P NMR spectra in which the individual lipid components are resolved on the basis of their chemical shift [@ bb0075 ]. The chemical shift observed provides information on the local electrostatic environment of the phosphate moiety of the lipid headgroup and is thus an excellent reporter on the interaction of the lipid headgroup with the protein [@ bb0080; @ bb0085; @ bb0090 ]. Typically the application of NMR to study lipid / protein interactions has proved challenging as exchange between the bulk lipid and annular sites occurs too rapidly on the NMR timescale to permit the observation of the bound lipids. Furthermore, interactions between the lipids and the proteins are typically hydrophobic in nature with little difference in electrostatic environment between the bound and free lipids resulting in only minor perturbafiPns in chemical shift. In contrast, phosphatidylglycerol bound at the non - annular binding site is predicted to experience a significantly different electrostatic environment from the annular / bulk lipids with higher - affinity interactions resulting in longer residency times within the binding site. Below we demonstrate that this results in a spectroscopically distinct species that we resolve in the ^ 31 ^ P MAS - NMR spectrum of KcsA reconstituted into lipid vesicles, providing insights into the interactions involved in lipid binding. Using site directed mutagenesis we have demonstrated that the perturbation in electrostatic environment arises through the interaction of the li9if head group with the positively charged sidechain of R64 and R89. Single - channel current recordings have been measured to ascertain the role that these residues play in determining the channel gating behaviour of KcsA. 2. Materials and methods {# s0010} = = = = = = = = = = = = = = = = = = = = = = = = 2. 1. Materials {# s0015} - - - - - - - - - - - - - - Palmitoyloleoyl - phosphatidylcholine (POPC) and palmitoyloleoyl - phosphatidylglycerol (POPG) were purchased from Avanti Polar Lipids (Alabaster, AL ). The pQE32 vector and M15 \ [PREP \] * Escherichia coli * strain were bought from Qiagen (UK ). The detergent, dodecylmaltoside (DDM) was from Anatrace (UK ). The other reagents for the purification were (btaiged from Sigma (UK ). 2. 2. Cloning and mutagenesis of KcsA {# s0030} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The pQE32 vector containing the KcsA gene with a hexahistidine epitope at the N - terminus, kindly donated by Professor Lee (University of Southampton, UK ), was expressed in M15 cells. Three KcsA mutants were generated by site - directed mutagenesis using the Quik - change protocol from Stratagene (La Jolla, CA ). Three KcsA mutants were prepared replacing the arginine with the slightly smaller but uncharged leucine at residue 64 (R64L ), 89 (R89L) or at both sites (R64, 89L ). The ,urants were generated by PCR using synthetic oligonucleotide primers containing the desired mutations (Eurofins MWG, UK ). Complementary oligonuceotides, 5 ′ - AGCTGATCAC GTATCCGTTA GCGCTGTGGT GGTCC - 3 ′ and 5 ′ - GACCACCACA GCGCTAACGG ATACGTGATC AGCTG - 3 ′ were used as forward and reverse primers respectively to create the R64L mutation. Similarly, R89L was produced using the synthetic oligonucleotides 5 ′ - GTGACTCTGT GGGGCCTGC TCGTGGCCG TGGTGGTGA T - 3 ′ and 5 ′ - ATCACCACCA CGGCCACGA GCAGGCCCC ACAGAGTCA C - 3 ′ as primers. Po<yHerase chain reaction was used to generate the mutants and the resultant PCR product was Dpn1 - treated for 1 hr at 37 ° C to digest any methylated parental DNA. The DNA was udeV to transform competent M15 \ [PREP \] * E. coli * cells that were then plated onto agar plates supplemented with ampicillin. Mutations were confirmed by sequencing. 2. 3. Over expression and purification of KcsA {# s0035} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M15 * E. coli * cells transformed with KcsA or one of the KcsA mutants were used to inoculate 10 mL of Luria Broth (LB) medium containing 100 μg / mL of ampicillin. The overnight culture was then used to inoculate 1 L of LB containing 100 μg / mL of ampicillin and grown to an OD ~ 600 ~ of 0. 8 at 37 ° C. Over - expression of KcsA wild type and mutant protein was induced by the addition of IPTG to a final concentration of 1 mM and the culture was grown for a further 4 h at 37 ° C. The cells were harvested at 4 ° C by centrifugation at 12, 000 * g * for 20 min. The cell pellet was resuspended in buffer A (50 mM Tris, 150 mM NaCl, 150 mM KCl, pH 7. 4) with 1 mM PMSF and sonicated on ice for 5 min: 15 s on; 20 s off at power level 7 (Misonix sonicator ). The membrane fraction was clarified by ultracentrifugation at 420, 000 * g * for 40 min at 5 ° C. The membrane - containing pellet was homogenised and | 1. Introduction {#s0005} =============== Interactions of integral membrane proteins with their surrounding lipid environment play a key role in stabilising their structure and influencing their activity. To obtain insight into the nature and of interactions occurring between lipids and integral membrane proteins a of biophysical techniques including electron spin resonance [@bb0005; @bb0010] and fluorescence [@bb0145; @bb0020] spectroscopy have been used in numerous studies. This has provided a wealth of information the specificity of proteins for particular classes of and the affinity of these interactions [@bb0025; @bb0030]. have revealed that in addition to the 'bulk' lipids dynamic properties remain largely unchanged by the presence of integral proteins within the bilayer, there exist a of whose motional freedom constrained through their interaction with integral membrane proteins. The motionally restricted population of lipids can be into 'annular lipids' which exhibit affinity interactions with the hydrophobic surface of membrane proteins and 'non-annular lipids' which exert high affinity interactions at sites located in clefts on the surface or at the interface between protein subunits [@bb0025; @bb0035; @bb0040; @bb0045]. The interactions at both sites play an role in modulating the of integral membrane proteins, but it has recently become apparent that occupation of the non-annular binding site can be particularly critical for function [@bb0050]. the importance of these interactions a detailed atomic level description of the type and nature of interplay between lipids integral membrane proteins is limited as the lipids are often missing from high-resolution crystal structures of membrane proteins. One of the best-studied ion channels the potassium channel, KcsA from *Streptomyces This channel, common with other members this family, has an absolute requirement for anionic lipids such as phosphatidylglycerol, phosphatidylserine or cardiolipin for function; in their absence the exists in a non-conducting state [@bb0055; The binding of lipids to has been extensively investigated by fluorescence quenching studies that clearly identify annular and non-annular populations [@bb0035; @bb0050]. Binding of lipids the annular sites revealed marked differences between the inner and extracellular leaflets, the extracellular side showing similar affinities for anionic and zwitterionic lipids. In contrast intracellular showed a twofold higher affinity anionic lipids over phosphatidylcholine, presumably due to the clustering of charged residues on close to bilayer surface. Fluorescence quenching studies have revealed that the non-annular binding sites show a high degree of selectivity of binding lipids almost exclusively, albeit with moderate affinity. The crystal structure of KcsA in detergent has valuable into interaction of lipids the non-annular binding with electron density being seen at the interface between the protein indicating the presence of a diacylglycerol-like The structure revealed that the *sn*-1 chain of this lipid molecule was tightly the groove between the pore helix the M2 helix whilst the *sn*-2 chain was less intimately associated with the protein [@bb0055; @bb0065]. Subsequent studies have revealed the lipid in the KcsA crystals to be phosphatidylglycerol which with the KcsA [@bb0055] (pdb accession number suggesting that phosphatidylglycerol headgroup exhibits significant motion or disorder the crystal, in the absence of electron density. The absence of electron density in the region corresponding the lipid headgroup has precluded a detailed understanding of how the phosphatidylglycerol is recognised. The presence of key residues (R64 and R89) in close to proposed region suggested a putative role for electrostatic interactions between the sidechains and the anionic lipids within the binding site [@bb0055]. The proposed interactions between R64 and R89 have been investigated by revealing the formation of H-bonds between the headgroups of the anionic lipids phosphatidic acid and phosphatidylglycerol and the arginine residues [@bb0070]. Notably, these interactions were absent or reduced in bilayers containing the zwitterionic lipid [@bb0070]. demonstrate the feasibility of detecting interactions between non-annular binding sites and anionic lipids we have undertaken a 31P magic-angle NMR study of KcsA reconstituted into model lipid bilayer composed of phosphatidylcholine and the negatively charged The of MAS permits the acquisition of ^31^P NMR spectra in the individual components are resolved on the basis of chemical shift [@bb0075]. The chemical observed provides information on the local electrostatic environment of the phosphate moiety of the lipid headgroup and is an excellent reporter the interaction of the lipid headgroup with the protein [@bb0080; @bb0085; @bb0090]. Typically the application of NMR to study interactions has proved challenging as between the bulk lipid and annular sites occurs rapidly on the NMR timescale permit the observation the bound Furthermore, interactions the lipids and the proteins are typically hydrophobic in nature with little difference in electrostatic environment between the bound and free lipids resulting in only minor perturbations in chemical shift. contrast, phosphatidylglycerol at non-annular binding site is predicted to experience a significantly electrostatic environment from the annular/bulk lipids with higher-affinity interactions resulting in longer residency times within binding site. we that this results in a spectroscopically distinct species that we resolve in the ^31^P MAS-NMR spectrum of KcsA reconstituted into lipid vesicles, providing insights into the involved binding. Using site directed mutagenesis we have demonstrated that the perturbation in electrostatic environment arises through the interaction of the lipid head group with the positively charged sidechain of and R89. Single-channel recordings have been measured to ascertain the role that play in determining the channel gating behaviour of KcsA. 2. Materials and methods {#s0010} ======================== 2.1. Materials {#s0015} -------------- Palmitoyloleoyl-phosphatidylcholine (POPC) and palmitoyloleoyl-phosphatidylglycerol were from Avanti Polar Lipids (Alabaster, AL). pQE32 vector M15\[PREP\] *Escherichia coli* strain were bought Qiagen (UK). The detergent, (DDM) was from Anatrace (UK). The other reagents for the purification were obtained from Sigma (UK). 2.2. Cloning and mutagenesis of KcsA {#s0030} ------------------------------------ The pQE32 vector containing KcsA gene a hexahistidine epitope at the N-terminus, kindly donated by Professor Lee (University of Southampton, UK), was expressed in M15 cells. Three KcsA mutants generated by site-directed mutagenesis using Quik-change protocol from Stratagene (La Jolla, CA). Three KcsA mutants were prepared replacing the arginine with the slightly smaller but uncharged leucine at 64 (R64L), 89 (R89L) or at both sites (R64,89L). The mutants were PCR using oligonucleotide primers containing the desired mutations (Eurofins UK). Complementary oligonuceotides, 5′-AGCTGATCAC GTATCCGTTA GCGCTGTGGT GGTCC-3′ and 5′-GACCACCACA GCGCTAACGG ATACGTGATC AGCTG-3′ were as forward and reverse primers respectively to create the R64L mutation. was produced using synthetic oligonucleotides 5′-GTGACTCTGT GGGGCCTGC TCGTGGCCG TGGTGGTGA T-3′ and 5′-ATCACCACCA CGGCCACGA GCAGGCCCC ACAGAGTCA C-3′ as primers. Polymerase chain reaction was used to generate the and the resultant PCR product was Dpn1- treated for 1 hr at °C to any methylated parental DNA. DNA was used to transform competent M15 \[PREP\] *E.coli* that were then plated onto agar plates supplemented with ampicillin. Mutations confirmed by sequencing. 2.3. Over expression and purification of KcsA {#s0035} --------------------------------------------- M15 *E.coli* cells transformed with KcsA or one of the KcsA mutants were used to inoculate 10 mL of Luria Broth (LB) medium containing 100 μg/mL of ampicillin. The overnight culture was then used to inoculate L of LB containing 100 of ampicillin and grown to OD~600~ of 0.8 at 37 °C. of KcsA wild type and mutant protein was induced the addition of IPTG to a final concentration of 1 mM and the culture was grown a further 4 h °C. The cells were harvested at 4 °C by centrifugation at 12,000 *g* for 20 min. The cell pellet was resuspended in A (50 mM Tris, 150 mM NaCl, 150 KCl, 7.4) 1 mM PMSF and sonicated on ice for 5 min: 15 s on; 20 s off at level 7 (Misonix sonicator). The membrane fraction was by ultracentrifugation *g* for 40 min at 5 °C. membrane-containing pellet was homogenised and | 1. IntroduCtiOn {#S0005}
===============
INtEraCTions OF iNtegraL MemBRANe pRoTeIns wiTh tHeIR SurROUnDiNG LiPid enVirONMenT pLay a kEY ROLE IN stABiLisIng tHEIR STRuCtURe And influencING Their ACTIvITY. to oBtaiN InsiGHT iNTO THE NaTure anD typE Of inTEractioNS oCCurRINg BETwEeN LipIds aNd INTegrAl meMbrAne PrOTeiNs a RAnGe Of biophysiCal TeCHniqueS inCludiNg eLECTron sPIn rEsoNAncE [@Bb0005; @bb0010] And flUOrEscEncE [@bB0145; @BB0020] SPeCtroscOpy Have bEen Used In nuMEROus StUdIEs. this hAs pROvIDEd a weAlth oF Information on The SpeCifIcIty Of pROtEInS foR pArticUlAr claSses oF LIPids anD THe aFFINItY of THESE InTErACTiONS [@bb0025; @BB0030]. these stuDIeS haVE rEveALED THaT iN aDDitioN To the 'BulK' lIpIDS wHosE DYNAMiC PRoPErTiES REMain lArgELY uNChaNgeD by tHe PreSEnCe of iNTegrAl memBraNE pROTEins WitHIn ThE BiLaYEr, theRE eXisT A POPuLatIOn Of LIPidS WHoSe MOTiOnaL fReedOM IS CONsTRAiNeD tHROUgh ThEIR iNterACTIOn wIth INtegRAL MeMBRaNe proTeINs. ThE motIOnAlly ResTRIcTEd POpulAtiOn OF LiPiDS CAN Be seGregaTEd IntO 'aNnULar LIpIdS' wHICH eXHIBit Low aFFiNITy INteRACtioNS witH THe HyDroPhOBIC SuRFaCE oF MEmBrANe pRoTEINs ANd 'nOn-aNNUlAR lIPIds' wHich eXErT HiGH AfFinItY iNtEracTiONs AT SiTES lOcated IN CLEfTs on THe PROTEIN SURFace Or At tHE InteRFACe BETWeeN PROTEIn sUbuNiTS [@BB0025; @Bb0035; @bb0040; @bb0045]. THe inTERACtIOnS At Both SiTEs PlaY aN imPortAnt ROle in mOdUlAtinG thE FuncTIon of intEgraL mEMbraNe pROteIns, bUt it has ReCently BECoME AppArENT tHAT OccUPATiOn OF The non-aNnulAr Binding sITe cAN BE paRtiCulaRLY CRitiCaL FoR FuNctIon [@bb0050]. deSpItE THe iMporTAnce Of tHESe iNterACTIoNs A dETAILEd ATOMic LeVeL dESCRIpTIon OF the tYpe and NaTURE oF THe INTerpLAY BEtWeen lIPiDS aNd IntEgraL MEmbRANE prOTEIns iS LIMiTeD AS THe LIpIdS arE OFTeN MisSiNg FrOM hIGH-ResoLuTion cRYSTAl sTRuCtuRes Of MembRANE pRoTEINS.
oNe of The bEST-sTudIed iOn chaNneLs iS ThE POtaSsium ChANNEL, KcSA From *sTREPToMYceS lividans*. tHis potaSsIUm CHAnnEl, In ComMOn wiTH OTHer MEMBErS OF THIS FAMILY, hAs AN AbSolute rEquiremENt fOr aNIoNiC lIPiDS SUch As PhOspHatIDYlglycErOl, PhoSpHaTiDYlSERInE oR cArdioLiPIn FOr fuNction; iN THeIr aBsenCe The chAnnel eXiStS In a nON-cOndUcTiNg StaTe [@Bb0055; @bb0060]. THe biNdINg oF LIpiDs TO KcSA hAS beEn EXTENSiVelY INvESTigaTeD by fLUoreSCEnce QueNching stuDiES ThAT CLearLY IDentifY aNNuLAR AnD NoN-ANNUlAr POpULaTioNs of LIPiDs [@bB0035; @BB0050]. BINding of LIpIds To ThE AnnulaR siTEs ReveALEd marKeD dIfFEreNCeS bETWeeN THE iNNER anD ExtRACeLluLAr LEAFLETs, wiTH thE eXtRaceLluLAR sIdE sHowING siMiLAr aFfInITiEs FOr aNIoNIC anD ZWitTErIONIC lIpiDS. In coNTrAST thE iNTrAceLLUlAR Side ShOwEd A tWOFoLD HIgHER AfFInITy foR ANioNIC LIPidS oVeR phoSphatidYlChoLinE, PReSUmabLY dUe To The ClUsTERInG Of CHArGEd ReSidUeS On KcSA cLOse To ThE BiLAYer sURFAcE. fLUOrescEnCE queNChiNg STuDieS HavE rEveaLed THat thE noN-Annular BInDiNG sIteS SHow A HIGh DEGree oF SElECTIVity of BiNdInG ANioNic lIpIdS ALMosT eXcLusIvELY, alBEit WitH ModeraTe AfFiNiTY.
THe cRystAL StrUctURe OF KcSa iN deteRGeNT HaS PROVideD vAlUAble INsIghTS INto thE iNterACtioN oF Lipids WIth tHE NOn-aNnULar bINDiNG sITE WItH EleCTROn DENsITy Being seeN At THE iNtERfAcE bETWeeN The PROTEiN SubUnITs INdicaTiNg ThE PReseNcE OF A DiACylglYCErol-liKe moIETy. THe CRYStal STrUCturE REVeaLED THAt ThE *sn*-1 chain OF thiS lipID moleCULE Was TIGHTly bUrieD in ThE gRoOVe beTwEEN The POrE HElIX AnD The M2 hELiX wHilst thE *SN*-2 chaIn wAS LesS INTimAtelY ASSOciateD WItH thE ProtEIN [@Bb0055; @bB0065]. SUBSEQuENt StudIES Have revEaled ThE lIpID PReseNT in the kCsA CrysTALs To Be PHOSPhaTiDylGlyCeroL WHICh cO-pURiFiES wiTh the kCSa [@bB0055] (pdb accesSIoN NuMBer 1k4c), sUgGeStIng That tHE pHosPHaTidYLgLYcERoL HEadGroUp EXhIBiTS siGnificAnT MOtiON oR DisORder wITHIN tHE CRYSTAl, REsultIng iN The aBseNCe OF EleCtRON DeNSiTy. tHe aBseNcE oF eLECTron denSitY iN ThE regiOn cOrresPOnDINg To the lipiD HEAdGRouP hAS pReCLUdEd a DEtAIled uNDerstandiNG OF HOw ThE pHoSphaTIdylGlYcerol iS RecOgnIsed. The pReSenCE oF TWo kEY ArgiNiNe reSIDues (r64 anD R89) In cLOse proXimIty TO ThE PrOPosEd HeAdgROup RegION sUgGESTEd A putAtiVe rOle FOr eLEctrOsTAtic iNteRAcTIONS beTWEeN ThE SIDecHAINS ANd thE ANioniC lIpids WITHIN tHE BINdinG siTe [@bb0055]. The PrOPOsEd INTERacTiONS Between r64 ANd R89 hAve beEN inveSTIgatED By MOlECular dYNamICS, REVEALInG tHE ForMAtIOn of H-BoNdS BetwEEn thE HEADGROUPS oF THE AnioNic lIPiDS PHosPHaTiDIc aCID and pHosPhatiDYLgLYCEROl aNd thE ARginINe ReSIDueS [@BB0070]. NOtabLy, THeSE iNTerActIOns WerE ABSeNT OR reDUCed in BilAYErS cONtaINiNg ThE ZWiTtErIonIc lIpiD pHoSPhatIdyleThanOLamine [@Bb0070].
tO DEMONstRatE The FEasiBILITy Of DETeCtIng INtErACtiONS BeTwEEN nON-ANNulAR BInDiNG siTeS and AnIONiC lIpIds We have UNDertAKEN A 31p mAGIC-aNGle sPinnING (mas) NMR STudy of kCsa reCoNsTItUtED INtO a modeL lipID BIlayeR CoMPOSeD oF PHoSPhATIDYLcHOlIne aND ThE negativelY cHaRGEd pHosPhATidylGLyCErol. thE applicatiOn oF mAs PeRmItS THe ACqUISiTION oF ^31^p Nmr sPecTRa in wHICh tHe INDivIduAL lIpId ComponEnTs aRe RESoLVED On tHe Basis oF THEir ChEMicAl SHift [@Bb0075]. The chemicAl shiFT OBSERveD pROViDEs INFOrMAtioN ON THe locaL ELECTRoStaTic enVIRONmeNt of tHE phoSpHAte mOiety oF thE Lipid HEadGrouP And iS tHUs An ExcEllEnt RepoRtEr ON ThE INtERAcTiOn of The LIPid heAdgROuP WIth The proTEIn [@bb0080; @Bb0085; @bb0090]. tyPIcalLy thE applICatIoN of nmR TO StudY liPId/pRoTeIN InTeRacTIONS HAs ProVed ChaLlenGiNg aS exChange betwEEN The BULk liPID ANd ANNULaR sites OCcUrS tOO rapiDly ON tHe nMR TiMEScale TO PErMiT the ObSeRVatiON OF THE BOUnD lIpIDs. FUrTHermORe, InTeraCtIONS beTWEeN THE LIpiDS AND tHe PROTEINS ARe TYpIcALlY hydropHObiC In nATUrE WiTH littlE dIFFEREnCe In EleCtrOstaTIC EnVIRonment BeTweeN tHE BOund aND fREe lipiDs reSuLTINg iN Only MINoR peRturbATIonS In cHEmIcAL sHIFt. iN coNtrAst, pHosphatIdYLGlYCEroL boUnD AT tHe nOn-ANNuLar BIndiNg sIte Is PREDICTEd tO EXPERIence a SIgNIFicaNtLY DIfFerenT eLEctRoStaTIC enVironMent FrOM THE ANnuLAr/bULK lipIds wIth higHEr-aFFInITy inTerAcTionS rEsULTing iN longER reSiDeNCy timeS WitHIN thE bIndIng siTE. BElow we DeMOnstrATE THat thIS REsults In A SpECTRoSCoPIcAlLy DiSTINCT SpecIeS tHAt we ReSOlvE iN tHe ^31^P MAS-nmR sPEcTRum OF KCSA REconstituTEd INtO lIpid VEsiClES, pROvidING iNSIGhTS intO the inTEracTionS InVolVEd In LIPid biNdinG. uSing SITE DiRected mUTagENEsIS we haVe demOnstRatEd tHat The pertURBatIoN in eLectroStatIc EnvIRonmeNT ARiSeS THRoUGH ThE IntErACtiON OF tHE LIPID hEAd groUP witH the poSitIveLY cHARGed SiDeChaiN oF r64 ANd R89. SiNGlE-chAnNeL CUrREnt RECORDINGs HaVE BEEN MeASuRED To ASCertAiN THE rOle ThaT ThESE reSidUEs PLaY IN dETermiNINg thE chAnneL GATinG bEhaViOUR Of kcSA.
2. MaTERIaLs and mETHOds {#S0010}
========================
2.1. MATeRiALS {#s0015}
--------------
PAlmIToYLoLeOyL-pHosPhAtidylCHOlinE (pOpC) aND palMItOylOLEoyl-pHOSPhATidylGlyCErOl (popG) WERE PURchasEd fROM avAntI PoLaR LIpids (ALAbasTeR, al). THE pqe32 VECtoR aNd m15\[PrEp\] *eSCheRIcHIA cOli* STRaIN WeRe bOuGhT FroM QiAGEN (Uk). tHe dEtErGENT, DodeCYLmaLTosIDE (dDM) WaS FROm AnatRAce (UK). tHE oTHER ReAGeNTs fOR tHE pUrIFicaTiOn werE ObTainED frOM sIgma (uK).
2.2. ClOniNg and mutAgeneSIS of kCSa {#s0030}
------------------------------------
The pQe32 vEcTOR COntAINinG tHe kCsa gene wITH A HEXaHIStIdInE epitope aT thE n-tERmiNUs, kInDly DonatED bY pRoFeSsor leE (UNiVeRsiTy OF soUThampTOn, uK), was exprEsSED In M15 Cells. ThrEe KCSA mUtANTS werE genERATed By sIte-dIrECTeD MutAgeneSis UsiNG THE QUiK-CHAnGE PrOTOCoL FRom StrATagEnE (La JOllA, CA). Three kCsA MuTANts wErE PrepArEd rEPlaCiNg THe argiNiNe wITH tHE sLiGhtly SMaLlER bUT uncHaRGEd leUcINE At resiDuE 64 (R64l), 89 (R89l) OR aT BOTh sItEs (R64,89l). ThE MuTaNts WerE GeNERAted by pCr UsIng SYnTHeTIc oLIGOnUCLeOTIDE PRimerS cOntainING THE dESiReD mUTatioNS (eURofiNs mwG, UK). comPleMeNTarY OLIGOnucEoTideS, 5′-AGCTgATcAc gtATccgtta gCgCtGtggt gGtcC-3′ And 5′-GAccaCcaCA gCGctaaCGg aTAcGtgATc AGCtG-3′ wErE usED aS fORWarD AnD REVeRSe PRIMERs RESPEcTIvelY to CReate The r64l MUTatioN. simIlaRly, R89L wAS proDUcEd usInG THe sYntHETIC OligONuCLeotIDes 5′-gTgAcTctGT GgGGCctgC tCgtgGCCG tggTggTGa t-3′ anD 5′-atCAcCAcca CGGCcAcga gCaGGccCC aCagAgtcA C-3′ aS PriMERs. POlymeRAsE cHaIN reaCtion waS UsED To gENERAte THE mutaNTS aND THE rEsUlTant pcR PrOdUct wAs DpN1- TReAtED FoR 1 hR At 37 °c tO dIGeST Any mEthylATEd pARenTal Dna. tHe dNa was usEd to TRAnSFORm COMPETEnt M15 \[pREp\] *e.COli* cELls that were THen plATed onTo aGar PlateS SuppLeMenTED wITh AMpiciLLin. mUtaTIoNS weRE ConFirmEd bY sEqueNcInG.
2.3. OVEr exPressIOn and PurIfIcaTion Of KCSa {#S0035}
---------------------------------------------
m15 *e.coli* CellS trANSFOrmeD WIth Kcsa Or ONE of the KcsA MUtAnTs were uSED to inOcULAtE 10 mL Of lurIA bRoTh (lb) mediuM ContAiniNg 100 Μg/ML OF AmPICilLIN. thE overnIGht CULTuRE WaS THEn useD tO iNoCulaTe 1 l OF lB coNTaINing 100 μG/Ml OF amPiCiLliN ANd GrowN TO An od~600~ Of 0.8 At 37 °C. over-ExPRESSioN oF KCSA WilD typE anD MUTAnt prOTeIN waS InDuCEd bY ThE adDiTIOn oF ipTG To a fINAl cOncEntraTION OF 1 mm AnD THe CuLtURe Was groWN fOr a FurtHEr 4 H AT 37 °c. tHE cElls WErE hArVESted aT 4 °C bY ceNtRIfUgAtIOn At 12,000 *g* FOR 20 mIn. the ceLL peLLET wAS reSuspeNded in bUFfER A (50 mm TrIS, 150 Mm NACl, 150 MM kCL, PH 7.4) WITh 1 Mm PmSf AND soniCAtEd on ice FOR 5 MiN: 15 s On; 20 s off at Power leveL 7 (mISONiX SonIcAToR). tHE mEMBrANE FRAcTiON Was ClAriFIeD BY uLTracENTRIfUgatiON aT 420,000 *G* fOr 40 MIN at 5 °C. THE MeMBraNe-CONtAinING PEllET WAs HOMOGeNIsed anD | 1. Introduction {#s0005} =============== Interactions of integral membrane proteins withtheirsurrounding lipid environment play a key role in stabilising their structure and influencing their activity. To obtain insight into the natureand type ofinteractions occurring between lipids and integral membrane proteinsa range of biophysical techniques including electronspin resonance [@bb0005; @bb0010] and fluorescence [@bb0145; @bb0020] spectroscopy have beenused in numerous studies. This has provided a wealth ofinformation on the specificity ofproteins forparticular classes of lipids and theaffinity of these interactions [@bb0025; @bb0030]. These studies have revealed thatin addition to the 'bulk' lipids whose dynamic properties remain largelyunchanged by the presence ofintegral membrane proteinswithin the bilayer,there exist a population of lipids whose motional freedom is constrained through their interaction with integral membrane proteins. The motionally restricted population of lipids can be segregated into 'annular lipids' which exhibit low affinity interactionswith the hydrophobicsurface of membraneproteinsand 'non-annular lipids' which exert highaffinity interactions at siteslocated in clefts on the protein surface or at the interface between protein subunits[@bb0025; @bb0035; @bb0040;@bb0045].The interactions at bothsites play an important role in modulating the function of integral membrane proteins, but it has recently become apparentthat occupation of the non-annular binding site can be particularly critical for function [@bb0050]. Despitethe importance of these interactions a detailed atomic level description of the type andnature of the interplay between lipidsand integral membrane proteins is limited as the lipids are often missing from high-resolution crystal structures of membraneproteins. One of the best-studiedion channels isthe potassium channel, KcsA from *Streptomyceslividans*. This potassium channel, in common with other membersof this family, has an absolute requirement foranionic lipidssuch as phosphatidylglycerol, phosphatidylserine or cardiolipin forfunction; in their absence the channel existsin a non-conducting state [@bb0055; @bb0060]. The binding of lipids to KcsA hasbeen extensively investigated by fluorescence quenching studies thatclearly identify annular and non-annular populations oflipids [@bb0035; @bb0050]. Binding oflipids to the annular sites revealed marked differences between the inner and extracellular leaflets,with the extracellular side showing similar affinities for anionic and zwitterionic lipids. Incontrast theintracellular side showed a twofold higher affinity foranionic lipids overphosphatidylcholine, presumably due to theclustering of charged residues on KcsA close to the bilayer surface. Fluorescence quenching studies have revealed that the non-annularbindingsites show a high degree of selectivityof binding anioniclipidsalmost exclusively,albeit with moderate affinity. The crystal structure of KcsAin detergent has providedvaluable insights into the interaction oflipids with the non-annular binding site with electron density being seen at the interface between the protein subunits indicating the presence of a diacylglycerol-likemoiety. The crystalstructure revealed that the *sn*-1 chain of this lipid moleculewas tightly buried inthe groovebetween the pore helix and the M2 helix whilst the *sn*-2 chain was less intimately associated with the protein [@bb0055;@bb0065]. Subsequent studies have revealed thelipid present in the KcsA crystals to be phosphatidylglycerol which co-purifies withthe KcsA[@bb0055](pdb accession number 1K4C),suggesting that the phosphatidylglycerol headgroup exhibits significantmotion or disorderwithin the crystal, resulting in the absence of electron density. The absenceof electron densityin theregion corresponding to the lipid headgroup has precluded a detailedunderstanding of how the phosphatidylglycerol is recognised. The presence of two key arginine residues (R64 and R89) in close proximity to the proposed headgroupregion suggested a putative role for electrostatic interactions between the sidechainsandthe anionic lipids within thebinding site [@bb0055]. The proposedinteractions between R64 and R89 have been investigated bymoleculardynamics,revealing the formation of H-bonds between the headgroups of the anionic lipids phosphatidic acid and phosphatidylglycerol andthearginineresidues[@bb0070]. Notably,these interactions were absent or reduced in bilayers containing the zwitterioniclipid phosphatidylethanolamine [@bb0070]. To demonstrate the feasibilityof detectinginteractionsbetween non-annular binding sites andanionic lipids we have undertaken a 31P magic-angle spinning (MAS) NMR study of KcsAreconstituted into a model lipidbilayer composed of phosphatidylcholine and the negatively charged phosphatidylglycerol. The application of MASpermits the acquisition of ^31^PNMR spectra in which the individual lipid components are resolved on thebasis of their chemical shift [@bb0075]. The chemical shift observed provides information on the local electrostatic environment of the phosphate moiety of the lipid headgroupand is thusanexcellent reporteron the interaction of the lipid headgroup with theprotein [@bb0080; @bb0085; @bb0090]. Typically the application of NMR to studylipid/protein interactions has proved challengingas exchange between thebulk lipid and annular sites occurstoo rapidly on the NMR timescaleto permit the observation of the bound lipids. Furthermore, interactions between thelipids and the proteinsare typically hydrophobic in nature withlittle difference in electrostatic environment between theboundandfree lipidsresulting in only minor perturbations in chemicalshift.Incontrast, phosphatidylglycerol bound at the non-annular binding site ispredicted to experience a significantly different electrostatic environment from theannular/bulk lipids with higher-affinity interactions resulting in longer residencytimes within the binding site. Below we demonstrate that this results in aspectroscopically distinct species that weresolvein the ^31^PMAS-NMR spectrumof KcsA reconstituted into lipid vesicles, providing insights into the interactions involved in lipid binding. Using site directed mutagenesis we have demonstrated that theperturbation in electrostatic environment arises through the interaction of thelipid head groupwith the positively chargedsidechain of R64 and R89. Single-channel current recordings havebeen measured to ascertain the role that these residues play in determining the channel gating behaviour of KcsA. 2. Materials and methods {#s0010} ======================== 2.1. Materials {#s0015} -------------- Palmitoyloleoyl-phosphatidylcholine (POPC) and palmitoyloleoyl-phosphatidylglycerol (POPG) were purchased from AvantiPolar Lipids (Alabaster, AL). The pQE32 vector and M15\[PREP\] *Escherichia coli* strain were bought from Qiagen (UK).The detergent, dodecylmaltoside (DDM) was from Anatrace (UK). The other reagentsfor the purification were obtained from Sigma(UK). 2.2.Cloning and mutagenesisof KcsA {#s0030}------------------------------------ The pQE32 vector containing the KcsA gene with a hexahistidine epitope at the N-terminus, kindly donated by Professor Lee (University ofSouthampton, UK), was expressed in M15 cells.Three KcsAmutants were generated by site-directed mutagenesis using the Quik-change protocol from Stratagene (La Jolla, CA). Three KcsA mutants were prepared replacing the arginine with the slightly smaller but unchargedleucine at residue 64 (R64L), 89 (R89L) or at both sites (R64,89L). The mutants were generated by PCRusing syntheticoligonucleotide primers containing the desired mutations(Eurofins MWG, UK). Complementary oligonuceotides, 5′-AGCTGATCAC GTATCCGTTA GCGCTGTGGT GGTCC-3′ and 5′-GACCACCACA GCGCTAACGG ATACGTGATC AGCTG-3′ were used as forward and reverse primers respectively tocreate the R64L mutation.Similarly,R89L was produced using the syntheticoligonucleotides 5′-GTGACTCTGT GGGGCCTGCTCGTGGCCG TGGTGGTGA T-3′ and 5′-ATCACCACCA CGGCCACGA GCAGGCCCC ACAGAGTCA C-3′ as primers. Polymerase chain reaction was used to generate the mutantsand the resultant PCR product was Dpn1- treatedfor 1 hr at 37°C to digestany methylated parental DNA. The DNA was used to transform competent M15\[PREP\] *E.coli* cells that were then platedonto agar plates supplemented withampicillin. Mutations wereconfirmed by sequencing. 2.3. Over expression and purification of KcsA {#s0035} --------------------------------------------- M15 *E.coli* cells transformed with KcsA or one of the KcsA mutants were used to inoculate 10 mL ofLuria Broth (LB) medium containing 100μg/mLof ampicillin. The overnight culture was then used to inoculate 1 L ofLB containing100 μg/mL of ampicillin and grown to an OD~600~of 0.8 at 37 °C. Over-expressionof KcsA wildtype and mutant protein wasinduced by the addition of IPTG to a final concentration of 1 mM andthe culture was grown for a further 4 h at 37 °C. The cells were harvested at 4 °C by centrifugation at 12,000 *g* for 20 min.The cell pellet was resuspended in buffer A(50 mM Tris, 150 mM NaCl, 150 mM KCl, pH 7.4) with 1mM PMSF andsonicated on ice for 5 min: 15 s on; 20 s off at powerlevel7 (Misonixsonicator). The membrane fractionwas clarified by ultracentrifugation at 420,000 *g* for 40 min at 5°C. The membrane-containing pellet was homogenised and | 1. _Introduction_ {#s0005} =============== Interactions of integral membrane _proteins_ with their surrounding lipid _environment_ play a key role in stabilising their structure and influencing their activity. To obtain insight _into_ the _nature_ and type of interactions occurring between lipids and _integral_ membrane proteins a _range_ of biophysical techniques including _electron_ spin resonance _[@bb0005;_ @bb0010] and _fluorescence_ [@bb0145; @bb0020] spectroscopy have been used in numerous studies. This has provided a wealth _of_ information on the specificity of proteins for particular classes of lipids and the affinity of these interactions [@bb0025; @bb0030]. _These_ studies have revealed that in addition _to_ the _'bulk'_ lipids whose dynamic _properties_ remain _largely_ unchanged by the presence of integral membrane _proteins_ _within_ the bilayer, there exist _a_ population of _lipids_ whose motional freedom is _constrained_ through their interaction with integral membrane proteins. The motionally restricted population of lipids _can_ _be_ segregated _into_ _'annular_ _lipids'_ which exhibit low _affinity_ interactions with the hydrophobic _surface_ _of_ membrane proteins and _'non-annular_ lipids' _which_ exert _high_ affinity interactions _at_ sites _located_ in clefts on the _protein_ _surface_ or at the interface between protein subunits [@bb0025; @bb0035; @bb0040; @bb0045]. The interactions at both sites play an _important_ _role_ in modulating _the_ function of integral _membrane_ proteins, but it has recently become apparent that occupation _of_ the non-annular binding site can be _particularly_ critical for function [@bb0050]. Despite the importance of _these_ _interactions_ a detailed atomic level description of the type and nature of the interplay between _lipids_ and integral membrane proteins is _limited_ as the lipids are often missing from high-resolution crystal _structures_ of membrane proteins. One _of_ the best-studied ion channels is _the_ potassium channel, KcsA from *Streptomyces _lividans*._ This _potassium_ channel, in _common_ with other members of this family, has an absolute requirement for anionic _lipids_ such as _phosphatidylglycerol,_ _phosphatidylserine_ or _cardiolipin_ for function; in their absence the channel exists in _a_ _non-conducting_ _state_ [@bb0055; @bb0060]. The binding of lipids to KcsA has been extensively investigated by fluorescence quenching studies that clearly _identify_ _annular_ and non-annular _populations_ of lipids [@bb0035; @bb0050]. Binding of lipids _to_ _the_ _annular_ sites revealed _marked_ differences between the inner and extracellular leaflets, with _the_ extracellular side showing similar affinities for _anionic_ and zwitterionic lipids. In contrast the intracellular side _showed_ a twofold higher affinity for anionic lipids over phosphatidylcholine, _presumably_ due to _the_ clustering of charged residues on KcsA close to the bilayer surface. Fluorescence quenching studies have revealed _that_ the non-annular _binding_ _sites_ show a high degree of selectivity _of_ binding _anionic_ _lipids_ almost exclusively, albeit with moderate affinity. _The_ crystal structure of KcsA in _detergent_ has _provided_ _valuable_ insights into _the_ interaction of lipids _with_ the _non-annular_ binding site _with_ electron density being seen at the interface between the protein subunits _indicating_ the presence of _a_ _diacylglycerol-like_ moiety. The crystal structure revealed _that_ the *sn*-1 chain _of_ this _lipid_ molecule _was_ tightly buried in the groove between _the_ pore _helix_ and _the_ M2 helix whilst _the_ _*sn*-2_ chain _was_ less intimately associated with the protein _[@bb0055;_ @bb0065]. Subsequent studies have revealed the lipid present in the KcsA crystals to be _phosphatidylglycerol_ which co-purifies with the KcsA [@bb0055] (pdb accession number _1K4C),_ suggesting that the phosphatidylglycerol headgroup exhibits significant motion or _disorder_ within _the_ _crystal,_ resulting _in_ the absence of electron density. The absence _of_ electron _density_ in _the_ region corresponding to the lipid headgroup has _precluded_ _a_ detailed _understanding_ of how the phosphatidylglycerol is recognised. The presence of two key _arginine_ residues (R64 and R89) in _close_ _proximity_ to the proposed headgroup region suggested a putative role for electrostatic interactions between the sidechains _and_ the anionic lipids within the binding _site_ [@bb0055]. The proposed interactions between R64 and R89 have been investigated by molecular dynamics, revealing the formation of H-bonds between the headgroups of the _anionic_ lipids _phosphatidic_ acid and phosphatidylglycerol and the arginine residues [@bb0070]. Notably, _these_ interactions were _absent_ or reduced _in_ _bilayers_ _containing_ _the_ zwitterionic lipid _phosphatidylethanolamine_ _[@bb0070]._ To demonstrate the feasibility of detecting interactions between non-annular binding sites and anionic _lipids_ we have _undertaken_ a 31P magic-angle spinning (MAS) NMR study of KcsA reconstituted _into_ a _model_ lipid bilayer composed of phosphatidylcholine and the _negatively_ charged _phosphatidylglycerol._ The application of MAS _permits_ _the_ acquisition _of_ ^31^P _NMR_ spectra in which the individual lipid components are resolved on the _basis_ of their chemical shift [@bb0075]. The _chemical_ shift observed provides information on the local electrostatic _environment_ of the phosphate moiety of the lipid headgroup and is thus an _excellent_ _reporter_ on the interaction of _the_ lipid headgroup with the protein [@bb0080; @bb0085; @bb0090]. Typically the application of NMR to study lipid/protein interactions has _proved_ challenging as exchange _between_ the bulk lipid and annular sites _occurs_ too rapidly on the NMR timescale to permit the observation of the bound lipids. Furthermore, interactions _between_ the lipids and _the_ proteins _are_ typically hydrophobic in nature with little difference in electrostatic environment _between_ the _bound_ and free lipids resulting in only minor perturbations in chemical shift. In contrast, _phosphatidylglycerol_ _bound_ _at_ the _non-annular_ binding site is _predicted_ to experience a significantly different electrostatic environment from the annular/bulk lipids with higher-affinity interactions resulting in longer residency times within the binding _site._ Below we _demonstrate_ that this results in _a_ spectroscopically distinct species that we resolve in the ^31^P MAS-NMR _spectrum_ of KcsA reconstituted _into_ lipid vesicles, providing insights into the interactions involved in lipid binding. Using site directed mutagenesis we have demonstrated that the perturbation in electrostatic environment arises through the _interaction_ of _the_ lipid head group with _the_ positively charged sidechain of R64 and _R89._ Single-channel current recordings have been measured to ascertain the role that these residues play in determining the channel _gating_ behaviour of _KcsA._ 2. Materials and methods {#s0010} ======================== _2.1._ Materials {#s0015} _--------------_ Palmitoyloleoyl-phosphatidylcholine (POPC) and palmitoyloleoyl-phosphatidylglycerol (POPG) were purchased from _Avanti_ Polar _Lipids_ (Alabaster, AL). _The_ pQE32 vector _and_ _M15\[PREP\]_ *Escherichia _coli*_ strain were bought from Qiagen (UK). The detergent, dodecylmaltoside (DDM) was from _Anatrace_ _(UK)._ The other reagents _for_ the purification were _obtained_ from Sigma (UK). 2.2. Cloning and _mutagenesis_ of KcsA {#s0030} ------------------------------------ _The_ pQE32 vector containing the KcsA gene with a hexahistidine epitope at the _N-terminus,_ kindly _donated_ by Professor Lee (University of Southampton, UK), was expressed in M15 cells. Three KcsA mutants _were_ _generated_ by site-directed mutagenesis using the Quik-change protocol from Stratagene (La Jolla, _CA)._ Three KcsA mutants were prepared replacing the arginine with _the_ slightly smaller but uncharged leucine at residue _64_ (R64L), 89 (R89L) _or_ at both sites (R64,89L). The mutants were generated by PCR _using_ synthetic oligonucleotide primers containing the _desired_ mutations _(Eurofins_ MWG, UK). _Complementary_ oligonuceotides, 5′-AGCTGATCAC GTATCCGTTA GCGCTGTGGT GGTCC-3′ and 5′-GACCACCACA GCGCTAACGG ATACGTGATC AGCTG-3′ were used as forward and reverse _primers_ respectively to create the _R64L_ mutation. Similarly, R89L _was_ produced using the synthetic oligonucleotides 5′-GTGACTCTGT GGGGCCTGC TCGTGGCCG TGGTGGTGA T-3′ and 5′-ATCACCACCA CGGCCACGA GCAGGCCCC ACAGAGTCA _C-3′_ as primers. Polymerase chain reaction was used _to_ generate _the_ _mutants_ and the resultant PCR product was Dpn1- _treated_ for 1 hr at 37 °C to digest any methylated parental _DNA._ _The_ DNA was used _to_ _transform_ competent M15 _\[PREP\]_ *E.coli* cells that were then plated onto _agar_ plates supplemented with ampicillin. Mutations were _confirmed_ by sequencing. 2.3. Over _expression_ and purification of _KcsA_ {#s0035} --------------------------------------------- _M15_ _*E.coli*_ cells transformed with KcsA or one of _the_ KcsA mutants were used to _inoculate_ 10 _mL_ of Luria Broth (LB) medium containing 100 μg/mL of ampicillin. The overnight culture was then used to inoculate 1 L of LB containing 100 μg/mL of ampicillin _and_ grown to an OD~600~ of 0.8 at _37_ °C. _Over-expression_ of KcsA wild type and mutant protein was _induced_ _by_ the addition of _IPTG_ to a final concentration of 1 mM and _the_ culture was grown for a further 4 h _at_ 37 °C. The cells were harvested at 4 °C by _centrifugation_ at 12,000 *g* for 20 min. _The_ _cell_ pellet was resuspended in _buffer_ A _(50_ mM Tris, 150 mM NaCl, 150 mM _KCl,_ pH _7.4)_ with 1 _mM_ PMSF and _sonicated_ on ice for 5 _min:_ 15 _s_ on; 20 s off at power level 7 (Misonix sonicator). The membrane fraction _was_ _clarified_ by ultracentrifugation at 420,000 *g* _for_ _40_ min at 5 °C. _The_ membrane-containing pellet was _homogenised_ _and_ |
(J Am Heart Assoc. 2017;6:e007026 DOI: 10.1161/JAHA.117.007026.)29042422
Clinical PerspectiveWhat Is New?In this nonrandomized, retrospective, observational study of patients undergoing cardiac resynchronization therapy (CRT) with quadripolar (QUAD) and non‐QUAD left ventricular leads, programmed to biventricular, single‐site left ventricular pacing, QUAD was associated with a lower total mortality, cardiac mortality, and heart failure hospitalization.These benefits were observed after both CRT‐defibrillation and CRT‐pacing, after adjustment for heart failure etiology.Re‐interventions for left ventricular displacement or phrenic nerve stimulation, which were lower with QUAD, were associated with worse outcomes.What Are the Clinical Implications?The markedly better outcomes after CRT observed with QUAD supports their preferential use over non‐QUAD in clinical practice.The relative benefits of CRT‐defibrillation over CRT‐pacing requires further evaluation in the QUAD era.
Introduction {#jah32587-sec-0008}
============
Cardiac resynchronization therapy (CRT), with CRT‐defibrillation (CRT‐D) or without (CRT‐pacing \[CRT‐P\]) defibrillation, is a standard treatment for selected patients with heart failure (HF) with severe left ventricular (LV) dysfunction and a wide QRS complex.[1](#jah32587-bib-0001){ref-type="ref"} Since the first transvenous CRT implantations were undertaken in the 1990s,[2](#jah32587-bib-0002){ref-type="ref"}, [3](#jah32587-bib-0003){ref-type="ref"} improvements in delivery catheter and LV lead design, as well as implantation techniques using venoplasty and snaring, have helped to improve implantation success. Prominent among the challenges still encountered at implantation and thereafter is achieving acceptable LV pacing thresholds without phrenic nerve stimulation (PNS).[4](#jah32587-bib-0004){ref-type="ref"} Deactivation of the LV lead mainly occurs as a result of LV lead displacement which, in studies using unipolar and bipolar leads, occurs more frequently than with atrial or right ventricular leads.[5](#jah32587-bib-0005){ref-type="ref"}, [6](#jah32587-bib-0006){ref-type="ref"}, [7](#jah32587-bib-0007){ref-type="ref"}, [8](#jah32587-bib-0008){ref-type="ref"}, [9](#jah32587-bib-0009){ref-type="ref"}
Since their launch in 2010, quadripolar LV leads (QUAD) have been considered by implanters as a "game‐changer," even before robust clinical evidence emerged in their favor. Observational studies and a randomized, controlled trial[10](#jah32587-bib-0010){ref-type="ref"} have since shown that QUAD is associated with higher implant success rates and lower rates of re‐interventions for LV lead displacement or PNS.[11](#jah32587-bib-0011){ref-type="ref"}, [12](#jah32587-bib-0012){ref-type="ref"}
Some observational studies have suggested that CRT‐D using QUAD programmed to single‐site LV pacing also improves survival.[12](#jah32587-bib-0012){ref-type="ref"}, [13](#jah32587-bib-0013){ref-type="ref"}, [14](#jah32587-bib-0014){ref-type="ref"} These findings, however, are not consistent,[15](#jah32587-bib-0015){ref-type="ref"} and there is uncertainty as to whether they also apply to CRT‐P. Moreover, the possible influence of HF etiology and the effects of QUAD on HF hospitalization and mode of death remain largely unexplored.
Methods {#jah32587-sec-0009}
-------
This is a nonrandomized, retrospective, observational study comparing clinical outcomes of patients undergoing CRT‐D and CRT‐P device implantation using unipolar, bipolar, and quadripolar leads in a single center (Queen Elizabeth Hospital, Birmingham, United Kingdom) from February 2010 to January 2017. The study was approved by the Clinical Audit Department at the Queen Elizabeth Hospital, which does not require informed consent for audit of clinical care delivery. The study conforms to the Declaration of Helsinki.
Implantation {#jah32587-sec-0010}
------------
Device implantation was undertaken using standard techniques with patients under local anesthesia and intravenous sedation. Access was gained via subclavian, axillary, and cephalic veins. The LV pacing site was chosen by the implanter on the basis of lead stability, absence of PNS, and adequate pacing parameters. An implant was considered a failure in the event of failure to deploy all desired leads and device at the index procedure. The first QUAD was implanted in February 2010. The following QUAD leads were used: Quartet 1458Q (St. Jude Medical, Sylmar, CA), Attain Performa (Medtronic Inc, Minneapolis, MN), and Acuity X4 (Boston Scientific, Marlborough, MA). The choice of vector was made at implantation and was made on the basis of presence or absence of PNS.
Follow‐Up {#jah32587-sec-0011}
---------
Patients were followed up in dedicated device therapy clinics. Before 2013, patients underwent systematic echocardiographic optimization. To this end, patients in sinus rhythm underwent transmitral Doppler‐directed optimization of atrioventricular delay using an iterative technique before discharge and at every scheduled visit thereafter. In patients with sinus rhythm, atrial pacing was set at 60 beats/min, and the pacing mode was set to DDDR with an interventricular delay of 0 to 4 ms, according to the manufacturer. In patients with permanent atrial fibrillation, right ventricular and LV leads were implanted and a CRT generator was used, plugging the atrial port and programming the generator to a ventricular triggered mode. In patients with uncontrolled atrial fibrillation despite medical therapy with suboptimal biventricular pacing capture (\<98%), atrioventricular junction ablation was undertaken, according to the individual clinician\'s decision. After 2013, echocardiographic optimization was only undertaken in symptomatic nonresponders.
End Points {#jah32587-sec-0012}
----------
The primary end point was total mortality, which included cardiac transplantation. Secondary end points included cardiac mortality and unplanned HF hospitalization. The first event was included in the analysis. With respect to mode of death, sudden cardiac death was defined as a "natural, unexpected death due to cardiac causes, heralded by an abrupt loss of consciousness within 1 hour of the onset of acute symptoms,"[16](#jah32587-bib-0016){ref-type="ref"} whereas death from pump failure was defined as "death after a period of clinical deterioration in signs and symptoms of heart failure despite medical treatment"[17](#jah32587-bib-0017){ref-type="ref"} or cardiac transplantation. Mortality data were collected through medical records every 3 months by investigators who were blinded to all other patient data. Mortality and event data were collected by separate investigators who were blinded to all other data, except patient identifiers.
Statistical Analysis {#jah32587-sec-0013}
--------------------
In preliminary analyses, no differences in outcomes emerged between unipolar and bipolar LV leads (data not shown). On this basis, the latter were classified as "non‐QUAD" in statistical analyses. Normality was tested using the Shapiro--Wilk test. Continuous variables are expressed as mean (±SD) and compared using the Student *t* test. Categorical variables were compared using the χ^2^ tests. Kaplan--Meier curves and the log‐rank tests were used to assess observed cumulative survival and to test for differences in survival, respectively. Cox proportional hazard models were used to compare hazard rates of subgroups. Variables reaching a *P*\<0.10 on univariable analyses were entered in multivariable models, and further backward elimination was applied for the final multivariable models. Confounders included in final models were the following: quadripolar lead, sex (male), age at implantation, New York Heart Association (NYHA) class, creatinine, QRS duration, and medication of angiotensin‐converting enzyme inhibitors/angiotensin receptor blockers. Proportionality hypotheses were verified by visual examination of log (survival) graphs to ensure parallel slopes, and by examining Schoenfeld residuals. Statistical analyses were undertaken using Stata 14 (StataCorp, Houston, TX). A 2‐sided *P*≤0.05 was considered statistically significant.
Results {#jah32587-sec-0014}
=======
Baseline Characteristics {#jah32587-sec-0015}
------------------------
Over the study period of 6.9 years, 847 patients underwent CRT (CRT‐D: 436 \[51.5%\]; CRT‐P: 411 \[48.5%\]), using QUAD (287 \[33.9%\]), unipolar (63 \[7.43%\]), or bipolar (497 \[58.7%\]) leads. Implantations using unipolar and bipolar leads were classified as non‐QUAD. As shown in Table [1](#jah32587-tbl-0001){ref-type="table"}, the groups were well matched for age, sex, cause of cardiomyopathy, comorbidities, proportion of upgrades from pacemaker, atrial rhythm (sinus rhythm or atrial fibrillation), QRS morphology, QRS duration, and left ventricular ejection fraction. Compared with the non‐QUAD group, QUAD were more likely to be in NYHA class I and II (*P | ( j am heart assoc. 2017 ; isbn : e007026 doi : 10. 1161 / jaha. 117. 007026. ) 29042422 clinical perspectivewhat is new? in this nonrandomized, retrospective, observational study of patients undergoing automated resynchronization therapy ( crt ) with stable ( quad ) and non ‐ quad left ventricular leads, programmed repetitive biventricular, single ‐ site left ventricular pacing, quad was associated with a lower total mortality, cardiac stimulation, and heart failure hospitalization. these benefits were observed after both crt adaptive defibrillation and crt ‐ pacing, after treatment for heart failure complications. re ‐ interventions for left ventricular displacement or phrenic nerve stimulation, which were lower with quad, were associated with worse outcomes. what are the clinical implications? the significantly better outcomes after crt observed with quad supports their preferential use over non ‐ quad in clinical practice. the relative benefits of crt ‐ defibrillation over crt ‐ pacing requires further evaluation in the quad era. introduction { # jah32587 - sec - 0008 } = = = = = = = = = = = = cardiac resynchronization therapy ( crt ), with crt ‐ defibrillation ( crt ‐ d ) or without ( crt ‐ pacing \ [ crt ‐ p \ ] ) defibrillation, is a standard treatment for selected patients with heart failure ( hf ) with severe left ventricular ( lv ) dysfunction and a wide qrs complex. [ 1 ] ( # jah32587 - bib - 0001 ) { ref - type = " ref " } since the first transvenous crt implantations were undertaken in the 1990s, [ 2 ] ( # jah32587 - bib - 0002 ) { ref - type = " ref " }, [ 0 ] ( # jah32587 - bib - 0003 ) { ref - type = " ref " } improvements in delivery catheter and lv lead design, as well as implantation techniques using venoplasty and snaring, have helped to improve implantation success. prominent among the challenges still encountered at implantation and thereafter is achieving acceptable lv pacing thresholds without phrenic nerve stimulation ( pns ). [ 4 ] ( # jah ##32587 - bib - 0004 ) { ref - type = " ref " } deactivation of the lv lead mainly occurs as a result of lv lead displacement which, in studies using unipolar and bipolar leads, occurs more frequently than with atrial or right ventricular leads. [ 5 ] ( # jah32587 - bib - 0005 ) { ref - type = " ref " }, [ 6 ] ( # jah32587 - bib - 0006 ) { ref - type = " ref " }, [ 7 ] ( # jah32587 - bib - 0007 ) { ref - type = " ref " }, [ 8 ] ( # jah32587 - bib - 0008 ) { ref - type = " ref " }, [ 9 ] ( # jah32587 - bib - 0009 ) { ref - type = " ref " } since their launch in 2010, quadripolar lv leads ( quad ) have been considered by implanters as a " game ‐ changer, " even before robust clinical evidence emerged in their favor. observational studies and a randomized, controlled trial [ 10 ] ( # jah32587 - bib - 0010 ) { ref - type = " ref " } have since shown that quad is associated with higher implant success rates and lower rates of re ‐ interventions for lv lead displacement or pns. [ 11 ] ( # jah32587 - bib - 0011 ) { ref - type = " ref " }, [ 12 ] ( # jah32587 - bib - 0012 ) { ref - type = " ref " } some observational studies have suggested that crt ‐ d using quad programmed to single ‐ site lv pacing also improves survival. [ 12 ] ( # jah32587 - bib - 0012 ) { ref - type = " ref " }, [ 13 ] ( # jah32587 - bib - 0013 ) { ref - type = " ref " }, [ 14 ] ( # jah32587 - bib - 0014 ) { ref - type = " ref " } these findings, however, are not consistent, [ 15 ] ( # jah32587 - bib - 0015 ) { ref - type = " ref " } and there is uncertainty as to whether they also apply to crt ‐ p. moreover, the possible influence of hf etiology and the effects of quad on hf hospitalization and mode of death remain largely unexplored. methods { # jah32587 - sec - 0009 } - - - - - - - this is a nonrandomized, retrospective, observational study comparing clinical outcomes of patients undergoing crt ‐ d and crt ‐ p device implantation using unipolar, bipolar, and quadripolar leads in a single center ( queen elizabeth hospital, birmingham, united kingdom ) from february 2010 to january 2017. the study was approved by the clinical audit department at the queen elizabeth hospital, which does not require informed consent for audit of clinical care delivery. the study conforms to the declaration of helsinki. implantation { # jah32587 - sec - 0010 } - - - - - - - - - - - - device implantation was undertaken using standard techniques with patients under local anesthesia and intravenous sedation. access was gained via subclavian, axillary, and cephalic veins. the lv pacing site was chosen by the implanter on the basis of lead stability, absence of pns, and adequate pacing parameters. an implant was considered a failure in the event of failure to deploy all desired leads and device at the index procedure. the first quad was implanted in february 2010. the following quad leads were used : quartet 1458q ( st. jude medical, sylmar, ca ), attain performa ( medtronic inc, minneapolis, mn ), and acuity x4 ( boston scientific, marlborough, ma ). the choice of vector was made at implantation and was made on the basis of presence or absence of pns. follow ‐ up { # jah32587 - sec - 0011 } - - - - - - - - - patients were followed up in dedicated device therapy clinics. before 2013, patients underwent systematic echocardiographic optimization. to this end, patients in sinus rhythm underwent transmitral doppler ‐ directed optimization of atrioventricular delay using an iterative technique before discharge and at every scheduled visit thereafter. in patients with sinus rhythm, atrial pacing was set at 60 beats / min, and the pacing mode was set to dddr with an interventricular delay of 0 to 4 ms, according to the manufacturer. in patients with permanent atrial fibrillation, right ventricular and lv leads were implanted and a crt generator was used, plugging the atrial port and programming the generator to a ventricular triggered mode. in patients with uncontrolled atrial fibrillation despite medical therapy with suboptimal biventricular pacing capture ( \ < 98 % ), atrioventricular junction ablation was undertaken, according to the individual clinician \ ' s decision. after 2013, echocardiographic optimization was only undertaken in symptomatic nonresponders. end points { # jah32587 - sec - 0012 } - - - - - - - - - - the primary end point was total mortality, which included cardiac transplantation. secondary end points included cardiac mortality and unplanned hf hospitalization. the first event was included in the analysis. with respect to mode of death, sudden cardiac death was defined as a " natural, unexpected death due to cardiac causes, heralded by an abrupt loss of consciousness within 1 hour of the onset of acute symptoms, " [ 16 ] ( # jah32587 - bib - 0016 ) { ref - type = " ref " } whereas death from pump failure was defined as " death after a period of clinical deterioration in signs and symptoms of heart failure despite medical treatment " [ 17 ] ( # jah32587 - bib - 0017 ) { ref - type = " ref " } or cardiac transplantation. mortality data were collected through medical records every 3 months by investigators who were blinded to all other patient data. mortality and event data were collected by separate investigators who were blinded to all other data, except patient identifiers. statistical analysis { # jah32587 - sec - 0013 } - - - - - - - - - - - - - - - - - - - - in preliminary analyses, no differences in outcomes emerged between unipolar and bipolar lv leads ( data not shown ). on this basis, the latter were classified as " non ‐ quad " in statistical analyses. normality was tested using the shapiro - - wilk test. continuous variables are expressed as mean ( ±sd ) and compared using the student * t * test. categorical variables were compared using the χ ^ 2 ^ tests. kaplan - - meier curves and the log ‐ rank tests were used to assess observed cumulative survival and to test for differences in survival, respectively. cox proportional hazard models were used to compare hazard rates of subgroups. variables reaching a * p * \ < 0. 10 on univariable analyses were entered in multivariable models, and further backward elimination was applied for the final multivariable models. confounders included in final models were the following : quadripolar lead, sex ( male ), age at implantation, new york heart association ( nyha ) class, creatinine, qrs duration, and medication of angiotensin ‐ converting enzyme inhibitors / angiotensin receptor blockers. proportionality hypotheses were verified by visual examination of log ( survival ) graphs to ensure parallel slopes, and by examining schoenfeld residuals. statistical analyses were undertaken using stata 14 ( statacorp, houston, tx ). a 2 ‐ sided * p * ≤0. 05 was considered statistically significant. results { # jah32587 - sec - 0014 } = = = = = = = baseline characteristics { # jah32587 - sec - 0015 } - - - - - - - - - - - - - - - - - - - - - - - - over the study period of 6. 9 years, 847 patients underwent crt ( crt ‐ d : 436 \ [ 51. 5 % \ ] ; crt ‐ p : 411 \ [ 48. 5 % \ ] ), using quad ( 287 \ [ 33. 9 % \ ] ), unipolar ( 63 \ [ 7. 43 % \ ] ), or bipolar ( 497 \ [ 58. 7 % \ ] ) leads. implantations using unipolar and bipolar leads were classified as non ‐ quad. as shown in table [ 1 ] ( # jah32587 - tbl - 0001 ) { ref - type = " table " }, the groups were well matched for age, sex, cause of cardiomyopathy, comorbidities, proportion of upgrades from pacemaker, atrial rhythm ( sinus rhythm or atrial fibrillation ), qrs morphology, qrs duration, and left ventricular ejection fraction. compared with the non ‐ quad group, quad were more likely to be in nyha class i and ii ( * p | ( J Am Heart Assoc. 2017; 6: e007026 DOI: 10. 1161 / JAHA. 117. 007026.) 29042422 Clinical PerspectiveWhat Is New? In this nonrandomized, retrospective, observational study of patients undergoing cardiac resynchronization therapy (CRT) with quadripolar (QUAD) and non ‐ QjZD left ventricular leads, programmed to biventricular, single ‐ site left ventricular pacing, QUAD was associated with a lower total mortality, cardiac mortality, and heart failure hospitalization. These benefits were observed after both CRT ‐ defibrillation and CRT ‐ pacing, after adjustment for heart failure etiology. Re ‐ interventions for left ventricular displacement or phrenic nerve stimulation, which were lower with QUAD, were associated with worse outcomes. What Are the Clinical ImplicWtiogs? The markedly better outcomes after CRT observed with QUAD supports their preferential use over non ‐ QUAD in clinical practice. The relative benefits of CRT ‐ defibrillation over CRT ‐ pacing requires further evaluation in the QUAD era. Introduction {# jah32587 - sec - 0008} = = = = = = = = = = = = Cardiac resynchronization therapy (CRT ), with CRT ‐ defibrillation (CRT ‐ D) or without (CRT ‐ pacing \ [CRT ‐ P \] ) defibrillation, is a standard treatment for selected patients with heart failure (HF) with severe left ventricular (LV) dysfunction and a wide QRS complex. [1] (# jah32587 - bib - 0001) {ref - type = " ref "} Since the first transvenous CRT implantations were undertaken in the 1990s, [2] (# jah32587 - bib - 0002) {ref - type = " ref " }, [3] (# jah32587 - bib - 0003) {ref - type = " ref "} improvements in delivery catheter and LV lead design, as well as implantation techniques using venoplasty and snaring, have helped to improve implantation success. Prominent among the challenges still encountered at implantation and thereafter is achieving acceptable LV pacing thresholds without phrenic nerve stimulation (PNS ). [4] (# jah32587 - bib - 0004) {ref - type = " ref "} Deactivation of the LV lead mainly occ*$s as a result of LV lead displacement which, in studies using unipolar and bipolar leads, occurs more frequently than with atrial or right ventricular leads. [5] (# jah32587 - bib - 0005) {ref - type = " ref " }, [6] (# jah32587 - bib - 0006) {ref - type = " ref " }, [7] (# jah32587 - bib - 0007) {ref - type = " ref " }, [8] (# jah32587 - bib - 0008) {ref - type = " ref " }, [9] (# jah32587 - bib - 0009) {ref - type = " ref "} Since their launch in 2010, quadripolar LV leads (QUAD) have been considered by implanters as a " game ‐ changer, " even bsgore robust clinical evidence emerged in their favor. Observational studi#X and a randomized, controlled trial [10] (# jah32587 - bib - 0010) {ref - type = " ref "} have since shown that QUAD is associated with higher implant success rates and lower rates of re ‐ interventions for LV lead displacement or PNS. [11] (# jah32587 - bib - 0011) {ref - type = " ref " }, [12] (# jah32587 - bib - 0012) {ref - type = " ref "} Some observational studies have suggested tmxt CRT ‐ D using QUAD programmed to single ‐ site LV pacing also improves survival. [12] (# jah32587 - bib - 0012) {ref - type = " ref " }, [13] (# jah32587 - bib - 0013) {ref - type = " ref " }, [14] (# jah32587 - bib - 0014) {ref - type = " ref "} These findings, however, are not consistent, [15] (# jah32587 - bib - 0015) {ref - type = " ref "} and there is uncertainty as to whether they also apply to CRT ‐ P. Moreover, the possible influence of HF etiology and the effects of QUAD on HF hospitalization and mode of death remain largely unexplored. Methods {# jah32587 - sec - 0009} - - - - - - - This is a nonrandomized, retrospective, observational study comparing clinical outcomes of patients undergoing CRT ‐ D and CRT ‐ P device implantation using unipolar, bipolar, and quadripolar leads in a single center (Queen Elizabeth Hospital, Birmingham, United Kingdom) from February 2010 to January 2017. The study was approved by the Clinical Audit Department at the Queen Elizabeth Hospital, which does not require informed consent for audit of clinical care delivery. The study conforms to the Declaration of Helsinki. Implantation {# jah32587 - sec - 0010} - - - - - - - - - - - - Device implantation was undertaken using standard techniques with patients under local anesthesia and intravenous sedation. Access was gained via subclavian, axillary, and cephalic veins. The LV pacing site was xhPsen by the implanter on the basis of lead stability, absence of PNS, and adequate pacing parameters. An implant was considered a failure in the event of failure to deploy all desired leads and device at the index procedure. The first QUAD was implanted in February 2010. The following QUAD leads were used: Quartet 1458Q (St. Jude Medical, Sylmar, CA ), Attain Performa (Medtronic Inc, Minneapolis, MN ), and Acuity X4 (Boston Scientific, Marlborough, MA ). The choice of vector was made at implantation and was made on the basis of presence or absence of PNS. Follow ‐ Up {# jah32587 - sec - 0011} - - - - - - - - - Patients were followed up in dedicated device therapy clinics. Before 2013, patients underwent systematic echocardiographic optimization. To this end, patients in sinus rhythm underwent transmitral Doppler ‐ directed optimization of atrioventricular delay using an iterative technique before discharge and at every scheduled visit thereafter. In patients with sinus rhythm, atrial pacing was set at 60 beats / min, and the pacing mode was set to DDDR with an interventricular delay of 0 to 4 ms, according to the manufacturer. In patients with permanent atrial fibrillation, right ventricular and LV leads were implanted and a CRT generator was used, plugging the atrial port and programming the generator to a ventricular triggered mode. In patients with uncontrolled atrial fibrillation des)i6e medical therapy with suboptimal biventricular pacing capture (\ <98% ), atrioventricular junction ablation was undertaken, according to the individual clinician \ ' s decision. After 2013, echocardiographic optimization was only undertaken in symptomatic nonresponders. End Points {# jah32587 - sec - 0012} - - - - - - - - - - The primary end point was total mortality, which included cardiac transplantation. Secondary end points included cardiac mortality and unplanned HF hospitalization. The first event was included in the analysis. WOgh respect to mode of death, sudden cardiac death was defined as a " natural, unexpected death due to cardiac causes, heralded by an abrupt loss of consciousness within 1 hour of the onset of acute symptoms, " [16] (# jah32587 - bib - 0016) {ref - type = " ref "} whereas death from pump failure was defined as " death after a period of clinical deterioration in signs and symptoms of heart failure despite medical treatment " [17] (# jah32587 - bib - 0017) {ref - type = " ref "} or cardiac transplantation. Mortality data were collected through medical records every 3 months by investigators who were blinded to all other patient data. Mortality and event data were collected by separate investigators who were blinded to all other data, except patient identifiers. Statistical Analysis {# jah32587 - sec - 0013} - - - - - - - - - - - - - - - - - - - - In preliminary analyses, no differences in outcomes emerged between unipolar and bipolar LV leads (data not shown ). On this basis, the latter were classified as " non ‐ QUAD " in statistical analyses. Normality was tested using the Shapiro - - Wilk test. Continuous variables are expressed as mean (± SD) and compared using the Student * t * test. Categorical variables were compared using the χ ^ 2 ^ tests. Kaplan - - Meier curves and the log ‐ rank tests were used to assess observed cumulative survival and to test for differences in survival, respectively. Cox proportional hazard models were used to compare hazard rates of subgroups. Variables reaching a * P * \ <0. 10 on univariable analyses were entered in multivariable models, and further backward elimination was applied for the final multivariable models. Confounders included in final models were the following: quadripolar lead, sex (male ), age at implantation, New York Heart Association (NYHA) class, creatinine, QRS duration, and medication of angiotensin ‐ converting enzyme inhibitors / angiotensin receptor blockers. Proportionality hypotheses were verified by visual examination of log (survival) graphs to ensure parallel slopes, and by examining Schoenfeld residuals. Statistical analyses were undertaken using StaYA 14 (StataCorp, Houston, TX ). A 2 ‐ sided * P * ≤ 0. 05 was considered statistically significant. Results {# jah32587 - sec - 0014} = = = = = = = Baseline Characteristics {# jah32587 - sec - 0015} - - - - - - - - - - - - - - - - - - - - - - - - Over the study period of 6. 9 years, 847 patients underwent CRT (CRT ‐ D: 436 \ [51. 5% \ ]; CRT ‐ P: 411 \ [48. 5% \] ), using QUAD (287 \ [33. 9% \] ), unipolar (63 \ [7. 43% \] ), or bipolar (497 \ [58. 7% \] ) leads. Implantations using unipolar and bipolar leads were classified as non ‐ QUAD. As shown in Table [1] (# jah32587 - tbl - 0001) {ref - type = " table " }, the groups were well matched for age, sex, cause of cardiomyopathy, comorbidities, proportion of upgrades from pacemaker, atrial rhythm (sinus rhythm or atrial fibrillation ), QRS morphology, QRS duration, and left ventricular ejection fraction. Compared with the non ‐ QUAD group, QUAD were more likely to be in NYHA class I and II (* P | (J Am Heart Assoc. DOI: 10.1161/JAHA.117.007026.)29042422 PerspectiveWhat Is New?In this nonrandomized, retrospective, observational study of patients undergoing cardiac resynchronization therapy (CRT) with quadripolar (QUAD) and non‐QUAD left ventricular leads, programmed to biventricular, single‐site left ventricular pacing, was associated a lower total mortality, cardiac mortality, and heart hospitalization.These observed after both CRT‐defibrillation and CRT‐pacing, after adjustment for heart failure etiology.Re‐interventions for left ventricular displacement or phrenic nerve which were lower with QUAD, were associated with worse outcomes.What Are the Clinical Implications?The markedly better outcomes CRT with supports their preferential use over non‐QUAD in clinical practice.The benefits over CRT‐pacing requires further evaluation in the QUAD era. Introduction {#jah32587-sec-0008} ============ Cardiac resynchronization therapy (CRT), with CRT‐defibrillation (CRT‐D) or without (CRT‐pacing defibrillation, is a standard treatment for selected patients with heart failure (HF) with severe left (LV) dysfunction and a wide QRS complex.[1](#jah32587-bib-0001){ref-type="ref"} Since the first transvenous CRT were in the 1990s,[2](#jah32587-bib-0002){ref-type="ref"}, [3](#jah32587-bib-0003){ref-type="ref"} in delivery catheter and LV lead design, as well as implantation techniques using venoplasty and snaring, have helped to improve implantation success. Prominent among the challenges encountered at implantation thereafter is achieving LV pacing thresholds without phrenic nerve stimulation (PNS).[4](#jah32587-bib-0004){ref-type="ref"} Deactivation of LV lead mainly occurs as a result of LV lead displacement which, in studies using unipolar bipolar leads, occurs more than with atrial or ventricular leads.[5](#jah32587-bib-0005){ref-type="ref"}, [6](#jah32587-bib-0006){ref-type="ref"}, [8](#jah32587-bib-0008){ref-type="ref"}, [9](#jah32587-bib-0009){ref-type="ref"} Since their launch in 2010, quadripolar LV leads (QUAD) have been by implanters as "game‐changer," even robust clinical evidence emerged in their favor. Observational and a controlled trial[10](#jah32587-bib-0010){ref-type="ref"} have shown that QUAD is associated with higher implant success rates and lower of re‐interventions for LV lead displacement or PNS.[11](#jah32587-bib-0011){ref-type="ref"}, [12](#jah32587-bib-0012){ref-type="ref"} Some observational studies have suggested that using QUAD programmed single‐site LV pacing also improves survival.[12](#jah32587-bib-0012){ref-type="ref"}, [13](#jah32587-bib-0013){ref-type="ref"}, [14](#jah32587-bib-0014){ref-type="ref"} These findings, however, are not consistent,[15](#jah32587-bib-0015){ref-type="ref"} and there is uncertainty to whether they also apply to CRT‐P. Moreover, the possible of HF etiology the effects of QUAD on HF hospitalization mode of death remain largely Methods {#jah32587-sec-0009} ------- This is a nonrandomized, retrospective, observational study comparing clinical of patients undergoing CRT‐D and CRT‐P device implantation using unipolar, bipolar, and quadripolar leads in a single center (Queen Elizabeth Hospital, Birmingham, United Kingdom) from February 2010 to January 2017. The study was approved by the Clinical Audit Department at the Queen Elizabeth Hospital, which does not require informed consent for audit clinical care delivery. The conforms to the Declaration of Helsinki. Implantation {#jah32587-sec-0010} ------------ Device implantation was undertaken using standard with patients under local anesthesia and intravenous sedation. Access was gained via subclavian, axillary, and cephalic veins. The LV pacing site was by the implanter on the basis of lead stability, absence of PNS, and pacing parameters. An implant was considered a failure in the event of failure to deploy all desired and device at the index procedure. The first QUAD was in February 2010. The following QUAD leads were used: Quartet 1458Q (St. Jude Medical, Sylmar, CA), Attain Performa Minneapolis, MN), and Acuity X4 (Boston Marlborough, MA). The choice of vector was made at implantation and was on the basis of presence or PNS. Follow‐Up {#jah32587-sec-0011} --------- Patients were followed up in dedicated device therapy clinics. Before 2013, patients underwent systematic echocardiographic To this end, patients in sinus rhythm underwent transmitral Doppler‐directed optimization of atrioventricular delay using an iterative technique before discharge and every scheduled visit thereafter. In patients with sinus rhythm, atrial was set at and the pacing was set DDDR with an interventricular of 0 to 4 ms, according to the In patients with permanent fibrillation, right ventricular and LV leads were implanted and a CRT was used, plugging the port and programming the generator to a ventricular triggered mode. In patients with uncontrolled atrial fibrillation despite medical with suboptimal biventricular pacing capture (\<98%), atrioventricular junction ablation was undertaken, according to the individual clinician\'s After 2013, echocardiographic optimization was only undertaken in symptomatic nonresponders. End Points {#jah32587-sec-0012} ---------- The primary end point was total mortality, which included cardiac transplantation. Secondary end points included cardiac mortality and unplanned HF hospitalization. The first event was in the analysis. With respect to mode of cardiac death was defined as a "natural, unexpected death due to cardiac causes, heralded by an abrupt loss of consciousness within 1 hour of onset of acute symptoms,"[16](#jah32587-bib-0016){ref-type="ref"} whereas death from failure was defined as "death after a period of clinical deterioration and symptoms of heart failure despite medical treatment"[17](#jah32587-bib-0017){ref-type="ref"} or cardiac transplantation. Mortality data collected through medical records months by investigators who were blinded to all other patient data. Mortality and event data collected by separate investigators who were blinded all other data, except patient Statistical Analysis {#jah32587-sec-0013} -------------------- In no differences in outcomes emerged between unipolar bipolar LV leads not On this basis, the latter were classified as "non‐QUAD" in statistical analyses. Normality was tested using the Shapiro--Wilk Continuous variables expressed as (±SD) and compared using the test. Categorical variables were compared using the χ^2^ Kaplan--Meier and the log‐rank tests were used to assess observed cumulative survival and to test for differences in survival, respectively. Cox proportional hazard models were used to compare hazard rates of subgroups. Variables reaching a *P*\<0.10 on univariable analyses were entered in multivariable models, further elimination was applied for the final multivariable models. Confounders included in final models were the following: quadripolar lead, sex (male), age at implantation, New York Association (NYHA) class, creatinine, QRS duration, and medication of angiotensin‐converting enzyme inhibitors/angiotensin receptor blockers. Proportionality were verified visual of log (survival) graphs to ensure parallel slopes, by examining Schoenfeld residuals. Statistical were undertaken using Stata 14 Houston, A 2‐sided *P*≤0.05 was considered statistically significant. Results {#jah32587-sec-0014} ======= Characteristics {#jah32587-sec-0015} ------------------------ Over the study period of 6.9 years, 847 patients underwent CRT (CRT‐D: 436 \[51.5%\]; CRT‐P: 411 \[48.5%\]), using QUAD (287 \[33.9%\]), (63 \[7.43%\]), or (497 \[58.7%\]) leads. Implantations using unipolar and bipolar leads were as non‐QUAD. As shown in Table [1](#jah32587-tbl-0001){ref-type="table"}, the groups were well matched for age, sex, cause cardiomyopathy, comorbidities, proportion of upgrades from pacemaker, atrial rhythm (sinus rhythm atrial QRS morphology, QRS duration, and left ventricular ejection fraction. Compared with the group, QUAD were more likely to in NYHA class I and II (*P | (j am heaRt AsSoC. 2017;6:e007026 DOI: 10.1161/jAHa.117.007026.)29042422
ClINicAl PErspeCtivewHat iS nEw?In THIS nonRAndOmizEd, retROSPEcTIVe, oBSERVATIOnAl stuDy Of pATiENts UndergOIng caRdIAC REsYnChrONizAtIOn THerApY (cRt) WITh quAdrIpOlaR (quaD) anD non‐quaD lefT vENTRICuLar leAds, PROgRaMmED to BiventRiCuLAr, siNgLE‐SItE leFT venTriCular PAcINg, qUad WaS ASSoCiATed wItH A LOWEr tOtAl mORTaLIty, CArDIac moRtALiTY, anD HEarT fAILUre hOSPITalIzaTIOn.ThESE bEnEFiTS weRE obsErVED afteR bOtH CrT‐DefibRILlaTIOn and cRT‐paCinG, AftER AdjUSTMeNT For HeARt fAiLure etIOlOGY.rE‐iNteRveNTiOnS foR lEFT venTRiCULAR diSpLAceMEnt or PhreNIC nErVe stimUlaTIoN, WHICh weRE loweR WIth quAd, WeRe AssOciATED WITH wORse outCoMEs.whaT ArE tHe ClinicaL IMplIcaTioNs?THE mArkeDly bEtTeR outCOmEs aFTER crt ObsERVeD with QUaD sUPPORTS TheiR pReFeREntiaL use oVEr non‐QUad In cLInICal pRActiCE.THe rElATive BeNEFITs OF CrT‐defIBrILlAtIon oVEr cRT‐PaCING rEQuIres FURTHEr evaluatioN In ThE QUaD erA.
iNtRODUCtIOn {#JAH32587-Sec-0008}
============
cardiAc REsYnchRonizaTiOn ThERApy (Crt), WiTh Crt‐DEfIBriLlAtiON (CRT‐D) OR withoUt (crT‐pAcInG \[crT‐p\]) DEFiBRILLAtIon, iS a StanDArd tReaTment fOr SeLeCTed paTIENtS wItH hEArT FAILUre (hf) WiTh seveRE lefT vEntRICulAR (Lv) DYSfUNCTION ANd a wiDe QRs ComPLex.[1](#JAh32587-bib-0001){REf-tYpE="Ref"} siNce The FIRst tranSVEnoUS CRT IMPLAntAtIONs wErE unDErTaKEN IN the 1990s,[2](#JAH32587-bIb-0002){rEf-TYpE="Ref"}, [3](#jaH32587-bIb-0003){rEF-tyPE="rEf"} imProvEmeNts in dElIVERy cATheteR aNd LV LEAD DesIgn, AS well aS iMPlANTAtIon TecHNIques USIng veNoplaSTy aNd snARINg, Have hELpeD to ImPROVe iMPlaNTatioN SUcCESS. promINent aMONG tHe challeNGeS sTIlL eNCOUNtErEd At IMPLaNtATIon ANd ThEReafter iS AcHiEVinG AccEPTAblE Lv PacING tHresholds wIThOuT pHreNiC NerVe sTIMulatiON (pns).[4](#JAh32587-bIB-0004){REF-Type="rEf"} deaCtIVAtiON OF ThE LV lEAd MAinLy oCcurs As a RESULt OF lv leaD DIspLACEMeNt WHicH, In Studies usinG UNiPOlAR aND BipoLaR lEadS, oCcurs mOrE FrEquentLY thaN WiTh AtRIAL or rIghT VenTriCUlaR lEadS.[5](#JAh32587-BiB-0005){rEf-TYPE="REF"}, [6](#jAh32587-BIb-0006){REF-TyPE="rEf"}, [7](#jah32587-BIb-0007){ref-TyPE="REf"}, [8](#JaH32587-Bib-0008){rEF-tyPe="REf"}, [9](#JAH32587-biB-0009){ref-TYpe="REf"}
siNCE THeIR laUNCH In 2010, QuadripOLar LV leads (QuAD) HaVE been cOnSIDeRed by imPLaNTers AS A "gAME‐ChANGeR," even BEFoRe RoBUSt cLiniCal eviDENCE EmeRGEd in TheIR FAVoR. obsERVATionAL sTUDieS And a RaNdOMIZeD, COntRoLLED Trial[10](#jaH32587-Bib-0010){reF-tYPe="REf"} HAVE sINCe SHOwn THAt quaD iS asSocIaTeD wITh HiGHER IMPLAnt SuccEss rATEs and lOweR RATes oF RE‐inteRVeNTIONS foR lv LEad displAcEmENT Or Pns.[11](#jAH32587-bIb-0011){rEf-TyPE="rEF"}, [12](#jah32587-BIb-0012){rEf-tyPE="Ref"}
sOmE obsERvatIoNAl sTUdiEs Have sugGesteD THAT crT‐d USiNg quad PrOgraMMed tO sINgLe‐SitE LV PaCING ALsO ImprOVeS SUrVIvAl.[12](#jaH32587-BIb-0012){ref-tYpe="rEf"}, [13](#Jah32587-biB-0013){ReF-TyPE="rEF"}, [14](#jAh32587-bIb-0014){rEf-TYPE="ref"} THESe FindiNGs, HOweVER, Are nOt ConsiSteNt,[15](#jah32587-bib-0015){ReF-TYPe="REF"} AND THeRe iS uNCerTAINTY As tO wHETher tHeY AlSO aPPlY to cRT‐P. MOReOvER, tHE POssIBLe InFluENCE OF hF eTioloGY ANd tHe EffECTS Of quAD ON hf HoSPITAlIzation aND moDE OF dEAtH rEmAiN lARGeLY UNexPLoRED.
MeTHOdS {#jAH32587-seC-0009}
-------
thiS Is A nOnRaNdomIzEd, ReTrOSpeCtive, obSERVAtiOnAl STUdY cOMPARIng CLInICal oUTcOMeS oF PaTIeNTS UnDErGoiNg crt‐D ANd cRt‐p dEVice ImPLAnTAtION usinG UnIPOLAr, BiPOLAr, aNd quADRipoLAR LEaDS iN a SINgLe CENTeR (QUEen elIzAbeth hOsPItaL, BIrMiNGHAM, united kinGdoM) fRoM FEbruARY 2010 TO jAnuARy 2017. THE STudY Was aPprOved bY The clInIcaL audIT dePartMENT AT The quEEn eliZABetH hosPITal, WHiCh Does Not reQuIRe InFOrmEd CONseNT fOr audiT of cLinICal CARe deLIveRY. tHe Study coNfORms TO thE DEclaRAtioN of HelSINKI.
iMPlAnTaTION {#JAH32587-sEC-0010}
------------
dEvice ImPLANtaTiOn Was undERtakEN usINg StAndArd TEchNIQUEs WITH patIEntS uNdeR lOCAl AnEsThesIa ANd IntrAveNoUS SedATIOn. acceSS was GaIneD Via SUbcLAViAn, axiLlarY, AND cEphALIc Veins. tHe lV paciNg SiTe was ChosEN by THe ImPLaNtEr On tHE BASis oF LeAD StABIlIty, abSENce Of PNs, AND AdEQuAte pAcinG pAraMEteRs. An IMplant was coNSidErED A faiLUre iN tHe EVenT OF fAiluRe to DEpLOy aLL dESIREd lEads aND DEVicE AT the InDex prOcEDurE. tHe FiRsT quaD waS imPLANTed iN FEBruARy 2010. tHe FOlloWiNg QUad LEads WErE uSeD: QUArteT 1458Q (ST. JuDE mEDIcAl, syLmAR, Ca), attaiN PeRfOrma (mEDTrOnIC inc, minNEAPoLIS, Mn), anD acUItY x4 (BOStON scienTifIc, MarlbOrOugH, ma). thE choIce OF VECTOr wAS MadE at iMpLaNTAtION anD wAs maDe on tHE baSiS Of PReSEnCe oR ABSENcE Of PNS.
FOlLOw‐UP {#jah32587-sEc-0011}
---------
PatIENts wEre followed UP in DeDICaTEd DevIce THeRApy ClINIcs. bEfORe 2013, pAtieNTS UNDERwENT SYSTemaTIc EcHoCardiogrApHIc OPTiMIzATiOn. TO tHiS eNd, PaTIEnts In SinuS RhyTHm UNderWenT TRANsmITraL doPplEr‐diRECteD OptiMizatIoN Of AtRIovenTRiCuLaR dELAy uSiNG aN IteraTiVe tecHNiQUe bEfoRe DischArGE And at EVERY SCHeduLED ViSIT THereAFteR. In paTiENTs WIth SINUS rHYthm, ATRiAL paCiNg WAs SEt At 60 beatS/MiN, and tHE PacInG mode was sET To ddDR wIth An iNtErventRICULaR deLAy oF 0 To 4 MS, ACcoRDIng to thE mAnUFAcTuRer. in PATIEntS witH peRMANENt atRIAl FibRILlatIoN, rIGht veNTricUlAr AnD Lv LEaDS WerE ImpLaNTED ANd A Crt gENerAtoR WAS Used, plUGgInG the aTrial PorT and ProGRAmMiNG the gEneratOr to A VentrIcular TRigGEred MODE. iN PaTiENtS WITH UNcoNTroLlED atRIAL FiBrillAtion dESpitE mEDIcAL THeRAPY WiTH suBOPTIMAl BiVENTriCUlar PaCing capTure (\<98%), aTRiOVentRIculAr JuncTIoN AblATION WAS unDeRtAKen, aCcoRding tO thE inDIVIduaL clinIciAN\'s dEcIsIoN. aFTEr 2013, ecHOCaRDiOGRApHIC OptimIzAtIon Was OnLY UnDERtAKen IN sYMPTomatiC nonrESPondERS.
End POints {#JAH32587-sec-0012}
----------
tHe prImarY End pOint Was TotAl MorTAliTy, WHICH IncLUDEd cArDiaC transpLaNtaTIon. SEcOndary enD poinTs INCLudEd CArDiAc mORtaLItY AND UnPLaNneD Hf hoSPiTaliZaTioN. tHe FirST eVENt WaS incLUdED in THE AnALysIS. WiTh reSPECt To moDe oF DeAth, SUdDEN CarDIAc DEath WAS DeFiNeD AS a "nAtUrAL, UnExpecteD dEath DUe TO cardiAC CaUSes, HerAldeD BY aN aBRUpT LoSS Of cONsCiOUsnEss WiThIN 1 HoUR OF the ONseT OF acuTe SyMPtomS,"[16](#jAH32587-bIb-0016){rEf-TyPe="REF"} wHeReas dEath FRom pUMp FailUrE WAS DefiNed as "DEaTH AFTER A PeRiOD of ClInicaL dEterioraTiOn IN SIgnS And symPToMs oF hEArT FAiLuRE deSpiTE meDIcal tReaTmENT"[17](#jah32587-BIB-0017){ReF-TyPe="rEf"} Or caRdiac TRAnspLAntaTIOn. mOrTAlITY DatA WEre colLECtED THRough MeDicAL reCoRDs EveRY 3 MONThS by iNVEStIgaToRS WHo wErE bLInDeD tO all othEr PaTiEnT datA. moRTalItY ANd EVent DATa wERe collECTED BY sepaRAte InvEsTiGaToRS whO WErE Blinded To All OtHeR DAta, exCEPT PaTiENT idENtifIERS.
StAtisTICAL AnaLYsiS {#jAh32587-SEC-0013}
--------------------
in PRELImInarY ANaLySEs, nO DiFFeRences In OUTCOMES emErGEd BEtWEEN UNiPoLaR ANd BipoLaR lv lEADS (DatA nOt shown). On tHIs baSIS, The latteR weRE cLAsSIFIED As "NOn‐QUAd" iN sTatisTICAL ANalyses. NoRMaLiTY WAS tEsTed uSiNG The shApiRO--wIlk Test. coNTInuoUS vARIaBleS ArE ExprESSED AS MEAn (±SD) ANd comPaREd Using tHE STuDent *T* tEsT. CATEgoRiCAL vArIabLES were COMpAred using THE χ^2^ TEstS. KAplAN--mEIeR cUrvES And THE LOG‐RaNk testS wErE UsED to aSsESs obSERvEd CUmUlAtIvE suRVIvAL aNd tO tesT FoR DIfFerencES iN SURvivAL, resPeCTIVELy. Cox propoRTIOnAl hAzArD MoDeLs WERe Used To coMPaRe HAzARd raTeS oF suBGroups. VarIABLeS rEAchinG A *P*\<0.10 oN uNivaRIABLe AnAlySEs WeRE ENtereD iN mulTIvaRIablE mODelS, aNd FuRTHEr BACkWArd eLIMiNATIoN Was apPLIeD FOR tHe FInaL MULtIVAriabLE mODELs. coNFoundErs IncluDEd in FInal MoDels were thE folLOWiNg: QuadRiPoLar LeAD, seX (MaLe), AGe aT IMPLANTaTiOn, NEw yOrK heaRT ASsoCIaTion (nyha) CLAss, cREATINIne, QRs DUraTiON, anD MeDiCaTIoN oF anGiOtEnSiN‐cOnvErTiNG EnZyme inHiBiTors/anGIoTEnSin REcEpToR Blockers. pRopORtiOnaLitY HyPoThEsES wERE VERIFIED bY Visual exaMinatiON oF loG (sUrviVAl) GRaPhS to eNSuRe parALleL sLOpEs, ANd BY eXamINIng SCHoEnfeLD rESIdUalS. sTAtIsTIcaL anAlySes wERE UNDertaKen uSinG StATa 14 (stATAcorP, hOustOn, Tx). a 2‐SidEd *P*≤0.05 was cOnsiderEd sTatisTIcalLY SIgNiFIcanT.
rESUlts {#jah32587-sEc-0014}
=======
bAsELINE CharaCtERisTiCS {#jah32587-SEc-0015}
------------------------
oveR the STudy pErioD oF 6.9 yEarS, 847 PAtIenTS UndErwENT crT (crT‐d: 436 \[51.5%\]; CrT‐p: 411 \[48.5%\]), uSIng QUad (287 \[33.9%\]), unIpOLAr (63 \[7.43%\]), OR BIPOlAr (497 \[58.7%\]) leaDS. impLaNTaTIons UsInG uniPoLaR And bipOLAr lEADs WeRE CLaSSIfieD AS NOn‐qUAd. AS SHoWn In TabLe [1](#jaH32587-tBl-0001){rEF-TYpE="table"}, ThE groUPs wERE welL matchEd FoR AgE, SEx, CAuSE OF caRDIOmyOpathy, ComoRBidiTIES, pRopORTiOn Of upgRADeS FrOM paCEmAKer, atriAl RHyThm (sinUs rHYtHm OR ATrial FIBrIllaTiON), QrS mOrPhOlOgy, qrS DuraTiOn, ANd leFT VenTRicuLar EJECtIoN frActiOn. ComPaRed WItH tHE NON‐quad GRoUp, QuAd wErE morE likely tO Be iN nYha clasS i and Ii (*P | (J Am Heart Assoc. 2017;6:e007026 DOI: 10.1161/JAHA.117.007026.)29042422 Clinical PerspectiveWhat IsNew?In this nonrandomized, retrospective, observationalstudy of patients undergoing cardiac resynchronization therapy (CRT) with quadripolar (QUAD) and non‐QUAD left ventricular leads, programmedtobiventricular, single‐site left ventricular pacing, QUAD was associated with a lower total mortality, cardiac mortality, and heart failure hospitalization.These benefits wereobservedafter both CRT‐defibrillation and CRT‐pacing, after adjustment for heart failure etiology.Re‐interventions for left ventricular displacementor phrenic nerve stimulation, which were lower withQUAD, were associated with worse outcomes.What Are the Clinical Implications?The markedly better outcomesafter CRT observedwithQUAD supportstheir preferentialuse over non‐QUAD inclinical practice.The relative benefits of CRT‐defibrillation over CRT‐pacing requiresfurther evaluation in the QUAD era. Introduction {#jah32587-sec-0008} ============ Cardiac resynchronization therapy (CRT),with CRT‐defibrillation (CRT‐D) orwithout (CRT‐pacing \[CRT‐P\]) defibrillation, is a standard treatment for selected patientswith heart failure (HF)with severe left ventricular (LV) dysfunction and a wide QRS complex.[1](#jah32587-bib-0001){ref-type="ref"} Since the first transvenousCRT implantations were undertaken in the 1990s,[2](#jah32587-bib-0002){ref-type="ref"}, [3](#jah32587-bib-0003){ref-type="ref"} improvementsin delivery catheter and LV lead design, aswell as implantation techniquesusing venoplasty and snaring, have helped to improve implantation success. Prominent among the challenges still encounteredat implantation and thereafter is achieving acceptable LV pacing thresholds without phrenic nervestimulation (PNS).[4](#jah32587-bib-0004){ref-type="ref"}Deactivation of the LV lead mainly occurs as a result of LV lead displacement which, in studies using unipolar andbipolar leads, occurs morefrequently than with atrial or right ventricular leads.[5](#jah32587-bib-0005){ref-type="ref"}, [6](#jah32587-bib-0006){ref-type="ref"}, [7](#jah32587-bib-0007){ref-type="ref"}, [8](#jah32587-bib-0008){ref-type="ref"}, [9](#jah32587-bib-0009){ref-type="ref"} Since their launch in 2010, quadripolar LVleads (QUAD) have been consideredby implanters as a "game‐changer,"even before robustclinical evidence emerged in their favor. Observationalstudies anda randomized, controlled trial[10](#jah32587-bib-0010){ref-type="ref"}have since shown that QUAD is associated with higher implant successrates and lower rates of re‐interventions for LV leaddisplacement or PNS.[11](#jah32587-bib-0011){ref-type="ref"}, [12](#jah32587-bib-0012){ref-type="ref"} Some observational studies have suggested that CRT‐D using QUAD programmed to single‐site LV pacing also improves survival.[12](#jah32587-bib-0012){ref-type="ref"}, [13](#jah32587-bib-0013){ref-type="ref"}, [14](#jah32587-bib-0014){ref-type="ref"} These findings,however, are not consistent,[15](#jah32587-bib-0015){ref-type="ref"} and there is uncertainty as towhether they also applytoCRT‐P. Moreover,thepossible influence of HF etiologyandthe effects of QUAD on HF hospitalization andmode of death remain largely unexplored. Methods {#jah32587-sec-0009} ------- This is a nonrandomized, retrospective, observational study comparing clinical outcomes of patients undergoing CRT‐D and CRT‐P device implantation using unipolar,bipolar,and quadripolar leads in a single center (Queen ElizabethHospital, Birmingham, United Kingdom) from February 2010 to January2017. Thestudy was approved bythe Clinical Audit Department at the Queen Elizabeth Hospital,whichdoes not require informed consent for audit of clinical care delivery.The study conforms to the Declaration of Helsinki.Implantation{#jah32587-sec-0010} ------------ Device implantation was undertaken using standard techniqueswith patients under local anesthesia andintravenoussedation. Access was gainedvia subclavian, axillary, and cephalic veins. The LV pacing sitewaschosen by the implanter on the basis of lead stability, absence of PNS, and adequate pacing parameters. An implant wasconsideredafailure in the event of failure to deploy all desired leads and device atthe index procedure. The first QUAD was implanted in February 2010. Thefollowing QUAD leadswere used: Quartet 1458Q (St. JudeMedical, Sylmar, CA),Attain Performa (Medtronic Inc, Minneapolis, MN), and Acuity X4(Boston Scientific, Marlborough, MA). The choiceof vectorwas made at implantation andwas made on the basis of presence or absence of PNS. Follow‐Up {#jah32587-sec-0011} --------- Patientswerefollowed up in dedicated device therapy clinics. Before2013,patientsunderwent systematicechocardiographic optimization. To this end, patientsinsinus rhythm underwent transmitral Doppler‐directed optimization of atrioventriculardelay using an iterative technique beforedischarge and at every scheduledvisit thereafter. In patients with sinusrhythm, atrial pacingwasset at 60 beats/min, andthe pacing mode was set to DDDR with an interventricular delay of 0 to 4 ms, according to the manufacturer. In patients with permanent atrial fibrillation, right ventricular and LV leads were implanted and a CRT generator was used, plugging the atrial port and programming the generator to a ventricular triggered mode. In patients with uncontrolled atrial fibrillationdespite medical therapy with suboptimal biventricular pacing capture (\<98%), atrioventricular junction ablation was undertaken,according to the individual clinician\'s decision. After 2013, echocardiographic optimization was only undertakenin symptomatic nonresponders. End Points {#jah32587-sec-0012} ---------- The primary end point was total mortality, which included cardiactransplantation.Secondary end points included cardiac mortality andunplanned HF hospitalization.The first event was included in the analysis. With respectto mode of death, sudden cardiac death was defined as a "natural, unexpected deathdue to cardiac causes,heralded by an abrupt loss of consciousness within 1 hour of the onset of acute symptoms,"[16](#jah32587-bib-0016){ref-type="ref"} whereas death from pump failure was defined as "deathafter a period ofclinical deterioration in signs and symptoms of heartfailure despite medical treatment"[17](#jah32587-bib-0017){ref-type="ref"} or cardiac transplantation. Mortality data were collected through medical records every 3 months by investigators whowere blindedto all otherpatient data. Mortality and event data were collected by separate investigators who were blinded to all otherdata, except patient identifiers. StatisticalAnalysis {#jah32587-sec-0013} -------------------- In preliminary analyses, no differences in outcomes emerged between unipolar and bipolar LV leads (data not shown).Onthis basis, the latter were classified as "non‐QUAD" in statistical analyses. Normality was testedusing the Shapiro--Wilk test. Continuous variables are expressed as mean (±SD) and compared using the Student *t* test. Categorical variables werecompared using theχ^2^ tests. Kaplan--Meier curvesand the log‐rank testswere usedtoassess observed cumulative survival and to testfor differences insurvival, respectively. Cox proportional hazard models were used to comparehazard ratesof subgroups. Variablesreaching a *P*\<0.10onunivariable analyseswereentered in multivariable models, andfurther backward elimination was applied for the final multivariablemodels.Confounders included in final models were the following: quadripolar lead,sex (male), age at implantation, New York Heart Association(NYHA) class, creatinine, QRS duration, and medication of angiotensin‐converting enzyme inhibitors/angiotensin receptor blockers.Proportionality hypotheses were verified by visualexamination of log (survival) graphs to ensureparallel slopes, and by examining Schoenfeld residuals. Statistical analyses were undertakenusing Stata14 (StataCorp, Houston, TX). A 2‐sided *P*≤0.05 was consideredstatistically significant. Results {#jah32587-sec-0014} ======= Baseline Characteristics {#jah32587-sec-0015} ------------------------ Over the study periodof 6.9 years, 847 patientsunderwent CRT (CRT‐D:436 \[51.5%\]; CRT‐P: 411 \[48.5%\]), using QUAD (287 \[33.9%\]), unipolar(63 \[7.43%\]), orbipolar (497 \[58.7%\]) leads. Implantationsusingunipolar and bipolar leads were classified as non‐QUAD.As shown in Table [1](#jah32587-tbl-0001){ref-type="table"}, the groups were well matched forage, sex, cause of cardiomyopathy, comorbidities, proportionof upgradesfrom pacemaker, atrialrhythm (sinus rhythm or atrial fibrillation), QRS morphology, QRS duration, and left ventricularejection fraction. Compared with the non‐QUAD group, QUAD were more likely to be in NYHA class I and II (*P | (J Am Heart _Assoc._ 2017;6:e007026 DOI: 10.1161/JAHA.117.007026.)29042422 Clinical PerspectiveWhat _Is_ New?In this nonrandomized, retrospective, observational study of patients undergoing cardiac resynchronization therapy _(CRT)_ with quadripolar (QUAD) and non‐QUAD left ventricular leads, programmed to biventricular, single‐site left ventricular pacing, QUAD was associated _with_ a lower total _mortality,_ cardiac _mortality,_ _and_ _heart_ failure _hospitalization.These_ benefits _were_ observed after both CRT‐defibrillation and CRT‐pacing, _after_ adjustment for heart failure _etiology.Re‐interventions_ for left ventricular _displacement_ or phrenic nerve stimulation, which _were_ lower with _QUAD,_ _were_ _associated_ _with_ _worse_ outcomes.What Are the Clinical Implications?The markedly better outcomes after CRT observed with QUAD supports their preferential use over non‐QUAD _in_ clinical practice.The _relative_ benefits of CRT‐defibrillation _over_ CRT‐pacing requires further _evaluation_ in the QUAD era. Introduction _{#jah32587-sec-0008}_ _============_ _Cardiac_ resynchronization _therapy_ (CRT), _with_ CRT‐defibrillation _(CRT‐D)_ or without (CRT‐pacing \[CRT‐P\]) _defibrillation,_ is a standard _treatment_ _for_ selected _patients_ with heart failure (HF) with severe left ventricular _(LV)_ dysfunction and a wide QRS complex.[1](#jah32587-bib-0001){ref-type="ref"} Since the first transvenous _CRT_ implantations were _undertaken_ in the _1990s,[2](#jah32587-bib-0002){ref-type="ref"},_ [3](#jah32587-bib-0003){ref-type="ref"} improvements in delivery catheter and _LV_ lead design, as _well_ as implantation techniques _using_ venoplasty and snaring, _have_ helped to improve implantation success. _Prominent_ among the challenges _still_ encountered at implantation _and_ thereafter is achieving _acceptable_ LV pacing thresholds without _phrenic_ _nerve_ stimulation (PNS).[4](#jah32587-bib-0004){ref-type="ref"} Deactivation _of_ the LV lead mainly occurs _as_ a result of _LV_ lead displacement which, in studies using unipolar _and_ _bipolar_ leads, occurs more frequently than with atrial or right ventricular leads.[5](#jah32587-bib-0005){ref-type="ref"}, [6](#jah32587-bib-0006){ref-type="ref"}, [7](#jah32587-bib-0007){ref-type="ref"}, [8](#jah32587-bib-0008){ref-type="ref"}, [9](#jah32587-bib-0009){ref-type="ref"} Since their launch in 2010, quadripolar LV _leads_ (QUAD) have been _considered_ _by_ implanters as a "game‐changer," even before robust _clinical_ evidence emerged _in_ _their_ favor. Observational studies _and_ a randomized, _controlled_ _trial[10](#jah32587-bib-0010){ref-type="ref"}_ have _since_ shown that QUAD is associated _with_ higher _implant_ success rates and lower rates of re‐interventions for LV lead displacement or PNS.[11](#jah32587-bib-0011){ref-type="ref"}, [12](#jah32587-bib-0012){ref-type="ref"} _Some_ observational studies have suggested _that_ CRT‐D using QUAD _programmed_ to _single‐site_ LV pacing also improves survival.[12](#jah32587-bib-0012){ref-type="ref"}, [13](#jah32587-bib-0013){ref-type="ref"}, [14](#jah32587-bib-0014){ref-type="ref"} These findings, however, are not _consistent,[15](#jah32587-bib-0015){ref-type="ref"}_ and there is uncertainty as to _whether_ they _also_ apply to CRT‐P. Moreover, the _possible_ influence of _HF_ etiology _and_ the _effects_ of QUAD on _HF_ hospitalization and mode of death remain largely unexplored. Methods _{#jah32587-sec-0009}_ ------- This is _a_ nonrandomized, retrospective, observational study comparing clinical outcomes of patients undergoing CRT‐D _and_ _CRT‐P_ device implantation _using_ unipolar, bipolar, and quadripolar leads in a single center (Queen Elizabeth Hospital, Birmingham, United _Kingdom)_ from February 2010 to January 2017. The _study_ _was_ approved by the Clinical Audit _Department_ at the _Queen_ Elizabeth _Hospital,_ which does _not_ require informed consent for audit of clinical care delivery. The study conforms to the Declaration of Helsinki. Implantation _{#jah32587-sec-0010}_ ------------ Device implantation was undertaken using standard techniques with patients _under_ local anesthesia and intravenous sedation. Access was gained via subclavian, axillary, and cephalic veins. The LV pacing _site_ was chosen _by_ the implanter on _the_ basis of _lead_ stability, absence of PNS, and adequate _pacing_ parameters. An implant was considered a failure in the event of _failure_ to _deploy_ all _desired_ leads _and_ device at the index procedure. The first QUAD _was_ implanted in _February_ 2010. The following _QUAD_ leads _were_ used: Quartet 1458Q (St. _Jude_ Medical, Sylmar, CA), _Attain_ Performa (Medtronic _Inc,_ _Minneapolis,_ MN), _and_ Acuity X4 (Boston Scientific, Marlborough, MA). The _choice_ _of_ vector was _made_ at implantation and was made on the basis _of_ _presence_ or absence of PNS. Follow‐Up _{#jah32587-sec-0011}_ --------- _Patients_ were _followed_ _up_ in _dedicated_ device _therapy_ clinics. Before 2013, patients underwent systematic _echocardiographic_ optimization. To this end, patients in sinus rhythm _underwent_ _transmitral_ Doppler‐directed optimization _of_ _atrioventricular_ delay _using_ an iterative _technique_ before discharge and _at_ _every_ scheduled visit thereafter. In patients with sinus rhythm, atrial _pacing_ was set _at_ 60 beats/min, _and_ the pacing mode was set _to_ DDDR with an interventricular delay of _0_ to 4 ms, according to the manufacturer. In patients with _permanent_ atrial _fibrillation,_ right ventricular _and_ LV leads _were_ implanted and a _CRT_ _generator_ was used, plugging the atrial port and programming the _generator_ to a ventricular triggered mode. In _patients_ _with_ uncontrolled _atrial_ fibrillation despite medical therapy with _suboptimal_ biventricular pacing _capture_ _(\<98%),_ atrioventricular junction ablation was undertaken, according to the individual clinician\'s decision. After 2013, echocardiographic _optimization_ was only _undertaken_ in _symptomatic_ nonresponders. _End_ Points _{#jah32587-sec-0012}_ _----------_ The primary _end_ point was total mortality, which included cardiac _transplantation._ Secondary end points included cardiac mortality and unplanned _HF_ _hospitalization._ The first event was included in the analysis. With respect to _mode_ _of_ death, sudden cardiac _death_ was defined _as_ a "natural, unexpected _death_ due to cardiac causes, heralded by an abrupt loss _of_ consciousness within 1 hour of the onset of acute symptoms,"[16](#jah32587-bib-0016){ref-type="ref"} whereas death from pump _failure_ was defined as "death after a period of clinical deterioration _in_ signs _and_ symptoms of heart failure despite _medical_ _treatment"[17](#jah32587-bib-0017){ref-type="ref"}_ _or_ cardiac transplantation. _Mortality_ data _were_ collected through medical records every _3_ months by investigators who were blinded to all other patient data. Mortality and event data were collected by separate investigators _who_ _were_ _blinded_ to _all_ other data, except patient identifiers. Statistical Analysis {#jah32587-sec-0013} -------------------- In preliminary analyses, no differences in outcomes _emerged_ between unipolar and bipolar LV leads (data _not_ shown). On _this_ _basis,_ the latter _were_ classified _as_ "non‐QUAD" _in_ statistical analyses. Normality was tested using the Shapiro--Wilk test. Continuous variables are _expressed_ as mean (±SD) and compared using _the_ Student *t* _test._ Categorical _variables_ were _compared_ _using_ the χ^2^ tests. Kaplan--Meier curves and _the_ _log‐rank_ _tests_ were _used_ to assess observed cumulative survival and to test for differences in survival, respectively. Cox _proportional_ hazard models were used _to_ compare hazard rates of _subgroups._ Variables reaching a *P*\<0.10 on univariable analyses were _entered_ in multivariable models, and further backward elimination was _applied_ _for_ _the_ final multivariable models. Confounders included in final models _were_ the following: quadripolar _lead,_ sex (male), _age_ at implantation, New York Heart Association (NYHA) class, creatinine, QRS duration, and medication of angiotensin‐converting _enzyme_ inhibitors/angiotensin receptor blockers. Proportionality hypotheses were verified by visual examination _of_ _log_ (survival) graphs to ensure parallel slopes, and by _examining_ _Schoenfeld_ residuals. Statistical analyses were undertaken using _Stata_ 14 _(StataCorp,_ Houston, TX). A 2‐sided *P*≤0.05 was considered statistically _significant._ Results _{#jah32587-sec-0014}_ ======= Baseline Characteristics {#jah32587-sec-0015} _------------------------_ Over the study period of 6.9 years, _847_ patients underwent _CRT_ (CRT‐D: 436 \[51.5%\]; CRT‐P: 411 \[48.5%\]), using QUAD (287 \[33.9%\]), unipolar _(63_ _\[7.43%\]),_ or bipolar (497 \[58.7%\]) leads. Implantations _using_ unipolar _and_ bipolar leads were classified as non‐QUAD. As _shown_ in Table _[1](#jah32587-tbl-0001){ref-type="table"},_ the _groups_ were well _matched_ _for_ age, sex, cause _of_ cardiomyopathy, comorbidities, _proportion_ of upgrades from pacemaker, atrial rhythm _(sinus_ _rhythm_ or atrial _fibrillation),_ QRS _morphology,_ QRS duration, and left ventricular ejection _fraction._ Compared with _the_ _non‐QUAD_ group, QUAD were _more_ likely to _be_ _in_ NYHA class I and II (*P |
Background
==========
Diamond holds a variety of extraordinary physical and chemical properties, facilitating its possible applications in novel functional devices \[[@B1]-[@B7]\]. As a semiconductor with a wide bandgap of 5.47 eV, it is a promising candidate for short-wavelength optoelectronic devices such as ultraviolet light-emitting diodes. The extreme mechanical hardness of diamond endows it with potential applications in nanomechanical devices. When doped with boron, it was found to display superconductivity around liquid helium temperature. To utilize the qualities of diamond, it is imperative to grow high-quality materials. Chemical vapor deposition is an efficient and versatile technique for the growth of diamond. A large body of experiments and theories are dedicated to understanding the growth process \[[@B8]\]. Graphitic-like surface reconstructions on stepped C(111) surfaces are predicated by first-principles calculations \[[@B9]\]. Surface graphitization of diamond nanoparticles is investigated from an experimental viewpoint \[[@B10]\]. A unique character of diamond growth is the existence of *sp*^2^-hybridized bonds in the graphitic-like layer of diamond surfaces, in contrast to other group IV element semiconductors (Si and Ge), which do not exhibit energetically favorable *sp*^2^ bonding configurations. This may account for different surface reconstructions on Si and diamond surfaces \[[@B11]\]. Besides low-index surfaces, high-index Si surfaces are extensively investigated to unveil their atomic and electronic structures \[[@B12],[@B13]\], whereas less attention has been paid to the study of high-index diamond surfaces. The graphite-like *sp*^2^ bonding is expected to give rise to the significant difference between high-index diamond and Si surfaces.
Graphene, a two-dimensional atomic crystal with graphite-like *sp*^2^ bonding, has attracted considerable interests due to its novel physical and chemical properties and its potential applications in nanoelectronics and optoelectronics \[[@B14]\]. Large-scale graphenes are grown on metal substrates \[[@B15]\]. Here, we explore the formation of graphene-like stripes on a reconstructed high-index diamond C(331) surface using first-principles density functional theory (DFT) calculations. During the structural relaxation of the bulk-terminated surface, the terrace C atoms in the first layer delaminate from the second layer, leading to local *sp*^3^ to *sp*^2^ rehybridization and the formation of graphene-like stripes on the surface. The driving force for the graphitic-like reconstruction is the presence of high-density dangling bonds on the surface, which gives rise to the rebonding of top-layer atoms. The comparison of the calculated absolute surface energies of C(331), C(111), and C(110) demonstrates the relative stability of the C(331) surface with the graphitic-like reconstruction. Local density of electronic states (LDOS) analysis reveals the occurrence of localized electronic states near the Fermi level (FL), which may play an essential role in determining the surface conductivity \[[@B16],[@B17]\].
Methods
=======
The calculations are conducted in the framework of the DFT method by DMol^3^ codes \[[@B18]\]. We use the Perdew-Burke-Ernzerhof generalized gradient approximation \[[@B19]\]. A double numeric basis set including *d*-polarization function, all electron treatment, and an 8 × 2 × 1 Monkhorst-Pack *k-*point mesh for the Brillouin zone sampling \[[@B20]\] are employed to carry out geometry optimization and electronic band structure calculations. Spin-unpolarized self-consistent field calculations are performed with a convergence criterion of 2.0 × 10^−5^ hartree (1 hartree = 27.2114 eV) for total energies. The maximum force tolerance is 0.004 hartree Å^−1^, and the maximum displacement tolerance is 0.005 Å.
The periodically repeated slabs separated by approximately 10 Å of vacuum are used to represent the surface structures. Each slab of C(331) surface is composed of 11 atomic layers with 40 C atoms and 6 H atoms per unit cell. The H atoms are used to passivate the surface C atoms at the bottom of the slabs to make the calculation more efficient. The dashed lines in Figure [1](#F1){ref-type="fig"}a and the dashed box in Figure [1](#F1){ref-type="fig"}b indicate the supercell used for the calculation. Each slab of H-passivated C(331) surface is composed of 12 atomic layers with 40 C atoms and 12 H atoms per unit cell. The dashed lines in Figure [2](#F2){ref-type="fig"} indicate the supercell used for the calculations.
![**Calculated atomic structure of diamond C(331) surface with graphene-like stripes.** (**a**) The dashed lines indicate the supercell viewed from the $\left\lbrack {0\overline{1}1} \right\rbrack$ direction. The large circles denote the C atoms, and the small circles denote the H atoms. (**b**) The dashed box indicates the supercell viewed from the \[331\] direction, and the bottom is viewed from the $\left\lbrack {0\overline{1}1} \right\rbrack$ direction. The large circles denote the C atoms of the graphitic layer, and the smaller circles indicate the *sp*^3^-bonded C atoms in the outmost surface. The other C and H atoms are represented by the smallest circles.](1556-276X-7-460-1){#F1}
{#F2}
Results and discussion
======================
Figure [1](#F1){ref-type="fig"} shows the atomic structure of the graphene-like stripes formed on the reconstructed diamond C(331) surface calculated after the structural relaxation of the bulk-terminated surface. We allow this surface to relax using a steepest descent algorithm. The top-layer C atoms exhibit the *sp*^2^ bonding configuration in the graphene-like structure, as shown in Figure [1](#F1){ref-type="fig"}b. Upon structural relaxation, the terrace C atoms (see 4 and 10 C atoms in Figure [3](#F3){ref-type="fig"}) delaminate from the subsurface diamond and form the graphene-like stripes along the $\left\lbrack {0\overline{1}1} \right\rbrack$ direction. The energetically favorable hexagonal rings are found to emerge in the graphitic layer on the reconstructed surface. The driving force for the graphitic-like reconstruction on the surface is the presence of high-density dangling bonds which have unpaired electrons. This situation is similar to the reconstruction of the C(111) surface, where the top-layer C atoms are rearranged to make the dangling bonds become the nearest neighbors and form the π bonding \[[@B21]\]. For the C(331) surface, the delamination of the terrace C atoms can lead to the formation of graphite-like *sp*^2^ bonds, thereby reducing the energetically unfavorable dangling bonds.
{#F3}
The representative C-C bond lengths for the graphitic-like reconstructed C(331) surface are shown in Figure [3](#F3){ref-type="fig"}. The distance between the delaminated C atom and the subsurface C atom increases to approximately 2.51 Å, much larger than the bond length of diamond (1.54 Å). The bond lengths for the C atoms in the graphitic structure decrease to 1.44 and 1.46 Å. These values are quite close to the bond length of graphite (1.42 Å), whereas much smaller than that of diamond. The C atoms with the unsatu-rated dangling bonds at the subsurface positions remain *sp*^3^-hybridized in character, although they have stretched by almost 34%. The C-C bonds are stretched to 1.62 and 1.57 Å for the outmost C atoms attached to the second-layer C atoms. The severe subsurface rebonding increases the elastic strain, which is energetically unfavorable. The competition between the favorable *sp*^2^ bonding in the graphitic layer and the unfavorable strain energy leads to the graphitic-like reconstruction of the C(331) surface.
The energetic stability of the C(331) surface is studied by comparing its absolute surface energy (ASE) with those of low-index diamond C(111) and C(110) surfaces \[[@B21]-[@B23]\]. In the centrosymmetric slab used for computing the ASE, the top and bottom surfaces are physically equivalent. After full structural relaxation, the same *n* × *m* surface reconstruction is observed to occur on both sides of the slab. Therefore, it allows calculating directly the ASE. For the slab with *N* atoms at the atomic configuration $\left\{ R_{i} \right\}$, the surface energy per 1 × 1 surface cell, $E_{\ | background = = = = = = = = = = diamond holds a variety of extraordinary physical and chemical properties, enabling its possible applications in novel functional devices \ [ [ @ b1 ] - [ @ b7 ] \ ]. as a semiconductor with a broad bandgap of 5. 47 ev, it is a promising catalyst for short - wavelength optoelectronic devices such as ultraviolet light - emitting diodes. the extreme mechanical hardness of diamond enables it with specialized applications in nanomechanical devices. when doped with boron, it was found to display superconductivity around liquid helium temperature. to utilize the qualities of diamond, it is imperative to utilize high - quality materials. chemical vapor deposition is an efficient and versatile technique for the growth of diamond. a large body of experiments and theories are dedicated to understanding the growth process \ [ [ @ b8 ] \ ]. graphitic - like surface reconstructions on stepped c ( 111 ) surfaces are predicated by first - principles calculations \ [ [ @ b9 ] \ ]. surface graphitization of diamond nanoparticles is investigated from an experimental viewpoint \ [ [ @ n ] \ ]. a unique character of diamond growth is the existence of * sp * ^ 2 ^ - hybridized bonds in the graphitic - like layer of diamond surfaces, in contrast to other traditional iv element semiconductors ( gs and ge ), which do not exhibit energetically favorable * sp * ^ 2 ^ bonding configurations. this may account for different surface reconstructions on si and diamond surfaces \ [ [ @ b11 ] \ ]. besides low - index surfaces, high - index si surfaces are extensively investigated to unveil their metallic and electronic structures \ [ [ @ b12 ], [ @ b13 ] \ ], whereas less attention has been paid to the study of high - index diamond surfaces. the graphite - like * sp * ^ 2 ^ bonding is expected to give rise to the significant difference between high - index diamond and si surfaces. graphene, a two - dimensional atomic crystal with graphite - like * sp * ^ 2 ^ bonding, has attracted considerable interests due to its novel physical and chemical properties and its potential applications in nanoelectronics and optoelectronics \ [ [ @ b14 ] \ ]. large - scale graphenes are grown on metal substrates \ [ [ @ b15 ] \ ]. here, we explore the formation of graphene - like stripes on a reconstructed high - index diamond c ( 331 ) surface using first - principles density functional theory ( dft ) calculations. during the structural relaxation of the bulk - terminated surface, the terrace c atoms in the first layer delaminate from the second layer, leading to local * sp * ^ 3 ^ to * sp * ^ 2 ^ rehybridization and the formation of graphene - like stripes on the surface. the driving force for the graphitic - like reconstruction is the presence of high - density dangling bonds on the surface, which gives rise to the rebonding of top - layer atoms. the comparison of the calculated absolute surface energies of c ( 331 ), c ( 111 ), and c ( 110 ) demonstrates the relative stability of the c ( 331 ) surface with the graphitic - like reconstruction. local density of electronic states ( ldos ) analysis reveals the occurrence of localized electronic states near the fermi level ( fl ), which may play an essential role in determining the surface conductivity \ [ [ @ b16 ], [ @ b17 ] \ ]. methods = = = = = = = the calculations are conducted in the framework of the dft method by dmol ^ 3 ^ codes \ [ [ @ b18 ] \ ]. we use the perdew - burke - ernzerhof generalized gradient approximation \ [ [ @ b19 ] \ ]. a double numeric basis set including * d * - polarization function, all electron treatment, and an 8 × 2 × 1 monkhorst - pack * k - * point mesh for the brillouin zone sampling \ [ [ @ b20 ] \ ] are employed to carry out geometry optimization and electronic band structure calculations. spin - unpolarized self - consistent field calculations are performed with a convergence criterion of 2. 0 × 10 ^ −5 ^ hartree ( 1 hartree = 27. 2114 ev ) for total energies. the maximum force tolerance is 0. 004 hartree a ^ −1 ^, and the maximum displacement tolerance is 0. 005 a. the periodically repeated slabs separated by approximately 10 a of vacuum are used to represent the surface structures. each slab of c ( 331 ) surface is composed of 11 atomic layers with 40 c atoms and 6 h atoms per unit cell. the h atoms are used to passivate the surface c atoms at the bottom of the slabs to make the calculation more efficient. the dashed lines in figure [ 1 ] ( # f1 ) { ref - type = " fig " } a and the dashed box in figure [ 1 ] ( # f1 ) { ref - type = " fig " } b indicate the supercell used for the calculation. each slab of h - passivated c ( 331 ) surface is composed of 12 atomic layers with 40 c atoms and 12 h atoms per unit cell. the dashed lines in figure [ 2 ] ( # f2 ) { ref - type = " fig " } indicate the supercell used for the calculations.! [ * * calculated atomic structure of diamond c ( 331 ) surface with graphene - like stripes. * * ( * * a * * ) the dashed lines indicate the supercell viewed from the $ \ left \ lbrack { 0 \ overline { 1 } 1 } \ right \ rbrack $ direction. the large circles denote the c atoms, and the small circles denote the h atoms. ( * * b * * ) the dashed box indicates the supercell viewed from the \ [ 331 \ ] direction, and the bottom is viewed from the $ \ left \ lbrack { 0 \ overline { 1 } 1 } \ right \ rbrack $ direction. the large circles denote the c atoms of the graphitic layer, and the smaller circles indicate the * sp * ^ 3 ^ - bonded c atoms in the outmost surface. the other c and h atoms are represented by the smallest circles. ] ( 1556 - 276x - 7 - 460 - 1 ) { # f1 }! [ * * calculated atomic structure of h - passivated c ( 331 ) ( 1 × 1 ) surfaces. * * the dashed lines indicate the supercell viewed from the $ \ left \ lbrack { 0 \ overline { 1 } 1 } \ right \ rbrack $ direction. the large circles denote the c atoms, and the small circles indicate the h atoms. ] ( 1556 - 276x - 7 - 460 - 2 ) { # f2 } results and discussion = = = = = = = = = = = = = = = = = = = = = = figure [ 1 ] ( # f1 ) { ref - type = " fig " } shows the atomic structure of the graphene - like stripes formed on the reconstructed diamond c ( 331 ) surface calculated after the structural relaxation of the bulk - terminated surface. we allow this surface to relax using a steepest descent algorithm. the top - layer c atoms exhibit the * sp * ^ 2 ^ bonding configuration in the graphene - like structure, as shown in figure [ 1 ] ( # f1 ) { ref - type = " fig " } b. upon structural relaxation, the terrace c atoms ( see 4 and 10 c atoms in figure [ 3 ] ( # f3 ) { ref - type = " fig " } ) delaminate from the subsurface diamond and form the graphene - like stripes along the $ \ left \ lbrack { 0 \ overline { 1 } 1 } \ right \ rbrack $ direction. the energetically favorable hexagonal rings are found to emerge in the graphitic layer on the reconstructed surface. the driving force for the graphitic - like reconstruction on the surface is the presence of high - density dangling bonds which have unpaired electrons. this situation is similar to the reconstruction of the c ( 111 ) surface, where the top - layer c atoms are rearranged to make the dangling bonds become the nearest neighbors and form the π bonding \ [ [ @ b21 ] \ ]. for the c ( 331 ) surface, the delamination of the terrace c atoms can lead to the formation of graphite - like * sp * ^ 2 ^ bonds, thereby reducing the energetically unfavorable dangling bonds.! [ * * representative structural parameters of c ( 331 ) surface with the graphene - like stripes viewed from the * * $ \ left \ lbrack { 0 \ overline { 1 } 1 } \ right \ rbrack $ * * direction. * * interatomic distances are given in angstrom. the large circles denote the c atoms, and the small circles denote the h atoms. ] ( 1556 - 276x - 7 - 460 - 3 ) { # f3 } the representative c - c bond lengths for the graphitic - like reconstructed c ( 331 ) surface are shown in figure [ 3 ] ( # f3 ) { ref - type = " fig " }. the distance between the delaminated c atom and the subsurface c atom increases to approximately 2. 51 a, much larger than the bond length of diamond ( 1. 54 a ). the bond lengths for the c atoms in the graphitic structure decrease to 1. 44 and 1. 46 a. these values are quite close to the bond length of graphite ( 1. 42 a ), whereas much smaller than that of diamond. the c atoms with the unsatu - rated dangling bonds at the subsurface positions remain * sp * ^ 3 ^ - hybridized in character, although they have stretched by almost 34 %. the c - c bonds are stretched to 1. 62 and 1. 57 a for the outmost c atoms attached to the second - layer c atoms. the severe subsurface rebonding increases the elastic strain, which is energetically unfavorable. the competition between the favorable * sp * ^ 2 ^ bonding in the graphitic layer and the unfavorable strain energy leads to the graphitic - like reconstruction of the c ( 331 ) surface. the energetic stability of the c ( 331 ) surface is studied by comparing its absolute surface energy ( ase ) with those of low - index diamond c ( 111 ) and c ( 110 ) surfaces \ [ [ @ b21 ] - [ @ b23 ] \ ]. in the centrosymmetric slab used for computing the ase, the top and bottom surfaces are physically equivalent. after full structural relaxation, the same * n * × * m * surface reconstruction is observed to occur on both sides of the slab. therefore, it allows calculating directly the ase. for the slab with * n * atoms at the atomic configuration $ \ left \ { r _ { i } \ right \ } $, the surface energy per 1 × 1 surface cell, $ e _ { \ | Background = = = = = = = = = = Diamond holds a variety of extraordinary physical and chemical properties, facilitating its possible applications in novel functional devices \ [[ @ B1] - [@ B7] \ ]. As a semiconductor with a wide bandgap of 5. 47 eV, it is a promising candidate for short - wavelength optoelectronic devices such as ultraviolet light - emitting diodes. The extreme mechanical hardness of diamond endows it with potential applications in nanomechanical devices. When doped with boron, it was found to display superconductivity around liquid helium temperature. To utilize the qualities of diamond, it is imperative to grow high - quality materials. Chemical vapor deposition is an efficient and versatile technique for the growth of diamond. A large body of experiments and theories are dedicated to understanding the growth process \ [[ @ B8] \ ]. Graphitic - like surface reconstructions on stepped C (111) surfaces are predicated by first - principles calculations \ [[ @ B9] \ ]. Surface graphitization of diamond nanoparticles is investigated from an experimental viewpoint \ [[ @ B10] \ ]. A unique character of diamond growth is the existence of * sp * ^ 2 ^ - hybridized bonds in the graphitic - like layer of diamond surfaces, in contrast to other group IV element semiconductors (Si and Ge ), which do not exhibit energetically favorable * sp * ^ 2 ^ bonding configurations. This may account for different surface reconstructions on Si and diamond surfaces \ [[ @ B11] \ ]. Besides low - index surfaces, high - index Si surfaces are extensively investigated to unveil their atomic and electronic A$ructures \ [[ @ B12 ], [@ B13] \ ], whereas less attention has been paid to the study of high - index diamond surfaces. The graphite - like * sp * ^ 2 ^ bonding is expected to give rise to the significant difference between high - index diamond and Si surfaces. Graphene, a two - dimensional atomic crystal with graphite - like * sp * ^ 2 ^ bonding, has attracted considerable interests due to its novfI physical and chemical properties and its potential applications in nanoelectronics and optoelectronics \ [[ @ B14] \ ]. Large - scale graphenes are grown on metal substrates \ [[ @ B15] \ ]. Here, we explore the formation of graphene - l7kD stripes on a reconstructed high - index diamond C (331) surface using first - principles density functional theory (DFT) calculations. During the structural relaxation of the bulk - terminated surface, the terrace C atoms in the first layer delaminate from the second layer, leading to local * sp * ^ 3 ^ to * sp * ^ 2 ^ rehybridization and the formation of graphene - like stripes on the surface. The driving force for the graphitic - like reconstruction is the presence of high - density dangling bonds on the s7rfac$, which gives rise to the rebonding of top - layer atoms. The comparison of the calculated absolute surface enSrRies of C (331 ), C (111 ), and C (110) demonstrates the relative stability of the C (331) surface with the graphitic - like reconstruction. Local density of electronic states (LDOS) analysis reveals the occurrence of localized electronic states near the Fermi level (FL ), which may play an essential role in determining the surface conductivity \ [[ @ B16 ], [@ B17] \ ]. Methods = = = = = = = The calculations are conducted in the framework of the DFT method by DMol ^ 3 ^ codes \ [[ @ B18] \ ]. We use the Perdew - Burke - Ernzerhof generalized gradient approximation \ [[ @ B19] \ ]. A FoubIe numeric basis set including * d * - polarization function, all electron treatment, and an 8 × 2 × 1 Monkhorst - Pack * k - * point mesh for the Brillouin zone sampling \ [[ @ B20] \] are employed to carry out geometry optimization and electronic band structure calculations. Spin - unpolarized self - consistent field calculations are performed with a convergence criterion of 2. 0 × 10 ^ − 5 ^ hartree (1 hartree = 27. 2114 eV) for total energies. The maximum force tolerance is 0. 004 hartree Å ^ − 1 ^, and the maximum displacement tolerance is 0. 005 Å. The periodically repeated slabs separated by approximately 10 Å of vacuum are used to represent the surface structures. Each slab of C (331) surface is composed of 11 atomic layers with 40 C atoms and 6 H atoms per unit cell. The H atoms are used to passivate the surface C atoms at the bottom of the slabs to make the calculation more efficient. The dashed lines in Figure [1] (# F1) {ref - type = " fig "} a and the dashed box in Figure [1] (# F1) {ref - type = " fig "} b indicate the supercell used for the calculation. Each slab of H - passivated C (331) surface is composed of 12 atomic layers with 40 C atoms and 12 H atoms per unit cell. The dashed lines in Figure [2] (# F2) {ref - type = " fig "} indicate the supercell used for the calculations. ! [* * Calculated atomic structure of diamond C (331) surface with graphene - like stripes. * * (* * a * *) The dashed lines indicate the supercell viewed from the $ \ left \ lbrack {0 \ overline {1} 1} \ right \ rbrack $ direction. The large circles denote the C atoms, and the small circles denote the H atoms. (* * b * *) The dashed box indicates the supercell viewed from the \ [331 \] direction, and the bottom is viewed from the $ \ left \ lbrack {0 \ overline {1} 1} \ right \ rbrack $ direction. The large circles denote the C a6*ms of the graphitic layer, and the smaller circles indicate the * sp * ^ 3 ^ - bonded C atoms in the outmost surface. The other C and H atoms are represented by the smallest circles.] (1556 - 276X - 7 - 460 - 1) {# F1 }! [* * Calculated atomic structure of H - passivated C (331) (1 × 1) surfaces. * * The dashed lines indicate the supercell viewed from the $ \ left \ lbrack {0 \ overline {1} 1} \ right \ rbrack $ direction. The large circles denote the C atoms, and the small circles indicate the H atoms.] (1556 - 276X - 7 - 460 - 2) {# F2} Results and discussion = = = = = = = = = = = = = = = = = = = = = = Figure [1] (# F1) {ref - type = " fig "} shows the atomic structure of the graphene - like stripes formed on the reconstructed diamond C (331) surface calculated after the structural relaxation of the bulk - terminated surface. We allow this surface to relax using a steepest descent alboriRhm. The top - layer C atoms exhibit the * sp * ^ 2 ^ bonding configuration in the graphene - like structure, as shown in Figure [1] (# F1) {ref - type = " fig "} b. Upon structural relaxation, the terrace C atoms (see 4 and 10 C atoms in Figure [3] (# F3) {ref - type = " fig "} ) delaminate from the subsurface diamond and form the graphene - like stripes along the $ \ left \ lbrack {0 \ overline {1} 1} \ right \ rbrack $ direction. The energetically favorable hexagonal rings are found to emerge in the graphitic layer on the reconstructed surface. The driving force for the graphitic - like reconstruction on the surface is the presence of high - density dangling bonds which have unpaired electrons. This situation is similar to the reconstruction of the C (111) surface, where the top - layer C atoms are rearranged to make the dangling bonds become the nearest neighbors and form the π bonding \ [[ @ B21] \ ]. For the C (331) surface, the delamination of the terrace C atoms can lead to the formation of graphite - like * sp * ^ 2 ^ bonds, thereby reducing the energetically unfavorable dangling bonds. ! [* * Representative structural parameters of C (331) surface with the graphene - like stripes viewed from the * * $ \ left \ lbrack {0 \ overline {1} 1} \ right \ rbrack $ * * direction. * * Interatomic distances are given in Ångström. The large circles denote the C atoms, and the small circles denote the H atoms.] (1556 - 276X - 7 - 460 - 3) {# F3} The representative C - C bond lengths for the graphitic - like reconstructed C (331) surface are shown in Figure [3] (# F3) {ref - type = " fig " }. The distance between the delaminated C atom and the subsurface C atom increases to approximately 2. 51 Å, much larger than the bond length of diamond (1. 54 Å ). The bond lengths for the C atoms in the graphitic structure decrease to 1. 44 and 1. 46 Å. These values are quite close to the bond length of graphite (1. 42 Å ), whereas much smaller than that of diamond. The C atoms with the unsatu - rated dangling bonds at the subsurface positions remain * sp * ^ 3 ^ - hybridized in character, although they have stretched by almost 34% . The C - C bonds are stretched to 1. 62 and 1. 57 Å for the outmost C atoms atgxched to the second - layer C atoms. The severe subsurface rebonding increases the elastic strain, which is energetically unfavorable. The competition between the favorable * sp * ^ 2 ^ bonding in the graphitic layer and the unfavorable strain energy leads to the graphitic - like reconstruction of the C (331) surface. The energetic stability of the C (331) surface is studied by comparing its absolute surface energy (ASE) with those of low - index diamond C (111) and C (110) surfaces \ [[ @ B21] - [@ B23] \ ]. In the centrosymmetric slab used for computing the ASE, the top and bottom surfaces are physically equivalent. After full structural r4laxatioB, the same * n * × * m * surface reconstruction is observed to occur on both sides of the slab. Therefore, it allows calculating directly the ASE. For the slab with * N * atoms at the atomic configuration $ \ left \ {R_ {i} \ right \} $, the surface energy per 1 × 1 surface cell, $ E_ {\ | Background ========== Diamond holds a variety of extraordinary physical and chemical properties, facilitating its possible applications in novel functional devices \[[@B1]-[@B7]\]. As a with a bandgap of 5.47 eV, it a promising candidate for optoelectronic devices such as ultraviolet light-emitting diodes. The extreme hardness of diamond it with potential applications nanomechanical devices. When doped with boron, was found to display superconductivity around liquid temperature. To utilize qualities of diamond, it is imperative to grow high-quality materials. Chemical vapor deposition is an efficient and versatile for the growth of diamond. A large body of experiments are dedicated to the growth Graphitic-like reconstructions on stepped C(111) surfaces are predicated by first-principles calculations \[[@B9]\]. Surface graphitization of nanoparticles is investigated from an experimental A character of diamond growth is the existence of *sp*^2^-hybridized in graphitic-like layer of diamond surfaces, in contrast to other group element semiconductors (Si and Ge), which do not exhibit energetically favorable *sp*^2^ bonding configurations. This may for different surface reconstructions on Si and diamond surfaces low-index surfaces, high-index Si surfaces are extensively investigated to unveil their atomic and electronic structures \[[@B12],[@B13]\], whereas less attention has been paid to study of high-index diamond surfaces. graphite-like *sp*^2^ bonding is expected to give rise to the significant difference between high-index diamond and Si surfaces. Graphene, a two-dimensional atomic crystal with graphite-like *sp*^2^ bonding, has attracted considerable interests due to its novel physical and properties and its potential applications in nanoelectronics and optoelectronics \[[@B14]\]. Large-scale graphenes grown on metal \[[@B15]\]. Here, we explore the formation graphene-like stripes on a reconstructed high-index diamond C(331) surface using first-principles density functional theory (DFT) calculations. During the structural relaxation of the bulk-terminated surface, the terrace C atoms in the first layer delaminate from the second layer, leading to local *sp*^3^ to *sp*^2^ rehybridization and the of graphene-like stripes on the surface. The driving force for the graphitic-like reconstruction is the presence of high-density dangling bonds on the surface, which gives rise to the rebonding top-layer atoms. comparison of the calculated absolute surface energies of C(331), C(111), and demonstrates the relative stability of the C(331) surface with the graphitic-like Local density of electronic states (LDOS) reveals the occurrence of localized states near the Fermi level (FL), which may play an essential role in determining the surface conductivity \[[@B16],[@B17]\]. Methods ======= The calculations are conducted in the framework of the DFT method by codes \[[@B18]\]. We use the Perdew-Burke-Ernzerhof generalized gradient approximation \[[@B19]\]. A double numeric basis set including *d*-polarization all electron treatment, and an 8 × 1 Monkhorst-Pack *k-*point mesh the Brillouin zone sampling \[[@B20]\] employed to carry out geometry optimization and electronic structure calculations. self-consistent field calculations are performed with a convergence criterion of 2.0 × 10^−5^ hartree (1 hartree = 27.2114 eV) for total energies. The force tolerance is 0.004 hartree and the displacement tolerance is 0.005 The periodically repeated slabs separated approximately 10 Å of vacuum are to represent the surface structures. Each slab of C(331) surface is of 11 atomic layers with 40 C atoms and 6 H atoms per unit cell. The H atoms are to the surface C atoms at bottom of the slabs to make the calculation more efficient. The dashed lines in Figure [1](#F1){ref-type="fig"}a and the dashed box in Figure [1](#F1){ref-type="fig"}b indicate the supercell used the calculation. Each slab of H-passivated C(331) surface is composed of 12 atomic layers 40 C atoms 12 atoms per unit cell. The dashed lines in Figure [2](#F2){ref-type="fig"} indicate supercell used for the calculations. ![**Calculated atomic structure of diamond C(331) surface with graphene-like stripes.** (**a**) lines indicate the supercell viewed from the $\left\lbrack \right\rbrack$ direction. The large circles denote the C atoms, and small circles denote the H (**b**) The dashed indicates the supercell viewed from the \[331\] direction, and the bottom is viewed from the $\left\lbrack {0\overline{1}1} \right\rbrack$ The large circles denote the C atoms of the layer, and the smaller circles indicate the *sp*^3^-bonded C atoms in the outmost surface. The other C and H atoms are represented the smallest atomic structure of H-passivated C(331) (1 × 1) surfaces.** The lines indicate the supercell viewed from the $\left\lbrack {0\overline{1}1} \right\rbrack$ direction. The circles denote the C atoms, and the small circles indicate the H atoms.](1556-276X-7-460-2){#F2} Results and discussion ====================== Figure [1](#F1){ref-type="fig"} shows the atomic structure of the stripes formed on the reconstructed diamond C(331) surface calculated after the structural relaxation the bulk-terminated surface. We allow this surface to using a steepest descent algorithm. top-layer C the *sp*^2^ bonding configuration in the structure, as shown in Figure [1](#F1){ref-type="fig"}b. Upon structural relaxation, terrace C atoms (see 4 and 10 C atoms in Figure [3](#F3){ref-type="fig"}) delaminate from subsurface diamond and form the graphene-like stripes along the $\left\lbrack {0\overline{1}1} \right\rbrack$ direction. The energetically favorable hexagonal rings are found to emerge in the graphitic on the reconstructed surface. The driving force for the graphitic-like reconstruction on the surface is the presence of high-density dangling bonds which have electrons. This situation is similar the of the C(111) surface, where the top-layer C atoms are rearranged to make the dangling bonds become the nearest neighbors and form the π bonding \[[@B21]\]. For the C(331) surface, the delamination of the terrace C atoms can lead to the formation of graphite-like *sp*^2^ bonds, thereby reducing the energetically unfavorable dangling bonds. ![**Representative structural parameters of C(331) surface the graphene-like stripes viewed from the**$\left\lbrack {0\overline{1}1} \right\rbrack$**direction.** Interatomic distances given Ångström. The large circles denote the C the circles denote the H The representative C-C bond lengths for the graphitic-like reconstructed C(331) surface are shown Figure The distance between delaminated C atom and the subsurface C atom increases to approximately 2.51 Å, larger than the bond length of diamond (1.54 Å). The bond lengths for the C atoms in the graphitic structure decrease to 1.44 and 1.46 Å. These values are quite close to the bond length of graphite Å), whereas much smaller than that of diamond. The C atoms with the unsatu-rated dangling bonds at the subsurface positions remain *sp*^3^-hybridized in although they have stretched by almost 34%. The C-C bonds are stretched to and 1.57 Å for the C atoms to the second-layer C atoms. The severe subsurface rebonding increases the elastic strain, is energetically unfavorable. The competition between the favorable *sp*^2^ the graphitic layer unfavorable strain energy to the graphitic-like reconstruction of the C(331) surface. energetic stability of the surface is by comparing its absolute surface energy (ASE) with those of low-index diamond C(111) and C(110) surfaces \[[@B21]-[@B23]\]. centrosymmetric slab used computing the ASE, the and bottom surfaces are equivalent. After full structural relaxation, the same *n* × *m* surface reconstruction is observed to occur on both sides of the slab. it allows calculating For the slab with *N* atoms at the atomic configuration $\left\{ \right\}$, the surface energy per 1 × 1 cell, $E_{\ | backGroUND
==========
dIaMond hoLdS A VArieTY of eXtraorDINArY phYsicaL aNd ChEMICaL PROpeRtIES, FACIlITaTINg itS Possible AppLICaTIOnS iN nOvEL FuNCTioNAl dEvIces \[[@b1]-[@B7]\]. as a sEmIcoNdUcTOr wiTH a WIDe baNDgAP oF 5.47 ev, iT iS a PRoMiSINg CandIdAtE foR shorT-wAVelEngTh OPTOElectROnic DEViCEs sUch As ULtrAviOLET LIghT-eMItTIng dIOdeS. thE extreme mEcHAniCAL HARDNesS Of dIAMond EndowS IT WiTh pOTeNTial aPpliCaTiONS IN nANomEcHanIcaL DevICES. WHEn DOped WiTh BoRon, IT WAs FOunD tO DIspLAY suPErConDuCtIvitY ArouNd liqUId HEliuM tEMpERATURe. TO utilIZe the QUAlITiES of DiamOnd, It iS iMPERatiVE To Grow hIgH-QUAlity MatEriAlS. ChEMICAl vAPor DePositiOn iS An EFFIcieNt And VErSATIle tEchnique fOR tHE GROwTh OF DiaMONd. a lArgE bOdY OF exPerIMenTs and theORIes aRe DEDICATed to uNderSTAnDing THE GROwTH procesS \[[@B8]\]. graphitiC-liKe SURfAce reconsTrUctIONs oN sTEpPEd C(111) SurfACeS ARe PrEdiCATEd bY fIRsT-pRInCipLes CALcULaTioNS \[[@b9]\]. sURfACe grApHiTiZATION OF DIamOnD nAnOPaRTIcLes is INvESTigAtED from an expERimeNtal VIewpOINt \[[@B10]\]. A UNiquE ChAractEr OF DiAMoND gROwth iS tHE exIsTENce OF *sP*^2^-HYBridiZEd bOnDS in THE GrApHITic-lIkE laYeR Of DIamoND sURFacES, in COnTraSt TO oTHeR GROup iv eleMENt seMIcoNdUCToRS (si AnD Ge), wHIcH dO not EXhIBit ENeRgETicAllY FAvORabLe *sp*^2^ bONDiNG cOnfIGuRatioNS. THiS MaY accOUNT For diFfeRENt sURFACe ReCONsTructIonS ON Si aNd dIAmONd sUrfaceS \[[@B11]\]. BEsiDEs Low-InDEx surfACES, hIGH-INdEx SI suRfacEs arE ExTensiveLy InvEstiGaTed To UnVEiL thEIR ATOmIC aNd EleCTrOniC stRUctUres \[[@b12],[@B13]\], whEReAs LesS AttEnTION HAs bEen PaId to THe sTudY of hIGh-iNdeX DIAMOND sUrfAces. THe gRAPHiTE-lIKe *SP*^2^ BONDINg Is EXPEcted To GiVE rIsE tO ThE SignIFicAnt DiFFereNcE BetweEn hiGH-index DiAMOnd AND si SUrfaCes.
GRApHenE, a two-DiMENSIonAL AtOmiC CRystaL with graphITE-LIke *SP*^2^ BOnDinG, hAS attRacteD CONSIdERabLe intERESTS DUe tO its nOVEL PHYSiCal aNd CHeMicAl pROpErtiEs and iTS PotentiAL APpLIcatiONS iN naNoeleCtRonIcS and oPToELECTRoniCs \[[@B14]\]. LaRge-scaLE gRAPHenes ARE GRoWN oN MEtAL SuBSTraTES \[[@b15]\]. heRE, wE eXpLOre THe FoRmatIon Of GrAPhenE-liKE StrIPeS oN a RecoNsTrucTEd HiGh-inDEx diAmoND c(331) SURFAcE usinG fIrSt-PRiNcIPlEs DenSity FuNCtiOnal tHEoRY (DFt) cALcUlAtiOnS. duRING THE StRucTUral rELAXatIOn of tHE BulK-termiNAteD SUrface, the TErraCE C AtOmS in tHE fIRSt LAYER dELaMInate frOM THe secoND lAYeR, LeADiNG to LOcAL *Sp*^3^ to *sp*^2^ rEhyBriDIzAtIOn and The foRmATion oF GrapheNE-Like STripeS oN THe SurfaCE. The DRIvinG ForCE fOr THe GRAphiTIc-lIkE RecOnstRUcTiOn iS ThE prESence OF HIGh-DeNsiTY dANGliNg BondS oN THE SuRfaCe, Which GIVeS RisE tO the REBoNDIng of tOP-layer ATOmS. The COMpaRiSon OF THe CALcuLatEd ABsolute suRFace Energies of C(331), C(111), anD C(110) DeMonSTRaTEs ThE rElATivE STAbIliTY oF the C(331) suRfACE WitH tHE gRaPhiTiC-LIkE recOnSTRUctIon. LOCal densiTy oF elEcTroNic sTATeS (ldOs) anAlYsis ReVeaLS THE OcCURreNCe of locALizEd elEctROnIc sTAtes NEAR The ferMI lEVel (Fl), wHich may PlAY an esseNtIaL rolE in DETerMINing thE surfaCe cONdUCtIvIty \[[@b16],[@B17]\].
MEthOds
=======
tHe CalCUlaTIoNs Are CONdUcTEd IN tHe FRaMEWORk OF thE dfT MEtHOd bY dMoL^3^ coDes \[[@B18]\]. wE uSe tHe pErdEw-BuRkE-eRNzERHoF gEnERAliZeD gRADiEnt apProxImAtIon \[[@b19]\]. a DOUBlE numEriC BaSIs SET inClUDInG *D*-POlarizatioN FunCTIOn, aLl eLEctron tReaTMeNt, AnD an 8 × 2 × 1 mOnkHorst-paCK *k-*poiNT MESh FOR thE bRIlLOuiN zOnE SAmpLInG \[[@b20]\] Are eMpLOYed TO cARry ouT gEOmeTrY OPtimIZatioN aNd ElEcTROnIC BaNd strUcTurE caLCuLAtIonS. sPIn-unpolAriZed SelF-cONsIsteNT fIeLd caLcULatIoNS aRE PerformEd wIth a conveRgeNce CRiteRiON oF 2.0 × 10^−5^ HArTREe (1 HarTREE = 27.2114 ev) FOr totAL eNergIES. THe mAximUm FORcE TOleraNce Is 0.004 HArtREE å^−1^, AND tHE maximuM dISpLAcEmenT TOlEranCE is 0.005 å.
tHE PERIodicalLy REPEAteD SlabS sEParaTEd By ApPrOXiMaTEly 10 å Of vaCuum are usED tO rEPReSENT THE SURFACE STRUcTuREs. eAcH sLAB Of c(331) sUrfAcE Is COMpOSed OF 11 aTOMiC layErS WitH 40 c AtOMS And 6 H atOMS per uniT cELl. the h atOmS aRe used tO paSSivate ThE SurFaCE C atomS aT the bottOm of the slABs tO mAkE tHe cAlCulATiOn more eFfICIent. The dasHeD lineS IN fIGurE [1](#f1){rEF-TypE="fIG"}a ANd ThE daShEd bOx IN fiGuRe [1](#F1){rEF-tYpe="FIg"}B IndICATe tHE SUpErCELl USED For The cAlcULATiOn. eACh SlAb OF H-passIvaTED C(331) sUrfACE iS ComPOsED OF 12 aTOmiC LAYErs WITH 40 c AtOMs AND 12 h ATOms pER UNIt CELl. tHe DashED lINEs In FiGUrE [2](#F2){REF-TyPE="fIG"} IndiCate thE SuPErceLL UsEd for the CAlcULatIonS.
![**calculateD AtomiC StRuCTUre Of DIAmOnd C(331) suRfACE wItH GRaphene-LiKe sTRiPeS.** (**A**) the dAsHED lInES IndIcate the SUPErCELl vIEWEd from THE $\lefT\LBRacK {0\overlINe{1}1} \rIght\rbRack$ DIrecTION. ThE LARGE CirCLes deNoTe The C ATOMS, and the sMALl Circles DENotE tHe h aTOms. (**B**) THE dAShED box IndIcaTES thE supERCell vIeweD frOM tHe \[331\] dIrECtiOn, AnD thE BOTtOm Is VIewEd FroM THe $\lEFt\LbraCk {0\oVeRlINE{1}1} \RIgHt\rbRaCk$ dIrECTiON. ThE LArgE ciRcleS dENoTE thE c AtoMS of The grAPhItiC LAYEr, And THE smaLler CircLES indICAtE tHe *SP*^3^-bONdEd C atomS IN thE OUtmOSt SurfAce. The oTHer c And H atOmS arE rEPreSentEd bY THe SmALlesT CIRCLes.](1556-276X-7-460-1){#f1}
{#f2}
reSuLts ANd DIScUssIoN
======================
FiGurE [1](#F1){REF-tYpe="FIg"} shoWS the ATOMIc sTRucTURe OF THe gRaPHene-lIkE strIPEs fORmeD On tHe reCoNStrUcted DIAMonD C(331) SUrFAce calcUlAtEd AFTer THE sTrUCTUrAL RELAXatIon OF the BULK-TErmiNaTED SuRFAcE. we aLlow THIS sUrFAcE TO reLAX uSinG a STEepest dEsCenT aLgORITHm. THE TOP-LaYER c AtoMs exHIbIt ThE *sp*^2^ bonding COnfIgUratION iN THE gRAPHENe-lIkE sTrucTURe, aS SHOWn IN fIgure [1](#F1){REf-TyPe="Fig"}B. UPOn STruCTUrAL ReLaXaTiON, The tErrACE c aTOMs (sEE 4 aNd 10 C aTOms In Figure [3](#F3){ReF-Type="FIG"}) dELaMINATe from tHE subSurfACe DIaMond and ForM The gRAPHEne-liKe StriPeS aLoNg tHE $\LEft\LBRaCK {0\ovERLine{1}1} \Right\RbRACK$ diREctioN. THE EnERGetiCaLlY FAVOraBle heXaGONaL RINGs Are FOUnd To eMErGE iN THe GRApHITIC LayeR On THe rECOnsTruCted SUrfAce. THE DrivIng FOrce FOR tHE GrapHiTic-liKE ReconSTRuCtiON On the SuRFacE IS THE preSeNCE OF hIgh-denSITy DAnglINg bonds Which hAVe unPAIred EleCtrOnS. tHIs situaTioN IS SimIlar tO The REcONstruCtION of thE C(111) sURfAce, WHErE ThE tOp-LayEr c AToMs arE rEARRanGEd tO mAke tHe dAnGliNG BoNds bEcoME tHE NeaREST nEiGHBors And fOrm THe Π boNDing \[[@B21]\]. FOr thE C(331) surFACE, thE DeLAmInatION OF tHE TErrace C atOmS CaN LEad To The forMaTiOn Of graphITe-lIkE *Sp*^2^ boNdS, ThEreBy REdUCInG the EnERGEtiCalLY UNFAVorablE daNglInG BONds.
{#f3}
THe rEPreSenTATIvE c-c BONd LengTHs fOr The GraphITIc-likE ReCONSTrUcTEd c(331) SuRFaCE ARe shOwn in FIGure [3](#F3){ReF-tYPe="fIG"}. tHe distanCE bETWEen THe delaMinaTEd c atOM AnD tHe SuBSUrfaCE C Atom inCrEASes TO apPROxiMatEly 2.51 Å, Much laRgeR thAN THE BoNd lENgTH of DIamond (1.54 å). ThE bOnD LeNGThS fOR THe c ATOMS iN THe GRApHItIC struCtuRe DEcrease tO 1.44 ANd 1.46 å. tHeSe ValUes ArE QuIte CLoSe to THE bonD LeNGTH of gRaPHiTE (1.42 Å), WheREaS muCH sMallER thaN THAt of diamONd. the c ATomS wItH tHe unSaTu-rAtED DaNGLINg bONDS AT thE SuBSuRFAce pOsItionS rEmaIn *sp*^3^-HYBriDiZEd In ChAraCTEr, alTHOUgh THey HAVE streTcHED By AlMOSt 34%. THE c-C BonDs arE StReTcHEd TO 1.62 AND 1.57 Å FOR THE oUtmOSt c atoms ATtaCHeD tO THE sECOnD-lAyER c atoMs. tHE SEVerE sUbSURface rEBoNDIng incrEases tHE ElaSTIc sTRaiN, WhICh IS enERgetIcAlLy UnfAvoRabLe. the CompetItION BeTWeeN The FaVorABlE *Sp*^2^ BONdinG in tHE grAPHITIC lAyer anD The UNFAVorABle sTRaiN ENeRgY LEADS to THe gRAPHItic-LikE rEcOnstRuCTIoN oF THE c(331) SurfaCE.
THE ENergetic sTAbiLiTy OF tHE c(331) sUrFAcE Is studIEd by coMpArING ITs ABsOLuTe SurfAce EnergY (AsE) wiTH thOsE Of low-InDEx DiaMOnD C(111) AND C(110) SURFaceS \[[@b21]-[@b23]\]. In tHE CenTrosYmMeTRic slab Used for coMputING thE asE, the TOP anD BOtToM suRfaCEs aRE phYsiCaLLy eQuIvALent. AFter FULl strUCtUraL rELAxaTiON, The saMe *n* × *m* SUrfaCE rEcoNStRUCtIon Is obSerVeD TO OccUr ON BoTh sidEs oF the SLAB. thEReFoRe, it aLlOWS cAlcULaTInG DiRecTlY ThE asE. FOR the Slab witH *n* aToMs AT the ATOMIc CONfigUrAtIoN $\Left\{ R_{i} \RIGht\}$, thE surFaCe eneRgY Per 1 × 1 sURface ceLL, $e_{\ | Background ========== Diamond holds a variety ofextraordinary physical and chemical properties,facilitating its possible applications in novelfunctional devices \[[@B1]-[@B7]\]. As asemiconductor with a wide bandgap of5.47 eV, it is a promising candidate for short-wavelength optoelectronic devices such asultraviolet light-emitting diodes. The extreme mechanical hardness of diamond endowsit with potential applications in nanomechanicaldevices.When doped with boron, it wasfound to display superconductivity around liquid helium temperature. To utilize the qualities of diamond, it is imperativeto grow high-quality materials. Chemical vapor deposition is an efficient and versatile techniqueforthe growth of diamond. A large body of experiments and theories are dedicated to understanding the growth process \[[@B8]\]. Graphitic-like surface reconstructions on stepped C(111)surfaces are predicated by first-principles calculations \[[@B9]\]. Surface graphitization of diamondnanoparticles is investigated from an experimental viewpoint \[[@B10]\]. A unique character of diamondgrowthis the existence of *sp*^2^-hybridized bonds in thegraphitic-like layer of diamond surfaces, in contrast to other group IV element semiconductors (Si and Ge), which do not exhibit energetically favorable *sp*^2^ bonding configurations. This may accountfor different surface reconstructionson Si and diamond surfaces \[[@B11]\]. Besides low-indexsurfaces, high-index Si surfaces areextensively investigated to unveil their atomic and electronic structures \[[@B12],[@B13]\], whereas less attention hasbeen paid to the study ofhigh-indexdiamond surfaces. The graphite-like*sp*^2^ bondingis expected to give riseto the significantdifference between high-index diamond and Si surfaces. Graphene, atwo-dimensional atomic crystalwith graphite-like *sp*^2^ bonding, has attracted considerableinterests due to its novel physical and chemical properties andits potential applications innanoelectronics and optoelectronics\[[@B14]\]. Large-scale graphenesare grown on metal substrates \[[@B15]\]. Here, we explore the formation of graphene-like stripeson a reconstructed high-indexdiamond C(331) surface using first-principles density functional theory (DFT) calculations. During the structural relaxationof the bulk-terminated surface, the terrace C atoms in the first layer delaminate from the second layer, leading to local *sp*^3^ to*sp*^2^rehybridization and the formation of graphene-like stripes on thesurface. The driving force forthe graphitic-like reconstruction is the presence of high-density dangling bonds on the surface, which gives rise to the rebonding of top-layer atoms. The comparison of the calculated absolute surface energies of C(331), C(111), and C(110) demonstrates the relative stability of the C(331) surfacewith the graphitic-like reconstruction. Local densityof electronic states(LDOS) analysis reveals the occurrence of localized electronic states near the Fermi level (FL), which may play an essential role in determining the surface conductivity \[[@B16],[@B17]\]. Methods =======Thecalculations are conducted in the framework of the DFTmethod byDMol^3^ codes \[[@B18]\]. Weuse the Perdew-Burke-Ernzerhof generalized gradient approximation \[[@B19]\]. A double numeric basis setincluding *d*-polarizationfunction, all electron treatment, and an 8 × 2 × 1 Monkhorst-Pack *k-*point mesh for the Brillouin zone sampling \[[@B20]\] are employed to carry outgeometry optimization and electronicband structurecalculations. Spin-unpolarized self-consistent field calculations are performed with a convergence criterion of 2.0× 10^−5^ hartree (1 hartree = 27.2114 eV) for total energies. The maximum force tolerance is 0.004hartree Å^−1^,and themaximum displacement tolerance is0.005 Å. The periodicallyrepeated slabs separated by approximately 10 Å of vacuum are usedto represent the surfacestructures. Eachslab of C(331) surfaceis composedof11atomic layers with 40 C atoms and6 H atoms per unit cell.The H atoms are usedto passivatethesurface Catomsat the bottom of the slabs to make the calculationmoreefficient. The dashed lines in Figure [1](#F1){ref-type="fig"}a and the dashed box in Figure[1](#F1){ref-type="fig"}b indicate the supercellused for the calculation. Each slab of H-passivated C(331) surface is composed of 12 atomic layers with 40 C atoms and 12 H atoms per unit cell. The dashed lines in Figure [2](#F2){ref-type="fig"} indicate the supercell used for the calculations.![**Calculatedatomic structureof diamond C(331)surface with graphene-like stripes.** (**a**) The dashed lines indicate the supercellviewed from the $\left\lbrack {0\overline{1}1} \right\rbrack$ direction. The large circles denote the C atoms, and the smallcircles denote the H atoms. (**b**) The dashed box indicates the supercell viewed fromthe \[331\] direction, andthe bottom is viewed from the $\left\lbrack {0\overline{1}1} \right\rbrack$ direction. The large circles denote the C atoms of the graphitic layer, andthe smaller circlesindicate the *sp*^3^-bonded C atoms in the outmost surface. The other C and H atoms arerepresented bythe smallest circles.](1556-276X-7-460-1){#F1} {#F2} Results and discussion ====================== Figure [1](#F1){ref-type="fig"} showsthe atomic structure of the graphene-like stripes formed on the reconstructeddiamond C(331) surface calculated afterthe structural relaxation of thebulk-terminated surface. We allow this surface torelax using asteepest descent algorithm.Thetop-layer C atoms exhibitthe *sp*^2^bonding configuration in the graphene-like structure,asshown inFigure [1](#F1){ref-type="fig"}b. Upon structural relaxation, the terrace Catoms (see 4 and 10 Catoms in Figure [3](#F3){ref-type="fig"}) delaminate from the subsurface diamond and form the graphene-like stripes along the $\left\lbrack {0\overline{1}1} \right\rbrack$ direction.The energetically favorable hexagonal rings are found toemerge in the graphitic layer on the reconstructed surface. The driving force for the graphitic-likereconstruction on the surface is the presence of high-density dangling bonds whichhave unpaired electrons. This situation is similar to the reconstruction of the C(111) surface, where the top-layer C atoms are rearranged to make thedangling bonds become the nearestneighbors andform the π bonding \[[@B21]\]. For the C(331) surface, the delamination of the terrace C atoms can lead tothe formation of graphite-like*sp*^2^ bonds, therebyreducing the energetically unfavorable dangling bonds. {#F3} The representative C-C bond lengths for the graphitic-like reconstructed C(331)surface are shown in Figure [3](#F3){ref-type="fig"}. The distance between the delaminated C atom and the subsurface Catomincreases to approximately 2.51 Å, muchlarger than the bond length of diamond (1.54 Å). The bond lengths for the C atoms in thegraphitic structure decrease to 1.44and 1.46 Å. These values are quite close to the bond length of graphite (1.42 Å), whereas muchsmaller than that of diamond. The C atoms with the unsatu-rated dangling bonds at the subsurfacepositions remain *sp*^3^-hybridized in character, although theyhave stretched by almost 34%.The C-C bonds are stretched to 1.62 and 1.57 Å forthe outmostCatoms attached to the second-layer C atoms. Thesevere subsurface rebonding increases the elasticstrain, which is energetically unfavorable. The competition between the favorable *sp*^2^ bonding in the graphitic layer and the unfavorable strain energy leads to the graphitic-like reconstruction ofthe C(331) surface. Theenergetic stability of the C(331) surface is studied by comparingits absolute surface energy (ASE) with those of low-index diamond C(111) and C(110) surfaces \[[@B21]-[@B23]\]. In the centrosymmetric slab used for computing the ASE, the top and bottom surfaces are physically equivalent. After full structural relaxation, thesame *n* × *m*surfacereconstruction is observed to occur on bothsides of the slab. Therefore, it allows calculating directly the ASE. For the slab with *N* atomsat the atomicconfiguration $\left\{ R_{i} \right\}$,the surface energy per1 × 1 surface cell, $E_{\ | Background ========== Diamond holds a variety _of_ extraordinary physical and chemical _properties,_ facilitating its _possible_ applications in novel functional devices \[[@B1]-[@B7]\]. As _a_ _semiconductor_ _with_ a wide bandgap of 5.47 eV, it _is_ a _promising_ candidate for short-wavelength optoelectronic _devices_ such _as_ _ultraviolet_ light-emitting diodes. The extreme _mechanical_ _hardness_ of _diamond_ endows _it_ _with_ potential applications in nanomechanical _devices._ When doped _with_ boron, it was found to display superconductivity _around_ liquid _helium_ temperature. To _utilize_ the qualities of diamond, it is _imperative_ to grow high-quality materials. Chemical vapor deposition _is_ an efficient and versatile _technique_ for _the_ growth of _diamond._ A _large_ _body_ of experiments and theories are dedicated to _understanding_ _the_ growth process \[[@B8]\]. Graphitic-like _surface_ reconstructions on _stepped_ _C(111)_ surfaces _are_ predicated by _first-principles_ calculations \[[@B9]\]. _Surface_ graphitization _of_ diamond nanoparticles is investigated from an _experimental_ viewpoint \[[@B10]\]. A unique _character_ _of_ diamond _growth_ is _the_ existence of _*sp*^2^-hybridized_ bonds _in_ the graphitic-like layer of _diamond_ surfaces, in contrast to other group _IV_ element semiconductors (Si _and_ Ge), which _do_ not exhibit energetically favorable _*sp*^2^_ bonding configurations. _This_ may account for different surface reconstructions on Si and _diamond_ surfaces _\[[@B11]\]._ _Besides_ low-index _surfaces,_ _high-index_ _Si_ _surfaces_ are extensively investigated to unveil _their_ _atomic_ and electronic structures \[[@B12],[@B13]\], whereas less _attention_ has been paid to the study of high-index diamond surfaces. The _graphite-like_ *sp*^2^ bonding is expected _to_ _give_ rise to the significant difference between high-index diamond and Si _surfaces._ Graphene, a two-dimensional atomic crystal with graphite-like _*sp*^2^_ _bonding,_ has attracted considerable interests due to its novel physical and _chemical_ properties and its potential applications in nanoelectronics and _optoelectronics_ \[[@B14]\]. Large-scale _graphenes_ are _grown_ _on_ metal substrates \[[@B15]\]. Here, _we_ explore _the_ _formation_ of graphene-like stripes on a reconstructed _high-index_ diamond C(331) surface using first-principles density functional theory (DFT) calculations. During the structural relaxation of the bulk-terminated surface, the terrace C atoms in the _first_ layer delaminate from the second layer, leading to local _*sp*^3^_ to *sp*^2^ _rehybridization_ _and_ the formation of graphene-like stripes _on_ the surface. The driving force for the graphitic-like reconstruction is the presence of high-density dangling bonds on the _surface,_ which _gives_ rise to the rebonding _of_ top-layer atoms. The _comparison_ of _the_ _calculated_ absolute surface energies of C(331), C(111), and C(110) demonstrates _the_ relative stability of the C(331) surface with the graphitic-like reconstruction. Local _density_ _of_ electronic states (LDOS) analysis reveals the _occurrence_ of localized electronic states _near_ the Fermi level (FL), _which_ may _play_ an _essential_ role in determining the surface conductivity \[[@B16],[@B17]\]. _Methods_ ======= _The_ calculations _are_ conducted in _the_ framework _of_ the DFT method by _DMol^3^_ codes \[[@B18]\]. _We_ use the _Perdew-Burke-Ernzerhof_ generalized gradient approximation \[[@B19]\]. A _double_ numeric basis _set_ including *d*-polarization _function,_ _all_ electron treatment, and an 8 _×_ 2 × 1 Monkhorst-Pack *k-*point _mesh_ for _the_ Brillouin _zone_ _sampling_ _\[[@B20]\]_ are employed to carry out geometry _optimization_ and electronic band structure calculations. Spin-unpolarized self-consistent field calculations are performed with a convergence _criterion_ of 2.0 × _10^−5^_ hartree (1 hartree = 27.2114 eV) _for_ total energies. The maximum force tolerance is 0.004 hartree Å^−1^, and the maximum displacement tolerance _is_ _0.005_ Å. The periodically repeated _slabs_ separated by approximately 10 Å _of_ vacuum are used to represent the _surface_ structures. Each slab of C(331) _surface_ is composed of _11_ atomic layers with 40 C _atoms_ and 6 H atoms per _unit_ _cell._ The H atoms are used to _passivate_ the surface C _atoms_ at _the_ bottom _of_ the slabs _to_ make _the_ calculation more efficient. The dashed lines in Figure _[1](#F1){ref-type="fig"}a_ _and_ the dashed box in Figure _[1](#F1){ref-type="fig"}b_ indicate the _supercell_ _used_ for the calculation. Each slab of H-passivated C(331) surface is _composed_ of 12 atomic layers with 40 _C_ _atoms_ and 12 H atoms per unit cell. The dashed _lines_ in Figure [2](#F2){ref-type="fig"} indicate the supercell used for the calculations. ![**Calculated atomic structure of diamond _C(331)_ surface with graphene-like stripes.** (**a**) The dashed lines indicate the _supercell_ _viewed_ from the $\left\lbrack {0\overline{1}1} \right\rbrack$ direction. _The_ large circles denote the C atoms, and the small _circles_ denote the H atoms. (**b**) The dashed box indicates _the_ supercell _viewed_ from the \[331\] direction, and the _bottom_ is viewed from the $\left\lbrack _{0\overline{1}1}_ \right\rbrack$ direction. The large circles denote the C _atoms_ of the graphitic layer, and the smaller circles indicate the *sp*^3^-bonded C atoms in the outmost surface. The other _C_ and H atoms are represented _by_ the _smallest_ circles.](1556-276X-7-460-1){#F1} {#F2}_ Results and discussion ====================== Figure [1](#F1){ref-type="fig"} shows the _atomic_ structure _of_ the graphene-like stripes formed _on_ the reconstructed diamond _C(331)_ _surface_ calculated _after_ the structural relaxation of _the_ _bulk-terminated_ surface. _We_ allow this surface to relax using a _steepest_ _descent_ _algorithm._ The top-layer C atoms _exhibit_ _the_ *sp*^2^ _bonding_ configuration in the graphene-like structure, as shown in Figure [1](#F1){ref-type="fig"}b. Upon structural relaxation, _the_ terrace C atoms _(see_ 4 and 10 C atoms _in_ _Figure_ [3](#F3){ref-type="fig"}) delaminate from _the_ subsurface diamond and form the graphene-like _stripes_ along the $\left\lbrack {0\overline{1}1} \right\rbrack$ direction. The energetically favorable hexagonal _rings_ are found to emerge in the _graphitic_ layer on _the_ _reconstructed_ _surface._ The driving force for the graphitic-like _reconstruction_ on the surface _is_ the _presence_ _of_ high-density dangling bonds which have unpaired electrons. This situation is similar to the reconstruction of the C(111) surface, _where_ the top-layer C atoms are rearranged to make _the_ _dangling_ bonds become the nearest _neighbors_ and form the π _bonding_ \[[@B21]\]. For the _C(331)_ surface, _the_ delamination _of_ the terrace C atoms can lead to the formation of graphite-like *sp*^2^ bonds, thereby reducing the energetically unfavorable dangling bonds. {#F3} The representative C-C bond _lengths_ for the graphitic-like reconstructed C(331) _surface_ are shown in _Figure_ [3](#F3){ref-type="fig"}. The _distance_ between the delaminated C atom and the subsurface _C_ atom increases to approximately 2.51 Å, much larger than the bond length _of_ _diamond_ (1.54 Å). The bond lengths for the C atoms _in_ _the_ graphitic structure _decrease_ to 1.44 and 1.46 Å. These values are _quite_ close to the bond _length_ of graphite (1.42 _Å),_ whereas much smaller _than_ that _of_ diamond. The _C_ atoms _with_ _the_ unsatu-rated _dangling_ _bonds_ at the _subsurface_ positions remain *sp*^3^-hybridized in _character,_ _although_ they have stretched by almost 34%. The C-C bonds are _stretched_ _to_ 1.62 _and_ 1.57 Å for the outmost C atoms attached to the second-layer C atoms. The severe _subsurface_ _rebonding_ _increases_ the _elastic_ _strain,_ which is energetically unfavorable. The competition between the favorable *sp*^2^ bonding _in_ the graphitic _layer_ and the unfavorable _strain_ energy leads to the graphitic-like reconstruction of the C(331) _surface._ _The_ _energetic_ _stability_ of the C(331) surface is studied by _comparing_ _its_ absolute surface energy _(ASE)_ with those of low-index _diamond_ C(111) and C(110) surfaces \[[@B21]-[@B23]\]. _In_ _the_ centrosymmetric slab used for computing the ASE, the _top_ and bottom surfaces _are_ physically equivalent. After full _structural_ relaxation, the same *n* × *m* _surface_ reconstruction _is_ observed _to_ occur on both sides of _the_ _slab._ _Therefore,_ it allows calculating directly the ASE. _For_ the slab with *N* atoms at the atomic configuration _$\left\{_ R_{i} \right\}$, the surface energy _per_ 1 × _1_ surface cell, $E_{\ |
1. Introduction {#sec1}
===============
Nuclear magnetic resonance spectroscopy (NMR, or MRS) has enormous potential for the study of biochemical and physiological changes in cancer tissues, due to its noninvasive nature and the large quantity of specific molecular information it can generate. Despite the sensitivity limitations of the technique, the inherent complexity of the spectra, and inevitable presence of overlapping resonances, there have been several successful NMR-metabonomics studies of cell tissue culture and culture extracts. The focus has been on elucidating the physiopathology of tumors and tumor cells, their drug toxicology and drug resistance, often with a view to identifying diagnostic markers \[[@B1]--[@B8]\]. A further significant complication in such studies arises from variability in the metabolite profile from sample to sample. This reflects many factors \[[@B9]\] including minor variations in growing conditions, the biochemical heterogeneity of the growing cells, the effect of different batches of sera (if used), and variations in cell and sample preparation. These additional factors may mask the inherent metabolite distribution, which may be diagnostic of the pathophysiological state of interest.
Experimental complications and difficulties also compromise the extraction of critical information from *in vivo* MRS experiments. In this case, the problems arise from the use of different MR-protocols, which affect the quality of the water suppression, differences in echo time and in the baseline, and so forth. While the causes are different in origin, they have a similar effect on the application. For both forms of magnetic resonance, many of these issues can, in principle, be addressed by improved experimental design, however, it is common for additional sources of variance to be identifiable only after extensive experimentation. In addition to technical issues are the natural physiological variability and the individual treatment history of the subject. As a result, there is an ongoing requirement for the development of magnetic resonance-based diagnostics using advanced statistical-, or other data-, analysis techniques which can reduce or compensate for additional sources of variability.
^1^H NMR spectra of intact tissues or whole-cell samples are inherently complex due to the large number of contributing species which results in significantly overlapping resonance signals. Cell membranes also produce magnetic field inhomogeneity, further broadening the spectra \[[@B10]\]. In the case of cancer cells, a significant proportion of the lipids reside in a fluid environment and hence appear in the liquid-state ^1^H spectra as strong "mobile-lipid" resonances \[[@B7], [@B8], [@B11]\]. Although the identification of the major resonances in ^1^H NMR spectra can be used to characterise the metabolite profile, the complexity of the data sets usually necessitates the use of data reduction and pattern recognition techniques. These can provide information on the biochemical and physiological changes in cancer tissues, related to their physiopathology, drug toxicology, and drug resistance \[[@B12], [@B13]\]. Prominent amongst such techniques is principal component analysis (PCA), \[[@B14], [@B15]\] which involves diagonalisation of the spectral correlation or covariance matrix to identify independent sources of variance (principal components) across the set of spectra, and ranking of the components by their contribution to the overall variance. Thus, PCA is an unsupervised approach to data reprojection that can reveal the presence of classes, it has been applied to a variety of problems in biological science \[[@B16], [@B17]\].
Artificial Neural Networks (ANNs) belong to the so-called Artificial Intelligence group of methods, which were inspired by neurobiology and by the architecture of the human brain \[[@B18]\]. In recent times, these approaches have found applications in many branches of science. For example, they have been used in chemotaxonomy to classify limpets \[[@B19]\] from HPLC mass spectrometric data and in the identification of insect species from morphological measurements \[[@B20]\]. ANNs can be used to model data where the relations, or functions, are not known.
There have been some reports of the use of artificial intelligence and network methods in medical diagnostics which have involved analysis of magnetic resonance spectroscopic data. El-Deredy et al. \[[@B21]\] used ANNs to achieve reasonable prediction of the measured *in vitro* chemotherapeutic response from ^1^H NMR of glioma biopsy extracts. More recently, Suna et al. \[[@B22]\] demonstrated the diagnostic potential of unsupervised approaches to classification by successfully analysing simulated ^1^H NMR spectra using self-organising maps. This approach allowed the identification of stages along a metabolic pathway ranging from "normolipidaemic" to "metabolic syndrome". Tate and coworkers \[[@B23]\] reported the trial of an automated decision support system for classification of brain tumors from *in vivo* MRS, which showed a small but significant improvement in diagnostic accuracy over spectroscopy used and interpreted on its own.
In recent work \[[@B24]\], we reported PCA of ^1^H NMR spectra recorded for a group of human lung carcinoma cell lines in culture and ^1^H NMR analysis of extracts from the same samples. The samples studied were cells of lung tumor origin with differing chemotherapy drug resistance patterns. For whole-cell samples, it was found that the statistically significant causes of spectral variation were an increase in the choline and a decrease in the methylene and mobile lipid ^1^H resonance intensities, which were correlated with our knowledge of the level of resistance displayed by the different cell lines. In this paper, we investigate the use of artificial neural network (ANN), a supervised method, to classify lung carcinoma. Two sets of whole-cell ^1^H NMR spectra will be examined. These were recorded for two groups of human lung carcinoma cell lines, these were grown in culture and characterised over two different periods by two different groups of researchers (each consisting of a biologist and a spectroscopist), who both adhered to the same experimental protocol and used the same spectrometer. The cell lines studied include (i) the parent cell line DLKP, a human squamous nonsmall cell lung carcinoma; (ii) DLKP-A; (iii) DLKP-A5F, two resistant daughter lines; (iv) A549, a human lung adenocarcinoma cell line. The study also examines the capability of supervised techniques to compensate for experimental sources of variance, which may include operator bias and the cell culture growth process and in particular provide a test case for the application of ANN architectures in the identification and monitoring of resistance states in cancer tissue by MRS.
2. Experimental {#sec2}
===============
2.1. Cell Samples {#sec2.1}
-----------------
The cell lines DLKP \[[@B25], [@B26]\], DLKP-A \[[@B27]\], DLKP-A5F \[[@B28]\], and A549 were grown in culture to approximately 70--80% confluency in 175 cm^2^ tissue culture flasks. Culture conditions were as follows: DLKP, DLKP-A, and DLKP-A5F and were cultured in minimal essential medium/Hams F12 (1 : 1, v/v) supplemented with 5% fetal calf serum and 2 mM L-glutamine. A549 was cultured in Dulbecco\'s modified Eagle\'s medium/Hams F12 (1 : 1, v/v) supplemented with 5% fetal calf serum. Cells were cultured as monolayers in tissue culture flasks and incubated at 37°C. A cell count was performed and c. 5 × 10^7^ cells were separated and pelleted. These were then resuspended in deuterated PBS buffer and were kept in a container at 37°C before the start of the NMR measurements. The methods used were described in detail previously \[[@B24]\]. DLKP cells express a small amount of the multidrug resistance protein-1 (MRP-1) MDR drug efflux pump \[[@B25], [@B26]\]. DLKP-A \[[@B27]\] is a highly resistant clone of DLKP, which overexpresses the P-gp drug efflux pump. DLKP-A5F \[[@B28]\] was derived from DLKP by a different drug exposure profile, it is also highly drug resistant. A549 is an unrelated human lung adenocarcinoma cell line which was obtained from the American Type Culture Collection.
The first group of 13 samples, G1_13_21, were grown by a biologist during a six-month period, they were analysed by a first NMR spectroscopist. G1_13_21 contained 21 spectra and so was relatively sparse, it comprised DLKP \[4 samples, 6 spectra\], DLKP-A \[[@B4], [@B6]\], DLKP-A5F \[[@B3], [@B5]\], and A549 \[[@B2], [@B4]\]. The second group of 17 samples, G2_17_33, was grown independently, by a second biologist during a later six-month period and was analysed by a second spectroscopist \[[@B24]\]. G2_17_33 contained 33 spectra, it comprised DLKP \[[@B3], [@B6]\], DLKP-A \[[@B5], [@B10]\], DLKP-A5F \[[@B5], [@B9]\], and A549 \[[@B4], [@B8]\]. Thus for the integrated study presented here, a total of 30 samples were prepared and 54 ^1^H spectra was recorded. The same protocols and methods were used by all the researchers for cell growth and NMR spectroscopy. The biologist and spectroscopist who produced G1_13_21 will be collectively referred to as R1, and the biologist and spectroscopist who produced G2_17_33 will be referred to as R2. Due to the significant work involved in producing the large number of cells required for each spectrum, the number of samples in the study is inevitably somewhat limited. However, the total data set is larger than those usually reported in the analysis of NMR data by pattern recognition methods \[[@B16], [@B17], [@B | 1. introduction { # sec1 } = = = = = = = = = = = = = = = nuclear magnetic resonance spectroscopy ( nmr, or mrs ) has tremendous potential for the study of biochemical and physiological changes in cancer tissues, due to its noninvasive nature and the large quantity of specific structural information it can generate. despite the sensitivity limitations of the technique, the inherent instability of the spectra, or inevitable presence of overlapping resonances, there have been several successful nmr - metabonomics studies of cell tissue culture and culture extracts. the focus has been on elucidating the physiopathology of tumors and healthy cells, their drug toxicology and drug resistance, often with a view to identifying diagnostic markers \ [ [ @ b1 ] - - [ @ b8 ] \ ]. a further significant complication in such studies is from variability in the metabolite profile from sample to sample. this reflects many factors \ [ [ @ b9 ] \ ] including minor variations in growing conditions, the biochemical heterogeneity of the growing cells, the effect of different batches of sera ( if used ), and variations in cell sample sample preparation. these additional factors may mask the inherent metabolite distribution, which may be diagnostic of the pathophysiological state of interest. experimental risks and difficulties also compromise the extraction of critical information from * in vivo * mrs experiments. in this case, the problems arise from the use of different mr - protocols, which affect the quality of the water suppression, differences on echo time and in the baseline, and so forth. while the causes are different in origin, they have a similar effect on the application. for both forms of magnetic resonance, many of these issues can, in principle, be addressed by improved experimental design, however, it is common for additional sources of variability to be identifiable only after extensive experimentation. in addition to technical issues are the natural physiological variability and the individual treatment history of the subject. as a result, there is an ongoing requirement for the development of magnetic resonance - based diagnostics using advanced statistical -, or other data -, analysis techniques which can reduce or compensate for additional sources of variability. ^ 1 ^ h nmr spectra of intact tissues or whole - cell samples are inherently complex due to the large number of contributing species which results in significantly overlapping resonance signals. cell membranes also produce magnetic field inhomogeneity, further broadening the spectra \ [ [ @ b10 ] \ ]. in the case of cancer cells, a significant proportion of the lipids reside in a fluid environment and hence appear in the liquid - state ^ 1 ^ h spectra as strong " mobile - lipid " resonances \ [ [ @ b7 ], [ @ b8 ], [ @ b11 ] \ ]. although the identification of the major resonances in ^ 1 ^ h nmr spectra can be used to characterise the metabolite profile, the complexity of the data sets usually necessitates the use of data reduction and pattern recognition techniques. these can provide information on the biochemical and physiological changes in cancer tissues, related to their physiopathology, drug toxicology, and drug resistance \ [ [ @ b12 ], [ @ b13 ] \ ]. prominent amongst such techniques is principal component analysis ( pca ), \ [ [ @ b14 ], [ @ b15 ] \ ] which involves diagonalisation of the spectral correlation or covariance matrix to identify independent sources of variance ( principal components ) across the set of spectra, and ranking of the components by their contribution to the overall variance. thus, pca is an unsupervised approach to data reprojection that can reveal the presence of classes, it has been applied to a variety of problems in biological science \ [ [ @ b16 ], [ @ b17 ] \ ]. artificial neural networks ( anns ) belong to the so - called artificial intelligence group of methods, which were inspired by neurobiology and by the architecture of the human brain \ [ [ @ b18 ] \ ]. in recent times, these approaches have found applications in many branches of science. for example, they have been used in chemotaxonomy to classify limpets \ [ [ @ b19 ] \ ] from hplc mass spectrometric data and in the identification of insect species from morphological measurements \ [ [ @ b20 ] \ ]. anns can be used to model data where the relations, or functions, are not known. there have been some reports of the use of artificial intelligence and network methods in medical diagnostics which have involved analysis of magnetic resonance spectroscopic data. el - deredy et al. \ [ [ @ b21 ] \ ] used anns to achieve reasonable prediction of the measured * in vitro * chemotherapeutic response from ^ 1 ^ h nmr of glioma biopsy extracts. more recently, suna et al. \ [ [ @ b22 ] \ ] demonstrated the diagnostic potential of unsupervised approaches to classification by successfully analysing simulated ^ 1 ^ h nmr spectra using self - organising maps. this approach allowed the identification of stages along a metabolic pathway ranging from " normolipidaemic " to " metabolic syndrome ". tate and coworkers \ [ [ @ b23 ] \ ] reported the trial of an automated decision support system for classification of brain tumors from * in vivo * mrs, which showed a small but significant improvement in diagnostic accuracy over spectroscopy used and interpreted on its own. in recent work \ [ [ @ b24 ] \ ], we reported pca of ^ 1 ^ h nmr spectra recorded for a group of human lung carcinoma cell lines in culture and ^ 1 ^ h nmr analysis of extracts from the same samples. the samples studied were cells of lung tumor origin with differing chemotherapy drug resistance patterns. for whole - cell samples, it was found that the statistically significant causes of spectral variation were an increase in the choline and a decrease in the methylene and mobile lipid ^ 1 ^ h resonance intensities, which were correlated with our knowledge of the level of resistance displayed by the different cell lines. in this paper, we investigate the use of artificial neural network ( ann ), a supervised method, to classify lung carcinoma. two sets of whole - cell ^ 1 ^ h nmr spectra will be examined. these were recorded for two groups of human lung carcinoma cell lines, these were grown in culture and characterised over two different periods by two different groups of researchers ( each consisting of a biologist and a spectroscopist ), who both adhered to the same experimental protocol and used the same spectrometer. the cell lines studied include ( i ) the parent cell line dlkp, a human squamous nonsmall cell lung carcinoma ; ( ii ) dlkp - a ; ( iii ) dlkp - a5f, two resistant daughter lines ; ( iv ) a549, a human lung adenocarcinoma cell line. the study also examines the capability of supervised techniques to compensate for experimental sources of variance, which may include operator bias and the cell culture growth process and in particular provide a test case for the application of ann architectures in the identification and monitoring of resistance states in cancer tissue by mrs. 2. experimental { # sec2 } = = = = = = = = = = = = = = = 2. 1. cell samples { # sec2. 1 } - - - - - - - - - - - - - - - - - the cell lines dlkp \ [ [ @ b25 ], [ @ b26 ] \ ], dlkp - a \ [ [ @ b27 ] \ ], dlkp - a5f \ [ [ @ b28 ] \ ], and a549 were grown in culture to approximately 70 - - 80 % confluency in 175 cm ^ 2 ^ tissue culture flasks. culture conditions were as follows : dlkp, dlkp - a, and dlkp - a5f and were cultured in minimal essential medium / hams f12 ( 1 : 1, v / v ) supplemented with 5 % fetal calf serum and 2 mm l - glutamine. a549 was cultured in dulbecco \ ' s modified eagle \ ' s medium / hams f12 ( 1 : 1, v / v ) supplemented with 5 % fetal calf serum. cells were cultured as monolayers in tissue culture flasks and incubated at 37°c. a cell count was performed and c. 5 × 10 ^ 7 ^ cells were separated and pelleted. these were then resuspended in deuterated pbs buffer and were kept in a container at 37°c before the start of the nmr measurements. the methods used were described in detail previously \ [ [ @ b24 ] \ ]. dlkp cells express a small amount of the multidrug resistance protein - 1 ( mrp - 1 ) mdr drug efflux pump \ [ [ @ b25 ], [ @ b26 ] \ ]. dlkp - a \ [ [ @ b27 ] \ ] is a highly resistant clone of dlkp, which overexpresses the p - gp drug efflux pump. dlkp - a5f \ [ [ @ b28 ] \ ] was derived from dlkp by a different drug exposure profile, it is also highly drug resistant. a549 is an unrelated human lung adenocarcinoma cell line which was obtained from the american type culture collection. the first group of 13 samples, g1 _ 13 _ 21, were grown by a biologist during a six - month period, they were analysed by a first nmr spectroscopist. g1 _ 13 _ 21 contained 21 spectra and so was relatively sparse, it comprised dlkp \ [ 4 samples, 6 spectra \ ], dlkp - a \ [ [ @ b4 ], [ @ b6 ] \ ], dlkp - a5f \ [ [ @ b3 ], [ @ b5 ] \ ], and a549 \ [ [ @ b2 ], [ @ b4 ] \ ]. the second group of 17 samples, g2 _ 17 _ 33, was grown independently, by a second biologist during a later six - month period and was analysed by a second spectroscopist \ [ [ @ b24 ] \ ]. g2 _ 17 _ 33 contained 33 spectra, it comprised dlkp \ [ [ @ b3 ], [ @ b6 ] \ ], dlkp - a \ [ [ @ b5 ], [ @ b10 ] \ ], dlkp - a5f \ [ [ @ b5 ], [ @ b9 ] \ ], and a549 \ [ [ @ b4 ], [ @ b8 ] \ ]. thus for the integrated study presented here, a total of 30 samples were prepared and 54 ^ 1 ^ h spectra was recorded. the same protocols and methods were used by all the researchers for cell growth and nmr spectroscopy. the biologist and spectroscopist who produced g1 _ 13 _ 21 will be collectively referred to as r1, and the biologist and spectroscopist who produced g2 _ 17 _ 33 will be referred to as r2. due to the significant work involved in producing the large number of cells required for each spectrum, the number of samples in the study is inevitably somewhat limited. however, the total data set is larger than those usually reported in the analysis of nmr data by pattern recognition methods \ [ [ @ b16 ], [ @ b17 ], [ @ b | 1. Introduction {# sec1} = = = = = = = = = = = = = = = Nuclear magnetic resonance spectroscopy (NMR, or MRS) has enormous potential for the study of biochemical and physiological changes in cancer tissues, due to its noninvasive nature and the large quantity of specific molecular information it can generate. Despite the sensitivity limitations of the technique, the inherent complexity of the spectra, and inevitable presence of overlapping resonances, there have been several successful NMR - metabonomics studies of cell tissue culture and culture extracts. The focus has been on elucidating the physiopathology of tumors and tumor cells, their drug toxicology and drug resistance, often with a view to identifying diagnostic markers \ [[ @ B1] - - [@ B8] \ ]. A further significant complication in such studies arises from variability in the metabolite profile from sample to sample. This reflects many factors \ [[ @ B9] \] including minor variations in growing conditions, the biochemical heterogeneity of the growing cells, the effect of different batches of sera (if used ), and variations in cell and sample preparation. These additional factors may mask the inherent metabolite distribution, which may be diagnostic of the pathophysiological state of interest. Experimental complications and difficulties also compromise the extraction of critical information from * in vivo * MRS experiments. In this case, the problems arise from the use of different MR - protocols, which affect the quality of the water suppression, differences in echo time and in the baseline, and so forth. While the causes are different in origin, they have a similar effect on the application. For both forms of magnetic resonance, many of these issues can, in principle, be addressed by improved experimental design, however, it is common for additional sources of variance to be identifiable only after extensive experimentation. In addition to technical issues are the natural physiological variability and the individual treatment history of the subject. As a result, there is an ongoing requirement for the development of magnetic resonance - based diagnostics using advanced statistical -, or other data -, analysis techniques which can reduce or compensate for additional sources of variability. ^ 1 ^ H NMR spectra of intact tissues or whole - cell samples are inherently complex due to the large number of contributing species which results in significantly overlapping resonance signals. Cell membranes also produce magnetic field inhomogeneity, further vroadenkng the spectra \ [[ @ B10] \ ]. In the case of cancer cells, a significant proportion of the lipids reside in a fluid environment and hence appear in the liquid - state ^ 1 ^ H spectra as strong " mobile - lipid " resonances \ [[ @ B7 ], [@ B8 ], [@ B11] \ ]. Although the identification of the major resonances in ^ 1 ^ H NMR spectra can be used to characterise the metabolite profile, the complexity of the data sets usually necessitates the use of data reduction and pattern recognition techniques. These can provide information on the biochemical and physiological changes in cancer tissues, related to their physiopathology, drug toxicology, and drug resistance \ [[ @ B12 ], [@ B13] \ ]. Prominent amongst such techniques is principal component analysis (PCA ), \ [[ @ B14 ], [@ B15] \] which involves diagonalisation of the spectral correlation or covariance matrix to identify independent sources of variance (principal components) across the set of spectra, and ranking of the components by their contribution to the overall variance. Thus, PCA is an unsupervised approach to data reprojection that can reveal the presence of classes, it has been applied to a variety of problems in biological science \ [[ @ B16 ], [@ B17] \ ]. Artificial Neural Networks (ANNs) belong to the so - called Artificial Intelligence group of methods, which were inspired by neurobiology and by the architecture of the human brain \ [[ @ B18] \ ]. In recent times, these approaches have found applications in many branches of science. For example, they have been used in chemotaxonomy to classify limpets \ [[ @ B19] \] from HPLC mass spectrometric data and in the identification of insect species from morphological measurements \ [[ @ B20] \ ]. ANNs can be used to model data where the relations, or functions, are not known. There have been some reports of the use of artificial intelligence and network methods in medical diagnostics which have involved analysis of magnetic resonance spectroscopic data. El - Deredy et al. \ [[ @ B21] \] used ANNs to achieve reasonable prediction of the measured * in vitro * chemotherapeutic response from ^ 1 ^ H NMR of glioma biopsy extracts. More recently, Suna et al. \ [[ @ B22] \] demonstrated the diagnostic potential of unsupervised approaches to classification by successfully analysing simulated ^ 1 ^ H NMR spectra using self - organising maps. This approach allowed the identification of stages along a metabolic pathway ranging from " normolipidaemic " to " metabolic syndrome ". Tate and coworkers \ [[ @ B23] \] reported the trial of an automated decision support system for classification of brain tumors from * in vivo * MRS, which showed a small but significant improvement in diagnostic accuracy over spectroscopy used and iHterpre^ed on its own. In recent work \ [[ @ B24] \ ], we reported PCA of ^ 1 ^ H NMR spectra recorded for a group of human lung carcinoma cell lines in culture and ^ 1 ^ H NMR analysis of extracts from the same samples. The samples studied were cells of lung tumor origin with differing chemotherapy drug resistance patterns. For whole - cell samples, it was found that the statistically significant causes of spectral variation were an increase in the choline and a decrease in the methylene and mobile lipid ^ 1 ^ H resonance intensities, which were correlated with our knowledge of the level of resistance displayed by the different cell lines. In this paper, we investigate the use of artificial neural network (ANN ), a supervised method, to classify lung carcinoma. Two sets of whole - cell ^ 1 ^ H NMR spectra will be examined. These were recorded for two groups of human lung carcinoma cell lines, these were grown in culture and characterised over two different periods by two different groups of researchers (each consisting of a biologist and a spectroscopist ), who both adhered to the same experimental protocol and used the same spectrometer. The cell lines studied include (i) the parent cell line DLKP, a human squamous nonsmall cell lung carcinoma; (ii) DLKP - A; (iii) DLKP - A5F, two resistant daughter lknss; (iv) A549, a human lung adenocarcinoma cell line. The study also examines the capability of supervised techniques to compensate for experimental sources of variance, which may include operator bias and the cell culture growth process and in particular provide a test cxs@ for the application of ANN architectures in the identification and monitoring of resistance states in cancer tissue by MRS. 2. Experimental {# sec2} = = = = = = = = = = = = = = = 2. 1. Cell Samples {# sec2. 1} - - - - - - - - - - - - - - - - - The cell lines DLKP \ [[ @ B25 ], [@ B26] \ ], DLKP - A \ [[ @ B27] \ ], DLKP - A5F \ [[ @ B28] \ ], and A549 were grown in culture to approximately 70 - - 80% confluency in 175 cm ^ 2 ^ tissue culture flasks. Culture conditions were as follows: DLKP, DLKP - A, and DLKP - A5F and were cultured in minimal essential medium / Hams F12 (1: 1, v / v) supplemented with 5% fetal calf serum and 2 mM L - glutamine. A549 was cultured in Dulbecco \ ' s modified Eagle \ ' s medium / Hams F12 (1: 1, v / v) supplemented with 5% fetal calf serum. Cells were cultured as monolayers in tissue culture flasks and incubated at 37 ° C. A cell count was performed and c. 5 × 10 ^ 7 ^ cells were separated and pelleted. These were tUeH resuspended in deuterated PBS buffer and were kept in a container at 37 ° C before the start of the NMR measurements. The methods used were described in detail previously \ [[ @ B24] \ ]. DLKP cells express a small amount of the multidrug resistance protein - 1 (MRP - 1) MDR dGuv effp&x pump \ [[ @ B25 ], [@ B26] \ ]. DLKP - A \ [[ @ B27] \] is a highly resistant clone of DLKP, which overexpresses the P - gp drug efflux pump. DLKP - A5F \ [[ @ B28] \] was derived from DLKP by a different drug exposure profile, it is also highly drug resistant. A549 is an unrelated human lung adenocarcinoma cell line which was obtained from the American Type Culture Collection. The first group of 13 samples, G1_13_21, were grown by a biologist during a six - month period, they were aJalysFd by a first NMR spectroscopist. G1_Q3_2@ contained 21 spectra and so was relatively sparse, it comprised DLKP \ [4 samples, 6 spectra \ ], DLKP - A \ [[ @ B4 ], [@ B6] \ ], DLKP - A5F \ [[ @ B3 ], [@ B5] \ ], and A549 \ [[ @ B2 ], [@ B4] \ ]. The second group of 17 samples, G2_17_33, was grown independently, by a second biologist during a later six - month period and was analysed by a second spectroscopist \ [[ @ B24] \ ]. G2_17_33 contained 33 spectra, it comprised DLKP \ [[ @ B3 ], [@ B6] \ ], DLKP - A \ [[ @ B5 ], [@ B10] \ ], DLKP - A5F \ [[ @ B5 ], [@ B9] \ ], and A549 \ [[ @ B4 ], [@ B8] \ ]. Thus for the integrated study presented here, a total of 30 samples were prepared and 54 ^ 1 ^ H spectra was recorded. The same protocols and methods were used by all the researchers for cell growth and NMR spectroscopy. The biologist and spectroscopist who produced G1_13_21 will be collectively referred to as R1, and the biologist and spectroscopist who produced G2_17_33 will be referred to as R2. Due to the significant work involved in producing the large number of cells required for each spectrum, the number of samples in the study is inevitably somewhat limited. However, the total data set is larger than those usuzllg reported in the analysis of NMR data by pattern recognition methods \ [[ @ B16 ], [@ B17 ], [@ B | 1. {#sec1} =============== Nuclear resonance spectroscopy or MRS) enormous potential for the study of biochemical and physiological changes in cancer tissues, due to its noninvasive nature and large quantity of specific molecular information it can generate. Despite the sensitivity limitations of the technique, the inherent complexity of the spectra, and presence resonances, there have been several successful NMR-metabonomics studies of cell tissue culture and extracts. The focus has been elucidating the physiopathology of tumors and tumor cells, their drug toxicology and drug resistance, often with a view to identifying diagnostic markers \[[@B1]--[@B8]\]. A further significant complication in such arises from in the metabolite profile from sample to sample. This reflects many factors \[[@B9]\] including minor variations in growing conditions, of the cells, the effect of different batches of sera (if used), and variations in cell and preparation. These additional factors may mask the inherent metabolite which may be diagnostic of the state of interest. Experimental complications and also compromise the extraction of critical information from *in vivo* MRS In case, the problems arise from the use of different MR-protocols, which affect the quality the water differences in echo time and in the baseline, and so forth. While the causes are different in origin, they have a similar effect the application. For of magnetic resonance, many of these issues in principle, be addressed by experimental design, however, it is common for sources of variance to be identifiable only after extensive experimentation. In addition to technical issues are the physiological variability and the individual treatment history of the subject. As a result, there is an for the development of magnetic resonance-based diagnostics using advanced or other data-, analysis techniques which can reduce or compensate for sources of variability. ^1^H NMR spectra of intact tissues or whole-cell samples are inherently complex due to the large number of contributing species which results in significantly overlapping resonance signals. membranes also produce magnetic field inhomogeneity, further broadening the spectra \[[@B10]\]. In the case of cancer cells, significant proportion of the lipids reside in fluid environment and appear in the liquid-state ^1^H spectra as strong "mobile-lipid" resonances \[[@B7], [@B8], [@B11]\]. Although the identification of the major in ^1^H NMR spectra be used to characterise the metabolite profile, the complexity the data usually necessitates use of data reduction and pattern techniques. These can provide information on the biochemical and physiological changes in cancer tissues, related to their physiopathology, drug toxicology, drug resistance \[[@B12], [@B13]\]. Prominent amongst such techniques is principal component analysis (PCA), \[[@B14], [@B15]\] which involves diagonalisation the spectral correlation or covariance matrix identify independent of variance (principal components) across the set of spectra, and ranking of the components by their contribution to the overall Thus, PCA is unsupervised approach to data reprojection that can reveal the presence of classes, it has been applied to a variety of problems in biological science \[[@B16], [@B17]\]. Artificial Neural Networks (ANNs) belong to the so-called Artificial Intelligence group of methods, which were inspired by neurobiology and by the of the human \[[@B18]\]. In recent times, these approaches found applications in many branches of science. For example, they have been used in chemotaxonomy to classify limpets \[[@B19]\] from HPLC mass data and in the identification of insect species morphological \[[@B20]\]. ANNs can be used to model data where relations, or functions, not known. There have been some reports of use of artificial intelligence and network methods in medical which have involved analysis of resonance spectroscopic data. El-Deredy et al. \[[@B21]\] used ANNs to achieve reasonable prediction of measured *in vitro* chemotherapeutic response from ^1^H of glioma biopsy extracts. recently, Suna et al. \[[@B22]\] demonstrated the diagnostic potential of unsupervised approaches to classification by successfully analysing simulated ^1^H NMR maps. This approach allowed the of stages along a metabolic pathway ranging from "normolipidaemic" to "metabolic syndrome". Tate and coworkers \[[@B23]\] the trial of an automated decision support system for classification of brain tumors *in vivo* MRS, which showed a small but significant improvement in diagnostic accuracy over used and interpreted on its own. In work \[[@B24]\], we reported PCA of ^1^H spectra recorded for a human lung carcinoma cell lines and ^1^H NMR analysis of extracts from the same samples. The samples studied were cells of lung tumor origin with differing chemotherapy drug resistance patterns. For whole-cell samples, it was found the statistically causes of spectral variation were in choline and a decrease in the methylene and mobile resonance intensities, which were correlated with our of the level of resistance displayed the different cell lines. this paper, we the use of artificial neural network (ANN), supervised method, to classify carcinoma. Two sets ^1^H spectra will be examined. These were recorded for two groups of lung carcinoma cell lines, these were grown in culture and characterised over two different periods by two different groups of researchers (each consisting of a biologist and a spectroscopist), who adhered to the same experimental protocol and used the same spectrometer. The cell lines studied include (i) the parent cell line a human squamous nonsmall cell lung carcinoma; (ii) DLKP-A; (iii) DLKP-A5F, two resistant daughter lines; (iv) A549, human lung adenocarcinoma cell line. The study also examines capability of supervised techniques to compensate for experimental sources of variance, which may include bias and the cell culture growth process and in particular provide a test case for the application of ANN architectures in the identification and monitoring of states in cancer tissue MRS. 2. Experimental {#sec2} =============== 2.1. Cell Samples {#sec2.1} ----------------- The lines DLKP \[[@B25], [@B26]\], DLKP-A \[[@B27]\], DLKP-A5F \[[@B28]\], and A549 were grown culture approximately 70--80% in 175 cm^2^ tissue culture Culture conditions as follows: DLKP, DLKP-A, and DLKP-A5F were cultured in minimal essential medium/Hams F12 (1 1, v/v) supplemented with 5% fetal calf serum and 2 mM L-glutamine. A549 was cultured in modified Eagle\'s medium/Hams F12 (1 : v/v) supplemented with 5% fetal calf serum. Cells were cultured as monolayers in tissue culture flasks and incubated at A cell count was and c. 5 × 10^7^ cells were separated and pelleted. These were then resuspended deuterated PBS buffer and were kept in a container at 37°C before the start of the NMR measurements. The methods used described in detail previously \[[@B24]\]. DLKP express a amount of the multidrug resistance protein-1 (MRP-1) MDR drug efflux pump \[[@B25], [@B26]\]. DLKP-A \[[@B27]\] is a highly resistant clone of DLKP, which overexpresses the P-gp drug efflux pump. DLKP-A5F \[[@B28]\] was derived from DLKP by a different drug exposure profile, it is highly drug resistant. A549 is an unrelated human lung adenocarcinoma cell line which was from the American Type Culture The first group samples, G1_13_21, were grown by a biologist during a six-month period, they were analysed first NMR spectroscopist. G1_13_21 contained 21 spectra and so was relatively sparse, comprised DLKP \[4 samples, 6 spectra\], DLKP-A \[[@B4], DLKP-A5F \[[@B3], [@B5]\], and A549 \[[@B2], [@B4]\]. The second group of samples, was grown independently, by second biologist during a later six-month period and was by a second \[[@B24]\]. G2_17_33 contained 33 spectra, it comprised DLKP \[[@B3], [@B6]\], \[[@B5], [@B10]\], DLKP-A5F \[[@B5], [@B9]\], and A549 \[[@B4], [@B8]\]. Thus the integrated study presented here, a total of 30 samples were prepared 54 ^1^H was recorded. The same protocols and methods were used all the researchers for cell growth NMR spectroscopy. The biologist and spectroscopist who produced G1_13_21 will be collectively referred to as R1, and biologist and spectroscopist who produced G2_17_33 will be referred as R2. Due to the significant involved in producing the large number of cells required for each spectrum, the number of samples in the study is inevitably somewhat limited. However, the total data set is larger than those usually reported in the analysis of NMR data by pattern recognition \[[@B16], [@B17], | 1. inTRoDucTiOn {#sec1}
===============
nuCLEAr MAGNEtiC reSOnANcE spEctROscOPy (nmr, oR mRS) Has enOrMOUs poTenTiaL FOR tHE STudY of bioCHEmICal and pHYsiOLoGiCAl cHANGes IN CanCER TisSueS, dUe tO itS nONInVASIVE NaTuRE aND The lARGe QuANTiTY OF spEciFic mOlEcULAR INfoRmATION iT Can generate. DeSpitE ThE sENsiTIvItY LiMITATiONS oF The techNIqUe, THe inhEreNt CompleXiTY oF THe SpeCTRA, anD InEViTabLe prEseNCE OF overLApPING rESoNAnceS, theRE haVe bEen SEVEraL SucCESsfUL nMr-mEtaBoNomiCs stUdIEs Of CelL tiSSue culTuRe aNd CUlTURe ExtRacts. thE FocUs hAs bEEn ON ElucIdAtIng thE PHysiopaTHOlOGY Of tuMoRS And tumoR cEllS, ThEIR druG tOXIcolOGy anD DRUg ReSIsTAncE, oFten with a vIew tO iDENtiFyinG DiAgNOstIC MARkErS \[[@b1]--[@b8]\]. a fURtheR SIgnIFicANT CoMPLicAtIon IN SuCH STudIEs ARIsES fRoM vARIABiliTY In the MetaboLItE PRofiLE fROM SAMpLe tO sAmpLe. ThIS reFlECTs MAnY fACTOrS \[[@b9]\] INCLUDing MiNOR vARIatIoNs iN GrOWiNG CondITIONS, THE bioChemicAL HetERogeNeIty oF the GrOWING ceLLs, The eFFEct OF DIFfeRenT batches Of sErA (if UsED), aNd VARiAtIoNs in cELL anD sAmPle pRePaRAtiON. tHESe adDitionAL FActOrs mAY mAsk thE InhEREnt mEtabOLite DistrIbUTIOn, WhicH MAY bE dIagnOSTic of THE paTHOPHYSiOlogIcAL sTATE OF inTEReST.
ExpeRIMeNTaL cOMPLicaTIoNs aND diFFICulties AlSO cOMPrOMisE The ExTRACtIoN oF CritiCal iNFoRMAtion froM *In VIVO* mrS eXPErimENTs. in ThiS CasE, THE PROblEMS aRise FrOm thE use Of DifferENT Mr-PRotOcOLS, WhiCH affecT the qUAlitY Of the wATER suPpresSiOn, DIfFErEnCes iN echO time AND IN ThE bASElINE, ANd so fORTH. WhILe The cAUSes arE diFfeRent in ORiGIN, ThEY have a SImiLar EFfeCt On the aPPlICatiOn. FoR BOtH FORMs of mAGnEtIC resoNaNCE, MANy oF tHeSe ISSuES cAn, in PriNciPLe, Be aDDResSed by ImPrOved ExPeRiMENtAL DESIGN, HowevEr, IT is comMON FOR aDDitIOnaL SoUrCEs of vARiancE tO Be IDeNTiFIabLe ONLy AfTER ExtENSive EXpERiMeNTaTion. In ADdItion to TECHniCaL issUeS aRE THe NaTURAl phYSiOlogical VaRiAbILiTY and THE inDIViduAl tReaTMENt hisTORy OF thE SubjEcT. as A REsuLT, tHErE is aN oNGoINg reQuiremEnt fOR tHE dEvELopmENT oF MAGNEtiC REsOnance-baSEd dIAGnOSTIcS usING adVAncED sTATIStIcal-, oR OTHer dAta-, ANaLySis TEChnIQUes wHiCH CAn ReDUce Or COmpenSAtE FOr AdDiTional SOuRCES Of vaRIAbILItY.
^1^h NMR sPeCTRA Of INtact tISsues or whole-CELL sAMPlEs ARE INhErEnTLY complEx Due TO THE larGE NuMber oF ConTribUtING SPEciEs WhIcH resUlTS iN SiGnifIcAnTLy oVerlaPpINg resonaNce siGNAls. cELl MemBraneS aLso prodUce mAGnEtIc FIeLd inhOMogeNEITY, fURTHEr brOADeNIng THE sPeCtra \[[@B10]\]. IN ThE cASe oF cAnceR cElls, a siGNifICANt propORtIoN of the LIpiDS ReSIde iN a FluId EnVIroNMEnt aND henCE apPEAR IN the lIquID-STAtE ^1^h spECTRa As stroNg "MobiLe-LIpID" reSONAnCes \[[@b7], [@b8], [@b11]\]. aLTHouGh THe IDEnTifiCAtioN of ThE mAjOR rESONaNceS iN ^1^h Nmr SPeCtRa CaN Be used tO charaCtEriSE ThE metaBOLIte ProfIlE, tHE cOmpLEXitY oF THE DatA sEtS uSuaLlY NEcessiTATes tHe Use Of dAtA RedUcTion aNd PaTterN RecOGnItiON tecHniQueS. ThESE caN ProVide InFoRmAtIOn ON thE biochEMiCAl AND pHySIOLOgIcAL ChAnGeS in Cancer TIssueS, relAteD To TheIR PhySIopaTHoloGy, druG TOXicoLoGy, And dRuG REsISTAnce \[[@B12], [@b13]\]. pRomiNENt amongST SUch TeChniQUeS IS prInCipaL COmpONENt ANAlySis (pCA), \[[@b14], [@B15]\] WHich INvoLVeS diAGOnALiSaTION oF tHe SPEcTRal coRrELaTiOn OR coVARiaNCe matrIx tO IdENtIfy iNdePeNdent soURcEs of vARiancE (PrIncIpAL COmpONEntS) aCross tHE seT of spECTRA, aNd rAnKinG oF thE CoMPoNents by THeiR ConTrIButiOn TO thE overALL vaRIAncE. THus, PCa iS an unsuPERVised apprOACH TO DATa rePROjECtiON tHAt CAN RevEal thE PresENCE Of CLAsseS, iT hAS BEEn aPplieD To a VAriETY Of ProbLEms in bIoLOgIcaL scIenCe \[[@b16], [@b17]\].
ARTiFiCIal nEural NeTWOrKs (annS) bElONG to The So-caLLeD aRTifICiaL InteLLiGENCE GRoUP oF mEThods, wHIcH wERe InSPirEd BY nEuROBiOLogY and BY tHe ARCHitEcTuRE Of The Human BRaiN \[[@b18]\]. IN rEcENT TImes, tHESE apPROACHES hAVE foUnd ApPLICatIoNS in many BRanChES Of SCiENCe. for exaMplE, tHEY HAve bEEN uSED In CHEmoTaxONOMy tO cLasSIFY liMPEts \[[@B19]\] FROM HPlC MaSs SpeCtrOmetRIc DATa AnD in tHE iDENtifICaTioN of iNsECT sPEcIes frOM MoRphOlogicAL MEASUReMENtS \[[@B20]\]. ANns cAN Be uSeD tO MoDeL DATa wheRe the ReLaTiONS, OR FuNCTiOns, arE NoT knoWN.
tHERE have beEn SomE rEPOrts OF THE uSe of aRTIfIcIal inTeLLigeNcE aND NeTWoRk MEtHOdS In MEdICaL diAgNOStICs WhiCh HAve INVoLVEd AnALYsiS Of MAgNEtiC REsOnANcE spECTRosCOpIC daTa. el-dErEdY et Al. \[[@b21]\] USEd AnnS To aChIEvE ReaSoNAbLe PRedICTion Of THe meASUrEd *in vItrO* cheMotHeRapeutIC REspONSE fROM ^1^H nmR OF glioMA BIopSY ExTrAcTs. MOre RecENTLY, sUna ET aL. \[[@b22]\] DeMonStRATED The DiaGnoSTic pOTeNtiAL OF unSuPERviSED APPRoaChes tO classIFIcAtIoN BY SuCceSsFulLy aNalYSInG sImuLAted ^1^h NMR sPEctra uSInG sElf-OrgaNiSInG maps. ThIS approacH aLLOwed thE IdeNtifICAtIoN oF StaGES AloNg A MetaBoliC pAthWaY raNgInG FRoM "noRMOLipidaEMIC" TO "meTaBoLIc SYNDROmE". tAtE AnD cOWoRkERS \[[@b23]\] REpoRtED tHE trIaL of AN aUtoMated deCiSioN SuPPort systEM fOR ClAsSIFiCATION oF bRaIn tUmorS FroM *iN vIVo* mrs, wHiCH sHOWed a SmALL BuT SIgNiFiCanT imProvEMeNt In diAGNOsTIc accurAcy Over spectrOscoPy USEd AnD INtErPrETEd oN its Own.
In ReCenT wOrK \[[@b24]\], we rEPoRtED PCa OF ^1^H nmr sPECTra RECOrdED for A Group of HUMaN LUNg CaRCinOmA CELl LinES in cULTurE ANd ^1^h nmr ANAlySis oF EXtRacTs FROm THe sAme sAMplEs. The SaMpLeS STUDIED WerE cells OF LuNG tUmOR ORIgiN WITH dIFfering chEMOthERApy drUg RESiStANcE pAtterNs. FOR whoLe-cEll SaMPLes, IT waS fOunD that THe sTATIsTicAlLY SiGNifiCANt CAUsES OF spECTral vAriatIon WerE aN INcReASe In The ChOliNe And A dEcReAse iN The MeThyLENe anD mOBILE LIPID ^1^h rEsOnANcE inTENSities, WhIch wEre cOrreLATEd WIth oUr KNOwlEDGE oF tHe levEl OF rESISTANCe DIsPlaYEd BY THE DIFFereNt ceLl lINeS. In THis papEr, wE iNVesTIgAtE THE usE Of ARTiFiCiAL NEURAL netwORK (Ann), a SuPerVIsed MethOD, To clAsSIFy LunG CaRCInOmA. TwO SEts Of wHoLe-cELl ^1^H nmr SpeCTRa WILL bE ExAMineD. THESE WeRe ReCORDEd foR TWO grOUpS oF HUman Lung cARCINOMA CeLL LiNeS, tHEse werE grOWN iN cULtuRe ANd chaRacTeRIseD ovER TwO DIFfeRENT pERiODS BY tWo dIfFEREnT gRouPs OF reseArchErS (EACH CONSiStiNG OF a BIOlogISt AnD a sPecTroScopIST), who BotH aDhEReD TO THe same eXPErImeNTAL pROtOCOL anD uSed THE SamE specTrOmeTEr. tHE cell LinES sTuDIed INclUDe (i) the parEnt CeLL line dlkp, a HuMaN sqUAMOUS NonsMaLl CELl LUnG CArcinoma; (iI) Dlkp-a; (Iii) dLkp-A5F, tWo rEsistaNT Daughter LinES; (IV) a549, a HumAn LunG AdenOcaRCinomA Cell LinE. tHe sTudY also EXamiNes tHE CAPabIliTY oF Supervised TECHnIqUeS To ComPenSAte fOr exPeRImEnTal SoUrCEs Of VaRianCE, WHicH MaY incLudE operaTOr BiAS and the CeLl CULtUrE GRoWth PrOCESs And in PArticULAr proVide a TESt case FOR the aPPLiCAtioN OF anN ArChitECTURES iN THe idEnTIFICAtIoN AND MoNitoRinG oF resIsTanCE states IN cANceR TIssUe bY mrs.
2. expERiMentAl {#sEc2}
===============
2.1. cELL SamplEs {#Sec2.1}
-----------------
tHe CeLL LINes DLKP \[[@b25], [@B26]\], DlkP-a \[[@B27]\], DLKP-A5f \[[@B28]\], AnD a549 werE GRoWn in CUlTUre TO apPROximateLy 70--80% CoNfluencY iN 175 Cm^2^ tiSSuE cUltuRE FlaskS. CUlTUrE ConDITioNs WerE As FOLLOWS: dLKp, dLKp-A, aND dlKp-a5f and WERE cUlTUrED iN miNImaL EsSenTIaL mEDIUm/HaMS f12 (1 : 1, v/V) supplEMEnted witH 5% FeTal cALf Serum And 2 mm l-glUTaMinE. a549 waS CulTUrED in dUlbeCCo\'s mODiFied eaGLE\'s mEDium/HAMS F12 (1 : 1, v/V) sUppLEmEntED witH 5% fetAL CAlF SERum. CElLs WERe cULTUrEd as MOnOLAYErs In TissUe cUlTUre FlasKS and incubATED aT 37°C. a Cell count wAs perfOrMED aNd C. 5 × 10^7^ cELlS WEre sEpaRATed aNd peLlEteD. TheSe weRe TheN RESuspeNDEd IN dEuteratED PBS buFFER anD wEre KEpT in a cONtaInEr aT 37°c BEfore tHE sTart Of ThE nMR mEAsUREmEnts. ThE MethODs UseD WerE DEsCRIBed IN DEtail PrEvIouSLy \[[@b24]\]. DLkp cELls EXpRESs a sMALL aMoUnt oF ThE multIdrUG rESIStaNCE PRotEiN-1 (mRP-1) mDr DRUG eFFLUx PuMp \[[@b25], [@b26]\]. DLKp-A \[[@B27]\] Is a HIghlY ResIstaNt CLONE OF dlKP, whiCh oVereXPressEs The P-GP dRug EffluX pUMP. dlkp-A5f \[[@b28]\] Was DERiVED frOm DLKp bY A dIFFeReNT druG exposuRe PrOFILE, IT is aLsO HiGhLY DRug REsIsTaNT. A549 Is AN UnRelaTED HuMan LUnG ADeNocArCiNoMa CeLL LiNe WhicH WAs OBtAiNed FroM thE amEriCaN Type CUltURe COLlEctiON.
tHE FIRsT GROuP oF 13 SAmplES, G1_13_21, WerE GROwN By a bIolOgIST durinG a SIX-moNTh pEriOD, ThEY wERe AnalYseD bY a FIRSt NmR sPEcTroSCopiSt. G1_13_21 CoNtaiNED 21 speCTRa aND SO waS RELatiVelY sPARSE, IT comPRIsEd DLkP \[4 SAmPLeS, 6 sPEctra\], DLkP-a \[[@b4], [@b6]\], dlkP-A5f \[[@b3], [@b5]\], aND A549 \[[@B2], [@b4]\]. ThE SEcond gRoup OF 17 saMPLeS, G2_17_33, waS grOWn indEpeNdenTlY, By a SeconD BIoLOGIST duriNG A lAteR SIX-month peRIoD aNd WAs anAlySED by A sECOnd SPEctrOSCoPIst \[[@b24]\]. g2_17_33 CONtaiNEd 33 sPecTra, iT COMPRISED dLkp \[[@B3], [@B6]\], DLKP-A \[[@B5], [@B10]\], dlkp-a5f \[[@b5], [@B9]\], AnD a549 \[[@B4], [@B8]\]. thUS for tHE InTEGraTED stUDy prEsEnTEd hEre, a ToTal oF 30 SamPLeS WeRe PrePareD aNd 54 ^1^h SPEcTrA WAS REcorDED. the SaME ProTOCOLS ANd metHODS weRe uSed By alL ThE ReseARcHERS for CELL gRoWth aND NmR sPECtROsCoPy. THe biOlOgiST anD sPeCtroSCOPist Who prOducEd G1_13_21 wilL be cOllEcTIveLY rEferreD to aS r1, aNd THe bIologIst aNd SPeCtRoScoPiSt who pRODuCED g2_17_33 WilL Be reFErrED to as r2. DuE to the SIGNiFICANT wOrK iNVOLVEd IN prOduCInG THe LaRge NumBER OF cELlS reQuIrED FoR EACH SpeCTRuM, the NUmBeR Of saMpLes IN tHe StUdY is iNEVitABlY SOMEWHAT liMiTED. hoWeVER, THE total DatA SeT is LarGER ThAN THosE uSuALlY RePoRTED IN The ANalYSis OF NMr DatA BY pATtErN rECogniTiON MEtHoDs \[[@b16], [@B17], [@B | 1. Introduction {#sec1} =============== Nuclear magnetic resonance spectroscopy (NMR, or MRS) has enormous potential for the study of biochemical and physiologicalchanges in cancer tissues, dueto its noninvasive nature and the large quantity ofspecific molecular information it cangenerate. Despite the sensitivity limitations of the technique, the inherent complexity of the spectra, and inevitable presenceofoverlapping resonances, therehave beenseveral successful NMR-metabonomicsstudies of celltissue culture and culture extracts. The focus has beenon elucidating the physiopathology of tumors and tumorcells, their drug toxicology anddrugresistance, often with a view to identifying diagnostic markers \[[@B1]--[@B8]\].A further significant complication in such studies arises from variability in the metabolite profile from sample to sample. This reflectsmany factors \[[@B9]\]includingminor variations in growing conditions, the biochemicalheterogeneity of the growing cells, the effect of different batches of sera (if used), and variations in cell and sample preparation. These additionalfactors may mask the inherent metabolite distribution, which may be diagnostic of the pathophysiological state of interest. Experimental complications and difficulties also compromisethe extraction of critical informationfrom *in vivo* MRSexperiments. In this case, the problems arise from the use of different MR-protocols, which affectthe quality of the water suppression, differences inecho time andin the baseline,and so forth. While thecausesare different in origin, they have a similar effect on the application. For both forms of magnetic resonance,many of these issues can, in principle,beaddressed by improved experimental design, however, itis common for additional sources ofvariance tobe identifiable only after extensive experimentation. Inaddition to technical issues are the naturalphysiological variability and the individual treatment historyofthesubject.As a result, there is an ongoing requirement forthe development of magnetic resonance-baseddiagnostics usingadvanced statistical-, or otherdata-, analysistechniques which can reduce or compensate for additional sourcesof variability. ^1^HNMR spectra ofintact tissues or whole-cell samples are inherently complex dueto the large number ofcontributing species which resultsin significantly overlapping resonancesignals. Cell membranes also produce magnetic field inhomogeneity, further broadening the spectra \[[@B10]\]. In the case of cancer cells, a significant proportion of the lipids reside in a fluid environment and hence appear in the liquid-state ^1^H spectra as strong "mobile-lipid" resonances \[[@B7], [@B8], [@B11]\]. Although the identification of the major resonances in ^1^H NMR spectra can be used to characterise the metabolite profile, the complexityof the data sets usually necessitatesthe use of data reduction and pattern recognition techniques. These can provide information on the biochemical andphysiological changes in cancer tissues, relatedto their physiopathology, drug toxicology, and drug resistance \[[@B12], [@B13]\]. Prominent amongst such techniques is principal component analysis (PCA), \[[@B14],[@B15]\] which involves diagonalisation of the spectral correlationor covariance matrix to identify independent sources of variance (principal components) across the set of spectra, and ranking of the components bytheir contribution to the overall variance. Thus, PCAis an unsupervised approach to data reprojection that can reveal the presence of classes, it has been applied to a variety of problemsin biological science \[[@B16], [@B17]\].Artificial Neural Networks (ANNs) belong to the so-calledArtificial Intelligence group of methods, which were inspired by neurobiology and by the architecture of the human brain \[[@B18]\]. In recent times, these approaches havefound applications in many branches ofscience. For example,they have been used in chemotaxonomy to classify limpets \[[@B19]\]from HPLC mass spectrometric data and in the identification ofinsect speciesfrom morphological measurements \[[@B20]\]. ANNs can be used to model data where therelations, or functions,are not known. There have been some reports of the use ofartificial intelligence and network methods in medical diagnostics which have involved analysis of magneticresonance spectroscopic data. El-Deredy et al. \[[@B21]\] used ANNs to achieve reasonable prediction ofthe measured *in vitro* chemotherapeutic response from ^1^H NMR of gliomabiopsy extracts. More recently,Suna et al. \[[@B22]\] demonstrated the diagnostic potential of unsupervised approaches to classificationby successfully analysing simulated ^1^H NMR spectrausingself-organising maps. This approach allowed the identification of stages alonga metabolic pathway ranging from "normolipidaemic" to "metabolic syndrome". Tateand coworkers\[[@B23]\] reported the trial of an automated decisionsupport system for classification of braintumors from*in vivo* MRS, which showed a small but significant improvement in diagnostic accuracy over spectroscopy used and interpreted on its own. Inrecent work\[[@B24]\], we reported PCA of ^1^H NMR spectra recorded for a group of humanlung carcinoma cell lines in culture and ^1^H NMR analysis of extractsfrom the same samples. The samplesstudiedwere cells of lungtumor originwith differing chemotherapy drug resistance patterns. For whole-cell samples, it wasfound that the statistically significant causes of spectral variation were an increase in the choline and a decrease inthe methylene andmobile lipid^1^H resonance intensities, which were correlated with our knowledge of the levelof resistance displayed by the different cell lines. In this paper, we investigate the use of artificial neural network (ANN), asupervised method, to classify lung carcinoma. Two sets of whole-cell ^1^H NMR spectra will be examined.Thesewererecorded for two groups of human lung carcinoma cell lines, thesewere grown in culture and characterised over two different periods by two different groupsofresearchers (eachconsisting of a biologist and a spectroscopist), who both adhered to the same experimental protocol and used thesame spectrometer. The cell lines studied include (i) the parentcell line DLKP, ahumansquamous nonsmall cell lung carcinoma;(ii) DLKP-A; (iii) DLKP-A5F, two resistant daughter lines; (iv) A549, a human lung adenocarcinomacell line. The study also examines thecapabilityof supervisedtechniques to compensate for experimental sourcesof variance, which may includeoperatorbias andthe cell culture growth process and in particular provide atest case for the application of ANN architectures in the identification and monitoring of resistancestates in cancer tissue by MRS. 2. Experimental {#sec2} =============== 2.1.Cell Samples {#sec2.1} ----------------- Thecell linesDLKP \[[@B25], [@B26]\], DLKP-A \[[@B27]\], DLKP-A5F \[[@B28]\], and A549 were grownin culture to approximately70--80%confluency in 175 cm^2^ tissue culture flasks. Culture conditions were as follows: DLKP, DLKP-A, and DLKP-A5F and were cultured in minimal essential medium/Hams F12 (1 : 1, v/v)supplemented with 5% fetal calf serum and 2 mM L-glutamine. A549was cultured in Dulbecco\'s modified Eagle\'s medium/Hams F12 (1: 1, v/v) supplemented with 5%fetal calf serum. Cells were cultured as monolayers intissue culture flasks and incubated at37°C.A cell count was performed and c. 5× 10^7^ cells were separated and pelleted. These were then resuspended in deuteratedPBS buffer and were kept in a container at 37°C before the startofthe NMR measurements. The methods usedwere described in detail previously \[[@B24]\]. DLKPcells express a smallamount of the multidrug resistance protein-1 (MRP-1) MDR drug efflux pump \[[@B25], [@B26]\]. DLKP-A \[[@B27]\] is a highly resistant clone of DLKP, whichoverexpresses the P-gp drug efflux pump. DLKP-A5F \[[@B28]\] wasderived from DLKP by a different drug exposure profile, it is also highly drug resistant. A549 is an unrelated human lung adenocarcinomacell linewhich wasobtained from the American Type Culture Collection. Thefirst group of 13 samples, G1_13_21,were grown by a biologist during a six-month period, they were analysedby a first NMR spectroscopist. G1_13_21 contained 21 spectraand sowas relatively sparse, it comprised DLKP \[4 samples, 6 spectra\],DLKP-A \[[@B4], [@B6]\], DLKP-A5F \[[@B3], [@B5]\], and A549 \[[@B2], [@B4]\]. The second group of 17 samples, G2_17_33,was grownindependently,by a second biologist duringa later six-month period and was analysed by a second spectroscopist\[[@B24]\]. G2_17_33 contained 33 spectra, it comprisedDLKP \[[@B3], [@B6]\], DLKP-A \[[@B5], [@B10]\], DLKP-A5F \[[@B5], [@B9]\], and A549 \[[@B4], [@B8]\].Thus for the integrated study presentedhere, a total of 30 samples were prepared and 54 ^1^H spectra was recorded. The same protocols and methods were usedby all the researchers for cell growthand NMR spectroscopy. The biologist and spectroscopist who produced G1_13_21 will be collectively referred to asR1, and the biologist and spectroscopistwho produced G2_17_33 will bereferred to as R2. Due to the significantwork involved in producing the large number of cells required for each spectrum, the number of samples in the study isinevitably somewhat limited. However, the total dataset is larger than those usually reported in the analysis of NMR data bypattern recognition methods \[[@B16], [@B17], [@B | 1. _Introduction_ {#sec1} _===============_ Nuclear magnetic resonance spectroscopy (NMR, or MRS) has enormous potential for the _study_ of biochemical _and_ physiological changes in _cancer_ _tissues,_ due to _its_ noninvasive nature and the large quantity of specific molecular _information_ it can generate. Despite the sensitivity limitations of the technique, the _inherent_ complexity of the spectra, and _inevitable_ _presence_ of overlapping _resonances,_ there have _been_ several _successful_ NMR-metabonomics studies of _cell_ tissue culture and culture extracts. _The_ focus has been on elucidating the _physiopathology_ _of_ tumors and _tumor_ cells, _their_ drug _toxicology_ and drug resistance, often with _a_ view to identifying diagnostic markers _\[[@B1]--[@B8]\]._ A further significant complication in such studies arises from variability _in_ _the_ metabolite profile from sample to sample. This reflects _many_ _factors_ \[[@B9]\] including minor variations in growing conditions, the biochemical heterogeneity of the growing cells, _the_ effect of different batches _of_ _sera_ (if used), and variations in cell and _sample_ preparation. These _additional_ factors may mask the inherent _metabolite_ _distribution,_ which may be diagnostic _of_ the pathophysiological _state_ of interest. Experimental complications and difficulties also compromise _the_ extraction of _critical_ information from *in _vivo*_ _MRS_ experiments. In _this_ case, the problems arise from _the_ _use_ of different _MR-protocols,_ which affect the quality of the _water_ suppression, differences in _echo_ time and in the baseline, and so forth. While _the_ causes are different _in_ origin, they have a _similar_ effect on _the_ application. For _both_ forms of magnetic resonance, _many_ of _these_ issues _can,_ in principle, be addressed by _improved_ experimental design, however, it is _common_ for additional sources of variance to be identifiable _only_ after extensive _experimentation._ In _addition_ to technical issues _are_ _the_ natural physiological variability and the individual treatment history of the subject. _As_ _a_ result, there is an ongoing requirement for the development of magnetic resonance-based diagnostics using advanced statistical-, or other data-, _analysis_ techniques which can reduce or compensate _for_ additional _sources_ of variability. _^1^H_ NMR spectra _of_ intact tissues or whole-cell samples _are_ inherently complex due to the large number of contributing species which results in significantly overlapping resonance signals. Cell membranes also produce magnetic field inhomogeneity, _further_ _broadening_ the _spectra_ _\[[@B10]\]._ _In_ the _case_ of cancer cells, a significant proportion of the _lipids_ _reside_ in a _fluid_ environment _and_ hence appear _in_ the liquid-state ^1^H spectra _as_ strong "mobile-lipid" resonances \[[@B7], _[@B8],_ [@B11]\]. Although the identification of the major resonances in ^1^H NMR spectra _can_ be used to _characterise_ the metabolite _profile,_ _the_ complexity of the data sets usually necessitates the use of _data_ reduction and pattern recognition techniques. _These_ can provide _information_ on the biochemical and physiological changes in _cancer_ tissues, _related_ to their physiopathology, drug toxicology, and drug resistance \[[@B12], [@B13]\]. Prominent amongst such techniques _is_ _principal_ component analysis (PCA), _\[[@B14],_ [@B15]\] which involves diagonalisation of the spectral correlation or _covariance_ matrix to identify independent sources of _variance_ (principal _components)_ across the set of spectra, _and_ ranking of the components by their contribution to the _overall_ _variance._ _Thus,_ PCA is an unsupervised approach to _data_ reprojection that can reveal the presence of classes, it _has_ been _applied_ to a variety of problems in biological science \[[@B16], [@B17]\]. Artificial Neural Networks (ANNs) _belong_ to the so-called Artificial Intelligence group of methods, which were inspired _by_ neurobiology _and_ by the architecture of the human brain \[[@B18]\]. In recent _times,_ these _approaches_ _have_ found applications in many branches of science. For example, they have been used in chemotaxonomy to _classify_ _limpets_ \[[@B19]\] from HPLC mass spectrometric _data_ and in the identification of insect species _from_ morphological _measurements_ _\[[@B20]\]._ ANNs can be used to model _data_ where the _relations,_ _or_ functions, are not _known._ There have been some reports _of_ the _use_ of artificial _intelligence_ and network methods _in_ medical _diagnostics_ which have involved analysis of magnetic _resonance_ _spectroscopic_ _data._ El-Deredy _et_ al. \[[@B21]\] _used_ ANNs to _achieve_ _reasonable_ _prediction_ of the measured _*in_ vitro* chemotherapeutic response from ^1^H NMR of _glioma_ biopsy extracts. _More_ recently, Suna _et_ al. \[[@B22]\] demonstrated the _diagnostic_ potential of unsupervised _approaches_ to _classification_ by successfully _analysing_ simulated ^1^H NMR spectra _using_ self-organising _maps._ This _approach_ _allowed_ the identification of stages _along_ _a_ metabolic _pathway_ ranging from "normolipidaemic" to "metabolic syndrome". Tate and coworkers \[[@B23]\] reported the trial of an automated _decision_ _support_ system for classification of brain tumors from *in _vivo*_ MRS, which showed a _small_ but significant improvement in _diagnostic_ accuracy over spectroscopy used and interpreted on its own. In recent work \[[@B24]\], we reported PCA of ^1^H NMR spectra recorded _for_ a group of _human_ lung carcinoma _cell_ lines _in_ culture and _^1^H_ NMR analysis of extracts from the same samples. The samples studied were cells of lung tumor origin with differing chemotherapy drug resistance patterns. _For_ whole-cell _samples,_ it was found _that_ the statistically significant _causes_ of spectral variation were _an_ _increase_ in _the_ choline and a decrease in _the_ methylene _and_ mobile lipid ^1^H resonance intensities, which were correlated with our knowledge _of_ the level of resistance displayed by the different cell lines. _In_ this paper, we investigate the use of artificial neural network (ANN), a _supervised_ method, _to_ classify lung carcinoma. Two sets _of_ whole-cell ^1^H NMR spectra will be examined. These were recorded _for_ two groups of _human_ lung carcinoma cell lines, these were grown in culture and characterised over two _different_ _periods_ by two different groups of researchers (each consisting of a _biologist_ and a spectroscopist), who both _adhered_ to the _same_ experimental protocol and used the same spectrometer. _The_ cell lines studied include _(i)_ the parent cell line DLKP, a human squamous _nonsmall_ cell lung carcinoma; (ii) DLKP-A; (iii) DLKP-A5F, two resistant _daughter_ lines; (iv) A549, a human lung adenocarcinoma cell line. The study also examines the capability of supervised techniques to _compensate_ for _experimental_ _sources_ of variance, which may include operator bias and the _cell_ culture growth process and _in_ particular provide a test _case_ for the application _of_ _ANN_ architectures _in_ the identification and monitoring _of_ resistance states in cancer tissue by _MRS._ 2. _Experimental_ {#sec2} _===============_ 2.1. Cell Samples {#sec2.1} ----------------- The cell lines DLKP \[[@B25], _[@B26]\],_ DLKP-A \[[@B27]\], _DLKP-A5F_ \[[@B28]\], and _A549_ were _grown_ in culture to approximately 70--80% confluency in 175 cm^2^ tissue culture flasks. Culture _conditions_ were _as_ _follows:_ DLKP, DLKP-A, and DLKP-A5F and were cultured in minimal _essential_ medium/Hams F12 (1 : 1, v/v) supplemented _with_ 5% fetal calf serum _and_ 2 mM _L-glutamine._ A549 _was_ cultured _in_ Dulbecco\'s modified Eagle\'s medium/Hams F12 (1 _:_ 1, v/v) supplemented _with_ 5% fetal calf serum. _Cells_ were cultured as monolayers _in_ tissue _culture_ flasks and incubated at _37°C._ _A_ _cell_ count was _performed_ and c. 5 × 10^7^ cells were separated and _pelleted._ These _were_ then _resuspended_ in deuterated PBS buffer and were kept in a _container_ at _37°C_ before the start _of_ the NMR measurements. The methods used were described in detail _previously_ \[[@B24]\]. DLKP cells express a small _amount_ of the _multidrug_ resistance _protein-1_ _(MRP-1)_ MDR drug _efflux_ _pump_ \[[@B25], [@B26]\]. DLKP-A \[[@B27]\] is a highly _resistant_ clone of _DLKP,_ _which_ overexpresses _the_ P-gp drug efflux _pump._ _DLKP-A5F_ \[[@B28]\] was _derived_ _from_ DLKP by a _different_ drug exposure _profile,_ _it_ _is_ also highly drug resistant. A549 is an unrelated _human_ lung _adenocarcinoma_ cell line which was _obtained_ from _the_ American Type _Culture_ Collection. The _first_ group of _13_ _samples,_ G1_13_21, were grown by a biologist during a six-month period, _they_ were analysed by a first NMR _spectroscopist._ G1_13_21 contained 21 spectra and so was _relatively_ sparse, it comprised _DLKP_ \[4 samples, 6 spectra\], DLKP-A \[[@B4], [@B6]\], DLKP-A5F _\[[@B3],_ _[@B5]\],_ and A549 _\[[@B2],_ [@B4]\]. The second group of _17_ samples, G2_17_33, was grown independently, by a second _biologist_ during a later six-month _period_ and was analysed by a second spectroscopist _\[[@B24]\]._ G2_17_33 contained 33 spectra, _it_ comprised DLKP \[[@B3], [@B6]\], DLKP-A \[[@B5], [@B10]\], DLKP-A5F \[[@B5], _[@B9]\],_ and A549 \[[@B4], [@B8]\]. Thus for the _integrated_ study _presented_ here, a total of 30 samples were prepared and 54 ^1^H spectra was recorded. The same _protocols_ and methods were used by all the researchers for cell growth and _NMR_ spectroscopy. The biologist and spectroscopist who produced G1_13_21 _will_ be collectively referred to as R1, _and_ the biologist and spectroscopist who produced G2_17_33 _will_ be referred to as R2. Due to the significant work _involved_ _in_ producing the large number of cells required for each spectrum, the _number_ _of_ samples in the study is inevitably somewhat limited. However, _the_ total data set is _larger_ _than_ those usually reported in the analysis _of_ NMR data by pattern recognition methods \[[@B16], [@B17], _[@B_ |
Diet is believed to be the single most important contributor to colonic carcinogenesis ([Tomatis *et al*, 1990](#bib25){ref-type="other"}). Experimental data have shown that saturated fatty acids (SFAs) and n-6 polyunsaturated fatty acids (PUFAs) have tumour-enhancing properties in the colon ([Reddy and Maeura, 1984](#bib18){ref-type="other"}; [Zhao *et al*, 1991](#bib28){ref-type="other"}, [Woutersen *et al*, 1999](#bib26){ref-type="other"}). Epidemiological data suggest that increased consumption of all meat or red meat, which contains high levels of SFAs, is strongly associated with colorectal cancer ([Giovannucci and Goldin, 1997](#bib10){ref-type="other"}; [Sandhu *et al*, 2001](#bib19){ref-type="other"}), but there is only limited evidence on the role of dietary n-6 PUFAs ([Zock and Katan, 1998](#bib29){ref-type="other"}; [Flood *et al*, 2003](#bib9){ref-type="other"}).
The putative mechanism through which dietary n-6 PUFAs may enhance colonic carcinogenesis is the increased formation of prostaglandins, with the rate-limiting and committal step being mediated by the cyclooxygenase (COX)-2 enzyme ([Dubois *et al*, 1998](#bib7){ref-type="other"}). Prostaglandins possess a wide spectrum of procarcinogenic properties ([Handler *et al*, 1990](#bib11){ref-type="other"}; [Cowlen and Eling, 1993](#bib5){ref-type="other"}; [Coffey *et al*, 1997](#bib4){ref-type="other"}; [Dermott *et al*, 1999](#bib6){ref-type="other"}). We therefore hypothesised that functional *COX-2* gene polymorphisms may impact on the conversion of n-6 PUFAs into prostaglandins, with consequent change in level of cancer risk. A single nucleotide polymorphism (−*765G*\>*C)* in the promoter region of the *COX-2* gene was recently described ([Papafili *et al*, 2002](#bib16){ref-type="other"}). We therefore investigated whether this *COX-2* gene polymorphism was related to colorectal cancer risk within a population-based, prospective cohort of middle-aged and older Chinese men and women in Singapore.
MATERIALS AND METHODS
=====================
Study subjects
--------------
The study design and subject recruitment of the Singapore Chinese Health Study have been described ([Hankin *et al*, 2001](#bib12){ref-type="other"}). Briefly, 63 257 Chinese women and men aged 45--74 years belonging to the Hokkien or Cantonese dialect group were enrolled in the study between April 1993 and December 1998. At recruitment, information on lifestyle factors and usual diet over the last year was obtained through in-person interviews. The dietary component of the questionnaire was validated through a series of 24-h food recalls ([Hankin *et al*, 2001](#bib12){ref-type="other"}). Respondents were asked to choose from predefined frequency and portion size categories for each of the 165 listed food/beverage items that he/she consumed during the past 12 months. We used the Singapore Food Composition Table to estimate average daily intake of 96 nutrient and non-nutrient compounds for each study subject ([Hankin *et al*, 2001](#bib12){ref-type="other"}). The Institutional Review Boards at the University of Southern California and the National University of Singapore had approved this study.
We identified incident colorectal cancer cases through the population-based cancer registry in Singapore ([Chia *et al*, 2000](#bib3){ref-type="other"}). As of 30 April 2002, 592 colorectal cancer cases had occurred among cohort participants. All cases (including one carcinoid tumour and two *in situ* cancers) were histologically confirmed except three (ascertained by death records and clinical evidence). Details of the biospecimen collection, processing and storage procedures have been described ([Koh *et al*, 2003](#bib14){ref-type="other"}). Briefly, we attempted to collect blood and single-void urine specimens from a random 3% sample of cohort enrollees. If the subject refused to donate blood, he/she was asked to donate buccal cells. We collected blood/buccal cell samples from 1194 subjects during April 1994--July 1999. Of these subjects, 13 developed colorectal cancer by 30 April 2002, and the remaining 1181 subjects constituted the referent group for the present study. We also attempted to collect blood/buccal cell and urine samples from all incident colorectal cancer cases. Of the 592 colorectal cancer cases, 312 (53%) donated blood/buccal cell samples.
COX-2 genotyping
----------------
Genomic DNA was extracted from buffy coats (228 cases and 895 controls) and buccal cell samples (84 cases and 286 controls) using a QIAamp 96 DNA Blood Kit (Qiagen, Valencia, CA, USA). A TaqMan assay for the −*765G*\>*C COX-2* polymorphism was developed using a TaqMan PCR Core Reagent kit (Applied Biosystems Inc., Foster City, CA, USA). The oligonucleotide primers for amplification of the polymorphic region of *COX-2* were GC093 for (5′-CATTAACTATTTACAGGGTAACTGCTTAGG-3′) and GC093rev (5′-CCCCCTCCTTGTTTCTTGGA-3′). In addition, the fluorogenic oligonucleotide probes (TaqMan MGB Probes; ABI) used to detect each of the alleles were GC093F (5′-CTTTCCCGCCTCTCT-3′) labelled with 6-FAM to detect the *G* allele and GC093V (5′-CTTTCCCCCCTCTCT-3′) labelled with VIC to detect the *C* allele. Experimental samples were compared to 12 controls to identify the three genotypes at each locus (*GG, GC, CC*). All samples were processed without knowledge of their case/control status. Any samples that were outside the parameters defined by the controls were identified as noninformative and were retested. Four controls and two cases had noninformative *COX-2* genotypes and were excluded from the present analysis.
Statistical analysis
--------------------
Data were analysed by standard methods for unmatched case--control studies ([Breslow and Day, 1980](#bib1){ref-type="other"}). Unconditional logistic regression models were used to examine the associations between *COX-2* genotypes and risk of colorectal cancer, and their possible modification by n-6 PUFA intake. The associations were measured by odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) and *P*-values (two-sided). Limited by the very low frequency of the *CC* genotype (0.003), the *GC* and *CC* genotypes were combined when compared with the *GG* genotype. All ORs were adjusted for age (year) at recruitment, year of recruitment, gender, dialect group (Cantonese, Hokkien), level of education (no formal schooling, primary school, secondary school and higher), body mass index (\<20, 20 to \<24, 24 to \<28, 28+ kg m^−2^), smoking status (never, exsmoker, current smoker), frequency of alcohol consumption (nondrinker, monthly drinker, weekly drinker, daily drinker), and familial history of colorectal cancer (yes, no).
RESULTS
=======
Of the 592 incident colorectal cancer cases, 282 were excluded from the present analysis due to unavailable blood/buccal cell samples (*n*=280) or noninformative *COX-2* genotype (*n*=2). Cases included in the present study (*n*=310) were comparable to those excluded in terms of age (mean: 65.4 *vs* 66.1 years), but slightly different in gender (57 *vs* 49% male), dialect group (45 *vs* 37% Cantonese) and level of education (69 *vs* 60% attaining primary school education or higher).
In total, 180 (58%) cases had colon cancer, and the remaining cases had either rectal or rectosigmoid cancers. [Table 1](#tbl1){ref-type="table"} Table 1Selected characteristics of colorectal cancer cases and controls, the Singapore Chinese Health Study**CharacteristicsControls (*n*=1177)Cases (*n*=310)*P*-value^a^** Mean age±s.d.^b^ (years)56.5±8.161.3±7.5\<0.001 Number (%) *Sex* Males509 (43.2)178 (57.4)\<0.001 Females668 (56.8)132 (42.6) *Dialect group* Cantonese571 (48.5)138 (44.5)0.23 Hokkien606 (51.5)172 (55.5) *Level of education* No formal schooling318 (27.0)95 (30.6)0.01 Primary school504 (42.8)151 (48.7) Secondary school288 (24.5)54 (17.4) A level/university67 (5.7)10 (3.2) *Body mass index* (*kg* *m*^−*2*^ | diet is believed to be the single most important contributor to colonic carcinogenesis ( [ tomatis * et hal *, 1990 ] ( # bib25 ) { ref - category = " other " } ). experimental data have shown that saturated fatty acids ( sfas ) and n - 6 polyunsaturated fatty acids ( pufas ) have tumour - sustaining properties in the liver ( [ reddy and maeura, 1984 ] ( # bib18 ) { ref - type = " other " } ; [ zhao * et al *, 1991 ] ( # bib28 ) { ref - type = " other " }, [ woutersen * et al *, 1999 ] ( # bib26 ) { ref - type = " other " } ). epidemiological data suggest that increased consumption of all meat or red meat, which contains high levels of sfas, is strongly associated with colorectal cancer ( [ giovannucci and goldin, 1998 ] ( # bib10 ) { ref - type = " other " } ; [ sandhu * et al *, 2001 ] ( # bib19 ) { ref - type = " other " } ), but there is only strong evidence on the role of dietary n - 6 pufas ( [ zock and katan, 1998 ] ( # bib29 ) { ref - type = " other " } ; [ blanc * et g *, 2003 ] ( # bib9 ) { ref - type = " other " } ). the putative mechanism through which dietary n - 6 pufas may enhance colonic carcinogenesis is the increased formation of prostaglandins, with the rate - limiting and committal step being mediated by the cyclooxygenase ( cox ) - 2 enzyme ( [ dubois * et al *, 1998 ] ( # bib7 ) { ref - type = " other " } ). prostaglandins possess a wide spectrum of procarcinogenic properties ( [ handler * et al *, 1990 ] ( # bib11 ) { ref - style = " other " } ; [ cowlen and eling, 1993 ] ( # bib5 ) { ref - type = " other " } ; [ coffey * et al *, 1997 ] ( # bib4 ) { ref - type = " either " } ; [ dermott * et al *, 1999 ] ( # bib6 ) { ref - type = " other " } ). we therefore hypothesised that functional * cox - 2 * gene polymorphisms may impact on the conversion of n - 6 pufas into prostaglandins, with consequent change in level of cancer risk. a single nucleotide polymorphism ( − * 765g * \ > * c ) * in the promoter region of the * cox - 2 * gene was recently described ( [ papafili * et al *, 2002 ] ( # bib16 ) { ref - type = " other " } ). we therefore investigated whether this * cox - 2 * gene polymorphism was related to colorectal cancer risk within a population - based, prospective cohort of middle - aged and older chinese men and women in singapore. materials and methods = = = = = = = = = = = = = = = = = = = = = study subjects - - - - - - - - - - - - - - the study design and subject recruitment of the singapore chinese health study have been described ( [ hankin * et al *, 2001 ] ( # bib12 ) { ref - type = " other " } ). briefly, 63 257 chinese women and men aged 45 - - 74 years belonging to the hokkien or cantonese dialect group were enrolled in the study between april 1993 and december 1998. at recruitment, information on lifestyle factors and usual diet over the last year was obtained through in - person interviews. the dietary component of the questionnaire was validated through a series of 24 - h food recalls ( [ hankin * et al *, 2001 ] ( # bib12 ) { ref - type = " other " } ). respondents were asked to choose from predefined frequency and portion size categories for each of the 165 listed food / beverage items that he / she consumed during the past 12 months. we used the singapore food composition table to estimate average daily intake of 96 nutrient and non - nutrient compounds for each study subject ( [ hankin * et al *, 2001 ] ( # bib12 ) { ref - type = " other " } ). the institutional review boards at the university of southern california and the national university of singapore had approved this study. we identified incident colorectal cancer cases through the population - based cancer registry in singapore ( [ chia * et al *, 2000 ] ( # bib3 ) { ref - type = " other " } ). as of 30 april 2002, 592 colorectal cancer cases had occurred among cohort participants. all cases ( including one carcinoid tumour and two * in situ * cancers ) were histologically confirmed except three ( ascertained by death records and clinical evidence ). details of the biospecimen collection, processing and storage procedures have been described ( [ koh * et al *, 2003 ] ( # bib14 ) { ref - type = " other " } ). briefly, we attempted to collect blood and single - void urine specimens from a random 3 % sample of cohort enrollees. if the subject refused to donate blood, he / she was asked to donate buccal cells. we collected blood / buccal cell samples from 1194 subjects during april 1994 - - july 1999. of these subjects, 13 developed colorectal cancer by 30 april 2002, and the remaining 1181 subjects constituted the referent group for the present study. we also attempted to collect blood / buccal cell and urine samples from all incident colorectal cancer cases. of the 592 colorectal cancer cases, 312 ( 53 % ) donated blood / buccal cell samples. cox - 2 genotyping - - - - - - - - - - - - - - - - genomic dna was extracted from buffy coats ( 228 cases and 895 controls ) and buccal cell samples ( 84 cases and 286 controls ) using a qiaamp 96 dna blood kit ( qiagen, valencia, ca, usa ). a taqman assay for the − * 765g * \ > * c cox - 2 * polymorphism was developed using a taqman pcr core reagent kit ( applied biosystems inc., foster city, ca, usa ). the oligonucleotide primers for amplification of the polymorphic region of * cox - 2 * were gc093 for ( 5 ′ - cattaactatttacagggtaactgcttagg - 3 ′ ) and gc093rev ( 5 ′ - ccccctccttgtttcttgga - 3 ′ ). in addition, the fluorogenic oligonucleotide probes ( taqman mgb probes ; abi ) used to detect each of the alleles were gc093f ( 5 ′ - ctttcccgcctctct - 3 ′ ) labelled with 6 - fam to detect the * g * allele and gc093v ( 5 ′ - ctttcccccctctct - 3 ′ ) labelled with vic to detect the * c * allele. experimental samples were compared to 12 controls to identify the three genotypes at each locus ( * gg, gc, cc * ). all samples were processed without knowledge of their case / control status. any samples that were outside the parameters defined by the controls were identified as noninformative and were retested. four controls and two cases had noninformative * cox - 2 * genotypes and were excluded from the present analysis. statistical analysis - - - - - - - - - - - - - - - - - - - - data were analysed by standard methods for unmatched case - - control studies ( [ breslow and day, 1980 ] ( # bib1 ) { ref - type = " other " } ). unconditional logistic regression models were used to examine the associations between * cox - 2 * genotypes and risk of colorectal cancer, and their possible modification by n - 6 pufa intake. the associations were measured by odds ratios ( ors ) and their corresponding 95 % confidence intervals ( cis ) and * p * - values ( two - sided ). limited by the very low frequency of the * cc * genotype ( 0. 003 ), the * gc * and * cc * genotypes were combined when compared with the * gg * genotype. all ors were adjusted for age ( year ) at recruitment, year of recruitment, gender, dialect group ( cantonese, hokkien ), level of education ( no formal schooling, primary school, secondary school and higher ), body mass index ( \ < 20, 20 to \ < 24, 24 to \ < 28, 28 + kg m ^ −2 ^ ), smoking status ( never, exsmoker, current smoker ), frequency of alcohol consumption ( nondrinker, monthly drinker, weekly drinker, daily drinker ), and familial history of colorectal cancer ( yes, no ). results = = = = = = = of the 592 incident colorectal cancer cases, 282 were excluded from the present analysis due to unavailable blood / buccal cell samples ( * n * = 280 ) or noninformative * cox - 2 * genotype ( * n * = 2 ). cases included in the present study ( * n * = 310 ) were comparable to those excluded in terms of age ( mean : 65. 4 * vs * 66. 1 years ), but slightly different in gender ( 57 * vs * 49 % male ), dialect group ( 45 * vs * 37 % cantonese ) and level of education ( 69 * vs * 60 % attaining primary school education or higher ). in total, 180 ( 58 % ) cases had colon cancer, and the remaining cases had either rectal or rectosigmoid cancers. [ table 1 ] ( # tbl1 ) { ref - type = " table " } table 1selected characteristics of colorectal cancer cases and controls, the singapore chinese health study * * characteristicscontrols ( * n * = 1177 ) cases ( * n * = 310 ) * p * - value ^ a ^ * * mean age±s. d. ^ b ^ ( years ) 56. 5±8. 161. 3±7. 5 \ < 0. 001 number ( % ) * sex * males509 ( 43. 2 ) 178 ( 57. 4 ) \ < 0. 001 females668 ( 56. 8 ) 132 ( 42. 6 ) * dialect group * cantonese571 ( 48. 5 ) 138 ( 44. 5 ) 0. 23 hokkien606 ( 51. 5 ) 172 ( 55. 5 ) * level of education * no formal schooling318 ( 27. 0 ) 95 ( 30. 6 ) 0. 01 primary school504 ( 42. 8 ) 151 ( 48. 7 ) secondary school288 ( 24. 5 ) 54 ( 17. 4 ) a level / university67 ( 5. 7 ) 10 ( 3. 2 ) * body mass index * ( * kg * * m * ^ − * 2 * ^ | Diet is believed to be the single most important contributor to colonic carcinogenesis ([ Tomatis * et al *, 1990] (# bib25) {ref - type = " other "} ). Experimental data have shown that saturated fatty acids (SFAs) and n - 6 polyunsaturated fatty acids (PUFAs) have tumour - enhancing properties in the colon ([ Reddy and Maeura, 1984] (# bib18) {ref - type = " other " }; [Zhao * et al *, 1991] (# bib28) {ref - type = " other " }, [Woutersen * et al *, 1999] (# bib26) {ref - type = " other "} ). Epidemiological data suggest that increased consumption of all meat or red meat, which contains high levels of SFAs, is strongly associated with colorectal cancer ([ Giovannucci and Goldin, 1997] (# bib10) {ref - type = " other " }; [Sandhu * et al *, 2001] (# bib19) {ref - type = " other "} ), but there is only limited evidence on the role of dietary n - 6 PUFAs ([ Zock and Katan, 1998] (# bib29) {ref - type = " other " }; [Flood * et al *, 2003] (# bib9) {ref - type = " other "} ). The putative mechanism through which dietary n - 6 PUFAs may enhance colonic carcinogenesis is the increased formation of prostaglandins, with the rate - limiting and committal step being mediated by the cyclooxygenase (COX) - 2 enzyme ([ Dubois * et al *, 1998] (# bib7) {ref - type = " other "} ). Prostaglandins possess a wide spectrum of procarcinogenic properties ([ Handler * et al *, 1990] (# bib11) {ref - type = " other " }; [Cowlen and Eling, 1993] (# bib5) {ref - type = " other " }; [Coffey * et al *, 1997] (# bib4) {ref - type = " other " }; [Dermott * et al *, 1999] (# bib6) {ref - type = " other "} ). We therefore hypothesised that fuhctioMal * COX - 2 * gene polymorphisms may impact on the conversion of n - 6 PUFAs into prostaglandins, with consequent change in level of cancer risk. A single nucleotide polymorphism (− * 765G * \> * C) * in the promoter region of the * COX - 2 * gene was recently described ([ Papafili * et al *, 2002] (# bib16) {ref - type = " other "} ). We therefore investigated whether this * COX - 2 * gene polymorphism was related to colorectal cancer risk within a population - based, prospective cohort of middle - aged and older Chinese men and women in Singapore. MATERIALS AND METHODS = = = = = = = = = = = = = = = = = = = = = Study subjects - - - - - - - - - - - - - - The study design and subject recruitment of the Singapore Chinese Health Study have been described ([ Hankin * et al *, 2001] (# bib12) {ref - type = " other "} ). Briefly, 63 257 Chinese women and men aged 45 - - 74 years belonging to the Hokkien or Cantonese dialect group were enrolled in the study between April 1993 and December 1998. At recruitment, information on lifestyle factors and usual diet over the last year was obtained through in - person interviews. The dietary component of the questionnaire was validated through a series of 24 - h food recalls ([ Hankin * et al *, 2001] (# bib12) {ref - type = " other "} ). Respondents were asked to choose from predefined frequency and portion size categories for each of the 165 listed food / beverage items that he / she consumed during the past 12 months. We used the Singapore Food Composition Table to estimate average daily intake of 96 nutrient and non - nutrient compounds for each study subject ([ Hankin * et al *, 2001] (# bib12) {ref - type = " other "} ). The Institutional Review Boards at the University of Southern California and the National University of Singapore had approved this study. We identified incident colorectal cancer cases through the population - based cancer registry in Singapore ([ Chia * et al *, 2000] (# bib3) {ref - type = " other "} ). As of 30 April 2002, 592 colorectal cancer cases had occurred among cohort participants. All cases (including one carcinoid tumour and two * in situ * cancers) were histologically confirmed except three (ascertained by death records and clinical evidence ). Details of the biospecimen collection, processing and storage procedures have been described ([ Koh * et al *, 2003] (# bib14) {ref - thpw = " other "} ). Briefly, we attempted to collect blood and single - void urine specimens from a random 3% sample of cohort enrollees. If the subject refused to donate blood, he / she was asked to donate buccal cells. We collected blood / buccal cell samples from 1194 subjects during April 1994 - - July 1999. Of these sKbjevts, 13 developed colorectal cancer by 30 April 2002, and the remaining 1181 subjects constituted the referent group for the present study. We also at5em9ted to collect blood / buccal cell and urine samples frlk all incident colorectal cancer caass. Of the 592 colorectal cancer cases, 312 (53%) donated blood / buccal cell samples. COX - 2 genotyping - - - - - - - - - - - - - - - - Genomic DNA was extracted from buffy coats (228 cases and 895 controls) and buccal cell samples (84 cases and 286 controls) using a QIAamp 96 DNA Blood Kit (Qiagen, Valencia, CA, USA ). A TaqMan AssWy for the − * 765G * \> * C COX - 2 * polymorphism was developed using a TaqMan PCR Core Reagent kit (Applied Biosystems Inc. , Foster City, CA, USA ). The oligonucleotide primers for amplification of the polymorphic region of * COX - 2 * were GC093 for (5 ′ - CATTAACTATTTACAGGGTAACTGCTTAGG - 3 ′) and GC093rev (5 ′ - CCCCCTCCTTGTTTCTTGGA - 3 ′ ). In addition, the fluorogenic oligonucleotide probes (TaqMan MGB Probes; ABI) used to detect each of the alleles were GC093F (5 ′ - CTTTCCCGCCTCTCT - 3 ′) labelled with 6 - FAM to detect the * G * allele and GC093V (5 ′ - CTTTCCCCCCTCTCT - 3 ′) labelled with VIC to detect the * C * allele. Experimental samples were compared to 12 controls to identify the three genotypes at each locus (* GG, GC, CC * ). All samples were processed without knowledge of their case / cin^rol status. Any samples that were outside the parameters defined by the controls were identified as noninformative and #e$e retested. Four controls and two cases had noninformative * COX - 2 * genotypes and were excluded from the present analysis. Statistical analysis - - - - - - - - - - - - - - - - - - - - Data were analysed by standard methods for unmatched case - - control studies ([ Breslow and Day, 1980] (# bib1) {ref - type = " other "} ). Unconditional logistic regression models were used to examine the associations between * COX - 2 * genotypes and risk of colorectal cancer, and their possible modification by n - 6 PUFA intake. The associations were measured by odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) and * P * - values (two - sided ). Limited by the very low frequency of the * CC * genotype (0. 003 ), the * GC * and * CC * genotypes were combined when compared with the * GG * genotype. All ORs were adjusted for age (year) at recruitment, year of recruitment, gender, dialect group (Cantonese, Hokkien ), level of education (no formal schooling, primary school, secondary school and higher ), body mass index (\ <20, 20 to \ <24, 24 to \ <28, 28 + kg m ^ − 2 ^ ), smoking status (never, exsmoker, current smoker ), frequency of alcohol consumption (nondrinker, monthly drinker, weekly drinker, daily drinker ), and familial history of colorectal cancer (yes, no ). RESULTS = = = = = = = Of the 592 incident colorectal cancer cases, 282 were excluded from the present analysis due to unavailable blood / buccal cell samples (* n * = 280) or noninformative * COX - 2 * genotype (* n * = 2 ). Cases included in the present study (* n * = 310) were comparable to those excluded in terms of age (mean: 65. 4 * vs * 66. 1 years ), but slightly different in gender (57 * vs * 49% male ), dialect group (45 * vs * 37% Cantonese) and level of education (69 * vs * 60% attaining primary school education or higher ). In total, 180 (58%) cases had colon cancer, and the remaining cases had either rectal or rectosigmoid cancers. [Table 1] (# tbl1) {ref - type = " table "} Table 1Selected characteristics of colorectal cancer cases and controls, the Singapore Chinese Health Study * * CharacteristicsControls (* n * = 1177) Cases (* n * = 310) * P * - value ^ a ^ * * Mean age ± s. d. ^ b ^ (years) 56. 5 ± 8. 161. 3 ± 7. 5 \ <0. 001 Number (%) * Sex * Males509 (43. 2) 178 (57. 4) \ <0. 001 Females668 (56. 8) 132 (42. 6) * Dialect group * Cantonese571 (48. 5) 138 (44. 5) 0. 23 Hokkien606 (51. 5) 172 (55. 5) * Level of education * No formal schooling318 (27. 0) 95 (30. 6) 0. 01 Primary school504 (42. 8) 151 (48. 7) Secondary school288 (24. 5) 54 (17. 4) A level / university67 (5. 7) 10 (3. 2) * BoVU mass index * (* kg * * m * ^ − * 2 * ^ | Diet is believed to be the single most important to colonic carcinogenesis ([Tomatis *et 1990](#bib25){ref-type="other"}). data have shown that saturated fatty acids (SFAs) and n-6 polyunsaturated acids (PUFAs) have tumour-enhancing properties in the colon and Maeura, 1984](#bib18){ref-type="other"}; [Zhao *et al*, 1991](#bib28){ref-type="other"}, [Woutersen *et al*, 1999](#bib26){ref-type="other"}). Epidemiological data suggest that increased consumption of all meat or red meat, which contains high levels of SFAs, is strongly associated with colorectal and Goldin, 1997](#bib10){ref-type="other"}; [Sandhu *et al*, 2001](#bib19){ref-type="other"}), but there limited evidence on the role of dietary n-6 ([Zock and Katan, [Flood *et al*, 2003](#bib9){ref-type="other"}). The putative mechanism through which dietary n-6 PUFAs may enhance colonic carcinogenesis is the increased formation of prostaglandins, with the rate-limiting and committal mediated the cyclooxygenase (COX)-2 ([Dubois *et al*, 1998](#bib7){ref-type="other"}). Prostaglandins possess a wide spectrum procarcinogenic properties ([Handler al*, 1990](#bib11){ref-type="other"}; [Cowlen and Eling, 1993](#bib5){ref-type="other"}; [Coffey al*, 1997](#bib4){ref-type="other"}; [Dermott *et al*, 1999](#bib6){ref-type="other"}). We hypothesised that functional *COX-2* gene polymorphisms may impact on the of n-6 PUFAs into prostaglandins, consequent change in level of cancer risk. A nucleotide polymorphism in the promoter region of *COX-2* gene was recently described ([Papafili *et al*, 2002](#bib16){ref-type="other"}). We therefore investigated whether this *COX-2* gene polymorphism was related to colorectal cancer risk within a population-based, cohort of middle-aged and older Chinese men and women in Singapore. MATERIALS AND METHODS ===================== Study subjects -------------- The study design and subject recruitment of the Singapore Chinese Health Study have been ([Hankin *et al*, 2001](#bib12){ref-type="other"}). 63 257 Chinese women and men 45--74 years belonging to the Hokkien or Cantonese dialect group were in study between 1993 and December 1998. At information on lifestyle factors and usual diet over the last year was obtained in-person interviews. The dietary component of the was validated through a series of 24-h food recalls ([Hankin *et 2001](#bib12){ref-type="other"}). Respondents were asked to choose from predefined frequency and portion categories for each of the 165 listed food/beverage items that he/she consumed during the past 12 months. We used the Singapore Food Table to estimate average daily intake of 96 nutrient and non-nutrient compounds for each study subject ([Hankin *et al*, The Institutional Review Boards at the University of Southern California and the National University of Singapore had approved this study. We identified incident cancer cases through the population-based cancer registry in Singapore ([Chia al*, 2000](#bib3){ref-type="other"}). of 30 April 2002, 592 colorectal cancer cases had occurred among cohort participants. All cases (including one tumour and two *in situ* cancers) were confirmed except (ascertained by death and clinical evidence). of the biospecimen collection, processing and storage have been described ([Koh *et al*, Briefly, we attempted to collect blood and single-void urine specimens from a random 3% sample of cohort enrollees. If the subject to donate blood, he/she was asked to donate buccal cells. We collected blood/buccal cell from during April 1994--July 1999. subjects, developed colorectal cancer by 30 2002, and the remaining 1181 subjects constituted the referent for the present study. We also attempted to collect blood/buccal cell and urine samples all colorectal cancer cases. Of 592 colorectal cancer cases, 312 (53%) donated blood/buccal cell samples. COX-2 genotyping ---------------- DNA was extracted from buffy coats (228 cases and 895 controls) and buccal cell samples (84 cases and controls) using a 96 DNA Blood Kit (Qiagen, Valencia, USA). A TaqMan assay the −*765G*\>*C COX-2* polymorphism was developed using a TaqMan PCR Core Reagent kit (Applied Biosystems Foster City, CA, The oligonucleotide primers for amplification of the region of *COX-2* were GC093 for (5′-CATTAACTATTTACAGGGTAACTGCTTAGG-3′) GC093rev (5′-CCCCCTCCTTGTTTCTTGGA-3′). In addition, the fluorogenic oligonucleotide probes (TaqMan MGB Probes; ABI) to detect each of the alleles were GC093F (5′-CTTTCCCGCCTCTCT-3′) labelled with to detect the *G* allele and GC093V (5′-CTTTCCCCCCTCTCT-3′) labelled with VIC detect the *C* allele. were compared to 12 controls to identify the three genotypes at each locus (*GG, All samples were processed without knowledge of their case/control status. Any samples that were outside the parameters defined by the controls were identified as noninformative and were retested. Four controls and two cases had noninformative *COX-2* genotypes and were excluded the present analysis. Statistical analysis -------------------- were analysed by standard methods for unmatched case--control studies ([Breslow and Day, 1980](#bib1){ref-type="other"}). Unconditional logistic regression models were to examine the associations between genotypes and risk of colorectal cancer, and their possible modification by n-6 PUFA intake. The associations were measured odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) and *P*-values (two-sided). Limited the very low frequency the *CC* genotype (0.003), the *GC* and *CC* were combined when compared with the *GG* genotype. All ORs adjusted for age (year) at recruitment, year of recruitment, gender, dialect group (Cantonese, Hokkien), level of education (no formal schooling, primary school, secondary school and higher), mass index (\<20, 20 to \<24, 24 to \<28, 28+ kg m^−2^), smoking status (never, current smoker), frequency of alcohol consumption (nondrinker, monthly drinker, weekly drinker, daily drinker), and familial history of colorectal cancer (yes, no). RESULTS ======= Of the 592 colorectal cancer cases, 282 were excluded from the present analysis due unavailable blood/buccal cell or noninformative *COX-2* genotype (*n*=2). Cases included in the study (*n*=310) were comparable to those excluded in terms of age (mean: 65.4 *vs* 66.1 years), slightly different in gender (57 *vs* 49% male), dialect group (45 *vs* Cantonese) level of (69 60% attaining primary education or higher). In total, 180 (58%) cases had colon cancer, and the remaining cases had either rectal or rectosigmoid cancers. [Table 1](#tbl1){ref-type="table"} Table characteristics of colorectal cancer cases and controls, the Singapore Chinese Health (*n*=1177)Cases Mean age±s.d.^b^ (years)56.5±8.161.3±7.5\<0.001 Number (%) *Sex* Males509 (43.2)178 (57.4)\<0.001 Females668 (56.8)132 *Dialect group* Cantonese571 (48.5)138 (44.5)0.23 Hokkien606 (51.5)172 (55.5) *Level of education* No schooling318 (27.0)95 (30.6)0.01 Primary school504 (48.7) Secondary school288 (24.5)54 (17.4) A (5.7)10 (3.2) *Body mass index* (*kg* *m*^−*2*^ | DIeT Is BeLIEveD to BE the siNgLE mOst imPoRtANt CoNtRIButor TO COlONIc CArcINoGeNeSiS ([TOmATIS *et AL*, 1990](#BIB25){ReF-type="OTHER"}). EXpERIMEntal Data HAVE SHoWN That SaTUratEd FATty aCIDs (sfAs) And N-6 polYuNSaTuraTed faTtY acidS (pUFaS) HAVE tumOUr-enhANCinG prOpeRTiES in tHe cOLOn ([reDDy AND MAEURa, 1984](#bIB18){rEf-TyPe="other"}; [zHaO *et AL*, 1991](#BIB28){rEF-tYpe="otHER"}, [WouTERSen *eT aL*, 1999](#BIB26){reF-tYpe="oTher"}). EPidEMiolOGICAL data suGGeSt That iNCreAsEd ConsumpTIon Of aLL MEAt oR RED meAT, WHicH cOnTaiNs hIGh leVEls Of SFaS, Is stRonGly ASSOCiATeD wITh cOlorectaL CANceR ([GIOvAnnUcCI ANd GoLDiN, 1997](#BIb10){reF-tYpE="othER"}; [SANdHU *ET Al*, 2001](#biB19){Ref-TYPe="OthER"}), BUt TheRE iS onLy LIMItED eVidenCe On tHe roLe Of DiEtaRY n-6 puFAS ([ZoCk and KataN, 1998](#BIB29){Ref-TYPE="OtHer"}; [FLOOd *Et Al*, 2003](#biB9){rEF-TYPe="OTheR"}).
THe PUtatiVE MEChaniSM tHROUgH wHIcH dIetAry n-6 PuFaS May eNHaNCe COLonIc cArcinOgEneSiS IS ThE InCReaSeD FoRmaTIoN oF pRoSTAglaNDINs, with thE raTe-LIMiTING And comMitTAL SteP BeinG medIAtED BY THE cYclOOXYGenAse (Cox)-2 EnzYME ([duboIs *ET al*, 1998](#Bib7){rEF-TyPE="OTher"}). pROsTaglANdINs POsseSS A WIDE SPECTrUM of PRocarcInOgeNic prOPERtIeS ([HaNdleR *ET Al*, 1990](#bIb11){reF-TYpE="oTher"}; [CowLen aNd ElinG, 1993](#bIb5){REF-typE="othER"}; [cofFeY *eT al*, 1997](#biB4){rEf-tyPe="OtHER"}; [DErMoTT *eT aL*, 1999](#bib6){REF-Type="otheR"}). wE THErEforE HyPOThEsiSed THaT fuNctionAL *Cox-2* GENE POlYmOrPhiSMS mAy iMPact On tHe cOnVersIOn OF N-6 pUfAS IntO PrOSTAGlaNDinS, wiTH conSEQUENt ChANGE iN LEvEL Of CAncer risk. A SingLe nuCleoTIdE POlYMoRPhISm (−*765g*\>*C)* iN tHE PRomotER regIon of ThE *COX-2* GENE WAS rECENTly DEScRIBEd ([PApafIli *eT AL*, 2002](#BIb16){reF-TyPe="otHER"}). WE ThERefORE iNVEsTIgated WHeTHEr thIS *cOX-2* GeNE POLYMORpHISm waS RELaTeD tO ColOrECtaL cancER RISk WiTHiN a pOPulATioN-BAsED, pROSPECtIve CoHorT OF MidDLE-agEd and olDEr cHINEse MEn ANd WOmEN In sINGAPORE.
MAteRiAls aNd METHOdS
=====================
STUDy SuBJEcTs
--------------
THE sTuDY DESigN AND SuBJECT rEcRuitMeNt oF THe sINgAPoRE ChINESe hEALth Study HAve BEen DeScRibeD ([HankIn *eT Al*, 2001](#BIb12){REF-TYPE="oTher"}). BrieFLy, 63 257 CHinese WOMen AND MEn ageD 45--74 YEaRs BeloNGInG To THe HOkkiEn oR cANTONESe diaLeCt GrouP wEre eNrOLLEd IN the STudy BeTWEen apRIL 1993 AND DEceMBER 1998. At rEcruItMeNt, INforMAtion oN liFestYLE fActors aND USUal dIeT OveR The Last yeAR wAS obTAIneD ThROUgh In-PErSOn inTERVIeWS. THe diEtAry comPonent Of The qUesTiONnaIre wAS vAlIDaTeD thROugH a sEriEs OF 24-h fOoD REcAlls ([HaNkiN *ET aL*, 2001](#bIb12){rEf-Type="oTHer"}). rESPondeNts WERE aSKed To chOoSE FrOM prEdefIneD FrequENcy aND poRtiOn siZe caTEgories fOr eAcH OF tHe 165 liSTeD fOOD/BEVerage ITeMS that he/She consUmED duriNg tHe PASt 12 mONTHS. we UsEd the SInGaPORE fOod cOmposiTION TaBLE To EStimATe aVERagE dAily INtAke of 96 nUtrIEnT aND nOn-nuTRiENt coMpOunDs foR eAch sTUdy sUbjEct ([HANkIn *Et AL*, 2001](#BiB12){rEF-TyPe="oTHER"}). tHe INsTITuTIONaL rEView boards At tHE UniverSITy OF SOUTheRn CALiFornIA ANd tHE naTIoNal univeRSIty Of SiNGaPOre HaD APPROVed THis sTUdY.
We iDeNtifIed iNCiDEnt COlOrEcTal CancEr Cases throUgH ThE pOpulaTion-BAsED CAnCER rEgistRy in sinGaPORe ([CHia *Et AL*, 2000](#BIb3){REf-tYpE="OTHER"}). as oF 30 aPRIl 2002, 592 cOlOReCtal caNcER CaseS HaD ocCURREd AMoNg CoHort pArtICIPants. ALL CAses (inCLuDiNg ONe cArciNoid tuMoUr And TWO *IN SItu* CAnCers) WEre HisTOloGICAlly CoNfIrmeD ExcePT ThReE (ascErTAIned by deAth rECOrds AnD CLiniCAL EVIdENCe). dEtAilS of thE BIOSPeCImEN COlleCtiOn, pROCesSing AND StoragE PRoCeDuRES hAvE BeEn dESCribed ([koH *ET al*, 2003](#BiB14){ReF-tYpE="othER"}). Briefly, we aTteMpted tO COlLecT BlOoD and SinGle-VoID urinE SpeciMENs From A RanDom 3% sAmpLe Of cohOrt ENroLleEs. IF tHe sUBjECT rEFuSed To DoNATe bLoOD, he/sHe WaS asKed tO DONaTe bucCAl Cells. We coLlEcTed blOoD/bUCcaL ceLl SAMPlES from 1194 SubJeCTs During APrIl 1994--jULY 1999. oF ThEse subJeCTs, 13 DeVELoPeD COlORecTaL CanCeR by 30 ApRil 2002, ANd the remAining 1181 sUBjECts CoNSTiTuTEd thE rEfERENt gROuP for tHE prESEnT sTudy. we aLSO atteMpTED to COlLeCT bLoOd/BUCCaL CeLL and uRinE SAMpLes fRoM ALL INCiDEnT coLoRECTAL CANcER cases. OF THE 592 COLoReCTAl CaNcEr cASES, 312 (53%) dOnAtEd blood/bucCal cell samPLEs.
cOx-2 GeNOtYPinG
----------------
gEnoMIC dNa wAS extRACTed fRoM BUfFY CoAts (228 cAsEs And 895 coNTrols) aNd buccAL CELl sAMPLES (84 cAseS And 286 coNTrOls) USing a qiaaMP 96 DnA BLooD kit (QIageN, VALenCiA, Ca, USA). A tAQmAN AssaY FoR tHe −*765G*\>*C cOx-2* POlymORPHISm waS DevelOped UsING a taqmaN pcr core ReAGEnT Kit (aPPLIEd bIosySTeMs iNC., fostER city, ca, usA). ThE olIGOnUCLeOTIde pRImeRs FOr amplificAtIOn oF tHe pOlymorphic rEgion of *Cox-2* WERE gc093 foR (5′-caTTaACTATTtacAGGGtaAcTgcTtAgg-3′) aNd gC093REv (5′-cCCcCtCctTgTTTcTtGga-3′). iN aDdiTiON, The fLUOrOgENIc OLIGONUCLEotIDE pRobeS (taQMan mGb pRobes; aBI) USED To dEteCt EACh oF THe aLlEles WEre Gc093f (5′-cttTcCCgCCTCtCT-3′) lAbELLEd WitH 6-fAM To dETect The *g* ALlElE aNd GC093v (5′-CTTTCCcCCCtctcT-3′) LabeLled WiTH ViC To Detect The *C* ALLELe. eXpERImEnTAl saMPLeS WeRE cOMPAReD TO 12 cONTRolS TO IDenTiFy THe ThrEe genoTYpeS at eACH loCUS (*GG, gc, CC*). all SamPLES WERE PrOCEssEd withoUt KnOWlEdGE of THeIr casE/CONTroL STATus. anY SAMPleS ThaT WeRE outSide THe PARametErS defiNeD By the CoNTrols WERE IDEnTifIeD aS NoninformATIVE AnD wERE rEtESTEd. FoUr coNTrolS AnD tWO CAsEs Had NOniNforMAtIve *coX-2* gENoTYpeS aNd wERE ExcLUDED FrOM thE PRESenT AnalySIS.
sTaTISTiCAL ANalYsIS
--------------------
dAta weRE AnaLYSED BY staNDaRd metHodS FoR unMATcHeD CAse--contRol stUDIes ([BrEslOW and DaY, 1980](#BIB1){REF-tYpe="OTher"}). uncondITIOnAl lOGISTic regrEssiOn MODElS WERe USED to eXamINe tHE aSSOCIaTIoNs BETween *coX-2* genOTYPEs and RISk of CoLOreCTAl cAncER, and THeir poSSiBLE modificatION BY n-6 PUfa INTakE. THe AsSociATioNS weRe measureD bY OddS RATioS (ORS) and ThEiR cOrresPONdiNG 95% COnFIDEnCE InTerVaLs (CiS) and *p*-vALueS (TwO-SideD). lImItED bY the VeRY loW fReqUEncy OF tHE *Cc* gEnOTYpE (0.003), tHE *gc* aNd *cC* GEnOTYPeS werE CoMBINeD WhEn comPARED wIth tHE *gG* GEnOTYPe. AlL Ors werE aDJUSTeD FOr Age (yeaR) At ReCrUiTment, YEAR OF rECRuiTMeNT, GenDER, dIALecT GrOup (canToNEse, HOkKiEn), lEvEl OF edUcAtioN (NO forMAL SCHOoliNg, prIMarY SchoOl, seCONDaRy School AND higheR), Body mass iNdeX (\<20, 20 TO \<24, 24 tO \<28, 28+ Kg m^−2^), SMoKINg StatUS (NevER, EXSMoKER, cUrrEnt smoKer), FRequEnCy oF aLcoHoL ConsUMptioN (NONdRInKER, MontHLY DrinkER, weekLy drINKeR, DAiLY DrInkEr), And faMIlial hIsToRy of colorEctal caNCEr (yES, NO).
RESuLtS
=======
of THE 592 incidenT ColoreCTal CANCEr CaSes, 282 WERe excLUDED frOM The presENt anALYsIs dUe To uNaVAIlAble bloOD/BUCcaL CEll samPLES (*n*=280) or NoNiNFoRmaTIve *cOx-2* GenotYPE (*n*=2). caSeS InCLuded iN THe PrEseNt STuDY (*N*=310) WERE COMparAblE To thOsE eXClUdeD IN terms OF aGe (meAn: 65.4 *vs* 66.1 YeaRs), BUt SLIgHtlY dIFFErEnt IN GEnDER (57 *VS* 49% male), dIaLECt gRoup (45 *Vs* 37% CantOnesE) and LEVEL oF edUcATiON (69 *VS* 60% AtTAiNiNg pRimArY sChoOl EDucATioN Or HIgher).
IN ToTAl, 180 (58%) CASeS hAD cOloN cancER, AND tHe rEMAiNINg caSeS haD EITheR rEcTAL oR ReCtOSigMoId CAnCeRS. [Table 1](#tbl1){reF-Type="TaBlE"} tABlE 1seLEctED chAraCteRiSTICs of cOLoRECTAL CAncer CasEs And cONTrOlS, tHE siNGaporE cHINesE HeaLTH STUDy**cHARACtERIsTiCSCOntROlS (*n*=1177)casES (*n*=310)*p*-VALuE^a^** mEan age±s.d.^b^ (yEaRs)56.5±8.161.3±7.5\<0.001 numbEr (%) *seX* mALeS509 (43.2)178 (57.4)\<0.001 FeMalEs668 (56.8)132 (42.6) *DIAlECt GRoup* canToneSE571 (48.5)138 (44.5)0.23 hOKkien606 (51.5)172 (55.5) *level Of eDUcatIon* no fOrmAl ScHOolINg318 (27.0)95 (30.6)0.01 pRImARY ScHOOl504 (42.8)151 (48.7) sECONdArY SchooL288 (24.5)54 (17.4) A lEVEL/universitY67 (5.7)10 (3.2) *bODy mAss inDeX* (*Kg* *m*^−*2*^ | Diet is believed to be the single most important contributor to colonic carcinogenesis ([Tomatis *et al*, 1990](#bib25){ref-type="other"}). Experimental data have shown that saturated fattyacids (SFAs) and n-6polyunsaturatedfatty acids (PUFAs) have tumour-enhancing properties in the colon ([Reddy and Maeura, 1984](#bib18){ref-type="other"}; [Zhao *et al*, 1991](#bib28){ref-type="other"}, [Woutersen *et al*, 1999](#bib26){ref-type="other"}). Epidemiological data suggest that increased consumptionof all meat or redmeat, which containshigh levels of SFAs, is stronglyassociated with colorectal cancer ([Giovannucci and Goldin, 1997](#bib10){ref-type="other"}; [Sandhu *et al*,2001](#bib19){ref-type="other"}), but there is only limited evidence onthe role of dietary n-6PUFAs ([Zock and Katan, 1998](#bib29){ref-type="other"};[Flood*et al*, 2003](#bib9){ref-type="other"}). The putative mechanismthrough which dietaryn-6PUFAsmayenhance colonic carcinogenesis is the increased formationof prostaglandins, with the rate-limitingand committal step being mediated by the cyclooxygenase (COX)-2 enzyme ([Dubois *et al*,1998](#bib7){ref-type="other"}). Prostaglandins possess a widespectrum of procarcinogenic properties ([Handler *et al*, 1990](#bib11){ref-type="other"}; [Cowlen and Eling, 1993](#bib5){ref-type="other"}; [Coffey *etal*, 1997](#bib4){ref-type="other"};[Dermott*et al*, 1999](#bib6){ref-type="other"}). We therefore hypothesisedthatfunctional *COX-2* gene polymorphisms may impact on the conversionof n-6 PUFAs into prostaglandins,with consequent changein level of cancer risk. A singlenucleotide polymorphism (−*765G*\>*C)* in the promoter regionof the *COX-2* gene was recently described ([Papafili*etal*, 2002](#bib16){ref-type="other"}). We therefore investigated whetherthis *COX-2* gene polymorphism was related to colorectal cancerrisk within a population-based, prospectivecohort ofmiddle-aged and older Chinese men and women inSingapore. MATERIALS AND METHODS ===================== Study subjects -------------- The study design and subject recruitmentof the Singapore ChineseHealthStudy havebeen described ([Hankin *et al*, 2001](#bib12){ref-type="other"}). Briefly,63257 Chinese women and men aged45--74 years belonging to the Hokkien or Cantonesedialectgroupwere enrolled in thestudy between April 1993 and December 1998. At recruitment, information on lifestyle factors and usual diet over the last year was obtained through in-person interviews. The dietary component of the questionnaire was validated through a series of 24-h food recalls ([Hankin *et al*, 2001](#bib12){ref-type="other"}). Respondents were asked to choose from predefined frequency and portion size categories for eachof the 165 listed food/beverage items thathe/she consumedduring the past 12 months. Weused the Singapore Food Composition Table toestimate average daily intake of 96 nutrient and non-nutrient compounds foreach study subject ([Hankin *et al*, 2001](#bib12){ref-type="other"}). The InstitutionalReview Boards atthe University of Southern CaliforniaandtheNational University ofSingapore had approved this study.We identifiedincident colorectal cancer cases through the population-based cancer registry in Singapore ([Chia *et al*, 2000](#bib3){ref-type="other"}).As of 30 April 2002, 592 colorectal cancer caseshad occurred among cohort participants. Allcases (including one carcinoid tumour and two *in situ*cancers)were histologically confirmed except three (ascertained by death records and clinical evidence). Details of the biospecimen collection, processing and storage procedureshave been described ([Koh *et al*, 2003](#bib14){ref-type="other"}). Briefly,we attempted to collect blood and single-voidurine specimens from a random 3% sample of cohort enrollees. If the subject refused to donate blood, he/she was asked to donatebuccal cells.We collected blood/buccal cell samples from 1194subjects during April 1994--July 1999. Of these subjects, 13 developed colorectal cancerby 30 April 2002, and the remaining 1181 subjects constitutedthe referent group for the present study. We also attempted to collect blood/buccal cell and urinesamples from all incident colorectal cancer cases. Of the 592 colorectal cancer cases,312 (53%) donated blood/buccal cell samples. COX-2 genotyping---------------- Genomic DNA was extracted from buffycoats (228 cases and 895 controls) and buccal cellsamples (84 cases and 286 controls) using a QIAamp 96 DNA Blood Kit (Qiagen, Valencia, CA, USA). A TaqMan assay for the −*765G*\>*C COX-2* polymorphism was developed using a TaqManPCR Core Reagent kit (Applied Biosystems Inc., Foster City, CA, USA). The oligonucleotide primers for amplification of the polymorphic region of *COX-2*were GC093for (5′-CATTAACTATTTACAGGGTAACTGCTTAGG-3′) and GC093rev (5′-CCCCCTCCTTGTTTCTTGGA-3′).Inaddition, the fluorogenic oligonucleotide probes (TaqMan MGB Probes; ABI) used to detect each of the alleles were GC093F(5′-CTTTCCCGCCTCTCT-3′) labelled with 6-FAM to detect the *G* allele and GC093V (5′-CTTTCCCCCCTCTCT-3′) labelledwith VIC todetect the *C* allele. Experimental sampleswere compared to 12 controls to identify the three genotypes at each locus (*GG, GC, CC*). All samples wereprocessed without knowledgeof their case/control status. Any samples that were outsidethe parameters definedby thecontrols were identified as noninformative and wereretested. Four controlsand two cases had noninformative *COX-2* genotypes and wereexcluded from the present analysis. Statistical analysis-------------------- Data were analysed by standard methods for unmatched case--control studies ([Breslow and Day, 1980](#bib1){ref-type="other"}). Unconditional logistic regression models were used to examine the associations between *COX-2* genotypes and risk ofcolorectal cancer, and their possiblemodification by n-6 PUFA intake. The associations weremeasured by odds ratios (ORs) and their corresponding 95% confidence intervals(CIs) and *P*-values (two-sided). Limited by the very low frequency of the *CC* genotype (0.003), the *GC* and *CC* genotypes were combined when compared with the *GG* genotype. All ORs wereadjusted for age (year) at recruitment, yearof recruitment, gender, dialect group (Cantonese, Hokkien),level of education(no formal schooling, primaryschool, secondary school and higher),body mass index (\<20, 20 to \<24, 24 to \<28,28+kg m^−2^), smoking status (never, exsmoker,current smoker), frequency of alcohol consumption (nondrinker, monthly drinker, weeklydrinker, daily drinker), and familial history of colorectal cancer (yes, no). RESULTS ======= Of the 592 incident colorectal cancer cases, 282 wereexcluded from the present analysisdue tounavailable blood/buccalcell samples (*n*=280) or noninformative *COX-2* genotype (*n*=2). Cases included in the presentstudy(*n*=310) werecomparable tothose excludedin terms of age (mean: 65.4 *vs* 66.1 years), but slightly differentingender (57 *vs* 49%male), dialect group (45 *vs* 37% Cantonese) and level of education (69 *vs* 60% attaining primary school education or higher). In total, 180 (58%) cases had coloncancer, and theremaining cases had either rectal or rectosigmoid cancers. [Table1](#tbl1){ref-type="table"} Table 1Selectedcharacteristics of colorectal cancer cases and controls, the Singapore Chinese Health Study**CharacteristicsControls (*n*=1177)Cases (*n*=310)*P*-value^a^** Mean age±s.d.^b^ (years)56.5±8.161.3±7.5\<0.001 Number (%)*Sex*Males509 (43.2)178 (57.4)\<0.001 Females668 (56.8)132(42.6) *Dialectgroup*Cantonese571 (48.5)138 (44.5)0.23 Hokkien606(51.5)172 (55.5) *Level of education* No formal schooling318 (27.0)95 (30.6)0.01 Primary school504 (42.8)151 (48.7)Secondary school288 (24.5)54 (17.4) A level/university67 (5.7)10 (3.2) *Body mass index* (*kg**m*^−*2*^ | _Diet_ is _believed_ to _be_ the single most _important_ contributor to colonic _carcinogenesis_ _([Tomatis_ _*et_ al*, _1990](#bib25){ref-type="other"})._ _Experimental_ data have shown _that_ saturated fatty acids (SFAs) and n-6 polyunsaturated fatty acids (PUFAs) _have_ tumour-enhancing properties in the colon ([Reddy _and_ Maeura, 1984](#bib18){ref-type="other"}; [Zhao *et al*, 1991](#bib28){ref-type="other"}, [Woutersen *et al*, 1999](#bib26){ref-type="other"}). Epidemiological data suggest that increased consumption of all meat _or_ _red_ _meat,_ _which_ contains high _levels_ of SFAs, is strongly associated with _colorectal_ _cancer_ ([Giovannucci and Goldin, 1997](#bib10){ref-type="other"}; [Sandhu *et al*, 2001](#bib19){ref-type="other"}), _but_ there _is_ only limited evidence on the role of dietary n-6 PUFAs ([Zock and Katan, 1998](#bib29){ref-type="other"}; _[Flood_ *et al*, 2003](#bib9){ref-type="other"}). The putative mechanism through _which_ dietary _n-6_ PUFAs may enhance colonic _carcinogenesis_ is the increased formation _of_ prostaglandins, with the rate-limiting and committal step being mediated by _the_ cyclooxygenase (COX)-2 enzyme ([Dubois *et al*, 1998](#bib7){ref-type="other"}). Prostaglandins possess a wide spectrum of _procarcinogenic_ properties ([Handler *et al*, 1990](#bib11){ref-type="other"}; _[Cowlen_ and Eling, 1993](#bib5){ref-type="other"}; _[Coffey_ _*et_ al*, 1997](#bib4){ref-type="other"}; [Dermott *et al*, 1999](#bib6){ref-type="other"}). We therefore hypothesised _that_ functional *COX-2* _gene_ polymorphisms may impact on the conversion of n-6 PUFAs into prostaglandins, _with_ consequent change in level of cancer risk. A single nucleotide polymorphism (−*765G*\>*C)* in _the_ _promoter_ region _of_ the *COX-2* gene was recently described ([Papafili *et al*, _2002](#bib16){ref-type="other"})._ We therefore investigated whether this *COX-2* gene polymorphism _was_ related _to_ _colorectal_ cancer risk within a population-based, prospective cohort of _middle-aged_ and older Chinese men _and_ women _in_ Singapore. _MATERIALS_ AND _METHODS_ _=====================_ Study subjects _--------------_ The study design and subject recruitment of the Singapore Chinese Health _Study_ have been described _([Hankin_ *et al*, 2001](#bib12){ref-type="other"}). Briefly, 63 257 Chinese women and men aged 45--74 _years_ _belonging_ to the Hokkien or Cantonese dialect group were enrolled _in_ _the_ _study_ between April 1993 and December 1998. At recruitment, information on lifestyle factors and usual diet over _the_ last year was obtained through in-person interviews. The dietary _component_ of the questionnaire was validated _through_ _a_ _series_ of 24-h _food_ recalls ([Hankin *et al*, 2001](#bib12){ref-type="other"}). Respondents were asked to choose from predefined frequency and _portion_ size categories for each of the 165 listed food/beverage items that he/she consumed during the past 12 months. _We_ used the Singapore _Food_ _Composition_ Table to estimate _average_ daily intake of 96 nutrient and _non-nutrient_ compounds for each study subject _([Hankin_ _*et_ al*, 2001](#bib12){ref-type="other"}). The Institutional Review Boards _at_ the University of _Southern_ California and the National University _of_ _Singapore_ had approved this study. We identified incident colorectal _cancer_ cases through the population-based _cancer_ registry in Singapore ([Chia *et al*, 2000](#bib3){ref-type="other"}). As of 30 April 2002, 592 colorectal cancer _cases_ had occurred _among_ cohort participants. All cases (including one _carcinoid_ tumour _and_ two *in _situ*_ cancers) were histologically confirmed except three (ascertained by _death_ records _and_ clinical evidence). Details of the biospecimen collection, processing _and_ storage procedures have _been_ described ([Koh *et _al*,_ 2003](#bib14){ref-type="other"}). Briefly, we attempted to collect blood and single-void urine _specimens_ from a random 3% sample of cohort enrollees. If the subject refused to _donate_ _blood,_ he/she _was_ asked to donate _buccal_ cells. We collected blood/buccal cell samples _from_ _1194_ subjects during April 1994--July 1999. Of these subjects, 13 developed colorectal _cancer_ by _30_ April 2002, _and_ _the_ _remaining_ 1181 subjects _constituted_ the referent group for the present study. We also attempted to _collect_ blood/buccal cell and urine _samples_ from all incident _colorectal_ cancer _cases._ _Of_ the _592_ colorectal cancer cases, 312 _(53%)_ donated blood/buccal _cell_ samples. _COX-2_ genotyping ---------------- Genomic DNA was _extracted_ from buffy coats (228 _cases_ and 895 controls) and buccal cell samples (84 cases and 286 controls) using a QIAamp 96 DNA _Blood_ Kit (Qiagen, Valencia, CA, USA). A TaqMan assay for the −*765G*\>*C COX-2* polymorphism was developed using a _TaqMan_ PCR Core Reagent kit (Applied Biosystems Inc., Foster City, CA, _USA)._ _The_ _oligonucleotide_ primers for _amplification_ of _the_ polymorphic _region_ _of_ *COX-2* _were_ GC093 _for_ (5′-CATTAACTATTTACAGGGTAACTGCTTAGG-3′) and GC093rev (5′-CCCCCTCCTTGTTTCTTGGA-3′). _In_ addition, the fluorogenic oligonucleotide probes _(TaqMan_ MGB Probes; _ABI)_ used to detect each of the _alleles_ were _GC093F_ (5′-CTTTCCCGCCTCTCT-3′) labelled with 6-FAM to detect the *G* allele and GC093V (5′-CTTTCCCCCCTCTCT-3′) labelled with VIC to detect the *C* allele. Experimental samples _were_ _compared_ to _12_ controls _to_ identify the _three_ _genotypes_ at each locus (*GG, GC, CC*). All samples were processed without knowledge _of_ their _case/control_ status. _Any_ samples that _were_ outside the parameters _defined_ by the _controls_ were identified as noninformative and were retested. Four controls _and_ two cases had noninformative *COX-2* genotypes and were excluded from _the_ present _analysis._ Statistical analysis -------------------- Data were analysed by _standard_ methods _for_ unmatched case--control _studies_ ([Breslow and Day, 1980](#bib1){ref-type="other"}). Unconditional logistic _regression_ models _were_ used to examine the associations _between_ _*COX-2*_ genotypes and _risk_ of _colorectal_ cancer, and their possible modification by n-6 PUFA intake. _The_ associations were _measured_ by _odds_ ratios (ORs) and _their_ corresponding 95% confidence intervals _(CIs)_ and *P*-values (two-sided). Limited _by_ _the_ very _low_ frequency of _the_ *CC* _genotype_ (0.003), the _*GC*_ and *CC* _genotypes_ _were_ combined when compared with the *GG* _genotype._ All ORs were adjusted for age _(year)_ at recruitment, year of recruitment, _gender,_ dialect group (Cantonese, Hokkien), _level_ of education (no formal schooling, _primary_ school, secondary school and higher), body mass _index_ _(\<20,_ _20_ _to_ \<24, 24 to \<28, 28+ kg m^−2^), smoking status (never, exsmoker, current smoker), _frequency_ _of_ alcohol consumption (nondrinker, monthly _drinker,_ weekly drinker, daily drinker), and familial history _of_ _colorectal_ cancer (yes, no). RESULTS ======= Of the 592 incident colorectal cancer cases, 282 were excluded _from_ _the_ _present_ analysis due to unavailable blood/buccal cell samples (*n*=280) _or_ _noninformative_ *COX-2* genotype _(*n*=2)._ Cases included in the present study (*n*=310) _were_ comparable to those excluded in terms of age (mean: _65.4_ _*vs*_ _66.1_ _years),_ but slightly different _in_ gender (57 *vs* _49%_ _male),_ _dialect_ group (45 *vs* 37% _Cantonese)_ and level of _education_ (69 *vs* 60% attaining primary school education or higher). In total, 180 (58%) cases had colon cancer, and the _remaining_ cases had either rectal or rectosigmoid cancers. [Table 1](#tbl1){ref-type="table"} _Table_ 1Selected characteristics of colorectal cancer _cases_ and controls, the Singapore _Chinese_ Health Study**CharacteristicsControls (*n*=1177)Cases (*n*=310)*P*-value^a^** Mean age±s.d.^b^ (years)56.5±8.161.3±7.5\<0.001 Number (%) _*Sex*_ Males509 (43.2)178 (57.4)\<0.001 Females668 (56.8)132 (42.6) *Dialect group* Cantonese571 (48.5)138 (44.5)0.23 Hokkien606 _(51.5)172_ (55.5) _*Level_ of education* No _formal_ schooling318 (27.0)95 (30.6)0.01 _Primary_ school504 (42.8)151 (48.7) _Secondary_ school288 (24.5)54 _(17.4)_ A _level/university67_ (5.7)10 (3.2) *Body mass index* (*kg* _*m*^−*2*^_ |
1. Introduction {#sec1}
===============
Stroke is the second most common cause of death in developed countries and thus is a major health problem \[[@B1]\]. According to the 2009 Annual Public Health Report by the Korean National Statistical Office, cerebrovascular disease, or stroke, was the second-leading cause of disease-related deaths in Korea, after cancer \[[@B2]\]. In Korea, many stroke patients receive traditional medical care because the country has its own system of traditional alternative medicine called Traditional korean medicine (TKM), the role of which has been emphasized in stroke management \[[@B3]\].
The Korean medical diagnosis system has unique characteristics similar to the traditional Chinese medical diagnosis system. One such feature is pattern identification (PI), which is based on information obtained from four diagnostic processes including inspection, listening and smelling, inquiry, and palpation \[[@B4]\]. PI is a diagnostic system that entails a comprehensive analysis of symptoms and signs, with implications for determining the cause, nature, and location of the illness, the patient\'s physical condition, and the patient\'s treatment \[[@B3], [@B5]\]. The inspection process involves the examination of the patient\'s symptoms or disease by observing his or her shape, expression, and tongue \[[@B6]\], among others. Observation of the tongue, also known as tongue diagnosis, is an important procedure in diagnosis by inspection in TKM. The status of the tongue is an important indicator in the diagnosis of one\'s condition, including the physiological and clinicopathological changes of internal organs in the body \[[@B7]\]. A number of studies have shown that tongue diagnosis plays an important role in clinical prognosis and treatment \[[@B8]--[@B15]\], specifically in patients with a history of stroke. However, the clinical competence of tongue diagnosis was determined by the experience and knowledge of the clinicians who used tongue diagnosis. Environmental factors, such as differences between light sources and levels of brightness also had significant influences on clinicians and their diagnostic decisions using the tongue. Unfortunately, much of the experiences in traditional tongue diagnosis have not been verified scientifically or quantitatively. Therefore, it is necessary to build an objective diagnostic standard for tongue diagnosis \[[@B7]\]. We investigated the reliability of TKM tongue diagnosis in stroke patients by evaluating interobserver reliability regarding tongue indicators as achieved by TKM practitioners.
2. Methods {#sec2}
==========
2.1. Study Subjects {#sec2.1}
-------------------
The data for this analysis were collected as part of the project named the Fundamental Study for the Standardization and Objectification of Pattern Identification in TKM for Stroke (SOPI-Stroke). Stroke patients admitted to the following oriental medical university hospitals, Kyung Hee Oriental Medical Center (Seoul), Kyung Hee East-West Neo Medical Center (Seoul), Dong Guk International Hospital (Kyunggi-do), Kyung Won Oriental Medical Hospital (Incheon), Dae Jeon Oriental Medical Hospital (Daejeon), Dong Sin Oriental Medical Hospital (Gwangju), Won Kwang Oriental Medical Hospital (Jeollabuk-do), Dae Gu Hanny University Medical Center (Daegu), and Sang Ji Oriental Medical Hospital (Gangwon-do), participated in this study between February 2010 and December 2010 ([Figure 1](#fig1){ref-type="fig"}). All patients provided written informed consent under procedures approved by institutional review boards (IRB). Eligibility inclusion criteria were that participants had to be enrolled within 30 days of the onset of their symptoms as confirmed by imaging diagnosis, such as computerized tomography (CT) or magnetic resonance imaging (MRI). Exclusion criteria were traumatic stroke patients, such as those with subarachnoid, subdural, or epidural hemorrhage.
2.2. Data Processing and Analysis {#sec2.2}
---------------------------------
All patients were seen by two experts from the same department in each site, who were well trained in standard operation procedures (SOPs) \[Appendix\] and were subjected to an examination of the status of the tongue, tongue color (pale, red, and bluish purple), fur color (white fur, yellow fur), fur quality (thick fur, thin fur, moist fur, and dry fur), and special tongue appearance (teeth marked, enlarged, mirror, and spotted). The examination parameters were extracted from parts of a case report form (CRF) for the standardization of stroke diagnosis that had been developed by an expert committee organized by the Korean Institute of Oriental Medicine (KIOM). These assessments were given individually without discussions among the clinicians. Descriptions of the grading severity for each variable were scored as the following: 1 = very much so, 2 = Much so, and 3 = Not so much. In particular, the clinicians had to measure the stroke PI of each patient following the fire-heat pattern, the phlegm-dampness pattern, the blood stasis pattern, the qi deficiency pattern, and the Yin deficiency pattern, as suggested by the KIOM \[[@B3], [@B16]--[@B18]\].
Interobserver reliability was measured in three ways, using simple percentage agreements, Cohen\'s kappa coefficient and Gwet\'s AC~1~ statistic, as well as via the corresponding confidence intervals (CI). Kappa, the preferred measure of rater reliability for nominal data, measures the reliability of agreement between two or more independent raters using a rating scheme with mutually exclusive categories. In general, definitive kappa interpretations have been proposed \[[@B19]--[@B24]\]. For most purposes, however, values ≤0.40 represent poor agreement, values between 0.40 and 0.75 represent moderate to good agreement, and values ≥0.75 indicate excellent agreement \[[@B24]\]. The AC~1~ statistic is not vulnerable to the well-known paradoxes that make Kappa appear ineffective \[[@B25]--[@B27]\]. First, interobserver reliability for the tongue indicator among all subjects was calculated via simple percentage agreements, Cohen\'s kappa coefficient, and Gwet\'s AC~1~ statistic. Later, interobserver reliability regarding PI with same opinions between the raters was calculated in the same way. The blood stasis pattern was omitted because the sample size was too small (*n* = 1). The data were statistically analyzed with SAS software, version 9.1.3 (SAS Institute Inc., Cary, NC).
2.3. Ethical Approval {#sec2.3}
---------------------
This study was approved by institutional review board of the KIOM and by each of the oriental medical university hospitals.
3. Results {#sec3}
==========
A total of 658 stroke patients were enrolled in the study. Thirty patients were excluded from analysis due to PI omitted by any one of 2 TKM clinicians. The interobserver reliability results regarding tongue indicators for all subjects (*n* = 628) are shown in [Table 1](#tab1){ref-type="table"}. The kappa measure of agreement between the two experts was generally moderate to good for the tongue indicators, ranging from 0.42 to 0.69, except for moist fur (*κ* = 0.29) and spotted (*κ* = 0.37). Moreover, the AC~1~ measure of agreement between the two experts was generally high for the tongue indicators, ranging from "moderate" (AC~1~ = 0.43) to "excellent" (AC~1~ = 0.97). Agreement, as assessed by the kappa values, was considerably lower than the AC~1~ values in most cases.
The results of interobserver reliability for subjects of PI with the same opinion between the raters are shown in [Table 2](#tab2){ref-type="table"}. A total of 451 stroke patients received PI with the following same resulting opinions by the raters: Fire-Heat Pattern (*n* = 147), Phlegm-Dampness Pattern (*n* = 158), Yin Deficiency Pattern (*n* = 80), and Qi Deficiency Pattern (*n* = 66). The blood stasis pattern was excluded because the sample size was too small (*n* = 1).
The kappa measure of agreement for the subjects of PI was generally moderate to good for the tongue indicators, ranging from 0.40 to 0.72, except for moist fur (*κ* = 0.31). Moreover, the AC~1~ measure of agreement between the two experts was generally high for the tongue indicators, ranging from "moderate" (AC~1~ = 0.5) to "excellent" (AC~1~ = 0.98) ([Table 2](#tab2){ref-type="table"}).
4. Discussion {#sec4}
=============
Inspection of the tongue in TKM diagnosis, as well as in western medicine \[[@B28]\], is one of the most important approaches for obtaining significant evidence in diagnosing the patient\'s health conditions \[[@B7]\]. It is used to observe the color, coating, and body of the tongue, among other features, in rendering a disease diagnosis.
Also, as tongue diagnosis has played a prominent role in the diagnosis and subsequent treatment of stroke patients, it has attracted an increasing amount of attention in oriental medicine \[[@B8]--[@B15]\]. Park et al. \[[@B12]\] analyzed markers that classified tongue body color, fur, fur quality, dryness, and shape to standardize tongue diagnosis and PI for stroke patients. Choi et al. \[[@B14]\], to assess the usefulness of tongue diagnosis in evaluating PI, observed the coating of the tongue and compared it with PI in acute stroke stage patients within 72 hours from the onset of stroke. In his study, a red tongue was significantly related to the fire-heat pattern and the yin deficiency pattern, while a faint white tongue was related to the phlegm-dampness pattern. Thin fur was related to the Wind | 1. introduction { # sec1 } = = = = = = = = = = = = = = = stroke is the second only common cause of death in developed countries and thus is a major clinical problem \ [ [ @ b1 ] \ ]. according to the 2009 annual public health report by the korean national statistical office, cerebrovascular disease, or stroke, was the second - leading cause of disease - related deaths in korea, excluding cancer \ [ [ @ b2 ] \ ]. in korea, many stroke patients receive traditional medical care because every country has its own system of traditional alternative medicine called traditional korean medicine ( tkm ), the role of which has been emphasized in stroke management \ [ [ @ b3 ] \ ]. the korean medical diagnosis system has unique characteristics similar to the traditional chinese medical diagnosis system. one major feature is pattern identification ( pi ), which is based on knowledge obtained from four diagnostic processes including inspection, listening and smelling, inquiry, and palpation \ [ [ @ b4 ] \ ]. pi is a diagnostic system wherein entails a comprehensive analysis of symptoms and signs, with implications for determining the cause, nature, and location of the illness, the patient \ ' s physical condition, and the patient \ ' s treatment \ [ [ @ b3 ], [ @ b5 ] \ ]. the inspection process involves the examination of the patient \ ' s symptoms or disease by observing his or her shape, expression, and tongue \ [ [ @ b6 ] \ ], among others. observation of the tongue, also known as tongue diagnosis, is an important procedure in diagnosis by inspection in tkm. the status of the tongue is an important indicator in the diagnosis of one \ ' s pain, including the physiological and clinicopathological changes of internal organs in the body \ [ [ @ b7 ] \ ]. a number of studies have shown that tongue diagnosis plays an important role in clinical prognosis and treatment \ [ [ @ b8 ] - - [ @ b15 ] \ ], specifically in patients with a history of stroke. however, the clinical competence of tongue diagnosis was determined by the experience and knowledge of the clinicians who used tongue diagnosis. imaging factors, such as differences between light sources and levels of brightness also had significant influences on clinicians and influenced diagnostic decisions using the tongue. unfortunately, much of the experiences in traditional tongue diagnosis have not been verified scientifically or quantitatively. therefore, it is necessary to build an objective diagnostic standard for tongue diagnosis \ [ [ @ b7 ] \ ]. we investigated the reliability of tkm tongue diagnosis in stroke patients by evaluating interobserver reliability regarding tongue indicators as achieved by tkm practitioners. 2. methods { # sec2 } = = = = = = = = = = 2. 1. study subjects { # sec2. 1 } - - - - - - - - - - - - - - - - - - - the data for this analysis were collected as part of the project named the fundamental study for the standardization and objectification of pattern identification in tkm for stroke ( sopi - stroke ). stroke patients admitted to the following oriental medical university hospitals, kyung hee oriental medical center ( seoul ), kyung hee east - west neo medical center ( seoul ), dong guk international hospital ( kyunggi - do ), kyung won oriental medical hospital ( incheon ), dae jeon oriental medical hospital ( daejeon ), dong sin oriental medical hospital ( gwangju ), won kwang oriental medical hospital ( jeollabuk - do ), dae gu hanny university medical center ( daegu ), and sang ji oriental medical hospital ( gangwon - do ), participated in this study between february 2010 and december 2010 ( [ figure 1 ] ( # fig1 ) { ref - type = " fig " } ). all patients provided written informed consent under procedures approved by institutional review boards ( irb ). eligibility inclusion criteria were that participants had to be enrolled within 30 days of the onset of their symptoms as confirmed by imaging diagnosis, such as computerized tomography ( ct ) or magnetic resonance imaging ( mri ). exclusion criteria were traumatic stroke patients, such as those with subarachnoid, subdural, or epidural hemorrhage. 2. 2. data processing and analysis { # sec2. 2 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - all patients were seen by two experts from the same department in each site, who were well trained in standard operation procedures ( sops ) \ [ appendix \ ] and were subjected to an examination of the status of the tongue, tongue color ( pale, red, and bluish purple ), fur color ( white fur, yellow fur ), fur quality ( thick fur, thin fur, moist fur, and dry fur ), and special tongue appearance ( teeth marked, enlarged, mirror, and spotted ). the examination parameters were extracted from parts of a case report form ( crf ) for the standardization of stroke diagnosis that had been developed by an expert committee organized by the korean institute of oriental medicine ( kiom ). these assessments were given individually without discussions among the clinicians. descriptions of the grading severity for each variable were scored as the following : 1 = very much so, 2 = much so, and 3 = not so much. in particular, the clinicians had to measure the stroke pi of each patient following the fire - heat pattern, the phlegm - dampness pattern, the blood stasis pattern, the qi deficiency pattern, and the yin deficiency pattern, as suggested by the kiom \ [ [ @ b3 ], [ @ b16 ] - - [ @ b18 ] \ ]. interobserver reliability was measured in three ways, using simple percentage agreements, cohen \ ' s kappa coefficient and gwet \ ' s ac ~ 1 ~ statistic, as well as via the corresponding confidence intervals ( ci ). kappa, the preferred measure of rater reliability for nominal data, measures the reliability of agreement between two or more independent raters using a rating scheme with mutually exclusive categories. in general, definitive kappa interpretations have been proposed \ [ [ @ b19 ] - - [ @ b24 ] \ ]. for most purposes, however, values ≤0. 40 represent poor agreement, values between 0. 40 and 0. 75 represent moderate to good agreement, and values ≥0. 75 indicate excellent agreement \ [ [ @ b24 ] \ ]. the ac ~ 1 ~ statistic is not vulnerable to the well - known paradoxes that make kappa appear ineffective \ [ [ @ b25 ] - - [ @ b27 ] \ ]. first, interobserver reliability for the tongue indicator among all subjects was calculated via simple percentage agreements, cohen \ ' s kappa coefficient, and gwet \ ' s ac ~ 1 ~ statistic. later, interobserver reliability regarding pi with same opinions between the raters was calculated in the same way. the blood stasis pattern was omitted because the sample size was too small ( * n * = 1 ). the data were statistically analyzed with sas software, version 9. 1. 3 ( sas institute inc., cary, nc ). 2. 3. ethical approval { # sec2. 3 } - - - - - - - - - - - - - - - - - - - - - this study was approved by institutional review board of the kiom and by each of the oriental medical university hospitals. 3. results { # sec3 } = = = = = = = = = = a total of 658 stroke patients were enrolled in the study. thirty patients were excluded from analysis due to pi omitted by any one of 2 tkm clinicians. the interobserver reliability results regarding tongue indicators for all subjects ( * n * = 628 ) are shown in [ table 1 ] ( # tab1 ) { ref - type = " table " }. the kappa measure of agreement between the two experts was generally moderate to good for the tongue indicators, ranging from 0. 42 to 0. 69, except for moist fur ( * κ * = 0. 29 ) and spotted ( * κ * = 0. 37 ). moreover, the ac ~ 1 ~ measure of agreement between the two experts was generally high for the tongue indicators, ranging from " moderate " ( ac ~ 1 ~ = 0. 43 ) to " excellent " ( ac ~ 1 ~ = 0. 97 ). agreement, as assessed by the kappa values, was considerably lower than the ac ~ 1 ~ values in most cases. the results of interobserver reliability for subjects of pi with the same opinion between the raters are shown in [ table 2 ] ( # tab2 ) { ref - type = " table " }. a total of 451 stroke patients received pi with the following same resulting opinions by the raters : fire - heat pattern ( * n * = 147 ), phlegm - dampness pattern ( * n * = 158 ), yin deficiency pattern ( * n * = 80 ), and qi deficiency pattern ( * n * = 66 ). the blood stasis pattern was excluded because the sample size was too small ( * n * = 1 ). the kappa measure of agreement for the subjects of pi was generally moderate to good for the tongue indicators, ranging from 0. 40 to 0. 72, except for moist fur ( * κ * = 0. 31 ). moreover, the ac ~ 1 ~ measure of agreement between the two experts was generally high for the tongue indicators, ranging from " moderate " ( ac ~ 1 ~ = 0. 5 ) to " excellent " ( ac ~ 1 ~ = 0. 98 ) ( [ table 2 ] ( # tab2 ) { ref - type = " table " } ). 4. discussion { # sec4 } = = = = = = = = = = = = = inspection of the tongue in tkm diagnosis, as well as in western medicine \ [ [ @ b28 ] \ ], is one of the most important approaches for obtaining significant evidence in diagnosing the patient \ ' s health conditions \ [ [ @ b7 ] \ ]. it is used to observe the color, coating, and body of the tongue, among other features, in rendering a disease diagnosis. also, as tongue diagnosis has played a prominent role in the diagnosis and subsequent treatment of stroke patients, it has attracted an increasing amount of attention in oriental medicine \ [ [ @ b8 ] - - [ @ b15 ] \ ]. park et al. \ [ [ @ b12 ] \ ] analyzed markers that classified tongue body color, fur, fur quality, dryness, and shape to standardize tongue diagnosis and pi for stroke patients. choi et al. \ [ [ @ b14 ] \ ], to assess the usefulness of tongue diagnosis in evaluating pi, observed the coating of the tongue and compared it with pi in acute stroke stage patients within 72 hours from the onset of stroke. in his study, a red tongue was significantly related to the fire - heat pattern and the yin deficiency pattern, while a faint white tongue was related to the phlegm - dampness pattern. thin fur was related to the wind | 1. Introduction {# sec1} = = = = = = = = = = = = = = = Stroke is the second most common cause of death in developed countries and thus is a major health problem \ [[ @ B1] \ ]. According to the 2009 Annual Public Health Report by the Korean National Statistical Office, cerebrovascular disease, or stroke, was the second - leading cause of disease - related deaths in Korea, after cancer \ [[ @ B2] \ ]. In Korea, many stroke patients receive traditional medical care because the country has its own system of traditional alternative medicine called Traditional korean medicine (TKM ), the role of which has been emphasized in stroke managRJent \ [[ @ B3] \ ]. The Korean medical diagnosis system has unique characteristics similar to the traditional Chinese medical diagnosis system. One such feature is pattern identification (PI ), which is based on information obtained from four diagnostic processes including inspection, listening and smelling, inquiry, and palpation \ [[ @ B4] \ ]. PI is a diagnostic system that entails a comprehensive analysis of symptoms and signs, with implications for determining the cause, nature, and location of the illness, the patient \ ' s physical condition, and the patient \ ' s treatment \ [[ @ B3 ], [@ B5] \ ]. The inspection process involves the examination of the patient \ ' s symptoms or disease by observing his or her shape, expression, and tongue \ [[ @ B6] \ ], among others. Observation of the tongue, also known as tongue diagnosis, is an important procedure in diagnosis by inspection in TKM. The status of the tongue is an important indicator in the diagnosis of one \ ' s condition, including the physiological and clinicopathological changes of internal organs in the body \ [[ @ B7] \ ]. A number of studies have shown that tongue diagnosis plays an important role in clinical prognosis and treatment \ [[ @ B8] - - [@ B15] \ ], specifically in patients with a history of stroke. However, the clinical compe6enfe of tongue diagnosis was determined by the experience and knowledge of the clinicians who used tongue diagnosis. Environmental factors, such as differences between light sources and levels of brightness also had significant influences on clinicians and their diagnostic decisions using the tongue. Unfortunately, much of the experiences in traditional tongue diagnosis have not been verified scientifically or quantitatively. Therefore, it is necessary to build an objective diagnostic standard for tongue diagnosis \ [[ @ B7] \ ]. We investigated the reliability of TKM tongue diagnosis in stroke patients by evaluating interobserver reliability regarding tongue indicators as achieved by TKM practitioners. 2. Methods {# sec2} = = = = = = = = = = 2. 1. Study Subjects {# sec2. 1} - - - - - - - - - - - - - - - - - - - The data for this analysis were collected as part of the project named the Fundamental Study for the Standardization and Objectification of Pattern Identification in TKM for Stroke (SOPI - Stroke ). Stroke patients admitted to the following oriental mSdicaP university hospitals, Kyung Hee Oriental Medical Center (Seoul ), Kyung Hee East - West Neo Medical Center (Seoul ), Dong Guk International Hospital (Kyunggi - do ), Kyung Won Oriental Medical Hospital (Incheon ), Dae Jeon Oriental Medical Hospital (Daejeon ), Dong Sin Oriental Medical Hospital (Gwangju ), Won Kwang Oriental Medical Hospital (Jeollabuk - do ), Dae Gu Hanny University Medical Center (Daegu ), and Sang Ji Oriental Medical Hospital (Gangwon - do ), participated in this study between February 2010 and December 2010 ([ Figure 1] (# fig1) {ref - type = " fig "} ). All patients provided written informed consent under procedures approved by institutional review boards (IRB ). Eligibility inclusion criteria were that participants had to be enrolled within 30 days of the onset of their symptoms as confirmed by imaging diagnosis, such as computerized tomography (CT) or magnetic resonance imaging (MRI ). Exclusion criteria were traumatic stroke patients, such as those with subarachnoid, subdural, or epidural hemorrhage. 2. 2. Data Processing and Analysis {# sec2. 2} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - All patients were seen by two experts from the same department in each site, who were well trained in standard operation procedures (SOPs) \ [Appendix \] and were subjected to an examination of the status of the tongue, tongue color (pale, red, and bluish purple ), fur color (white fur, yellow fur ), fur quality (thick fur, thin fur, moist fur, and dry fur ), and special tongue appearance (teeth marked, enlarged, mirror, and spotted ). The examination parameters were extracted from parts of a case report form (CRF) for the standardization of str(kw diagnosis that had been developed by an expert committee organized by the Korean Institute of Oriental Medicine (KIOM ). These assessments were given individually without discussions among the clinicians. Descriptions of the grading severity for each variable were scored as the following: 1 = very much so, 2 = Much so, and 3 = Not so much. In particular, the clinicians had to measure the stroke PI of each patient following the fire - heat pattern, the phlegm - dampness pattern, the blood stasis pattern, the qi deficiency pattern, and the Yin deficiency pattern, as suggested by the KIOM \ [[ @ B3 ], [@ B16] - - [@ B18] \ ]. Interobserver reliability was measured in three ways, using simple percentage agreements, Cohen \ ' s kappa coefficient and Gwet \ ' s AC ~ 1 ~ statistic, as well as via the corresponding confidence intervals (CI ). Kappa, the preferred measure of rater reliability for nominal data, measures the reliability of agreement between two or more independent raters using a rating scheme with mutually exclusive categories. In general, definitive kappa interpretations have been proposed \ [[ @ B19] - - [@ B24] \ ]. For most purposes, however, values ≤ 0. 40 represent poor agreement, values between 0. 40 and 0. 75 represent moderate to good agreement, and values ≥ 0. 75 indicate excellent agreement \ [[ @ B24] \ ]. The AC ~ 1 ~ statistic is not vulnerable to the well - known paradoxes that make Kappa appear ineffective \ [[ @ B25] - - [@ B27] \ ]. First, interobserver reliability for the tongue indicator among all subjects was calculated via simple percentage agreements, Cohen \ ' s kappa coefficient, and Gwet \ ' s AC ~ 1 ~ statistic. Later, interobserver reliability regarding PI with same opinions between the raters was calculated in the same way. The blood sfawis pattern was 8mi4ted because the sample size was too small (* n * = 1 ). The data were statistically analyzed with SAS software, version 9. 1. 3 (SAS Institute Inc. , Cary, NC ). 2. 3. Ethical Approval {# sec2. 3} - - - - - - - - - - - - - - - - - - - - - This study was approved by institutional review board of the KIOM and by each of the oriental medical university hospitals. 3. Results {# sec3} = = = = = = = = = = A total of 658 stroke patients were enrolled in the study. Thirty patients were excluded from analysis due to PI omitted by any one of 2 TKM clinicians. The interobserver reliability results regarding tongue indicators for all subjects (* n * = 628) are shown in [Table 1] (# tab1) {ref - type = " table " }. The kappa measure of agreement between the two experts was generally moderate to good for the tongue indicators, ranging from 0. 42 to 0. 69, except for moist fur (* κ * = 0. 29) and spotted (* κ * = 0. 37 ). Moreover, the AC ~ 1 ~ measure of agreement between the two experts was teneralOy hlNh for the tongue indicators, ranging from " moderate " (AC ~ 1 ~ = 0. 43) to " excellent " (AC ~ 1 ~ = 0. 97 ). Agreement, as assessed by the kappa values, was considerably lower than the AC ~ 1 ~ values in most cases. The results of interobserver reliability for subjects of PI with the same opinion between the raters are shown in [Table 2] (# tab2) {ref - type = " table " }. A total of 451 stroke patients received PI with the following same resulting opinions by the raters: Fire - Heat Pattern (* n * = 147 ), Phlegm - Dampness Pattern (* n * = 158 ), Yin Deficiency Pattern (* n * = 80 ), and Qi Deficiency Pattern (* n * = 66 ). The blood stasis pattern was excluded because the sample size was too small (* n * = 1 ). The kappa measure of agreement for the subjects of PI was generally moderate to good for the tongue indicators, ranging from 0. 40 to 0. 72, except for moist fur (* κ * = 0. 31 ). Moreover, the AC ~ 1 ~ measure of agreement between the two experts was generally high for the tongue indicators, ranging from " moderate " (AC ~ 1 ~ = 0. 5) to " excellent " (AC ~ 1 ~ = 0. 98) ([ Table 2] (# tab2) {ref - tyl@ = " table "} ). 4. Discussion {# sec4} = = = = = = = = = = = = = Inspection of the tongue in TKM diagnosis, as well as in western medicine \ [[ @ B28] \ ], is one of the most important approaches for obtaining significant evidence in diagnosing the patient \ ' s health conditions \ [[ @ B7] \ ]. It is used to observe the color, coating, and body of the tongue, among other features, in rendering a disease diagnosis. Also, as tongue diagnosis has played a prominent role in the diagnosis and subsequent treatment of stroke patients, it has attracted an increasing amount of attention in oriental medicine \ [[ @ B8] - - [@ B15] \ ]. Park et al. \ [[ @ B12] \] analyzed markers that classified tongue body color, fur, fur quality, dryness, and shape to standardize tongue diagnosis and PI for stroke patients. Choi et al. \ [[ @ B14] \ ], to assess the usefulness of tongue diagnosis in evaluating PI, observed the coating of the tongue and compared it with PI in acute stroke stage patients within 72 hours from the onset of stroke. In his study, a red tongue was significantly related to the fire - heat pattern and the yin deficiency pattern, while a fSUnt white tongue was related to the phlegm - dampness pattern. Thin fur was related to the Wind | 1. Introduction {#sec1} Stroke is the second common cause death in developed countries and thus is a major health problem \[[@B1]\]. According to the 2009 Annual Public Health Report by the Korean National Statistical Office, cerebrovascular disease, was the second-leading cause of disease-related deaths in Korea, after cancer \[[@B2]\]. In Korea, many stroke receive traditional medical care because the country has its system of traditional alternative medicine called Traditional medicine (TKM), the role which has been emphasized in stroke \[[@B3]\]. The Korean medical diagnosis system characteristics similar to the traditional Chinese medical diagnosis One such feature is pattern identification (PI), which is on information obtained four diagnostic processes including inspection, listening and smelling, inquiry, and palpation \[[@B4]\]. PI is a diagnostic system that entails a comprehensive analysis of symptoms and with implications for determining the cause, nature, and location of the illness, the patient\'s physical condition, and patient\'s treatment \[[@B3], [@B5]\]. The inspection process involves the of the patient\'s symptoms or disease by observing his or her shape, expression, and tongue \[[@B6]\], among others. Observation of the tongue, also known as tongue diagnosis, is an important procedure in diagnosis by inspection in TKM. The status of the tongue is important indicator in the diagnosis one\'s condition, including the physiological and clinicopathological changes of internal organs in the \[[@B7]\]. A studies have shown that tongue diagnosis plays an important role in clinical and treatment \[[@B8]--[@B15]\], in patients with a history of stroke. However, clinical competence of tongue diagnosis was determined by the experience and knowledge of the clinicians who used tongue Environmental factors, such as differences light sources and levels of brightness also had significant influences on clinicians and their diagnostic decisions using the tongue. Unfortunately, much of the experiences in traditional tongue diagnosis have not been verified scientifically or quantitatively. Therefore, it is necessary to build an objective diagnostic standard for tongue diagnosis \[[@B7]\]. We investigated the reliability of tongue diagnosis in stroke patients by evaluating interobserver reliability regarding tongue indicators as achieved by TKM practitioners. 2. {#sec2} ========== 2.1. Study Subjects {#sec2.1} ------------------- The data for this analysis were collected as part the project named Study for the Standardization and Objectification of Pattern TKM for Stroke (SOPI-Stroke). Stroke patients admitted to the following oriental medical university hospitals, Kyung Hee Oriental Center (Seoul), Kyung Hee East-West Neo Medical Center (Seoul), Dong Guk International Hospital (Kyunggi-do), Kyung Won Oriental Medical Hospital (Incheon), Dae Jeon Oriental Medical Hospital Dong Medical Hospital Won Kwang Oriental Medical Hospital (Jeollabuk-do), Dae Hanny University Medical Center (Daegu), and Sang Ji Oriental Medical Hospital (Gangwon-do), participated in this study between 2010 December 2010 ([Figure 1](#fig1){ref-type="fig"}). All patients provided written informed consent under procedures approved by institutional review boards Eligibility inclusion criteria were to be enrolled within 30 days of the onset of as confirmed by imaging diagnosis, such computerized tomography (CT) magnetic resonance imaging (MRI). Exclusion criteria were traumatic stroke patients, such as with subarachnoid, subdural, or epidural hemorrhage. 2.2. Data Processing and Analysis {#sec2.2} --------------------------------- All patients were seen by two experts from the same department in each site, who were well trained in standard operation procedures (SOPs) \[Appendix\] and subjected to an examination of the status of the tongue color (pale, and bluish purple), fur color (white fur, yellow fur), (thick fur, thin fur, moist fur, and dry fur), and special tongue appearance marked, enlarged, mirror, and spotted). The examination parameters were extracted from parts of a case (CRF) for the standardization of stroke diagnosis that had been developed by an expert committee organized by the Korean Institute Oriental Medicine (KIOM). These assessments were given individually without discussions among clinicians. Descriptions of the grading severity for each variable were scored as the following: 1 = very much so, 2 = so, and = Not so much. In particular, the clinicians to measure the stroke PI of each patient following fire-heat the phlegm-dampness pattern, the blood stasis pattern, the qi deficiency pattern, the Yin deficiency pattern, as suggested by the \[[@B3], [@B16]--[@B18]\]. Interobserver reliability was measured in three using simple percentage agreements, Cohen\'s kappa coefficient and Gwet\'s AC~1~ statistic, as well as via the confidence intervals (CI). Kappa, the preferred of rater reliability for nominal the agreement between two or more independent raters using a rating scheme with mutually exclusive categories. In definitive kappa interpretations have been proposed \[[@B19]--[@B24]\]. For most purposes, however, values ≤0.40 represent poor agreement, values between 0.40 and 0.75 represent moderate to good agreement, and values ≥0.75 indicate excellent agreement \[[@B24]\]. AC~1~ statistic is not vulnerable to well-known paradoxes that make Kappa appear \[[@B25]--[@B27]\]. First, interobserver for the tongue indicator among all subjects was calculated via simple percentage agreements, Cohen\'s kappa coefficient, and Gwet\'s AC~1~ statistic. Later, interobserver regarding PI with same opinions between the raters calculated in same way. The blood stasis pattern was because the sample size was small (*n* = 1). The data were statistically analyzed with SAS software, version 9.1.3 (SAS Institute Inc., Cary, NC). 2.3. Approval {#sec2.3} --------------------- This study was approved by institutional review board the KIOM and by each of the oriental medical university hospitals. 3. Results {#sec3} ========== A total of 658 stroke patients were enrolled in the study. Thirty patients were excluded from analysis due to PI omitted by any one of 2 TKM clinicians. The reliability regarding indicators for all (*n* 628) are shown in [Table 1](#tab1){ref-type="table"}. The kappa measure of agreement the two experts was generally moderate to good the indicators, ranging from 0.42 to 0.69, except for fur (*κ* = 0.29) and spotted (*κ* = 0.37). Moreover, the AC~1~ measure of agreement between the two experts was generally high for the tongue ranging from "moderate" (AC~1~ = 0.43) to "excellent" = 0.97). Agreement, as assessed by the kappa values, considerably lower than the AC~1~ values in most cases. The results of interobserver reliability for of PI with the same opinion between the raters are shown in A total of 451 stroke patients received PI with the following same resulting opinions the raters: Fire-Heat (*n* = 147), Phlegm-Dampness Pattern = 158), Yin Deficiency (*n* = and Qi Deficiency Pattern (*n* = 66). The blood pattern was excluded because the sample size small = 1). kappa measure of agreement for the subjects of PI was generally moderate to for the tongue indicators, ranging from 0.40 to 0.72, except for fur (*κ* 0.31). the measure agreement between two experts was generally high for the tongue indicators, ranging from "moderate" (AC~1~ = 0.5) (AC~1~ = 0.98) ([Table 2](#tab2){ref-type="table"}). Discussion {#sec4} ============= Inspection of the tongue in TKM diagnosis, as well as western medicine \[[@B28]\], is one of the important approaches obtaining significant evidence in diagnosing the patient\'s health conditions \[[@B7]\]. It is to observe the color, coating, and body of the among other features, in rendering a disease diagnosis. Also, as tongue diagnosis has played a prominent role in the diagnosis subsequent of stroke patients, it has attracted an increasing amount of attention oriental medicine \[[@B8]--[@B15]\]. Park et al. analyzed markers that classified body fur, fur quality, dryness, and shape to standardize tongue diagnosis and for stroke patients. et al. \[[@B14]\], to assess the usefulness of tongue diagnosis in evaluating PI, the coating of the tongue and compared it with PI in acute stroke stage patients within 72 hours from the onset of stroke. In his study, a red tongue was significantly related to fire-heat pattern and the yin deficiency while a faint white tongue was related to the phlegm-dampness pattern. Thin fur was related to the Wind | 1. inTRoductION {#Sec1}
===============
StROkE is THe secONd Most ComMon CAuse OF DEAtH In deVELOpEd cOunTries And THUS Is A mAJOr HEALtH ProBLEM \[[@B1]\]. AcCOrDiNG to THE 2009 Annual PubliC HealtH RePOrT by The koRean naTIoNal sTaTISTical offIcE, ceREbROvAscuLAr DisEAse, OR STRokE, wAS the SECoNd-LeADIng cauSE oF DisEasE-rElAted DeATHs IN KOrEa, AFter CANCER \[[@B2]\]. iN KoReA, MaNy sTRoKE pAtiEnts ReceIve TrAdiTiONal medicaL caRE BECauSe THE CounTrY has ITs OWn sYSteM Of tradItiONAL alteRNaTIVe MEdICIne calLeD TRadItioNAl kOREAN medIcINE (TKm), tHE role OF whIch haS BeeN EMPhaSiZEd IN stRoke ManagemENt \[[@B3]\].
tHe KOReaN mediCAl diagnOsIs SYsTem haS UNiquE ChARACtERISTICS SiMiLAr tO tHe tRaDiTIONAL chineSe meDIcAl DIagnOsiS System. ONe such feATurE IS PaTterN ideNTIfiCAtIoN (pi), WhIcH IS BaSeD ON InfoRmAtIOn ObTainEd FROM Four dIaGNOsTiC pROCesses inclUDing InSPeCtIoN, liStening AnD smELLING, INQuIRy, aND PALpaTiOn \[[@b4]\]. pI IS a diAgNostic syStem ThaT ENTaILs a CoMpreHENsIVe aNalYsIs oF symPTomS and SIGns, wIth iMpLicaTioNS For dETErMINiNg THe CAuSE, NaTURE, And LoCATIon of The IlLnEss, the PAtieNt\'S PHysiCal conDItioN, anD thE PATiENt\'S TrEatmeNT \[[@B3], [@B5]\]. the INSPeCTIoN prOcess InvolVES ThE EXaminaTioN Of tHE PAtiEnt\'S syMpToms OR disEAsE by ObSERVInG his OR heR ShaPe, exprEsSion, and tOnGUE \[[@B6]\], aMoNg oTHers. OBSerVaTiOn oF the tonGue, alSo KNOWn As TonGuE DiAGnoSiS, IS AN ImpoRTanT PrOCEDure In DiAgNoSiS by iNsPectioN IN TKm. THE StatUs Of THe TOngue Is AN iMPoRTant INdICatoR In THE diAGnOsIS oF ONe\'s CONDItIon, INcLUdiNG the PhysIOlogIcAL AND cLINicoPAthOLOgICAl ChAngES of inTErnaL oRgANs In THe bODY \[[@B7]\]. A numBER OF STUdIeS HAVE SHOWn ThAt TONgUE DiaGNOSiS PlaYS An IMpORtAnT ROle in cliNiCal pROgnosIS And tREaTmENt \[[@b8]--[@b15]\], specifICaLlY IN PATIEnTs wItH a HIsTOry Of STROke. hoWEVer, ThE cLINICAl cOmpEtence Of tonguE diAgnosis WaS DetErMInED By THe EXPErIeNcE anD KnoWlEdgE OF The cliNiciANS wHo uSEd TONGUe dIAgnOsiS. eNviRoNMeNTAL factoRS, such as diFferENcES bETWeEN LIGht sourceS And lEVELs of bRIgHtnESs ALso haD sigNifIcaNt iNfLUEnCeS on clIniCiAns AnD ThEir dIAgnOSTIC dEcISiONs uSIng tHE ToNGue. uNFOrTuNAteLy, mucH oF thE eXpeRIENCes iN TrAdItIONaL tOngUe dIAGnoSiS hAve NOT BEEN veRiFIED SCIeNTifiCALLy Or qUAntItAtivEly. tHEreFORE, IT is NECesSary To bUIld aN oBjEcTIve DiagNoStIc sTandaRD fOr TOngue dIagNoSiS \[[@B7]\]. we InVeSTIgatEd THE ReliAbILIty oF tKM TOnGUe DiAGnosiS in STrOkE pATIEnTs BY eValUatInG inteRobsErveR ReLiaBIlity reGARDiNg tongue INDIcatORS As AChiEVEd by tKM PRACtITIOnERs.
2. MeTHods {#SEC2}
==========
2.1. stuDy SUBjECts {#sEC2.1}
-------------------
ThE DatA fOr thIs anALysIs werE cOLlEcTED aS PaRT OF THE projEct NAmeD tHe FunDAMeNtal StudY fOR ThE STAnDARdiZaTION ANd objeCtIFICaTIon of PATTerN iDentiFICatIoN In tKM foR STROKE (SOPI-sTRoKE). sTRoKE PATiEnTs adMitteD to the FOLloWING ORIentaL mEdicAL univeRsITy hospitaLs, kyUng HEE OrIentAl mEDiCAl centeR (SeouL), KyunG HEe EAsT-WeSt neO mEDicaL CENtER (sEOuL), dong guk InTernAtiONAl HOSPItAL (KyuNggI-do), KyUng WON OrieNtAL MeDICal hoSPiTAl (InChEOn), dAe jeOn ORieNtAl meDicAl hOspiTal (daejEON), Dong sIN ORieNTAL MedIcal hosPITAl (GwANgJU), Won kwAng OrIENtAl mEdiCAL HOspiTAl (jEoLlAbUK-Do), DAE GU hAnNY unIVeRSitY MEDical CENTEr (DAEgu), ANd sAng ji ORIeNTal MedIcAL hOSPitAl (gAnGWon-Do), particIpATed in THIS StUdy BETWeeN FEbrUARY 2010 anD deCeMber 2010 ([FIguRe 1](#FiG1){ref-TYpE="FIG"}). All pATIEnts PROVIdEd writtEn infoRMED COnsEnT uNDer pROCeDuREs aPProvED by iNSTitUTioNal REvIew BoARDs (Irb). ELIGiBIliTy iNcLusioN CRITerIa weRe thAt PARTiCiPants HAD To Be EnRolleD wIthin 30 dAYS oF THe onSET oF THEiR sYMptoMS AS COnFiRMED BY ImaGIng DIaGnoSiS, sUCh aS CompUTERiZed toMoGrAPHy (cT) oR mAgNETiC REsONaNce iMaGiNG (MRI). EXCLUSiON CrITeRIA WEre TRaumaTiC STrOkE paTiEnTS, sUcH As THosE With SubARaCHNOiD, SuBdural, Or EpIduRaL HEMORRHAGe.
2.2. DAtA pROcEssing And anAlysIS {#sEC2.2}
---------------------------------
all paTiEnts werE sEEN By tWO eXperTs frOm THE saMe dePaRTMEnT iN eACH SITE, WhO WERe WEll TraiNeD IN sTANdArD opERAtIOn prOCeDUReS (SoPs) \[APPEnDIx\] And wERE SUBjECTeD To AN eXAmination of tHe StatuS OF THe ToNgUE, tongUe CoLOr (palE, rED, ANd blUISH PUrPLe), FUR cOlOr (WhITe Fur, yEllOw fuR), fuR QUALItY (thIcK fUr, thin FuR, moIsT fUR, And dry fUr), anD speCIal ToNguE appeaRAnce (TEEtH marKed, eNLarGEd, MIrRoR, And SpOttED). THe exAmiNATIoN pARamETers WerE exTrACted frOM parTs oF a CaSE rEPorT Form (CRf) fOR tHE sTanDardIZaTIOn oF sTRoKe dIagnoSiS thaT had beEN dEVElOpED bY An ExpErt coMMItTEe ORganized by the KOreaN InstITUte Of oRiENtaL MEDiCInE (kIOM). THeSe aSSEssmentS Were givEn INDIVIDUAllY WithoUT DIScuSSionS amONg tHE CLINIciANs. DesCrIptIONs of THe graDINg seVeRITY foR EAch variaBlE WERE sCorED as tHe fOLLoWINg: 1 = vEry muCh sO, 2 = MUCH so, aND 3 = NOt So Much. IN PARTicUlar, THE ClInicians HaD tO meaSuRe tHE stRoKE PI OF eACh pAtIEnt fOLlOWinG tHe fiRE-HEAt PAtTeRn, tHe PHlEGM-DAmpNESS pattERN, the bLOOD STASis PaTtern, ThE QI DeFIcIenCy PATteRn, anD ThE yIn DeficiEnCY PAtTeRN, aS SUGgeSTed By tHE kiOm \[[@b3], [@b16]--[@b18]\].
inTErObserVER rEliaBiLITy WaS mEAsUREd in THRee WAys, usINg sIMplE pERceNTage AgreEmENtS, COhEN\'s kappa CoefFiCiEnt and gWeT\'s Ac~1~ sTATIstIC, AS WElL as vIA THe CoRReSponDING cONfiDence inTerVALS (Ci). kaPpA, thE PrEfERRED mEaSuRe Of rater rELiABIliTY foR NomInAL dAta, MeAsURes tHe reLIAbilitY of AgREemenT beTwEEn two Or moRE iNdEpEnDENT RaTERs USInG a ratiNG sCHEme wiTH MutuALLy exClusivE CaTEGOrIEs. IN genErAl, DefinItIVe KAppA interpREtatioNS hAvE bEen proPOSed \[[@B19]--[@B24]\]. FoR MoST PURposES, HOWEveR, vALueS ≤0.40 RePReseNT Poor aGREEMent, VAlUeS BETWEeN 0.40 aNd 0.75 rEprESEnT modeRAtE To gOOD AgREeMeNt, AND VALUeS ≥0.75 IndIcatE EXcElLENt aGrEEmeNT \[[@b24]\]. the aC~1~ staTistIC is Not VulNeraBLE To tHE WELl-KnOwN pAradOXES THaT mAKe KaPPa ApPEaR InEffeCtive \[[@B25]--[@B27]\]. FIrSt, iNtERoBsERVEr reliaBILITy FoR THe TonGue InDicator AmONG all SUBJecTs wAS calcULaTed viA siMPlE PerceNTaGE AgReEMEnTs, coheN\'s KApPa coEffICienT, anD gwET\'S Ac~1~ staTIstIC. LaTeR, INTEroBSErVEr RELiAbIlITy REGARDing pI WItH SaMe OPINIONS BETwEEN the raTers WAS caLCULaTED In THe Same Way. THE BLooD STaSis paTteRN WAS OMItTED BeCAUse ThE SAMplE sIZE wAs TOo SmaLl (*n* = 1). tHe dAta Were statISticAllY AnALyzeD wItH saS sOFtWARe, VErsIoN 9.1.3 (saS InSTITuTe InC., CARy, Nc).
2.3. EThIcal AppRovAl {#sEC2.3}
---------------------
THiS sTUDY WAs aPproVeD By INSTITuTioNal rEVIeW bOArD Of tHE KIom and By EAcH Of the oRIeNTaL mEdICal UNiVeRsITY hOsPITAls.
3. reSULtS {#sec3}
==========
A tOTal Of 658 STRoke PATIENTS WErE ENROllEd iN the study. thIrtY paTIEnts werE ExCLUDeD FROm anAlYSiS Due TO PI omITted by aNY ONE OF 2 TKm clInicIanS. ThE INTerObsErVER ReLiAbiLITY RESULts ReGaRDinG ToNGue INdicatoRS fOR All SUbjeCts (*N* = 628) ArE SHOwN iN [TabLe 1](#TAb1){REF-tyPE="taBLE"}. tHe kappa meAsure Of AgREeMEnt bETWEEN The twO ExPErts wAS GEneRALly MOderaTe TO goOd FOR THE TongUe inDicatorS, RanGING From 0.42 to 0.69, EXCePt For MOiSt FUr (*κ* = 0.29) aNd SpoTteD (*κ* = 0.37). MOrEovEr, tHE ac~1~ MeaSure Of AGrEeMenT BetWeEn THe twO ExpERTs WAs geNErally HIgH For THe toNgUe INdicaToRS, raNgINg frOm "MoDERatE" (Ac~1~ = 0.43) tO "EXCeLlenT" (Ac~1~ = 0.97). agREeMenT, as asSESsED by tHE KApPa VAluEs, WaS CoNSidErAbLY lOwEr THAN ThE aC~1~ vaLuEs IN MOST CASES.
the REsUlts OF INterobsErver RELIAbIliTy FOr SuBJects of Pi wIth ThE Same opiNioN betWeen tHe rATeRs ArE sHown IN [tABLe 2](#tAB2){Ref-tYPe="tAbLE"}. a tOTAL oF 451 strOkE PaTIents rECEiVed Pi WiTh THe FOlLOWINg saME ReSUlTING opINioNs bY ThE raTers: FiRE-heAT paTteRN (*N* = 147), pHLEGm-DAMpnEss PATtERN (*n* = 158), Yin dEfICIENcY PatTern (*N* = 80), anD qi DefICieNCy PaTTErN (*n* = 66). THe blOOD STASiS PATtern wAS eXcLuDeD BECauSE THE SamPLe SiZe WaS tOo Small (*n* = 1).
The kappA MeAsURe oF AGReement fOR The subJectS of Pi WAs geNEralLy modEraTe to Good for ThE TongUe IndicAToRS, RanGing from 0.40 tO 0.72, exCept FoR MOIsT fur (*κ* = 0.31). moReOVer, The ac~1~ MEAsure OF AGreement beTWeen THe Two eXPeRtS WAs GeneRaLly higH fOR THe tonGuE iNDIcATorS, RangIng FRoM "mOderATe" (ac~1~ = 0.5) TO "eXCeLlEnt" (aC~1~ = 0.98) ([tAblE 2](#TAb2){ReF-typE="tABLe"}).
4. dIsCussiON {#sEc4}
=============
iNsPectiON of THE TOnGuE IN tkm diAGNOsIs, As welL as in WeStErn meDICinE \[[@b28]\], iS ONE of tHe moST ImPortANt aPproAcHeS for ObTAinInG SiGNifIcAnt EvidEnce iN DiAGnOSING THe PATienT\'S heALtH cOndItIOnS \[[@B7]\]. IT iS Used To obSeRve thE COLoR, coatIng, and BODy oF THE TONgUE, AmONg oThER fEatUreS, IN renDerING a DisEasE diagNOSiS.
alsO, as TONguE diAGNoSis HAS pLaYED a PromIneNt RoLE iN tHE DiagNOsIs and SUBseqUenT treaTmeNt OF StRokE PAtIENts, iT hAs AttRacteD AN inCrEAsINg AmOuNt of attENTioN IN OrienTAl mediCINe \[[@b8]--[@b15]\]. pArk et al. \[[@b12]\] aNalYzed MaRKeRs ThAt cLaSSIfIEd ToNGuE bODY cOlOr, fUr, fur QuAlitY, DrYness, anD SHaPe to STANdArDIzE tOngUE DIaGNoSIS aND Pi FoR stRoke PatIENTS. CHOI Et AL. \[[@b14]\], tO asseSS The useFuLnEss Of Tongue DiAgNoSIs iN EvalUatIng PI, oBserVeD tHe CoAtinG of the TONgUe and CoMParEd It With Pi IN aCutE StrOKe stAge patienTs WItHIn 72 hOuRS FROM ThE oNsET oF StROkE. iN HiS STUDY, a rED tONGUe was SIgniFicAnTLy rELateD tO tHe FIRe-hEat patTErN anD thE yiN DefiCiEnCY PATTErn, WhiLE a fainT WhITE TONGUe WAS RElated To ThE PHleGM-daMPneSs PattErN. THiN fUr WAs RELated TO THe wInD | 1. Introduction {#sec1} =============== Stroke is the second most commoncause of death indeveloped countries and thus is a major health problem\[[@B1]\]. According to the2009 AnnualPublic Health Report by the Korean National Statistical Office, cerebrovascular disease, or stroke, was the second-leading causeof disease-related deathsin Korea, after cancer\[[@B2]\].In Korea, manystroke patients receive traditional medical care because the country has its own system of traditional alternative medicine called Traditional korean medicine (TKM), the role of which has been emphasized in stroke management\[[@B3]\]. The Koreanmedical diagnosis system has unique characteristics similar to thetraditional Chinese medical diagnosis system. One suchfeature ispattern identification (PI), which is based oninformation obtained from four diagnostic processes including inspection, listeningandsmelling, inquiry, and palpation \[[@B4]\].PI is adiagnostic system that entails a comprehensive analysis ofsymptoms and signs,with implications for determining the cause, nature, andlocation of the illness, the patient\'s physical condition, and the patient\'s treatment \[[@B3], [@B5]\]. The inspection process involves the examination of the patient\'ssymptoms or disease by observing his or her shape, expression, and tongue \[[@B6]\], among others. Observation ofthe tongue, also known as tongue diagnosis, isan important procedure in diagnosis by inspection in TKM. The status of the tongue is an important indicatorin the diagnosis of one\'s condition, including the physiological and clinicopathologicalchanges of internal organs in the body \[[@B7]\]. A number of studies have shown that tonguediagnosis plays an important role in clinical prognosis and treatment \[[@B8]--[@B15]\], specifically inpatients with a history of stroke. However, theclinical competence of tongue diagnosis wasdetermined by the experienceand knowledge of the clinicianswho used tonguediagnosis.Environmentalfactors, such as differences between light sources and levels of brightness also had significant influenceson clinicians and their diagnostic decisions using the tongue. Unfortunately, much of the experiences in traditional tonguediagnosis have not been verified scientifically or quantitatively. Therefore, it is necessary to build an objective diagnosticstandard for tongue diagnosis \[[@B7]\]. We investigated the reliability of TKM tongue diagnosis in stroke patients by evaluating interobserver reliability regarding tongue indicators as achieved by TKM practitioners. 2. Methods {#sec2} ========== 2.1. Study Subjects {#sec2.1} ------------------- Thedata forthis analysis were collected aspartof the project namedthe Fundamental Study for the Standardization andObjectification of Pattern Identification in TKM for Stroke(SOPI-Stroke). Stroke patients admitted to the following oriental medical universityhospitals,Kyung Hee OrientalMedical Center (Seoul), Kyung Hee East-West NeoMedical Center (Seoul), DongGuk International Hospital (Kyunggi-do), KyungWon Oriental Medical Hospital(Incheon), Dae Jeon Oriental MedicalHospital (Daejeon), Dong Sin Oriental MedicalHospital(Gwangju), Won Kwang OrientalMedical Hospital (Jeollabuk-do), Dae Gu Hanny University Medical Center (Daegu), and Sang Ji Oriental Medical Hospital (Gangwon-do), participated in this study between February 2010 and December2010 ([Figure 1](#fig1){ref-type="fig"}). All patients provided written informed consent under procedures approved by institutional review boards (IRB). Eligibilityinclusion criteria were that participantshad to be enrolledwithin30 days of the onset of theirsymptoms as confirmedby imaging diagnosis, such as computerized tomography (CT) or magnetic resonance imaging (MRI). Exclusioncriteria were traumatic stroke patients, such as those withsubarachnoid, subdural, or epidural hemorrhage. 2.2. Data Processing and Analysis {#sec2.2} --------------------------------- All patients were seen by two experts from the same department in eachsite,who werewell trained in standard operation procedures (SOPs) \[Appendix\] and were subjectedto an examination of the status of the tongue, tongue color(pale, red, and bluish purple),fur color (white fur, yellow fur), fur quality(thick fur,thin fur, moist fur, and dry fur), and special tongue appearance (teethmarked, enlarged, mirror,and spotted). Theexaminationparameterswere extracted from partsof a case report form (CRF) for the standardizationof strokediagnosis that hadbeen developed by anexpert committee organized by theKorean Instituteof Oriental Medicine(KIOM). These assessmentswere given individually without discussions among the clinicians. Descriptionsof the gradingseverity foreach variable were scored as the following: 1 = very much so, 2 = Much so, and3 = Not so much. In particular, the clinicians had to measurethe stroke PI of eachpatient following the fire-heat pattern, thephlegm-dampness pattern, the blood stasis pattern, the qi deficiencypattern, andthe Yindeficiency pattern, as suggested by the KIOM \[[@B3],[@B16]--[@B18]\]. Interobserver reliability was measured in three ways, using simple percentage agreements, Cohen\'s kappa coefficient and Gwet\'s AC~1~ statistic, as wellas via the corresponding confidence intervals(CI). Kappa, the preferred measure of rater reliability for nominal data,measuresthe reliability of agreement between two or more independent raters using a rating scheme with mutually exclusive categories. In general,definitivekappa interpretations have been proposed \[[@B19]--[@B24]\]. For most purposes, however, values ≤0.40 represent poor agreement, valuesbetween 0.40 and 0.75 represent moderate to good agreement,and values ≥0.75 indicate excellent agreement \[[@B24]\]. The AC~1~statistic is not vulnerable to the well-known paradoxes that make Kappa appear ineffective \[[@B25]--[@B27]\]. First, interobserver reliability for thetongue indicator amongall subjectswas calculated via simple percentage agreements, Cohen\'s kappa coefficient, and Gwet\'sAC~1~ statistic. Later, interobserver reliability regardingPI with same opinionsbetween the raters was calculated in the same way. The bloodstasis pattern was omitted because the sample size was too small (*n* = 1). The data were statistically analyzed with SAS software,version 9.1.3 (SAS InstituteInc., Cary, NC). 2.3. EthicalApproval{#sec2.3} ---------------------This study was approved by institutional review board of the KIOM and byeach of the oriental medical university hospitals.3. Results{#sec3}========== A total of 658 stroke patients were enrolled in the study. Thirtypatients wereexcluded from analysis dueto PI omitted by any oneof 2 TKM clinicians. The interobserver reliability results regarding tongue indicators forall subjects (*n* = 628)are shown in [Table 1](#tab1){ref-type="table"}. The kappa measure of agreement between the two experts was generally moderate to good for the tongue indicators, ranging from 0.42 to 0.69, except for moistfur(*κ* = 0.29) and spotted (*κ* = 0.37). Moreover, theAC~1~ measureof agreement between the two experts was generally high for the tongue indicators, ranging from "moderate" (AC~1~ =0.43) to "excellent" (AC~1~ = 0.97). Agreement, asassessed by thekappa values, was considerably lower than the AC~1~ values inmost cases. The results of interobserver reliability forsubjects of PIwiththe same opinion between the raters are shown in [Table2](#tab2){ref-type="table"}. A total of 451 stroke patientsreceived PI with the following same resultingopinions by the raters: Fire-Heat Pattern (*n* = 147),Phlegm-Dampness Pattern (*n* = 158), Yin DeficiencyPattern (*n* = 80),and Qi Deficiency Pattern (*n* = 66). The bloodstasis pattern was excluded because the sample size was too small (*n* = 1).The kappa measure of agreement for the subjects ofPI was generally moderate to goodfor the tongue indicators, ranging from 0.40 to 0.72, except for moist fur (*κ* =0.31).Moreover, the AC~1~ measure of agreement between the two experts was generally high for the tongue indicators, rangingfrom "moderate" (AC~1~ = 0.5) to "excellent" (AC~1~ = 0.98) ([Table 2](#tab2){ref-type="table"}). 4. Discussion {#sec4} ============= Inspection of the tongue in TKM diagnosis, as well as inwestern medicine \[[@B28]\], is oneof the most important approaches for obtaining significant evidence in diagnosing the patient\'s health conditions \[[@B7]\]. It isused to observe the color, coating, and bodyof the tongue, among other features,in renderinga disease diagnosis. Also, as tongue diagnosis has played aprominent role in the diagnosisand subsequent treatment of stroke patients, it has attracted an increasing amount of attention in oriental medicine \[[@B8]--[@B15]\]. Park etal. \[[@B12]\] analyzed markers that classified tongue body color,fur, furquality, dryness,and shape to standardize tongue diagnosis and PI for stroke patients. Choi et al. \[[@B14]\], to assess the usefulness of tongue diagnosis inevaluating PI, observed the coating of thetongue and compared it with PI in acute stroke stage patientswithin 72 hours from the onsetof stroke. In his study, a red tongue was significantly related to the fire-heat pattern and the yin deficiencypattern,while afaint white tongue was related to the phlegm-dampness pattern. Thin furwas related to the Wind | 1. _Introduction_ {#sec1} =============== Stroke is the second most common cause of death in developed countries and thus is a _major_ health problem \[[@B1]\]. According to the 2009 Annual Public Health Report _by_ the Korean National Statistical Office, _cerebrovascular_ disease, or stroke, was the second-leading cause of disease-related deaths in Korea, _after_ cancer \[[@B2]\]. In Korea, many stroke patients receive _traditional_ medical care because _the_ country has its own system _of_ traditional alternative medicine _called_ Traditional korean medicine (TKM), the _role_ _of_ which has been emphasized _in_ stroke management \[[@B3]\]. The _Korean_ medical _diagnosis_ system has unique characteristics _similar_ to the _traditional_ _Chinese_ _medical_ diagnosis system. One such feature is _pattern_ identification (PI), _which_ is based _on_ information obtained from four diagnostic processes including inspection, listening and smelling, inquiry, and palpation \[[@B4]\]. PI is _a_ _diagnostic_ system that entails a comprehensive analysis of symptoms and signs, with implications for determining the cause, nature, and location of the illness, the _patient\'s_ physical condition, and _the_ _patient\'s_ treatment \[[@B3], [@B5]\]. The inspection process _involves_ the examination of the patient\'s _symptoms_ _or_ disease _by_ observing his or her shape, expression, and tongue \[[@B6]\], among _others._ Observation of the tongue, also known as tongue diagnosis, _is_ an important procedure in diagnosis by inspection in _TKM._ The status of the tongue is an important indicator in the diagnosis of _one\'s_ condition, including the physiological _and_ _clinicopathological_ _changes_ of internal organs in the body _\[[@B7]\]._ A number _of_ studies have shown that _tongue_ diagnosis plays an _important_ role in clinical prognosis _and_ treatment \[[@B8]--[@B15]\], specifically _in_ patients with a history of stroke. _However,_ _the_ _clinical_ competence of tongue diagnosis was determined by the _experience_ and knowledge of the _clinicians_ who used _tongue_ diagnosis. Environmental factors, such as differences between light sources _and_ _levels_ of _brightness_ also had significant influences on clinicians _and_ their diagnostic decisions using the tongue. Unfortunately, much of the experiences in traditional tongue diagnosis have not been verified scientifically _or_ quantitatively. Therefore, it is necessary _to_ build an objective diagnostic standard for _tongue_ _diagnosis_ \[[@B7]\]. We investigated _the_ reliability _of_ TKM _tongue_ _diagnosis_ _in_ stroke patients _by_ evaluating _interobserver_ reliability regarding tongue indicators _as_ achieved by TKM practitioners. 2. _Methods_ {#sec2} ========== 2.1. Study Subjects {#sec2.1} ------------------- _The_ data _for_ this analysis _were_ collected _as_ part _of_ the project named the Fundamental Study for the Standardization _and_ Objectification of _Pattern_ Identification _in_ TKM for _Stroke_ (SOPI-Stroke). _Stroke_ patients admitted to the _following_ oriental _medical_ _university_ hospitals, Kyung Hee Oriental Medical _Center_ (Seoul), Kyung Hee East-West Neo Medical Center (Seoul), Dong Guk _International_ Hospital (Kyunggi-do), Kyung Won Oriental _Medical_ Hospital (Incheon), Dae _Jeon_ Oriental Medical Hospital (Daejeon), _Dong_ Sin Oriental Medical Hospital (Gwangju), Won Kwang Oriental _Medical_ Hospital (Jeollabuk-do), Dae Gu Hanny University Medical _Center_ (Daegu), and Sang Ji Oriental Medical Hospital (Gangwon-do), _participated_ in this study between _February_ 2010 _and_ December _2010_ ([Figure _1](#fig1){ref-type="fig"})._ All patients provided written _informed_ _consent_ _under_ _procedures_ approved by institutional _review_ boards _(IRB)._ Eligibility _inclusion_ criteria were _that_ participants had to _be_ enrolled within 30 days of the onset of their symptoms _as_ confirmed _by_ _imaging_ diagnosis, such as computerized tomography (CT) or magnetic resonance imaging (MRI). Exclusion criteria were traumatic stroke patients, such as _those_ with subarachnoid, subdural, or epidural hemorrhage. 2.2. Data Processing _and_ Analysis {#sec2.2} --------------------------------- All patients were _seen_ by _two_ experts from the same department in _each_ site, who were well trained _in_ _standard_ operation procedures (SOPs) \[Appendix\] and were subjected _to_ an examination of _the_ status of the tongue, tongue color _(pale,_ _red,_ and bluish _purple),_ fur color _(white_ _fur,_ yellow fur), fur quality _(thick_ _fur,_ thin fur, moist fur, and dry fur), and special tongue appearance (teeth marked, enlarged, mirror, _and_ spotted). The examination parameters _were_ _extracted_ from parts of _a_ case _report_ _form_ (CRF) for the _standardization_ of _stroke_ diagnosis that had been developed by an expert _committee_ organized _by_ _the_ _Korean_ Institute of Oriental _Medicine_ (KIOM). These assessments were given individually without discussions among the clinicians. _Descriptions_ of _the_ _grading_ severity for _each_ variable were scored as the following: 1 = _very_ much so, 2 = _Much_ _so,_ and 3 = Not so much. In _particular,_ the clinicians had to measure the stroke _PI_ _of_ each patient _following_ the fire-heat pattern, the phlegm-dampness pattern, the blood stasis pattern, the qi deficiency _pattern,_ and the Yin _deficiency_ pattern, as suggested by the KIOM \[[@B3], _[@B16]--[@B18]\]._ _Interobserver_ reliability was measured _in_ _three_ ways, _using_ simple percentage _agreements,_ Cohen\'s kappa coefficient and Gwet\'s AC~1~ statistic, _as_ well as via the corresponding confidence _intervals_ _(CI)._ Kappa, the preferred measure of rater _reliability_ _for_ nominal _data,_ measures _the_ reliability of agreement between two or more independent raters using a rating scheme _with_ mutually exclusive categories. In _general,_ _definitive_ kappa interpretations have _been_ _proposed_ _\[[@B19]--[@B24]\]._ For most purposes, however, values _≤0.40_ represent poor agreement, values between 0.40 and 0.75 represent moderate to good agreement, and values ≥0.75 indicate excellent agreement \[[@B24]\]. The AC~1~ statistic is _not_ vulnerable to _the_ _well-known_ _paradoxes_ that _make_ Kappa appear _ineffective_ \[[@B25]--[@B27]\]. First, interobserver reliability _for_ the tongue indicator among all subjects _was_ calculated via simple percentage agreements, _Cohen\'s_ kappa coefficient, _and_ Gwet\'s AC~1~ statistic. Later, interobserver reliability regarding PI with same opinions between the _raters_ was calculated in _the_ same way. The blood stasis pattern was omitted _because_ the sample _size_ was _too_ small (*n* = _1)._ The data were statistically _analyzed_ _with_ SAS software, _version_ _9.1.3_ _(SAS_ _Institute_ Inc., Cary, NC). 2.3. _Ethical_ Approval {#sec2.3} --------------------- This study was approved by institutional review board of _the_ KIOM and by each of the oriental medical _university_ hospitals. 3. Results {#sec3} ========== A total _of_ 658 stroke patients were enrolled _in_ _the_ study. Thirty patients were excluded from analysis due to _PI_ omitted by any one of 2 TKM clinicians. _The_ interobserver reliability _results_ regarding tongue _indicators_ _for_ all subjects (*n* = 628) _are_ _shown_ in _[Table_ 1](#tab1){ref-type="table"}. _The_ kappa _measure_ _of_ agreement between the two experts _was_ generally _moderate_ _to_ good _for_ the tongue indicators, ranging from 0.42 _to_ 0.69, except for moist fur _(*κ*_ = 0.29) and spotted (*κ* = 0.37). _Moreover,_ the AC~1~ _measure_ of agreement between _the_ two experts was generally high for the tongue indicators, ranging from "moderate" (AC~1~ = 0.43) _to_ "excellent" (AC~1~ _=_ 0.97). _Agreement,_ as _assessed_ _by_ the kappa values, was considerably lower than the AC~1~ values in most cases. The results of interobserver reliability for subjects of PI with the same opinion between the raters are shown in [Table 2](#tab2){ref-type="table"}. A _total_ of 451 stroke patients received PI with the following same resulting _opinions_ by the raters: Fire-Heat Pattern (*n* = 147), Phlegm-Dampness Pattern _(*n*_ = 158), Yin _Deficiency_ Pattern (*n* = 80), and Qi Deficiency Pattern _(*n*_ = 66). The blood _stasis_ pattern _was_ excluded because the sample size was too small _(*n*_ = 1). The kappa measure of agreement for the _subjects_ of PI _was_ generally moderate to good for the tongue indicators, ranging from 0.40 to 0.72, except for _moist_ fur (*κ* = 0.31). Moreover, _the_ _AC~1~_ _measure_ _of_ _agreement_ between the two experts was _generally_ high for the _tongue_ indicators, ranging from "moderate" (AC~1~ = _0.5)_ to "excellent" _(AC~1~_ _=_ 0.98) ([Table 2](#tab2){ref-type="table"}). 4. Discussion _{#sec4}_ ============= Inspection of _the_ tongue _in_ TKM _diagnosis,_ as _well_ as in western medicine \[[@B28]\], is _one_ of _the_ most important approaches _for_ obtaining significant evidence in diagnosing the patient\'s health _conditions_ \[[@B7]\]. It is used to observe the color, coating, and body of the tongue, _among_ other features, in rendering a disease diagnosis. Also, as tongue diagnosis has played a prominent role _in_ the diagnosis and subsequent treatment _of_ stroke patients, _it_ has attracted an _increasing_ _amount_ of attention in oriental medicine \[[@B8]--[@B15]\]. Park et _al._ \[[@B12]\] analyzed _markers_ _that_ classified _tongue_ body _color,_ fur, fur quality, dryness, and _shape_ to standardize _tongue_ diagnosis and _PI_ for stroke patients. Choi _et_ al. \[[@B14]\], to assess the _usefulness_ of tongue diagnosis in evaluating PI, observed _the_ coating of the tongue and compared it with _PI_ in acute stroke stage patients within _72_ hours from the onset of stroke. In _his_ study, _a_ red _tongue_ was significantly related _to_ the fire-heat pattern and the yin deficiency pattern, _while_ _a_ _faint_ _white_ tongue was related to the phlegm-dampness _pattern._ Thin fur was related _to_ _the_ Wind |
Introduction {#Sec1}
============
Small heat-shock proteins (sHSPs) are a diverse family of proteins that share a conserved ≈ 90-residue α-crystallin domain (ACD) that is flanked by variable N- and C-terminal regions (Basha et al. [@CR3]; Hilton et al. [@CR17]; McHaourab et al. [@CR28]). Although sHSPs are relatively small as monomers (12 to 42 kDa), the majority assemble into large oligomers. These range in size from 12 to \> 40 subunits, with some family members being monodisperse and others forming polydisperse ensembles (Basha et al. [@CR3]; Hilton et al. [@CR17]; McHaourab et al. [@CR28]). Found in all kingdoms of life, many sHSPs have been demonstrated in vitro to act as ATP-independent molecular chaperones with the ability to capture denaturing proteins in a partially unfolded form such that they can be reactivated by the cell's ATP-dependent chaperones. Recent reviews have described models for this canonical mechanism of sHSP chaperone action; however, details are derived primarily from in vitro studies with recombinant proteins and model interactors from non-homologous organisms (Haslbeck and Vierling [@CR16]; Treweek et al. [@CR40]). Thus, a major gap in our understanding of sHSP mechanism is the considerable lack of information about which substrates they protect in the cell.
In order to investigate the properties of proteins that are sHSP interactors, we identified HSP16.6 from the single-celled cyanobacterium *Synechocystis* sp. PCC 6803 (hereafter *Synechocystis*) as an ideal system to interrogate. HSP16.6 is the only sHSP in *Synechocystis* (Giese and Vierling [@CR12]; Lee et al. [@CR25]). It is strongly induced at high temperature, and cells deleted for HSP16.6 (Δ16.6) grow normally at optimal growth temperature but are sensitive to heat stress (Giese and Vierling [@CR12], [@CR13]). The temperature-sensitivity phenotype of Δ16.6 cells has enabled studies of sHSP properties required for activity in vivo in a homologous system. Crucially, point mutations in the N-terminal domain were found to decrease heat tolerance in vivo, but to have no effect on the efficiency of chaperone function in assays with model substrates in vitro (Giese et al. [@CR14]). This observation emphasizes the need to identify native interactors of sHSPs and renders *Synechocystis* an excellent system with which to do so.
We previously used immunoprecipitation and mass spectrometry (MS)-based proteomics to identify 13 proteins associated in vivo with HSP16.6 from *Synechocystis* cells that had been heat-stressed prior to cell lysis (Basha et al. [@CR2]). Notably, these 13 proteins were not detected in equivalent pull-downs from cells that had not been heat-stressed, or when recombinant HSP16.6 was added to heat-stressed Δ16.6 cells before lysis (to control for sHSP-protein interactions that might occur in the lysate, as opposed to during heat stress in vivo). Although these proteins were associated with the sHSP in the soluble cell fraction, they were also found in the insoluble cell fraction after heat stress (Basha et al. [@CR2]). All of these proteins, whose functions span a variety of cellular processes, including translation, transcription, secondary metabolism, and cell signaling, could be released from the immunoprecipitate by addition of DnaK, co-chaperones, and ATP (Basha et al. [@CR2]). In addition, one of these interactors, a serine esterase, when purified, was shown to be heat sensitive and to associate with HSP16.6 and thereby be protected from insolubilization (Basha et al. [@CR2]). While these data identified 13 proteins as potential interactors for canonical sHSP chaperone function, their relatively small number meant it was not possible to derive any common protein features that might dictate interaction with the sHSP.
Here, we have extended the identification of HSP16.6-interactors to a total of 83 proteins by performing an affinity pull-down from heat-stressed *Synechocystis*. By performing rigorous bioinformatic analyses, we provide new insights into the primary and secondary structural properties of proteins that interact with sHSPs in the soluble cell fraction during stress. We also catalogue the functions of the interactors and compare these to sHSP interactors previously identified in two other prokaryotes, *Escherichia coli* and *Deinococcus radiodurans* (Bepperling et al. [@CR4]; Fu et al. [@CR10]). Our combined results indicate that sHSPs protect a specific yet diverse set of proteins from aggregation in the cell.
Methods {#Sec2}
=======
Affinity isolation of HSP16.6-interacting proteins {#Sec3}
--------------------------------------------------
Isogenic *Synechocystis* strains were used in which the wild-type HSP16.6 gene had been replaced with a spectinomycin resistance gene (*aadA* gene) (ΔHSP16.6 strain) or with the spectinomycin gene and HSP16.6 carrying a Strep-tag II affinity tag (WSHPQFEK) on the C-terminus (HSP16.6-Strep strain) (Basha et al. [@CR2]). This HSP16.6-Strep strain had been shown previously to behave like wild type in assays of heat tolerance (Basha et al. [@CR2]), and recombinant HSP16.6-strep protein was equivalent to untagged protein in assays of chaperone activity in vitro (Friedrich et al. [@CR9]).
Cells were grown in 50-mL cultures at 30 °C as described previously to *A*~730~ ≈ 0.2 (Basha et al. [@CR2]) and then subjected to treatment at 42 °C for 2 h followed by 1 h recovery at 30 °C, to allow accumulation of HSP16.6-Strep protein. Control samples were prepared directly after this treatment, while heat-stressed samples were treated for an additional 30 min at 46 °C. To control for interaction of HSP16.6-Strep protein during sample processing, recombinant HSP16.6-Strep protein was added to heat-stressed samples of the ΔHSP16.6 strain directly after heat treatment at a concentration matching that in heat-stressed cells. Cells were harvested, suspended in 1.5 mL lysis buffer (25 mM HEPES-KOH, 0.2 M NaCl, 0.5% Triton X-100, 5 mM ϵ-aminocaproic acid, 1 mM benzamidine, 1 μg mL^−1^ leupeptin, and 1 mM EDTA, pH 7.5), and opened as described previously (Basha et al. [@CR2]). The soluble fraction was mixed with 30 μL of Strep-Tactin resin (Sigma) at 4 °C for 2 h. Resin was washed six times in lysis buffer, and bound proteins were eluted using either sample buffer (for SDS-PAGE) or isoelectric focusing (IEF) rehydration buffer (for 2D gels) (7.0 M urea, 2.5 M thiourea, 2% CHAPS, 2% IPG buffer pH 3--10 NL (Amersham Biotech), and 3 mg mL^−1^ dithiothreitol).
For 2D gel analysis, pH 3--10 NL first dimension strips (18 cm; Amersham Biotech) were rehydrated overnight at room temperature using 600 μL of sample in IEF rehydration buffer. IEF was carried out for 2 h at 150 V, 2 h at 300 V, 5 h at 500 V, and 7 h at 3500 V. The second dimension was separated by 11--17% SDS-PAGE for 30 min at 15 mA and then for 7 h at 25 mA. Samples were also separated by SDS-PAGE according to standard protocols, using 8% acrylamide gels in order to afford good separation of proteins above 100 kDa, which are typically not well resolved on the 2D system. Gels were silver stained according to a previous protocol (Rabilloud [@CR33])*.*
Protein identification by means of mass spectrometry {#Sec4}
----------------------------------------------------
Proteins unique to the heat-stressed HSP16.6-Strep sample were excised from 1D or 2D gels and digested with trypsin, and peptides were prepared for MS as described previously (Basha et al. [@CR2]). Peptide extracts were introduced onto a 100-μm I.D. × 5-cm C18 column using an autosampler and separated with a 25-min gradient of 2--100% acetonitrile in 0.5% formic acid. The column eluate was directed into a Thermo Finnigan LCQ Deca ion trap mass spectrometer. The mass range scanned was 400 to 1500 *m*/*z*, and data-dependent scanning was used to select the three most abundant ions in each parent scan for tandem MS. Peptides were searched using SEQUEST and allowed for static modification of Cys (57 Da; iodoacetamidation), and differential modification of Met (16 | introduction { # sec1 } = = = = = = = = = = = = small heat - shock proteins ( shsps ) are most diverse family of proteins which share a conserved ≈ 90 - residue α - crystallin domain ( acd ) that is flanked by variable n - and c - terminal regions ( basha et al. [ @ cr3 ] ; hilton et al. [ @ cr17 ] ; mchaourab chat al. [ @ cr28 ] ). although shsps are relatively small as monomers ( 12 to 42 kda ), the majority assemble into large oligomers. groups range in size of 12 to \ > 40 subunits, with some family members being monodisperse and others forming polydisperse ensembles ( basha et al. [ @ cr3 ] ; hilton et al. [ @ cr17 ] ; mchaourab et al. [ @ 20 ] ). found in all kingdoms of life, many shsps have been demonstrated in humans to act as atp - independent molecular chaperones with the ability to capture denaturing proteins in a partially unfolded form such that they can be reactivated by the cell ' s atp - dependent chaperones. recent reviews have described models for this canonical mechanism of shsp chaperone action ; however, details are derived primarily from in vitro studies with recombinant proteins and model interactors from non - homologous organisms ( haslbeck and vierling [ @ cr16 ] ; treweek et r. [ @ cr40 ] ). thus, a major gap in our understanding of shsp mechanism is the considerable lack of information about which substrates they protect to the cell. in order to investigate the sequence of proteins that are shsp interactors, we identified hsp16. 6 from the single - celled cyanobacterium * synechocystis * sp. pcc 6803 ( hereafter * synechocystis * ) as an ideal system to interrogate. hsp16. 6 is the only shsp in * synechocystis * ( giese and vierling [ @ cr12 ] ; lee et al. [ @ cr25 ] ). it is strongly induced at high temperature, and cells deleted for hsp16. 6 ( δ16. 6 ) grow normally at optimal growth temperature but are sensitive to heat stress ( giese and vierling [ @ cr12 ], [ @ cr13 ] ). the temperature - sensitivity phenotype of δ16. 6 cells has enabled studies of shsp properties required for activity in vivo in a homologous system. crucially, point mutations in the n - terminal domain were found to decrease heat tolerance in vivo, but to have no effect on the efficiency of chaperone function in assays with model substrates in vitro ( giese et al. [ @ cr14 ] ). this observation emphasizes the need to identify native interactors of shsps and renders * synechocystis * an excellent system with which to do so. we previously used immunoprecipitation and mass spectrometry ( ms ) - based proteomics to identify 13 proteins associated in vivo with hsp16. 6 from * synechocystis * cells that had been heat - stressed prior to cell lysis ( basha et al. [ @ cr2 ] ). notably, these 13 proteins were not detected in equivalent pull - downs from cells that had not been heat - stressed, or when recombinant hsp16. 6 was added to heat - stressed δ16. 6 cells before lysis ( to control for shsp - protein interactions that might occur in the lysate, as opposed to during heat stress in vivo ). although these proteins were associated with the shsp in the soluble cell fraction, they were also found in the insoluble cell fraction after heat stress ( basha et al. [ @ cr2 ] ). all of these proteins, whose functions span a variety of cellular processes, including translation, transcription, secondary metabolism, and cell signaling, could be released from the immunoprecipitate by addition of dnak, co - chaperones, and atp ( basha et al. [ @ cr2 ] ). in addition, one of these interactors, a serine esterase, when purified, was shown to be heat sensitive and to associate with hsp16. 6 and thereby be protected from insolubilization ( basha et al. [ @ cr2 ] ). while these data identified 13 proteins as potential interactors for canonical shsp chaperone function, their relatively small number meant it was not possible to derive any common protein features that might dictate interaction with the shsp. here, we have extended the identification of hsp16. 6 - interactors to a total of 83 proteins by performing an affinity pull - down from heat - stressed * synechocystis *. by performing rigorous bioinformatic analyses, we provide new insights into the primary and secondary structural properties of proteins that interact with shsps in the soluble cell fraction during stress. we also catalogue the functions of the interactors and compare these to shsp interactors previously identified in two other prokaryotes, * escherichia coli * and * deinococcus radiodurans * ( bepperling et al. [ @ cr4 ] ; fu et al. [ @ cr10 ] ). our combined results indicate that shsps protect a specific yet diverse set of proteins from aggregation in the cell. methods { # sec2 } = = = = = = = affinity isolation of hsp16. 6 - interacting proteins { # sec3 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - isogenic * synechocystis * strains were used in which the wild - type hsp16. 6 gene had been replaced with a spectinomycin resistance gene ( * aada * gene ) ( δhsp16. 6 strain ) or with the spectinomycin gene and hsp16. 6 carrying a strep - tag ii affinity tag ( wshpqfek ) on the c - terminus ( hsp16. 6 - strep strain ) ( basha et al. [ @ cr2 ] ). this hsp16. 6 - strep strain had been shown previously to behave like wild type in assays of heat tolerance ( basha et al. [ @ cr2 ] ), and recombinant hsp16. 6 - strep protein was equivalent to untagged protein in assays of chaperone activity in vitro ( friedrich et al. [ @ cr9 ] ). cells were grown in 50 - ml cultures at 30 °c as described previously to * a * ~ 730 ~ ≈ 0. 2 ( basha et al. [ @ cr2 ] ) and then subjected to treatment at 42 °c for 2 h followed by 1 h recovery at 30 °c, to allow accumulation of hsp16. 6 - strep protein. control samples were prepared directly after this treatment, while heat - stressed samples were treated for an additional 30 min at 46 °c. to control for interaction of hsp16. 6 - strep protein during sample processing, recombinant hsp16. 6 - strep protein was added to heat - stressed samples of the δhsp16. 6 strain directly after heat treatment at a concentration matching that in heat - stressed cells. cells were harvested, suspended in 1. 5 ml lysis buffer ( 25 mm hepes - koh, 0. 2 m nacl, 0. 5 % triton x - 100, 5 mm [UNK] - aminocaproic acid, 1 mm benzamidine, 1 μg ml ^ −1 ^ leupeptin, and 1 mm edta, ph 7. 5 ), and opened as described previously ( basha et al. [ @ cr2 ] ). the soluble fraction was mixed with 30 μl of strep - tactin resin ( sigma ) at 4 °c for 2 h. resin was washed six times in lysis buffer, and bound proteins were eluted using either sample buffer ( for sds - page ) or isoelectric focusing ( ief ) rehydration buffer ( for 2d gels ) ( 7. 0 m urea, 2. 5 m thiourea, 2 % chaps, 2 % ipg buffer ph 3 - - 10 nl ( amersham biotech ), and 3 mg ml ^ −1 ^ dithiothreitol ). for 2d gel analysis, ph 3 - - 10 nl first dimension strips ( 18 cm ; amersham biotech ) were rehydrated overnight at room temperature using 600 μl of sample in ief rehydration buffer. ief was carried out for 2 h at 150 v, 2 h at 300 v, 5 h at 500 v, and 7 h at 3500 v. the second dimension was separated by 11 - - 17 % sds - page for 30 min at 15 ma and then for 7 h at 25 ma. samples were also separated by sds - page according to standard protocols, using 8 % acrylamide gels in order to afford good separation of proteins above 100 kda, which are typically not well resolved on the 2d system. gels were silver stained according to a previous protocol ( rabilloud [ @ cr33 ] ) *. * protein identification by means of mass spectrometry { # sec4 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - proteins unique to the heat - stressed hsp16. 6 - strep sample were excised from 1d or 2d gels and digested with trypsin, and peptides were prepared for ms as described previously ( basha et al. [ @ cr2 ] ). peptide extracts were introduced onto a 100 - μm i. d. × 5 - cm c18 column using an autosampler and separated with a 25 - min gradient of 2 - - 100 % acetonitrile in 0. 5 % formic acid. the column eluate was directed into a thermo finnigan lcq deca ion trap mass spectrometer. the mass range scanned was 400 to 1500 * m * / * z *, and data - dependent scanning was used to select the three most abundant ions in each parent scan for tandem ms. peptides were searched using sequest and allowed for static modification of cys ( 57 da ; iodoacetamidation ), and differential modification of met ( 16 | Introduction {# Sec1} = = = = = = = = = = = = Small heat - shock proteins (sHSPs) are a diverse family of proteins that share a conserved ≈ 90 - residue α - crystallin domain (ACD) that is flanked by variable N - and C - terminal regions (Basha et al. [@ CR3 ]; Hilton et al. [@ CR17 ]; McHaourab et al. [@ CR28] ). Although sHSPs are relatively small as monomers (12 to 42 kDa ), the majority assemble into large oligomers. These range in size from 12 to \> 40 subunits, with some family members being monodisperse and others forming polydisperse ensembles (Basha et al. [@ CR3 ]; Hilton et al. [@ vd17 ]; McHaourab et al. [@ CR28] ). Found in all kingdoms of life, many sHSPs have been demonstrated in vitro to act as ATP - independent molecular chaperones with the ability to capture denaturing proteins in a partially unfolded form such that they can be reactivated by the cell ' s ATP - dependent chaperones. Recent reviews have described models for this canonical mechanism of sHSP chaperone action; however, details are derived primarily f48m in vitro studies with recombinant proteins and model interactors from non - homologous organisms (Haslbeck and Vierling [@ CR16 ]; Treweek et al. [@ CR40] ). Thus, a major gap in our understanding of sHSP mechanism is the considerable lack of information about which substrates they protect in the cell. In order to investigate the properties of proteins that are sHSP interactors, we identified HSP16. 6 from the single - celled cyanobacterium * Synechocystis * sp. PCC 6803 (hereafter * Synechocystis *) as an ideal system to interrogate. HSP16. 6 is the pnlJ sHSP in * Synechocystis * (Giese and Vierling [@ CR12 ]; Lee et al. [@ CR25] ). It is strongly inducrS at high temperature, and cells deleted for HSP16. 6 (Δ16. 6) grow normally at optimal growth temperature but are sensitive to heat stress (Giese and Vierling [@ CR12 ], [@ CR13] ). The temperature - sensitivity phenotype of Δ16. 6 cells has enabled studies of sHSP properties required for activity in vivo in a homologous system. Crucially, point mutations in the N - terminal domain were found to decrease heat tolerance in vivo, but to have no effect on the efficiency of chaperone function in assays with model substrates in vitro (Giese et al. [@ CR14] ). This observation emphasizes the need to identify native interactors of sHSPs and renders * Synechocystis * an excellent system with which to do so. We previously used immunoprecipitation and mass spectrometry (MS) - based proteomics to identify 13 proteins associated in vivo with HSP16. 6 f%oH * Synechocystis * cells that had been heat - stressed prior to cell lysis (Basha et al. [@ CR2] ). Notably, these 13 proteins were not detected in equivalent pull - downs from cells tUar had not been heat - stressed, or when recombinant HSP16. 6 was added to heat - stressed Δ16. 6 cells before lysis (to control for sHSP - protein interactions that might occur in the lysate, as opposed to during heat stress in vivo ). AlthI*gh these proteins were associated with the sHSP in the soluble cell fraction, they were also found in the insoluble cell fraction after heat stress (Basha et al. [@ CR2] ). All of these proteins, whose functions span a variety of cellular processes, including translation, transcription, secondary metabolism, and cell signaling, could be released from the immunoprecipitate by addition of DnaK, co - chaperones, and ATP (Basha et al. [@ CR2] ). In addition, one of these interactors, a serine esterase, when purified, was shown to be heat sensitive and to associate with HSP16. 6 and thereby be protected from insolubilization (Basha et al. [@ CR2] ). While these data identified 13 proteins as potential interactors for canonical sHSP chaperone function, their relatively small number meant it was not possible to derive any common protein features that might EicRate interaction with the sHSP. Here, we have extended the identification of HSP16. 6 - interactors to a total of 83 proteins by performing an affinity pull - down from heat - stressed * Synechocystis *. By performing rigorous bioinformatic analyses, we provide new insights into the primary and secondary structural properties of proteins that interact with sHSPs in the soluble cell fraction during stress. We also catalogue the functions of the interactors and compare these to sHSP interactors previously identified in two other prokaryotes, * Escherichia coli * and * Deinococcus radiodurans * (Bepperling et al. [@ CR4 ]; Fu et al. [@ CR10] ). Our combined results indicate that sHSPs protect a specific yet diverse set of proteins from aggregation in the cell. Methods {# Sec2} = = = = = = = Affinity isolation of HSP16. 6 - interacting proteins {# Sec3} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Isogenic * Synechocystis * strains were used in which the wild - type HSP16. 6 gene had been replaced with a spectinomycin resistance gene (* aadA * gene) (ΔHSP16. 6 strain) or with the spectinomycin gene and HSP16. 6 carrying a Strep - tag II affinity tag (WSHPQFEK) on the C - terminus (HSP16. 6 - Strep strain) (Basha et al. [@ CR2] ). This HSP16. 6 - Strep strain had been shown previously to behave like 1jld type in assays of heat tolerance (Basha et al. [@ CR2] ), and recombinant HSP16. 6 - strep protein was equivalent to untagged protein in assays of chaperone activity in vitro (Friedrich et al. [@ CR9] ). Cells were grown in 50 - mL cultures at 30 ° C as described previously to * A * ~ 730 ~ ≈ 0. 2 (Basha et al. [@ CR2] ) and then subjected to treatment at 42 ° C for 2 h followed by 1 h recovery at 30 ° C, to allow accumulation of HSP16. 6 - Strep protein. Control samples were prepared directly after this treatment, while heat - stressed samples were treated for an additional 30 min at 46 ° C. To control for interaction of HSP16. 6 - Strep protein during sample processing, recombinant HSP16. 6 - Strep protein was added to heat - stressed samples of the ΔHSP16. 6 strain directly after heat treatment at a concentration matching that in h2zt - stressed cells. Cells were harvested, suspended in 1. 5 mL lysis buffer (25 mM HEPES - KOH, 0. 2 M NaCl, 0. 5% Triton X - 100, 5 mM ϵ - aminocaproic acid, 1 mM benzamidine, 1 μg mL ^ − 1 ^ leupeptin, and 1 mM EDTA, pH 7. 5 ), and opened as described previously (Basha et al. [@ CR2] ). The soluble fraction was mixed with 30 μL of Strep - Tactin resin (Sigma) at 4 ° C for 2 h. Resin was washed six times in lysis buffer, and bound proteins were eluted using either sample buffer (for SDS - PAGE) or isoelectric focusing (IEF) rehydration buffer (for 2D gels) (7. 0 M urea, 2. 5 M thiourea, 2% CHAPS, 2% IPG buffer pH 3 - - 10 NL (Amersham Biotech ), and 3 mg mL ^ − 1 ^ dithiothreitol ). For 2D gel analysis, pH 3 - - 10 NL first dimension strips (18 cm; Amersham Biotech) were rehydrated overnight at room temperature using 600 μL of sample in IEF rehydration buffer. IEF was carried out for 2 h at 150 V, 2 h at 300 V, 5 h at 500 V, and 7 h at 3500 V. The second dimension was separated by 11 - - 17% SDS - PAGE for 30 min at 15 mA and then for 7 h at 25 mA. Samples were also separated by SDS - PAGE according to standard protocols, using 8% acrylamide gels in order to afford good separation of proteins above 100 kDa, which are typically not well resolved on the 2D system. Gels were silver stained according to a previous protocol (Rabilloud [@ CR33] ) *. * Protein identification by means of mass spectrometry {# Sec4} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Proteins unique to the heat - stressed HSP16. 6 - Strep sample were excised from 1D or 2D gels and digested with trypsin, and peptides were prepared for MS as described previously (Basha et al. [@ CR2] ). Peptide extracts were introduced onto a 100 - μm I. D. × 5 - cm C18 column using an autosampler and separated with a 25 - min gradient of 2 - - 100% acetonitrile in 0. 5% formic acid. The column eluate was directed into a Thermo Finnigan LCQ Deca ion trap mass spectrometer. The mass range scanned was 400 to 1500 * m * / * z *, and data - dependent scanning was used to select the three most abundant ions in each parent scan for tandem MS. Peptides were searched using SEQUEST and allowed for static modification of Cys (57 Da; iodoacetamidation ), and differential modification of Met (16 | Introduction {#Sec1} ============ Small heat-shock proteins (sHSPs) are a family of proteins that share a conserved ≈ α-crystallin domain (ACD) that is flanked by variable N- and C-terminal (Basha et [@CR3]; Hilton al. [@CR17]; McHaourab et al. [@CR28]). sHSPs are small as monomers (12 to 42 kDa), the majority assemble into large oligomers. These range in size 12 to \> 40 subunits, with some family members being monodisperse and others forming polydisperse ensembles (Basha et [@CR3]; Hilton et al. [@CR17]; McHaourab et al. [@CR28]). Found all kingdoms of life, many sHSPs have demonstrated vitro to act as ATP-independent molecular chaperones with the ability capture denaturing proteins in a partially unfolded form such that they can be reactivated by the cell's chaperones. Recent reviews have models for this canonical mechanism of sHSP chaperone action; however, details are derived primarily from in vitro studies with recombinant proteins and model interactors from non-homologous organisms (Haslbeck and Vierling Treweek et al. [@CR40]). Thus, a major gap in our understanding of sHSP mechanism is the considerable lack of about substrates they protect in the cell. In order to investigate the properties of proteins that are sHSP interactors, we identified HSP16.6 from single-celled cyanobacterium *Synechocystis* sp. PCC 6803 (hereafter *Synechocystis*) as an ideal system to interrogate. HSP16.6 is the only sHSP in *Synechocystis* (Giese Vierling [@CR12]; Lee et al. It is strongly induced at high temperature, and for HSP16.6 (Δ16.6) grow normally at optimal growth temperature but sensitive heat stress (Giese and Vierling [@CR12], The temperature-sensitivity phenotype of Δ16.6 cells has enabled studies of sHSP properties required for activity in vivo in a homologous system. Crucially, point mutations in the N-terminal domain were found to decrease heat tolerance in vivo, but to have no effect on the of chaperone function in assays with in (Giese et al. [@CR14]). This observation emphasizes need to identify native interactors of sHSPs and *Synechocystis* an excellent system with which do so. We previously immunoprecipitation and mass spectrometry (MS)-based proteomics to identify 13 proteins associated in vivo with HSP16.6 from *Synechocystis* cells that been heat-stressed prior to cell (Basha et al. [@CR2]). these 13 proteins were not detected in equivalent pull-downs from cells that had not been heat-stressed, or when recombinant HSP16.6 was added to heat-stressed Δ16.6 cells before lysis (to control for sHSP-protein interactions that occur in the lysate, to heat stress in Although these proteins associated with the sHSP in the soluble cell fraction, they were also found in the insoluble cell fraction after heat stress (Basha et al. All of proteins, whose functions a variety processes, including transcription, secondary metabolism, and cell signaling, could be from the immunoprecipitate by addition of co-chaperones, and ATP (Basha et al. [@CR2]). In addition, one of these interactors, a serine esterase, when purified, was shown be heat sensitive and to associate with HSP16.6 and thereby be protected from insolubilization (Basha et [@CR2]). While these data identified 13 proteins as potential for canonical sHSP function, their relatively small meant it was not possible derive any common protein features that might dictate with the sHSP. Here, we have the identification of HSP16.6-interactors to a total of 83 proteins by performing an affinity pull-down from heat-stressed *Synechocystis*. By performing rigorous bioinformatic analyses, we provide insights into the primary and secondary of with sHSPs in the soluble cell stress. We also catalogue the of the interactors and compare these to sHSP interactors previously in two other prokaryotes, *Escherichia and *Deinococcus (Bepperling et [@CR4]; Fu et al. [@CR10]). Our combined results indicate that sHSPs protect a specific yet diverse set of proteins from aggregation in the cell. Methods {#Sec2} ======= Affinity isolation of HSP16.6-interacting proteins {#Sec3} -------------------------------------------------- Isogenic *Synechocystis* strains were used in which the wild-type HSP16.6 gene had been with a spectinomycin resistance gene (*aadA* gene) (ΔHSP16.6 with the gene and HSP16.6 a Strep-tag II affinity tag (WSHPQFEK) on the C-terminus (HSP16.6-Strep strain) (Basha et al. This HSP16.6-Strep strain had been shown previously to behave like wild type assays of heat tolerance (Basha et al. [@CR2]), and recombinant HSP16.6-strep protein equivalent to untagged protein in of chaperone activity in vitro (Friedrich al. [@CR9]). Cells grown in 50-mL cultures at 30 °C described previously to *A*~730~ ≈ 0.2 (Basha et al. [@CR2]) and to treatment at 42 °C for 2 h followed by 1 h recovery at 30 °C, to allow accumulation of HSP16.6-Strep protein. Control prepared directly after this treatment, while heat-stressed samples were treated for an additional min at 46 °C. To control for interaction of HSP16.6-Strep protein during sample processing, recombinant HSP16.6-Strep protein was added to heat-stressed samples of the ΔHSP16.6 strain directly after heat treatment at a concentration matching that in heat-stressed cells. were harvested, in 1.5 mL lysis buffer (25 mM HEPES-KOH, M NaCl, 0.5% Triton 5 mM ϵ-aminocaproic 1 benzamidine, 1 μg mL^−1^ leupeptin, and 1 EDTA, pH 7.5), and opened as described previously (Basha et al. [@CR2]). The soluble fraction was mixed 30 μL of Strep-Tactin resin (Sigma) at 4 °C for 2 h. Resin was washed times in lysis buffer, and bound proteins were eluted using either sample (for SDS-PAGE) or isoelectric focusing (IEF) (for 2D gels) (7.0 M urea, 2.5 M thiourea, 2% CHAPS, 2% IPG buffer pH 3--10 NL (Amersham Biotech), and 3 mg mL^−1^ dithiothreitol). For 2D gel analysis, pH NL first strips (18 cm; Amersham Biotech) were overnight room temperature using μL of sample in rehydration buffer. IEF was carried for 2 h at 150 V, 2 h at 300 V, 5 h at 500 V, and 7 h at 3500 V. The second dimension separated by 11--17% SDS-PAGE for 30 min at 15 mA and then for 7 h at 25 mA. Samples were also separated by SDS-PAGE according to standard protocols, using 8% acrylamide gels in order to afford good separation of proteins above 100 kDa, which are not well resolved on the 2D system. were silver stained according to a previous protocol (Rabilloud [@CR33])*.* Protein identification by means of mass {#Sec4} ---------------------------------------------------- Proteins unique to the heat-stressed HSP16.6-Strep sample were excised from 1D or 2D and with trypsin, and peptides were prepared for MS as described previously (Basha et al. [@CR2]). Peptide extracts were introduced onto a 100-μm I.D. × 5-cm C18 column an autosampler separated with a 25-min of 2--100% acetonitrile in 0.5% formic acid. column eluate was directed into a Thermo Finnigan LCQ Deca ion mass spectrometer. The range scanned was 400 to 1500 *m*/*z*, and data-dependent scanning was used select the three most abundant in each parent scan for tandem MS. Peptides were using SEQUEST and allowed for static modification of Cys (57 Da; iodoacetamidation), and differential modification of Met (16 | INtRoDuCtIoN {#Sec1}
============
SmAlL hEat-sHOcK pROTeiNS (shSPS) aRe a DivErsE FaMily oF PROteiNs THAt shArE A coNServeD ≈ 90-REsidUE Α-cRYsTallIN DomaiN (AcD) THAt iS fLANkEd by vAriAble N- and c-tERmINAL rEgIoNs (bASHa ET Al. [@Cr3]; Hilton ET aL. [@cR17]; mChAoURab et aL. [@cR28]). ALthoUGh ShsPs aRE RELaTivELY SmAlL aS MOnOmeRS (12 tO 42 KdA), the majoRITy ASsEmBLe inTo largE oLigOMERs. these rANge In SIZe frOM 12 TO \> 40 SubunITs, wIth some FAmiLY membErS bEINg MOnoDiSPErse AND oThers fOrMing POLyDISpERse eNSembLES (basHA eT AL. [@Cr3]; HIlTon eT al. [@Cr17]; mcHaOuRAb et AL. [@cR28]). fOUnd in alL kingDOMs OF Life, MaNy shspS have BEEn dEMonsTraTED iN vitRO tO AcT As aTP-iNdEpEnDenT MOlECULAR cHaPErOnes WItH ThE abILITY To CaptURe dEnAturing proTEiNS iN a pArTiAlly unFoLded form sUCh THAT ThEy Can BE reACtIvAtED by ThE celL'S atp-DEPENDeNt ChAPEronES. reCeNT REvieWS HavE DescriBEd ModElS For thIS cAnONICAl mechAnISm Of ShSP cHAPEroNE aCTiON; hOWeVeR, DetAiLs are dErIVED pRiMarIlY fRom in VitRO StUdIes WITH RECOMbINANt PrOteINS And mODel inTeracTORs FROM non-hOMOloGOUs ORgAnismS (HaSLBecK And vIerlING [@Cr16]; tReWeek Et aL. [@cr40]). THuS, a maJOr gap iN oUR unDerstAnding of Shsp mECHANisM IS tHe cOnsiDerAblE lAcK oF iNformATioN ABOUT whicH sUBstratEs tHeY PRoTEct iN THE cEll.
in orDeR to inVEsTigATe THE prOPErTieS Of pROteiNs thaT ArE shSP INteRactORS, we iDenTifieD hsp16.6 froM tHe SinGle-CelLed CyaNobACterIUm *sYnechocySTis* sp. pcc 6803 (heReAfteR *SyNEcHocyStis*) As AN iDEaL SySTem tO inTerROGATe. hsP16.6 Is thE oNLy sHsp in *synEchOCYsTIS* (GiEse AND VIErLINg [@CR12]; lEe Et Al. [@cr25]). iT iS sTRonGLy inDUced at HIgH TEmpeRaTuRe, aND CelLS DElEted FOr hsp16.6 (δ16.6) gROW NOrMaLLY aT oPTIMal grOWTH teMPeratUrE BUt arE SENsItivE tO heat StrEsS (gIesE ANd VieRLiNg [@CR12], [@Cr13]). the TemPEraTurE-SEnSiTIvITY PheNoType Of δ16.6 cELlS HAs EnAbLED StUdieS of ShSp prOPeRtIeS requiREd FoR ACtiVItY In vIvo In A HomologOUs sYsTEm. CRuCiaLlY, PoInt MUtaTionS iN tHe n-TErMINal dOmAiN WErE FoUND To DECreasE HEat tolERAnCe IN VIVo, BUt TO have no efFeCT On thE EfFIcienCy of cHApErone fuNctiON iN ASsAYs WItH MODel SuBSTRaTES IN vitrO (gIESe ET al. [@cR14]). tHIs OBSeRvaTioN eMphASIZEs The NEeD tO identiFY NAtiVE INtEraCtOrS Of sHsPs AnD rENDERS *syNeCHocYSTIs* an EXceLlENt SySTEM wiTh WHICh To DO So.
WE PReVioUSly UsEd immuNOprecIpitATIon And MAss SPeCTrOMEtRY (MS)-BAsEd PrOteomIcS tO IdEnTifY 13 pRotEiNS ASsocIaTed In Vivo WiTH HSp16.6 FrOm *synechOcysTis* CElls ThAT Had bEEn HEaT-stRESSed PrIor to cELL lYsIs (baSHA Et Al. [@cr2]). NotaBly, THESe 13 PROtEIns WerE nOt dETECtED in EqUivalENt PulL-dOWns FrOM cELls thaT haD nOt bEeN heAT-StREssed, OR WHEN recombInaNt hsp16.6 wAs addEd To hEAT-STReSsEd Δ16.6 ceLlS bEFOre LYSIs (to conTroL FoR sHsP-PROTEIN INterAcTions That mIGHt OCcur IN The lYSaTe, As OPpoSeD TO duRinG Heat stresS in vIVo). alTHouGH tHese PrOTeiNs WERE ASSoCiAted WItH ThE sHsP IN the SoluBLE cELl fractiOn, tHEy wERE ALsO fOUNd In tHE inSoLublE CELl fRaCTion aftER HEat stResS (baSHA eT aL. [@CR2]). aLl Of THESe proteIns, WHOsE funcTIoNS SpaN a VArieTy Of celLuLar PROcESSES, INCLuDiNG tRANslATIon, tranSCrIPtION, SecoNDARy mEtaBOLiSM, anD cELL sigNAliNG, COuld BE reLeASED fROm ThE IMmUNopRecIPITatE BY AdDiTiOn OF dnak, co-cHApeROnES, ANd atP (bAShA ET aL. [@cr2]). IN ADDitIOn, oNe Of thESE iNTEractOrs, a SERiNe EsTERasE, WheN PUrIFIed, WaS sHowN tO Be HEaT SeNsitiVE anD tO aSsoCiatE WIth hsP16.6 AND tHeREby be PROTEcteD frOm iNSOLuBilIZATIOn (baSha et AL. [@CR2]). WHIlE THESe dATA iDENTifIEd 13 PROTeiNS AS pOtEnTIaL InTErACTORS for CANonICAl Shsp CHAPeroNe FuNCTiOn, thEIR RElATiVELY smAll NuMBeR MeaNT it was NOt possIBLe tO DerIVE ANY COmMoN pRoTeIn FeaTuRES that MIGHt dICtatE INterAcTIon WITh the shSp.
hErE, wE HaVe eXTeNDeD THE IdentIFICaTIOn Of Hsp16.6-interACtOrs to a TOtAL oF 83 pROTEIns by peRFORMING AN AffiNity PuLL-DoWN froM hEaT-streSsED *SynECHocYSTIS*. By peRFoRMing riGOrous BIOiNFoRmAtic anAlySeS, WE pROviDE new iNSiGHtS INTO thE PRImaRy aNd sEcOnDary sTrUcTuRAl pROpERtieS oF PRotEiNS ThAT intEract wItH ShSPs IN THe SOlUble ceLl FraCtIOn DuRing STrESs. wE aLsO catAlOGUE the fUnCTIOns OF the iNteraCTORS aND COmPARE thEsE tO shsP InTerACTORs pREvIoUsly idEntifiEd iN TwO OThEr prOKaryOTeS, *escHERiChiA CoLi* AND *DEinoCOccUs rAdIoduRAnS* (BEPPERLINg ET AL. [@Cr4]; fU Et Al. [@cr10]). oUR COmBINED rESUlTs InDiCaTE THaT shSps PROTeCt A speCIfiC YET DiVErse SET OF proTeiNs FRom AgGREgAtIoN iN thE CeLl.
MeThods {#sec2}
=======
AFFINItY isolaTIon Of HSP16.6-INTErAcTinG pRotEins {#SeC3}
--------------------------------------------------
isOgENic *SynEchoCYsTIS* stRains WErE useD iN WHiCh thE wild-TyPE Hsp16.6 GeNe had Been rEplaced wITH A sPeCtINOmyCiN RESisTAnCE GENE (*AaDa* GENe) (ΔHSP16.6 STrAin) or WItH the SpEcTiNOMYCIN gEne ANd hSP16.6 CaRRYING A sTrEp-taG ii afFiNitY tAG (wshPQfEk) on thE c-TeRmiNUs (hsp16.6-strEP STraIN) (baSha et AL. [@cR2]). ThiS hsp16.6-STrEP strAIn HAD BeEN sHOWn PREvIOUSLy to BEHaVe LikE wILd tyPE iN aSsayS OF heAT toLeranCe (BAshA ET Al. [@CR2]), ANd rEcombInant Hsp16.6-stRep proTeIN WAS EqUIVaLEnt To unTAgGED protEIn In aSsAYS of CHApERONe acTIViTy In VitRO (fRIedrICh ET al. [@CR9]).
cELls weRE GROwn in 50-ml CuLTUreS AT 30 °C AS dESCrIbeD PRevIouSlY To *A*~730~ ≈ 0.2 (bAsha eT al. [@cr2]) aNd TheN SUBjeCteD tO TrEatMEnt At 42 °c fOr 2 h FolLoWEd By 1 H rECovERy AT 30 °C, to ALlOW aCcUMULATioN OF hSP16.6-sTREP PRoteiN. CONtRol SAMpLeS WErE PrEPared DIrECTly aFTER ThIS TREATmENT, WHILe hEaT-StrEssEd SamPlES Were tReATEd For AN ADdItiOnAL 30 min aT 46 °C. tO CoNtrOl fOr iNtERACtioN of HsP16.6-sTReP proTEin duriNg sampLe PROCESSING, REcOMbiNanT Hsp16.6-STRep PrOTeIn Was ADdEd to HeAt-stResseD SaMPlES oF the ΔHsP16.6 strAIN dIRECTlY AftEr heAT TREAtMeNt aT a cOnCentRAtIOn MaTcHInG tHAt In HeaT-StrEssED CELLS. celLS werE hARvEsTED, suSPeNDed IN 1.5 Ml lYsiS BuffEr (25 MM HePeS-KOH, 0.2 M nAcl, 0.5% tRitOn X-100, 5 mm Ε-aMinocAProiC aCiD, 1 mM benzamidIne, 1 μg ML^−1^ lEUPEPTIn, ANd 1 mm eDtA, pH 7.5), aNd opENEd aS dEsCRiBED pReVIOuslY (BAsHA ET aL. [@cR2]). THe SolubLE fractIOn waS MiXed wiTH 30 Μl OF STreP-taCTin resIn (sIgma) at 4 °C FOr 2 H. RESin waS wAshed sIx TImEs in lySIs BUFFer, anD BOUND ProTeinS werE eluTeD uSiNG EItHER SAMpLE buFFeR (fOR SDs-pAGe) oR ISOELectrIC FocUsInG (IEF) RehyDRATIoN bUFFER (for 2d geLs) (7.0 M ureA, 2.5 M tHIOureA, 2% chAPS, 2% ipG bUFFEr PH 3--10 NL (amErsHAm BIoTecH), aND 3 Mg mL^−1^ DiThiOthrEIToL).
for 2D GEl aNaLYsiS, ph 3--10 nL fiRsT DIMEnsIon sTRips (18 cM; AMERSHaM BioTEcH) wEre REhydrated OVerniGHt AT rOoM tEmpERatuRe USinG 600 ΜL OF sAMPLe iN iEF REhyDRatiOn BUffeR. IeF Was CarrIED ouT FOR 2 H At 150 V, 2 H AT 300 V, 5 H At 500 v, aNd 7 H AT 3500 V. The seConD dimENSiOn WaS sepaRateD By 11--17% sDS-paGE foR 30 mIn at 15 ma ANd ThEn FOr 7 h at 25 ma. SAMplEs wErE ALsO SEParAted BY Sds-pagE AcCorDINg To StAndarD pROTocOls, uSIng 8% ACRylamIde Gels IN Order to AfFoRD gOoD SePARaTION OF pRoteIns aboVe 100 kdA, wHiCH are TYpicAlLY not weLL ReSoLvEd ON thE 2D sYSTEm. Gels wERE siLVeR stAinED aCCordiNG TO A PrEVioUs protOcol (rAbILloUd [@Cr33])*.*
proTein IdEnTIfICATioN By MeAnS of masS SpeCtRometRY {#sec4}
----------------------------------------------------
prOteinS UnIQUE tO thE hEAT-STRESSed hsP16.6-sTREp sAMPle WERe eXcISeD FROM 1D Or 2D gelS AND DiGEstEd wiTH tryPSIN, ANd pEpTIdEs WeRE prEPARed FOR ms as dESCRIbeD PReViOUSLy (BaSHA eT aL. [@cR2]). PEPtide ExtrACTS Were inTROdUCEd Onto A 100-Μm i.d. × 5-cM C18 coluMN UsING AN AUToSamplER AND sepaRaTeD WIth A 25-miN graDIenT OF 2--100% AcetONItRilE iN 0.5% fORMIC aCiD. THE COlUmN eLuaTe was dIrEcTed iNTo A tHeRmo fiNniGAn lcQ dECa ioN trap mAss sPeCtRomEteR. tHe mAss rAnGE sCAnNed Was 400 To 1500 *m*/*z*, ANd datA-DEpENDeNt sCaNnINg waS USeD TO SELeCT ThE ThrEE MoST abundant IoNS In EACh PArenT ScAN FOR TAndEM ms. pEPtIdEs wERE seaRCHed uSinG SEQUESt AND aLloWed for STATic ModifIcatioN oF cYs (57 Da; IodOAcETamIdAtIon), aNd dIffeREntiAl modifICATioN of met (16 | Introduction {#Sec1} ============ Small heat-shock proteins (sHSPs) are a diverse family of proteins thatshare a conserved ≈ 90-residue α-crystallin domain (ACD) that is flanked byvariable N-and C-terminalregions (Basha et al. [@CR3]; Hilton et al. [@CR17]; McHaourab et al. [@CR28]). Although sHSPs are relatively small as monomers (12 to 42 kDa), the majority assemble into large oligomers.These range insize from 12 to \> 40subunits, with some family members being monodisperse and others forming polydisperse ensembles (Bashaet al. [@CR3]; Hilton et al. [@CR17]; McHaourab et al. [@CR28]). Found in all kingdoms of life,many sHSPs have been demonstrated in vitro toact as ATP-independent molecular chaperones with the ability to capture denaturing proteins in apartially unfoldedform such that they can bereactivated by thecell's ATP-dependent chaperones. Recent reviews have described models for this canonical mechanism of sHSP chaperone action; however, detailsare derived primarily from in vitro studies with recombinant proteins andmodel interactors from non-homologous organisms (Haslbeck and Vierling [@CR16]; Treweek et al. [@CR40]). Thus, a major gap in ourunderstanding of sHSP mechanism is the considerable lack of information aboutwhichsubstrates they protect in the cell. Inorder to investigate the properties of proteins that are sHSP interactors, weidentifiedHSP16.6 from the single-celled cyanobacterium*Synechocystis* sp. PCC 6803 (hereafter *Synechocystis*) as an ideal system to interrogate. HSP16.6 is the onlysHSP in *Synechocystis* (Giese and Vierling [@CR12]; Lee et al. [@CR25]). It isstrongly inducedat high temperature,and cells deleted for HSP16.6 (Δ16.6) grow normally at optimal growth temperature but aresensitive to heat stress (Giese and Vierling [@CR12], [@CR13]). The temperature-sensitivityphenotype of Δ16.6 cells hasenabled studies of sHSPproperties required for activity in vivo ina homologous system. Crucially,point mutationsin the N-terminal domain were foundto decrease heat tolerance in vivo, but tohave no effect on the efficiency of chaperone functioninassayswith model substratesin vitro (Giese et al. [@CR14]). This observation emphasizes theneed to identify native interactors of sHSPs and renders *Synechocystis* anexcellent system with which todo so. We previously usedimmunoprecipitation and mass spectrometry (MS)-based proteomics to identify 13 proteins associatedinvivo with HSP16.6 from*Synechocystis* cells that had been heat-stressedprior to celllysis (Basha et al.[@CR2]). Notably, these 13 proteins were notdetected in equivalent pull-downsfrom cells thathadnot beenheat-stressed, or when recombinantHSP16.6 was added to heat-stressedΔ16.6 cells before lysis(to control for sHSP-protein interactions that might occurin the lysate, as opposed to during heat stress in vivo). Although these proteins were associated withthe sHSP in the soluble cell fraction, they werealsofound in the insoluble cell fraction after heat stress (Basha et al. [@CR2]). All of these proteins, whose functionsspan a variety of cellular processes, including translation, transcription, secondary metabolism, and cell signaling, could be released from the immunoprecipitateby addition of DnaK,co-chaperones, and ATP (Basha et al. [@CR2]). In addition, one of these interactors, a serine esterase, when purified,was shown to be heat sensitive and to associate with HSP16.6 and thereby be protected frominsolubilization (Basha et al. [@CR2]). While these data identified13 proteinsas potential interactors forcanonical sHSP chaperone function, their relatively small number meant it was notpossible to derive any common protein features that might dictate interaction with the sHSP. Here, we have extended the identificationof HSP16.6-interactors to a total of 83 proteins by performing an affinity pull-down from heat-stressed *Synechocystis*. Byperformingrigorous bioinformaticanalyses, we provide new insights into the primaryand secondary structural properties of proteins that interact with sHSPs in the solublecell fraction during stress. We also catalogue the functions of the interactors and compare these to sHSP interactors previously identified in two other prokaryotes, *Escherichiacoli* and*Deinococcus radiodurans* (Bepperling et al. [@CR4];Fu et al. [@CR10]). Our combined results indicate thatsHSPs protect a specific yet diverse set of proteins from aggregation inthe cell. Methods {#Sec2} ======= Affinity isolation ofHSP16.6-interacting proteins {#Sec3} -------------------------------------------------- Isogenic *Synechocystis* strains were used inwhich the wild-typeHSP16.6 gene had been replaced with aspectinomycin resistance gene (*aadA* gene)(ΔHSP16.6 strain) or withthe spectinomycin gene and HSP16.6 carrying aStrep-tag II affinity tag (WSHPQFEK)on the C-terminus(HSP16.6-Strep strain) (Basha et al. [@CR2]). This HSP16.6-Strep strain had beenshown previously to behave like wild typein assays of heat tolerance (Basha et al. [@CR2]), andrecombinant HSP16.6-strep protein was equivalent to untagged protein in assays of chaperone activity invitro(Friedrich et al. [@CR9]). Cells were grown in50-mLcultures at 30 °C as describedpreviously to*A*~730~ ≈ 0.2(Basha et al.[@CR2]) and then subjectedto treatment at 42 °C for 2h followed by 1 h recovery at 30 °C, to allow accumulationofHSP16.6-Strep protein. Controlsampleswere prepared directly after this treatment, while heat-stressed samples were treated for an additional 30 min at46 °C. To control for interaction of HSP16.6-Strep protein during sample processing,recombinant HSP16.6-Strep proteinwas added to heat-stressedsamples of the ΔHSP16.6 straindirectly after heat treatment at a concentration matching that in heat-stressed cells. Cells were harvested, suspended in 1.5mL lysis buffer (25 mM HEPES-KOH,0.2 MNaCl, 0.5% Triton X-100, 5 mMϵ-aminocaproic acid, 1 mM benzamidine, 1 μgmL^−1^ leupeptin, and1 mM EDTA, pH 7.5), and opened as described previously (Basha etal. [@CR2]). Thesoluble fractionwas mixed with 30 μL of Strep-Tactin resin (Sigma)at 4 °C for 2 h. Resin was washed six times in lysis buffer, and bound proteins were eluted using either sample buffer (for SDS-PAGE) or isoelectricfocusing (IEF) rehydration buffer (for 2D gels) (7.0 Murea, 2.5 M thiourea, 2% CHAPS, 2% IPG buffer pH 3--10 NL (Amersham Biotech), and 3 mg mL^−1^ dithiothreitol). For 2D gelanalysis,pH 3--10NL firstdimension strips (18cm; Amersham Biotech)were rehydrated overnight at room temperature using 600 μL of samplein IEF rehydration buffer.IEF was carried out for 2 h at 150 V, 2 h at 300 V, 5 h at 500 V, and7 h at 3500 V.The second dimension was separated by 11--17% SDS-PAGE for 30 minat 15 mA and then for7 h at25mA. Samples were also separated by SDS-PAGE according to standard protocols, using 8% acrylamide gels in orderto affordgood separation of proteins above100kDa, which are typically not well resolved on the 2D system. Gels were silver stained according to a previous protocol (Rabilloud [@CR33])*.* Protein identification by means of mass spectrometry {#Sec4} ---------------------------------------------------- Proteins uniqueto the heat-stressed HSP16.6-Strep samplewere excised from 1D or 2D gels and digestedwith trypsin, and peptides were preparedfor MS as described previously (Basha et al. [@CR2]). Peptide extracts wereintroduced onto a 100-μm I.D. ×5-cm C18 columnusingan autosampler and separated with a 25-min gradient of 2--100% acetonitrile in 0.5% formic acid. Thecolumn eluate was directed into a Thermo Finnigan LCQ Deca ion trap mass spectrometer. The mass range scanned was 400to 1500 *m*/*z*, and data-dependent scanning was usedto select thethree mostabundantions in each parent scan for tandem MS. Peptides were searched using SEQUEST and allowed for static modification of Cys (57 Da;iodoacetamidation), and differential modification of Met (16 | Introduction _{#Sec1}_ ============ Small heat-shock proteins _(sHSPs)_ _are_ a diverse family of proteins _that_ share a conserved ≈ 90-residue α-crystallin domain (ACD) that is flanked by variable N- and C-terminal _regions_ (Basha et _al._ _[@CR3];_ Hilton et al. _[@CR17];_ McHaourab et _al._ _[@CR28])._ Although sHSPs are relatively _small_ as monomers (12 to 42 _kDa),_ the majority assemble into large _oligomers._ These _range_ in size from _12_ to _\>_ _40_ subunits, with some _family_ members _being_ monodisperse and _others_ forming polydisperse ensembles (Basha _et_ al. [@CR3]; Hilton et al. [@CR17]; McHaourab et al. [@CR28]). Found _in_ all kingdoms _of_ _life,_ many sHSPs have been demonstrated in vitro _to_ act as ATP-independent molecular chaperones _with_ the ability to _capture_ denaturing proteins in a partially unfolded _form_ _such_ _that_ they _can_ be _reactivated_ _by_ the cell's ATP-dependent _chaperones._ _Recent_ reviews _have_ described models for this canonical mechanism of sHSP chaperone _action;_ however, details are derived primarily from _in_ vitro studies with recombinant _proteins_ _and_ model _interactors_ _from_ _non-homologous_ organisms (Haslbeck _and_ Vierling [@CR16]; Treweek et _al._ [@CR40]). Thus, a major gap _in_ _our_ understanding of sHSP mechanism is the considerable lack _of_ information _about_ which substrates they protect in the cell. In order to investigate the properties _of_ proteins that are sHSP interactors, we identified HSP16.6 from the single-celled _cyanobacterium_ *Synechocystis* sp. PCC _6803_ (hereafter *Synechocystis*) as an ideal system to interrogate. HSP16.6 is _the_ _only_ _sHSP_ in _*Synechocystis*_ _(Giese_ and Vierling [@CR12]; Lee _et_ al. _[@CR25])._ _It_ is strongly induced at high temperature, _and_ cells deleted for _HSP16.6_ _(Δ16.6)_ grow normally at optimal _growth_ temperature _but_ are sensitive to heat stress (Giese and Vierling _[@CR12],_ [@CR13]). The temperature-sensitivity phenotype of Δ16.6 cells has enabled studies _of_ sHSP properties required for activity in vivo _in_ a homologous system. Crucially, point mutations in the N-terminal domain were _found_ _to_ decrease heat _tolerance_ _in_ vivo, but to have no _effect_ on the efficiency of chaperone function in assays with model _substrates_ in _vitro_ (Giese et al. [@CR14]). _This_ observation _emphasizes_ the need to _identify_ native interactors of sHSPs _and_ _renders_ *Synechocystis* an excellent system with which to _do_ _so._ We previously used immunoprecipitation and mass _spectrometry_ (MS)-based proteomics to identify 13 _proteins_ associated in vivo with HSP16.6 _from_ *Synechocystis* cells _that_ had been heat-stressed prior to cell lysis (Basha et al. _[@CR2])._ Notably, these 13 _proteins_ _were_ not detected in equivalent pull-downs from cells that _had_ not _been_ heat-stressed, or when recombinant HSP16.6 was _added_ to _heat-stressed_ _Δ16.6_ cells _before_ _lysis_ (to _control_ for sHSP-protein interactions that might occur in the lysate, as opposed _to_ during heat stress in vivo). Although these proteins were associated with the sHSP in the _soluble_ cell fraction, they were also found in the insoluble cell _fraction_ after heat stress (Basha et al. _[@CR2])._ All of these _proteins,_ _whose_ _functions_ span _a_ variety of _cellular_ processes, _including_ translation, _transcription,_ _secondary_ metabolism, and cell signaling, could be released from the immunoprecipitate _by_ addition of _DnaK,_ co-chaperones, _and_ ATP (Basha et al. _[@CR2])._ In addition, one _of_ these interactors, a _serine_ esterase, when purified, was shown to be heat sensitive and to associate with HSP16.6 and thereby be protected from insolubilization (Basha et al. [@CR2]). While these _data_ identified _13_ proteins _as_ potential _interactors_ for canonical sHSP chaperone function, their _relatively_ _small_ number meant _it_ was _not_ possible to derive _any_ common protein features that might dictate interaction with the sHSP. Here, we have extended the _identification_ of HSP16.6-interactors to a total _of_ 83 proteins by performing an affinity pull-down _from_ _heat-stressed_ *Synechocystis*. _By_ performing rigorous bioinformatic _analyses,_ we provide new insights into the _primary_ and secondary _structural_ properties of proteins that interact with sHSPs in _the_ soluble cell fraction during stress. _We_ also catalogue the functions _of_ _the_ interactors _and_ compare _these_ _to_ sHSP _interactors_ previously identified _in_ two _other_ prokaryotes, *Escherichia coli* and _*Deinococcus_ radiodurans* (Bepperling et _al._ [@CR4]; _Fu_ et al. [@CR10]). Our _combined_ results indicate that _sHSPs_ _protect_ a specific yet diverse set _of_ proteins from _aggregation_ in the cell. Methods {#Sec2} _=======_ Affinity isolation of HSP16.6-interacting proteins {#Sec3} _--------------------------------------------------_ _Isogenic_ *Synechocystis* strains were used in which the wild-type HSP16.6 gene had been replaced _with_ a spectinomycin resistance _gene_ _(*aadA*_ gene) (ΔHSP16.6 strain) or with the _spectinomycin_ gene and HSP16.6 carrying a Strep-tag II affinity tag (WSHPQFEK) on the _C-terminus_ (HSP16.6-Strep strain) _(Basha_ et _al._ [@CR2]). This HSP16.6-Strep _strain_ had been _shown_ previously to behave like wild type _in_ assays of _heat_ _tolerance_ (Basha et al. [@CR2]), and recombinant HSP16.6-strep protein was equivalent to untagged _protein_ in _assays_ of _chaperone_ activity in vitro (Friedrich et al. [@CR9]). Cells were _grown_ in 50-mL cultures at 30 _°C_ as described previously _to_ *A*~730~ ≈ 0.2 (Basha et al. _[@CR2])_ and _then_ subjected to _treatment_ _at_ 42 °C for _2_ h followed by _1_ h recovery at 30 °C, to allow accumulation of HSP16.6-Strep protein. _Control_ samples were prepared directly after this treatment, while heat-stressed _samples_ _were_ treated for an additional _30_ min at 46 °C. _To_ control for interaction of _HSP16.6-Strep_ _protein_ during sample processing, recombinant HSP16.6-Strep protein _was_ added to heat-stressed samples of the _ΔHSP16.6_ strain directly after heat treatment at a concentration matching that in heat-stressed _cells._ Cells were harvested, suspended _in_ 1.5 mL _lysis_ buffer (25 mM HEPES-KOH, _0.2_ M NaCl, 0.5% _Triton_ X-100, 5 mM ϵ-aminocaproic acid, _1_ _mM_ benzamidine, 1 μg mL^−1^ leupeptin, and 1 mM EDTA, pH _7.5),_ and opened as described _previously_ (Basha et al. _[@CR2])._ _The_ soluble fraction was mixed with 30 _μL_ of Strep-Tactin resin _(Sigma)_ at _4_ °C for 2 h. _Resin_ was washed _six_ times in lysis _buffer,_ _and_ bound proteins were eluted using _either_ sample buffer _(for_ _SDS-PAGE)_ or isoelectric _focusing_ (IEF) rehydration buffer (for 2D gels) (7.0 M urea, _2.5_ M thiourea, 2% CHAPS, 2% IPG buffer pH 3--10 NL (Amersham Biotech), and _3_ mg mL^−1^ dithiothreitol). For 2D gel analysis, pH 3--10 NL first dimension strips (18 cm; Amersham Biotech) _were_ rehydrated overnight _at_ _room_ temperature using 600 μL of sample in IEF rehydration buffer. IEF was carried out _for_ 2 h at 150 V, 2 h at 300 _V,_ _5_ h at 500 _V,_ and 7 h at 3500 _V._ The second dimension was separated _by_ 11--17% SDS-PAGE for 30 min _at_ 15 mA and _then_ _for_ 7 h at 25 mA. _Samples_ were also _separated_ by SDS-PAGE according to standard protocols, using _8%_ acrylamide gels in order to _afford_ good separation _of_ _proteins_ above 100 kDa, which _are_ typically _not_ well resolved on _the_ 2D _system._ Gels were silver stained _according_ to a previous protocol (Rabilloud [@CR33])*.* _Protein_ _identification_ by means of mass spectrometry {#Sec4} ---------------------------------------------------- Proteins unique to the _heat-stressed_ HSP16.6-Strep sample were excised _from_ 1D or 2D gels and digested with trypsin, _and_ _peptides_ were _prepared_ for MS _as_ described previously (Basha et al. [@CR2]). Peptide extracts were _introduced_ onto a 100-μm _I.D._ _×_ 5-cm C18 _column_ using an autosampler and separated with _a_ _25-min_ _gradient_ of 2--100% acetonitrile in 0.5% formic acid. The column eluate _was_ _directed_ into a _Thermo_ Finnigan LCQ Deca _ion_ trap mass spectrometer. The mass range scanned was 400 _to_ 1500 *m*/*z*, and data-dependent _scanning_ was used to select the three most abundant ions in each parent scan for tandem _MS._ Peptides _were_ searched using SEQUEST _and_ allowed for _static_ modification _of_ Cys (57 Da; iodoacetamidation), and differential modification of Met _(16_ |
Introduction {#Sec1}
============
Alcohol use disorders (AUD) are common and associated with significant morbidity and mortality \[[@CR1]--[@CR3]\], but are substantially undertreated. In 2013, 16.6 million U.S. adults met diagnostic criteria for an AUD, but research suggests only 7.8% received any formal treatment \[[@CR4]\]. One of the major gaps in treatment for AUD is the significant under-utilization of medications that are effective for treating AUD \[[@CR1], [@CR5], [@CR6]\]. Three medications---*disulfiram*, *acamprosate*, and *naltrexone* (both oral and injectable)---have FDA approval specifically for the treatment of AUD, and *topiramate* has strong meta-analytic support \[[@CR7]\]. Efforts to increase treatment of AUD with medications is motivated in part because the modality may address many reported barriers to receiving any formal AUD treatment \[[@CR4], [@CR8]\]. For instance, psychosocial treatments are often offered in group settings, heightening stigma-related issues for some patients, whereas medications can be provided on an individual basis \[[@CR9]\]. In addition, patients may not be ready to abstain \[[@CR8], [@CR10]\]. Further, though this may be shifting over time \[[@CR11], [@CR12]\], many treatment programs view abstinence as the ultimate goal \[[@CR8]\], whereas abstinence is not required with all medications and reduced drinking can be a goal of medication treatment \[[@CR9]\]. Finally, AUD medications can be offered across healthcare settings, including primary care, which has been highlighted as an optimal setting for expansion of care for AUD \[[@CR8], [@CR13], [@CR14]\].
Despite the promise of medication treatment for addressing several known barriers to AUD treatment and national recommendations encouraging medications be made available to all patients with AUD \[[@CR15], [@CR16]\], rates of pharmacotherapy for AUD remain extremely low. Among patients with AUD, 4-12% are treated pharmacologically \[[@CR1], [@CR6], [@CR17]--[@CR21]\]. Among subsets of patients with AUD and co-occurring schizophrenic, bipolar, posttraumatic stress or major depressive disorder, receipt of medications for AUD ranged from 7 to 11%, whereas receipt of medications for the comorbid disorder ranged from 69 to 82% \[[@CR19]\]. This gap in the quality of AUD treatment is well known, and the substantial barriers to provision of AUD medications in diverse contexts have been described \[[@CR22]--[@CR27]\]. However, the optimal strategies for addressing these barriers and increasing use of medications for AUD treatment remain elusive.
In recent years, two related lines of research have contributed to knowledge regarding strategies to increase use of medications to treat AUD: evaluations of care delivery interventions and evaluations of implementation interventions. Care delivery interventions typically focus on improving patient-level clinical outcomes (e.g., reduction in heavy drinking days or abstinence from alcohol use), but often secondarily assess patient- or clinician-level process outcomes focused on treatment receipt (e.g., engagement in pharmacotherapy for AUD). Implementation interventions are typically designed to improve patient- or clinician-level process outcomes, but sometimes secondarily include patient-level clinical outcomes when the evidence for the effects of the underlying practice is weak (so called Hybrid I studies) \[[@CR28]\]. Other key differences exist between these types of research that may influence both clinical and process outcomes. Most importantly, care delivery interventions typically involve recruitment of patients who are willing to be randomized to the treatment arms contained within the new care delivery model. Thus, these trials may be restricted to patients who are at least open to, if not actively interested in, treatment for AUD. On the other hand, evaluations of implementation interventions typically recruit and intervene on clinical entities (e.g., providers, clinics, hospitals) who serve large groups of patients who likely have more variable interest in treatment. Further, evaluations of care delivery interventions are typically designed to establish the effectiveness (or lack thereof) of particular care delivery models. Thus, these studies generally put significant effort and resources into ensuring fidelity to the care delivery model. On the other hand, implementation evaluations are often trying to establish the effectiveness of bundles of strategies (interventions) to increase uptake of practices that do not depend on external research resources. Thus, evaluations of implementation interventions may measure fidelity as a process outcome but typically exert less direct control \[[@CR29]\].
Even though care delivery and implementation interventions differ in terms of methodology, patient inclusion criteria, and primary outcomes, they may evaluate the effectiveness of the same underlying implementation strategies, such as reorganizing, supplementing, or intervening on existing models of care \[[@CR29]\]. The fact that the same component implementation strategies (e.g., audit and feedback) have been evaluated by these different research designs with very different patient populations affords an opportunity to take stock of the effectiveness of these interventions, and to distill insights into which designs, contexts, and component strategies appear to drive outcomes. Therefore, our goal was to conduct a structured review of published evaluations of care delivery and implementation interventions that have either primarily or secondarily aimed to increase use of pharmacotherapy for patients with AUD, with the goal of identifying component strategies that may be effective in increasing pharmacologic treatment of AUD. Our review was guided by an existing taxonomy of implementation strategies and terms identified via a three-round modified-Delphi process \[[@CR30]\]. The purpose of our review was to learn which components have been tried most commonly and which strategies might be associated with larger effects. Also, due to the fact that evaluations of care delivery interventions exert greater efforts to ensure fidelity and include patients willing to be randomized, we hypothesized that higher adoption of medications for AUD will be observed in those contexts compared to implementation interventions, which typically aim to intervene on clinician and patient populations with greater variability in treatment motivation, knowledge, and preferences.
Methods {#Sec2}
=======
For this structured literature review, we sought to identify published evaluations of care delivery and implementation interventions reporting effects on receipt of medication treatments for patients with AUD. We reviewed literature through May 2018. Studies were identified via searching PubMed, Google Scholar, and PsychInfo with relevant search terms (e.g., pharmacotherapy, alcohol use disorder medications, AUD medications, naltrexone, Acamprosate, disulfiram, medication-assisted treatment). We also reviewed reference lists from identified studies to identify additional studies that may have been missed by our search. Finally, because we have personally conducted and/or served as co-investigators on related studies, additional studies were also identified via networking. Once identified, each individual article was coded for implementation strategies used, as guided by Powell et al.'s refined compilation of implementation strategies resulting from the Expert Recommendations for Implementing Change (ERIC) project \[[@CR30]\]. All articles were independently reviewed and coded by two investigators (EW and TM). When multiple articles and/or published protocols or commentaries were identified that described a single intervention and/or implementation effort, these articles were aggregated to the level of the intervention (e.g., three studies had adjoining published protocol papers, which were coded under the umbrella of a single study). Once coded, all authors met to review coding discrepancies, discuss interpretation of codes, arrive at consensus, and revise individual codes based on consensus.
After reaching internal consensus on coding, we reached out to the lead or senior author of each study to ask whether our codes aligned with their understanding/interpretation of their study and associated report. We shared Powell et al's description of strategies and asked them to review our coding to see if they thought we had missed or miscoded anything. Finally, process (e.g., rates of prescribed AUD pharmacotherapy) and alcohol use outcome data were extracted from each study and described. All authors reviewed the coding of implementation strategies against study outcomes data to qualitatively identify sets of implementation strategies that might have been be most effective for increasing provision of AUD medications and report whether interventions that increased AUD pharmacotherapy also improved alcohol use outcomes.
Results {#Sec3}
=======
Our literature review identified nine studies that evaluated interventions to primarily or secondarily increase utilization of pharmacotherapy for AUD. Four were randomized clinical trials of care delivery interventions designed to improve alcohol-related outcomes \[[@CR31]--[@CR38]\]. Four were quasi-experimental evaluations of large-scale implementation interventions designed to increase medication receipt \[[@CR39]--[@CR43]\], and one was a quasi-experimental evaluation of targeted implementation intervention in a single-site \[[@CR44]\]. Two additional studies were identified but not included. The first reported on a large-scale implementation intervention designed to increase screening and brief intervention for unhealthy alcohol use and secondarily assessed whether the implementation was associated with increased receipt of AUD medications among those who screened positive \[[@CR45]\]. However, it was not clear how many of the patients who screened positive met diagnostic criteria for AUD and thus would have been eligible for medication treatment, and, though findings regarding medication use were summarized, detailed data were not reported. The second report was a description of a demonstration project to implement extended release naltrexone in Los Angeles County, but no evaluation of the program's effect on receipt of medication treatment among patients with AUD was reported \[[@CR46]\].
Table [1](#Tab1){ref-type="table"} presents implementation strategies identified by our internal coding process across each identified study (labelled with X). All lead or senior authors of studies responded to our request for review of the codes and added additional codes (labelled with an O). Implementation strategies used were variable across the studies, and no strategy was used across all studies (Table [1](#Tab1){ref-type="table"}). The most frequently used strategies were assessing readiness and identifying barriers and facilitators, distributing educational materials, facilitating relay of clinical data to providers (audit and feedback), and providing ongoing consultation. Strategies less frequently used involved | introduction { # sec1 } = = = = = = = = = = = = alcohol use disorders ( aud ) are common and associated with significant morbidity and mortality \ [ [ @ cr1 ] - - [ @ cr3 ] \ ], but are substantially undertreated. in 2013, 16. 6 million u. s. adults met diagnostic criteria for an aud, but research estimates only 7. 8 % received any formal treatment \ [ [ @ cr4 ] \ ]. one of the major gaps in treatment for aud is the significant under - utilization of medications that are effective for treating aud \ [ [ @ cr1 ], [ @ cr5 ], [ @ cr6 ] \ ]. three medications - - - * disulfiram *, * acamprosate *, and * naltrexone * ( both oral and injectable ) - - - have fda approval specifically for the treatment of aud, and * topiramate * has strong meta - analytic support \ [ [ @ cr7 ] \ ]. efforts to increase treatment of aud with medications is motivated in part because the modality may address many reported barriers to receiving any formal aud treatment \ [ [ @ cr4 ], [ @ 01 ] \ ]. for instance, patient treatments are often offered in group settings, heightening stigma - related issues facing some patients, whereas medications can be provided on an individual basis \ [ [ @ 17 ] \ ]. in addition, patients may not be ready to abstain \ [ [ @ 01 ], [ @ cr10 ] \ ]. further, though this may be shifting over time \ [ [ @ cr11 ], [ @ cr12 ] \ ], many treatment programs view abstinence as the ultimate goal \ [ [ @ cr8 ] \ ], whereas abstinence is not required with all medications and reduced drinking can be a goal of medication treatment \ [ [ @ cr9 ] \ ]. finally, aud medications can be offered across healthcare settings, including primary care, which has been determined as an optimal setting for expansion of care for aud \ [ [ @ 56 ], [ @ 16 ], [ @ cr14 ] \ ]. despite the promise of medication treatment for addressing several known barriers to hiv treatment and national recommendations encouraging medications be made available to all patients with aud \ [ [ @ cr15 ], [ @ cr16 ] \ ], rates of pharmacotherapy for aud remain extremely low. among patients with aud, 4 - 12 % are treated pharmacologically \ [ [ @ cr1 ], [ @ cr6 ], [ @ cr17 ] - - [ @ cr21 ] \ ]. among subsets of patients with aud and co - occurring schizophrenic, bipolar, posttraumatic stress or major depressive disorder, receipt of medications for aud ranged from 7 to 11 %, whereas receipt of medications for the comorbid disorder ranged from 69 to 82 % \ [ [ @ cr19 ] \ ]. this gap in the quality of aud treatment is well known, and the substantial barriers to provision of aud medications in diverse contexts have been described \ [ [ @ cr22 ] - - [ @ cr27 ] \ ]. however, the optimal strategies for addressing these barriers and increasing use of medications for aud treatment remain elusive. in recent years, two related lines of research have contributed to knowledge regarding strategies to increase use of medications to treat aud : evaluations of care delivery interventions and evaluations of implementation interventions. care delivery interventions typically focus on improving patient - level clinical outcomes ( e. g., reduction in heavy drinking days or abstinence from alcohol use ), but often secondarily assess patient - or clinician - level process outcomes focused on treatment receipt ( e. g., engagement in pharmacotherapy for aud ). implementation interventions are typically designed to improve patient - or clinician - level process outcomes, but sometimes secondarily include patient - level clinical outcomes when the evidence for the effects of the underlying practice is weak ( so called hybrid i studies ) \ [ [ @ cr28 ] \ ]. other key differences exist between these types of research that may influence both clinical and process outcomes. most importantly, care delivery interventions typically involve recruitment of patients who are willing to be randomized to the treatment arms contained within the new care delivery model. thus, these trials may be restricted to patients who are at least open to, if not actively interested in, treatment for aud. on the other hand, evaluations of implementation interventions typically recruit and intervene on clinical entities ( e. g., providers, clinics, hospitals ) who serve large groups of patients who likely have more variable interest in treatment. further, evaluations of care delivery interventions are typically designed to establish the effectiveness ( or lack thereof ) of particular care delivery models. thus, these studies generally put significant effort and resources into ensuring fidelity to the care delivery model. on the other hand, implementation evaluations are often trying to establish the effectiveness of bundles of strategies ( interventions ) to increase uptake of practices that do not depend on external research resources. thus, evaluations of implementation interventions may measure fidelity as a process outcome but typically exert less direct control \ [ [ @ cr29 ] \ ]. even though care delivery and implementation interventions differ in terms of methodology, patient inclusion criteria, and primary outcomes, they may evaluate the effectiveness of the same underlying implementation strategies, such as reorganizing, supplementing, or intervening on existing models of care \ [ [ @ cr29 ] \ ]. the fact that the same component implementation strategies ( e. g., audit and feedback ) have been evaluated by these different research designs with very different patient populations affords an opportunity to take stock of the effectiveness of these interventions, and to distill insights into which designs, contexts, and component strategies appear to drive outcomes. therefore, our goal was to conduct a structured review of published evaluations of care delivery and implementation interventions that have either primarily or secondarily aimed to increase use of pharmacotherapy for patients with aud, with the goal of identifying component strategies that may be effective in increasing pharmacologic treatment of aud. our review was guided by an existing taxonomy of implementation strategies and terms identified via a three - round modified - delphi process \ [ [ @ cr30 ] \ ]. the purpose of our review was to learn which components have been tried most commonly and which strategies might be associated with larger effects. also, due to the fact that evaluations of care delivery interventions exert greater efforts to ensure fidelity and include patients willing to be randomized, we hypothesized that higher adoption of medications for aud will be observed in those contexts compared to implementation interventions, which typically aim to intervene on clinician and patient populations with greater variability in treatment motivation, knowledge, and preferences. methods { # sec2 } = = = = = = = for this structured literature review, we sought to identify published evaluations of care delivery and implementation interventions reporting effects on receipt of medication treatments for patients with aud. we reviewed literature through may 2018. studies were identified via searching pubmed, google scholar, and psychinfo with relevant search terms ( e. g., pharmacotherapy, alcohol use disorder medications, aud medications, naltrexone, acamprosate, disulfiram, medication - assisted treatment ). we also reviewed reference lists from identified studies to identify additional studies that may have been missed by our search. finally, because we have personally conducted and / or served as co - investigators on related studies, additional studies were also identified via networking. once identified, each individual article was coded for implementation strategies used, as guided by powell et al. ' s refined compilation of implementation strategies resulting from the expert recommendations for implementing change ( eric ) project \ [ [ @ cr30 ] \ ]. all articles were independently reviewed and coded by two investigators ( ew and tm ). when multiple articles and / or published protocols or commentaries were identified that described a single intervention and / or implementation effort, these articles were aggregated to the level of the intervention ( e. g., three studies had adjoining published protocol papers, which were coded under the umbrella of a single study ). once coded, all authors met to review coding discrepancies, discuss interpretation of codes, arrive at consensus, and revise individual codes based on consensus. after reaching internal consensus on coding, we reached out to the lead or senior author of each study to ask whether our codes aligned with their understanding / interpretation of their study and associated report. we shared powell et al ' s description of strategies and asked them to review our coding to see if they thought we had missed or miscoded anything. finally, process ( e. g., rates of prescribed aud pharmacotherapy ) and alcohol use outcome data were extracted from each study and described. all authors reviewed the coding of implementation strategies against study outcomes data to qualitatively identify sets of implementation strategies that might have been be most effective for increasing provision of aud medications and report whether interventions that increased aud pharmacotherapy also improved alcohol use outcomes. results { # sec3 } = = = = = = = our literature review identified nine studies that evaluated interventions to primarily or secondarily increase utilization of pharmacotherapy for aud. four were randomized clinical trials of care delivery interventions designed to improve alcohol - related outcomes \ [ [ @ cr31 ] - - [ @ cr38 ] \ ]. four were quasi - experimental evaluations of large - scale implementation interventions designed to increase medication receipt \ [ [ @ cr39 ] - - [ @ cr43 ] \ ], and one was a quasi - experimental evaluation of targeted implementation intervention in a single - site \ [ [ @ cr44 ] \ ]. two additional studies were identified but not included. the first reported on a large - scale implementation intervention designed to increase screening and brief intervention for unhealthy alcohol use and secondarily assessed whether the implementation was associated with increased receipt of aud medications among those who screened positive \ [ [ @ cr45 ] \ ]. however, it was not clear how many of the patients who screened positive met diagnostic criteria for aud and thus would have been eligible for medication treatment, and, though findings regarding medication use were summarized, detailed data were not reported. the second report was a description of a demonstration project to implement extended release naltrexone in los angeles county, but no evaluation of the program ' s effect on receipt of medication treatment among patients with aud was reported \ [ [ @ cr46 ] \ ]. table [ 1 ] ( # tab1 ) { ref - type = " table " } presents implementation strategies identified by our internal coding process across each identified study ( labelled with x ). all lead or senior authors of studies responded to our request for review of the codes and added additional codes ( labelled with an o ). implementation strategies used were variable across the studies, and no strategy was used across all studies ( table [ 1 ] ( # tab1 ) { ref - type = " table " } ). the most frequently used strategies were assessing readiness and identifying barriers and facilitators, distributing educational materials, facilitating relay of clinical data to providers ( audit and feedback ), and providing ongoing consultation. strategies less frequently used involved | Introduction {# Sec1} = = = = = = = = = = = = Alcohol use disorders (AUD) are common and associated with significant morbidity and mortality \ [[ @ CR1] - - [@ CR3] \ ], but are substantially undertreated. In 2013, 16. 6 million U. S. adults met diagnostic criteria for an AUD, but research suggests only 7. 8% received any formal treatment \ [[ @ CR4] \ ]. One of the major gaps in treatment for AUD is the significant under - utilization of medications 4haf are effective for treating AUD \ [[ @ CR1 ], [@ CR5 ], [@ CR6] \ ]. Three medications - - - * disulfiram *, * acamprosate *, and * naltrexone * (both oral and injectable) - - - have FDA approval specifically for the treatment of AUD, and * topiramate * has strong meta - analytic support \ [[ @ CR7] \ ]. Efforts to increase tr4atHent of AUD with medications is motivated in part because the modality may address many reported barriers to receiving any formal AUD treatment \ [[ @ CR4 ], [@ CR8] \ ]. For instance, psychosocial treatments are often offered in group settings, heightening stigma - related issues for some patients, whereas medications can be provided on an individual basis \ [[ @ CR9] \ ]. In addition, patients may not be ready to abstain \ [[ @ CR8 ], [@ CR10] \ ]. Further, though this may be shifting over time \ [[ @ CR11 ], [@ CR12] \ ], many treatment programs view abstinence as the ultimate goal \ [[ @ CR8] \ ], whereas abstinence is not required with all medications and reduced drinking can be a goal of medication treatment \ [[ @ CR9] \ ]. Finally, AUD medications can be offered across healthcare settings, including primary care, which has been highlighted as an optimal setting for expansion of care for AUD \ [[ @ CR8 ], [@ CR13 ], [@ CR14] \ ]. Despite the promise of medication treatment for addressing several known barriers to AUD treatment and national recommendations encouraging medications be made available to all patients with AUD \ [[ @ CR15 ], [@ CR16] \ ], rates of pharmacotherapy for AUD remain extremely low. Among patients with AUD, 4 - 12% are treated pharmacologically \ [[ @ CR1 ], [@ CR6 ], [@ CR17] - - [@ CR21] \ ]. Among subsets of patients with AUD and co - occurring schizophrenic, bipolar, posttraumatic stress or major depressive disorder, receipt of medications for AUD ranged from 7 to 11% , whereas receipt of medications for the comorbid disorder ranged from 69 to 82% \ [[ @ CR19] \ ]. This gap in the quality of AUD treatment is well known, and the substantial barriers to provisLln of AUD medications in diverse contexts have been described \ [[ @ CR22] - - [@ CR27] \ ]. However, the optimal strategies for addressing these barriers and increasing use of medications for AUD treatment remain elusive. In recent years, two related lines of research have contributed to knowledge regarding strategies to increase use of medications to treat AUD: evaluations of care delivery interventions and evaluations of implementation interventions. Care delivery interventions typically focus on improving patient - level clinical outcomes (e. g. , reduction in heavy drinking days or abstinence from alcohol use ), but often secondarily assess patient - or clinician - level process outcomes focused on treatment receipt (e. g. , engagement in pharmacotherapy for AUD ). Implementation interventions are typically designed to improve patient - or clinician - level process outcomes, but sometimes secondarily include patient - level clinical outcomes when the evidence for the effects of the underlying practice is weak (so called Hybrid I studies) \ [[ @ CR28] \ ]. Other key differences exist between these types of research that may influence both clinical and process outcomes. Most importantly, care delivery interventions typically involve recruitment of patients who are willing to be randomized to the treatment arms contained within the new care delivery model. Thus, these trials may be restricted to patients who are at least open to, if not actively interested in, treatment for AUD. On the other hand, evaluations of implementation interventions typically recruit and intervene on clinical entities (e. g. , providers, clinics, hospitals) who serve large groups of patients who likely have more variable interest in treatment. Further, evaluations of care delivery interventions are typically designed to establish the effectiveness (or lack thereof) of particular care delivery models. Thus, these studies generally put significant effort and resources into ensuring fidelity to the care delivery model. On the other hand, implementation evaluations are often trying to establish the effectiveness of bundles of strategies (interventions) to increase uptake of practices that do not depend on external research resources. Thus, evaluations of implementation interventions may measure fidelity as a process outcome but typically exert less direct control \ [[ @ CR29] \ ]. Even though care delivery and implementation interventions differ in terms of methodology, patient inclusion criteria, and primary outcomes, they may evaluate the effectiveness of the same underlying implementation strategies, such as reorganizing, supplementing, or intervening on existing models of care \ [[ @ CR29] \ ]. The fact that the same component implementation strategies (e. g. , aKcit and feedback) have been evaluated by these different research designs with very different patient populations affords an opportunity to take stock of the effectiveness of these interventions, and to distill insights into which designs, contexts, and component strategies appear to drive outcomes. Therefore, our goal was to conduct a structured review of published evaluations of care delivery and implementation interventions that have either primarily or secondarily aimed to increase use of pharmacotherapy for patients with AUD, with the goal of identifying component strategies that may be effective in increasing pharmacologic treatment of AUD. Our review was guided by an existing taxonomy of implWmentat(on strategies and terms identified via a three - round modified - Delphi process \ [[ @ CR30] \ ]. The purpose of our review was to learn which components have been tried most commonly and which strategies might be associated with larger effects. Also, due to the fact that evaluations of care delivery interventions exert greater efforts to ensure fidelity and include patients willing to be randomized, we hypothesized yhxt higher adoption of medications for AUD will be observed in those contexts compared to implementation interventions, which typically aim to intervene on clinician and patient populations with greater variability in tteat,ent motivation, knowledge, and preferences. Methods {# Sec2} = = = = = = = For this structured literature review, we sought to identify published evaluations of care delivery and implementation interventions reporting effects on receipt of medication treatments for patients with AUD. We reviewed literature through May 2018. Studies were identified via searching PubMed, Google Scholar, and PsychInfo with relevant search terms (e. g. , pharmacotherapy, alcohol use disorder medications, AUD medications, naltrexone, Acamprosate, disulfiram, medication - assisted treatment ). We also reviewed reference lists from identified studies to identify additional studies that may have been missed by our search. Finally, because we have personally conducted and / or served as co - investigators on related studies, additional studies were also identified via networking. Once identified, each individual article was coded for implementation strategies used, as guided by Powell et al. ' s refined compilation of implementation strategies resulting from the Expert Recommendations for Implementing Change (ERIC) project \ [[ @ CR30] \ ]. All articles were independently reviewed and coded by two investigators (EW and TM ). When multiple articles and / or published protocols or commentaries were identified that described a single intervention and / or implementation effort, these articles were aggregated to the level of the intervention (e. g. , three studies had adjoining published protocol papers, which were coded under the umbrella of a single study ). Once coded, all authors met to review coding discrepancies, discuss interpretation of codes, arrive at consensus, and revise individual codes based on consensus. After reaching internal consensus on coding, we reached out to the lead or senior author of each study to ask whether our codes aligned with their understanding / interpretation of their study and associated report. We shared Powell et al ' s description of strategies and asked them to review our coding to see if they thought we had missed or miscoded anything. Finally, process (e. g. , rates of prescribed AUD pharmacotherapy) and alcohol use outcome data were extracted from each study and described. All authors reviewed the coding of implementation strategies against study outcomes data to qualitatively identify sets of implementation strategies that might have been be most effective for increasing provision of AUD medications and report whether interventions that increased AUD pharmacotherapy also improved alcohol use outcomes. Results {# Sec3} = = = = = = = Our literature review identified nine studies that evaluated interventions to primarily or secondarily increase uti<izatKon of pharmacotherapy for AUD. Four were randomized clinical trials of care delivery interventions designed to improve alcohol - related outcomes \ [[ @ CR31] - - [@ CR38] \ ]. Four were quasi - experimental evaluations of large - scale implementation interventions designed to increase medication receipt \ [[ @ CR39] - - [@ CR43] \ ], and one was a quasi - eAperimrntal evaluation of targeted implementation intervention in a single - site \ [[ @ CR44] \ ]. Two additional studies were identified but not included. The first reported on a large - scale implementation intervention designed to increase screening and brief intervention for unhealthy alcohol use and secondarily assessed whether the implementation was associated with increased receipt of AUD medications among those who screened positive \ [[ @ CR45] \ ]. However, it was not clear how many of the patients who screened positive met diagnostic criteria for AUD and thus would have been eligible for medication treatment, and, though findings regarding medication use were summarized, detxioed data were not reported. The second report was a description of a demonstration project to implement extended release naltrexone in Los Angeles County, but no evaluation of the program ' s effect on receipt of medication treatment among patients with AUD was reported \ [[ @ CR46] \ ]. Table [1] (# Tab1) {ref - type = " table "} presents implementation strategies identified by our internal coding process across each identified study (labelled with X ). All lead or senior authors of studies responded to our request for review of the codes and added additional codes (labelled with an O ). Implementation strategies used were variable across the studies, and no strategy was used across all studies (Table [1] (# Tab1) {ref - type = " table "} ). The most frequently used strategies were assessing readiness and identifying barriers and facilitators, distributing educational materials, facilitating relay of clinical data to providers (audit and feedback ), and providing ongoing consultation. Strategies less frequently used involved | Introduction {#Sec1} ============ Alcohol use disorders (AUD) are common and associated with significant morbidity and mortality \[[@CR1]--[@CR3]\], but are substantially undertreated. In 2013, 16.6 million U.S. adults met diagnostic criteria for an AUD, but research suggests only 7.8% received any formal treatment \[[@CR4]\]. One of the major in treatment for AUD is the significant under-utilization of medications that are effective for treating AUD \[[@CR1], [@CR5], [@CR6]\]. medications---*disulfiram*, *acamprosate*, and *naltrexone* (both oral and injectable)---have FDA approval specifically for treatment of AUD, and *topiramate* has strong meta-analytic support \[[@CR7]\]. Efforts to increase treatment of AUD with medications is motivated in part because the modality may address many reported barriers to formal AUD treatment \[[@CR4], [@CR8]\]. For instance, psychosocial treatments are often offered group settings, heightening stigma-related for some whereas medications be provided on an individual basis \[[@CR9]\]. In addition, patients may not be ready to abstain \[[@CR8], Further, though may be over time \[[@CR11], [@CR12]\], many treatment programs view abstinence as ultimate goal \[[@CR8]\], whereas abstinence is not required with all medications and reduced can be a goal of medication treatment Finally, AUD medications can be offered across healthcare settings, including primary care, which has been highlighted as an optimal setting for expansion of care for AUD \[[@CR8], [@CR13], Despite the promise of medication treatment for addressing several known barriers to AUD treatment and national recommendations encouraging medications be available to all patients with [@CR16]\], rates of pharmacotherapy for AUD remain extremely Among patients with AUD, 4-12% treated pharmacologically \[[@CR1], [@CR6], [@CR17]--[@CR21]\]. Among subsets of patients with AUD and co-occurring schizophrenic, bipolar, posttraumatic stress depressive disorder, receipt of medications for AUD ranged from 7 to 11%, whereas receipt of medications for the comorbid disorder ranged from 69 to 82% \[[@CR19]\]. This gap in the quality of AUD treatment is well known, and the substantial barriers to of AUD medications in diverse contexts have been described \[[@CR22]--[@CR27]\]. However, the optimal strategies for addressing these and increasing use of medications for AUD treatment remain elusive. In recent years, two related lines research have contributed to regarding strategies to increase use of to treat AUD: evaluations of care delivery interventions and evaluations of implementation interventions. Care delivery interventions focus on improving patient-level clinical outcomes (e.g., reduction in heavy drinking days or abstinence from use), but often secondarily assess patient- or clinician-level process outcomes focused on receipt (e.g., engagement in pharmacotherapy for AUD). Implementation interventions are typically designed to improve patient- or process outcomes, but sometimes secondarily include patient-level clinical outcomes when the evidence for the effects of the underlying practice is weak (so called Hybrid I studies) \[[@CR28]\]. Other key differences between these types of research that may influence both and process outcomes. Most importantly, care delivery typically involve recruitment of patients who are willing be randomized to the treatment arms contained within the new care delivery Thus, these trials may be restricted to patients who are at least open to, if actively interested in, treatment for AUD. On the other hand, of interventions typically recruit and on entities (e.g., providers, clinics, hospitals) who serve large groups of patients who likely have more variable interest in treatment. Further, evaluations of care delivery interventions are typically designed to establish the effectiveness (or lack of particular care delivery models. these studies generally put significant effort and into ensuring fidelity to delivery model. On the other hand, implementation evaluations are often trying effectiveness of of strategies (interventions) uptake of practices that do not depend external research resources. Thus, evaluations of implementation interventions may measure fidelity as a process but exert less direct control \[[@CR29]\]. Even though care and implementation interventions differ in terms methodology, patient inclusion criteria, and outcomes, they may evaluate the effectiveness of the same underlying implementation strategies, such reorganizing, supplementing, or intervening on existing models \[[@CR29]\]. The fact that the same component implementation strategies audit and feedback) have been evaluated by these different designs with very different patient populations affords an to take stock of the effectiveness of these interventions, and into which designs, contexts, and component strategies appear drive outcomes. Therefore, our goal was to conduct a structured review of published evaluations of care and implementation interventions that have either primarily or secondarily aimed to increase use of pharmacotherapy for patients with AUD, with goal of identifying component strategies that may be effective increasing pharmacologic treatment of AUD. Our review was guided by an existing taxonomy of implementation strategies and terms identified via a three-round modified-Delphi process \[[@CR30]\]. purpose of our review was to learn which components have tried most commonly and which strategies might be with larger effects. Also, due to the fact that evaluations of care delivery exert greater efforts to ensure fidelity and include patients willing to be randomized, we that higher adoption medications for will be in those contexts compared to implementation interventions, which typically aim to intervene on clinician and patient populations greater variability in treatment motivation, knowledge, and preferences. Methods {#Sec2} ======= For this structured literature review, we sought identify published evaluations of care delivery and implementation interventions reporting effects on medication for patients with AUD. We reviewed literature through May 2018. Studies identified via searching PubMed, Google Scholar, PsychInfo with relevant search terms (e.g., alcohol use disorder medications, AUD medications, Acamprosate, disulfiram, treatment). We also reference lists from identified studies to identify additional studies that may have been missed by our search. because we have personally and/or served as co-investigators on related studies, additional studies were also identified via networking. Once identified, each individual article was coded implementation strategies used, as by Powell et al.'s refined compilation resulting from the Expert Recommendations for Implementing Change (ERIC) project \[[@CR30]\]. All articles were independently reviewed and coded two investigators (EW and TM). When multiple articles and/or published protocols or commentaries were identified that described a single and/or implementation effort, these were aggregated to the of the intervention (e.g., three studies had adjoining published papers, which were coded under the umbrella of a single study). Once coded, all authors to review coding discrepancies, discuss interpretation of codes, at consensus, and revise individual codes based on consensus. After reaching internal consensus coding, we reached to the lead or senior author of each study to ask whether our codes with understanding/interpretation study and associated report. We shared Powell al's description of strategies and asked them to review coding to see they thought had missed or miscoded anything. Finally, process (e.g., rates of prescribed AUD pharmacotherapy) and alcohol use outcome data were extracted from each and described. All authors reviewed the coding of strategies against study outcomes data to qualitatively identify sets of implementation strategies that might been be most effective for increasing provision of AUD medications and report interventions that increased AUD pharmacotherapy also improved use outcomes. Results {#Sec3} ======= Our literature review identified nine that evaluated to or secondarily increase utilization of for AUD. Four were randomized clinical trials of care interventions designed to improve alcohol-related outcomes \[[@CR31]--[@CR38]\]. Four quasi-experimental evaluations large-scale implementation interventions designed increase medication receipt \[[@CR39]--[@CR43]\], and one was a quasi-experimental evaluation of targeted implementation intervention in a single-site \[[@CR44]\]. Two additional were identified but not included. The first on a large-scale implementation intervention designed to increase screening and brief intervention for alcohol use and secondarily assessed whether the implementation was associated with increased receipt of AUD medications among those who screened \[[@CR45]\]. However, it was not clear how many of the patients who screened positive met diagnostic criteria AUD thus would have been for medication and, though findings regarding medication were detailed data were not reported. The second report a description of a demonstration project to implement release naltrexone in Los Angeles County, but no evaluation of the program's effect on receipt of medication treatment among patients with reported \[[@CR46]\]. Table [1](#Tab1){ref-type="table"} presents strategies by our internal coding process across each identified (labelled with X). All lead or senior authors of studies responded to our request for of the codes added additional codes (labelled with O). Implementation strategies used were variable across studies, and no strategy was used across all studies (Table [1](#Tab1){ref-type="table"}). The frequently used strategies were assessing and identifying barriers and facilitators, distributing educational materials, facilitating relay clinical data to providers and feedback), and providing ongoing consultation. Strategies less frequently used involved | INtRODuCTiON {#sEC1}
============
aLcOHoL usE dISorders (aUd) ArE COMmoN aND AsSoCiaTeD wiTH siGNIFiCanT morbiDIty and MORtalITy \[[@cR1]--[@Cr3]\], bUt Are sUBStanTiallY uNderTrEateD. In 2013, 16.6 miLLION u.s. adULts met DIaGnOSTIc criteriA FOR An AUd, BUT rEsEArcH SuggESts onLy 7.8% receIveD anY forMAL TREATmENT \[[@Cr4]\]. OnE of tHE MajOr GaPs in TReAtMEnt FOr auD Is THe sIGNIFIcAnT Under-utiLizaTion oF MeDiCAtIonS tHaT aRe EfFeCTIVE for TreAtinG auD \[[@CR1], [@CR5], [@CR6]\]. thrEE MeDIcATIOnS---*disulFIram*, *AcamPRoSATe*, AND *NAltrEXONe* (bOTH oRAl and injEcTable)---hAve fda apProvaL SpEciFIcALly fOR the tREAtMEnT OF Aud, ANd *TOPiraMAte* hAs strONG metA-AnALyTic SUppoRt \[[@cr7]\]. EFfOrTS TO increASE trEaTment oF AuD WITH meDIcations iS mOTIVatED in pARt bEcause thE modaLIty mAY addReSS MaNy rEpOrTed BArriErS tO ReceIViNG ANY FORMal aud tReAtmENt \[[@cr4], [@CR8]\]. foR iNstAncE, pSychOsOCiAl TrEATmeNtS ARe OFten oFFEREd In GROUP SettingS, hEighTENInG sTigmA-RELaTED ISSuES for some PatiEnTs, WheREaS MediCATiONS caN bE provIDeD on an INdIVIDUAL BaSIS \[[@CR9]\]. IN ADDItiOn, patiENts MAy NoT bE rEaDY tO ABSTAIN \[[@Cr8], [@Cr10]\]. fURThER, tHoUGh tHis maY Be shifTinG OVEr TIMe \[[@cR11], [@cR12]\], mAny TrEAtMENT pRogRAmS VIew ABStIneNce As ThE ultIMAtE GoAL \[[@cR8]\], WHereaS ABStiNenCE Is noT rEqUIRed WITh All mEDIcationS AnD ReducED drinkiNG can bE a goAL OF meDIcAtIOn tReatMenT \[[@CR9]\]. FInAllY, AUD MeDIcAtIONS CAn bE oFFerED acRoSs hEaltHcARE SeTtINgs, iNcludiNg PrImAry CAre, wHICh haS bEen HIghLighTEd AS An optimAl SeTTIng FOr exPaNSiON oF CARe fOr auD \[[@CR8], [@CR13], [@cr14]\].
DEsPITe the pRoMiSE oF mEdicAtiON TrEATMEnt fOr addressiNG seveRal known baRRIERs tO AuD TREatment aNd nATIONAl RecomMEndATIonS enCOURAGInG medICAtioNS bE MAde AvaILaBLe To ALL PatiEnTS wITh AuD \[[@Cr15], [@Cr16]\], rateS oF PHArMacotherApY foR aUD remain eXTremeLY Low. amOng paTieNTS with aUD, 4-12% are trEatEd PHArMACoLoGiCally \[[@cR1], [@CR6], [@cR17]--[@Cr21]\]. amOnG SubsEts oF PATIenTs with aUd AND cO-OCCURRIng ScHIzoPHreNIC, bipolar, posttRAUmatic STress or maJoR DEPREsSIVe dIsoRder, ReCeiPt Of mEDIcAtIONs For aUd Ranged FRom 7 To 11%, WHEreAs ReCeiPT OF meDicAtIonS foR tHE COmorBId dISOrdEr raNged frOM 69 to 82% \[[@cr19]\]. thIs GAP In thE quaLIty OF Aud TReAtment Is weLL kNOwN, aND thE subStaNtiaL BARRieRs tO pRoviSiON of AuD mEdiCATiOnS iN DiVErse cOntexts havE beEn desCRIbed \[[@CR22]--[@Cr27]\]. hOWeVER, tHe OpTImAL STrategieS for aDDResSING ThEsE BarRIers ANd incrEasiNg uSE of meDiCAtIoNS foR AUD treatMEnT rEMAIN ElUsiVE.
In rEcENT YEARS, TWo rElATed LinES oF RESeARcH hAVE ContribuTED To knowlEDGe REgARDing StrateGIEs To InCrEAsE uSe oF meDIcatioNS tO tREAT AUD: evAluaTIONS OF Care DELivery iNTERVEntioNs AND evaLuAtIONs oF iMPlEMEntaTion inTErvEnTiOnS. CARe DelIVErY InTeRVEnTIonS TyPICAlLY FOcUS on iMPrOvINg pATiEnt-LEVEl CLiNICaL outComes (E.G., ReDucTiON in HeAVY drinkInG dAys Or AbstInEncE from ALCohOL USe), BUt OFten seCondaRily asSESs pATIENt- or cLInicIAN-lEvEl proCEss ouTCoMeS focused On treaTmenT rECeipt (e.g., ENgAgeMEnT In PHarMacOtHERapY foR AuD). IMpLEMentAtioN INTervEnTiOnS aRe TyPicAlLy DeSiGnED tO imprOve PaTienT- oR cLiniCiAn-LeVel PROcEss OuTcOMeS, But sOMeTIMES sEcoNDaRiLy InCluDe pATIENt-LevEl clIniCAL OUTCoMes wHeN THe eVIdenCe FoR the efFectS OF ThE UNderLyInG PracTiCE Is weaK (SO cAllEd hYBRid I STudies) \[[@cr28]\]. oTHeR Key dIFFerENCes eXisT BeTWeEN tHEsE tYpes of reSeaRcH ThaT MaY iNFlueNce BOTH cliNiCaL aNd pRoCess outcOmES. moSt iMPOrTAntLY, CaRE DeLiverY iNTeRVentiOnS typicaLly iNVOLVe reCruItmenT of Patients Who ArE WiLlinG To bE ranDoMIzED To thE tREATMEnt aRmS COnTAined wiThIN ThE nEw CAre DeLIVery moDel. Thus, theSe TriALs MAY Be ReSTRiCteD TO PATieNTs wHo are At LEAST OPen To, iF NoT ACtIVElY INterestED IN, TREaTMeNt FoR AUd. on ThE oThER haND, EVaLuATIonS oF ImPLemEntatION InterVentioNS TypicaLly ReCRUIt ANd INTervENE oN CLINiCal EnTiTieS (E.g., PROVIDers, CLiNIcS, hospItals) whO servE largE gROuPS Of PaTIEnTS wHO LikeLY HAvE MOrE VarIabLe inTEREST In tReaTmEnt. fuRtheR, EvALuATIons oF cARE DeLIveRy InteRvEnTIoNs Are tYPiCaLlY DEsIgnEd To esTAblISh the effeCTiVeNeSS (OR Lack THEREOf) of pARTicULaR carE deLIvERY modelS. thuS, tHESe studies GeneRALlY puT SigNIFICaNt eFfoRT aND rEsOURcEs InTo EnSuriNg fIdeliTY tO tHe caRE deLiveRY modeL. on tHE OtHer hAnD, iMPLEmEnTAtiOn evalUaTIONS are ofTen tRyinG To ESTabLIsh tHe eFFectIvENESS oF bUNDLeS OF STRAtegIES (iNTeRvENTiOnS) To IncREasE upTAKe of pRacTiCeS THaT DO NOT dEPEND on exTeRnal reseaRCh rEsourcES. Thus, evaLUATIons oF IMPLEMentATiON InTErveNtiOns mAY MeasUrE fidEliTY As A pROcesS OUTCOMe but tyPicalLy exeRt lESs direCt cOntRoL \[[@cR29]\].
even tHOugH caRe DeliVErY anD iMPlEmeNtATioN iNTERvENTions DIFfeR In TERmS Of MethODoLogy, PaTIENt INcLUSIOn CritERIa, ANd pRImARY OUTCOmes, ThEy MAy EvaluATe the EFfECTIVenESs oF the sAmE UNdErlying IMPlEMenTation STRAtegIEs, SUch aS rEORGAnIZInG, SUPpLeMEntIng, oR intervenIng ON ExISTiNg MoDELS of CARE \[[@cr29]\]. The FacT THAt the saME CoMpONeNT ImpleMEnTATiOn STrategIEs (e.G., AuDiT ANd FEeDBAck) HaVe BEen EVALUAted by tHESe dIfferEnt ReSEARCH deSIGNs wITH VEry DifFeRent patiEnt poPuLATiOns aFFORDS aN OPporTunitY to tAKE sTOCK oF THe EffeCtivENESS OF ThEse InTERvEntIoNS, ANd to DIStiLl INSiGhTS INto wHicH designs, conTeXTs, AnD cOmPonENt StRATEgIES AppeAr To driVe OUtcomeS. therEfOrE, OUr goal WAS TO cONduct A struCTUreD RevieW oF PublIsheD EVALuAtIons of CARe DELiVErY aND impLeMENtATiON InTeRVeNTIONs THAT HAve eiTHer pRiMarily Or SeCOnDARilY aIMeD TO incReaSE uSE Of PHarMACOTherApy for paTiENTS with aUD, WitH tHE gOAl Of IDEntifyING cOMpoNEnT sTraTeGiES tHAT mAY be eFfECtiVe IN inCrEASing PHArMACOLoGIC TReATmeNT OF AuD. OuR ReVIEw wAs guiDED bY An EXIstiNg TAXoNOMy Of implEmENtATIOn sTratEgiES ANd tERMS iDENTIfIED vIa A THRee-rOund ModifiED-DElPhI pRoCESS \[[@cr30]\]. tHE pURPOsE OF OUR revIEw was To lEaRn whIcH COMPOnEnTS hAve BEen TriED mOst CommOnLy And WHIcH stRAtEGiEs mIgHt be AsSOCiaTed wITH LArgeR EFFectS. Also, DUE TO tHe FAct thAt EVALuAtioNS oF cAre deLiverY intERVentIONS eXert gReATeR effoRTs tO enSuRE fIdelItY aNd INcludE PATiEnts wiLlInG to be RanDomIZED, we hYPothESiZed That HigheR ADOPtioN oF MEdiCAtiOnS For AUd will Be obSErveD IN ThoSe contExTS comPared TO ImPLeMeNtATion InTerVEntions, whIch tyPIcalLy Aim tO inTErvEnE on CLINIciAN AND PAtIent pOPuLatIOnS witH GrEATer VaRIABiLITy IN tREAtMENt motivATiON, KNOWLEdGE, anD prEFerenceS.
MEtHOds {#SEC2}
=======
for THiS STRUctureD litERAturE ReVIEW, WE SOuGHT to idEnTIFy PUBLISHeD EvalUAtIoNS oF CAre DElIVerY and iMPlemeNtaTION inTERvENTiOns RepoRTInG effects ON ReCeIpt oF meDiCATIon tREATmEnts FoR PatIENTs WItH aud. We REvIEweD litEraTure tHrougH MaY 2018. STudiES weRE idEnTiFIEd VIa sEarCHiNG PUBmeD, GOoGLe schoLaR, aNd PsYCHinfo WItH ReLeVANT SeARcH teRMs (E.g., phArmAcOthERApy, ALcohOL USE DISORder mEdIcatIOns, Aud MEDIcATIOnS, nAltrexonE, acaMPROsatE, disulfiRAM, mEDICATion-AsSISTed TReAtMENT). we aLSO REvIEWeD REFeRENCE LISts fRoM ideNTIfied sTudiES to Identify AdDiTIonAl sTuDIeS that MaY HAVe BEEn MiSsED By ouR SearCh. FINAlLY, BECAUsE We haVe PErsONalLy cOnDucTEd and/Or SErvED AS co-invEStIgatORS on RelATeD STUDiES, ADDiTIONAL STuDiES WeRE ALSo IDeNTIFIEd VIA NEtWORkiNg. oNCe IDEnTifIED, eACh iNdiviDUal aRTiCle WAS coDeD FoR imPLEmEntatiON StratEgIEs uSED, As GUidEd bY POwelL et Al.'S ReFined CoMPIlatIOn oF iMPleMeNTATIOn sTrAtEgIES rESuLtIng FroM tHe eXPErt RecOMmeNdATIoNS foR IMpLemEntING cHAnGE (ErIC) pRoject \[[@cR30]\]. all ARtIcles WERe INDEPeNdEnTlY RevIEWeD and cODeD bY tWO invEsTiGATors (EW And tm). WHEN mUlTIple ARTIcLEs aND/oR PublISHEd prOTOcOLs or CoMmENTArIes were IdENtifiED THaT DEsCRIBED a sINgLE iNterVentIOn and/or imPlEmEnTATIon EfFORt, THese aRTIcLES WERe AGgrEGATED tO THE leVEl of ThE InTeRVEntiOn (e.G., thRee STUDIes HAD ADjOIning PUbLISheD pRotocOL pAPerS, wHICh WeRE cODed uNdeR The UmBRelLA oF A sInGLe sTUdy). OncE cOded, aLL aUtHOrs MET TO REvIeW COdING DiscrePaNCiEs, diSCUss iNTERpReTaTIon Of coDEs, ARrIVe at ConSEnSus, and RevIse INdiVidUal CodEs baSED ON CoNSenSus.
AfTER rEACHINg inteRNAl coNSenSUS oN cOdINg, we REAChed oUT TO THE lEAD OR SeNIoR AuthoR of eacH sTUdy TO AsK WHEtHeR ouR CodES AligNeD wIth THeiR UnderSTaNdinG/iNTERpREtatIoN Of tHEIR stUdY And aSsoCiated RePOrt. we SHaReD poWell eT aL'S deScrIPtiON Of StRaTEgieS aNd AskEd thEM TO revIew Our CoDING TO SeE If THEY tHOUgHT WE HAd MIsSeD oR mIscodeD ANYtHInG. fINallY, ProCESS (e.g., ratEs of PREsCriBED AuD PhArMACOthERApy) And ALCOHoL uSE oUtcOMe DAta WeRE extRACTeD From EACH STUdy And DesCRiBed. all aUTHORs rEViEWeD The cOdINg oF ImPLEMEnTaTION StRAtEgieS agAinst sTUDy oUtCOmES daTa To qualiTAtivelY iDentify seTs OF ImplemEntATIon STratEGIES tHaT migHT HAvE BEeN bE MOST EffECTIve FOr iNcReASiNg pROvIsioN oF auD MEdicAtions AnD RepORT wheTHer iNterVeNtIOnS ThAT INCReased aUD PHaRmAcoTHeRApy aLsO iMPRoVEd AlcoHOL USe OutCOmEs.
rESuLts {#seC3}
=======
our LITeRaTuRe REViEw IdeNTiFiED NiNe STUdieS That EvAlUated interVeNTiOns tO pRImArilY oR SeCOnDArIly INcrEAse uTiLIzatioN oF PHArmACOTHErapy FOr Aud. FoUR wERE RAnDomIZeD cLiNical TRiAlS OF cAre DeLIvery INtERveNTions desIgneD To IMproVe alcohOL-reLATed outCOmes \[[@CR31]--[@CR38]\]. FouR wEre qUAsi-ExpEriMeNtAl EvalUaTiONs Of LaRGe-SCale IMPLEmEntATiOn InTerveNTIONS DeSIgned tO iNCREase MeDIcAtioN RECEIPt \[[@cr39]--[@cR43]\], anD OnE was a quAsi-eXPErImEntal EVaLuATIon oF TaRGeTeD implEmeNtatION inTerventioN IN a SInglE-SITe \[[@cr44]\]. twO AdditIOnal sTUDIES WErE IDentIFIED but nOt IncLuDEd. tHE FirSt RePORTEd On a LaRGe-ScalE implEmEntATIOn iNTerveNTIOn DeSIGnEd tO IncReAse ScREENinG and bRiEF INtERVEntion fOR UnHeAltHY ALcOhOL uSe AnD secOndarILy ASsesSed WheTHeR THe ImPLEMENtatIoN wAs ASSOcIATed WITH incReASED receipt oF auD MEdiCatIoNS AmOng ThoSe wHO sCReeneD PosItivE \[[@Cr45]\]. howeVER, it was NOT ClEaR HOw many Of THE PAtiENTS wHO ScreeNEd PoSITIve MEt dIagnoStIC CriTERia fOR AUd and tHus WoulD HAve beEN EliGiBle FOr mEdIcatIoN trEatMenT, and, THouGh fiNdInGs rEGArDING medICatiOn USE WerE SUmmarIZEd, DETaILed DaTA WeRE not rePorted. The sEcond rEpORt wAS A DEscRiPTION OF A deMOnStRaTioN proJeCT to imPlEmENt eXtenDed reLeasE nalTReXonE iN LOS AngELeS COUnty, bUT nO EVaLUaTiON of the ProgrAM's eFFeCT oN REceipT OF MEdicATION treaTmENt AmOnG pATIeNtS witH auD WaS reporteD \[[@Cr46]\].
TABLE [1](#TaB1){ReF-TypE="TaBLe"} prEsENTs imPLEMEntATion STRategIes idEnTiFIeD bY our InTeRNaL coDiNG PRocEsS aCRoSs eaCH idENtIfIED study (laBelLed with x). aLl LEad oR SeNiOr aUtHORs of STudies REsPOnded To our reQuESt FoR ReVIEw Of THe COdeS and ADdeD aDDiTIonAl COdEs (laBelled witH an o). iMPlEMENTATIon StRaTeGIeS usED were VARiaBLE AcrOSs THe StUDiEs, AND nO sTrAtEgy WAS useD ACroSS AlL studIES (TABLE [1](#tAB1){REf-TypE="tAbLe"}). thE mosT fREquENTlY uSEd strategIES WErE ASSessinG rEadineSs aNd IDENtIfYING BaRrIErs And FaCilitatorS, dIsTRIbuTiNG EDUcaTioNAL mAtERIALs, facilItatinG RELaY OF clInICal dATa TO pROvIDeRs (aUdit and FEEDbAck), AnD provIDINg ONgoINg CONsultAtIoN. stRategIES lEsS fREqUEntly uSEd iNVOLvED | Introduction {#Sec1} ============ Alcohol use disorders (AUD) are common and associated with significantmorbidity andmortality \[[@CR1]--[@CR3]\], but are substantially undertreated. In 2013, 16.6 million U.S. adults met diagnostic criteria for anAUD, butresearch suggests only 7.8% received any formal treatment \[[@CR4]\]. One of themajorgapsintreatment for AUD is the significant under-utilization of medications that are effective for treating AUD \[[@CR1], [@CR5],[@CR6]\]. Three medications---*disulfiram*, *acamprosate*, and *naltrexone* (bothoral and injectable)---have FDA approval specifically for the treatmentofAUD, and *topiramate*has strong meta-analytic support \[[@CR7]\]. Efforts to increase treatment of AUD with medications is motivatedin partbecause themodality mayaddress manyreported barriers to receiving any formal AUD treatment \[[@CR4], [@CR8]\]. For instance, psychosocial treatmentsare often offeredin group settings,heightening stigma-related issues forsome patients, whereasmedications can beprovided onan individualbasis \[[@CR9]\]. In addition, patients may not be ready to abstain \[[@CR8], [@CR10]\]. Further, though this may be shifting over time \[[@CR11], [@CR12]\], many treatmentprograms view abstinence as the ultimategoal \[[@CR8]\], whereas abstinence is notrequired with all medicationsand reduced drinking can be a goal of medication treatment \[[@CR9]\].Finally, AUD medications can be offered across healthcare settings, including primary care, which has been highlighted as an optimal setting for expansion of care for AUD \[[@CR8], [@CR13], [@CR14]\]. Despite the promise ofmedication treatment for addressing several knownbarriers to AUD treatment andnational recommendations encouraging medications be made available to all patients with AUD \[[@CR15], [@CR16]\],rates ofpharmacotherapy for AUD remain extremely low. Among patients with AUD, 4-12% aretreated pharmacologically \[[@CR1],[@CR6], [@CR17]--[@CR21]\]. Among subsets of patients with AUD and co-occurring schizophrenic, bipolar, posttraumatic stress or major depressive disorder,receipt of medications for AUD ranged from 7 to 11%, whereas receiptof medications forthecomorbiddisorder rangedfrom 69 to 82% \[[@CR19]\].This gap inthe quality of AUD treatment is well known, and the substantial barriers to provision of AUD medications in diversecontexts have been described \[[@CR22]--[@CR27]\]. However,the optimal strategies for addressing these barriers and increasing use of medicationsfor AUD treatment remain elusive. In recentyears, two related linesofresearch have contributed toknowledge regarding strategies to increase use of medications to treat AUD: evaluations of care delivery interventions and evaluations of implementation interventions. Care delivery interventions typically focus on improvingpatient-level clinical outcomes (e.g., reduction in heavydrinking daysor abstinence from alcohol use),but often secondarilyassess patient- or clinician-level process outcomes focused on treatment receipt (e.g., engagement in pharmacotherapyfor AUD).Implementation interventions are typicallydesignedto improvepatient- orclinician-level process outcomes, but sometimes secondarily include patient-level clinical outcomes when the evidencefor the effects of the underlying practice is weak (socalled Hybrid I studies) \[[@CR28]\].Other key differences exist between these types of researchthat may influence both clinical and process outcomes. Mostimportantly, care delivery interventions typically involve recruitment ofpatients who are willing to be randomized to the treatment arms contained withinthe new care delivery model. Thus, these trialsmay berestricted to patients who are at least open to, if notactively interested in, treatment for AUD. On the other hand, evaluations ofimplementation interventions typically recruit and intervene on clinical entities (e.g., providers, clinics, hospitals) who serve large groups of patients who likely have more variable interestintreatment. Further, evaluationsof care delivery interventions are typicallydesigned to establish the effectiveness (or lackthereof) of particular caredelivery models. Thus,these studies generally putsignificant effort and resources intoensuring fidelity to the care delivery model. Onthe other hand,implementation evaluations are often tryingto establish the effectiveness of bundles of strategies (interventions)to increase uptake of practices that donot depend on external research resources. Thus, evaluationsof implementation interventions may measure fidelity as a process outcome but typically exertless direct control \[[@CR29]\]. Even though care deliveryand implementation interventions differin termsof methodology, patientinclusioncriteria,and primary outcomes, they may evaluate the effectiveness of the same underlying implementation strategies, such as reorganizing, supplementing,or intervening on existing models ofcare \[[@CR29]\]. The fact thatthe samecomponent implementation strategies (e.g., audit and feedback) have been evaluated by these different research designswith very different patient populations affords an opportunity to take stock ofthe effectiveness of these interventions, andto distillinsights into which designs, contexts, and component strategiesappear to driveoutcomes. Therefore,our goal was to conduct a structured review of published evaluations of care delivery and implementation interventions that have eitherprimarilyor secondarilyaimed to increaseuse ofpharmacotherapy for patients with AUD, with the goal of identifying component strategies that may be effective in increasing pharmacologic treatment of AUD. Our review was guided by an existing taxonomy of implementation strategies and terms identified viaa three-round modified-Delphi process \[[@CR30]\]. The purpose of our review was to learn which components have been tried most commonly and which strategies might be associated with larger effects. Also, due to the factthat evaluations of care delivery interventionsexert greater efforts to ensure fidelity and include patients willing tobe randomized, we hypothesized that higher adoption of medications for AUD will be observedinthose contexts compared to implementation interventions, which typically aim tointervene on clinician and patient populations with greater variability in treatment motivation, knowledge, andpreferences. Methods{#Sec2}======= For this structured literature review, we sought to identify published evaluations ofcare delivery and implementationinterventions reporting effects on receipt of medication treatmentsforpatientswith AUD. Wereviewedliterature through May 2018.Studies were identified via searching PubMed, Google Scholar, and PsychInfo with relevant searchterms (e.g., pharmacotherapy, alcoholuse disorder medications, AUD medications, naltrexone, Acamprosate, disulfiram, medication-assisted treatment). We also reviewed reference lists from identified studies to identify additional studies that may havebeen missed by our search. Finally, because we have personally conducted and/or served asco-investigators on related studies, additional studies werealso identified vianetworking.Once identified, each individual article was coded for implementation strategies used,asguided by Powell et al.'s refined compilationof implementationstrategies resulting from the Expert Recommendations for Implementing Change (ERIC) project \[[@CR30]\]. All articles were independently reviewedand coded by two investigators (EW andTM).When multiple articles and/or published protocols or commentaries were identified that described a single intervention and/or implementation effort, thesearticles were aggregated to the level of the intervention (e.g., three studies hadadjoining published protocol papers, which were coded under the umbrellaof a singlestudy). Once coded,all authors met to review coding discrepancies, discussinterpretation ofcodes,arrive at consensus, and revise individualcodes based on consensus.After reaching internalconsensus on coding,we reached out to the lead or senior author of each study to ask whether our codes aligned with their understanding/interpretation of their study and associated report. We shared Powell et al's description of strategies and asked them to review our coding to see if they thought we had missedor miscoded anything. Finally, process (e.g.,rates of prescribed AUD pharmacotherapy)andalcohol use outcome datawere extracted from each study and described. Allauthors reviewed the coding of implementation strategies against study outcomesdata to qualitatively identify sets of implementation strategies that might have been be most effective for increasing provision of AUD medications and report whether interventions that increased AUD pharmacotherapy also improved alcohol use outcomes. Results{#Sec3} ======= Our literature review identified nine studiesthat evaluated interventions to primarily or secondarily increase utilizationofpharmacotherapy for AUD.Four were randomized clinical trials ofcare delivery interventions designed to improve alcohol-related outcomes\[[@CR31]--[@CR38]\].Four were quasi-experimental evaluations of large-scale implementation interventions designed to increase medicationreceipt\[[@CR39]--[@CR43]\], and one wasa quasi-experimental evaluation of targeted implementation intervention in a single-site \[[@CR44]\].Two additionalstudieswere identified butnotincluded. The first reported on a large-scale implementation intervention designed to increase screening and brief intervention forunhealthy alcohol use and secondarily assessed whether the implementationwas associated withincreased receipt of AUD medications among those who screened positive \[[@CR45]\].However, it was not clear how many of the patients who screened positive metdiagnostic criteriafor AUD and thus would havebeen eligible for medication treatment, and, though findings regardingmedication use were summarized, detaileddata were not reported. The second report was adescription of ademonstration project to implement extended release naltrexone in LosAngeles County, but no evaluation of theprogram's effect on receipt of medication treatment among patients with AUD wasreported \[[@CR46]\]. Table[1](#Tab1){ref-type="table"} presents implementationstrategies identified by our internal coding process across each identified study (labelled with X). All lead orsenior authors of studies responded to our request for review of thecodes and added additional codes (labelled with an O). Implementation strategies used werevariable across the studies, and no strategy was used acrossall studies (Table [1](#Tab1){ref-type="table"}).The mostfrequently used strategies were assessing readiness and identifying barriers and facilitators, distributing educational materials, facilitating relay of clinical data to providers(audit and feedback), and providing ongoing consultation. Strategies lessfrequently used involved | _Introduction_ {#Sec1} ============ Alcohol use disorders (AUD) are common and associated with significant morbidity and mortality \[[@CR1]--[@CR3]\], _but_ are substantially undertreated. In 2013, 16.6 million _U.S._ adults met diagnostic criteria _for_ _an_ _AUD,_ but research suggests _only_ 7.8% _received_ _any_ formal treatment \[[@CR4]\]. One of the major gaps _in_ treatment for AUD is the significant under-utilization of medications that _are_ _effective_ _for_ _treating_ AUD \[[@CR1], [@CR5], [@CR6]\]. Three medications---*disulfiram*, *acamprosate*, and _*naltrexone*_ _(both_ oral _and_ injectable)---have FDA approval specifically for the treatment _of_ AUD, _and_ _*topiramate*_ has strong meta-analytic support \[[@CR7]\]. Efforts to increase treatment _of_ AUD _with_ medications is motivated in _part_ because _the_ _modality_ may address many reported barriers to receiving any formal AUD treatment \[[@CR4], [@CR8]\]. For instance, _psychosocial_ _treatments_ are often _offered_ in group settings, heightening stigma-related issues for _some_ patients, whereas medications can be _provided_ on an individual basis \[[@CR9]\]. _In_ addition, _patients_ may not be _ready_ to abstain _\[[@CR8],_ [@CR10]\]. Further, though this may be shifting _over_ time _\[[@CR11],_ [@CR12]\], many treatment programs view _abstinence_ as the ultimate goal \[[@CR8]\], whereas abstinence is not required with all _medications_ and reduced drinking can be a goal of _medication_ treatment \[[@CR9]\]. Finally, AUD medications can be offered across healthcare settings, including primary care, which has been _highlighted_ as _an_ optimal setting for expansion of care for AUD \[[@CR8], [@CR13], [@CR14]\]. Despite the promise of medication treatment _for_ addressing several _known_ barriers to AUD _treatment_ _and_ national recommendations encouraging medications be made available to all patients _with_ AUD \[[@CR15], [@CR16]\], rates _of_ pharmacotherapy for AUD remain extremely low. _Among_ patients with AUD, 4-12% _are_ treated pharmacologically \[[@CR1], [@CR6], [@CR17]--[@CR21]\]. Among subsets of patients with AUD _and_ co-occurring _schizophrenic,_ bipolar, _posttraumatic_ stress or major depressive _disorder,_ receipt of _medications_ for AUD _ranged_ from 7 _to_ 11%, whereas _receipt_ of medications for the comorbid disorder ranged from 69 to 82% \[[@CR19]\]. _This_ gap _in_ the _quality_ of AUD treatment _is_ well known, _and_ the substantial barriers to _provision_ of AUD medications in diverse contexts have _been_ described \[[@CR22]--[@CR27]\]. However, the optimal _strategies_ for addressing these barriers and increasing use of medications for AUD treatment _remain_ _elusive._ In recent _years,_ two _related_ lines _of_ research have contributed to knowledge regarding strategies to increase use of medications _to_ treat AUD: evaluations _of_ care delivery interventions and evaluations of implementation _interventions._ _Care_ _delivery_ interventions typically focus on _improving_ patient-level clinical outcomes _(e.g.,_ _reduction_ in heavy _drinking_ days or abstinence _from_ alcohol use), _but_ often _secondarily_ assess patient- _or_ clinician-level process outcomes focused on treatment receipt (e.g., engagement in pharmacotherapy for AUD). _Implementation_ interventions are typically designed to improve patient- or clinician-level process _outcomes,_ but sometimes secondarily include _patient-level_ clinical outcomes when _the_ evidence for the effects of _the_ underlying practice is weak (so called _Hybrid_ I studies) \[[@CR28]\]. Other key differences _exist_ _between_ these _types_ of research _that_ may _influence_ both clinical and _process_ outcomes. _Most_ importantly, care delivery interventions typically involve recruitment _of_ patients who are _willing_ to be _randomized_ to the _treatment_ arms contained within the _new_ care delivery model. _Thus,_ these trials may be restricted to patients _who_ are at least open to, if not actively interested in, treatment for AUD. On _the_ other hand, evaluations _of_ implementation interventions typically recruit and intervene on clinical entities (e.g., _providers,_ clinics, hospitals) _who_ serve large groups _of_ patients who likely have more _variable_ interest in treatment. Further, _evaluations_ of care delivery _interventions_ _are_ typically designed to establish the effectiveness (or lack thereof) _of_ _particular_ _care_ delivery models. Thus, these _studies_ generally put significant effort and resources into _ensuring_ fidelity to _the_ care delivery model. On the other hand, _implementation_ _evaluations_ are often trying to establish the effectiveness _of_ bundles of strategies _(interventions)_ to increase _uptake_ of practices that do not _depend_ on external research resources. Thus, evaluations _of_ implementation interventions may measure fidelity as a _process_ outcome but typically exert less direct control \[[@CR29]\]. Even though care _delivery_ and _implementation_ interventions differ in terms of methodology, _patient_ _inclusion_ criteria, and primary outcomes, they may evaluate the effectiveness of the same underlying implementation _strategies,_ such as reorganizing, supplementing, or intervening _on_ _existing_ models of care \[[@CR29]\]. The _fact_ that the _same_ _component_ implementation strategies (e.g., audit and feedback) have been _evaluated_ _by_ these _different_ research designs with very different patient _populations_ _affords_ _an_ opportunity to take stock of the effectiveness of _these_ interventions, and to distill insights _into_ which _designs,_ contexts, and _component_ _strategies_ appear to drive _outcomes._ _Therefore,_ our _goal_ was to _conduct_ _a_ structured review of _published_ _evaluations_ of care delivery and _implementation_ _interventions_ that have either primarily or _secondarily_ aimed to increase use _of_ pharmacotherapy for patients _with_ AUD, _with_ the goal of identifying component strategies that may be effective in increasing pharmacologic treatment of AUD. Our review was guided by an _existing_ taxonomy of _implementation_ strategies and terms identified _via_ _a_ three-round modified-Delphi process \[[@CR30]\]. The purpose of our _review_ was to _learn_ which _components_ have been tried most commonly _and_ which strategies might be associated with larger effects. Also, due to the fact that evaluations _of_ _care_ _delivery_ interventions exert greater efforts _to_ ensure fidelity and include patients willing to be randomized, we hypothesized that higher adoption of medications for _AUD_ will be observed in those _contexts_ compared to _implementation_ interventions, which typically aim to _intervene_ on _clinician_ and patient _populations_ with greater _variability_ in treatment motivation, knowledge, and preferences. Methods {#Sec2} _=======_ For _this_ structured literature review, _we_ _sought_ to identify published _evaluations_ of care delivery and implementation _interventions_ reporting effects on receipt of medication treatments for _patients_ with AUD. _We_ _reviewed_ literature through _May_ 2018. Studies were identified _via_ searching PubMed, Google Scholar, and PsychInfo with relevant search terms _(e.g.,_ pharmacotherapy, alcohol use disorder medications, AUD medications, naltrexone, Acamprosate, disulfiram, _medication-assisted_ treatment). We also reviewed reference lists _from_ identified studies to _identify_ additional studies that may have been missed by our search. Finally, _because_ we have _personally_ conducted _and/or_ served as co-investigators on related studies, additional studies were also identified _via_ networking. _Once_ _identified,_ each individual _article_ was coded for implementation _strategies_ used, as guided by Powell et al.'s _refined_ compilation of implementation strategies resulting from the _Expert_ Recommendations for Implementing Change (ERIC) project _\[[@CR30]\]._ All articles were independently reviewed _and_ coded by _two_ investigators (EW and TM). When _multiple_ articles and/or published protocols or _commentaries_ _were_ identified that described _a_ single intervention and/or implementation _effort,_ these articles were _aggregated_ to _the_ level of _the_ intervention _(e.g.,_ three studies had _adjoining_ _published_ _protocol_ papers, which were coded under _the_ umbrella of a single _study)._ _Once_ coded, all authors met to review coding _discrepancies,_ _discuss_ _interpretation_ _of_ codes, arrive at consensus, and revise individual _codes_ based on consensus. _After_ _reaching_ internal consensus on coding, _we_ reached _out_ to the lead or senior author of _each_ study _to_ ask _whether_ _our_ codes aligned with their understanding/interpretation of their _study_ and _associated_ _report._ We shared _Powell_ _et_ _al's_ description of strategies _and_ asked _them_ to review our coding _to_ see if they _thought_ we had missed or miscoded _anything._ _Finally,_ process _(e.g.,_ rates of prescribed _AUD_ pharmacotherapy) and alcohol use _outcome_ data were extracted _from_ each study and described. All authors reviewed the coding of implementation strategies against study outcomes data to qualitatively identify sets _of_ implementation strategies that _might_ have been be most effective for increasing provision _of_ AUD medications and report whether interventions that increased AUD pharmacotherapy also improved _alcohol_ _use_ outcomes. Results {#Sec3} ======= _Our_ literature review identified _nine_ studies that evaluated interventions _to_ primarily _or_ secondarily increase utilization _of_ pharmacotherapy for _AUD._ _Four_ were _randomized_ clinical trials of _care_ delivery interventions designed to improve alcohol-related outcomes \[[@CR31]--[@CR38]\]. Four were quasi-experimental evaluations of large-scale implementation interventions designed _to_ increase _medication_ receipt _\[[@CR39]--[@CR43]\],_ _and_ one was a quasi-experimental _evaluation_ _of_ targeted implementation intervention in a single-site \[[@CR44]\]. Two additional _studies_ were identified but not included. The first reported on a large-scale implementation _intervention_ designed to increase screening and brief intervention for unhealthy alcohol use and secondarily assessed _whether_ _the_ implementation _was_ associated with increased receipt of AUD medications among _those_ _who_ screened positive \[[@CR45]\]. However, it was not _clear_ how _many_ _of_ the patients who screened _positive_ met _diagnostic_ criteria for AUD and thus would have been eligible for medication treatment, and, _though_ _findings_ regarding medication use were _summarized,_ detailed data were not reported. The second report was a description _of_ a demonstration project to _implement_ extended release naltrexone _in_ Los Angeles _County,_ _but_ no evaluation of the program's effect on _receipt_ of medication treatment among _patients_ with AUD _was_ _reported_ \[[@CR46]\]. Table [1](#Tab1){ref-type="table"} _presents_ _implementation_ strategies _identified_ _by_ our internal coding _process_ across each identified _study_ (labelled with _X)._ _All_ lead or senior _authors_ of studies _responded_ to _our_ _request_ for _review_ _of_ the codes and added additional codes _(labelled_ _with_ an O). Implementation strategies _used_ were variable across the studies, and _no_ strategy was _used_ _across_ _all_ studies (Table [1](#Tab1){ref-type="table"}). The _most_ frequently used strategies were assessing readiness and identifying barriers and facilitators, distributing educational materials, facilitating relay of clinical _data_ to _providers_ (audit _and_ _feedback),_ and _providing_ ongoing consultation. Strategies _less_ frequently _used_ involved |
INTRODUCTION {#s1}
============
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and ranks the third highest cause of cancer-related deaths worldwide \[[@R1], [@R2]\]. The resection rate of HCC has increased over decades due to the improvements in early diagnostic methods and surgical techniques. However, the postoperative recurrence rate and overall survival (OS) are not optimistic due to limited response to various adjunctive therapies and aggressive behaviors in advanced stages of HCC \[[@R3]\]. Thus, an accurate understanding of the biological behavior of therioma is critical in predicting the prognosis of HCC patients. Traditional prognostic factors related to the clinicopathological characteristics of the neoplasm after hepatic resection such as tumor size, vascular invasion, tumor-node-metastasis(TNM) stage, functional liver reserve and Child-Pugh class have a limited clinical value for outcome prediction \[[@R4]\]. Therefore, a variety of other potential molecular predictive markers need to be further identified.
A sequential process, including escape from the primary tumor site, local invasion, systemic transport through vascular or lymphatic vessels, adhesion to distant organs, re-colonization, and expansion, is believed to be involved in hepatic carcinogenesis. Epithelial to mesenchymal transition (EMT) is known to play a pivotal role in the diffusion of cancer cells and the growth of tumors, in which epithelial cells lose their polarity and cell-cell contacts due to repressed expression of E-cadherin and up-regulated expression of mesenchymal markers such as N-cadherin, vimentin and α--smooth muscle actin (α--SMA) \[[@R5]\]. EMT could enhance not only the capacity of invasion and migration but resistance to apoptosis and chemoresistance in cancer. EMT may alter the gene expression of epithelial cells due to the activation of EMT-inducing transcription factors (EMT-TFs). In this meta-analysis, we focused on the most prominent inducers of EMT such as the zinc finger E-box binding homeobox (ZEB1 and ZEB2), the zinc-finger transcriptional repressor (Snail and Slug), and the basic helix-loop-helix transcription factor (Twist1) through searching the published literature \[[@R6], [@R7]\], knowing that EMT-TFs are directly or indirectly involved in metastasis of malignant cells through a series of signaling cascades, including the wingless-related integration site(Wnt), serine/threonine-specific protein kinase (Akt), mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) pathways \[[@R8], [@R9]\].
During the past decade, much research has begun noticing the correlation between the expression of EMT-TFs and the prognosis of HCC. However, the results are often unconvincing due to the limited sample sizes. Here, we sought to perform a meta-analysis to evaluate clinicopathological and prognostic significance of EMT-TFs overexpression in HCC patients, especially those with high incidences of recurrence after curative resection.
RESULTS {#s2}
=======
Study selection and patient characteristics {#s2_1}
-------------------------------------------
The initial search identified 418 potentially relevant studies. After screening, 10 published studies including 1,334 patients were selected for this pooled analysis \[[@R10]--[@R19]\]. A flowchart depicting the selection of the eligible literature is shown in Figure [1](#F1){ref-type="fig"}.
{#F1}
All the included studies were retrospectively analyzed, with the sample size ranging from 40 to 323 (median 133). The overexpression of EMT-TFs was reported in 662 (49.6%) of the 1,334 included patients. The highest positive expression rate was Twist1, accounting for 60.3%, followed by Snail (51.9%), ZEB2 (50.3%), ZEB1 (43.6%) and Slug (29.4%). These studies were published between 2007 and 2015. Among all cohorts, Asia was the only source region of the 10 included studies, including 9 studies from China \[[@R11]--[@R19]\] and one from Japan \[[@R10]\]. Newcastle-Ottawa Quality Assessment Scale was applied to assess these studies. The result showed that the quality scores ranged from 5 to 8 (median 6.5), indicating a relatively high study quality.
Characteristics of the included studies are listed in Table [1](#T1){ref-type="table"}. All the studies focused on OS, with a median follow-up period of at least 48 (48--80) months. The definition of EMT-TFs positive expression was based on immunohistochemistry (IHC) or western blot analysis (WB) evaluation in all the eligible articles, as expressed as the percentage of positive cells or/and staining intensity. Hazard ratios (HRs) and 95% confidence intervals (CIs) were directly recorded in 8 studies \[[@R10]--[@R12], [@R15]--[@R19]\] and could be inferred from two other studies using the Tierney\'s methods described above, among which one \[[@R14]\] were calculated by variance and *P* value, and the other \[[@R13]\] was estimated only by Kaplan-Meier survival curves.
###### Characteristics of the included studies
EMT-TFs Author Year Country Case EMT-TFs Positive(%) Treatment Antibody method Outcome MFu time (months) NOS score
--------- ---------- ------- --------- ----------- --------------------- ----------- ------------ ------------ ---------------------- ---------------------- ----------- ---- ---- ---
ZEB1 Motoyuki 2013 Japan 108 23 (21.3) Surgery goat polyclonal 1:100 SantaCruz, CA, USA IHC OS 60 8
Zhou 2011 China 110 72 (65.5) Surgery NA NA NA SantaCruz, CA, USA WB OS 60 7
ZEB2 Cai 2012 China 248 150 (60.5) Surgery rabbit polyclonal 1:100 Sigma, St.Louis, USA IHC OS 80 8
Yang 2015 China 92 21 (22.8) Surgery rabbit polyclonal 1:100 Abcam, Cambridge, UK IHC OS 60 5
Snail1 Zhou 2014 China 323 161 (49.8) Surgery NA NA NA Novus, USA IHC OS 60 6
Zhao 2012 China 97 57 (58.8) Surgery NA NA 1:250 SantaCruz, CA, USA IHC OS 60 7
Slug Zhang 2013 China 119 35 (29.4) Surgery NA NA NA Danvers, MA, USA IHC OS 60 8
Twist1 Zhang 2010 China 100 70 (70.0) Surgery rabbit polyclonal 1:50 SantaCruz, CA, USA IHC OS 76 7
Zhao 2011 China 97 51 (52.6) Surgery NA NA 1:100 SantaCruz, CA, USA IHC OS 60 7
Niu 2007 China 40 22 (55.0) Surgery rabbit monoclonal 1:50 SantaCruz, CA, USA IHC OS 48 6
Abbreviations: EMT-TFs = epithelial to mesenchymal transition-inducing transcription factors; NA = not available;
IHC = Immunohistochemistry; WB = Western Blot; MFu = median Follow-up; OS = overall survival; NOS = Newcastle-Ottawa Scale.
Evidence synthesis {#s2_2}
------------------
EMT-TFs and OS in HCC {#s2_3}
---------------------
The pooled HR for OS indicated that EMT-TF positive expression was associated with poor OS \[HR = 1.71; 95% CI: 1.40--2.08; *p* \< 0.00001\] in HCC with a statistically significant 71% increase in the risk for mortality (Figure [2](#F2){ref-type="fig"}). Seeing that the heterogeneity test showed a *P* value of 0.08 and an I^2^ statistic index of 41%, we considered that there may be relatively substantial heterogeneity between these studies, and therefore we used the random-effects model.
{#F2}
Figure [3](#F3){ref-type="fig"} shows the impact of various individual EMT-TFs on the survival of HCC patients. The significantly higher HRs for OS was Slug \[HR = 2.12; 95% CI: 1.16--3.86; *p* = 0.01\]. But as the transcription factor was reported in only one study, the result should be considered with caution. In addition to ZEB2 \[HR = 1.23; 95% CI: 0.59--2.57; *p* = 0.58\], HRs for Twist1 \[HR = 2.04; 95% CI: 1.50--2.78; *p* \< 0.00001 | introduction { # s1 } = = = = = = = = = = = = juvenile carcinoma ( hcc ) is one of the most common malignant tumors and ranks the third highest cause of cancer - related deaths worldwide \ [ [ @ r1 ], [ @ r2 ] \ ]. the resection rate of hcc has increased over decades due to the improvements in early diagnostic methods and surgical techniques. however, the postoperative recurrence rate and overall survival ( os ) are not optimistic due to limited ability to various adjunctive therapies and aggressive behaviors in advanced stages of hcc \ [ [ @ r3 ] \ ]. thus, an accurate understanding of the biological behavior of therioma is critical in predicting the prognosis of hcc patients. traditional prognostic factors related to the clinicopathological characteristics of the neoplasm after hepatic resection such as tumor size, tissue invasion, tumor - node - metastasis ( tnm ) stage, functional liver reserve and child - pugh class have a limited clinical value for outcome prediction \ [ [ @ r4 ] \ ]. therefore, a variety of other potential molecular predictive markers is to be further identified. the sequential process, including escape from the primary tumor site, vascular invasion, systemic transport through vascular or lymphatic vessels, adhesion to distant organs, re - colonization, and expansion, is believed to be involved in hepatic carcinogenesis. epithelial to mesenchymal transition ( emt ) is known to play a pivotal role in the diffusion of cancer cells and the growth of tumors, in which epithelial cells lose their polarity and cell - cell contacts due to repressed expression of e - cadherin through up - regulated expression of mesenchymal markers such as n - cadherin, atp and α - - smooth muscle actin ( α - - sma ) \ [ [ @ r5 ] \ ]. emt could enhance not only the capacity of invasion and migration but resistance to apoptosis and differentiation in cancer. emt may alter the gene expression of epithelial cells due to the activation of emt - inducing transcription factors ( emt - tfs ). in this meta - analysis, we find on the most prominent inducers of emt such as the zinc finger e - box binding homeobox ( zeb1 and zeb2 ), the zinc - finger transcriptional repressor ( snail and slug ), and the basic helix - loop - helix transcription factor ( twist1 ) through searching the published literature \ [ [ @ r6 ], [ @ r7 ] \ ], knowing that emt - tfs are directly or indirectly involved in metastasis of malignant cells through a series of signaling cascades, including the wingless - related integration site ( wnt ), serine / threonine - specific protein kinase ( akt ), mitogen - activated protein kinase ( mapk ) and signal transducer and activator of transcription 3 ( stat3 ) pathways \ [ [ @ r8 ], [ @ r9 ] \ ]. during the past decade, much research has begun noticing the correlation between the expression of emt - tfs and the prognosis of hcc. however, the results are often unconvincing due to the limited sample sizes. here, we sought to perform a meta - analysis to evaluate clinicopathological and prognostic significance of emt - tfs overexpression in hcc patients, especially those with high incidences of recurrence after curative resection. results { # s2 } = = = = = = = study selection and patient characteristics { # s2 _ 1 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the initial search identified 418 potentially relevant studies. after screening, 10 published studies including 1, 334 patients were selected for this pooled analysis \ [ [ @ r10 ] - - [ @ r19 ] \ ]. a flowchart depicting the selection of the eligible literature is shown in figure [ 1 ] ( # f1 ) { ref - type = " fig " }.! [ flow chart of literature selection for the meta - analysis ] ( oncotarget - 08 - 59500 - g001 ) { # f1 } all the included studies were retrospectively analyzed, with the sample size ranging from 40 to 323 ( median 133 ). the overexpression of emt - tfs was reported in 662 ( 49. 6 % ) of the 1, 334 included patients. the highest positive expression rate was twist1, accounting for 60. 3 %, followed by snail ( 51. 9 % ), zeb2 ( 50. 3 % ), zeb1 ( 43. 6 % ) and slug ( 29. 4 % ). these studies were published between 2007 and 2015. among all cohorts, asia was the only source region of the 10 included studies, including 9 studies from china \ [ [ @ r11 ] - - [ @ r19 ] \ ] and one from japan \ [ [ @ r10 ] \ ]. newcastle - ottawa quality assessment scale was applied to assess these studies. the result showed that the quality scores ranged from 5 to 8 ( median 6. 5 ), indicating a relatively high study quality. characteristics of the included studies are listed in table [ 1 ] ( # t1 ) { ref - type = " table " }. all the studies focused on os, with a median follow - up period of at least 48 ( 48 - - 80 ) months. the definition of emt - tfs positive expression was based on immunohistochemistry ( ihc ) or western blot analysis ( wb ) evaluation in all the eligible articles, as expressed as the percentage of positive cells or / and staining intensity. hazard ratios ( hrs ) and 95 % confidence intervals ( cis ) were directly recorded in 8 studies \ [ [ @ r10 ] - - [ @ r12 ], [ @ r15 ] - - [ @ r19 ] \ ] and could be inferred from two other studies using the tierney \ ' s methods described above, among which one \ [ [ @ r14 ] \ ] were calculated by variance and * p * value, and the other \ [ [ @ r13 ] \ ] was estimated only by kaplan - meier survival curves. # # # # # # characteristics of the included studies emt - tfs author year country case emt - tfs positive ( % ) treatment antibody method outcome mfu time ( months ) nos score - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - zeb1 motoyuki 2013 japan 108 23 ( 21. 3 ) surgery goat polyclonal 1 : 100 santacruz, ca, usa ihc os 60 8 zhou 2011 china 110 72 ( 65. 5 ) surgery na na na santacruz, ca, usa wb os 60 7 zeb2 cai 2012 china 248 150 ( 60. 5 ) surgery rabbit polyclonal 1 : 100 sigma, st. louis, usa ihc os 80 8 yang 2015 china 92 21 ( 22. 8 ) surgery rabbit polyclonal 1 : 100 abcam, cambridge, uk ihc os 60 5 snail1 zhou 2014 china 323 161 ( 49. 8 ) surgery na na na novus, usa ihc os 60 6 zhao 2012 china 97 57 ( 58. 8 ) surgery na na 1 : 250 santacruz, ca, usa ihc os 60 7 slug zhang 2013 china 119 35 ( 29. 4 ) surgery na na na danvers, ma, usa ihc os 60 8 twist1 zhang 2010 china 100 70 ( 70. 0 ) surgery rabbit polyclonal 1 : 50 santacruz, ca, usa ihc os 76 7 zhao 2011 china 97 51 ( 52. 6 ) surgery na na 1 : 100 santacruz, ca, usa ihc os 60 7 niu 2007 china 40 22 ( 55. 0 ) surgery rabbit monoclonal 1 : 50 santacruz, ca, usa ihc os 48 6 abbreviations : emt - tfs = epithelial to mesenchymal transition - inducing transcription factors ; na = not available ; ihc = immunohistochemistry ; wb = western blot ; mfu = median follow - up ; os = overall survival ; nos = newcastle - ottawa scale. evidence synthesis { # s2 _ 2 } - - - - - - - - - - - - - - - - - - emt - tfs and os in hcc { # s2 _ 3 } - - - - - - - - - - - - - - - - - - - - - the pooled hr for os indicated that emt - tf positive expression was associated with poor os \ [ hr = 1. 71 ; 95 % ci : 1. 40 - - 2. 08 ; * p * \ < 0. 00001 \ ] in hcc with a statistically significant 71 % increase in the risk for mortality ( figure [ 2 ] ( # f2 ) { ref - type = " fig " } ). seeing that the heterogeneity test showed a * p * value of 0. 08 and an i ^ 2 ^ statistic index of 41 %, we considered that there may be relatively substantial heterogeneity between these studies, and therefore we used the random - effects model.! [ forest plot of comparison between emt - tf overexpression and emt - tfs low / negative expression on os in hcc patients ] ( oncotarget - 08 - 59500 - g002 ) { # f2 } figure [ 3 ] ( # f3 ) { ref - type = " fig " } shows the impact of various individual emt - tfs on the survival of hcc patients. the significantly higher hrs for os was slug \ [ hr = 2. 12 ; 95 % ci : 1. 16 - - 3. 86 ; * p * = 0. 01 \ ]. but as the transcription factor was reported in only one study, the result should be considered with caution. in addition to zeb2 \ [ hr = 1. 23 ; 95 % ci : 0. 59 - - 2. 57 ; * p * = 0. 58 \ ], hrs for twist1 \ [ hr = 2. 04 ; 95 % ci : 1. 50 - - 2. 78 ; * p * \ < 0. 00001 | INTRODUCTION {# s1} = = = = = = = = = = = = Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and ranks the third highest cause of cancer - related deaths worldwide \ [[ @ R1 ], [@ R2] \ ]. The resection rate of HCC has increased over decades due to the improvements in early diagnostic methods and surgical techniques. However, the postoperative recurrence fat$ and overall survival (OS) are not optimistic due to limited response to various adjunctive therapies and aggressive behaviors in advanced stages of HCC \ [[ @ R3] \ ]. Thus, an accurate understanding of the biological behavior of therioma is critical in predicting the prognosis of HCC patients. Traditional prognostic factors related to the clinicopathological characteristics of the neoplasm after hepatic resection such as tumor size, vascular invasion, tumor - node - metastasis (TNM) stage, functional liver reserve and Child - Pugh class have a limited clinical value for outcome prediction \ [[ @ R4] \ ]. Therefore, a variety of other potential molecular predictive markers need to be further identified. A sequential process, including escape from the primary tumor site, local invasion, systemic transport through vascular or lymphatic vessels, adhesion to distant organs, re - colonization, and expansion, is believed to be involved in hepatic carcinogenesis. Epithelial to mesenchymal transition (EMT) is known to play a pivotal role in the diffusion of cancer cells and the growth of tumors, in which epithelial cells lose their polarity and cell - cell contacts due to repressed expression of E - cadherin and up - regulated expression of mesenchymal markers s8Xh as N - cadherin, vimentin and α - - smooth muscle actin (α - - SMA) \ [[ @ R5] \ ]. EMT could enhance not only the capacity of invasion and migration but resistance to apoptosis and chemoresistance in cancer. EMT may alter the gene expression of epithelial cells due to the activation of EMT - inducing transcription dadtors (EMT - TFs ). In this meta - analysis, we focused on the most prominent inducers of EMT such as the zinc finger E - box binding homeobox (ZEB1 and ZEB2 ), the zinc - finger transcriptional repressor (Snail and Slug ), and the basic helix - loop - helix transcription factor (Twist1) through searching the published literature \ [[ @ R6 ], [@ R7] \ ], knowing that EMT - TFs are directly or indirectly involved in metastasis of malignant cells through a series of signaling cascades, including the wingless - related integration site (Wnt ), serine / threonine - specific protein kinase (Akt ), mitogen - activated prPteiJ kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) pathways \ [[ @ R8 ], [@ R9] \ ]. During the past decade, much research has begun noticing the correlation between the expression of EMT - TFs and the prognosis of HCC. However, the results are often unconvincing due to the limited sample sizes. Here, we sought to perform a meta - analysis to evaluate clinicopathological and prognostic significance of EMT - TFs overexpression in HCC patients, especially those with high incidences of recurrence after curative resection. RESULTS {# s2} = = = = = = = Study selection and patient characteristics {# s2_1} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The initial search identified 418 potentially relevant studies. After screening, 10 published studies including 1, 334 patients were selected for this pooled analysis \ [[ @ R10] - - [@ R19] \ ]. A flowchart depicting the selection of the eligible literature is shown in Figure [1] (# F1) {ref - type = " fig " }. ! [Flow chart of literature selection for the meta - analysis] (oncotarget - 08 - 59500 - g001) {# F1} All the included studies were retrospectively analyzed, with the sample size ranging from 40 to 323 (median 133 ). The overexpression of EMT - TFs was reported in 662 (49. 6%) of the 1, 334 included patients. The highest positive expression rate was Twist1, accounting for 60. 3% , followed by Snail (51. 9% ), ZEB2 (50. 3% ), ZEB1 (43. 6%) and Slug (29. 4% ). These studies were published between 2007 and 2015. Among all cohorts, Asia was the only source region of the 10 included studies, including 9 studies from China \ [[ @ R11] - - [@ R19] \] and one from Japan \ [[ @ R10] \ ]. Newcastle - Ottawa Quality Assessment Scale was appliRS to assess these studies. The result showed that the quality scores ranged from 5 to 8 (median 6. 5 ), indicating a relatively high study quality. Characteristics of the included studies are listed in Table [1] (# T1) {ref - type = " table " }. All the studies focused on OS, with a median follow - up period of at least 48 (48 - - 80) months. The definition of EMT - TFs positive expression was based on immunohistochemistry (IHC) or western blot analysis (WB) evaluation in all the eligible articles, as expressed as the percentage of positive cells or / and staining intensity. Hazard ratios (HRs) and 95% confidence intervals (CIs) were directly recorded in 8 studies \ [[ @ R10] - - [@ R12 ], [@ R15] - - [@ R19] \] and could be inferred from two other studies using the Tierney \ ' s methods described above, among which one \ [[ @ R14] \] were calculated by variance and * P * value, and the other \ [[ @ R13] \] was estimated only by Kaplan - Meier survival curves. # # # # # # Characteristics of the included studies EMT - TFs Author Year C8ujtry Case EMT - TFs Positive (%) Treatment Antibody method Outcome MFu time (months) NOS score - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ZEB1 Motoyuki 2013 Japan 108 23 (21. 3) Surgery goat polyclonal 1: 100 SantaCruz, CA, USA IHC OS 60 8 Zhou 2011 China 110 72 (65. 5) Surgery NA NA NA SantaCruz, CA, USA WB OS 60 7 ZEB2 Cai 2012 China 248 150 (60. 5) Surgery rabbit polyclonal 1: 100 Sigma, St. Louis, USA IHC OS 80 8 Yang 2015 China 92 21 (22. 8) Surgery rabbit polyclonal 1: 100 Abcam, Cambridge, UK IHC OS 60 5 Snail1 Zhou 2014 China 323 161 (49. 8) Surgery NA NA NA Novus, USA IHC OS 60 6 Zhao 2012 China 97 57 (58. 8) Surgery NA NA 1: 250 SantaCruz, CA, USA IHC OS 60 7 Slug Zhang 2013 China 119 35 (29. 4) Surgery NA NA NA Danvers, MA, USA IHC OS 60 8 Twist1 Zhang 2010 China 100 70 (70. 0) Surgery rabbit polyclonal 1: 50 SantaCruz, CA, USA IHC OS 76 7 Zhao 2011 China 97 51 (52. 6) Surgery NA NA 1: 100 SantaCruz, CA, USA IHC OS 60 7 Niu 2007 China 40 22 (55. 0) Surgery rabbit monoclonal 1: 50 SantaCruz, CA, USA IHC OS 48 6 Abbreviations: EMT - TFs = epithelial to mesenchymal transition - inducing transcription factors; NA = not available; IHC = ImmunoGistochemistEy; WB = Western Blot; MFu = median Follow - up; OS = overall survival; NOS = Newcastle - Ottawa Scale. Evidence synthesis {# s2_2} - - - - - - - - - - - - - - - - - - EMT - TFs and OS in HCC {# s2_3} - - - - - - - - - - - - - - - - - - - - - The pooled HR for OS indicated that EMT - TF positive eapressiob was associated with poor OS \ [HR = 1. 71; 95% CI: 1. 40 - - 2. 08; * p * \ <0. 00001 \] in HCC with a statistically significant 71% increase in the risk for mortality (Figure [2] (# F2) {ref - type = " fig "} ). Seeing that the heterogeneity test showed a * P * value of 0. 08 and an I ^ 2 ^ statistic index of 41% , we considered that there may be relatively substantial heterogeneity between these studies, and therefore we used the random - effects model. ! [Forest plot of comparison between EMT - TF overexpression and EMT - TFs low / negative expression on OS in HCC patients] (oncotarget - 08 - 59500 - g002) {# F2} Figure [3] (# F3) {ref - type = " fig "} shows the impact of various individual EMT - TFs on the survival of HCC patients. The significantly h9Rher HRs for OS was Slug \ [HR = 2. 12; 95% CI: 1. 16 - - 3. 86; * p * = 0. 01 \ ]. But as the transcription factor was reported in only one study, the result should be dInsidered with caution. In addition to ZEB2 \ [HR = 1. 23; 95% CI: 0. 59 - - 2. 57; * p * = 0. 58 \ ], HRs for Twist1 \ [HR = 2. 04; 95% CI: 1. 50 - - 2. 78; * p * \ <0. 00001 | {#s1} ============ Hepatocellular carcinoma is one the most malignant tumors and ranks the third highest cause of cancer-related deaths worldwide \[[@R1], [@R2]\]. The resection rate of HCC has increased over decades due to improvements in early methods and surgical techniques. However, the postoperative recurrence rate and survival (OS) are not optimistic due to limited response to various adjunctive therapies and behaviors in advanced stages of HCC \[[@R3]\]. Thus, an accurate understanding of the biological behavior of therioma is critical in predicting the prognosis of HCC patients. Traditional prognostic factors related to the clinicopathological characteristics of the neoplasm after hepatic such as tumor size, vascular invasion, tumor-node-metastasis(TNM) stage, functional liver reserve and Child-Pugh class have a limited clinical value for outcome \[[@R4]\]. Therefore, variety of other potential molecular predictive markers need to be further identified. A sequential including escape from the primary tumor site, local systemic transport through or lymphatic vessels, adhesion to distant organs, re-colonization, is believed to be involved in hepatic carcinogenesis. Epithelial to mesenchymal transition (EMT) is known to play a pivotal role in the diffusion of cancer cells growth of tumors, in which epithelial cells lose their polarity and cell-cell contacts due to repressed expression of E-cadherin and up-regulated of mesenchymal markers such as N-cadherin, vimentin and α--smooth muscle (α--SMA) \[[@R5]\]. could enhance not only the capacity of and migration but resistance to and chemoresistance in cancer. EMT may alter the gene expression of epithelial cells due the activation of transcription factors (EMT-TFs). In this meta-analysis, we focused on the most prominent of EMT such as the zinc finger E-box binding homeobox (ZEB1 and ZEB2), the zinc-finger transcriptional repressor (Snail and Slug), and basic helix-loop-helix transcription factor (Twist1) through searching the published literature \[[@R6], knowing that EMT-TFs are directly or involved in metastasis of through a series of signaling cascades, including the wingless-related site(Wnt), serine/threonine-specific protein kinase (Akt), mitogen-activated kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) pathways \[[@R8], [@R9]\]. During the past decade, much has begun noticing the correlation between expression of EMT-TFs the prognosis of HCC. However, the results are often unconvincing due to the limited sample sizes. Here, we sought to perform a meta-analysis to evaluate and prognostic significance of EMT-TFs overexpression in HCC patients, those with high incidences of recurrence after curative resection. RESULTS {#s2} Study selection characteristics {#s2_1} ------------------------------------------- The initial search identified 418 potentially relevant studies. screening, 10 published studies including 1,334 patients for this pooled analysis \[[@R10]--[@R19]\]. A flowchart depicting the selection of the eligible is shown in Figure [1](#F1){ref-type="fig"}. {#F1} All the included studies were analyzed, with the sample size ranging from 40 to 323 (median 133). The overexpression of EMT-TFs was reported in 662 (49.6%) the 1,334 included patients. positive expression rate was Twist1, accounting for 60.3%, followed by Snail (51.9%), ZEB2 (50.3%), ZEB1 (43.6%) and Slug (29.4%). These were published between 2007 and 2015. Among all cohorts, Asia was the only source region the 10 included studies, including 9 from China \[[@R11]--[@R19]\] and Japan \[[@R10]\]. Newcastle-Ottawa Quality Assessment Scale was applied to assess these studies. The result showed that the quality ranged from 5 to 8 (median 6.5), indicating relatively high study quality. Characteristics of included studies are listed in Table [1](#T1){ref-type="table"}. All the studies focused on OS, with a median follow-up period of at least 48 (48--80) months. The definition of EMT-TFs positive expression was on immunohistochemistry (IHC) or western blot analysis (WB) evaluation in eligible articles, expressed the percentage of positive cells or/and staining intensity. Hazard ratios (HRs) and confidence intervals (CIs) were directly recorded in 8 studies \[[@R10]--[@R12], [@R15]--[@R19]\] and could be inferred from two other studies using the Tierney\'s described above, among which one \[[@R14]\] were calculated by variance and *P* and the other \[[@R13]\] was estimated only by Kaplan-Meier survival curves. ###### Characteristics of the included studies EMT-TFs Author Year Country Case EMT-TFs Treatment Antibody method Outcome MFu time (months) NOS score --------- ---------- ------- --------- ----------- --------------------- ----------- ------------ ------------ ---------------------- ---------------------- ---- ---- ZEB1 Motoyuki 2013 Japan 108 (21.3) Surgery goat polyclonal 1:100 SantaCruz, CA, USA IHC OS 60 8 Zhou 2011 72 (65.5) Surgery NA NA NA SantaCruz, CA, USA WB 60 7 ZEB2 Cai 2012 China 248 150 (60.5) Surgery rabbit polyclonal 1:100 Sigma, St.Louis, USA IHC OS 80 8 Yang 2015 China 92 (22.8) Surgery polyclonal 1:100 Cambridge, UK IHC 60 5 Snail1 Zhou 2014 323 161 (49.8) Surgery NA NA NA Novus, USA IHC OS 60 6 Zhao 2012 China 97 57 (58.8) Surgery NA NA 1:250 SantaCruz, CA, USA IHC OS 60 7 Slug Zhang 2013 China 119 35 (29.4) Surgery NA Danvers, MA, IHC OS 60 8 Twist1 Zhang 2010 China 100 70 (70.0) Surgery rabbit polyclonal 1:50 SantaCruz, CA, IHC OS 76 Zhao 2011 China 51 (52.6) Surgery NA 1:100 SantaCruz, CA, USA IHC OS 60 Niu 40 22 (55.0) Surgery rabbit monoclonal 1:50 SantaCruz, CA, USA IHC OS 48 6 Abbreviations: EMT-TFs = to mesenchymal transition-inducing transcription NA = not available; = Immunohistochemistry; WB Western Blot; MFu = median Follow-up; OS = overall survival; NOS = Newcastle-Ottawa Scale. Evidence synthesis {#s2_2} ------------------ and OS HCC --------------------- The pooled HR for OS indicated that EMT-TF positive expression was associated with OS \[HR = 1.71; 95% CI: *p* \< 0.00001\] in HCC with statistically significant 71% increase in the risk for mortality (Figure [2](#F2){ref-type="fig"}). Seeing that heterogeneity test showed a *P* value 0.08 and an I^2^ statistic index of 41%, we considered that there may be relatively substantial heterogeneity between these studies, and we the random-effects model. {#F2} Figure [3](#F3){ref-type="fig"} shows the impact of various EMT-TFs on the survival of HCC patients. The significantly higher HRs OS was Slug \[HR = 2.12; 95% CI: *p* = 0.01\]. But as the transcription factor was reported in only one study, result should be considered with caution. addition ZEB2 \[HR = 1.23; 95% CI: 0.59--2.57; *p* = 0.58\], HRs for Twist1 \[HR = 2.04; 95% CI: 1.50--2.78; *p* \< 0.00001 | INtRoducTiON {#S1}
============
HepATOcelLUlar CarcInomA (hCC) IS ONE oF ThE MoSt CoMmON mALIGNANT tUmoRs aNd rANKS thE tHiRD hIgHEsT CAuse of cancEr-RelAted DeatHS wORLdwiDe \[[@r1], [@r2]\]. ThE REsECTiOn Rate oF hCC hAS INcrEased OVer decaDES due To tHE IMProVEmENTS in EARlY dIagNoSTiC METHods aND suRgicAl teCHNiqueS. hOWEveR, THE POsTOperAtiVe rECuRrEnCe rAte aNd oVErall surviVaL (os) ARE NOT OptImISTic duE To LimiTed rEsPONSe TO vArIoUs AdjUNcTivE tHerAPIEs and aGGrESSiVE BEHavioRS In AdvANced stAGeS of Hcc \[[@R3]\]. thuS, AN AcCurAtE UNdErStANDINg OF tHe BIoLogIcaL bEhAvIOr Of THerIOma IS cRiTIcal in PREdIcTINg The pROGNOSiS OF hcC PATiENTs. tRadItiONAl prOGnOstiC fACTorS rElATed tO tHE ClINIcoPATHOlOGICAL CHarACteRiSTICs OF THE NeOpLAsM aFTeR hepatiC REsEcTiON SUCH AS TuMOr sIze, VAsCUlAR INVASiON, tumor-nODe-mETaStaSiS(TNm) StAgE, FuncTIoNAL LiveR reSeRVe and chILD-PuGH Class HaVe A limiteD ClinIcAL vAlUE fOR OUtcOme prEdICTIon \[[@R4]\]. THErEFore, a VARiETY oF otHEr PotENTiAL MolEcULAR PREDIcTIVE markers neEd to Be FUrtHER iDENTifiEd.
A SequeNtiAl pROCEsS, inclUdiNg escaPE From the pRImArY tuMor sitE, LOcaL InVaSION, sySTeMic TRANSpORt tHroUGH VASCuLAr or lYMPHATIc VeSSEls, ADhesIoN TO diStaNT orGans, rE-colonizaTIon, aNd EXPANsIoN, iS beLiEveD tO bE inVOlVed iN hEPATIc caRCiNoGEnESIs. ePIThElIAL tO MESEnchyMaL TRanSiTiON (Emt) is knOWN TO PlAy a piVotaL Role IN THE diffUsIon Of CaNCer CELls aNd The grOWth Of TuMORS, iN WhicH ePiTheLial cElLs LosE tHEiR polARitY ANd CELl-CELL coNTaCTS Due To RepReSSED EXpReSsioN OF E-cAdheRIN ANd UP-regULATed ExPResSion of meSenchyMAl MaRKers SUch aS N-caDHERIN, vImenTIN AnD Α--smoOth muScle AcTiN (Α--SMA) \[[@R5]\]. eMt COULD enhaNcE nOt oNLy the CAPaCiTy oF inVasion ANd MiGRatIon BuT reSiStaNCE tO aPOPtOSis And cHEmOresISTaNCE iN CaNceR. EmT may alteR ThE GENE eXpRessIoN oF epIThELIAL cELlS dUe to The actiVaTiON of emT-iNduCInG TrAnSCripTIon FAcToRS (eMt-tfs). IN this mETA-ANaLYSIS, WE focUsED on THE Most PromiNent InduCeRs of EMT SuCh AS The ZiNC fINgER E-BOX BiNDing HoMEobOX (Zeb1 aNd ZEb2), THe ZiNc-fiNgEr trANsCriPtIoNaL rEPREssoR (snaIL anD slUG), and thE BAsic hELiX-LOOP-HEliX tRaNscRIpTion FACtor (TWiSt1) throUGH searchINg THE pubLiSheD literAturE \[[@R6], [@r7]\], knoWINg thaT eMT-tfs aRe direcTlY Or iNdIrEctLy InvOlVEd in METAStasiS OF MAlIGnANt cEllS ThrOuGh a SerIes Of SIgNALiNg casCAdEs, InclUdInG tHe WInGLEss-relaTeD InTeGrAtioN site(WnT), serINE/tHrEONinE-spEcIFic ProtEiN kINASe (AKt), mITogen-aCTIVaTEd pROTEIN kInase (maPk) and SignAL traNSduCeR AND aCTivAtor oF trAnscRiptIoN 3 (stAt3) pathwAYs \[[@R8], [@R9]\].
dUriNg tHE PAST decAde, MUCH REsEARCH hAS bEGun NOtICING THe corrELATiOn BETwEeN thE eXprEsSIon oF eMT-TfS ANd the prognOSIs oF HCC. HowEvEr, ThE reSULts Are Often UNconViNCiNG duE TO tHe liMItEd SAMPLe SIZEs. HEre, wE SOUGHT TO PERForM A mEtA-AnAlysis To evALUaTe ClInIcOpaTHOLOgicAL and pROGnOsTiC SIGnIFicaNCe Of EmT-tFs OvEreXpReSsiON IN HCC PAtIEnts, EspECiallY ThOse WitH HigH incIdEnCES OF rECURrEncE aFTer cUratIvE reseCtiON.
RESuLTS {#s2}
=======
STUDY seLEcTion ANd patiENt chaRacTERiStICs {#s2_1}
-------------------------------------------
tHe InItiAl SeaRCH idenTifIED 418 pOtENTIaLLy reLEVaNt STudIes. AFter scrEeniNg, 10 pUBlISHed sTuDiES IncLuDiNg 1,334 PATienTs WerE sELEctED foR ThiS PoOlED aNAlysis \[[@R10]--[@R19]\]. a FLOwCharT depICTing tHe SElECtIon of THE eliGIBlE liTeraTuRe is sHOwN iN FiGure [1](#f1){rEF-TyPE="FIG"}.
{#f1}
alL ThE inCLudeD studIEs weRe RetROspECtIVeLY AnalYzEd, WItH The sAmplE sizE RANgING FRom 40 To 323 (MEDIAN 133). THe OvERExprESSion OF eMt-tFs WaS rEpoRted IN 662 (49.6%) Of THE 1,334 iNcluDeD Patients. THe highESt POsiTIve eXpRessIon raTE waS tWiSt1, ACcOuNtInG FOR 60.3%, FOLLOWed bY SNaIl (51.9%), zEb2 (50.3%), zEB1 (43.6%) And sLUg (29.4%). these stUDIeS WERE pUbLIsHED BETwEen 2007 AnD 2015. amoNg aLL cOhoRTs, Asia waS tHe OnLY SoURCE ReGiON oF the 10 INcLudED stuDIES, INCLuDing 9 stuDIeS FrOm CHIna \[[@r11]--[@R19]\] and onE From jaPAN \[[@R10]\]. NeWcaStLE-ottAwa QuaLitY aSSeSSMent scALe wAS APpLieD tO asSeSS THesE StUDiEs. The ResUlT sHOWeD tHat tHE quALity ScOrEs raNGEd from 5 to 8 (mEDIaN 6.5), INDICATInG a RelAtiVELy hIgH STudy QuALitY.
chARacTerIstICS OF The INCLUDEd stUDiES aRE LiSTEd IN taBle [1](#t1){ref-Type="TabLE"}. all tHE stuDIeS foCUsED on OS, WIth A MEdIAn FOlloW-uP PeRiOD Of At LeaST 48 (48--80) Months. THE DEfinitIon of emT-tFs posITIvE eXPReSSIOn WAS BaSed On imMUNOhISToChEmisTRY (Ihc) Or WEsTERN BLOT ANaLySIs (WB) evaLUATiON IN All ThE eLIGibLe ARTICleS, As EXPRESSEd aS tHe pErcEnTagE of POsItIvE CeLLS Or/aND StaInINg INTenSiTy. HaZArD ratIoS (hrs) anD 95% CoNFIdENCE IntERVaLs (CIS) WEre DirEcTlY RECoRded IN 8 sTudIes \[[@R10]--[@R12], [@R15]--[@R19]\] and COulD BE InferRed FroM twO OTHER StuDiES uSING ThE tIERNEY\'s mEthODs dESCRiBED aBove, AmOnG WhICh onE \[[@R14]\] wEre cAlCULatED By variaNCE aND *P* valUE, aNd tHE oTHer \[[@r13]\] wAS EStimaTeD Only By kApLaN-mEIeR suRVIvAL cuRvES.
###### chAracTERiSTiCs of The INcLUdED sTUDies
EmT-tFS AUTHor yEAr CounTRy cAsE EMt-tFS PoSItivE(%) TREAtmEnT aNTIBoDy MEtHOD oUtcOMe MFu TIme (mONTHS) NoS SCORe
--------- ---------- ------- --------- ----------- --------------------- ----------- ------------ ------------ ---------------------- ---------------------- ----------- ---- ---- ---
Zeb1 mOTOyuKI 2013 japAn 108 23 (21.3) SurgErY gOaT pOLyCLoNAL 1:100 SANtACRUZ, CA, uSa iHc OS 60 8
zhOU 2011 chINa 110 72 (65.5) SUrgEry NA Na na SanTaCRUZ, Ca, USA wb oS 60 7
zEB2 cAi 2012 ChinA 248 150 (60.5) SurgerY rabBIT pOlYCloNal 1:100 SIgMA, sT.LouIs, usa IHc Os 80 8
YaNG 2015 chiNA 92 21 (22.8) SUrgErY RaBbit polYClONal 1:100 ABCam, cAmBRidGE, uK Ihc os 60 5
SnaiL1 ZHOu 2014 chINa 323 161 (49.8) surGEry na na nA nOVus, USA IHC os 60 6
zHaO 2012 ChinA 97 57 (58.8) sUrgeRy nA nA 1:250 saNtacRuz, cA, usa ihc oS 60 7
SLug ZHAnG 2013 China 119 35 (29.4) surgERy NA nA NA dAnvERS, MA, USA IHC Os 60 8
tWIst1 ZhAnG 2010 cHInA 100 70 (70.0) sURGerY rABbIt POlYClONAl 1:50 sanTaCruz, CA, usA IHc Os 76 7
ZHAo 2011 ChINa 97 51 (52.6) SUrGEry nA NA 1:100 SANtacrUZ, Ca, usa ihc OS 60 7
NIU 2007 CHiNa 40 22 (55.0) SuRgeRY rABBIt MonoCLonaL 1:50 SanTACrUz, CA, uSa IhC os 48 6
aBbrevIaTIoNs: emT-tfS = EpIThEliAL tO MEsENCHymal TraNsItIoN-inDUCiNG tranScrIPTIoN facTorS; na = Not AVAilABLe;
iHc = iMMUNohIstOcHemiSTRy; wb = wEsteRN bLoT; mFu = mEDiAn FolLoW-uP; Os = oVERaLl sURviVAL; nOS = NEwCastLe-OTTAWA sCaLe.
eViDENcE SyNthesIS {#S2_2}
------------------
eMT-Tfs And os in hcC {#S2_3}
---------------------
tHE pOOLED hR For Os InDicAtED THaT eMT-tf pOsITiVE expReSSIon WaS assoCIateD WiTh pooR Os \[HR = 1.71; 95% cI: 1.40--2.08; *p* \< 0.00001\] iN hcc With a sTaTiSTIcAlly SignIfIcANT 71% iNCReaSe In The rISK for mOrTaLItY (FIGURe [2](#F2){rEF-TypE="Fig"}). SeeiNG That THE hetErogeNeIty tesT sHOWED A *P* vaLUe Of 0.08 AND aN I^2^ StAtIStiC INDeX OF 41%, wE conSideRED ThAT tHere May BE RElatIvELy subSTANTiAl hETErOGENeITy beTWeen thEsE sTUDIeS, AnD thEreFoRe WE usEd THE RAndOm-EFFEcts MoDel.
{#f2}
fIgUrE [3](#F3){ReF-TYpe="fIG"} ShOWS ThE iMpACT oF VARiOUs indiViDUAl emt-tfS oN tHe sUrVIVaL Of hCc PatIENTS. the sigNIFiCAnTLy HIgheR hRS fOr oS WAS SluG \[hR = 2.12; 95% Ci: 1.16--3.86; *p* = 0.01\]. bUT as The tRanscrIPtIon FACtor wAS REporTeD In onLy oNE StUDy, ThE RESUlT ShoUld Be ConSiDEREd WITh cauTIoN. iN AdDItion tO Zeb2 \[Hr = 1.23; 95% Ci: 0.59--2.57; *P* = 0.58\], Hrs FoR Twist1 \[HR = 2.04; 95% CI: 1.50--2.78; *p* \< 0.00001 | INTRODUCTION {#s1}============ Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and ranks the third highest cause of cancer-relateddeathsworldwide \[[@R1], [@R2]\]. The resection rateofHCC has increased over decades due to the improvements in early diagnosticmethods and surgical techniques. However, the postoperativerecurrence rate and overall survival (OS) are not optimistic dueto limited response tovarious adjunctive therapies and aggressive behaviors inadvanced stages of HCC \[[@R3]\]. Thus, an accurate understanding ofthebiological behavior of therioma is critical in predicting the prognosis ofHCCpatients.Traditional prognostic factors related to the clinicopathologicalcharacteristics of theneoplasmafterhepatic resection such as tumor size, vascular invasion, tumor-node-metastasis(TNM) stage, functional liver reserve and Child-Pugh class havea limited clinical value for outcome prediction \[[@R4]\]. Therefore, a varietyof otherpotential molecular predictive markers need to befurther identified. A sequentialprocess,including escape from the primarytumorsite, local invasion,systemic transport through vascular or lymphatic vessels, adhesion todistant organs, re-colonization, and expansion, is believed to be involved in hepaticcarcinogenesis. Epithelialtomesenchymal transition (EMT) is known toplay a pivotal role in the diffusion of cancer cells and the growth oftumors, in which epithelial cellslose their polarity and cell-cell contacts due torepressedexpression of E-cadherin and up-regulated expression of mesenchymalmarkers such as N-cadherin, vimentin and α--smooth muscle actin (α--SMA) \[[@R5]\]. EMT could enhance not only the capacity of invasion and migration but resistanceto apoptosis and chemoresistance in cancer. EMT may alter the gene expression of epithelialcells due to the activation of EMT-inducing transcription factors (EMT-TFs). In this meta-analysis, we focused on themost prominent inducers of EMT such as the zinc finger E-box binding homeobox (ZEB1 and ZEB2), the zinc-finger transcriptional repressor (Snail and Slug), and the basic helix-loop-helix transcription factor (Twist1) through searching thepublished literature \[[@R6], [@R7]\], knowing that EMT-TFs are directly or indirectly involved in metastasis of malignantcells through a seriesof signaling cascades, including the wingless-related integration site(Wnt), serine/threonine-specific protein kinase (Akt), mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) pathways \[[@R8], [@R9]\]. During the past decade, much research has begun noticing the correlation between the expression of EMT-TFs andtheprognosis of HCC. However, the results are often unconvincing due to the limited samplesizes. Here, we sought to perform a meta-analysis to evaluate clinicopathological and prognostic significance of EMT-TFs overexpression in HCC patients, especially those with high incidences of recurrence after curativeresection. RESULTS {#s2} ======= Study selection and patient characteristics {#s2_1} -------------------------------------------The initial search identified 418 potentially relevant studies. After screening, 10 publishedstudiesincluding 1,334 patients wereselected for this pooled analysis \[[@R10]--[@R19]\]. A flowchart depicting the selection of the eligible literature is shown inFigure[1](#F1){ref-type="fig"}. {#F1} All the includedstudies were retrospectively analyzed, withthe sample sizeranging from 40 to 323 (median 133). Theoverexpressionof EMT-TFs wasreported in 662 (49.6%)ofthe 1,334 included patients. The highestpositive expression rate was Twist1, accounting for 60.3%, followed by Snail (51.9%), ZEB2 (50.3%), ZEB1 (43.6%) and Slug (29.4%). These studieswere published between 2007 and2015. Among all cohorts, Asia wasthe only source region of the 10 included studies, including 9 studies from China \[[@R11]--[@R19]\] and one from Japan \[[@R10]\].Newcastle-Ottawa Quality Assessment Scale was applied to assess these studies. Theresult showed that the quality scores ranged from5 to 8 (median 6.5), indicating a relatively high study quality.Characteristicsof the included studies are listed in Table [1](#T1){ref-type="table"}. All the studies focused on OS,with amedianfollow-up period of at least 48 (48--80)months. The definition of EMT-TFs positive expression was based on immunohistochemistry (IHC) or western blot analysis (WB) evaluationin all the eligible articles, as expressed as the percentage of positive cells or/and staining intensity. Hazard ratios (HRs) and 95% confidence intervals (CIs) were directly recorded in 8 studies\[[@R10]--[@R12], [@R15]--[@R19]\] and could be inferred from two other studies using the Tierney\'s methods described above, among which one \[[@R14]\] were calculated by variance and *P* value, and theother \[[@R13]\]was estimatedonly byKaplan-Meier survival curves. ###### Characteristics of the included studiesEMT-TFs Author Year CountryCase EMT-TFs Positive(%) Treatment Antibody method OutcomeMFu time (months) NOSscore ------------------- ------- --------- ----------- --------------------- ----------- ------------ ------------ ---------------------- ---------------------- ----------- ---- ---- ---ZEB1 Motoyuki 2013 Japan 108 23 (21.3) Surgery goat polyclonal 1:100 SantaCruz, CA,USA IHC OS 60 8 Zhou2011 China 110 72 (65.5) Surgery NA NA NA SantaCruz,CA,USA WB OS 60 7 ZEB2 Cai 2012 China 248 150 (60.5)Surgery rabbit polyclonal 1:100 Sigma,St.Louis, USA IHC OS80 8 Yang 2015 China 92 21 (22.8) Surgery rabbit polyclonal 1:100 Abcam, Cambridge, UK IHC OS 60 5 Snail1 Zhou 2014 China 323 161 (49.8) Surgery NANA NA Novus, USA IHC OS 606 Zhao 2012 China 97 57 (58.8) Surgery NA NA 1:250 SantaCruz, CA, USA IHC OS 60 7Slug Zhang 2013 China 119 35 (29.4) Surgery NA NA NA Danvers, MA, USA IHC OS 60 8 Twist1 Zhang 2010 China 100 70 (70.0) Surgery rabbit polyclonal 1:50 SantaCruz, CA, USA IHC OS76 7 Zhao 2011 China 97 51 (52.6)SurgeryNA NA 1:100 SantaCruz, CA, USA IHC OS 60 7 Niu 2007 China 40 22 (55.0)Surgery rabbit monoclonal 1:50SantaCruz,CA, USA IHC OS 48 6 Abbreviations: EMT-TFs= epithelial tomesenchymaltransition-inducingtranscription factors; NA = not available;IHC=Immunohistochemistry; WB = Western Blot; MFu = median Follow-up; OS = overall survival; NOS = Newcastle-Ottawa Scale. Evidence synthesis{#s2_2} ------------------ EMT-TFsand OS inHCC {#s2_3} ---------------------The pooled HRfor OS indicated that EMT-TF positive expressionwas associated with poorOS \[HR = 1.71;95%CI: 1.40--2.08; *p* \< 0.00001\] in HCC with astatistically significant71% increase in the risk for mortality (Figure [2](#F2){ref-type="fig"}).Seeing that the heterogeneity test showed a *P* valueof 0.08 and an I^2^statisticindex of 41%, we considered that theremaybe relatively substantial heterogeneity between these studies, and therefore we used the random-effectsmodel. {#F2} Figure [3](#F3){ref-type="fig"} shows the impact of various individual EMT-TFs on the survival of HCC patients. The significantly higherHRs for OS was Slug \[HR = 2.12; 95% CI: 1.16--3.86; *p* = 0.01\]. But as thetranscription factor was reportedin only one study, theresult shouldbe considered with caution. In additionto ZEB2\[HR = 1.23; 95% CI: 0.59--2.57; *p* = 0.58\], HRs forTwist1 \[HR = 2.04; 95% CI: 1.50--2.78; *p* \<0.00001 | _INTRODUCTION_ {#s1} ============ Hepatocellular carcinoma (HCC) _is_ _one_ of the most common malignant _tumors_ _and_ ranks the third highest cause _of_ _cancer-related_ deaths worldwide \[[@R1], [@R2]\]. _The_ _resection_ _rate_ _of_ _HCC_ has increased over decades due _to_ the improvements in early diagnostic methods and surgical techniques. However, the postoperative recurrence rate and overall _survival_ (OS) are not optimistic due to limited response _to_ various adjunctive _therapies_ _and_ aggressive behaviors in _advanced_ stages of HCC \[[@R3]\]. _Thus,_ an accurate _understanding_ of _the_ biological behavior of _therioma_ is critical _in_ predicting the prognosis of HCC _patients._ Traditional prognostic _factors_ related to the clinicopathological characteristics of the neoplasm _after_ _hepatic_ resection such as tumor size, _vascular_ invasion, tumor-node-metastasis(TNM) stage, functional _liver_ reserve and _Child-Pugh_ class have a limited clinical value for outcome _prediction_ \[[@R4]\]. _Therefore,_ a variety of other potential molecular predictive markers _need_ to be further identified. _A_ _sequential_ process, including escape _from_ the primary tumor _site,_ local _invasion,_ systemic transport through _vascular_ or lymphatic vessels, adhesion _to_ distant organs, re-colonization, and expansion, is _believed_ to be involved _in_ _hepatic_ _carcinogenesis._ Epithelial to _mesenchymal_ transition (EMT) is known _to_ play a pivotal _role_ in the _diffusion_ of cancer _cells_ _and_ _the_ _growth_ of tumors, _in_ _which_ epithelial cells lose their polarity and cell-cell _contacts_ due _to_ repressed expression of _E-cadherin_ _and_ up-regulated _expression_ of mesenchymal markers _such_ as _N-cadherin,_ vimentin and α--smooth muscle actin (α--SMA) \[[@R5]\]. EMT could enhance not only the capacity of invasion and migration but _resistance_ to apoptosis and _chemoresistance_ in cancer. EMT may _alter_ the gene _expression_ _of_ _epithelial_ _cells_ due to the activation of EMT-inducing transcription factors (EMT-TFs). In this meta-analysis, we focused on the _most_ prominent inducers of EMT such as the zinc finger E-box _binding_ homeobox (ZEB1 and ZEB2), the _zinc-finger_ transcriptional repressor (Snail and Slug), _and_ the basic helix-loop-helix _transcription_ factor (Twist1) through _searching_ the _published_ _literature_ \[[@R6], [@R7]\], _knowing_ _that_ EMT-TFs are directly or indirectly _involved_ in metastasis of malignant cells _through_ a series of signaling cascades, including the wingless-related integration site(Wnt), _serine/threonine-specific_ protein kinase _(Akt),_ mitogen-activated _protein_ kinase (MAPK) and _signal_ transducer _and_ activator of transcription _3_ (STAT3) pathways _\[[@R8],_ [@R9]\]. During the past _decade,_ _much_ research has begun noticing the correlation between _the_ expression of EMT-TFs and _the_ prognosis _of_ HCC. However, _the_ _results_ are often unconvincing due to the limited _sample_ sizes. Here, we _sought_ to perform a _meta-analysis_ to evaluate clinicopathological and _prognostic_ _significance_ of EMT-TFs overexpression in HCC patients, especially those with high _incidences_ _of_ recurrence after _curative_ resection. _RESULTS_ {#s2} ======= _Study_ selection and patient characteristics {#s2_1} ------------------------------------------- The _initial_ search identified 418 _potentially_ relevant _studies._ _After_ screening, 10 published studies including 1,334 patients were selected for this pooled analysis \[[@R10]--[@R19]\]. _A_ flowchart depicting the _selection_ of the _eligible_ literature is shown in Figure _[1](#F1){ref-type="fig"}._ {#F1} All the included studies were retrospectively _analyzed,_ with _the_ sample size _ranging_ from 40 _to_ 323 (median 133). The overexpression of EMT-TFs was reported _in_ 662 (49.6%) _of_ the _1,334_ _included_ patients. The highest positive expression rate was Twist1, accounting for _60.3%,_ followed by Snail _(51.9%),_ ZEB2 (50.3%), _ZEB1_ (43.6%) and _Slug_ _(29.4%)._ _These_ studies were published _between_ 2007 _and_ 2015. Among all cohorts, Asia _was_ the only source _region_ of _the_ 10 included studies, including 9 _studies_ from China \[[@R11]--[@R19]\] and one from Japan _\[[@R10]\]._ _Newcastle-Ottawa_ Quality _Assessment_ Scale _was_ applied _to_ assess these _studies._ The result showed that the quality scores ranged from 5 to 8 _(median_ 6.5), _indicating_ _a_ _relatively_ high _study_ quality. Characteristics of the included studies _are_ listed in Table [1](#T1){ref-type="table"}. _All_ the studies focused on OS, with a _median_ follow-up period _of_ at least _48_ (48--80) _months._ The definition of _EMT-TFs_ positive expression was based on _immunohistochemistry_ (IHC) or western blot analysis (WB) evaluation in all the eligible articles, as expressed as the percentage _of_ _positive_ cells or/and _staining_ intensity. Hazard ratios (HRs) and 95% confidence intervals (CIs) were _directly_ _recorded_ _in_ 8 _studies_ \[[@R10]--[@R12], [@R15]--[@R19]\] and could be inferred from two _other_ studies using the _Tierney\'s_ methods described above, _among_ which one \[[@R14]\] were calculated by variance and _*P*_ value, and the other \[[@R13]\] _was_ estimated only by Kaplan-Meier survival _curves._ ###### Characteristics of the included studies EMT-TFs Author Year Country Case _EMT-TFs_ Positive(%) Treatment Antibody _method_ Outcome MFu time (months) NOS score --------- ---------- ------- --------- ----------- --------------------- ----------- ------------ ------------ _----------------------_ ---------------------- ----------- _----_ ---- --- _ZEB1_ Motoyuki 2013 _Japan_ 108 23 (21.3) Surgery goat polyclonal 1:100 SantaCruz, CA, USA IHC OS 60 8 Zhou 2011 China 110 72 (65.5) _Surgery_ _NA_ NA NA SantaCruz, CA, USA _WB_ OS 60 7 _ZEB2_ Cai 2012 _China_ 248 150 (60.5) Surgery rabbit polyclonal 1:100 _Sigma,_ St.Louis, _USA_ IHC OS _80_ 8 Yang _2015_ _China_ 92 21 (22.8) _Surgery_ rabbit polyclonal 1:100 Abcam, Cambridge, UK _IHC_ OS 60 _5_ Snail1 _Zhou_ 2014 China 323 _161_ (49.8) Surgery _NA_ NA _NA_ Novus, USA IHC OS _60_ 6 _Zhao_ 2012 China 97 _57_ (58.8) Surgery NA NA 1:250 _SantaCruz,_ CA, _USA_ IHC OS _60_ 7 Slug _Zhang_ 2013 China 119 35 (29.4) Surgery NA _NA_ NA _Danvers,_ MA, USA IHC OS _60_ 8 Twist1 _Zhang_ 2010 China _100_ _70_ (70.0) Surgery rabbit polyclonal 1:50 SantaCruz, CA, USA IHC OS _76_ _7_ _Zhao_ 2011 _China_ 97 51 (52.6) Surgery NA NA 1:100 SantaCruz, CA, USA IHC OS 60 _7_ Niu 2007 China 40 22 (55.0) Surgery rabbit monoclonal 1:50 _SantaCruz,_ CA, USA IHC _OS_ 48 6 Abbreviations: EMT-TFs = epithelial to _mesenchymal_ transition-inducing transcription factors; NA = _not_ available; IHC = _Immunohistochemistry;_ WB = Western Blot; MFu = median _Follow-up;_ OS = overall survival; NOS = Newcastle-Ottawa Scale. Evidence synthesis _{#s2_2}_ ------------------ EMT-TFs _and_ OS in HCC {#s2_3} _---------------------_ The pooled HR for OS indicated that EMT-TF positive expression was _associated_ with _poor_ OS \[HR _=_ 1.71; 95% _CI:_ 1.40--2.08; _*p*_ \< 0.00001\] _in_ HCC with a statistically significant 71% _increase_ in the risk for mortality (Figure _[2](#F2){ref-type="fig"})._ Seeing that the heterogeneity _test_ _showed_ a *P* value of 0.08 and an I^2^ statistic _index_ of 41%, we considered that there may _be_ relatively _substantial_ heterogeneity between these studies, _and_ therefore we used the random-effects model. {#F2} _Figure_ [3](#F3){ref-type="fig"} shows the impact of various individual _EMT-TFs_ on _the_ survival of HCC patients. _The_ significantly higher _HRs_ for OS was Slug _\[HR_ = 2.12; 95% CI: 1.16--3.86; *p* = 0.01\]. _But_ as the transcription factor was reported _in_ only one study, the result should be _considered_ with _caution._ In addition to ZEB2 _\[HR_ = 1.23; 95% _CI:_ 0.59--2.57; *p* = 0.58\], HRs for _Twist1_ \[HR = 2.04; 95% _CI:_ 1.50--2.78; *p* \< 0.00001 |
Introduction
============
Better adjuvant therapy, improved metal implants, and innovative surgical techniques have led surgeons to consider limb salvage surgery as an alternative treatment for malignant bone tumour other than amputation. Orthopaedic oncology patients have a chance for an active, disease-free life after limb salvage surgery. In the first evidence-based study, Simon *et al.* had reported the benefits of limb-salvaging procedures for bone tumours.[@b1-rado-46-03-189] Their multicentre study reported the rates of local recurrence, metastasis and survival in 227 patients with osteosarcoma in the distal femur and suggested that the Kaplan-Meier curves of the patients without recurrence were not statistically different between limb-salvaging surgery and amputation patients during a 5.5-year follow-up. Limb-salvage surgery was considered as safe as an amputation in the management of patients with high-grade osteosarcoma.
The goal of limb-salvaging surgery is to preserve the function of limbs, prevent tumour recurrence, and enable the rapid administration of chemotherapy or radiotherapy.[@b2-rado-46-03-189] It can be reached with meticulous technique, detailed operative planning, and the use of endoprosthetic replacements and/or bone grafting. For a successful limb-salvage surgery in high-grade malignant tumour, such as sarcomas, a wide margin is necessary to obtain a local control.[@b3-rado-46-03-189]--[@b5-rado-46-03-189] Since marginal and intralesional margins are related to local recurrence, the reconstruction with limb-salvaging options should be carefully considered. The clinical outcome of the limb-salvage surgery with arthroplasty is closely related to the accuracy of the surgical procedure. To improve the final outcome, one must take into account the length of the osteotomy plane, as well as the alignment of the prosthesis with respect to the mechanical axis in order to keep the balance of the soft tissues. Furthermore, the parameters measured with the 3D imagine must be used during the individual manufacture of implant in order to reconstruct the skeletal structure accurately. Therefore, geometric data (such as length of leg, offset) and morphologic data are required.
Magnetic resonance imaging (MRI) was beneficial for tumour detection and consequently staging of musculoskeletal neoplasia. MRI became an ideal imaging modality for musculoskeletal neoplasia because of superior soft-tissue resolution and multiplanar imaging capabilities and had a significant impact on the ability to appropriately stage lesions and adequately plan for limb-salvage surgery.[@b6-rado-46-03-189],[@b7-rado-46-03-189] In contrast, multi-slice spiral computed tomography (CT) could provide super three-dimensional morphological delineation of the diseased bone. Theoretically, the complimentary use of these two imaging modalities could give the surgeon a more accurate way to implement preoperative planning than the conventional application of 2D images.
The purpose of this prospective study was to report our initial experience with limb salvage surgery for orthopaedic oncology patients by using both MR imaging and multi-slice spiral CT for preoperative planning.
Patients and methods
====================
Patients and preparation
------------------------
The study protocol has complied with all relevant national regulations and institutional policies and has been approved by the local institutional ethics committee. Informed consent was obtained from all patients before the procedure. Patients with malignant bone tumours of lower/upper limb were enrolled in the study.
Preoperative work-up consisted of history and clinical examination, routine laboratory tests and an aesthetic assessment, plain radiography of the limb, 64-slice spiral CT scan of the limb and chest, Technetium-99m bone scan, and, in all of the cases, MRI of the affected limb. Antibiotics were administrated before the surgery. Biopsy was performed for pathological examination. Chemotherapy was commenced 6 weeks before the surgery in those cases which were diagnosed as osteosarcoma and dedifferentiated chondrosarcoma. Patients were classified according to the Enneking staging system.[@b8-rado-46-03-189],[@b9-rado-46-03-189]
The patients received a detailed narrative of conventional, surgical and amputation options after the limb salvage surgery at their own request. Nine consecutive patients with lower/upper limb malignant tumour of bone (5 women, 4 man, mean age 28.6 years, range: 19--52 years) were treated with limb-salvaging procedures. Lesion size (longitudinal direction), location and histology are summarized in [Table 1](#t1-rado-46-03-189){ref-type="table"}.
MR imaging
----------
MR images were performed at a 1.5-T superconductive unit (Gyroscan Intera, Philips Medical Systems, Netherlands) and a synergy surface coil was used. The sequences included transverse, sagittal and coronal turbo spin echo T1- and fat-suppressed T2-weighted images. The parameters of these sequences were TR/TE=400 /20 ms for T1-weighted imaging, TR/TE=3500 /120 ms for T2-weighted imaging and a field of view of 480 mm×480 mm for sagittal imaging and 40 mm×40 mm for transverse imaging and 480 mm×480 mm for coronal imaging with a matrix of 512×512, 4--6 signals acquisition and a slice thickness/gap = 5/0.5 mm. Contrast enhanced sagittal, coronal and transverse T1-weighted imaging were obtained after the intravenous injection of gadopentetate dimeglumine (Magnevist, Schering, Berlin, Germany) with a dosage of 0.2 mmol/kg of body weight.
Multi-slice spiral CT
---------------------
CT scan was performed by using a 64-slice spiral CT (Sensation 64, Siemens Medical Systems, Germany). The raw data obtained using an axial collimation of 64×0.6 mm, a pitch of 1.0, a tubular voltage of 120 KV and a tubular current of 360 mAs, were reconstructed into contiguous 1-mm thick slices with an increment of 0.5 mm and a field of view of 376 mm × 376 mm and a matrix of 512 × 512 by using the standard soft tissue and bone algorithm. These thin-slice images were postprocessed by using the techniques of multiplanar reformation (MPR) and volume rendering (VR) to demonstrate the lesion details and perform related measurements.
Preoperative planning
---------------------
All preoperative radiographs were evaluated by one radiologist and two consultant orthopaedic surgeons, who were members of the surgical team performing the operations. First, the osteotomy plane was determined separately on CT and MRI. On orthogonal coronal enhanced MR images and CT MPR images, the bulk margin of the tumour in the medullary cavity was defined according to the different signal characteristics or attenuation of the tumour itself and the marrow oedema around the tumour. Then, the maximum distance from the top of the greater trochanter to this tumour margin was measured on orthogonal coronal T1-weigthted MRI images, if the tumour was located in the proximal part of femur. The maximum distance from the knee joint line to the tumour margin was measured for the tumours located in the distal part of femur. The maximum distance was defined as the intramedullary extension of the primary tumour and subsequently was used as a reference for the CT measurement. The osteotomy plane on CT MPR images was defined 30 mm distal from the margin of tumour. This distance was also used to determine the length of the extra medullary part of the prosthesis. After the osteotomy plane had been determined, the detailed shape of the medullary cavity of the preserved part of the femur was assessed using the orthogonal MPR technique for determining the diameter and length of the intra medullary part of prosthesis. Diameters of the medullary cavity at the level of the osteotomy plane and the level of the narrowest plane were measured to determine the diameter of the intra medullary stem of the prosthesis. The length of the intra- medullary stem of the prosthesis should be well matched to the length of medullary cavity of the preserved part of femur, which would be optimal if it had an equal length to the extra medullary part of prosthesis. Finally, the centre axis of the femoral shaft measured on CT was used as a reference. Offset, the distance from the central axis of the femoral shaft and the rotation centre of the femoral head, was the index used to determine the neck length of the prosthesis.
Surgery
-------
All patients underwent en bloc resection and customized prosthetic reconstruction. An anterolateral incision encircling the biopsy scar was used. Limb-salvage surgery consisted of intentional marginal excision, preserving important structures such as major neurovascular bundles, tendons, and ligaments. The osteotomy plane, 30 mm distal from the primary tumour was confirmed based on MRI for all patients. For patients with lesion in the proximal part of femur/humerus, the customized prosthesis was secured using methylmethacrylate cement after the resection. For patients with the tumour in the distal part of the femur, en bloc resection including the tibial plateau was performed and the customized prosthesis was secured using methylmethacrylate cement in both the tibia and femur after the resection. The extensor mechanism was reconstructed by reattachment of the patellar tendon to the slot on the tibial component. After surgery, functional rehabilitation and neoadjuvant chemotherapy were performed.
Postoperative measurement
-------------------------
After surgery, the patients were followed with a mean of 13 months (range, 9 to 20 months). The postoperative assessment of prosthesis was performed on plain radiography. The central axis of the femoral or humeral shaft and offset were defined. The vertical distance from the line between the top of bilateral ischial tuberosities to the femoral | introduction = = = = = = = = = = = = better adjuvant therapy, improved metal implants, and innovative surgical techniques have led surgeons can consider limb salvage surgery as an alternative treatment for malignant bone tumour other than amputation. orthopaedic oncology patients have a chance for an active, disease - free life after limb salvage surgery. in the first evidence - based study, simon * et al. * again reported the benefits of limb - salvaging procedures for bone tumours. [ @ b1 - rado - 46 - 03 - 189 ] their multicentre study reported the rates of local recurrence, disability and survival in 227 diagnosed with osteosarcoma in the distal femur and suggested that the kaplan - meier curves of the patients without recurrence were positively statistically different between limb - salvaging surgery and amputation patients during a 5. 5 - year follow - up. limb - salvage surgery was considered as valid as an amputation in effective management of patients with high - grade osteosarcoma. the goal of limb - salvaging surgery is to preserve minimal function of tissues, prevent tumour recurrence, and enable the rapid administration of chemotherapy or radiotherapy. [ @ b2 - rado - 46 - 03 - 189 ] it can be reached with meticulous technique, detailed operative planning, and the use of endoprosthetic replacements and / or bone grafting. for a successful limb - salvage surgery in high - grade malignant tumour, such as sarcomas, a wide margin is necessary to obtain a local control. [ @ b3 - rado - 46 - 03 - 189 ] - - [ @ b5 - rado - 46 - 03 - 189 ] since marginal and intralesional margins are related to local recurrence, the reconstruction with limb - salvaging options should be carefully considered. the clinical outcome of the limb - salvage surgery with arthroplasty is closely related to the accuracy of the surgical procedure. to improve the final outcome, one must take into account the length of the osteotomy plane, as well as the alignment of the prosthesis with respect to the mechanical axis in order to keep the balance of the soft tissues. furthermore, the parameters measured with the 3d imagine must be used during the individual manufacture of plastic in order to reconstruct the skeletal structure accurately. therefore, geometric data ( such as length of leg, offset ) and morphologic data are required. magnetic resonance imaging ( mri ) was beneficial for tumour detection and consequently staging of musculoskeletal neoplasia. mri became an ideal imaging modality for musculoskeletal neoplasia because of superior soft - tissue resolution and multiplanar imaging capabilities and had a significant impact on the ability to appropriately stage lesions and adequately plan for limb - salvage surgery. [ @ b6 - rado - 46 - 03 - 189 ], [ @ b7 - rado - 46 - 03 - 189 ] in contrast, multi - slice spiral computed tomography ( ct ) could provide super three - dimensional morphological delineation of the diseased bone. theoretically, the complimentary use of these two imaging modalities could give the surgeon a more accurate way to implement preoperative planning than the conventional application of 2d images. the purpose of this prospective study was to report our initial experience with limb salvage surgery for orthopaedic oncology patients by using both mr imaging and multi - slice spiral ct for preoperative planning. patients and methods = = = = = = = = = = = = = = = = = = = = patients and preparation - - - - - - - - - - - - - - - - - - - - - - - - the study protocol has complied with all relevant national regulations and institutional policies and has been approved by the local institutional ethics committee. informed consent was obtained from all patients before the procedure. patients with malignant bone tumours of lower / upper limb were enrolled in the study. preoperative work - up consisted of history and clinical examination, routine laboratory tests and an aesthetic assessment, plain radiography of the limb, 64 - slice spiral ct scan of the limb and chest, technetium - 99m bone scan, and, in all of the cases, mri of the affected limb. antibiotics were administrated before the surgery. biopsy was performed for pathological examination. chemotherapy was commenced 6 weeks before the surgery in those cases which were diagnosed as osteosarcoma and dedifferentiated chondrosarcoma. patients were classified according to the enneking staging system. [ @ b8 - rado - 46 - 03 - 189 ], [ @ b9 - rado - 46 - 03 - 189 ] the patients received a detailed narrative of conventional, surgical and amputation options after the limb salvage surgery at their own request. nine consecutive patients with lower / upper limb malignant tumour of bone ( 5 women, 4 man, mean age 28. 6 years, range : 19 - - 52 years ) were treated with limb - salvaging procedures. lesion size ( longitudinal direction ), location and histology are summarized in [ table 1 ] ( # t1 - rado - 46 - 03 - 189 ) { ref - type = " table " }. mr imaging - - - - - - - - - - mr images were performed at a 1. 5 - t superconductive unit ( gyroscan intera, philips medical systems, netherlands ) and a synergy surface coil was used. the sequences included transverse, sagittal and coronal turbo spin echo t1 - and fat - suppressed t2 - weighted images. the parameters of these sequences were tr / te = 400 / 20 ms for t1 - weighted imaging, tr / te = 3500 / 120 ms for t2 - weighted imaging and a field of view of 480 mm×480 mm for sagittal imaging and 40 mm×40 mm for transverse imaging and 480 mm×480 mm for coronal imaging with a matrix of 512×512, 4 - - 6 signals acquisition and a slice thickness / gap = 5 / 0. 5 mm. contrast enhanced sagittal, coronal and transverse t1 - weighted imaging were obtained after the intravenous injection of gadopentetate dimeglumine ( magnevist, schering, berlin, germany ) with a dosage of 0. 2 mmol / kg of body weight. multi - slice spiral ct - - - - - - - - - - - - - - - - - - - - - ct scan was performed by using a 64 - slice spiral ct ( sensation 64, siemens medical systems, germany ). the raw data obtained using an axial collimation of 64×0. 6 mm, a pitch of 1. 0, a tubular voltage of 120 kv and a tubular current of 360 mas, were reconstructed into contiguous 1 - mm thick slices with an increment of 0. 5 mm and a field of view of 376 mm × 376 mm and a matrix of 512 × 512 by using the standard soft tissue and bone algorithm. these thin - slice images were postprocessed by using the techniques of multiplanar reformation ( mpr ) and volume rendering ( vr ) to demonstrate the lesion details and perform related measurements. preoperative planning - - - - - - - - - - - - - - - - - - - - - all preoperative radiographs were evaluated by one radiologist and two consultant orthopaedic surgeons, who were members of the surgical team performing the operations. first, the osteotomy plane was determined separately on ct and mri. on orthogonal coronal enhanced mr images and ct mpr images, the bulk margin of the tumour in the medullary cavity was defined according to the different signal characteristics or attenuation of the tumour itself and the marrow oedema around the tumour. then, the maximum distance from the top of the greater trochanter to this tumour margin was measured on orthogonal coronal t1 - weigthted mri images, if the tumour was located in the proximal part of femur. the maximum distance from the knee joint line to the tumour margin was measured for the tumours located in the distal part of femur. the maximum distance was defined as the intramedullary extension of the primary tumour and subsequently was used as a reference for the ct measurement. the osteotomy plane on ct mpr images was defined 30 mm distal from the margin of tumour. this distance was also used to determine the length of the extra medullary part of the prosthesis. after the osteotomy plane had been determined, the detailed shape of the medullary cavity of the preserved part of the femur was assessed using the orthogonal mpr technique for determining the diameter and length of the intra medullary part of prosthesis. diameters of the medullary cavity at the level of the osteotomy plane and the level of the narrowest plane were measured to determine the diameter of the intra medullary stem of the prosthesis. the length of the intra - medullary stem of the prosthesis should be well matched to the length of medullary cavity of the preserved part of femur, which would be optimal if it had an equal length to the extra medullary part of prosthesis. finally, the centre axis of the femoral shaft measured on ct was used as a reference. offset, the distance from the central axis of the femoral shaft and the rotation centre of the femoral head, was the index used to determine the neck length of the prosthesis. surgery - - - - - - - all patients underwent en bloc resection and customized prosthetic reconstruction. an anterolateral incision encircling the biopsy scar was used. limb - salvage surgery consisted of intentional marginal excision, preserving important structures such as major neurovascular bundles, tendons, and ligaments. the osteotomy plane, 30 mm distal from the primary tumour was confirmed based on mri for all patients. for patients with lesion in the proximal part of femur / humerus, the customized prosthesis was secured using methylmethacrylate cement after the resection. for patients with the tumour in the distal part of the femur, en bloc resection including the tibial plateau was performed and the customized prosthesis was secured using methylmethacrylate cement in both the tibia and femur after the resection. the extensor mechanism was reconstructed by reattachment of the patellar tendon to the slot on the tibial component. after surgery, functional rehabilitation and neoadjuvant chemotherapy were performed. postoperative measurement - - - - - - - - - - - - - - - - - - - - - - - - - after surgery, the patients were followed with a mean of 13 months ( range, 9 to 20 months ). the postoperative assessment of prosthesis was performed on plain radiography. the central axis of the femoral or humeral shaft and offset were defined. the vertical distance from the line between the top of bilateral ischial tuberosities to the femoral | Introduction = = = = = = = = = = = = Better adjuvant therapy, improved metal implants, and innovative surgical techniques have led surgeons to consider limb salvage surgery as an alternative treatment for malignant bone tumour other than amputation. Orthopaedic oncology patients have a chance for an active, disease - free life after limb salvage surgery. In the first evidence - based study, Simon * et al. * had reported the benefits of limb - salvaging procedures for bone tumours. [@ b1 - rado - 46 - 03 - 189] Their multicentre study reported the rates of local recurrence, metastasis and survival in 227 patients with osteosarcoma in the distal femur and suggested that the Kaplan - Meier curves of the patients without recurrence were not statistically different between limb - salvaging surgery and amputation patients during a 5. 5 - year follow - up. Limb - salvage surgery was considered as safe as an amputation in the management of patients with high - grade osteosarcoma. The goal of limb - salvaging surgery is to preserve the function of limbs, prevent tumour recurrence, and enable the rapid administration of chemotherapy or radiotherapy. [@ b2 - rado - 46 - 03 - 189] It can be reached with meticulous technique, detailed operative planning, and the use of endoprosthetic replacements and / or bone grafting. For a successful limb - salvage surgery in high - grade malignant tumour, such as sarcomas, a wide margin is necessary to obtain a local control. [@ b3 - rado - 46 - 03 - 189] - - [@ b5 - rado - 46 - 03 - 189] Since marginal and intralesional margins are related to local recurrence, the reconstruction with limb - salvaging options should be carefully considered. The clinical outcome of the limb - salvage surgery with arthroplasty is closely related to the accuracy of the surgical procedure. To improve the final outcome, one must take into account the length of the osteotomy plane, as well as the alignment of the prosthesis with respect to the mechanical axis in order to keep the balance of the soft tissues. Furthermore, the parameters measured with the 3D imagine must be used during the individual manufacture of implant in ord$g to reconstruct the skeletal structure accurately. Therefore, geometric data (such as length of leg, offset) and morphologic data are required. Magnetic resonance imaging (MRI) was beneficial for tumour detection and consequently staging of musculoskeletal neoplasia. MRI became an ideal imaging modality for musculoskeletal neoplasia because of superior soft - tissue resolution and multiplanar imaging capabilities and had a significant impact on the ability to appropriately stage lesions and adequately plan for limb - salvage surgery. [@ b6 - rado - 46 - 03 - 189 ], [@ b7 - rado - 46 - 03 - 189] In contrast, multi - slice spiral computed tomography (CT) could provide super three - dimensional morphological delineation of the diseased bone. Theoretically, the complimentary use of these two imaging modalities could give the surgeon a more accurate way to implement preoperative planning than the conventional application of 2D images. The purpose of this prospective study was to report our initial experience with limb salvage surgery for orthopaedic oncology patients by using both MR imaging and multi - slice spiral CT for preoperative planning. Patients and methods = = = = = = = = = = = = = = = = = = = = Patients and preparation - - - - - - - - - - - - - - - - - - - - - - - - The study protocol has complied with all relevant national regulations and institutional policies and has been approved by the local institutional ethics committee. Informed consent was obtained from all patients before the procedure. Patients with malignant bone tumours of lower / upper limb were enrolled in the study. Preoperative work - up consisted of history and clinical examination, routine laboratory tests and an aesthetic assessment, plain radiography of the limb, 64 - slice spiral CT scan of the limb and chest, Technetium - 99m bone scan, and, in all of the cases, MRI of the affected limb. Antibiotics were administrated before the surgery. Biopsy was performed for pathological examination. Chemotherapy was commenced 6 sSeks before the surgery in those cases which were diagnosed as osteosarcoma and dedifferentiated chondrosarcoma. Patients were classified according to the Enneking stagOnf system. [@ b8 - rado - 46 - 03 - 189 ], [@ b9 - rado - 46 - 03 - 189] The patients received a detailed narrative of conventional, surgical and amputation options aEHer the limb salvage surgery at their own request. Nine consecutive patients with lower / upper limb malignant tumour of bone (5 women, 4 man, mean age 28. 6 years, range: 19 - - 52 years) were treated with limb - salvaging procedures. Lesion size (longitudinal direction ), location and histology are summarized in [Table 1] (# t1 - rado - 46 - 03 - 189) {ref - type = " table " }. MR imaging - - - - - - - - - - MR images were performed at a 1. 5 - T superconductive unit (Gyroscan Intera, Philips Medical Systems, Netherlands) and a synergy surface coil was used. The sequences included transverse, sagittal and coronal turbo spin echo T1 - and fat - suppressed T2 - weighted images. The parameters of these sequences were TR / TE = 400 / 20 ms for T1 - weighted imaging, TR / TE = 3500 / 120 ms for T2 - weighted imaging and a field of view of 480 mm × 480 mm for sagittal imaging and 40 mm × 40 mm for transverse imaging and 480 mm × 480 mm for coronal imaging with a matrix of 512 × 512, 4 - - 6 signals acquisition and a slice thickness / gap = 5 / 0. 5 mm. Contrast enhanced sagittal, cpr)nal and transverse T1 - weighted imaging were obtained after the intravenous injection of gadopentetate dimeglumine (Magnevist, Schering, Berlin, Germany) with a dosage of 0. 2 mmol / kg of body weight. Multi - slice spiral CT - - - - - - - - - - - - - - - - - - - - - CT scan was performed by using a 64 - slice spiral CT (Sensation 64, Siemens Medical Systems, Germany ). The raw data obtained using an axial collimation of 64 × 0. 6 mm, a pitch of 1. 0, a tubular voltage of 120 KV and a tubular current of 360 mAs, were reconstructed into contiguous 1 - mm thick slices with an increment of 0. 5 mm and a field of view of 376 mm × 376 mm and a matrix of 512 × 512 by using the standard soft tissue and bone algorithm. These thin - slice images were postprocessed by using the techniques of multiplanar reformation (MPR) and volume rendering (VR) to demonstrate the lesion details and perform related measurements. Preoperative planning - - - - - - - - - - - - - - - - - - - - - All preoperative radiographs were evaluated by one radiologist and two consultant orthopaedic surgeons, who were members of the surgical team performing the operations. First, the osteotomy plane was determined separately on CT and MRI. On orthogonal coronal enhanced MR images and CT MPR images, the bulk margin of the tumour in the medullary cavity was defined according to the different signal characteristics or attenuation of the tumour itself and the marrow oedema around the tumour. Then, the maximum distance from the top of the greater trochanter to this tumour margin was measured on orthogonal coronal T1 - weigthted MRI images, if the tumour was located in the proximal part of femur. The maximum distance from the knee joint line to the tumour margin was measured for the tumours located in the distal part of femur. The maximum distance was defined as the intramedullary extension of the primary tumour and subsequently was used as a reference for the CT measurement. The osteotomy plane on CT MPR images was defined 30 mm distal from the margin of tumour. This distance was also used to determine the length of the extra medullary part of the prosthesis. After the osteotomy plane had been determined, the detailed shape of the medullary cavity of the preserved part of the femur was assessed using the orthogonal MPR technique for determining the diameter and length of the intra medullary part of prosthesis. Diameters of the medullary cavity at the level of the osteotomy plane and the level of the narrowest plane were measured to determine the diameter of the intra medullary stem of the prosthesis. The length of the intra - medullary stem of the prosthesis should be well matched to the length of medullary cavity of the preserved part of femur, which would be optimal if it had an equal length to the extra medullary part of proDtheWis. Finally, the centre axis of the femoral shaft measured on CT was used as a reference. Offset, the distance from the central axis of the f#horal shaft and the rotation centre of the femoral head, was the index tsdd to determine the neck length of the proshhes&s. Surgery - - - - - - - All patients underwent en bloc resection and customized prosthetic reconstruction. An anterolateral incision encircling the biopsy scar was used. Limb - salvage surgery consisted of intentional marginal excision, preserving important structures such as major neurovascular bundles, tendons, and ligaments. The osteotomy plane, 30 mm distal from the primary tumour was confirmed based on MRI for all patients. For patients with lesion in the proximal part of femur / humerus, the customized prosthesis was secured using methylmethacrylate cement after the resection. For patients with the tumour in the distal part of the femur, en bloc resection including the tibial plateau was performed and the customized prosthesis was secured using methylmethacrylate cement in b*tN the tibia and femur after the resection. The extensor mechanism was reconstructed by reattachment of the patellar tendon to the slot on the tibial component. After surgery, functional rehabilitation and neoadjuvant chemotherapy were performed. Postoperative measurement - - - - - - - - - - - - - - - - - - - - - - - - - After surgery, the patients were followed with a mean of 13 months (range, 9 to 20 months ). The postoperative assessment of prosthesis was performed on plain radiography. The central axis of the femoral or humeral shaft and offset were defined. The vertical distance from the line between the top of bilateral ischial tuberosities to the femoral | Introduction ============ Better adjuvant therapy, improved metal and innovative surgical techniques have led surgeons to consider limb salvage surgery as an alternative treatment for malignant bone tumour other than amputation. Orthopaedic oncology patients a chance for an active, disease-free life after limb salvage surgery. the first study, *et al.* had reported the benefits of procedures for bone tumours.[@b1-rado-46-03-189] Their multicentre study reported the rates of local recurrence, metastasis and survival in 227 patients with osteosarcoma in the distal femur and suggested that the Kaplan-Meier curves of the patients recurrence were not statistically different between limb-salvaging and patients during a 5.5-year follow-up. Limb-salvage surgery was considered as safe as an amputation in the management of patients with high-grade osteosarcoma. The goal of surgery is to preserve the function of limbs, prevent tumour recurrence, and enable the rapid administration of chemotherapy or radiotherapy.[@b2-rado-46-03-189] It can be with meticulous technique, detailed operative and the use of endoprosthetic replacements bone grafting. For successful limb-salvage surgery high-grade malignant tumour, such as sarcomas, a wide margin is necessary to obtain a local control.[@b3-rado-46-03-189]--[@b5-rado-46-03-189] Since marginal and intralesional margins are related to the reconstruction limb-salvaging options should be considered. The clinical outcome of the limb-salvage surgery with arthroplasty is closely related to the accuracy of the surgical procedure. To improve the outcome, one take into account the of the osteotomy plane, as well as the alignment of the prosthesis with respect to the mechanical axis in order to keep the balance of soft tissues. Furthermore, the parameters with the imagine must be during the individual manufacture implant in order to the skeletal structure accurately. Therefore, geometric data (such as length of leg, and morphologic data are required. imaging (MRI) was beneficial for tumour and consequently staging musculoskeletal neoplasia. MRI became an ideal modality for musculoskeletal neoplasia because of superior soft-tissue resolution and multiplanar imaging capabilities and had a significant impact on ability to appropriately stage lesions and adequately plan for limb-salvage surgery.[@b6-rado-46-03-189],[@b7-rado-46-03-189] In contrast, multi-slice computed tomography (CT) could super morphological delineation of the diseased bone. the complimentary use of these modalities could give the a accurate way to implement preoperative than the application of 2D images. The purpose of this prospective was to report our initial experience limb salvage surgery for orthopaedic oncology patients by using both MR imaging and multi-slice spiral CT for Patients and methods ==================== Patients and preparation ------------------------ The study protocol has complied with all relevant national and institutional policies and has been approved by the local institutional ethics committee. Informed consent was obtained from all patients before the procedure. Patients with malignant bone tumours of lower/upper limb were enrolled in the study. Preoperative work-up consisted of history and clinical examination, routine laboratory tests and aesthetic assessment, plain radiography of the limb, 64-slice spiral scan of the limb and chest, Technetium-99m bone scan, and, in all of the cases, MRI of the affected limb. Antibiotics were administrated before the surgery. Biopsy performed for pathological examination. Chemotherapy commenced 6 weeks before surgery in those cases which were diagnosed as osteosarcoma and dedifferentiated chondrosarcoma. Patients were to the Enneking staging system.[@b8-rado-46-03-189],[@b9-rado-46-03-189] The received a detailed narrative of conventional, surgical and amputation options after the limb salvage surgery at their own request. Nine consecutive patients with lower/upper limb tumour of bone (5 women, 4 mean age 28.6 years, range: 19--52 years) were treated with limb-salvaging procedures. Lesion (longitudinal direction), location and histology summarized in [Table 1](#t1-rado-46-03-189){ref-type="table"}. MR imaging MR images were performed at a 1.5-T superconductive unit (Gyroscan Intera, Philips Medical Systems, and a synergy surface coil was used. The transverse, sagittal and coronal turbo spin echo T1- and fat-suppressed T2-weighted images. The parameters these sequences were TR/TE=400 /20 ms for T1-weighted imaging, TR/TE=3500 ms for T2-weighted and a field of view of 480 mm×480 mm for imaging and 40 mm×40 mm for transverse imaging and 480 mm×480 mm for coronal imaging a matrix of 512×512, 4--6 signals acquisition and a slice = 5/0.5 mm. enhanced sagittal, coronal and transverse T1-weighted imaging were obtained the intravenous injection of gadopentetate dimeglumine Schering, Berlin, Germany) with a dosage of 0.2 mmol/kg of body weight. Multi-slice spiral CT --------------------- CT scan performed by a 64-slice spiral (Sensation 64, Siemens Medical Systems, Germany). raw data obtained an axial collimation of mm, a pitch of 1.0, a tubular of 120 KV a tubular current of 360 mAs, were reconstructed into contiguous 1-mm thick slices with an increment of 0.5 mm and a field of of 376 mm × 376 mm and a matrix of 512 × by using the standard soft tissue bone These thin-slice images were postprocessed by the of multiplanar reformation (MPR) and rendering (VR) to demonstrate the lesion details and perform measurements. Preoperative planning --------------------- All preoperative radiographs were evaluated by radiologist and two consultant orthopaedic surgeons, who were members of the surgical team performing the operations. First, the osteotomy plane was determined separately CT and MRI. On orthogonal coronal enhanced MR and MPR images, the bulk margin of the tumour in medullary cavity was defined according to the different signal characteristics attenuation of the tumour itself and the marrow oedema around the tumour. Then, the maximum distance from the of the greater trochanter to this tumour margin was measured orthogonal coronal T1-weigthted MRI images, if the tumour was located in the proximal part of femur. The maximum distance from the knee joint line to the margin was measured for the located in the distal part of femur. The maximum distance was defined the intramedullary extension of the primary tumour and subsequently was used a reference for the CT measurement. The osteotomy plane on CT MPR was defined 30 mm distal from the margin of tumour. This distance was also used to determine length of the extra medullary part of the prosthesis. After the osteotomy plane had the detailed shape of the medullary cavity of the preserved part the femur was assessed using the orthogonal MPR technique for determining the diameter and length of the intra medullary part of prosthesis. Diameters of the medullary cavity at the level of the osteotomy plane and the of the narrowest plane were measured to determine the the intra medullary stem of the prosthesis. The of the medullary stem of the should be well matched to the length of medullary cavity of the preserved part of femur, which would be optimal if it had an equal length to the extra part of prosthesis. Finally, the centre axis of femoral shaft measured on was used as a reference. Offset, the distance from the central axis of the femoral shaft and the centre of the femoral head, was the index used to determine the neck of the prosthesis. Surgery ------- patients underwent en and customized prosthetic reconstruction. An anterolateral incision encircling the biopsy scar was used. Limb-salvage consisted of intentional marginal excision, preserving important structures as major neurovascular bundles, tendons, ligaments. The plane, 30 mm distal from the primary tumour was confirmed based MRI for all patients with lesion in the proximal part of femur/humerus, the prosthesis was secured using methylmethacrylate after the resection. patients with the tumour in the distal part of the femur, en bloc resection including the tibial plateau was performed the customized prosthesis was secured using methylmethacrylate in both the tibia and femur after the resection. extensor mechanism was reconstructed by of the patellar tendon to the slot the component. After surgery, functional rehabilitation and neoadjuvant chemotherapy were performed. Postoperative ------------------------- After surgery, the patients were followed a mean of 13 months (range, 9 to 20 The postoperative assessment of prosthesis was on plain radiography. The central axis of the femoral or humeral shaft and offset were defined. vertical distance from the line between the top of bilateral ischial tuberosities to the femoral | IntrODuction
============
BETteR aDJuVant thERaPY, imPRoved mEtal IMPlants, And INnOVAtiVE surgICaL TecHNIQUeS HaVE LED sUrGeonS To ConSider LImb SalVAgE SURGeRY AS AN ALtErNatIVe tReatmENT FOR MAliGnANt bone TuMOUr oTHeR THAN AmPUTATION. ORTHOPaEDiC ONCOLogy pATieNts HaVE A cHaNce foR AN ACTIVe, DISEase-FREe LIfe aFTeR limB sALVAgE SURgerY. IN The fiRST EVIDence-bASeD sTuDy, sIMOn *Et Al.* HAD repORted THE benEFiTS OF lIMb-sALvagiNG ProCEDures fOr bOne tUmOuRS.[@b1-RADO-46-03-189] ThEIr MULtICEntRe STuDy RePoRted thE RaTes Of lOcAL ReCUrREncE, mEtasTASiS AND sURvival in 227 paTIEnts with OsTEOsaRcoMa In ThE DistAl fEmuR And sUGgestEd ThaT The kapLAN-MEieR CURvES Of ThE paTIentS WITHoUt ReCurRenCE WERe noT StAtiSTIcalLY DIFFeReNt BetweeN LImb-sAlVaGING SURgerY anD amPUtatION paTIEnts DUrinG a 5.5-YEAR fOLloW-Up. LiMB-SAlvAge surGERY wAS conSIdeRED AS SAfE As an amPutAtion In The MaNAGement of PATiENts WiTh hIgh-gRadE OsTEOsarCOmA.
The goAL OF LimB-saLVagiNG sUrgErY iS tO pREsERVE tHE FunCtiOn OF limBs, PreVeNT TuMOur ReCUrrencE, And EnaBLE ThE RAPId ADMINIsTRAtIon Of CHEmOThErApY oR rADIOtHerapY.[@b2-RaDO-46-03-189] It cAN BE reaCHed WitH mEtICULOUs TEChNIquE, DEtAILED OPeraTIVe PlANnING, aND the UsE oF eNDopRoStHeTiC rEPLaceMeNtS AnD/OR boNe grAftINg. fOr A SuCcEsSful LImB-SAlVAGe suRGeRY IN hIGh-gRADe mAliGnAnt TUMoUr, sUch As sArCoMAS, A widE MaRgIn iS NeCeSsARY To OBtaIN a lOcAL COnTroL.[@B3-rADO-46-03-189]--[@b5-raDO-46-03-189] SiNCe margINAl And intraLESiONaL mArGiNs arE reLaTEd to loCaL recurRenCe, The REconsTRUCtion with LImB-SaLVagING OptionS shOuLd be cAreFULLy CoNsIDeREd. THe ClinICAL OuTCOme OF THe lIMb-SalVAgE SurgerY WiTh aRTHRoPlaSTY iS CloSEly rELateD tO tHe aCcURaCy Of tHE suRGicaL PrOCEdure. TO imPROVE tHe fInal OUTCOmE, oNe MuSt tAkE inTO ACcOUNT THe lEnGtH oF THe osTEoTOMY plANE, AS wElL aS THe aLignmenT OF The prostHEsIs wITh ReSpECT tO The mECHanIcAl AXis iN oRDeR tO keep ThE balAnCE OF tHE SOFt TissUEs. fuRTHermoRE, The PARameTERs MeasURED wITH THE 3D IMAgInE MusT Be USEd dURiNg tHe iNdiViduAL ManuFaCTuRe Of IMplanT iN oRDEr tO REconsTRuCt THE sKELETaL stRucTURE AcCUratEly. THerefOrE, GEOMeTRIc DaTA (sUCH as LenGTH of leg, ofFSeT) anD MORpHOlogic dAta arE rEquIReD.
MaGNETIC reSonaNCe imaGiNg (mRi) wAs BEnEFICiAl FOr tUmOuR DeTEctiOn and cOnSEQueNTlY STaGING OF muscUloskeletaL NEopLAsIa. MRI BeCAME An IDEAL ImaGiNG mODALiTY fOR MuScuLOsKELEtal nEopLaSiA BeCaUse oF SUpeRior soFt-tiSsUE reSoLUTIon anD muLtIPLaNar iMAginG cApaBIlitiEs aNd haD a sigNIFiCANt impAcT On The aBiLitY To aPpROPRiATeLY STage leSioNs and aDeqUAtEly PlAn For lIMb-SALvaGE sUrGeRy.[@b6-RADO-46-03-189],[@b7-RAdO-46-03-189] In cOntRaSt, mULTi-slICe SPIrAL COmPUTed toMOGrapHy (ct) cOUld provIDe supEr tHreE-dIMeNsIOnAl MORPHOLOgiCAL deLINeaTiON Of tHe DIseAsED boNE. THeOREticAllY, THE CoMpLiMeNTarY Use oF THESe tWo IMaGiNG mODALItiES COUlD gIve thE surGeON a MOrE AccuRATe waY to IMPLEmENt preOPEratIVe PlanNiNg Than thE CoNventIOnAL APplIcaTiON oF 2d ImageS.
the PuRPOse of This ProsPecTIvE STudy waS TO RePorT OUr INitIaL eXPErIENcE wItH Limb saLvaGE surgERY FoR OrthoPaedic ONCOlOGY PATiEnTS by UsIng bOTH mr IMAginG aNd mUlTi-SLiCE spIrAL CT FOR PReOpErATIVe PLANNING.
pATientS and mETHOdS
====================
pAtIEntS ANd PrepAraTIon
------------------------
thE STuDY PROTOCoL HAS ComPLIEd WITH aLl relEVAnt NaTionAl rEGuLATiONS aND iNSTITUtIOnAl pOlICIes AND has BEEN aPProVEd By the lOcal iNstITUTIOnAL etHiCS cOMmiTTEe. InfORmeD CONSenT WAS oBTAinED fROm ALL pAtIeNTs BEfoRe thE PRoCEdURe. PATIENtS WIth MalIgNANt bonE TUmoUrS OF LOWEr/UPpEr LImb wErE eNRoLlEd in ThE StuDy.
pReOpeRaTive WORK-UP cOnsIstEd OF HiSTorY aND cLiNiCAl ExAMINAtioN, RoUTine LaBOrAtoRy tEsts anD aN AEStHETic aSSesSMENT, pLAIn rAdIOgRapHY oF tHE LimB, 64-sliCe SpIrAl ct SCan oF ThE limb AND cHEsT, TEChnEtIuM-99m BoNe scan, And, In ALL Of THE caSes, mri of THe aFFeCTEd LImB. ANTiBIOticS wERE aDmiNiStrATeD bEFoRe THE suRgery. BiOpSy WAs pErForMED For PathOLOGICAl eXAmiNAtiOn. CHEMothEraPy WaS comMencED 6 Weeks BeFOrE tHe sURGErY iN THose cAses WhicH WERE diAgNOseD As OSTEosArCoMa anD deDifFErentIAteD cHonDrOSarcOMA. pATieNTS WErE ClaSsiFIeD ACcoRding to The eNnEKINg StagING SysTem.[@b8-rAdo-46-03-189],[@B9-rAdo-46-03-189]
The paTiENTs ReCeIVeD A DETaiLed nARratIVE OF convenTIOnal, SUrgICAl aND AmPuTATioN oPTIOns aFTeR THe limB sALVAge suRGERy AT tHEiR oWn REQUEst. ninE CoNseCuTIvE PATIenTs WITh LoWEr/UPPEr LImb maLIGnAnT TUmoUr OF BoNE (5 wOMeN, 4 mAN, MeaN Age 28.6 yEaRS, RANgE: 19--52 YeArs) were tReAtEd WITh liMB-SAlVaGING pRocedUREs. lesION sIze (LongitUdinaL diRECTiON), LoCATiON anD hIStoloGY aRe sUMMaRiZeD in [tAblE 1](#t1-rADo-46-03-189){ReF-tYPe="tABLE"}.
mR ImagiNG
----------
mR ImaGeS WerE pErfOrmed AT A 1.5-t suPercONDucTivE uNiT (GYROsCAN inTerA, phIliPS MedIcaL SYSteMS, NEtHerlAnDs) aND A syNERGy sUrFACE cOIL wAs USed. THe sEQuenCEs INCLUdEd trAnSverSE, saGITtAL AnD coRoNAL TUrbO spiN ECho T1- aNd fAt-SUpPReSSeD t2-WeIgHtED IMAGES. The PAramETERs OF ThEsE seqUEnces WeRe tR/Te=400 /20 MS FoR t1-WEIgHteD IMaGinG, Tr/tE=3500 /120 ms foR t2-weIghTeD iMAgIng And A FiEld oF VIEw oF 480 Mm×480 Mm FOR SAgiTTAl iMaginG AND 40 mm×40 Mm for traNSveRSE IMAging aND 480 mm×480 mm FOr Coronal iMagIng wITh a maTrIx oF 512×512, 4--6 SIGnALS acqUIsitION ANd A Slice ThIcknESS/GAP = 5/0.5 mm. coNTRAST enHAnced sAGItTaL, CorOnAl And TrANSveRsE t1-weiGHTeD imaging wERE oBtaiNeD AFtEr The IntrAvenoUS InjECtiON OF gadoPeNtEtATe DIMeGluMIne (mAGneVISt, scHerInG, BErlIN, GERMAny) With a doSAGE OF 0.2 MMoL/kg oF boDY WEIgHT.
MuLTI-slicE SPIral ct
---------------------
ct ScaN WAS PErFoRMed BY UsInG A 64-SLicE SPIrAl Ct (seNsAtIOn 64, sIEmeNS MEDical sYsTems, GeRMany). THe rAw DAta OBTaiNED UsInG AN AXial COllIMATiON OF 64×0.6 mm, A PitcH Of 1.0, a Tubular voltaGE Of 120 kV ANd A TubulAr CUrReNt OF 360 mAS, weRe reCOnSTRuCTeD INtO cOntiguoUs 1-mM thIck sliCes witH an InCreMEnt of 0.5 Mm AnD a fIelD Of VIeW OF 376 mM × 376 MM aND A MaTrIX Of 512 × 512 BY UsiNG thE StANDarD soFT TiSSUE and BOne AlGORITHM. ThesE ThIN-SlIcE imaGes WeRE postprocESsED by UsiNG tHe TEChniquEs OF MUltIpLanAR RefOrMATIon (MPR) aND VOLuME ReNDerINg (vr) tO DEMonSTRATE thE lEsiOn deTAIls And PeRfoRm RelATeD measurEMENtS.
PreopeRativE PLANNING
---------------------
alL pReOPerativE RaDIoGRaphS weRe evalUATED bY onE rAdiOLOGisT aNd tWO ConSuLtant oRTHOPAeDiC SurGEoNs, wHo wERe MembErS OF THE SurgIcal teAm peRfORMINg ThE opeRATIoNS. FirsT, thE oSTEOtOmy PLane WaS deTErMinED SeparaTEly oN CT and mRI. on orTHOgONAl corONAl enhAnCed mr imAGES anD Ct Mpr imaGES, tHe BULk MaRGin of The tumoUR iN THE MEDullary cAVity wAS deFinEd aCCOrDIng to tHe DIffeReNT sigNAl cHarAcTeristICS OR ATteNUaTiOn of tHE tumOUr iTseLF anD tHe MArrow OeDEMA aRounD THE tUmOuR. ThEn, THe MAXiMum DiSTanCE fROM the Top Of ThE GREATer TrOChaNTEr to ThIs tUmoUr MArgIn WAS mEASURED on ORTHoGoNaL CoroNAL t1-wEIGtHTED mrI iMaGeS, if ThE tuMour WaS LOcATeD IN THE pRoXImAl PaRT OF FeMUR. The mAxIMUM DistaNce frOM tHE KNEE JoINT lInE tO THe TumoUr maRGin WaS measUreD For tHE TUMouRS Located IN THE dIStaL PART oF fEmur. THe maximUm dIStaNce waS dEfINed as tHe InTRAMEDullarY exTEnSiOn OF THe PrIMary tumoUr AND sUBseqUENtly WAs usEd AS A REfErENCe foR THE cT MeasurEMent. ThE oStEoTomY plAnE On cT mPr ImaGeS Was defINEd 30 mm dIStAL FROM The MaRgIN OF TUmOUR. this distaNCE WAS also used To DEtERminE tHE LEnGtH Of thE eXtrA MeDUlLARy ParT oF tHe PROstHEsIs. AfteR The OSTeotomy PlaNE had beEN DetermiNED, tHe detAiLED ShapE Of THe MEdULLaRy CAVItY Of the pREsERvEd PArT of The fEmuR waS aSSesSed USiNG tHe OrtHoGonAL MpR TecHNIQuE FOr dETERMiNING tHE dIAmetEr And LENGtH Of ThE INTra meDULLAry PaRt Of PrOstHesis. diaMeTErS of ThE MEDULLaRy CAviTY AT THe lEvEl OF the oStEOTomY plaNE aND tHe lEvEL OF The NArrowEsT PLANE WerE MeasuREd TO dEterMINE THe DiamETer oF tHE INTRa MedULlARy stEm OF THE PRoSthEsis. THE LeNgTh Of tHe inTRa- MeDulLarY STEm OF tHE PrOstHEsiS SHoUld be WElL maTcHEd To the LENgTH Of mEDuLlaRY CAVItY oF The PreseRVeD parT OF fEMur, WHIch WoUlD bE opTIMAL If it HAD An EQUal lEngtH tO The ExtrA meduLLAry pART OF PRosthESIs. fInALlY, The centRE aXIS Of THE femOraL ShAFt MeASUrEd ON CT was uSEd aS A rEferENce. oFfsET, The DisTaNcE From the cenTRAL axIS oF the femorAL shAFt ANd tHe RoTAtION CentRE oF ThE FEMOrAl Head, Was ThE iNDex used to deTeRMine ThE NECk lENgtH of tHe PRoSThesIs.
SuRGery
-------
AlL PATIentS UnderwEnt En BlOc reSeCTIOn And CusTOMIzed PrOstheTIC reConsTrUcTIon. An AnteROLatERal INciSioN encircLing THE bIOPsY ScaR WaS UsEd. liMB-sALVagE surgERy cOnSisTed oF IntENtIOnaL maRgiNAL excIsiOn, PrESERving IMpOrTAnT stRuCtUrES SUCH AS MaJoR neURovascuLar BuNdLEs, TEnDonS, aNd lIgaMeNTs. THE osTeOtOMy pLaNe, 30 MM DiStAl FRoM tHE PRImAry TumouR waS CONFIrMED basEd oN MRi FOR all patIeNtS. for patiENTS WIth Lesion in The prOximal Part of FEMUr/hUmerUs, ThE CUsToMiZed pROstHeSIs Was sEcured uSinG MeThYLmEThAcrYlATe cEMenT AFter THE REsectioN. foR PATientS WiTH THE TUmoUr In tHe DistaL paRt oF THe fEMUR, eN BlOC REsEctiOn INClUdInG THE tibiaL plATeAU waS pErfOrmED AND thE CusTOMIZED proSTHESiS wAS SeCUReD uSINg METhylMEthaCrYlatE CEMenT iN bOTh thE tIBiA aNd fEmUR AFTEr the RESeCTIoN. THe EXTeNsOR MEcHAnISm Was recOnstRUCTED By REaTTachment OF thE paTeLLAR TEndON tO tHE SLOT ON the TiBIaL ComPonEnt. AFTEr sUrGErY, fUNcTIoNAl ReHabIlItatIoN anD nEoADjuVant chEMoThEraPY weRe perfoRMeD.
postoPERATive meaSUREmENt
-------------------------
aftEr sURgEry, thE PaTiENTS were follOwed witH A MeAN of 13 MonthS (ranGe, 9 tO 20 mOnTHs). thE PosToPERAtivE ASSeSSment oF proStheSIS WAs PErfORMED on plAiN RadiogrAphY. THe CEntraL AxiS OF THe femOraL Or hUmeRaL sHAFt And OFFset Were DeFINed. THE veRTiCaL diStaNce From the LIne BEtwEen THE TOP oF biLaTeRAL isChiaL tuBErositIes TO the femoRAL | Introduction ============ Better adjuvant therapy, improved metal implants, andinnovative surgical techniques have led surgeons to considerlimb salvagesurgery as an alternative treatmentfor malignant bone tumourother than amputation. Orthopaedic oncology patients have achance foran active, disease-free life after limb salvagesurgery. In the first evidence-based study, Simon *et al.* had reportedthe benefits of limb-salvagingproceduresfor bone tumours.[@b1-rado-46-03-189]Theirmulticentre study reported therates oflocal recurrence, metastasis and survival in227 patients with osteosarcoma in the distal femur and suggested that theKaplan-Meier curves ofthe patients without recurrence were not statisticallydifferent between limb-salvaging surgery and amputation patients during a 5.5-year follow-up. Limb-salvage surgery was considered as safe asan amputation in the management of patients with high-grade osteosarcoma. The goal of limb-salvaging surgery is to preserve thefunction of limbs, preventtumour recurrence, and enable the rapid administration of chemotherapyor radiotherapy.[@b2-rado-46-03-189] It can be reached with meticuloustechnique, detailed operative planning, and the use of endoprosthetic replacements and/or bone grafting. For a successful limb-salvage surgery inhigh-grade malignant tumour, such as sarcomas, a wide margin is necessary to obtain alocal control.[@b3-rado-46-03-189]--[@b5-rado-46-03-189] Since marginal and intralesional margins are related to localrecurrence, the reconstruction with limb-salvaging options shouldbe carefully considered. The clinical outcome of the limb-salvagesurgery with arthroplasty is closely related to the accuracyofthe surgicalprocedure. To improve the final outcome, one must take into account thelength of the osteotomy plane, as well as the alignment of the prosthesis with respect to the mechanical axis in order to keep thebalance of the soft tissues. Furthermore, the parameters measured with the 3D imagine mustbe used during theindividual manufacture of implant in order to reconstruct theskeletalstructureaccurately. Therefore, geometric data (such as length of leg, offset) and morphologic data are required. Magnetic resonance imaging (MRI) was beneficial fortumour detection and consequently staging of musculoskeletal neoplasia. MRI became anideal imaging modality for musculoskeletal neoplasia because of superiorsoft-tissue resolution and multiplanar imaging capabilities and had a significantimpact on the ability to appropriately stage lesions and adequately plan for limb-salvage surgery.[@b6-rado-46-03-189],[@b7-rado-46-03-189] In contrast, multi-slice spiral computed tomography (CT) could provide super three-dimensional morphologicaldelineation of the diseased bone. Theoretically, the complimentary use of these two imaging modalities could give the surgeon a more accurate way to implement preoperative planning than the conventionalapplication of2D images. The purpose of this prospective study was to report our initial experience with limb salvage surgery for orthopaedic oncology patients by using bothMR imaging and multi-slice spiral CT for preoperative planning. Patients and methods ==================== Patients and preparation ------------------------ The study protocol has complied withall relevant national regulations and institutional policies and has been approvedby the local institutional ethics committee. Informed consent was obtained from all patients beforethe procedure. Patients with malignant bone tumours of lower/upperlimb were enrolled in the study. Preoperative work-up consisted of history and clinical examination, routine laboratory tests and an aestheticassessment, plainradiography of the limb,64-slicespiral CT scan of the limb and chest, Technetium-99m bone scan, and, in all of the cases, MRI of the affected limb. Antibiotics were administrated before the surgery. Biopsy was performed forpathological examination. Chemotherapy was commenced 6 weeks beforethe surgery in those caseswhich were diagnosed as osteosarcoma and dedifferentiated chondrosarcoma.Patients wereclassified according to theEnneking staging system.[@b8-rado-46-03-189],[@b9-rado-46-03-189] The patients received a detailed narrative of conventional, surgical and amputationoptions after the limb salvage surgery at their own request. Nineconsecutive patients with lower/upper limb malignant tumour of bone (5women, 4 man, mean age 28.6 years, range: 19--52 years) were treated with limb-salvaging procedures. Lesion size (longitudinal direction), location and histology are summarizedin [Table 1](#t1-rado-46-03-189){ref-type="table"}. MR imaging ---------- MR images were performedat a 1.5-T superconductive unit (Gyroscan Intera, Philips Medical Systems, Netherlands) and a synergy surface coil was used.Thesequences included transverse, sagittal and coronal turbospin echo T1- and fat-suppressed T2-weightedimages. The parameters of these sequenceswere TR/TE=400/20 ms for T1-weighted imaging, TR/TE=3500/120 ms for T2-weighted imaging and a field of view of480mm×480 mm for sagittal imaging and 40mm×40 mm for transverse imaging and 480 mm×480 mm for coronal imaging with a matrix of 512×512, 4--6signals acquisition and a slice thickness/gap = 5/0.5 mm. Contrast enhanced sagittal, coronal and transverse T1-weighted imaging were obtained afterthe intravenous injection of gadopentetate dimeglumine (Magnevist, Schering, Berlin, Germany) with a dosage of 0.2 mmol/kg of body weight. Multi-slicespiral CT--------------------- CT scan was performedby using a 64-slice spiral CT (Sensation 64, Siemens Medical Systems, Germany). The raw data obtained usingan axial collimation of 64×0.6 mm, a pitchof 1.0, atubularvoltage of 120KV and a tubular current of 360 mAs, were reconstructed intocontiguous 1-mm thick slices with an increment of 0.5 mm and a field of view of 376 mm × 376 mm and a matrix of 512 ×512 by using the standard soft tissue and bone algorithm. These thin-slice images were postprocessed by using the techniques of multiplanar reformation (MPR) and volume rendering (VR) todemonstrate the lesion details and performrelated measurements. Preoperative planning--------------------- All preoperative radiographs were evaluatedby one radiologist andtwo consultant orthopaedic surgeons, who weremembersof thesurgical team performing the operations. First, the osteotomy plane was determined separately on CT and MRI. On orthogonal coronal enhanced MRimages and CT MPR images, thebulk margin of thetumour in the medullary cavity was defined accordingto the different signal characteristicsor attenuationof the tumour itself and the marrow oedema aroundthe tumour. Then, the maximum distance from the topof the greater trochanter to this tumour marginwas measured on orthogonal coronal T1-weigthtedMRI images, ifthe tumour waslocated in the proximal part of femur. The maximum distance from theknee joint line tothe tumour margin was measured for the tumours located in the distal part of femur. The maximum distance was definedas the intramedullary extension of the primary tumour and subsequently was used asa reference for the CT measurement. The osteotomy plane on CT MPR images was defined 30 mm distal from themargin of tumour.This distance was also used todetermine the length of the extra medullary part of the prosthesis. After the osteotomy plane had been determined, the detailed shape of the medullary cavity of the preserved part of thefemur was assessedusingthe orthogonal MPR technique for determining the diameter and lengthof the intra medullary partof prosthesis. Diameters of themedullarycavity at the level of the osteotomy planeandthe level of the narrowest plane were measured to determine the diameter of the intra medullary stem of the prosthesis. The length ofthe intra- medullary stem of the prosthesis should be well matched to the length of medullary cavityof the preserved part of femur, which would be optimalif it had an equal length to the extra medullary part of prosthesis. Finally, the centre axis of the femoralshaftmeasured on CT was usedas a reference. Offset, the distance from the central axis of the femoralshaftandthe rotation centre of the femoral head, was the index used to determine the neck length of the prosthesis. Surgery ------- All patients underwent en bloc resection andcustomized prosthetic reconstruction. An anterolateral incision encircling the biopsy scar was used.Limb-salvage surgery consisted of intentional marginal excision, preserving important structures such as major neurovascularbundles, tendons, and ligaments. The osteotomy plane, 30 mm distal from the primary tumour was confirmed based on MRI for all patients.For patients with lesion in the proximal partoffemur/humerus, the customized prosthesis was secured using methylmethacrylate cement after the resection. For patients with the tumour in the distalpart of the femur,enbloc resectionincluding the tibialplateau was performedand the customized prosthesis was secured using methylmethacrylate cement in both the tibia and femur after the resection. The extensor mechanism was reconstructed by reattachment of thepatellar tendon to the slot on the tibial component.After surgery, functional rehabilitation andneoadjuvantchemotherapy were performed. Postoperative measurement ------------------------- After surgery, thepatients were followed with a mean of 13 months (range, 9 to 20 months). Thepostoperative assessment of prosthesis was performed on plain radiography. The centralaxis ofthefemoral or humeral shaft andoffset were defined. Thevertical distance from the line between the top of bilateral ischial tuberosities to thefemoral | Introduction ============ Better adjuvant therapy, improved metal _implants,_ and innovative surgical techniques have _led_ surgeons to consider limb _salvage_ surgery as an _alternative_ treatment _for_ malignant bone tumour other than amputation. Orthopaedic oncology patients _have_ _a_ chance _for_ an _active,_ disease-free life _after_ limb _salvage_ _surgery._ In the _first_ evidence-based _study,_ _Simon_ _*et_ al.* had reported _the_ benefits _of_ limb-salvaging _procedures_ for bone tumours.[@b1-rado-46-03-189] Their multicentre study reported the rates _of_ _local_ recurrence, metastasis and survival in 227 patients with osteosarcoma in _the_ distal femur and suggested _that_ the Kaplan-Meier curves of the _patients_ without recurrence were not _statistically_ different between _limb-salvaging_ surgery and amputation patients during _a_ 5.5-year follow-up. Limb-salvage surgery was _considered_ as _safe_ _as_ an amputation in the management of patients with high-grade osteosarcoma. The _goal_ of _limb-salvaging_ surgery is to preserve the function of _limbs,_ prevent _tumour_ recurrence, and enable the rapid administration of chemotherapy _or_ radiotherapy.[@b2-rado-46-03-189] It can _be_ reached with meticulous technique, detailed _operative_ planning, _and_ the use of endoprosthetic replacements and/or bone grafting. For a successful limb-salvage surgery in high-grade malignant tumour, such as sarcomas, a wide margin is necessary to _obtain_ a local control.[@b3-rado-46-03-189]--[@b5-rado-46-03-189] _Since_ marginal and intralesional margins are _related_ to _local_ recurrence, the reconstruction _with_ limb-salvaging options should be carefully considered. _The_ _clinical_ outcome of the _limb-salvage_ _surgery_ with arthroplasty is closely related to the _accuracy_ of the surgical procedure. _To_ improve the _final_ outcome, one must take _into_ account _the_ length of the osteotomy plane, as well as the alignment of _the_ prosthesis with respect to _the_ mechanical axis _in_ _order_ to _keep_ the _balance_ of the soft tissues. _Furthermore,_ the parameters measured _with_ the _3D_ _imagine_ _must_ be _used_ _during_ the individual manufacture of implant in order to reconstruct the skeletal _structure_ accurately. Therefore, _geometric_ data (such as length of leg, offset) and morphologic data are required. Magnetic _resonance_ imaging (MRI) was beneficial for tumour detection and _consequently_ staging of musculoskeletal neoplasia. _MRI_ became _an_ ideal imaging modality _for_ musculoskeletal neoplasia because of superior soft-tissue resolution and multiplanar imaging capabilities and had a significant impact on the _ability_ _to_ _appropriately_ _stage_ lesions and adequately plan for _limb-salvage_ surgery.[@b6-rado-46-03-189],[@b7-rado-46-03-189] In contrast, multi-slice _spiral_ computed tomography (CT) _could_ provide super three-dimensional morphological delineation of the diseased bone. Theoretically, the complimentary _use_ of these two imaging modalities could give the surgeon a _more_ _accurate_ way _to_ implement preoperative planning than the _conventional_ application of 2D images. The purpose of this prospective _study_ was to report our _initial_ experience with limb salvage surgery for _orthopaedic_ _oncology_ patients _by_ using both MR imaging and multi-slice spiral CT for preoperative planning. _Patients_ and methods _====================_ Patients _and_ preparation ------------------------ The study protocol has _complied_ with all relevant national _regulations_ and institutional policies and _has_ been approved by the local institutional ethics committee. Informed consent _was_ obtained from all patients before _the_ procedure. Patients with _malignant_ bone tumours of lower/upper _limb_ were enrolled in _the_ study. _Preoperative_ work-up consisted of history and _clinical_ examination, _routine_ laboratory tests _and_ an _aesthetic_ _assessment,_ plain radiography of the limb, 64-slice spiral CT scan of the _limb_ and chest, Technetium-99m bone scan, and, _in_ all of _the_ cases, _MRI_ of the affected limb. Antibiotics were administrated before the surgery. Biopsy was performed for _pathological_ examination. Chemotherapy was commenced _6_ weeks _before_ the surgery in those cases which _were_ _diagnosed_ as osteosarcoma and dedifferentiated chondrosarcoma. Patients _were_ classified according to the Enneking staging system.[@b8-rado-46-03-189],[@b9-rado-46-03-189] The patients received _a_ detailed narrative of conventional, surgical and amputation options after the limb salvage surgery at their _own_ request. Nine _consecutive_ _patients_ with _lower/upper_ _limb_ malignant tumour of _bone_ (5 women, _4_ man, mean age 28.6 _years,_ range: _19--52_ years) were treated with limb-salvaging _procedures._ Lesion size (longitudinal direction), location _and_ histology are summarized in [Table _1](#t1-rado-46-03-189){ref-type="table"}._ MR imaging ---------- MR _images_ _were_ performed at a 1.5-T superconductive unit _(Gyroscan_ _Intera,_ Philips Medical Systems, Netherlands) and a synergy surface coil was used. The sequences included transverse, sagittal and coronal turbo spin echo T1- _and_ fat-suppressed T2-weighted images. The parameters of these sequences were _TR/TE=400_ /20 ms for T1-weighted imaging, _TR/TE=3500_ /120 ms _for_ T2-weighted imaging and a field of view of 480 mm×480 mm for _sagittal_ imaging _and_ _40_ mm×40 mm for transverse imaging and 480 mm×480 mm for _coronal_ _imaging_ with a _matrix_ _of_ 512×512, 4--6 signals acquisition and a slice thickness/gap = 5/0.5 mm. Contrast enhanced sagittal, _coronal_ and transverse T1-weighted imaging _were_ obtained _after_ the _intravenous_ _injection_ of gadopentetate dimeglumine (Magnevist, _Schering,_ Berlin, Germany) with a dosage of 0.2 _mmol/kg_ of body _weight._ Multi-slice spiral CT _---------------------_ CT scan was _performed_ _by_ _using_ a 64-slice spiral CT (Sensation _64,_ Siemens Medical Systems, _Germany)._ _The_ raw _data_ obtained using an axial collimation of 64×0.6 mm, a pitch _of_ 1.0, _a_ tubular voltage of 120 KV _and_ a tubular _current_ of 360 _mAs,_ _were_ reconstructed _into_ contiguous 1-mm thick slices with an increment of 0.5 mm and _a_ field of view _of_ 376 mm × 376 mm and a matrix of 512 × _512_ _by_ using _the_ standard _soft_ tissue and bone algorithm. These thin-slice images were _postprocessed_ by using the _techniques_ of multiplanar reformation _(MPR)_ _and_ volume rendering (VR) to demonstrate the _lesion_ details _and_ perform related measurements. Preoperative _planning_ --------------------- _All_ preoperative radiographs were evaluated by one radiologist and two _consultant_ orthopaedic surgeons, who were members of the surgical team performing the _operations._ First, the osteotomy plane _was_ determined separately on CT and MRI. On orthogonal coronal enhanced MR images and CT MPR images, the bulk margin _of_ the _tumour_ _in_ _the_ _medullary_ _cavity_ _was_ defined according to _the_ different signal characteristics or attenuation of the tumour itself and the marrow oedema around the tumour. Then, _the_ _maximum_ distance from the _top_ of _the_ greater trochanter to _this_ tumour margin was measured _on_ orthogonal _coronal_ _T1-weigthted_ MRI images, if the tumour was located in the _proximal_ part of femur. The maximum _distance_ from _the_ knee joint _line_ to the tumour _margin_ was measured for the tumours located in _the_ distal _part_ _of_ femur. The maximum distance was defined as _the_ _intramedullary_ extension of the primary tumour and _subsequently_ _was_ used as a reference for _the_ CT measurement. The osteotomy plane on CT MPR images _was_ defined 30 mm distal from _the_ margin of tumour. _This_ distance _was_ also used to determine the _length_ of the extra medullary part of the _prosthesis._ After the osteotomy plane had been determined, the _detailed_ shape of the medullary _cavity_ of the _preserved_ part of the _femur_ was assessed using the _orthogonal_ _MPR_ technique for _determining_ the _diameter_ and length of _the_ intra medullary part _of_ prosthesis. Diameters of the medullary cavity at the _level_ _of_ the _osteotomy_ plane and the level of the narrowest plane were _measured_ to determine the diameter of the intra medullary stem of the prosthesis. _The_ length of _the_ _intra-_ medullary stem of the prosthesis should _be_ well _matched_ to the length of medullary cavity _of_ _the_ preserved part of femur, _which_ would be optimal if it _had_ an equal length to the extra _medullary_ _part_ _of_ prosthesis. _Finally,_ the centre _axis_ _of_ the femoral shaft measured on CT was used as a reference. Offset, the distance from the _central_ _axis_ of the femoral shaft and _the_ rotation centre _of_ _the_ femoral head, was _the_ index used to determine _the_ neck length _of_ _the_ prosthesis. Surgery ------- All patients underwent en _bloc_ resection and customized prosthetic reconstruction. An anterolateral incision encircling _the_ biopsy scar was _used._ Limb-salvage surgery consisted of _intentional_ marginal excision, preserving important structures such as major neurovascular bundles, tendons, _and_ ligaments. The osteotomy plane, 30 mm distal from the primary tumour was confirmed _based_ on MRI for all patients. For _patients_ with lesion in the proximal part of femur/humerus, the customized prosthesis was _secured_ using methylmethacrylate _cement_ after the _resection._ _For_ patients with the tumour in the distal part of the _femur,_ en bloc resection _including_ _the_ tibial plateau was performed _and_ the customized prosthesis was secured using methylmethacrylate cement in both the _tibia_ and femur after the resection. _The_ _extensor_ _mechanism_ _was_ reconstructed by reattachment of the patellar _tendon_ _to_ the slot on _the_ tibial component. After surgery, functional rehabilitation _and_ neoadjuvant chemotherapy were performed. Postoperative measurement ------------------------- _After_ surgery, the _patients_ _were_ followed with a mean of 13 months (range, 9 to _20_ _months)._ _The_ postoperative _assessment_ of prosthesis was performed on plain radiography. The central _axis_ of the femoral or humeral shaft and offset were defined. The vertical distance from the line between the top _of_ _bilateral_ ischial tuberosities to the femoral |
Building upon the pioneering work of Vicsek *et al.*[@b1], physicists, mathematicians and biologists have contemplated the self-organization of living-organism groups into flocks as an emergent process stemming from simple interaction rules at the individual level[@b2][@b3][@b4]. This idea has been supported by quantitative trajectory analysis in animal groups[@b5][@b6][@b7], together with a vast number of numerical and theoretical models[@b3][@b4], and more recently by the observations of flocking behaviour in ensembles of non-living motile particles such as shaken grains, active colloids, and mixtures of biofilaments and molecular motors[@b8][@b9][@b10][@b11][@b12]. From a physicist\'s perspective, these various systems are considered as different instances of polar active matter, which encompasses any ensemble of motile bodies endowed with local velocity--alignment interactions. The current paradigm for flocking physics is the following. Active particles are persistent random walkers, which when dilute form a homogeneous isotropic gas. Upon increasing density, collective motion emerges in the form of spatially localized swarms that may cruise in a sea of randomly moving particles; further increasing density, a homogeneous polar liquid forms and spontaneously flows along a well-defined direction[@b1][@b13][@b14]. This picture is the outcome of experiments, simulations and theories mostly performed in unbounded or periodic domains.
Beyond this picture, significant attention has been devoted over the last five years to confined active matter[@b3][@b12][@b15][@b16][@b17][@b18][@b19][@b20][@b21][@b22][@b23][@b24][@b25][@b26]. Confined active particles have consistently, yet not systematically, been reported to self-organize into vortex-like structures. However, unlike for our understanding of flocking, we are still lacking a unified picture to account for the emergence and structure of such vortex patterns. This situation is mostly due to the extreme diversity in the nature and symmetries of the interactions between the active particles that have been hitherto considered. Do active vortices exist only in finite-size systems as in the case of bacterial suspensions[@b17], which lose this beautiful order and display intermittent turbulent dynamics[@b27] when unconfined? What are the necessary interactions required to observe and/or engineer bona fide stationary swirling states of active matter?
In this paper, we answer these questions by considering the impact of geometrical boundaries on the collective behaviour of motile particles endowed with velocity--alignment interactions. Combining quantitative experiments on motile colloids, numerical simulations and analytical theory, we elucidate the phase behaviour of *polar* active matter restrained by geometrical boundaries. We use colloidal rollers, which, unlike most of the available biological self-propelled bodies, interact via well-established dynamical interactions[@b11]. We first exploit this unique model system to show that above a critical concentration populations of motile colloids undergo a non-equilibrium phase transition from an isotropic gaseous state to a novel ordered state where the entire population self-organizes into a single heterogeneous steadily rotating vortex. This self-organization is *not* the consequence of the finite system size. Rather, this emergent vortex is a genuine state of polar active matter lying on the verge of a macroscopic phase separation. This novel state is the only ordered phase found when unidirectional directed motion is hindered by convex isotropic boundaries. We then demonstrate theoretically that a competition between alignment, repulsive interactions and confinement is necessary to yield large-scale vortical motion in ensembles of motile particles interacting via alignment interactions, thereby extending the relevance of our findings to a broad class of active materials.
Results
=======
Experiments
-----------
The experimental setup is fully described in the *Methods* section and in [Fig. 1a,b](#f1){ref-type="fig"}. Briefly, we use colloidal rollers powered by the Quincke electrorotation mechanism as thoroughly explained in ref. [@b11]. An electric field **E**~**0**~ is applied to insulating colloidal beads immersed in a conducting fluid. Above a critical field amplitude *E*~Q~, the symmetry of the electric charge distribution at the bead surface is spontaneously broken. As a result, a net electric torque acts on the beads causing them to rotate at a constant rate around a random axis transverse to the electric field[@b28][@b29][@b30]. When the colloids sediment, or are electrophoretically driven, onto one of the two electrodes, rotation is converted into a net rolling motion along a random direction. Here, we use poly(methyl methacrylate) (PMMA) spheres of radius *a*=2.4 μm immersed in a hexadecane solution.
As sketched in [Fig. 1a](#f1){ref-type="fig"}, the colloids are handled and observed in a microfluidic device made of double-sided scotch tape and of two glass slides coated with an indium-tin-oxide layer. The ITO layers are used to apply a uniform DC field in the *z*-direction, with *E*~0~=1.6 V μm^−1^ (*E*~0~=1.1*E*~Q~). Importantly, the electric current is nonzero solely in a disc-shaped chamber at the centre of the main channel. As exemplified by the trajectories shown in [Fig. 1b](#f1){ref-type="fig"} and in [Supplementary Movie 1](#S1){ref-type="supplementary-material"}, Quincke rotation is hence restrained to this circular region in which the rollers are trapped. We henceforth characterize the collective dynamics of the roller population for increasing values of the colloid packing fraction *φ*~0~.
Individual self-propulsion
--------------------------
For area fractions smaller than , the ensemble of rollers uniformly explores the circular confinement as illustrated by the flat profile of the local packing fraction averaged along the azimuthal direction *φ*(*r*) in [Fig. 2a](#f2){ref-type="fig"}. The rollers undergo uncorrelated persistent random walks as demonstrated in [Fig. 2b,c](#f2){ref-type="fig"}. The probability distribution of the roller velocities is isotropic and sharply peaked on the typical speed *v*~0~=493±17 μm s^−1^. In addition, the velocity autocorrelation function decays exponentially at short time as expected from a simple model of self-propelled particles having a constant speed *v*~0~ and undergoing rotational diffusion with a rotational diffusivity *D*^−1^=0.31±0.02 s that hardly depends on the area fraction (see [Supplementary Note 1](#S1){ref-type="supplementary-material"}). These quantities correspond to a persistence length of that is about a decade smaller than the confinement radius *R*~c~ used in our experiments: 0.9 mm\<*R*~c~\<1.8 mm.
At long time, because of the collisions on the disc boundary, the velocity autocorrelation function sharply drops to 0 as seen in [Fig. 2c](#f2){ref-type="fig"}. Unlike swimming cells[@b26][@b31], self-propelled grains[@b8][@b22][@b23] or autophoretic colloids[@b32], dilute ensembles of rollers do not accumulate at the boundary. Instead, they bounce off the walls of this virtual box as shown in a close-up of a typical roller trajectory in [Fig. 2d](#f2){ref-type="fig"}, and in the [Supplementary Movie 1](#S1){ref-type="supplementary-material"}. As a result, the outer region of the circular chamber is depleted, and the local packing fraction vanishes as *r* goes to *R*~c~, [Fig. 2a](#f2){ref-type="fig"}. The repulsion from the edges of the circular hole in the microchannel stems from another electrohydrodynamic phenomenon[@b33]. When an electric field is applied, a toroidal flow sketched in [Fig. 1a](#f1){ref-type="fig"} is osmotically induced by the transport of the electric charges at the surface of the insulating adhesive films. Consequently, a net inward flow sets in at the vicinity of the bottom electrode. As the colloidal rollers are prone to reorient in the direction of the local fluid velocity[@b11], this vortical flow repels the rollers at a distance typically set by the channel height *H* while leaving unchanged the colloid trajectories in the centre of the disc. This electrokinetic flow will be thoroughly characterized elsewhere.
Collective motion in confinement
--------------------------------
As the area fraction is increased above , collective motion emerges spontaneously at the entire population level. When the electric field is applied, large groups of rollers akin to the band-shaped swarms reported in[@b11] form and collide. However, unlike what was observed in periodic geometries, the colloidal swarms are merely transient and ultimately self-organize into a single vortex pattern spanning the entire confining disc as shown in [Fig. 3a](#f3){ref-type="fig"} and [Supplementary Movie 2](#S1){ref-type="supplementary-material"}. Once formed, the vortex is very robust, rotates steadily and retains an axisymmetric shape. To go beyond this qualitative picture, we measured the local colloid velocity field **v**(**r**, *t*) and use it to define the polarization field **Π**(**r**, *t*)≡**v**/*v*~0~, which quantifies local orientational ordering. The spatial average of **Π** vanishes when a coherent vortex forms, therefore we use its projection along the azimuthal direction as a macroscopic order parameter to probe the transition from an isotropic gas to a polar-v | building upon the pioneering work of vicsek * et al. * [ @ c2 ], physicists, mathematicians and biologists have contemplated the self - organization of living - organism groups into flocks as an emergent process stemming from simple interaction rules at the individual level [ @ b2 ] [ @ b3 ] [ @ b4 ]. this idea has been supported by spatial trajectory analysis in animal groups [ @ b5 ] [ @ b6 ] [ @ b7 ], together with a vast number of numerical and theoretical models [ @ b3 ] [ @ b4 ], and more recently by the observations of flocking behaviour in ensembles of non - living motile particles such as shaken grains, active colloids, and mixtures of biofilaments and molecular motors [ @ b8 ] [ @ b9 ] [ @ b10 ] [ @ b11 ] [ @ b12 ]. from a physicist \ ' s perspective, these various systems are considered as different instances of polar active matter, which encompasses any ensemble of motile bodies endowed with local velocity - - particle interactions. the current paradigm for flocking physics is the following. active particles are persistent random walkers, which when dilute form a homogeneous isotropic gas. upon increasing density, collective motion emerges from the form of spatially localized swarms that may cruise in a sea of randomly moving particles ; further increasing density, a homogeneous polar liquid forms and spontaneously flows along a well - defined direction [ @ b1 ] [ @ b13 ] [ @ b14 ]. this picture is the outcome of experiments, simulations and theories being performed in unbounded or periodic domains. beyond this picture, significant attention has been devoted over the ensuing five years to confined active matter [ @ b3 ] [ @ b12 ] [ @ b15 ] [ @ b16 ] [ @ b17 ] [ @ b18 ] [ @ b19 ] [ @ b20 ] [ @ b21 ] [ @ c2 ] [ @ b23 ] [ @ b24 ] [ @ b25 ] [ @ b26 ]. confined active particles have consistently, yet not systematically, been reported to re - organize into funnel - like structures. however, unlike for our understanding of flocking, we are still lacking a unified picture to account for the emergence and structure of such vortex patterns. this situation is mostly due to the vast diversity in the nature and symmetries of the interactions between the active particles that have been hither ##to considered. do active vortices exist only in finite - size systems as in the case of bacterial suspensions [ @ b17 ], which lose this beautiful order and display intermittent turbulent dynamics [ @ b27 ] when unconfined? what are the necessary interactions required to observe and / or engineer bona fide stationary swirling states of active matter? in this paper, we answer these questions by considering the impact of geometrical boundaries on the collective behaviour of motile particles endowed with velocity - - alignment interactions. combining quantitative experiments on motile colloids, numerical simulations and analytical theory, we elucidate the phase behaviour of * polar * active matter restrained by geometrical boundaries. we use colloidal rollers, which, unlike most of the available biological self - propelled bodies, interact via well - established dynamical interactions [ @ b11 ]. we first exploit this unique model system to show that above a critical concentration populations of motile colloids undergo a non - equilibrium phase transition from an isotropic gaseous state to a novel ordered state where the entire population self - organizes into a single heterogeneous steadily rotating vortex. this self - organization is * not * the consequence of the finite system size. rather, this emergent vortex is a genuine state of polar active matter lying on the verge of a macroscopic phase separation. this novel state is the only ordered phase found when unidirectional directed motion is hindered by convex isotropic boundaries. we then demonstrate theoretically that a competition between alignment, repulsive interactions and confinement is necessary to yield large - scale vortical motion in ensembles of motile particles interacting via alignment interactions, thereby extending the relevance of our findings to a broad class of active materials. results = = = = = = = experiments - - - - - - - - - - - the experimental setup is fully described in the * methods * section and in [ fig. 1a, b ] ( # f1 ) { ref - type = " fig " }. briefly, we use colloidal rollers powered by the quincke electrorotation mechanism as thoroughly explained in ref. [ @ b11 ]. an electric field * * e * * ~ * * 0 * * ~ is applied to insulating colloidal beads immersed in a conducting fluid. above a critical field amplitude * e * ~ q ~, the symmetry of the electric charge distribution at the bead surface is spontaneously broken. as a result, a net electric torque acts on the beads causing them to rotate at a constant rate around a random axis transverse to the electric field [ @ b28 ] [ @ b29 ] [ @ b30 ]. when the colloids sediment, or are electrophoretically driven, onto one of the two electrodes, rotation is converted into a net rolling motion along a random direction. here, we use poly ( methyl methacrylate ) ( pmma ) spheres of radius * a * = 2. 4 μm immersed in a hexadecane solution. as sketched in [ fig. 1a ] ( # f1 ) { ref - type = " fig " }, the colloids are handled and observed in a microfluidic device made of double - sided scotch tape and of two glass slides coated with an indium - tin - oxide layer. the ito layers are used to apply a uniform dc field in the * z * - direction, with * e * ~ 0 ~ = 1. 6 v μm ^ −1 ^ ( * e * ~ 0 ~ = 1. 1 * e * ~ q ~ ). importantly, the electric current is nonzero solely in a disc - shaped chamber at the centre of the main channel. as exemplified by the trajectories shown in [ fig. 1b ] ( # f1 ) { ref - type = " fig " } and in [ supplementary movie 1 ] ( # s1 ) { ref - type = " supplementary - material " }, quincke rotation is hence restrained to this circular region in which the rollers are trapped. we henceforth characterize the collective dynamics of the roller population for increasing values of the colloid packing fraction * φ * ~ 0 ~. individual self - propulsion - - - - - - - - - - - - - - - - - - - - - - - - - - for area fractions smaller than, the ensemble of rollers uniformly explores the circular confinement as illustrated by the flat profile of the local packing fraction averaged along the azimuthal direction * φ * ( * r * ) in [ fig. 2a ] ( # f2 ) { ref - type = " fig " }. the rollers undergo uncorrelated persistent random walks as demonstrated in [ fig. 2b, c ] ( # f2 ) { ref - type = " fig " }. the probability distribution of the roller velocities is isotropic and sharply peaked on the typical speed * v * ~ 0 ~ = 493±17 μm s ^ −1 ^. in addition, the velocity autocorrelation function decays exponentially at short time as expected from a simple model of self - propelled particles having a constant speed * v * ~ 0 ~ and undergoing rotational diffusion with a rotational diffusivity * d * ^ −1 ^ = 0. 31±0. 02 s that hardly depends on the area fraction ( see [ supplementary note 1 ] ( # s1 ) { ref - type = " supplementary - material " } ). these quantities correspond to a persistence length of that is about a decade smaller than the confinement radius * r * ~ c ~ used in our experiments : 0. 9 mm \ < * r * ~ c ~ \ < 1. 8 mm. at long time, because of the collisions on the disc boundary, the velocity autocorrelation function sharply drops to 0 as seen in [ fig. 2c ] ( # f2 ) { ref - type = " fig " }. unlike swimming cells [ @ b26 ] [ @ b31 ], self - propelled grains [ @ b8 ] [ @ b22 ] [ @ b23 ] or autophoretic colloids [ @ b32 ], dilute ensembles of rollers do not accumulate at the boundary. instead, they bounce off the walls of this virtual box as shown in a close - up of a typical roller trajectory in [ fig. 2d ] ( # f2 ) { ref - type = " fig " }, and in the [ supplementary movie 1 ] ( # s1 ) { ref - type = " supplementary - material " }. as a result, the outer region of the circular chamber is depleted, and the local packing fraction vanishes as * r * goes to * r * ~ c ~, [ fig. 2a ] ( # f2 ) { ref - type = " fig " }. the repulsion from the edges of the circular hole in the microchannel stems from another electrohydrodynamic phenomenon [ @ b33 ]. when an electric field is applied, a toroidal flow sketched in [ fig. 1a ] ( # f1 ) { ref - type = " fig " } is osmotically induced by the transport of the electric charges at the surface of the insulating adhesive films. consequently, a net inward flow sets in at the vicinity of the bottom electrode. as the colloidal rollers are prone to reorient in the direction of the local fluid velocity [ @ b11 ], this vortical flow repels the rollers at a distance typically set by the channel height * h * while leaving unchanged the colloid trajectories in the centre of the disc. this electrokinetic flow will be thoroughly characterized elsewhere. collective motion in confinement - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - as the area fraction is increased above, collective motion emerges spontaneously at the entire population level. when the electric field is applied, large groups of rollers akin to the band - shaped swarms reported in [ @ b11 ] form and collide. however, unlike what was observed in periodic geometries, the colloidal swarms are merely transient and ultimately self - organize into a single vortex pattern spanning the entire confining disc as shown in [ fig. 3a ] ( # f3 ) { ref - type = " fig " } and [ supplementary movie 2 ] ( # s1 ) { ref - type = " supplementary - material " }. once formed, the vortex is very robust, rotates steadily and retains an axisymmetric shape. to go beyond this qualitative picture, we measured the local colloid velocity field * * v * * ( * * r * *, * t * ) and use it to define the polarization field * * π * * ( * * r * *, * t * ) ≡ * * v * * / * v * ~ 0 ~, which quantifies local orientational ordering. the spatial average of * * π * * vanishes when a coherent vortex forms, therefore we use its projection along the azimuthal direction as a macroscopic order parameter to probe the transition from an isotropic gas to a polar - v | Building upon the pioneering work of Vicsek * et al. * [@ b1 ], physicists, mathematicians and biologists have contemplated the CSlf - 9rgaBization of living - organism groups into flocks as an emergent process stemming from simple interaction rules at the individual level [@ b2] [@ b3] [@ b4 ]. This idea has been supported by quantitative trajectory analysis in animal groups [@ b5] [@ b6] [@ b7 ], together with a vast number of numerical and theoretical node.s [@ b3] [@ b4 ], and more recently by the observations of flocking behaviour in ensembles of non - living motile particles such as shaken grains, active colloids, and mixtures of biofilaments and molecular motors [@ b8] [@ b9] [@ b10] [@ b11] [@ b12 ]. From a physicist \ ' s perspective, these various systems are considered as different instances of polar active matter, which encompasses any ensemble of motile bodies endowed with local velocity - - alignment interactions. The current paradigm for f;ockiMg physics is the following. Active particles are persistent random walkers, which when dilute form a homogeneous isotropic gas. Upon increasing density, collective motion emerges in the form of spatially localized swarms that may cruise in a sea of randomly moving particles; further increasing density, a homogeneous polar liquid forms and spontaneously flows along a well - defined direction [@ b1] [@ b13] [@ b14 ]. This picture is the outcome of experiments, simulations and theories mostly performed in unbounded or periodic domains. Beyond this picture, significant attention has been devoted over the last five years to confined active matter [@ b3] [@ b12] [@ b15] [@ b16] [@ b17] [@ b18] [@ b19] [@ b20] [@ b21] [@ b22] [@ b23] [@ b24] [@ b25] [@ b26 ]. Confined active particles have consistently, yet not systematically, been reported to self - organize into vortex - like structures. However, unlike for our understanding of flocking, we are still lacking a unified picture to account for the emergence and structure of such vortex patterns. This situation is mostly due to the extreme diversity in the nature and symmetries of the interactions between the active particles that have been hitherto considered. Do active vortices exist only in finite - size systems as in the case of bacterial suspensions [@ b17 ], which lose this beautiful order and display intermittent turbulent dynamics [@ b27] when unconfined? What are the necessary interactions required to observe and / or engineer bona fide stationary swirling states of active matter? In this paper, we answer these questions by considering the impact of geometrical boundaries on the collective behaviour of motile particles endowed with velocity - - alignment interactions. Combining quantitative experiments on motile colloids, numerical simulations and analytical theory, we elucidate the phase behaviour of * polar * active matter restrained by geometrical boundaries. We use colloidal rollers, which, unlike most of the available biological self - propelled bodies, interact via well - established dynamical interactions [@ b11 ]. We first exploit this unique model system to show that above a critical concentration populations of motile colloids undergo a non - equilibrium phase transition from an isotropic gaseous state to a novel ordered state where the entire population self - organizes into a single heterogeneous steadily rotating vortex. This self - organization is * not * the consequence of the finite system size. Rather, this emergent vortex is a genuine state of polar active matter lying on the verge of a macroscopic phase separation. This novel state is the only ordered phase found when unidirectional directed mitioJ is hindered by convex isotropic boundaries. We then demonstrate theoretically that a competition between alignment, repulsive interactions and confinement is necessary to yield large - scale vortical motion in ensembles of motile particles interacting via alignment interactions, thereby extending the relevance of our findings to a broad class of active materials. Results = = = = = = = Experiments - - - - - - - - - - - The experimental setup is fully described in the * Methods * section and in [Fig. 1a, b] (# f1) {ref - type = " fig " }. Briefly, we use colloidal rollers powered by the Quincke electrorotation mechanism as thoroughly explained in ref. [@ b11 ]. An electric field * * E * * ~ * * 0 * * ~ is applied to insulating colloidal beads immersed in a conducting fluid. Above a critical field amplitude * E * ~ Q ~, the symmetry of the electric charge distribution at the bead surface is spontaneously broken. As a result, a net electric torque acts on the beads causing them to rotate at a constant rate around a random axis transverse to the electric field [@ b28] [@ b29] [@ b30 ]. qhwn the colloids sediment, or are electrophoretically driven, onto one of the two electrodes, rotation is converted into a net rolling motion along a random direction. Here, we use poly (methyl methacrylate) (PMMA) spheres of radius * a * = 2. 4 μm immersed in a hexadecane solution. As sketched in [Fig. 1a] (# f1) {ref - type = " fig " }, the colloids are handled and observed in a microfluidic device made of double - sided scotch tape and of two glass slides coated with an indium - tin - oxide layer. The ITO layers are used to apply a uniform DC field in the * z * - direction, with * E * ~ 0 ~ = 1. 6 V μm ^ − 1 ^ (* E * ~ 0 ~ = 1. 1 * E * ~ Q ~ ). Importantly, the electric current is nonzero solely in a disc - shaped chamber at the centre of the main channel. As exemplified by the trajectories shown in [Fig. 1b] (# f1) {ref - type = " fig "} and in [Supplementary Movie 1] (# S1) {ref - type = " supplementary - material " }, Quincke rotation is hence restrained to this circular region in which the rollers are trapped. We henceforth characterize the collective dynamics of the roller population for increasing values of the colloid packing fraction * φ * ~ 0 ~. Individual self - propulsion - - - - - - - - - - - - - - - - - - - - - - - - - - For area fractions smaller than, the ensemble of r(lPers uniformly explores the c9rxular confinement as illustrated by the flat profile of the local packing fraction averaged along the azimuthal direction * φ * (* r *) in [Fig. 2a] (# f2) {ref - type = " fig " }. The rollers undergo uncorrelated persistent random walks as demonstrated in [Fig. 2b, c] (# f2) {ref - type = " fig " }. The probability distribution of the roller velocities is isotropic and sharply peaked on the typical speed * v * ~ 0 ~ = 493 ± 17 μm s ^ − 1 ^. In addition, the velocity autocorrelation function decays exponentially at short time as expected from a simple model of self - propelled particles having a constant speed * v * ~ 0 ~ and undergoing rotational diffusion with a rotational diffusivity * D * ^ − 1 ^ = 0. 31 ± 0. 02 s that hardly depends on the area fraction (see [Supplementary Note 1] (# S1) {ref - type = " supplementary - material "} ). These quantities correspond to a persistence length of that is about a decade smaller than the confinement radius * R * ~ c ~ used in our experiments: 0. 9 mm \ <* R * ~ c ~ \ <1. 8 mm. At long time, because of the collisions on the disc boundary, the velocity autocorrelation function sharply drops to 0 as seen in [Fig. 2c] (# f2) {ref - type = " fig " }. Unlike swimming cells [@ b26] [@ b31 ], self - propelled grains [@ b8] [@ b22] [@ b23] or autophoretic colloids [@ b32 ], dilute ensembles of rollers do not accumulate at the boundary. Instead, they bounce off the walls of this virtual box as shown in a close - up of a typical roller trajectory in [Fig. 2d] (# f2) {ref - type = " fig " }, and in the [Supplementary Movie 1] (# S1) {ref - type = " supplementary - material " }. As a result, the outer region of the circular chamber is depleted, and the local packing fraction vanishes as * r * goes to * R * ~ c ~, [Fig. 2a] (# f2) {ref - type = " fig " }. The repulsion from the edges of the circular hole in the microchannel stems from another electrohydrodynamic phenomenon [@ b33 ]. When an electric field is applied, a toroidal flow sketched in [Fig. 1a] (# f1) {ref - type = " fig "} is osmotically induced by the transport of the electric charges at the surface of the insulating adhesive films. Consequently, a net inward flow sets in at the vicinity of the bottom elfStrode. As the colloidal rollers are prone to reorient in the direction of the local fluid velocity [@ b11 ], this vortical flow repels the rollers at a distance typically set by the channel height * H * while leaving unchanged the colloid trajectories in the centre of the disc. This electrokinetic flow will be thoroughly characterized elsewhere. Collective motion in confinement - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - As the area fraction is increased above, collective motion emerges spontaneously at the entire population level. When the electric field is applied, large groups of rollers akin to the band - shaped swarms reported in [@ b11] form and collide. However, unlike what was observed in periodic geometries, the colloidal swarms are merely transient and ultimately self - organize into a single vortex pattern spanning the entire confining d7Zc as shown in [Fig. 3a] (# f3) {ref - type = " fig "} and [Supplementary Movie 2] (# S1) {ref - type = " supplementary - material " }. Once formed, the vortex is very robust, rotates steadily and retains an axisymmetric shape. To go beyond this qualitative picture, we measured the local colloid velocity field * * v * * (* * r * *, * t *) and use it to define the polarization field * * Π * * (* * r * *, * t *) ≡ * * v * * / * v * ~ 0 ~, which quantifies local orientational ordering. The spatial average of * * Π * * vanishes when a coherent vortex forms, therefore we use its projection along the azimuthal direction as a macroscopic order parameter to probe the transition from an isotropic gas to a polar - v | Building upon the pioneering work of Vicsek *et al.*[@b1], physicists, mathematicians and biologists have contemplated the of living-organism groups into flocks as an emergent process stemming from simple interaction rules at the individual level[@b2][@b3][@b4]. This idea has been supported by quantitative trajectory analysis in animal groups[@b5][@b6][@b7], together with a vast number of numerical theoretical models[@b3][@b4], and more by the observations of flocking behaviour in ensembles of non-living motile particles such as grains, active colloids, and mixtures of biofilaments motors[@b8][@b9][@b10][@b11][@b12]. a physicist\'s systems are considered as instances of polar matter, which encompasses any ensemble of motile bodies endowed with local velocity--alignment interactions. The current paradigm for flocking physics is the following. Active particles are persistent random which when form a isotropic gas. Upon increasing density, collective motion emerges the form of localized swarms that may cruise in a sea of randomly particles; further increasing density, a homogeneous liquid forms and spontaneously flows along well-defined direction[@b1][@b13][@b14]. This picture is the outcome of experiments, simulations theories mostly performed in unbounded or periodic domains. Beyond this picture, attention has been devoted over the last five years to active Confined active particles have consistently, yet not systematically, been reported to self-organize vortex-like structures. However, unlike our understanding of flocking, we are still unified picture account for the emergence and structure of such vortex patterns. This situation is mostly due the extreme diversity the and symmetries of the interactions between the active particles that been hitherto considered. active vortices exist only in finite-size systems as the case of bacterial suspensions[@b17], lose this beautiful order and intermittent turbulent dynamics[@b27] when unconfined? What are the necessary interactions required to observe and/or engineer bona fide stationary swirling states of active matter? In this paper, we answer these by considering the impact of geometrical boundaries on the collective behaviour of motile particles endowed with velocity--alignment interactions. quantitative experiments on motile colloids, numerical simulations and analytical theory, we elucidate the phase behaviour of *polar* active matter restrained geometrical boundaries. We use rollers, which, unlike most of the available biological self-propelled bodies, interact via well-established dynamical interactions[@b11]. We first exploit unique model system to show that above a critical concentration populations of motile colloids undergo a non-equilibrium phase from an gaseous state to a novel ordered state where the entire population self-organizes into a single heterogeneous steadily rotating vortex. This self-organization is *not* the consequence of the finite system size. Rather, this emergent vortex is a genuine state of polar active matter lying on verge of a macroscopic phase This novel state is the only ordered found when unidirectional directed motion is hindered by convex isotropic We then demonstrate that a competition between alignment, repulsive interactions and is necessary to yield large-scale vortical motion in ensembles of motile particles interacting via alignment interactions, thereby extending the relevance of our findings to a class active materials. Results ======= Experiments The experimental fully described in the *Methods* section and [Fig. Briefly, we use colloidal rollers powered by the Quincke electrorotation mechanism as thoroughly explained in ref. [@b11]. An electric field **E**~**0**~ is applied to insulating colloidal beads immersed in a conducting fluid. Above a critical amplitude *E*~Q~, the symmetry the electric charge distribution at surface is spontaneously broken. As a result, a net electric torque acts the beads causing to rotate at constant rate a random transverse to the electric When colloids sediment, or are electrophoretically driven, onto one of the two rotation converted into a net motion along a random direction. Here, we use poly(methyl methacrylate) (PMMA) spheres of radius *a*=2.4 μm immersed in a hexadecane solution. As sketched in [Fig. 1a](#f1){ref-type="fig"}, the colloids handled and observed in device made of double-sided scotch tape and of two glass slides coated with an indium-tin-oxide layer. The ITO layers are used to apply a uniform field in the *z*-direction, with *E*~0~=1.6 V μm^−1^ (*E*~0~=1.1*E*~Q~). Importantly, the electric current is nonzero solely in a disc-shaped chamber at the centre of the main channel. As exemplified by the trajectories [Fig. 1b](#f1){ref-type="fig"} and in Movie 1](#S1){ref-type="supplementary-material"}, rotation is hence restrained to this circular in which rollers are trapped. We henceforth characterize the collective dynamics of the roller population for increasing values of colloid packing fraction *φ*~0~. Individual self-propulsion -------------------------- area fractions smaller than , the ensemble of rollers uniformly explores the circular confinement as illustrated by the flat profile of the local packing averaged along the azimuthal direction in [Fig. 2a](#f2){ref-type="fig"}. The rollers undergo uncorrelated persistent random walks as demonstrated in [Fig. 2b,c](#f2){ref-type="fig"}. The probability distribution the roller velocities is isotropic and sharply peaked on the speed *v*~0~=493±17 s^−1^. In addition, the velocity autocorrelation function decays exponentially short time as expected from a simple of self-propelled particles having a constant speed *v*~0~ and undergoing rotational diffusion with a rotational diffusivity *D*^−1^=0.31±0.02 s that hardly depends on fraction (see [Supplementary Note 1](#S1){ref-type="supplementary-material"}). These quantities correspond a persistence length of that about a decade smaller than the confinement radius *R*~c~ used in our experiments: 0.9 mm\<*R*~c~\<1.8 mm. At long because of the collisions on the disc boundary, the velocity autocorrelation function sharply drops to 0 as seen in [Fig. 2c](#f2){ref-type="fig"}. Unlike cells[@b26][@b31], self-propelled grains[@b8][@b22][@b23] or autophoretic colloids[@b32], dilute ensembles rollers not accumulate at the boundary. they bounce off the walls this virtual box as in a close-up of a typical roller trajectory in [Fig. 2d](#f2){ref-type="fig"}, and in the [Supplementary Movie 1](#S1){ref-type="supplementary-material"}. As result, the outer region of the circular chamber is depleted, and local packing vanishes as *r* goes to *R*~c~, 2a](#f2){ref-type="fig"}. The repulsion from the edges of the circular hole in the microchannel stems from another electrohydrodynamic phenomenon[@b33]. When an electric field applied, toroidal flow sketched in [Fig. 1a](#f1){ref-type="fig"} is osmotically induced by the of the electric charges at the surface of the insulating adhesive films. Consequently, a net inward flow sets in at the vicinity of the bottom electrode. the rollers are prone to reorient in the direction of the local fluid velocity[@b11], this flow repels the rollers at a typically set by the channel height *H* leaving unchanged colloid trajectories in the centre of the disc. This electrokinetic flow be thoroughly characterized elsewhere. Collective motion in confinement -------------------------------- As the area fraction is increased above , collective motion emerges spontaneously at the entire population level. When the electric field is applied, large groups of akin to the band-shaped swarms reported in[@b11] form and collide. However, unlike what was observed in periodic geometries, colloidal swarms are merely transient and ultimately self-organize into single vortex pattern the entire confining disc shown in [Fig. and [Supplementary Movie 2](#S1){ref-type="supplementary-material"}. Once formed, the vortex is very robust, rotates steadily and retains an axisymmetric shape. go beyond qualitative picture, we measured the local colloid field **v**(**r**, *t*) and use it to define the polarization field **Π**(**r**, *t*)≡**v**/*v*~0~, which quantifies local orientational ordering. spatial average **Π** vanishes when a coherent vortex forms, therefore we use its projection the azimuthal direction as a order parameter to probe the transition from an isotropic gas to a polar-v | BuildING uPOn tHe pIoneeRInG WOrk OF ViCsek *ET AL.*[@B1], pHySICiSTS, matHeMaTICIanS AND biOLOGIsTS hAVE CoNTempLaTED tHE sELF-orGanIZAtIOn OF liViNG-OrGaNIsm gROUps INto floCkS as AN EmeRgEnT PROceSS STEmmIng froM SiMPLe iNTerAcTiOn rULES at THE INdIvIdual LEvEL[@B2][@B3][@b4]. tHIs idea HAs BeEN SUppoRteD by QUANtItaTIVE trajEcTOrY ANalySIs In aNImAl GROups[@b5][@b6][@b7], TOGethEr wItH a vast nuMbEr of NUMeRIcaL and thEorETiCAL MoDELS[@B3][@b4], anD MOrE rECENtlY By The oBsERVATiONS of fLoCkInG BEhaVIOUR iN eNSEmBLEs Of Non-liViNg MOtiLE pARTICLes sUch As shakEn graiNs, ActIvE cOlLoIdS, aNd MIXTuRes Of bIoFIlaMEnTs and mOlECULAR MoTORs[@B8][@B9][@b10][@b11][@b12]. fRom A PHYsiCIsT\'S PERSPECtIvE, tHeSE VaRioUs SyStEms ArE conSIDerED AS dIFFEReNT InstAnCES Of POlAr ACtIVe mAttER, Which encomPaSSes ANy eNsEMbLE OF mOtIlE bOdiES EndowED WIth lOcAL vELoCiTY--aligNmenT IntERaCtIOns. tHE cUrREnt PARadiGM fOr FLOcKiNG PhysicS IS ThE folLowiNg. aCTivE pARTiCleS Are PErsisTent RaNdOm walKeRS, WHiCH WhEn DiLUte fORm A hOmOGeNEOUS iSOtrOPIc Gas. UpOn INCReAsinG denSIty, CoLLeCtIVe moTIon emErgeS iN tHe fORM oF SPatially lOcaLIZED SWaRMS ThaT maY CruISE in a sEA Of RanDOMLY moviNG paRtICLeS; furThER inCREasiNG DENsity, a hoMogeNEOUS pOlar liquid FORms aND sPoNTAneOuslY FlOws Along a wELl-DeFINED dirEctioN[@b1][@B13][@b14]. thIS PictUrE is The oUTcome of expeRimeNTs, SiMULaTionS ANd theoRies MostlY PeRFoRmED in UNbOUNDed or pERioDIc DoMainS.
BEYOnD tHis piCTURe, SigniFiCAnt atteNtiON Has Been dEvoTeD ovER THe lAST FIVe yeARS To CoNfineD acTIVe MAtTeR[@B3][@b12][@b15][@b16][@b17][@B18][@B19][@B20][@b21][@B22][@b23][@b24][@B25][@b26]. COnFIned acTiVe PArTICLEs haVe cOnSIsTeNTLY, Yet noT SYstEMaTiCaLly, bEen REPOrTED To sElf-ORGaNIze INtO VorTeX-LIKE StRuCTUreS. hoWeVER, uNliKE fOr our unDeRSTandINg of fLOCKiNg, we aRE stIll lacKinG A unIfiED PIctUre TO acCoUNT fOr the EMeRgEncE aND strUcTuRE OF SUcH vORtEx pAttERns. THiS SITUATIon Is mOStLY Due tO tHe eXtREMe DIVERsity in The NaTURE AnD sYmmETRIES OF THe INTeRACtIoNS bEtweEN The AcTIVE partIClES thAT hAve beEN hiTHErTO COnSIderED. DO active VOrtIcES eXIST oNlY in FinIte-SizE systemS aS in The cASe OF bACtErIAl susPENsIonS[@B17], wHIch LOsE THis BeAuTIfUL ordeR anD DISplaY intErMIttent TURbUleNt dynaMICs[@b27] WHen unconfineD? WHaT ARE thE nEcEssary iNtERacTiOnS REqUIreD to obsErVE AnD/Or eNGInEeR Bona fIdE StAtIonARY swiRlINg STaTES Of aCtiVE MaTTEr?
IN tHiS papeR, we ansWeR these QuestIoNS By cONsIDERInG thE impaCt oF gEoMETrIcal bOUnDaRiES on tHE colleCTiVe bEHaVioUR oF mOTiLe PARticlEs ENdowED WiTh vELOCITy--ALIGnMENt inTeRaCTions. coMBiNING QUAnTITaTIVE eXpERImenTs ON mOtiLe coLLoIds, nUmErICal siMUlATIONs anD aNaLYtiCAL tHeORy, We EluciDATe the PHaSE beHaVIoUr OF *poLar* actIvE MATTEr REStrAINeD By GEOMeTriCaL bOuNdariES. WE uSE coLloIDAL RoLLErs, wHiCH, UnLIKe MosT Of the AvAiLAbLe BIologICal selF-PRoPELlEd bODieS, iNtEraCT ViA wElL-EstABlISheD DyNAmical INteraCtiOns[@B11]. WE fIRsT ExPloIt this Unique MODeL sySTEM tO SHoW ThAT ABovE a CRiTICaL ConcentraTioN poPULatIoNs Of MOtiLe COLlOIDS undergo A NOn-equIlIbriUM PhaSE TraNsitIOn frOM An isOtRopiC gAseOus STatE TO A nOveL ORDerEd staTe WHeRE The EntiRe pOpULAtion sELF-orgaNizeS iNTo A siNGle HETeROgENeOuS STEAdilY rOTAting vORTeX. THIs SelF-orGAniZaTIon IS *NOT* The CoNsEQUEnCe of tHE FinIte SYstem sIZE. raTheR, this eMERGEnt vORTex Is a GENUiNE statE Of POLAR AcTivE mAtTeR LyINg On the VeRGe OF A MaCroScOpiC pHasE SEPAraTION. THiS nOveL sTaTE Is tHe oNlY oRdErEd phASE foUND wHEN unIdiRECtIoNal dIreCtED mOtiON iS HiNdErED by cONvEX iSotRoPIC BOundarIEs. WE theN DEMoNSTrATE THeORetIcalLY THAt A COMpEtiTIOn BETweeN aLiGNment, RepuLSiVE iNTeRAcTIONs AnD cONFiNeMent is nEcESsAry To YIeld LArgE-sCAlE VortIcAL MOtion IN ENSEmBleS of motile PArtiClES iNtERactINg VIA AligNMEnt InTerActIONs, Thereby eXtENdINg THE RELEvAnce OF ouR fiNDiNGS tO A bRoAD class of aCtivE MatErials.
ReSulTS
=======
EXpERimeNTS
-----------
THE eXPERImENtaL SetUP Is fullY DEsCribeD in The *MeTHods* SEcTION aNd In [FiG. 1A,b](#F1){Ref-tYPE="FiG"}. brieFLY, we use COLLoidAL RolleRS poweREd BY thE qUiNCke eLeCtRorotatiON MecHanISM AS THoROugHLy EXPlAIneD IN REf. [@B11]. An ElecTrIc fieLD **e**~**0**~ iS aPPLieD To insUlatIng COLLOIDAl BEaDS imMeRSED IN a coNductINg fLUid. abOve A cRiTICAL fielD AMPlITudE *e*~q~, tHe symMETrY Of thE elEcTRiC CharGE diStRIbUtION aT tHe beAd sUrFACE is spontANEOusLy Broken. as A REsulT, A NEt elEctRic TOrque ACTS ON tHe BEaDs CAUSINg THem TO ROtAtE at a coNStAnT RaTe aRounD a raNDOm axIS TrAnSvErse To thE ElECtric fiEld[@B28][@B29][@b30]. WHEN The COLLOidS SEDIMeNt, oR aRe ElectrOpHOreticAlLy DRiVEN, onTo oNe of thE tWO eLEcTrODEs, rOTatIon is cONVeRTeD iNto A Net ROLLINg MoTiON AlonG a RAnDOm dirECtIoN. hERE, we use POly(mEThYl MEtHacRyLate) (pMmA) sPhEreS oF RadIUs *a*=2.4 Μm ImMERsed In a HeXADeCANE SoLuTion.
as SketCHed IN [Fig. 1A](#f1){reF-tYPE="fIG"}, tHE COLloidS aRe haNDlEd aNd OBSErvEd in a MIcRofLuidIC deVicE MAdE OF DOUBle-siDeD scotch TaPe AnD Of twO GlAsS SLIdeS cOATEd witH aN inDIuM-TiN-oxIDE Layer. thE ItO LaYERs are UsED To ApPlY a UNIFORm dC FiEld in THe *Z*-dirECtION, wItH *e*~0~=1.6 v μM^−1^ (*E*~0~=1.1*e*~Q~). ImpOrTanTLy, tHe ELEcTRIc cUrRENT Is NonzeRO sOlely iN A dISC-shaPeD ChAMbEr At The cenTrE oF thE mAIn ChANNEl. aS exemPlIfIEd bY THE TraJECtORies ShOwN In [FiG. 1B](#f1){reF-Type="FIG"} aND In [supPLEmENTary movIe 1](#s1){Ref-tyPe="sUPPlEmeNTaRY-mateRiaL"}, QuINcKE RotatION Is Hence rEStRAinEd to tHIS CirCulAR Region in wHiCH the RollERs ARE TRapPeD. WE hENceFoRth chARacteriZE the CoLLECtiVe dYNamICs OF ThE rOlLER PopulAtion FoR iNCREASiNg VALUes of ThE Colloid pAckING frAcTioN *Φ*~0~.
INdIvIDUAl Self-PropuLsion
--------------------------
For area fractIoNs sMallEr than , tHe eNSEMbLE OF RoLlerS uniforMly EXplOres tHE CirCuLar CoNFineMENt As IlluStRATED BY THE FLaT PROFile oF the LOCAL paCKING fRACTiOn aveRaGED alOnG THE AZiMuthAl DIRECTIOn *φ*(*R*) in [fIG. 2A](#f2){ReF-type="Fig"}. ThE rollERS UNderGo UNcORrelateD PeRSisTeNt RANdom walkS AS DEMONStRaTeD in [Fig. 2b,c](#f2){REf-typE="Fig"}. tHE probABilITY dIStRiBuTiOn of ThE roLLer VeLociTIES is IsOtrOPiC And SHarPly pEAkEd On thE TYpical spEeD *v*~0~=493±17 Μm S^−1^. IN AdDiTiOn, THE VELocITy aUtoCOrReLATiON fUNcTIoN Decays exPOneNtialLY At shoRt TimE AS expEcTED FROM A sIMPle moDel of sElF-PrOpeLLed PaRTIClES haVInG a CONsTAnT sPEEd *v*~0~ ANd UNDErgoInG RoTATIonaL DIfFUSIoN WitH A RotATiOnaL DifFuSIVity *D*^−1^=0.31±0.02 S ThAT hARdLY dEPeNDs ON tHE ArEa frACtiOn (SeE [suPpLEmeNtARy note 1](#S1){REf-TypE="sUPPLEMeNTArY-MaTeRiAl"}). THese quaNTItieS cORRESpoNd To A PeRSISteNCE LenGTH oF ThAT iS aBoUt a Decade sMALlEr tHAn tHe COnFiNemeNt raDIuS *r*~C~ USeD IN OuR ExpErIMenTS: 0.9 mm\<*R*~c~\<1.8 mm.
at lONG timE, BEcaUSe of The coLlIsIONS on tHe diSC BOundARY, thE vELOcIty AUtoCORreLaTiOn FUNCTIOn shaRPLy DROpS tO 0 aS sEen in [fIG. 2C](#F2){ref-tyPe="fIG"}. uNlike swImMIng cELlS[@b26][@B31], SElf-pROPellEd gRains[@B8][@b22][@b23] or aUtOphoReTIc cOllOIDs[@B32], diluTe EnSEMbleS oF ROlLErs dO nOT aCcumUlaTe AT ThE boundAry. inStEAD, TheY bOunCE Off The WaLls of tHIS virTuaL boX aS SHOwN IN A cloSe-up OF a tYpiCAL ROller trajeCtory IN [fIG. 2d](#f2){reF-typE="fIg"}, And IN the [SuPplemEnTArY moVie 1](#s1){reF-TYPE="supPLEMentARY-maTeriAL"}. AS A resuLt, tHe outer ReGioN OF tHE ciRcuLAR cHaMber IS DepLETed, AnD THe lOCaL pAcKiNg FRacTIoN vAnIsHES aS *r* gOes to *r*~C~, [Fig. 2a](#F2){ref-Type="fiG"}. The RepuLSioN FROm ThE edGES oF THe CiRculAR holE IN tHe MICroChaNNeL sTEmS FrOm AnothER EleCtrohyDRodynAMIc pHENOMenon[@b33]. wheN an electRiC FIeld iS APPlIEd, a tOroIDAL FlOw skETcHED in [fIG. 1a](#F1){reF-tYpe="fiG"} is oSMotIcALLy IndUced by The tRANsPOrt Of tHE ELECTrIC chArgEs aT tHe surfacE oF thE insuLating ADhEsive filmS. CONsEQUentlY, A net iNWARD FlOW sETs in aT THe viCINity OF THe bOTtOm eLEctroDe. As ThE COLLoIdaL rollErs Are prOnE to rEoRIENT In ThE dIrECtioN of thE LocaL flUID VElocItY[@B11], ThIS VorTIcAl fLow RePElS thE rOLleRS At A DIstAnce TYPIcAlLy SET by tHE cHanNel HeiGHT *H* whIlE LEAvInG UNchaNgED THE COLloID TRAjectORIeS In the CeNTre OF thE dISc. ThIS ELEctrOkiNEtic fLoW wIlL BE ThoRoUgHlY ChARaCTErIzed ELsewheRE.
CoLLectIve MoTIoN in confINemeNT
--------------------------------
aS ThE arEa FractIOn IS InCrEasED ABOve , CoLLECTIve MOtIon EmeRGeS SpONTANeoUSlY AT tHe eNtiRE pOPulatiOn LEveL. WHEn ThE EleCtRic FielD is APpLIEd, laRge gRoUps of ROLLErs Akin tO THE Band-sHapeD SwarMs RePORTED iN[@b11] FOrM AND coLlIde. HoWeveR, UnLike WHaT WAS oBseRVED in PErIODiC gEomETries, thE coLLOIdAL SWaRMS arE merELy tRAnsIeNT And ulTimatELy sElf-OrgAnIze INtO a sIngLE vORTEX pAtTErn sPaNNINg the EnTirE CoNfInInG disC as shOWN IN [fig. 3a](#F3){Ref-TYPE="fIG"} AnD [sUpplemENtArY moVIe 2](#S1){REf-tYpE="SupPLeMentAry-MATERIaL"}. oNcE fOrMed, the vorTeX iS vErY RoBUST, ROTATeS sTEAdiLY AND REtaIns An AXISYmMETRic SHaPe. TO GO BEyOND ThiS quAlItAtIVe PictuRe, wE MeaSurEd thE LOCal ColloiD VeLOCITY FIeLd **V**(**R**, *t*) AnD Use IT TO dEFINE ThE PolaRIZatIOn fiELd **π**(**R**, *T*)≡**v**/*V*~0~, WHIcH QUanTIfIES local oRIENtATIonal oRDEring. tHE SpAtIAl aVErAGe OF **π** vaNiSHes WhEN a CoHereNT vOrtEX foRMs, tHereFORE We UsE iTS PROjecTION aloNG THe aZiMuthaL DIrECtIOn aS a mACroScoPic ORdeR pARAmetER TO proBE The TRANSITion FRoM AN isOtrOpic GaS To A POlar-V | Building upon thepioneering work of Vicsek *et al.*[@b1], physicists, mathematicians and biologists have contemplated the self-organization of living-organismgroups into flocks as an emergent process stemming from simple interaction rulesat the individual level[@b2][@b3][@b4]. This idea has been supported by quantitative trajectory analysis in animal groups[@b5][@b6][@b7], together with a vast number of numericaland theoretical models[@b3][@b4], and more recently by the observations of flockingbehaviour in ensembles of non-living motileparticles suchas shaken grains,active colloids,and mixtures of biofilaments and molecular motors[@b8][@b9][@b10][@b11][@b12]. From a physicist\'s perspective,thesevarioussystemsare considered as different instances of polar active matter, which encompasses any ensemble of motile bodiesendowed withlocal velocity--alignment interactions. The current paradigm forflocking physics is the following. Activeparticles are persistentrandom walkers, which when dilute form a homogeneous isotropic gas.Upon increasing density, collective motion emerges in the form of spatially localized swarms that may cruise ina sea of randomly moving particles; further increasingdensity, a homogeneouspolar liquid forms and spontaneously flows along a well-defined direction[@b1][@b13][@b14]. This picture is theoutcomeof experiments, simulations and theories mostly performedin unbounded or periodicdomains. Beyond thispicture,significantattentionhas been devoted over thelast five years toconfinedactive matter[@b3][@b12][@b15][@b16][@b17][@b18][@b19][@b20][@b21][@b22][@b23][@b24][@b25][@b26]. Confined active particles have consistently, yet not systematically, been reported to self-organize intovortex-like structures. However,unlikefor our understanding offlocking, we are still lacking aunified picture to account for the emergence and structureof such vortexpatterns. This situation is mostly due to the extreme diversity in thenatureand symmetries of the interactions between the active particles that have been hitherto considered. Do active vorticesexist only in finite-size systems as in the case of bacterial suspensions[@b17], which losethis beautiful order and display intermittent turbulent dynamics[@b27] when unconfined? What are the necessaryinteractions required to observe and/or engineer bona fide stationary swirling states of activematter? Inthis paper, we answer these questionsby considering the impact of geometrical boundarieson the collective behaviour ofmotileparticles endowed with velocity--alignment interactions. Combining quantitative experiments on motile colloids, numericalsimulations and analytical theory, we elucidate thephase behaviour of *polar* active matterrestrained by geometrical boundaries. We use colloidalrollers, which,unlike mostof the availablebiological self-propelled bodies, interactviawell-established dynamical interactions[@b11]. We first exploit this unique model system to showthatabove a critical concentrationpopulations of motilecolloidsundergo a non-equilibrium phase transition from an isotropic gaseous state to a novel ordered state wheretheentire population self-organizes into asingle heterogeneous steadily rotatingvortex. This self-organization is *not* the consequence of the finite system size. Rather, this emergentvortex is a genuine state ofpolaractive matterlying on the vergeof a macroscopic phase separation. This novel state is the only ordered phase found when unidirectional directed motion is hindered by convex isotropic boundaries. We then demonstrate theoretically that a competition between alignment,repulsive interactions and confinementisnecessary to yield large-scale vortical motion in ensembles ofmotile particles interacting via alignment interactions, therebyextending the relevance of our findings to a broadclass of active materials. Results ======= Experiments ----------- The experimental setupis fully described in the *Methods* section and in [Fig. 1a,b](#f1){ref-type="fig"}. Briefly, we use colloidal rollers powered by the Quincke electrorotation mechanismas thoroughlyexplained in ref. [@b11]. An electric field **E**~**0**~ is applied to insulating colloidal beads immersedin a conducting fluid. Above a critical field amplitude *E*~Q~, the symmetry of the electric charge distribution at the bead surface is spontaneously broken.Asa result, a net electric torque actson thebeads causing them torotate at a constant rate around a random axistransverseto the electric field[@b28][@b29][@b30]. When the colloids sediment, or are electrophoretically driven, onto one of the twoelectrodes, rotation isconverted intoa net rolling motion along a random direction. Here,we use poly(methyl methacrylate) (PMMA) spheresof radius *a*=2.4μm immersed in a hexadecane solution. Assketched in [Fig. 1a](#f1){ref-type="fig"}, the colloids are handled and observed ina microfluidic devicemade of double-sided scotch tape and of two glass slides coated with an indium-tin-oxide layer. The ITOlayers are used to apply a uniform DCfield in the*z*-direction, with *E*~0~=1.6 V μm^−1^ (*E*~0~=1.1*E*~Q~). Importantly, theelectric currentis nonzero solely ina disc-shaped chamber at the centre of the main channel. As exemplified by the trajectories shown in [Fig. 1b](#f1){ref-type="fig"} and in [Supplementary Movie 1](#S1){ref-type="supplementary-material"}, Quincke rotation is hence restrained to this circularregion inwhich therollers are trapped. We henceforthcharacterize the collective dynamics of the roller population for increasing values of the colloid packing fraction *φ*~0~. Individual self-propulsion-------------------------- For area fractions smaller than , the ensemble of rollers uniformly explores the circular confinement as illustrated by the flat profile ofthe local packing fraction averaged along the azimuthal direction *φ*(*r*) in [Fig. 2a](#f2){ref-type="fig"}. Therollers undergo uncorrelated persistent random walks asdemonstratedin [Fig.2b,c](#f2){ref-type="fig"}. Theprobability distribution of the roller velocities is isotropic and sharplypeaked on the typical speed *v*~0~=493±17 μms^−1^. In addition, the velocity autocorrelationfunction decays exponentially at short time as expected from a simple model of self-propelled particles having a constant speed *v*~0~ and undergoing rotational diffusionwith a rotational diffusivity *D*^−1^=0.31±0.02s that hardly depends onthe area fraction (see [Supplementary Note 1](#S1){ref-type="supplementary-material"}). These quantities correspond toa persistence length of that isabout a decadesmaller than the confinement radius *R*~c~ used in our experiments:0.9 mm\<*R*~c~\<1.8 mm. At long time, because of the collisions on the disc boundary, the velocity autocorrelation function sharply dropsto 0 as seen in [Fig. 2c](#f2){ref-type="fig"}. Unlike swimming cells[@b26][@b31], self-propelled grains[@b8][@b22][@b23] or autophoreticcolloids[@b32], dilute ensembles of rollers donotaccumulate atthe boundary. Instead, they bounce off the walls of this virtual box as shown in a close-upofa typical roller trajectory in[Fig. 2d](#f2){ref-type="fig"}, and in the [Supplementary Movie 1](#S1){ref-type="supplementary-material"}. As a result, the outer region of the circular chamber is depleted, and the local packing fraction vanishes as *r* goes to *R*~c~, [Fig. 2a](#f2){ref-type="fig"}. The repulsion from the edges of thecircular hole in the microchannel stems from another electrohydrodynamic phenomenon[@b33]. Whenan electric field is applied, a toroidal flow sketched in [Fig. 1a](#f1){ref-type="fig"} is osmotically induced by the transport of the electric charges at thesurface of the insulating adhesivefilms.Consequently, a net inwardflow sets in at the vicinity of the bottomelectrode. As the colloidalrollers are prone to reorient in the direction of the local fluid velocity[@b11], this vortical flow repels the rollersat a distancetypically set by the channel height *H* whileleaving unchanged the colloid trajectories in the centre of the disc. This electrokinetic flow willbe thoroughly characterized elsewhere. Collective motion inconfinement -------------------------------- Asthe area fraction is increased above, collective motion emerges spontaneously at the entire population level. When theelectric fieldis applied, largegroups of rollers akin to the band-shaped swarms reported in[@b11] form and collide. However, unlikewhatwas observed in periodic geometries, the colloidal swarms are merelytransient and ultimately self-organize intoa single vortex pattern spanning the entire confining disc asshown in [Fig. 3a](#f3){ref-type="fig"} and [Supplementary Movie 2](#S1){ref-type="supplementary-material"}. Once formed, thevortex is very robust, rotates steadilyand retains anaxisymmetric shape. To go beyond this qualitative picture, we measured the local colloidvelocityfield**v**(**r**, *t*) and use it to define the polarization field **Π**(**r**, *t*)≡**v**/*v*~0~, which quantifies local orientational ordering. The spatial average of **Π** vanishes when a coherentvortex forms, therefore we use its projection along the azimuthal direction asa macroscopicorderparameter to probe the transitionfrom an isotropic gas to apolar-v | Building upon the pioneering _work_ of _Vicsek_ *et al.*[@b1], physicists, mathematicians _and_ biologists have _contemplated_ the self-organization _of_ living-organism groups into flocks as an _emergent_ process _stemming_ from _simple_ interaction rules _at_ the individual _level[@b2][@b3][@b4]._ This idea has _been_ supported by quantitative trajectory analysis in animal groups[@b5][@b6][@b7], together with _a_ vast number of numerical and theoretical models[@b3][@b4], _and_ _more_ recently by _the_ observations of _flocking_ behaviour in _ensembles_ of non-living motile particles such as shaken grains, active colloids, _and_ mixtures of biofilaments and _molecular_ motors[@b8][@b9][@b10][@b11][@b12]. From a physicist\'s _perspective,_ these various systems are considered as different instances of _polar_ active matter, which _encompasses_ _any_ ensemble of _motile_ bodies endowed with local velocity--alignment interactions. _The_ current paradigm for flocking physics is the following. Active particles _are_ persistent random walkers, _which_ when dilute form _a_ homogeneous isotropic gas. Upon increasing _density,_ collective motion emerges in the form of spatially localized swarms _that_ may cruise in _a_ sea of randomly moving particles; further increasing density, _a_ homogeneous _polar_ liquid _forms_ and _spontaneously_ flows along _a_ _well-defined_ direction[@b1][@b13][@b14]. This picture is the _outcome_ of experiments, _simulations_ and theories mostly performed _in_ _unbounded_ or periodic domains. Beyond this picture, _significant_ attention has _been_ devoted over the last _five_ _years_ to confined active matter[@b3][@b12][@b15][@b16][@b17][@b18][@b19][@b20][@b21][@b22][@b23][@b24][@b25][@b26]. Confined _active_ particles have consistently, yet not _systematically,_ been reported to self-organize into vortex-like structures. However, unlike _for_ our understanding _of_ flocking, _we_ are still lacking _a_ unified picture _to_ account for the _emergence_ and structure _of_ such vortex patterns. This situation is mostly due to the extreme diversity in _the_ nature and symmetries of the interactions between the active particles that have been hitherto considered. Do active vortices _exist_ only in _finite-size_ _systems_ as in _the_ case of bacterial suspensions[@b17], which lose this _beautiful_ _order_ and display intermittent turbulent dynamics[@b27] _when_ _unconfined?_ What _are_ _the_ necessary interactions _required_ to observe and/or engineer bona fide stationary swirling states of active matter? In this paper, _we_ answer these questions _by_ considering the impact of geometrical boundaries on _the_ collective behaviour of motile particles endowed _with_ _velocity--alignment_ interactions. Combining quantitative experiments on motile colloids, numerical simulations and analytical theory, we _elucidate_ _the_ phase _behaviour_ of *polar* active _matter_ _restrained_ _by_ geometrical boundaries. _We_ use colloidal rollers, _which,_ unlike most _of_ the available biological _self-propelled_ bodies, interact via well-established dynamical interactions[@b11]. _We_ first exploit this _unique_ model system to show _that_ _above_ a critical concentration _populations_ of motile _colloids_ undergo _a_ non-equilibrium phase transition from an isotropic gaseous state to _a_ novel _ordered_ state where the entire population self-organizes into a single _heterogeneous_ steadily rotating _vortex._ _This_ self-organization is *not* the _consequence_ of the finite system size. _Rather,_ this emergent vortex is a _genuine_ state of polar active matter _lying_ on the verge of a macroscopic phase separation. This novel state is the only _ordered_ phase found when _unidirectional_ directed motion is hindered by _convex_ isotropic boundaries. We then demonstrate _theoretically_ _that_ _a_ _competition_ between alignment, _repulsive_ _interactions_ and confinement is necessary to _yield_ large-scale vortical motion in ensembles of motile particles interacting via _alignment_ interactions, _thereby_ _extending_ _the_ relevance of _our_ findings _to_ a _broad_ _class_ of active materials. Results ======= _Experiments_ ----------- The experimental setup is _fully_ described in the *Methods* section and in [Fig. 1a,b](#f1){ref-type="fig"}. Briefly, _we_ use _colloidal_ rollers powered by _the_ Quincke electrorotation _mechanism_ as thoroughly explained in ref. _[@b11]._ An electric field **E**~**0**~ is applied to insulating colloidal beads immersed in _a_ conducting _fluid._ _Above_ a critical field amplitude _*E*~Q~,_ the _symmetry_ of the electric charge distribution _at_ the bead surface is spontaneously broken. As a result, _a_ net electric torque acts on the _beads_ causing _them_ to rotate at a _constant_ _rate_ around a random _axis_ transverse _to_ _the_ electric field[@b28][@b29][@b30]. _When_ _the_ _colloids_ sediment, or _are_ electrophoretically driven, onto one of the two electrodes, rotation is _converted_ into a net _rolling_ motion along a _random_ direction. Here, we use poly(methyl methacrylate) (PMMA) spheres of radius *a*=2.4 μm _immersed_ in a hexadecane solution. As sketched in [Fig. 1a](#f1){ref-type="fig"}, the colloids are handled and observed _in_ a microfluidic _device_ made of double-sided scotch tape _and_ of two glass slides coated _with_ an indium-tin-oxide layer. _The_ _ITO_ layers are used to apply a uniform DC field in _the_ _*z*-direction,_ _with_ _*E*~0~=1.6_ V _μm^−1^_ (*E*~0~=1.1*E*~Q~). Importantly, the electric current is _nonzero_ solely in _a_ _disc-shaped_ chamber at the _centre_ of the main channel. As exemplified by the trajectories shown in [Fig. _1b](#f1){ref-type="fig"}_ and _in_ [Supplementary Movie 1](#S1){ref-type="supplementary-material"}, Quincke rotation is hence restrained to this circular region in which the rollers are _trapped._ We henceforth characterize the collective dynamics _of_ the roller _population_ for increasing values _of_ the colloid packing _fraction_ *φ*~0~. _Individual_ self-propulsion -------------------------- For area fractions smaller than _,_ the _ensemble_ of rollers uniformly explores the _circular_ confinement as illustrated by the flat profile of the _local_ _packing_ fraction averaged along the _azimuthal_ direction *φ*(*r*) _in_ [Fig. 2a](#f2){ref-type="fig"}. The _rollers_ undergo uncorrelated persistent random walks as demonstrated in [Fig. _2b,c](#f2){ref-type="fig"}._ The probability distribution _of_ the roller velocities is isotropic and _sharply_ peaked on the typical speed _*v*~0~=493±17_ μm s^−1^. _In_ addition, the velocity autocorrelation function decays exponentially at short time _as_ expected from a simple _model_ of self-propelled particles having a constant speed _*v*~0~_ _and_ undergoing rotational diffusion _with_ a rotational diffusivity *D*^−1^=0.31±0.02 s that _hardly_ depends on the area fraction _(see_ [Supplementary Note 1](#S1){ref-type="supplementary-material"}). These quantities correspond to a persistence length of that is about a _decade_ smaller than the confinement _radius_ *R*~c~ used in our _experiments:_ 0.9 mm\<*R*~c~\<1.8 mm. At long time, _because_ of the collisions on the _disc_ boundary, the velocity autocorrelation function sharply drops to 0 as _seen_ in [Fig. 2c](#f2){ref-type="fig"}. Unlike swimming cells[@b26][@b31], self-propelled _grains[@b8][@b22][@b23]_ or autophoretic _colloids[@b32],_ dilute ensembles _of_ rollers _do_ not accumulate at the _boundary._ Instead, they bounce off the walls _of_ this virtual _box_ _as_ _shown_ _in_ a close-up of a typical roller _trajectory_ in [Fig. 2d](#f2){ref-type="fig"}, _and_ in the [Supplementary Movie 1](#S1){ref-type="supplementary-material"}. As a result, _the_ _outer_ region of _the_ circular chamber is depleted, and the local packing fraction _vanishes_ as *r* goes _to_ *R*~c~, [Fig. _2a](#f2){ref-type="fig"}._ The repulsion from _the_ edges of _the_ circular hole in the microchannel stems from another electrohydrodynamic phenomenon[@b33]. When _an_ _electric_ _field_ _is_ _applied,_ _a_ toroidal flow _sketched_ _in_ [Fig. 1a](#f1){ref-type="fig"} _is_ osmotically induced by the _transport_ of the electric charges at the surface of _the_ _insulating_ adhesive films. Consequently, a net inward flow sets in _at_ the vicinity _of_ the bottom electrode. As the colloidal rollers _are_ prone _to_ reorient in the direction of the local fluid velocity[@b11], this vortical flow _repels_ the rollers at a distance typically set _by_ the _channel_ _height_ *H* while leaving unchanged the colloid trajectories in the _centre_ of the disc. This _electrokinetic_ flow will be thoroughly _characterized_ _elsewhere._ Collective motion in confinement -------------------------------- As the area fraction is increased above _,_ collective _motion_ emerges spontaneously at _the_ entire population level. When the electric field is applied, large groups of rollers akin _to_ the band-shaped swarms reported in[@b11] form and collide. However, unlike _what_ was observed in _periodic_ geometries, _the_ colloidal swarms are merely transient _and_ ultimately self-organize into _a_ single vortex pattern spanning _the_ entire confining disc _as_ shown _in_ [Fig. _3a](#f3){ref-type="fig"}_ and [Supplementary Movie 2](#S1){ref-type="supplementary-material"}. Once formed, the vortex is _very_ robust, rotates steadily and retains _an_ _axisymmetric_ shape. To go beyond this _qualitative_ picture, we measured the local _colloid_ velocity field **v**(**r**, *t*) and _use_ _it_ to _define_ the polarization field _**Π**(**r**,_ *t*)≡**v**/*v*~0~, which quantifies local orientational ordering. _The_ _spatial_ _average_ _of_ **Π** vanishes _when_ _a_ coherent vortex forms, therefore we use its projection _along_ _the_ azimuthal _direction_ as a _macroscopic_ _order_ parameter to _probe_ the transition from an _isotropic_ gas to a polar-v |
{#sp1 .253}
{#sp2 .254}
| ! [ ] ( indmedgaz71974 - 23 ) { # 236. 247 }! [ ] ( dec - 0020 ) { # 232. 235 } | ! [] (indmWdgAz71974 - 00WI) {# sp1. 253 }! [] (&nd<edgaz71974 - 00W0) {# sp2. 254 } | {#sp1 .253} {#sp2 | {#sp1 .253}
{#SP2 .254}
| {#sp1 .253} {#sp2 .254} | {#sp1 .253} {#sp2 .254} |
1. Introduction {#sec1}
===============
Health care is changing dynamically in the 2010s. The economic recession and problems with recruiting professionals \[[@B1], [@B2]\], staff retention \[[@B3]\], creating healthy work environments \[[@B4], [@B5]\], and a growing demand for customer orientation \[[@B6]\] pose challenges for nurse managers\' work. More expertise in management is needed to respond to these issues.
One essential area of nurse manager\'s management skills is the use of different leadership styles \[[@B7]\]. Leadership styles can be seen as different combinations of tasks and transaction behaviours that influence people in achieving goals \[[@B8]\]. Earlier studies indicate that nurse manager\'s effective leadership style is affiliated to staff retention \[[@B5]\], work unit climate \[[@B4]\], nurses\' job satisfaction \[[@B9], [@B10]\], nurses\' commitment \[[@B11]\], and patient satisfaction \[[@B12]\].
Transformational leadership style \[[@B5], [@B6], [@B13], [@B14]\] and transactional leadership \[[@B7]\] help to respond to these issues. Transformational leadership refers to the leader\'s skills to influence others towards achieving goals by changing the followers\' beliefs, values, and needs \[[@B7]\]. Transactional leadership complements and enhances the effects of transformational leadership outcomes \[[@B15]\].
There are certain skills required from nurse managers so as to be able to use these effective leadership styles. The skills include the ability to create an organization culture that combines high-quality health care and patient/employee safety and highly developed collaborative and team-building skills \[[@B1]\]. Nurse managers also need to have the readiness to observe their own behaviour \[[@B16]\] and its effects on the work unit; as a result, employees can adjust to a better leadership style. These kinds of skills are related to manager\'s emotional intelligence (EI).
EI is an ability to lead ourselves and our relationships effectively \[[@B17]\]. It has been defined as the ability to observe one\'s own and others\' feelings and emotions, to discriminate among them and to use this information to direct one\'s thinking and actions \[[@B18]\]. EI is composed of personal competence and social competence. Self-awareness and self-management are reflections of personal competence, influencing the way the leader manages him/herself. Social awareness and relationship management reflect social competence, which affects how the leader manages relationships with others \[[@B17]\].
Nurse managers with that skill can easily form relationships with others, read employees\' feelings and responses accurately, and lead successfully \[[@B19]--[@B21]\]. Emotionally intelligent leaders\' behaviour also stimulates the creativity of their employees \[[@B22]\]. Goleman et al. \[[@B23]\] have identified visionary, coaching, affiliate, and democratic styles as resonant, and pacesetting and commanding styles as dissonant leadership styles. Most leaders use both resonant and dissonant leadership styles. The leadership styles of Goleman et al. are applied as the basis of this study because earlier studies refer to the significance of these styles, especially that of EI in manager\'s work. In addition, these leadership styles are one way of aiming to carry out transformational leadership. Especially visionary, coaching, affiliate, and democratic styles include elements that promote transformational leadership. Such elements are for example the leader being visionary and empowering staff \[[@B4]\].
This paper focuses on Finnish nurse managers\' leadership styles. The Finnish health care system is a strong institution where health care services are offered to all citizens and funded by taxes \[[@B24]\]. It has widely recognized that health care services in Finland are of high-quality Despite recent concerns about equity issues, Finns are in general very satisfied with their health care services. \[[@B25]\]. Consequently it is important to explore nurse managers\' leadership styles especially in this context.
2. Materials and Methods {#sec2}
========================
2.1. Aim of the Study {#sec2.1}
---------------------
The intention of this study was to explore nurses\' and supervisors\' perceptions of nurse leaders\' leadership styles. The research questions were as follows: what kind of leadership styles do nurse managers use and what are the factors affected by their leadership styles.
2.2. Participants {#sec2.2}
-----------------
To achieve the aim of this study data were collected through open interviews. The majority of Finnish nurse managers, nurses, and supervisors work in hospitals or long-term facilities. Selection of participants was performed in convenience sampling \[[@B26]\]. Participants were selected paying attention to the fact they were of different ages, working in different wards and units (e.g., psychiatry, internal diseases, gerontology) in either hospitals or long-term facilities, and had worked with more than one nurse manager.
The researcher contacted the participants and asked whether they were interested in taking part in the study. The participants were informed about the aim of the study. Participation was voluntary. Prior to the interviews each participant signed a form where they gave their consent to participate in the study. A total of 11 nurses and 10 supervisors, 20 women and one man, from eight Finnish hospitals and five long-term care facilities participated in the study. The age of the nurses varied between 30 and 53 and their experience in health care between 7 and 25 years. The age of the supervisors varied between 38 and 59 and their experience as supervisors between 5 and 21 years. Both nurses and supervisors had worked with many nurse leaders and they were interviewed about nurse managers in general. They thus had experience of different nurse managers on different wards and they were able to describe leadership styles from various aspects.
2.3. Data Collection and Analysis {#sec2.3}
---------------------------------
Semistructured interviews were used to gather data on the perceptions of nurse managers\' leadership styles and factors affected by leadership styles. Interviews were usually carried out in the office in the participants\' workplace. All interviews were recorded with individual consent. Participants were initially asked to describe their work and earlier study and work history. They were subsequently asked about their perception of leadership styles and asked to describe the leadership styles used by their nurse managers. After that they were asked about factors affected by leadership styles. Each interview was approached individually, guided by participants\' responses. The interview sessions lasted between 30 and 85 minutes. Every interview was transcribed word for word from the recordings. Interviewing was continued until saturation of the data was achieved \[[@B27]\].
Because nurses and supervisors might have differed in their perceptions of leadership styles, the data were first analysed separately in two separate groups, following the same process for each group. Content analysis was chosen because it is a research method for making valid inferences from data to the contexts of their use \[[@B28]\].
The interview texts were read through multiple times, based on the author\'s empirical and theoretical preunderstanding of the professional area of the participating nurses and nurse managers. A structured categorization matrix of leadership styles was developed based on the primal leadership model \[[@B23]\] and research of Vesterinen et al. \[[@B29]\]. When using a structured matrix of analysis, an item of the data that does not fit the categorization frame is used to create its own concept, based on the principles of inductive content analysis \[[@B30]\]. When both the data of nurses and superiors were analysed, the results were compared. The categories and subthemes were congruent and therefore the results are presented together, albeit paying attention to differences and similarities of the perceptions of nurses and superiors.
The data analysis of the factors affected by leadership styles was inductive. All the data of nurses and supervisors were analysed together. This process included open coding, creating categories, and abstraction. A classification framework of the factors was formed inductively by defining categories and sub-themes. The criteria for allocating a unit to a category were formed by asking questions if the unit was suitable to the category. The sub-themes were named using descriptive concepts and classified as "belonging" to a particular category. After that, the categories were given names \[[@B31]\].
2.4. Trustworthiness {#sec2.4}
--------------------
The trustworthiness of this study has been ensured by confirming truth value, consistency, neutrality, and transferability of this study \[[@B32]\].
When considering this study from the viewpoint of trustworthiness, there are some threats that should be taken into consideration. The researcher collected the data and performed the analysis alone and the interpretation could have been affected by her professional history \[[@B33]\]. With interviews there is a risk that respondents try to please the interviewer by reporting things they assume s/he wants to hear. The researcher confirmed the truth value of the study by selecting participants in convenience sampling. The respondents\' age distribution was wide and they worked in different units. Their perspectives and descriptions were broad and gave a diverse picture.
The truth value of this study was also confirmed by analysing data as they emerged based on the interviews. To ensure the trustworthiness of the study quotes from interviews are included in the results.
In view of consistency, the research process is described so that it can be repeated if necessary. This gives a possibility to understand the limitations of the process of data collection and analysis. To ensure neutrality in this study, interpretations were based on original data. This is confirmed by citations from the interview data.
In this study the sample was small, consisting of Finnish nurses and supervisors, and the results only reflect their perceptions of leadership styles. As a result, transferability of results is limited. However, when considering the main objective in this study, it was not transferability of research results, but it was to enhance understanding of leadership styles and use it for future studies.
2.5. Ethical Considerations {#sec2.5}
---------------------------
The data for this study were collected following approval from the administrations of the organizations. All participants were informed of the | 1. introduction { # sec1 } = = = = = = = = = = = = = = = health care is changing dynamically for the 2010s. the economic climate and problems with recruiting professionals \ [ [ @ b1 ], [ @ b2 ] \ ], staff retention \ [ [ @ b3 ] \ ], creating healthy work environments \ [ [ @ b4 ], [ @ b5 ] \ ], and a growing demand for customer orientation \ [ [ @ b6 ] \ ] pose challenges for nurse managers \ ' work. more expertise in planning is needed to respond to these issues. one essential area of nurse manager \ ' s management skills is the use of different leadership styles \ [ [ @ b7 ] \ ]. leadership styles can be seen as different combinations of tasks and transaction behaviours that influence people in achieving goals \ [ [ @ b8 ] \ ]. earlier studies indicate that nurse manager \ ' s effective leadership style is affiliated to staff retention \ [ [ @ b5 ] \ ], work unit climate \ [ [ @ b4 ] \ ], nurses \ ' customer satisfaction \ [ [ @ b9 ], [ @ b10 ] \ ], nurses \ ' commitment \ [ [ @ b11 ] \ ], and patient satisfaction \ [ [ @ b12 ] \ ]. transformational leaders style \ [ [ @ b5 ], [ @ b6 ], [ @ b13 ], [ @ b14 ] \ ] and transactional leadership \ [ [ @ b7 ] \ ] help to react to these issues. transformational leadership refers to the leader \ ' s skills to influence others towards achieving goals by changing the followers \ ' beliefs, values, and needs \ [ [ @ b7 ] \ ]. transactional leadership complements and enhances the effects of transformational leadership style \ [ [ @ b15 ] \ ]. there are certain skills required from nurse managers so as to be able into use these effective leadership styles. the skills include the ability to create an organization culture that combines high - quality health care and patient / employee safety and highly developed collaborative and team - building skills \ [ [ @ b1 ] \ ]. nurse managers also choose to have the readiness to evaluate their own behaviour \ [ [ @ b16 ] \ ] and its effects on the work unit ; as a result, employees can adjust to a better leadership style. these kinds of skills are related to manager \ ' s emotional intelligence ( ei ). ei is an ability to lead ourselves and our relationships effectively \ [ [ @ b17 ] \ ]. it has been defined as the ability to observe one \ ' s own and others \ ' feelings and emotions, to discriminate among them and to use this information to direct one \ ' s thinking and actions \ [ [ @ b18 ] \ ]. ei is composed of personal competence and social competence. self - awareness and self - management are reflections of personal competence, influencing the way the leader manages him / herself. social awareness and relationship management reflect social competence, which affects how the leader manages relationships with others \ [ [ @ b17 ] \ ]. nurse managers with that skill can easily form relationships with others, read employees \ ' feelings and responses accurately, and lead successfully \ [ [ @ b19 ] - - [ @ b21 ] \ ]. emotionally intelligent leaders \ ' behaviour also stimulates the creativity of their employees \ [ [ @ b22 ] \ ]. goleman et al. \ [ [ @ b23 ] \ ] have identified visionary, coaching, affiliate, and democratic styles as resonant, and pacesetting and commanding styles as dissonant leadership styles. most leaders use both resonant and dissonant leadership styles. the leadership styles of goleman et al. are applied as the basis of this study because earlier studies refer to the significance of these styles, especially that of ei in manager \ ' s work. in addition, these leadership styles are one way of aiming to carry out transformational leadership. especially visionary, coaching, affiliate, and democratic styles include elements that promote transformational leadership. such elements are for example the leader being visionary and empowering staff \ [ [ @ b4 ] \ ]. this paper focuses on finnish nurse managers \ ' leadership styles. the finnish health care system is a strong institution where health care services are offered to all citizens and funded by taxes \ [ [ @ b24 ] \ ]. it has widely recognized that health care services in finland are of high - quality despite recent concerns about equity issues, finns are in general very satisfied with their health care services. \ [ [ @ b25 ] \ ]. consequently it is important to explore nurse managers \ ' leadership styles especially in this context. 2. materials and methods { # sec2 } = = = = = = = = = = = = = = = = = = = = = = = = 2. 1. aim of the study { # sec2. 1 } - - - - - - - - - - - - - - - - - - - - - the intention of this study was to explore nurses \ ' and supervisors \ ' perceptions of nurse leaders \ ' leadership styles. the research questions were as follows : what kind of leadership styles do nurse managers use and what are the factors affected by their leadership styles. 2. 2. participants { # sec2. 2 } - - - - - - - - - - - - - - - - - to achieve the aim of this study data were collected through open interviews. the majority of finnish nurse managers, nurses, and supervisors work in hospitals or long - term facilities. selection of participants was performed in convenience sampling \ [ [ @ b26 ] \ ]. participants were selected paying attention to the fact they were of different ages, working in different wards and units ( e. g., psychiatry, internal diseases, gerontology ) in either hospitals or long - term facilities, and had worked with more than one nurse manager. the researcher contacted the participants and asked whether they were interested in taking part in the study. the participants were informed about the aim of the study. participation was voluntary. prior to the interviews each participant signed a form where they gave their consent to participate in the study. a total of 11 nurses and 10 supervisors, 20 women and one man, from eight finnish hospitals and five long - term care facilities participated in the study. the age of the nurses varied between 30 and 53 and their experience in health care between 7 and 25 years. the age of the supervisors varied between 38 and 59 and their experience as supervisors between 5 and 21 years. both nurses and supervisors had worked with many nurse leaders and they were interviewed about nurse managers in general. they thus had experience of different nurse managers on different wards and they were able to describe leadership styles from various aspects. 2. 3. data collection and analysis { # sec2. 3 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - semistructured interviews were used to gather data on the perceptions of nurse managers \ ' leadership styles and factors affected by leadership styles. interviews were usually carried out in the office in the participants \ ' workplace. all interviews were recorded with individual consent. participants were initially asked to describe their work and earlier study and work history. they were subsequently asked about their perception of leadership styles and asked to describe the leadership styles used by their nurse managers. after that they were asked about factors affected by leadership styles. each interview was approached individually, guided by participants \ ' responses. the interview sessions lasted between 30 and 85 minutes. every interview was transcribed word for word from the recordings. interviewing was continued until saturation of the data was achieved \ [ [ @ b27 ] \ ]. because nurses and supervisors might have differed in their perceptions of leadership styles, the data were first analysed separately in two separate groups, following the same process for each group. content analysis was chosen because it is a research method for making valid inferences from data to the contexts of their use \ [ [ @ b28 ] \ ]. the interview texts were read through multiple times, based on the author \ ' s empirical and theoretical preunderstanding of the professional area of the participating nurses and nurse managers. a structured categorization matrix of leadership styles was developed based on the primal leadership model \ [ [ @ b23 ] \ ] and research of vesterinen et al. \ [ [ @ b29 ] \ ]. when using a structured matrix of analysis, an item of the data that does not fit the categorization frame is used to create its own concept, based on the principles of inductive content analysis \ [ [ @ b30 ] \ ]. when both the data of nurses and superiors were analysed, the results were compared. the categories and subthemes were congruent and therefore the results are presented together, albeit paying attention to differences and similarities of the perceptions of nurses and superiors. the data analysis of the factors affected by leadership styles was inductive. all the data of nurses and supervisors were analysed together. this process included open coding, creating categories, and abstraction. a classification framework of the factors was formed inductively by defining categories and sub - themes. the criteria for allocating a unit to a category were formed by asking questions if the unit was suitable to the category. the sub - themes were named using descriptive concepts and classified as " belonging " to a particular category. after that, the categories were given names \ [ [ @ b31 ] \ ]. 2. 4. trustworthiness { # sec2. 4 } - - - - - - - - - - - - - - - - - - - - the trustworthiness of this study has been ensured by confirming truth value, consistency, neutrality, and transferability of this study \ [ [ @ b32 ] \ ]. when considering this study from the viewpoint of trustworthiness, there are some threats that should be taken into consideration. the researcher collected the data and performed the analysis alone and the interpretation could have been affected by her professional history \ [ [ @ b33 ] \ ]. with interviews there is a risk that respondents try to please the interviewer by reporting things they assume s / he wants to hear. the researcher confirmed the truth value of the study by selecting participants in convenience sampling. the respondents \ ' age distribution was wide and they worked in different units. their perspectives and descriptions were broad and gave a diverse picture. the truth value of this study was also confirmed by analysing data as they emerged based on the interviews. to ensure the trustworthiness of the study quotes from interviews are included in the results. in view of consistency, the research process is described so that it can be repeated if necessary. this gives a possibility to understand the limitations of the process of data collection and analysis. to ensure neutrality in this study, interpretations were based on original data. this is confirmed by citations from the interview data. in this study the sample was small, consisting of finnish nurses and supervisors, and the results only reflect their perceptions of leadership styles. as a result, transferability of results is limited. however, when considering the main objective in this study, it was not transferability of research results, but it was to enhance understanding of leadership styles and use it for future studies. 2. 5. ethical considerations { # sec2. 5 } - - - - - - - - - - - - - - - - - - - - - - - - - - - the data for this study were collected following approval from the administrations of the organizations. all participants were informed of the | 1. Introduction {# sec1} = = = = = = = = = = = = = = = Health care is changing dynamically in the 2010s. The economic recession and problems with recruiting professionals \ [[ @ B1 ], [@ B2] \ ], staff retention \ [[ @ B3] \ ], creating healthy work environments \ [[ @ B4 ], [@ B5] \ ], and a growing demXmd for customer orientation \ [[ @ B6] \] pose challenges for nurse managers \ ' work. More expertise in management is needed to respond to these issues. One essential area of nurse manager \ ' s management skills is the use of different leadership styles \ [[ @ B7] \ ]. Leadership styles can be seen as different combinations of tasks and transaction behavLoHrs that influence people in achieving goals \ [[ @ B8] \ ]. Earlier studies indicate that nurse manager \ ' s effective leadership style is affiliated to staff retention \ [[ @ B5] \ ], work unit climate \ [[ @ B4] \ ], nurses \ ' job satisfaction \ [[ @ B9 ], [@ B10] \ ], nurses \ ' commitment \ [[ @ B11] \ ], and patient satisfaction \ [[ @ B12] \ ]. Transformational leadership style \ [[ @ B5 ], [@ B6 ], [@ B13 ], [@ B14] \] and transactional leadership \ [[ @ B7] \] help to respond to these issues. Transformational leadership refers to the leader \ ' s skills to influence others towards achieving goals by changing the followers \ ' beliefs, values, and needs \ [[ @ B7] \ ]. Transactional leadership complements and enhances the effects of transformational leadership outcomes \ [[ @ B15] \ ]. There are certain skills required from nurse managers so as to be able to use these effective leadership styles. The skills include the ability to create an organization culture that combines high - quality health care and patient / employee safety and highly developed collaborative and team - building skills \ [[ @ B1] \ ]. Nurse managers also need to have the readiness to observe thDiT own behaviour \ [[ @ B16] \] and its effects on the work unit; as a result, employees can adjust to a better leadership style. These kinds of skills are related to manager \ ' s emotional intelligence (EI ). EI is an ability to lead ourselves and our relationships effectively \ [[ @ B17] \ ]. It has been defined as the ability to observe one \ ' s own and others \ ' feelings and emotions, to discriminate among them and to use this information to direct one \ ' s thinking and actions \ [[ @ B18] \ ]. EI is composed of personal competence and social competence. Self - awareness and self - management are reflections of perQonXl competence, influencing the way the leader manages him / herself. Social awareness and relationship management reflect social competence, which affects how the leader manages relationships with others \ [[ @ B17] \ ]. Nurse managers with that skill can easily form relationships with others, read employees \ ' feelings and responses accurately, and lead successfully \ [[ @ B19] - - [@ B21] \ ]. Emotionally intelligent leaders \ ' behaviour also stimulates the creativity of their employees \ [[ @ B22] \ ]. Goleman et al. \ [[ @ B23] \] have identified visionary, coaching, affiliate, and democratic styles as resonant, and pacesetting and commanding styles as dissonant leadership styles. Most leaders use both resonant and dissonant leadership styles. The leadership styles of Goleman et al. are applied as the basis of this study because earlier studies refer to the significance of these styles, especially that of EI in manager \ ' s work. In addition, these leadership styles are one way of aiming to carry out transformational leadership. Especially visionary, coaching, affiliate, and democratic styles include elements that promote transformational leadership. Such elements are for example the leader being visionary and empowering staff \ [[ @ B4] \ ]. This paper focuses on Finnish nurse managers \ ' leadership styles. The Finnish health care system is a strong institution where health care services are offered to all citizens and funded by taxes \ [[ @ B24] \ ]. It has widely recognized that health care services in Finland are of high - quality Despite recent concerns aHouy equity issues, Finns are in general very satisfied with their health care services. \ [[ @ B25] \ ]. Consequently it is important to explore nurse managers \ ' lFWdership styles especially in this context. 2. Materials and Methods {# sec2} = = = = = = = = = = = = = = = = = = = = = = = = 2. 1. Aim of the Study {# sec2. 1} - - - - - - - - - - - - - - - - - - - - - The intention of this study was to explore nurses \ ' and supervisors \ ' perceptions of nurse leaders \ ' leadership styles. The research questions were as follows: what kind of leadership styles do nurse managers use and what are the factors affected by their leadership styles. 2. 2. Participants {# sec2. 2} - - - - - - - - - - - - - - - - - To achieve the aim of this study data were collected through open interviews. The majority of Finnish nurse managers, nurses, and supervisors work in hospitals or long - term facilities. Selection of participants was performed in convenience sampling \ [[ @ B26] \ ]. Participants were selected paying attention to the fact they were of different ages, working in different wards and units (e. g. , psychiatry, internal diseases, gerontology) in either hospitals or long - term facilities, and had worked with more than one nurse manager. The researcher contacted the participants and asked whether they were interested in taking part in the study. The participants were informed about the aim of the study. Participation was voluntary. Prior to the interviews each participant signed a form where they gave their consent to participate in the study. A total of 11 nurses and 10 supervisors, 20 women and one man, from eight Finnish hospitals and five long - term care facilities participated in the study. The age of the nurses varied between 30 and 53 and their experience in health care between 7 and 25 years. The age of the supervisors varied between 38 and 59 and their experience as supervisors between 5 and 21 years. Both nurses and supervisors had worked with many nurse leaders and they were interviewed about nurse managers in general. They thus had experience of different nurse managers on different wards and they were able to describe leadership styles from various aspects. 2. 3. Data Collection and Analysis {# sec2. 3} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Semistructured interviews were used to gather data on the perceptions of nurse managers \ ' leadership styles and factors affected by leadership styles. Interviews were usually carried out in the office in the participants \ ' workplace. All interviews were recorded with individual consent. Participants were initially asked to describe their work and earlier study and work history. They were subsequently asked about their perception of leadership styles and asked to describe the leadership styles used by their nurse managers. qftet that they were asked about factors affected by leadership styles. Each interview was approached individually, guided by participants \ ' responses. The interview sessions lasted between 30 and 85 minutes. Every interview was transcribed word for word from the recordings. Interviewing was continued until saturation of the data was achieved \ [[ @ B27] \ ]. Because nurses and supervisors might have differed in their perceptions of leadership styles, the data were first analysed separately in two separate groups, following the same process for each group. Content analysis was chosen because it is a research method for making valid inferences from data to the contexts of their use \ [[ @ B28] \ ]. The interview texts were read through multiple times, based on the author \ ' s empirical and theoretical preunderstanding of the professional area of the participating nurses and nurse managers. A structured categorization matrix of leadership styles was developed based on the primal leadership model \ [[ @ B23] \] and research of Vesterinen et al. \ [[ @ B29] \ ]. When using a structured matrix of analysis, an item of the data that does not fit the categorization frame is used to create its own concept, based on the principles of inductive content analysis \ [[ @ B30] \ ]. When both the data of nurses and superiors were analysed, the results were compared. The categories and subthemes were c0ngruen% and therefore the results are presented together, albeit paying attention to differences and similarities of the perceptions of nurses and superiors. The data analysis of the factors affected by leadership styles was inductive. All the data of nurses and supervisors were analysed together. This process included open coding, creating categories, and abstraction. A classification framework of the factors was formed inductively by defining categories and sub - themes. The criteria for allocating a unit to a category were formed by asking questions if the unit was suitable to the category. The sub - themes were named using descriptive concepts and classified as " belonging " to a particular category. After that, the categories were given names \ [[ @ B31] \ ]. 2. 4. Trustworthiness {# sec2. 4} - - - - - - - - - - - - - - - - - - - - The trustworthiness of this study has been ensured by confirming truth value, consistency, neutrality, and transferability of this study \ [[ @ B32] \ ]. When considering this study from the viewpoint of trustworthiness, there are some threats that should be taken into consideration. The researcher collected the data and performed the analysis alone and the interpretation could have been affected by her professional history \ [[ @ B33] \ ]. With interviews there is a risk that respondents try to please the interviewer by reporting things they assume s / he wants to hear. The researcher confirmed the truth value of the study by selecting participants in c0bvenience sampling. The respondents \ ' age distribution was wide and they worked in different units. Their perspectives and descriptions were broad and gave a diverse picture. The truth value of this study was also confirmed by analysing data as they emerged based on the interviews. To ensure the trustworthiness of the study quotes from interviews are included in the results. In view of consistency, the research process is described so that it can be repeated if necessary. This gives a possibility to understand the limitations of the process of data collection and analysis. To ensure neutrality in this study, interpretations were based on original data. This is confirmed by citations from the interview data. In this study the sample was small, consisting of Finnish nurses and supervisors, and the results only reflect their perceptions of leadership styles. As a result, transferability of results is limited. However, when considering the main objective in this study, it was not transferability of research results, but it was to enhance understanding of leadership styles and use it for future studies. 2. 5. Ethical Considerations {# sec2. 5} - - - - - - - - - - - - - - - - - - - - - - - - - - - The data for this study were collected following approval from the administrations of the orNanlzations. All participants were informed of the | Introduction {#sec1} =============== Health care is changing dynamically in the 2010s. The economic and problems with recruiting professionals \[[@B1], [@B2]\], staff retention \[[@B3]\], creating healthy work environments \[[@B4], [@B5]\], a growing demand for customer orientation \[[@B6]\] challenges for nurse managers\' work. More expertise in management needed to respond to these issues. One area of nurse manager\'s management skills is the use of different leadership styles Leadership styles can be seen as different combinations of tasks and transaction behaviours that influence people in achieving goals \[[@B8]\]. Earlier studies indicate that nurse manager\'s effective leadership style is affiliated to staff retention \[[@B5]\], work unit climate \[[@B4]\], nurses\' job satisfaction \[[@B9], [@B10]\], nurses\' commitment \[[@B11]\], and patient satisfaction Transformational style \[[@B5], [@B6], [@B13], [@B14]\] and transactional leadership \[[@B7]\] help to respond to these Transformational leadership refers to leader\'s skills to influence others towards achieving goals by changing the followers\' beliefs, values, and needs \[[@B7]\]. Transactional leadership complements and enhances the effects of transformational outcomes \[[@B15]\]. There are certain skills required from nurse managers as to be able to use these effective leadership styles. The skills include the ability to create an culture that high-quality health care and safety and highly developed collaborative and team-building skills \[[@B1]\]. Nurse managers also need to have the readiness to observe their own behaviour and its effects on the work unit; as a result, employees can to a better leadership style. These kinds skills are related to manager\'s emotional intelligence EI is an ability to lead and our relationships effectively \[[@B17]\]. It has been defined as the ability to one\'s and others\' feelings and emotions, to discriminate among them and use this information to direct thinking and actions \[[@B18]\]. EI is composed personal competence social competence. Self-awareness and self-management are reflections of personal competence, influencing the way the leader him/herself. Social awareness relationship management social competence, which affects how the leader manages relationships others \[[@B17]\]. Nurse managers with that skill can easily form with others, read feelings and accurately, and lead successfully Emotionally intelligent leaders\' behaviour also the creativity of their employees \[[@B22]\]. Goleman et al. \[[@B23]\] have identified visionary, coaching, affiliate, and democratic styles as and pacesetting commanding styles as dissonant leadership styles. Most leaders both resonant and styles. The leadership of Goleman et al. are applied as the basis of this study because earlier refer to the of these especially that of EI in manager\'s work. these leadership styles are one way of aiming to carry transformational leadership. Especially visionary, coaching, affiliate, and democratic styles include elements that promote transformational leadership. Such elements are for example the leader being and empowering staff \[[@B4]\]. This paper focuses on Finnish nurse managers\' leadership styles. The Finnish health care system is a strong institution where health care services are offered to all citizens and funded by taxes \[[@B24]\]. It has widely recognized that care services in Finland are of high-quality Despite recent concerns about equity issues, Finns are in general satisfied with their health care services. \[[@B25]\]. Consequently is important to explore nurse managers\' leadership styles especially in this context. 2. Materials and Methods {#sec2} ======================== 2.1. Aim of Study {#sec2.1} --------------------- The of this study to explore nurses\' and supervisors\' of nurse leaders\' leadership styles. The research questions were as follows: what kind of leadership styles do nurse and what are the factors affected by their leadership styles. 2.2. Participants ----------------- To achieve the aim of data were collected through open interviews. of Finnish nurse managers, nurses, and supervisors work in hospitals or long-term facilities. of participants was performed in convenience sampling \[[@B26]\]. were selected paying attention to the fact they were of different ages, working in different wards and units (e.g., psychiatry, internal diseases, gerontology) in either hospitals or long-term facilities, and had worked with than one nurse manager. The researcher contacted the participants and asked whether they in taking part in the study. The participants were the aim of the study. Participation was voluntary. Prior to the interviews each signed a where they gave their consent to participate in the study. A total of 11 nurses and 10 supervisors, 20 women and one man, from Finnish hospitals and five long-term care facilities participated in the study. The age of the nurses varied between 30 53 and their experience in health care between 7 25 years. The of supervisors varied between 59 and their experience as supervisors between 5 and years. Both nurses and supervisors had worked many nurse leaders and they were interviewed about nurse managers in general. had experience of different nurse managers on different wards and they were able describe leadership styles from various aspects. 2.3. Data and Analysis {#sec2.3} --------------------------------- Semistructured interviews were used to gather data on perceptions of nurse managers\' leadership styles and affected by leadership styles. Interviews were usually carried out in the in the participants\' workplace. All interviews were with individual consent. Participants were initially asked to describe work and earlier study and work history. They were subsequently asked about their perception of leadership styles and asked to describe the leadership styles used by their nurse managers. After that they were asked about factors affected by leadership styles. Each interview was approached individually, guided by participants\' responses. The interview sessions lasted between 30 and 85 Every interview was transcribed word for word from the recordings. was continued until saturation of the data was achieved Because nurses and supervisors might differed in their perceptions of leadership styles, the data were first analysed separately in two separate groups, following same process each group. analysis was chosen because it is research method for making inferences from data to the contexts of their use \[[@B28]\]. The interview were read through multiple times, based on the author\'s and theoretical preunderstanding of the professional area of the participating nurses and nurse A structured categorization matrix of leadership styles was developed based on the primal leadership model and research of Vesterinen et al. \[[@B29]\]. When structured of analysis, an item the data that does not fit the categorization frame is used create its own concept, based on the principles of inductive content analysis \[[@B30]\]. When both the data of nurses and superiors were analysed, the results were compared. The categories and subthemes were congruent and therefore the results are presented together, albeit paying attention to differences and of the perceptions of nurses and superiors. The data analysis of the factors affected by leadership styles was inductive. All data of nurses and supervisors analysed together. This process included open coding, creating categories, and abstraction. A framework of factors was formed inductively by defining categories and sub-themes. The criteria for allocating a unit to a category were formed by asking questions if the was suitable to the category. The sub-themes were named using descriptive concepts and classified as "belonging" a particular category. that, the categories were given names \[[@B31]\]. Trustworthiness {#sec2.4} -------------------- The trustworthiness this study has been ensured by confirming truth value, consistency, neutrality, and transferability of this study \[[@B32]\]. When considering study the viewpoint of trustworthiness, there are some threats that should be taken into consideration. collected the data and performed the analysis alone and the interpretation been affected by her professional history \[[@B33]\]. With interviews there is a risk that respondents try to please interviewer by reporting things they assume wants to hear. The researcher confirmed the value of the by selecting participants convenience sampling. The respondents\' age distribution was wide and they worked in different units. perspectives and descriptions were broad and a diverse picture. The truth value this was also confirmed by analysing data as emerged based on interviews. To ensure the trustworthiness of the study from interviews are included in the results. In view of process is described so that it can be repeated if This gives a possibility to understand the limitations of the process of data collection and analysis. To ensure neutrality in this study, interpretations were based on original data. is confirmed by citations from the interview data. In this study sample small, consisting of Finnish nurses and supervisors, and the results only reflect their perceptions of leadership styles. a result, of results is limited. However, when considering the main objective in this was transferability of research results, but it was to enhance understanding of leadership styles and use it for 2.5. Ethical Considerations {#sec2.5} --------------------------- The for this study were collected following approval from administrations of the participants were informed the | 1. inTroDUcTIoN {#SeC1}
===============
hEaltH CaRe iS ChANGiNG dYnamICaLLy In THE 2010S. thE econOmic recEsSioN aNd PRoBlems With rECRuITINg proFESsionaLs \[[@B1], [@b2]\], stAfF rEteNTiOn \[[@B3]\], CrEaTInG HealTHy woRk envIROnMentS \[[@b4], [@b5]\], And A gRoWinG dEMANd fOr CuSTOmER orIENtAtiON \[[@B6]\] poSE ChAlLENGEs FoR nUrsE maNAgErs\' work. MoRE EXpERtIse in MAnAgement Is nEEDED TO REspond TO THese ISSues.
oNe esSENTiaL Area Of nurSe mANagEr\'S MaNaGemENT skIlLS is tHE use Of DiFfEReNT LeaDeRSHiP StyLes \[[@b7]\]. LeAdership sTyLeS can BE sEeN as DIfFerEnt COMBInaTiONs of TasKS ANd TRaNsactIon bEhAViOurs tHAT InflUencE PeOpLE In AchieVinG GOals \[[@b8]\]. EArlIER StUDies iNDiCatE THaT nURSe mAnAgeR\'S effecTiVE LEadErsHIp sTyLe IS AffILiATed to staff rETEnTIOn \[[@B5]\], work uNiT Climate \[[@B4]\], nUrSEs\' JOB SATIsfACtioN \[[@b9], [@b10]\], NURSEs\' COmmitmeNt \[[@B11]\], anD pAtIEnT sAtiSfACtION \[[@b12]\].
tRanSfORmAtIonaL LEAderSHiP sTyLE \[[@b5], [@B6], [@B13], [@B14]\] aND trAnsActIoNaL LeADERshIP \[[@B7]\] Help to RespoNd TO tHESE ISsUes. TrANsFORmaTiOnal LeaDERsHiP REfeRS To the LEadEr\'s SKills TO inflUENCE othERS TOwARds AcHievINg gOALS BY cHANging thE FOllOWeRS\' bELIefS, valueS, AnD nEEDs \[[@b7]\]. tRanSACtIonaL LEADerSHiP coMpLeMENtS AND enHancEs the EFFeCtS OF trAnsFOrmationaL lEadeRsHip OUTcomes \[[@b15]\].
tHEre arE CErTaIn sKiLLs requiReD FrOm NUrse MaNaGeRs so as to be ABle To uSe ThESE EffEcTIVe lEadERship sTYLeS. THE SkIlLs incluDE tHE aBILIty TO CReaTE aN orGAniZAtIOn culturE That comBiNEs HiGH-quAliTY HeaLTH CARE and PAtienT/EMPLoYEe SaFETy AND HighLy dEvelOped collabOrATiVE aNd tEaM-buildInG SkIllS \[[@b1]\]. nUrse manAGErS aLsO nEeD TO hAVe THE reAdINesS to ObSERvE THeiR oWn BEhAViour \[[@b16]\] AND iTs EFfECtS ON THe work UnIt; As A ReSULT, EmpLOYees CaN ADJust TO A BEtTer leaDERshIp sTYLE. THESE kiNdS Of skiLlS Are ReLated to manaGer\'s EMotiOnal IntElLiGeNCE (Ei).
ei IS aN aBiLITy tO Lead oURSelvEs And ouR ReLatioNShIPS efFEcTiVELY \[[@B17]\]. IT HAs beEn dEfIned As The aBILitY To oBSERvE ONe\'s OWn AND oTHErS\' FeelInGS And EMOTiOnS, to disCriMInate AmOng tHEM and TO uSe THIS INfORmatIoN tO DIRect oNe\'s tHinKINg and actIONs \[[@B18]\]. eI iS COmpoSed of PerSonal CoMpetenCE AND SOcIaL cOmpeTencE. seLf-awaREnESS ANd sElf-MAnAGeMEnT ArE ReFLEcTions Of pERSonal cOmpETEncE, INfluEnCING ThE wAY The LEADeR MaNaGES him/heRselF. SoCiaL aWArenEss And rELATIOnSHIP MANagEment rEFLeCT sociaL COmpeTEnCe, wHIcH AFfeCTs How tHE lEADEr mANAGeS RELATionShipS WiTH othERs \[[@B17]\].
NURSE MANaGErS wIth tHat sKilL can eASILy FORm rELATiONsHips wiTh otheRs, reaD EmPlOYeeS\' feEliNgS ANd reSPonsES AccUraTeLY, anD LEAD SuCcesSfuLly \[[@b19]--[@B21]\]. eMoTIOnAlly inTellIGEnT LeADERs\' bEhavioUr aLSo stImuLATes thE CreATivitY oF THeiR eMpLoyeES \[[@b22]\]. gOLEMAN et aL. \[[@b23]\] haVE ideNTiFieD VIsiOnarY, CoachING, AfFIlIATE, AnD DeMoCratiC STYLEs AS resoNaNT, anD PaCesEttInG And cOmmanDing stYLes as DissoNAnT lEADeRshIp STYlES. moST LEAdErs usE BoTh RESonant aNd DISSoNaNT leADERship stYLeS. ThE leAdersHIp sTyLES Of GOlemAn ET AL. aRe AppliED as tHE bASIs of thIS STUdY beCauSE EArLier Studies reFER TO the signIfICAnce Of THeSE styleS, EspEciALly tHaT Of eI IN maNaGER\'S WORK. IN AdDItiON, THese LEADerShiP stYleS arE ONE way oF aImINg to cARRy ouT tRansformAtioNAL leadeRship. esPeCIAllY ViSiOnARy, cOaCHiNG, afFiliAte, ANd DEmOcRATIC sTYLeS iNcLUDe eLeMENtS ThaT PROMoTE TrANsFoRMATIoNAl lEaDersHip. SuCh eleMeNtS aRE for eXamPLE ThE LEADEr bEING vISionArY and eMpOweriNg STaFf \[[@b4]\].
THis PapEr foCUsEs ON FINNisH NUrSE maNAGErS\' leADERSHIp StYLEs. thE FiNNiSH HeAlTH caRE sySTEM is A sTRoNg iNsTitUTiOn whERe hEaLTh cARe sErVIceS aRE OFFeRed TO aLL citIZeNs AND fundeD BY TAxes \[[@B24]\]. iT HAs WIdelY RecoGnIzED thAt healTh caRe seRviCES in fiNLAnd ARE oF HIgH-quAlIty deSPITe ReCenT COnceRnS AbOUt EqUiTY isSUes, FinNS arE in GeNeraL VERY SATIsFied WitH Their Health caRe SeRViCeS. \[[@b25]\]. CoNseqUEnTLY it Is IMPORtanT To EXPLORE nuRSe MaNAGERs\' lEAdERsHIp STYles esPEcIAllY in ThIs CoNTeXt.
2. MATeRIaLS And methoDs {#sEc2}
========================
2.1. AIm Of THe StUdY {#Sec2.1}
---------------------
ThE INTENTiOn Of thIs sTudY was To ExpLoRE NuRsEs\' AnD SuPeRviSORS\' PErCEptIons of nuRSE lEAdERs\' LeAdersHip sTylES. tHE REsEarch QuestIONs WeRE As fOLlows: whaT KinD OF LEadERSHiP sTylEs do NuRsE MAnAgErs USe AnD WhAT ARe THe fAcTORs AfFEcted by THEiR LEaDersHIp sTYLEs.
2.2. PaRTICipanTs {#seC2.2}
-----------------
tO ACHIEVe ThE aiM oF thIS StUDY dAtA wERe COlLECtEd THroUgh Open iNteRvIeWS. thE MaJORiTY of finniSH NURse mANageRs, nuRSes, aNd sUpErVISORs wORK IN hoSPITals oR lonG-tERm faCILiTiES. sElEcTiON of pArTIcIpaNTS WAs PeRfOrMEd In CoNvenieNCE samPLinG \[[@B26]\]. paRTicipANtS WErE SeLECtED paYing aTTEntIOn to tHE FacT They WERE oF DIfFeREnT aGES, WORkinG iN DIfFerenT WArdS ANd UniTS (E.g., PsycHIATRY, INtERNaL diSeASeS, GeronTOLOgY) in EitHER hospiTaLS or lOnG-tERm FaciLItIeS, anD hAD wORkeD wITH MOre THaN oNE NuRsE MANAGer.
THE ReseaRChEr cONTaCtED tHe PaRTICIpANTS AND AskED WHeTHER THEY were iNTeRESted in TaKING pArT In THE sTUdy. The ParTIcIPaNTs wErE iNforMED abOut the aim oF ThE sTUDY. pARtIcipaTION WaS voLUNTaRY. PrIor TO THE INTERVIews eACH PARTicIPaNT SiGNEd A fOrM WhErE ThEY gAvE THEiR consenT To PArtiCIPaTE In tHE stUdy. a toTAL OF 11 nURses aND 10 sUPeRVisOrs, 20 WoMEn AND oNE Man, FRom eIghT finNISH hOspItALs aND fivE lOng-TeRM CARe faCiLiTIES paRTIcipateD In the sTuDY. thE Age oF thE nuRSes VARied betWeEN 30 AND 53 AND Their eXpEriEnCE IN hEAlth CarE bEtWEeN 7 and 25 yEaRS. THE age oF the SUPeRViSORS vAriED beTWeeN 38 ANd 59 ANd tHEIr ExpErIEnCe as SUPERVIsors bETween 5 and 21 YeaRs. boTh nurseS ANd sUPERVIsors had WorkEd WItH ManY NurSe leAdErS AnD ThEy weRe InTeRViewED ABoUt nURSE mAnagers In General. THey THus haD expERieNcE of DIFfErent nUrSE MANaGERs on diffeRENt WaRdS aNd THey WeRe aBLE to DescrIbE LEAdERshiP sTyLeS frOm VarioUS aSpECts.
2.3. data COlLeCTIon AnD aNalYsiS {#seC2.3}
---------------------------------
SeMiStRUctuREd INtERViEWs WERE usED to gAThEr DaTA On the perCEpTIOnS of NuRSe mAnaGerS\' lEADership StYLEs anD fACTORs AFFEctEd BY LEaDerShIP STylEs. IntervIeWs werE UsUaLLY carrIEd out IN thE OFFIcE iN thE pArTiCIpAnts\' WORKpLAce. alL intERvIEwS wEre rECoRDed WIth indIvidUAl COnseNt. PARTiCipaNTs WERe iNiTIaLlY aSked To dEScRiBE THeiR woRK AnD EARlIer sTUdY ANd wOrK hIStOry. thEy wErE sUbseqUeNTlY ASKEd AbOUt TheiR pERCEptiON OF leAdERshIp stYleS ANd ASKEd To DEscRiBe thE LeaDeRShip STyLES usED By thEiR NURSE MAnaGeRS. AFtEr tHAT TheY WeRe aSkED AbOut FActOrs AffECtEd by LeaderShIP StYLES. Each iNtErVIEW wAs ApPrOAChEd inDIViduAlLy, guidEd bY PArTiCIPAnTS\' RESPonsEs. ThE intervIew SeSsiOns lasteD betWEEN 30 aNd 85 mINUtes. EVeRY intErvIew waS TRANScRibed worD foR wORD fROM The recORDINGs. INTErvIeWINg WAs CONTInUeD UnTiL sAtuRaTIon of tHE data WAs achievEd \[[@B27]\].
beCAuSE NURsEs AND SupErvISoRs mIghT haVe DIFFEred in tHeIr percepTionS Of leaDeRshiP StYLes, THe DatA weRE firSt AnALYsED sePaRAtELY in TWo SePArAtE groups, FoLLoWInG the Same pROcesS fOR eACh gRoUP. ContENT aNAlySIS waS choSEn beCAUSe it is a rEseArcH mEThoD For mAKINg ValiD INFerencEs froM dATa TO THE CONTEXts oF THeir uSE \[[@b28]\].
thE INtErVIew tExts WEre READ THROuGH mULtiple TIMEs, BASeD On the AuthOR\'s EMpiRIcAl aNd ThEorEtiCal PReuNDErStANdINg of The PrOFEsSIONal aREa oF THe PARTicIpatinG NuRSEs ANd nurse mAnaGers. A STRuctuRed cateGORIZAtiOn MaTrIX of lEadERSHIP stYLes waS DEVEloPed BaseD on The primaL LeADErShIp moDeL \[[@B23]\] AnD REseARch Of VesterINen eT aL. \[[@b29]\]. wheN UsiNg a sTruCtUreD mATrIX OF aNAlYsiS, aN ITem OF thE dAta ThaT does noT FiT THE cAteGORIzATiOn frAme Is USED to CrEate iTs Own cONcEPT, BaSED oN thE pRinCiPlEs OF INdUctive coNteNt ANalYSIS \[[@b30]\]. WhEN BOTh tHe daTa OF nurSES ANd sUPErIOrs WERE ANaLYSeD, tHe reSUlTS wERe coMpAREd. tHE CaTegORiES And SuBTHEMeS weRe CongRuEnT AnD tHeREfore The rEsULtS ArE PreSenTED TOGetheR, ALBEiT PaYING aTTentION tO DIffeREnceS and sImILARITIES oF The PercEpTIoNS OF nuRSes and suPerIORs.
THe dATA analYSIs oF The FaCtors AfFEctEd by lEadeRsHIp StyLES Was INDUCtIVE. all tHe DaTA OF NurseS And suPeRVIsors were aNaLYsEd ToGetHER. tHIS pRocesS iNCluDEd OpeN CoDIng, CreAtiNg categoRIeS, And aBSTrActIOn. A claSsIficaTiOn fRAmEWoRK OF tHe fActoRs WAs ForMeD iNDuctiVeLy by DefInING categoRiEs ANd Sub-tHEMEs. THe CriTeRiA FOr ALLOCAtINg a unIt tO A caTEgorY weRe FoRmEd By askInG quEsTions iF The uNIT WaS suiTABLE to tHE cATEgORy. THe sUB-THEMEs wERe nAMed USIng DescRIpTIvE CONCeptS ANd clAsSIfIEd AS "beloNGinG" to a PARtiCUlar CateGORY. AFtER THaT, THE CATEgOrIes wEre GivEn names \[[@B31]\].
2.4. tRUSTworThiNesS {#SEc2.4}
--------------------
ThE truSTwOrThiNESS Of ThIS stuDy has BEEn EnSuRED BY conFirMing TRuth ValUe, COnSiSTeNCY, neUTRALITy, AnD TransFERabiliTY OF THIs sTUdy \[[@b32]\].
wHEN cOnSIdErIng this STuDY FroM tHe VIEWpOInt oF tRUSTwORTHiNess, ThERE ARE SOMe ThREAtS THAt shOULd bE taKEN inTO CONsideraTION. thE RESeaRChER COllecTEd The Data aNd PerfoRmED The AnALysiS aLOnE aNd tHE iNteRprEtatioN cOulD haVe beeN afFeCTeD By hEr pRofEssIonaL HIsTorY \[[@B33]\]. with iNTerVIewS thEre IS A riSk ThAT reSpondentS TRY TO pleAsE THE INTervIeWER BY REPoRTing thiNgs THey ASSume s/He wAntS to hEAR. tHE rESeARcHer coNFirmed The trUtH VaLUE OF THe STudY by sELEcTiNg paRTICiPaNTs in conveNIence SampLing. tHE rEspONdEnTs\' AGE diStribUtIoN WAs WiDE aNd tHey WORKeD iN DIfFereNt UNItS. ThEIr PErSpectIves AnD dESCRIpTionS wERe BrOAD aNd GavE a divErSe piCtURE.
tHE TRutH vAlUE OF ThIS stuDY was ALsO ConfiRmED BY ANalySInG dATA aS THeY emERgEd bAseD on thE iNTERViEWs. TO ENSurE The TRUStWoRTHIneSS oF the studY qUOTES FrOm intervieWS ARE iNCluDeD in tHE ResUltS.
In vieW oF cONsISteNcY, the ReSeaRcH PrOceSs iS DEscrIbEd SO thAT iT CAN bE RePeAtED if NEcESsarY. tHiS givES A POsSibiLITY TO uNdeRstAnd tHe LiMITAtIOnS Of tHe proCesS Of dAtA colleCtiOn And ANaLYSIs. TO ensuRe neutRAlitY IN this stUDy, inTErPRetaTIONS wEre based oN OrIgINal DaTA. ThiS Is conFIRMEd BY CItaTionS fROM ThE INTerVIEw DaTA.
iN ThIs StudY THE samplE WAs SMall, cONsisting oF fINNish nurSES ANd sUpErViSors, And thE resuLts oNly rEfLEcT thEIR PercepTiOns Of lEadERShIP stYlES. aS A RESULt, tRanSFEraBIliTY OF resULTS is limIteD. HowEvEr, WHEn CoNsiDeRiNG the mAIn oBJeCtivE in ThiS StUdY, it wAS not tRANsFerABiLIty OF researCH Results, BUT It Was To EnhancE UnDERSTaNDING OF lEADeRShip STylES aND UsE iT for FUTUrE STuDiEs.
2.5. ETHicAl coNsiDeRaTioNs {#SeC2.5}
---------------------------
thE DATa fOr This STUdY WERE colLEcted foLLOWinG aPProvAl fROM thE aDMInIstrAtIONS OF tHE OrGANIzaTIONs. alL paRTIciPaNTs werE inFORmed OF THe | 1. Introduction {#sec1} ===============Health careis changing dynamicallyin the 2010s. The economic recession and problems with recruiting professionals \[[@B1], [@B2]\], staff retention \[[@B3]\],creating healthy work environments\[[@B4], [@B5]\],and a growing demand for customer orientation \[[@B6]\] pose challenges for nurse managers\' work. More expertise in management is needed to respond to theseissues. One essential area ofnurse manager\'s management skills is the use ofdifferent leadership styles \[[@B7]\]. Leadership stylescan be seen as differentcombinations of tasks and transactionbehaviours that influence people in achieving goals \[[@B8]\]. Earlier studies indicate that nurse manager\'seffective leadershipstyle is affiliated to staff retention \[[@B5]\], work unitclimate \[[@B4]\], nurses\' job satisfaction\[[@B9],[@B10]\], nurses\' commitment \[[@B11]\], and patient satisfaction \[[@B12]\]. Transformational leadership style\[[@B5], [@B6], [@B13], [@B14]\]andtransactional leadership \[[@B7]\] help torespondto these issues. Transformational leadershiprefersto the leader\'sskills to influence others towardsachievinggoals by changing the followers\' beliefs, values, and needs \[[@B7]\]. Transactionalleadershipcomplements and enhances the effects of transformational leadership outcomes \[[@B15]\]. There arecertain skills required from nurse managers so asto be able to use these effective leadership styles. The skills include theability tocreate an organizationculture that combines high-quality health care and patient/employee safety and highly developed collaborativeand team-building skills \[[@B1]\]. Nurse managers also need to havethe readiness toobserve their own behaviour \[[@B16]\] and its effects on the work unit;as a result, employees can adjust to a better leadership style.These kinds of skills are relatedto manager\'semotional intelligence (EI). EI is an ability to lead ourselves and ourrelationshipseffectively \[[@B17]\]. It has been defined as the ability to observe one\'s own and others\' feelings and emotions, to discriminate among them and touse this information to direct one\'sthinking and actions \[[@B18]\]. EI is composed of personal competence and socialcompetence. Self-awareness and self-management are reflections of personal competence, influencingthe way the leadermanages him/herself. Socialawareness and relationship management reflectsocial competence, which affects how the leader manages relationships with others \[[@B17]\]. Nurse managers with that skill can easily form relationships withothers, reademployees\' feelings andresponses accurately, andlead successfully\[[@B19]--[@B21]\]. Emotionally intelligent leaders\' behaviour also stimulates thecreativityoftheir employees \[[@B22]\]. Golemanet al. \[[@B23]\] have identified visionary, coaching, affiliate, and democraticstyles as resonant, and pacesetting and commanding styles as dissonant leadership styles. Most leaders use both resonant and dissonant leadership styles.The leadership styles of Golemanet al. are applied asthe basis of this studybecause earlier studies refer to the significance of these styles, especially that ofEI in manager\'s work. Inaddition,these leadership styles are one wayof aiming to carry outtransformationalleadership.Especiallyvisionary,coaching, affiliate,and democratic styles include elements thatpromote transformational leadership. Such elements are for example the leader being visionary andempowering staff \[[@B4]\]. This paper focuses on Finnish nurse managers\' leadership styles. The Finnish health care system is astrong institution where health care services are offered to all citizensand funded bytaxes \[[@B24]\].It haswidely recognized that healthcare services in Finland are of high-quality Despite recent concerns about equity issues, Finns are in general very satisfied with their health care services. \[[@B25]\]. Consequently itis important to explore nurse managers\'leadership styles especially in this context. 2. Materials and Methods {#sec2} ======================== 2.1. Aim ofthe Study {#sec2.1} ---------------------The intentionof this studywas toexplorenurses\' and supervisors\' perceptions of nurse leaders\' leadership styles. The researchquestions were as follows: whatkind of leadership styles do nurse managers use and what are the factors affected by theirleadershipstyles.2.2. Participants {#sec2.2} ----------------- To achieve the aim of this study data were collectedthrough open interviews. The majority ofFinnish nurse managers, nurses, and supervisors work inhospitals or long-term facilities. Selectionofparticipants was performed in convenience sampling \[[@B26]\]. Participants were selected paying attention to the fact they were of different ages, working in differentwards and units (e.g.,psychiatry, internal diseases, gerontology) in either hospitals or long-term facilities,and hadworked with more than one nurse manager.The researchercontacted the participants and asked whether they were interestedin taking part in the study. The participants were informed about the aim of thestudy. Participationwas voluntary. Prior to theinterviews each participant signed a form where they gave their consent to participate in the study.A total of11 nurses and 10 supervisors, 20 women and one man, from eight Finnish hospitals and five long-term carefacilities participated in the study. The age of the nursesvaried between 30 and53 and their experience in health care between 7 and 25years. The age of the supervisors varied between 38and 59 and theirexperienceas supervisors between 5 and 21 years. Both nursesand supervisors hadworkedwith many nurse leaders and they were interviewed about nurse managers in general. Theythus had experience of different nurse managers ondifferent wardsand theywere able to describe leadership stylesfrom various aspects. 2.3. Data Collectionand Analysis {#sec2.3} --------------------------------- Semistructured interviews were used to gather data on the perceptions of nurse managers\'leadership styles and factors affected by leadership styles.Interviews were usually carried out inthe office in the participants\' workplace. All interviews were recorded with individual consent. Participantswere initially asked to describe their work and earlier study and work history. Theywere subsequently asked about their perceptionof leadership styles and asked to describe the leadership styles used by their nurse managers. After that they wereasked about factors affected by leadership styles. Eachinterviewwas approached individually, guided by participants\' responses. The interview sessions lasted between 30 and 85 minutes. Every interview was transcribed word for word from the recordings. Interviewing was continued until saturation of the data was achieved \[[@B27]\]. Because nursesand supervisors might have differed in their perceptions ofleadership styles, the data were first analysed separately in two separategroups, following the same process for each group.Contentanalysis was chosen because it is a research methodformaking valid inferences from data to the contextsof their use \[[@B28]\]. Theinterview texts wereread through multipletimes, based on the author\'s empirical and theoretical preunderstanding ofthe professional area of theparticipating nurses and nurse managers. Astructured categorization matrix of leadership styles wasdevelopedbased on the primal leadership model \[[@B23]\] andresearchof Vesterinen et al. \[[@B29]\]. When using a structured matrix of analysis, an item of the datathat does notfit the categorizationframeisused tocreate its own concept, based on theprinciples of inductive content analysis \[[@B30]\]. When both the data of nurses and superiors wereanalysed, the results were compared. The categories and subthemes were congruent and therefore the results are presented together, albeit paying attentionto differences andsimilarities ofthe perceptions of nurses and superiors. The data analysisofthe factors affected by leadership styles wasinductive. Allthe data of nurses and supervisors wereanalysed together. This process included open coding, creating categories, andabstraction. A classification framework of the factors was formed inductively by defining categories and sub-themes. The criteria for allocating a unit to a category were formed by asking questions if the unitwassuitable to thecategory. Thesub-themes were named using descriptiveconcepts and classifiedas "belonging" to a particular category. After that, the categories were given names \[[@B31]\]. 2.4. Trustworthiness {#sec2.4}-------------------- The trustworthiness of thisstudy has been ensured byconfirming truth value, consistency, neutrality, and transferability of this study \[[@B32]\]. Whenconsidering this studyfrom the viewpoint of trustworthiness, there are some threats that should be takeninto consideration. The researcher collected the data and performed the analysis alone and the interpretation could have been affected by her professional history \[[@B33]\].With interviews there isa risk that respondents try to please the interviewer by reporting things they assume s/hewants to hear. The researcher confirmed thetruth value of the study byselecting participants in convenience sampling. The respondents\' age distribution was wide and they workedin different units. Their perspectivesand descriptions were broad and gave a diverse picture. The truthvalueof this study was also confirmedby analysingdataas they emergedbased on the interviews. To ensure the trustworthiness of the study quotes from interviews are included in the results. In view ofconsistency, the research process is described so that it can be repeated if necessary. This gives a possibilityto understand the limitationsof the process of data collection and analysis. To ensure neutrality in this study, interpretations were based on original data. This is confirmedby citations from the interview data. In this study thesample was small, consisting of Finnish nursesand supervisors, andthe results only reflect their perceptions of leadershipstyles. As a result, transferability of results is limited. However, when considering the main objective in this study, it was not transferability of research results, but it was to enhance understanding ofleadership styles and use itfor future studies. 2.5. Ethical Considerations {#sec2.5} --------------------------- The data for thisstudywere collected following approval from the administrations of the organizations. All participants were informed of the | 1. Introduction {#sec1} =============== Health care is changing dynamically in the 2010s. _The_ economic recession and _problems_ with recruiting _professionals_ _\[[@B1],_ [@B2]\], staff retention \[[@B3]\], _creating_ healthy work environments \[[@B4], [@B5]\], and a growing demand for customer orientation _\[[@B6]\]_ _pose_ challenges _for_ nurse managers\' _work._ More expertise _in_ management _is_ needed to respond to _these_ issues. _One_ essential area of nurse manager\'s management skills _is_ the _use_ of _different_ _leadership_ styles \[[@B7]\]. Leadership _styles_ can _be_ seen as different _combinations_ of tasks _and_ transaction _behaviours_ that influence _people_ in _achieving_ goals \[[@B8]\]. Earlier studies indicate that nurse manager\'s effective leadership style is affiliated _to_ staff retention \[[@B5]\], work unit climate \[[@B4]\], _nurses\'_ job satisfaction \[[@B9], [@B10]\], nurses\' commitment \[[@B11]\], _and_ patient satisfaction \[[@B12]\]. Transformational leadership _style_ \[[@B5], [@B6], [@B13], _[@B14]\]_ _and_ transactional leadership \[[@B7]\] _help_ to _respond_ to these _issues._ Transformational leadership refers to the leader\'s skills _to_ influence others towards achieving goals _by_ changing the followers\' _beliefs,_ values, and needs \[[@B7]\]. _Transactional_ leadership complements and enhances the effects of transformational leadership _outcomes_ \[[@B15]\]. There are certain skills _required_ from nurse _managers_ so as to be able to use these _effective_ leadership styles. The skills include the _ability_ to create _an_ organization culture that _combines_ _high-quality_ health care _and_ patient/employee _safety_ _and_ highly developed _collaborative_ _and_ team-building skills \[[@B1]\]. Nurse managers also need _to_ have the _readiness_ to observe their _own_ behaviour \[[@B16]\] and its effects on the work unit; _as_ a result, employees _can_ adjust to a _better_ leadership style. These _kinds_ of skills are related to manager\'s emotional intelligence (EI). EI is an ability to lead ourselves and our relationships _effectively_ \[[@B17]\]. It has been defined as the ability _to_ observe one\'s _own_ _and_ others\' feelings and emotions, to discriminate among them and to use _this_ _information_ to _direct_ _one\'s_ thinking and actions _\[[@B18]\]._ EI is _composed_ _of_ personal competence _and_ _social_ competence. Self-awareness _and_ _self-management_ are reflections of personal competence, influencing _the_ way the leader manages him/herself. _Social_ awareness and _relationship_ management reflect social competence, which affects how the leader manages relationships with others _\[[@B17]\]._ _Nurse_ managers with that _skill_ can easily form relationships with others, _read_ employees\' feelings and responses accurately, and lead _successfully_ _\[[@B19]--[@B21]\]._ Emotionally intelligent leaders\' _behaviour_ also stimulates the creativity of their employees \[[@B22]\]. Goleman et al. \[[@B23]\] have identified visionary, coaching, _affiliate,_ and democratic styles as resonant, and pacesetting and commanding styles as dissonant leadership styles. Most leaders use both resonant and _dissonant_ _leadership_ styles. The leadership styles of Goleman _et_ al. are _applied_ _as_ the _basis_ of this study _because_ earlier studies refer to the significance of these styles, _especially_ that of EI in manager\'s work. In addition, these leadership styles are one way of aiming to carry out transformational _leadership._ Especially _visionary,_ coaching, affiliate, and democratic _styles_ include elements that _promote_ transformational leadership. Such elements are for example the leader being visionary and _empowering_ staff \[[@B4]\]. This paper focuses on Finnish nurse _managers\'_ leadership _styles._ The Finnish health _care_ system is a strong institution where health care _services_ are offered to all _citizens_ and funded _by_ taxes \[[@B24]\]. It _has_ widely recognized that health care _services_ in Finland are of high-quality Despite _recent_ concerns about equity issues, Finns are in general _very_ satisfied with their health _care_ services. \[[@B25]\]. Consequently _it_ is important _to_ explore _nurse_ _managers\'_ leadership styles especially in this context. 2. Materials and Methods {#sec2} _========================_ 2.1. Aim of the _Study_ _{#sec2.1}_ --------------------- The intention _of_ this study was _to_ explore nurses\' and supervisors\' perceptions of nurse leaders\' _leadership_ styles. The research questions were as follows: what kind of leadership styles do nurse _managers_ _use_ and what are the factors affected by their leadership styles. 2.2. _Participants_ _{#sec2.2}_ ----------------- _To_ achieve the aim of _this_ study _data_ were _collected_ through open interviews. The majority _of_ Finnish nurse managers, nurses, and _supervisors_ _work_ _in_ hospitals or long-term facilities. Selection _of_ participants was performed in convenience sampling \[[@B26]\]. Participants were selected paying attention to the fact _they_ were of different ages, working in different wards and _units_ (e.g., psychiatry, internal diseases, _gerontology)_ in either hospitals or long-term facilities, and had _worked_ with _more_ _than_ one nurse manager. The researcher contacted the participants and asked whether they were interested in taking _part_ in the _study._ The participants were informed about the _aim_ of the study. Participation _was_ voluntary. Prior to the interviews each participant signed a form where they gave their consent _to_ participate in _the_ study. A _total_ of 11 _nurses_ and 10 supervisors, 20 women and _one_ man, from eight Finnish hospitals and five long-term care facilities participated in the study. The age of the _nurses_ varied _between_ 30 and 53 and _their_ _experience_ in health care between 7 and 25 _years._ The age of the supervisors varied between 38 and 59 and their experience as supervisors between 5 _and_ _21_ _years._ Both nurses _and_ supervisors had worked _with_ _many_ nurse leaders _and_ they were interviewed about nurse managers in general. They thus had experience of different nurse managers on different wards and _they_ were able to _describe_ leadership styles from various _aspects._ 2.3. _Data_ Collection _and_ Analysis {#sec2.3} --------------------------------- Semistructured interviews _were_ used to _gather_ _data_ on the perceptions _of_ nurse managers\' _leadership_ styles and factors affected by leadership styles. Interviews _were_ usually _carried_ out _in_ the office _in_ the participants\' workplace. All _interviews_ were recorded _with_ _individual_ consent. Participants were _initially_ asked to _describe_ their work and _earlier_ _study_ and _work_ history. They were subsequently asked about their perception of leadership styles and asked to _describe_ the leadership styles _used_ _by_ _their_ nurse _managers._ After that _they_ were asked about _factors_ _affected_ by leadership styles. Each interview _was_ _approached_ individually, guided by participants\' responses. The interview sessions lasted between 30 and 85 minutes. _Every_ interview _was_ transcribed word _for_ word from the recordings. Interviewing _was_ continued until saturation of _the_ data was achieved \[[@B27]\]. Because nurses _and_ supervisors might have differed in their perceptions of leadership _styles,_ the _data_ were _first_ _analysed_ separately in _two_ separate groups, following the same process _for_ each group. Content _analysis_ was chosen _because_ _it_ is a research method for _making_ valid inferences from _data_ _to_ the contexts _of_ their use \[[@B28]\]. The interview texts were read through multiple _times,_ based on _the_ author\'s empirical _and_ theoretical preunderstanding of the professional area _of_ _the_ participating _nurses_ and nurse managers. A structured categorization matrix of leadership styles _was_ developed based _on_ the _primal_ leadership model \[[@B23]\] _and_ research of Vesterinen _et_ al. \[[@B29]\]. When using a structured matrix _of_ analysis, an item of the data that does not fit the _categorization_ frame is used to create its own _concept,_ based on the principles of inductive _content_ analysis _\[[@B30]\]._ _When_ both the data of nurses _and_ superiors were analysed, the _results_ were compared. _The_ categories and subthemes were congruent and therefore the _results_ are presented together, albeit paying _attention_ to differences and similarities of the perceptions of nurses and superiors. The _data_ analysis of the factors affected by _leadership_ styles was inductive. All the _data_ _of_ nurses and supervisors were analysed together. This process included open coding, creating _categories,_ and abstraction. A classification _framework_ of _the_ factors was formed _inductively_ by defining categories and sub-themes. The criteria for allocating a unit to a _category_ were _formed_ by _asking_ questions _if_ the unit was _suitable_ _to_ the category. _The_ sub-themes _were_ named using descriptive _concepts_ and classified as "belonging" to a particular category. After that, the categories were given names _\[[@B31]\]._ 2.4. Trustworthiness {#sec2.4} _--------------------_ _The_ trustworthiness of this study has been ensured by confirming _truth_ value, _consistency,_ neutrality, and transferability of this study \[[@B32]\]. When considering this study from the viewpoint of trustworthiness, _there_ are some threats that should be _taken_ into consideration. The researcher collected _the_ _data_ and performed the analysis alone _and_ _the_ interpretation _could_ have been affected by her professional history \[[@B33]\]. _With_ interviews there is a risk that respondents _try_ to please _the_ interviewer by reporting things they assume s/he _wants_ to hear. The researcher _confirmed_ _the_ truth value of the study by selecting participants in convenience sampling. _The_ respondents\' age distribution was wide and they worked _in_ different _units._ _Their_ perspectives and descriptions were broad and _gave_ a diverse picture. The truth value of this study was also confirmed by _analysing_ data as they emerged based on the _interviews._ To _ensure_ _the_ trustworthiness of _the_ study _quotes_ from _interviews_ are included in the _results._ _In_ _view_ of consistency, the research process _is_ _described_ so that it _can_ be repeated if necessary. This gives _a_ possibility to _understand_ the limitations of the _process_ of data collection and analysis. To ensure neutrality in this _study,_ interpretations were based on original data. _This_ is confirmed _by_ citations from the interview data. In this study the sample was small, consisting of Finnish nurses and supervisors, _and_ _the_ results only reflect their perceptions of leadership styles. As a result, transferability of results is limited. However, when considering the _main_ objective in this _study,_ it was not transferability of research results, but it was to _enhance_ understanding of leadership styles and _use_ it _for_ future studies. _2.5._ _Ethical_ _Considerations_ _{#sec2.5}_ --------------------------- The data _for_ this study _were_ collected following approval _from_ _the_ administrations of the organizations. All participants were informed of the |
1. Introduction {#sec1}
===============
Soil organic C (SOC) and total N (TN) are very important C and N pools in the terrestrial ecosystems \[[@B1], [@B2]\]. As the components of labile C and N pools in soils, dissolved organic C (DOC) and N (DON) and soil ammonium and nitrate N (NH~4~ ^+^-N and NO~3~ ^−^-N) play crucial roles in the biogeochemistry of C and N and in the nutrient transformation \[[@B3]--[@B5]\]. With the context of climatic warming, how SOC, TN, DOC, DON, NH~4~ ^+^-N, and NO~3~ ^−^-N respond is vital to global C and N cycling \[[@B1], [@B2]\]. However, inconsistent results on the responses of these C and N pools to climatic warming have been observed with respect to vegetation types and initial soil characteristics \[[@B2], [@B3], [@B6]--[@B14]\]. For example, He et al. \[[@B2]\] demonstrated that six-year warming (\~1.4°C increase of 10 cm soil temperature) significantly decreased soil C by 129.3 g m^−2^ in a temperate steppe of Inner Mongolia. In contrast, Li et al. \[[@B7]\] found that two-year warming significantly increased SOC in an alpine meadow (\~2.1°C increase of air temperature) but significantly reduced TN in an alpine swamp meadow (\~2.3°C increase of air temperature) on the Tibetan Plateau. Hagedorn et al. \[[@B13]\] indicated that one-growing-season warming (\~4°C increase of 5 cm soil temperature) did not significantly influence DOC. Song et al. \[[@B1]\] pointed out that six-year warming (\~1.2°C increase of 10 cm soil temperature) significantly reduced DOC in a temperate steppe in Inner Mongolia. Biasi et al. \[[@B15]\] indicated that two-year warming (\~0.9°C increase of 5 cm soil temperature) did not have obvious effects on DON, NH~4~ ^+^-N, NO~3~ ^−^-N, and N~min~ in a lichen-rich dwarf shrub tundra in Siberia. Bai et al. \[[@B14]\] stated that experimental warming (\~0.6--6.7°C in soil temperature) had a significant positive effect on N~min~ but not on TN across all biomes. Therefore, how climatic warming acts on C and N cycling still remains unclear.
More than 70% of the Tibetan Plateau is covered with grasslands \[[@B16]\]. The alpine grasslands of this Plateau are one of the systems most sensitive to global change \[[@B17], [@B18]\]. In alpine grasslands, understanding the responses of SOC, DOC, TN, DON, NH~4~ ^+^-N, and NO~3~ ^−^-N to climatic warming are crucial for predicting future changes in soil fertility and C sequestration. The alpine meadow is one of the most typical grasslands types on the Tibetan Plateau being subjected to climatic warming \[[@B19]\]. Information on how these C and N pools along an elevation gradient respond to climatic warming is scarce on the Tibetan Plateau. Here we set up a warming experiment in an alpine meadow at three elevations (i.e., 4313 m, 4513 m, and 4693 m) on the Northern Tibetan Plateau.
The main objective was to investigate the effects of short-term experimental warming on SOC, TN, DOC, DON, NH~4~ ^+^-N, and NO~3~ ^−^-N. Our previous study indicated that short-term experimental warming could not affect soil microbial biomass \[[@B20]\] and soil microbial activity regulated the balances of soil C and N pools in the alpine meadow \[[@B21]\]. We hypothesized that experimental warming may not affect these C and N pools in this study.
2. Materials and Methods {#sec2}
========================
2.1. Study Area, Experimental Design, and Soil Sampling {#sec2.1}
-------------------------------------------------------
A detailed description of the study area, the warming experimental design, the measurements of microclimate factors (including soil temperature and soil moisture), and the soil sampling are given in Fu et al. \[[@B20], [@B22]\].
Briefly, three alpine meadow sites were established at three elevations (i.e., a low (30°30′N, 91°04′E, and 4313 m), mid- (30°31′N, 91°04′E, and 4513 m), and high (30°32′N, 91°03′E, and 4693 m) elevation) at Damxung Grassland Observation Station of Tibet Autonomous Region in China in May 2010.
Annual mean air temperature and precipitation is 1.3°C and \~476.8 mm, respectively \[[@B20], [@B21]\]. The vegetation is*Kobresia*-dominated alpine meadow and roots are mainly concentrated in the topsoil layer (0--20 cm) \[[@B21], [@B22]\]. The soil is classified as sandy loam, with pH of 6.0--6.7, organic matter of 0.3--11.2%, and total N of 0.03--0.49% \[[@B20], [@B22]\].
Open top chambers (OTCs, 3 mm thick polycarbonate) were used to enhance temperature \[[@B22], [@B23]\]. The bottom and top diameters and the height of OTCs were 1.45 m and 1.00 m and 0.40 m, respectively \[[@B20], [@B22]\]. For each site, four OTCs and their paired control plots (1 m × 1 m) were randomly established in May 2010. There was \~3 m distance between plots.
Daily mean soil temperature (*T* ~*s*~) during the study period of July-September in 2011 inside the OTCs increased by 1.26°C, 0.98°C, and 1.37°C at the low, mid-, and high elevation, respectively, compared to control plots \[[@B20]\]. In contrast, experimental warming decreased daily mean soil moisture (SM) by 0.04 m^3^ m^−3^ in all sites \[[@B20]\]. Daily mean *T* ~*s*~ decreased with increasing elevation from the low to high elevation \[[@B20]\].
We collected topsoil samples (0--20 cm depth) inside each plot using a probe 3.0 cm in diameter on July 7, August 9, and September 10, 2011 \[[@B20]\]. Five soil subsamples were randomly sampled and composited into one soil sample for each plot \[[@B20]\]. Subsamples of the fresh soil were used to measure DOC, DON, NH~4~ ^+^-N, and NO~3~ ^−^-N and other subsamples of the fresh soil were air-dried for the measurements of SOC and TN.
2.2. Soil Analysis {#sec2.2}
------------------
A more detailed description of measurements of soil inorganic N (N~min~, i.e., sum of NH~4~ ^+^-N and NO~3~ ^−^-N), DON, and DOC can be found in Fu et al. \[[@B21]\]. Briefly, soil inorganic N in 20 g fresh soil sample was extracted with 100 mL K~2~SO~4~, filtered through 0.45 *μ*m membrane, and analyzed on a LACHAT Quikchem Automated Ion Analyzer. Dissolved organic C and TN (DTN) in another 20 g fresh soil sample was extracted with 100 mL ultrapure water and filtered through 0.45 membrane. The extractable SOC and TN concentrations in the ultrapure water extracts were measured using a Liqui TOC II elementar analyzer (Elementar Liqui TOC, Elementar Co., Hanau, Germany) and a UV-1700 PharmaSpec visible spectrophotometer (220 nm and 275 nm), respectively. We also analyzed dissolved inorganic N (DIN) in the ultrapure water extracts on a LACHAT Quikchem Automated Ion Analyzer. Then DON was calculated as the difference between DTN and DIN. The potassium dichromate method was used to determine SOC \[[@B24]\]. Soil TN was measured on a CN analyzer (Elementar Variomax CN). Soil microbial biomass (MBC) and N (MBN) data were obtained from Fu et al. \[[@B20]\].
2.3. Statistical Analysis {#sec2.3}
-------------------------
In order to examine the elevation effect, repeated-measures ANOVA with experimental warming and elevation as the between subject factors and with sampling date as the within subject factor was performed for a specific soil property (i.e., SOC, TN, DOC, DON, ratio of DOC to DON (DOC/DON), NH~4~ ^+^-N, NO~3~ ^−^-N, ratio of NH~4~ ^+^-N to NO~3~ ^−^-N(NH~4~ ^+^-N/NO~3~ ^−^-N), and N~min~). At each site, repeated-measures ANOVA with experimental warming (i.e., OTCs versus control) as the between subject factor and with sampling date as the within subject factor was conducted for each soil property. Single factor linear reg | 1. introduction { # sec1 } = = = = = = = = = = = = = = = soil organic c ( soc ) and total n ( tn ) are very important c and n pools in the terrestrial wetland \ [ [ @ b1 ], [ @ b2 ] \ ]. as the components of labile c and n pools in soils, dissolved organic c ( doc ) and n ( don ) and soil ammonium and nitrate n ( nh ~ 4 ~ ^ + ^ - n and no ~ 3 ~ ^ − ^ - n ) play key roles in ecosystem biogeochemistry of c and n and in the nutrient transformation \ [ [ @ b3 ] - - [ @ b5 ] \ ]. with the context of climatic warming, how soc, tn, doc, don, nh ~ 4 ~ ^ + ^ - n, and no ~ 3 ~ ^ − ^ - n respond is vital to global c and dynamic cycling \ [ [ @ b1 ], [ @ b2 ] \ ]. however, inconsistent results on the responses of these c and n pools to climatic warming have been observed with respect to vegetation types and initial soil characteristics \ [ [ @ b2 ], [ @ b3 ], [ @ b6 ] - - [ @ b14 ] \ ]. for example, he et al. \ [ [ @ b2 ] \ ] demonstrated a six - year warming ( \ ~ 1. 4°c increase of 10 cm soil temperature ) significantly decreased soil c by 129. 3 g m ^ −2 ^ in a temperate steppe and upper mongolia. in contrast, li et al. \ [ [ @ b7 ] \ ] found that two - year warming significantly increased soc in an alpine meadow ( \ ~ 2. 1°c increase of air temperature ) but significantly reduced ecology in an alpine swamp meadow ( \ ~ 2. 3°c increase of air temperature ) on the tibetan plateau. hagedorn et al. \ [ [ @ b13 ] \ ] indicated that one - growing - season warming ( \ ~ 4°c increase of 5 cm soil temperature ) did not significantly influence doc. song it al. \ [ [ @ b1 ] \ ] pointed notes that six - year warming ( \ ~ 1. 2°c increase of 10 cm soil temperature ) significantly reduced doc in a temperate steppe in inner mongolia. biasi et al. \ [ [ @ b15 ] \ ] indicated that two - year warming ( \ ~ 0. 9°c increase of 5 cm soil temperature ) did not have obvious effects on don, nh ~ 4 ~ ^ + ^ - n, no ~ 3 ~ ^ − ^ - n, and n ~ min ~ in a lichen - rich dwarf shrub tundra in siberia. bai et al. \ [ [ @ b14 ] \ ] stated that experimental warming ( \ ~ 0. 6 - - 6. 7°c in soil temperature ) had a significant positive effect on n ~ min ~ but not on tn across all biomes. therefore, how climatic warming acts on c and n cycling still remains unclear. more than 70 % of the tibetan plateau is covered with grasslands \ [ [ @ b16 ] \ ]. the alpine grasslands of this plateau are one of the systems most sensitive to global change \ [ [ @ b17 ], [ @ b18 ] \ ]. in alpine grasslands, understanding the responses of soc, doc, tn, don, nh ~ 4 ~ ^ + ^ - n, and no ~ 3 ~ ^ − ^ - n to climatic warming are crucial for predicting future changes in soil fertility and c sequestration. the alpine meadow is one of the most typical grasslands types on the tibetan plateau being subjected to climatic warming \ [ [ @ b19 ] \ ]. information on how these c and n pools along an elevation gradient respond to climatic warming is scarce on the tibetan plateau. here we set up a warming experiment in an alpine meadow at three elevations ( i. e., 4313 m, 4513 m, and 4693 m ) on the northern tibetan plateau. the main objective was to investigate the effects of short - term experimental warming on soc, tn, doc, don, nh ~ 4 ~ ^ + ^ - n, and no ~ 3 ~ ^ − ^ - n. our previous study indicated that short - term experimental warming could not affect soil microbial biomass \ [ [ @ b20 ] \ ] and soil microbial activity regulated the balances of soil c and n pools in the alpine meadow \ [ [ @ b21 ] \ ]. we hypothesized that experimental warming may not affect these c and n pools in this study. 2. materials and methods { # sec2 } = = = = = = = = = = = = = = = = = = = = = = = = 2. 1. study area, experimental design, and soil sampling { # sec2. 1 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - a detailed description of the study area, the warming experimental design, the measurements of microclimate factors ( including soil temperature and soil moisture ), and the soil sampling are given in fu et al. \ [ [ @ b20 ], [ @ b22 ] \ ]. briefly, three alpine meadow sites were established at three elevations ( i. e., a low ( 30°30 ′ n, 91°04 ′ e, and 4313 m ), mid - ( 30°31 ′ n, 91°04 ′ e, and 4513 m ), and high ( 30°32 ′ n, 91°03 ′ e, and 4693 m ) elevation ) at damxung grassland observation station of tibet autonomous region in china in may 2010. annual mean air temperature and precipitation is 1. 3°c and \ ~ 476. 8 mm, respectively \ [ [ @ b20 ], [ @ b21 ] \ ]. the vegetation is * kobresia * - dominated alpine meadow and roots are mainly concentrated in the topsoil layer ( 0 - - 20 cm ) \ [ [ @ b21 ], [ @ b22 ] \ ]. the soil is classified as sandy loam, with ph of 6. 0 - - 6. 7, organic matter of 0. 3 - - 11. 2 %, and total n of 0. 03 - - 0. 49 % \ [ [ @ b20 ], [ @ b22 ] \ ]. open top chambers ( otcs, 3 mm thick polycarbonate ) were used to enhance temperature \ [ [ @ b22 ], [ @ b23 ] \ ]. the bottom and top diameters and the height of otcs were 1. 45 m and 1. 00 m and 0. 40 m, respectively \ [ [ @ b20 ], [ @ b22 ] \ ]. for each site, four otcs and their paired control plots ( 1 m × 1 m ) were randomly established in may 2010. there was \ ~ 3 m distance between plots. daily mean soil temperature ( * t * ~ * s * ~ ) during the study period of july - september in 2011 inside the otcs increased by 1. 26°c, 0. 98°c, and 1. 37°c at the low, mid -, and high elevation, respectively, compared to control plots \ [ [ @ b20 ] \ ]. in contrast, experimental warming decreased daily mean soil moisture ( sm ) by 0. 04 m ^ 3 ^ m ^ −3 ^ in all sites \ [ [ @ b20 ] \ ]. daily mean * t * ~ * s * ~ decreased with increasing elevation from the low to high elevation \ [ [ @ b20 ] \ ]. we collected topsoil samples ( 0 - - 20 cm depth ) inside each plot using a probe 3. 0 cm in diameter on july 7, august 9, and september 10, 2011 \ [ [ @ b20 ] \ ]. five soil subsamples were randomly sampled and composited into one soil sample for each plot \ [ [ @ b20 ] \ ]. subsamples of the fresh soil were used to measure doc, don, nh ~ 4 ~ ^ + ^ - n, and no ~ 3 ~ ^ − ^ - n and other subsamples of the fresh soil were air - dried for the measurements of soc and tn. 2. 2. soil analysis { # sec2. 2 } - - - - - - - - - - - - - - - - - - a more detailed description of measurements of soil inorganic n ( n ~ min ~, i. e., sum of nh ~ 4 ~ ^ + ^ - n and no ~ 3 ~ ^ − ^ - n ), don, and doc can be found in fu et al. \ [ [ @ b21 ] \ ]. briefly, soil inorganic n in 20 g fresh soil sample was extracted with 100 ml k ~ 2 ~ so ~ 4 ~, filtered through 0. 45 * μ * m membrane, and analyzed on a lachat quikchem automated ion analyzer. dissolved organic c and tn ( dtn ) in another 20 g fresh soil sample was extracted with 100 ml ultrapure water and filtered through 0. 45 membrane. the extractable soc and tn concentrations in the ultrapure water extracts were measured using a liqui toc ii elementar analyzer ( elementar liqui toc, elementar co., hanau, germany ) and a uv - 1700 pharmaspec visible spectrophotometer ( 220 nm and 275 nm ), respectively. we also analyzed dissolved inorganic n ( din ) in the ultrapure water extracts on a lachat quikchem automated ion analyzer. then don was calculated as the difference between dtn and din. the potassium dichromate method was used to determine soc \ [ [ @ b24 ] \ ]. soil tn was measured on a cn analyzer ( elementar variomax cn ). soil microbial biomass ( mbc ) and n ( mbn ) data were obtained from fu et al. \ [ [ @ b20 ] \ ]. 2. 3. statistical analysis { # sec2. 3 } - - - - - - - - - - - - - - - - - - - - - - - - - in order to examine the elevation effect, repeated - measures anova with experimental warming and elevation as the between subject factors and with sampling date as the within subject factor was performed for a specific soil property ( i. e., soc, tn, doc, don, ratio of doc to don ( doc / don ), nh ~ 4 ~ ^ + ^ - n, no ~ 3 ~ ^ − ^ - n, ratio of nh ~ 4 ~ ^ + ^ - n to no ~ 3 ~ ^ − ^ - n ( nh ~ 4 ~ ^ + ^ - n / no ~ 3 ~ ^ − ^ - n ), and n ~ min ~ ). at each site, repeated - measures anova with experimental warming ( i. e., otcs versus control ) as the between subject factor and with sampling date as the within subject factor was conducted for each soil property. single factor linear reg | 1. Introduction {# sec1} = = = = = = = = = = = = = = = Soil organic C (SOC) and total N (TN) are very important C and N pools in the terrestrial ecosystems \ [[ @ B1 ], [@ B2] \ ]. As the components of labile C and N pools in soils, dissolved organic C (DOC) and N (DON) and soil ammomiJm and nitrate N (NH ~ 4 ~ ^ + ^ - N and NO ~ 3 ~ ^ − ^ - N) play crucial roles in the biogeochemistry of C and N and in the nutrient transformation \ [[ @ B3] - - [@ B5] \ ]. With the context of climatic warming, how SOC, TN, DOC, DON, NH ~ 4 ~ ^ + ^ - N, and NO ~ 3 ~ ^ − ^ - N respond is vital to global C and N cycling \ [[ @ B1 ], [@ B2] \ ]. However, inconsistent results on the responses of these C and N pools to climatic warming have been observed with respect to vegetation types and initial soil characteristics \ [[ @ B2 ], [@ B3 ], [@ B6] - - [@ B14] \ ]. For example, He et al. \ [[ @ B2] \] demonstrated that six - year warming (\ ~ 1. 4 ° C increase of 10 cm soil temperature) significantly decreased soil C by 129. 3 g m ^ − 2 ^ in a temperate steppe of Inner Mongolia. In contrast, Li et al. \ [[ @ B7] \] found that two - year warming significantly increased SOC in an alpine meadow (\ ~ 2. 1 ° C increase of air temperature) but significantly reduced TN in an alpine swamp meadow (\ ~ 2. 3 ° C increase of air temperature) on the Tibetan Plateau. Hagedorn et al. \ [[ @ B13] \] indicated that one - growing - season warming (\ ~ 4 ° C increase of 5 cm soil temperature) did not significantly influence DOC. Song et al. \ [[ @ B1] \] pointed out that six - year warming (\ ~ 1. 2 ° C increase of 10 cm soil temperature) significantly reduced DOC in a temperate steppe in Inner Mongolia. Biasi et al. \ [[ @ B15] \] indicated that two - year warming (\ ~ 0. 9 ° C increase of 5 cm soil temperature) did not have obvious effects on DON, NH ~ 4 ~ ^ + ^ - N, NO ~ 3 ~ ^ − ^ - N, and N ~ min ~ in a lichen - rich dwarf shrub tundra in Siberia. Bai et al. \ [[ @ B14] \] stated that experimental warming (\ ~ 0. 6 - - 6. 7 ° C in soil temperature) had a significant positive effect on N ~ min ~ but not on TN across all biomes. Therefore, how climatic warming acts on C and N cycling still remains unclear. More than 70% of the Tibetan Plateau is covered with grasslands \ [[ @ B16] \ ]. The alpine grasslands of this Plateau are one of the systems most sensitive to global change \ [[ @ B17 ], [@ B18] \ ]. In alpine grasslands, understanding the responses of SOC, DOC, TN, DON, NH ~ 4 ~ ^ + ^ - N, and NO ~ 3 ~ ^ − ^ - N to climatic warming are crucial for predicting future changes in soil fertility and C sequestration. The alpine meadow is one of the most typical grasslands types on the Tibetan Plateau being wubjexted to climatic warming \ [[ @ B19] \ ]. Information on how these C and N pools along an elevation graSOent respond to climatic warming is scarce on the Tibetan Plateau. Here we set up a warming experiment in an alpine meadow at three elevations (i. e. , 4313 m, E5!3 m, and 4693 m) on the Northern Tibetan Plateau. The main objective was to investigate the effects of short - term experimental warming on SOC, TN, DOC, DON, NH ~ 4 ~ ^ + ^ - N, and NO ~ 3 ~ ^ − ^ - N. Our previous study indicated that short - term experimental warming could not affect soil microbial biomass \ [[ @ B20] \] and soil microbial activity regulated the balances of soil C and N pools in the alpine meadow \ [[ @ B21] \ ]. We hypothesized that experimental warming may not affect these C and N pools in this study. 2. Materials and Methods {# sec2} = = = = = = = = = = = = = = = = = = = = = = = = 2. 1. Wfudy Area, Experimental Design, and Soil Sampling {# e2c2. 1} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A detailed description of the study area, the warming experimental design, the measurements of microclimate factors (including soil temperature and soil moisture ), and the soil sampling are given in Fu et al. \ [[ @ B20 ], [@ B22] \ ]. Briefly, three alpine meadow sites were established at three elevations (i. e. , a low (30 ° 30 ′ N, 91 ° 04 ′ E, and 4313 m ), mid - (30 ° 31 ′ N, 91 ° 04 ′ E, and 4513 m ), and high (30 ° 32 ′ N, 91 ° 03 ′ E, and 4693 m) elevation) at Damxung Grassland Observation Station of Tibet Autonomous Region in China in May 2010. Annual mean air temperature and precipitation is 1. 3 ° C and \ ~ 476. 8 mm, respectively \ [[ @ B20 ], [@ B21] \ ]. The vegetation is * Kobresia * - dominated alpine meadow and roots are mainly concentrated in the topsoil layer (0 - - 20 cm) \ [[ @ B21 ], [@ B22] \ ]. The soil is classified as sandy loam, with pH of 6. 0 - - 6. 7, organic matter of 0. 3 - - 11. 2% , and total N of 0. 03 - - 0. 49% \ [[ @ B20 ], [@ B22] \ ]. Open top chambers (OTCs, 3 mm thick polycarbonate) were used to enhance temperature \ [[ @ B22 ], [@ B23] \ ]. The bottom and top diameters and the height of OTCs were 1. 45 m and 1. 00 m and 0. 40 m, respectively \ [[ @ B20 ], [@ B22] \ ]. For each site, four OTCs and their paired control plots (1 m × 1 m) were randomly established in May 2010. There was \ ~ 3 m distance between plots. Daily mean soil temperature (* T * ~ * s * ~) during the study period of July - September in 2011 inside the OTCs increased by 1. 26 ° C, 0. 98 ° C, and 1. 37 ° C at the low, mid -, and high elevation, respectively, compared to control plots \ [[ @ B20] \ ]. In contrast, experimental warming decreased daily mean soil moisture (SM) by 0. 04 m ^ 3 ^ m ^ − 3 ^ in all sites \ [[ @ B20] \ ]. Daily mean * T * ~ * s * ~ decreased with increasing elevation from the low to high elevation \ [[ @ B20] \ ]. We collected topsoil samples (0 - - 20 cm depth) inside each plot using a probe 3. 0 cm in diameter on July 7, August 9, and September 10, 2011 \ [[ @ B20] \ ]. Five soil subsamples were randomly sampled and composited into one soil saKp?e for each plot \ [[ @ B20] \ ]. Subsamples of the fresh soil were used to measure DOC, DON, NH ~ 4 ~ ^ + ^ - N, and NO ~ 3 ~ ^ − ^ - N and other subsamples of the fresh soil were air - dried for the measurements of SOC and TN. 2. 2. Soil Analysis {# sec2. 2} - - - - - - - - - - - - - - - - - - A more detailed description of measurements of soil inorganic N (N ~ min ~, i. e. , sum of NH ~ 4 ~ ^ + ^ - N and NO ~ 3 ~ ^ − ^ - N ), DON, and DOC can be found in Fu et al. \ [[ @ B21] \ ]. Briefly, soil inorganic N in 20 g fresh soil sample was extracted with 100 mL K ~ 2 ~ SO ~ 4 ~, filtered through 0. 45 * μ * m membrane, and analyzed on a LACHAT Quikchem Automa5wd Ion Analyzer. Dissolved organic C and TN (DTN) in another 20 g fresh soil sample was extracted with 100 mL ultrapure water and filtered through 0. 45 membrane. The extractable SOC and TN concentrations in the ultrapure water extracts were measured using a Liqui TOC II elementar analyzer (Elementar Liqui TOC, Elementar Co. , Hanau, Germany) and a UV - 1700 PharmaSpec visible spectrophotometer (220 nm and 275 nm ), respectively. We also analyzed dissolved inorganic N (DIN) in the ultrapure water extracts on a LACHAT Quikchem Automated Ion Analyzer. Then DON was calculated as the difference between DTN and DIN. The potassium dichromate method was used to determine SOC \ [[ @ B24] \ ]. Eokl TN was measured on a CN analyzer (Elementar Variomax CN ). Soil microbial biomass (MBC) and N (MBN) data were obtained from Fu et al. \ [[ @ B20] \ ]. 2. 3. Statistical Analysis {# sec2. 3} - - - - - - - - - - - - - - - - - - - - - - - - - In order to examine the elevation effect, repeated - measures ANOVA with experimental warming and elevation as the between subject factors and with sampling date as the within subject factor was performed for a specific soil property (i. e. , SOC, TN, DOC, DON, ratio of DOC to DON (DOC / DON ), NH ~ 4 ~ ^ + ^ - N, NO ~ 3 ~ ^ − ^ - N, ratio of NH ~ 4 ~ ^ + ^ - N to NO ~ 3 ~ ^ − ^ - N (NH ~ 4 ~ ^ + ^ - N / NO ~ 3 ~ ^ − ^ - N ), and N ~ min ~ ). At each site, repeated - measures ANOVA with experimental warming (i. e. , OTCs versus control) as the between s^bjec6 factor and with sampling date as the within subject factor was conducted for each soil property. Single factor linear reg | 1. {#sec1} =============== Soil organic (SOC) and total N (TN) are very important C and N pools the terrestrial ecosystems \[[@B1], the components of labile C and N pools in soils, dissolved organic C (DOC) and N (DON) and soil ammonium and nitrate N (NH~4~ ^+^-N and NO~3~ play crucial in the biogeochemistry of C and N in the nutrient transformation \[[@B3]--[@B5]\]. With the context of climatic warming, how SOC, TN, DOC, NH~4~ ^+^-N, and NO~3~ ^−^-N respond vital to global C and N cycling \[[@B1], results on the responses of C and N pools to climatic warming have been observed with respect to vegetation and soil characteristics \[[@B2], [@B3], [@B6]--[@B14]\]. For example, He al. \[[@B2]\] demonstrated that warming increase of 10 cm soil temperature) significantly decreased soil C by 129.3 g m^−2^ in a temperate steppe of Inner Mongolia. In contrast, Li et al. \[[@B7]\] that two-year warming significantly increased in an alpine meadow increase of air temperature) but significantly in an alpine meadow (\~2.3°C of air temperature) the Tibetan Plateau. Hagedorn et al. \[[@B13]\] indicated that warming (\~4°C increase of 5 cm soil temperature) did not significantly influence DOC. et al. \[[@B1]\] pointed out that six-year (\~1.2°C increase of 10 cm temperature) significantly reduced DOC in temperate steppe in Inner Biasi et al. \[[@B15]\] indicated that two-year warming (\~0.9°C of 5 cm soil did not have obvious effects on DON, NH~4~ ^+^-N, NO~3~ ^−^-N, N~min~ in a lichen-rich dwarf tundra in Siberia. Bai et al. \[[@B14]\] stated that experimental (\~0.6--6.7°C in soil temperature) had a significant positive effect on N~min~ not on TN across all biomes. Therefore, how climatic warming acts on C and N cycling still remains unclear. than 70% of the Tibetan Plateau is covered with grasslands \[[@B16]\]. alpine grasslands of Plateau are one of the systems most sensitive global change \[[@B17], [@B18]\]. In alpine grasslands, understanding the responses SOC, DOC, TN, DON, NH~4~ and NO~3~ ^−^-N to climatic warming are crucial for predicting future changes in soil fertility and C sequestration. The alpine meadow one of the most typical grasslands types on the Tibetan Plateau being subjected to climatic warming \[[@B19]\]. Information how these C and N pools an gradient respond to climatic warming is scarce on the Tibetan Plateau. we set up a warming experiment in an meadow at three elevations (i.e., 4313 m, 4513 m, and 4693 m) on the Northern Tibetan Plateau. The main objective was to investigate the effects of short-term experimental warming on TN, DON, NH~4~ ^+^-N, and NO~3~ ^−^-N. Our previous study indicated that short-term experimental warming could affect soil biomass \[[@B20]\] and soil microbial activity regulated the balances of soil C and N pools in the alpine meadow \[[@B21]\]. We hypothesized that warming may not affect these C and N in this study. 2. Materials and Methods {#sec2} ======================== 2.1. Study Area, Design, Soil Sampling {#sec2.1} A detailed description the area, the warming experimental design, the measurements of microclimate factors (including soil temperature and soil moisture), and the soil sampling are given in Fu et al. \[[@B20], [@B22]\]. Briefly, three alpine meadow sites were established at three elevations (i.e., a low (30°30′N, 91°04′E, and 4313 m), mid- (30°31′N, 91°04′E, and 4513 m), high (30°32′N, 91°03′E, and m) at Damxung Grassland Observation Station Tibet Autonomous Region in China in May 2010. Annual mean air temperature and precipitation is 1.3°C and \~476.8 mm, respectively \[[@B20], [@B21]\]. vegetation alpine meadow and roots mainly concentrated in the topsoil layer (0--20 cm) \[[@B21], [@B22]\]. The soil is classified as sandy loam, with pH of 6.0--6.7, organic matter of 0.3--11.2%, and N 0.03--0.49% \[[@B20], [@B22]\]. Open chambers (OTCs, 3 thick polycarbonate) were used to enhance temperature \[[@B22], [@B23]\]. The bottom and top diameters and height of were 1.45 m and 1.00 and 0.40 m, respectively \[[@B20], [@B22]\]. For each site, four OTCs and their control plots (1 m × 1 m) were randomly established in May 2010. There was \~3 distance between plots. mean soil temperature (*T* ~*s*~) during the study period of July-September 2011 inside the OTCs by 0.98°C, and at low, mid-, and high elevation, respectively, compared plots \[[@B20]\]. In contrast, experimental warming decreased mean soil moisture (SM) 0.04 m^3^ m^−3^ all sites \[[@B20]\]. Daily mean ~*s*~ decreased with increasing elevation the low high elevation \[[@B20]\]. We collected topsoil samples (0--20 cm inside each plot using a probe 3.0 cm in diameter on July 7, August and September 10, \[[@B20]\]. Five soil subsamples were randomly sampled and composited into one soil sample for each plot \[[@B20]\]. Subsamples of the fresh soil were used to measure DOC, DON, NH~4~ ^+^-N, and NO~3~ ^−^-N and other subsamples the fresh soil were for the measurements of SOC TN. 2.2. Soil Analysis {#sec2.2} ------------------ A more detailed description of measurements of soil inorganic N (N~min~, i.e., sum of NH~4~ and ^−^-N), DON, and DOC can be found in Fu al. \[[@B21]\]. Briefly, soil inorganic N in 20 g fresh soil sample was extracted with 100 mL K~2~SO~4~, through 0.45 *μ*m membrane, and on a LACHAT Quikchem Automated Ion Analyzer. Dissolved organic C and TN (DTN) in another 20 g fresh soil sample was extracted with 100 mL ultrapure water and filtered through 0.45 membrane. extractable SOC and TN concentrations in the ultrapure water extracts were measured using a Liqui II elementar analyzer (Elementar Liqui TOC, Elementar Co., Hanau, Germany) and a UV-1700 PharmaSpec visible spectrophotometer (220 nm and 275 nm), respectively. We also analyzed dissolved inorganic N (DIN) in the water extracts on a LACHAT Quikchem Automated Ion Analyzer. Then DON was calculated as the difference between DTN and DIN. The potassium dichromate method was used to determine SOC \[[@B24]\]. Soil TN was measured on a analyzer (Elementar Variomax CN). Soil microbial biomass (MBC) and N (MBN) data were obtained from Fu et al. \[[@B20]\]. Statistical Analysis {#sec2.3} ------------------------- In order to examine the elevation effect, repeated-measures ANOVA with experimental warming and elevation as the between subject factors and with sampling date as the within subject was performed for a specific soil property (i.e., SOC, TN, DOC, DON, ratio of DOC DON NH~4~ ^+^-N, NO~3~ ^−^-N, ratio of NH~4~ ^+^-N to NO~3~ ^−^-N(NH~4~ ^+^-N/NO~3~ ^−^-N), and At each site, ANOVA with experimental warming (i.e., OTCs versus control) as the between subject factor and sampling the within factor was conducted for each soil property. Single factor linear reg | 1. iNTRodUCtiOn {#Sec1}
===============
soIL oRGAniC c (SoC) and Total N (tN) ArE vERY IMPortANT c AnD n poOlS In THe TERrESTRIAL ECoSYSteMs \[[@B1], [@b2]\]. AS the cOmpOnentS Of lABIle c aND N pooLS In sOilS, disSOlved OrgaNIC c (Doc) ANd N (dON) and sOiL AMMoniUm AnD nITraTE N (nH~4~ ^+^-N anD No~3~ ^−^-n) PLaY CrUCiAl ROLeS In THE bIogEoCHeMisTRY oF C anD n aND iN THE NutrIENT trAnSFOrmaTiON \[[@b3]--[@b5]\]. WitH the ContexT OF ClImAtiC wArMiNG, hoW soC, tn, Doc, Don, nH~4~ ^+^-N, And No~3~ ^−^-n reSpONd is VitAl to global C anD n CyclInG \[[@B1], [@b2]\]. hOwevEr, inconSisteNT REsultS On ThE reSpoNSeS Of TheSE C AnD n pOols to ClimAtIc wARmiNG haVE been ObSERvEd wiTH rESpEcT tO vegetATiON tYpEs aNd INitIAl SoiL chaRAcTerISTIcS \[[@B2], [@B3], [@b6]--[@b14]\]. FoR exAmPle, hE Et al. \[[@b2]\] DEMoNsTrateD tHaT sIx-YeAR WarmING (\~1.4°C increASe of 10 CM soIL tEmPeRAtuRE) sIGniFiCaNTLY DECReASed soil c BY 129.3 g M^−2^ iN a TEMperAtE steppe oF innER mOnGoliA. In ContRASt, Li eT aL. \[[@b7]\] fOUND tHat TwO-yeaR WArmiNg siGNifICANTly IncReasED sOC IN An AlPINE mEaDoW (\~2.1°c IncReaSE OF aiR TEMPErAtuRe) bUT siGNiFicanTlY reDUCeD Tn in an ALPInE SWAMp meADOW (\~2.3°C INCRease Of aiR tEMpeRaTure) On tHe TiBetAN plAtEAu. hagedoRn Et aL. \[[@b13]\] inDIcAtEd THAt ONE-GrOwing-sEaSon wArmINg (\~4°C inCREaSE of 5 Cm SoIL TemPeraTUrE) DId nOt sIgNiFicantLy iNFLuENcE dOC. SOnG Et Al. \[[@b1]\] POIntED OuT THaT SIx-YeAr WArMinG (\~1.2°C iNcRease oF 10 Cm SOIL temperATURE) sIgNIfiCanTlY REduced dOC In A TeMPERATE sTepPE In InNER mONGoliA. BIasI eT al. \[[@b15]\] inDiCAtED THAt TwO-yEAr Warming (\~0.9°c iNcreAsE OF 5 CM SOiL tEmPErATUrE) did nOT hAvE ObViOuS eFfEctS oN DoN, nh~4~ ^+^-N, no~3~ ^−^-n, aNd N~Min~ iN A LICheN-ricH dWarF SHRUb tUNdRa iN sibeRiA. Bai et AL. \[[@b14]\] Stated tHat EXpERimENtAL WarMINg (\~0.6--6.7°c In sOil tEmPeRATUrE) hAD A SIgnIFiCAnt POsItIvE EffEcT oN n~MiN~ BUt nOT ON tn acRoss alL BIomEs. tHEreFOrE, hoW CLimATIc WarMInG AcTs On c AnD n cyCLiNg sTiLL rEmAINs unClEaR.
MorE ThaN 70% oF THe tiBetAN PLATeaU is CovERED WiTh grAsSlANds \[[@B16]\]. thE ALPIne gRasSLaNDS oF THis plAtEaU arE oNE of THE sYstEms MOST sENSiTive To gLOBaL CHANGE \[[@b17], [@B18]\]. in ALPIne grAsSLANds, UnDerstaNding tHe RespoNsEs Of soC, Doc, Tn, DON, NH~4~ ^+^-n, and no~3~ ^−^-n To clImAtic warming ArE CrUCIal For pREdictInG fuTuRE ChANgEs in SoiL feRtIliTY and c SEQUesTRAtiOn. thE alPIne mEADow is OnE oF ThE MOST typICAL gRaSSlands tYpEs ON ThE tibetan plateau being SuBjECTed tO ClimATIC waRMiNg \[[@b19]\]. inFormaTIOn ON hoW tHese C And n PooLS alOng aN ElEVATIon gRadIENT rESPOnd To ClIMAtIc wArmINg is SCaRCE on ThE TiBETAN PLAteAU. hEre we Set Up a WaRMiNg EXPeriMenT iN AN AlpINe meADOW at thrEe eLEVAtiOns (I.E., 4313 M, 4513 M, and 4693 M) On the NorThern TIbeTAN PlAtEau.
tHE maIn OBjECTIve Was TO InvEsTiGAte THe EffECTS Of ShoRT-teRM exPerImEntaL wArMing oN soC, tN, DOC, Don, NH~4~ ^+^-n, aNd no~3~ ^−^-N. oUr pReVIOUs STuDY iNdIcaTeD THAT sHoRt-tERm ExpeRImentAl WARMInG cOULD nOt AFfEct sOil mICROBIAL BIomass \[[@b20]\] And sOiL MiCrOBIAl acTivIty rEGULATED ThE balancEs of SoIl c AnD n PoOLs IN THE aLpiNE mEaDOW \[[@b21]\]. we HypoThEsizeD THat experIMENtAl warMInG May nOT aFfeCT ThESE c And n pOols in thiS Study.
2. MaTEriaLs anD MeTHODS {#SeC2}
========================
2.1. STUdy AreA, EXPeRimeNTAL DEsIgn, aND SoIl sAMpLiNG {#SEC2.1}
-------------------------------------------------------
A dEtAIlED DescRipTiON OF thE StUDY AreA, THE WarMInG expERiMentAL DesiGN, The meAsuREmenTs Of MICroclImatE faCToRs (incLuDINg soil TEmpERatUre ANd sOIl moIsture), AnD thE SoIl SamPliNG ARe gIVen iN fu et aL. \[[@b20], [@B22]\].
brIEFLy, THRee alpINe MEaDOw sItEs WEre EstABLIshEd at THree ELevaTIonS (i.E., A lOW (30°30′n, 91°04′E, ANd 4313 m), mID- (30°31′n, 91°04′E, aNd 4513 M), anD High (30°32′N, 91°03′e, AND 4693 m) eLEVaTion) aT dAmxuNg graSSland OBSeRVatioN sTATIOn Of tiBeT autoNomoUS ReGioN in ChInA In May 2010.
annUal meaN aiR TEMpEratuRE AND PREcIPItAtiOn iS 1.3°C anD \~476.8 Mm, rESPecTiVEly \[[@b20], [@b21]\]. thE veGEtAtIon Is*KObrESia*-dOMiNaTed aLpine meadow and RoOtS are maINlY cOnCeNTrATeD In ThE toPSOil layEr (0--20 CM) \[[@b21], [@B22]\]. THe soIL Is ClAssiFied As Sandy loam, with Ph oF 6.0--6.7, organIC maTter Of 0.3--11.2%, AND toTAL n Of 0.03--0.49% \[[@B20], [@B22]\].
opeN TOp ChAMbers (OTCS, 3 mM thicK PoLYcaRboNaTE) WERe UsED To enHaNce TeMPeratuRe \[[@B22], [@B23]\]. tHe bOTtOm and TOP DiAMETers ANd THe Height oF oTcS wErE 1.45 M AnD 1.00 M anD 0.40 m, ResPeCtIVely \[[@b20], [@B22]\]. FOr EaCH site, FOuR OtcS anD theiR PairEd COnTRol PlOTS (1 m × 1 M) wErE RaNDoMLy ESTABLIsHeD iN MAy 2010. tHERe WaS \~3 M DiStANCE bEtwEEn pLOTs.
DAIly MEAN soiL tEmPEraTUrE (*T* ~*S*~) DURiNG tHe sTUdY PeRiod of JUly-sEpTEmBEr In 2011 INSIDE thE otcs INcREAsed bY 1.26°C, 0.98°c, anD 1.37°C at THe lOW, mID-, AnD hIGh ELevatiON, REsPEcTIVElY, coMPARED TO cOntroL PLOtS \[[@b20]\]. iN CoNtRAsT, eXPerImentAl wARmiNG deCReASed DAily mEAN soiL moIStUre (sM) by 0.04 M^3^ m^−3^ In aLL siteS \[[@b20]\]. DAILy meAn *t* ~*s*~ decreaseD wITh INCrEaSing EleVATion fRom tHE lOw TO hIgh ELEVatioN \[[@B20]\].
we CoLLectEd TOpsOIl SAMpLES (0--20 CM dEPTH) InsiDE EaCH ploT UsINg a PRobE 3.0 cm IN DIAmetER oN jULy 7, aUgust 9, aNd SEPTEMBer 10, 2011 \[[@B20]\]. fivE soiL sUBsAmPlES wEre rAndOmly SAmPLeD aNd COMpOSITeD InTo ONE Soil SAmpLe FOr EAch pLot \[[@b20]\]. SuBsAmples OF tHe frEsh SoIl WEre USEd tO MeAsuRe DoC, dON, NH~4~ ^+^-n, AND no~3~ ^−^-N ANd OtHEr suBSAMPles Of The FrEsh SoiL WERe air-DrieD For tHe meAsUREmEnTs Of Soc ANd tn.
2.2. soil anaLySis {#Sec2.2}
------------------
a MorE DeTAIlEd deSCripTion OF MeASUReMentS oF soiL INoRGanIC N (N~mIn~, I.E., Sum of Nh~4~ ^+^-N and NO~3~ ^−^-n), DON, AND DOc CAN bE fOUnD in fu et Al. \[[@b21]\]. brIEfLY, soil inOrGaNIC n In 20 g fReSh SoIl SAMplE waS eXTrAcTED wITh 100 Ml K~2~SO~4~, fIlteRED THRouGh 0.45 *Μ*M MembrAnE, and aNalYzeD ON A LacHAt qUiKChEM aUToMated ION aNALyZEr. diSsolVeD ORGaniC c anD tN (DTn) IN AnOtHeR 20 G FrESh SOIl SAMplE WaS EXtrAcTed wiTH 100 ML uLtRaPurE WatEr anD fILteRED ThrOUgH 0.45 MembRANE. thE eXtrACtable SoC and tn COncEnTRatIOns in The uLTrApURe wAteR extRaCTS wEre mEasuRed uSING a LIQui ToC Ii elemeNtAr AnALyzeR (ELEMeNtaR lIQUi tOC, eLemENtAr cO., hAnAU, GERMAny) aNd a Uv-1700 PHArmAsPEC vIsiBle SPEcTRophOTOmeTEr (220 NM anD 275 Nm), rESPeCTIVelY. we ALsO ANAlyZEd disSOlVED inorGANic N (dIN) In The UltrapURE wateR eXtRAcTs oN A LaChAT QUiKchEM automaTED IOn analyzEr. tHen DoN waS cALCulATEd aS ThE diFfErence BEtWeen DtN AND dIN. The PotaSSIum DiCHROmatE MEtHod waS usEd to DEtErmINe sOc \[[@b24]\]. SoiL tn WAs measureD On A cN ANAlYzeR (ElEMENTaR VaRiOMax Cn). soIL mIcROBIal BIOmass (MbC) and n (MBn) dATA WERE oBtaINEd froM fu et al. \[[@B20]\].
2.3. sTATISTIcAL aNAlYsiS {#sec2.3}
-------------------------
iN Order to EXamINe ThE eLEVATiON eFFeCt, REPEateD-MEAsuRES ANovA with experImeNtaL WArming aNd ElEVAtiOn aS tHe BetWEen sUBJeCT FaCToRs AND wITH SAMpLiNg daTe AS ThE wITHin SUBJEcT FaCToR Was pErformED fOR a sPeCIfiC sOIl proPErTy (i.e., sOc, Tn, DoC, DoN, RatIO Of dOC TO don (dOc/don), NH~4~ ^+^-n, No~3~ ^−^-n, RatIO Of Nh~4~ ^+^-n tO no~3~ ^−^-n(nh~4~ ^+^-n/No~3~ ^−^-N), and N~mIn~). At EAch siTe, rEPeated-MEaSuRES anoVa wiTh EXpERImenTAl WaRmING (I.e., oTCS Versus controL) as tHe beTWEEN SUbjecT fActOr aND wITH sampLINg daTE aS thE withIN suBJEcT fACToR waS CONDUcTed fOr EacH sOiL PrOPERTY. SiNGle fACtOr LiNeaR REg | 1. Introduction {#sec1}=============== Soil organic C (SOC) and total N (TN) arevery important C and N pools in the terrestrialecosystems \[[@B1], [@B2]\].As the components of labile CandNpools in soils, dissolved organicC (DOC) and N(DON) and soil ammonium and nitrateN (NH~4~ ^+^-N and NO~3~ ^−^-N)play crucialroles in the biogeochemistry of Cand N and in the nutrient transformation \[[@B3]--[@B5]\]. With the context of climatic warming, how SOC, TN, DOC,DON, NH~4~ ^+^-N, and NO~3~ ^−^-N respond is vital to global C and N cycling \[[@B1],[@B2]\]. However, inconsistent results on the responses of these C and N poolsto climatic warming have been observedwith respect tovegetation types and initial soil characteristics \[[@B2], [@B3], [@B6]--[@B14]\]. For example, Heetal. \[[@B2]\]demonstrated that six-year warming (\~1.4°C increase of10 cm soiltemperature) significantly decreased soilCby 129.3 g m^−2^ina temperate steppeof Inner Mongolia. In contrast, Li et al. \[[@B7]\] foundthattwo-year warming significantlyincreased SOC inanalpine meadow (\~2.1°C increase of air temperature) but significantly reduced TN inanalpine swamp meadow (\~2.3°C increase of air temperature) on the Tibetan Plateau.Hagedorn et al. \[[@B13]\] indicated that one-growing-season warming (\~4°C increase of 5 cm soil temperature) did not significantly influence DOC. Song et al. \[[@B1]\] pointed out that six-year warming (\~1.2°C increase of10 cm soil temperature) significantly reduced DOC in a temperate steppe in Inner Mongolia. Biasi et al.\[[@B15]\] indicated that two-year warming (\~0.9°C increase of 5 cm soil temperature) did not have obvious effects on DON, NH~4~ ^+^-N,NO~3~ ^−^-N, and N~min~ in a lichen-rich dwarf shrub tundrain Siberia. Bai et al. \[[@B14]\] stated that experimental warming (\~0.6--6.7°C in soil temperature) had a significant positiveeffect on N~min~ butnot on TN across all biomes. Therefore, how climatic warming acts on C and N cycling still remains unclear.More than 70% of the Tibetan Plateau is covered with grasslands \[[@B16]\]. The alpine grasslands of this Plateau are one of the systems most sensitive to global change \[[@B17], [@B18]\]. Inalpine grasslands, understanding the responses of SOC, DOC, TN, DON, NH~4~ ^+^-N, and NO~3~ ^−^-Nto climatic warming are crucial for predicting future changes in soil fertility and C sequestration. The alpine meadow isone of the most typical grasslands types onthe Tibetan Plateaubeing subjected to climatic warming \[[@B19]\]. Information on how theseCand Npools alongan elevation gradient respond to climatic warming is scarce on the Tibetan Plateau. Here weset up a warming experiment in an alpine meadowat three elevations (i.e., 4313 m, 4513m, and4693m) on the Northern Tibetan Plateau. The main objective was to investigate the effects of short-term experimental warming on SOC, TN, DOC, DON, NH~4~ ^+^-N, and NO~3~ ^−^-N. Our previous study indicated that short-term experimental warmingcould notaffect soil microbial biomass \[[@B20]\] and soil microbial activity regulated the balancesofsoil CandN pools in the alpine meadow \[[@B21]\]. We hypothesized that experimental warming may not affect theseC and N pools inthis study. 2. Materials andMethods {#sec2}======================== 2.1. Study Area, Experimental Design, and Soil Sampling {#sec2.1} -------------------------------------------------------A detaileddescriptionof the study area, the warming experimental design, the measurements ofmicroclimate factors (includingsoiltemperatureand soil moisture), and the soil sampling are given in Fu et al. \[[@B20], [@B22]\]. Briefly, threealpine meadow siteswere established at three elevations (i.e., a low (30°30′N, 91°04′E, and 4313 m), mid- (30°31′N, 91°04′E, and 4513 m),and high (30°32′N, 91°03′E, and 4693 m) elevation)at Damxung Grassland ObservationStationof Tibet Autonomous Region in China in May 2010. Annual mean air temperature and precipitation is 1.3°Cand\~476.8 mm, respectively \[[@B20], [@B21]\]. Thevegetation is*Kobresia*-dominatedalpine meadow and roots are mainly concentratedin the topsoil layer (0--20cm) \[[@B21], [@B22]\]. The soilis classified as sandy loam,withpH of 6.0--6.7, organic matter of 0.3--11.2%,and total Nof0.03--0.49% \[[@B20], [@B22]\]. Open top chambers (OTCs, 3 mm thickpolycarbonate) were used to enhance temperature \[[@B22], [@B23]\]. The bottom and top diameters and the heightofOTCs were 1.45 m and 1.00 m and 0.40 m,respectively \[[@B20], [@B22]\].For each site, four OTCs and their paired control plots (1 m ×1 m)wererandomly established in May 2010. There was \~3 m distance between plots. Dailymean soil temperature (*T* ~*s*~) during the study periodof July-September in 2011 inside the OTCsincreased by1.26°C, 0.98°C, and 1.37°C at the low, mid-,and highelevation, respectively, compared to control plots \[[@B20]\]. In contrast, experimental warming decreased daily mean soil moisture (SM) by 0.04 m^3^ m^−3^ in allsites \[[@B20]\]. Daily mean *T* ~*s*~ decreased with increasing elevation from the low tohigh elevation \[[@B20]\]. We collected topsoilsamples (0--20 cm depth)inside each plotusing a probe 3.0 cm in diameter on July 7,August9, and September 10, 2011 \[[@B20]\]. Five soil subsamples wererandomly sampled and composited intoone soil sample for each plot \[[@B20]\]. Subsamples of the fresh soil were used tomeasure DOC, DON, NH~4~^+^-N, and NO~3~ ^−^-N andother subsamples of the fresh soil were air-dried for themeasurements of SOC and TN. 2.2. Soil Analysis {#sec2.2}------------------ A more detailed description of measurements of soil inorganic N(N~min~,i.e.,sum of NH~4~ ^+^-N and NO~3~ ^−^-N),DON, and DOC can be foundin Fu et al.\[[@B21]\].Briefly, soilinorganicN in 20g fresh soilsamplewas extracted with 100 mL K~2~SO~4~, filtered through 0.45 *μ*m membrane, andanalyzed on a LACHAT Quikchem Automated Ion Analyzer. Dissolved organic C andTN (DTN) inanother 20 g fresh soil sample was extractedwith 100 mL ultrapurewaterand filtered through 0.45 membrane. The extractable SOC and TN concentrations in the ultrapure water extractswere measured using a Liqui TOC II elementar analyzer (Elementar Liqui TOC, Elementar Co., Hanau, Germany) anda UV-1700PharmaSpecvisible spectrophotometer (220 nm and 275nm), respectively. We also analyzed dissolved inorganic N (DIN) in the ultrapure waterextracts on a LACHAT Quikchem Automated Ion Analyzer. Then DON was calculatedas thedifference between DTNand DIN. The potassium dichromate method was used to determine SOC \[[@B24]\].Soil TN was measured on a CN analyzer (Elementar Variomax CN). Soil microbial biomass (MBC) and N(MBN) data were obtained from Fuet al. \[[@B20]\]. 2.3. Statistical Analysis {#sec2.3} ------------------------- In order to examine the elevation effect, repeated-measures ANOVAwithexperimental warming and elevation as the between subjectfactorsand with sampling date as the within subject factorwas performedfor a specificsoil property (i.e., SOC,TN,DOC, DON, ratio of DOC to DON(DOC/DON), NH~4~ ^+^-N, NO~3~ ^−^-N, ratio of NH~4~ ^+^-N to NO~3~ ^−^-N(NH~4~ ^+^-N/NO~3~ ^−^-N), and N~min~). Ateach site, repeated-measures ANOVA with experimental warming (i.e., OTCs versus control)as the between subject factor andwith sampling date as the within subject factorwas conducted for each soil property. Singlefactorlinear reg | 1. Introduction {#sec1} =============== Soil organic _C_ (SOC) and total N (TN) are very important C and N _pools_ in the _terrestrial_ _ecosystems_ \[[@B1], _[@B2]\]._ As the components of labile C and _N_ pools in soils, dissolved organic C (DOC) and N (DON) and soil ammonium and _nitrate_ N (NH~4~ ^+^-N _and_ NO~3~ _^−^-N)_ _play_ crucial roles in the biogeochemistry of C and N and in _the_ nutrient transformation \[[@B3]--[@B5]\]. With _the_ context of climatic warming, how SOC, _TN,_ DOC, DON, NH~4~ ^+^-N, and NO~3~ _^−^-N_ respond is vital to _global_ C and N cycling _\[[@B1],_ [@B2]\]. However, inconsistent results on the responses of these _C_ _and_ N _pools_ to climatic warming have _been_ observed _with_ respect to vegetation types and initial soil characteristics \[[@B2], _[@B3],_ [@B6]--[@B14]\]. _For_ example, He et al. _\[[@B2]\]_ demonstrated that six-year warming (\~1.4°C _increase_ of 10 _cm_ _soil_ temperature) significantly decreased soil C by 129.3 g m^−2^ in a temperate steppe of Inner Mongolia. _In_ _contrast,_ _Li_ _et_ al. \[[@B7]\] found that two-year _warming_ significantly _increased_ SOC _in_ an alpine meadow (\~2.1°C increase of air temperature) but significantly _reduced_ TN in an alpine swamp meadow (\~2.3°C _increase_ of air temperature) on the Tibetan _Plateau._ Hagedorn et _al._ \[[@B13]\] indicated that one-growing-season _warming_ (\~4°C increase of 5 cm soil temperature) did not significantly _influence_ DOC. Song et al. \[[@B1]\] pointed out _that_ six-year warming (\~1.2°C increase _of_ 10 cm soil _temperature)_ significantly reduced DOC in a temperate steppe in Inner Mongolia. Biasi et al. \[[@B15]\] indicated that two-year warming (\~0.9°C increase of _5_ cm soil temperature) _did_ not have obvious _effects_ _on_ DON, NH~4~ ^+^-N, NO~3~ ^−^-N, and N~min~ in a lichen-rich dwarf _shrub_ tundra in Siberia. _Bai_ _et_ al. \[[@B14]\] stated that experimental warming (\~0.6--6.7°C in soil temperature) _had_ a significant positive effect on _N~min~_ _but_ not on _TN_ _across_ all _biomes._ Therefore, _how_ climatic _warming_ acts on _C_ and N cycling still remains unclear. More than 70% of the Tibetan Plateau is covered with grasslands \[[@B16]\]. The alpine _grasslands_ _of_ this Plateau are one of the systems most _sensitive_ to _global_ change _\[[@B17],_ [@B18]\]. _In_ alpine grasslands, _understanding_ the _responses_ of SOC, DOC, TN, DON, NH~4~ ^+^-N, and NO~3~ ^−^-N _to_ climatic warming are _crucial_ for predicting future changes in soil fertility and C sequestration. The alpine meadow is one of _the_ most _typical_ grasslands types on the Tibetan Plateau being subjected to _climatic_ _warming_ \[[@B19]\]. Information on how these C and _N_ pools _along_ an elevation gradient respond to _climatic_ warming is scarce on the Tibetan Plateau. Here we _set_ up _a_ warming experiment in an alpine meadow at three _elevations_ (i.e., 4313 m, 4513 m, and _4693_ m) on _the_ Northern Tibetan _Plateau._ The main objective was to investigate the effects of short-term experimental _warming_ on SOC, TN, _DOC,_ DON, NH~4~ _^+^-N,_ and NO~3~ ^−^-N. _Our_ previous study _indicated_ _that_ short-term _experimental_ warming could _not_ _affect_ _soil_ _microbial_ biomass \[[@B20]\] and soil _microbial_ activity regulated the balances _of_ soil C and N pools in _the_ alpine meadow _\[[@B21]\]._ _We_ hypothesized that _experimental_ warming may not affect these C and N _pools_ in _this_ study. 2. Materials and Methods {#sec2} ======================== 2.1. Study Area, _Experimental_ _Design,_ and Soil Sampling _{#sec2.1}_ ------------------------------------------------------- A detailed description of the study area, the warming experimental design, the _measurements_ of microclimate factors _(including_ soil temperature _and_ soil _moisture),_ _and_ the soil sampling are given in Fu et al. \[[@B20], [@B22]\]. Briefly, three alpine meadow sites were established at three elevations (i.e., a _low_ (30°30′N, _91°04′E,_ and 4313 m), mid- _(30°31′N,_ 91°04′E, _and_ 4513 _m),_ _and_ high (30°32′N, 91°03′E, and 4693 _m)_ _elevation)_ at Damxung Grassland Observation Station of Tibet Autonomous Region in China in May _2010._ Annual mean air _temperature_ and precipitation is _1.3°C_ _and_ \~476.8 mm, respectively \[[@B20], [@B21]\]. The vegetation is*Kobresia*-dominated alpine meadow _and_ _roots_ are mainly concentrated in _the_ topsoil layer (0--20 _cm)_ \[[@B21], [@B22]\]. The soil _is_ classified as sandy _loam,_ with pH _of_ 6.0--6.7, organic matter _of_ 0.3--11.2%, and total _N_ _of_ 0.03--0.49% \[[@B20], [@B22]\]. Open top chambers (OTCs, _3_ mm _thick_ polycarbonate) _were_ used to enhance _temperature_ _\[[@B22],_ _[@B23]\]._ The bottom _and_ top diameters and _the_ height of OTCs were 1.45 m and 1.00 m _and_ 0.40 m, respectively \[[@B20], [@B22]\]. _For_ each site, four OTCs and their _paired_ _control_ plots _(1_ _m_ × 1 m) were _randomly_ established _in_ May 2010. There was \~3 m _distance_ between plots. Daily mean _soil_ temperature _(*T*_ _~*s*~)_ during the _study_ period _of_ July-September in 2011 inside _the_ OTCs _increased_ by 1.26°C, 0.98°C, and 1.37°C _at_ the _low,_ mid-, and high elevation, respectively, compared to control plots \[[@B20]\]. _In_ contrast, experimental warming _decreased_ _daily_ mean _soil_ moisture (SM) by _0.04_ m^3^ m^−3^ in _all_ sites \[[@B20]\]. Daily mean *T* ~*s*~ decreased with increasing elevation from the _low_ to high elevation \[[@B20]\]. We collected topsoil samples (0--20 cm depth) inside each plot using _a_ probe 3.0 cm in diameter on July 7, August _9,_ and September _10,_ 2011 \[[@B20]\]. _Five_ soil subsamples _were_ randomly sampled and composited into one soil _sample_ for each plot \[[@B20]\]. Subsamples of the fresh soil were _used_ _to_ measure DOC, DON, NH~4~ ^+^-N, and NO~3~ ^−^-N and other subsamples _of_ the fresh soil were air-dried for the measurements of SOC _and_ TN. 2.2. Soil Analysis _{#sec2.2}_ ------------------ A more detailed description of measurements of soil inorganic N (N~min~, i.e., sum of NH~4~ ^+^-N and NO~3~ ^−^-N), _DON,_ and DOC _can_ be found in Fu et al. \[[@B21]\]. Briefly, soil inorganic _N_ in 20 g fresh soil sample was _extracted_ with 100 mL K~2~SO~4~, filtered _through_ 0.45 *μ*m membrane, and analyzed on a LACHAT Quikchem Automated _Ion_ Analyzer. Dissolved organic C and TN (DTN) in another 20 g fresh soil sample was extracted with 100 mL ultrapure water and filtered _through_ 0.45 membrane. The extractable _SOC_ and TN concentrations in the ultrapure water _extracts_ _were_ measured using a Liqui TOC II _elementar_ analyzer (Elementar _Liqui_ TOC, Elementar Co., Hanau, Germany) and a UV-1700 PharmaSpec visible spectrophotometer (220 _nm_ and 275 nm), respectively. We also analyzed dissolved inorganic N _(DIN)_ in the _ultrapure_ water extracts on a LACHAT Quikchem Automated Ion Analyzer. Then _DON_ _was_ _calculated_ as the difference between DTN and DIN. The potassium dichromate method _was_ _used_ to _determine_ SOC _\[[@B24]\]._ _Soil_ TN _was_ measured on _a_ CN analyzer (Elementar Variomax CN). Soil microbial biomass _(MBC)_ and N (MBN) data were obtained from Fu et al. \[[@B20]\]. 2.3. Statistical Analysis {#sec2.3} ------------------------- In order to _examine_ the elevation effect, repeated-measures ANOVA with experimental warming and elevation as the _between_ subject factors and with _sampling_ date _as_ the _within_ subject _factor_ _was_ performed _for_ a _specific_ soil property (i.e., _SOC,_ TN, DOC, DON, ratio of DOC _to_ _DON_ (DOC/DON), NH~4~ ^+^-N, NO~3~ ^−^-N, ratio _of_ NH~4~ ^+^-N to NO~3~ ^−^-N(NH~4~ ^+^-N/NO~3~ ^−^-N), and N~min~). At each site, repeated-measures ANOVA with experimental warming _(i.e.,_ OTCs versus control) as the between subject factor _and_ with sampling _date_ as the within subject factor was conducted _for_ each soil _property._ Single factor linear _reg_ |
The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.
Introduction {#s1}
============
Mechanical ventilation (MV) has been used in critical care patients for decades. In spite of its life-saving potential, it has several shortcomings. A number of experimental studies have shown that mechanical ventilation may result in the appearance of inflammatory mediators in the lung [@pone.0114247-Uhlig1] and subsequently in oedema. [@pone.0114247-Dreyfuss1] Ventilator-Induced Lung Injury (VILI) causes macro and microscopic unspecific changes[@pone.0114247-Katzenstein1] similar to those found in patients with Acute Respiratory Distress Syndrome (ARDS). As it happens with ARDS, VILI is basically the result of important changes in the permeability of the alveolar-capillary membrane. [@pone.0114247-Dreyfuss2] The potential of mechanical ventilation for triggering or worsening pulmonary damages has been shown in animal models where the application of non-physiological ventilatory parameters (mostly very high tidal volumes) aggravated the condition of animals with a previously injured lung [@pone.0114247-Corbridge1], and even caused an injury in those without a previous pulmonary pathology. [@pone.0114247-Dreyfuss1] The use of low tidal volumes has proved to be a better approach in ARDS patients, survival being improved in strategies based on its usage. [@pone.0114247-Amato1]--[@pone.0114247-Villar1] Interestingly, recent experimental and clinical work has demonstrated that MV with low tidal volume can induce similar pulmonary changes to those noticed for VILI [@pone.0114247-Cobelens1]--[@pone.0114247-Wolthuis1] and that its appearance may be related to MV exposure time. [@pone.0114247-Hegeman1]
Aquaporins are a family of small transmembrane proteins that help water to move fast, selectively and bi-directionally through lipid bi-layers. [@pone.0114247-Kozono1], [@pone.0114247-Ma1] 13 different types have been identified in mammals, [@pone.0114247-Verkman1] from which the lung is known to express four: AQP-1, in the pulmonary capillary endothelium (especially alveolar), and the visceral pleura; AQP-3, in the tracheal epithelium; AQP-4, in the tracheal and bronchial epithelium; and AQP-5, on type I pneumocyte cells of the alveoli, on the membrane adjoining to the alveolar lumen. [@pone.0114247-King1] Their role in the development and resolution of pulmonary oedema gives rise to controversy, although it does seem to play a part in VILI. [@pone.0114247-Hales1]
This research aimed to verify if MV with low or moderately high tidal volumes (10 ml/Kg) sustained over time results in lung injury, subsequently altering pulmonary water content and microvascular permeability, as observed in VILI, and to objectivize what happens with AQP 1 and 5 expression, both types mainly involved in the formation of lung oedema, under the same ventilation conditions.
Material and Methods {#s2}
====================
1. Ethics statement {#s2a}
-------------------
The project was carried out after approval from the Ethics Committee for Animal Experimentation and Wellbeing of the Research Foundation of Valencia\'s Hospital Clínico Universitario.
2. Animal model and monitoring {#s2b}
------------------------------
A total of 30 rats were anaesthetised by intraperitoneal injection of ketamine 80 mg/kg and xylazine 5 mg/kg. 5 rats (group C or controls) were sacrificed by intravenous injection of 100 mg/Kg thiopental. The rest of the animals (n = 25) were performed a surgical tracheostomy, using a teflon cannula (Surflo, 16G). Rats were randomly allocated into two groups. 12 rats were ventilated for 2 hours (group 2H) with a Harvard Rodent Ventilator, model 683 (Harvard Apparatus) with a tidal volume of 10 ml/kg and a respiratory rate of 90 breaths/minute. 13 rats were ventilated with exactly the same parameters for 4 hours (group 4H). The cervical vascular bundle was dissected, and the right internal jugular vein and the right carotid artery were catheterized to continuously monitor heart rate (HR) and mean arterial pressure (MAP). Peak inspiratory pressure and respiratory system compliance were continuously recorded.
Anaesthesia was maintained by continuous intravenous infusion of ketamine and cisatracurium using dosis of 100 mcg/Kg/min and 2--3 mcg/Kg/min, respectively (20 ml of ketamine 5%, 10 ml of cisatracurium 0,2% and 20 ml of saline solution 0,9% at a rate of approximately 0,1 ml/h in the internal jugular vein). Anesthesia was supplemented, in cases in which it was necessary, by administration of an intravenous bolus of 0.1 ml of the mixture. Gasometric samples were taken in all animals in groups 2H and 4H at the beginning of MV and 30 minutes before the end of MV.
Rats were sacrificed by intravenous injection of sodium thiopental. The left lung was used for the determination of lung water content. The right lung of 6 rats from groups 2H and 4H was used for determining AQP 1 and AQP 5 expression. Lungs were either frozen in liquid nitrogen or paraffin-embedded for immunohistochemical sectioning and marking. 2 rats in group C and 4 rats from groups 2H and 4H were used to establish pulmonary macrovascular permeability.
3. Measuring lung oedema {#s2c}
------------------------
Lungs were dried with filter paper and placed on a Petri dish with known weight to obtain lung wet weight (LWW). They were then placed in a drying chamber at 80°C for 96 hours and their dry weight (LDW) was determined.
Two indicators of the amount of oedema were obtained: Lung WW/DW ratio and the proportion of pulmonary water, expressed in percentages (%~water~). The latter parameter was estimated using this formula: % ~water~ = (LWW - LDW)/LWW \* 100
4. Measuring microvascular permeability {#s2d}
---------------------------------------
Microvascular permeability was quantified using Evans Blue Dye. 0.5 ml Evans Blue was injected intravenously (30 mg/Kg) 30 minutes before sacrificing the animal. Rats were sacrificed by exsanguination from the carotid artery, but saline was simultaneously infused via the jugular vein in the same amount as that of the blood extracted.
After death, the right lung was separated and immersed in formamide (5 ml) and homogenised for 2 min. The resulting suspension was incubated at 37°C/18 h and then centrifuged at 5000xg/30 minutes, and the supernatant was measured. Concentration of Evans Blue in the supernatant was spectrophotometrically determined.
5. Study of aquaporin expression {#s2e}
--------------------------------
### 5.1 Western blot {#s2e1}
Proteins were extracted from previously frozen lungs. A Compartmental Protein Extraction Kit (Chemicon International, Temecula CA) was used. 200--400 mg tissue was homogenised in cold buffer C (1 ml/g tissue) and Ultra Turrax (KA, Staufen, Germany). Two protein fractions were obtained for each sample: cytoplasm and membrane, and they were quantified. Proteins in each fraction (100 mg) were separately run on a Tris-HCl/SDS gel, 8% acrylamide, and they were transferred onto a nitrocellulose membrane (Hybond-ECL, Amersham). After washing the membrane with distilled water, it was blocked with a PBS/Tween solution, 0.2%, with 5% skimmed milk. It was then incubated with the primary antibody during 2 hours at room temperature. The antibodies used were Anti-Rat AQP1 (Alpha Diagnostic, San Antonio, TX) and Anti-Rat AQP5 (Alpha Diagnostic, San Antonio, TX), both with a 2 mg/ml concentration. After several washes with PBS/Tween 0.2%, it was incubated with the secondary antibody Anti-Rabbit IgG (DAKO, Glostrup, Denmark) in 1∶2000 dilution. β-actin expression was detected as an internal control, and relative protein content was analysed using the enhanced chemoluminiscence method.
### 5.2 Real-time polymerase chain reaction with reverse transcriptase {#s2e2}
Lungs were cut with a microtome, three sections being obtained for each sample for total RNA extraction with TRIZOL (Reagent InvitrogenTM Life Technologies) as in the phenol extraction method described by Chomczynsky. [@pone.0114247-Chomczynski1] Microsections were added 1 ml TRIZOL and homogenized (Polytron PT 1200, Kinematica AG) and centrifuged at 10.000 rpm/10 min at 4°C. The supernatant was removed and RNA was precipitated by adding 0.1 volumes of sodium acetate 3 M, 2.5 volumes cold ethanol and 0.5 µl glycogen (20 mg/ml). RNA was centrifuged again, air-dried and resuspended in 20 µl Tris/EDTA buffer. RNA was reversely transcribed to cDNA with Superscript II (Invitrogen), by incubation | the authors confirm and all data underlying the findings are fully available without restriction. all relevant data are within the paper. introduction { # s1 } = = = = = = = = = = = = mechanical ventilation ( sv ) has been used against critical care patients for decades. in spite of actual life - saving potential, it has several shortcomings. a number of experimental studies have shown that mechanical ventilation may result in the disruption of inflammatory mediators in the lung [ @ pone. 0114247 - uhlig1 ] and subsequently in oedema. [ @ pone. com - dreyfuss1 ] ventilator - acute lung injury ( vili ) causes macro and microscopic unspecific changes [ @ pone. 0114247 - katzenstein1 ] similar to those found in patients with acute limb distress syndrome ( ards ). as it happens with ards, vili is basically the result of important changes in the permeability of the alveolar - capillary membrane. [ @ pone. 0114247 - dreyfuss2 ] the potential of mechanical ventilation for triggering or worsening pulmonary damages has been shown in animal models where the application of non - physiological ventilatory parameters ( mostly very high tidal volumes ) aggravated the condition of animals with a previously injured lung [ @ pone. 0114247 - corbridge1 ], and even caused an injury in those without a previous pulmonary pathology. [ @ pone. 0114247 - dreyfuss1 ] the use of low tidal volumes has proved to be a better approach in ards patients, survival being improved in strategies based on mv usage. [ @ pone. 0114247 - amato1 ] - - [ @ pone. 0114247 - villar1 ] interestingly, recent experimental and clinical work has demonstrated that mv with low tidal volume can induce similar pulmonary changes to those noticed for vili [ @ pone. 0114247 - cobelens1 ] - - [ @ pone. 0114247 - wolthuis1 ] and that its appearance may be related to mv exposure time. [ @ pone. 0114247 - hegeman1 ] there are a family of small transmembrane proteins that help water to move fast, selectively and bi - directionally through lipid bi - layers. [ @ po ##ne. 0114247 - kozono1 ], [ @ pone. 0114247 - ma1 ] 13 different types have been identified in mammals, [ @ pone. 0114247 - verkman1 ] from which the lung is known to express four : aqp - 1, in the pulmonary capillary endothelium ( especially alveolar ), and the visceral pleura ; aqp - 3, in the tracheal epithelium ; aqp - 4, in the tracheal and bronchial epithelium ; and aqp - 5, on type i pneumocyte cells of the alveoli, on the membrane adjoining to the alveolar lumen. [ @ pone. 0114247 - king1 ] their role in the development and resolution of pulmonary oedema gives rise to controversy, although it does seem to play a part in vili. [ @ pone. 0114247 - hales1 ] this research aimed to verify if mv with low or moderately high tidal volumes ( 10 ml / kg ) sustained over time results in lung injury, subsequently altering pulmonary water content and microvascular permeability, as observed in vili, and to objectivize what happens with aqp 1 and 5 expression, both types mainly involved in the formation of lung oedema, under the same ventilation conditions. material and methods { # s2 } = = = = = = = = = = = = = = = = = = = = 1. ethics statement { # s2a } - - - - - - - - - - - - - - - - - - - the project was carried out after approval from the ethics committee for animal experimentation and wellbeing of the research foundation of valencia \ ' s hospital clinico universitario. 2. animal model and monitoring { # s2b } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - a total of 30 rats were anaesthetised by intraperitoneal injection of ketamine 80 mg / kg and xylazine 5 mg / kg. 5 rats ( group c or controls ) were sacrificed by intravenous injection of 100 mg / kg thiopental. the rest of the animals ( n = 25 ) were performed a surgical tracheostomy, using a teflon cannula ( surflo, 16g ). rats were randomly allocated into two groups. 12 rats were ventilated for 2 hours ( group 2h ) with a harvard rodent ventilator, model 683 ( harvard apparatus ) with a tidal volume of 10 ml / kg and a respiratory rate of 90 breaths / minute. 13 rats were ventilated with exactly the same parameters for 4 hours ( group 4h ). the cervical vascular bundle was dissected, and the right internal jugular vein and the right carotid artery were catheterized to continuously monitor heart rate ( hr ) and mean arterial pressure ( map ). peak inspiratory pressure and respiratory system compliance were continuously recorded. anaesthesia was maintained by continuous intravenous infusion of ketamine and cisatracurium using dosis of 100 mcg / kg / min and 2 - - 3 mcg / kg / min, respectively ( 20 ml of ketamine 5 %, 10 ml of cisatracurium 0, 2 % and 20 ml of saline solution 0, 9 % at a rate of approximately 0, 1 ml / h in the internal jugular vein ). anesthesia was supplemented, in cases in which it was necessary, by administration of an intravenous bolus of 0. 1 ml of the mixture. gasometric samples were taken in all animals in groups 2h and 4h at the beginning of mv and 30 minutes before the end of mv. rats were sacrificed by intravenous injection of sodium thiopental. the left lung was used for the determination of lung water content. the right lung of 6 rats from groups 2h and 4h was used for determining aqp 1 and aqp 5 expression. lungs were either frozen in liquid nitrogen or paraffin - embedded for immunohistochemical sectioning and marking. 2 rats in group c and 4 rats from groups 2h and 4h were used to establish pulmonary macrovascular permeability. 3. measuring lung oedema { # s2c } - - - - - - - - - - - - - - - - - - - - - - - - lungs were dried with filter paper and placed on a petri dish with known weight to obtain lung wet weight ( lww ). they were then placed in a drying chamber at 80°c for 96 hours and their dry weight ( ldw ) was determined. two indicators of the amount of oedema were obtained : lung ww / dw ratio and the proportion of pulmonary water, expressed in percentages ( % ~ water ~ ). the latter parameter was estimated using this formula : % ~ water ~ = ( lww - ldw ) / lww \ * 100 4. measuring microvascular permeability { # s2d } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - microvascular permeability was quantified using evans blue dye. 0. 5 ml evans blue was injected intravenously ( 30 mg / kg ) 30 minutes before sacrificing the animal. rats were sacrificed by exsanguination from the carotid artery, but saline was simultaneously infused via the jugular vein in the same amount as that of the blood extracted. after death, the right lung was separated and immersed in formamide ( 5 ml ) and homogenised for 2 min. the resulting suspension was incubated at 37°c / 18 h and then centrifuged at 5000xg / 30 minutes, and the supernatant was measured. concentration of evans blue in the supernatant was spectrophotometrically determined. 5. study of aquaporin expression { # s2e } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - # # # 5. 1 western blot { # s2e1 } proteins were extracted from previously frozen lungs. a compartmental protein extraction kit ( chemicon international, temecula ca ) was used. 200 - - 400 mg tissue was homogenised in cold buffer c ( 1 ml / g tissue ) and ultra turrax ( ka, staufen, germany ). two protein fractions were obtained for each sample : cytoplasm and membrane, and they were quantified. proteins in each fraction ( 100 mg ) were separately run on a tris - hcl / sds gel, 8 % acrylamide, and they were transferred onto a nitrocellulose membrane ( hybond - ecl, amersham ). after washing the membrane with distilled water, it was blocked with a pbs / tween solution, 0. 2 %, with 5 % skimmed milk. it was then incubated with the primary antibody during 2 hours at room temperature. the antibodies used were anti - rat aqp1 ( alpha diagnostic, san antonio, tx ) and anti - rat aqp5 ( alpha diagnostic, san antonio, tx ), both with a 2 mg / ml concentration. after several washes with pbs / tween 0. 2 %, it was incubated with the secondary antibody anti - rabbit igg ( dako, glostrup, denmark ) in [UNK] dilution. β - actin expression was detected as an internal control, and relative protein content was analysed using the enhanced chemoluminiscence method. # # # 5. 2 real - time polymerase chain reaction with reverse transcriptase { # s2e2 } lungs were cut with a microtome, three sections being obtained for each sample for total rna extraction with trizol ( reagent invitrogentm life technologies ) as in the phenol extraction method described by chomczynsky. [ @ pone. 0114247 - chomczynski1 ] microsections were added 1 ml trizol and homogenized ( polytron pt 1200, kinematica ag ) and centrifuged at 10. 000 rpm / 10 min at 4°c. the supernatant was removed and rna was precipitated by adding 0. 1 volumes of sodium acetate 3 m, 2. 5 volumes cold ethanol and 0. 5 µl glycogen ( 20 mg / ml ). rna was centrifuged again, air - dried and resuspended in 20 µl tris / edta buffer. rna was reversely transcribed to cdna with superscript ii ( invitrogen ), by incubation | The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Introduction {# s1} = = = = = = = = = = = = Mechanical ventilation (MV) has been used in critical care patients for decades. In spite of its life - saving potential, it has several shortcomings. A number of experimental studies have shown that mechanical ventilation may result in the appearance of inflammatory mediators in the lung [@ pone. 0114247 - Uhlig1] and subsequently in oedema. [@ pone. 0114247 - Dreyfuss1] Ventilator - Induced Lung Injury (VILI) causes macro and microscopic unspecific changes [@ pone. 0114247 - Katzenstein1] sLmi>ar to those found in patients with Acute Respiratory Distress Syndrome (ARDS ). As it happens with ARDS, VILI is basically the result of important changes in the permeability of the alveolar - capillary membrane. [@ pone. 0114247 - Dreyfuss2] The potential of mechanical ventilation for triggering or worsening pulmonary damages has been shown in animal models where the application of non - physiological ventilatory parameters (mostly very high tidal volumes) aggravated the condition of animals with a previously injured lung [@ pone. 0114247 - Corbridge1 ], and even caused an injury in those without a previous pulmonary pathology. [@ pone. 0114247 - Dreyfuss1] The use of low tidal volumes has proved to be a better approach in ARDS patients, survival being improved in strategies bsZed on its usage. [@ pone. 0114247 - Amato1] - - [@ pone. 0114247 - Villar1] Interestingly, recent experimental and clinical work has demonstrated that MV with low tidal volume can induce similar pulmonary changes to those nltuced for VILI [@ pone. 0114247 - Cobelens1] - - [@ pone. 0114247 - Wolthuis1] and that its appearance may be related to MV exposure time. [@ pone. 0114247 - Hegeman1] Aqua08rins are a family of small transmembrane proteins that help water to move fast, selectively and bi - directionally through lipid bi - layers. [@ pone. 0114247 - Kozono1 ], [@ pone. 0114247 - Ma1] 13 different types have been identified in mammals, [@ pone. 0114247 - Verkman1] from which the lung is known to express four: AQP - 1, in the pulmonary capillary endothelium (especially alveolar ), and the visceral pleura; AQP - 3, in the tracheal epithelium; AQP - 4, in the tracheal and bronchial epithelium; and AQP - 5, on type I pneumocyte cells of the alveoli, on the membrane adjoining to the alveolar lumen. [@ pone. 0114247 - King1] Their role in the development and resolution of pulmonary oedema gives rise to controversy, although it does seem to play a part in VILI. [@ pone. 0114247 - Hales1] This research aimed to verify if MV with low or moderately high tidal volumes (10 ml / Kg) sustained over time results in lung injury, subsequently altering pulmonary water content and microvascular permeability, as observed in VILI, and to objectivize what happens with AQP 1 and 5 expression, both types mainly involved in the formation of lung oedema, under the same ventilation conditions. Material and Methods {# s2} = = = = = = = = = = = = = = = = = = = = 1. Ethics statement {# s2a} - - - - - - - - - - - - - - - - - - - The project was carried out after approval from the Ethics Committee for Animal Experimentation and Wellbeing of the Research Foundation of Valencia \ ' s Hospital Clínico Universitario. 2. Animal model and monitoring {# s2b} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A total of 30 rats were anaesthetised by intrapeDitKneal injection of ketamine 80 mg / kg and xylazine 5 mg / kg. 5 rats (group C or controls) were sacrificed by intravenous injection of 100 mg / Kg thiopental. The rest of the animals (n = 25) were performed a surgical tracheostomy, using a teflon cannula (Surflo, 16G ). Rats were randomly allocated into two groups. 12 rats were ventilated for 2 hours (group 2H) with a Harvard Rodent Ventilator, model 683 (Harvard Apparatus) with a tidal volume of 10 ml / kg and a respiratory rate of 90 breaths / minute. 13 rats were ventilated with exactly the same parameters for 4 hours (group 4H ). The cervical vascular bundle was dissected, and the right internal jugular vein and the right carotid artery were catheterized to continuously monitor heart rate (HR) and mean arterial pressure (MAP ). Peak inspiratory pressure and respiratory system compliance were continuously recorded. Anaesthesia was maintained by continuous intravenous infusion of ketamine and cisatracurium using dosis of 100 mcg / Kg / min and 2 - - 3 mcg / Kg / min, respectively (20 ml of ketamine 5% , 10 ml of cisatracurium 0, 2% and 20 ml of saline solution 0, 9% at a rate of approximately 0, 1 ml / h in the internal jugular vein ). Anesthesia was supplemented, in cases in which it was necessary, by administration of an intravenous bolus of 0. 1 ml of the mixture. Gasometric samples were taken in all animals in groups 2H and 4H at the beginning of MV and 30 minutes before the end of MV. Rats were sacrificed by intravenous injection of sodium thiopental. The left lung was used for the determination of lung water content. The right lung of 6 rats from groups 2H and 4H was used for determining AQP 1 and AQP 5 expression. Lungs were either frozen in liquid nitrogen or paraffin - embedded for immunohistochemical sectioning and marking. 2 rats in group C and 4 rats from groups 2H and 4H were used to establish pulmonary macrovascular permeability. 3. Measuring lung oedema {# s2c} - - - - - - - - - - - - - - - - - - - - - - - - Lungs were dried with filter paper and placed on a Petri dish with known weight to obtain lung wet weight (LWW ). They were then placed in a drying chamber at 80 ° C for 96 hours and their dry weight (LDW) was determined. Two indicators of the amount of oedema were obtained: Lung WW / DW ratio and the proportion of pulmonary water, expressed in percentages (% ~ water ~ ). The latter parameter was estimated using this formula: % ~ water ~ = (LWW - LDW) / LWW \ * 100 4. Measuring microvascular permeability {# s2d} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Microvascular permeability was quantified using Evans Blue Dye. 0. 5 ml Evans Blue was injected intravenously (30 mg / Kg) 30 minutes before sacrificing the animal. Rats were sacrificed by exsanguination from the carotid artery, but saline was simultaneously infused via the jugKlaG vein in the same amount as that of the blood extracted. After death, the right lung was separated and immersed in formamide (5 ml) and homogenised for 2 min. The resulting suspension was incubated at 37 ° C / 18 h and then centrifuged at 5000xg / 30 minutes, and the supernatant was measured. Concentration of Evans Blue in the supernatant was spectrophotometrically determined. 5. Study of aquaporin expression {# s2e} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - # # # 5. 1 Western blot {# s2e1} Proteins were extracted from previously frozen lungs. A Compartmental Protein Extraction Kit (Chemicon International, Temecula CA) was used. 200 - - 400 mg tissue was homogenised in cold buffer C (1 ml / g tissue) and Ultra Turrax (KA, Staufen, Germany ). Two protein fractions were obtained for each sample: cytoplasm and membrane, and they were quantified. Proteins in each fraction (100 mg) w@r$ separately run on a Tris - HCl / SDS gel, 8% acrylamide, and they Sede transferred onto a nitrocellulose membrane (Hybond - ECL, Amersham ). After washing the membrane with distilled water, it was blocked with a PBS / Tween solution, 0. 2% , with 5% skimmed milk. It was then incubated with the primary antibody during 2 hours at room temperature. The antibodies used were Anti - Rat AQP1 (Alpha Diagnostic, San Antonio, TX) and Anti - Rat AQP5 (Alpha Diagnostic, San Antonio, TX ), both with a 2 mg / ml concentration. After several washes with PBS / Tween 0. 2% , it was incubated with the secondary antibody Anti - Rabbit IgG (DAKO, Glostrup, Denmark) in 1 ∶ 2000 dilution. β - actin expression was detected as an internal control, and relative protein content was analysed using the enhanced chemoluminiscence method. # # # 5. 2 Real - 4i,e polymerase chain reaction with reverse transcriptase {# s2e2} Lungs were cut with a microtome, three sections being obtained for each sample for total RNA extraction with TRIZOL (Reagent InvitrogenTM Life Technologies) as in the phenol extraction method described by Chomczynsky. [@ pone. 0114247 - Chomczynski1] Microsections were added 1 ml TRIZOL and homogenized (Polytron PT 1200, Kinematica AG) and centrifuged at 10. 000 rpm / 10 min at 4 ° C. The supernatant was removed and RNA was precipitated by adding 0. 1 volumes of sodium acetate 3 M, 2. 5 v*l Tmes cold ethanol and 0. 5 µl glycogen (20 mg / ml ). RNA was centrifuged again, air - dried and resuspended in 20 µl Tris / EDTA buffer. RNA was reversely transcribed to cDNA with Superscript II (Invitrogen ), by incubation | The authors confirm that all data findings are fully available restriction. All relevant data are within the paper. Introduction {#s1} ============ Mechanical ventilation (MV) has been used in critical care patients for decades. In spite of its life-saving potential, it has several shortcomings. A number experimental studies have shown that mechanical ventilation may result in appearance of inflammatory mediators in the lung [@pone.0114247-Uhlig1] and subsequently in oedema. [@pone.0114247-Dreyfuss1] Lung Injury (VILI) causes macro and microscopic unspecific changes[@pone.0114247-Katzenstein1] similar to those found in patients with Respiratory Distress Syndrome (ARDS). As it happens with ARDS, VILI is basically the result of important changes in the permeability of the alveolar-capillary membrane. [@pone.0114247-Dreyfuss2] The potential of mechanical ventilation for triggering or pulmonary damages been shown in animal models where the application of non-physiological ventilatory parameters very high tidal aggravated the condition of animals with a previously injured lung [@pone.0114247-Corbridge1], even caused an injury in those without a previous pulmonary pathology. The use of volumes proved to be a better approach in ARDS patients, survival being improved in strategies based on its usage. [@pone.0114247-Amato1]--[@pone.0114247-Villar1] Interestingly, recent experimental and clinical work has demonstrated that MV with tidal volume similar pulmonary changes to those noticed for VILI [@pone.0114247-Cobelens1]--[@pone.0114247-Wolthuis1] and that its appearance may be related to MV exposure time. [@pone.0114247-Hegeman1] Aquaporins are a family of small transmembrane proteins that help water to move fast, and bi-directionally through lipid bi-layers. [@pone.0114247-Kozono1], [@pone.0114247-Ma1] 13 different types been in mammals, [@pone.0114247-Verkman1] which the lung is known to express four: in the pulmonary endothelium (especially alveolar), and the visceral pleura; AQP-3, in the tracheal epithelium; AQP-4, in the tracheal bronchial epithelium; and AQP-5, on type I pneumocyte cells of the on the membrane adjoining to the alveolar lumen. [@pone.0114247-King1] Their role in the and resolution of oedema gives rise to controversy, although it does seem to play a part in VILI. [@pone.0114247-Hales1] This research aimed to verify if MV with low or moderately high tidal volumes ml/Kg) sustained over time results lung subsequently altering microvascular permeability, as observed in VILI, and to objectivize what happens with AQP 1 and 5 both types involved in the formation of lung oedema, under the same ventilation conditions. Material and Methods {#s2} ==================== 1. Ethics statement {#s2a} ------------------- The project carried out after approval from the Committee for Animal Experimentation Wellbeing of the Research Foundation of Valencia\'s Clínico Universitario. 2. model and monitoring {#s2b} ------------------------------ A total of 30 rats were anaesthetised by injection of ketamine 80 mg/kg and xylazine 5 mg/kg. 5 rats (group C or controls) were sacrificed by intravenous of 100 mg/Kg thiopental. The of the animals (n = were performed a surgical tracheostomy, a cannula (Surflo, 16G). were randomly allocated into two groups. 12 rats were ventilated 2 hours (group 2H) a Harvard Rodent Ventilator, 683 (Harvard with a tidal volume of 10 ml/kg and a rate of 90 breaths/minute. 13 rats were ventilated with exactly the same parameters for hours (group 4H). The vascular bundle was dissected, and internal jugular vein and the right carotid artery were catheterized to continuously monitor heart rate (HR) and mean arterial (MAP). Peak pressure and respiratory system compliance were continuously recorded. Anaesthesia was by continuous intravenous infusion of ketamine and cisatracurium using dosis of 100 and 2--3 mcg/Kg/min, respectively (20 ml of ketamine 5%, 10 ml of cisatracurium 0,2% and 20 ml of solution 0,9% at a of 0,1 ml/h in the internal jugular vein). Anesthesia was supplemented, in cases in which it was necessary, administration of an intravenous bolus of 0.1 ml of the mixture. Gasometric samples were taken in all animals in groups 2H and 4H at the beginning of MV and 30 minutes before the end of MV. Rats were sacrificed by intravenous injection of thiopental. The left lung was used for the determination of lung water content. The right lung of 6 rats from groups 2H and 4H was used for determining AQP 1 and AQP 5 expression. Lungs were frozen in liquid nitrogen or for immunohistochemical and marking. 2 rats in group C and 4 rats from groups 2H and 4H were used to establish pulmonary macrovascular permeability. oedema {#s2c} ------------------------ Lungs were dried with filter paper and placed on a Petri dish with known to obtain lung wet weight (LWW). They were then placed in a chamber at 80°C for hours and their dry weight (LDW) was Two indicators of the amount of oedema were obtained: Lung WW/DW ratio and the proportion of pulmonary expressed in percentages (%~water~). The latter parameter was estimated using formula: % = (LWW - LDW)/LWW \* 100 4. Measuring microvascular {#s2d} --------------------------------------- Microvascular permeability was quantified using Evans Blue Dye. 0.5 Evans was injected intravenously (30 mg/Kg) 30 minutes before sacrificing the animal. Rats were sacrificed by exsanguination from carotid artery, but saline was simultaneously infused via the jugular vein in the same amount that of the blood extracted. After death, the right lung was separated and immersed in (5 ml) and homogenised for 2 min. resulting suspension was incubated at 37°C/18 h and then centrifuged 5000xg/30 minutes, and the supernatant was measured. Concentration Blue the supernatant was spectrophotometrically determined. 5. Study of aquaporin {#s2e} -------------------------------- ### 5.1 Western blot extracted from previously frozen lungs. A Compartmental Protein Extraction Kit (Chemicon International, Temecula CA) was used. 200--400 mg tissue was homogenised in cold buffer C (1 ml/g tissue) and Ultra (KA, Staufen, Germany). Two protein fractions were obtained for each sample: cytoplasm and membrane, and they were quantified. Proteins in each fraction (100 mg) run on a Tris-HCl/SDS acrylamide, and they were transferred onto a nitrocellulose membrane (Hybond-ECL, Amersham). After washing the membrane with distilled water, it was PBS/Tween 0.2%, 5% skimmed milk. It then incubated with primary antibody during 2 hours room temperature. antibodies used were Anti-Rat AQP1 (Alpha Diagnostic, San Antonio, TX) and AQP5 (Alpha Diagnostic, San Antonio, TX), both with a 2 mg/ml After several washes with PBS/Tween 0.2%, it was incubated with the secondary Anti-Rabbit IgG (DAKO, Glostrup, Denmark) in 1∶2000 dilution. β-actin expression was detected as an internal control, and relative protein content was analysed using the enhanced chemoluminiscence method. ### 5.2 Real-time polymerase chain with reverse {#s2e2} Lungs were cut a microtome, three sections being obtained for each sample for RNA extraction with TRIZOL (Reagent InvitrogenTM Technologies) as in phenol extraction method described by Chomczynsky. [@pone.0114247-Chomczynski1] Microsections were added 1 TRIZOL and homogenized (Polytron PT 1200, AG) and centrifuged at 10.000 min at 4°C. supernatant was removed and RNA was precipitated by adding 0.1 volumes of sodium acetate 3 M, 2.5 volumes cold ethanol and 0.5 glycogen (20 mg/ml). RNA was centrifuged again, air-dried and resuspended in 20 Tris/EDTA buffer. RNA was reversely transcribed to cDNA with Superscript II (Invitrogen), by incubation | thE AutHors CoNFirM tHat ALL daTa uNDERlyInG THE FinDINgS arE FULLy AvaILablE WithoUt REStrIctiOn. ALL RELEVant DAtA are wITHin tHe paPeR.
iNTRoDUCtion {#S1}
============
mECHANiCal vENTILATion (MV) Has beEn USed in cRItical caRe PaTiEnTs fOR DecAdeS. iN spitE oF iTs LiFE-sAVing PoteNTiAL, it hAs seVErAL ShOrTcOMiNgS. a nuMbEr OF EXPERimEntal StUDies Have sHowN thAt mEcHaNICAl VeNTilatioN MaY RESuLt In thE AppEaraNCE of iNflaMMaToRy MEdiaTORs iN THE lung [@PoNE.0114247-UHLIG1] AnD SUBsEqUEntlY In OeDEMA. [@pONE.0114247-DREyFusS1] VENtilAtor-iNDUceD lUNG INJURY (VIlI) CAUSES MACro anD micRoScopic uNsPEcIfIc chANgEs[@Pone.0114247-KaTzenStein1] SImilar tO thosE FoUND IN PaTIeNts wiTh ACUTe REsPiRatory DiStreSS sYndrome (aRDS). AS it hAppeNS WitH ArDS, ViLi is BaSIcALly ThE ReSuLT OF IMPORTaNT ChangES In tHe PerMEaBilITy of The aLVeoLAr-CApiLlARy MEMbRANe. [@PONe.0114247-DReyFuss2] tHE PoTEntIaL Of MeChaNIcAL vENTILATIon for TrIgGERing oR WOrSeNINg pULMOnaRY DAmAGEs hAS beEn SHOwN iN animAl mODeLs wHErE tHE ApPlIcAtiON of NoN-phYSIologiCAL VENtiLATOrY PaRaMEteRS (mostLY VeRY High Tidal voLuMEs) AGGRAVAted the coNdiTion Of ANIMALS wiTh A pRevIoUsLY inJURED lUng [@poNe.0114247-CorBridge1], AND even CAuseD an INJury in ThosE wITHoUT a PREviOUs PulMoNARy paThOLoGy. [@PONE.0114247-drEYfusS1] ThE usE oF lOw TiDal vOLUMEs HAS PROVEd To BE A BeTTEr aPproACh in arDS PAtiENTS, SUrvIVaL bEiNG ImProVED IN StRaTEgIes basED oN Its uSagE. [@PoNE.0114247-AmATo1]--[@pONE.0114247-viLlar1] interestIngLY, reCENT EXpEriMentaL And CLiNICAL work Has DeMOnstrAteD THat mv wITh lOw tIDaL vOluME can indUcE SiMILaR PULMonArY ChanGES to ThOse notiCED FOr vILi [@POne.0114247-CObelEnS1]--[@PONe.0114247-WoLthuIS1] aND ThaT iTS APpeArAnCe MAY Be reLatED TO MV EXPoSuRe TImE. [@pOnE.0114247-heGemaN1]
aQuaporins aRE A fAMIly of sMaLL TRANsmemBrAnE prOTeInS ThAT HELp WatEr to moVe fast, SelEcTIVELy And bi-diRecTiOnAlLY ThROuGh lIPId bI-LayErs. [@POne.0114247-KOZonO1], [@PonE.0114247-Ma1] 13 DIFFeREnt TypEs HavE bEEn identIfIED In MAmMalS, [@pONE.0114247-VErKMAn1] FrOM WhicH The LUNg is kNoWN To EXPReSs Four: aqp-1, in thE puLMOnaRY CaPiLlARy eNdOTheLiUm (esPeCiAlLy aLvEOlAR), AND thE VisCeRal pleuRA; aqP-3, in THe TRaCHeAl EpithElium; AqP-4, in THe TrACHeal And bRONCHIal ePitheLiUm; And AQp-5, On TyPe i PnEUmocYtE cELLS OF THe alveolI, on tHe MEmBRANE adjOiniNg tO The aLVeolAR luMeN. [@pOne.0114247-KinG1] TheiR RoLe IN ThE dEVEloPMeNt AnD rEsolUTion Of PULMOnAry oedemA givES Rise TO contrOVERsy, aLthoUGH It doES SeEM tO PLAy a PArT IN vIlI. [@pone.0114247-HALes1]
tHis rEsEArCH AimeD tO VerIFY if mV wItH LOw oR ModERaTElY High TIdaL voLUMes (10 Ml/Kg) SUSTAINed oVER tIME rEsULtS iN LUNg INjuRY, sUbsEquENtLY ALteRiNg pUlMonArY WateR COntenT aND MicrOvAscuLAr PErmEAbiLITy, aS ObSERvEd IN viLi, and to objeCTIviZe What HAPPEnS wITH aQp 1 aNd 5 exPrEsSioN, Both typES mAINLY InVOlVEd In ThE foRMAtioN of luNG oEdeMa, UNDeR THe sAME vENTIlAtIoN COnDITionS.
mateRial AnD meThOdS {#S2}
====================
1. eThiCS stAteMEnt {#S2a}
-------------------
thE PrOject Was caRRIEd ouT after aPpROVal From THE EtHiCs COMMiTtEE for ANIMal ExperimeNTaTION aND WELlBEING oF tHE ResEARCh fOundAtIoN OF VaLenCIA\'s HosPitAl cLíniCO UNiVersITARio.
2. ANIMAl moDeL AND mONitOrinG {#S2b}
------------------------------
a TOTAl oF 30 raTs WERE AnaEsTHetIsed by INTRApERITOneAl INJeCtIoN of KEtAMIne 80 MG/kG And xyLaZINe 5 mg/KG. 5 rATS (gRoUp C OR cOnTRoLS) WeRE sACrifIcED By IntRAvENoUS injECtion OF 100 Mg/Kg THiOpEnTAL. THE rESt of The AnImALs (N = 25) were pERforMed A surgIcAL tRAChEosTOmy, usING A teFLoN CaNNulA (suRflO, 16G). Rats WErE rAndOmLY ALloCaTEd iNtO Two GroUps. 12 RAtS wERE vEnTilAtED fOR 2 HOUrs (GrOUp 2h) witH A hArVaRd rodeNT VenTiLATor, moDEl 683 (hARVArd APpARATUS) WItH A TiDAL vOlUmE Of 10 Ml/kg aND a REsPiRAtoRY RATe oF 90 BrEATHs/mINUTe. 13 raTS WEre VenTILATEd wITh exACTly The samE PARAMeTERs FOr 4 hOurS (groUp 4H). tHe cerVicaL VASCULaR bUnDLE WaS DISSecteD, AND tHE riGHT InTERNAL JUgular VEIn AnD tHE RighT CArOTId artEry weRe CAthEterizeD TO CONTInUoUSly mONITOr HeaRT RaTe (hR) aNd meAn ARteriAL PRESSURe (Map). pEAk iNsPIraToRY pressurE AND RESpIraTORy System cOmplIaNcE WEre CoNTinUOuSLy recordEd.
ANAESTHESiA waS maInTAINeD By CONTInUoUS inTRaVeNOUs infusION of KeTAMInE anD CISAtraCuriuM usInG dosiS oF 100 mcG/kg/min AND 2--3 mCg/KG/MiN, reSpecTIveLy (20 mL of KEtAMInE 5%, 10 ML OF cIsaTraCUrium 0,2% anD 20 ml of salInE Solution 0,9% aT a ratE of aPPROximatElY 0,1 ml/H iN the InTeRnaL jUgulAR VeIn). anESThESiA was sUppLementED, in Cases in whIch it wAs nECesSArY, by AdmIniStRaTiON of AN inTrAvenOus BOlUS of 0.1 ML Of THe MIXTUre. GASOmeTric sAMPLeS WERe tAken In All AnImAlS IN grOUps 2H aND 4h at The Beginning oF MV And 30 miNUtes BEForE THE eNd oF mV.
ratS WERe SAcrIFiced By iNtraVENOUs INjECtIoN OF SOdiUM THiOpeNTAl. THe LeFt lung WAs UsEd FOR thE DeTeRmINatioN OF lUNg WAtER cONTENT. ThE rIghT luNG Of 6 rAts from GRoUps 2H AnD 4h Was UsED For dETermInINg aQp 1 ANd aqP 5 eXPReSSiOn. LuNgS wEre EITHeR fROZEN IN LIQUiD nitROGeN OR parAffIn-EMBeDdeD fOR ImmUnOhiSTOcheMiCAL SeCTioninG anD maRkInG. 2 rATS IN Group C ANd 4 rAts FROm GROupS 2h aNd 4H WERE USeD to EsTabLISh PuLmonAry mACrovascuLAr PErmeaBiLity.
3. MEASuriNG lung oEdEMA {#S2C}
------------------------
LuNGs Were DrIEd wItH fILTEr PAper aNd plaCED on A pETri DIsH wITh KNOwN weigHT To OBTAIn luNG wEt WEight (Lww). tHEy wErE tHeN PLaCed iN A dryInG CHAmbeR AT 80°C foR 96 HOURs AND thEIr DRY wEighT (LDw) waS dEtERmiNEd.
twO INDicATORs Of The Amount oF oedEmA WERE ObTaINEd: luNg Ww/dw rATIO AnD thE pRopOrTiON Of PuLmoNAry wATER, expREsseD iN PeRcEnTages (%~water~). THE LAtTeR pARAmeteR was EstiMAted uSing this fORMUla: % ~wAtEr~ = (lwW - lDw)/lWW \* 100
4. MEASuRiNG MiCROvAscuLAr peRMEabILITY {#s2d}
---------------------------------------
miCROvAscUlAr peRMEabiLITy wAs QuantifIED UsiNG EVANS bLue dYE. 0.5 Ml EVANS bLUE WAs injeCted inTRAveNOusLY (30 Mg/KG) 30 minutEs BEfOre sACriFiCing the ANImal. RaTS WeRE sACRifICEd bY eXSaNGuINAtIon fRoM ThE caRotId ArtERY, BuT sALinE Was siMULtaneOuSlY inFUSED ViA THE JuGULar vEIN iN the SaME AMoUNT aS That Of tHe blooD eXtRaCTEd.
AFTEr dEAth, THe rigHT LUnG waS SEpaRATeD aND iMmERSEd iN FOrMAmIdE (5 mL) AnD homOgenisED For 2 min. The ReSulTInG SUsPEnsioN waS incUbaTED At 37°c/18 h AND THEn CEntrIfuGEd at 5000XG/30 miNutES, AND the SUpErnaTanT Was meaSUReD. concENTraTIon oF EvanS BLuE iN tHe supeRNAtAnt wAs SPEcTrophoToMeTRIcALly deTERmineD.
5. sTUDY Of aqUAPorIN EXPREssiOn {#S2E}
--------------------------------
### 5.1 WeStERn blot {#s2E1}
PROtEiNs werE exTrACTed frOM pReVIously FrOzEn luNGs. A COMpartmEntAL proTeiN exTRActIOn kIT (ChemiCOn InTERNATiOnAl, teMeCULa CA) WaS uSed. 200--400 MG tiSSUE waS hOmogeNisEd In cold BuFfer c (1 mL/G tISsUE) AND UlTrA TURraX (Ka, STAuFEN, gERmanY). Two prOTEIn fRAcTIONs Were OBtainED fOR eAch SaMPLE: CyToPlaSM aND MembRanE, ANd tHey WerE qUaNTIFIed. prOtEiNS iN EAch frACtION (100 mG) weRe separATELy Run ON A TrIs-HCl/SDS Gel, 8% AcRyLAmIde, and THey WeRE tRaNsFErRed OnTO A NITrOCeLlulOSE MeMBrAne (hybONd-eCL, aMeRsham). AfteR waShiNg THE membRanE wItH DISTILleD WAtEr, It WaS bLOCKED wIth a PBS/tWeEn SolUtiON, 0.2%, WITh 5% SKImMED MiLK. IT WAs theN InCUBatEd wITh the pRiMARY ANtibODY DURiNG 2 HOurS at ROom teMpErATure. THE AntIbOdIeS uSED WeRe ANti-rat aqP1 (alphA DIagnOStic, sAn ANTonio, tx) AnD aNTI-RAT Aqp5 (Alpha DIAgnoSTIc, San aNtOniO, TX), Both WITH A 2 MG/Ml COncENtRAtiOn. AFTER SEvEraL WaSheS wITh PBs/tweEN 0.2%, IT WAS iNcuBaTed WiTH THE SeCoNdarY aNtiBOdy AnTi-rAbbiT IgG (dAkO, GloStrup, DEnmaRK) iN 1∶2000 Dilution. β-ActiN exPREsSion WAs deTeCTed AS An iNTErNAl CONtRoL, anD RELAtIVe PrOTEiN coNteNt WAS ANALYSED usING THe eNhanCeD chemOLUMInIScEnCe mEthOd.
### 5.2 Real-tIME pOLyMerASE CHAiN reaCtIOn witH reveRsE trANScrIPTaSE {#s2E2}
lungS WerE Cut WIth A MicRoToME, three SecTIoNS bEIng ObtAIneD fOr eaCh sAMple foR toTal rNA ExtRacTion WITH TriZoL (REAgeNT INVItrogenTM LiFe tecHnoLOGIeS) aS IN thE phEnOl EXtRaCTIoN MethoD DescRIBed BY cHoMCZYnskY. [@PoNe.0114247-cHoMCzYnSkI1] MicrosEctIoNS wERe AddeD 1 ml trizol anD hoMOgenIZed (PolYtROn Pt 1200, kinEmATicA ag) anD CEntrifUgEd AT 10.000 rpM/10 mIN aT 4°C. tHe SuPErNatAnt WAS RemOVeD anD RnA was pREcipItaTED by AdDiNg 0.1 VOlumEs of SODiuM ACEtAte 3 M, 2.5 VOlUmes cOLd EthaNOL AND 0.5 ΜL gLyCOGen (20 MG/ML). rNa wAs centrifuGed AgaIN, air-Dried aND REsUSPENDed In 20 Μl TriS/EDTa BUFfER. RNA was reveRseLy TRANscRIBeD tO CDnA wItH SupeRscRIpt iI (InviTrOGen), bY incUBAtiOn | The authorsconfirmthatalldata underlying the findings are fully available without restriction. All relevant data are within the paper. Introduction {#s1} ============ Mechanical ventilation (MV) has been used in critical care patientsfor decades. Inspite ofits life-saving potential, it has several shortcomings. Anumber of experimentalstudies have shown that mechanical ventilation may result in theappearance of inflammatory mediators in the lung [@pone.0114247-Uhlig1] and subsequently in oedema. [@pone.0114247-Dreyfuss1] Ventilator-Induced Lung Injury (VILI) causes macro and microscopic unspecific changes[@pone.0114247-Katzenstein1]similar to those foundin patients with Acute Respiratory Distress Syndrome (ARDS). As ithappens with ARDS,VILI isbasicallythe resultof important changes in the permeability of thealveolar-capillarymembrane.[@pone.0114247-Dreyfuss2] The potential of mechanical ventilation fortriggering or worsening pulmonary damageshas been shown in animal models wherethe application of non-physiological ventilatory parameters (mostly very high tidal volumes) aggravated the conditionof animals witha previously injured lung [@pone.0114247-Corbridge1], and even caused aninjury in those without a previous pulmonary pathology. [@pone.0114247-Dreyfuss1]The use of low tidal volumes has provedto be a better approachin ARDS patients, survival being improved in strategiesbasedon its usage. [@pone.0114247-Amato1]--[@pone.0114247-Villar1] Interestingly,recentexperimental andclinical work has demonstrated thatMV with low tidal volume can induce similar pulmonary changes to those noticed for VILI [@pone.0114247-Cobelens1]--[@pone.0114247-Wolthuis1] and that its appearance may berelated to MV exposure time. [@pone.0114247-Hegeman1] Aquaporins are a family of small transmembraneproteins that help water to move fast, selectively and bi-directionally throughlipid bi-layers. [@pone.0114247-Kozono1], [@pone.0114247-Ma1] 13 different types have been identified in mammals, [@pone.0114247-Verkman1] from which the lung is known to express four: AQP-1, in the pulmonary capillary endothelium (especially alveolar), and the visceral pleura;AQP-3,in the tracheal epithelium;AQP-4, inthe tracheal and bronchial epithelium; and AQP-5,on typeI pneumocyte cells of the alveoli, on themembrane adjoining to the alveolar lumen. [@pone.0114247-King1] Their role in the development and resolution of pulmonary oedema gives rise to controversy, although it does seemto playa part in VILI. [@pone.0114247-Hales1] Thisresearch aimed to verify if MVwith low or moderately high tidal volumes(10 ml/Kg) sustained overtime results in lung injury, subsequently altering pulmonary water content and microvascular permeability, as observed in VILI,and to objectivize what happens with AQP 1 and 5 expression, both types mainly involvedin the formationof lung oedema, under the same ventilation conditions. Materialand Methods {#s2} ==================== 1. Ethics statement {#s2a} ------------------- Theproject wascarried out after approval from the Ethics Committee for Animal Experimentation and Wellbeing of the Research Foundation of Valencia\'s Hospital Clínico Universitario. 2. Animal modeland monitoring {#s2b} ------------------------------ A totalof 30 rats were anaesthetised by intraperitoneal injection of ketamine 80 mg/kgandxylazine 5 mg/kg. 5 rats (groupC or controls)were sacrificed by intravenous injection of 100 mg/Kg thiopental. The rest of the animals (n = 25) were performed a surgical tracheostomy, using a teflon cannula (Surflo, 16G). Rats were randomly allocated into two groups. 12 ratswere ventilated for 2 hours (group 2H) with a Harvard RodentVentilator, model683 (HarvardApparatus) with a tidal volume of 10 ml/kg and a respiratory rateof 90 breaths/minute. 13 rats were ventilated with exactly the same parameters for 4 hours (group 4H). The cervical vascular bundle was dissected, and the right internal jugularvein and the right carotid artery werecatheterized to continuously monitor heart rate (HR)and meanarterial pressure (MAP). Peakinspiratory pressure and respiratory system compliance were continuously recorded. Anaesthesia was maintained by continuous intravenous infusion of ketamine and cisatracurium using dosis of 100 mcg/Kg/min and 2--3 mcg/Kg/min, respectively (20 mlof ketamine5%, 10ml of cisatracurium0,2% and 20 ml of saline solution 0,9% ata rate of approximately 0,1 ml/h in theinternal jugular vein). Anesthesia was supplemented,in cases inwhich it was necessary, by administrationof an intravenous bolus of 0.1 ml of the mixture. Gasometric samples were taken inall animals in groups 2H and4H at the beginning of MV and 30minutes before the end ofMV.Rats were sacrificed by intravenous injection of sodium thiopental.Theleft lung was used for thedetermination of lung water content. The right lungof 6 rats fromgroups 2Hand 4H was used for determining AQP 1 and AQP 5 expression. Lungs were either frozen in liquid nitrogen or paraffin-embedded for immunohistochemical sectioning and marking. 2rats in group C and 4 rats from groups 2H and 4H were usedto establish pulmonarymacrovascular permeability. 3. Measuring lung oedema {#s2c} ------------------------Lungswere dried with filter paper and placed on a Petri dish with known weight toobtain lung wet weight (LWW). They were then placed in a drying chamber at 80°C for 96 hours and their dry weight (LDW) was determined. Two indicators of theamountof oedema were obtained: Lung WW/DW ratio and the proportion of pulmonary water, expressed in percentages (%~water~). The latter parameter was estimated using this formula: % ~water~=(LWW - LDW)/LWW \* 100 4. Measuring microvascular permeability {#s2d} --------------------------------------- Microvascular permeability was quantified using Evans Blue Dye. 0.5 ml Evans Blue was injected intravenously (30 mg/Kg) 30minutes before sacrificing the animal.Rats were sacrificedby exsanguination from the carotidartery, butsalinewas simultaneouslyinfused via the jugular vein in the same amount as thatof the blood extracted. After death,the right lung was separated and immersed in formamide (5 ml) and homogenisedfor 2 min. The resultingsuspension was incubated at 37°C/18 h and then centrifuged at5000xg/30 minutes, and the supernatant was measured. Concentration of Evans Blue in the supernatant wasspectrophotometricallydetermined.5. Study of aquaporin expression {#s2e} -------------------------------- ### 5.1 Western blot {#s2e1} Proteins were extractedfrom previouslyfrozenlungs. ACompartmental ProteinExtraction Kit (Chemicon International, Temecula CA) was used. 200--400 mg tissuewas homogenised in cold buffer C (1 ml/g tissue) and UltraTurrax(KA, Staufen, Germany). Two protein fractions were obtained for eachsample:cytoplasm and membrane, and they were quantified.Proteinsin eachfraction (100 mg) were separately run on a Tris-HCl/SDSgel, 8% acrylamide, and they were transferred onto a nitrocellulose membrane (Hybond-ECL, Amersham). After washingthe membrane with distilledwater, it was blocked with a PBS/Tween solution, 0.2%, with 5%skimmed milk. It was then incubated withthe primary antibody during2hours at room temperature. Theantibodies used were Anti-RatAQP1(AlphaDiagnostic, San Antonio, TX) and Anti-Rat AQP5 (AlphaDiagnostic, San Antonio, TX), both with a 2 mg/ml concentration. After several washes with PBS/Tween 0.2%, it was incubated with the secondary antibody Anti-Rabbit IgG (DAKO, Glostrup, Denmark) in 1∶2000 dilution.β-actin expression was detected as an internal control,and relative protein content was analysed usingthe enhanced chemoluminiscence method. ### 5.2 Real-time polymerase chainreactionwith reverse transcriptase {#s2e2} Lungs were cut with a microtome, three sections being obtained for each sample for total RNA extraction with TRIZOL (Reagent InvitrogenTM Life Technologies) asin the phenol extraction method describedby Chomczynsky. [@pone.0114247-Chomczynski1] Microsectionswere added 1ml TRIZOL and homogenized (Polytron PT 1200, Kinematica AG) and centrifuged at10.000rpm/10 min at4°C. Thesupernatant was removed and RNA was precipitated by adding 0.1 volumesof sodium acetate 3 M, 2.5 volumes cold ethanol and 0.5 µl glycogen (20 mg/ml). RNA was centrifuged again,air-dried and resuspended in 20 µl Tris/EDTA buffer.RNA wasreversely transcribed to cDNA with SuperscriptII (Invitrogen), byincubation | The authors confirm that _all_ data underlying the findings are fully available without _restriction._ All relevant data are within _the_ paper. _Introduction_ {#s1} ============ Mechanical _ventilation_ (MV) _has_ been _used_ in critical care patients for decades. In _spite_ _of_ its life-saving potential, it _has_ several shortcomings. A _number_ of experimental _studies_ have shown that mechanical ventilation may result in the _appearance_ of _inflammatory_ mediators in the lung [@pone.0114247-Uhlig1] and subsequently in _oedema._ _[@pone.0114247-Dreyfuss1]_ _Ventilator-Induced_ _Lung_ Injury (VILI) causes _macro_ and microscopic unspecific _changes[@pone.0114247-Katzenstein1]_ similar to _those_ found _in_ patients with Acute Respiratory _Distress_ Syndrome (ARDS). As it happens _with_ ARDS, _VILI_ is basically the result of important changes _in_ the permeability of _the_ alveolar-capillary membrane. [@pone.0114247-Dreyfuss2] The potential of _mechanical_ ventilation for triggering or worsening pulmonary damages has been shown in animal models where the _application_ of non-physiological ventilatory parameters (mostly very high tidal volumes) aggravated the condition of animals with a previously injured lung [@pone.0114247-Corbridge1], and _even_ caused an injury _in_ those _without_ a previous pulmonary pathology. [@pone.0114247-Dreyfuss1] The _use_ of low tidal volumes has proved to be a better approach in ARDS patients, _survival_ being improved in strategies based _on_ its usage. _[@pone.0114247-Amato1]--[@pone.0114247-Villar1]_ Interestingly, recent _experimental_ and clinical work has demonstrated that MV with _low_ _tidal_ volume can induce similar pulmonary changes to those noticed for VILI [@pone.0114247-Cobelens1]--[@pone.0114247-Wolthuis1] _and_ that _its_ _appearance_ may _be_ related to MV _exposure_ time. [@pone.0114247-Hegeman1] Aquaporins are a family of small transmembrane proteins that help _water_ to move fast, selectively and bi-directionally _through_ _lipid_ bi-layers. [@pone.0114247-Kozono1], [@pone.0114247-Ma1] _13_ different types have been identified in mammals, [@pone.0114247-Verkman1] _from_ which the lung is _known_ _to_ express four: AQP-1, in the pulmonary capillary _endothelium_ (especially alveolar), and the _visceral_ pleura; AQP-3, in the tracheal _epithelium;_ AQP-4, in the _tracheal_ and bronchial _epithelium;_ and AQP-5, on type I pneumocyte cells of _the_ alveoli, on the _membrane_ adjoining _to_ the alveolar lumen. [@pone.0114247-King1] _Their_ role in the development and _resolution_ _of_ pulmonary oedema gives rise _to_ controversy, although _it_ does seem to play a part in VILI. [@pone.0114247-Hales1] This _research_ aimed to _verify_ if MV with low or moderately high _tidal_ volumes (10 ml/Kg) sustained over time results in lung injury, subsequently altering pulmonary water content and microvascular permeability, as observed in VILI, and to objectivize what happens with AQP 1 _and_ 5 _expression,_ both types mainly involved _in_ the formation of lung oedema, under the same ventilation conditions. Material and Methods {#s2} ==================== 1. Ethics statement _{#s2a}_ ------------------- _The_ project _was_ carried out after approval from the Ethics Committee _for_ Animal _Experimentation_ and Wellbeing of the _Research_ Foundation of Valencia\'s _Hospital_ Clínico Universitario. 2. Animal model and _monitoring_ {#s2b} _------------------------------_ A total _of_ 30 _rats_ were anaesthetised by _intraperitoneal_ _injection_ of ketamine 80 mg/kg and _xylazine_ 5 mg/kg. 5 _rats_ _(group_ C or controls) were sacrificed by intravenous injection of 100 _mg/Kg_ thiopental. The rest of the animals (n = 25) were performed a surgical _tracheostomy,_ using a teflon cannula (Surflo, _16G)._ _Rats_ were randomly allocated into two groups. 12 rats were ventilated for _2_ _hours_ (group 2H) with a Harvard Rodent _Ventilator,_ model 683 _(Harvard_ Apparatus) _with_ a tidal volume of 10 ml/kg and a respiratory _rate_ of _90_ breaths/minute. _13_ rats were ventilated _with_ exactly the same parameters for 4 hours (group 4H). The _cervical_ vascular _bundle_ was dissected, and the right internal jugular vein and the right carotid artery were catheterized to continuously monitor heart _rate_ _(HR)_ and mean arterial pressure (MAP). Peak inspiratory pressure _and_ _respiratory_ system compliance _were_ continuously recorded. Anaesthesia was maintained by _continuous_ intravenous infusion of ketamine and cisatracurium using dosis of 100 mcg/Kg/min and 2--3 _mcg/Kg/min,_ respectively (20 ml _of_ ketamine _5%,_ 10 ml of cisatracurium 0,2% and _20_ ml of saline solution 0,9% at a rate of approximately 0,1 _ml/h_ in the internal jugular vein). Anesthesia was supplemented, in cases in which _it_ _was_ necessary, by administration of an _intravenous_ bolus of 0.1 ml of _the_ mixture. Gasometric _samples_ were taken in all _animals_ in groups 2H and 4H at the beginning of MV and 30 minutes before the end of MV. Rats were sacrificed _by_ intravenous injection of sodium thiopental. The left lung _was_ used for _the_ _determination_ of _lung_ water content. The right lung _of_ 6 rats from _groups_ _2H_ and 4H was used for determining _AQP_ _1_ _and_ AQP 5 _expression._ _Lungs_ were either frozen _in_ liquid _nitrogen_ or paraffin-embedded for _immunohistochemical_ sectioning _and_ marking. 2 rats in _group_ C _and_ 4 rats _from_ groups 2H and 4H were used to _establish_ _pulmonary_ macrovascular permeability. 3. _Measuring_ lung oedema {#s2c} ------------------------ Lungs were dried with filter paper _and_ placed on _a_ _Petri_ dish with _known_ weight to obtain lung wet weight _(LWW)._ They were then placed in a _drying_ _chamber_ at 80°C for 96 _hours_ _and_ their dry weight (LDW) _was_ determined. Two _indicators_ of the amount of oedema were obtained: Lung WW/DW ratio and _the_ proportion of pulmonary water, expressed in percentages _(%~water~)._ The latter _parameter_ was estimated using this formula: % ~water~ = _(LWW_ - _LDW)/LWW_ \* 100 4. Measuring microvascular _permeability_ {#s2d} --------------------------------------- Microvascular permeability was quantified using Evans Blue Dye. 0.5 ml Evans Blue was _injected_ intravenously _(30_ mg/Kg) 30 minutes before sacrificing the animal. Rats were sacrificed by exsanguination _from_ the carotid artery, but _saline_ _was_ simultaneously infused via the jugular vein in the same amount _as_ that _of_ the blood extracted. After death, the right lung was separated and _immersed_ in formamide (5 ml) and _homogenised_ for 2 _min._ The resulting suspension was incubated at _37°C/18_ h and _then_ centrifuged at _5000xg/30_ _minutes,_ and the supernatant was _measured._ _Concentration_ of Evans Blue in the supernatant was spectrophotometrically determined. 5. Study _of_ aquaporin expression _{#s2e}_ _--------------------------------_ ### 5.1 Western blot _{#s2e1}_ _Proteins_ were extracted from previously _frozen_ lungs. A Compartmental Protein Extraction Kit (Chemicon International, Temecula CA) was used. 200--400 mg tissue was homogenised in _cold_ buffer C _(1_ ml/g tissue) and Ultra Turrax (KA, Staufen, Germany). Two protein fractions _were_ _obtained_ for each sample: cytoplasm and membrane, and they _were_ quantified. _Proteins_ in each fraction (100 mg) _were_ _separately_ run on a _Tris-HCl/SDS_ gel, 8% _acrylamide,_ and they were transferred onto _a_ nitrocellulose membrane (Hybond-ECL, Amersham). After washing _the_ _membrane_ with distilled water, it was _blocked_ with a PBS/Tween solution, _0.2%,_ _with_ 5% skimmed milk. It was then incubated with the primary antibody during 2 hours at room temperature. _The_ antibodies used _were_ Anti-Rat AQP1 (Alpha Diagnostic, San Antonio, TX) and Anti-Rat AQP5 (Alpha Diagnostic, _San_ Antonio, TX), both _with_ a 2 mg/ml concentration. After several washes _with_ _PBS/Tween_ _0.2%,_ it _was_ incubated with _the_ secondary antibody _Anti-Rabbit_ IgG _(DAKO,_ Glostrup, Denmark) in _1∶2000_ dilution. β-actin _expression_ _was_ detected as an internal control, and _relative_ protein content was _analysed_ using the enhanced _chemoluminiscence_ method. ### 5.2 Real-time _polymerase_ chain reaction with reverse transcriptase {#s2e2} Lungs _were_ cut _with_ a microtome, three _sections_ _being_ obtained for each sample for total RNA extraction _with_ _TRIZOL_ (Reagent InvitrogenTM Life Technologies) as in the phenol extraction method described _by_ Chomczynsky. [@pone.0114247-Chomczynski1] _Microsections_ were added 1 ml TRIZOL _and_ homogenized (Polytron PT 1200, Kinematica _AG)_ and _centrifuged_ at 10.000 rpm/10 min at 4°C. The supernatant was removed and RNA _was_ precipitated by adding 0.1 volumes of sodium acetate 3 M, 2.5 _volumes_ cold ethanol _and_ 0.5 µl glycogen (20 mg/ml). RNA _was_ centrifuged again, air-dried and resuspended in 20 µl Tris/EDTA buffer. RNA was reversely transcribed to cDNA with Superscript II (Invitrogen), _by_ incubation |
Introduction {#s1}
============
Usher syndrome (USH) is an autosomal recessive disorder characterized by hearing loss (HL), retinitis pigmentosa (RP) and vestibular dysfunction. Three clinical subtypes can be distinguished. USH type 1 (USH1) is the most severe among them because of profound HL, absent vestibular responses, and prepubertal onset RP. USH type 2 (USH2) is characterized by congenital moderate to severe HL, with a high-frequency sloping configuration. The vestibular function is normal and onset of RP is in the first or second decade. The onset of the visual symptoms such as night blindness in USH usually occurs several years later than in USH1. USH type 3 (USH3) is characterized by variable onset of progressive HL, variable onset of RP, and variable impairment of vestibular function (normal to absent) [@pone.0090688-Kimberling1], [@pone.0090688-Yan1].
To date, nine genetic loci for USH1(*USH1B-H*, *J*, and *K*) have been mapped to chromosomes 11q13.5, 11p15.1, 10q22.1, 21q21, 10q21-q22, 17q24-q25, 15q22-q23 (*USH1H* and *J*), and 10p11.21--q21.1 [@pone.0090688-Yan1], [@pone.0090688-Jaworek1], [@pone.0090688-Riazuddin1]. Six of the corresponding genes have been identified: the actin-based motor protein myosin VIIa (*MYO7A*, USH1B) [@pone.0090688-Weil1]; two cadherin-related proteins, cadherin 23 (*CDH23*, USH1D) [@pone.0090688-Bork1] and protocadherin 15 (*PCDH15*, USH1F) [@pone.0090688-Ahmed1]; and two scaffold proteins, harmonin (*USH1C*) [@pone.0090688-Verpy1] and sans (*USH1G*) [@pone.0090688-Mustapha1]; the Ca^2+^- and integrin-binding protein (*CIB2*, USH1J) [@pone.0090688-Riazuddin1]. In Caucasian USH1 patients, previous studies showed that mutations in *MYO7A*, *USH1C*, *CDH23*, *PCDH15*, and *USH1G*, were found in 39--55%, 7--14%, 7--35%, 7--11%, and 0--7%, respectively (the frequency of *CIB2* is still unknown) [@pone.0090688-Ouyang1], [@pone.0090688-Bonnet1], [@pone.0090688-LeQuesneStabej1]. In Japanese, Nakanishi et al. showed that *MYO7A* and *CDH23* mutations are present in USH1 patients [@pone.0090688-Nakanishi1], however, the frequency is not yet known. In addition, mutations in three corresponding genes (usherin *USH2A* [@pone.0090688-Eudy1], G protein-coupled receptor 98; *GPR98* [@pone.0090688-Weston1], and deafness, autosomal recessive 31; *DFNB31* [@pone.0090688-Aller1]) have been reported so far in USH2, and USH3 is caused by mutations in the clarin 1 (*CLRN1*) [@pone.0090688-Joensuu1] gene.
Comprehensive molecular diagnosis of USH has been hampered both by genetic heterogeneity and the large number of exons for most of the USH genes. The six USH1 genes collectively contain 180 coding exons [@pone.0090688-Riazuddin1], [@pone.0090688-Mustapha1], [@pone.0090688-Ouyang1] the three USH2 genes comprise 175 coding exons [@pone.0090688-Weston1], [@pone.0090688-Aller1], [@pone.0090688-Nakanishi2], and the USH3 gene has five coding exons [@pone.0090688-Joensuu1]. In addition some of these genes are alternatively spliced ([@pone.0090688-Riazuddin1], [@pone.0090688-Ahmed1], [@pone.0090688-Verpy1], [@pone.0090688-Aller1], [@pone.0090688-Joensuu1] and NCBI database: <http://www.ncbi.nlm.nih.gov/nuccore/>). Thus far, large-scale mutation screening has been performed using direct sequence analysis, but that is both time-consuming and expensive. We thought that targeted exon sequencing of selected genes using the Massively Parallel DNA Sequencing (MPS) technology would enable us to systematically tackle previously intractable monogenic disorders and improve molecular diagnosis.
Therefore, in this study, we have conducted genetic analysis using MPS-based genetic screening to find mutations in nine causative USH genes (except *CIB2*) in Japanese USH1 patients.
Results {#s2}
=======
Mutation analysis of the nine USH genes in 17 unrelated USH1 patients revealed 19 different probable pathogenic variants, of which 14 were novel ([Table 1](#pone-0090688-t001){ref-type="table"}).
10.1371/journal.pone.0090688.t001
###### Possible pathogenic variants found in this study.
{#pone-0090688-t001-1}
Gene Mutation type Nucleotide change Amino acid change exon/intron number Domain control (in 384 alleles) SIFT Score PolyPhen Score Reference
---------- --------------- ------------------- ------------------- -------------------- -------------- -------------------------- ------------ ---------------- --------------------------------
*MYO7A* Frameshift c.1623dup p.Lys542GlnfsX5 Exon 14 \- N/A \- \- Le Quesne Stabej et al. (2012)
c.4482_4483insTG p.Trp1495CysfsX55 Exon 34 \- N/A \- \- This study
c.6205_6206delAT p.Ile2069ProfsX6 Exon 45 \- N/A \- \- This study
Nonsense c.1477C\>T p.Gln493X Exon 13 \- N/A \- \- This study
c.1708C\>T p.Arg570X Exon 15 \- N/A \- \- This study
c.2115C\>A p.Cys705X Exon 18 \- N/A \- \- This study
c.6321G\>A p.Trp2107X Exon 46 \- N/A \- \- This study
Missense c.2074G\>A p.Val692Met Exon 17 Motor domain 0 0.09 0.982 This study
c.2311G\>T p.Ala771Ser Exon 20 IQ 2 0.0026 0.01 0.825 Nakanishi et al. (2010)
c.6028G\>A p.Asp2010Asn Exon 44 FERM 2 0 0 0.925 Jacobson et al. (2009)
*CDH23* Frameshift c.3567delG p.Arg1189ArgfsX5 Exon 30 \- N/A \- \- This study
c.5780_5781delCT p.Ser1927Cysfs16 Exon 44 \- N/A \- \- This study
Splicing c.5821-2A\>G ? Intron 44 \- N/A \- \- This study
Nonsense c.6319C\>T p.Arg2107X Exon 48 \- N/A \- \- Nakanishi et al. (2010)
*PCDH15* Splicing c.158-1G\>A ? Intron 3 \- N/A \- \- This study
Nonsense c.1006C\>T p.Arg336X Exon 10 \- N/A \- \- This study
c.2971C\>T p.Arg991X Exon 22 \- N/A \- \- Roux et al. (2006)
c.3337G\>T p.Glu1113X Exon 25 \- N/A \- \- This study
Missense c.3724G\>A p.Val1242Met Exon 28 Cadherin 11 0 | h { # s1 } = = = = = = = = = = = = usher syndrome ( ush ) presents an autosomal recessive disorder characterized by hearing loss ( hl ), retinitis pigmentosa ( rp ) and vestibular dysfunction. three clinical subtypes can be distinguished. ush type 1 ( ush1 ) is the most severe among them because of profound hl, absent vestibular responses, and prepubertal onset rp. ush type 2 ( ush2 ) is characterized by congenital moderate to severe hl, with a high - frequency sloping configuration. the vestibular function is normal and onset of rp is in the first or second decade. the onset of the visual symptoms such as night blindness in ush usually occurs several days later than in ush1. ush type 3 ( ush3 ) is characterized by variable onset of progressive hl, variable onset of rp, and variable impairment of vestibular function ( normal to absent ) [ @ pone. 0090688 - kimberling1 ], [ @ at. 0090688 - yan1 ]. to date, nine genetic loci for ush1 ( * ush1b - h *, * j *, and * k * ) have been altered to chromosomes 11q13. 5, 11p15. 1, 10q22. 1, 01, 10q21 - q22, 17q24 - q25, 15q22 - q23 ( * ush1h * and * j * ), and 10p11. 21 - - q21. 1 [ @ pone. 0090688 - yan1 ], [ @ pone. 0090688 - jaworek1 ], [ @ pone. 0090688 - riazuddin1 ]. six of their corresponding genes have been identified : the actin - based motor protein myosin viia ( * cr *, ush1b ) [ @ pone. 0090688 - weil1 ] ; two cadherin - related proteins, cadherin 23 ( * cb *, ush1d ) [ @ pone. 0090688 - bork1 ] and chromosome 15 ( * pcdh15 *, ush1f ) [ @ po ##ne. 0090688 - ahmed1 ] ; and two scaffold proteins, harmonin ( * ush1c * ) [ @ pone. 0090688 - verpy1 ] and sans ( * ush1g * ) [ @ pone. 0090688 - mustapha1 ] ; the ca ^ 2 + ^ - and integrin - binding protein ( * cib2 *, ush1j ) [ @ pone. 0090688 - riazuddin1 ]. in caucasian ush1 patients, previous studies showed that mutations in * myo7a *, * ush1c *, * cdh23 *, * pcdh15 *, and * ush1g *, were found in 39 - - 55 %, 7 - - 14 %, 7 - - 35 %, 7 - - 11 %, and 0 - - 7 %, respectively ( the frequency of * cib2 * is still unknown ) [ @ pone. 0090688 - ouyang1 ], [ @ pone. 0090688 - bonnet1 ], [ @ pone. 0090688 - lequesnestabej1 ]. in japanese, nakanishi et al. showed that * myo7a * and * cdh23 * mutations are present in ush1 patients [ @ pone. 0090688 - nakanishi1 ], however, the frequency is not yet known. in addition, mutations in three corresponding genes ( usherin * ush2a * [ @ pone. 0090688 - eudy1 ], g protein - coupled receptor 98 ; * gpr98 * [ @ pone. 0090688 - weston1 ], and deafness, autosomal recessive 31 ; * dfnb31 * [ @ pone. 0090688 - aller1 ] ) have been reported so far in ush2, and ush3 is caused by mutations in the clarin 1 ( * clrn1 * ) [ @ pone. 0090688 - joensuu1 ] gene. comprehensive molecular diagnosis of ush has been hampered both by genetic heterogeneity and the large number of exons for most of the ush genes. the six ush1 genes collectively contain 180 coding exons [ @ pone. 0090688 - riazuddin1 ], [ @ pone. 0090688 - mustapha1 ], [ @ pone. 0090688 - ouyang1 ] the three ush2 genes comprise 175 coding exons [ @ pone. 0090688 - weston1 ], [ @ pone. 0090688 - aller1 ], [ @ pone. 0090688 - nakanishi2 ], and the ush3 gene has five coding exons [ @ pone. 0090688 - joensuu1 ]. in addition some of these genes are alternatively spliced ( [ @ pone. 0090688 - riazuddin1 ], [ @ pone. 0090688 - ahmed1 ], [ @ pone. 0090688 - verpy1 ], [ @ pone. 0090688 - aller1 ], [ @ pone. 0090688 - joensuu1 ] and ncbi database : < http : / / www. ncbi. nlm. nih. gov / nuccore / > ). thus far, large - scale mutation screening has been performed using direct sequence analysis, but that is both time - consuming and expensive. we thought that targeted exon sequencing of selected genes using the massively parallel dna sequencing ( mps ) technology would enable us to systematically tackle previously intractable monogenic disorders and improve molecular diagnosis. therefore, in this study, we have conducted genetic analysis using mps - based genetic screening to find mutations in nine causative ush genes ( except * cib2 * ) in japanese ush1 patients. results { # s2 } = = = = = = = mutation analysis of the nine ush genes in 17 unrelated ush1 patients revealed 19 different probable pathogenic variants, of which 14 were novel ( [ table 1 ] ( # pone - 0090688 - t001 ) { ref - type = " table " } ). 10. 1371 / journal. pone. 0090688. t001 # # # # # # possible pathogenic variants found in this study.! [ ] ( pone. 0090688. t001 ) { # pone - 0090688 - t001 - 1 } gene mutation type nucleotide change amino acid change exon / intron number domain control ( in 384 alleles ) sift score polyphen score reference - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - * myo7a * frameshift c. 1623dup p. lys542glnfsx5 exon 14 \ - n / a \ - \ - le quesne stabej et al. ( 2012 ) c. 4482 _ 4483instg p. trp1495cysfsx55 exon 34 \ - n / a \ - \ - this study c. 6205 _ 6206delat p. ile2069profsx6 exon 45 \ - n / a \ - \ - this study nonsense c. 1477c \ > t p. gln493x exon 13 \ - n / a \ - \ - this study c. 1708c \ > t p. arg570x exon 15 \ - n / a \ - \ - this study c. 2115c \ > a p. cys705x exon 18 \ - n / a \ - \ - this study c. 6321g \ > a p. trp2107x exon 46 \ - n / a \ - \ - this study missense c. 2074g \ > a p. val692met exon 17 motor domain 0 0. 09 0. 982 this study c. 2311g \ > t p. ala771ser exon 20 iq 2 0. 0026 0. 01 0. 825 nakanishi et al. ( 2010 ) c. 6028g \ > a p. asp2010asn exon 44 ferm 2 0 0 0. 925 jacobson et al. ( 2009 ) * cdh23 * frameshift c. 3567delg p. arg1189argfsx5 exon 30 \ - n / a \ - \ - this study c. 5780 _ 5781delct p. ser1927cysfs16 exon 44 \ - n / a \ - \ - this study splicing c. 5821 - 2a \ > g? intron 44 \ - n / a \ - \ - this study nonsense c. 6319c \ > t p. arg2107x exon 48 \ - n / a \ - \ - nakanishi et al. ( 2010 ) * pcdh15 * splicing c. 158 - 1g \ > a? intron 3 \ - n / a \ - \ - this study nonsense c. 1006c \ > t p. arg336x exon 10 \ - n / a \ - \ - this study c. 2971c \ > t p. arg991x exon 22 \ - n / a \ - \ - roux et al. ( 2006 ) c. 3337g \ > t p. glu1113x exon 25 \ - n / a \ - \ - this study missense c. 3724g \ > a p. val1242met exon 28 cadherin 11 0 | Introduction {# s1} = = = = = = = = = = = = Usher syndrome (USH) is an autosomal recessive disorder characterized by hearing loss (HL ), retinitis pigmentosa (RP) and vestibular dysfunction. Three clinical subtypes can be distinguished. USH type 1 (USH1) is the most severe among them because of profound HL, absent vestibular responses, and prepubertal onset RP. USH type 2 (USH2) is characterized by congenital moderate to sebsre HL, with a high - frequency sloping configuration. The vestibular function is normal and onset of RP is in the first or second decade. The onset of the visual symptoms such as night blindness in USH usually occurs several years later than in USH1. USH type 3 (USH3) is characterized by variable onset of progressive HL, variable onset of RP, and variable impairment of vestibular function (normal to absent) [@ pone. 0090688 - Kimberling1 ], [@ pone. 0090688 - Yan1 ]. To date, nine genetic loci for UaHw (* USH1B - H *, * J *, and * K *) have hwen mapped to chromosomes 11q13. 5, 11p15. 1, 10q22. 1, 21q21, 10q21 - q22, 17q24 - q25, 15q22 - q23 (* USH1H * and * J * ), and 10p11. 21 - - q21. 1 [@ pone. 0090688 - Yan1 ], [@ pone. 0090688 - Jaworek1 ], [@ pone. 0090688 - Riazuddin1 ]. Six of the corresponding genes have been identified: the actin - baC@d motor protein myosin VIIa (* MYO7A *, USH1B) [@ pone. 0090688 - Weil1 ]; two cadherin - related proteins, cadherin 23 (* CDH23 *, USH1D) [@ pone. 0090688 - Bork1] and protocadherin 15 (* PCDH15 *, USH1F) [@ pone. 0090688 - Ahmed1 ]; and two scaffold proteins, harmonin (* USH1C *) [@ pone. 0090688 - Verpy1] and sans (* USH1G *) [@ pone. 0090688 - Mustapha1 ]; the Ca ^ 2 + ^ - and integrin - binding protein (* CIB2 *, USH1J) [@ pone. 0090688 - Riazuddin1 ]. In Caucasian USH1 paGienrs, previous studies showed that mutations in * MYO7A *, * USH1C *, * CDH23 *, * PCDH15 *, and * USH1G *, were found in 39 - - 55% , 7 - - 14% , 7 - - 35% , 7 - - 11% , and 0 - - 7% , respectively (the frequency of * CIB2 * is still unknown) [@ pone. 0090688 - Ouyang1 ], [@ pone. 0090688 - Bonnet1 ], [@ pone. 0090688 - LeQuesneStabej1 ]. In Japanese, Nakanishi et al. showed that * MYO7A * and * CDH23 * mutations are present in USH1 patients [@ pone. 0090688 - Nakanishi1 ], however, the frequency is not yet known. In addition, mutations in three corresponding genes (usherin * USH2A * [@ pone. 0090688 - Eudy1 ], G protein - coupled receptor 98; * GPR98 * [@ pone. 0090688 - Weston1 ], and deafness, autosomal recessive 31; * DFNB31 * [@ pone. 0090688 - Aller1] ) have been reported so far in USH2, and USH3 is caused by mutations in the clarin 1 (* CLRN1 *) [@ pone. 0090688 - Joensuu1] gene. Comprehensive molecular diagnosis of USH has been hampered both by genetic heterogeneity and the large number of exons for most of the USH genes. The six USH1 genes collectively contain 180 coding exons [@ pone. 0090688 - Riazuddin1 ], [@ pone. 0090688 - Mustapha1 ], [@ pone. 0090688 - Ouyang1] the three USH2 VeJes comprise 175 coding exons [@ pone. 0090688 - Weston1 ], [@ pone. 0090688 - Aller1 ], [@ pone. 0090688 - Nakanishi2 ], and the USH3 BenS has five coding exons [@ pone. 0090688 - Joensuu1 ]. In addition some of these genes are alternatively spliced ([ @ pone. 0090688 - Riazuddin1 ], [@ pone. 0090688 - Ahmed1 ], [@ pone. 0090688 - Verpy1 ], [@ pone. 0090688 - Aller1 ], [@ pone. 0090688 - Joensuu1] and NCBI database: <http: / / www. ncbi. nlm. nih. gov / nuccore /> ). Thus far, lary2 - scale mutation screening has been performed using direct sequence analysis, but that is both time - consuming and expensive. We thought that targeted exon sequencing of selected genes using the Massively Parallel DNA Sequencing (MPS) technology would enable us to systematically tackle previously intractable monogenic disorders and improve molecular diagnosis. Therefore, in this study, we have conducted genetic analysis using MPS - based genetic screening to find mutations in nine causative USH genes (excwp4 * CIB2 *) in Japanese USH1 patients. Results {# s2} = = = = = = = Mutation analysis of the nine USH genes in 17 unrelated USH1 patients revealed 19 different probable pathogenic variants, of which 14 were novel ([ Table 1] (# pone - 0090688 - t001) {ref - type = " table "} ). 10. 1371 / journal. pone. 0090688. t001 # # # # # # Possible pathogenic variants found in this study. ! [] (pone. 0090688. t001) {# pone - 0090688 - t001 - 1} Gene Mutation type Nucleotide change Amino acid change exon / intron number Domain control (in 384 alleles) SIFT Score PolyPhen Score Reference - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - * MYO7A * Frameshift c. 1623dup p. Lys542GlnfsX5 Exon 14 \ - N / A \ - \ - Le Quesne Stabej et al. (2012) c. 4482_4483insTG p. Trp1495CysfsX55 Exon 34 \ - N / A \ - \ - This study c. 6205_6206delAT p. Ile2069ProfsX6 Exon 45 \ - N / A \ - \ - This study Nonsense c. 1477C \> T p. Gln493X Exon 13 \ - N / A \ - \ - This study c. 1708C \> T p. Arg570X Exon 15 \ - N / A \ - \ - This study c. 2115C \> A p. Cys705X Exon 18 \ - N / A \ - \ - This study c. 6321G \> A p. Trp2107X Exon 46 \ - N / A \ - \ - This study Missense c. 2074G \> A p. Val692Met Exon 17 Motor domain 0 0. 09 0. 982 This study c. 2311G \> T p. Ala771Ser Exon 20 IQ 2 0. 0026 0. 01 0. 825 Nakanishi et al. (2010) c. 6028G \> A p. Asp2010Asn Exon 44 FERM 2 0 0 0. 925 Jacobson et al. (2009) * CDH23 * Frameshift c. 3567delG p. Arg1189ArgfsX5 Exon 30 \ - N / A \ - \ - This study c. 5780_5781delCT p. Ser1927Cysfs16 Exon 44 \ - N / A \ - \ - This study Splicing c. 5821 - 2A \> G? Intron 44 \ - N / A \ - \ - This study Nonsense c. 6319C \> T p. Agg2@07X Exon 48 \ - N / A \ - \ - Nakanishi et al. (2010) * PCDH15 * Splicing c. 158 - 1G \> A? Intron 3 \ - N / A \ - \ - This study Nonsense c. 1006C \> T p. Arg336X Exon 10 \ - N / A \ - \ - This study c. 2971C \> T p. Arg991X Exon 22 \ - N / A \ - \ - Roux et al. (2006) c. 3337G \> T p. Glu1113X Exon 25 \ - N / A \ - \ - This study Missense c. 3724G \> A p. Val1242Met Exon 28 Cadherin 11 0 | Introduction ============ Usher syndrome (USH) is an recessive disorder characterized by hearing loss (HL), retinitis pigmentosa (RP) and vestibular dysfunction. Three clinical subtypes can be distinguished. type 1 is the most among them because of profound HL, absent vestibular responses, and prepubertal onset RP. USH 2 (USH2) is by congenital moderate to severe HL, with a high-frequency sloping configuration. The vestibular function is normal and onset of RP is in the first or second decade. The onset of the visual such as night blindness in usually occurs several years later in USH1. USH type (USH3) is characterized by variable onset of progressive HL, variable onset of and variable impairment of vestibular function (normal to absent) [@pone.0090688-Yan1]. To date, nine genetic loci for USH1(*USH1B-H*, *J*, and *K*) have to chromosomes 11q13.5, 11p15.1, 10q22.1, 21q21, 10q21-q22, 17q24-q25, 15q22-q23 (*USH1H* and *J*), and 10p11.21--q21.1 [@pone.0090688-Yan1], [@pone.0090688-Jaworek1], Six of the corresponding genes been identified: the motor protein myosin VIIa (*MYO7A*, USH1B) [@pone.0090688-Weil1]; two cadherin-related proteins, cadherin 23 USH1D) [@pone.0090688-Bork1] and 15 (*PCDH15*, USH1F) [@pone.0090688-Ahmed1]; proteins, harmonin (*USH1C*) [@pone.0090688-Verpy1] and sans (*USH1G*) [@pone.0090688-Mustapha1]; the Ca^2+^- and integrin-binding protein (*CIB2*, USH1J) [@pone.0090688-Riazuddin1]. In Caucasian USH1 patients, previous studies showed that in *USH1C*, *CDH23*, *PCDH15*, and *USH1G*, were found in 39--55%, 7--14%, 7--11%, and 0--7%, respectively (the frequency *CIB2* is [@pone.0090688-Ouyang1], [@pone.0090688-Bonnet1], [@pone.0090688-LeQuesneStabej1]. In Japanese, Nakanishi et al. showed that *MYO7A* and *CDH23* mutations are present in USH1 patients [@pone.0090688-Nakanishi1], however, the is not yet known. In addition, mutations three corresponding genes (usherin *USH2A* [@pone.0090688-Eudy1], G protein-coupled receptor 98; *GPR98* [@pone.0090688-Weston1], and deafness, autosomal recessive 31; *DFNB31* [@pone.0090688-Aller1]) have been reported so far USH2, and USH3 is by in the clarin 1 (*CLRN1*) [@pone.0090688-Joensuu1] gene. Comprehensive molecular diagnosis of USH been hampered both by genetic heterogeneity the large number of exons for of the USH genes. The six USH1 genes collectively contain 180 coding exons [@pone.0090688-Riazuddin1], [@pone.0090688-Ouyang1] the three USH2 genes comprise 175 coding exons [@pone.0090688-Weston1], [@pone.0090688-Aller1], [@pone.0090688-Nakanishi2], and the USH3 gene has five coding exons [@pone.0090688-Joensuu1]. In addition some of genes are alternatively spliced ([@pone.0090688-Riazuddin1], [@pone.0090688-Ahmed1], [@pone.0090688-Verpy1], [@pone.0090688-Aller1], and NCBI database: <http://www.ncbi.nlm.nih.gov/nuccore/>). Thus far, large-scale mutation screening has been performed using direct analysis, but that is time-consuming and expensive. We thought that targeted exon sequencing of selected genes using the Parallel DNA Sequencing (MPS) technology would enable us to tackle previously intractable monogenic disorders and improve molecular diagnosis. Therefore, in this study, we conducted genetic analysis using MPS-based genetic screening to find mutations in nine causative USH genes (except *CIB2*) in Japanese USH1 patients. Results ======= Mutation of the nine genes in 17 unrelated USH1 patients revealed 19 different probable pathogenic of which 14 were novel ([Table 1](#pone-0090688-t001){ref-type="table"}). 10.1371/journal.pone.0090688.t001 ###### Possible variants found in this study. Gene Mutation type Nucleotide change Amino acid change exon/intron number Domain control (in 384 alleles) SIFT Score PolyPhen Score Reference ---------- --------------- ------------------- ------------------- -------------------- -------------- -------------------------- ------------ ---------------- *MYO7A* Frameshift c.1623dup p.Lys542GlnfsX5 Exon \- N/A \- Le Quesne et al. (2012) c.4482_4483insTG p.Trp1495CysfsX55 Exon 34 \- N/A \- This study p.Ile2069ProfsX6 Exon 45 N/A \- This study Nonsense c.1477C\>T p.Gln493X Exon 13 \- N/A \- \- This study c.1708C\>T p.Arg570X 15 \- N/A \- \- This study c.2115C\>A p.Cys705X Exon 18 \- \- \- This study c.6321G\>A p.Trp2107X 46 \- N/A \- \- This study Missense c.2074G\>A p.Val692Met Exon 17 Motor domain 0 0.09 0.982 This study Exon 20 IQ 2 0.0026 0.01 0.825 Nakanishi et al. (2010) c.6028G\>A Exon 44 FERM 2 0 0 Jacobson al. (2009) *CDH23* Frameshift c.3567delG Exon 30 \- N/A \- \- This study c.5780_5781delCT p.Ser1927Cysfs16 Exon 44 \- N/A \- \- This study c.5821-2A\>G ? Intron 44 \- N/A \- \- This Nonsense c.6319C\>T p.Arg2107X Exon 48 \- N/A \- \- Nakanishi et al. (2010) *PCDH15* Splicing c.158-1G\>A ? Intron 3 \- N/A \- \- study Nonsense c.1006C\>T p.Arg336X Exon 10 \- N/A \- \- study c.2971C\>T p.Arg991X 22 \- N/A \- \- Roux et al. c.3337G\>T p.Glu1113X Exon 25 \- N/A \- \- This study Missense c.3724G\>A p.Val1242Met Exon 28 Cadherin 11 0 | intRODUCTiON {#S1}
============
uSher syndrOME (UsH) is an autoSoMAL ReCeSsivE DiSoRDEr cHarACteRiZED By hEarIng LoSs (hL), RETIniTIs pIGMENTOsa (RP) AND VEsTIbULAr DySfunCtiOn. THrEE clinIcAl SUBTypEs Can be DISTiNguiShED. usH Type 1 (Ush1) IS THE moST SevERe AmOng tHem BeCAuSe OF PrOFoUnd Hl, absEnt vEsTiBUlAr ResPoNseS, ANd PREpubERTAl ONsET Rp. UsH tyPe 2 (UsH2) Is chArActERIzEd bY coNGeNitaL mODERAte tO severe Hl, WiTH a hIgh-frEQueNCy sLOPINg CONFIGuRaTIOn. THe veStiBULAr fuNctioN IS normAl aND Onset of rP iS IN tHe FiRSt OR secoNd DEcADe. The ONSet oF ThE visuaL SYmPTOmS SUCh as NIGht bLiNdneSs in uSH uSUALlY ocCuRS sEVerAL YeARs lAteR ThAn in uSH1. uSh typE 3 (usH3) Is ChaRACteRIZeD BY VaRiAblE oNSet OF prOgressIVE hl, VaRiAblE ONSeT oF rp, AnD vARIaBLe impAiRmEnt Of VEstiBULAR fUnCtIoN (noRmal To aBseNT) [@poNe.0090688-KIMBeRLIng1], [@pOnE.0090688-yAn1].
TO DatE, nINE genEtic LOcI For uSh1(*USh1B-H*, *j*, ANd *K*) hAve been mapPeD to CHRoMosoMEs 11Q13.5, 11p15.1, 10q22.1, 21Q21, 10q21-Q22, 17q24-q25, 15Q22-Q23 (*USh1h* AND *J*), AnD 10p11.21--q21.1 [@PonE.0090688-yan1], [@pOnE.0090688-JAwoRek1], [@poNE.0090688-riazUdDin1]. SiX OF tHe CorREspOnDInG geNes HAVE bEeN IDeNTIFieD: tHE actiN-bAsed MoTor PrOTein MYoSIn VIiA (*mYo7A*, uSh1B) [@pOne.0090688-WeIL1]; TwO cADHeRiN-reLaTED prOTeinS, cADHERIN 23 (*cDH23*, uSH1D) [@pone.0090688-Bork1] ANd prOtoCaDhERin 15 (*Pcdh15*, uSH1F) [@pone.0090688-AHMEd1]; aNd two sCAfFold PROtEINs, HaRMonIn (*usH1C*) [@PONE.0090688-VeRpY1] aND sAnS (*USh1G*) [@pone.0090688-muSTapHA1]; the Ca^2+^- ANd INTeGrIN-BinDINg pRoTEin (*cIb2*, uSH1J) [@PoNE.0090688-RIazuddin1]. iN CAucAsIan USH1 paTIENTS, pReVioUs STUDIeS ShoWed thaT mUTAtIoNS in *Myo7A*, *USH1c*, *CdH23*, *PcDh15*, And *uSH1G*, wErE FOuND IN 39--55%, 7--14%, 7--35%, 7--11%, anD 0--7%, RespECtIvElY (thE fREQuEnCy Of *ciB2* IS StiLL unKNOwN) [@ponE.0090688-OUYanG1], [@PoNE.0090688-bONNeT1], [@poNe.0090688-lEquesneSTABej1]. iN JapAnesE, NakAnIShi ET AL. ShOwED THaT *MYO7a* aND *cdh23* MuTAtIonS are PrEsenT In uSH1 pATieNtS [@PoNe.0090688-NaKanISHI1], however, THe FREquENcY Is NOt yET KNoWN. iN addItioN, mUTaTions iN THREe CoRreSpONdINg geNes (UShEriN *USH2A* [@ponE.0090688-eudY1], G prOTeIN-COuPleD REcePtoR 98; *GpR98* [@poNE.0090688-westOn1], ANd DeAFNEsS, aUToSomal rECesSIVe 31; *dFNb31* [@poNE.0090688-ALlER1]) HaVE BEEn REpOrTeD sO FAr In Ush2, AnD ush3 Is CaUsEd By MutAtIONS iN The clArIN 1 (*clRN1*) [@poNE.0090688-jOenSuU1] GeNE.
coMprehENSIVE MOLecuLAR DiAGNoSis OF uSh haS Been HAMpERED bOTH by genetiC hEteROGENEiTY And ThE laRgE Number OF exOns FOR MOST oF THE ush GENES. thE sIx uSH1 GeNEs cOLLEctivELY CoNTaIn 180 codING Exons [@PONE.0090688-RiaZuddIN1], [@poNE.0090688-mUStApHA1], [@POne.0090688-oUYaNG1] thE tHREE uSH2 geNES CoMpriSE 175 COdInG ExonS [@POne.0090688-WEsTOn1], [@PoNE.0090688-ALleR1], [@POne.0090688-nAKaNisHI2], AND THe USh3 gEne HAs fIve CodinG eXoNs [@POne.0090688-jOensuU1]. iN aDditiON soME oF ThEse GENes Are altERNativELy sPlIceD ([@pOne.0090688-riazUDdiN1], [@pone.0090688-aHMed1], [@PoNE.0090688-VerPy1], [@pone.0090688-alLER1], [@pONE.0090688-JOENSUu1] ANd nCBI daTAbAse: <hTtp://wWW.NCbi.nlm.nih.GOv/nuCCoRe/>). ThUs Far, larGE-SCAlE mutaTiOn SCreENiNG hAS BEEn pErForMEd Using dIReCt SequeNcE aNaLYSis, bUt tHat IS BOth TiME-ConsUMING AnD ExpenSIVE. WE tHouGHt THAt taRgETed EXOn SEquencIng oF sElECTED gEnes UsING THE MaSsIvEly paRAllel dnA SeQUenCIng (Mps) tEchNOlOgY woUlD eNaBle US TO sysTEMAticaLLy tACKlE pREviOuSLY inTRaCTAbLe MOnOGeniC dISORDerS aND iMproVE MolEcUlAR DiagnOSis.
therefORE, iN THis sTUdY, we hAVE cONDuCTed gEnETiC AnALYSis uSiNG mps-BasEd geNeTic ScREeNiNG tO fiND muTAtIOns iN NINe CAUSativE uSH GenES (eXcEPT *Cib2*) iN JapanesE usH1 pAtients.
RESuLts {#s2}
=======
mUtAtion ANALYsIS of tHe niNe uSh gENes iN 17 UNRelATEd uSh1 pAtieNts rEVEAleD 19 DIFfEreNT proBaBlE PATHogeniC varIAntS, oF WhIch 14 WERe nOvEL ([TABLE 1](#pone-0090688-t001){REf-TyPe="taBLe"}).
10.1371/jOURnAL.PONE.0090688.T001
###### PoSSible pAThOgeNiC VaRiAnTs FoUnD iN This STudy.
{#pONe-0090688-t001-1}
GEnE MutaTiON tYpE nUCLEOTIDE ChANGe amINo acID Change eXoN/inTRoN nUmBEr dOmain CoNtROL (in 384 ALleles) SifT scoRe polYphEN scORe ReFEreNCE
---------- --------------- ------------------- ------------------- -------------------- -------------- -------------------------- ------------ ---------------- --------------------------------
*myO7A* frAmEsHIfT c.1623dup p.LYs542glnFsX5 eXoN 14 \- N/a \- \- Le qUesNE sTabEj Et aL. (2012)
c.4482_4483iNsTG p.TRp1495cYSfsx55 eXoN 34 \- n/a \- \- ThIs StUdy
C.6205_6206DeLaT p.ilE2069PROfSX6 Exon 45 \- N/a \- \- thIs studY
nonSeNSE C.1477C\>t p.gLn493X eXon 13 \- N/a \- \- THIs STUDY
C.1708c\>t P.ArG570x exoN 15 \- n/A \- \- thIs sTUdy
c.2115c\>a P.cys705X exoN 18 \- N/A \- \- THIS StUDy
C.6321g\>A P.TRp2107X exon 46 \- n/A \- \- ThIS stUdY
MISseNse C.2074g\>a p.val692MeT ExoN 17 MoTOR dOmain 0 0.09 0.982 ThIS stuDy
c.2311g\>t P.Ala771SeR EXoN 20 Iq 2 0.0026 0.01 0.825 NaKANishi ET al. (2010)
C.6028g\>a p.ASP2010Asn exON 44 FERM 2 0 0 0.925 jACobsON Et Al. (2009)
*cdh23* FrAMEshift c.3567dELg p.arg1189ArGFSx5 ExON 30 \- N/A \- \- ThiS stUdY
c.5780_5781DELct p.sER1927cySfS16 eXoN 44 \- n/A \- \- THIs STUDY
spLICing c.5821-2A\>g ? InTrON 44 \- n/A \- \- ThiS sTUDy
noNsENSe c.6319c\>t p.Arg2107X eXOn 48 \- n/a \- \- NaKANISHi eT al. (2010)
*PCDh15* spLicinG c.158-1G\>A ? INTRON 3 \- N/a \- \- tHIs StUDY
nONseNSe c.1006C\>T P.Arg336X exon 10 \- N/A \- \- This sTUDY
c.2971c\>T p.aRG991x EXon 22 \- n/a \- \- rOUx et AL. (2006)
C.3337g\>t p.gLu1113x exoN 25 \- N/a \- \- thIS study
mIsSeNSe C.3724G\>a P.VAL1242Met exon 28 cAdherIN 11 0 | Introduction{#s1} ============ Usher syndrome (USH) is an autosomal recessive disorder characterized by hearing loss(HL), retinitis pigmentosa (RP) and vestibular dysfunction. Threeclinical subtypescan be distinguished. USH type 1 (USH1) isthe most severe among them because of profound HL, absent vestibular responses, and prepubertal onset RP. USH type 2 (USH2) ischaracterized by congenital moderate to severe HL, with ahigh-frequency sloping configuration. The vestibular function is normal and onset of RP is in the first or second decade. Theonset of the visualsymptoms such as nightblindness inUSH usually occurs severalyears later than inUSH1. USHtype 3 (USH3) is characterized by variable onset of progressive HL, variable onsetof RP, and variable impairment of vestibular function (normal to absent) [@pone.0090688-Kimberling1], [@pone.0090688-Yan1]. To date, nine genetic loci for USH1(*USH1B-H*, *J*, and *K*) have been mappedto chromosomes11q13.5, 11p15.1, 10q22.1, 21q21, 10q21-q22, 17q24-q25, 15q22-q23 (*USH1H*and *J*), and10p11.21--q21.1 [@pone.0090688-Yan1], [@pone.0090688-Jaworek1],[@pone.0090688-Riazuddin1].Six of the corresponding genes have been identified: theactin-based motor protein myosin VIIa(*MYO7A*, USH1B)[@pone.0090688-Weil1]; two cadherin-related proteins, cadherin 23 (*CDH23*, USH1D) [@pone.0090688-Bork1] and protocadherin 15 (*PCDH15*, USH1F) [@pone.0090688-Ahmed1];and two scaffold proteins,harmonin (*USH1C*) [@pone.0090688-Verpy1] and sans (*USH1G*) [@pone.0090688-Mustapha1]; the Ca^2+^- and integrin-binding protein (*CIB2*, USH1J) [@pone.0090688-Riazuddin1]. InCaucasian USH1 patients, previousstudies showed thatmutations in *MYO7A*, *USH1C*, *CDH23*,*PCDH15*, and *USH1G*, were found in 39--55%, 7--14%, 7--35%,7--11%, and 0--7%, respectively (the frequencyof*CIB2*is still unknown) [@pone.0090688-Ouyang1], [@pone.0090688-Bonnet1], [@pone.0090688-LeQuesneStabej1]. In Japanese, Nakanishi et al. showed that *MYO7A* and*CDH23* mutations arepresent in USH1 patients [@pone.0090688-Nakanishi1], however,the frequency is not yet known. In addition, mutations inthree corresponding genes (usherin *USH2A* [@pone.0090688-Eudy1], G protein-coupled receptor 98; *GPR98* [@pone.0090688-Weston1], and deafness, autosomal recessive 31; *DFNB31* [@pone.0090688-Aller1]) have been reported so far inUSH2, and USH3 iscausedby mutations in theclarin 1(*CLRN1*) [@pone.0090688-Joensuu1] gene. Comprehensive moleculardiagnosis of USH has been hampered both by genetic heterogeneity and the large number of exons for most of theUSHgenes. The six USH1 genes collectively contain 180 coding exons [@pone.0090688-Riazuddin1], [@pone.0090688-Mustapha1],[@pone.0090688-Ouyang1] the three USH2 genes comprise 175 codingexons [@pone.0090688-Weston1], [@pone.0090688-Aller1], [@pone.0090688-Nakanishi2], andthe USH3gene has five coding exons [@pone.0090688-Joensuu1]. Inaddition some ofthesegenes are alternatively spliced ([@pone.0090688-Riazuddin1], [@pone.0090688-Ahmed1], [@pone.0090688-Verpy1], [@pone.0090688-Aller1], [@pone.0090688-Joensuu1] and NCBI database: <http://www.ncbi.nlm.nih.gov/nuccore/>).Thus far, large-scale mutation screening hasbeenperformed using direct sequence analysis, but that isbothtime-consuming and expensive.We thoughtthat targeted exon sequencing of selected genes using the Massively Parallel DNASequencing (MPS) technology would enableus tosystematically tackle previously intractable monogenic disorders and improvemolecular diagnosis. Therefore, in this study, we have conductedgenetic analysis usingMPS-based geneticscreening to find mutations in nine causative USH genes (except *CIB2*) inJapanese USH1patients. Results {#s2}======= Mutation analysis ofthe nine USHgenes in17 unrelatedUSH1patients revealed 19different probable pathogenic variants, of which14 were novel ([Table1](#pone-0090688-t001){ref-type="table"}). 10.1371/journal.pone.0090688.t001 ###### Possible pathogenic variants found inthis study. {#pone-0090688-t001-1} Gene Mutation type Nucleotide change Amino acid change exon/intron number Domain control (in 384 alleles) SIFT Score PolyPhen Score Reference ---------- --------------- ------------------- ------------------- -------------------- -------------- -------------------------- ------------ ---------------- -------------------------------- *MYO7A* Frameshift c.1623dup p.Lys542GlnfsX5 Exon 14 \- N/A \- \- Le Quesne Stabej et al. (2012) c.4482_4483insTG p.Trp1495CysfsX55 Exon 34 \- N/A \- \- This study c.6205_6206delAT p.Ile2069ProfsX6 Exon 45 \- N/A\- \- This studyNonsense c.1477C\>T p.Gln493X Exon 13 \- N/A \-\- This study c.1708C\>T p.Arg570X Exon 15\- N/A \- \- Thisstudy c.2115C\>A p.Cys705X Exon 18 \- N/A \- \- This study c.6321G\>A p.Trp2107XExon 46 \- N/A \- \- This study Missense c.2074G\>A p.Val692Met Exon17 Motor domain 0 0.09 0.982 This study c.2311G\>T p.Ala771Ser Exon 20 IQ 2 0.0026 0.01 0.825Nakanishi et al. (2010) c.6028G\>A p.Asp2010Asn Exon 44 FERM2 0 0 0.925 Jacobson et al.(2009) *CDH23*Frameshift c.3567delG p.Arg1189ArgfsX5 Exon 30 \-N/A \- \- This studyc.5780_5781delCTp.Ser1927Cysfs16Exon 44 \- N/A \- \- This study Splicing c.5821-2A\>G ? Intron 44 \- N/A \- \- This study Nonsense c.6319C\>Tp.Arg2107XExon48 \- N/A\- \- Nakanishi et al. (2010)*PCDH15* Splicing c.158-1G\>A ?Intron 3 \- N/A \- \- This study Nonsense c.1006C\>T p.Arg336XExon 10\- N/A \- \- Thisstudy c.2971C\>T p.Arg991X Exon 22 \- N/A \- \- Roux et al.(2006) c.3337G\>T p.Glu1113X Exon25 \- N/A \- \- This study Missense c.3724G\>A p.Val1242Met Exon 28 Cadherin 11 0 | Introduction {#s1} ============ _Usher_ syndrome (USH) is an autosomal recessive _disorder_ characterized by hearing loss (HL), retinitis pigmentosa _(RP)_ and _vestibular_ _dysfunction._ Three clinical _subtypes_ _can_ be _distinguished._ USH type 1 (USH1) is the most severe among them because of profound _HL,_ absent vestibular responses, and prepubertal onset RP. USH type _2_ (USH2) _is_ characterized by _congenital_ moderate to severe HL, _with_ a high-frequency sloping configuration. The _vestibular_ function is normal and onset of RP is _in_ the first or second _decade._ The _onset_ of _the_ visual symptoms such as night blindness in USH _usually_ occurs several years later _than_ in USH1. USH type 3 _(USH3)_ _is_ characterized by _variable_ onset of progressive HL, variable _onset_ of RP, and variable impairment _of_ vestibular function _(normal_ to absent) [@pone.0090688-Kimberling1], [@pone.0090688-Yan1]. To _date,_ _nine_ genetic loci for USH1(*USH1B-H*, *J*, and *K*) _have_ been _mapped_ to chromosomes _11q13.5,_ 11p15.1, 10q22.1, 21q21, 10q21-q22, 17q24-q25, 15q22-q23 (*USH1H* and *J*), _and_ 10p11.21--q21.1 [@pone.0090688-Yan1], [@pone.0090688-Jaworek1], [@pone.0090688-Riazuddin1]. Six _of_ _the_ _corresponding_ genes have been identified: the actin-based _motor_ protein myosin VIIa (*MYO7A*, _USH1B)_ _[@pone.0090688-Weil1];_ _two_ cadherin-related proteins, cadherin _23_ _(*CDH23*,_ USH1D) [@pone.0090688-Bork1] and protocadherin 15 _(*PCDH15*,_ USH1F) [@pone.0090688-Ahmed1]; and _two_ scaffold proteins, harmonin _(*USH1C*)_ _[@pone.0090688-Verpy1]_ and sans _(*USH1G*)_ [@pone.0090688-Mustapha1]; the Ca^2+^- _and_ integrin-binding protein (*CIB2*, _USH1J)_ [@pone.0090688-Riazuddin1]. In Caucasian USH1 patients, previous studies showed that _mutations_ in *MYO7A*, _*USH1C*,_ *CDH23*, _*PCDH15*,_ and *USH1G*, were found in 39--55%, 7--14%, 7--35%, 7--11%, _and_ _0--7%,_ respectively _(the_ frequency of *CIB2* _is_ _still_ unknown) [@pone.0090688-Ouyang1], [@pone.0090688-Bonnet1], _[@pone.0090688-LeQuesneStabej1]._ In Japanese, Nakanishi et al. showed that *MYO7A* _and_ *CDH23* _mutations_ are present in _USH1_ patients [@pone.0090688-Nakanishi1], _however,_ the frequency is _not_ _yet_ known. In addition, mutations in three corresponding genes (usherin _*USH2A*_ [@pone.0090688-Eudy1], G protein-coupled _receptor_ 98; *GPR98* [@pone.0090688-Weston1], and _deafness,_ autosomal recessive 31; _*DFNB31*_ _[@pone.0090688-Aller1])_ have been reported so far _in_ _USH2,_ and USH3 is caused by mutations in the clarin 1 _(*CLRN1*)_ [@pone.0090688-Joensuu1] gene. Comprehensive molecular _diagnosis_ of USH has been hampered both by genetic _heterogeneity_ and _the_ large _number_ of _exons_ for most _of_ the USH _genes._ _The_ six USH1 genes collectively contain 180 coding exons _[@pone.0090688-Riazuddin1],_ [@pone.0090688-Mustapha1], [@pone.0090688-Ouyang1] the three USH2 genes _comprise_ 175 coding exons [@pone.0090688-Weston1], [@pone.0090688-Aller1], [@pone.0090688-Nakanishi2], and the USH3 gene has five _coding_ exons [@pone.0090688-Joensuu1]. In addition some of these genes are alternatively spliced ([@pone.0090688-Riazuddin1], _[@pone.0090688-Ahmed1],_ [@pone.0090688-Verpy1], [@pone.0090688-Aller1], _[@pone.0090688-Joensuu1]_ and NCBI _database:_ <http://www.ncbi.nlm.nih.gov/nuccore/>). Thus far, large-scale mutation screening has been performed using _direct_ sequence analysis, but _that_ _is_ _both_ time-consuming and expensive. We thought _that_ targeted exon _sequencing_ of _selected_ genes _using_ the Massively Parallel DNA _Sequencing_ (MPS) technology would enable _us_ to _systematically_ _tackle_ previously _intractable_ _monogenic_ _disorders_ and improve _molecular_ diagnosis. _Therefore,_ _in_ this _study,_ we have conducted genetic analysis using MPS-based genetic screening to find mutations in nine causative USH genes (except *CIB2*) in Japanese USH1 patients. Results {#s2} ======= _Mutation_ analysis of the _nine_ _USH_ genes in 17 unrelated USH1 patients revealed 19 _different_ _probable_ pathogenic _variants,_ _of_ _which_ 14 were _novel_ ([Table 1](#pone-0090688-t001){ref-type="table"}). 10.1371/journal.pone.0090688.t001 ###### Possible pathogenic variants found in this study. _{#pone-0090688-t001-1}_ Gene _Mutation_ type _Nucleotide_ change Amino acid change _exon/intron_ number Domain control (in 384 alleles) SIFT _Score_ _PolyPhen_ _Score_ _Reference_ _----------_ --------------- _-------------------_ ------------------- -------------------- -------------- -------------------------- ------------ ---------------- _--------------------------------_ *MYO7A* Frameshift c.1623dup p.Lys542GlnfsX5 Exon 14 \- N/A \- \- Le Quesne Stabej _et_ _al._ (2012) c.4482_4483insTG p.Trp1495CysfsX55 Exon 34 \- N/A \- \- This study c.6205_6206delAT _p.Ile2069ProfsX6_ Exon _45_ \- N/A \- \- This study Nonsense c.1477C\>T p.Gln493X _Exon_ 13 \- N/A \- \- This study c.1708C\>T p.Arg570X Exon 15 \- N/A \- \- This study c.2115C\>A _p.Cys705X_ Exon 18 \- N/A _\-_ \- This study c.6321G\>A p.Trp2107X Exon 46 \- N/A \- _\-_ This _study_ Missense _c.2074G\>A_ p.Val692Met Exon 17 Motor domain 0 0.09 0.982 This study c.2311G\>T p.Ala771Ser Exon 20 IQ 2 0.0026 0.01 0.825 Nakanishi et al. _(2010)_ _c.6028G\>A_ p.Asp2010Asn Exon 44 FERM 2 0 0 0.925 Jacobson et al. _(2009)_ *CDH23* _Frameshift_ _c.3567delG_ p.Arg1189ArgfsX5 Exon 30 \- N/A \- \- This study c.5780_5781delCT p.Ser1927Cysfs16 Exon _44_ \- N/A \- \- _This_ study Splicing c.5821-2A\>G _?_ _Intron_ _44_ \- N/A \- \- This study Nonsense _c.6319C\>T_ p.Arg2107X Exon 48 \- _N/A_ \- \- Nakanishi et al. (2010) *PCDH15* Splicing c.158-1G\>A ? Intron _3_ \- N/A \- _\-_ This study Nonsense _c.1006C\>T_ p.Arg336X _Exon_ 10 _\-_ N/A _\-_ \- This study _c.2971C\>T_ _p.Arg991X_ Exon 22 _\-_ N/A \- \- Roux et _al._ (2006) c.3337G\>T p.Glu1113X Exon 25 \- N/A \- \- This study Missense c.3724G\>A p.Val1242Met Exon 28 _Cadherin_ _11_ 0 |
1. Introduction {#sec1-polymers-08-00159}
===============
Phenol-formaldehyde (PF) is a high-performance resin that is synthesized by the copolymerization of phenol with formaldehyde. It is widely applied for industrial uses, including adhesives, impregnating resins, and plastics. The excellent properties of PF resin include high mechanical, thermal, and weather stability \[[@B1-polymers-08-00159]\]. However, the lower curing rate and required higher curing temperature compared to other thermosetting adhesives limit the application of PF resins for use in impregnating resins or adhesives \[[@B2-polymers-08-00159],[@B3-polymers-08-00159]\]. Many attempts have been made to accelerate the curing rate or lower the curing temperature, including testing of various catalysts or additives to alter the reaction kinetics, such as carboxylic acid esters \[[@B4-polymers-08-00159],[@B5-polymers-08-00159]\], anhydrides \[[@B6-polymers-08-00159]\], amides \[[@B7-polymers-08-00159]\], carbonate \[[@B8-polymers-08-00159]\], and metallic ions \[[@B9-polymers-08-00159]\]. Additionally, the effects of the condensation condition on the PF resin structure and properties have been well studied by conventional analytical techniques. For example, various mechanisms of PF resin hardening accelerated by catalysts or additives have been reported \[[@B10-polymers-08-00159]\]. Some additives, such as sodium carbonate, act solely to accelerate the curing reaction, but other additives, such as propylene carbonate, both accelerate the reaction and also increase the average functionality of the PF reaction system to allow a tighter final network \[[@B10-polymers-08-00159]\]. The properties of basic catalysts, such as the valence and ionic radius of hydrated cations, affected the mechanisms and kinetics of PF resin condensation and, thus, the composition of the final products \[[@B9-polymers-08-00159]\]. Some studies also reported that an alkaline catalyst promoted the formation of dimethylene ethers in the polymerization reaction, and that *ortho* to *ortho* (*o*,*o*′) ethers were more stable \[[@B11-polymers-08-00159]\]. However, there has been no comprehensive study about the action of catalysts to increase or decrease the ratio of *ortho*/*para* reaction position or analysis of the corresponding physicochemical properties of the accelerated PF resins.
The aromatic ring of phenol has *ortho* and *para* positions capable of reaction with formaldehyde under certain conditions, but the *para* position has higher reactivity than the *ortho* position. The presence of two *ortho* positions and one *para* position in an aromatic ring generally could lead to a PF resin containing mostly *ortho* hydroxymethyl groups \[[@B2-polymers-08-00159]\]. However, in the process of PF resin synthesis, some catalysts could make more formaldehyde or methylol toward phenol *ortho* positions to increase the ratio of *ortho*/*para* substituted positions \[[@B12-polymers-08-00159]\], leading to more reactive functional groups or more unreacted *para* positions at the curing stage, which may shorten the curing time and increase the cross-linking degree of cured PF resin.
Since metal ions can accelerate the curing of PF resins, we tested the ability of barium hydroxide (Ba(OH)~2~), sodium carbonate (Na~2~CO~3~), lithium hydroxide (LiOH), and zinc acetate ((CH~3~COO)~2~Zn) to decrease curing temperature and accelerate the curing rate of PF resins. To elucidate the chemical structure of the cure-accelerated PF resins, we performed quantitative liquid ^13^C NMR to analyze the structural features. Finally, possible synthesis mechanism of metal-mediated polymerization of PF resins was proposed based on the chemical structure analysis and thermogravimetric (DTG) curve.
2. Materials and Methods {#sec2-polymers-08-00159}
========================
2.1. Materials {#sec2dot1-polymers-08-00159}
--------------
Phenol, formaldehyde (37%), Ba(OH)~2~, Na~2~CO~3~, LiOH, and (CH~3~COO)~2~Zn were obtained from Zhong'an Chemical Industries, Beijing, China and were used directly without further purification, and all other chemicals were AR grade and obtained from Beijing Chemical Industries, Beijing, China.
2.2. Preparation of PF Resins {#sec2dot2-polymers-08-00159}
-----------------------------
The catalyst-accelerated PF resin was synthesized by batch polymerization with phenol and formaldehyde at a molar ratio of 1:2.2, and the additive amount of catalyst was 6% based on the total mass of PF resin. In the first step, phenol was mixed in a flask with one third of the formaldehyde and one third of the catalyst. The mixture was quickly heated to 70 °C, and then the heater was turned off. The temperature of the mixture increased to 90 °C due to the heat produced by polymerization reaction and remained at 93--95 °C for 1 h. In the second step, the remaining formaldehyde and two thirds of the catalyst were added to the flask and the mixture was heated to 90 °C and kept at that temperature for 0.5 h. Finally, the mixture was cooled to 40 °C to yield PF resin. PF resins with different catalysts were synthesized with the same procedure.
2.3. Preparation of Plywood {#sec2dot3-polymers-08-00159}
---------------------------
Three-layer plywood (400 mm × 400 mm × 4.8 mm) was prepared with a single poplar veneer in the middle and two poplar veneers on the top and bottom simulating actual industrial parameters. The middle poplar veneer was coated with 125--150 g/m^2^ resin on each side. Four pieces of three-layer plywood for each catalyst-accelerated resin (including control) were hot-pressed under 1.2 MPa at 100, 110, 120, and 130 °C, respectively. The hot-press time was 7 min, including the first one minute and the last one minute to load and unload the pressure, respectively.
2.4. Characterization of PF Resins {#sec2dot4-polymers-08-00159}
----------------------------------
The solid (non-volatile) content of resol resin was determined in accordance with ASTM standard D4426-01. The viscosity of resin was measured using a Brookfield DV-II viscometer (AMETEK-BROOKFIELD Corporation, Middleboro, MA, USA) using 61\# rotor with spinning rate of 100 rpm. Gel time was defined as the time period from the immersion of the test tube into the oil bath (135 °C) to the beginning of the resin gelation (resin forming a string when a glass rod was lifted from the resin).
2.5. Characterization of the Plywood {#sec2dot5-polymers-08-00159}
------------------------------------
The shear strength was measured as per ASTM D906-98.
2.6. FT-IR Analysis of PF Resins {#sec2dot6-polymers-08-00159}
--------------------------------
The resins were placed in 0.01 MPa vacuum at 60 °C for 4 h to dry to non-volatility. FT-IR spectra of vacuum-dried PF resins were performed in a Nicolet IS10 instrument (Thermo Fisher Scientific Corporation, Waltham, MA, USA). Each spectrum was recorded with 32 scans in a frequency range of 600--4000 cm^−1^ at a spectral resolution of 4 cm^−1^.
2.7. Contact Angle Measurement {#sec2dot7-polymers-08-00159}
------------------------------
The contact angle measurements of the PF resins were performed on the tangential surfaces of wood samples with an optical contact angle apparatus (OCA 20 DataPhysics Instruments GmnH, Filderstadt, Germany). Sessile droplets (3 μL, measured with a microsyringe) of liquid resin were placed on the wood surface. The right and left angles of the drops on the surface were collected at intervals of 0.1 s for a total duration of 60 s, and the average angle was calculated.
2.8. Quantitative Liquid ^13^C NMR Measurement {#sec2dot8-polymers-08-00159}
----------------------------------------------
All of the resins were characterized by quantitative ^13^C NMR spectroscopy with a VARIAN INOUR-300 (JEOL Corporation, Tokyo, Japan) spectrometer with a frequency of 75.51 MHz using the inverse-gated decoupling method. All of the spectra were recorded at room temperature with a delay time of 8 s, a 13 h acquisition time and a 15.4 μs pulse width (90°). About 8000 scans were accumulated to obtain spectra for each spectrum. The chemical shifts of each spectrum were accurate to 0.1 ppm and all the resin samples were directly used for ^13^C NMR measurement.
2.9. Thermogravimetric Analysis (TG) of Resins {#sec2dot9-polymers-08-00159}
----------------------------------------------
Samples were dried at 120 °C for 2 h to evaporate the moisture and then TG was performed in a nitrogen atmosphere within a temperature range from room temperature to 700 °C, with a heating rate of 10 °C/min.
3. Results and Discussion {#sec3-polymers-08-00159}
=========================
3.1. Performance of the Catalyst-Accelerated PF Resin {#sec3dot1- | 1. introduction { # sec1 - polymers - 08 - 00159 } = = = = = = = = = = = = = = = phenol - formaldehyde ( pf ) is a high - performance resin this is synthesized by the copolymerization of phenol with formaldehyde. it is widely applied for industrial uses, including adhesives, impregnating resins, and plastics. the chemical properties of pf resin include high mechanical, thermal, and weather stability \ [ [ @ b1 - polymers - 08 - 00159 ] \ ]. however, the lower curing rate and required higher curing temperature compared to other thermosetting adhesives limit the application of pf resins for use in impregnating resins or adhesives \ [ [ @ b2 - polymers - 08 - 00159 ], [ @ b3 - polymers - 08 - 00159 ] \ ]. many attempts have been made to accelerate the curing rate or lower the required temperature, including testing of various catalysts or inhibitors to alter the reaction kinetics, such the carboxylic acid esters \ [ [ @ b4 - polymers - 08 - 00159 ], [ @ b5 - polymers - 08 - 00159 ] \ ], anhydrides \ [ [ @ b6 - polymers - 08 - 00159 ] \ ], amides \ [ [ @ b7 - polymers - 08 - 00159 ] \ ], carbonate \ [ [ @ b8 - polymers - 08 - 00159 ] \ ], and metallic ions \ [ [ @ b9 - polymers - 08 - 00159 ] \ ]. additionally, the effects of the condensation condition on the pf resin structure and properties have been well studied by conventional analytical techniques. for example, various mechanisms of pf resin hardening accelerated acid catalysts or enzymes have been reported \ [ [ @ b10 - polymers - 08 - 00159 ] \ ]. some additives, such as sodium carbonate, act solely to accelerate the curing reaction, but other additives, such as propylene carbonate, both accelerate organic reaction and also increase the average functionality of the pf reaction system to allow a tighter final network \ [ [ @ b10 - polymers - 08 - 00159 ] \ ]. the incorporation of basic catalysts, such as the valence and ionic radius using hydrated cations, affected the mechanisms and kinetics of pf resin condensation and, thus, the composition of the final products \ [ [ @ b9 - polymers - 08 - 00159 ] \ ]. some studies also reported that an alkaline catalyst promoted the formation of dimethylene ethers in the polymerization reaction, and that * ortho * to * ortho * ( * o *, * o * ′ ) ethers were more stable \ [ [ @ b11 - polymers - 08 - 00159 ] \ ]. however, there has been no comprehensive study about the action of catalysts to increase or decrease the ratio of * ortho * / * para * reaction position or analysis of the corresponding physicochemical properties of the accelerated pf resins. the aromatic ring of phenol has * ortho * and * para * positions capable of reaction with formaldehyde under certain conditions, but the * para * position has higher reactivity than the * ortho * position. the presence of two * ortho * positions and one * para * position in an aromatic ring generally could lead to a pf resin containing mostly * ortho * hydroxymethyl groups \ [ [ @ b2 - polymers - 08 - 00159 ] \ ]. however, in the process of pf resin synthesis, some catalysts could make more formaldehyde or methylol toward phenol * ortho * positions to increase the ratio of * ortho * / * para * substituted positions \ [ [ @ b12 - polymers - 08 - 00159 ] \ ], leading to more reactive functional groups or more unreacted * para * positions at the curing stage, which may shorten the curing time and increase the cross - linking degree of cured pf resin. since metal ions can accelerate the curing of pf resins, we tested the ability of barium hydroxide ( ba ( oh ) ~ 2 ~ ), sodium carbonate ( na ~ 2 ~ co ~ 3 ~ ), lithium hydroxide ( lioh ), and zinc acetate ( ( ch ~ 3 ~ coo ) ~ 2 ~ zn ) to decrease curing temperature and accelerate the curing rate of pf resins. to elucidate the chemical structure of the cure - accelerated pf resins, we performed quantitative liquid ^ 13 ^ c nmr to analyze the structural features. finally, possible synthesis mechanism of metal - mediated polymerization of pf resins was proposed based on the chemical structure analysis and thermogravimetric ( dtg ) curve. 2. materials and methods { # sec2 - polymers - 08 - 00159 } = = = = = = = = = = = = = = = = = = = = = = = = 2. 1. materials { # sec2dot1 - polymers - 08 - 00159 } - - - - - - - - - - - - - - phenol, formaldehyde ( 37 % ), ba ( oh ) ~ 2 ~, na ~ 2 ~ co ~ 3 ~, lioh, and ( ch ~ 3 ~ coo ) ~ 2 ~ zn were obtained from zhong ' an chemical industries, beijing, china and were used directly without further purification, and all other chemicals were ar grade and obtained from beijing chemical industries, beijing, china. 2. 2. preparation of pf resins { # sec2dot2 - polymers - 08 - 00159 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the catalyst - accelerated pf resin was synthesized by batch polymerization with phenol and formaldehyde at a molar ratio of 1 : 2. 2, and the additive amount of catalyst was 6 % based on the total mass of pf resin. in the first step, phenol was mixed in a flask with one third of the formaldehyde and one third of the catalyst. the mixture was quickly heated to 70 °c, and then the heater was turned off. the temperature of the mixture increased to 90 °c due to the heat produced by polymerization reaction and remained at 93 - - 95 °c for 1 h. in the second step, the remaining formaldehyde and two thirds of the catalyst were added to the flask and the mixture was heated to 90 °c and kept at that temperature for 0. 5 h. finally, the mixture was cooled to 40 °c to yield pf resin. pf resins with different catalysts were synthesized with the same procedure. 2. 3. preparation of plywood { # sec2dot3 - polymers - 08 - 00159 } - - - - - - - - - - - - - - - - - - - - - - - - - - - three - layer plywood ( 400 mm × 400 mm × 4. 8 mm ) was prepared with a single poplar veneer in the middle and two poplar veneers on the top and bottom simulating actual industrial parameters. the middle poplar veneer was coated with 125 - - 150 g / m ^ 2 ^ resin on each side. four pieces of three - layer plywood for each catalyst - accelerated resin ( including control ) were hot - pressed under 1. 2 mpa at 100, 110, 120, and 130 °c, respectively. the hot - press time was 7 min, including the first one minute and the last one minute to load and unload the pressure, respectively. 2. 4. characterization of pf resins { # sec2dot4 - polymers - 08 - 00159 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the solid ( non - volatile ) content of resol resin was determined in accordance with astm standard d4426 - 01. the viscosity of resin was measured using a brookfield dv - ii viscometer ( ametek - brookfield corporation, middleboro, ma, usa ) using 61 \ # rotor with spinning rate of 100 rpm. gel time was defined as the time period from the immersion of the test tube into the oil bath ( 135 °c ) to the beginning of the resin gelation ( resin forming a string when a glass rod was lifted from the resin ). 2. 5. characterization of the plywood { # sec2dot5 - polymers - 08 - 00159 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the shear strength was measured as per astm d906 - 98. 2. 6. ft - ir analysis of pf resins { # sec2dot6 - polymers - 08 - 00159 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the resins were placed in 0. 01 mpa vacuum at 60 °c for 4 h to dry to non - volatility. ft - ir spectra of vacuum - dried pf resins were performed in a nicolet is10 instrument ( thermo fisher scientific corporation, waltham, ma, usa ). each spectrum was recorded with 32 scans in a frequency range of 600 - - 4000 cm ^ −1 ^ at a spectral resolution of 4 cm ^ −1 ^. 2. 7. contact angle measurement { # sec2dot7 - polymers - 08 - 00159 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the contact angle measurements of the pf resins were performed on the tangential surfaces of wood samples with an optical contact angle apparatus ( oca 20 dataphysics instruments gmnh, filderstadt, germany ). sessile droplets ( 3 μl, measured with a microsyringe ) of liquid resin were placed on the wood surface. the right and left angles of the drops on the surface were collected at intervals of 0. 1 s for a total duration of 60 s, and the average angle was calculated. 2. 8. quantitative liquid ^ 13 ^ c nmr measurement { # sec2dot8 - polymers - 08 - 00159 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - all of the resins were characterized by quantitative ^ 13 ^ c nmr spectroscopy with a varian inour - 300 ( jeol corporation, tokyo, japan ) spectrometer with a frequency of 75. 51 mhz using the inverse - gated decoupling method. all of the spectra were recorded at room temperature with a delay time of 8 s, a 13 h acquisition time and a 15. 4 μs pulse width ( 90° ). about 8000 scans were accumulated to obtain spectra for each spectrum. the chemical shifts of each spectrum were accurate to 0. 1 ppm and all the resin samples were directly used for ^ 13 ^ c nmr measurement. 2. 9. thermogravimetric analysis ( tg ) of resins { # sec2dot9 - polymers - 08 - 00159 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - samples were dried at 120 °c for 2 h to evaporate the moisture and then tg was performed in a nitrogen atmosphere within a temperature range from room temperature to 700 °c, with a heating rate of 10 °c / min. 3. results and discussion { # sec3 - polymers - 08 - 00159 } = = = = = = = = = = = = = = = = = = = = = = = = = 3. 1. performance of the catalyst - accelerated pf resin { # sec3dot1 - | 1. Introduction {# sec1 - polymers - 08 - 00159} = = = = = = = = = = = = = = = Phenol - formaldehyde (PF) is a high - performance resin that is synthesized by the copolymerization of phenol with formaldehyde. It is widely applied for industrial uses, including adhesives, impregnating resins, and plastics. The excellent properties of PF resin include high mechanical, thermal, and weQ^her stability \ [[ @ B1 - polymers - 08 - 00159] \ ]. However, the lower curing rate and req*irDd higher curing temperature compared to other thermosetting adhesives limit the application of PF resins for use in impregnating resins or adhesives \ [[ @ B2 - polymers - 08 - 00159 ], [@ B3 - polymers - 08 - 00159] \ ]. Many attempts have been made to accelerate the curing rate or lower the curing temperature, including testing of various catalysts or additives to alter the reaction kinetics, such as carboxylic acid esters \ [[ @ B4 - polymers - 08 - 00159 ], [@ B5 - polymers - 08 - 00159] \ ], anhydrides \ [[ @ B6 - polymers - 08 - 00159] \ ], amides \ [[ @ B7 - polymers - 08 - 00159] \ ], carbonate \ [[ @ B8 - polymers - 08 - 00159] \ ], and metallic ions \ [[ @ B9 - polymers - 08 - 00159] \ ]. Additionally, the effects of the condensation condition on the PF resin structure and properties have been well studied by conventional analytical techniques. For example, various mechanisms of PF resin hardening accelerated by catalysts or additives have been reported \ [[ @ B10 - polymers - 08 - 00159] \ ]. Some additives, such as sodium carbonate, act solely to accelerate the curing reaction, but other additives, such as propylene carbonate, both accelerate the reaction and also increase the average functionality of the PF reaction system to allow a tighter final network \ [[ @ B10 - polymers - 08 - 00159] \ ]. The properties of basic catalysts, such as the valence and ionic radius of hydrated cations, affected the mechanisms and kinetics of PF resin condensation and, thus, the composition of the final products \ [[ @ B9 - polymers - 08 - 00159] \ ]. Some studies also reported that an alkaline catalyst promoted the formation of dimethylene ethers in the polymerization reaction, and that * ortho * to * ortho * (* o *, * o * ′) ethers were more stable \ [[ @ B11 - polymers - 08 - 00159] \ ]. However, there has been no comprehensive wtudu about the action of catalysts to increase or decrease the ratio of * ortho * / * para * reaction position or analysis of the corresponding physicochemical properties of the accelerated PF resins. The aromatic ring of phenol has * ortho * and * para * positions capable of reaction with formaldehyde under certain conditions, but the * para * position has higher reactivity than the * ortho * position. The presence of two * orFhP * positions and one * para * position in an aromatic ring generally could lead to a PF resin containing mostly * ortho * hydroxymethyl gdPups \ [[ @ B2 - polymers - 08 - 00159] \ ]. However, in the process of PF resin synthesis, some catalysts could make more formaldehyde or methylol toward phenol * ortho * positions to increase the ratio of * ortho * / * para * substituted positions \ [[ @ B12 - polymers - 08 - 00159] \ ], leading to more reactive functional groups or more unreacted * para * positions at the curing stage, which may shorten the curing time and increase the cross - linking degree of cured PF resin. Since metal ions can accelerate the curing of PF resins, we tested the ability of barium hydroxide (Ba (OH) ~ 2 ~ ), sodium carbonate (Na ~ 2 ~ CO ~ 3 ~ ), lithium hydroxide (LiOH ), and zinc acetate (( CH ~ 3 ~ COO) ~ 2 ~ Zn) to decrease curing temperature and accelerate the curing rate of PF resins. To elucidate the chemical structure of the cure - accelerated PF resins, we performed quantitative liquid ^ 13 ^ C NMR to analyze the structural features. Finally, possible synthesis mechanism of metal - mediated polymerization of PF resins was proposed based on the chemical structure analysis and thermogravimetric (DTG) curve. 2. Materials and Methods {# sec2 - polymers - 08 - 00159} = = = = = = = = = = = = = = = = = = = = = = = = 2. 1. Materials {# sec2dot1 - polymers - 08 - 00159} - - - - - - - - - - - - - - Phenol, formaldehyde (37% ), Ba (OH) ~ 2 ~, Na ~ 2 ~ CO ~ 3 ~, LiOH, and (CH ~ 3 ~ COO) ~ 2 ~ Zn were obtained from Zhong ' an Chemical Industries, Beijing, China and were used directly without further purification, and all other chemicals were AR grade and obtained from Beijing Chemical Industries, Beijing, China. 2. 2. Preparation of PF Resins {# sec2dot2 - polymers - 08 - 00159} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The catalyst - accelerated PF resin was synthesized by batch polymerization with phenol and formaldehyde at a molar ratio of 1: 2. 2, and the additive amount of catalyst was 6% based on the total mass of PF resin. In the first step, phenol was mixed in a flask with one third of the formaldehyde and one third of the catalyst. The mixture was quickly heated to 70 ° C, and then the heater was turned off. The temperature of the mixture increased to 90 ° C due to the heat produced by polymerization reaction and remained at 93 - - 95 ° C for 1 h. In the second step, the remaining fo#maldeMyde and two thirds of the catalyst were added to the flask and the mixture was heated to 90 ° C and kept at that temperature for 0. 5 h. Finally, the mixture was cooled to 40 ° C to yield PF resin. PF resins with different catalysts were synthesized with the same procedure. 2. 3. Preparation of Plywood {# sec2dot3 - polymers - 08 - 00159} - - - - - - - - - - - - - - - - - - - - - - - - - - - Three - layer plywood (400 mm × 400 mm × 4. 8 mm) was prepared with a single poplar veneer in the middle and two poplar veneers on the top and bottom simulating actual industrial parameters. The middle poplar veneer was coated with 125 - - 150 g / m ^ 2 ^ resin on each side. Four pieces of three - layer plywood for each catalyst - accelerated resin (including control) were hot - pressed under 1. 2 MPa at 100, 110, 120, and 130 ° C, respectively. The hot - press time was 7 min, including the first one minute and the last one minute to load and unload the pressure, respectively. 2. 4. Characterization of PF Resins {# sec2dot4 - polymers - 08 - 00159} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The solid (non - volatile) content of resol resin was determined in accordance with ASTM standard D4426 - 01. The viscosity of resin was measured using a Brookfield DV - II viscometer (AMETEK - BROOKFIELD Corporation, Middleboro, MA, USA) using 61 \ # rotor with spinning rate of 100 rpm. Gel time was defined as the time period from the immersion of the test tube into the oil bath (135 ° C) to the beginning of the resin gelation (resin forming a string when a glass rod was lifted from the resin ). 2. 5. Characterization of the Plywood {# sec2dot5 - polymers - 08 - 00159} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The shear strength was measured as per ASTM D906 - 98. 2. 6. FT - IR Analysis of PF Resins {# sec2dot6 - polymers - 08 - 00159} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The resins were placed in 0. 01 MPa vacuum at 60 ° C for 4 h to dry to non - volatility. FT - IR spectra of vacuum - dried PF resins were performed in a Nicolet IS10 instrument (Thermo Fisher Scientific Corporation, Waltham, MA, USA ). Each spectrum was recorded with 32 scans in a frequency range of 600 - - 4000 cm ^ − 1 ^ at a spectral resolution of 4 cm ^ − 1 ^. 2. 7. Contact Angle Measurement {# sec2dot7 - polymers - 08 - 00159} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The contact angle measurements of the PF resins were performed on the tangential surfaces of wood samples with an optical contact angle apparatus (OCA 20 DataPhysics Instruments GmnH, Filderstadt, Germany ). Sessile droplets (3 μL, measured with a microsyringe) of liquid resin were placed on the wood surface. The right and left angles of the drops on the surface were collected at intervals of 0. 1 s for a total duration of 60 s, and the average angle was calculated. 2. 8. Quantitative Liquid ^ 13 ^ C NMR Measurement {# sec2dot8 - polymers - 08 - 00159} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - All of the resins were characterized by quantitative ^ 13 ^ C NMR spectroscopy with a VARIAN INOUR - 300 (JEOL Corporation, Tokyo, Japan) spectrometer with a frequency of 75. 51 MHz using the inverse - gated decoupling method. All of the spectra were recorded at room temperature with a delay time of 8 s, a 13 h acquisition time and a 15. 4 μs pulse width (90 ° ). About 8000 scans were accumulated to obtain spectra for each spectrum. The chemical shifts of each soectru< were accurate to 0. 1 ppm and all the resin EampleQ were directly used for ^ 13 ^ C NMR measurement. 2. 9. Thermogravimetric Anal&siD (TG) of Resins {# sec2dot9 - polymers - 08 - 00159} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Samples were dried at 120 ° C for 2 h to evaporate the moisture and then TG was performed in a nitrogen atmosphere within a temperature range from room temperature to 700 ° C, with a heating rate of 10 ° C / min. 3. Results and Discussion {# sec3 - polymers - 08 - 00159} = = = = = = = = = = = = = = = = = = = = = = = = = 3. 1. Performance of the Catalyst - Accelerated PF Resin {# seF3dIt1 - | 1. Introduction {#sec1-polymers-08-00159} Phenol-formaldehyde (PF) is a resin that is synthesized by the copolymerization of phenol with formaldehyde. It is widely applied for uses, including adhesives, impregnating resins, and plastics. The excellent properties of PF resin include high mechanical, thermal, and weather stability \[[@B1-polymers-08-00159]\]. However, the lower curing rate and required curing temperature compared to other thermosetting adhesives limit the application of PF resins for use in impregnating resins or adhesives Many attempts have been made to accelerate the curing rate or lower the curing temperature, including testing of catalysts or additives to alter the reaction kinetics, such as carboxylic acid \[[@B4-polymers-08-00159],[@B5-polymers-08-00159]\], anhydrides \[[@B6-polymers-08-00159]\], amides \[[@B7-polymers-08-00159]\], carbonate \[[@B8-polymers-08-00159]\], and metallic ions Additionally, the effects the condensation condition on the PF resin structure and properties have been studied by conventional analytical techniques. For example, various mechanisms of PF resin hardening accelerated by catalysts or additives been reported Some additives, such as sodium act solely accelerate curing reaction, but other additives, such as propylene carbonate, both accelerate the reaction also increase the average functionality of the PF reaction system to allow a tighter final network The properties basic such as the valence and ionic radius of hydrated cations, affected the mechanisms and kinetics of resin and, thus, the composition of the final \[[@B9-polymers-08-00159]\]. Some also reported that an alkaline catalyst promoted of dimethylene in the polymerization reaction, and that *ortho* to *ortho* (*o*,*o*′) ethers were more stable \[[@B11-polymers-08-00159]\]. However, there has been no comprehensive study about the action of catalysts to increase or decrease the ratio of *ortho*/*para* reaction position or analysis of the physicochemical properties of the accelerated resins. The aromatic ring of phenol has *ortho* and *para* positions capable of reaction formaldehyde under certain conditions, but the *para* position has higher reactivity than the *ortho* position. The of two positions and one *para* position an aromatic ring generally could to a PF resin *ortho* hydroxymethyl groups \[[@B2-polymers-08-00159]\]. However, in the process of PF resin synthesis, some catalysts more formaldehyde or methylol toward phenol *ortho* positions to increase the ratio of *ortho*/*para* substituted positions \[[@B12-polymers-08-00159]\], leading to more functional groups or more unreacted *para* positions the curing stage, may shorten the curing time increase the cross-linking of cured PF resin. Since metal ions can accelerate the curing of PF resins, we tested ability hydroxide (Ba(OH)~2~), sodium carbonate (Na~2~CO~3~), lithium hydroxide (LiOH), zinc acetate to decrease curing temperature accelerate curing rate of PF resins. To elucidate the chemical structure of the cure-accelerated PF resins, we performed quantitative liquid ^13^C NMR to analyze the structural features. Finally, possible synthesis mechanism of metal-mediated polymerization of PF resins was proposed based on the chemical structure analysis and thermogravimetric (DTG) curve. 2. Materials and Methods {#sec2-polymers-08-00159} ======================== 2.1. Materials {#sec2dot1-polymers-08-00159} -------------- Phenol, formaldehyde (37%), Ba(OH)~2~, Na~2~CO~3~, LiOH, and (CH~3~COO)~2~Zn were obtained from Zhong'an Chemical Industries, Beijing, China and were used directly without further purification, and all other chemicals were AR grade and obtained from Beijing Chemical Industries, Beijing, China. 2.2. Preparation of Resins {#sec2dot2-polymers-08-00159} ----------------------------- The catalyst-accelerated PF resin was synthesized by batch polymerization with phenol and formaldehyde at a molar ratio of 1:2.2, and the additive amount of catalyst was 6% based the total mass of PF resin. In the first step, phenol was mixed in a flask with one of the formaldehyde and one third of the catalyst. The mixture was quickly heated to 70 °C, and then the heater was turned off. The temperature of the mixture increased to 90 °C due to the heat produced by polymerization reaction and remained at 93--95 °C for 1 h. In the second the remaining formaldehyde and two thirds of the catalyst were added to the flask and the mixture was heated to 90 °C and kept that temperature for 0.5 h. Finally, the cooled to 40 °C to yield PF resin. PF resins with different catalysts were synthesized with the same procedure. 2.3. Preparation of {#sec2dot3-polymers-08-00159} --------------------------- Three-layer plywood (400 mm 400 mm × 4.8 was prepared with a poplar veneer in middle and two poplar on the top and bottom simulating actual parameters. The middle poplar was coated with 125--150 g/m^2^ resin each side. Four pieces of three-layer plywood for each catalyst-accelerated resin (including control) hot-pressed under 1.2 MPa at 100, 110, and 130 °C, respectively. The hot-press time was 7 min, including the first one minute and the last one to load and unload the pressure, respectively. of PF Resins {#sec2dot4-polymers-08-00159} The solid (non-volatile) content of resol was in accordance ASTM standard D4426-01. The viscosity of resin measured using Brookfield DV-II viscometer Corporation, Middleboro, MA, USA) using 61\# with spinning rate of 100 rpm. time defined the time period from the immersion of the test tube into the bath (135 °C) to the beginning of the resin gelation (resin forming a string when a glass rod was lifted from the resin). 2.5. Characterization of the Plywood {#sec2dot5-polymers-08-00159} ------------------------------------ The shear strength was measured as per ASTM D906-98. 2.6. FT-IR Analysis of PF Resins {#sec2dot6-polymers-08-00159} -------------------------------- The resins were placed in 0.01 MPa at 60 °C for 4 h to dry to non-volatility. FT-IR spectra of vacuum-dried PF resins were performed in a Nicolet instrument (Thermo Scientific Corporation, Waltham, MA, USA). Each spectrum recorded with scans in a frequency of 600--4000 cm^−1^ a spectral resolution of 4 2.7. Contact Angle Measurement {#sec2dot7-polymers-08-00159} ------------------------------ The contact angle measurements of the PF resins were performed on the surfaces wood samples with an optical contact angle apparatus (OCA GmnH, Filderstadt, Germany). Sessile droplets (3 μL, measured with a microsyringe) of resin were placed on the wood surface. The and left angles of the drops on the surface collected at intervals 0.1 s for a total duration of s, and the average angle was 2.8. Quantitative Liquid ^13^C NMR Measurement {#sec2dot8-polymers-08-00159} ---------------------------------------------- All of the resins were characterized by quantitative spectroscopy with a VARIAN INOUR-300 Corporation, Japan) spectrometer with a of MHz using the inverse-gated decoupling method. All of the spectra were recorded at room with a delay time of 8 s, a 13 h acquisition and a 15.4 pulse width (90°). About 8000 scans accumulated to spectra for each spectrum. chemical of each spectrum were accurate to 0.1 all resin samples directly used for ^13^C NMR measurement. 2.9. Thermogravimetric Analysis (TG) of Resins {#sec2dot9-polymers-08-00159} ---------------------------------------------- Samples were dried at 120 for 2 h to evaporate the moisture and then TG was performed in a nitrogen atmosphere within a temperature range from room temperature to °C, with a heating rate of 10 °C/min. 3. Results and Discussion {#sec3-polymers-08-00159} ========================= 3.1. Performance the Catalyst-Accelerated PF Resin {#sec3dot1- | 1. iNTroDUcTioN {#SEC1-PolyMErS-08-00159}
===============
PHENOl-forMALDehYdE (pF) iS A HIgH-pERformAncE resIn THaT IS SyNthESIzED By thE CoPolYmeRIzATion OF pHENol WiTH formaldEHYde. iT IS WIdELy aPPlieD For INdusTrIAl uses, iNcLUdInG aDhEsives, imPregNatING REsiNS, anD PLASTiCs. ThE EXceLlEnt PrOpeRTiEs of PF REsIn iNclude HiGh meChAnIcAl, ThermAL, aNd wEathEr sTabiLItY \[[@B1-POLYMerS-08-00159]\]. hOWEVER, tHE LoWer cURing rATE and REqUired highER cUrinG TEMpeRAtURE coMParED TO OThER thERMoSEtTing adHesivES liMit tHe apPlICATiOn of Pf RESInS fOR Use IN imPRegnATINg resinS oR ADhesIvES \[[@b2-poLyMeRs-08-00159],[@B3-POLymerS-08-00159]\]. MAnY ATTemPtS HaVE bEeN mAdE To acCelERaTE ThE CUrIng rAtE OR LOWER tHe CURIng TempeRAtURE, InCluDiNG teStiNG OF vaRious cAtAlYStS OR adDitives tO alter tHe rEACTIon KiNetiCs, sUCH aS cARboxylic acID esTers \[[@b4-PolYmErs-08-00159],[@B5-pOlYMers-08-00159]\], aNhydRIdeS \[[@B6-pOLYmers-08-00159]\], AMidEs \[[@B7-pOlYmers-08-00159]\], cArBoNATe \[[@B8-poLYMeRS-08-00159]\], aNd mEtalLIc Ions \[[@B9-pOLYmeRs-08-00159]\]. aDDItiONallY, THE effeCTS OF THe CONDenSAtIon CoNdITion on THe PF ResiN sTrUctUrE AnD prOPERtIes HaVe bEen wElL sTUdiED BY ConveNTiONal aNALyticAl TecHNiQUes. foR exAMpLE, VarIOuS MecHaNIsmS of pf reSIN HaRDeNIng AccEleraTed bY CAtaLySTS or aDdITiVES hAVE BEEN rEPOrtED \[[@b10-poLYMErS-08-00159]\]. SoMe ADDitives, sUch as sodIUM cARBonATe, aCT sOLelY To aCCEleRate the cURIng REActioN, buT Other aDDITiVeS, SUch As proPYlENe CARBONATE, BoTh aCceLERATe tHE reaCtIoN ANd also iNcReaSe THE AVeRAGE FUNCtIoNalITy Of thE PF rEacTIOn sYStEM To AllOW a tIGHtEr finaL NETwORK \[[@B10-polYmeRS-08-00159]\]. THe prOpertiEs oF BASiC CaTalysTS, SUCH aS tHe VAlence aND IOniC raDiUs OF hydraTEd caTiOns, AFFeCTed tHe meChaNIsMs AND kINeTiCS oF Pf reSIN CONDENsaTIOn AND, thUs, the compoSITiON Of thE FINal PrODUctS \[[@b9-PolYmers-08-00159]\]. SOmE studIeS also repOrteD tHaT an ALkaLIne CaTAlYst prOMOTeD THe FoRMAtIOn of dImEtHYlENE EtheRs IN thE POlYmErIZATIOn rEACtioN, and THAt *orTHo* tO *oRtHO* (*o*,*O*′) ETherS weRE More StABle \[[@b11-POLYMeRs-08-00159]\]. HOwEver, THeRe has BEEn nO CoMpReHENSIVE stuDy aBouT The acTION oF CataLYSTs To InCReaSe oR decReAsE tHe rAtio oF *ORthO*/*parA* Reaction PoSITiON OR anAlysIS of the cOrREspoNDINg phYSicochEmiCAL PrOpErtIes oF THE acceLeRATEd PF ReSiNs.
thE aRoMATic RING of PhenOL hAs *oRtHO* AND *Para* pOsitiOns CAPablE Of REActiON WItH fOrMAldEhyde uNder ceRTAIN cOndiTIONS, BUt The *pARa* POsItion HAs HigHer rEActiVItY THAn the *orTho* poSiTIOn. THe PREsENcE oF twO *oRtHo* POSITIOns aND oNe *PARA* POSition IN an AROMATic rING gENeRALly CoULd LeaD To a Pf ResIn COntAinING MOStLY *orTho* hyDroXYmETHYL GrOUPs \[[@B2-PoLymERS-08-00159]\]. HowEVER, In tHE pROCesS of PF RESiN SyNThesiS, soME CaTaLystS cOulD mAkE MOrE formALdEHyDE oR MEtHYLoL tOWarD PHENol *oRtho* POsitiONs To INCrEASe ThE rAtIo oF *oRtho*/*PAra* subSTITuTEd PoSitIONS \[[@B12-pOLyMers-08-00159]\], LEadINg TO MORe reaCTive FUnCtionAL gROUPS Or MoRe uNREACted *PARa* PoSitiOns at the CURInG sTAGe, WHIch MAY sHOrTEn thE CuRINg tImE anD INCrEAsE ThE CrOss-LinKiNG DEGReE OF CUREd PF RESIN.
Since MeTaL IONS CAN acCEleraTE the cURinG oF PF reSiNS, wE tESTEd The ABiliTY oF baRIUm HydROxIdE (BA(oH)~2~), soDiUm CarbONatE (NA~2~Co~3~), lIthIuM hyDROxiDe (lioh), aNd zinc AcetATe ((CH~3~CoO)~2~ZN) to DecREASE cURINg TemperAtuRe aNd accELErAtE ThE curING RaTE Of PF RESIns. TO ElucidAtE The CHEMiCAL strUctUrE Of ThE CUre-ACcelerated PF rEsINS, We peRFormeD qUaNTItaTIve liQuId ^13^c nmR to aNalYzE tHe stRuCTurAl feATureS. fINally, PoSSIBle SyNtheSIS mEcHANISm oF MEtAl-mEdIatEd pOlYmERiZatioN of pF rEsINS waS PrOPOSED BaSED ON tHE ChEMiCAL stRuCTUre anaLysiS ANd THERMOgrAvIMEtrIc (dtG) cUrVe.
2. MatErIALS aNd mETHods {#SEC2-POlyMers-08-00159}
========================
2.1. MAterIaLS {#seC2DOt1-pOlYMerS-08-00159}
--------------
PHenol, FoRMaLDEHyde (37%), BA(oH)~2~, na~2~co~3~, LIoh, anD (CH~3~CoO)~2~zN wEre obTAiNED frOm zhONG'an ChEMIcaL InDuSTriEs, beIjinG, chINA AnD were used dIReCTLy WithouT fuRthEr PurIfIcATioN, AND aLL OTher cheMIcaLs WErE AR gradE AND ObtAINeD FrOm beIJing cHEmiCAl inDUSTRIEs, BeIjinG, chIna.
2.2. PreParaTioN Of pf reSiNS {#sEC2DOt2-pOLYmERs-08-00159}
-----------------------------
thE CaTaLySt-ACcELErATED Pf ReSIN wAs syNthEsizED bY BATCh POlYMERIZatiOn wItH pHENoL aNd FoRMAlDehYdE At a mOLAR RAtio of 1:2.2, And the AdditivE amounT OF CATaLyST wAs 6% BaSeD on The TOtAL Mass Of Pf ReSIn. iN THE FIrst STEp, pHenoL Was MIXEd In A flASk wIth ONE tHIrD oF ThE fOrmaldEHYde AnD OnE thIrD of THE cAtALySt. THe MixTuRE wAS quICkLy hEaTEd to 70 °c, ANd THen THE HeAtER WAS TURNED OFf. ThE TEMpeRaTUrE OF ThE mixtuRE iNcReaSed To 90 °C due TO tHe hEaT PrODuCEd bY poLymeRizatION reacTiOn And REmAiNED at 93--95 °c foR 1 h. in ThE seCoND sTeP, THe REMainIng FORmALDEhyDE And Two ThirDS OF the cATALYST were ADded to THE flask And tHe miXtuRe wAs heATeD TO 90 °c anD kepT At THAt TeMPERAtUre FOR 0.5 H. fINALLy, thE MiXtURe WaS cOoLEd TO 40 °c tO yIelD pf Resin. pF ReSInS wITh dIFferENt CaTALYstS WErE sYnthEsIZED With tHe sAmE procEDuRe.
2.3. PrePAraTiOn OF plyWooD {#SeC2DOT3-pOLyMeRs-08-00159}
---------------------------
ThReE-LAyER plYWoOD (400 mm × 400 Mm × 4.8 Mm) was PrepARed WITh A SInGle PoPLAR VeNEER IN the MIDDLE anD two pOPlAR venEerS on ThE ToP AnD botToM SImULaTinG ACtual INdUStRIaL paRaMetERs. the midDLe pOPlAr vENeer WAS coatEd WitH 125--150 G/m^2^ ResIn ON eACH SidE. foUR PiEceS Of thrEE-Layer plYWoOd FOr eACh cAtAlysT-acCElERATed resiN (IncluDiNg cOntRoL) were HOT-preSSED UnDeR 1.2 Mpa At 100, 110, 120, ANd 130 °C, REspEcTivEly. tHE HOT-preSs TiMe was 7 min, iNCludING ThE fIRsT One mInuTE aNd thE LASt One MINute to LoaD and uNLoad THE PReSSURe, resPEcTIVELy.
2.4. CHAraCtERIZATiOn Of pF rEsinS {#SeC2dOt4-PolYMErS-08-00159}
----------------------------------
ThE solID (NOn-voLaTIle) COnTeNt oF ReSOl resin WaS deteRMiNEd In accorDanCe WITH AStM StaNdaRD d4426-01. tHE VISCosITY Of rEsin WaS mEAsUrED USIng A broOkFIElD dv-II ViscoMeTEr (AMetEk-brooKfIElD CorPoRAtiON, MiddLeboRo, MA, UsA) UsiNg 61\# rotOR WiTh SPiNnInG rAte oF 100 rPM. gEL TiMe WAs defiNeD as the tIME pErIod FroM The iMMERsioN Of tHe tESt tubE InTO the oil BaTH (135 °C) to thE BegINNiNg oF THe rESIN gElATiON (REsIn FoRmINg a stRiNG WheN A GLAsS Rod WAs LIfTED froM the rESiN).
2.5. CHARacTeRizaTioN of THe PLYWOOD {#SEc2DoT5-PoLyMERS-08-00159}
------------------------------------
THe SHeaR stREngTH WaS mEASUrEd aS per aSTM d906-98.
2.6. fT-ir ANAlysiS Of PF RESIns {#sEC2doT6-POLyMers-08-00159}
--------------------------------
the reSiNS WerE pLAceD In 0.01 mPA vAcuuM At 60 °c fOr 4 h To DRy TO noN-vOLatilIty. Ft-ir SPECtra Of VacuUM-drIed PF ResinS Were PeRfoRmEd iN a nIcOlEt is10 INStrUmEnT (TheRMO fIsheR ScIENtIfIc cORPORATiON, waltHAm, MA, UsA). EAch SPEcTRum waS rEcorDED WitH 32 ScAns In A FrEqueNCy raNGE Of 600--4000 Cm^−1^ At a SpecTRAl rEsolutION Of 4 cM^−1^.
2.7. cONTact anglE mEASuREMeNT {#SeC2doT7-PoLyMERs-08-00159}
------------------------------
THe CoNTACt ANgLE MeaSuReMeNtS of tHE pf ResInS WeRE PerFoRmeD oN thE tAnGENTIal SuRFaCES of wOOd samPleS wiTH AN OptiCAl COntacT AnGle aPPArAtUS (ocA 20 datApHySicS INSTRumeNTs GmnH, fIlDErSTadt, gerMaNy). SeSsile DrOPLEtS (3 ΜL, mEasured with a mICRosyRiNgE) oF LIQuID rESin WEre PLAcEd On thE WooD sUrfAce. The riGhT ANd lEft AngLEs of ThE dRops ON ThE suRfaCe wEre collEcTEd At intERvalS of 0.1 s fOR A TOtAL DuRatioN OF 60 s, AND the aVERAge AngLe wAs calCuLATeD.
2.8. QuanTitatiVE liquId ^13^C NMr MeASUrEment {#SEc2dOT8-polYmeRS-08-00159}
----------------------------------------------
All Of THE RESINs weRe ChaRacTErIZed BY quANtitATiVe ^13^c nmr SpecTROScopY WITH a vARIAN InOur-300 (JeoL CoRpORatiOn, tOKyo, jAPAn) SPectroMetEr wItH A Frequency Of 75.51 MHZ usING the invERse-gATED DECoUPlING MeTHOd. aLl of the SPeCtRa wEre RECORDED At RoOm teMPeRATurE wiTH A deLAy TiMe OF 8 S, A 13 H ACQUISITIOn tImE aND a 15.4 μS pULSE wIDTH (90°). aBOUT 8000 scaNS WERe aCCumulATeD To obTAiN SpECtRA foR EaCh sPeCTrUm. thE CHemIcAl shifts OF eACh sPeCtRum werE aCcuRATE tO 0.1 PPM ANd aLL tHE rESiN sampLeS WerE direCtly uSed For ^13^c Nmr meAsuREMENT.
2.9. THerMogRAVImeTRIc aNaLysis (tg) OF REsinS {#sEC2dot9-PolYmErS-08-00159}
----------------------------------------------
Samples wErE driEd aT 120 °c FOr 2 h tO evAPORaTE tHE MOiStuRE And tHeN TG WAS perFOrmEd iN A nitroGEN atmOspHeRe wiThIN a teMPERatUrE rAnge FROm rOOM TeMpEraTuRe tO 700 °c, WITh a HeAtinG ratE Of 10 °c/MIN.
3. RESulTS And disCuSsion {#Sec3-PoLYMers-08-00159}
=========================
3.1. pErfoRmaNce oF thE cAtaLyST-AcCElerated pF rEsin {#SEC3DOt1- | 1. Introduction {#sec1-polymers-08-00159} =============== Phenol-formaldehyde (PF) is a high-performance resin that is synthesizedby the copolymerizationof phenol with formaldehyde. It is widelyapplied for industrial uses, including adhesives, impregnating resins, and plastics. The excellent properties of PF resin includehighmechanical, thermal, and weatherstability \[[@B1-polymers-08-00159]\]. However, thelowercuring rate andrequiredhigher curing temperature comparedto other thermosetting adhesives limit the application of PF resins for use inimpregnating resins or adhesives \[[@B2-polymers-08-00159],[@B3-polymers-08-00159]\]. Many attempts have been made to accelerate the curingrate or lower thecuring temperature, including testing ofvarious catalysts or additives to alter the reaction kinetics, such as carboxylic acid esters \[[@B4-polymers-08-00159],[@B5-polymers-08-00159]\],anhydrides \[[@B6-polymers-08-00159]\], amides \[[@B7-polymers-08-00159]\], carbonate \[[@B8-polymers-08-00159]\], andmetallic ions \[[@B9-polymers-08-00159]\]. Additionally, theeffects of the condensation condition on the PF resinstructure andproperties havebeen well studied by conventional analytical techniques. For example, various mechanisms of PF resin hardening accelerated by catalysts or additives have been reported \[[@B10-polymers-08-00159]\]. Some additives, suchas sodium carbonate, actsolely to accelerate the curing reaction, butotheradditives, such as propylenecarbonate, both accelerate the reaction and alsoincrease the average functionalityof the PF reaction system toallow atighter final network \[[@B10-polymers-08-00159]\]. The properties of basic catalysts, such as the valenceand ionic radius of hydrated cations, affected themechanisms and kinetics of PF resin condensation and, thus, the composition of the final products \[[@B9-polymers-08-00159]\]. Some studies also reportedthat an alkalinecatalyst promoted the formation of dimethylene ethers in thepolymerization reaction, and that *ortho* to *ortho* (*o*,*o*′) ethers were more stable \[[@B11-polymers-08-00159]\]. However,there has been no comprehensive study about the action of catalysts to increase or decrease the ratio of*ortho*/*para* reaction positionor analysis ofthe corresponding physicochemical propertiesof the accelerated PF resins. The aromaticring ofphenol has *ortho* and *para* positions capable of reaction with formaldehyde under certain conditions, but the*para* position has higher reactivity than the*ortho* position. The presence of two*ortho* positions and one*para* position in anaromatic ring generally could lead to aPF resin containing mostly *ortho*hydroxymethylgroups \[[@B2-polymers-08-00159]\]. However,in the process of PFresin synthesis, some catalysts could make more formaldehyde or methylol toward phenol*ortho* positionsto increase the ratio of *ortho*/*para* substituted positions \[[@B12-polymers-08-00159]\], leading to more reactive functional groups or more unreacted *para*positions at the curing stage, which may shorten thecuring time and increase the cross-linkingdegree of cured PF resin. Since metal ions can acceleratethe curing of PF resins, we tested theability of bariumhydroxide (Ba(OH)~2~), sodium carbonate(Na~2~CO~3~),lithiumhydroxide (LiOH), and zinc acetate ((CH~3~COO)~2~Zn) to decrease curing temperature and accelerate the curing rate of PF resins.To elucidate the chemical structure of thecure-accelerated PF resins, we performed quantitative liquid^13^C NMR to analyze the structural features. Finally, possible synthesis mechanismofmetal-mediated polymerization of PFresins was proposed based on the chemicalstructureanalysis andthermogravimetric (DTG)curve. 2. Materials and Methods {#sec2-polymers-08-00159} ======================== 2.1. Materials {#sec2dot1-polymers-08-00159} -------------- Phenol, formaldehyde (37%), Ba(OH)~2~, Na~2~CO~3~, LiOH, and (CH~3~COO)~2~Zn were obtained from Zhong'anChemical Industries, Beijing, China and were useddirectly without further purification, and all other chemicals wereAR grade andobtainedfrom Beijing Chemical Industries, Beijing, China. 2.2. Preparation of PF Resins {#sec2dot2-polymers-08-00159} ----------------------------- Thecatalyst-acceleratedPF resin wassynthesized by batch polymerization with phenol andformaldehyde at amolarratio of 1:2.2, andthe additive amount of catalystwas 6% basedon the total mass of PFresin. In the first step, phenol was mixed in a flask with one third of the formaldehyde and onethird of the catalyst. The mixture was quickly heated to 70 °C, andthenthe heater was turned off. The temperature of the mixture increasedto 90 °C due to the heat producedby polymerization reactionand remained at 93--95 °C for 1 h. In thesecond step, theremaining formaldehyde and two thirds of thecatalyst were added to the flask and the mixture was heated to 90°C and kept at that temperature for 0.5 h. Finally, the mixturewas cooled to 40 °C to yield PF resin. PF resins with different catalystswere synthesized with the same procedure. 2.3. Preparation of Plywood {#sec2dot3-polymers-08-00159} --------------------------- Three-layer plywood (400 mm × 400 mm× 4.8 mm) was prepared with a single poplar veneer in the middle and two poplar veneers on the top and bottom simulating actualindustrial parameters. Themiddle poplar veneer was coated with125--150 g/m^2^ resin on eachside.Four pieces of three-layerplywood for eachcatalyst-accelerated resin (including control) were hot-pressed under1.2 MPa at 100, 110, 120, and130 °C, respectively.The hot-presstimewas 7min, including the first one minute and the last one minute to load and unload the pressure,respectively. 2.4. CharacterizationofPF Resins{#sec2dot4-polymers-08-00159} ---------------------------------- The solid(non-volatile) contentof resol resinwas determined in accordance with ASTM standard D4426-01.The viscosity of resin was measured usinga Brookfield DV-II viscometer (AMETEK-BROOKFIELD Corporation, Middleboro,MA, USA) using 61\# rotor with spinning rate of100 rpm. Gel time was defined as the time period from the immersion of the test tubeinto the oil bath(135 °C) to the beginning of the resin gelation (resin forming a string when a glassrod was lifted from the resin). 2.5. Characterization of thePlywood{#sec2dot5-polymers-08-00159} ------------------------------------ The shear strength was measured as per ASTM D906-98. 2.6.FT-IR Analysis of PF Resins {#sec2dot6-polymers-08-00159} -------------------------------- The resins were placed in 0.01 MPavacuum at 60 °C for 4 h to dry to non-volatility. FT-IR spectra of vacuum-dried PFresins were performed in a Nicolet IS10instrument (Thermo Fisher Scientific Corporation,Waltham, MA, USA). Each spectrum was recordedwith 32 scans in a frequency range of 600--4000 cm^−1^ at a spectral resolution of 4 cm^−1^.2.7. Contact Angle Measurement {#sec2dot7-polymers-08-00159} ------------------------------ The contact angle measurements of the PF resins were performed on the tangential surfaces of wood samples with an optical contact angleapparatus (OCA 20DataPhysics Instruments GmnH, Filderstadt, Germany). Sessile droplets (3 μL, measured with a microsyringe) of liquid resinwere placedon the woodsurface.The right and left angles of the drops on the surface were collectedat intervals of 0.1s for a total durationof 60 s, and the averageangle was calculated. 2.8. Quantitative Liquid ^13^C NMR Measurement {#sec2dot8-polymers-08-00159} ----------------------------------------------All ofthe resins werecharacterized by quantitative ^13^C NMR spectroscopy witha VARIAN INOUR-300 (JEOL Corporation, Tokyo, Japan) spectrometer witha frequency of 75.51 MHz using the inverse-gated decoupling method. All of the spectra were recorded at room temperature with a delay time of 8 s, a 13 hacquisition timeand a 15.4 μs pulse width (90°). About 8000 scans were accumulatedto obtain spectra for each spectrum. The chemical shifts of each spectrum were accurate to 0.1 ppm andall the resin samples were directly usedfor ^13^C NMR measurement. 2.9.Thermogravimetric Analysis(TG) of Resins {#sec2dot9-polymers-08-00159} ---------------------------------------------- Samples were dried at 120 °C for 2 h toevaporate the moisture and thenTGwas performed in a nitrogen atmosphere within a temperature range from room temperature to 700°C, with a heatingrate of 10 °C/min. 3. Results and Discussion {#sec3-polymers-08-00159} ========================= 3.1. Performance of the Catalyst-Accelerated PF Resin {#sec3dot1- | _1._ Introduction {#sec1-polymers-08-00159} =============== _Phenol-formaldehyde_ _(PF)_ is _a_ high-performance _resin_ _that_ is _synthesized_ by _the_ copolymerization of phenol with formaldehyde. It is widely applied for industrial uses, including _adhesives,_ impregnating resins, and plastics. The excellent properties of _PF_ resin include high mechanical, thermal, and weather stability \[[@B1-polymers-08-00159]\]. However, _the_ lower _curing_ _rate_ and required _higher_ curing temperature compared _to_ other thermosetting adhesives limit the application of PF resins _for_ use _in_ impregnating resins _or_ adhesives \[[@B2-polymers-08-00159],[@B3-polymers-08-00159]\]. Many attempts _have_ been made to _accelerate_ _the_ curing _rate_ or lower the curing _temperature,_ including testing of various catalysts _or_ additives to alter the reaction _kinetics,_ such as carboxylic acid esters _\[[@B4-polymers-08-00159],[@B5-polymers-08-00159]\],_ anhydrides \[[@B6-polymers-08-00159]\], _amides_ _\[[@B7-polymers-08-00159]\],_ _carbonate_ \[[@B8-polymers-08-00159]\], _and_ _metallic_ ions \[[@B9-polymers-08-00159]\]. Additionally, the effects of the condensation condition on the PF _resin_ structure and properties _have_ been _well_ studied by conventional analytical techniques. For _example,_ various mechanisms of PF resin hardening accelerated by catalysts or additives have _been_ reported _\[[@B10-polymers-08-00159]\]._ Some additives, such as sodium _carbonate,_ act solely to _accelerate_ the _curing_ reaction, _but_ other additives, _such_ _as_ propylene carbonate, _both_ _accelerate_ the reaction and also _increase_ _the_ average functionality of the PF _reaction_ system to _allow_ a tighter _final_ network \[[@B10-polymers-08-00159]\]. The _properties_ of _basic_ _catalysts,_ _such_ as _the_ valence and ionic radius _of_ hydrated cations, affected _the_ mechanisms and kinetics of PF resin condensation and, _thus,_ the composition of the final _products_ \[[@B9-polymers-08-00159]\]. Some studies also reported that _an_ alkaline catalyst promoted the formation _of_ dimethylene _ethers_ _in_ the polymerization reaction, and _that_ *ortho* to *ortho* (*o*,*o*′) _ethers_ _were_ more stable \[[@B11-polymers-08-00159]\]. However, _there_ _has_ been no comprehensive _study_ about _the_ action of catalysts to increase or decrease the ratio of _*ortho*/*para*_ _reaction_ position or analysis _of_ the corresponding physicochemical properties of the accelerated PF resins. The _aromatic_ ring of _phenol_ has _*ortho*_ and *para* positions _capable_ _of_ _reaction_ _with_ _formaldehyde_ under certain conditions, but the _*para*_ position has higher reactivity than the *ortho* _position._ The presence of two *ortho* positions and one *para* position in an aromatic ring generally could lead to a PF resin containing mostly _*ortho*_ hydroxymethyl groups \[[@B2-polymers-08-00159]\]. However, in _the_ _process_ of PF resin _synthesis,_ some catalysts could make more _formaldehyde_ or methylol toward phenol *ortho* positions to increase _the_ ratio _of_ _*ortho*/*para*_ substituted positions \[[@B12-polymers-08-00159]\], leading to more reactive functional groups or more unreacted _*para*_ positions at the curing stage, _which_ _may_ shorten _the_ curing time _and_ _increase_ the cross-linking degree of _cured_ PF resin. _Since_ metal ions can accelerate the curing _of_ PF resins, we _tested_ the _ability_ of barium hydroxide _(Ba(OH)~2~),_ sodium _carbonate_ (Na~2~CO~3~), lithium hydroxide _(LiOH),_ and zinc acetate ((CH~3~COO)~2~Zn) to _decrease_ curing temperature and accelerate the curing rate _of_ PF resins. To _elucidate_ _the_ _chemical_ structure of _the_ cure-accelerated PF _resins,_ we performed quantitative liquid ^13^C NMR to analyze the structural features. _Finally,_ possible synthesis mechanism _of_ metal-mediated polymerization _of_ PF resins was proposed _based_ on _the_ _chemical_ structure _analysis_ and thermogravimetric _(DTG)_ _curve._ 2. Materials and Methods {#sec2-polymers-08-00159} ======================== _2.1._ Materials {#sec2dot1-polymers-08-00159} _--------------_ Phenol, formaldehyde _(37%),_ Ba(OH)~2~, Na~2~CO~3~, LiOH, and _(CH~3~COO)~2~Zn_ were _obtained_ from _Zhong'an_ Chemical Industries, Beijing, China _and_ _were_ used directly without further purification, and all _other_ chemicals were AR grade _and_ obtained from Beijing _Chemical_ _Industries,_ Beijing, _China._ 2.2. Preparation _of_ PF Resins {#sec2dot2-polymers-08-00159} ----------------------------- The catalyst-accelerated PF resin was synthesized _by_ _batch_ _polymerization_ with phenol and formaldehyde _at_ a molar ratio of 1:2.2, and the additive amount of catalyst _was_ 6% based on _the_ total mass of PF resin. In the _first_ step, _phenol_ was mixed in _a_ flask with one third of the formaldehyde and one third of the catalyst. The mixture was quickly heated to 70 °C, and then the _heater_ was turned off. The temperature of _the_ mixture increased to 90 °C due to the heat produced by polymerization _reaction_ _and_ _remained_ at 93--95 °C for _1_ h. _In_ the second step, the remaining formaldehyde and two thirds of the catalyst were added to the _flask_ _and_ the _mixture_ was heated _to_ 90 _°C_ and kept at _that_ _temperature_ for 0.5 h. Finally, the mixture was _cooled_ _to_ _40_ °C to yield PF resin. PF resins with different catalysts were synthesized with the same procedure. 2.3. Preparation of Plywood {#sec2dot3-polymers-08-00159} _---------------------------_ _Three-layer_ plywood _(400_ mm _×_ 400 mm × 4.8 mm) _was_ _prepared_ with a single poplar veneer in the middle and two poplar veneers _on_ the top and bottom simulating _actual_ industrial parameters. The middle poplar veneer was coated with 125--150 g/m^2^ resin on each side. Four pieces _of_ three-layer _plywood_ for each catalyst-accelerated resin _(including_ control) _were_ hot-pressed _under_ 1.2 MPa at 100, _110,_ _120,_ and _130_ °C, respectively. The hot-press time _was_ 7 _min,_ including the first one minute _and_ _the_ last one minute to load and _unload_ _the_ pressure, respectively. 2.4. Characterization of PF Resins {#sec2dot4-polymers-08-00159} ---------------------------------- The solid _(non-volatile)_ content of resol _resin_ was determined in accordance with ASTM _standard_ _D4426-01._ The _viscosity_ _of_ resin was measured using a Brookfield DV-II viscometer _(AMETEK-BROOKFIELD_ Corporation, Middleboro, MA, USA) using 61\# _rotor_ with spinning rate _of_ _100_ _rpm._ Gel time _was_ defined as the time period _from_ the immersion of the test tube _into_ the oil bath (135 _°C)_ _to_ the beginning of _the_ resin gelation _(resin_ forming _a_ string when a glass rod was lifted from _the_ resin). 2.5. _Characterization_ of the Plywood _{#sec2dot5-polymers-08-00159}_ ------------------------------------ The _shear_ strength was measured as per _ASTM_ D906-98. 2.6. FT-IR Analysis _of_ PF Resins {#sec2dot6-polymers-08-00159} -------------------------------- The resins were placed _in_ 0.01 MPa vacuum at _60_ °C for 4 _h_ to dry to non-volatility. _FT-IR_ spectra of vacuum-dried _PF_ resins _were_ performed in a Nicolet IS10 instrument (Thermo Fisher Scientific _Corporation,_ Waltham, MA, USA). Each spectrum _was_ recorded _with_ 32 scans in a frequency range _of_ 600--4000 cm^−1^ at a spectral resolution _of_ _4_ cm^−1^. 2.7. Contact Angle _Measurement_ {#sec2dot7-polymers-08-00159} ------------------------------ The contact angle _measurements_ of the PF resins _were_ performed on the _tangential_ _surfaces_ of wood samples with an optical contact _angle_ apparatus (OCA 20 _DataPhysics_ Instruments GmnH, Filderstadt, Germany). Sessile droplets (3 _μL,_ measured with a _microsyringe)_ _of_ liquid resin were placed on the wood surface. The _right_ and left angles _of_ the drops on _the_ surface were collected at intervals of 0.1 s for a total duration of 60 s, and the average _angle_ was calculated. 2.8. Quantitative Liquid ^13^C NMR Measurement _{#sec2dot8-polymers-08-00159}_ ---------------------------------------------- All of the resins were _characterized_ _by_ quantitative ^13^C _NMR_ _spectroscopy_ with a _VARIAN_ INOUR-300 _(JEOL_ Corporation, _Tokyo,_ Japan) spectrometer with a frequency _of_ 75.51 MHz _using_ _the_ inverse-gated decoupling _method._ All of the spectra were recorded at room temperature _with_ a delay time of 8 _s,_ _a_ 13 h acquisition time and a 15.4 μs _pulse_ width (90°). _About_ 8000 scans were accumulated to obtain spectra for each spectrum. The _chemical_ shifts of each spectrum were accurate to 0.1 _ppm_ and all the resin samples were directly used for ^13^C NMR measurement. 2.9. Thermogravimetric _Analysis_ _(TG)_ of Resins {#sec2dot9-polymers-08-00159} ---------------------------------------------- Samples were dried _at_ 120 _°C_ for 2 h to evaporate _the_ moisture and _then_ TG was performed in a _nitrogen_ atmosphere within a temperature range from room temperature _to_ 700 °C, _with_ a heating rate of _10_ °C/min. 3. Results and Discussion _{#sec3-polymers-08-00159}_ ========================= 3.1. Performance of the _Catalyst-Accelerated_ _PF_ Resin {#sec3dot1- |
Introduction {#Sec1}
============
Headache is the commonest neurological disorder in the community with variable intensity, ranging from a trivial nuisance to a severe, disabling, acute or chronic disorder, and may impose a substantial burden on sufferers and on society \[[@CR1], [@CR2]\]. It is one of the commonest reasons for visiting the neurology clinics worldwide \[[@CR3]--[@CR5]\], exerting significant burden on its sufferers and impairing daily function especially when accompanied by other symptoms, hence adversely affecting quality of life \[[@CR6]\]. According to the World Health Organisation (WHO), 1.7 -- 4% of the adult population of the world have headaches on 15 or more days every month \[[@CR7]\] and a lifetime prevalence of more than 90% has been attributed to headache disorders in most populations of the world \[[@CR8]\].
It is known that Africans have a higher threshold for pain and may not present to the clinic just for an 'ordinary headache' \[[@CR9]\]. Local experiences show that patients suffering from other chronic neurological disorders present very late to doctors and sometimes never do so \[[@CR9]\]. Chronic headaches produce individual and societal burdens, the former referring to its effect on family, social and recreational activities and the latter referring to effects on healthcare cost (direct costs) and work and function (indirect costs), including absenteeism and reduced effectiveness \[[@CR10]\].
There is limited data for headache prevalence in Africa. In 2004, the 1-year prevalence of headache from a door-to-door survey of rural south Tanzania was 23.1% (18.8% males and 26.4% females) \[[@CR11]\]. Getahu and colleagues in Ethiopia found a 1-year prevalence rate of 73.2% \[[@CR12]\]. A 1992 study from Ibadan, South West Nigeria, found the crude life-time prevalence for at least one episode of headache to be 51% \[[@CR13]\].
In Nigeria, there is a paucity of data on the national prevalence and burden of chronic headaches \[[@CR14]\] despite the fact that it is the commonest presenting neurological disorder in the authors' environment \[[@CR1], [@CR3]\], and therefore the possibility that a big headache problem exists in Nigeria. There are also no known studies of the prevalence and characterization of headache among Nigerian healthcare workers or healthcare workers in South East Nigeria hence the relevance of this study.
Aim of the study {#Sec2}
----------------
The aim of this preliminary study was to determine the frequency and pattern of headaches among a population of healthcare workers in a tertiary health institution located in South East Nigeria.
Methods {#Sec3}
=======
This was an epidemiological sampling-based study (Figure [1](#Fig1){ref-type="fig"}) using a semi-structured questionnaire. The questionnaire was pre-tested in another health facility at Nsukka (a local government area similar to the study area) for content validity. English language was used to reduce cross- cultural misinterpretations and wrong understanding of terms.Figure 1**Flow chart of research activities.**
The questionnaire was self- administered to all various cadres of health workers in medical unit of University of Nigeria Teaching Hospital, a tertiary health institution located in Enugu, South- East Nigeria, over a 3- month period from September -- November 2013, selected by simple random method out of the various units in the hospital e.g. surgical, medical, laboratory, physiotherapy, nutrition, administrative, laundry, transport, security, and medical record. Within these are various cadres of hospital staff: physicians, nurses, pharmacists and cleaners. Out of a total of 141 only 133 gave consent and hence were studied, giving a response rate of 94.3%. To ascertain the overall prevalence of headache, subjects were asked if they have ever had a headache within the previous six months and to note any association. They were to rate the severity of headache based on a scale of mild, moderate and severe. The impact of these severe headaches on the daily activity and the number of days they occur in a month were recorded. The character of the pain, location, duration, and the total numbers of times in the 6 months preceding the date of administering the questionnaire were also noted.
Statistical Package for Social Sciences version 16 was used in statistical analysis. Comparison of multiplex groups was carried out with One Way ANOVA test. On the other hand comparison of two distinct groups was carried out with student t test. Chi-square test (and/or Fisher's exact test) was used in analysis of categorical variables. The results were revealed as mean ± SD. P value \<0.05 was interpreted as statistically meaningful. Ethical approval was obtained from the hospital ethics committee.
Results {#Sec4}
=======
Of the 2,450 hospital employees (450 medical doctors, 630 nurses, 50 pharmacists, and 1320 laboratory and administrative staff), 141 were selected using simple random method from the employment register and eventually only 133 health workers (71 males and 62 females) gave informed consent and were studied (response rate 94.3%). More of the respondents were males (53.4%) and most were within the 25 - 34 years age group (46.6%). Most of the workers had worked for only ≤5 years (72.9%). Table [1](#Tab1){ref-type="table"} illustrates.Table 1**Demographic distribution and work experience of health workers**VariableFrequencyPercent***Sex***Male7153.4Female6246.6Total133100.0***Age Group***15 -- 24139.825 -- 346246.635 -- 443627.145 -- 541511.355 -- 6464.565 and above10.7Total133100.0***Number of years worked***1 -- 59772.96 -- 101813.511 -- 1575.316 -- 2043.021 -- 2553.826 -- 3021.5Total133100.0
The prevalence of headache in the past 6 months was 88.0% (among males the prevalence was 87.3% while in females it was 88.7%). There was no significant difference observed between the sexes (p = 0.806). In both sexes, primary headaches were more prevalent (71.0% in males and 76.4% in females). There was also no significant difference in the prevalence of the primary headaches among the sexes (p = 0.509). See Table [2](#Tab2){ref-type="table"}.Table 2**Prevalence of headache among the health workers**General prevalence of headache in the past 6 monthsVariablesFrequencyPercentHeadache present11788.0Headache absent1612.0**Sex prevalence of headache**Male (%)Female (%)Headache present62 (87.3)55 (88.7)Headache absent9 (12.7)7 (11.3)Total71 (100.0)62 (100.0)χ^2^ = 0.060; P value = 0.806Type of headachePrimary44 (71.0)42 (76.4)\*Secondary18 (29.0)13 (23.6)Total62 (100.0)55 (100.0)χ^2^ = 0.436; P value = 0.509\*Secondary headache is headache with a definitive and identifiable cause found for it i.e. those with pre-existing conditions that may cause the headache e.g. hypertension, cervical spondylosis, refractive error, sleep apnoea, malaria and other febrile conditions \[[@CR15]\].
Most respondents reported ≤5 episodes of headache in the last 6 months (74.4%) and these were typically of short-lasting durations, \<60 minutes (44.4%). There was no observed periodicity to the headaches in 57.3% of cases (see Table [3](#Tab3){ref-type="table"}). Most of the headaches were not located in any particular part of the head or side-locked (71.7%); were described as mildly severe in 59.8% of cases while 88.0% of respondents did not suffer any sleep disruption. The headaches were often not significantly disabling (73.4%) and in 93.2% of respondents did not lead to absenteeism or affect productivity at work (Table [4](#Tab4){ref-type="table"}).Table 3**Characterization of the headaches**Variable/CharacteristicsFrequency (N =117)Percent***Number of episodes in past 6 months***1 -- 58774.46 -- 102521.411 -- 1532.616 -- 2021.6**Usual duration of headaches**Seconds1916.2Minutes5244.4Hours3630.9Days108.5***Usual time of day of the headache***Morning1815.4Afternoon1512.8Night1311.1Continuous43.4No particular time6757.3***Is the headache becoming stronger, last longer or occur more frequent?***Yes2218.8No9581.2**What is the commonest nature of the headache?**Throbbing/exploding4336.8Sharp43.4Tightness54.3Dull65.1Aching2420.5Pressure in head3227.3Grinding32.6Table 4**Usual location and severity of the headache*Usual Location of headache***FrequencyPercentLeft side32.6Forehead97.7Around the head/ill-defined119.4Right side21.7Both Temples21.7Top of the head10.9Neck21.7Back of head32.6No particular side8471.7***Severity of headache***Mild7059.8Moderate4538.5severe21.7***Is the headache strong enough to wake you from sleep?*** | introduction { # sec1 } = = = = = = = = = = = = headache is the commonest neurological disorder in the community with variable intensity, ranging from a trivial nuisance to a serious, disabling, acute or chronic disorder, and may impose a substantial burden on sufferers and on society \ [ [ @ cr1 ], [ @ cr2 ] \ ]. it is one of the commonest reasons for visiting the neurology clinics worldwide \ [ [ @ cr3 ] - - [ @ cr5 ] \ ], exerting significant burden on its sufferers and impairing daily function especially when accompanied by other symptoms, hence adversely affecting quality of life \ [ [ @ cr6 ] \ ]. according to the world health organisation ( sense ), 1. 7 - - 4 % of the adult population within the world have headaches on 15 or 16 days every month \ [ [ @ cr7 ] \ ] and a lifetime prevalence of more than 90 % has been attributed to headache disorders in most populations of the world \ [ [ @ cr8 ] \ ]. it is known that africans have a higher threshold for pain and may not present to the clinic just for an ' ordinary headache ' \ [ [ @ cr9 ] \ ]. local experiences show that patients suffering from other chronic neurological disorders present very late to doctors and sometimes never do so \ [ [ @ cr9 ] \ ]. chronic headaches produce individual and societal burdens, the former referring to its effect on family, social and recreational activities and the latter referring to effects on healthcare cost ( direct costs ) and comfort and function ( indirect benefits ), including absenteeism and reduced effectiveness \ [ [ @ cr10 ] \ ]. there is limited justification for headache prevalence in africa. in 2004, the 1 - year prevalence of headache from a door - to - door survey of rural south africa was 23. 1 % ( 18. 8 % blacks and 26. 4 % females ) \ [ [ @ cr11 ] \ ]. getahu and colleagues in ethiopia found a 3 - year prevalence rate of 73. 2 % \ [ [ @ cr12 ] \ ]. a 1992 study from ibadan, south west nigeria, found the crude life - time prevalence for at least one episode of headache to be 51 % \ [ [ @ cr13 ] \ ]. in nigeria, there is a paucity of data on the national prevalence and burden of chronic headaches \ [ [ @ cr14 ] \ ] despite the fact that it is the commonest presenting neurological disorder in the authors ' environment \ [ [ @ cr1 ], [ @ cr3 ] \ ], and therefore the possibility that a big headache problem exists in nigeria. there are also no known studies of the prevalence and characterization of headache among nigerian healthcare workers or healthcare workers in south east nigeria hence the relevance of this study. aim of the study { # sec2 } - - - - - - - - - - - - - - - - the aim of this preliminary study was to determine the frequency and pattern of headaches among a population of healthcare workers in a tertiary health institution located in south east nigeria. methods { # sec3 } = = = = = = = this was an epidemiological sampling - based study ( figure [ 1 ] ( # fig1 ) { ref - type = " fig " } ) using a semi - structured questionnaire. the questionnaire was pre - tested in another health facility at nsukka ( a local government area similar to the study area ) for content validity. english language was used to reduce cross - cultural misinterpretations and wrong understanding of terms. figure 1 * * flow chart of research activities. * * the questionnaire was self - administered to all various cadres of health workers in medical unit of university of nigeria teaching hospital, a tertiary health institution located in enugu, south - east nigeria, over a 3 - month period from september - - november 2013, selected by simple random method out of the various units in the hospital e. g. surgical, medical, laboratory, physiotherapy, nutrition, administrative, laundry, transport, security, and medical record. within these are various cadres of hospital staff : physicians, nurses, pharmacists and cleaners. out of a total of 141 only 133 gave consent and hence were studied, giving a response rate of 94. 3 %. to ascertain the overall prevalence of headache, subjects were asked if they have ever had a headache within the previous six months and to note any association. they were to rate the severity of headache based on a scale of mild, moderate and severe. the impact of these severe headaches on the daily activity and the number of days they occur in a month were recorded. the character of the pain, location, duration, and the total numbers of times in the 6 months preceding the date of administering the questionnaire were also noted. statistical package for social sciences version 16 was used in statistical analysis. comparison of multiplex groups was carried out with one way anova test. on the other hand comparison of two distinct groups was carried out with student t test. chi - square test ( and / or fisher ' s exact test ) was used in analysis of categorical variables. the results were revealed as mean ± sd. p value \ < 0. 05 was interpreted as statistically meaningful. ethical approval was obtained from the hospital ethics committee. results { # sec4 } = = = = = = = of the 2, 450 hospital employees ( 450 medical doctors, 630 nurses, 50 pharmacists, and 1320 laboratory and administrative staff ), 141 were selected using simple random method from the employment register and eventually only 133 health workers ( 71 males and 62 females ) gave informed consent and were studied ( response rate 94. 3 % ). more of the respondents were males ( 53. 4 % ) and most were within the 25 - 34 years age group ( 46. 6 % ). most of the workers had worked for only ≤5 years ( 72. 9 % ). table [ 1 ] ( # tab1 ) { ref - type = " table " } illustrates. table 1 * * demographic distribution and work experience of health workers * * variablefrequencypercent * * * sex * * * male7153. 4female6246. 6total133100. 0 * * * age group * * * 15 - - 24139. 825 - - 346246. 635 - - 443627. 145 - - 541511. 355 - - 6464. 565 and above10. 7total133100. 0 * * * number of years worked * * * 1 - - 59772. 96 - - 101813. 511 - - 1575. 316 - - 2043. 021 - - 2553. 826 - - 3021. 5total133100. 0 the prevalence of headache in the past 6 months was 88. 0 % ( among males the prevalence was 87. 3 % while in females it was 88. 7 % ). there was no significant difference observed between the sexes ( p = 0. 806 ). in both sexes, primary headaches were more prevalent ( 71. 0 % in males and 76. 4 % in females ). there was also no significant difference in the prevalence of the primary headaches among the sexes ( p = 0. 509 ). see table [ 2 ] ( # tab2 ) { ref - type = " table " }. table 2 * * prevalence of headache among the health workers * * general prevalence of headache in the past 6 monthsvariablesfrequencypercentheadache present11788. 0headache absent1612. 0 * * sex prevalence of headache * * male ( % ) female ( % ) headache present62 ( 87. 3 ) 55 ( 88. 7 ) headache absent9 ( 12. 7 ) 7 ( 11. 3 ) total71 ( 100. 0 ) 62 ( 100. 0 ) χ ^ 2 ^ = 0. 060 ; p value = 0. 806type of headacheprimary44 ( 71. 0 ) 42 ( 76. 4 ) \ * secondary18 ( 29. 0 ) 13 ( 23. 6 ) total62 ( 100. 0 ) 55 ( 100. 0 ) χ ^ 2 ^ = 0. 436 ; p value = 0. 509 \ * secondary headache is headache with a definitive and identifiable cause found for it i. e. those with pre - existing conditions that may cause the headache e. g. hypertension, cervical spondylosis, refractive error, sleep apnoea, malaria and other febrile conditions \ [ [ @ cr15 ] \ ]. most respondents reported ≤5 episodes of headache in the last 6 months ( 74. 4 % ) and these were typically of short - lasting durations, \ < 60 minutes ( 44. 4 % ). there was no observed periodicity to the headaches in 57. 3 % of cases ( see table [ 3 ] ( # tab3 ) { ref - type = " table " } ). most of the headaches were not located in any particular part of the head or side - locked ( 71. 7 % ) ; were described as mildly severe in 59. 8 % of cases while 88. 0 % of respondents did not suffer any sleep disruption. the headaches were often not significantly disabling ( 73. 4 % ) and in 93. 2 % of respondents did not lead to absenteeism or affect productivity at work ( table [ 4 ] ( # tab4 ) { ref - type = " table " } ). table 3 * * characterization of the headaches * * variable / characteristicsfrequency ( n = 117 ) percent * * * number of episodes in past 6 months * * * 1 - - 58774. 46 - - 102521. 411 - - 1532. 616 - - 2021. 6 * * usual duration of headaches * * seconds1916. 2minutes5244. 4hours3630. 9days108. 5 * * * usual time of day of the headache * * * morning1815. 4afternoon1512. 8night1311. 1continuous43. 4no particular time6757. 3 * * * is the headache becoming stronger, last longer or occur more frequent? * * * yes2218. 8no9581. 2 * * what is the commonest nature of the headache? * * throbbing / exploding4336. 8sharp43. 4tightness54. 3dull65. 1aching2420. 5pressure in head3227. 3grinding32. 6table 4 * * usual location and severity of the headache * usual location of headache * * * frequencypercentleft side32. 6forehead97. 7around the head / ill - defined119. 4right side21. 7both temples21. 7top of the head10. 9neck21. 7back of head32. 6no particular side8471. 7 * * * severity of headache * * * mild7059. 8moderate4538. 5severe21. 7 * * * is the headache strong enough to wake you from sleep? * * * | Introduction {# Sec1} = = = = = = = = = = = = Headache is the commonest neurological disorder in the community with variable intensity, ranging from a trivial nuisance to a severe, disabling, acute or chronic disorder, and may impose a substantial burden on sufferers and on society \ [[ @ CR1 ], [@ CR2] \ ]. It is one of the commonest reasons for visiting the neurology clinics worldwide \ [[ @ CR3] - - [@ CR5] \ ], exerting significant burden on its sufferers and impairing daul^ function especially when accompanied by other symptoms, hence adversely affecting quality of life \ [[ @ CR6] \ ]. According to the World Health Organisation (WHO ), 1. 7 - - 4% of the adult population of the world have headaches on 15 or more days every month \ [[ @ CR7] \] and a lifetime prevalence of more than 90% has been attributed to headache disorders in most populations of the world \ [[ @ CR8] \ ]. It is known that Africans have a higher threshold for pain and may not present to the clinic just for an ' ordinary headache ' \ [[ @ CR9] \ ]. Local experiences show that patients suffering from other chronic neurological disorders present very late to doctors and sometimes never do so \ [[ @ CR9] \ ]. Chronic headash@s produce individual and societal burdens, the former referring to its effect on family, social and recreational activities and the latter referring to effects on healthcare cost (direct costs) and work and function (indirect costs ), including absenteeism and reduced effectiveness \ [[ @ CR10] \ ]. There is limited data for headache prevalence in Africa. In 2004, the 1 - year prevalence of headache from a door - to - door survey of rural south Tanzania was 23. 1% (18. 8% mXled and 26. 4% females) \ [[ @ CR11] \ ]. Getahu and colleagues in Ethiopia found a 1 - year prevalence rate of 73. 2% \ [[ @ CR12] \ ]. A 1992 study from Ibadan, South West Nigeria, found the crude life - time prevalence for at least one episode of headache to be 51% \ [[ @ CR13] \ ]. In Nigeria, there is a paucity of data on the national prevalence and burden of chronic headaches \ [[ @ CR14] \] despite the fact that it is the commonest presenting neurological disorder in the authors ' environment \ [[ @ CR1 ], [@ CR3] \ ], and therefore the possibility that a big headache problem exists in Nigeria. The4$ are also no known studies of the prevalence and characterization of headache among Nigerian healthcare workers or healthcare workers in South East Nigeria hence the relevance of this study. Aim of the study {# Sec2} - - - - - - - - - - - - - - - - The aim of this preliminary study was to determine the frequency and pattern of headaches among a population of healthcare workers in a tertiary health institution located in South East Nigeria. Methods {# Sec3} = = = = = = = This was an epidemiological sampling - based study (Figure [1] (# Fig1) {ref - type = " fig "} ) using a semi - structured questionnaire. The questionnaire was pre - tested in another health facility at Nsukka (a local government area similar to the study area) for content validity. English language was used to reduce cross - cultural misinterpretations and wrong understanding of terms. Figure 1 * * Flow chart of research activities. * * The questionnaire was self - administered to all various cadres of health workers in medical unit of University of Nigeria Teaching Hospital, a tertiary health institution located in Enugu, South - East Nigeria, over a 3 - month period from September - - November 2013, selected by simple random method out of the various units in the hospital e. g. surgical, medical, laboratory, physiotherapy, nutrition, administrative, laundry, transport, security, and medical record. Within these are various cadres of hospital staff: physicians, nurses, pharmacists and cleaners. Out of a total of 141 only 133 gave consent and hence were studied, giving a response rate of 94. 3% . To ascertain the overall prevalence of headache, subjects were asked if they have eGe4 had a headache within the previous six months and to note any association. They were to rate the severity of headache based on a scale of mild, moderate and severe. The impact of these severe heaFacnes on the daily activity and the number of days they occur in a month were recorded. The character of the pain, location, duration, and the total numbers of times in the 6 months preceding the date of administering the questionnaire were also noted. Statistical Package for Social Sciences version 16 was used in statistical analysis. Comparison of multiplex groups was carried out with One Way ANOVA test. On the other hand comparison of two distinct groups was carried out with student t test. Chi - square test (and / or Fisher ' s exact test) was used in analysis of categorical variables. The results were revealed as mean ± SD. P value \ <0. 05 was interpreted as statistically meaningful. Ethical approval was obtained from the hospital ethics committee. Results {# Cec3} = = = = = = = Of the 2, 450 hospital employees (450 medical doctors, 630 nurses, 50 pharmacists, and 1320 laboratory and administrative staff ), 141 were selected using simple random method from the employment register and eventually only 133 health workers (71 males and 62 females) gave informed consent and were studied (response rate 94. 3% ). More of the respondents were males (53. 4%) and most were within the 25 - 34 years age group (46. 6% ). Most of the workers had worked for only ≤ 5 years (72. 9% ). Table [1] (# Tab1) {ref - type = " table "} illustrates. Table 1 * * Demographic distribution and work experience of health w(rkerX * * VariableFrequencyPercent * * * Sex * * * Male7153. 4Female6246. 6Total133100. 0 * * * Age Group * * * 15 - - 24139. 825 - - 346246. 635 - - 443627. 145 - - 541511. 355 - - 6464. 565 and above10. 7Total133100. 0 * * * Number of years worked * * * 1 - - 59772. 96 - - 101813. 511 - - 1575. 316 - - 2043. 021 - - 2553. 826 - - 3021. 5Total133100. 0 The prevalence of headache in the past 6 months was 88. 0% (among males the prevalence was 87. 3% while in females it was 88. 7% ). There was no significant difference observed between the sexes (p = 0. 806 ). In both sexes, primary headaches were more prevalent (71. 0% in males and 76. 4% in females ). There was also no significant difference in the prevalence of the primary headaches among the sexes (p = 0. 509 ). See Table [2] (# Tab2) {ref - type = " table " }. Table 2 * * Prevalence of headache among the health workers * * General prevalence of headache in the past 6 monthsVariablesFrequencyPercentHeadache present11788. 0Headache absent1612. 0 * * Sex prevalence of headache * * Male (%) Female (%) Headache present62 (87. 3) 55 (88. 7) Headache absent9 (12. 7) 7 (11. 3) Total71 (100. 0) 62 (100. 0) χ ^ 2 ^ = 0. 060; P value = 0. 806Type of headachePrimary44 (71. 0) 42 (76. 4) \ * Secondary18 (29. 0) 13 (23. 6) Total62 (100. 0) 55 (100. 0) χ ^ 2 ^ = 0. 436; P value = 0. 509 \ * Secondary headache is headache with a definitive and identifiable cause found for it i. e. those with pre - existing conditions that may cause the headache e. g. hypertension, cervical spondylosis, refractive error, sleep apnoea, malaria and other febrile conditions \ [[ @ CR15] \ ]. Most respondents reported ≤ 5 episodes of headache in the last 6 months (74. 4%) and these were typically of short - lasting durations, \ <60 minutes (44. 4% ). There was no observed periodicity to the headaches in 57. 3% of cases (see Table [3] (# Tab3) {ref - type = " table "} ). Most of the headaches were not located in any particular part of the head or side - locked (71. 7% ); were described as mildly Devfre in 59. 8% of cases while 88. 0% of respondents did not suffer any sleep disruption. The headaches were often not significantly disabling (73. 4%) and in 93. 2% of respondents did not lead to absenteeism or affect productivity at work (Table [4] (# Tab4) {ref - type = " table "} ). Table 3 * * Characterization of the headaches * * Variable / CharacteristicsFrequency (N = 117) Percent * * * Number of episodes in past 6 months * * * 1 - - 58774. 46 - - 102521. 411 - - 1532. 616 - - 2021. 6 * * Usual duration of headaches * * Seconds1916. 2Minutes5244. 4Hours3630. 9Days108. 5 * * * Usual time of day of the headache * * * Morning1815. 4Afternoon1512. 8Night1311. 1Continuous43. 4No particular time6757. 3 * * * Is the headache becoming stronger, last longer or occur more frequent? * * * Yes2218. 8No9581. 2 * * What is the commonest nature of the headache? * * Throbbing / exploding4336. 8Sharp43. 4Tightness54. 3Dull65. 1Aching2420. 5Pressure in head3227. 3Grinding32. 6Table 4 * * Usual location and severity of the headache * Usual Location of headache * * * FrequencyPercentLeft side32. 6Forehead97. 7Around the head / ill - defined119. 4Right side21. 7Both Temples21. 7Top of the head10. 9Neck21. 7Back of head32. 6No particular side74Y1. 7 * * * Severity of headache * * * Mild7059. 8Moderate4538. 5severe21. 7 * * * Is the headache strong enough to wake you from sleep? * * * | Introduction {#Sec1} ============ Headache is the commonest neurological disorder in the community with variable ranging from a trivial nuisance to a severe, disabling, acute or chronic and may impose a substantial burden on sufferers and on society \[[@CR1], [@CR2]\]. It is one of the commonest reasons for visiting the neurology clinics worldwide exerting burden its sufferers and impairing daily function especially when accompanied by other symptoms, hence adversely quality of life \[[@CR6]\]. According to the World Health Organisation (WHO), 1.7 -- 4% of the adult population of the world have on 15 or more days every month \[[@CR7]\] and a lifetime prevalence of more than 90% has been attributed to headache disorders in populations of the world \[[@CR8]\]. It is known that Africans have a higher threshold for pain and not present to the clinic just for 'ordinary headache' \[[@CR9]\]. Local experiences show that patients suffering from other chronic neurological disorders present very late to doctors and sometimes do so \[[@CR9]\]. Chronic headaches produce individual and societal burdens, the former referring to its on family, social and recreational activities and latter to on healthcare cost (direct costs) and work and function (indirect including absenteeism and reduced effectiveness \[[@CR10]\]. There is limited data for headache prevalence in Africa. In 2004, the 1-year prevalence headache from door-to-door survey of rural south Tanzania 23.1% males and 26.4% females) \[[@CR11]\]. Getahu and colleagues Ethiopia found a 1-year prevalence rate of \[[@CR12]\]. A 1992 study from Ibadan, South West Nigeria, found the crude life-time prevalence for at least episode of headache be 51% \[[@CR13]\]. In Nigeria, there is a paucity of data the national prevalence and chronic headaches \[[@CR14]\] despite the fact it is the commonest presenting neurological the authors' environment \[[@CR1], [@CR3]\], and therefore the possibility that a big headache problem exists in Nigeria. There are also known studies of the prevalence and characterization of headache among Nigerian healthcare workers or workers in Nigeria hence the relevance of this Aim of the study ---------------- aim of this preliminary study was to determine the frequency and pattern of headaches among a population of healthcare workers in a tertiary health institution located in South East Nigeria. Methods {#Sec3} ======= This was an epidemiological sampling-based study (Figure [1](#Fig1){ref-type="fig"}) using a semi-structured questionnaire. The questionnaire was in another health facility at Nsukka (a local area similar to the study area) for content validity. language was used to reduce cross- cultural misinterpretations and wrong of terms.Figure 1**Flow of research activities.** The questionnaire was self- administered all various cadres of health workers in medical unit of University of Teaching Hospital, a tertiary health institution located in Enugu, South- East Nigeria, over 3- month period from September -- November 2013, simple random method out of the various units in the hospital e.g. surgical, medical, laboratory, physiotherapy, nutrition, administrative, transport, security, and medical record. are various cadres of hospital physicians, nurses, pharmacists and cleaners. Out a total of 141 only 133 gave and hence were studied, giving a response rate of 94.3%. To ascertain the overall prevalence headache, subjects were asked if they have ever had a headache within the previous six months and to note any They were to rate the severity of headache based on a scale mild, moderate and severe. The impact of severe headaches on the daily activity and the number of days they occur in month recorded. The character of the pain, duration, and the total numbers of in 6 months preceding the date of administering the questionnaire were Statistical Package for Social version 16 was used in statistical analysis. Comparison of groups was carried out with ANOVA test. On the other hand comparison of distinct groups was carried out with student t test. Chi-square test (and/or Fisher's exact test) was used in analysis of categorical variables. The results were revealed as mean ± SD. P value \<0.05 was interpreted as meaningful. Ethical approval was obtained the hospital ethics committee. Results {#Sec4} Of the 2,450 hospital employees (450 medical doctors, 630 nurses, 50 pharmacists, and 1320 laboratory and administrative staff), 141 were selected using simple random method from the employment register and eventually only 133 health workers (71 males and 62 females) gave informed consent were studied (response rate 94.3%). More of the respondents were males (53.4%) and most were within the - years age group (46.6%). Most of the workers worked for only ≤5 years (72.9%). Table [1](#Tab1){ref-type="table"} illustrates.Table 1**Demographic distribution and work of health workers**VariableFrequencyPercent***Sex***Male7153.4Female6246.6Total133100.0***Age Group***15 -- 24139.825 -- 346246.635 -- -- 541511.355 -- 6464.565 above10.7Total133100.0***Number of years -- 59772.96 -- 101813.511 -- 1575.316 -- 2043.021 -- 2553.826 -- 3021.5Total133100.0 The of headache in the past 6 months 88.0% (among males the prevalence was 87.3% while in females it was 88.7%). There was no significant difference observed between the sexes (p = 0.806). In both sexes, primary headaches were more prevalent in males and 76.4% females). There was also no significant difference in the prevalence of the primary headaches among the (p = 0.509). See Table [2](#Tab2){ref-type="table"}.Table 2**Prevalence of headache among the health prevalence of headache in past 6 monthsVariablesFrequencyPercentHeadache present11788.0Headache absent1612.0**Sex prevalence of headache**Male (%)Female (%)Headache present62 (87.3)55 (88.7)Headache (12.7)7 (11.3)Total71 (100.0)62 (100.0)χ^2^ P value = of headachePrimary44 (71.0)42 (76.4)\*Secondary18 (29.0)13 (23.6)Total62 (100.0)55 0.436; P = 0.509\*Secondary headache is headache with a definitive and identifiable cause found for it i.e. those with pre-existing conditions that may cause the headache e.g. hypertension, spondylosis, refractive error, sleep apnoea, malaria and other febrile conditions \[[@CR15]\]. Most respondents reported episodes of headache in the last 6 months (74.4%) and were typically of durations, \<60 minutes (44.4%). There was no observed to the headaches in 57.3% of cases (see Table [3](#Tab3){ref-type="table"}). Most of the headaches not located in any particular part of or side-locked (71.7%); were described mildly severe in 59.8% of cases while 88.0% of respondents did not suffer disruption. The headaches were often not (73.4%) and in 93.2% of respondents did not to absenteeism or affect productivity at work (Table [4](#Tab4){ref-type="table"}).Table 3**Characterization of the headaches**Variable/CharacteristicsFrequency (N =117)Percent***Number of episodes 6 months***1 -- 58774.46 102521.411 -- 1532.616 2021.6**Usual duration of headaches**Seconds1916.2Minutes5244.4Hours3630.9Days108.5***Usual time of day of the headache***Morning1815.4Afternoon1512.8Night1311.1Continuous43.4No particular time6757.3***Is headache becoming stronger, last longer or occur frequent?***Yes2218.8No9581.2**What is the commonest nature of the headache?**Throbbing/exploding4336.8Sharp43.4Tightness54.3Dull65.1Aching2420.5Pressure in head3227.3Grinding32.6Table 4**Usual location and severity the Location of side32.6Forehead97.7Around the head/ill-defined119.4Right side21.7Both Temples21.7Top of the head10.9Neck21.7Back of head32.6No particular side8471.7***Severity of the headache strong enough to wake you from sleep?*** | INtroDuCTion {#sEc1}
============
HeADachE is THe CoMMOnESt NeUrOloGicAL dISORDer iN tHe CoMmuNIty WITH vARIABlE iNteNsitY, rANginG froM a TrIVIAL NUISANce TO A SEveRE, DIsaBLING, aCUtE or CHrOnic DISOrDER, ANd maY IMposE A sUBsTANTIAL burdEN oN sUfferErS And ON sOCietY \[[@cR1], [@CR2]\]. it iS oNE OF THe CoMmOneSt reaSOns foR VisiTIng The NeurOlOgY cLinIcS worlDWIde \[[@cR3]--[@Cr5]\], ExERtinG SiGNifiCaNt buRdEn On ItS SUFfererS And ImpaiRIng DAILY FunCTion ESpEcialLY WHEN accompanIed BY OtheR sYmPtOmS, hEncE aDverSeLy AFFectING qualIty oF LIfe \[[@cR6]\]. aCcordInG tO the woRLD heAlTH oRGanISATIoN (whO), 1.7 -- 4% Of thE aDULt POPulAtIoN of The WoRLD HaVe HeadachEs on 15 oR mORE DAys eVERY MONTH \[[@cR7]\] anD A LIfEtiME pREVALEnce oF More ThAn 90% hAS bEEn aTTRibuTed to headAChE diSorDErs In mOST POpUlaTIONs Of ThE woRLd \[[@cR8]\].
It is kNown THaT AFrIcanS hAVe a hiGHEr ThreSHOLd FOr PAin AnD mAy not PrEsENt To the CliniC JUst fOR AN 'oRDINAry HeaDAcHe' \[[@cR9]\]. lOCal eXPERIEnCes shOw that PAtiEnTs suffeRiNG FrOm OtHeR chroNic NEUrolOgIcAL DIsoRDErs presENT VEry LatE TO dOCtorS and sOmEtimeS NevEr Do SO \[[@cr9]\]. ChrOnIC HeadaChes prODuce InDIVIduaL ANd sOciETAL burDEnS, tHE FOrMeR REFeRring to its EffECT On faMily, SoCIAl And RecREATional ACTiVITIEs anD ThE LaTteR reFErrING To EfFeCtS On heALtHCarE Cost (dIReCT COSTs) aNd WorK aNd functIoN (INDIRect COsts), INClUDiNg ABSentEeisM aND rEDUced EffECTiVenESs \[[@cR10]\].
ThERe Is liMiTeD DatA FoR HEADACHe PrEValence iN AFRiCa. in 2004, tHe 1-yeaR pReVaLEnce Of HeaDacHe frOm a dOor-To-dOOR SuRveY of ruraL SOUtH tAnZania waS 23.1% (18.8% mAlEs AnD 26.4% femAleS) \[[@cr11]\]. GETAHU anD COlLeagUeS iN ETHiOPIa FOunD A 1-year Prevalence rate Of 73.2% \[[@CR12]\]. A 1992 STudY From iBADAn, SOUTH WEST nigerIa, founD tHe CruDE LiFe-Time prEVaLenCE for AT leASt ONE epiSOdE Of hEadAChE TO be 51% \[[@CR13]\].
IN NigERIA, ThEre IS a pAUciTy Of DAta on tHE natIonal PrEVAlEnCE ANd bUrDEN Of cHrOniC hEAdACHes \[[@cr14]\] dESPitE the FacT tHaT It IS The COmMONeST PReSEnTing NeURoLOGicAL dIsORDER In THe AuTHORS' eNVIROnmEnT \[[@cR1], [@cr3]\], anD thErefOre THE possIbiLITY tHAT A bIg headACHe PRObleM EXIstS in NiGEriA. THEre ARE alSO NO kNown stUdiEs OF THe prEvaleNcE ANd cHAracteRiZatioN oF HeAdaChE aMong NIGeriAN HEALThCAre WoRkers OR heALThCARe worKers In SouTH EAsT NIGERIA heNcE thE rElEVANcE of tHis sTUDy.
aiM OF tHe study {#SEC2}
----------------
THe AiM oF ThiS PRELimINARy STuDY WAS TO detERmiNE the fREQUencY aNd pAttern of hEadAChes aMoNG A PopUlaTiON OF HeaLTHCArE WorkErs in A TertiARY HEaLTH INstiTutioN LOcATeD In SouTH east nigeriA.
MethODS {#sEc3}
=======
ThIs was an epiDemIoLOGIcAl sAMplING-BasEd Study (FIGuRE [1](#fIg1){REf-TypE="Fig"}) UsinG a SeMi-STrUctURed qUestionNAiRe. thE qUesTiOnnaIre was PRe-TeSTED In aNotHER hEaLth facilitY aT nsukKA (a LOcaL gOvernMeNT aREa simIlAr to THe StUdy aREA) FoR cONTENT VaLiDiTY. eNglisH LaNguage Was USed TO ReDuCE Cross- CULTURAL mISInTERpREtAtiONs ANd WrOng UNDERStANDIng oF TeRms.fIgUre 1**FloW ChART of ReSEArch acTiviTIeS.**
thE qUeStIOnnAiRE WAS SELF- ADmINISTerED To all VARIOuS caDres of hEalTH wORKeRs In mEDICAL UniT Of uNivERsiTY OF NIgEria teAcHIng HoSPiTAl, A tErtiarY heaLTh iNSTitUTIon LocATED In enuGu, south- EAST NigErIa, OveR A 3- MontH PeRiOD From SEptembER -- NOVeMbER 2013, sElEcTed BY SiMpLE rANdoM MEthoD OuT of The VarIOUs UNits iN The hoSPItaL E.G. sURgiCAL, medICAl, laBoRatOrY, phYsioTheRapy, NuTrITIoN, ADMInISTRaTIVe, launDRy, TranspoRt, seCURitY, anD meDicAl REcOrd. WIThIn tHESE arE VaRious cadrES OF hosPiTaL STafF: PHYSICIaNs, NuRses, PHaRmACIStS anD CLEAnERs. oUt Of A TOtAl oF 141 ONLY 133 gavE CoNseNt And hEnce WerE sTudied, GivING a RespoNse RatE Of 94.3%. TO aSCERtAiN thE OvEralL PreVaLenCe OF heADAchE, suBjeCtS WerE asKeD If ThEY HavE EVER had a headachE wiThIn the PREviOUs siX mONTHS ANd TO noTE ANy AsSoCIatION. tHey WERe TO RAtE THE SEVErity Of Headache basEd on a ScaLE of mIlD, mODErAtE AnD SEvere. The imPAct OF ThesE sevERe HeAdAcHes oN THE DAiLY AcTIviTY aND thE NuMbEr Of dAYs ThEY ocCUr in A MoNTH WERE rECOrdeD. THe chaRacTEr OF THE paiN, lOcatIoN, DUraTIOn, and tHe ToTal NuMbers Of tImes In tHe 6 MontHs prECedINg the dAtE of aDmiNistEriNG the queStiOnNAirE WERe ALSo NOTEd.
STaTISticaL PaCKAge FOR socIaL ScIEnCEs VErsION 16 Was USeD in STatIstICAl aNalYsIS. CoMPaRISOn OF mUlTipleX GRoUPS was CarrIed Out WItH ONE Way AnovA tEst. On ThE otheR hand cOMPaRISon of two DisTiNcT gROupS waS cARRIed OuT WITh StuDENt t tesT. chi-Square teSt (ANd/oR FIsHer's exACt TEST) WAS UsED iN ANaLYsiS OF cAtEgoRiCAL VarIABLES. THe RESulTS werE REveALed as MEan ± SD. P vALuE \<0.05 Was iNterPrEtEd As stAtisTicALlY mEaNiNgfUL. ethICAL aPPRoVAl WaS OBTaiNeD from the hoSPiTal eTHIcs COmMiTTee.
results {#SEc4}
=======
of tHE 2,450 HoSpItaL EMPLOyEes (450 medicaL doCtorS, 630 nUrseS, 50 phArMACiSts, aNd 1320 LAbOraTORy AnD aDmINIstrAtiVE sTAfF), 141 weRE SEleCtED usiNg simple raNdoM methoD FrOM The EmPlOYment REgISTeR AnD evEnTUalLy oNLy 133 HEALTh worKERS (71 malES anD 62 FemALeS) GAvE InfORmeD CoNSEnT And Were stUdIEd (RESPoNsE rate 94.3%). MOrE of thE rESPoNdENts WERE MAleS (53.4%) aNd MOst werE WITHIN thE 25 - 34 yEaRS AGe gROUP (46.6%). Most oF the WORkerS hAd WOrkEd FOr onLy ≤5 YeARS (72.9%). TaBle [1](#TAb1){reF-TYpe="TablE"} ILluSTrAteS.taBLE 1**deMOGrAphiC diSTriButiOn And woRk exPeriENCe OF HEaLtH workErS**VaRiAbLEfrEQuEncYpeRcent***SeX***MAle7153.4FemAlE6246.6TotAL133100.0***agE GroUP***15 -- 24139.825 -- 346246.635 -- 443627.145 -- 541511.355 -- 6464.565 aND ABovE10.7total133100.0***NumBER oF YeARS WorkED***1 -- 59772.96 -- 101813.511 -- 1575.316 -- 2043.021 -- 2553.826 -- 3021.5TOtAl133100.0
the PREvaLenCe of hEadache in thE pAsT 6 monTHs WaS 88.0% (amONg MaLEs THE pRevalENCE WAS 87.3% WhiLE In fEMAlES it Was 88.7%). THErE waS nO sIGniFICAnT dIffErencE OBSERVed bEtweEN THe sEXES (P = 0.806). in BOTH SEXEs, PRIMARY HEAdacHes weRe MoRE PrEvaLent (71.0% in Males aNd 76.4% iN FEmALEs). tHere wAs aLso nO SIGNificant diFFerEnCe in thE prEVaLeNce OF tHe primarY hEaDaCHes AmoNG THE sEXeS (p = 0.509). sEe tabLE [2](#tAb2){ref-Type="TaBle"}.TABLE 2**prEvalEnce OF HEaDAchE aMOnG THe hEAlTH WORkERs**gEnerAL prevAlence OF heADAChE iN the Past 6 MOnThsVaRiablESfrequENcyPERcenTHEADAChe PReSent11788.0heADACHE ABsenT1612.0**sex preVaLence of HeADAChE**MaLe (%)FeMALe (%)HEaDacHE PrESENt62 (87.3)55 (88.7)HEaDachE absenT9 (12.7)7 (11.3)toTal71 (100.0)62 (100.0)Χ^2^ = 0.060; P vALUE = 0.806tYpe oF hEAdAchepriMaRY44 (71.0)42 (76.4)\*secONDary18 (29.0)13 (23.6)TOTaL62 (100.0)55 (100.0)Χ^2^ = 0.436; p vALuE = 0.509\*SEconDaRY HeadAche IS headAcHe wiTH a defINitIVe aND IDenTiFiABLE cauSE foUND fOR It i.e. Those WitH pRe-ExISTiNG coNDItIonS tHat May cAUSe tHe hEADacHE E.G. hYpERTeNSion, cervICAL SpONDYLOSIS, REfraCTiVe ErroR, SlEEP apNoeA, mALAriA aNd OtHEr fEbRilE CoNdITioNs \[[@Cr15]\].
Most RESpONDEnts reporteD ≤5 epISoDEs oF HEAdAcHe IN the LaSt 6 MOntHs (74.4%) AnD TheSE weRe TyPicALlY OF shORT-LaSTiNG duratioNS, \<60 MinUtes (44.4%). tHERe WAs NO oBSERVED perIOdiCitY TO thE HEadaCHEs In 57.3% of caSes (sEe TABLE [3](#Tab3){Ref-TYPe="TABle"}). mosT Of the headAches wEre Not lOcATeD in Any PArTICuLar PART oF THe hEaD oR Side-LockEd (71.7%); weRE dEsCRIBEd as MildLY sEvErE IN 59.8% oF CAsES whilE 88.0% oF respoNDENTS diD NOt SUFfER anY slEEP DIsrUPtion. the HeaDAcHEs WERE OFTEn noT SignIFiCANTly dISaBlING (73.4%) ANd in 93.2% OF RESPOnDents did NOT LEaD to aBsEnteEISm or affECT PROduCtIViTY aT WORK (TABle [4](#Tab4){REF-tYPE="taBlE"}).TaBle 3**CHaRacteriZaTioN Of The HEadAChES**VARiaBlE/chARacteRistIcSfRequEnCy (N =117)PERCEnT***NUmBEr Of EPiSOdeS in past 6 mOnTHs***1 -- 58774.46 -- 102521.411 -- 1532.616 -- 2021.6**USUal DURaTION OF heAdAcHes**SeCOnDS1916.2mInutEs5244.4hoUrS3630.9DAyS108.5***uSuAL tIMe Of DAY OF The hEAdaChe***MoRniNG1815.4AftErNooN1512.8nigHt1311.1cONtiNuOuS43.4No ParTiCuLAR Time6757.3***IS tHE hEaDAChE bECoMInG strongEr, LAsT LoNgER OR oCcur mOrE FReQuenT?***yEs2218.8No9581.2**wHat iS thE cOmmoNest naTURE OF The HeaDaChe?**THrobBINg/eXplOding4336.8sharp43.4TiGhtnesS54.3DUlL65.1AChing2420.5PressurE iN HEaD3227.3GRinding32.6TABLE 4**UsUAL LOcaTION AnD SEvERity OF thE heADaCHE*usuAl lOcAtIon of HeadaCHe***FReQUeNcypERcEntlEfT SiDe32.6foreHeAd97.7aRound ThE heAd/iLl-dEFINeD119.4RIGhT Side21.7BOTH teMPLeS21.7Top Of the Head10.9nEcK21.7baCk oF HeaD32.6nO parTIculAR SIDe8471.7***seVeriTY OF headaChE***MIld7059.8MODeratE4538.5sEVERe21.7***IS THE HEADACHE sTrOnG enOUgH TO waKe yOU froM sLeep?*** | Introduction{#Sec1} ============ Headache isthe commonest neurological disorder in the community with variable intensity, ranging from a trivial nuisance to a severe, disabling, acute or chronicdisorder,and may impose a substantial burden on sufferers and on society\[[@CR1], [@CR2]\]. It is one of the commonest reasons forvisiting theneurologyclinics worldwide\[[@CR3]--[@CR5]\], exerting significantburden on its sufferers andimpairing daily function especially when accompanied by other symptoms, hence adversely affecting quality of life \[[@CR6]\]. Accordingto theWorldHealth Organisation (WHO), 1.7 -- 4% of the adult population of the world have headaches on 15 or more days every month \[[@CR7]\]anda lifetime prevalence of more than 90%has been attributed toheadache disordersin most populations of the world \[[@CR8]\]. It isknown that Africanshaveahigher threshold for pain and may notpresent tothe clinic just for an 'ordinary headache' \[[@CR9]\]. Local experiences show that patientssuffering from other chronic neurological disorderspresent very late to doctors and sometimes never do so \[[@CR9]\]. Chronicheadachesproduce individual and societal burdens, the formerreferring to its effect on family, socialand recreational activities and the latter referring to effects on healthcare cost (direct costs) and work andfunction (indirect costs), includingabsenteeism and reduced effectiveness \[[@CR10]\]. There is limited data forheadache prevalence in Africa. In 2004, the 1-year prevalence of headache from a door-to-door surveyof rural south Tanzaniawas 23.1% (18.8% males and 26.4% females) \[[@CR11]\]. Getahu and colleagues in Ethiopia found a1-year prevalence rate of 73.2% \[[@CR12]\]. A 1992 study from Ibadan, South West Nigeria, found the crude life-time prevalence for atleastone episode of headache to be 51% \[[@CR13]\]. In Nigeria, there is a paucity of data on the national prevalence and burdenof chronic headaches \[[@CR14]\] despite the fact that it is the commonest presenting neurological disorderin the authors' environment \[[@CR1], [@CR3]\], and therefore the possibility that a bigheadache problem exists in Nigeria. There are also noknown studies of the prevalence and characterization of headacheamong Nigerian healthcare workers or healthcareworkersin South East Nigeria hence the relevance of this study. Aim of the study {#Sec2} ---------------- The aim of this preliminary study wasto determine the frequency and pattern of headachesamong a populationof healthcare workers in a tertiary healthinstitution located in South East Nigeria. Methods {#Sec3} ======= This wasan epidemiological sampling-based study (Figure [1](#Fig1){ref-type="fig"}) using a semi-structured questionnaire. Thequestionnaire waspre-tested in another healthfacility at Nsukka (a local government area similar to the study area) for content validity. English language was used to reduce cross-cultural misinterpretations and wrong understanding of terms.Figure 1**Flow chartof research activities.**The questionnaire was self- administered to all various cadres of healthworkers in medicalunit of University of NigeriaTeaching Hospital, a tertiary health institution located inEnugu, South- East Nigeria, over a3- monthperiod from September -- November 2013, selected by simplerandom methodoutofthevarious units in the hospital e.g. surgical, medical, laboratory, physiotherapy, nutrition, administrative, laundry,transport, security, andmedical record. Within these arevarious cadres of hospital staff: physicians,nurses, pharmacists and cleaners. Out of a total of 141 only 133 gaveconsent and hence were studied, giving a response rate of 94.3%. To ascertain the overall prevalence of headache, subjects were asked if they have ever had a headache within the previous six months and to note any association.They were torate theseverity of headache based on ascale ofmild, moderate andsevere.The impact of these severe headacheson the daily activity and thenumber of days they occur in a month were recorded. Thecharacter of the pain, location,duration, and the total numbers oftimes in the 6 months preceding thedate of administering the questionnaire were also noted. StatisticalPackage for Social Sciences version 16was usedin statisticalanalysis. Comparisonof multiplex groups wascarriedout with One Way ANOVA test. On theother hand comparison of two distinct groups was carried out with student ttest.Chi-square test (and/or Fisher's exact test)was used in analysis of categorical variables. The resultswere revealedas mean ± SD. P value \<0.05 was interpreted as statistically meaningful. Ethical approval was obtained from the hospital ethics committee. Results {#Sec4} ======= Of the 2,450 hospitalemployees (450 medical doctors, 630 nurses,50 pharmacists, and 1320 laboratory and administrative staff), 141were selected usingsimple random method from the employment register and eventually only 133 health workers (71 males and 62 females)gave informed consentand were studied (response rate 94.3%). More of therespondentswere males(53.4%) and most were within the 25 - 34years agegroup(46.6%). Most of the workers hadworkedfor only ≤5 years (72.9%). Table [1](#Tab1){ref-type="table"} illustrates.Table 1**Demographicdistribution and work experienceof health workers**VariableFrequencyPercent***Sex***Male7153.4Female6246.6Total133100.0***Age Group***15 -- 24139.825 -- 346246.635 --443627.145 -- 541511.355 -- 6464.565 andabove10.7Total133100.0***Number of years worked***1 -- 59772.96 -- 101813.511 -- 1575.316 -- 2043.021 -- 2553.826-- 3021.5Total133100.0 The prevalence of headache in the past 6 months was88.0% (among malesthe prevalence was 87.3% while in females it was 88.7%). Therewas nosignificantdifference observedbetween the sexes (p = 0.806). Inbothsexes, primary headaches were more prevalent (71.0% in males and 76.4% in females). There was alsono significant difference in the prevalence of theprimary headaches among the sexes (p =0.509). See Table [2](#Tab2){ref-type="table"}.Table 2**Prevalence ofheadache among the health workers**General prevalence of headache in the past 6 monthsVariablesFrequencyPercentHeadache present11788.0Headache absent1612.0**Sex prevalence of headache**Male (%)Female (%)Headachepresent62 (87.3)55 (88.7)Headache absent9 (12.7)7 (11.3)Total71 (100.0)62 (100.0)χ^2^ = 0.060; P value = 0.806Type of headachePrimary44 (71.0)42 (76.4)\*Secondary18 (29.0)13 (23.6)Total62 (100.0)55 (100.0)χ^2^= 0.436; P value = 0.509\*Secondary headache is headache with a definitive and identifiable cause found forit i.e. those with pre-existing conditions that may cause the headachee.g. hypertension, cervical spondylosis, refractive error, sleep apnoea, malaria and other febrile conditions \[[@CR15]\]. Most respondents reported ≤5 episodes ofheadache in the last 6 months (74.4%) and these were typically of short-lasting durations, \<60 minutes (44.4%). There was no observed periodicitytothe headachesin 57.3% of cases (see Table [3](#Tab3){ref-type="table"}). Most of the headacheswere not located in any particular part of the head or side-locked (71.7%); were described as mildly severe in 59.8% of cases while 88.0% of respondents did not suffer any sleep disruption. The headacheswere often not significantly disabling (73.4%) and in 93.2% of respondentsdid not lead to absenteeism oraffect productivity at work (Table [4](#Tab4){ref-type="table"}).Table 3**Characterization of the headaches**Variable/CharacteristicsFrequency (N =117)Percent***Numberof episodes in past6 months***1 -- 58774.46 -- 102521.411 -- 1532.616 -- 2021.6**Usual duration of headaches**Seconds1916.2Minutes5244.4Hours3630.9Days108.5***Usual timeofday of theheadache***Morning1815.4Afternoon1512.8Night1311.1Continuous43.4No particular time6757.3***Is the headache becoming stronger, lastlonger or occur more frequent?***Yes2218.8No9581.2**What is the commonest nature of the headache?**Throbbing/exploding4336.8Sharp43.4Tightness54.3Dull65.1Aching2420.5Pressure in head3227.3Grinding32.6Table 4**Usual location and severity of the headache*UsualLocation of headache***FrequencyPercentLeft side32.6Forehead97.7Around the head/ill-defined119.4Right side21.7Both Temples21.7Topof the head10.9Neck21.7Back of head32.6No particular side8471.7***Severity of headache***Mild7059.8Moderate4538.5severe21.7***Is the headache strongenough to wake you from sleep?*** | Introduction {#Sec1} ============ Headache is _the_ commonest neurological disorder in the _community_ with variable intensity, ranging from _a_ trivial nuisance to a severe, disabling, acute or chronic disorder, and may _impose_ a _substantial_ burden on sufferers and on _society_ \[[@CR1], [@CR2]\]. It is one _of_ the commonest _reasons_ _for_ _visiting_ _the_ _neurology_ clinics worldwide \[[@CR3]--[@CR5]\], _exerting_ significant _burden_ on its sufferers and _impairing_ _daily_ function especially _when_ accompanied by _other_ _symptoms,_ hence adversely _affecting_ quality of life \[[@CR6]\]. _According_ to the _World_ Health Organisation (WHO), 1.7 -- 4% of the adult population of the world have headaches on 15 _or_ more days every month \[[@CR7]\] and a lifetime prevalence of _more_ than 90% _has_ been attributed _to_ headache disorders _in_ most populations of the world \[[@CR8]\]. It _is_ known that Africans _have_ a higher threshold for _pain_ and may not present to the clinic _just_ for an 'ordinary headache' \[[@CR9]\]. _Local_ experiences show that patients suffering _from_ other chronic neurological disorders present _very_ late to doctors and _sometimes_ never _do_ so \[[@CR9]\]. Chronic headaches produce individual and societal burdens, the former referring to _its_ _effect_ on family, _social_ and recreational activities and the latter _referring_ to effects on _healthcare_ cost _(direct_ _costs)_ and _work_ and function _(indirect_ costs), including absenteeism and _reduced_ effectiveness \[[@CR10]\]. There is limited data _for_ headache prevalence in Africa. In 2004, the 1-year _prevalence_ of headache from a door-to-door survey of rural _south_ Tanzania was 23.1% (18.8% males and 26.4% females) \[[@CR11]\]. Getahu _and_ colleagues in Ethiopia found a 1-year _prevalence_ rate of 73.2% _\[[@CR12]\]._ A _1992_ study from Ibadan, South _West_ _Nigeria,_ found the crude life-time prevalence for at _least_ one episode of headache to be 51% _\[[@CR13]\]._ In Nigeria, there is a paucity of data on the national prevalence _and_ burden of chronic headaches \[[@CR14]\] despite the fact that it is the commonest presenting neurological disorder in the authors' environment \[[@CR1], [@CR3]\], and _therefore_ the _possibility_ that _a_ big headache _problem_ exists _in_ Nigeria. _There_ are also _no_ known studies of the prevalence _and_ characterization of headache among Nigerian _healthcare_ _workers_ or _healthcare_ _workers_ in _South_ _East_ _Nigeria_ hence the relevance of this _study._ _Aim_ of the study {#Sec2} ---------------- The aim _of_ _this_ preliminary _study_ was to determine the frequency and _pattern_ of headaches among _a_ _population_ of _healthcare_ workers _in_ a _tertiary_ health _institution_ located in South East Nigeria. Methods _{#Sec3}_ ======= This was an _epidemiological_ _sampling-based_ _study_ (Figure [1](#Fig1){ref-type="fig"}) _using_ a semi-structured questionnaire. The questionnaire was pre-tested in another _health_ _facility_ at Nsukka (a local government area similar to _the_ _study_ area) _for_ content validity. English language was _used_ to reduce _cross-_ cultural misinterpretations and _wrong_ _understanding_ of terms.Figure 1**Flow chart _of_ research activities.** The questionnaire was self- administered to _all_ _various_ cadres of health workers in medical _unit_ of University of Nigeria Teaching Hospital, _a_ tertiary health institution located _in_ Enugu, South- _East_ Nigeria, over a _3-_ month period from September -- _November_ 2013, _selected_ by simple _random_ method out _of_ the various _units_ in the hospital e.g. surgical, medical, laboratory, physiotherapy, _nutrition,_ administrative, laundry, transport, security, and medical record. Within these _are_ various cadres of _hospital_ staff: _physicians,_ nurses, pharmacists and _cleaners._ Out of a total of 141 only 133 gave consent and _hence_ were studied, giving _a_ response rate _of_ 94.3%. To ascertain the overall _prevalence_ _of_ _headache,_ subjects _were_ asked _if_ they have ever had a headache within _the_ previous six months and to note any association. They _were_ to rate _the_ severity of headache based on a scale of mild, _moderate_ and _severe._ The impact of these severe headaches on the daily activity and the number of days they _occur_ in a _month_ were recorded. The character of _the_ pain, location, _duration,_ and the total numbers of _times_ in the 6 months _preceding_ the date of _administering_ the questionnaire _were_ _also_ _noted._ Statistical Package for Social Sciences version 16 _was_ _used_ _in_ statistical analysis. Comparison of _multiplex_ groups was carried out with One Way ANOVA test. On the other hand comparison of two distinct groups was _carried_ out with student t test. Chi-square _test_ (and/or Fisher's exact test) was used in analysis _of_ categorical _variables._ _The_ results were revealed as _mean_ ± SD. _P_ value \<0.05 was interpreted as statistically meaningful. _Ethical_ approval was _obtained_ _from_ _the_ hospital ethics committee. Results {#Sec4} ======= Of the _2,450_ hospital employees (450 medical doctors, _630_ nurses, 50 pharmacists, _and_ 1320 laboratory _and_ administrative staff), 141 were selected using simple random method from the employment _register_ and eventually only 133 _health_ workers (71 males _and_ 62 females) gave _informed_ consent and _were_ studied (response rate 94.3%). _More_ of the respondents were males (53.4%) _and_ most were within the 25 - 34 years age group (46.6%). Most of the workers had _worked_ for only ≤5 _years_ (72.9%). _Table_ [1](#Tab1){ref-type="table"} illustrates.Table 1**Demographic distribution and work experience of _health_ workers**VariableFrequencyPercent***Sex***Male7153.4Female6246.6Total133100.0***Age Group***15 -- 24139.825 -- 346246.635 -- 443627.145 -- 541511.355 -- 6464.565 and above10.7Total133100.0***Number _of_ _years_ worked***1 -- 59772.96 -- _101813.511_ -- 1575.316 -- _2043.021_ -- 2553.826 -- 3021.5Total133100.0 The prevalence of _headache_ in the past 6 months was 88.0% (among males the prevalence was 87.3% while _in_ females it _was_ 88.7%). _There_ was no significant difference observed between _the_ sexes (p = 0.806). In both sexes, _primary_ headaches were more prevalent (71.0% in males and 76.4% _in_ females). There was also no significant difference in the _prevalence_ of the primary _headaches_ _among_ the _sexes_ (p = 0.509). See Table _[2](#Tab2){ref-type="table"}.Table_ _2**Prevalence_ of headache _among_ the health workers**General prevalence of headache in the past 6 monthsVariablesFrequencyPercentHeadache present11788.0Headache absent1612.0**Sex prevalence of headache**Male (%)Female (%)Headache present62 (87.3)55 _(88.7)Headache_ absent9 (12.7)7 (11.3)Total71 (100.0)62 (100.0)χ^2^ = _0.060;_ P value = 0.806Type of headachePrimary44 _(71.0)42_ (76.4)\*Secondary18 (29.0)13 (23.6)Total62 (100.0)55 _(100.0)χ^2^_ = 0.436; P value _=_ 0.509\*Secondary _headache_ is headache with a definitive and _identifiable_ cause found for it _i.e._ those with pre-existing conditions that may _cause_ the headache e.g. hypertension, cervical _spondylosis,_ refractive error, sleep apnoea, malaria and other febrile conditions \[[@CR15]\]. Most respondents reported ≤5 episodes of headache in the last _6_ _months_ (74.4%) and _these_ were typically of short-lasting _durations,_ \<60 minutes _(44.4%)._ _There_ _was_ no observed periodicity to the _headaches_ in 57.3% _of_ cases (see Table [3](#Tab3){ref-type="table"}). Most of _the_ headaches were not located in any particular part _of_ the head _or_ side-locked _(71.7%);_ _were_ described as mildly _severe_ in 59.8% of _cases_ while 88.0% _of_ respondents did _not_ suffer any sleep disruption. The headaches were _often_ not significantly disabling (73.4%) and in 93.2% of respondents did not _lead_ to absenteeism or affect productivity at work (Table [4](#Tab4){ref-type="table"}).Table 3**Characterization of the headaches**Variable/CharacteristicsFrequency (N =117)Percent***Number of _episodes_ _in_ _past_ 6 months***1 -- 58774.46 -- _102521.411_ -- 1532.616 -- 2021.6**Usual duration of headaches**Seconds1916.2Minutes5244.4Hours3630.9Days108.5***Usual time of day of the headache***Morning1815.4Afternoon1512.8Night1311.1Continuous43.4No particular time6757.3***Is _the_ headache becoming _stronger,_ last longer or occur more _frequent?***Yes2218.8No9581.2**What_ _is_ the _commonest_ nature of the headache?**Throbbing/exploding4336.8Sharp43.4Tightness54.3Dull65.1Aching2420.5Pressure in head3227.3Grinding32.6Table 4**Usual location and severity of the headache*Usual Location _of_ headache***FrequencyPercentLeft side32.6Forehead97.7Around the head/ill-defined119.4Right side21.7Both Temples21.7Top of the _head10.9Neck21.7Back_ of head32.6No particular side8471.7***Severity _of_ headache***Mild7059.8Moderate4538.5severe21.7***Is _the_ headache _strong_ enough to wake you from sleep?*** |
INTRODUCTION
============
Silicon (Si), in the form of dissolved silicic acid---often referred to as silicate and hereafter abbreviated as DSi---is an inorganic nutrient instrumental to ocean functioning. Its availability modulates processes as relevant as ocean primary productivity ([@R1]) and the exchange of CO~2~ with the atmosphere ([@R2]). Regional patterns of DSi availability largely result from the consumption of this nutrient by marine organisms (silicifiers) to build their skeletons of biogenic silica (BSi), with diatom utilization among the best known and quantified ([@R1], [@R3]). However, sponges, radiolarians, silicoflagellates, choanoflagellates, testate amoebae, and chrysophyceans, among others, also consume DSi in the ocean ([@R4]), but their activity remains poorly quantified and little understood from a physiological and molecular perspective. In the diatoms, the kinetics of DSi uptake have been investigated in a large variety of species, all of which were reported initially to follow a saturable Michaelis-Menten model. It is also that those saturable kinetics shift into nonsaturable uptake when DSi availability drastically increases and/or under particular physiological conditions ([@R5], [@R6]). Likewise, membrane silicon transporters (SITs) incorporating actively ambient DSi into the diatom cell have long been described and, although passive transporters have not been identified yet, a diffusion-based uptake has been described for at least some diatoms at high DSi availability \[reviewed in ([@R7])\]. In contrast, little is known about these physiological and molecular processes in other groups of marine silicifiers. Because diatoms are restricted to the photic zone of the ocean, a major gap in knowledge relative to DSi utilization in the dark ocean by nonphototrophic silicifiers persists. The discoveries that extensive aggregations of highly silicified sponges are common in the deep sea ([@R8]), that they can accumulate substantial amounts of BSi at the regional scale ([@R9], [@R10]), and that they trigger significant losses of BSi from the ocean ([@R11]) have raised considerable interest in deciphering how Si is processed in these singular, sponge-dominated, deep-sea systems. The lack of knowledge regarding these processes in sponges currently hinders the ability to understand the Si utilization in the dark ocean and makes it difficult to model adequately the role of the biological component in the marine biogeochemical cycle of silicon ([@R3], [@R11]).
Information on the physiology of DSi consumption by sponges is sparse and derived exclusively from shallow-water species in the class Demospongiae. These studies indicate Michaelis-Menten kinetics but with optimal DSi consumption attained at environmental DSi concentrations of \>100 μM ([@R12]--[@R16]). Because DSi concentrations higher than 100 µM are virtually never reached in the shallow waters of the modern ocean ([@R17]), the skeletal growth of all shallow-water demosponges investigated to date is therefore chronically limited by Si availability ([@R15], [@R16], [@R18]--[@R20]). Whether this kinetic limitation also applies to sponges in the class Hexactinellida---deep-sea specialists characterized by impressive siliceous skeletons---remains unknown. The physiology of DSi consumption and the molecular pathways of DSi uptake remain largely uninvestigated in hexactinellids, hampered by impediments to conducting in situ and laboratory experimentation with such relatively large and delicate deep-sea animals. Nevertheless, the interest is enormous. Major differences in the kinetics of DSi utilization between the two major lineages of siliceous sponges (i.e., Demospongiae and Hexactinellida) cannot be discarded, as silicification in demosponges revolves around the activity of the nonsoluble silicatein enzyme ([@R21]), while the process in hexactinellids appears to be governed by a phylogenetically unrelated, soluble enzyme, glassin ([@R22]). In addition, because hexactinellids have essentially syncytial organization while demosponges have a conventional cellular organization, the membrane transporters involved in Si utilization may not be shared but may be lineage specific instead. Very little is known on molecular Si transport in demosponges ([@R23], [@R24]) and nothing in hexactinellids. This lack of knowledge obscures the understanding of the evolution of the biosilicification process in the animal kingdom and its relationships to that in other organisms.
Here, we characterized experimentally the kinetics of DSi consumption in the hexactinellid sponge *Vazella pourtalesii* (Schmidt, 1870), a rosellid distributed from \~100 to 935 m in the northwest Atlantic that forms extensive monospecific aggregations on the deep continental shelf off Nova Scotia ([Fig. 1A](#F1){ref-type="fig"} and fig. S1), eastern Canada ([@R25]). We tested the laboratory-based kinetic model by comparing its predictions to both in situ determinations of DSi consumption rates using incubation chambers ([Fig. 1, B to D](#F1){ref-type="fig"}, and movies S1 and S2) and rates of BSi production derived from individuals of a known age grown in the wild on an artificial substrate ([Fig. 1, E and F](#F1){ref-type="fig"}). In combination with those physiological experiments, we conducted a quantitative large-scale assessment of gene expression as a function of DSi availability. The results of this study offer a mechanistic explanation for the kinetics of DSi utilization in hexactinellids and provide fresh insights into the molecular systems of Si transport and their evolution within sponges and across other silicifying organisms.
{#F1}
RESULTS
=======
Modeling and testing the physiology of DSi consumption
------------------------------------------------------
Live sponges were collected using the remotely operated vehicle (ROV) Remotely Operated Platform for Ocean Sciences (ROPOS) and taken to the laboratory for incubation in progressively increasing DSi concentrations (12, 30, 60, 100, 150, 200, and 250 μM DSi; see Materials and Methods). Initially, all 11 assayed individuals increased their DSi consumption rate in response to the progressive increase of DSi availability in the seawater ([Fig. 2, A and B](#F2){ref-type="fig"}, and tables S1 and S2). As also known for demosponges, the DSi consumption rate notably varied among individuals, with an average maximum consumption of 0.106 ± 0.050 μmol Si per milliliter of sponge tissue and per hour (hereafter given as μmol Si ml^−1^ hour^−1^) at an average DSi concentration of 150.9 ± 69.3 μM. Over that concentration threshold, the consumption rate of most individuals did not increase with increasing DSi availability, revealing that the Si transport system reaches the maximum speed (i.e., optimal utilization) at about 150 μM DSi and saturates at higher concentrations.
{#F2}
Unlike in all demosponges studied to date, the model best fitting the average DSi consumption rate of *V. pourtalesii* in response to DSi availability did not follow Michaelis-Menten kinetics (*r*^2^ = 0.841, *P* = 0.004; [Fig. 2A](#F2){ref-type="fig"}). An exponential rise to a maximum (ERM) model showed the best fit (*r*^2^ = 0.898, *P* = 0.002; [Fig. 2, A and B](#F2){ref-type="fig"}) to the empirical data on DSi consumption as a function of DSi availability, "consumption rate = *a* (1 − *b*^\[DSi\]^)". Although the difference in statistical fit between the "Michaelis-Menten" and "ERM" models was apparently small, the ERM model was built on parameters with higher statistical significance (*a* = 0.093 ± 0.008, *P* \< 0.001 | introduction = = = = = = = = = = = = silicon ( si ), in the form of dissolved silicic acid - - - often referred to as silicate and hereafter abbreviated as dsi - - - is an inorganic nutrient instrumental to ocean functioning. its availability modulates processes as relevant as ocean primary productivity ( [ @ r1 ] ) and the exchange of co ~ 2 ~ with the atmosphere ( [ @ r2 ] ). regional patterns of dsi availability largely arise from sustainable consumption of this nutrient by marine organisms ( silicifiers ) to build their skeletons of biogenic silica ( bsi ), with diatom utilization among the best known and quantified ( [ @ r1 ], [ @ r3 ] ). however, sponges, radiolarians, silicoflagellates, choanoflagellates, testate organisms, and bacteria, among others, also consume dsi in the ocean ( [ @ r4 ] ), but their activity remains poorly quantified and little understood from a physiological and molecular perspective. unlike the diatoms, the kinetics of dsi uptake have been investigated in a large variety animal species, all of which were reported initially to follow a saturable michaelis - menten model. it is also that those saturable kinetics shift into nonsaturable uptake when dsi availability drastically increases and / or under particular physiological conditions ( [ @ r5 ], [ @ r6 ] ). likewise, membrane silicon transporters ( sits ) incorporating actively ambient dsi into the diatom cell have long been described and, although passive transporters have not been identified yet, a diffusion - mediated uptake has been described for at least some diatoms at high dsi availability \ [ reviewed in ( [ @ r7 ] ) \ ]. in contrast, little is known about these physiological and molecular processes in other groups of marine silicifiers. because diatoms are restricted to the restricted zone of the ocean, a major gap in knowledge relative to dsi utilization in the dark ocean by nonphototrophic silicifiers persists. the perception that extensive aggregations of highly silicified sponges are common in the deep sea ( [ @ r8 ] ), that plants can accumulate substantial amounts of bsi at the regional scale ( [ @ r9 ], [ @ r10 ] ), and that they trigger significant losses of bsi from the ocean ( [ @ r11 ] ) have raised considerable interest in deciphering how si is processed in these singular, sponge - dominated, deep - sea systems. the lack of knowledge regarding these processes in sponges currently hinders the ability to understand the si utilization in the dark ocean and makes it difficult to model adequately the role of the biological component in the marine biogeochemical cycle of silicon ( [ @ r3 ], [ @ r11 ] ). information on the physiology of dsi consumption by sponges is sparse and derived exclusively from shallow - water species in the class demospongiae. these studies indicate michaelis - menten kinetics but with optimal dsi consumption attained at environmental dsi concentrations of \ > 100 μm ( [ @ r12 ] - - [ @ r16 ] ). because dsi concentrations higher than 100 µm are virtually never reached in the shallow waters of the modern ocean ( [ @ r17 ] ), the skeletal growth of all shallow - water demosponges investigated to date is therefore chronically limited by si availability ( [ @ r15 ], [ @ r16 ], [ @ r18 ] - - [ @ r20 ] ). whether this kinetic limitation also applies to sponges in the class hexactinellida - - - deep - sea specialists characterized by impressive siliceous skeletons - - - remains unknown. the physiology of dsi consumption and the molecular pathways of dsi uptake remain largely uninvestigated in hexactinellids, hampered by impediments to conducting in situ and laboratory experimentation with such relatively large and delicate deep - sea animals. nevertheless, the interest is enormous. major differences in the kinetics of dsi utilization between the two major lineages of siliceous sponges ( i. e., demospongiae and hexactinellida ) cannot be discarded, as silicification in demosponges revolves around the activity of the nonsoluble silicatein enzyme ( [ @ r21 ] ), while the process in hexactinellids appears to be governed by a phylogenetically unrelated, soluble enzyme, glassin ( [ @ r22 ] ). in addition, because hexactinellids have essentially syncytial organization while demosponges have a conventional cellular organization, the membrane transporters involved in si utilization may not be shared but may be lineage specific instead. very little is known on molecular si transport in demosponges ( [ @ r23 ], [ @ r24 ] ) and nothing in hexactinellids. this lack of knowledge obscures the understanding of the evolution of the biosilicification process in the animal kingdom and its relationships to that in other organisms. here, we characterized experimentally the kinetics of dsi consumption in the hexactinellid sponge * vazella pourtalesii * ( schmidt, 1870 ), a rosellid distributed from \ ~ 100 to 935 m in the northwest atlantic that forms extensive monospecific aggregations on the deep continental shelf off nova scotia ( [ fig. 1a ] ( # f1 ) { ref - type = " fig " } and fig. s1 ), eastern canada ( [ @ r25 ] ). we tested the laboratory - based kinetic model by comparing its predictions to both in situ determinations of dsi consumption rates using incubation chambers ( [ fig. 1, b to d ] ( # f1 ) { ref - type = " fig " }, and movies s1 and s2 ) and rates of bsi production derived from individuals of a known age grown in the wild on an artificial substrate ( [ fig. 1, e and f ] ( # f1 ) { ref - type = " fig " } ). in combination with those physiological experiments, we conducted a quantitative large - scale assessment of gene expression as a function of dsi availability. the results of this study offer a mechanistic explanation for the kinetics of dsi utilization in hexactinellids and provide fresh insights into the molecular systems of si transport and their evolution within sponges and across other silicifying organisms.! [ imagery depicting various aspects of field work. \ ( * * a * * ) general view of the aggregation of * v. pourtalesii * in the sambro bank sponge conservation area in emerald basin. ( * * b * * ) collected sponge transferred to the floor piece of the incubation chamber. ( * * c * * ) sponge enclosed in incubation unit, which is being clutched by the rov arm for deployment on the seabed. ( * * d * * ) incubation unit deployed on the sponge ground. ( * * e * * ) recovered ocean tracking network ( otn ) mooring with * v. pourtalesii * ( arrows ) recruitment. scale bar, 25 cm. ( * * f * * ) close - up of a sponge recruited on the mooring showing its protruding bsi skeleton. scale bar, 1 cm. pictures ( a ) to ( d ) are frames from movies extracted and processed by m. maldonado ( ceab - csic ). pictures ( e ) and ( f ) were taken by m. maldonado ( ceab - csic ). ] ( aba9322 - f1 ) { # f1 } results = = = = = = = modeling and testing the physiology of dsi consumption - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - live sponges were collected using the remotely operated vehicle ( rov ) remotely operated platform for ocean sciences ( ropos ) and taken to the laboratory for incubation in progressively increasing dsi concentrations ( 12, 30, 60, 100, 150, 200, and 250 μm dsi ; see materials and methods ). initially, all 11 assayed individuals increased their dsi consumption rate in response to the progressive increase of dsi availability in the seawater ( [ fig. 2, a and b ] ( # f2 ) { ref - type = " fig " }, and tables s1 and s2 ). as also known for demosponges, the dsi consumption rate notably varied among individuals, with an average maximum consumption of 0. 106 ± 0. 050 μmol si per milliliter of sponge tissue and per hour ( hereafter given as μmol si ml ^ −1 ^ hour ^ −1 ^ ) at an average dsi concentration of 150. 9 ± 69. 3 μm. over that concentration threshold, the consumption rate of most individuals did not increase with increasing dsi availability, revealing that the si transport system reaches the maximum speed ( i. e., optimal utilization ) at about 150 μm dsi and saturates at higher concentrations.! [ summary of dsi consumption as a function of experimental dsi availability. \ ( * * a * * ) dsi consumptions of 11 individuals of * v. pourtalesii * as a function of experimental silicic acid ( dsi ) concentration in the laboratory. the averaged response fits an erm model ( blue lines ) better than a michaelis - menten ( mm ) kinetics ( red lines ). ( * * b * * ) statistics of the average consumption ( ±sd ) best fitting to an erm model. note that dsi consumption calculated both from in situ incubations and from bsi produced under field conditions fall within the 95 % confidence band of the model. ] ( aba9322 - f2 ) { # f2 } unlike in all demosponges studied to date, the model best fitting the average dsi consumption rate of * v. pourtalesii * in response to dsi availability did not follow michaelis - menten kinetics ( * r * ^ 2 ^ = 0. 841, * p * = 0. 004 ; [ fig. 2a ] ( # f2 ) { ref - type = " fig " } ). an exponential rise to a maximum ( erm ) model showed the best fit ( * r * ^ 2 ^ = 0. 898, * p * = 0. 002 ; [ fig. 2, a and b ] ( # f2 ) { ref - type = " fig " } ) to the empirical data on dsi consumption as a function of dsi availability, " consumption rate = * a * ( 1 − * b * ^ \ [ dsi \ ] ^ ) ". although the difference in statistical fit between the " michaelis - menten " and " erm " models was apparently small, the erm model was built on parameters with higher statistical significance ( * a * = 0. 093 ± 0. 008, * p * \ < 0. 001 | INTRODUCTION = = = = = = = = = = = = Silicon (Si ), in the form of dissolved silicic acid - - - often referred to as silicate and hereafter abbreviated as DSi - - - is an inorganic nutrient instrumental to ocean functioning. Its availability modulates processes as relevant as ocean primary productivity ([ @ R1] ) and the exchange of CO ~ 2 ~ with the atmosphere ([ @ R2] ). Regional patterns of DSi availability largely result from the consumption of this nutrient by marine organisms (silicifiers) to build their skeletons of biogenic silica (BSi ), with diatom utilization among the best known and quantified ([ @ R1 ], [@ R3] ). However, sponges, radiolarians, silicoflagellates, choanoflagellates, testate amoebae, and chrysophyceans, among others, also consume DSi in the ocean ([ @ R4] ), but their activity remains poorly quantified and little understood from a physiological and molecular perspective. In the diatoms, the kinetics of DSi uptake have been investigated in a large variety of species, all of which were relorred initially to follow a saturable Michaelis - Menten model. It is also that those saturable kinetics shift into nonsaturable uptake when DSi availability drastically increases and / or unee3 particular physiological conditions ([ @ R5 ], [@ R6] ). Likewise, membrane silicon transporters (SITs) incorporating actively ambient DSi into the diatom cell have long been described and, although passive transporters have not been identified yet, a diffusion - based uptake has been described for at least some diatoms at high DSi availability \ [reviewed in ([ @ R7] ) \ ]. In contrast, little is known about these physiological and molecular processes in other groups of marine silicifiers. Because diatoms are restricted to the photic zone of the ocean, a major gap in knowledge relative to DSi utilization in the dark ocean by nonphototrophic silicifiers persists. The discoveries that extensive aggregations of highly silicified sponges are common in the deep sea ([ @ R8] ), that they can accumulate substantial amounts of BSi at the rdgiknal scale ([ @ R9 ], [@ R10] ), and that they trigger significant losses of BSi from the ocean ([ @ R11] ) have raised considerable interest in deciphering how Si is processed in these singular, sponge - dominated, deep - sea systems. The lack of knowledge regarding these processes in sponges currently hinders the ability to understand the Si utilization in the dark ocean and makes it difficult to model adequately the role of the biological component in the marine biogeochemical cycle of silicon ([ @ R3 ], [@ R11] ). Information on the physiology of DSi consumption by sponges is sparse and derived exclusively from shallow - water species in the class Demospongiae. These studies indicate Michaelis - Menten kinetics but with optimal DSi consumption attained at environmental DSi concentrations of \> 100 μM ([ @ R12] - - [@ R16] ). Because DSi concentrations higher than 100 µM are virtually never reached in the shallow waters of the modeGJ ocean ([ @ R17] ), the skeletal growth of all shallow - water demosponges investigated to date is therefore chronically limited by Si availability ([ @ R15 ], [@ R16 ], [@ R18] - - [@ R20] ). Whether this kinetic limitation also applies to sponges in the class Hexactinellida - - - deep - sea specialists characterized by impressive siliceous skeletons - - - remains unknown. The physiology of DSi consumption and the molecular pathways of DSi uptake remain largely uninvestigated in hexactinellids, hampered by impediments to conducting in situ and laboratory experimentation with such relatively large and delicate deep - sea animals. Nevertheless, the interest is enormous. Major differences in the kinetics of DSi utilization between the two major lineages of siliceous sponges (i. e. , Demospongiae and Hexactinellida) cannot be discarded, as silicification in demosponges revolves around the activity of the nonsoluble silicatein enzyme ([ @ R21] ), while the (roceZs in hexactinellids appears to be governed by a phylogenetically unrelated, soluble enzyme, glassin ([ @ R22] ). In addition, because hexactinellids have essentially syncytial organization while demosponges have a conventional cellular organization, the membrane transporters involved in Si utilization may not be shared but may be lineage specific instead. Very little is known on molecular Si transport in demosponges ([ @ R23 ], [@ R24] ) and nothing in hexactinellids. This lack of knowledge obscures the understanding of the evolution of the biosilicification process in the animal kingdom and its relationships to that in other organisms. Here, we characterized experimentally the kinetics of DSi consumption in the hexactinellid sponge * bazelia pourtalesii * (Schmidt, 1870 ), a rosellid distributed from \ ~ 100 to 935 m in the northwest Atlantic that forms extensive monospecific aggregations on the deep continental shelf off Nova Scotia ([ Fig. 1A] (# F1) {ref - type = " fig "} and fig. S1 ), eastern Canada ([ @ R25] ). We tested the laboratory - based kinetic model by comlAring its predictions to both in situ determinations of DSi consumption rates using incubation chambers ([ Fig. 1, B to D] (# F1) {ref - type = " fig " }, and movies S1 and S2) and rates of BSi production derived from individuals of a known age grown in the wild on an artificial substrate ([ Fig. 1, E and F] (# F1) {ref - type = " fig "} ). In combination with those physiological experiments, we conducted a quantitative large - scale assessment of gene expression as a function of DSi availability. The results of this study offer a mechanistic explanation for the kinetics of DSi utilization in hexactinellids and provide fresh insights into the molecular systems of Si transport and their evolution within sponges and across other silicifying organisms. ! [Imagery depicting various aspects of field work. \ (* * A * *) General view of the aggregation of * V. pourtalesii * in the Sambro Bank Sponge Conservation Area in EmerxlR Basin. (* * B * *) Collected sponge transferred to the floor piece of the incubation chamber. (* * C * *) Sponge enclosed in incubation unit, which is being clutched by the ROV arm for deployment on the seabed. (* * D * *) Incubation unit deployed on the sponge ground. (* * E * *) Recovered Ocean Tracking Network (OTN) mooring with * V. pourtalesii * (arrows) recruitment. Scale bar, 25 cm. (* * F * *) Close - up of a sponge recruited on the mooring showing its protruding BSi skeleton. Scale bar, 1 cm. Pictures (A) to (D) are frames from movies extracted and processed by M. Maldonado (CEAB - CSIC ). Pictures (E) and (F) were taken by M. Maldonado (CEAB - CSIC ).] (aba9322 - F1) {# F1} RESULTS = = = = = = = Modeling and testing the physiology of DSi consumption - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Live sponges were collected using the remotely operated vehicle (ROV) Remotely Operated Platform for Ocean Sciences (ROPOS) and taken to the laboratory for incubation in progressively increasing DSi concentrations (12, 30, 60, 100, 150, 200, and 250 μM DSi; see Materials and Methods ). Initially, all 11 assayed individuals increased their DSi consumption rate in response to the progressive increase of DSi availability in the seawater ([ Fig. 2, A and B] (# F2) {ref - type = " fig " }, and tables S1 and S2 ). As also known for demosponges, the DSi consumption rate notably varied among individuals, with an average maximum consumption of 0. 106 ± 0. 050 μmol Si per milliliter of sponge tissue and per hour (hereafter given as μmol Si ml ^ − 1 ^ hour ^ − 1 ^) at an average DSi concentration of 150. 9 ± 69. 3 μM. Over that concentration threshold, the consumption rate of most individuals did not increase with increasing DSi availability, revealing that the Si transport sysH3m reaches the maximum speed (i. e. , optimal utilization) at about 150 μM DSi and saturates at higher concentrations. ! [Summary of DSi consumption as a function of experimental DSi availability. \ (* * A * *) DSi consumptions of 11 individuals of * V. pourtalesii * as a function of experimental silicic acid (DSi) concentration in the laboratory. The averaged response fits an ERM model (blue lines) better than a Michaelis - Menten (MM) kinetics (red lines ). (* * B * *) Statistics of the average consumption (± SD) best fitting to an ERM model. Note that DSi consumption calculated both from in situ incubations and from BSi produced under field conditions fall within the 95% confidence band of the model.] (aba9322 - F2) {# F2} Unlike in all demosponges studied to date, the model best fitting the average DSi consumption rate of * V. pourtalesii * in response to DSi availability did not follow Michaelis - Menten kinetics (* r * ^ 2 ^ = 0. 841, * P * = 0. 004; [Fig. 2A] (# F2) {ref - type = " fig "} ). An exponenHiWl rise to a maximum (ERM) model showed the best fit (* r * ^ 2 ^ = 0. 898, * P * = 0. 002; [Fig. 2, A and B] (# F2) {ref - type = " fig "} ) to the empirical data on DSi consumption as a function of DSi availability, " consumption rate = * a * (1 − * b * ^ \ [DSi \] ^) ". Although the difference in statistical fit between the " Michaelis - Menten " and " ERM " models was apparently small, the ERM model was built on parameters with higher statistical significance (* a * = 0. 093 ± 0. 008, * P * \ <0. 001 | INTRODUCTION ============ Silicon (Si), in the form of dissolved silicic acid---often referred to as silicate and hereafter abbreviated as an inorganic nutrient instrumental to ocean functioning. Its availability modulates processes as relevant as ocean productivity and the exchange of with the atmosphere ([@R2]). Regional patterns of DSi availability largely result from the of this nutrient by organisms (silicifiers) to build their skeletons of biogenic silica (BSi), with diatom utilization among the known and quantified ([@R1], [@R3]). However, sponges, silicoflagellates, choanoflagellates, testate amoebae, and chrysophyceans, among others, also consume DSi in the ocean but activity remains poorly quantified and little understood a physiological and molecular perspective. In the diatoms, the kinetics of DSi uptake have been investigated in a large of species, all of which were reported initially to follow a saturable Michaelis-Menten model. It is also that those saturable kinetics shift into nonsaturable uptake when DSi availability drastically increases and/or under particular physiological conditions ([@R5], [@R6]). Likewise, membrane silicon transporters (SITs) incorporating actively ambient DSi into the diatom cell have long been described and, although passive transporters have not been identified yet, diffusion-based uptake been described for at least diatoms at high DSi availability \[reviewed In contrast, little is known about these physiological and molecular processes in of marine silicifiers. Because are restricted to the photic zone of the ocean, a major gap in relative to DSi utilization the dark ocean by nonphototrophic silicifiers persists. The discoveries that extensive aggregations of highly sponges are common in the deep sea ([@R8]), that they accumulate substantial amounts of BSi at the regional scale ([@R9], [@R10]), and that trigger losses of BSi from the ([@R11]) have raised considerable interest in deciphering how Si is processed these sponge-dominated, systems. The lack of knowledge regarding these processes in sponges currently hinders the ability to understand Si utilization in the dark ocean makes it difficult to model adequately the role the biological component in the biogeochemical cycle of silicon ([@R3], Information on the physiology of DSi consumption sponges is sparse derived exclusively from shallow-water species in the class Demospongiae. These studies indicate Michaelis-Menten kinetics but with optimal DSi consumption attained at environmental DSi concentrations of \>100 μM ([@R12]--[@R16]). Because DSi higher than 100 µM are virtually never reached in the shallow waters of the modern ocean the skeletal growth of all shallow-water demosponges investigated to date is therefore chronically limited by availability ([@R15], [@R16], [@R18]--[@R20]). Whether this kinetic also applies to sponges the class specialists characterized by impressive siliceous skeletons---remains unknown. physiology of DSi consumption and the molecular pathways of DSi uptake remain largely uninvestigated in hexactinellids, hampered by impediments conducting in situ and laboratory with such relatively delicate deep-sea animals. the interest is enormous. Major differences in the kinetics of DSi utilization between the two major lineages of siliceous sponges (i.e., Demospongiae and Hexactinellida) be as silicification in demosponges revolves around the activity of the nonsoluble enzyme ([@R21]), while the process in hexactinellids to be governed by a phylogenetically unrelated, soluble enzyme, glassin ([@R22]). In addition, because hexactinellids have essentially syncytial organization while demosponges have a conventional cellular organization, the membrane transporters involved in Si utilization may not be shared but may be lineage specific instead. is known on molecular Si transport in demosponges ([@R23], [@R24]) and in hexactinellids. This lack of knowledge obscures the understanding of of the biosilicification process in the animal kingdom and its relationships to that in other organisms. Here, we characterized experimentally the kinetics of DSi consumption in the hexactinellid sponge *Vazella pourtalesii* 1870), a rosellid distributed \~100 935 m in the northwest Atlantic forms extensive monospecific aggregations on the deep continental shelf off Nova Scotia ([Fig. 1A](#F1){ref-type="fig"} and fig. S1), eastern Canada ([@R25]). We tested the laboratory-based kinetic model by comparing its predictions to in situ determinations of DSi rates using incubation chambers ([Fig. B to D](#F1){ref-type="fig"}, and movies S1 and S2) and rates of BSi production derived from of a known age in wild on an artificial substrate ([Fig. E and F](#F1){ref-type="fig"}). In combination with those physiological experiments, we conducted a quantitative large-scale assessment of expression as function of availability. The results of this study offer a mechanistic explanation for kinetics of DSi utilization in provide fresh insights into the molecular systems of Si and evolution within sponges and other silicifying organisms. {#F1} RESULTS ======= Modeling and testing the physiology of DSi consumption ------------------------------------------------------ Live sponges were collected using the remotely vehicle (ROV) Operated Platform for Ocean Sciences (ROPOS) and taken to the laboratory for incubation in progressively increasing DSi concentrations (12, 30, 60, 100, 150, 200, and 250 μM see Materials and Methods). Initially, all 11 assayed increased their DSi rate in response to the progressive increase of DSi availability in the seawater ([Fig. 2, A and B](#F2){ref-type="fig"}, and S1 S2). As also known for demosponges, the DSi consumption rate varied among individuals, with an average maximum consumption of 0.106 ± μmol Si per milliliter of sponge tissue and hour given as μmol Si ml^−1^ hour^−1^) at an average DSi concentration of 150.9 ± 69.3 μM. Over that concentration threshold, the consumption rate of most individuals did not increase with increasing DSi availability, revealing that the Si transport system reaches the maximum speed (i.e., optimal utilization) at about 150 μM DSi and saturates at higher concentrations. {#F2} in all demosponges to date, the best fitting the average DSi rate of *V. in to DSi availability did not follow Michaelis-Menten kinetics (*r*^2^ = 0.841, *P* 0.004; [Fig. 2A](#F2){ref-type="fig"}). exponential rise to a (ERM) model showed the best fit (*r*^2^ = 0.898, *P* = [Fig. 2, A and data on DSi as function of DSi "consumption rate = (1 − *b*^\[DSi\]^)". Although the difference in statistical fit between the "Michaelis-Menten" and "ERM" models was apparently small, the ERM model was built on parameters with higher statistical significance (*a* = 0.093 ± 0.008, *P* \< 0.001 | INTRoducTIOn
============
SILICOn (si), iN The fORM oF dIsSolVEd SilicIC acId---OFTen rEFerrED TO As sILIcAtE aND hEREAfTeR AbBReViatED aS DsI---Is AN InORGAniC nutrieNt INSTRUmeNtaL TO OcEan fUnctiONing. iTS avAIlabILiTY ModuLaTeS pRoCeSSES as reLeVaNt AS oCean PRiMaRy pRoDuctIvITY ([@r1]) aND tHe eXCHAnge of Co~2~ WiTH thE ATmOSPHeRE ([@R2]). REgioNaL PatTErns of dsI AVaiLAbiliTy LARgeLY reSUlt fRoM ThE cOnSumpTiON of ThiS nuTRiEnT bY MaRInE OrganismS (SIlIcIfierS) to BUild theIR SkeLEtons OF biOgENic sIliCa (Bsi), WitH DIatOM UtILIZATioN aMONG THE bEST known and QUANTIfiED ([@R1], [@R3]). HOWeVeR, SpONgEs, radioLArians, SilIcoFLAGELlaTEs, cHoAnofLAGELlAtEs, tEstate AmoEbae, AND cHrYsophYCEaNS, AmonG OTHErS, AlsO CONSuMe dsi in thE oceaN ([@r4]), BUt THEiR actIViTY rEmAiNS poORlY quaNTIfIed aNd LITtLe UNDeRStOOD froM A pHYSIOlOgiCal And MOleCulaR perSpEctivE. iN tHE diaTOms, tHE KINEtICs of DSI UPTAke hAVE BeEN invEsTIGAtEd IN a lARGE varIETy oF sPeCiES, All oF WHIch weRE RePorTED initiAlLy tO FoLlow a sATUrABLE micHAelis-mENtEn MOdEl. It iS AlSO tHAT ThoSe saTURAbLE KINeTics shifT iNtO nOnSAtUraBLE uPTakE WhEn DsI aVAILaBiLity DrasticALLy IncreASeS AnD/Or uNdeR pARTICulAr PHYSiolOGical cONDITiONS ([@r5], [@R6]). LikEWIsE, MembRANe Silicon TRaNsPoRTeRs (siTs) IncorPoRAtInG ACTIVeLy ambiEnT dsI inTO the diATom CeLL HAVE lONg bEen DEscRiBed aND, aLThOUGh pASsiVE TrANSpOrTErS HavE not BEen iDeNtIfiEd yET, a DifFuSIon-basED UPtake HAs BEEn dEscRibED fOr AT lEAST sOME dIatomS At HiGH dsi AVailAbiliTY \[rEVIewEd In ([@r7])\]. In coNtraSt, LITtLe is knoWN About thEse pHysiolOGICal aND MOLECular PRocESsEs IN OtheR GRoUPS Of MarINe SiliciFiErS. BECAuSE DIaTOmS arE rEsTRIctEd tO tHE PHotIC ZoNe oF ThE OceaN, A MAJOR gAP in kNoWleDgE ReLatIve to dsI UTiLizAtION in tHe DaRK ocEAn by nOnphoTotROPhIC SIlICIfiers PerSiStS. ThE DiscoverIEs ThAT exTenSiVE aGGrEgatioNS of hIGhLY sILicIfIED sPOnGeS arE COMMOn in the deep Sea ([@R8]), thaT THeY caN AccUmulaTE sUbstaNtial amouNts oF BsI at The REgionAL scAlE ([@R9], [@R10]), AND thaT THEY triGGer SIGNIFIcaNt losSES oF bsI frOm ThE oCeaN ([@r11]) HaVe rAISED coNSIdeRABLe iNteReSt IN dEciPhErinG How SI iS PROCessEd In these siNGUlAR, SPONGE-DomInated, dEep-sEA SysTeMs. the lAcK oF KnoWlEdGe REgardiNG THese pRocesses In SPonGEs CURrenTLy hINDeRS THe ABILITy tO uNDErstaND ThE sI UtIlizAtion IN ThE DaRK OceaN anD MAKEs it dIFfIcULt TO modEl adeqUatELy The roLE Of The bioloGical coMPoNEnt iN THe MARINe BIOgEOChemICal CYclE oF SILiCoN ([@R3], [@r11]).
infORMATIon oN the pHYSIolOgy OF dsI coNSUMpTion by spoNgEs is Sparse and DERiveD ExcLUSIVElY FRom sHAlLOw-wAter SPeCIes in the clAsS DEMOSpONgIAe. tHESE sTUdieS indicate MICHaeLiS-MentEn KINetiCS BUt witH oPtIMaL dSI cOnsUMPTiON ATTaiNed at eNViroNMeNtAl DsI cONcENtRAtIONs of \>100 μM ([@r12]--[@R16]). BEcauSe dSi cOnceNtRaTioNs HIGheR THaN 100 ΜM Are viRTUALLY nEVer rEAcHED IN tHe sHallOW wAtERs Of ThE MOderN ocEAN ([@r17]), The skeLETAl GROwtH oF all shALLow-water DEmOSPOngeS InVESTiGAtEd To daTe Is THEREFoRe chrONiCalLY lImIteD By si aVaIlAbilitY ([@r15], [@r16], [@r18]--[@r20]). WhetHeR tHis kInETiC LIMITatION AlsO ApplIEs TO spONgeS In THE clASs HEXacTINeLliDa---DeEp-SEA SpecIaLiSTS CharActErIzEd By IMPREsSiVe sILICEouS SkELEtons---REMAInS UNknOwN. the PhysiOlOgY Of DSi cOnSuMPTION ANd thE MoLeCUlaR PathWAYS of dSI uPtaKE remAin LARgely uNiNVesTIGateD iN HeXAcTinEllIDS, HaMpERed By iMPedIMenTs to CoNdUcTiNG In SItu And lABORAtory EXPeRiMENTATiOn WIth sucH reLAtIVely larGe And DElICAte deEp-sEA anIMALs. neVErthELeSS, THe InteResT Is ENormoUs. mAJOr dIfFerEnCES iN THE kiNeTIcS of DSi UtilIZaTion bEtWEEN THe tWO MAjOR LineaGEs OF SiLICeous sponGES (I.E., DEmoSpOngiAE aNd hExActINeLlidA) cANnOT bE discarDEd, AS siliCIfICaTiOn IN DemOSPONGES REvOlVes AroUnD thE ACTiVITY of the NONsoluBLe SIlICaTeiN EnzYmE ([@R21]), WHilE ThE PRoceSS IN HeXACTiNeLLiDS APPeaRs To BE GOveRNeD by A phylOgENetiCaLLy unrELATed, SoluBlE EnZymE, GlassIN ([@r22]). iN addiTIoN, BECAUse hEXACtineLLiDs HAVe ESSenTIALLy sYncyTial ORGaNIZaTiOn whILe DEMoSpoNgES havE a ConVENtIOnal CElLUlaR oRGaNizAtIoN, tHE meMBrANe TranspOrTERS InVOLVeD iN SI UTilIzAtIOn MAY NOT bE ShAREd bUT MaY Be lIneAGe SpECiFIC inSteaD. veRy liTtLe iS KnoWn oN moLEcUlAr si traNspORT IN dEMOSpoNGEs ([@R23], [@R24]) AND NoThINg iN HEXACtinelLidS. tHis LAck of kNOwlEDGe oBsCUres tHe undERstANdING OF THE EvolutIoN OF ThE biOSIlicifiCATiOn PROcESs in THE AnImAL kiNGDoM ANd ITs reLATiOnShiPS TO tHAt in oTher ORGAnisms.
herE, we charaCtEriZEd expERImEnTALly THe kINETIcs Of dSI conSuMPtioN iN THe hexactinelLId spONGE *VAzELLa POUrTALesIi* (ScHmIdt, 1870), a RoSELLId dISTRiBuTED frOM \~100 to 935 m iN tHe nOrthWesT aTlAntiC THat fOrMs EXtenSiVE moNosPecIFIc aGGreGATiOns on tHe dEeP cONTINeNTaL ShElF Off nova sCOTIa ([FIG. 1A](#F1){rEf-TYPE="Fig"} ANd fIG. s1), eastern CAnAdA ([@R25]). WE TEsTeD THE LABoratorY-BasED KiNETiC MoDeL by cOmpAriNG ITs PreDICTIonS to both in Situ detErmInaTIonS OF dsI coNsuMPTioN rATeS USInG incUBAtiOn CHaMBERS ([Fig. 1, B to D](#F1){ReF-tyPe="FIg"}, and mOVIeS s1 aNd s2) anD rAteS Of bsi prODuCTion DERIvED FROm InDIViDUAls Of a knoWN AGE GRown in ThE Wild On an aRtiFICiAL sUbstrAte ([FiG. 1, E anD F](#f1){reF-Type="fig"}). IN CombinatION wITH ThosE PhYsiOLOGIcAL expErImentS, wE coNDuctEd A QUAntitATIVe LarGe-ScALe aSSessMENT of GENE exPreSSion AS A FUnCtIOn of Dsi AVaIlaBIlITY. the REsultS of tHis STUdy ofFeR a mEcHAnIsTIC eXplanaTiON fOr tHE kINeTIcs Of dsI UTiLiZATioN IN HExaCtInelliDS and PROViDe FrEsh INSighTS InTO ThE mOlEculAr systEmS Of Si TraNSPoRT AnD thEIR EvOLUTiON wItHIN SPoNGeS AND AcRoss oTHEr SILICifyiNg OrgaNIsMs.
{#f1}
rESULTs
=======
ModeLiNG AnD tEsTINg ThE PHySiolOgy oF dSi CoNSumption
------------------------------------------------------
lIve SpONGES WeRe coLLectED usinG THe ReMOTeLy opeRaTeD VEHiclE (roV) RemOTElY operATed PLAtform for OCean sciEnces (ROPOs) anD tAkEN tO THe LaBOraTOry fOR iNcuBAtiON in PROGresSiVElY INCreasING dSi ConcentrAtioNs (12, 30, 60, 100, 150, 200, AnD 250 ΜM DSI; SEE matERialS aND MEthOds). inITiaLLy, AlL 11 asSAYED indivIDuals INCREASEd tHeiR DSi ConsUMPtiOn RATe iN respONSE TO tHe prOgresSiVE inCreAsE Of DsI aVaiLAbIlity In THE sEaWAtER ([fIg. 2, a anD b](#F2){REf-tYPE="fig"}, and TABlEs s1 AND s2). as AlsO KNOwN foR dEMOSPONgeS, THe DsI cONsUmPTioN raTE NOTAbly vAried AMOnG InDIvIduAlS, wITh An AVERage maxIMUM cONsUmpTIon of 0.106 ± 0.050 μmOl si per milLiLITER of sPonGe TiSSue AnD pEr HOuR (heREAfteR GiVEn As ΜMoL si ML^−1^ Hour^−1^) at an aVeRagE dsi cOnCENTRATion Of 150.9 ± 69.3 μm. OVeR thaT CoNCeNtRaTion thReshoLD, tHE cONsuMptIoN Rate Of MOsT INDiViduAlS did NoT iNCrEase WITH iNCrEAsiNG Dsi avAiLabIlITY, rEvEAlinG tHAt THe sI TrANspOrT systeM ReacHEs thE MaxImuM spEEd (I.e., OPTImaL UTILIzaTIOn) At ABouT 150 Μm dsi anD sAturatEs At hIGHEr coNCenTRAtiOnS.
{#F2}
Unlike In AlL dEmoSpOnGes stuDIed tO DatE, the modEl best fItting THE AvErAgE DsI CONsumpTiON RaTe oF *v. PoURtALesii* iN RESpONsE TO DSI AVailAbIliTy did noT foLlOw mICHaELis-mENten KiNeticS (*r*^2^ = 0.841, *p* = 0.004; [fiG. 2a](#f2){Ref-tYPe="fiG"}). aN ExponenTiAL riSE to a maximUM (erm) MOdeL ShOwed The BeST FIt (*R*^2^ = 0.898, *P* = 0.002; [fIG. 2, a AND B](#f2){rEf-typE="fig"}) tO THe EMPIrIcal daTA On dsi cONsUmPtION as A FUNctIOn of DSi AvailaBiLiTY, "cOnSuMPTioN RaTE = *a* (1 − *B*^\[Dsi\]^)". AlTHough thE DiFFEreNCE in STAtiSTicAL FIT bETWEEn The "MICHaelIS-MENTen" aND "ERm" moDels wAS APPaRENTly smaLl, tHE erm MoDEL WAS BuiLt on ParAMetErs wITH hiGhEr StAtistiCAl sigNiFicaNce (*a* = 0.093 ± 0.008, *P* \< 0.001 | INTRODUCTION ============ Silicon (Si), in the form of dissolved silicic acid---oftenreferred to as silicate and hereafter abbreviated as DSi---is an inorganic nutrientinstrumental to ocean functioning. Its availabilitymodulates processesas relevantas ocean primary productivity ([@R1]) and the exchange of CO~2~ with the atmosphere ([@R2]). Regional patterns of DSi availabilitylargely result from the consumption of thisnutrient by marine organisms (silicifiers)to build their skeletons of biogenic silica (BSi), with diatom utilizationamong the best known and quantified ([@R1], [@R3]). However,sponges, radiolarians, silicoflagellates, choanoflagellates, testate amoebae, andchrysophyceans, among others,also consume DSi in the ocean([@R4]), but theiractivityremains poorly quantified and little understood from a physiological and molecularperspective. In the diatoms, the kinetics of DSi uptake have been investigated in a large variety of species, allof whichwere reportedinitially to follow a saturable Michaelis-Menten model. It is also that those saturable kinetics shift into nonsaturable uptakewhen DSi availability drasticallyincreases and/or underparticular physiological conditions([@R5], [@R6]).Likewise, membrane silicon transporters (SITs)incorporating actively ambient DSi into the diatom cell have long been described and, although passive transportershave not beenidentified yet,a diffusion-based uptake has been described for at leastsome diatoms at high DSi availability\[reviewed in ([@R7])\]. In contrast, little is known about these physiological and molecular processes in other groups of marine silicifiers.Because diatomsare restricted to the photic zone of the ocean,a major gapin knowledge relative to DSi utilization in the dark ocean by nonphototrophic silicifiers persists. The discoveries that extensive aggregations of highly silicified sponges are common in the deepsea([@R8]), thatthey can accumulate substantial amounts of BSi at the regionalscale ([@R9], [@R10]), and that they trigger significant losses of BSi from the ocean ([@R11]) have raised considerable interest in decipheringhow Si is processed in these singular, sponge-dominated,deep-sea systems.The lack of knowledge regarding these processes in sponges currently hinders the ability tounderstand theSi utilizationin the dark ocean and makes it difficult to modeladequatelythe role of the biological component in the marine biogeochemical cycleof silicon ([@R3], [@R11]).Information on the physiologyof DSiconsumption by sponges is sparse and derived exclusively from shallow-water species in the class Demospongiae.These studies indicate Michaelis-Menten kineticsbut with optimal DSi consumption attainedat environmentalDSiconcentrations of \>100 μM ([@R12]--[@R16]). Because DSi concentrations higher than100 µM are virtually neverreached in theshallow waters of the modern ocean ([@R17]), the skeletal growth of all shallow-water demosponges investigated to date is therefore chronically limited by Siavailability ([@R15], [@R16], [@R18]--[@R20]). Whether this kinetic limitation also applies to sponges in the class Hexactinellida---deep-sea specialists characterized by impressive siliceous skeletons---remainsunknown. The physiology of DSi consumption and the molecular pathways of DSi uptake remain largely uninvestigated inhexactinellids, hampered by impediments to conducting in situ and laboratory experimentation with such relatively large and delicate deep-sea animals. Nevertheless, the interest is enormous. Majordifferences in the kineticsof DSi utilizationbetweenthe two major lineages of siliceous sponges (i.e., DemospongiaeandHexactinellida) cannot be discarded,as silicification in demosponges revolves around theactivity of the nonsoluble silicatein enzyme ([@R21]), while the process in hexactinellids appears to be governedby aphylogenetically unrelated, soluble enzyme, glassin ([@R22]). In addition, because hexactinellids have essentiallysyncytial organization while demosponges have a conventional cellular organization, the membrane transporters involved in Si utilization may not be shared but maybe lineage specificinstead. Very little is known onmolecular Si transport in demosponges ([@R23], [@R24]) and nothing in hexactinellids. This lack of knowledge obscures the understanding of the evolution of the biosilicification process in the animal kingdom and its relationships to that inother organisms. Here, we characterized experimentally the kinetics of DSi consumption in the hexactinellid sponge *Vazella pourtalesii*(Schmidt, 1870), a roselliddistributed from \~100 to 935 m in thenorthwest Atlantic that forms extensive monospecific aggregations on the deep continental shelf off Nova Scotia ([Fig. 1A](#F1){ref-type="fig"} and fig. S1), eastern Canada ([@R25]). We testedthe laboratory-based kinetic model by comparing its predictions to both in situdeterminations of DSiconsumption rates using incubation chambers ([Fig. 1, B to D](#F1){ref-type="fig"}, and moviesS1and S2) and rates of BSi production derivedfrom individualsof a knownage grown inthe wild on an artificial substrate ([Fig. 1,E andF](#F1){ref-type="fig"}).In combination with those physiological experiments, we conducted a quantitative large-scale assessment of gene expression as a function of DSi availability. The results of this study offer a mechanistic explanation for the kinetics of DSi utilization in hexactinellids and provide fresh insights into the molecular systems of Si transport and their evolution within sponges and acrossother silicifying organisms. {#F1} RESULTS ======= Modeling and testing the physiology of DSi consumption ------------------------------------------------------Live spongeswere collected using the remotely operated vehicle (ROV) Remotely OperatedPlatform for Ocean Sciences (ROPOS) and taken to the laboratory forincubation in progressively increasing DSi concentrations (12, 30,60, 100, 150, 200, and 250 μMDSi; see Materialsand Methods). Initially,all 11 assayed individuals increased their DSi consumption rate in responseto the progressive increase of DSi availability in the seawater ([Fig. 2,A and B](#F2){ref-type="fig"}, and tables S1 and S2). As also knownfor demosponges,the DSi consumptionrate notably varied among individuals,with an average maximum consumption of 0.106 ± 0.050μmol Si permilliliter of sponge tissue and per hour (hereafter given as μmol Si ml^−1^ hour^−1^) at an average DSi concentration of 150.9 ± 69.3 μM. Over that concentration threshold, the consumption rate of mostindividuals did not increase withincreasing DSi availability, revealing that the Si transport system reachesthe maximum speed (i.e., optimal utilization) atabout 150 μM DSi and saturates at higherconcentrations. {#F2} Unlike in all demosponges studied to date, the model best fitting the average DSi consumption rate of *V.pourtalesii* in response to DSi availability did not follow Michaelis-Menten kinetics (*r*^2^ = 0.841, *P* = 0.004; [Fig. 2A](#F2){ref-type="fig"}). An exponential rise toa maximum (ERM) model showedthe best fit (*r*^2^ = 0.898,*P* = 0.002; [Fig. 2, Aand B](#F2){ref-type="fig"}) to the empirical data onDSiconsumption as a function of DSi availability, "consumption rate =*a* (1 − *b*^\[DSi\]^)". Although the differenceinstatistical fitbetween the"Michaelis-Menten" and "ERM" models was apparently small, the ERM model was built on parameters with higher statistical significance(*a* = 0.093 ±0.008, *P* \< 0.001 | _INTRODUCTION_ ============ Silicon (Si), in the form of _dissolved_ silicic acid---often referred to as silicate and _hereafter_ abbreviated as _DSi---is_ an inorganic nutrient instrumental to ocean functioning. Its availability modulates processes as relevant _as_ _ocean_ primary productivity ([@R1]) and the _exchange_ of CO~2~ with the _atmosphere_ _([@R2])._ Regional patterns of DSi availability largely _result_ from the consumption of this nutrient by marine organisms (silicifiers) to _build_ their skeletons of _biogenic_ silica (BSi), with diatom utilization among the best _known_ _and_ _quantified_ ([@R1], [@R3]). However, sponges, radiolarians, silicoflagellates, _choanoflagellates,_ testate amoebae, and chrysophyceans, among others, also _consume_ _DSi_ in the ocean ([@R4]), but their activity remains poorly quantified and little _understood_ from a physiological and _molecular_ _perspective._ In the diatoms, the kinetics _of_ DSi uptake have been investigated _in_ a large variety of species, all of _which_ were reported _initially_ to follow _a_ _saturable_ _Michaelis-Menten_ _model._ It is _also_ that those saturable kinetics shift into nonsaturable uptake when DSi availability drastically increases and/or _under_ _particular_ _physiological_ conditions ([@R5], [@R6]). _Likewise,_ _membrane_ silicon _transporters_ (SITs) incorporating actively ambient DSi into _the_ diatom cell have long been described and, although passive transporters have _not_ _been_ identified yet, a diffusion-based uptake has been _described_ for _at_ least some diatoms at high DSi availability \[reviewed in ([@R7])\]. In contrast, little _is_ known about _these_ physiological and molecular processes in _other_ groups of marine silicifiers. Because diatoms are restricted to _the_ photic _zone_ _of_ the ocean, a major gap in _knowledge_ _relative_ to _DSi_ utilization in the dark ocean by nonphototrophic silicifiers persists. The _discoveries_ that extensive aggregations of highly silicified sponges are _common_ in _the_ deep sea ([@R8]), that they _can_ accumulate _substantial_ _amounts_ of BSi at _the_ regional scale ([@R9], [@R10]), and that they _trigger_ significant losses of BSi _from_ the ocean ([@R11]) _have_ raised _considerable_ interest in deciphering _how_ Si is processed _in_ _these_ _singular,_ sponge-dominated, _deep-sea_ _systems._ _The_ lack of knowledge regarding these processes in sponges currently hinders the _ability_ to understand the Si utilization in the dark ocean and _makes_ it difficult to model _adequately_ the role _of_ the biological component in the marine biogeochemical _cycle_ _of_ silicon _([@R3],_ [@R11]). Information on _the_ physiology of DSi consumption by sponges is sparse _and_ _derived_ exclusively _from_ shallow-water species in _the_ class Demospongiae. These studies indicate _Michaelis-Menten_ kinetics but with _optimal_ DSi consumption attained _at_ environmental DSi concentrations of \>100 μM ([@R12]--[@R16]). _Because_ _DSi_ _concentrations_ higher than 100 µM _are_ _virtually_ never reached in _the_ shallow waters of _the_ modern _ocean_ ([@R17]), the skeletal _growth_ _of_ _all_ _shallow-water_ demosponges _investigated_ to date is _therefore_ chronically limited by Si _availability_ ([@R15], [@R16], [@R18]--[@R20]). _Whether_ this kinetic limitation also applies to _sponges_ in the class Hexactinellida---deep-sea _specialists_ _characterized_ by _impressive_ siliceous _skeletons---remains_ unknown. The physiology of DSi consumption and the molecular pathways of DSi uptake _remain_ largely uninvestigated in hexactinellids, _hampered_ by _impediments_ to conducting in situ and laboratory _experimentation_ with such relatively large and _delicate_ deep-sea animals. Nevertheless, the interest is enormous. Major differences in the kinetics _of_ DSi _utilization_ between _the_ two major lineages of siliceous sponges (i.e., Demospongiae and Hexactinellida) cannot be discarded, as _silicification_ in demosponges revolves around the activity _of_ the _nonsoluble_ silicatein enzyme ([@R21]), while the process in hexactinellids appears to _be_ governed by a _phylogenetically_ unrelated, soluble enzyme, glassin ([@R22]). In addition, _because_ hexactinellids have essentially syncytial _organization_ _while_ demosponges _have_ _a_ conventional cellular organization, _the_ membrane transporters involved in _Si_ utilization may _not_ be shared _but_ may be lineage specific _instead._ Very little is known on molecular Si _transport_ in demosponges ([@R23], [@R24]) and nothing in hexactinellids. This lack of _knowledge_ obscures the understanding of the _evolution_ _of_ the biosilicification process in the animal kingdom and its relationships to that _in_ other _organisms._ Here, we _characterized_ experimentally _the_ kinetics of DSi _consumption_ in the hexactinellid sponge *Vazella _pourtalesii*_ _(Schmidt,_ 1870), a _rosellid_ distributed from \~100 to 935 m in the northwest Atlantic that forms extensive monospecific aggregations on _the_ deep _continental_ _shelf_ off _Nova_ Scotia ([Fig. 1A](#F1){ref-type="fig"} and fig. S1), eastern Canada ([@R25]). _We_ tested the laboratory-based kinetic model _by_ comparing its _predictions_ to both in situ determinations of DSi _consumption_ rates using incubation chambers ([Fig. 1, B to D](#F1){ref-type="fig"}, _and_ movies S1 _and_ S2) and rates of BSi _production_ derived from individuals of a known age grown in the wild on an artificial substrate ([Fig. 1, E and _F](#F1){ref-type="fig"})._ In _combination_ with those physiological experiments, _we_ _conducted_ a quantitative large-scale assessment of gene expression as a function of DSi availability. The results of _this_ study offer a mechanistic explanation for _the_ kinetics of DSi utilization in _hexactinellids_ _and_ provide fresh insights into the molecular _systems_ _of_ Si transport and their evolution within _sponges_ and across _other_ silicifying organisms. {#F1} RESULTS ======= _Modeling_ and testing the physiology of DSi consumption ------------------------------------------------------ Live _sponges_ were _collected_ using _the_ remotely operated vehicle (ROV) _Remotely_ _Operated_ Platform for Ocean Sciences _(ROPOS)_ and taken to the laboratory for incubation _in_ progressively increasing DSi _concentrations_ (12, 30, 60, 100, 150, _200,_ and _250_ μM DSi; see Materials and Methods). Initially, all 11 assayed individuals increased their DSi consumption rate in response to the progressive increase of _DSi_ availability in the seawater ([Fig. 2, A _and_ B](#F2){ref-type="fig"}, and _tables_ S1 _and_ S2). As also known _for_ demosponges, the DSi consumption rate notably varied among _individuals,_ with _an_ average maximum consumption of 0.106 ± 0.050 μmol Si _per_ milliliter _of_ sponge tissue and per hour (hereafter given _as_ μmol Si ml^−1^ _hour^−1^)_ _at_ _an_ average DSi concentration _of_ 150.9 ± 69.3 μM. Over _that_ concentration _threshold,_ the _consumption_ rate of most individuals did not increase _with_ increasing DSi availability, revealing _that_ _the_ Si transport system _reaches_ the maximum speed _(i.e.,_ optimal _utilization)_ _at_ about _150_ μM DSi and saturates at higher concentrations. {#F2} Unlike in all demosponges _studied_ _to_ date, _the_ model best fitting the average DSi consumption _rate_ _of_ *V. pourtalesii* _in_ response to DSi availability did _not_ follow Michaelis-Menten kinetics (*r*^2^ = _0.841,_ *P* = 0.004; [Fig. 2A](#F2){ref-type="fig"}). An _exponential_ rise to a maximum (ERM) model showed the best fit (*r*^2^ _=_ _0.898,_ *P* = 0.002; [Fig. 2, A and B](#F2){ref-type="fig"}) to _the_ empirical data on DSi _consumption_ as a function _of_ DSi _availability,_ "consumption rate = *a* (1 − *b*^\[DSi\]^)". Although the difference in statistical fit between the "Michaelis-Menten" and "ERM" models _was_ _apparently_ small, _the_ ERM _model_ was _built_ on parameters with higher _statistical_ significance (*a* = 0.093 ± 0.008, _*P*_ \< 0.001 |
When Landauer argued in 1961 that any physical realisation of erasure of information has a fundamental thermodynamic work cost he irrevocably linked thermodynamics and information theory[@b1][@b2][@b3][@b4][@b5][@b6][@b7][@b8][@b9]. A practical consequence of this insight is that all computers must dissipate a minimal amount of heat in each irreversible computing step, a threshold that is becoming a concern with future computer chips entering atomic scales. The treatment of general *quantum* information processing tasks within the wider framework of quantum thermodynamics has only recently begun[@b13]. Quantum mechanics differs from classical mechanics in at least three central aspects: the special nature of measurement, the possibility of a quantum system to be in a superposition and the existence of quantum correlations. The thermodynamic energy needed to perform a (selective) measurement has been investigated[@b10] and the total work for a closed thermodynamic measurement cycle explored[@b11]. The catalytic role of quantum superposition states when used in thermal operations has been uncovered[@b12] and it has been shown that work can be drawn from quantum correlations[@b13][@b14] in a thermodynamic setting, see [Fig. 1](#f1){ref-type="fig"}. In particular, del Rio *et al.*[@b14] showed that contrary to Landauer's principle, it is possible to *extract* work while performing erasure of a system's state when the system is correlated to a memory. This can occur if and only if the initial correlations imply a negative conditional entropy, a uniquely quantum feature. The thermodynamic process does however now require operation on degrees of freedom external to the system, i.e. the memory's.
Results
=======
Projections and the optimal work value of removing coherences
-------------------------------------------------------------
Our motivation is here to shed light on the implications of performing a measurement on a quantum state that has coherences. We will consider this task in the thermodynamic setting of Landauer's erasure, involving a heat bath at fixed temperature *T* and operation on *N* → ∞ uncorrelated and identically prepared copies of the system (i.i.d. limit). This is of interest in the context of the quantum Jarzynski equality, for example, and will also be central for experiments testing quantum thermodynamic predictions in the future. To tackle this question we define the information-theoretic "projection" for a given initial quantum state *ρ* and a complete set of mutually orthogonal projectors . Such state transformation can be seen as analogous to the state transfer of erasure, , to a blank state . Physically, this projection can be interpreted as the result of an unread, or unselective[@b15], measurement of an observable that has eigenvector projectors . In an unselective measurement the individual measurement outcomes are not recorded and only the statistics of outcomes is known. In the literature the implementation of unselective measurements is often not specified, although it is typically thought of as measuring individual outcomes, e.g. with a Stern-Gerlach experiment, see [Fig. 2a](#f2){ref-type="fig"}, followed by mixing. The crux is that the information-theoretic projection can be implemented in many physical ways. The associated thermodynamic heat and work will differ depending on *how* the projection was done and we will refer to the various realisations as "thermodynamic projection processes". One possibility is decohering[@b16] the state in the so-called pointer basis, , a thermodynamic process where an environment removes coherences in an uncontrolled manner resulting in no associated work. In general it is possible to implement the state transfer in a finely controlled fashion achieving optimal thermodynamic heat and work values.
Of particular importance in thermodynamics is the projection of the system's initial state *ρ* onto the set of energy eigenstates of the system's Hamiltonian with *E*~*k*~ the energy eigenvalues. Here the state's off-diagonals with respect to the energy eigenbasis are removed - a state transformation that is frequently employed in quantum thermodynamic derivations and referred to as "dephasing" or "measuring the energy". Our key observation is that there exists a thermodynamic projection process realising this transformation and allowing to draw from the quantum system a non-trivial *optimal average work* of
Here *T* is the temperature of the heat bath with which the system is allowed to interact, see illustration [Fig. 1](#f1){ref-type="fig"}, *k*~*B*~ is the Boltzmann constant and *S* is the von Neumann entropy. Crucially, this work is strictly positive for quantum states with coherences. Extending the key observation to general projections one finds that optimal thermodynamic projection processes can be implemented that allow to draw an average work of
where an additional internal energy change term appears.
Physical interpretation and assumptions made to derive the optimal work
-----------------------------------------------------------------------
The optimal work values stated in Eqs. [(1](#eq12){ref-type="disp-formula"}) and ([2](#eq14){ref-type="disp-formula"}) are valid for processes applied to classical and quantum states alike. While for a classical ensemble the entropy change, , will be zero this is not so in the general quantum situation, where initial non-diagonal quantum states result in a strictly positive entropy change[@b17]. We note that while the optimal work values are in principle attainable, practical implementations may be suboptimal resulting in a reduced work gain or a higher work cost.
The physical meaning of can be grasped by considering a lower bound[@b18] on it, , see Supplement. Here *d* is the dimension of the system and denotes the Hilbert-Schmidt norm. The first factor quantifies the distance of the initial state from the fully mixed state, while the second factor, , quantifies the angle between the diagonal basis of *ρ* and the projection basis . These terms correspond to incoherent and coherent mixing contributions. The entropy change is non-trivially bounded only if the initial state is not an incoherent mixture with respect to that basis. The entropy bound is the largest for pure initial states whose basis is mutually unbiased with respect to . In this case the optimal entropy change is .
One may wonder where the work has gone to. There are two equivalent approaches to the accounting of work. In the present analysis the focus is on the work that the system exchanges, as done in statistical physics[@b5][@b19][@b20][@b21][@b22]. In this approach it is often not explicitly mentioned where the work goes to, but the only place work can go to are the externally controlled energy sources. Similarly, the heat, i.e. the energy change minus the work, is established implicitly. For example, in the experimental realisation of classical Landauer erasure with a colloidal silica bead trapped in an optical tweezer[@b21], the dissipated heat of erasure was calculated by knowing the applied tilting forces and integrating over the bead's dynamics. The second approach is to collect work in a separate work storage system[@b23], as illustrated by the weight in [Fig. 1](#f1){ref-type="fig"} and detailed in the Supplement. Both the implicit and the explicit treatment of work are equivalent in the sense that the results obtained in one approach can be translated into the other.
The thermodynamic assumptions made to prove Eq. [(2)](#eq14){ref-type="disp-formula"} are congruent with current literature[@b9][@b23][@b24][@b25]; specifically they are: (T0) an isolated system is a system that only exchanges work and not heat; (T1) the validity of the *first law* relating the internal energy change, Δ*U*, of the system during a process to its average heat absorbed and work drawn, ; (T2) the validity of the *second law* relating the system's entropy change to its average absorbed heat, , when interacting with a bath at temperature *T*, with equality attainable by an optimal process; (T3) the thermodynamic entropy to be equal to the von Neumann entropy in equilibrium as well as out-of-equilibrium, . In addition we make the following standard quantum mechanics assumptions: (Q0) an isolated system evolves unitarily; (Q1) control of a quantum system includes its coherences. Details of the proof are in the Methods Summary. We note that in the single-shot setting whole families of second laws apply[@b7][@b8] that differ from (T2) stated above. However, in the limit of infinitely many independent and identically prepared copies of the system these collapse to the standard second law, (T2), on the basis of which Eq. [(2)](#eq14){ref-type="disp-formula"} is derived.
From the information-theory point of view the projections considered here constitute just one example of the larger class of trace-preserving completely positive (TPCP) maps characterising quantum dynamics. Of course, all TPCP maps can be interpreted thermodynamically with the assumptions stated above, resulting in an optimal average work given by a free energy difference. Erasure is another such map whose study forged the link between information theory and thermodynamics. The benefit of discussing "projections" here lies in the insight that this focus provides: it uncovers that coherences offer the potential to draw work making it a genuine and testable quantum thermodynamic feature. This work is non-trivial even when the thermodynamic process is operated on the system alone, not involving any side-information[@b14] stored in other degrees of freedom.
Qubit example for drawing optimal work
--------------------------------------
To gain a detailed understanding of thermodynamic projection processes that give the optimal work stated in Eq. [(1)](#eq12){ref-type="disp- | when landauer argued in 1961 that any physical realisation of erasure of information has a fundamental thermodynamic work cost he irrevocably linked thermodynamics and information theory [ @ b1 ] [ @ b2 ] [ @ b3 ] [ @ b4 ] [ @ b5 ] [ @ b6 ] [ @ b7 ] [ @ b8 ] [ @ b9 ]. a practical consequence of this insight is that all computers must dissipate a minimal amount of heat in each irreversible computing step, a threshold that is becoming a concern with future computer chips entering atomic scales. the treatment of general * quantum * information processing tasks within the wider framework of quantum thermodynamics has only recently begun [ @ b13 ]. quantum mechanics differs from classical mechanics in at least three central aspects : finite exponential nature of measurement, the tendency of a quantum system to be in a superposition and the existence of quantum correlations. increased thermodynamic energy needed to enable a ( selective ) measurement has been investigated [ @ b10 ] and the total work for a closed thermodynamic measurement cycle explored [ @ b11 ]. the catalytic role of quantum superposition states when used in thermal operations has been uncovered [ @ b12 ] and it has been shown that work can be drawn from quantum correlations [ @ a2 ] [ @ b14 ] in a thermodynamic setting, see [ fig. 1 ] ( # f1 ) { ref - type = " fig " }. in particular, del rio * y al. * [ @ b14 ] showed that contrary to landauer ' s principle, it is possible to * extract * work while performing erasure of a system ' s state when the system is correlated to a memory. this can occur if and only if the initial correlations imply a negative conditional entropy, a uniquely quantum feature. the thermodynamic process does however explicitly require operation on degrees of freedom external to the system, u. e. the memory ' s. results = = = = = = = projections yield the optimal work value of removing coherences - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - our motivation is here to shed light on the implications of performing a measurement on a quantum state that has coherences. we will consider this task in the thermodynamic setting of landauer ' s erasure, involving a heat bath at fixed temperature * t * and operation on * n * → ∞ uncorrelated and identically prepared copies of the system ( i. i. d. limit ). this is of interest in the context of the quantum jarzynski equality, for example, and will also be central for experiments testing quantum thermodynamic predictions in the future. to tackle this question we define the information - theoretic " projection " for a given initial quantum state * ρ * and a complete set of mutually orthogonal projectors. such state transformation can be seen as analogous to the state transfer of erasure,, to a blank state. physically, this projection can be interpreted as the result of an unread, or unselective [ @ b15 ], measurement of an observable that has eigenvector projectors. in an unselective measurement the individual measurement outcomes are not recorded and only the statistics of outcomes is known. in the literature the implementation of unselective measurements is often not specified, although it is typically thought of as measuring individual outcomes, e. g. with a stern - gerlach experiment, see [ fig. 2a ] ( # f2 ) { ref - type = " fig " }, followed by mixing. the crux is that the information - theoretic projection can be implemented in many physical ways. the associated thermodynamic heat and work will differ depending on * how * the projection was done and we will refer to the various realisations as " thermodynamic projection processes ". one possibility is decohering [ @ b16 ] the state in the so - called pointer basis,, a thermodynamic process where an environment removes coherences in an uncontrolled manner resulting in no associated work. in general it is possible to implement the state transfer in a finely controlled fashion achieving optimal thermodynamic heat and work values. of particular importance in thermodynamics is the projection of the system ' s initial state * ρ * onto the set of energy eigenstates of the system ' s hamiltonian with * e * ~ * k * ~ the energy eigenvalues. here the state ' s off - diagonals with respect to the energy eigenbasis are removed - a state transformation that is frequently employed in quantum thermodynamic derivations and referred to as " dephasing " or " measuring the energy ". our key observation is that there exists a thermodynamic projection process realising this transformation and allowing to draw from the quantum system a non - trivial * optimal average work * of here * t * is the temperature of the heat bath with which the system is allowed to interact, see illustration [ fig. 1 ] ( # f1 ) { ref - type = " fig " }, * k * ~ * b * ~ is the boltzmann constant and * s * is the von neumann entropy. crucially, this work is strictly positive for quantum states with coherences. extending the key observation to general projections one finds that optimal thermodynamic projection processes can be implemented that allow to draw an average work of where an additional internal energy change term appears. physical interpretation and assumptions made to derive the optimal work - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the optimal work values stated in eqs. [ ( 1 ] ( # eq12 ) { ref - type = " disp - formula " } ) and ( [ 2 ] ( # eq14 ) { ref - type = " disp - formula " } ) are valid for processes applied to classical and quantum states alike. while for a classical ensemble the entropy change,, will be zero this is not so in the general quantum situation, where initial non - diagonal quantum states result in a strictly positive entropy change [ @ b17 ]. we note that while the optimal work values are in principle attainable, practical implementations may be suboptimal resulting in a reduced work gain or a higher work cost. the physical meaning of can be grasped by considering a lower bound [ @ b18 ] on it,, see supplement. here * d * is the dimension of the system and denotes the hilbert - schmidt norm. the first factor quantifies the distance of the initial state from the fully mixed state, while the second factor,, quantifies the angle between the diagonal basis of * ρ * and the projection basis. these terms correspond to incoherent and coherent mixing contributions. the entropy change is non - trivially bounded only if the initial state is not an incoherent mixture with respect to that basis. the entropy bound is the largest for pure initial states whose basis is mutually unbiased with respect to. in this case the optimal entropy change is. one may wonder where the work has gone to. there are two equivalent approaches to the accounting of work. in the present analysis the focus is on the work that the system exchanges, as done in statistical physics [ @ b5 ] [ @ b19 ] [ @ b20 ] [ @ b21 ] [ @ b22 ]. in this approach it is often not explicitly mentioned where the work goes to, but the only place work can go to are the externally controlled energy sources. similarly, the heat, i. e. the energy change minus the work, is established implicitly. for example, in the experimental realisation of classical landauer erasure with a colloidal silica bead trapped in an optical tweezer [ @ b21 ], the dissipated heat of erasure was calculated by knowing the applied tilting forces and integrating over the bead ' s dynamics. the second approach is to collect work in a separate work storage system [ @ b23 ], as illustrated by the weight in [ fig. 1 ] ( # f1 ) { ref - type = " fig " } and detailed in the supplement. both the implicit and the explicit treatment of work are equivalent in the sense that the results obtained in one approach can be translated into the other. the thermodynamic assumptions made to prove eq. [ ( 2 ) ] ( # eq14 ) { ref - type = " disp - formula " } are congruent with current literature [ @ b9 ] [ @ b23 ] [ @ b24 ] [ @ b25 ] ; specifically they are : ( t0 ) an isolated system is a system that only exchanges work and not heat ; ( t1 ) the validity of the * first law * relating the internal energy change, δ * u *, of the system during a process to its average heat absorbed and work drawn, ; ( t2 ) the validity of the * second law * relating the system ' s entropy change to its average absorbed heat,, when interacting with a bath at temperature * t *, with equality attainable by an optimal process ; ( t3 ) the thermodynamic entropy to be equal to the von neumann entropy in equilibrium as well as out - of - equilibrium,. in addition we make the following standard quantum mechanics assumptions : ( q0 ) an isolated system evolves unitarily ; ( q1 ) control of a quantum system includes its coherences. details of the proof are in the methods summary. we note that in the single - shot setting whole families of second laws apply [ @ b7 ] [ @ b8 ] that differ from ( t2 ) stated above. however, in the limit of infinitely many independent and identically prepared copies of the system these collapse to the standard second law, ( t2 ), on the basis of which eq. [ ( 2 ) ] ( # eq14 ) { ref - type = " disp - formula " } is derived. from the information - theory point of view the projections considered here constitute just one example of the larger class of trace - preserving completely positive ( tpcp ) maps characterising quantum dynamics. of course, all tpcp maps can be interpreted thermodynamically with the assumptions stated above, resulting in an optimal average work given by a free energy difference. erasure is another such map whose study forged the link between information theory and thermodynamics. the benefit of discussing " projections " here lies in the insight that this focus provides : it uncovers that coherences offer the potential to draw work making it a genuine and testable quantum thermodynamic feature. this work is non - trivial even when the thermodynamic process is operated on the system alone, not involving any side - information [ @ b14 ] stored in other degrees of freedom. qubit example for drawing optimal work - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - to gain a detailed understanding of thermodynamic projection processes that give the optimal work stated in eq. [ ( 1 ) ] ( # eq12 ) { ref - type = " disp - | When Landauer argued in 1961 that any physical realisation of erasure of information has a fundamental thermodynamic work SosR he irrevocably linked thermodynamics and information theory [@ b1] [@ b2] [@ b3] [@ b4] [@ b5] [@ b6] [@ b7] [@ b8] [@ b9 ]. A practical consequence of this insight is that all computers must dissipate a minimal amount of heat in each irreversible computing step, a threshold that is becoming a concern with future computer chips entering atomic scales. The treatment of general * quantum * information processing tasks within the wider framework of quantum thermodynamics has only recently begun [@ b13 ]. Quantum mechanics differs from classical mechanics in at least three central aspects: the special nature of measurement, the possibility of a quantum system to be in a superposition and the existence of quantum correlations. The thermodynamic energy needed to perform a (selective) measurement has been investigated [@ b10] and the total work for a cl(sew thermodynamic measurement cycle explored [@ b11 ]. The catalytic role of quantum superposition states when used in thermal operations has been uncovered [@ b12] and it has been shown that work can be drawn from quantum correlations [@ b13] [@ b14] in a thermodynamic setting, see [Fig. 1] (# f1) {ref - type = " fig " }. In particular, del Rio * et al. * [@ b14] showed that contrary to Landauer ' s principle, it is possible to * extract * work while performing erasure of a system ' s state when the system is correlated to a memory. This can occur if and only if the initial correlations imply a negative conditional entropy, a uniquely quantum feature. The thermodynamic process does however now require operation on degrees of freedom external to the system, i. e. the memory ' s. Results = = = = = = = Projections and the optimal work value of removing coherences - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Our motivation is here to shed light on the implications of performing a measurement on a quantum state that has coherences. We will consider this task in the thermodynamic setting of Landauer ' s erasure, involving a heat bath at fixed temperature * T * and operation on * N * → ∞ uncorrelated and identically prepared copies of the system (i. i. d. limit ). This is of interest in the context of the quantum Jarzynski equality, for example, and will also be central for experiments testing quantum thermodynamic predictions in the future. To tackle this question we define the information - theoretic " projection " for a given initial quantum state * ρ * and a complete set of mutually orthogonal projectors. Such state transformation can be seen as analogous to the state transfer of erasure, , to a blank state. Physically, this projection can be interpreted as the result of an unread, or unselective [@ b15 ], measurement of an observable that has eigenvector projectors. In an unselective measurement the individual measurement outcomes are not recorded and only the statistics of outcomes is known. In the literature the implementation of unselective measurements is often not specified, although it is typically thought of as measuring individual outcomes, e. g. with a Stern - Gerlach experiment, see [Fig. 2a] (# f2) {ref - type = " fig " }, followed by mixing. The crux is that the information - theoretic projection can be implemented in many physical ways. The associated thermodynamic heat and work will differ depending on * how * the projection was done and we will refer to the various reQlisahions as " thermodynamic projection processes ". One possibility is decohering [@ b16] the state in the so - called pointer basis, , a thermodynamic process 2Gere an environment removes coherences in an uncontrolled manner resulting in no associated work. In general it is possible to implement the state transfer in a finely controlled fashion achieving optimal thermodynamic heat and work values. Of particular importance in tTermodynamixs is the projection of the system ' s initial state * ρ * onto the set of energy eigenstates of the system ' s Hamiltonian with * E * ~ * k * ~ the energy eigenvalues. Here the state ' s off - diagonals with respect to the energy eigenbasis are removed - a state transformation that is frequently employed in quantum thermodynamic derivations and referred to as " dephasing " or " measuring the energy ". Our key observation is that there exists a thermodynamic projection process realising this transformation and allowing to draw from the quantum system a non - trivial * optimal average work * of Here * T * is the temperature of the heat bath with which the system is allowed to interact, see illustration [Fig. 1] (# f1) {ref - type = " fig " }, * k * ~ * B * ~ is the Boltzmann constant and * S * is the von GeumZnn entropy. Crucially, this work is strictly positive for quantum states with coherences. Extending the key observation to general projections one finds that optimal thermodynamic projection processes can be implemented that allow to draw an average work of where an additional internal energy change term appears. Physical interpretation and assumptions made to derive the optimal work - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The optimal work values stated in Eqs. [( 1] (# eq12) {ref - type = " disp - formula "} ) and ([ 2] (# eq14) {ref - type = " disp - formula "} ) are valid for processes applied to classical and quantum states alike. While for a classical ensemble the entropy change, , will be zero this is not so in the general quantum situation, where initial non - diagonal quantum sgateX result in a strictly positive entropy change [@ b17 ]. We note that while the optimal work values are in principle attainable, practical implementations may be suboptimal resulting in a reduced work gain or a higher work cost. The physical meaning of can be grasped by considering a lower bound [@ b18] on it, , see Supplement. Here * d * is the dimension of the system and denotes the Hilbert - Schmidt norm. The first factor quantifies the distance of the initial state from the fully mixed state, while the second factor, , quantifies the angle between the dJagInal basis of * ρ * and the projection basis. These terms correspond to incoherent and coherent mixing contributions. The entropy change is non - trivially bound@C only if the initial state is not an incoherent mixture with respect to that basis. The entropy bound is the largest for pure initial states whose basis is mutually unbiased with respect to. In this case the optimal entropy change is. One may wonder where the work has gone to. TnerF are two equivalent approaches to the accounting of work. In the present analysis the focus is on the work that the system exchanges, as done in statistical physics [@ b5] [@ b19] [@ b20] [@ b21] [@ b22 ]. In this approach it is often not explicitly mentioned where the work goes to, but the only place work can go to are the externally controlled energy sources. Similarly, the heat, i. e. the energy change minus the work, is established implicitly. For example, in the experimental realisation of classical Landauer erasure with a colloidal silica bead trapped in an optical tweezer [@ b21 ], the dissipated heat of erasure was calculated by knowing the applied tilting forces and integrating over the bead ' s dynamics. The second approach is to collect work in a separate work storage system [@ b23 ], as illustrated by the weight in [Fig. 1] (# f1) {ref - type = " fig "} and detailed in the Supplement. Both the implicit and the explicit treatment of work are equivalent in the sense that the results obtained in one approach can be translated into the other. The thermodynamic assumptions made to prove Eq. [( 2) ] (# eq14) {ref - type = " disp - formula "} are congruent with current literature [@ b9] [@ b23] [@ b24] [@ b25 ]; specifically they are: (T0) an isolated system is a system that only exchanges work and not heat; (T1) the validity of the * first law * relating the internal energy change, Δ * U *, of the system during a process to its average heat absorbed and work drawn, ; (T2) the validity of the * second law * relating the system ' s entropy change to its average absorbed heat, , when interacting with a bath at temperature * T *, with equality attainable by an optimal process; (T3) the thermodynamic entropy to be equal to the von Neumann entropy in equilibrium as well as out - of - equilibrium, . In addition we make the following standard quantum mechanics assumptions: (Q0) an isolated system evolves unitarily; (Q1) control of a quantum system includes its coherences. Details of the proof are in the Methods Summary. We note that in the single - shot setting whole families of second laws apply [@ b7] [@ b8] that differ from (T2) stated above. However, in the limit of infinitely many independent and identically prepared copies of the system these collapse to the standard second law, (T2 ), on the basis of which Eq. [( 2) ] (# eq14) {ref - type = " disp - formula "} is derived. From the information - theory point of view the projections considered here constitute just one example of the larger class of trace - preserving completely positive (TPCP) maps characterising quantum dynamics. Of course, all TPCP maps can be interpreted thermodynamically with the assumptions stated above, resulting in an optimal average work given by a free energy difference. Erasure is another such map whose study forged the link between information theory and thermodynamics. The benefit of discussing " projections " here lies in the insight that this focus provides: it uncovers that coherences offer the potential to draw work making it a genuine and testable quantum thermodynamic feature. This work is non - trivial even when the thermodynamic process is operated on the system alone, not involving any side - information [@ b14] stored in other degrees of freedom. Qubit example for drawing optimal work - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - To gain a detailed understanding of thermodynamic projection processes that give the optimal work stated in Eq. [( 1) ] (# eq12) {ref - type = " disp - | When Landauer argued in 1961 that physical realisation erasure of information has a fundamental thermodynamic work cost irrevocably linked thermodynamics and information theory[@b1][@b2][@b3][@b4][@b5][@b6][@b7][@b8][@b9]. A practical consequence of insight that computers must dissipate a minimal amount of heat in each irreversible computing step, a threshold that becoming a concern with future computer chips entering atomic scales. The treatment of general information processing tasks within the wider framework of quantum thermodynamics has only recently begun[@b13]. Quantum mechanics differs classical mechanics in at least three central aspects: the special nature measurement, of a quantum system to be in superposition and the existence of quantum correlations. thermodynamic energy needed to perform a (selective) measurement has been investigated[@b10] and the total work for a closed thermodynamic measurement cycle explored[@b11]. The catalytic role of quantum superposition states when used in thermal operations has been uncovered[@b12] and it has been shown work can be drawn from correlations[@b13][@b14] in a thermodynamic see [Fig. 1](#f1){ref-type="fig"}. In particular, del Rio *et al.*[@b14] showed that to Landauer's principle, it is to *extract* work while performing erasure of a when the system is correlated to a memory. This occur if and only if initial correlations imply a negative conditional entropy, a uniquely quantum feature. The thermodynamic process does however now require operation on degrees of freedom external to the system, the Results ======= Projections and the optimal work value of coherences ------------------------------------------------------------- Our motivation is here to shed light on the implications of performing a measurement on a quantum state that has coherences. will consider this task in the thermodynamic setting of Landauer's erasure, heat bath fixed temperature *T* and operation on *N* → ∞ and identically prepared copies of the system (i.i.d. limit). This is of interest in context of the quantum Jarzynski equality, for and will also be central for experiments testing quantum thermodynamic predictions in the future. To tackle this we define the information-theoretic "projection" for a given quantum state *ρ* and a complete of orthogonal projectors . Such state transformation can as analogous to the state transfer of to a blank . Physically, this projection can be interpreted as the result of an unread, or unselective[@b15], measurement of an observable that eigenvector projectors . In an unselective measurement the individual measurement outcomes are not recorded and only the statistics of outcomes is known. In the literature the implementation of unselective measurements is often not specified, it is typically thought of as measuring individual outcomes, e.g. with a Stern-Gerlach experiment, [Fig. followed by The crux is that the information-theoretic projection be implemented in many physical ways. The associated thermodynamic heat and work will differ depending on *how* the projection done and we will refer the various as "thermodynamic projection One possibility is decohering[@b16] the state in the so-called pointer basis, , a thermodynamic where an environment removes coherences in uncontrolled manner resulting in work. In general it is possible to the state in a finely controlled fashion achieving optimal thermodynamic heat and work values. Of particular importance in thermodynamics the projection of the system's state onto the set of energy of the system's Hamiltonian with *E*~*k*~ energy eigenvalues. the state's off-diagonals with respect the energy eigenbasis are removed - a state transformation that is frequently employed in quantum derivations and referred to as or "measuring energy". Our key observation is that there exists a thermodynamic projection this transformation and allowing to draw the quantum system a non-trivial *optimal average work* Here *T* the temperature of the heat bath with which the system is allowed to interact, see illustration [Fig. 1](#f1){ref-type="fig"}, *k*~*B*~ is Boltzmann constant and *S* is the von Neumann entropy. Crucially, this work is positive for quantum states with coherences. the key observation to general projections one finds that optimal thermodynamic projection processes can be implemented that allow to draw average work of where an additional internal energy change term appears. Physical interpretation and made to derive optimal work ----------------------------------------------------------------------- The optimal work values stated in Eqs. [(1](#eq12){ref-type="disp-formula"}) and ([2](#eq14){ref-type="disp-formula"}) are valid for processes applied to classical and quantum states While a classical ensemble the entropy change, , will be zero this is not so in the general quantum situation, where initial non-diagonal quantum states result in a positive entropy We note that while the optimal values are in principle attainable, practical implementations may be resulting in a reduced work gain or a higher work cost. The physical meaning of can be grasped by considering a lower bound[@b18] on , see Supplement. Here *d* is the dimension of the system and denotes the Hilbert-Schmidt norm. The first factor quantifies the distance of the initial state from the fully mixed the second factor, , quantifies the angle between diagonal basis of *ρ* and the projection basis . These terms correspond to incoherent and coherent mixing contributions. The entropy change is non-trivially bounded only if initial state is not an incoherent mixture with to that basis. The entropy bound is the largest for initial whose basis mutually unbiased with respect to . In this case the optimal change . One may wonder where the work has gone to. There two equivalent approaches to the accounting of work. In the present analysis the focus is on the work that the system exchanges, as done in statistical physics[@b5][@b19][@b20][@b21][@b22]. In this approach it is often not explicitly mentioned where the work goes but the only place work can go to are the externally controlled energy Similarly, the heat, i.e. the energy change minus the work, is established implicitly. For experimental realisation classical Landauer erasure with a silica bead trapped in an optical tweezer[@b21], the heat of erasure was calculated by knowing the applied tilting forces integrating over the bead's dynamics. The second approach is to collect work a separate work storage system[@b23], as by the weight in [Fig. 1](#f1){ref-type="fig"} and detailed in the Supplement. Both the implicit and the explicit treatment of work are equivalent in sense that the results obtained in one approach can be translated into the other. The thermodynamic assumptions made to prove Eq. [(2)](#eq14){ref-type="disp-formula"} are congruent with current literature[@b9][@b23][@b24][@b25]; specifically they are: (T0) an isolated system is system that only exchanges work and not heat; (T1) the validity of the law* the internal energy Δ*U*, of the during a process to its average heat absorbed and work drawn, ; (T2) validity of *second law* relating the system's entropy change to its average absorbed heat, , when interacting with a bath at temperature *T*, with equality attainable by an optimal process; (T3) the thermodynamic entropy to be to the von Neumann entropy in equilibrium as well as out-of-equilibrium, . addition we make the following standard quantum mechanics assumptions: (Q0) an isolated system evolves unitarily; (Q1) control of a system includes its coherences. Details of the proof in the Methods Summary. We note that in the single-shot setting whole families second laws apply[@b7][@b8] that differ from (T2) stated above. However, in the limit of many independent and identically copies the these collapse to the standard second law, on the basis which Eq. [(2)](#eq14){ref-type="disp-formula"} is derived. From the information-theory point of view projections considered constitute just example of the larger class of trace-preserving completely positive (TPCP) maps characterising quantum dynamics. Of all TPCP maps can be interpreted with the stated above, resulting in optimal average work given by free energy difference. Erasure is another such map whose study forged link between information theory The benefit of discussing "projections" here lies in the insight that this focus provides: it uncovers that coherences offer the potential to draw work making it a genuine and quantum thermodynamic feature. This work is non-trivial even when the thermodynamic process is on the system alone, not involving any stored in other degrees freedom. Qubit example for drawing optimal work -------------------------------------- gain a understanding thermodynamic projection processes that give the optimal work stated in Eq. [(1)](#eq12){ref-type="disp- | WhEn lanDaueR aRGuEd in 1961 that Any PHySICal RealisaTIoN of ERasuRE of INfoRMaTiON hAs a fuNdAmeNTaL theRmodyNaMIc WorK cOSt hE IRrEVOcAblY LInKeD THermoDyNaMiCs AND iNFoRmaTion THeoRy[@b1][@b2][@B3][@B4][@B5][@B6][@B7][@B8][@B9]. A pRActiCAl ConseQuencE OF thiS insigHT Is THAT all CoMPuters mUST dISSipATE A minIMaL amounT oF hEat IN eaCH iRrEvERSibLe cOmpUTing step, a thReShold THAt is bEcOMIng A coNcErN WiTh FUtuRE COMpUtER CHIpS ENTerIng atOMic ScaleS. the tReATMenT OF geNeral *QUANTuM* InfORmAtiON PRoCesSiNG TASks wIthIn THE wiDER fRAmeWORK of qUAntUM THermODYnaMIcS HaS ONlY REcENTLY bEgUn[@b13]. qUaNtuM mechaNIcS dIfFers FrOM CLassicaL MEcHAnICs IN AT least ThReE CentrAL ASpEcTs: THe sPeCIAL NaTURe oF meAsUREmENt, thE pOSsIbIliTY oF a QUaNTUm SysTem TO bE In a sUpeRPOsITioN and The EXiStENcE Of QUaNtUM coRRElAtiONs. ThE thermODyNAmIC eneRGy nEEDeD To PERfORm a (SElEctIvE) MeasuRemENt HaS bEEn INVesTiGaTed[@b10] aND THE toTal WOrK FOR a CLOsEd THErmODyNaMic MEaSUrEmEnt cYcle EXPLOREd[@b11]. thE CAtaLyTIc rOLe of QuAntUm sUPeRpoSItIOn StatES wHeN USeD in thErmAl opeRaTiOnS hAs beEn uNcovErEd[@b12] anD iT haS BEEn ShoWn thAT woRK CAN BE DrAWN FrOm quantuM correlations[@b13][@b14] In A therMoDYNAmiC sEttiNg, sEE [fIg. 1](#f1){Ref-tYpE="fiG"}. In pArTicular, deL RIO *eT al.*[@B14] sHowed tHAt COnTraRY TO lANdaUeR'S PrINCipLE, it iS posSIBle to *extRAcT* WORk WHiLe pERFORming erasURe OF A SySteM'S stAte WheN The SYSTEm Is coRrELateD to a MEMoRy. ThIS can ocCUR If anD OnLY IF ThE InitIal cORReLATIoNs ImpLy A neGatIVE COndiTioNAL EnTROPy, a uniQuEly qUantUM fEAtUrE. THe TheRmOdYnaMiC proCEss doES HowEvER NOW require OpeRATION On DegREeS OF frEedOM eXTErnAl TO the SYsTEm, i.e. tHE Memory'S.
rEsULTs
=======
PRojEcTionS and THE OptiMAl WORk vAlUe oF remOViNG coHeREnCES
-------------------------------------------------------------
OUR motiVAtioN Is herE To sHeD ligHt on tHE IMplICATIoNS Of perforMiNG a meASUremenT On a QuaNtum StATe THAT hAS COHErENCEs. WE Will COnsideR this TASk IN The THeRModynAMIC sETTinG OF lANDAueR's erasuRe, InVolVing a hEAt BATH At fiXed tEMPEratuRE *t* AnD oPERATiON ON *N* → ∞ uNCOrReLaTEd aNd iDentICAlLy PREParEd copIeS Of tHE sYsTeM (I.i.D. LIMIT). THiS is of InteREST iN The conText OF THE qUAntUm JaRzYnSKi eQuALiTy, FOr exaMplE, AnD wIlL ALso be CentRAL for expErimEntS TesTinG quANTUm thERmoDynaMIc PReDICtIons in THE FutuRE. to TAcKle THIs QUesTIoN WE dEfINE The INFOrmatiON-tHeoRetic "proJectION" FOr A giVen INiTial QuAntum StaTE *ρ* ANd A ComPleTe SeT of MutuAllY ORThogoNAl pROJEcTORS . sUCh stATe tRANSFOrmaTion CAn BE seEN As aNAlOgOus To ThE STaTe tRaNsfeR Of EraSUre, , TO a BlAnk STAte . pHYsIcALlY, tHis prOjEcTion cAN be iNTerPreTED aS The RESuLt oF An unrEad, Or uNsElECtIVE[@B15], MEAsUreMent of AN OBSERvABle THat has EIGeNvECTor PROJECtorS . in AN unSElEcTiVE measuremenT thE INdividuaL mEAsurEmENt ouTcoMES ARe noT recORDeD aND onLy tHE staTiSTics oF oUTcOmes Is known. iN tHe LIteRaTuRE ThE IMPLementAtIOn Of UNSelEcTIvE MEAsuReMeNtS is oFteN nOt specIfied, AlThOuGh IT is tyPIcally THoUGht of aS MeASuring indivIdual OUtCOmEs, E.g. wIth a STErN-geRlach ExPEriMenT, SEe [FIg. 2A](#F2){ReF-TYPE="FIg"}, FOLloWeD bY MiXing. tHe cRuX IS tHAT thE infoRMATion-theorETIC proJECtiOn caN be ImPlEMeNtEd In manY PHYsicAL WaYS. tHe ASsOCiAtEd THERmODYnamiC hEAT aNd WORK WILL DIFfeR dePenDING oN *HOw* tHe pROJecTioN WaS DonE aND we wIlL refER To the variOuS REaLiSATiOnS as "theRMoDyNamIC proJecTioN prOcesseS". One POsSIBILiTY is DecohErIng[@b16] The State IN THE So-cALLED poINteR basIS, , a thErMODYnAMic prOcess WHeRE AN EnvIRonMEnt REmoVes coheREnces in AN uNconTrollED MAnNER rESulTINg In no AsSOcIAtED wORk. iN GenERaL IT iS POSSIBLE To ImpLEmeNT The STAtE traNsfER in A FInelY CONTROLLed FASHiOn AcHIEviNG optiMAL ThERmoDyNAMiC HEaT aND wOrK vALUES.
OF paRtIcUlAR iMPOrTaNCE IN therMOdYnamIcs iS THE PROJeCtIon oF THe sYsteM's iNItIAl sTate *Ρ* ONTO THE seT oF EnErGY eIGeNSTATeS oF THE SYSTEM's HAmilToniAn wiTH *E*~*K*~ THe enERGy EIGeNVALUEs. hErE ThE StATe's Off-diAGONals wItH rEspEct To the enERGy eIGENbasis Are remoVeD - a StatE transfoRMaTioN THAt is FREQuENtly empLOyEd In qUanTUM ThErmoDyNaMiC deRIvaTioNs anD REFerrED To As "DepHAsInG" OR "MeaSURING thE EnERGY". our KEy ObsErvaTIOn IS that THere ExIsTS A TheRmODYNAMIc PRoJeCtIOn procEss REAlisIng THIs tRAnSFormAtiOn ANd ALlOWING TO DRaW fROM tHe QUAnTuM sYsTEM A NoN-trIVIaL *opTiMaL averaGe Work* OF
HeRe *T* IS the teMPeRATurE Of The heat bAth WitH WHIcH the sYStem Is ALlOWED tO IntERacT, sEE IllUsTraTIon [fIG. 1](#f1){rEf-TYPE="FIG"}, *k*~*B*~ iS tHe bOltZMaNN COnStANT anD *S* iS tHe VON neUmANN EntRopy. cRuCiAllY, ThIs WorK is stRiCtLy POsiTIvE FoR quAntum sTateS witH CoHerENceS. ExtEnDinG the kEY oBSErVatIOn to geNerAl PRoJEcTIOns ONE FInDS THat oPtIMaL THermOdYnAMic pRojecTION ProCEsSes cAn bE IMPlemENted ThAT aLloW to DRAw an AVeRAGE WOrK OF
wHerE AN addItIonAl INTErnal eNERGY CHanGE Term APpears.
PhysICAL InTeRpREtAtion aND aSsUmPtIonS MADe tO dErivE thE OPtimAl wOrK
-----------------------------------------------------------------------
THE OptiMaL WOrk VaLUEs staTED IN eQS. [(1](#EQ12){Ref-tYPE="dISp-FoRmuLA"}) ANd ([2](#EQ14){reF-TYPe="DISP-FoRmuLA"}) are valID for PrOcesseS apPliED To claSSICaL AnD QuAntuM sTaTEs AliKe. whiLe for A clASSICaL EnseMblE ThE enTrOPY chaNge, , WIlL BE ZerO This Is NOT SO iN ThE genErAl qUAnTUm SITUaTION, where iNItIal Non-dIAGonal QuantUm STatEs ReSuLT IN a StRICtlY poSItIVe enTRopy CHANgE[@B17]. WE NOTe thAt wHilE The oPtimAL WoRk vaLues aRe In PRInCIplE ATtAINable, PRactiCAL iMPlEMeNtaTiOns maY be SUbOPTImAl rEsULtIng in a REducED wOrk Gain OR a hIgHer wOrk cOST.
THE pHySiCal MEaNINg of Can be GrASPeD By COnSIDERiNg a LOwER Bound[@B18] on it, , seE SuppLeMeNt. heRe *d* IS tHe DImeNsIoN of THE sYStem anD DenOteS ThE HiLbERt-sChMiDT Norm. thE FiRSt fACtoR quaNTIFIes tHE DistAncE oF THe initiAL stATE FrOm THE fUllY MixEd state, WHiLE tHe sECoNd fAcTor, , QuAnTIfiES ThE ANGLE BEtweEn THE DiagonaL basis OF *Ρ* AnD tHE pROJECtIon BAsIs . THeSE TErMS cORreSpoNd tO iNCOhErenT aNd cohEreNT MixINg conTRIbUTIONS. The EnTROPY CHANgE IS noN-TriVIAlly bOUndeD OnLy IF thE inITIAl STATe Is nOt aN InCoHEreNt mIXtUrE wItH reSpECt To tHAt bASIs. The eNTroPy boUND is THe LaRGeST FoR purE IniTIal stateS whOSe baSIs IS MUTuaLly uNbiASEd witH ResPeCt to . in THiS cAsE THE OptIMAl EnTrOpY cHANge iS .
oNe May WoNDEr wHErE thE WoRK HaS gONE TO. thErE Are tWo eqUIvalEnT ApprOacHEs to tHe AcCouNTing oF WoRk. in tHE pRESEnt ANALysIs THE foCus iS ON THe wORk THAT THe sYsTEm excHAngEs, As DONe In StATIStiCaL PHYSicS[@B5][@b19][@b20][@b21][@b22]. In thIS ApprOAcH iT Is OfTeN NOt ExPLiCItly MEnTIoneD whERe THe WOrK GoEs tO, bUt THe Only pLACe WoRK CAn GO To ARE ThE extERNaLlY cONTROLLed enerGY SourcES. SimILARly, the HEat, i.e. tHe ENERGY CHANgE mINuS the WorK, IS EsTAblIsHEd ImPLiCiTLY. FoR exaMpLe, In the EXpErImentAL ReaLiSatiON Of clasSIcal laNdAUEr ErasURE wItH A cOlLOidAL sIlICa bEaD trAPPed in An oPtiCAL tweEZer[@B21], THE DisSIPatED HEAt OF ERaSurE WaS CAlCulAtEd By kNOwING ThE APPlIed tiLtIng fORcEs ANd iNTeGratING OVEr tHE bEad's DYnAMIcS. tHe sECOnD ApProaCh iS To cOlLeCt WorK In a SEPaRAtE WoRK STORage SYstem[@B23], aS ilLUSTrATED BY ThE weigHT in [Fig. 1](#F1){ReF-Type="FIg"} ANd DeTAiled In ThE suPpLEMEnt. BOtH The ImPlIcit aNd tHE eXPlIciT TreatMenT of work aRe equIvaleNT iN ThE senSe thAT ThE ResuLTS OBtaINed iN oNE ApPROAcH CAn bE transLated into the Other.
THE tHERModYnAmiC aSsuMPTiONs MaDE tO ProvE eQ. [(2)](#eQ14){REF-TYpe="disP-FormULa"} ArE congRuEnT wITH cuRReNT LITeRaTure[@B9][@b23][@B24][@B25]; SPecificAlLy they Are: (T0) aN ISOlAtED sYstEM Is A SystEm THat onLy eXchAngeS worK anD not heAt; (T1) The vAlIditY OF thE *First laW* RelaTING the InTeRNAl ENergY changE, δ*U*, Of ThE sYsTEm DurING a PrOCeSs To ITs aveRAGe HEaT aBSoRBEd anD woRK drawN, ; (T2) The ValiDItY oF THE *sEcoND lAw* ReLaTINg THE syStEm's entROPY chANgE To itS avERage AbSOrBed hEAt, , wHEN iNtERaCTInG WITh A Bath aT tEMPeRAtuRe *t*, wiTh eQuALItY aTtaINable BY aN optIMal prOCeSs; (T3) the tHErmODYnamIc enTROPY TO bE eQuAL To THE von NeUMAnN entROPy IN EQuilIbRIuM aS wEll as OUT-Of-eQUiLIbriUm, . In aDDiTIon wE MAKe tHE FoLLOwiNg STanDarD QUantum MECHaniCS ASSUMPTions: (Q0) An isOlATed sYsTeM EvOLveS uniTaRily; (q1) CONTROL OF A QUANTuM sYsteM IncLUDES ITS cOHerences. DetaIlS of thE ProOF are in tHe meThODs sumMArY. WE noTE tHat iN THE siNgLE-SHOT SeTTING WhoLe FaMIlieS of SECoND laWS appLY[@b7][@B8] ThaT DiFfEr FRoM (T2) sTaTed ABoVE. hoWeveR, In THE lImit OF iNfIniteLy mANy indepeNdENt and IDentIcALLY prEPaREd cOpiEs OF thE SysTEM ThEse CoLLapse tO tHe sTaNDArD SecONd LAW, (T2), oN thE BAsIs OF WHICH EQ. [(2)](#EQ14){rEf-tYpe="dISP-fORMUlA"} is dERiVeD.
froM The inFOrMAtIon-tHEORY poINT of VieW tHe prOjECtIOnS cOnSIDerED HeRe COnstItUte JUSt OnE EXamPLe OF The LARGeR ClasS oF tRaCE-PrESERvING cOMpleTeLY POSItiVe (TPcP) MAps CHAraCTERisIng qUantUM dyNAMiCs. oF cOURSe, alL TpCP MApS CaN be InTERPretED THeRMODYNaMiCalLY WITH ThE aSSuMPtiOnS sTateD aBoVe, RESULting in an OPTiMal AvErage WoRk GIVEn BY a FrEe enERgy diffErEnCE. erasuRE Is anOThER SuCh mAP whosE STUdy foRGed ThE LInk bETwEEn InFoRMatiOn THeoRy anD thErmodYNAmIcS. tHe bENEfIT OF disCUssiNg "pRojEctIoNs" HeRE LIES in THE INSiGht THaT thIs focUs PrOVideS: IT uNCoVErS That cOHEreNceS OfFeR tHE POTEntiAL TO DRaW wOrk mAKing it a genUinE ANd TEstabLE quANTuM ThERmoDYnAMIC FEatuRE. This woRk IS NoN-trIvIaL evEN WHen tHE TheRmODynAMic prOCeSs IS opErAted On thE sYsTEm ALoNE, NOT iNvOlvING Any SIDE-INformATion[@b14] STorED IN oTher dEGREEs Of FReEDOM.
QUbIt eXamPle fOr DRAwinG oPtimal work
--------------------------------------
To GAin A DEtAIlED unDeRStAndIng oF THeRMoDyNAMIC PROJECTIoN proCessEs ThaT GiVE tHE OpTiMal wORK sTAted in eQ. [(1)](#Eq12){reF-Type="dISP- | When Landauer argued in 1961 that any physical realisation of erasure of information has a fundamental thermodynamic work cost he irrevocably linked thermodynamics andinformation theory[@b1][@b2][@b3][@b4][@b5][@b6][@b7][@b8][@b9]. A practical consequence of thisinsightis that all computers must dissipate a minimal amount of heat in each irreversible computing step, a threshold that is becoming a concernwith futurecomputer chipsentering atomic scales. The treatment of general *quantum* informationprocessing tasks within the wider framework of quantum thermodynamicshas only recently begun[@b13]. Quantum mechanics differsfromclassicalmechanics inat least three central aspects: the special nature of measurement, the possibilityof a quantumsystem to be inasuperposition and the existenceof quantum correlations. The thermodynamic energy needed to perform a (selective) measurement hasbeen investigated[@b10] and the totalwork for a closed thermodynamic measurement cycle explored[@b11]. The catalytic role of quantum superpositionstates whenused in thermaloperations has beenuncovered[@b12] andit has been shown that work canbe drawnfromquantum correlations[@b13][@b14] in a thermodynamic setting, see [Fig. 1](#f1){ref-type="fig"}. In particular,del Rio *etal.*[@b14] showed that contrary to Landauer's principle, it is possible to *extract* work while performing erasure ofa system's state when the system is correlated to amemory. This can occur if and only if the initial correlations implya negativeconditional entropy, a uniquely quantum feature. The thermodynamic process does however now requireoperation on degrees of freedom external to the system, i.e. the memory's. Results ======= Projections and the optimal work value of removing coherences -------------------------------------------------------------Our motivation is here to shed light on the implications of performing a measurementon a quantum statethat hascoherences. We will consider this taskin the thermodynamicsettingof Landauer's erasure, involving a heat bath at fixed temperature *T* and operation on *N* →∞ uncorrelated and identically prepared copies ofthe system (i.i.d. limit). This is ofinterest in thecontextof the quantum Jarzynski equality,for example, and will also be central for experimentstesting quantum thermodynamicpredictions in the future. To tackle this question we define the information-theoretic "projection" for a given initial quantum state *ρ*and a complete set of mutuallyorthogonal projectors . Such state transformation can be seen as analogous to the state transfer of erasure, , to a blank state .Physically, this projectioncan be interpreted as the result of an unread,or unselective[@b15], measurement of an observablethat has eigenvector projectors. In an unselective measurement the individual measurement outcomes are not recorded andonly the statistics of outcomes isknown. In the literature the implementation of unselective measurements is often not specified,although it istypically thought of as measuring individual outcomes, e.g. with a Stern-Gerlach experiment, see [Fig. 2a](#f2){ref-type="fig"}, followed by mixing. Thecrux is that the information-theoretic projection can be implemented in many physical ways. The associated thermodynamic heatand work will differ depending on*how* the projection was done and wewill refer to the various realisationsas "thermodynamicprojection processes". One possibility isdecohering[@b16] the state in the so-called pointer basis, , a thermodynamic process where anenvironment removescoherences in an uncontrolled mannerresulting inno associated work. In general it is possible to implement the state transfer in a finely controlled fashion achievingoptimal thermodynamic heatand work values. Of particular importance inthermodynamics is the projectionof the system's initial state *ρ*onto the set of energy eigenstates of thesystem's Hamiltonian with *E*~*k*~ the energy eigenvalues. Here the state'soff-diagonals with respect to the energyeigenbasis are removed - a state transformation thatisfrequentlyemployed in quantum thermodynamic derivations and referred to as"dephasing" or "measuring the energy". Our key observationis that thereexists a thermodynamic projection process realising this transformation and allowing to draw from the quantum system anon-trivial *optimal average work* of Here *T* is the temperature of the heat bath with which the system is allowedto interact,see illustration [Fig. 1](#f1){ref-type="fig"}, *k*~*B*~ is the Boltzmann constant and *S* is the vonNeumannentropy. Crucially,this work isstrictly positivefor quantum states with coherences. Extending the key observation to general projections one finds that optimal thermodynamic projection processes canbe implemented that allow to draw an averagework of where an additional internal energy change term appears. Physicalinterpretation and assumptions made to derive the optimal work ----------------------------------------------------------------------- The optimal work values stated in Eqs.[(1](#eq12){ref-type="disp-formula"}) and ([2](#eq14){ref-type="disp-formula"}) are valid forprocesses applied to classical and quantum states alike. While for a classical ensemble the entropy change, , will bezerothis is not so in the general quantum situation, where initial non-diagonal quantum states result in astrictly positive entropy change[@b17]. We note that while the optimal work values are in principle attainable, practical implementations may be suboptimal resulting in areduced work gain or ahigher work cost. The physical meaning of canbe grasped byconsideringa lower bound[@b18] on it, , see Supplement. Here *d* is the dimension of thesystem and denotes the Hilbert-Schmidtnorm. The first factor quantifies the distance of the initial state fromthe fully mixed state, while the second factor, , quantifies the angle between the diagonal basis of *ρ* and the projection basis . These terms correspond to incoherent and coherent mixing contributions. The entropychange is non-trivially boundedonly if the initial state is not anincoherent mixture withrespect to that basis. The entropy bound is thelargest for pure initial states whose basis is mutuallyunbiased with respectto. In this case the optimal entropy change is . One may wonder wherethe workhas gone to. There are twoequivalent approaches to the accounting of work. In the present analysis the focusis on the work that the systemexchanges, as done in statistical physics[@b5][@b19][@b20][@b21][@b22]. In this approach it is often not explicitlymentioned where the work goes to, but the only place work can go to are the externally controlled energysources. Similarly, the heat, i.e.the energy change minus the work, is established implicitly. Forexample, in the experimental realisation of classical Landauer erasure with a colloidalsilica bead trapped in anoptical tweezer[@b21], the dissipated heat of erasure was calculatedby knowingtheapplied tiltingforces and integrating over the bead's dynamics.The secondapproach is to collect work in a separate work storage system[@b23], as illustrated by the weight in [Fig. 1](#f1){ref-type="fig"} and detailed in the Supplement. Boththe implicit and the explicit treatment of work are equivalent in the sensethatthe results obtained in one approach can be translated into the other. The thermodynamic assumptions made to prove Eq.[(2)](#eq14){ref-type="disp-formula"} are congruent with currentliterature[@b9][@b23][@b24][@b25];specifically they are: (T0) an isolated system is a system that only exchanges work and not heat; (T1) the validity of the *first law* relatingthe internal energy change, Δ*U*, of the system during a process to its average heat absorbed and work drawn, ;(T2) the validity of the*second law* relating the system's entropy change to its average absorbed heat, , when interacting with a bath at temperature *T*, with equality attainable by an optimal process; (T3) the thermodynamic entropy to be equal to the von Neumannentropy in equilibrium as wellas out-of-equilibrium, . In addition we makethefollowing standard quantum mechanics assumptions: (Q0) an isolated system evolves unitarily; (Q1)control of a quantum system includesits coherences.Details of the proof are in theMethods Summary. We notethat in the single-shotsetting whole families of second laws apply[@b7][@b8] that differ from (T2) stated above. However, in the limit of infinitely many independent and identically prepared copies of the system these collapse to the standard second law, (T2), on the basisofwhich Eq.[(2)](#eq14){ref-type="disp-formula"} is derived. From the information-theorypoint of view the projections considered here constitutejust one example of the larger class oftrace-preserving completelypositive(TPCP) mapscharacterising quantum dynamics. Ofcourse, all TPCP maps can be interpreted thermodynamically with the assumptions statedabove, resulting inan optimal average workgiven by a free energy difference. Erasure is another such map whose study forged the link betweeninformationtheory andthermodynamics. Thebenefit of discussing "projections" here lies inthe insight that this focus provides: it uncovers that coherences offerthe potential to draw work making it a genuine and testable quantum thermodynamic feature. This work is non-trivial even when the thermodynamic processis operated on the system alone, notinvolving any side-information[@b14]stored in other degrees of freedom. Qubit example fordrawing optimal work -------------------------------------- Togain a detailedunderstanding ofthermodynamic projection processes that give the optimalwork stated in Eq. [(1)](#eq12){ref-type="disp- | When Landauer argued in _1961_ that any physical realisation of erasure of information has a fundamental thermodynamic work cost he _irrevocably_ _linked_ thermodynamics and information _theory[@b1][@b2][@b3][@b4][@b5][@b6][@b7][@b8][@b9]._ A practical consequence of this insight is _that_ all computers must dissipate a minimal _amount_ of _heat_ in each irreversible computing step, a threshold that is becoming a concern with future _computer_ chips entering atomic scales. The treatment of _general_ *quantum* information processing tasks within the _wider_ framework of _quantum_ thermodynamics has only recently _begun[@b13]._ Quantum mechanics _differs_ from classical mechanics in at least three central aspects: the special nature of measurement, the _possibility_ of a quantum system to be in a superposition and the existence of quantum correlations. The thermodynamic energy needed to perform a (selective) _measurement_ has been investigated[@b10] and the total work for a closed _thermodynamic_ measurement cycle explored[@b11]. The catalytic _role_ _of_ quantum _superposition_ states when used in thermal operations has been uncovered[@b12] _and_ it has been shown that work can be _drawn_ from quantum _correlations[@b13][@b14]_ in a _thermodynamic_ setting, _see_ _[Fig._ 1](#f1){ref-type="fig"}. In particular, del Rio *et al.*[@b14] _showed_ that contrary to Landauer's principle, it is possible to *extract* work _while_ performing erasure of a system's state when the _system_ is correlated _to_ _a_ _memory._ This can occur _if_ and only if the initial correlations _imply_ a negative conditional entropy, a _uniquely_ _quantum_ feature. The thermodynamic process _does_ _however_ _now_ require operation on degrees _of_ freedom external _to_ _the_ system, i.e. the memory's. Results ======= Projections _and_ the optimal work _value_ of removing coherences ------------------------------------------------------------- Our motivation is here to shed light on the _implications_ of _performing_ a measurement on a quantum _state_ _that_ has coherences. We will consider this task in the thermodynamic _setting_ of Landauer's erasure, involving a _heat_ bath _at_ fixed temperature *T* and operation on _*N*_ → ∞ uncorrelated and identically _prepared_ copies of the system (i.i.d. limit). This is of interest in the context of the _quantum_ Jarzynski equality, for example, and will also be central for _experiments_ testing quantum thermodynamic predictions in the future. To tackle _this_ question we define the information-theoretic "projection" _for_ _a_ given initial quantum state *ρ* _and_ _a_ complete set of _mutually_ orthogonal _projectors_ . Such _state_ transformation _can_ be _seen_ as analogous to the state transfer of _erasure,_ , to _a_ blank state . _Physically,_ this projection can be _interpreted_ as the result of _an_ unread, _or_ unselective[@b15], _measurement_ of an observable that has eigenvector projectors . In an unselective measurement the individual measurement _outcomes_ are not recorded and only the statistics of outcomes _is_ known. _In_ _the_ literature the implementation of unselective measurements is often not specified, although it is typically thought of as _measuring_ individual outcomes, _e.g._ with a Stern-Gerlach _experiment,_ see [Fig. _2a](#f2){ref-type="fig"},_ followed by mixing. The crux _is_ that the information-theoretic _projection_ can be implemented in many physical ways. _The_ associated thermodynamic heat _and_ work will differ depending on _*how*_ the projection was done and we _will_ refer to the various realisations as _"thermodynamic_ projection _processes"._ One possibility is decohering[@b16] the state in the so-called pointer _basis,_ _,_ a thermodynamic process where an environment removes coherences in an uncontrolled manner resulting _in_ no associated work. In general it is possible to implement the state transfer in a finely _controlled_ fashion achieving optimal thermodynamic heat and work values. _Of_ particular _importance_ in thermodynamics is _the_ projection of the system's initial state *ρ* onto the set of energy eigenstates of the system's _Hamiltonian_ with *E*~*k*~ the _energy_ eigenvalues. Here the _state's_ off-diagonals with respect _to_ _the_ energy eigenbasis are removed - a state _transformation_ that is frequently employed in quantum thermodynamic _derivations_ and _referred_ to as _"dephasing"_ or "measuring the energy". Our key observation is that _there_ exists a _thermodynamic_ projection process realising _this_ transformation and allowing to draw from the quantum system a _non-trivial_ *optimal average work* of Here _*T*_ _is_ the temperature of the heat bath _with_ which the system is _allowed_ to interact, see illustration [Fig. 1](#f1){ref-type="fig"}, *k*~*B*~ _is_ _the_ Boltzmann _constant_ and *S* is _the_ von Neumann _entropy._ Crucially, _this_ _work_ is strictly positive for quantum states with coherences. Extending the _key_ observation _to_ general projections one finds _that_ optimal thermodynamic projection processes _can_ be implemented that allow _to_ draw an average work of where an additional internal energy change term appears. Physical interpretation and assumptions made to derive the optimal _work_ ----------------------------------------------------------------------- The optimal work values stated in Eqs. [(1](#eq12){ref-type="disp-formula"}) _and_ _([2](#eq14){ref-type="disp-formula"})_ _are_ valid _for_ processes applied to classical and quantum states alike. While _for_ a _classical_ ensemble the entropy change, , will be zero _this_ is not so in the general quantum _situation,_ _where_ initial _non-diagonal_ quantum states result in a strictly positive entropy change[@b17]. We note that while the optimal work values are _in_ _principle_ attainable, practical _implementations_ may be suboptimal resulting in a reduced work gain or a _higher_ work cost. The physical meaning _of_ can be grasped _by_ considering a lower bound[@b18] on it, , _see_ Supplement. _Here_ *d* _is_ the _dimension_ of the system and denotes the Hilbert-Schmidt norm. The first factor quantifies the distance of _the_ initial state _from_ the _fully_ mixed state, while _the_ second _factor,_ , quantifies the angle between the diagonal basis of *ρ* and the projection basis . These terms correspond to incoherent and coherent mixing contributions. The entropy change is non-trivially bounded only if the initial state is not an incoherent mixture with _respect_ to that basis. The entropy bound is the largest for pure initial states whose basis is _mutually_ unbiased _with_ respect to _._ In _this_ _case_ the _optimal_ entropy change is . One may wonder where the work has gone to. There are two equivalent approaches to _the_ _accounting_ _of_ work. In the _present_ analysis the _focus_ is on the work that the system exchanges, _as_ _done_ _in_ statistical physics[@b5][@b19][@b20][@b21][@b22]. In this approach _it_ is often not _explicitly_ mentioned where the work goes to, but the only place work can go to _are_ the _externally_ controlled energy _sources._ Similarly, the heat, _i.e._ the energy change minus the work, is _established_ implicitly. For example, _in_ the experimental realisation of classical _Landauer_ erasure _with_ a colloidal _silica_ bead _trapped_ in an optical tweezer[@b21], the dissipated _heat_ of erasure was calculated by knowing the applied _tilting_ forces and integrating over the bead's dynamics. The second _approach_ is to collect _work_ in a separate _work_ storage system[@b23], as illustrated by the weight _in_ [Fig. 1](#f1){ref-type="fig"} _and_ _detailed_ in _the_ Supplement. Both the implicit and the explicit treatment of work are equivalent in the sense that the results obtained in _one_ approach can be translated into the other. The thermodynamic _assumptions_ made to prove Eq. [(2)](#eq14){ref-type="disp-formula"} are _congruent_ with _current_ literature[@b9][@b23][@b24][@b25]; specifically they are: (T0) _an_ isolated _system_ is a system that only exchanges _work_ _and_ not heat; (T1) the validity of the _*first_ law* relating the internal energy change, Δ*U*, _of_ the system during a _process_ to its average heat absorbed and work _drawn,_ ; (T2) the validity _of_ _the_ *second _law*_ relating _the_ system's entropy _change_ to _its_ average _absorbed_ _heat,_ , when interacting with _a_ bath _at_ temperature *T*, with equality _attainable_ by an optimal process; (T3) the thermodynamic entropy to be equal to the von _Neumann_ entropy _in_ _equilibrium_ _as_ well as out-of-equilibrium, . In _addition_ we make the following standard _quantum_ mechanics _assumptions:_ (Q0) an _isolated_ system evolves unitarily; (Q1) control of a quantum system includes its coherences. _Details_ of the proof are in the Methods Summary. We note that _in_ the single-shot setting whole families _of_ second laws apply[@b7][@b8] that differ from (T2) stated above. However, _in_ the limit of _infinitely_ many _independent_ and identically prepared copies of the _system_ these collapse to _the_ standard second law, _(T2),_ _on_ the _basis_ of which Eq. [(2)](#eq14){ref-type="disp-formula"} _is_ derived. From the information-theory point of view the projections considered here _constitute_ just one example of the _larger_ class of _trace-preserving_ completely positive (TPCP) _maps_ characterising _quantum_ dynamics. Of course, all TPCP maps can be interpreted thermodynamically with the _assumptions_ stated above, resulting _in_ an optimal _average_ work _given_ by a free _energy_ difference. Erasure _is_ another such map whose study forged the _link_ between information theory and thermodynamics. The benefit _of_ discussing "projections" here lies in the insight that this focus provides: it uncovers that coherences offer the _potential_ to draw _work_ making it a genuine _and_ testable quantum thermodynamic feature. This work is _non-trivial_ even _when_ _the_ thermodynamic process is _operated_ on the system alone, not involving any side-information[@b14] stored in _other_ _degrees_ of freedom. Qubit example for drawing optimal work _--------------------------------------_ _To_ gain a detailed understanding _of_ thermodynamic projection processes that give the optimal _work_ _stated_ in Eq. [(1)](#eq12){ref-type="disp- |
Background {#Sec1}
==========
Congenital cataracts are diagnosed within the first year of life. These cataracts are one of the leading causes of blindness in children and are estimated to occur with a prevalence of 3--6 per 10,000 live births \[[@CR1]\]. Congenital cataracts may appear either in isolation or in association with other ocular or systemic anomalies. Up to 25 % of congenital cataracts are thought to be caused by genetic defects \[[@CR2]\]. The genetic landscape of mutations causing congenital cataract is extremely diverse; more than 40 genes and additional loci have been associated with nonsyndromic cataract \[[@CR2]--[@CR6]\].
*GCNT2* (glucosaminyl (N-acetyl) transferase 2, I-branching enzyme) was first identified in 2001 as the gene encoding for the glycosyltransferase responsible for the human blood group I antigen. Recessive mutations in *GCNT2* result in an adult i blood group phenotype, which is also associated with congenital cataracts in some cases \[[@CR7]\]. Alternative splicing of the *GCNT2* gene produces three transcripts (A, B, and C). The three transcripts share a common second and third coding exon with a unique first exon for each isoform; differing expression profiles were identified for the transcripts with only the *GCNT2B* isoform expressed in lens epithelial cells and only the *GCNT2C* isoform expressed in reticulocytes \[[@CR8]\]. To date, seven missense mutations, one nonsense mutation, and two large deletions have been reported; mutations in exon 1C, affecting only the *GCNT2C* isoform, cause the adult i blood group without cataracts while mutations/deletions affecting exons 2 and 3, shared by all isoforms, result in the adult i blood group along with congenital cataract \[[@CR7]--[@CR11]\].
Case presentation {#Sec2}
=================
Patient 1(individual II:1) is an 18-month old Pakistani female affected with bilateral dense central congenital cataract (Fig. [1a,](#Fig1){ref-type="fig"} Table [1](#Tab1){ref-type="table"}) which were visually significant and required extraction at 2 months of age, mild asymmetry of the palpebral fissures, and left nasolacrimal duct obstruction; her development is normal and growth parameters are generally normal with the exception of borderline microcephaly (length 83.8 cm, 75--90th centile; weight 10.2 kg, 25--50th centile; and head circumference 44 cm (3rd centile)). Physical exam at 4 months of age identified hypotelorism (familial) and mildly widely-spaced nipples. Her younger brother, age 6 months, was similarly affected with visually significant bilateral dense central congenital cataracts requiring extraction around 2 months of age; his length (67.5 cm, 25--50th centile), weight (6.8 cm, 5--10th centile), and head circumference (42.5 cm, 10--25th centile) are all within the normal range (Table [1](#Tab1){ref-type="table"}). Family history shows unaffected second-cousin parents with additional endogamous mating within the family. A double second-cousin to the proband is affected with bilateral non-syndromic anophthalmia/ microphthalmia with no additional details available.Fig. 1Patient photographs and pedigree. **a** Photograph of Patient 1's eyes at 2 months of age showing bilateral cataract. **b** Pedigree showing both affected siblings with a homozygous deletion of 6p24.3 while the unaffected parents are heterozygous carriers. WT: wild type; black arrow indicates probandTable 1Phenotype and genotype information of the affected patientsPatientLens phenotypeOther featuresDevelopmentDeletionGenes involvedPatient 1Bilateral dense central congenital cataracts; extraction at \~2 months of ageBorderline microcephaly (3rd centile), mild asymmetry of the palpebral fissures, left nasolacrimal duct obstruction, hypotelorism, somewhat widely-spaced nipplesWNL97.9 kb homozygous deletion of 6p24.3The first coding exons of *GCNT2A* and *GCNT2B,* two 5'noncoding exons of *GCNT2A*, and a part of the region upstream of *TFAP2A*Patient 2Bilateral dense central congenital cataracts; extraction at \~2 months of ageNoneWNL97.9 kb homozygous deletion of 6p24.3The first coding exons of *GCNT2A* and *GCNT2B,* two 5'noncoding exons of *GCNT2A*, and a part of the region upstream of *TFAP2A*
Materials and methods {#Sec3}
=====================
Whole exome sequencing was performed by Macrogen (previously Axeq) and analyzed as previously described \[[@CR12]\]; briefly, exome data from the proband was analyzed using the SNP & Variation Suite (SVS; Golden Helix, Bozeman, MT, USA) to identify/exclude mutations in the coding and splicing regions of 40 known nonsyndromic cataract genes and 7 additional crystallins \[[@CR3]--[@CR6]\]; synonymous variants and variants with a frequency of \>1 % in the general population ([http://exac.broadinstitute.org](http://exac.broadinstitute.org/), <http://evs.gs.washington.edu/EVS/>, <http://www.1000genomes.org/>) were considered to be benign variants. Copy number variation analysis was completed by screening exome sequencing data using the Copy Number Inference From Exome Reads (CoNIFER) v0.2.2 software package as previously outlined \[[@CR13]\]; regions of interest were further verified by independent quantitative PCR reactions using DNA samples from the proband and other available familial samples with SYBR Green PCR Master Mix (Applied Biosystems/Life Technologies, Carlsbad, CA, USA). qPCR reactions utilized three region-specific probes (Additional file [1](#MOESM1){ref-type="media"}: Table S1) and were performed as follows: primers located within regions of interest were designed using Primer3Plus software (<http://sourceforge.net/projects/primer3/>) using qPCR settings. Each reaction was comprised of five nanograms of DNA in a total reaction volume of 12uL. Each primer set was run three times in triplicate using patient, parental or control DNA on a Bio-Rad CFX Connect Real-Time PCR machine (Bio-Rad, Hercules, CA, USA). A primer set for the housekeeping gene *RPPH1* (ribonuclease P RNA component H1) was used to normalize all data. A probe located in *NDP* (Norrie disease (pseudoglioma)), located on the X-chromosome, was used as a copy-loss control. All experiments included a no-template control and an unaffected human DNA sample with presumably normal copy number at each region for comparison. Copy number changes were calculated using the 2^-ΔΔCt^ method as previously described \[[@CR14]\]. Following qPCR confirmation, the size and exact breakpoints of the deletion were determined using a series of regular PCR reactions that utilized primers located on both ends of the region (as defined by CoNIFER and qPCR analysis) and standard conditions (Additional file [1](#MOESM1){ref-type="media"}: Table S1). Since the patients were apparently homozygous for the deletion, no amplification product indicated that the primer(s) are located inside of the deleted region while the presence of a PCR product indicated primers outside of the deletion. Once sequences bordering the deleted region on the centromeric and telomeric sides were determined, the corresponding primers were used to amplify a 1.5 kb region across the breakpoints. The resultant product was cloned into pCRII-TOPO® (Life Technologies, Carlsbad, CA, USA) vector using the manufacturer's protocols and sequenced bidirectionally with M13 forward and reverse primers using Big Dye Terminator v3 chemistry and an ABI 3730XL sequencer (Applied Biosystems/Life Technologies, Carlsbad, CA, USA); the obtained sequences were compared with the corresponding reference sequence using BLAST (<http://blast.ncbi.nlm.nih.gov/Blast.cgi>).
Results and discussion {#Sec4}
======================
Review of the whole exome sequencing (WES) data from Patient 1 did not identify any potentially pathogenic variants (with only two synonymous variants) in known nonsyndromic cataract genes. The WES data was then analyzed for copy number variation which revealed a potential 208-kb deletion (6p24.3 chr6: 10,412,788-10,621,660) affecting *TFAP2A* and *GCNT2*. The deletion was verified using qPCR probes located in the first coding exon of *GCNT2* isoform A and the first exon of *TFAP2A*; the qPCR confirmed deletion of the *GCNT2* sequence in both unaffected parents (haploid, heterozygous) and affected children (complete loss, homozygous) while diploid copy of the *TFAP2A* sequence was identified in all family members. Further analysis of the region by a series of regular PCR reactions using affected DNA identified the centromeric breakpoint between chr6:10472330--10472606 (set 7; diploid) and chr6:10474759--10474901 (set 8; complete loss) and the telomeric breakpoint between chr6:10570580--10570905 (set 13, complete loss) and chr6: | background { # sec1 } = = = = = = = = = = congenital cataracts are diagnosed within the first year of life. these cataracts are one of the leading causes of blindness in children and are estimated to occur with a prevalence of 3 - - 6 × 10, 000 live births \ [ [ @ cr1 ] \ ]. congenital cataracts may appear either in isolation or in association with other ocular or systemic anomalies. up to 25 % reported congenital cataracts are thought to be caused by genetic defects \ [ [ @ cr2 ] \ ]. the genetic landscape of mutations causing congenital cataract is extremely diverse ; more than 40 genes and additional loci have been associated with nonsyndromic cataract \ [ [ @ cr2 ] - - [ @ cr6 ] \ ]. * gcnt2 * ( glucosaminyl ( n - acetyl ) transferase 2, i - branching enzyme ) was first identified in 2001 as the gene encoding for the glycosyltransferase responsible for the human blood group i antigen. recessive mutations in * rr * result in an adult i blood group phenotype, which is also associated from congenital cataracts in some cases \ [ [ @ cr7 ] \ ]. alternative splicing of the * gcnt2 * gene produces three transcripts ( a, b, and γ ). the three transcripts share a common second and third coding exon with a shorter first exon for each isoform ; differing expression profiles were identified for the mutation with only the * gcnt2b * isoform expressed in lens epithelial cells and causing the * gcnt2c * isoform expressed in ras \ [ [ @ cr8 ] \ ]. to date, seven missense mutations, two nonsense mutation, and two large deletions have been reported ; mutations in exon 1c, affecting only the * gcnt2c * isoform, cause the adult i blood group without cataracts while mutations / deletions affecting exons 2 and 3, shared by all isoforms, result in the adult i blood group along with congenital cataract \ [ [ @ cr7 ] - - [ @ cr11 ] \ ]. case presentation { # sec2 } = = = = = = = = = = = = = = = = = patient 1 ( individual ii : 1 ) is an 18 - month old pakistani female affected with bilateral dense central congenital cataract ( fig. [ 1a, ] ( # fig1 ) { ref - type = " fig " } table [ 1 ] ( # tab1 ) { ref - type = " table " } ) which were visually significant and required extraction at 2 months of age, mild asymmetry of the palpebral fissures, and left nasolacrimal duct obstruction ; her development is normal and growth parameters are generally normal with the exception of borderline microcephaly ( length 83. 8 cm, 75 - - 90th centile ; weight 10. 2 kg, 25 - - 50th centile ; and head circumference 44 cm ( 3rd centile ) ). physical exam at 4 months of age identified hypotelorism ( familial ) and mildly widely - spaced nipples. her younger brother, age 6 months, was similarly affected with visually significant bilateral dense central congenital cataracts requiring extraction around 2 months of age ; his length ( 67. 5 cm, 25 - - 50th centile ), weight ( 6. 8 cm, 5 - - 10th centile ), and head circumference ( 42. 5 cm, 10 - - 25th centile ) are all within the normal range ( table [ 1 ] ( # tab1 ) { ref - type = " table " } ). family history shows unaffected second - cousin parents with additional endogamous mating within the family. a double second - cousin to the proband is affected with bilateral non - syndromic anophthalmia / microphthalmia with no additional details available. fig. 1patient photographs and pedigree. * * a * * photograph of patient 1 ' s eyes at 2 months of age showing bilateral cataract. * * b * * pedigree showing both affected siblings with a homozygous deletion of 6p24. 3 while the unaffected parents are heterozygous carriers. wt : wild type ; black arrow indicates probandtable 1phenotype and genotype information of the affected patientspatientlens phenotypeother featuresdevelopmentdeletiongenes involvedpatient 1bilateral dense central congenital cataracts ; extraction at \ ~ 2 months of ageborderline microcephaly ( 3rd centile ), mild asymmetry of the palpebral fissures, left nasolacrimal duct obstruction, hypotelorism, somewhat widely - spaced nippleswnl97. 9 kb homozygous deletion of 6p24. 3the first coding exons of * gcnt2a * and * gcnt2b, * two 5 ' noncoding exons of * gcnt2a *, and a part of the region upstream of * tfap2a * patient 2bilateral dense central congenital cataracts ; extraction at \ ~ 2 months of agenonewnl97. 9 kb homozygous deletion of 6p24. 3the first coding exons of * gcnt2a * and * gcnt2b, * two 5 ' noncoding exons of * gcnt2a *, and a part of the region upstream of * tfap2a * materials and methods { # sec3 } = = = = = = = = = = = = = = = = = = = = = whole exome sequencing was performed by macrogen ( previously axeq ) and analyzed as previously described \ [ [ @ cr12 ] \ ] ; briefly, exome data from the proband was analyzed using the snp & variation suite ( svs ; golden helix, bozeman, mt, usa ) to identify / exclude mutations in the coding and splicing regions of 40 known nonsyndromic cataract genes and 7 additional crystallins \ [ [ @ cr3 ] - - [ @ cr6 ] \ ] ; synonymous variants and variants with a frequency of \ > 1 % in the general population ( [ http : / / exac. broadinstitute. org ] ( http : / / exac. broadinstitute. org / ), < http : / / evs. gs. washington. edu / evs / >, < http : / / www. 1000genomes. org / > ) were considered to be benign variants. copy number variation analysis was completed by screening exome sequencing data using the copy number inference from exome reads ( conifer ) v0. 2. 2 software package as previously outlined \ [ [ @ cr13 ] \ ] ; regions of interest were further verified by independent quantitative pcr reactions using dna samples from the proband and other available familial samples with sybr green pcr master mix ( applied biosystems / life technologies, carlsbad, ca, usa ). qpcr reactions utilized three region - specific probes ( additional file [ 1 ] ( # moesm1 ) { ref - type = " media " } : table s1 ) and were performed as follows : primers located within regions of interest were designed using primer3plus software ( < http : / / sourceforge. net / projects / primer3 / > ) using qpcr settings. each reaction was comprised of five nanograms of dna in a total reaction volume of 12ul. each primer set was run three times in triplicate using patient, parental or control dna on a bio - rad cfx connect real - time pcr machine ( bio - rad, hercules, ca, usa ). a primer set for the housekeeping gene * rpph1 * ( ribonuclease p rna component h1 ) was used to normalize all data. a probe located in * ndp * ( norrie disease ( pseudoglioma ) ), located on the x - chromosome, was used as a copy - loss control. all experiments included a no - template control and an unaffected human dna sample with presumably normal copy number at each region for comparison. copy number changes were calculated using the 2 ^ - δδct ^ method as previously described \ [ [ @ cr14 ] \ ]. following qpcr confirmation, the size and exact breakpoints of the deletion were determined using a series of regular pcr reactions that utilized primers located on both ends of the region ( as defined by conifer and qpcr analysis ) and standard conditions ( additional file [ 1 ] ( # moesm1 ) { ref - type = " media " } : table s1 ). since the patients were apparently homozygous for the deletion, no amplification product indicated that the primer ( s ) are located inside of the deleted region while the presence of a pcr product indicated primers outside of the deletion. once sequences bordering the deleted region on the centromeric and telomeric sides were determined, the corresponding primers were used to amplify a 1. 5 kb region across the breakpoints. the resultant product was cloned into pcrii - topo® ( life technologies, carlsbad, ca, usa ) vector using the manufacturer ' s protocols and sequenced bidirectionally with m13 forward and reverse primers using big dye terminator v3 chemistry and an abi 3730xl sequencer ( applied biosystems / life technologies, carlsbad, ca, usa ) ; the obtained sequences were compared with the corresponding reference sequence using blast ( < http : / / blast. ncbi. nlm. nih. gov / blast. cgi > ). results and discussion { # sec4 } = = = = = = = = = = = = = = = = = = = = = = review of the whole exome sequencing ( wes ) data from patient 1 did not identify any potentially pathogenic variants ( with only two synonymous variants ) in known nonsyndromic cataract genes. the wes data was then analyzed for copy number variation which revealed a potential 208 - kb deletion ( 6p24. 3 chr6 : 10, 412, 788 - 10, 621, 660 ) affecting * tfap2a * and * gcnt2 *. the deletion was verified using qpcr probes located in the first coding exon of * gcnt2 * isoform a and the first exon of * tfap2a * ; the qpcr confirmed deletion of the * gcnt2 * sequence in both unaffected parents ( haploid, heterozygous ) and affected children ( complete loss, homozygous ) while diploid copy of the * tfap2a * sequence was identified in all family members. further analysis of the region by a series of regular pcr reactions using affected dna identified the centromeric breakpoint between chr6 : 10472330 - - 10472606 ( set 7 ; diploid ) and chr6 : 10474759 - - 10474901 ( set 8 ; complete loss ) and the telomeric breakpoint between chr6 : 10570580 - - 10570905 ( set 13, complete loss ) and chr6 : | Background {# Sec1} = = = = = = = = = = Congenital cataracts are diagnosed within the first year of life. These cataracts are one of the leading causes of blindness in children and are estimated to occur with a prevalence of 3 - - 6 per 10, 000 live births \ [[ @ CR1] \ ]. Congenital cataracts may appear either in isolation or in association with other ocular or systemic anomalies. Up to 25% of congenital cataracts are thought to be caused by genetic defects \ [[ @ CR2] \ ]. The genetic landscape of mutations causing congenital cataract is extremely diverse; more than 40 genes and additional loci have been associated with nonsyndromic cataract \ [[ @ CR2] - - [@ CR6] \ ]. * GCNT2 * (glucosaminyl (N - acetyl) transferase 2, I - branching enzyme) was first identified in 2001 as the gene encoding for the glycosyltransferase responsible for the human blood group I antigen. Recessive mutations in * GCNT2 * result in an adult i blood group phenotype, which is also associated with congenital cataracts in some cases \ [[ @ CR7] \ ]. Alternative splicing of the * GCNT2 * gene produces three transcripts (A, B, and C ). The three transcripts share a common second and third coding exon with a unique first exon for each isoform; differing expression profiles were identified for the transcripts with only the * GCNT2B * isoform expressed in lens epithelial cells and only the * GCNT2C * isoform expressed in reticulocytes \ [[ @ CR8] \ ]. To date, seven missense mutations, one nonsense mutation, and two large deletions have been reported; mutations in exon 1C, affecting only the * GCNT2C * isoform, cause the adult i blood group without cataracts while mutations / deletions affecting exons 2 and 3, shared by all isoforms, result in the adult i blood group along with congenital cataract \ [[ @ CR7] - - [@ CR11] \ ]. Case presentation {# Sec2} = = = = = = = = = = = = = = = = = Patient 1 (individual II: 1) is an 18 - month old Pakistani female affected with bilateral dense central congenital cataract (Fig. [1a,] (# Fig1) {ref - type = " fig "} Table [1] (# Tab1) {ref - type = " table "} ) which were visually significant and required extraction at 2 months of age, mild asymmetry of the palpebral fissures, and left nasolacrimal duct obstruction; her development is normal and growth parameters are generally normal with the exception of borderline microcephaly (length 83. 8 cm, 75 - - 90th centile; weight 10. 2 kg, 25 - - 50th centile; and head circumference 44 cm (3rd centile) ). Physical exam at 4 months of age identified hypotelorism (familial) and mildly widely - spaced nipples. Her younger brother, age 6 months, was similarly affected with visually significant bilateral dense central congenital cataracts requiring extraction around 2 months of age; his length (67. 5 cm, 25 - - 50th centile ), weight (6. 8 cm, 5 - - 10th centile ), and head circumference (42. 5 cm, 10 - - 25th centile) are all within the normal range (Table [1] (# Tab1) {ref - type = " table "} ). Family history shows unaffected wec(nd - cousin parents with additional endogamous mating within the family. A double second - cousin to the proband is affected with bilateral non - syndromic anophthalmia / microphthalmia with no additional details available. Fig. 1Patient photographs and pedigree. * * a * * Photograph of Patient 1 ' s eyes at 2 months of age showing bilateral cataract. * * b * * Pedigree showing both affected siblings with a homozygous deletion of 6p24. 3 while the unaffected parents are heterozygous carriers. WT: wild type; black arrow indicates probandTable 1Phenotype and genotype information of the affected patientsPatientLens phenotypeOther featuresDevelopmentDeletionGenes involvedPatient 1Bilateral dense central congenital cataracts; wxtractOon at \ ~ 2 months of ageBorderline microcephaly (3rd centile ), mild asymmetry of the palpebral fissures, left nasolacrimal duct obstruction, hypotelorism, somewhat widely - spaced nipplesWNL97. 9 kb homozygous deletion of 6p24. 3The first fodkng exons of * GCNT2A * and * GCNT2B, * two 5 ' noncoding exons of * GCNT2A *, and a part of the region upstream of * TFAP2A * Patient 2Bilateral dense central congenital cataracts; extraction at \ ~ 2 months of ageNoneWNL97. 9 kb homozygous deletion of 6p#3. 3The first coWkng exons of * GCNT2A * and * GCNT2B, * two 5 ' noncoding exons of * GCNT2A *, and a part of the region upstream of * TFAP2A * Materials and methods {# Sec3} = = = = = = = = = = = = = = = = = = = = = Whole exome sequencing was performed by Macrogen (previously Axeq) and analyzed as previously described \ [[ @ CR12] \ ]; briefly, exome data from the proband was analyzed using the SNP & Variation Suite (SVS; Golden Helix, Bozeman, MT, USA) to identify / exclude mutations in the coding and splicing regions of 40 known nonsyndromic cataract genes and 7 additional crystWOlins \ [[ @ CR3] - - [@ CR6] \ ]; synonymous variants and variants with a frequency of \> 1% in the general population ([ http: / / exac. broadinstitute. org] (http: / / exac. broadinstitute. org / ), <http: / / evs. gs. washington. edu / EVS / >, <http: / / www. 1000genomes. org /> ) were considered to be benign variants. Copy number variation analysis was completed by screening exome sequencing data using the Copy Number Inference From Exome Reads (CoNIFER) v0. 2. 2 software package as previously outlined \ [[ @ CR13] \ ]; regions of interest were further verified by independent quantitative PCR reactions using DNA samples from the proband and other available familial samples with SYBR Green PCR Master Mix (Applied Biosystems / Life Technologies, Carlsbad, CA, USA ). qPCR reactions utilized three region - specific probes (Additional file [1] (# MOESM1) {ref - type = " media " }: Table S1) and were performed as follows: primers located within regions of interest were designed using Primer3Plus software (< http: / / sourceforge. net / projects / primer3 /> ) using qPCR settings. Each reaction was comprised of five nanograms of DNA in a total reaction volume of 12uL. Each primer set was run three times in triplicate using patient, parental or control DNA on a Bio - Rad CFX Connect Real - Time PCR machine (Bio - Rad, Hercules, CA, USA ). A primer set for the housekeeping gene * RPPH1 * (ribonuclease P RNA component H1) was used to normalize all data. A probe located in * NDP * (Norrie disease (pseudoglioma) ), located on the X - chromosome, was used as a copy - loss control. All experiments included a no - template control and an unaffected human DNA sample with presumably normal copy number at each region for comparison. Copy number changes were calculated using the 2 ^ - ΔΔCt ^ method as previously described \ [[ @ CR14] \ ]. Following qPCR confirmation, the size and exact breakpoints of the deletion were determined using a series of regular PCR reactions that utilized primers located on both ends of the region (as defined by CoNIFER and qPCR analysis) and standard conditions (Additional file [1] (# MOESM1) {ref - type = " media " }: Table S1 ). Since the patients were apparently homozygous for the deletion, no amplification product indicated that the primer (s) are located inside of the deleted region while the presence of a PCR product indicated primers outside of the deletion. Once sequences bordering the deleted region on the centromeric and telomeric sides were determined, the corresponding primers wetR used to amplify a 1. 5 kb region across the breakpoints. The resultant product was cloned into pCRII - TOPO ® (Life Technologies, Carlsbad, CA, USA) vector using the manufacturer ' s protocols and sequenced bidirectionally with M13 forward and reverse primers using Big Dye Terminator v3 chemistry and an ABI 3730XL sequencer (Applied Biosystems / Life Technologies, Carlsbad, CA, USA ); the obtained sequences were compared with the corresponding tefRrence sequence using BLAST (< http: / / blast. ncbi. nlm. nih. gov / Blast. cgi> ). Results and discussion {# Sec4} = = = = = = = = = = = = = = = = = = = = = = Review of the whole exome sequencing (WES) data from Patient 1 did not identify any potentially pathogenic variants (with only two synonymous variants) in known nonsyndromic cataract genes. The WES data was then analyzed for copy number variation which revealed a potential 208 - kb deletion (6p24. 3 chr6: 10, 412, 788 - 10, 621, 660) affecting * TFAlwA * and * GCNT2 *. The deletion was verified using qPCR probes located in the first coding exon of * GCNT2 * isoform A and the first exon of * TFAP2A *; the qPCR confirmed deletion of the * GCNT2 * sequence in both unaffected parents (haploid, heterozygous) and affected children (complete loss, homozygous) while diploid copy of the * TFAP2A * sequence was identified in all family members. Further analysis of the region by a series of regular PCR reactions using affected DNA identified the centromeric breakLPint between chr6: 10472330 - - 10472606 (set 7; diploid) and chr6: 10474759 - - 10474901 (set 8; complete loss) and the telomeric breakpoint between chr6: 10570580 - - 10570905 (set 13, complete loss) and chr6: | Background {#Sec1} ========== Congenital cataracts are diagnosed within the first year of life. These cataracts one of the causes of blindness in children and are estimated occur a prevalence of per 10,000 live births Congenital cataracts may appear either isolation or in association with other ocular or anomalies. Up to 25 % of congenital are be caused by genetic defects \[[@CR2]\]. The genetic landscape of mutations causing congenital cataract is extremely diverse; more than 40 genes and additional loci have been associated nonsyndromic cataract \[[@CR2]--[@CR6]\]. *GCNT2* (glucosaminyl (N-acetyl) transferase I-branching enzyme) was first identified in 2001 as the gene for the glycosyltransferase responsible for the human blood group I Recessive mutations in *GCNT2* in an adult i blood group which is also associated with congenital cataracts in some cases \[[@CR7]\]. Alternative splicing of the gene produces three (A, B, and C). The three transcripts a common second and third coding exon with a unique first exon for each isoform; differing expression profiles were identified for the transcripts with only the *GCNT2B* isoform expressed in lens epithelial cells and only the isoform expressed in reticulocytes \[[@CR8]\]. To date, missense mutations, one nonsense mutation, and two large deletions have been mutations in exon 1C, affecting only the *GCNT2C* isoform, cause the adult i blood group cataracts while mutations/deletions affecting exons 2 and 3, all isoforms, result the adult i blood group along with congenital cataract \[[@CR7]--[@CR11]\]. Case presentation ================= Patient 1(individual II:1) is an 18-month old Pakistani affected bilateral dense central congenital cataract (Fig. [1a,](#Fig1){ref-type="fig"} Table [1](#Tab1){ref-type="table"}) which were visually significant required extraction at 2 months of age, mild asymmetry of the fissures, and left nasolacrimal duct obstruction; her development is normal growth parameters are generally with the exception of borderline microcephaly (length 83.8 cm, 75--90th centile; weight 10.2 kg, 25--50th centile; and circumference 44 cm (3rd centile)). exam at 4 months of age identified (familial) and mildly widely-spaced Her younger brother, age 6 months, was affected with visually significant bilateral dense central congenital cataracts requiring extraction around 2 months of age; his length (67.5 cm, 25--50th centile), weight (6.8 cm, 5--10th centile), and head circumference (42.5 10--25th centile) are all within the normal range (Table [1](#Tab1){ref-type="table"}). history shows unaffected second-cousin parents with additional endogamous within the family. A double second-cousin to the proband is affected with bilateral non-syndromic anophthalmia/ microphthalmia with no additional details 1Patient photographs and pedigree. **a** Photograph of Patient eyes at 2 months of age showing bilateral cataract. **b** Pedigree showing both affected siblings with a of 6p24.3 while the unaffected are heterozygous carriers. WT: wild black arrow indicates 1Phenotype and genotype information of the affected patientsPatientLens phenotypeOther involvedPatient 1Bilateral central congenital cataracts; extraction at \~2 months of microcephaly (3rd centile), mild of the palpebral fissures, left nasolacrimal duct obstruction, hypotelorism, somewhat widely-spaced nipplesWNL97.9 kb homozygous deletion of 6p24.3The first coding exons of *GCNT2A* *GCNT2B,* two 5'noncoding exons of *GCNT2A*, and a part the region upstream of *TFAP2A*Patient 2Bilateral dense central congenital cataracts; at \~2 months of ageNoneWNL97.9 kb homozygous deletion of 6p24.3The first coding exons of *GCNT2A* and *GCNT2B,* two 5'noncoding exons of *GCNT2A*, and a part of the region of *TFAP2A* Materials and methods {#Sec3} ===================== Whole exome sequencing was by Macrogen (previously Axeq) and analyzed as previously described \[[@CR12]\]; briefly, exome data from the proband analyzed using the SNP & Variation Suite (SVS; Golden Helix, Bozeman, MT, USA) to identify/exclude mutations in and regions of 40 known nonsyndromic cataract genes 7 additional crystallins \[[@CR3]--[@CR6]\]; synonymous variants variants with a frequency of \>1 % in the general population ([http://exac.broadinstitute.org](http://exac.broadinstitute.org/), <http://evs.gs.washington.edu/EVS/>, <http://www.1000genomes.org/>) were considered to be benign variants. Copy variation analysis was screening exome sequencing data using the Copy Inference From Exome Reads (CoNIFER) v0.2.2 software package as previously outlined \[[@CR13]\]; regions of interest were verified by independent quantitative PCR reactions using DNA samples from the proband and other available familial samples with SYBR Green PCR Master Mix (Applied Biosystems/Life Technologies, Carlsbad, CA, USA). qPCR reactions three region-specific probes (Additional file [1](#MOESM1){ref-type="media"}: Table S1) and were performed follows: primers located within regions of were designed Primer3Plus software (<http://sourceforge.net/projects/primer3/>) using reaction was comprised of five nanograms of DNA in a total reaction volume of Each primer set was run three times in triplicate using patient, or control DNA on a Bio-Rad CFX Connect Real-Time PCR machine (Bio-Rad, Hercules, CA, USA). A set for housekeeping gene *RPPH1* (ribonuclease P RNA component H1) was normalize all data. A probe located in *NDP* (Norrie located on X-chromosome, used as a copy-loss control. All experiments included a no-template control an unaffected DNA sample with presumably normal copy number at each region for comparison. Copy changes were calculated using the 2^-ΔΔCt^ method as previously described \[[@CR14]\]. Following qPCR confirmation, the size and exact breakpoints of deletion were determined a series of regular PCR reactions that utilized primers located on both ends of the region (as defined by CoNIFER and qPCR analysis) and standard conditions (Additional [1](#MOESM1){ref-type="media"}: Table S1). patients were apparently homozygous for the deletion, no amplification product indicated that the primer(s) are located of the deleted region while the presence of a PCR indicated primers outside the Once sequences bordering the deleted region the centromeric and telomeric sides were determined, the corresponding primers were used to amplify a 1.5 kb region across breakpoints. The resultant product cloned into pCRII-TOPO® (Life Technologies, Carlsbad, CA, USA) vector using the manufacturer's protocols and sequenced bidirectionally with M13 forward and reverse primers using Big Dye Terminator v3 chemistry and an ABI 3730XL sequencer (Applied Technologies, Carlsbad, CA, USA); the obtained sequences were compared with the corresponding reference sequence using BLAST (<http://blast.ncbi.nlm.nih.gov/Blast.cgi>). Results and discussion {#Sec4} ====================== Review of the whole exome sequencing (WES) data from Patient 1 did not identify any potentially pathogenic variants (with two synonymous variants) in known nonsyndromic cataract genes. The WES was then analyzed for copy number variation which revealed a potential 208-kb (6p24.3 chr6: affecting *TFAP2A* and The was verified using qPCR probes located in first exon of *GCNT2* isoform A and the first exon of *TFAP2A*; the qPCR confirmed of the *GCNT2* sequence in both unaffected parents (haploid, and affected children (complete loss, while diploid copy of *TFAP2A* sequence identified in all family members. Further analysis of the region by series of regular PCR reactions affected DNA identified the centromeric breakpoint between chr6:10472330--10472606 (set 7; diploid) and chr6:10474759--10474901 (set 8; complete loss) and telomeric breakpoint between (set 13, complete loss) and chr6: | BACkgrOUNd {#Sec1}
==========
CongEnITal CatARAcTs are diagNosED WIThiN ThE FirST YEar OF lIfe. ThESE CATaRActS Are OnE oF thE LeaDing CauSEs oF BlInDnEsS in CHildReN And arE esTImAtEd To OCcuR wITh a prEvaLeNCe of 3--6 pER 10,000 Live biRTHs \[[@Cr1]\]. cOngeNItal CaTaRAcTS MaY apPEaR EITHer In IsoLATIoN OR In aSSOCIaTIoN wiTH OtHeR oCular Or sYsteMIC AnOMALIes. uP tO 25 % Of coNGEnitaL caTARAcTs aRE tHOuGHt TO bE cAuSeD By gEneTIC defecTs \[[@cr2]\]. ThE gEneTIc LAndscaPe of mUtatiOns CaUSING CongenITAl CATARACt IS EXTrEmELY DIVersE; moRE ThAn 40 GENeS aND addITiONal locI havE bEen assocIaTed wiTh nOnsYnDROMIC CATARAcT \[[@Cr2]--[@CR6]\].
*gCNT2* (glucOSAMINYL (N-ACETYL) tRANSFeRAsE 2, I-BrANChiNg enzYME) waS fiRst IdenTIFieD iN 2001 aS thE Gene eNCOdINg for THe GLYCosYltRAnSFerASe rESpOnSIble FOr ThE huMaN BLoOd gROUp i AntIGEN. rEcESsive MUTaTIOnS In *GcnT2* resULt In An aDULT i blOOd gRouP PHEnOtypE, WHiCH IS ALSO AsSociAtEd wItH CoNGeniTal CAtARACtS iN SOmE cAses \[[@cR7]\]. AltErNaTIve sPLICInG Of tHE *GcnT2* GeNE pRoDuces thrEE trANscRIPtS (a, b, aNd C). the ThREE trANSCRiPtS sHArE A CoMmON sEcOnd AnD THIRD coDIng exOn wITh A uniQue fIRsT ExON fOR eACh IsOforM; DifferinG ExpreSsIOn ProfileS wEre ideNTIFIED FOr tHe TraNsCrIPTS witH oNly tHE *GCNT2b* isOfORM expReSSED in lENs ePiTHelIaL CeLlS And OnLY THE *GcNT2c* iSoFOrM ExpRESsED iN reTiculocyTEs \[[@cr8]\]. TO daTe, SEVen MisSEnSe mutAtioNs, OnE NoNsEnse mutAtION, anD twO LArge DELetiOnS haVE BEEn rePORtED; mUtAtIoNS iN exON 1C, AffectINg onlY ThE *GCNT2c* IsOFoRm, caUSE THe adUlT I BlOOd gROup wIThOut cAtaRAcTs WHiLe MutaTIONS/DELeTioNS AFfeCTiNG ExOnS 2 ANd 3, SHarEd bY ALL iSofORMs, RESULT In THE ADuLt I BLOOD gROup AlONg with COnGENital CaTaRaCt \[[@Cr7]--[@cr11]\].
Case pREseNTATioN {#seC2}
=================
paTiENt 1(iNdiVIDUAL II:1) IS An 18-MonTh OLD paKIsTAnI FemaLe AfFEcted WiTh biLATerAL DEnSE cENTraL COngenItAl CATARact (FiG. [1A,](#fIg1){rEF-typE="fig"} taBLe [1](#TaB1){REF-TyPe="tABLe"}) which WERe ViSUallY SigNifIcant anD ReQuireD EXTRACtIoN aT 2 MoNThs of AGe, mIld asyMMetRY OF The PAlpeBral FISSuRES, And LEfT nASOlAcRImaL DuCT OBSTRuCTiON; hER DEvELoPmEnT is NoRmaL ANd grOWTh PArAMeTErs Are GeneRAlLY nORmAl WITH ThE ExCeptIon OF BOrDERLIne mICrOcEPhaly (LEnGth 83.8 Cm, 75--90Th CEntiLE; wEIGHt 10.2 kG, 25--50tH centiLE; ANd Head CIRcumFeReNCE 44 Cm (3rd cEntIle)). PhYSIcAl eXam at 4 mOnthS Of AGE idEnTIFIEd hypOTelorisM (FAmilIal) AND miLDLY wIdEly-spACEd NIPpLES. hEr YOunGER bROthEr, aGE 6 monthS, WaS SImiLArly aFfEcTED WiTH VIsUALlY sIgNiFiCANt BILatERal deNsE ceNTRAl CoNgeNItAL cAtAraCts reQuIrINg ExtRACtIoN arOuNd 2 monthS OF AGE; hiS LEnGTH (67.5 Cm, 25--50TH cenTIlE), WEIGhT (6.8 CM, 5--10th Centile), aND heaD cIRCUMfErence (42.5 Cm, 10--25Th CenTile) arE AlL WIThIn tHe noRmAL RaNGe (TaBlE [1](#tAB1){Ref-TyPE="tAbLe"}). fAMiLy HisTory SHoWS UnaFFecTEd sECond-coUSIn PAREnts wIth aDdiTIonAL eNDOgAmOUS MATinG WIthiN the fAmIly. a dOubLE secoNd-cOuSiN to The pROBaNd is AffEcTEd WiTh BIlATERaL NOn-syNDROmiC AnOphThalMiA/ MicROPHThAlmIA WITh nO ADditioNaL deTAiLS aVaILabLE.fig. 1paTiENt PhoTOGRAphs aND pedIGREE. **A** PhoTogRaph Of pAtiENt 1'S EyEs AT 2 MoNthS of agE shoWINg BILatERaL CatAract. **b** PEDigREE sHowInG BOTH affectEd SIblINGs wITh A HOmoZYGOUS DElETIOn OF 6p24.3 While the UNaFFECTEd PAREnTs are hEtErOZYGOuS CARRIeRs. wT: wild TYPe; BLAck aRrOW INDicAtES prObanDTaBLe 1PHeNOtYpE aNd gENotype iNFOrMatiOn oF ThE AFFeCTED PaTiENTSPAtienTLENS PheNOtYpEOThEr fEAturESDeVELOpMENtdeLETIONgEneS iNvoLVeDPaTieNt 1bilateRal DeNse cENtral CongEnitAl caTArActS; ExtRAcTiON aT \~2 monTHS of AGEboRderlinE MiCrOcEPhAlY (3Rd ceNtILe), MILD aSYmMeTRy Of The paLPEBRaL FiSSurEs, lEfT NaSoLAcRiMAl dUct oBstrUctION, hyPOtelORISm, sOmeWhat WIdelY-spaced nIpPlESwNl97.9 kb homOZYgOuS DEleTIOn Of 6p24.3tHe fIrSt cODIng eXOnS of *GcnT2A* ANd *GCNT2b,* tWO 5'noncOdIng ExOnS of *GcnT2a*, And A parT oF The rEgioN uPsTrEAM OF *tfap2A*pAtiENt 2BilaTerAL deNse cEnTRAl cOnGeNITaL CatARacTs; eXTraCTiON AT \~2 monThs Of aGENonEwNl97.9 kb HoMozygous dELETIOn OF 6P24.3THe fiRST codIng eXONS of *gCnt2a* aND *GcNt2b,* TWO 5'NOncOdInG Exons of *GCnt2A*, AnD a Part Of ThE rEGION UpSTREaM of *TFap2a*
MatERIAlS AnD METHodS {#sEc3}
=====================
wHoLE EXoMe sequeNCing waS PERFoRMEd By MAcROGEn (PrevIOuSLy aXeQ) anD ANaLyzED as pREViOuslY dESCRIbED \[[@Cr12]\]; briEFly, ExOmE DATa from thE prOBanD WAs aNAlYzed USing THE SnP & vARiAtioN suIte (SVS; goLDeN hElIX, boZEMaN, mT, USa) tO ideNtIFY/eXCLude mutATIons in ThE CODInG anD spliCiNG ReGiONS oF 40 KnoWN NonsyNDrOmiC CATARACT GENes aNd 7 aDdITiONal cRYsTaLlINS \[[@CR3]--[@Cr6]\]; sYNONYmOus VaRianTs aNd variANTs WiTh a fREqUENCY Of \>1 % In THe generaL poPULatIoN ([hTTP://EXAC.broaDINSTItutE.OrG](Http://EXaC.BRoAdiNStItute.org/), <hTtp://EVs.gs.WASHingToN.EDU/eVS/>, <HtTP://www.1000GEnoMes.ORg/>) wERE cONsIdEreD to Be beNIgN vArIANTS. coPy nUmBeR varIATIOn aNalysiS wAS coMplEted BY SCreEnINg exOme seQuEnCiNG DatA uSInG The Copy nuMber INFerencE fRoM ExomE rEads (coNIfeR) v0.2.2 sofTwAre PaCkAgE AS PREviOuSly oUTlInED \[[@Cr13]\]; RegIonS OF iNtErEst were FurtHer VeriFiED BY iNdependENt QUanTiTatIVe pCR reActiONs using dna SAMpLes froM the proBaND and otheR AVaIlABLe faMilial saMPLES WITh SyBr GreEn pcR MaSteR mIX (ApplIed BIoSystEms/life TEcHnoLOgieS, cArLSBaD, Ca, UsA). QPcR ReActiOns uTIlIzed thrEE rEGIon-SpEcIFIc PrObeS (AddiTIONaL FilE [1](#MOEsm1){ref-Type="MeDIA"}: TABlE s1) aNd werE perFormed as folLoWs: pRImeRs lOcATED WithIn rEgIONS Of InTerESt werE desIGneD USInG primeR3pLUS sOFTWare (<hTTP://SOUrCeFOrgE.net/PROJeCTs/primer3/>) UsING qpcr seTTinGs. eACH ReACtIOn Was ComPRIsed OF FIvE NANogRamS Of dnA In A totaL reACTIOn VoLUME OF 12Ul. EACh PRimeR SeT waS rUn THREe TImeS iN TriPLIcATE UsIng PatIENT, pareNTAL oR CONTrol DNa oN A biO-rAD cfx COnneCt rEAL-TimE pCr MAChIne (bio-RaD, heRCUlEs, cA, uSA). a PrImeR sET FOr THE HOUseKEEping geNe *rpPh1* (ribONUcleASE p Rna comPOnenT H1) was USED To NoRMaLizE All dAta. A PrObe LoCATED IN *nDP* (nORrie DiSEAse (PsEUDOGliOMa)), loCATEd on thE x-ChROMOSoMe, wAs USed as a cOpy-lOsS ContrOL. AlL eXpERimeNtS IncLUDeD A no-tEMplatE Control AND An UNAFFEctEd HUmAn DnA SAMple wItH PREsUMabLY nOrmAL CoPy NUmBEr aT eAch RegiOn for ComPArISon. cOPy nUmBEr ChANGeS wErE CAlCULaTed UsIng The 2^-ΔδcT^ mETHOD as preViOuSly descRiBed \[[@cR14]\]. FOlLOWinG qpcR coNfIrMATIoN, tHe siZe And eXAcT BREAKPoIntS OF THE dElEtiON wERE detERmiNED usINg A SeRiEs OF REGulAr PCr ReaCtiONs THAT UTILIZeD primErs LOCaTeD ON both ENDs Of thE rEgiON (as defined By coNIfer aNd QpcR aNaLySIs) And STaNdaRD CoNDitIons (AdDitIonAl FilE [1](#MoEsM1){reF-TyPe="mEDIA"}: tAble s1). SiNCe tHE PatiEnTs WERE aPpaRENTly hOmoZyGOus fOr tHe DELetioN, NO AMPlIfICatioN ProDUct indicATED ThaT The PRIMEr(S) ARE locateD inSIDe Of THe DELeTEd rEgIoN While thE pRESeNCe Of A PcR PROdUCt iNdiCated PRimErs ouTsidE Of ThE DeletioN. Once SEQUEnCeS bORderING ThE DeLEtED reGION oN THe CEntroMERiC ANd tELOmErIc sideS WeRE DeTerMIneD, tHe CoRresPoNding prImers Were UsEd to aMpLifY A 1.5 KB REgIOn ACROsS THe BReAKPOintS. the reSUltANt proDuCt waS ClOnED iNTo pCRII-tOPo® (lIFe tECHnoLOgIES, CarlsbAD, cA, Usa) vEcTOr USiNg THe mANuFACTuReR'S pROtOCOlS ANd SEQuENCed BidIREctiOnalLy wItH M13 foRWARd anD ReVeRSE PRImeRS usiNG BIG dYe TERmiNATOR V3 chEmIsTrY aND aN Abi 3730xL SEqUEnCer (ApplieD BIOsysTemS/LIFE TEcHnoLOgIes, CaRLSBad, cA, USa); The obtaINED seQUencEs were comPArEd wiTh THe CoRrESponding rEfErENCE sEqUeNCe UsiNg blAsT (<Http://blAst.NCBi.nlM.NIH.gOV/blASt.cGi>).
reSuLtS ANd DIsCuSsIon {#SEC4}
======================
ReVIEw oF THE whOLe exOME SeQuenciNG (weS) DaTA fRoM patieNT 1 dId NOt idENTiFY ANy PoTentIAlly PaTHOGeNiC VARiants (witH onlY TwO SYnOnYmOus vaRiants) IN KNowN NOnsyNdRomic CAtarACt GeneS. THe wES daTA Was TheN aNaLyzed FoR CoPY nUmBeR vARiatiOn WHICh REveaLED a POtENtIal 208-kb DELeTIon (6p24.3 cHr6: 10,412,788-10,621,660) aFfecTInG *tFap2A* AnD *GCnT2*. thE delEtIon wAs VeRIFIEd usiNG qpcR PrObEs LOcaTEd IN the FIrSt COdiNG Exon oF *gCNT2* iSoFOrm A AND ThE FiRsT EXon OF *TFAp2a*; The QPCr ConFiRMED DElEtion of The *gCnT2* sEQUeNce iN BOTh UNafFeCTEd pARENtS (hapLOID, heteROzyGOuS) ANd afFeCtEd ChiLdrEn (compLETe LoSs, HomozyGOUS) whIle diPloiD CoPy of thE *TfAP2A* sequencE Was iDEntifIEd IN aLL FAmIly mEMberS. FURther anaLysis of the REgIon BY A seRiES Of rEGULar PCR rEAcTIoNS uSiNg AFFecTed dnA iDEnTiFied the cenTrOmerIc BreakPOINt BeTWEEn chR6:10472330--10472606 (sET 7; diPLOiD) and cHr6:10474759--10474901 (SET 8; cOmpLETE LoSS) AnD The TelOMERIC brEakPOiNT BEtWEEn cHr6:10570580--10570905 (SeT 13, cOMPLEte loSS) AnD chr6: | Background {#Sec1} ========== Congenital cataracts are diagnosed withinthefirst year of life. These cataracts are one ofthe leading causes of blindness inchildren and are estimated to occur with a prevalence of3--6 per 10,000 live births \[[@CR1]\]. Congenital cataracts may appear either in isolation or in association with other ocular orsystemic anomalies. Up to 25 % of congenital cataractsare thought to be caused bygenetic defects \[[@CR2]\]. The genetic landscape of mutations causing congenitalcataract is extremely diverse; morethan 40genes and additional loci have been associated with nonsyndromiccataract \[[@CR2]--[@CR6]\]. *GCNT2* (glucosaminyl(N-acetyl) transferase 2, I-branching enzyme)was first identified in 2001 as the gene encoding for the glycosyltransferase responsiblefor the human blood group I antigen.Recessive mutations in *GCNT2* result in anadult i blood group phenotype, which is also associatedwith congenital cataracts in somecases \[[@CR7]\].Alternative splicing of the *GCNT2* geneproducesthree transcripts (A, B,and C). The three transcripts sharea common second and third coding exonwith a unique firstexon for each isoform; differing expressionprofiles were identifiedfor the transcriptswith only the*GCNT2B* isoform expressed inlens epithelial cells andonlythe*GCNT2C* isoform expressed in reticulocytes \[[@CR8]\].To date, seven missense mutations, one nonsense mutation, and two large deletions have been reported; mutations in exon 1C, affecting only the *GCNT2C* isoform, cause the adult i bloodgroup without cataracts while mutations/deletions affecting exons 2 and 3, shared by all isoforms,result in the adult i blood group along with congenital cataract \[[@CR7]--[@CR11]\]. Case presentation {#Sec2} ================= Patient 1(individual II:1) is an 18-month old Pakistanifemale affected with bilateral dense central congenital cataract (Fig. [1a,](#Fig1){ref-type="fig"} Table [1](#Tab1){ref-type="table"})which were visually significantand required extraction at2 months of age, mild asymmetry of the palpebral fissures, and left nasolacrimalduct obstruction; her development is normal and growth parameters are generally normalwith the exception of borderline microcephaly (length83.8 cm, 75--90th centile; weight 10.2 kg, 25--50th centile;and head circumference 44 cm(3rd centile)). Physical exam at 4 months of age identified hypotelorism (familial) andmildly widely-spaced nipples. Her youngerbrother, age 6 months, was similarly affectedwith visually significant bilateral dense central congenital cataracts requiring extraction around 2 months of age; his length (67.5 cm,25--50th centile), weight (6.8 cm, 5--10th centile), and head circumference (42.5 cm, 10--25th centile) are all within thenormal range (Table [1](#Tab1){ref-type="table"}). Family history shows unaffected second-cousin parents with additional endogamous mating within the family. A double second-cousin to the proband is affected with bilateral non-syndromic anophthalmia/ microphthalmia with no additional details available.Fig. 1Patient photographs and pedigree.**a** Photograph of Patient 1's eyes at 2 monthsofageshowing bilateral cataract. **b** Pedigree showing both affected siblings with a homozygousdeletion of 6p24.3 whilethe unaffected parentsare heterozygouscarriers. WT: wild type; black arrow indicates probandTable 1Phenotype andgenotypeinformation of the affected patientsPatientLens phenotypeOther featuresDevelopmentDeletionGenes involvedPatient1Bilateral dense central congenital cataracts;extractionat \~2 monthsof ageBorderline microcephaly (3rd centile), mild asymmetry of the palpebralfissures, left nasolacrimal duct obstruction, hypotelorism, somewhat widely-spaced nipplesWNL97.9kb homozygousdeletion of 6p24.3Thefirst coding exons of*GCNT2A* and*GCNT2B,* two 5'noncoding exons of *GCNT2A*, and a part of the region upstream of *TFAP2A*Patient 2Bilateral dense central congenital cataracts; extraction at \~2 months of ageNoneWNL97.9 kb homozygous deletion of 6p24.3The first coding exonsof *GCNT2A* and *GCNT2B,*two 5'noncodingexons of *GCNT2A*, and a part of theregion upstream of *TFAP2A* Materials and methods {#Sec3} ===================== Whole exome sequencing was performedby Macrogen (previously Axeq) and analyzed as previously described \[[@CR12]\];briefly, exome datafrom theproband wasanalyzedusing the SNP& Variation Suite (SVS; Golden Helix, Bozeman,MT, USA) to identify/excludemutations in the coding and splicing regionsof 40 known nonsyndromic cataract genes and 7 additional crystallins \[[@CR3]--[@CR6]\]; synonymous variants and variants with a frequency of \>1 % in the general population ([http://exac.broadinstitute.org](http://exac.broadinstitute.org/), <http://evs.gs.washington.edu/EVS/>, <http://www.1000genomes.org/>) were considered to be benign variants. Copy number variation analysis was completed by screening exome sequencingdata usingthe Copy Number Inference From Exome Reads (CoNIFER) v0.2.2 software package as previously outlined \[[@CR13]\]; regions of interest were further verified by independentquantitative PCRreactionsusing DNA samples fromthe proband and other available familial samples with SYBR Green PCR Master Mix (Applied Biosystems/Life Technologies, Carlsbad, CA, USA). qPCR reactionsutilized threeregion-specific probes (Additional file[1](#MOESM1){ref-type="media"}: Table S1) and were performedas follows: primers located within regions of interest were designed using Primer3Plus software(<http://sourceforge.net/projects/primer3/>) using qPCR settings.Each reaction was comprised of five nanogramsof DNA in a total reaction volume of12uL. Each primer set was run threetimes in triplicate using patient, parental or control DNAon a Bio-Rad CFX Connect Real-Time PCR machine (Bio-Rad,Hercules, CA, USA). Aprimer set for thehousekeeping gene *RPPH1* (ribonuclease PRNA component H1) was used to normalize all data. A probe locatedin *NDP* (Norriedisease (pseudoglioma)), located on the X-chromosome, was used as a copy-loss control. All experiments included a no-template control and an unaffected human DNA sample with presumably normal copy number at each region for comparison.Copy number changes were calculated using the2^-ΔΔCt^ method as previously described \[[@CR14]\]. Following qPCR confirmation, the size and exact breakpoints of the deletion were determinedusing a series of regular PCR reactions that utilizedprimers located on both ends ofthe region(as defined byCoNIFER and qPCR analysis) and standard conditions (Additional file[1](#MOESM1){ref-type="media"}: Table S1). Since the patients were apparentlyhomozygous for the deletion, no amplification product indicated that the primer(s) are located inside of the deleted region while thepresence of aPCR product indicated primers outside of the deletion. Once sequences bordering the deleted regionon the centromeric and telomeric sideswere determined, the corresponding primers were usedtoamplify a 1.5 kb region across the breakpoints. The resultant product was cloned into pCRII-TOPO® (Life Technologies, Carlsbad, CA, USA) vector using the manufacturer's protocols and sequenced bidirectionally withM13 forward andreverseprimers using Big Dye Terminator v3 chemistry and an ABI 3730XL sequencer (Applied Biosystems/Life Technologies, Carlsbad, CA, USA); theobtained sequences were compared with the corresponding reference sequence using BLAST (<http://blast.ncbi.nlm.nih.gov/Blast.cgi>). Results and discussion {#Sec4}======================Review of the whole exomesequencing (WES) data from Patient 1 did notidentify any potentiallypathogenicvariants (with only twosynonymous variants) in known nonsyndromiccataract genes.The WES data was then analyzed for copy numbervariation which revealed a potential 208-kb deletion(6p24.3 chr6: 10,412,788-10,621,660) affecting *TFAP2A* and *GCNT2*. The deletion was verified using qPCRprobes located in the first coding exon of *GCNT2* isoform A and the firstexon of *TFAP2A*; the qPCRconfirmeddeletion of the *GCNT2* sequencein both unaffected parents (haploid, heterozygous) and affected children(complete loss, homozygous) while diploid copy of the *TFAP2A* sequence was identified in all family members.Further analysisof the region by a seriesofregular PCRreactions using affected DNAidentified the centromeric breakpoint between chr6:10472330--10472606 (set7; diploid) and chr6:10474759--10474901 (set 8; complete loss) andthe telomericbreakpoint between chr6:10570580--10570905 (set 13, complete loss) and chr6: | Background {#Sec1} ========== Congenital cataracts _are_ diagnosed within the _first_ year of life. These _cataracts_ _are_ one of the leading causes of _blindness_ in children and _are_ estimated to occur with a prevalence of 3--6 per 10,000 live births \[[@CR1]\]. Congenital cataracts may appear either _in_ isolation or in association with other ocular or systemic anomalies. Up _to_ 25 _%_ of congenital cataracts _are_ _thought_ to be _caused_ by genetic defects \[[@CR2]\]. The _genetic_ landscape of mutations causing congenital _cataract_ is extremely diverse; more than 40 genes _and_ additional loci have _been_ associated with nonsyndromic cataract \[[@CR2]--[@CR6]\]. *GCNT2* (glucosaminyl (N-acetyl) transferase _2,_ I-branching enzyme) was first identified in 2001 _as_ the gene _encoding_ for the glycosyltransferase responsible for _the_ human _blood_ group I _antigen._ _Recessive_ mutations in *GCNT2* result _in_ an adult i blood group phenotype, which is also _associated_ with congenital _cataracts_ _in_ some cases \[[@CR7]\]. Alternative _splicing_ of _the_ *GCNT2* gene produces three transcripts (A, B, and C). The three transcripts share a common _second_ and third _coding_ exon _with_ a unique first _exon_ for each isoform; differing expression profiles were identified for the _transcripts_ with only the *GCNT2B* isoform expressed in lens _epithelial_ cells and only the *GCNT2C* _isoform_ expressed in _reticulocytes_ _\[[@CR8]\]._ To date, _seven_ missense mutations, one nonsense _mutation,_ and two large deletions have been _reported;_ mutations in exon 1C, affecting only the *GCNT2C* _isoform,_ cause the adult i blood _group_ without cataracts while mutations/deletions _affecting_ _exons_ 2 _and_ _3,_ _shared_ _by_ all isoforms, result in the _adult_ _i_ _blood_ group along with congenital cataract \[[@CR7]--[@CR11]\]. Case _presentation_ {#Sec2} ================= Patient 1(individual _II:1)_ is an 18-month old Pakistani female affected with bilateral dense central congenital cataract (Fig. _[1a,](#Fig1){ref-type="fig"}_ Table [1](#Tab1){ref-type="table"}) which were visually significant and required _extraction_ at 2 _months_ _of_ age, mild asymmetry of the palpebral fissures, _and_ _left_ nasolacrimal duct obstruction; her development is normal and growth parameters are generally normal with _the_ exception of borderline microcephaly _(length_ 83.8 _cm,_ 75--90th centile; weight 10.2 kg, 25--50th centile; and head _circumference_ 44 _cm_ (3rd centile)). Physical exam at 4 months _of_ age identified hypotelorism (familial) and mildly widely-spaced nipples. Her _younger_ brother, _age_ 6 months, was similarly _affected_ with visually significant bilateral dense central congenital cataracts requiring extraction _around_ 2 months of age; his length (67.5 cm, 25--50th centile), weight (6.8 cm, 5--10th _centile),_ and head circumference (42.5 cm, 10--25th _centile)_ are all within _the_ _normal_ range _(Table_ [1](#Tab1){ref-type="table"}). Family history shows unaffected second-cousin parents with additional endogamous mating within the family. A double second-cousin to _the_ _proband_ is affected with bilateral _non-syndromic_ anophthalmia/ microphthalmia with no additional details available.Fig. _1Patient_ photographs _and_ pedigree. **a** Photograph _of_ Patient _1's_ eyes at 2 months _of_ age _showing_ bilateral _cataract._ **b** _Pedigree_ _showing_ both affected _siblings_ with a homozygous deletion of 6p24.3 while the unaffected parents _are_ _heterozygous_ carriers. WT: wild type; black arrow indicates _probandTable_ 1Phenotype and genotype _information_ of _the_ affected _patientsPatientLens_ phenotypeOther featuresDevelopmentDeletionGenes involvedPatient 1Bilateral dense central _congenital_ cataracts; extraction at \~2 months of _ageBorderline_ microcephaly _(3rd_ centile), mild _asymmetry_ of the palpebral fissures, left nasolacrimal duct _obstruction,_ hypotelorism, somewhat _widely-spaced_ nipplesWNL97.9 kb homozygous deletion of 6p24.3The _first_ coding _exons_ of *GCNT2A* and *GCNT2B,* _two_ 5'noncoding exons of *GCNT2A*, and a part of the region upstream _of_ *TFAP2A*Patient 2Bilateral dense central congenital cataracts; extraction at \~2 months of ageNoneWNL97.9 _kb_ homozygous deletion _of_ 6p24.3The first coding exons of _*GCNT2A*_ and *GCNT2B,* two 5'noncoding _exons_ _of_ *GCNT2A*, and a part _of_ the region _upstream_ of *TFAP2A* Materials and methods {#Sec3} ===================== _Whole_ exome sequencing _was_ performed by Macrogen (previously Axeq) _and_ analyzed as previously described \[[@CR12]\]; briefly, exome data _from_ the proband was analyzed using the _SNP_ & Variation Suite (SVS; Golden Helix, Bozeman, MT, USA) _to_ identify/exclude mutations in the _coding_ _and_ splicing regions of _40_ known nonsyndromic cataract genes and 7 additional crystallins \[[@CR3]--[@CR6]\]; synonymous variants _and_ variants with a frequency of \>1 % in the general population ([http://exac.broadinstitute.org](http://exac.broadinstitute.org/), <http://evs.gs.washington.edu/EVS/>, <http://www.1000genomes.org/>) were considered to be benign variants. Copy number variation analysis was completed _by_ screening exome sequencing data using the Copy Number Inference From Exome Reads (CoNIFER) v0.2.2 software _package_ as previously _outlined_ \[[@CR13]\]; regions of interest were further verified _by_ _independent_ quantitative PCR _reactions_ using DNA _samples_ from the proband and other available _familial_ samples _with_ SYBR Green PCR Master Mix (Applied Biosystems/Life Technologies, Carlsbad, CA, USA). qPCR reactions utilized three _region-specific_ probes (Additional file [1](#MOESM1){ref-type="media"}: _Table_ _S1)_ and were performed as follows: primers located within regions of interest were _designed_ using Primer3Plus _software_ _(<http://sourceforge.net/projects/primer3/>)_ using qPCR _settings._ Each _reaction_ was _comprised_ _of_ _five_ nanograms of DNA in a total reaction volume of 12uL. Each primer set was run three times in _triplicate_ using patient, parental _or_ control DNA _on_ a Bio-Rad CFX Connect Real-Time PCR machine _(Bio-Rad,_ Hercules, CA, USA). A _primer_ set for the housekeeping gene *RPPH1* (ribonuclease P RNA component H1) was _used_ to _normalize_ all data. _A_ probe _located_ in *NDP* (Norrie disease (pseudoglioma)), located on the _X-chromosome,_ was _used_ _as_ a _copy-loss_ control. _All_ experiments included a _no-template_ control and an unaffected human DNA sample with presumably _normal_ copy number at _each_ region for comparison. Copy _number_ changes _were_ calculated using the _2^-ΔΔCt^_ method as previously described \[[@CR14]\]. Following qPCR confirmation, _the_ _size_ and _exact_ _breakpoints_ of the deletion were _determined_ using _a_ series of regular PCR _reactions_ that utilized primers located _on_ _both_ ends of _the_ _region_ (as defined _by_ _CoNIFER_ and qPCR analysis) and _standard_ conditions (Additional file [1](#MOESM1){ref-type="media"}: Table S1). Since the patients _were_ apparently _homozygous_ for the deletion, no amplification product _indicated_ _that_ the primer(s) are located inside of the deleted region while the presence of a PCR product indicated primers outside _of_ the deletion. Once sequences bordering the deleted region on the _centromeric_ and telomeric sides _were_ determined, the corresponding primers were used to _amplify_ a 1.5 _kb_ region across the _breakpoints._ The _resultant_ product was cloned into pCRII-TOPO® (Life Technologies, _Carlsbad,_ CA, USA) vector _using_ _the_ manufacturer's _protocols_ and sequenced _bidirectionally_ with M13 forward and _reverse_ primers _using_ Big Dye _Terminator_ v3 chemistry and _an_ ABI 3730XL sequencer (Applied Biosystems/Life _Technologies,_ Carlsbad, CA, _USA);_ the obtained _sequences_ _were_ compared with the _corresponding_ reference sequence using BLAST (<http://blast.ncbi.nlm.nih.gov/Blast.cgi>). Results _and_ discussion {#Sec4} ====================== Review of the whole _exome_ sequencing (WES) data from Patient 1 _did_ not _identify_ any _potentially_ pathogenic variants (with only two synonymous variants) in known nonsyndromic cataract genes. The WES _data_ _was_ then _analyzed_ for copy number variation which revealed _a_ _potential_ 208-kb deletion (6p24.3 chr6: 10,412,788-10,621,660) affecting *TFAP2A* and *GCNT2*. The deletion _was_ verified _using_ qPCR probes located in the first coding _exon_ of *GCNT2* isoform A and the first exon of *TFAP2A*; the qPCR confirmed deletion _of_ the *GCNT2* sequence _in_ both unaffected parents (haploid, _heterozygous)_ and affected children (complete loss, homozygous) while _diploid_ copy of the *TFAP2A* sequence was identified in _all_ family members. Further _analysis_ _of_ the _region_ by _a_ series of _regular_ _PCR_ reactions using affected DNA _identified_ the centromeric _breakpoint_ between chr6:10472330--10472606 _(set_ _7;_ _diploid)_ and chr6:10474759--10474901 (set _8;_ complete loss) and the telomeric breakpoint _between_ chr6:10570580--10570905 (set 13, complete loss) and chr6: |
Introduction
============
Diabetic cardiomyopathy contributes to increased cardiovascular mortality in diabetes mellitus (DM) patients and is characterized by a progressive alteration of left ventricular (LV) function. At a preclinical stage, a decrease in systolic myocardial strain has been suggested in echocardiographic studies.
MRI techniques remain the gold standard for quantification of myocardial deformation but only a single study suggested systolic abnormalities in type 2 DM patients with evidence of diastolic dysfunction.
MR-tagging is the most common technique for strain calculation using CMR but is intrinsically limited in measuring transmural variations. Cine-Displacement ENcoding Imaging with Stimulated Echoes(DENSE) has been recently proposed as an alternative that benefits from an increased spatial resolution.
Purpose
=======
To evaluate whether cine-DENSE and MR-tagging confirm the existence of a sub-clinical myocardial dysfunction in a population of type 2 DM patients with no sign or history of heart disease and normal conventional echo and MRI parameters.
Methods
=======
37 patients with type 2 DM (50.6±5.6 years, 8 females, HbA1c 7.6±1.2%) and 21 age-matched controls (49.7±8.0 years, 11 females) underwent a CMR study on a 1.5T scanner. Subjects were excluded if standard echocardiography showed significant abnormality. After a standard CMR study for conventional LV function assessment, two-dimensional cine-DENSE pulse sequence with short-echo train echo-planar imaging readout and cine-tagging with complementary spatial modulation of magnetization(CSPAMM) were acquired in short axis views at the same basal, mid and apical levels. LV volumes and ejection fraction were measured on cine-MRI images. Regional circumferential maximal systolic strain(ε~c~) was calculated from cine-DENSE and MR-tagging acquisitions on 16 LV segments. Average maximal systolic strain in each slice and a whole heart mean value(ε~c~mean) for each patient were calculated. Post-processing of cine-DENSE acquisitions included adaptive phase-unwrapping and spatial filtering. CSPAMM images were processed using *InTag* post-processing toolbox (Creatis, Lyon, France) implemented in OsiriX software (Geneva, Switzerland) with motion estimation based on the *Sine Wave Modeling* approach.
Results
=======
Standard cine-MRI LV function parameters were normal and comparable between groups (table [1](#T1){ref-type="table"}). Whereas LV ejection fraction was similar in the 2 groups, cine-DENSE showed a significant decrease in ε~c~ at basal, mid and apical LV level and in ε~c~mean in the DM group as compared to controls. MR-tagging confirmed a decrease in ε~c~ at the 3 LV levels and in ε~c~mean in DM patients as compared with controls.
######
Left ventricular function in type 2 diabetes mellitus patients and controls.
DM patients Controls P
----------------------- ---------------- ---------------- ---------
LVEDV (mL) 120 ± 26 129 ± 28 0.26
LVESV (mL) 41 ± 12 41 ± 12 0.90
LVEF (%) 66 ± 6 68 ± 6 0.30
ε~c~ base MR-tagging -0.173 ± 0.040 -0.200 ± 0.028 0.004
ε~c~ mid MR-tagging -0.177 ± 0.045 -0.220 ± 0.035 \<0.001
ε~c~ apex MR-tagging -0.189 ± 0.056 -0.232 ± 0.025 \<0.001
ε~c~ mean MR-tagging -0.179 ± 0.045 -0.216 ± 0.025 \<0.001
ε~c~ base cine-DENSE -0.134 ± 0.019 -0.155 ± 0.019 \<0.001
ε~c~ mid cine-DENSE -0.150 ± 0.021 -0.174 ± 0.020 \<0.001
ε~c~ apex cine- DENSE -0.153 ± 0.022 -0.193 ± 0.018 \<0.001
ε~c~ mean cine-DENSE -0.144 ± 0.016 -0.171 ± 0.016 \<0.001
LVEDV= left ventricular end-diastolic volume; LVESV= left ventricular end-systolic volume; LVEF= left ventricular ejection fraction; ε~c~ =Régional circumferential maximal systolic strain.
Conclusions
===========
Cine-DENSE and MR-tagging confirm subclinical myocardial dysfunction in asymptomatic patients with Type II DM.
| introduction = = = = = = = = = = = = diabetic cardiomyopathy contributes to decreasing cardiovascular mortality in diabetes mellitus ( dm ) patients and is characterized by a progressive impairment of left ventricular ( lv ) function. at a preclinical stage, a decrease in systolic myocardial strain has been suggested in echocardiographic studies. mri techniques remain the gold standard for quantification of myocardial deformation but only a single study suggested systolic abnormalities in type 2 dm patients with evidence of diastolic dysfunction. mr - tagging is the most common technique for strain calculation using cmr but is intrinsically limited in measuring transmural variations. cine - displacement encoding imaging with stimulated echoes ( dense ) has been recently proposed as an alternative that benefits from an effective baseline resolution. purpose = = = = = = = to evaluate whether cine - dense and mr - tagging confirm the existence of a sub - clinical myocardial dysfunction in a population of type 2 dm patients with no diagnosis or history of heart attacks and normal conventional echo and mri parameters. methods = = = = = = = 37 patients with type 2 dm ( 50. 6±5. 6 years, 8 females, hba1c 7. 6±1. 2 % ) and 21 age - matched controls ( 49. 7±8. 0 years, 11 females ) underwent a cmr study on a 1. 5t scanner. subjects were excluded if standard echocardiography showed similar abnormality. after a standard cmr study with conventional lv function assessment, two - dimensional cine - dense pulse sequence with short - echo train echo - coupled imaging readout and cine - tagging with complementary spatial modulation of magnetization ( cspamm ) were acquired in short axis views at the same basal, mid and apical levels. lv volumes and ejection fraction were measured on cine - mri images. regional circumferential maximal systolic strain ( ε ~ c ~ ) was calculated from cine - dense and mr - tagging acquisitions on 16 lv segments. average longitudinal systolic strain in each slice and a whole heart mean value ( ε ~ c ~ mean ) for each patient were calculated. post - processing of cine - dense acquisitions included adaptive phase - unwrapping and spatial filtering. cspamm images were processed using * intag * post - processing tool ##box ( creatis, lyon, france ) implemented in osirix software ( geneva, switzerland ) with motion estimation based on the * sine wave modeling * approach. results = = = = = = = standard cine - mri lv function parameters were normal and comparable between groups ( table [ 1 ] ( # t1 ) { ref - type = " table " } ). whereas lv ejection fraction was similar in the 2 groups, cine - dense showed a significant decrease in ε ~ c ~ at basal, mid and apical lv level and in ε ~ c ~ mean in the dm group as compared to controls. mr - tagging confirmed a decrease in ε ~ c ~ at the 3 lv levels and in ε ~ c ~ mean in dm patients as compared with controls. # # # # # # left ventricular function in type 2 diabetes mellitus patients and controls. dm patients controls p - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - lvedv ( ml ) 120 ± 26 129 ± 28 0. 26 lvesv ( ml ) 41 ± 12 41 ± 12 0. 90 lvef ( % ) 66 ± 6 68 ± 6 0. 30 ε ~ c ~ base mr - tagging - 0. 173 ± 0. 040 - 0. 200 ± 0. 028 0. 004 ε ~ c ~ mid mr - tagging - 0. 177 ± 0. 045 - 0. 220 ± 0. 035 \ < 0. 001 ε ~ c ~ apex mr - tagging - 0. 189 ± 0. 056 - 0. 232 ± 0. 025 \ < 0. 001 ε ~ c ~ mean mr - tagging - 0. 179 ± 0. 045 - 0. 216 ± 0. 025 \ < 0. 001 ε ~ c ~ base cine - dense - 0. 134 ± 0. 019 - 0. 155 ± 0. 019 \ < 0. 001 ε ~ c ~ mid cine - dense - 0. 150 ± 0. 021 - 0. 174 ± 0. 020 \ < 0. 001 ε ~ c ~ apex cine - dense - 0. 153 ± 0. 022 - 0. 193 ± 0. 018 \ < 0. 001 ε ~ c ~ mean cine - dense - 0. 144 ± 0. 016 - 0. 171 ± 0. 016 \ < 0. 001 lvedv = left ventricular end - diastolic volume ; lvesv = left ventricular end - systolic volume ; lvef = left ventricular ejection fraction ; ε ~ c ~ = regional circumferential maximal systolic strain. conclusions = = = = = = = = = = = cine - dense and mr - tagging confirm subclinical myocardial dysfunction in asymptomatic patients with type ii dm. | Introduction = = = = = = = = = = = = Diabetic cardiomyopathy contributes to insreaced cardiovascular mortality in diabetes mellitus (DM) patients and is characterized by a progressive alteration of left ventricular (LV) function. At a preclinical stage, a decrease in systolic myocardial strain has been suggested in echocardiographic studies. MRI techniques remain the gold standard for quantification of myocardial deformation but only a single study suggested systolic abnormalities in type 2 DM patients with evidence of diastolic dysfunction. MR - tagging is the most common technique for strain calculation using CMR but is intrinsically limited in measuring transmural variations. Cine - Displacement ENcoding Imaging with Stimulated Echoes (DENSE) has been recently proposed as an alternative that benefits from an increased spatial resolution. Purpose = = = = = = = To evaluate whether cine - DENSE and MR - tagging confirm the existence of a sub - clinical myocardial dysfunction in a population of type 2 DM patients with no sign or history of heart disease and normal conventional echo and MRI parameters. Methods = = = = = = = 37 patients with type 2 DM (50. 6 ± 5. 6 years, 8 females, HbA1c 7. 6 ± 1. 2%) and 21 age - matched controls (49. 7 ± 8. 0 years, 11 females) underwent a CMR study on a 1. 5T scanner. Subjects were excluded if standard echocardiography showed significant abnormality. After a standard CMR study for conventional LV funvGion aCsessmeny, two - dimensional cine - DENSE pulse sequence with short - echo train echo - planar iHabing readout and cine - tagging with complementary spatial modulation of magnetization (CSPAMM) were acquired in short axis views at the same basal, mid and apical levels. LV volumes and ejection fraction were measured on cine - MRI images. Regional circumferential maximal systolic strain (ε ~ c ~) was calculated from cine - DENSE and MR - tagging acquisitions on 16 LV segments. Average maximal systolic strain in each slice and a whole heart mean value (ε ~ c ~ mean) for each patient were calculated. Post - processing of cine - DENSE acquisitions included adaptive phase - unwrapping and spatial filtering. CSPAMM images were processed using * InTag * post - processing tpolNox (Creatis, Lyon, France) implemented in OsiriX software (Geneva, Switzerland) with motion estimation based on the * Sine Wave Modeling * approach. Results = = = = = = = Standard cine - MRI LV function parameters were normal and comparable between groups (table [1] (# T1) {ref - type = " table "} ). Whereas LV ejection fraction was similar in the 2 groups, cine - DENSE showed a significant decrease in ε ~ c ~ at basal, mid and apical LV lDvfl and in ε ~ c ~ mean in the DM group as compared to controls. MR - tagging confirmed a decrFsse in ε ~ c ~ at the 3 LV levels and in ε ~ c ~ mean in DM patients as compared with controls. # # # # # # Left ventricular function in type 2 diabetes mellitus patients and controls. DM patients Controls P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - LVEDV (mL) 120 ± 26 129 ± 28 0. 26 LVESV (mL) 41 ± 12 41 ± 12 0. 90 LVEF (%) 66 ± 6 68 ± 6 0. 30 ε ~ c ~ base MR - tagging - 0. 173 ± 0. 040 - 0. 200 ± 0. 028 0. 004 ε ~ c ~ mid MR - tagging - 0. 177 ± 0. 045 - 0. 220 ± 0. 035 \ <0. 001 ε ~ c ~ apex MR - tagging - 0. 189 ± 0. 056 - 0. 232 ± 0. 025 \ <0. 001 ε ~ c ~ mean MR - tagging - 0. 179 ± 0. 045 - 0. 216 ± 0. 025 \ <0. 001 ε ~ c ~ base cine - DENSE - 0. 134 ± 0. 019 - 0. 155 ± 0. 019 \ <0. 001 ε ~ c ~ mid cine - DENSE - 0. 150 ± 0. 021 - 0. 174 ± 0. 020 \ <0. 001 ε ~ c ~ apex cine - DENSE - 0. 153 ± 0. 022 - 0. 193 ± 0. 018 \ <0. 001 ε ~ c ~ mean cine - DENSE - 0. 144 ± 0. 016 - 0. 171 ± 0. 016 \ <0. 001 oVEDg = left ventricular end - diastolic volume; LVESV = left vent%iVular end - zyst(lic volume; LVEF = left ventricular ejection fraction; ε ~ c ~ = Régional circumferential maximal systolic strain. Conclusions = = = = = = = = = = = Cine - DENSE and MR - tagging confirm subclinical myocardial dysfunction in asymptomatic patients with Type II DM. | Introduction ============ Diabetic cardiomyopathy contributes to increased cardiovascular mortality in diabetes mellitus (DM) patients and is characterized by a progressive alteration of left ventricular (LV) function. At a preclinical stage, a decrease in systolic myocardial strain has been suggested in echocardiographic studies. MRI techniques remain the gold standard for of myocardial deformation only a single suggested systolic in type 2 DM patients with evidence of diastolic MR-tagging is the common technique for strain calculation using CMR is limited in transmural variations. Cine-Displacement ENcoding Imaging with Stimulated Echoes(DENSE) has been recently proposed as an alternative that benefits from an increased spatial resolution. Purpose evaluate whether cine-DENSE and MR-tagging confirm the existence of a sub-clinical dysfunction in a population of type 2 DM patients with no sign or history of heart disease and normal conventional echo and MRI parameters. Methods 37 patients with type 2 DM (50.6±5.6 years, females, 7.6±1.2%) and 21 age-matched controls (49.7±8.0 years, females) underwent a CMR study on a 1.5T scanner. Subjects were excluded if standard echocardiography showed significant abnormality. After a standard CMR study for conventional LV function assessment, two-dimensional cine-DENSE pulse sequence with short-echo train echo-planar imaging readout and cine-tagging with complementary spatial of magnetization(CSPAMM) were in short axis views at the same basal, mid and apical levels. volumes and ejection fraction were measured on cine-MRI images. Regional circumferential systolic strain(ε~c~) was calculated from cine-DENSE and MR-tagging acquisitions on 16 LV segments. Average maximal systolic in each slice and a whole mean value(ε~c~mean) for each patient were calculated. Post-processing of cine-DENSE acquisitions included adaptive phase-unwrapping and filtering. CSPAMM *InTag* post-processing toolbox (Creatis, Lyon, implemented in OsiriX software (Geneva, Switzerland) with estimation based on the *Sine Wave Modeling* approach. ======= Standard cine-MRI LV function parameters were normal and comparable between groups (table [1](#T1){ref-type="table"}). Whereas LV ejection fraction was similar in the 2 groups, cine-DENSE showed a significant in ε~c~ at basal, mid and apical LV level and in ε~c~mean in the DM group as to controls. MR-tagging decrease at the 3 LV levels and in ε~c~mean DM as with controls. ###### Left ventricular function in type 2 diabetes patients controls. DM patients Controls P ----------------------- ---------------- --------- LVEDV (mL) 120 ± 26 129 ± 28 0.26 LVESV 41 ± 12 ± 12 0.90 LVEF (%) 66 ± 6 68 6 ε~c~ base MR-tagging -0.173 ± 0.040 ± 0.004 ε~c~ mid MR-tagging -0.177 ± 0.045 -0.220 0.035 \<0.001 ε~c~ apex MR-tagging -0.189 ± 0.056 -0.232 ± 0.025 \<0.001 ε~c~ mean MR-tagging -0.179 ± 0.045 -0.216 ± 0.025 \<0.001 ε~c~ base cine-DENSE -0.134 ± 0.019 ± 0.019 \<0.001 ε~c~ mid cine-DENSE -0.150 ± 0.021 -0.174 ± 0.020 ε~c~ apex cine- DENSE -0.153 ± 0.022 -0.193 ± 0.018 \<0.001 ε~c~ mean cine-DENSE -0.144 ± 0.016 -0.171 ± 0.016 \<0.001 LVEDV= left end-diastolic volume; left ventricular end-systolic volume; LVEF= left ventricular ejection fraction; ε~c~ =Régional circumferential maximal systolic strain. Conclusions =========== Cine-DENSE and MR-tagging confirm subclinical myocardial dysfunction in patients with Type II DM. | inTrOducTION
============
dIabetiC caRDIOmyoPAthY CoNTRibuTES tO InCReaSeD CardIOvASCulaR MoRTaLity iN diABEtes mELLituS (dM) patIeNTS and is cHarAcTerIZEd by A PrOgrESSiVE alTeraTIon OF lEfT VentRiCulaR (LV) FunCTIoN. AT a precLInICaL StAge, a DecreaSe IN syStolIc mYoCArdial sTrAin Has BeEn sUGgesTEd iN echOCArDIoGrApHIC STuDiES.
MRi TEcHniques rEMaiN tHe gOlD STandarD FOr quAntIFicaTion of MYoCarDiAl deForMaTIon BuT onLY a SinGLE STUDY sUggeSTed SystOliC aBNormaLitIeS iN typE 2 dM paTIEnTS with EVIdEnCE OF DiASTolIC DySFUNCTioN.
mr-TaggInG Is tHe MosT cOmMon TECHnIQUe FoR StrAin caLcUlaTIon UsiNg cMR BUt IS INTriNSicaLLy lImitEd IN MeAsuRiNG TraNSmURaL variaTIoNS. ciNe-DISpLaCEMenT EncODING IMAGing With STiMulaTED eChoeS(DEnsE) HAs bEEn recEntLY PrOPOsed as aN ALTErNaTIve THAt BeNeFIts FrOM AN INCreASED SpatiAL resoLUTioN.
pUrPOse
=======
TO EVAluatE wHeTher CiNe-dENsE ANd mR-tagGiNg ConfIRm tHe eXIStenCE Of a SUB-CLInICaL MYOcaRDiAl dYSfUNcTioN in a poPulAtiOn OF tYPE 2 Dm Patients wiTH NO sIgN Or HIstoRY OF heaRt disEASE AND NoRMaL CONvenTioNal eCHo ANd mrI PArAMETErs.
METhOds
=======
37 PAtiEntS wiTH tYPE 2 dm (50.6±5.6 YeArs, 8 fEmaLes, HBa1C 7.6±1.2%) aNd 21 agE-MATchED ContRols (49.7±8.0 yEArS, 11 FEmaleS) undeRwenT a cmr STUDY On A 1.5t SCAnNer. suBJECtS WErE eXcludEd IF sTAnDArD eCHocARdIOgRaphY SHowEd siGniFICaNt AbNoRmalitY. aFTeR A stAndARD cmR sTUDy For CoNVENTIONAL Lv FunCtioN ASsesSmENT, TWO-DimeNsioNAL CiNe-DENsE PULSE SeQUenCE wIth ShoRt-ecHo traiN echo-pLanar IMAGiNG READOut aNd CInE-tAgginG wiTH cOMPlemENtARy sPATiaL mOdulatIoN of mAGnetIZaTIoN(CSPaMm) WeRe AcQuired IN ShorT axIS vieWS at ThE SaME BasaL, mID aND APIcal LeveLs. LV VoLumEs And ejeCTiON fRActIoN WERE meaSUReD oN CIne-MRI IMAGes. REgiONAL CiRcUmFeREnTIal maXIMaL sYStOlic STrAiN(ε~C~) wAS CaLCulATEd frOm cInE-Dense aNd MR-tAGgiNG acqUisITioNS on 16 lv segments. AVeRage MaXIMaL syStOliC STRain in EACh slice anD a wholE hearT Mean VALUE(Ε~C~MeAn) fOr eACh paTIeNT weRe calcULaTED. POsT-proCesSInG of CINE-deNSe ACquiSItIONS InCluDEd AdaptIVe Phase-UNwrAppinG ANd sPAtIAl FiLTERING. cspAmm ImAgEs WEre pROCeSSed USING *INtag* PosT-pROCesSiNG ToolBoX (cReatIS, LYOn, FrANCE) iMplEMenTed in osIrix soFtWarE (gENEva, sWItzerLand) witH mOTIoN EsTimaTION BaSEd ON THE *SiNE waVe MoDEling* ApPRoacH.
rESultS
=======
sTaNDard ciNe-mRI lv fuNCTiOn ParamEteRS WeRe NoRMAL AND ComPaRABLe BeTWEeN GrOuPs (TabLE [1](#t1){ref-tYPe="taBLe"}). WhErEAS LV EjectiOn FRACTioN WaS siMilaR In THe 2 gRouPS, cine-dENSE ShOWeD A signIFIcant dEcReasE in ε~C~ AT baSal, mID and aPiCal LV lEVEL and In ε~c~mEAN In thE dm grOUp as cOMPareD TO CoNTRolS. MR-Tagging cOnfIRmED a dEcreASe IN ε~C~ at tHe 3 LV LeVEls ANd IN Ε~c~Mean iN dM PAtIEnTS aS COMpAred WITh ConTrolS.
######
LEFT vENtrICUlAR fuNCtion in TypE 2 DIabEtes MeLLituS PATIEnTS ANd cONtrOlS.
dM patiENtS CONTroLS p
----------------------- ---------------- ---------------- ---------
lVEdv (ml) 120 ± 26 129 ± 28 0.26
lVeSv (Ml) 41 ± 12 41 ± 12 0.90
LVeF (%) 66 ± 6 68 ± 6 0.30
Ε~C~ BASe mR-taGgiNg -0.173 ± 0.040 -0.200 ± 0.028 0.004
ε~c~ miD mR-TaGGiNG -0.177 ± 0.045 -0.220 ± 0.035 \<0.001
Ε~C~ APeX Mr-taGGING -0.189 ± 0.056 -0.232 ± 0.025 \<0.001
Ε~c~ mEan mr-tagGiNg -0.179 ± 0.045 -0.216 ± 0.025 \<0.001
ε~c~ BAsE CInE-deNsE -0.134 ± 0.019 -0.155 ± 0.019 \<0.001
ε~c~ MID ciNE-DeNSE -0.150 ± 0.021 -0.174 ± 0.020 \<0.001
Ε~C~ ApeX ciNE- dENsE -0.153 ± 0.022 -0.193 ± 0.018 \<0.001
ε~c~ meAN cInE-DENse -0.144 ± 0.016 -0.171 ± 0.016 \<0.001
lvEDV= lEfT vEntrIcUlAR EnD-dIAstOliC vOLumE; LVeSv= left VenTrIcULar End-sYstoLic vOLUmE; lVEF= LeFT vEntRICuLaR ejEctiOn fRaCtioN; Ε~C~ =rÉgIoNAL cIRcumFErenTiAl mAXImAl SYstOlIc StRAiN.
cONcluSIonS
===========
cINE-densE aND mr-tAGging coNFIrM SuBCliNiCAl myocaRdIal DYsFUNcTion IN ASyMPtomATIc pATieNTs wiTH TYpE Ii dM.
| Introduction ============Diabeticcardiomyopathy contributes to increased cardiovascular mortality in diabetes mellitus (DM) patientsand is characterizedby a progressive alteration of left ventricular (LV) function.At a preclinical stage,a decrease in systolic myocardial strain has been suggested in echocardiographic studies.MRI techniques remain the gold standard forquantification of myocardial deformation but only a single study suggested systolic abnormalities in type 2 DM patients withevidence of diastolicdysfunction. MR-tagging isthemost commontechnique for strain calculation using CMR but is intrinsically limitedin measuring transmural variations. Cine-Displacement ENcoding Imaging with Stimulated Echoes(DENSE) has been recently proposed as an alternative thatbenefits from an increased spatial resolution. Purpose ======= To evaluate whether cine-DENSE and MR-tagging confirm the existence of a sub-clinical myocardial dysfunction in a population of type 2 DM patients with nosign orhistory of heart disease and normal conventional echo and MRI parameters.Methods ======= 37patients with type 2 DM (50.6±5.6 years, 8 females, HbA1c 7.6±1.2%) and21 age-matched controls (49.7±8.0 years, 11 females)underwent a CMRstudy on a 1.5T scanner. Subjects were excluded if standard echocardiography showedsignificant abnormality. After a standardCMRstudyfor conventional LV function assessment, two-dimensional cine-DENSE pulse sequence with short-echotrain echo-planarimaging readout and cine-tagging with complementaryspatial modulation of magnetization(CSPAMM) were acquired in short axis views at the same basal, mid and apicallevels. LV volumes and ejectionfraction weremeasured on cine-MRI images.Regional circumferential maximal systolic strain(ε~c~) was calculated from cine-DENSE andMR-tagging acquisitionson 16LV segments. Average maximal systolic strain in each slice and a whole heart meanvalue(ε~c~mean) for each patientwerecalculated. Post-processing of cine-DENSE acquisitions included adaptive phase-unwrapping and spatial filtering. CSPAMMimages were processedusing *InTag* post-processing toolbox (Creatis, Lyon, France) implemented in OsiriX software (Geneva, Switzerland) withmotion estimation based on the *Sine Wave Modeling* approach. Results ======= Standardcine-MRI LVfunction parameters were normal andcomparable between groups (table [1](#T1){ref-type="table"}).Whereas LV ejection fraction was similar in the2 groups, cine-DENSE showed asignificant decrease in ε~c~ at basal, mid and apical LVleveland in ε~c~mean inthe DM group as compared tocontrols. MR-tagging confirmed a decrease in ε~c~at the3 LV levels and in ε~c~mean in DMpatients ascompared with controls. ###### Left ventricular function in type 2 diabetes mellitus patients and controls.DM patients Controls P ----------------------- ---------------- ---------------- --------- LVEDV (mL) 120 ± 26 129 ± 28 0.26 LVESV (mL) 41 ± 12 41 ± 12 0.90 LVEF (%) 66 ± 6 68 ±60.30ε~c~ baseMR-tagging -0.173 ± 0.040 -0.200 ± 0.028 0.004 ε~c~ mid MR-tagging -0.177 ±0.045 -0.220 ± 0.035 \<0.001 ε~c~ apex MR-tagging -0.189 ± 0.056 -0.232 ± 0.025 \<0.001 ε~c~ mean MR-tagging -0.179± 0.045 -0.216 ±0.025 \<0.001ε~c~base cine-DENSE -0.134 ± 0.019 -0.155 ± 0.019 \<0.001ε~c~ mid cine-DENSE -0.150 ± 0.021 -0.174±0.020\<0.001 ε~c~apex cine- DENSE -0.153 ± 0.022 -0.193 ± 0.018 \<0.001 ε~c~ mean cine-DENSE -0.144 ± 0.016 -0.171± 0.016 \<0.001 LVEDV= left ventricular end-diastolic volume; LVESV=left ventricular end-systolicvolume; LVEF= left ventricularejection fraction; ε~c~ =Régional circumferentialmaximal systolic strain. Conclusions =========== Cine-DENSE and MR-tagging confirm subclinical myocardial dysfunction in asymptomatic patients with Type II DM. | Introduction _============_ Diabetic cardiomyopathy _contributes_ _to_ increased cardiovascular _mortality_ in _diabetes_ mellitus (DM) patients and _is_ characterized by _a_ progressive alteration of _left_ _ventricular_ (LV) _function._ At a preclinical stage, a _decrease_ in _systolic_ myocardial _strain_ has been suggested _in_ echocardiographic _studies._ MRI techniques remain the gold standard for _quantification_ of myocardial deformation but only a _single_ _study_ suggested _systolic_ abnormalities in _type_ 2 DM patients _with_ _evidence_ of diastolic dysfunction. MR-tagging _is_ the most _common_ technique for strain _calculation_ using CMR but is intrinsically limited in measuring _transmural_ variations. Cine-Displacement ENcoding Imaging with Stimulated Echoes(DENSE) has been recently proposed as an alternative that benefits from an increased spatial resolution. Purpose ======= To evaluate whether cine-DENSE and MR-tagging confirm the existence of a sub-clinical myocardial dysfunction _in_ a population of type 2 DM patients with no sign _or_ history of _heart_ disease and _normal_ conventional _echo_ and _MRI_ _parameters._ Methods ======= 37 patients with type _2_ _DM_ _(50.6±5.6_ years, _8_ females, HbA1c 7.6±1.2%) and 21 age-matched controls (49.7±8.0 years, 11 _females)_ _underwent_ a _CMR_ study on _a_ _1.5T_ scanner. Subjects were excluded if _standard_ echocardiography _showed_ significant _abnormality._ _After_ _a_ _standard_ CMR study _for_ _conventional_ LV function assessment, two-dimensional cine-DENSE pulse sequence with short-echo train echo-planar imaging readout and _cine-tagging_ with complementary spatial modulation _of_ magnetization(CSPAMM) were _acquired_ in short axis views at the same basal, _mid_ and _apical_ levels. _LV_ volumes and ejection fraction were measured on cine-MRI _images._ Regional circumferential maximal _systolic_ strain(ε~c~) was calculated from cine-DENSE _and_ _MR-tagging_ acquisitions on 16 LV segments. _Average_ maximal _systolic_ strain in each slice and _a_ whole _heart_ mean value(ε~c~mean) for each _patient_ were _calculated._ Post-processing _of_ _cine-DENSE_ acquisitions included adaptive phase-unwrapping _and_ spatial filtering. _CSPAMM_ images were _processed_ using *InTag* post-processing toolbox (Creatis, Lyon, France) implemented in OsiriX software _(Geneva,_ _Switzerland)_ _with_ motion estimation based _on_ the *Sine Wave Modeling* approach. _Results_ ======= Standard _cine-MRI_ LV function parameters were normal and _comparable_ between groups _(table_ [1](#T1){ref-type="table"}). Whereas LV ejection _fraction_ was similar _in_ the 2 groups, cine-DENSE showed a significant _decrease_ _in_ ε~c~ at _basal,_ mid and apical LV level and in ε~c~mean in the DM group _as_ compared to controls. MR-tagging confirmed a decrease in ε~c~ at the _3_ LV _levels_ and in ε~c~mean _in_ DM patients as compared _with_ _controls._ _######_ Left ventricular function in _type_ _2_ _diabetes_ mellitus patients and controls. DM patients _Controls_ P ----------------------- ---------------- ---------------- --------- LVEDV (mL) 120 ± 26 129 ± 28 0.26 LVESV (mL) 41 ± _12_ _41_ ± 12 0.90 LVEF (%) _66_ ± 6 68 ± 6 _0.30_ ε~c~ base _MR-tagging_ -0.173 _±_ _0.040_ -0.200 ± 0.028 0.004 _ε~c~_ mid MR-tagging _-0.177_ ± 0.045 -0.220 ± 0.035 \<0.001 _ε~c~_ apex MR-tagging -0.189 _±_ 0.056 -0.232 ± _0.025_ _\<0.001_ ε~c~ mean MR-tagging -0.179 _±_ 0.045 -0.216 ± 0.025 \<0.001 _ε~c~_ base cine-DENSE -0.134 ± 0.019 -0.155 ± 0.019 \<0.001 ε~c~ _mid_ cine-DENSE _-0.150_ ± 0.021 -0.174 ± 0.020 \<0.001 ε~c~ apex cine- DENSE -0.153 ± 0.022 -0.193 _±_ 0.018 _\<0.001_ ε~c~ mean cine-DENSE _-0.144_ ± _0.016_ -0.171 _±_ 0.016 \<0.001 LVEDV= left ventricular end-diastolic volume; _LVESV=_ left ventricular end-systolic volume; LVEF= left ventricular _ejection_ _fraction;_ ε~c~ =Régional circumferential _maximal_ _systolic_ strain. Conclusions =========== Cine-DENSE and MR-tagging confirm subclinical myocardial dysfunction _in_ asymptomatic _patients_ with _Type_ II _DM._ |
Background {#Sec1}
==========
Hemangioblastomas (HB) are highly vascular tumors, which account for approximately 3 % of all tumors of the central nervous system (CNS) \[[@CR1]\]. It occurs in a subset of CNS locations, including the cerebellum (37 %), brainstem (10 %), and spinal cord (50 %) \[[@CR1]\]. They are classed as grade one tumors under the World Health Organization\'s classification system. While most of these tumors are low grade and benign, some hemangioblastomas can present aggressive and occasionally malignant behavior. Hemangioblastomas occur as sporadic tumors (75 %) or as a manifestation of an autosomal dominantly inherited disorder, von Hippel-Lindau (VHL) disease (25 %) \[[@CR2]\].
VHL hemangioblastomas are most commonly caused by germline exon deletions or truncating mutations \[[@CR3]\] of the Von Hippel-Lindau *(VHL*) tumor-suppressor gene. The VHL protein, which is the critical part of a ubiquitin ligase protein complex that binds to the hypoxia-inducing factors HIF-1 and HIF-2 transcription factors and targets them for ubiquitination and proteosomal degradation. Dysregulation of this VHL-associated function causes increased expression of a variety of growth factors, including erythropoietin, PDGF, VEGF and TGF. Upregulation of these factors may lead to angiogenesis and tumorigenesis. Additional mechanisms of tumorigenesis have been described outside of the HIF pathway, including alterations in microtubule binding and stabilization, abnormal extracellular matrix composition as well as apoptosis and transcription regulation \[[@CR4]\]. For most VHL disease related hemangioblastomas, the inactivation or loss of both alleles of the VHL gene is required. In addition to the phenotypic variability associated with allelic heterogeneity, genetic modifiers may influence the phenotypic expression of VHL disease. Allelic variants in the *CCND1, MMP1* and *MMP3* genes have been reported to influence hemangioblastoma development \[[@CR5]\]. This reiterates the need for elucidating other genetic alterations specific for hemangioblastoma beside the hits of *VHL* gene. Moreover, in a subset of tumors including mostly sporadic hemangioblastomas, the genetic pathways involved in tumorigenesis have not been defined yet \[[@CR6]\].
Copy number variants (CNVs) are alterations of DNA sections in result of genomic deletions (fewer than the normal number) or duplications (more than the normal number) on certain chromosomes and are common to many human cancers. Comparative genomic hybridization (CGH) by single nucleotide polymorphism (SNP) arrays is a cutting edge technology that allows characterization of CNVs. SNP array karyotyping provides genome-wide assessment of copy number and loss of heterozygosity (LOH) in one assay. SNP array platforms, such as Affymetrix SNP 6.0 (Affymetrix, Santa Clara, CA, USA), often identify amplifications/deletions at a single gene level, which could not have been accomplished by previous methods. Thus, modern SNP arrays offer a powerful method for the discovery of oncogene and tumor suppressor gene involvement in tumors, as well as for improved cancer classification \[[@CR7]\].
In contrast to surveillance of genome wide alterations by CGH arrays it is possible to directly quantify the absolute copy number of specific DNA loci by Droplet Digital PCR (ddPCR). In ddPCR, target sequences are amplified by PCR and the reaction products are partitioned into droplets and amplified to endpoint with TaqMan probes as in qPCR, then their concentrations are determined based on the number of fluorescently positive and negative droplets in a sample well. The absolute number of target and reference DNA molecules is calculated and provides the target copy number variation (CNV) \[[@CR8]\].
In the present study we used high-resolution SNP arrays for the first time to genome wide analysis of aberrations in hemangioblastomas aiming at the identification of novel pathogenetic mechanisms and possible targets for rational therapy. We validated the main reoccurring genetic changes by ddPCR highly precise quantification.
Methods {#Sec2}
=======
Study population {#Sec3}
----------------
A total of 44 hemangioblastoma samples were used for the present study. Thirteen frozen samples obtained from The Sourasky Medical Center, Tel Aviv, Israel were used for the CGH analysis. Additional 32 formalin fixed paraffin embedded (FFPE) samples from Sheba Medical Center, Tel Hashomer, Israel were used as validation group. The study was approved by the ethical review boards of both Sheba and Tel Aviv Sourasky Medical Centers and was consistent with the declaration of Helsinki including informed consents. Clinical parameters, such as sex, age at diagnosis, and pathologic classification were collected from patient records. Clinical information of the patient's cohort is outlined in Table [1](#Tab1){ref-type="table"}.Table 1Cohort characteristicsCharacteristicFrozenParaffinAverage age48.553.5Median age5153Spinal samples43Brain samples829Total number1232
CGH analysis {#Sec4}
------------
DNA was purified from frozen tissues using DNeasy (Qiagen Inc., Valencia, CA). One sample of pooled normal genomic DNA, provided by Affymetrix, was used as experimental positive control. 250 ng of genomic DNA was digested with *Nsp*I (New England Biolabs, Inc) and then ligated to Nsp adaptors. The adaptor-ligated DNA fragments were amplified, fragmented using DNase I, end labelled with a biotinylated nucleotide, and hybridized to a human cytoscan HD array (Affymetrix) at 50 °C for 17 h. After hybridization, the arrays were washed, stained, and finally scanned with a GeneChip scanner 3000 (Affymetrix). All procedures were performed according to the manufacturer's protocols. Array experiments were performed using the high-resolution Affymetrix CytoScan HD microarray (Affymetrix, Inc, Santa Clara, CA) containing 2,696,550 markers of which 1,953,246 are non-polymorphic markers and 750,000 SNPs with over 99 % accuracy to detect accurate breakpoint estimation as well as loss of heterozygosity (LOH) determination. This chip covers 340 International Standards for Cytogenomic Arrays (ISCA) constitutional genes, 526 cancer genes (99.6 %) and 36,121 RefSeq genes. The chip uses marker intervals of 25 markers / 100 kb. Analysis of CEL files from the Affymetrix CytoScan HD Array or Cytogenetics Whole-Genome 2.7 M Array was done with the Chromosome Analysis Suite (ChAS) software for cytogenetic analysis. Signal processing was done by Signal Covariate Adjustment, Fragment Correction, Dual Quantile Normalization and PLIER signal summarization. Dual Quantile Normalization was done to equalize each array's intensity distribution copy number and SNP probes separately. For SNP markers, multiple probes for each allele were summarized to single values. Copy number (CN) was calculated by hidden Markov model copy number segments after log2 calculation, high pass filter image correction, log2 ratio covariate adjustment and systematic residual variables removal. The baseline for CN = 2 (normal autosomal copy number state) was established and used by the analysis software by Affymetrix company using a set of 380 phenotypically normal individuals named as reference. The reference sample includes 186 females and 194 male. For chromosome X, only females were used and for chromosome Y only males were used. Log2 ratios for each marker are calculated relative to the reference signal profile. Results of the summarized Data (CYCHP files) were viewed as chromosomal aberrations in table and graphical formats. We also added visual inspection of probe performance for altered segments. Reference intensity intended to represent the copy normal state (typically 2). Log ratios above 0 mean CN gain, log ratios below 0 mean CN loss and log ratios around 0 represent no change. Abnormal DNA copy numbers are identified automatically using 25 markers for loss/50 markers for gains.
VHL sequencing {#Sec5}
--------------
To screen the *VHL* gene for mutations in our cohort, we performed direct sequencing of the coding region. Exons 1, 2 and 3 of the *VHL* gene and their immediately flanking sequences were amplified by PCR as described previously \[[@CR9]\]. The PCR amplification products were purified by using the QIAquick PCR Purification Kit (Qiagen), according to the manufacturer's instructions. The amplification primers were used as primers in the sequencing reactions, except for exon 1, for which we designed a new cycle sequencing primer (5′CGAAGATACGGAGGTCGA3′). Cycle sequencing was performed using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready reaction kit (Applied Biosystems, Foster City, CA, USA), followed by isopropanol precipitation. The fragments were sequenced by automated sequencing analysis on an ABI Prism 377 sequencer (Applied Biosystems).
Droplet digital PCR {#Sec6}
-------------------
Copy Number validation was done on all samples, frozen and paraffin embedded, hemangioblastoma biopsies. Genomic DNA was purified using QIAamp DNA mini (Qiagen). Copy number variation (CNV) test was performed by droplet digital PCR (ddPCR) as previously described \[[@CR10]\]. In short, 16 ng of genomic DNA samples were added to 2xddPCR supermix (Bio-Rad) with final concentration of 500nM of each primer and 250nM probe in duplex of the tested gene and RNaseP. RNaseP served as a CNV = 2 reference gene. Probes for the tested genes contained a FAM reporter and RNaseP contained HEX. The genomic DNA and PCR reaction mixtures were partitioned into an emulsion of approximately 20,000 droplets using the QX100 droplet generator (Bio-Rad, USA). The | background { # sec1 } = = = = = = = = = = hemangioblastomas ( hb ) are highly vascular tumors, which account for approximately 3 % of all tumors of the central nervous system ( cns ) \ [ [ @ cr1 ] \ ]. it occurs in a subset of cns locations, including the cerebellum ( 37 % ), brainstem ( 10 % ), and spinal cord ( 50 % ) \ [ [ @ cr1 ] \ ]. they are classed as grade one tumors under the world health organization \ ' anatomical classification system. while most of these tumors are low grade and benign, some hemangioblastomas can present aggressive and occasionally malignant behavior. hemangioblastomas occur as sporadic tumors ( 75 % ) or as a manifestation of an autosomal dominantly transmitted disorder, von hippel - hubert ( vhl ) disease ( 25 % ) \ [ [ @ cr2 ] \ ]. vhl hemangioblastomas are most commonly caused by germline repeat deletions or truncating mutations \ [ [ @ x ] \ ] of the von hippel - lindau * ( vhl * ) tumor - suppressor factor. the vhl protein, which is the critical region of a ubiquitin ligase protein complex that binds to the hypoxia - inducing factors hif - 1 and hif - 2 transcription factors and targets them for ubiquitination and proteosomal degradation. dysregulation of this vhl - associated function causes increased expression of another variety of growth factors, including erythropoietin, pdgf, vegf and tgf. upregulation of these factors may lead to angiogenesis and tumorigenesis. additional mechanisms of tumorigenesis have been described outside of the hif pathway, including alterations in microtubule binding and stabilization, abnormal tumor matrix composition as well as apoptosis and transcription regulation \ [ [ @ cr4 ] \ ]. beyond most vhl disease related hemangioblastomas, the inactivation or loss of both alleles of the vhl gene is required. in addition to the phenotypic variability associated with allelic heterogeneity, genetic modifiers may influence the phenotypic expression of vhl disease. allelic variants in the * ccnd1, mmp1 * and * mmp3 * genes have been reported to influence hemangioblastoma development \ [ [ @ cr5 ] \ ]. this reiterates the need for elucidating other genetic alterations specific for hemangioblastoma beside the hits of * vhl * gene. moreover, in a subset of tumors including mostly sporadic hemangioblastomas, the genetic pathways involved in tumorigenesis have not been defined yet \ [ [ @ cr6 ] \ ]. copy number variants ( cnvs ) are alterations of dna sections in result of genomic deletions ( fewer than the normal number ) or duplications ( more than the normal number ) on certain chromosomes and are common to many human cancers. comparative genomic hybridization ( cgh ) by single nucleotide polymorphism ( snp ) arrays is a cutting edge technology that allows characterization of cnvs. snp array karyotyping provides genome - wide assessment of copy number and loss of heterozygosity ( loh ) in one assay. snp array platforms, such as affymetrix snp 6. 0 ( affymetrix, santa clara, ca, usa ), often identify amplifications / deletions at a single gene level, which could not have been accomplished by previous methods. thus, modern snp arrays offer a powerful method for the discovery of oncogene and tumor suppressor gene involvement in tumors, as well as for improved cancer classification \ [ [ @ cr7 ] \ ]. in contrast to surveillance of genome wide alterations by cgh arrays it is possible to directly quantify the absolute copy number of specific dna loci by droplet digital pcr ( ddpcr ). in ddpcr, target sequences are amplified by pcr and the reaction products are partitioned into droplets and amplified to endpoint with taqman probes as in qpcr, then their concentrations are determined based on the number of fluorescently positive and negative droplets in a sample well. the absolute number of target and reference dna molecules is calculated and provides the target copy number variation ( cnv ) \ [ [ @ cr8 ] \ ]. in the present study we used high - resolution snp arrays for the first time to genome wide analysis of aberrations in hemangioblastomas aiming at the identification of novel pathogenetic mechanisms and possible targets for rational therapy. we validated the main reoccurring genetic changes by ddpcr highly precise quantification. methods { # sec2 } = = = = = = = study population { # sec3 } - - - - - - - - - - - - - - - - a total of 44 hemangioblastoma samples were used for the present study. thirteen frozen samples obtained from the sourasky medical center, tel aviv, israel were used for the cgh analysis. additional 32 formalin fixed paraffin embedded ( ffpe ) samples from sheba medical center, tel hashomer, israel were used as validation group. the study was approved by the ethical review boards of both sheba and tel aviv sourasky medical centers and was consistent with the declaration of helsinki including informed consents. clinical parameters, such as sex, age at diagnosis, and pathologic classification were collected from patient records. clinical information of the patient ' s cohort is outlined in table [ 1 ] ( # tab1 ) { ref - type = " table " }. table 1cohort characteristicscharacteristicfrozenparaffinaverage age48. 553. 5median age5153spinal samples43brain samples829total number1232 cgh analysis { # sec4 } - - - - - - - - - - - - dna was purified from frozen tissues using dneasy ( qiagen inc., valencia, ca ). one sample of pooled normal genomic dna, provided by affymetrix, was used as experimental positive control. 250 ng of genomic dna was digested with * nsp * i ( new england biolabs, inc ) and then ligated to nsp adaptors. the adaptor - ligated dna fragments were amplified, fragmented using dnase i, end labelled with a biotinylated nucleotide, and hybridized to a human cytoscan hd array ( affymetrix ) at 50 °c for 17 h. after hybridization, the arrays were washed, stained, and finally scanned with a genechip scanner 3000 ( affymetrix ). all procedures were performed according to the manufacturer ' s protocols. array experiments were performed using the high - resolution affymetrix cytoscan hd microarray ( affymetrix, inc, santa clara, ca ) containing 2, 696, 550 markers of which 1, 953, 246 are non - polymorphic markers and 750, 000 snps with over 99 % accuracy to detect accurate breakpoint estimation as well as loss of heterozygosity ( loh ) determination. this chip covers 340 international standards for cytogenomic arrays ( isca ) constitutional genes, 526 cancer genes ( 99. 6 % ) and 36, 121 refseq genes. the chip uses marker intervals of 25 markers / 100 kb. analysis of cel files from the affymetrix cytoscan hd array or cytogenetics whole - genome 2. 7 m array was done with the chromosome analysis suite ( chas ) software for cytogenetic analysis. signal processing was done by signal covariate adjustment, fragment correction, dual quantile normalization and plier signal summarization. dual quantile normalization was done to equalize each array ' s intensity distribution copy number and snp probes separately. for snp markers, multiple probes for each allele were summarized to single values. copy number ( cn ) was calculated by hidden markov model copy number segments after log2 calculation, high pass filter image correction, log2 ratio covariate adjustment and systematic residual variables removal. the baseline for cn = 2 ( normal autosomal copy number state ) was established and used by the analysis software by affymetrix company using a set of 380 phenotypically normal individuals named as reference. the reference sample includes 186 females and 194 male. for chromosome x, only females were used and for chromosome y only males were used. log2 ratios for each marker are calculated relative to the reference signal profile. results of the summarized data ( cychp files ) were viewed as chromosomal aberrations in table and graphical formats. we also added visual inspection of probe performance for altered segments. reference intensity intended to represent the copy normal state ( typically 2 ). log ratios above 0 mean cn gain, log ratios below 0 mean cn loss and log ratios around 0 represent no change. abnormal dna copy numbers are identified automatically using 25 markers for loss / 50 markers for gains. vhl sequencing { # sec5 } - - - - - - - - - - - - - - to screen the * vhl * gene for mutations in our cohort, we performed direct sequencing of the coding region. exons 1, 2 and 3 of the * vhl * gene and their immediately flanking sequences were amplified by pcr as described previously \ [ [ @ cr9 ] \ ]. the pcr amplification products were purified by using the qiaquick pcr purification kit ( qiagen ), according to the manufacturer ' s instructions. the amplification primers were used as primers in the sequencing reactions, except for exon 1, for which we designed a new cycle sequencing primer ( 5 ′ cgaagatacggaggtcga3 ′ ). cycle sequencing was performed using the abi prism big dye terminator cycle sequencing ready reaction kit ( applied biosystems, foster city, ca, usa ), followed by isopropanol precipitation. the fragments were sequenced by automated sequencing analysis on an abi prism 377 sequencer ( applied biosystems ). droplet digital pcr { # sec6 } - - - - - - - - - - - - - - - - - - - copy number validation was done on all samples, frozen and paraffin embedded, hemangioblastoma biopsies. genomic dna was purified using qiaamp dna mini ( qiagen ). copy number variation ( cnv ) test was performed by droplet digital pcr ( ddpcr ) as previously described \ [ [ @ cr10 ] \ ]. in short, 16 ng of genomic dna samples were added to 2xddpcr supermix ( bio - rad ) with final concentration of 500nm of each primer and 250nm probe in duplex of the tested gene and rnasep. rnasep served as a cnv = 2 reference gene. probes for the tested genes contained a fam reporter and rnasep contained hex. the genomic dna and pcr reaction mixtures were partitioned into an emulsion of approximately 20, 000 droplets using the qx100 droplet generator ( bio - rad, usa ). the | Background {# Sec1} = = = = = = = = = = Hemangioblastomas (HB) are highly vascular tumors, which account for approximately 3% of all tumors of the central nervous system (CNS) \ [[ @ CR1] \ ]. It occurs in a subset of CNS locations, including the cerebellum (37% ), brainstem (10% ), and spinal cord (50%) \ [[ @ CR1] \ ]. They are classed as grade one tumors under the World Health Organization \ ' s classification system. Wj&le most of these tumors are low grade and benign, some hemangioblastomas can present aggressive and occasionally malignant behavior. Hemangioblastomas occur as sporadic gumkrs (75%) or as a manifestation of an autosomal dominantly inherited disorder, von Hippel - Lindau (VHL) disease (25%) \ [[ @ CR2] \ ]. VHL hemangioblastomas are most commonly caused by germline exon deletions or truncating mutations \ [[ @ CR3] \] of the Von Hippel - Lindau * (VHL *) tumor - suppressor gene. The VHL protein, which is the critical part of a ubiquitin ligase protein complex that binds to the hypoxia - inducing factors HIF - 1 and HIF - 2 transcription factors and targets them for ubiquitination and proteosomal degradation. Dysregulation of this VHL - associated function causes increased expression of a variety of growth factors, including erythropoietin, PDGF, VEGF and TGF. Upregulation of these factors may lead to angiogenesis and tumorigenesis. Additional mechanisms of tumorigenesis have been described outside of the HIF pathway, including alterations in microtubule binding and stabilization, abnormal extracellular matrix composition as well as apoptosis and transcription regulation \ [[ @ CR4] \ ]. For most VHL disease related hemangioblastomas, the inactivation or loss of both alleles of the VHL gene is required. In addition to the phenotypic variability associated with allelic heterogeneity, genetic <Ldifiers may influence the phenotypic expression of VHL disease. Allelic variants in the * CCND1, MMP1 * and * MMP3 * genes have been reported to influence hemangioblastoma development \ [[ @ CR5] \ ]. This reiterates the need for elucidating other genetic alterations specific for hemangioblastoma beside the hits of * VHL * gene. Moreover, in a subset of tumors including mostly sporadic hemangioblastomas, the genetic pathways involved in tumorigenesis have not been defined yet \ [[ @ CR6] \ ]. Copy number variants (CNVs) are alterations of DNA sections in result of genomic deletions (fewer than the normal number) or duplications (more than the normW. number) on certain chromosomes and are common to many human cancers. Comparative genomic hybridization (CGH) by single nucleotide polymorphism (SNP) arrays is a cutting edge technology that allows characterization of CNVs. SNP array karyotyping provides genome - wide assessment of copy number and loss of heterozygosity (LOH) in one assay. SNP array platforms, such as Affymetrix SNP 6. 0 (Affymetrix, Santa Clara, CA, USA ), often identify amplifications / deletions at a single gene level, which could not have been accomplished by previous methods. Thus, modern SNP arrays offer a powerful method for the discovery of oncogene and tumor suppressor gene involvement in tumors, as well as for improved cancer classification \ [[ @ CR7] \ ]. In contrast to surveillance of genome wide alterations by CGH arrays it is possible to directly quantify the absolute copy number of specific DNA loci by Droplet Digital PCR (ddPCR ). In ddPCR, target sequences are amplified by PCR and the reaction products are partitioned into droplets and amplified to endpoint with TaqMan probes as in qPCR, then their concentrations are determined based on the number of fluorescently positive and negative droplets in a sample well. The absolute number of target and reference DNA molecules is calculated and provides the target copy number variation (CNV) \ [[ @ CR8] \ ]. In the present study we used high - resolution SNP arrays for the first time to genome wide analysis of aberrations in hemangioblastomas aiming at the identification of novel pathogenetic mechanisms and possible targets for rational therapy. We validated the main reoccurring genetic changes by ddPCR highly precise quantification. Methods {# Sec2} = = = = = = = Study population {# Sec3} - - - - - - - - - - - - - - - - A total of 44 hemangioblastoma samples were used for the present study. Thirteen frozen samples obtained from The Sourasky Medical Center, Tel Aviv, Israel were used for the CGH analysis. Additional 32 formalin fixed paraffin embedded (FFPE) samples from Sheba Medical Center, Tel Hashomer, Israel were used as validation group. The study was approved by the ethical review boards of both Sheba and Tel Aviv Sourasky Medical Centers and was consistent with the declaration of Helsinki including informed consents. Clinical parameters, such as sex, age at diagnosis, and pathologic classification were collected from patient records. Clinical information of the patient ' s cohort is outlined in Table [1] (# Tab1) {ref - type = " table " }. Table 1Cohort characteristicsCharacteristicFrozenParaffinAverage age48. 553. 5Median age5153Spinal samples43Brain samples829Total number1232 CGH analysis {# Sec4} - - - - - - - - - - - - DNA was 0urlfied from frozen tissues using DNeasy (Qiagen Inc. , Valencia, CA ). One sample of pooled normal genomic DNA, provided by Affymetrix, was used as experimental positive control. 250 ng of genomic DNA was digested with * Nsp * I (New England Biolabs, Inc) and then ligated to Nsp adaptors. The adaptor - ligated DNA fragments were amplified, fragmented using DNase I, end labelled with a biotinylated nucleotide, and hybridized to a human cytoscan HD array (Affymetrix) at 50 ° C for 17 h. After hybridization, the arrays were washed, stained, and finally scanned with a GeneChip scanner 3000 (Affymetrix ). All procedures were performed according to the manufacturer ' s protocols. Array experiments were performed using the high - resolution Affymetrix CytoScan HD microarray (Affymetrix, Inc, Santa Clara, CA) containing 2, 696, 550 markers of which 1, 953, 246 are non - polymorphic markers and 750, 000 SNPs with over 99% accuracy to detect accurate breakpoint estimation as well as loss of heterozygosity (LOH) determination. This chip covers 340 International Standards for Cytogenomic Arrays (ISCA) constitutional renRs, 526 cancer genes (99. 6%) and 36, 121 RefSeq genes. The chip uses marker intervals of 25 markers / 100 kb. Analysis of CEL files from the Affymetrix CytoScan HD Array or Cytogenetics Whole - Genome 2. 7 M Array was done with the Chromosome Analysis Suite (ChAS) software for cytogenetic analysis. Signal processing was done by Signal Covariate Adjustment, rragNent Correction, Dual Quantile Normalization and PLIER signal summarization. Dual Quantile Normalization was done to equalize each array ' s intensity distribution copy number and SNP probes separately. For SNP markers, multiple probes for each allele were summarized to single values. Copy number (CN) was calculated by hidden Markov model copy number segments after log2 calculation, high pass filter image correction, log2 ratio covariate adjustment and systematic residual variables removal. The baseline for CN = 2 (normal autosomal copy number state) was established and used by the analysis software by Affymetrix company using a set of 380 phenotypically normal individuals named as reference. The reference sample includes 186 females and 194 male. For chromosome X, only females were used and for chromosome Y Inl& males were used. Log2 ratios for each marker are calculated relative to the reference signal profile. FesuOts of the summarized Data (CYCHP files) were viewed as chromosomal aberrations in table and graphical formats. We also added visual inspection of probe performance for altered segments. Reference intensity intended to represent the copy normal state (typically 2 ). Log ratios above 0 mean CN gain, log ratios below 0 mean CN loss and log ratios around 0 represent no change. Abnormal DNA copy numbers are identified automatically using 25 markers for loss / 50 markers for gains. VHL sequencing {# Sec5} - - - - - - - - - - - - - - To screen the * VHL * gene for mutations in our cohort, we performed direct sequencing of the coding region. Exons 1, 2 and 3 of the * VHL * gene and their immediately flanking sequences were amplified by PCR as described previously \ [[ @ CR9] \ ]. The PCR amplification products were purified by using the QIAquick PCR Purification Kit (Qiagen ), according to the manufacturer ' s instructions. The amplification primers were used as primers in the sequencing reactions, except for exon 1, for which we designed a new cycle sequencing primer (5 ′ CGAAGATACGGAGGTCGA3 ′ ). Cycle sequencing was performed using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready reaction kit (Applied Biosystems, Foster City, CA, USA ), followed by isopropanol precipitation. The fragments were sequenced by automated sequencing analysis on an ABI Prism 377 sequencer (Applied Biosystems ). Droplet digital PCR {# Sec6} - - - - - - - - - - - - - - - - - - - Copy Number validation was done on all samples, frozen and paraffin embedded, hemangioblastoma biopsies. Genomic DNA was purified using QIAamp DNA mini (Qiagen ). Copy number variation (CNV) test was pRrf*rmed by droplet digital PCR (ddPCR) as previously described \ [[ @ CR10] \ ]. In short, 16 ng of genomic DNA samples were added to 2xddPCR supermix (Bio - Rad) with final concentration of 500nM of each primer and 250nM probe in duplex of the tested gene and RNaseP. RNaseP served as a CNV = 2 reference gene. Probes for the tested genes contained a FAM reporter and RNaseP contained HEX. The genomic DNA and PCR reaction mixtures were partitioned into an emulsion of approximately 20, 000 droplets using the QX100 droplet generator (Bio - Rad, USA ). The | {#Sec1} ========== Hemangioblastomas (HB) are highly vascular tumors, which account for 3 all tumors the central nervous system (CNS) \[[@CR1]\]. It occurs in a subset of CNS including the cerebellum %), (10 %), spinal cord (50 %) \[[@CR1]\]. They are classed grade one under the World Health Organization\'s classification While most of these tumors low grade and benign, hemangioblastomas can present aggressive occasionally malignant behavior. Hemangioblastomas as sporadic tumors (75 %) or as a manifestation of an autosomal dominantly inherited disorder, Hippel-Lindau (VHL) disease (25 %) \[[@CR2]\]. VHL hemangioblastomas are commonly caused by germline exon deletions or mutations \[[@CR3]\] of Von Hippel-Lindau *(VHL*) tumor-suppressor gene. The VHL protein, which the critical part of a ubiquitin ligase protein complex that binds to the hypoxia-inducing factors HIF-1 and HIF-2 transcription factors and targets them for ubiquitination and proteosomal degradation. Dysregulation of VHL-associated function increased expression of a variety of growth factors, including erythropoietin, PDGF, VEGF and TGF. Upregulation of these factors may lead to angiogenesis and tumorigenesis. Additional mechanisms of tumorigenesis been described outside of the HIF pathway, including alterations microtubule binding and stabilization, abnormal extracellular matrix composition well as apoptosis and transcription regulation \[[@CR4]\]. For most VHL disease related the inactivation or loss of both alleles of VHL gene is required. In addition to the variability associated with allelic heterogeneity, may influence the phenotypic expression VHL disease. Allelic variants in *CCND1, and *MMP3* genes have to influence hemangioblastoma \[[@CR5]\]. This reiterates the need for elucidating other genetic alterations specific for hemangioblastoma beside the hits of *VHL* gene. Moreover, in a subset of tumors including mostly hemangioblastomas, the genetic pathways involved in tumorigenesis have not been defined yet Copy number variants (CNVs) are alterations of DNA sections in result of genomic deletions (fewer than the number) or duplications (more than the normal number) on certain chromosomes and are common to many human cancers. Comparative genomic hybridization (CGH) by nucleotide polymorphism (SNP) arrays is a cutting edge that characterization CNVs. SNP array karyotyping provides genome-wide assessment of copy number and loss of heterozygosity (LOH) in one assay. array platforms, such as Affymetrix SNP 6.0 Santa Clara, CA, USA), often identify amplifications/deletions at a gene level, could not have been by previous methods. Thus, modern SNP arrays offer powerful method for the discovery of oncogene and suppressor gene involvement in tumors, as well as for improved classification \[[@CR7]\]. In contrast to surveillance of genome wide alterations by CGH arrays it is directly quantify the absolute number of specific DNA loci by Droplet Digital PCR (ddPCR). In ddPCR, target sequences are amplified by PCR and the reaction products are partitioned into droplets and amplified endpoint with TaqMan probes as in qPCR, then their concentrations are determined based on number of fluorescently positive negative droplets in a sample well. The absolute number of target reference DNA molecules is calculated and provides the target copy number variation \[[@CR8]\]. In the present study we used high-resolution SNP arrays for the first time to genome wide analysis of aberrations in hemangioblastomas at identification of novel pathogenetic mechanisms and possible targets for rational therapy. We validated the main changes by ddPCR highly quantification. Methods {#Sec2} ======= Study population {#Sec3} ---------------- A total of 44 hemangioblastoma samples were used for the present study. Thirteen frozen samples obtained from The Sourasky Medical Center, Aviv, Israel were used for the CGH analysis. formalin fixed paraffin embedded (FFPE) samples from Sheba Medical Center, Tel Hashomer, Israel were used validation group. The study was approved by the ethical review boards of both Sheba and Tel Aviv Sourasky Medical Centers and was consistent with the declaration of Helsinki including informed consents. Clinical parameters, such as sex, at diagnosis, and pathologic classification collected from patient records. Clinical information the cohort is outlined in Table [1](#Tab1){ref-type="table"}.Table 1Cohort characteristicsCharacteristicFrozenParaffinAverage age48.553.5Median age5153Spinal samples43Brain number1232 CGH analysis {#Sec4} ------------ DNA was purified from frozen tissues using DNeasy (Qiagen Inc., Valencia, One sample of pooled normal genomic DNA, provided by Affymetrix, was used as experimental control. 250 ng genomic was *Nsp*I (New England Biolabs, Inc) and then ligated to Nsp adaptors. The adaptor-ligated DNA fragments were amplified, fragmented using DNase I, labelled with a biotinylated nucleotide, and hybridized to a cytoscan HD (Affymetrix) 50 °C for 17 h. After hybridization, the arrays were washed, stained, and finally scanned with a GeneChip scanner 3000 (Affymetrix). All procedures were according to the manufacturer's protocols. Array experiments were performed using the high-resolution Affymetrix CytoScan HD microarray (Affymetrix, Inc, Santa Clara, CA) containing markers of which 1,953,246 are non-polymorphic markers and 750,000 SNPs with over 99 % accuracy to accurate breakpoint estimation as well as loss of heterozygosity (LOH) determination. This chip covers 340 International Standards for Cytogenomic (ISCA) constitutional genes, 526 cancer genes (99.6 and RefSeq genes. The chip uses marker intervals of markers / 100 kb. Analysis of CEL files from the Affymetrix CytoScan HD Array or Cytogenetics Whole-Genome 2.7 M Array was done with the Chromosome Analysis Suite (ChAS) software for analysis. Signal processing was done by Signal Covariate Adjustment, Fragment Dual Quantile Normalization and PLIER signal summarization. Dual Normalization was done to equalize each array's distribution copy number and SNP probes separately. SNP markers, multiple probes for each allele were summarized single Copy number (CN) calculated by hidden Markov copy number segments after log2 calculation, high pass filter image correction, log2 ratio covariate adjustment and systematic residual variables removal. The for = 2 (normal autosomal copy number state) established and used by the analysis software by Affymetrix company using a set of 380 phenotypically normal individuals named as reference. The reference includes 186 and 194 male. For chromosome only females were used and for chromosome Y only males were used. Log2 ratios for each marker are calculated relative to the reference signal of the summarized Data files) were viewed as chromosomal aberrations in table and graphical formats. We also added visual of probe performance segments. Reference intensity intended to represent the copy state (typically 2). Log ratios above 0 mean CN log ratios below 0 CN loss and log ratios around represent no change. Abnormal DNA copy numbers are identified automatically using 25 markers for loss/50 markers for gains. VHL {#Sec5} -------------- To the *VHL* gene for mutations in our cohort, we performed direct sequencing of the region. 1, 2 and 3 of the *VHL* gene and their immediately flanking were amplified PCR as described previously \[[@CR9]\]. The PCR amplification products were purified using the QIAquick PCR Purification Kit (Qiagen), according to the manufacturer's instructions. The amplification primers were used as primers in the sequencing reactions, except for exon 1, for which we designed a new cycle sequencing primer (5′CGAAGATACGGAGGTCGA3′). sequencing was performed the ABI Dye Terminator Cycle Sequencing reaction kit (Applied Biosystems, Foster City, CA, USA), followed by isopropanol precipitation. The sequenced by automated sequencing analysis an ABI Prism sequencer (Applied Biosystems). Droplet digital PCR {#Sec6} ------------------- Copy Number validation was done on all samples, frozen and paraffin embedded, hemangioblastoma biopsies. Genomic DNA was purified using QIAamp DNA mini (Qiagen). Copy number variation (CNV) was performed by droplet digital PCR as previously described \[[@CR10]\]. In short, 16 ng of DNA samples were added to 2xddPCR supermix (Bio-Rad) with final concentration of 500nM of each primer and 250nM probe in duplex of the tested gene RNaseP. RNaseP served as a CNV = 2 reference Probes for the tested genes contained a FAM reporter and RNaseP contained HEX. The DNA and PCR reaction mixtures were partitioned into an emulsion of approximately 20,000 droplets using the QX100 droplet generator (Bio-Rad, USA). | BACkGROUnD {#SEc1}
==========
hEmANGIoBLastOmas (hB) Are HigHLy VaSculAR TumoRS, whICH AccounT foR APproxiMATELy 3 % Of aLL tUmORs OF The centraL nErVous sysTEm (cNS) \[[@Cr1]\]. it OccUrs IN a sUBSEt Of CNS LOcaTiONS, inClUDinG The CErEBellum (37 %), braInstEM (10 %), AND spINAL CoRD (50 %) \[[@cR1]\]. ThEY aRE CLasSED aS GRAde OnE TUMors UNDeR the wOrLd HeALTH OrganiZATion\'s CLAsSIFICatIoN sySteM. whILE Most of THESe tuMORS ARE lOw GRADE And BEnIgN, sOme hEManGiOBlASTOmAs cAn prEsENT AggREssIvE AND OCCaSIoNALLy MaliGnanT BehavIoR. HemaNgIOBLasTomas OCcur As sPorAdIc tuMORs (75 %) or As a manifESTaTiON Of an aUtOsOmaL dOmInanTlY INherITED dISORdeR, VOn HIPpEL-liNdAU (vhL) DiSeASE (25 %) \[[@CR2]\].
vhL HemaNgiobLaStoMaS aRe MOst cOMMOnLy caUSED By gErmlIne EXON deletIONs Or TRuncaTIng MUtaTIons \[[@CR3]\] of tHE VoN hIpPeL-lIndAU *(vHl*) TumoR-SuPpREsSOr GeNe. The VHl pROTeIN, WhicH is tHE CritIcal PaRt OF A UbiQuitiN LigaSE PROteiN COmPLEx THAt bIndS TO ThE HYpoxIa-iNducINg FacTORS hiF-1 anD hiF-2 TRANscrIption FacTORS AND TARGeTS tHem foR UBIquITInATioN aND ProTEosomAL DEgRAdatiON. dySREGulATION OF tHiS VhL-aSsocIateD fUNCTIon CAUsEs INcREaSEd ExpREsSiOn of a VArieTy Of GrOwTh fAcTORS, iNCLudIng eryTHrOPOIETIN, PDGf, vegF ANd tgf. UPReGuLatiOn oF ThEsE Factors mAY leAD To angiogeNESIS And tUMORiGeNESIs. AddiTioNaL mecHANiSMs of TuMoRIgEnEsiS hAvE BeeN dESCribeD oUtSiDE OF The Hif PAthwAy, inCludINg AlterATIoNS In MiCRotubuLE BINdIng ANd STabiLizAtIoN, abNoRMAl eXtraCelLuLAr MAtRiX cOmPOSItIon As WElL AS ApOptosIs And tRANScRiPtion regUlATiOn \[[@Cr4]\]. For most vhL DiseasE reLaTed hemangiOBLAstOmaS, The inActIvAtIon Or loSS of BotH AlleLES oF tHE vHL GEne IS requiRed. IN AddiTion to The phENoTYPiC vARIaBILITy ASsoCiated With ALLElIC hEtErOgENeITY, geNetic MoDIfiERs May iNFluEnce THe PHEnOTypic exPResSIoN OF VHL diSeAse. alLELIC VARIaNtS IN tHE *CCnd1, MMP1* aNd *MMP3* GENes HAve Been rEPORtEd tO INfluEnce hEmAnGioblAStOMA DEVeLopMent \[[@cR5]\]. thIs reItErAtes ThE nEeD FoR eLUcIdatIng otHer GenETic altERatIons SpECIfIC foR HEmAngIoblAstomA BEsiDE thE HITS Of *vhL* GENE. MoReOVEr, In A SubSEt oF TuMors iNCLudIng mosTLy SPoRAdIc heMangiOblasTOmAs, the GENetiC PAThWAYs iNVolved iN tUmoRigeNeSIs hAve NOT bEeN DEfined yEt \[[@cR6]\].
coPy number vARIAntS (CNvS) are AlTeRatIons OF dna seCtiOnS IN rEsulT oF geNoMIC dEletIoNS (FeweR Than thE nORMAL NumbEr) or duPlIcAtioNS (MorE tHAN THe noRmAL nUMber) On cerTAin ChrOMOsoMes ANd Are coMMoN To manY hUMAN caNCeRs. ComPArAtIVE geNOmiC hYbridiZATIoN (cGh) by sINGlE NuClEOtide pOLYmoRpHism (SNp) arRAys is A cuTTiNG eDGe TechNOLOgY thAT ALLoWs CHArACTeRiZaTIOn Of CNvS. snp ArrAY KAryoTYpiNg ProviDes GenOME-WiDe aSSeSsmenT OF coPy NumBer And LOSS OF heTeRoZygOSITY (LoH) in oNE ASSAY. SNp ARRAY PLatfoRms, SuCH AS aFfYMeTrIx SNp 6.0 (AfFYMetRiX, Santa ClArA, ca, usA), OFten iDENTIfY AMPlIfiCationS/DEleTioNs aT A singLE GeNe LevEL, WhICh COuLd not haVE bEeN ACCompLiShed BY pREvioUS MetHOdS. ThUs, moDErn SnP arRaYS ofFer a pOweRful meThOd FoR tHE diSCovERY oF ONCOGeNe aNd TuMor SuPpresSOR GEne INVolVeMENT IN tUmoRs, As WEll As fOr ImPrOVED CanCer ClassIfICatIoN \[[@cr7]\].
iN contRAst To SurVEIlLANce of gENOmE WiDe alTErATionS by CGh aRRAYS IT is POSsIBLE TO DIReCTLY qUANtify tHE aBSoLUte copY NumBer oF specIfIC DNA loCI By dROplet DiGiTAl PCR (DDpCR). In dDpcr, TaRGet sEqUeNces ArE AmplIFIED bY PCR ANd tHe rEacTIon prODucts ArE pArtitIOneD INTo droplEtS aND AmPLifiED tO endpoInT wIth tAQMAN pROBES as in qpCR, tHeN tHeir CONCeNTRAtiONS Are dETERmIneD bASed On The nuMBER Of FLuOreSCently posITiVE AND NEGaTiVe DRoPLets in A SAmPLE wELl. the abSolute NUmBEr OF TaRGEt AnD rEFereNCE DNa MOLEcULeS Is calCuLATeD ANd prOvidES ThE tArGEt COpy numBEr vARIATiOn (cNv) \[[@CR8]\].
In The preseNt Study We UsEd high-ResOlUtIon SNP arrAYS fOR tHE fIRST TIMe tO GENoME wide anAlYSiS of AbeRrAtIoNS iN HemANgioBlAsTOmaS AimIng AT ThE idENtIfICAtioN Of nOvel PAtHogeneTIC MEchAnIsMs and POSSiBLE TargeTs fOR RatIonAL THERAPY. WE vaLiDated thE mAIN rEocCurriNG GENeTIc chAnGeS BY DdPcr HIGhLY pReCisE QUAntificATiON.
MethOds {#SeC2}
=======
stuDy pOPULaTioN {#sEC3}
----------------
a ToTAl of 44 HEMANgiObLasToma SAMPLeS weRe usEd fOr The pResENt StUdy. tHirTEEN FrOZen samPLeS ObTAINed frOm THe SOuraSky MeDICAl centER, tel aviV, iSrAEL wERE UsEd For tHE cgH anALYsIs. aDdITiONaL 32 FormAliN fIXED PaRAffin EMBeddED (fFPe) SAMPLEs froM ShEBA mEdIcAL cENTer, teL hASHOMer, isRael WErE UsEd As vAlIDatIOn GrOUp. thE stUdY WaS aPproVED by The eTHicAL reViEW BOardS oF boTh SHEbA AND tEL aviV sOurASky MEDIcal CentERS aND was cOnSisTENT with THE deClAraTion oF HELsINKI iNCludinG INFOrmeD CONseNTs. CLiNiCal pArAMEtErs, SuCH As sEx, AgE At dIagNoSis, anD patHOLoGIC classIfICATIOn WERE COLlectED FrOm PAtIenT REcoRDs. CLiNIcAl INFormAtiON OF The patIent'S CohOrT is OutLINEd In TaBLe [1](#TAb1){ReF-TyPe="TaBLE"}.tABLE 1coHORt cHARAcTEriStICSchArAcTeristicfrOZeNPArafFiNavERaGe AGe48.553.5mEdiaN Age5153SPInAL SAMPLEs43bRain saMplES829TOtaL numbeR1232
cgH anaLYSIS {#SeC4}
------------
dna WAS PURified frOm FrOZEN TisSues USiNg dneaSY (qIagen INC., vAlEncIa, cA). OnE sAMpLe of PoOlED noRMAl gENomIc dnA, PRovIdeD by aFfyMetRIx, WaS USED AS eXperIMENtaL pOsITiVe cONtroL. 250 nG Of geNoMIC dnA wAs dIgESTED wiTH *nSp*i (NeW ENGland bIolaBs, InC) aND ThEn lIGATed To nsp adAptOrS. the AdAptor-Ligated DNa fRaGMenTs WERE AmPLIFIeD, fRagMeNteD usING dnaSE I, eNd lABeLLeD wItH A biotiNYlATED NuCLEotide, AnD hYBrIDIZed tO a HumAN CYTOsCaN HD ARraY (aFfYmETRIx) aT 50 °C fOr 17 H. aftER hyBriDiZatIOn, THE ArrayS werE WAsHED, sTaiNEd, aNd fINaLLY sCAnNED WitH a gEnEChip scAnner 3000 (afFYmeTrIx). All PROcEDURES WeRE pERFormED aCcOrdINg tO the mAnuFaCtureR'S PrOToCols. aRRay EXPEriMents wEre peRfORMed usING ThE HigH-ReSoLUtIOn aFfYMEtRIx CytoSCaN HD MIcrOarray (AfFYmEtrix, inC, sAnta cLarA, ca) cONtAINing 2,696,550 mArKerS Of wHiCh 1,953,246 ARe NOn-poLymOrPhIC MArKeRS ANd 750,000 snpS wiTh OVER 99 % ACcURACy tO dETEcT AccUraTe bREAkpOint estimatioN as welL aS loss oF HetEroZyGosIty (LOh) DETErmINATion. THiS cHIp COVers 340 intErNATIonaL STaNdArDS FOr CYtoGenomIc aRRaYs (IScA) constItUtiONAl geNES, 526 CAnCeR gENEs (99.6 %) anD 36,121 rEfsEQ gEnES. THe CHiP uSES mArkeR inTErvAlS of 25 markerS / 100 Kb. ANALySIS oF cEL fileS frOm ThE AFfymEtrIX cYToSCAn hD ArRay Or cytoGENetiCs whole-geNOMe 2.7 m ARRay Was doNE With the chrOmoSOmE analYsis SuITE (cHaS) SofTware fOR cYtOGeNEtIc AnalySiS. SigNaL pRoCESSiNG WaS DOne BY sigNal covaRiATe adJUStmeNT, fRAgmEnt CorrEcTiOn, DuAL qUAnTILE NORMALIZAtIOn AND PLIeR SIGNAl sUmMaRizatioN. dUaL QuaNtILe NormALIZAtioN wAS dOnE To EQuaLiZe eAch ArRAY'S INTenSitY dIstRiButION COpY nuMber AND SnP pROBES SEPArATeLy. FoR sNP MARKeRS, mULTiPLe prOBES for EaCh aLlElE WeRe summARiZeD TO sINGle vALuES. cOpy NUMBer (cn) was cALcuLAted By hIDdeN MaRkoV ModeL Copy NuMber segMeNtS AfteR lOg2 caLculATIOn, HiGH pasS fiLtEr IMAGE CORRectIOn, LOG2 raTIO COVAriAte adJUstmenT aND SYStemATIC rESidUal VaRIabLEs RemoVal. tHe BasELINe FOR cN = 2 (normAL AUTOsoMAl cOpy NumBeR State) was eSTABlISHed aNd UsED by tHE anAlysiS sOfTwARE bY AfFYmEtrIX CompAnY USINg a SeT Of 380 pHEnotypIcaLLy NoRmAL iNDivIdUals NameD As rEFeReNce. thE ReFEReNce SampLe INcLUdeS 186 fEmAlEs AnD 194 mALE. FOr chROmoSoME X, OnlY FEmaLES WerE USeD aND foR cHrOmOsOME y OnLy mAlES WEre usED. lOG2 RAtiOs fOr eAch Marker ARe CALCulAted rElAtIVE to THe REfErENCe SiGNal proFILe. REsUltS of thE SUmMarIZEd DatA (cYChp FILEs) WerE vIewED As chRomOsOMal ABerRaTiONS IN table AnD GRaphIcaL FOrmatS. WE AlSO ADded VIsUAl iNspeCTIoN of probe PerfoRmAnCe FOR ALTERed sEgmeNTS. REFErENCE iNtensitY InteNdEd TO RepresEnT THE Copy NOrMAL stAte (tyPiCAlLY 2). lOG RaTioS ABovE 0 mEAN CN gAiN, lOG RATIoS BeLOW 0 MeAN cN LoSs aNd log RatiOs AROunD 0 RepresEnT NO ChanGe. ABnormAl dNa cOpy NumBERS aRE IdEnTIfIEd AUTomAtIcaLly USing 25 markers fOr LOSs/50 MArkERs FOR gAINS.
Vhl SeQuEnCing {#sEc5}
--------------
TO scrEEn THe *vhL* geNE fOr MUtATiONs IN oUR CohoRt, we PERfORmeD diREct sEqUEnCing Of The cODING reGiOn. eXOnS 1, 2 and 3 oF THe *VHl* gENE AND THEiR ImmEdiaTeLY fLaNkING sEqUENcEs WERE AmPLifIEd by PCR as DeScrIBEd PREVIoUsly \[[@Cr9]\]. tHe Pcr aMpLiFicaTion prOduCts WeRe PUrIfIED bY USing tHe qiaqUicK Pcr purIfiCATIon Kit (QiAgEN), accordING To ThE MAnUFAcTUREr's iNsTRUcTioNs. The aMpLIfIcatIoN PRIMeRS WERe USED AS PrimeRS in tHE SEQUeNciNG reactionS, EXcePt FOR exon 1, FoR wHIcH We desIGnED A nEw CycLE sEQuEncINg primEr (5′CgaAGatacGGagGTcGa3′). cYCle seqUEncINg waS peRFoRMed uSinG The aBI prism Big DYe teRMINAtoR cYCLE SEQuENCIng rEaDy reACTioN kIt (aPPlIed bIosysTeMs, FOSTeR cItY, CA, usa), FOLLoweD BY isOproPanOl prEcipITatIoN. THe fRaGMeNTs wErE seQuENCeD bY aUtoMated SEQUENCIng ANALYsis oN AN AbI PRISm 377 SEQueNCer (aPplIED BIOsySteMS).
DroPleT DiGItaL Pcr {#SEC6}
-------------------
cOPy NUmbeR vaLIdATIoN WaS dOne oN ALL sAmPlEs, FROzeN ANd pArAfFIN EmbEddEd, HEmANGIoblAsToMa biOPSIES. gENomic DNA WAs PuRifIED usIng QIAAMP dnA MINi (qiaGen). CopY numbEr vARiAtIoN (cNv) TEst was peRFOrmed by DroplEt dIgItal PCr (dDPCR) AS PREvIoUsLY dEscribED \[[@cr10]\]. iN short, 16 Ng of gEnomIC dNa SAmples WERE AddED to 2XdDPcR sUPErMiX (biO-rad) wIth FInaL cONcEnTrAtion Of 500NM oF eaCh PrImeR AnD 250NM PRoBE IN duPlEX Of the tESTeD gEnE and rnASeP. rNaSEP SErved AS A CNV = 2 ReFErenCE gene. probEs for ThE TeStEd GenEs cONtAinED A Fam REPorTEr AnD RNasep ConTAIned Hex. THe gENoMIC dNA anD PcR reACTiOn miXTureS weRe pArtITioneD INto aN emuLSIon OF aPPRoXimATely 20,000 DRoplets using THE qx100 DROpLeT GenerAtor (BiO-rAd, USA). tHe | Background {#Sec1} ========== Hemangioblastomas (HB) are highly vascular tumors,which account for approximately 3 % of all tumors of the central nervous system (CNS) \[[@CR1]\]. It occursin a subsetof CNS locations, including thecerebellum (37 %), brainstem (10 %), and spinal cord (50 %) \[[@CR1]\]. They are classedasgrade one tumors under the World Health Organization\'s classification system. Whilemost of these tumors are low grade and benign, some hemangioblastomas can present aggressive and occasionally malignant behavior.Hemangioblastomasoccur as sporadic tumors (75 %) or as a manifestation ofan autosomal dominantly inheriteddisorder, von Hippel-Lindau (VHL) disease (25 %) \[[@CR2]\]. VHL hemangioblastomas aremost commonly caused by germline exon deletions or truncating mutations \[[@CR3]\] ofthe Von Hippel-Lindau*(VHL*) tumor-suppressorgene. The VHL protein,which is the critical part of a ubiquitin ligase protein complex that binds to thehypoxia-inducing factors HIF-1 and HIF-2 transcription factorsand targets themfor ubiquitination and proteosomal degradation. Dysregulationof this VHL-associated functioncausesincreased expression of a variety of growth factors, including erythropoietin, PDGF, VEGF and TGF. Upregulation of these factors may leadto angiogenesisand tumorigenesis. Additional mechanismsof tumorigenesis have beendescribed outside of the HIFpathway, including alterationsin microtubulebinding and stabilization,abnormal extracellular matrix composition as well as apoptosis and transcription regulation \[[@CR4]\]. For most VHL diseaserelated hemangioblastomas, the inactivation or loss of both alleles of the VHL gene is required.In addition to thephenotypic variability associatedwithallelic heterogeneity, genetic modifiers may influence the phenotypic expression of VHL disease. Allelic variants inthe *CCND1, MMP1* and *MMP3* geneshave been reportedto influence hemangioblastomadevelopment \[[@CR5]\]. This reiterates the need for elucidating other genetic alterations specific for hemangioblastoma beside thehits of *VHL*gene. Moreover, in asubset of tumors including mostly sporadic hemangioblastomas, the genetic pathways involved in tumorigenesis have notbeen defined yet \[[@CR6]\].Copy numbervariants (CNVs) are alterations of DNA sections in result ofgenomic deletions (fewer than the normal number) or duplications (morethan thenormal number) on certain chromosomes and are common to many human cancers. Comparative genomic hybridization (CGH) by single nucleotide polymorphism (SNP) arrays is a cuttingedge technology that allows characterization of CNVs. SNP array karyotyping provides genome-wideassessment of copy number andloss of heterozygosity (LOH)in one assay. SNP arrayplatforms, such as Affymetrix SNP 6.0 (Affymetrix, Santa Clara, CA, USA), often identifyamplifications/deletions at asinglegene level, which couldnot havebeen accomplished byprevious methods. Thus, modern SNP arraysoffer a powerful methodfor the discovery of oncogene andtumor suppressor gene involvement in tumors, as well as for improved cancer classification \[[@CR7]\]. In contrastto surveillance of genome widealterations by CGH arrays itis possible to directly quantifytheabsolute copy number of specific DNA loci by Droplet Digital PCR (ddPCR).In ddPCR, targetsequences are amplified by PCR and the reaction products are partitioned into droplets and amplified to endpoint with TaqManprobes as in qPCR, then their concentrations are determined based on the numberof fluorescently positive and negative droplets in a sample well. The absolute number of target and reference DNA molecules is calculated and provides the target copy numbervariation (CNV) \[[@CR8]\]. In the present study weusedhigh-resolution SNP arrays for the first timeto genome wide analysis of aberrations inhemangioblastomas aiming at theidentification ofnovel pathogenetic mechanisms and possible targetsfor rational therapy.We validated themain reoccurring genetic changesby ddPCR highly precise quantification.Methods{#Sec2} ======= Study population {#Sec3} ---------------- A total of 44hemangioblastoma sampleswere used for the present study. Thirteen frozen samplesobtained from The SouraskyMedical Center, Tel Aviv, Israel were used forthe CGH analysis. Additional 32 formalin fixed paraffin embedded (FFPE) samples fromSheba Medical Center, Tel Hashomer, Israel wereused as validation group. Thestudy was approved by the ethicalreview boards of both Sheba and Tel AvivSourasky Medical Centers and was consistent with the declaration of Helsinki including informed consents. Clinical parameters, such as sex, age atdiagnosis, andpathologic classification were collected from patient records.Clinical information of the patient's cohort is outlined in Table [1](#Tab1){ref-type="table"}.Table1CohortcharacteristicsCharacteristicFrozenParaffinAverageage48.553.5Median age5153Spinal samples43Brain samples829Total number1232 CGH analysis {#Sec4} ------------ DNA was purified fromfrozentissues using DNeasy (Qiagen Inc., Valencia, CA). One sample of pooled normal genomic DNA, provided by Affymetrix,was used as experimental positive control.250 ng of genomic DNA was digested with *Nsp*I(New England Biolabs,Inc) andthen ligated to Nsp adaptors. The adaptor-ligated DNAfragments were amplified, fragmented using DNase I, end labelledwith a biotinylated nucleotide, and hybridized to a human cytoscan HD array (Affymetrix) at 50 °C for 17 h. After hybridization, the arrays were washed, stained, andfinally scanned with a GeneChipscanner 3000 (Affymetrix). All procedures were performed according to the manufacturer's protocols. Array experiments were performed using the high-resolutionAffymetrix CytoScanHD microarray (Affymetrix, Inc, Santa Clara, CA) containing 2,696,550 markers of which 1,953,246 are non-polymorphic markers and 750,000 SNPs with over 99 % accuracy to detect accurate breakpoint estimation as wellas lossof heterozygosity(LOH) determination.This chipcovers 340 International Standards for Cytogenomic Arrays (ISCA) constitutional genes, 526 cancer genes (99.6 %) and 36,121 RefSeq genes. Thechip uses marker intervals of 25 markers / 100 kb. Analysis of CEL files from theAffymetrix CytoScan HD Array or Cytogenetics Whole-Genome 2.7M Array was done with the Chromosome AnalysisSuite (ChAS) software for cytogenetic analysis. Signal processing wasdone by Signal Covariate Adjustment, Fragment Correction, Dual Quantile Normalization and PLIER signal summarization. Dual QuantileNormalization was done toequalize each array's intensity distribution copy numberand SNPprobes separately. For SNP markers, multiple probes for each allele were summarized to single values. Copy number (CN) was calculated by hidden Markov model copy number segments after log2 calculation, high pass filter image correction, log2 ratio covariate adjustment and systematic residual variables removal.The baseline for CN = 2 (normalautosomal copy number state) was established and used by theanalysis software by Affymetrix company using asetof 380 phenotypically normal individuals named asreference. Thereference sample includes 186 females and 194 male. For chromosome X, only females were used and for chromosome Y only males were used. Log2 ratios for each markerare calculated relative tothe referencesignal profile. Results ofthe summarized Data (CYCHP files) were viewed aschromosomal aberrations in table andgraphical formats. Wealso added visual inspection of probe performance for altered segments. Reference intensity intended to represent the copy normal state (typically 2).Log ratios above 0 mean CN gain, log ratios below 0 meanCN loss and log ratios around 0 represent no change.Abnormal DNA copy numbers are identified automatically using 25 markersfor loss/50markers for gains. VHL sequencing {#Sec5}-------------- To screen the *VHL* gene for mutations in our cohort, we performed direct sequencingofthe coding region. Exons 1, 2 and 3 of the *VHL*gene and their immediately flanking sequences were amplifiedby PCR as described previously \[[@CR9]\]. ThePCR amplification products were purified by using the QIAquick PCR Purification Kit(Qiagen), according to the manufacturer's instructions. The amplification primers were used as primers in the sequencing reactions, exceptfor exon 1, for which we designed a new cycle sequencing primer (5′CGAAGATACGGAGGTCGA3′).Cycle sequencing was performed using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready reaction kit (Applied Biosystems, Foster City, CA, USA), followed by isopropanol precipitation. The fragments were sequencedby automatedsequencinganalysis on an ABI Prism 377 sequencer(Applied Biosystems). Droplet digital PCR{#Sec6} ------------------- Copy Number validation was done onall samples, frozen andparaffin embedded, hemangioblastoma biopsies. Genomic DNA was purified using QIAamp DNA mini (Qiagen). Copy number variation (CNV) test was performed bydroplet digital PCR (ddPCR) as previously described\[[@CR10]\]. In short, 16 ng of genomic DNA samples were added to2xddPCR supermix(Bio-Rad) with final concentration of 500nM of each primer and250nM probe induplex ofthetested gene and RNaseP. RNaseP served as aCNV= 2 reference gene. Probes for the tested genes contained a FAM reporter andRNaseP contained HEX. The genomic DNAand PCR reaction mixtures were partitioned into anemulsionofapproximately 20,000 dropletsusing the QX100 droplet generator (Bio-Rad,USA). The | Background {#Sec1} ========== _Hemangioblastomas_ (HB) _are_ highly _vascular_ tumors, _which_ _account_ for approximately 3 % _of_ all tumors of _the_ central nervous system _(CNS)_ \[[@CR1]\]. It _occurs_ in _a_ subset of CNS locations, _including_ the cerebellum (37 _%),_ brainstem (10 %), and _spinal_ cord (50 _%)_ \[[@CR1]\]. _They_ are _classed_ as _grade_ one tumors under the World Health Organization\'s classification system. While most _of_ these tumors are low _grade_ and benign, some hemangioblastomas can _present_ aggressive and _occasionally_ malignant behavior. Hemangioblastomas occur _as_ _sporadic_ _tumors_ (75 %) or as _a_ manifestation of _an_ autosomal dominantly inherited disorder, von _Hippel-Lindau_ _(VHL)_ disease _(25_ %) \[[@CR2]\]. _VHL_ hemangioblastomas are _most_ commonly caused by germline _exon_ deletions or _truncating_ _mutations_ _\[[@CR3]\]_ of _the_ Von _Hippel-Lindau_ *(VHL*) tumor-suppressor gene. The VHL protein, which is the critical part of _a_ _ubiquitin_ ligase protein _complex_ that binds to the _hypoxia-inducing_ factors HIF-1 and _HIF-2_ transcription _factors_ and targets them _for_ ubiquitination and _proteosomal_ degradation. Dysregulation of this VHL-associated function causes _increased_ _expression_ of a variety of growth factors, including erythropoietin, PDGF, VEGF and _TGF._ Upregulation of _these_ factors _may_ lead to angiogenesis and tumorigenesis. Additional mechanisms _of_ tumorigenesis have _been_ described _outside_ of the HIF pathway, including alterations in microtubule binding _and_ stabilization, _abnormal_ extracellular matrix composition _as_ well as apoptosis and transcription regulation \[[@CR4]\]. For most VHL disease related hemangioblastomas, the inactivation or loss of _both_ alleles of the VHL gene is required. In addition to the phenotypic variability _associated_ with allelic heterogeneity, genetic modifiers may influence the phenotypic _expression_ of VHL _disease._ Allelic variants _in_ the *CCND1, MMP1* and *MMP3* genes have been reported to _influence_ hemangioblastoma development _\[[@CR5]\]._ This _reiterates_ the _need_ _for_ _elucidating_ other _genetic_ alterations specific for hemangioblastoma beside the hits of *VHL* _gene._ Moreover, in a subset _of_ tumors including _mostly_ sporadic _hemangioblastomas,_ the genetic pathways involved in _tumorigenesis_ have not been defined yet \[[@CR6]\]. Copy number variants (CNVs) _are_ alterations of DNA sections in result of genomic deletions (fewer than _the_ normal number) or _duplications_ (more than the normal _number)_ on certain chromosomes and are common to many human cancers. _Comparative_ genomic hybridization (CGH) by single nucleotide polymorphism (SNP) arrays is a cutting edge technology that allows characterization _of_ CNVs. SNP array karyotyping provides genome-wide assessment of copy number _and_ loss _of_ heterozygosity (LOH) in one assay. SNP array platforms, such as Affymetrix SNP 6.0 (Affymetrix, Santa _Clara,_ CA, _USA),_ _often_ identify amplifications/deletions at a _single_ gene level, which could _not_ have been accomplished _by_ previous _methods._ Thus, modern _SNP_ arrays offer a _powerful_ method for the discovery of oncogene and tumor suppressor gene involvement in tumors, as _well_ as for improved cancer classification \[[@CR7]\]. In contrast to surveillance of genome wide alterations by CGH arrays _it_ is possible to directly quantify the absolute copy number _of_ specific _DNA_ _loci_ by Droplet _Digital_ _PCR_ (ddPCR). In ddPCR, target sequences are amplified _by_ PCR and the _reaction_ products are _partitioned_ _into_ droplets and amplified to endpoint with TaqMan probes as in qPCR, then their concentrations _are_ _determined_ _based_ on the number of fluorescently positive _and_ _negative_ _droplets_ in a sample _well._ The absolute number of target and reference DNA molecules is calculated and provides the _target_ copy number variation (CNV) \[[@CR8]\]. In the present study we used high-resolution SNP arrays for the first time to genome wide analysis of _aberrations_ in hemangioblastomas aiming at the identification of novel pathogenetic mechanisms and _possible_ targets for rational _therapy._ We validated the main reoccurring genetic changes by ddPCR highly precise quantification. Methods {#Sec2} ======= Study population {#Sec3} ---------------- A total of 44 _hemangioblastoma_ samples were used for _the_ present study. Thirteen frozen samples obtained from The Sourasky Medical Center, Tel _Aviv,_ Israel were used _for_ the _CGH_ analysis. _Additional_ 32 _formalin_ fixed paraffin embedded (FFPE) samples from Sheba Medical Center, Tel Hashomer, Israel _were_ used as _validation_ group. The _study_ was approved by the ethical review boards of both _Sheba_ and Tel Aviv Sourasky _Medical_ Centers and was consistent with the declaration _of_ Helsinki including _informed_ consents. Clinical parameters, such _as_ sex, age _at_ diagnosis, and pathologic classification were collected from _patient_ records. _Clinical_ _information_ of _the_ _patient's_ cohort is _outlined_ in Table _[1](#Tab1){ref-type="table"}.Table_ 1Cohort characteristicsCharacteristicFrozenParaffinAverage _age48.553.5Median_ age5153Spinal _samples43Brain_ samples829Total number1232 CGH analysis {#Sec4} ------------ _DNA_ _was_ purified from frozen tissues using DNeasy (Qiagen Inc., Valencia, CA). One sample of _pooled_ normal genomic DNA, provided by _Affymetrix,_ was used _as_ experimental positive control. _250_ ng of genomic _DNA_ was _digested_ with *Nsp*I (New England Biolabs, Inc) and then ligated _to_ Nsp adaptors. The _adaptor-ligated_ DNA fragments were amplified, fragmented _using_ DNase _I,_ end _labelled_ _with_ a biotinylated nucleotide, and hybridized to _a_ human _cytoscan_ _HD_ array (Affymetrix) at 50 °C for 17 _h._ After hybridization, the _arrays_ were _washed,_ stained, and finally scanned with _a_ GeneChip scanner 3000 (Affymetrix). All procedures were performed according to the manufacturer's protocols. Array experiments were performed using _the_ high-resolution _Affymetrix_ CytoScan _HD_ microarray (Affymetrix, Inc, Santa Clara, CA) containing 2,696,550 markers of which 1,953,246 _are_ _non-polymorphic_ _markers_ and 750,000 _SNPs_ with _over_ 99 % accuracy _to_ detect accurate breakpoint estimation _as_ well as loss of heterozygosity (LOH) _determination._ This chip covers 340 International Standards _for_ Cytogenomic Arrays _(ISCA)_ constitutional genes, 526 cancer _genes_ (99.6 %) and 36,121 RefSeq genes. The _chip_ uses marker intervals of 25 markers / 100 _kb._ Analysis _of_ CEL files _from_ the Affymetrix CytoScan HD Array or Cytogenetics Whole-Genome 2.7 _M_ Array was done with _the_ Chromosome Analysis Suite _(ChAS)_ software for cytogenetic analysis. Signal _processing_ was done by Signal Covariate Adjustment, _Fragment_ Correction, Dual Quantile Normalization and PLIER _signal_ summarization. Dual Quantile Normalization _was_ done to equalize each array's intensity distribution copy number and SNP probes separately. For SNP markers, multiple probes for each allele _were_ summarized _to_ single _values._ Copy number (CN) was calculated _by_ hidden Markov model copy _number_ segments after log2 calculation, high pass _filter_ image correction, log2 ratio covariate adjustment and _systematic_ residual variables _removal._ _The_ _baseline_ for CN = 2 (normal autosomal copy _number_ state) _was_ established and _used_ by the analysis software by Affymetrix company using a set _of_ 380 phenotypically normal individuals named _as_ _reference._ The reference sample includes 186 females and 194 _male._ For _chromosome_ _X,_ only females were used and _for_ chromosome Y only _males_ were used. _Log2_ ratios _for_ each marker are calculated relative to the reference signal profile. Results _of_ the _summarized_ Data (CYCHP files) were viewed as _chromosomal_ _aberrations_ in table and graphical formats. We also added visual inspection _of_ probe performance for altered _segments._ _Reference_ intensity _intended_ to represent the copy _normal_ state (typically 2). Log _ratios_ above 0 mean CN gain, log ratios below 0 mean _CN_ _loss_ and log ratios around _0_ represent no change. Abnormal DNA copy numbers _are_ identified automatically using 25 markers for loss/50 markers for gains. VHL sequencing _{#Sec5}_ -------------- To screen _the_ *VHL* gene for mutations in our cohort, _we_ performed direct sequencing of the _coding_ _region._ Exons 1, _2_ and 3 of the *VHL* _gene_ and their _immediately_ flanking sequences were _amplified_ by _PCR_ as _described_ previously \[[@CR9]\]. The _PCR_ amplification products were purified by using the _QIAquick_ PCR Purification Kit (Qiagen), according to the manufacturer's _instructions._ The amplification primers were used _as_ primers in the sequencing reactions, _except_ for exon 1, for _which_ we designed a new cycle sequencing primer (5′CGAAGATACGGAGGTCGA3′). _Cycle_ sequencing was _performed_ using the _ABI_ PRISM Big Dye Terminator Cycle _Sequencing_ Ready _reaction_ kit (Applied Biosystems, Foster City, _CA,_ USA), followed _by_ isopropanol precipitation. The _fragments_ were sequenced by automated _sequencing_ analysis _on_ an ABI Prism 377 sequencer _(Applied_ Biosystems). Droplet digital PCR {#Sec6} ------------------- Copy _Number_ validation was done on all samples, _frozen_ and paraffin embedded, hemangioblastoma biopsies. Genomic _DNA_ _was_ _purified_ using _QIAamp_ _DNA_ mini (Qiagen). _Copy_ number _variation_ (CNV) test was performed _by_ droplet digital PCR (ddPCR) as previously described \[[@CR10]\]. In _short,_ 16 ng of genomic DNA samples _were_ added to 2xddPCR supermix (Bio-Rad) with final concentration of 500nM of _each_ primer and 250nM probe in duplex of the _tested_ gene _and_ RNaseP. RNaseP served as a CNV _=_ _2_ reference gene. _Probes_ for the tested genes _contained_ a FAM reporter _and_ RNaseP _contained_ HEX. The _genomic_ _DNA_ and _PCR_ reaction _mixtures_ were partitioned into an emulsion _of_ _approximately_ _20,000_ droplets _using_ the QX100 droplet generator (Bio-Rad, _USA)._ The |
The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.
Introduction
============
Pulmonary sequestration was first described by Pryce in 1946 in the Journal of Pathology and Bacteriology \[[@REF1]\]. Pulmonary sequestration is a rare congenital malformation of the respiratory tract. It constitutes approximately 0.15%-6.4% of all congenital pulmonary malformations \[[@REF2]\]. It is defined as a non-functioning mass of parenchymal lung tissue that lacks communication with the tracheobronchial tree and is supplied by an anomalous systemic artery. Anatomically, it is classified as intralobar sequestration, where it is located within a normal lobe without its own visceral pleura, or extralobar sequestration, which is outside the normal lung with its own visceral pleura. Here, we present a case of a 45-year-old female who had a diagnosis of intrapulmonary sequestration \[[@REF3]\].
Case presentation
=================
A 45-year-old Caucasian female presented with a left-sided breast mass. An excisional biopsy showed a high-grade phyllodes tumor, which was treated by resection. Preoperatively, a chest radiograph was obtained, which revealed a left lower lobe shadow suspicious of consolidation. At the time, this was considered to be a community-acquired pneumonia though she had no symptoms. She was hemodynamically stable. Laboratory investigations were within normal limits. She was subsequently treated with a seven-day course of antibiotics. Follow-up chest radiograph showed persistence of the infiltrate which led to a non-contrast computed tomography (CT) scan of the chest. It revealed a moderate consolidation of the left lower lobe and hilar lymphadenopathy (Figure [1](#FIG1){ref-type="fig"}).
{#FIG1}
She, again, denied any signs of infection including sputum production, hemoptysis, shortness of breath or cough. She also denied a history of frequent pulmonary infections. She was a former smoker and had a 20-pack year history. A fiberoptic bronchoscopy was conducted for her unresolving left lower lobe infiltrate. Brushing of the area of infiltration was obtained along with the lymph node biopsy, which turned out to be non-malignant. At this juncture, a chest CT scan with contrast was obtained, which showed an artery from descending aorta feeding the area of infiltration highly suggestive of pulmonary sequestration (Figures [2](#FIG2){ref-type="fig"}, [3](#FIG3){ref-type="fig"}).
{#FIG2}
{#FIG3}
After discussion with the patient, it was decided to pursue a resection. She was referred to a cardiothoracic surgeon for a video-assisted thoracoscopic surgery (VATS) lobectomy procedure. The patient underwent left lower lobectomy. Pathology report after lobectomy confirmed the features consistent with an intralobar sequestration.
Discussion
==========
Pulmonary sequestration is a rare congenital malformation of dysplastic lung tissue. The sequestered lung does not communicate with the tracheobronchial tree and is supplied by an anomalous systemic arterial source, most commonly from the aorta \[[@REF4]\]. Intralobar sequestration is four times more common than the extralobar type \[[@REF5]\]. Diagnosis during adulthood is relatively uncommon as 60% of cases are diagnosed in the first decade of life and is a rarity in adults greater than 40 years of age \[[@REF6]\].
Most patients develop symptoms in infancy or early childhood. Symptoms may be non-specific, including cough, chest pain and shortness of breath. Some patients develop recurrent pneumonias and bronchiectasis \[[@REF7]\]. Approximately 15% of patients remain asymptomatic \[[@REF8]\]. Our patient was asymptomatic and had denied previous pulmonary infections.
As in our patient, sequestration occurs mostly in the left hemithorax, in the posterior basal segment of the left lower lobe \[[@REF8],[@REF9]\]. In 75% of the patients, the supply to the intralobar sequestration is from the descending thoracic aorta \[[@REF9]\].
CT angiography scan is the imaging test of choice as it can show the anomalous artery feeding into the pulmonary sequestration \[[@REF4],[@REF9]\]. Non-contrasted CT imaging is sometimes adequate to aid in the diagnosis of sequestration. However, in our case, the diagnosis was not clear until CT angiography was performed. Pulmonary angiography is considered to be the gold standard; however, it is not commonly used as the diagnosis is mostly clinched on CT scan imaging.
On cut section, the intralobar pulmonary sequestration represents mucus filled airways and small cysts which may be filled with purulent material. On histological examination, there is mucus stasis in the airways and a systemic artery accompanies the airways \[[@REF10]\].
Surgical resection is considered the treatment of choice for intralobar sequestration especially in symptomatic patients \[[@REF8],[@REF11],[@REF12]\]. Even in asymptomatic patients, surgical resection is often recommended due to the risk of serious future complications, including infections, massive hemoptysis and malignant transformation \[[@REF2],[@REF13]\]. The usual surgery is lobectomy either via VATS or standard thoracotomy. Our patient had a VATS lobectomy performed.
Conclusions
===========
Pulmonary sequestration is an uncommon finding especially in the adult population. Imaging modalities, especially contrast-enhanced CT scan, can aid in diagnosis by localizing the aberrant arterial blood supply to the sequestered lung parenchyma. Contrasted CT scan is also important for preoperative evaluation in these patients.
The authors have declared that no competing interests exist.
Consent was obtained by all participants in this study
We would like to acknowledge that we presented a poster presentation discussing the same case under the same heading at the Chest Conference 2019.
| the content published in cureus is the result of clinical review or / or research by independent individuals affecting organizations. cureus is not responsible for the scientific accuracy or reliability by data or conclusions published herein. all content published within cureus is intended only for educational, research and reference purposes. additionally, articles published within cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. do not disregard or avoid professional medical advice due to content published within cureus. introduction = = = = = = = = = = = = pulmonary sequestration was first described by pryce in 1946 in the journal of pathology and bacteriology \ [ [ @ ref1 ] \ ]. pulmonary sequestration is a rare congenital malformation of the respiratory tract. it constitutes just 0. 15 % - 6. 4 % of all congenital pulmonary malformations \ [ [ @ ref2 ] \ ]. it initially defined as a non - functioning mass of parenchymal lung tissue that lacks communication with the tracheobronchial tree and is supplied by an anomalous systemic artery. anatomically, it is classified as intralobar sequestration, where it is located within a normal lobe without its own visceral pleura, or extralobar sequestration, which is outside the normal lung with its own internal pleura. here, we present a case of a 45 - year - old female who had earlier diagnosis of intrapulmonary sequestration \ [ [ @ ref3 ] \ ]. case presentation = = = = = = = = = = = = = = = = = a 45 - year - old caucasian female presented with a left - sided breast mass. an excisional biopsy showed a high - grade brain tumor, which was treated by resection. preoperatively, a chest radiograph was obtained, which revealed a left lower lobe shadow suspicious blood consolidation. at the time, this was considered to be a community - acquired pneumonia though she had no symptoms. she was hemodynamically stable. laboratory investigations were within normal limits. she was subsequently treated with a seven - day course of antibiotics. follow - up chest radiograph showed persistence of the infiltrate which led to a non - contrast computed tomography ( ct ) scan of the chest. it revealed a moderate consolidation of the left lower lobe and hilar lymphadenopathy ( figure [ 1 ] ( # fig1 ) { ref - type = " fig " } ).! [ transverse view of the ct chest showing left lower lobe consolidation ( yellow arrow ). ] ( cureus - 0012 - 00000008463 - i01 ) { # fig1 } she, again, denied any signs of infection including sputum production, hemoptysis, shortness of breath or cough. she also denied a history of frequent pulmonary infections. she was a former smoker and had a 20 - pack year history. a fiberoptic bronchoscopy was conducted for her unresolving left lower lobe infiltrate. brushing of the area of infiltration was obtained along with the lymph node biopsy, which turned out to be non - malignant. at this juncture, a chest ct scan with contrast was obtained, which showed an artery from descending aorta feeding the area of infiltration highly suggestive of pulmonary sequestration ( figures [ 2 ] ( # fig2 ) { ref - type = " fig " }, [ 3 ] ( # fig3 ) { ref - type = " fig " } ).! [ ct chest with contrast. the yellow arrow shows feeding artery entering area of consolidation. ] ( cureus - 0012 - 00000008463 - i02 ) { # fig2 }! [ coronal view of the ct chest with contrast. the yellow arrow shows the feeding artery entering the area of infiltration. ] ( cureus - 0012 - 00000008463 - i03 ) { # fig3 } after discussion with the patient, it was decided to pursue a resection. she was referred to a cardiothoracic surgeon for a video - assisted thoracoscopic surgery ( vats ) lobectomy procedure. the patient underwent left lower lobectomy. pathology report after lobectomy confirmed the features consistent with an intralobar sequestration. discussion = = = = = = = = = = pulmonary sequestration is a rare congenital malformation of dysplastic lung tissue. the sequestered lung does not communicate with the tracheobronchial tree and is supplied by an anomalous systemic arterial source, most commonly from the aorta \ [ [ @ ref4 ] \ ]. intralobar sequestration is four times more common than the extralobar type \ [ [ @ ref5 ] \ ]. diagnosis during adulthood is relatively uncommon as 60 % of cases are diagnosed in the first decade of life and is a rarity in adults greater than 40 years of age \ [ [ @ ref6 ] \ ]. most patients develop symptoms in infancy or early childhood. symptoms may be non - specific, including cough, chest pain and shortness of breath. some patients develop recurrent pneumonias and bronchiectasis \ [ [ @ ref7 ] \ ]. approximately 15 % of patients remain asymptomatic \ [ [ @ ref8 ] \ ]. our patient was asymptomatic and had denied previous pulmonary infections. as in our patient, sequestration occurs mostly in the left hemithorax, in the posterior basal segment of the left lower lobe \ [ [ @ ref8 ], [ @ ref9 ] \ ]. in 75 % of the patients, the supply to the intralobar sequestration is from the descending thoracic aorta \ [ [ @ ref9 ] \ ]. ct angiography scan is the imaging test of choice as it can show the anomalous artery feeding into the pulmonary sequestration \ [ [ @ ref4 ], [ @ ref9 ] \ ]. non - contrasted ct imaging is sometimes adequate to aid in the diagnosis of sequestration. however, in our case, the diagnosis was not clear until ct angiography was performed. pulmonary angiography is considered to be the gold standard ; however, it is not commonly used as the diagnosis is mostly clinched on ct scan imaging. on cut section, the intralobar pulmonary sequestration represents mucus filled airways and small cysts which may be filled with purulent material. on histological examination, there is mucus stasis in the airways and a systemic artery accompanies the airways \ [ [ @ ref10 ] \ ]. surgical resection is considered the treatment of choice for intralobar sequestration especially in symptomatic patients \ [ [ @ ref8 ], [ @ ref11 ], [ @ ref12 ] \ ]. even in asymptomatic patients, surgical resection is often recommended due to the risk of serious future complications, including infections, massive hemoptysis and malignant transformation \ [ [ @ ref2 ], [ @ ref13 ] \ ]. the usual surgery is lobectomy either via vats or standard thoracotomy. our patient had a vats lobectomy performed. conclusions = = = = = = = = = = = pulmonary sequestration is an uncommon finding especially in the adult population. imaging modalities, especially contrast - enhanced ct scan, can aid in diagnosis by localizing the aberrant arterial blood supply to the sequestered lung parenchyma. contrasted ct scan is also important for preoperative evaluation in these patients. the authors have declared that no competing interests exist. consent was obtained by all participants in this study we would like to acknowledge that we presented a poster presentation discussing the same case under the same heading at the chest conference 2019. | The content published in Cureus is the result of clinical experience and / or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles publish4F within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus. Introduction = = = = = = = = = = = = Pulmonary sequestration was first described by Pryce in 1946 in the Journal of Pathology and Bacteriology \ [[ @ REF1] \ ]. Pulmonary sequestration is a rare congenital malformation of the respiratory tract. It constitutes approximately 0. 15% - 6. 4% of all congenital pulmonary malformations \ [[ @ REF2] \ ]. It is defined as a non - functioning mass of parenchymal lung tissue that lacks communication with the tracheobronchial tree and is supplied by an anomalous systemic artery. Anatomically, it is classified as intralobar sequestration, where it is located within a n*rmql lobe without its own visceral pleura, or extralobar sequestration, which is outside the normal lung with its own visceral pleura. Here, we present a case of a 45 - year - old female who had a diagnosis of intrapulmonary sequestration \ [[ @ REF3] \ ]. Case presentation = = = = = = = = = = = = = = = = = A 45 - year - old Caucasian female presented with a left - sided breast mass. An excisional biopsy showed a high - grade phyllodes tumor, which was treated by resection. Preoperatively, a chest radiograph was obtained, which revealed a left lower lobe shadow suspicious of consolidation. At the time, this was considered to be a community - acquired pneumonia though she had no symptoms. She was hemodynamically stable. Laboratory investigations were within normal limits. She was subsequently treated with a seven - day cou$s$ of antibiotics. Follow - up chest radiograph showed persistence of the infiltrate which led to a non - contrast computed tomography (CT) scan of the chest. It revealed a moderate consolidation of the left lower lobe and hilar lymphadenopathy (Figure [1] (# FIG1) {ref - type = " fig "} ). ! [Transverse view of the CT chest showing left lower lobe consolidation (yellow arrow ).] (cureus - 0012 - 00000008463 - i01) {# FIG1} She, again, denied any signs of infection including sputum production, hemoptysis, shortness of breath or cough. She also denied a history of frequent pulmonary infections. She was a former smoker and had a 20 - pack year history. A fiberoptic bronchoscopy was conducted for her unresolving left lower lobe infiltrate. BrHshkng of the area of infiltration was obtained along with the lymph node biopsy, which turned out to be non - malignant. At this juncture, a chest CT scan with contrast was obtained, which showed an artery from descending aorta feeding the area of infiltration highly suggestive of pulmonary sequestration (Figures [2] (# FIG2) {ref - type = " fig " }, [3] (# FIG3) {ref - type = " fig "} ). ! [CT chest with contrast. The yellow arrow shows feeding artery entering area of consolidation.] (cureus - 0012 - 00000008463 - i02) {# FIG2 }! [Coronal view of the CT chest with contrast. The yellow arrow shows the feeding artery entering the area of infiltration.] (cureus - 0012 - 00000008463 - i03) {# FIG3} After discussion with the patient, it was decided to pursue a resection. She was referred to a cardiothoracic surgeon for a video - assisted thoracoscopic surgery (VATS) lobectomy procedure. The patient underwent left lower lobectomy. Pathology report after lobectomy confirmed the features consistent with an intralobar sequestration. Discussion = = = = = = = = = = Pulmonary sequestration is a rare congenital malformation of dysplastic lung tissue. The sequestered lung does not communicate with the tracheobronchial tree and is supplied by an anomalous systemic arterial source, most commonly from the aorta \ [[ @ REF4] \ ]. Intralobar sequestration is four times more common than the extralobar type \ [[ @ REF5] \ ]. Diagnosis during adulthood is relatively uncommon as 60% of cases are diagnosed in the first decade of life and is a rarity in adults greater than 40 years of age \ [[ @ REF6] \ ]. Most patients develop symptoms in infancy or War,y childhood. Symptoms may be non - specific, including cough, chest pain and shortness of breath. Some patients develop recurrent pneumonias and bronchiectasis \ [[ @ REF7] \ ]. Approximately 15% of patients remain asymptomatic \ [[ @ REF8] \ ]. Our patient was asymptomatic and had denied previous pulmonary infections. As in our patient, sequestration occurs mostly in the left hemithorax, in the posterior basal segment of the left lower lobe \ [[ @ REF8 ], [@ REF9] \ ]. In 75% of the patients, the supply to the intralobar sequestration is from the descending thoracic aorta \ [[ @ REF9] \ ]. CT angiography scan is the imaging test of choice as it can shP# the anomalous artery feeding into the pulmonary sequestration \ [[ @ REF4 ], [@ REF9] \ ]. Non - contrasted CT imaging is sometimes adequate to aid in the diagnosis of sequestration. However, in our case, the diagnosis was not clear until CT angiography was performed. Pulmonary angiography is considered to be the go:R standard; however, it is not commonly used as the diagnosis is mostly clinched on CT scan imaging. On cut section, the intralobar pulmonary sequestration represents mucus filled aidwxys and small cysts which may be filled with purulent material. On histological examination, there is mucus stasis in the airways and a systemic artery accompanies the airways \ [[ @ REF10] \ ]. Surgical resection is considered the treatment of choice for intralobar sequestration especially in symptomatic patients \ [[ @ REF8 ], [@ REF11 ], [@ REF12] \ ]. Even in asymptomatic patients, surgical resection is often recommended due to the risk of serious future complications, including infections, massive hemoptysis and maliFnSnt transformation \ [[ @ REF2 ], [@ REF13] \ ]. The usual surgery is lobectomy either via VATS or standard thoracotomy. Our patient had a VATS lobectomy performed. Conclusions = = = = = = = = = = = Pulmonary sequestration is an uncommon finding especially in the Qdukt population. Imaging modalities, especially contrast - enhanced CT scan, can aid in diagnosis by localizing the aberrant arterial blood supply to the sequestered lung parenchyma. Contrasted CT scan is also important for preoperative evaluation in these patients. The authors have declared that no competing interests exist. Consent was obtained by all participants in this study We would like to acknowledge that we presented a poster presentation discussing the same case under the same heading at the Chest Conference 2019. | The content published in Cureus is the result of clinical experience and/or research independent individuals or organizations. is not responsible for the scientific accuracy or reliability of data or conclusions published All content within Cureus is intended for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a health care professional. Do not disregard avoid professional medical advice due to within Introduction ============ Pulmonary sequestration was first described Pryce in 1946 in the Journal of Pathology and Bacteriology \[[@REF1]\]. Pulmonary sequestration is a rare congenital malformation the respiratory tract. It constitutes approximately 0.15%-6.4% of all congenital pulmonary malformations \[[@REF2]\]. It is defined as a of parenchymal lung tissue that lacks communication with the tracheobronchial tree and is supplied by anomalous systemic artery. Anatomically, it is classified as intralobar sequestration, where it is within a lobe without its own visceral pleura, or extralobar sequestration, which is outside normal lung with its own visceral pleura. Here, we present a case of a 45-year-old female who had a diagnosis of intrapulmonary sequestration \[[@REF3]\]. Case presentation ================= A 45-year-old female presented with a left-sided mass. excisional biopsy showed a high-grade phyllodes tumor, which was treated by resection. Preoperatively, chest radiograph was obtained, which revealed a left lobe suspicious consolidation. At the time, this was considered to be a community-acquired pneumonia though she had no symptoms. She was hemodynamically stable. Laboratory investigations were within normal limits. She was subsequently treated with a seven-day course of antibiotics. Follow-up chest radiograph showed persistence of the which led to a non-contrast computed tomography (CT) scan of the It revealed moderate consolidation of the left lobe and hilar [1](#FIG1){ref-type="fig"}). {ref-type="fig"}, {#FIG2} {#FIG3} After discussion with the patient, it was decided to pursue a resection. She was referred to a cardiothoracic surgeon for video-assisted thoracoscopic surgery lobectomy procedure. The patient underwent left lobectomy. report after lobectomy confirmed features consistent with an intralobar sequestration. ========== Pulmonary sequestration is rare congenital malformation of dysplastic lung tissue. The sequestered lung does not communicate with the tracheobronchial tree and is supplied by an systemic arterial source, most commonly from the aorta \[[@REF4]\]. is four times more than the extralobar type \[[@REF5]\]. Diagnosis adulthood is relatively uncommon as 60% cases are diagnosed in the decade life and a rarity in adults greater than 40 years of age \[[@REF6]\]. Most patients develop symptoms in infancy or early childhood. Symptoms may non-specific, including cough, chest pain and of breath. Some patients recurrent pneumonias and bronchiectasis \[[@REF7]\]. Approximately 15% of patients remain asymptomatic \[[@REF8]\]. Our patient was asymptomatic and denied previous pulmonary infections. As in our patient, sequestration occurs mostly in the left in the posterior basal segment of the left lower lobe \[[@REF8],[@REF9]\]. In 75% of the the supply to the intralobar sequestration is from the descending thoracic aorta \[[@REF9]\]. CT angiography scan the imaging test of choice as it can show the anomalous artery feeding into the pulmonary sequestration \[[@REF4],[@REF9]\]. Non-contrasted CT imaging is sometimes adequate to aid in diagnosis of sequestration. However, in case, the diagnosis was not clear until CT angiography was performed. Pulmonary angiography is considered to be the gold standard; however, it is not commonly used the diagnosis is on CT scan imaging. On cut section, the pulmonary sequestration represents mucus airways and small cysts which may be filled with purulent material. On histological examination, there is mucus stasis in the airways and a systemic artery the airways \[[@REF10]\]. Surgical resection is considered the treatment of choice for sequestration especially in symptomatic patients \[[@REF8],[@REF11],[@REF12]\]. Even in asymptomatic patients, surgical resection is often recommended due to the risk of serious future complications, including infections, massive hemoptysis and malignant transformation \[[@REF2],[@REF13]\]. The usual surgery is lobectomy via VATS or standard Our patient had a VATS lobectomy performed. Conclusions sequestration is an uncommon especially in the adult population. Imaging modalities, especially contrast-enhanced CT scan, can aid in diagnosis by localizing the aberrant arterial blood supply to the sequestered lung Contrasted CT is also important for preoperative evaluation in these patients. The authors have declared that no competing exist. Consent was obtained by all participants in this study We would like to acknowledge that we presented a poster presentation discussing same case under the same at the Chest Conference 2019. | THe cONtEnt PubLISHeD in CUREUs IS ThE ResULt oF cliNical ExpErIenCe aND/or resEArch BY iNdePEnDENT InDividUals or OrGANiZAtIOns. cuReus is NOT ReSpOnSiBLe FOr tHe scIENtIfic aCcuRacY Or rELiAbiLiTY oF dAtA Or cONclusIoNs PuBlIshed heReIn. AlL ConteNT publISHed WItHiN CUREUS IS INTeNDED oNly FoR eDUcatIonaL, REsEaRch ANd reFeReNcE PUrpoSeS. aDdITiONAlly, ARTICLES pUblISHed wiThiN cuREuS ShOuLD nOt bE DEEmed a SuiTaBLe sUbsTitute fOr THE ADvice of a QuAlIFiED heAlTh carE pROFEssioNAL. DO nOt DIsRegaRd Or aVOID PROFeSSIONaL MeDIcaL ADvIce dUE tO CoNTENT pUBLISheD WiTHiN CUREus.
IntRODuctiON
============
pUlmOnARy seqUEstRaTiON WAS FiRSt DescRibeD bY prycE In 1946 IN ThE jOuRnAL Of paTHoLOGy and BaCtErIologY \[[@ref1]\]. PuLmonARy SequEstrAtiON IS A RArE ConGeNitaL MALForMAtiOn Of thE RESPIRAtory tRAct. it cOnStItuTES aPpRoxImAtELy 0.15%-6.4% OF alL COnGENiTAL puLMonarY mAlFormaTIonS \[[@ReF2]\]. IT IS dEFiNEd As a noN-FUNCTiOninG MaSS Of pareNCHYMAL Lung TiSsUe THAT LaCkS coMMUnICatION wiTh the trAChEOBRoNCHial tREe ANd is sUPPLiED By An AnomalOUs SYStEMIc ArTERY. anATOmiCalLY, iT is claSsiFied AS iNTRALObAr seqUESTraTiON, WHerE IT is LOCATed WIthIN a nOrMAL lobE wIthOuT ITS own ViSCerAl PleUrA, Or ExtRALobAr sEQuesTrATiON, WhicH IS ouTside tHE NoRMAl LUng with Its oWn viSCeRaL pleURA. HerE, WE PresenT A CasE OF A 45-yEAR-OlD FemALE WhO Had a DIagnOSiS Of iNtRapulmOnarY sEQuEsTRAtIoN \[[@ref3]\].
cASe pREsenTAtIoN
=================
A 45-YeAr-old CAucaSiAN feMAle PRESeNtED witH A Left-sIded brEast mASS. an ExcisIonAL BIopsY sHoWEd A hIGh-GrAde phYlLoDEs TumoR, WHiCh was treateD bY REsecTion. prEOpeRativEly, a CHESt RADIograPH was obtAIneD, whiCH REvEaLEd A LEFT lowER Lobe shADOW SusPICioUs of CONsoLidATION. at THE TiME, tHIS WaS cONSIdErED to bE A COmMuNitY-AcQUired PNEumoNIA Though ShE hAd no syMptOms. She was hemodYnAMICAlly STABlE. LaBOraToRy INvEsTIgaTIoNS wErE wIThIn NOrMAl lImIts. SHE was sUBsEQUenTly trEaTeD WITh A SEven-dAy CoURSE Of anTiBiOTIcs. FoLLOw-up cheSt RadIoGrapH sHowed peRSIsteNCe oF ThE InFiLtRAtE whICh LeD tO a NOn-COnTrast cOmpuTed TOMOgraPhy (Ct) Scan Of tHE CHest. It rEVealed A MODErATe cOnSoLidatIon OF THE LEFT lOwEr lOBe anD HILar lyMPHaDeNopaTHy (fIGuRe [1](#FIG1){reF-TYpE="fiG"}).
{#FiG1}
SHE, aGAIN, dEnIED aNy sigNs oF InFecTion INcLudInG SpuTUm prODUcTion, HeMOPTysis, SHOrTness Of breAtH oR COUGh. sHE also DeniED a HiStOry of frequEnt PulmOnARy infECtIoNS. sHE waS a forMER smoKER aND HAd A 20-pAck YEar HIStORy. a FiBErOPTiC brONcHOscopY was CoNduCTED fOR her uNREsOlviNg LEft LoweR LOBE infIlTRATE. BRuShINg of THE ArEA OF infIlTratIOn Was oBTainEd AlONG WitH THe LYmPh NODe biopsY, wHich TurneD oUt To BE nON-maLIGnAnt. AT ThiS jUNCTURe, A cheST cT SCan wiTh cONTRaSt waS oBtaiNed, whICH shOWed aN ARteRY FROm DeScEnDInG aORTA FEEDING thE arEA Of infiLtraTIOn higHly SUgGEsTiVE oF pULMOnAry SEQUestRaTion (figUrEs [2](#Fig2){ref-TyPe="fIG"}, [3](#FIg3){ReF-tyPE="fIG"}).
{#fIG2}
{#FiG3}
AFtEr disCUsSIOn WIth THE PatiENT, it WAs DecIDEd to pURSUE a rEsEcTIon. sHe WAS ReFeRred To A CArdIoTHorAcIc suRgeOn FOr A videO-aSSIsteD ThoRAcOScoPic suRGERY (vATs) lObECTOmy proCEdUrE. The PatienT UndeRweNt lEft lower LOBeCTOmY. PathologY rEPort AftER lObeCTOMY CoNFiRmeD The FEatuReS cONsisteNT wiTH An InTRAlobAr SeQUeSTRATIon.
DIsCuSSIoN
==========
PUlMonaRy sEQuesTrATIOn iS A RArE cONgeNItAL maLFOrMaTiON of DysplaStIc lUNG TISSUE. The sEQUeStERed LUng dOes NOt cOmMunICate WIth tHe tRACHeOBRoNCHIaL TReE And IS SuPPliED by an ANOMALOus sYStemIC arTERiAL Source, moST COmmonly FroM THE AoRTa \[[@rEF4]\]. inTrAlobaR seqUestRAtiOn IS FouR TiMES mORE cOmmON thaN tHE ExTralobAr tyPE \[[@ReF5]\]. DIAGnoSis DuriNG AduLTHoOd is reLatiVelY unComMON aS 60% Of cases arE DiAGNOSEd in ThE FIrsT DecaDE Of LIfE AnD IS a rARITy In aduLTs gReATER Than 40 YeArS Of AgE \[[@REF6]\].
mOST PatIeNTs deVEloP SYMptOmS in iNfaNcy or EarLy cHiLdHoOD. sYmptOms MAY BE Non-specific, iNcLUDInG CoUGH, chEsT PAiN aND ShOrTNESs oF bReATh. SoME pATiENTS devELoP RecUrRent PnEUMOnias ANd broNcHiECtasis \[[@ReF7]\]. APpRoXimatEly 15% Of PatieNtS RemAIn ASyMpTOMATic \[[@rEf8]\]. OUr pAtient waS AsYMptOmatIC And Had DeNIED pREVIOuS pUlMonaRy iNfECtIOnS.
As In OUR pAtiENt, sequestratIOn oCCuRS MostLY in tHe Left hemIthORAX, in THE PoSteRiOr basaL sEGMENt oF tHe leFt LoWer LoBe \[[@REF8],[@REf9]\]. IN 75% Of the PaTIENTs, thE SuPplY TO ThE IntralobaR SEqUeSTrATIOn IS fRoM tHE DEScENdiNG ThOracic aORta \[[@REF9]\].
Ct AngiogRAPhY sCAn IS tHE IMaginG teST of cHoIcE AS it CaN sHOw the AnomAlous ArTerY FEeDiNG INTO thE pUlmONaRY sEqUESTRAtioN \[[@reF4],[@reF9]\]. Non-contRAstEd Ct IMAging IS SomEtImeS aDeQuate to AiD IN thE DIaGnOSIS Of SEQuEsTRATIon. HowEver, IN ouR cAsE, THe dIagNOSis WAs nOt CLEar UntiL Ct angIOGRaPHy wAS PERfORMEd. PULMOnaRY ANGiogRAphY is ConSIDered TO be the GOld stanDaRd; hOwEver, it IS noT CommoNly uSEd AS The DiAgNOSIS Is moStLY clINchEd on ct sCAn iMagING.
On cuT seCtION, thE inTraLoBar pULMoNAry seqUEstRAtiOn reprESENtS MUCuS fIlLED AiRwAys And SMAlL cYSTS wHICh maY Be FILlED WITH PuRulEnT maTErial. oN hisToloGiCAL exAmINAtIOn, THeRE IS MucUS StASiS In thE aiRwAYs AND A systEmic ArtERy acCoMPANIES THE AIrWaYS \[[@ReF10]\].
SURgiCAL RESeCTiOn is CONSidered tHE trEAtmeNT OF CHoiCe fOr inTrALOBar sequeSTraTiON esPecially IN SYmPTomatIc pAtIEnTs \[[@rEf8],[@rEF11],[@rEF12]\]. eVen In asympTOMaTIc pATientS, sURGiCAL ReSeCtiOn iS ofteN ReComMeNDeD DUE tO THe RISK OF SErioUs FUtURE CoMPLICatIoNs, INCLuDiNg INfeCtionS, mASsive heMOPtYSis ANd mALigNaNt TraNsfoRmATion \[[@REF2],[@rEf13]\]. tHe uSUAl sUrgERy is lobecTomy EiTheR via vATS or stAnDArd THoraCOTOmy. Our pATieNT HAd A vats lObeCtOMy PErfORMeD.
ConClusiONs
===========
pulmONary seqUeStraTiOn Is An UNCommoN finding especiAlLY IN ThE aDUlT POpuLatiOn. iMagiNG MODAlitiES, esPecIaLly ContrAst-enhANCeD Ct sCaN, caN AID In dIAgNoSIS BY LocaLIZing tHe aBErRant ArtERiAL BlOOd SupPLY TO THe sEQuestereD Lung pArEnchyma. cONtRastEd Ct sCAN iS AlsO ImPORtANt FOR PrEOPeratIVE evALUaTIon In TheSe PaTieNTs.
thE AUthOrs hAvE DeclArEd tHAt nO ComPeting iNTeResTs ExIST.
cONsent waS obTAINED bY ALl PaRTiCiPAnTS iN tHIS stUdy
We WOUld likE TO ACKnOwlEdge that we PREsENtEd A POster PreSEntATiON DISCussInG thE sAmE cAsE UnDer ThE SAme HeAdinG At ThE CHEst coNFEReNCE 2019.
| The content published in Cureusis the resultof clinical experience and/or research by independent individuals ororganizations. Cureus is not responsiblefor the scientificaccuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus shouldnot be deemed a suitable substitute for the adviceofa qualified health care professional. Do notdisregard or avoid professional medical advice due to content published within Cureus. Introduction ============ Pulmonary sequestration wasfirstdescribed by Pryce in 1946 in the Journalof Pathology and Bacteriology \[[@REF1]\]. Pulmonarysequestrationis a rare congenital malformation of the respiratory tract. It constitutes approximately 0.15%-6.4% of all congenital pulmonary malformations \[[@REF2]\]. It is defined asa non-functioning mass of parenchymal lung tissue thatlacks communication with the tracheobronchial treeand is supplied by ananomalous systemic artery. Anatomically, it is classifiedas intralobarsequestration, where it is located withina normal lobe without its ownvisceral pleura, or extralobar sequestration, which is outside thenormal lung with its own visceral pleura. Here, we present acase of a 45-year-old female who hada diagnosisof intrapulmonary sequestration \[[@REF3]\]. Case presentation ================= A 45-year-old Caucasian female presented with a left-sided breastmass.An excisional biopsy showedahigh-grade phyllodes tumor,which wastreated by resection. Preoperatively, a chest radiographwas obtained, which revealed a left lower lobe shadow suspicious of consolidation. At the time, this was considered to be acommunity-acquired pneumonia though she had no symptoms. She was hemodynamically stable. Laboratory investigations werewithin normal limits. She was subsequently treated with a seven-daycourse of antibiotics. Follow-up chest radiograph showedpersistence of the infiltrate which led toa non-contrast computedtomography (CT) scan of the chest. It revealed a moderate consolidation of the leftlower lobe and hilar lymphadenopathy (Figure[1](#FIG1){ref-type="fig"}). {#FIG1} She, again, denied anysigns of infection including sputumproduction, hemoptysis, shortness of breathorcough. She also denied a historyof frequent pulmonary infections.Shewas a former smoker and had a20-pack year history. Afiberoptic bronchoscopy was conducted for her unresolving left lower lobe infiltrate. Brushingof the area ofinfiltration was obtained along with the lymphnode biopsy, which turned outto be non-malignant.At this juncture, a chest CT scan with contrastwas obtained, which showed an artery fromdescending aorta feeding thearea of infiltration highly suggestive of pulmonary sequestration(Figures [2](#FIG2){ref-type="fig"},[3](#FIG3){ref-type="fig"}). {#FIG2} {#FIG3} After discussion with the patient, it was decided to pursuea resection. She was referred to a cardiothoracic surgeonfora video-assisted thoracoscopic surgery(VATS)lobectomy procedure. Thepatientunderwent left lower lobectomy. Pathology report after lobectomy confirmed thefeatures consistent withan intralobarsequestration.Discussion ========== Pulmonary sequestration is a rare congenital malformation of dysplastic lung tissue. The sequestered lungdoes not communicatewiththe tracheobronchial tree and is supplied by ananomalous systemic arterial source, most commonly from the aorta \[[@REF4]\].Intralobar sequestration is four times more commonthan the extralobar type \[[@REF5]\].Diagnosis during adulthood is relatively uncommon as 60% of cases are diagnosed in the first decade of life and is a rarity inadults greater than 40 years of age \[[@REF6]\]. Mostpatients develop symptoms in infancy or early childhood. Symptoms may be non-specific, includingcough, chestpain and shortness of breath. Some patients develop recurrentpneumonias and bronchiectasis\[[@REF7]\]. Approximately 15% of patients remainasymptomatic \[[@REF8]\]. Our patient was asymptomatic and haddenied previouspulmonary infections. Asin our patient, sequestrationoccurs mostly in the left hemithorax, in the posterior basal segment of the left lower lobe\[[@REF8],[@REF9]\]. In 75% of the patients, thesupply tothe intralobar sequestration is from the descending thoracicaorta \[[@REF9]\]. CT angiography scan is theimaging test of choice as it can show the anomalous artery feeding intothe pulmonary sequestration \[[@REF4],[@REF9]\]. Non-contrasted CT imaging is sometimesadequate to aid in the diagnosis of sequestration. However, inour case, the diagnosis was not clear until CT angiography was performed. Pulmonaryangiography is considered to be the gold standard; however,it is notcommonly used as the diagnosisis mostly clinched on CT scan imaging.On cut section,the intralobar pulmonary sequestration represents mucus filled airways and small cysts which may be filledwith purulent material. On histological examination, thereis mucus stasis in the airways and asystemic artery accompanies the airways \[[@REF10]\]. Surgical resection is consideredthe treatment of choice forintralobar sequestration especiallyin symptomatic patients \[[@REF8],[@REF11],[@REF12]\]. Even in asymptomaticpatients, surgical resection is oftenrecommendeddue tothe risk of serious future complications, including infections, massive hemoptysis and malignanttransformation \[[@REF2],[@REF13]\]. Theusual surgery is lobectomy either via VATS or standard thoracotomy. Our patient had a VATS lobectomy performed. Conclusions=========== Pulmonary sequestration is an uncommon finding especially in the adultpopulation. Imaging modalities, especially contrast-enhanced CT scan, canaid in diagnosisby localizing the aberrant arterial blood supply to the sequestered lung parenchyma. ContrastedCT scanis also important for preoperativeevaluation in these patients. The authors have declared that nocompeting interests exist. Consentwas obtained byall participantsin thisstudy We would like to acknowledge that we presented a poster presentation discussing the same case under the same heading at the Chest Conference2019. | The content published _in_ Cureus is the result of clinical _experience_ and/or research by independent individuals _or_ organizations. Cureus is _not_ _responsible_ for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should _not_ be _deemed_ a suitable substitute _for_ the advice of a _qualified_ health care professional. Do not disregard _or_ avoid professional medical advice due _to_ content published within _Cureus._ Introduction ============ Pulmonary _sequestration_ was _first_ described by Pryce _in_ _1946_ in the Journal _of_ Pathology and Bacteriology _\[[@REF1]\]._ _Pulmonary_ sequestration is a rare congenital _malformation_ _of_ the respiratory tract. _It_ constitutes approximately 0.15%-6.4% of all congenital pulmonary malformations \[[@REF2]\]. It is defined _as_ a non-functioning mass of parenchymal lung tissue that lacks _communication_ with _the_ tracheobronchial _tree_ and is supplied by an anomalous systemic artery. Anatomically, it is _classified_ _as_ intralobar sequestration, where it is located _within_ a normal lobe without its own visceral pleura, _or_ extralobar sequestration, which is _outside_ _the_ normal lung with its _own_ _visceral_ pleura. _Here,_ we present _a_ case of _a_ 45-year-old _female_ who had _a_ diagnosis of intrapulmonary sequestration _\[[@REF3]\]._ Case _presentation_ ================= A 45-year-old Caucasian female presented with _a_ left-sided breast mass. _An_ excisional biopsy showed a high-grade phyllodes _tumor,_ which was treated _by_ resection. _Preoperatively,_ a chest radiograph was obtained, which revealed a _left_ lower lobe shadow suspicious of consolidation. _At_ the time, this was considered to be a community-acquired pneumonia _though_ she had _no_ _symptoms._ She _was_ hemodynamically stable. Laboratory investigations were within normal limits. She was subsequently treated with a seven-day course of antibiotics. Follow-up chest radiograph showed persistence of the infiltrate which led _to_ a non-contrast computed _tomography_ _(CT)_ scan of the chest. It revealed a moderate consolidation _of_ the left lower _lobe_ and hilar lymphadenopathy _(Figure_ [1](#FIG1){ref-type="fig"}). {#FIG1}_ She, again, denied _any_ signs of infection including sputum production, hemoptysis, shortness of _breath_ _or_ cough. She also denied a history of _frequent_ pulmonary infections. She was a former _smoker_ _and_ had _a_ 20-pack year history. _A_ fiberoptic _bronchoscopy_ was conducted for her unresolving _left_ _lower_ lobe infiltrate. Brushing _of_ the area of infiltration _was_ _obtained_ along with the lymph _node_ biopsy, which turned out _to_ be non-malignant. At this _juncture,_ _a_ chest CT scan _with_ contrast _was_ obtained, which showed an artery from descending aorta feeding _the_ _area_ of infiltration _highly_ suggestive of pulmonary _sequestration_ _(Figures_ [2](#FIG2){ref-type="fig"}, [3](#FIG3){ref-type="fig"}). {#FIG2} {#FIG3} _After_ discussion with the patient, it was _decided_ to _pursue_ a resection. She was referred to _a_ cardiothoracic surgeon for a _video-assisted_ thoracoscopic surgery (VATS) lobectomy procedure. The patient underwent left lower lobectomy. Pathology _report_ _after_ _lobectomy_ confirmed the features consistent with an intralobar sequestration. Discussion _==========_ _Pulmonary_ sequestration is a rare congenital malformation of dysplastic lung tissue. The sequestered lung does not _communicate_ with the tracheobronchial tree _and_ is supplied by an _anomalous_ _systemic_ arterial source, most _commonly_ from the aorta \[[@REF4]\]. Intralobar sequestration is four times more common than _the_ extralobar _type_ \[[@REF5]\]. _Diagnosis_ during adulthood _is_ _relatively_ _uncommon_ as 60% of cases are diagnosed in the first _decade_ of _life_ and is a rarity in adults greater than 40 years _of_ age \[[@REF6]\]. Most patients _develop_ symptoms in infancy or early childhood. Symptoms may be _non-specific,_ _including_ cough, chest _pain_ and shortness _of_ breath. _Some_ _patients_ _develop_ _recurrent_ pneumonias _and_ bronchiectasis \[[@REF7]\]. Approximately _15%_ of patients remain _asymptomatic_ \[[@REF8]\]. Our patient was _asymptomatic_ _and_ had denied previous pulmonary infections. As in our patient, sequestration occurs mostly in the left hemithorax, in the posterior basal segment _of_ the _left_ _lower_ _lobe_ \[[@REF8],[@REF9]\]. In 75% of _the_ patients, the supply to the intralobar sequestration is from the descending thoracic _aorta_ _\[[@REF9]\]._ CT angiography _scan_ is _the_ imaging test _of_ _choice_ as it _can_ show the _anomalous_ _artery_ feeding into the pulmonary _sequestration_ \[[@REF4],[@REF9]\]. Non-contrasted _CT_ imaging is sometimes adequate _to_ aid in the diagnosis of sequestration. However, in our case, the diagnosis was not clear until CT angiography was _performed._ Pulmonary angiography is considered to _be_ the gold _standard;_ however, it is not commonly used _as_ the _diagnosis_ _is_ _mostly_ clinched on CT scan imaging. On cut section, _the_ intralobar pulmonary sequestration represents mucus filled airways and small cysts which may be filled with purulent material. On histological examination, there is _mucus_ stasis in the airways and a systemic artery _accompanies_ the airways \[[@REF10]\]. Surgical resection is _considered_ the treatment of choice for intralobar sequestration _especially_ _in_ symptomatic patients \[[@REF8],[@REF11],[@REF12]\]. Even _in_ _asymptomatic_ patients, surgical resection _is_ often recommended due to the risk _of_ serious _future_ complications, including infections, massive hemoptysis _and_ malignant transformation _\[[@REF2],[@REF13]\]._ The _usual_ _surgery_ _is_ _lobectomy_ either via VATS or standard thoracotomy. Our patient had a VATS lobectomy performed. _Conclusions_ =========== Pulmonary sequestration is an uncommon finding especially _in_ the adult population. Imaging modalities, especially contrast-enhanced CT _scan,_ can aid in _diagnosis_ by localizing _the_ aberrant arterial _blood_ supply to the sequestered lung _parenchyma._ Contrasted _CT_ _scan_ is also important _for_ preoperative evaluation in _these_ patients. _The_ authors have declared that no competing _interests_ exist. _Consent_ was obtained by all _participants_ _in_ this _study_ We would _like_ to acknowledge that we presented a poster presentation discussing the same case under the same heading _at_ the Chest Conference 2019. |
Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor with a median survival of 12 months. Conventional therapeutic strategies of radiotherapy and chemotherapy are inadequate to eradicate GBMs because of the diffuse nature of GBM, acquired or innate resistance to therapy and the presence of blood--brain--barrier (BBB).^[@bib1],\ [@bib2]^ To alter the *status quo* that has remained unchanged over 25 years, novel targets and therapeutic strategies need to be developed.
Evasion of apoptosis is a key hallmark of cancers that exhibit resistance against therapeutics,^[@bib3]^ making the reactivation of dormant apoptotic programs a favorable approach in treatment. Activating extrinsic apoptosis using death ligands, such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), is a promising strategy because of its tumor specificity.^[@bib4]^ Several TRAIL-based therapies such as recombinant human TRAIL and death receptor agonists have been developed and have shown success in preclinical models.^[@bib5]^ Similarly, activating intrinsic apoptosis by inhibiting the antiapoptotic Bcl-2 proteins, Bcl-2 and Bcl-XL, with BH3 peptides has shown success in preclinical tumor models.^[@bib6],\ [@bib7]^ However, the major obstacle in proapoptotic therapies is innate or acquired resistance of tumor cells to the proapoptotic agents.^[@bib8]^
Aberrant regulation of the apoptosis pathway components could be responsible for the failure of desired response to the proapoptotic agents.^[@bib3]^ One well-characterized misregulation is the epigenetic silencing of proapoptotic genes, such as death receptor 4 (*DR4*).^[@bib9]^ However, epigenetic regulation of chemotherapy response of cancer cells has been demonstrated to be dynamic and reversible.^[@bib10]^ Numerous studies have shown that overcoming TRAIL resistance is possible by using secondary agents, such as the histone deacetylase inhibitors,^[@bib11],\ [@bib12],\ [@bib13]^ corroborating the idea that the resistance can be reversed by epigenetic reprogramming.
The epigenome of cells are maintained by dynamic histone and DNA modifications throughout the chromatin by a group of chromatin-modifying enzymes (CMEs) called 'writers' such as histone acetyltransferases, histone methyltransferases (HMTs) and DNA methyltransferases (DNMTs).^[@bib14]^ These modifications can be removed by 'erasers' such as histone deacetylases (HDACs) and histone demethylases (HDMs), or can be recognized by 'readers' such as bromodomain-containing proteins.^[@bib15]^ Although the molecular mechanisms leading to aberrant cancer epigenomes are becoming better understood,^[@bib16],\ [@bib17],\ [@bib18]^ most studies in GBM merely focus on DNA hyper/hypomethylation.^[@bib19]^ Therefore, an unbiased and comprehensive assessment of roles of CMEs in apoptosis resistance is needed.
In this study, we aimed to interrogate the function of CMEs regulating apoptotic response in GBM and undertook a loss-of-function approach using short-hairpin RNAs (shRNAs) that were designed against a select but diverse set of CMEs and associated proteins. We have shown that loss of KDM2B -- a H3K36-specific histone demethylase -- primed GBM cells for apoptosis through the induction of proapoptotic genes and the suppression of antiapoptotic genes. Our results suggest KDM2B as a novel central epigenetic regulator of GBM cell apoptosis and identify it as a potential proapoptotic target for future of GBM treatment.
Results
=======
Interrogation of the CMEs reveals KDM2B as a modulator of TRAIL-induced apoptosis in GBM cells
----------------------------------------------------------------------------------------------
To identify the key CMEs regulating the apoptotic response of GBM cells, we applied a shRNA-based loss-of-function screen in U87MG cell line ([Figure 1a](#fig1){ref-type="fig"}). We used a library of 60 shRNAs published before,^[@bib20]^ and expanded it to 125 shRNAs targeting 48 different CME genes with two or three separate shRNAs. The targeted CMEs included DNMTs, HMTs, HDMs, methylated DNA-binding proteins, polycomb-group proteins (PRCs) and a few transcription factors ([Figure 1b](#fig1){ref-type="fig"}). We first assessed the effects of shRNAs alone on U87MG cell viability, to identify shRNAs displaying intrinsic toxicity before TRAIL treatment. Two of the 125 shRNAs were eliminated from further analysis in the screen as they caused \~100% cell death after puromycin selection, possibly due to problems with viral packaging. Accordingly, 6 out of 125 shRNAs reduced cell viability more than 2 S.D. compared with shControl ([Figure 1c](#fig1){ref-type="fig"}). These were shRNAs targeting KDM5C (68±1%), KDM4C (71±1%), KDM4A (75±1%), KDM4A (75±1%), Set1A (77±1%) and KDM3B (77±1%). Five out of 125 shRNAs targeting MeCP2 (115±1%), MBD1 (115±1%), AHCY (117±5%), TCF3 (117±1%) and EZH1 (121±2%) caused minor increases in viability. However, the majority of the shRNAs did not significantly alter cell viability ([Figure 1c](#fig1){ref-type="fig"}). To identify shRNAs that modulated TRAIL sensitivity, we assessed the percent viability changes after TRAIL treatment. Accordingly, we categorized CMEs into two groups based on the phenotype observed upon their silencing compared with shControl cells: (1) suppressors of apoptosis, if their knockdown sensitized the cells to TRAIL, (2) enhancers of apoptosis, if their knockdown conferred cells more TRAIL resistant ([Figure 1d](#fig1){ref-type="fig"}). While TRAIL caused 25±4% reduction in the viability of shControl cells, there were seven shRNAs that sensitized cells to TRAIL. These were targeting five genes, namely *Suv39H2* (41±2%), *G9A* (42±2%), *NR2F2* (45±4%), *RING1A* (48±2 or 50±3%) and *KDM2B* (41±2 or 44±2%) ([Figure 1e](#fig1){ref-type="fig"}). We then focused on the genes *RING1A* and *KDM2B*, whose knockdown led to a phenotype with two independent shRNAs ([Figure 1e](#fig1){ref-type="fig"}). Taken together, our screen identified KDM2B and RING1, an H3K36-specific demethylase and an E3 ubiquitin-protein ligase of H2AK119, respectively, as novel regulators of TRAIL response.
Loss of KDM2B cooperates with TRAIL to reduce GBM cell viability
----------------------------------------------------------------
To assess the function of the novel apoptosis-modulating CMEs we identified in GBMs, we first checked the knockdown efficiency of the shRNAs and observed silencing down to 17--40% ([Supplementary Figure 1](#sup1){ref-type="supplementary-material"}). We then focused on KDM2B, as its protumorigenic functions have been demonstrated in various solid and hematological malignancies;^[@bib21],\ [@bib22]^ however, its function in GBMs was not defined. Quantitative RT-PCR (qRT-PCR) analysis of *KDM2B* levels revealed that both shRNAs reduced *KDM2B* mRNA levels down to \~50% ([Figure 2a](#fig2){ref-type="fig"}). However, shKDM2B-2 led to a more robust reduction at protein levels ([Figure 2b](#fig2){ref-type="fig"}). To assess whether the shRNAs targeting KDM2B is specific, we checked the levels of other KDM family members upon KDM2B knockdown and observed no major alterations in their levels ([Supplementary Figure 2](#sup1){ref-type="supplementary-material"}).
To further validate the screen results, we conducted ATP-based cell viability analysis of cells transduced with both shKDM2B vectors and verified that cells with reduced KDM2B exhibit cell death significantly more than the controls ([Figure 2c](#fig2){ref-type="fig"}). To examine these differences in cell death further, we used an assay that measures cell growth in real time, where cells' electrical impedance in a well is measured and transformed into a cell index. Accordingly, silencing of KDM2B not only augmented TRAIL response but also accelerated the process of cell death ([Figure 2d](#fig2){ref-type="fig"}). There was no observable difference between untreated cells, showing that KDM2B knockdown has no significant effects on short-term (24 h) proliferation dynamics. This phenotype was also validated by live-cell imaging, where the morphology of individual cells, as well as cell death processes, was observed in real time ([Figure 2e](#fig2){ref-type="fig"} and [Supplementary Videos 1--4](#sup1){ref-type="supplementary-material"}). As shown by automated quantification of cellular blebs that were indicative of apoptotic bodies, the process of apoptosis was accelerated in shKDM2B cells compared with controls ([Figure 2f](#fig2){ref-type="fig"}). The number of apoptotic bodies per frame reached its maxima within 6 h of TRAIL treatment in shKDM2B cells, earlier and significantly in higher numbers than shControl cells. To examine whether KDM2B effects can be recapitulated in additional GBM cell lines, we used a more TRAIL-sensitive line, T98G in parallel. There, KDM2B | glioblastoma multiforme ( gbm ) is the most common and aggressive primary brain tumor with a median survival of 12 months. conventional therapeutic strategies of combination and chemotherapy are inadequate to eradicate gbms because of the diffuse nature of gbm, intrinsic or innate resistance to therapy and the presence of blood - - brain - - barrier ( bbb ). ^ [ @ bib1 ], \ [ @ bib2 ] ^ to alter the * status quo * that has remained unchanged over 25 years, novel targets and therapeutic strategies start to be developed. evasion of apoptosis is a key hallmark of cancers that exhibit resistance against therapeutics, ^ [ @ bib3 ] ^ making the reactivation of dormant apoptotic programs a favorable approach in treatment. activating extrinsic apoptosis using death ligands, such as tumor necrosis factor - related apoptosis - inducing ligand ( trail ), is a promising strategy because of its tumor specificity. ^ [ @ bib4 ] ^ several trail - based therapies such as advanced human trail and death receptor agonists have been developed and have shown success in preclinical models. ^ [ @ bib5 ] ^ similarly, activating intrinsic apoptosis effectively inhibiting the antiapoptotic bcl - 2 proteins, bcl - 1b and bcl - xl, with bh3 peptides has shown success in preclinical tumor models. ^ [ @ bib6 ], \ [ @ bib7 ] ^ however, the major obstacle in proapoptotic therapies is innate or acquired recruitment of tumor cells to the chemotherapy agents. ^ [ @ bib8 ] ^ aberrant regulation of the apoptosis pathway components could be responsible for evolutionary failure of desired response to the proapoptotic agents. ^ [ @ bib3 ] ^ one well - characterized misregulation is the epigenetic silencing of proapoptotic genes, such as death receptor 4 ( * dr4 * ). ^ [ @ bib9 ] ^ however, epigenetic regulation of chemotherapy response of cancer cells has been demonstrated to be dynamic and reversible. ^ [ @ bib10 ] ^ numerous studies have shown that overcoming trail resistance is possible by using secondary agents, such for the histone deacetylase inhibitors, ^ [ @ bib ##11 ], \ [ @ bib12 ], \ [ @ bib13 ] ^ corroborating the idea that the resistance can be reversed by epigenetic reprogramming. the epigenome of cells are maintained by dynamic histone and dna modifications throughout the chromatin by a group of chromatin - modifying enzymes ( cmes ) called ' writers ' such as histone acetyltransferases, histone methyltransferases ( hmts ) and dna methyltransferases ( dnmts ). ^ [ @ bib14 ] ^ these modifications can be removed by ' erasers ' such as histone deacetylases ( hdacs ) and histone demethylases ( hdms ), or can be recognized by ' readers ' such as bromodomain - containing proteins. ^ [ @ bib15 ] ^ although the molecular mechanisms leading to aberrant cancer epigenomes are becoming better understood, ^ [ @ bib16 ], \ [ @ bib17 ], \ [ @ bib18 ] ^ most studies in gbm merely focus on dna hyper / hypomethylation. ^ [ @ bib19 ] ^ therefore, an unbiased and comprehensive assessment of roles of cmes in apoptosis resistance is needed. in this study, we aimed to interrogate the function of cmes regulating apoptotic response in gbm and undertook a loss - of - function approach using short - hairpin rnas ( shrnas ) that were designed against a select but diverse set of cmes and associated proteins. we have shown that loss of kdm2b - - a h3k36 - specific histone demethylase - - primed gbm cells for apoptosis through the induction of proapoptotic genes and the suppression of antiapoptotic genes. our results suggest kdm2b as a novel central epigenetic regulator of gbm cell apoptosis and identify it as a potential proapoptotic target for future of gbm treatment. results = = = = = = = interrogation of the cmes reveals kdm2b as a modulator of trail - induced apoptosis in gbm cells - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - to identify the key cmes regulating the apoptotic response of gbm cells, we applied a shrna - based loss - of - function screen in u87mg cell line ( [ figure 1a ] ( # fig1 ) { ref - type = " fig " } ). we used a library of 60 shrnas published before, ^ [ @ bib20 ] ^ and expanded it to 125 shrnas targeting 48 different cme genes with two or three separate shrnas. the targeted cmes included dnmts, hmts, hdms, methylated dna - binding proteins, polycomb - group proteins ( prcs ) and a few transcription factors ( [ figure 1b ] ( # fig1 ) { ref - type = " fig " } ). we first assessed the effects of shrnas alone on u87mg cell viability, to identify shrnas displaying intrinsic toxicity before trail treatment. two of the 125 shrnas were eliminated from further analysis in the screen as they caused \ ~ 100 % cell death after puromycin selection, possibly due to problems with viral packaging. accordingly, 6 out of 125 shrnas reduced cell viability more than 2 s. d. compared with shcontrol ( [ figure 1c ] ( # fig1 ) { ref - type = " fig " } ). these were shrnas targeting kdm5c ( 68±1 % ), kdm4c ( 71±1 % ), kdm4a ( 75±1 % ), kdm4a ( 75±1 % ), set1a ( 77±1 % ) and kdm3b ( 77±1 % ). five out of 125 shrnas targeting mecp2 ( 115±1 % ), mbd1 ( 115±1 % ), ahcy ( 117±5 % ), tcf3 ( 117±1 % ) and ezh1 ( 121±2 % ) caused minor increases in viability. however, the majority of the shrnas did not significantly alter cell viability ( [ figure 1c ] ( # fig1 ) { ref - type = " fig " } ). to identify shrnas that modulated trail sensitivity, we assessed the percent viability changes after trail treatment. accordingly, we categorized cmes into two groups based on the phenotype observed upon their silencing compared with shcontrol cells : ( 1 ) suppressors of apoptosis, if their knockdown sensitized the cells to trail, ( 2 ) enhancers of apoptosis, if their knockdown conferred cells more trail resistant ( [ figure 1d ] ( # fig1 ) { ref - type = " fig " } ). while trail caused 25±4 % reduction in the viability of shcontrol cells, there were seven shrnas that sensitized cells to trail. these were targeting five genes, namely * suv39h2 * ( 41±2 % ), * g9a * ( 42±2 % ), * nr2f2 * ( 45±4 % ), * ring1a * ( 48±2 or 50±3 % ) and * kdm2b * ( 41±2 or 44±2 % ) ( [ figure 1e ] ( # fig1 ) { ref - type = " fig " } ). we then focused on the genes * ring1a * and * kdm2b *, whose knockdown led to a phenotype with two independent shrnas ( [ figure 1e ] ( # fig1 ) { ref - type = " fig " } ). taken together, our screen identified kdm2b and ring1, an h3k36 - specific demethylase and an e3 ubiquitin - protein ligase of h2ak119, respectively, as novel regulators of trail response. loss of kdm2b cooperates with trail to reduce gbm cell viability - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - to assess the function of the novel apoptosis - modulating cmes we identified in gbms, we first checked the knockdown efficiency of the shrnas and observed silencing down to 17 - - 40 % ( [ supplementary figure 1 ] ( # sup1 ) { ref - type = " supplementary - material " } ). we then focused on kdm2b, as its protumorigenic functions have been demonstrated in various solid and hematological malignancies ; ^ [ @ bib21 ], \ [ @ bib22 ] ^ however, its function in gbms was not defined. quantitative rt - pcr ( qrt - pcr ) analysis of * kdm2b * levels revealed that both shrnas reduced * kdm2b * mrna levels down to \ ~ 50 % ( [ figure 2a ] ( # fig2 ) { ref - type = " fig " } ). however, shkdm2b - 2 led to a more robust reduction at protein levels ( [ figure 2b ] ( # fig2 ) { ref - type = " fig " } ). to assess whether the shrnas targeting kdm2b is specific, we checked the levels of other kdm family members upon kdm2b knockdown and observed no major alterations in their levels ( [ supplementary figure 2 ] ( # sup1 ) { ref - type = " supplementary - material " } ). to further validate the screen results, we conducted atp - based cell viability analysis of cells transduced with both shkdm2b vectors and verified that cells with reduced kdm2b exhibit cell death significantly more than the controls ( [ figure 2c ] ( # fig2 ) { ref - type = " fig " } ). to examine these differences in cell death further, we used an assay that measures cell growth in real time, where cells ' electrical impedance in a well is measured and transformed into a cell index. accordingly, silencing of kdm2b not only augmented trail response but also accelerated the process of cell death ( [ figure 2d ] ( # fig2 ) { ref - type = " fig " } ). there was no observable difference between untreated cells, showing that kdm2b knockdown has no significant effects on short - term ( 24 h ) proliferation dynamics. this phenotype was also validated by live - cell imaging, where the morphology of individual cells, as well as cell death processes, was observed in real time ( [ figure 2e ] ( # fig2 ) { ref - type = " fig " } and [ supplementary videos 1 - - 4 ] ( # sup1 ) { ref - type = " supplementary - material " } ). as shown by automated quantification of cellular blebs that were indicative of apoptotic bodies, the process of apoptosis was accelerated in shkdm2b cells compared with controls ( [ figure 2f ] ( # fig2 ) { ref - type = " fig " } ). the number of apoptotic bodies per frame reached its maxima within 6 h of trail treatment in shkdm2b cells, earlier and significantly in higher numbers than shcontrol cells. to examine whether kdm2b effects can be recapitulated in additional gbm cell lines, we used a more trail - sensitive line, t98g in parallel. there, kdm2b | Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor with a median survival of 12 months. Conventional therapeutic strategies of radiotherapy and chemotherapy are inadequate to eradicate GBMs because of the diffuse nature of GBM, acquired or innate resistance to therapy and the presence of blood - - brain - - barrier (BBB ). ^ [@ bib1 ], \ [@ bib2] ^ To alter the * status quo * that has remained unchanged over 25 years, novel targets and therapeutic strategies need to be developed. Evasion of apoptosis is a key hallmark of cancers that exhibit resistance against therapeutics, ^ [@ bib3] ^ making the reactivation of dormant apoptotic programs a favorable approach in treatment. Activating extrinsic apoptosis using death ligands, such as tumor necrosis factor - related apoptosis - inducing ligand (TRAIL ), is a promising strategy because of its tumor specificity. ^ [@ bib4] ^ Several TRAIL - based therapies such as recombinant human TRAIL and death receptor agonists have been developed and have shown success in preclinical models. ^ [@ bib5] ^ Similarly, activating intrinsic apoptosis by inhibiting the antiapoptotic Bcl - 2 proteins, Bcl - 2 and Bcl - XL, with BH3 peptides has shown success in preclinical tumor models. ^ [@ bib6 ], \ [@ bib7] ^ However, the major obstacle in proapoptotic therapies is innate or acquired resistance of tumor cells to the proapoptotic agents. ^ [@ bib8] ^ Aberrant regulation of the apoptosis pathway components could be responsible for the failure of desired response to the proapoptotic agents. ^ [@ bib3] ^ One well - characterized misregulation is the epigenetic silencing of proapoptotic genes, such as death receptor 4 (* DR4 * ). ^ [@ bib9] ^ However, epigenetic regulation of chemotherapy response of cancer cells has been demonstrated to be dynamic and reversible. ^ [@ bib10] ^ Numerous studies have shown that overcoming TRAIL resistance is possible by using secondary agents, such as the his6on$ deacetylase inhibitors, ^ [@ bib11 ], \ [@ bib12 ], \ [@ b9b!3] ^ corroborating the idea that the resistance can be reversed by epigenetic reprogramming. The epigenome of cells are maintained by dynamic histone and DNA modifications throughout the chromatin by a group of chromatin - modifying enzymes (CMEs) called ' writers ' such as histone acetyltransferases, histone methyltransferases (HMTs) and DNA methyltransferases (DNMTs ). ^ [@ bib14] ^ These modifications can be removed by ' erasers ' such as histone deacetylases (HDACs) and histone demethylases (HDMs ), or can be recognized by ' readers ' such as bromodomain - containing proteins. ^ [@ bib15] ^ Although the molecular mechanisms leading to aberrant cancer epigenomes are becoming better understood, ^ [@ bib16 ], \ [@ bib17 ], \ [@ bib18] ^ most studies in GBM merely focus on DNA hyper / hypomethylation. ^ [@ bib19] ^ Therefore, an unbiased and comprehensive assessment of roles of CMEs in apoptosis resistance is needed. In this study, we aimed to interrogate the function of CMEs regulating apoptotic response in GBM and undertook a loss - of - function approach using short - hairpin RNAs (shRNAs) that were designed against a select but diverse set of CMEs and associated proteins. We have shown that loss of KDM2B - - a H3K36 - specific histone demethylase - - primed GBM cells for apoptosis through the induction of proapoptotic genes and the suppression of antiapoptotic genes. Our results suggest KDM2B as a novel central epigenetic regulator of GBM cell apoptosis and identify it as a potential proapoptotic target for future of GBM treatment. Results = = = = = = = Interrogation of the CMEs reveals KDM2B as a modulator of TRAIL - induced apoptosis in GBM cells - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - To identify the key CMEs regulating the apoptotic response of GBM cells, we applied a shRNA - based loss - of - function screen in U87MG cell line ([ Figure 1a] (# fig1) {ref - type = " fig "} ). We used a library of 60 shRNAs publoshSd before, ^ [@ bib20] ^ and expanded it to 125 shRNAs targeting 48 different CME genes with two or three separate shRNAs. The targeted CMEs included DNMTs, HMTs, HDMs, methylated DNA - binding proteins, polycomb - group proteins (PRCs) and a few transcription factors ([ Figure 1b] (# fig1) {ref - t%pS = " fig "} ). We first assessed the effects of shRNAs alone on U87MG cell viability, to identify shRNAs displaying intrinsic toxicity before TRAIL treatment. Two of the 125 shRNAs were eliminated from further analysis in the screen as they caused \ ~ 100% cell death after puromycin selection, possibly due to problems with viral packaging. Accordingly, 6 out of 125 shRNAs reduced cell viability more than 2 S. D. compared DUth shControl ([ Figure 1c] (# fig1) {ref - type = " fig "} ). These were shRNAs targeting KDM5C (68 ± 1% ), KDM4C (71 ± 1% ), KDM4A (75 ± 1% ), KDM4A (75 ± 1% ), Set1A (77 ± 1%) and KDM3B (77 ± 1% ). Five out of 125 shRNAs targeting MeCP2 (115 ± 1% ), MBD1 (115 ± 1% ), AHCY (117 ± 5% ), TCF3 (117 ± 1%) and EZH1 (121 ± 2%) caused minor increases in viability. However, the majority of the shRNAs did not significantly alter cell viability ([ Figure 1c] (# fig1) {ref - type = " fig "} ). To identify shRNAs that modulated TRAIL sensitivity, we assessed the percent viability changes after TRAIL treatment. Accordingly, we categorized CMEs into two groups based on the phenotype observed upon their silencing compared with shControl cells: (1) suppressors of apoptosis, if their knockdown sensitized the cells to TRAIL, (2) enhancers of apoptosis, if their knockdown conferred cells <(re TRAIL resistant ([ Figure 1d] (# fig1) {ref - type = " fig "} ). While TRAIL caused 25 ± 4% reduction in the viability of shControl cells, there were seven shRNAs that sensitized cells to TRAIL. These were targeting five genes, namely * Suv39H2 * (41 ± 2% ), * G9A * (42 ± 2% ), * NR2F2 * (45 ± 4% ), * RING1A * (48 ± 2 or 50 ± 3%) and * KDM2B * (41 ± 2 or 44 ± 2%) ([ Figure 1e] (# fig1) {ref - type = " fig "} ). We then focused on the genes * RING1A * and * KDM2B *, whose knockdown led to a phenotype with two independent shRNAs ([ Figure 1e] (# fig1) {ref - type = " fig "} ). Taken together, our screen identified KDM2B and RING1, an H3K36 - specific demethylase and an E3 ubiquitin - protein ligase of H2AK119, respectively, as novel regulators of TRAIL response. Loss of KDM2B cooperates with TRAIL to reduce GBM cell viability - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - To assess the function of the novel apoptosis - modulating CMEs we identified in GBMs, we first checked the knockdown efficiency of the shRNAs and observed silencing down to 17 - - 40% ([ Supplementary Figure 1] (# sup1) {ref - type = " supplementary - material "} ). We then focused on KDM2B, as its protumorigenic functions have been demonstrated in various solid and hematological malignancies; ^ [@ bib21 ], \ [@ bib22] ^ however, its function in GBMs was not defined. Quantitative RT - PCR (qRT - PCR) analysis of * KDM2B * levels revealed that both shRNAs reduced * KDM2B * mRNA levels down to \ ~ 50% ([ Figure 2a] (# fig2) {ref - type = " fig "} ). However, shKDM2B - 2 led to a more robust reduction at protein levels ([ Figure 2b] (# fig2) {ref - type = " fig "} ). To assess whether the shRNAs targeting KDM2B is specific, we checked the levels of other KDM family members upon KDM2B knockdown and observed no major alterations in their levels ([ Supplementary Figure 2] (# sup1) {ref - type = " supplementary - material "} ). To further validate the screen results, we conducted ATP - based cell viability analysis of cells transduced with both shKDM2B vectors and verified that cells with reduced KDM2B exhibit cell death significantly more than the controls ([ Figure 2c] (# fig2) {ref - type = " fig "} ). To examine these differences in cell death further, we used an assay that measures cell growth in real time, where cells ' electrical impedance in a well is measured and transformed into a X@ll index. Accordingly, silencing of KDM2B not only augmented TRAIL response but also accelerated the process of cell death ([ Figure 2d] (# fig2) {ref - type = " fig "} ). There was no observable difference between untreated cells, showing that LDK2B knockdown has no significant effects on short - term (24 h) proliferation dynamics. rhls phenotype was also validated by live - cell imaging, where the morphology of individual cells, as well as cell death processes, was observed in real time ([ Figure 2e] (# fig2) {ref - type = " fig "} and [Supplementary Videos 1 - - 4] (# sup1) {ref - type = " supplementary - material "} ). As shown by automated quantification of cellular blebs that were indicative of apoptotic bodies, the process of apoptosis was accelerated in shKDM2B cells compared with controls ([ Figure 2f] (# fig2) {ref - type = " fig "} ). The number of apoptotic bodies per frame reached its maxima within 6 h of TRAIL treatment in shKDM2B cells, earlier and significantly in higher numbers than shDKntrol cells. To examine whether KDM2B effects can be recapitulated in additional GBM cell lines, we used a more TRAIL - sensitive line, T98G in parallel. There, KDM2B | Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor with a median survival of 12 months. Conventional therapeutic of radiotherapy and chemotherapy are inadequate to eradicate GBMs because of the diffuse nature of GBM, acquired or innate resistance to therapy and the presence of blood--brain--barrier (BBB).^[@bib1],\ [@bib2]^ To alter the *status quo* that has remained unchanged over 25 years, novel targets and therapeutic need be developed. Evasion of apoptosis is a key hallmark of that resistance against therapeutics,^[@bib3]^ the reactivation of dormant programs a favorable approach in treatment. Activating extrinsic apoptosis using death ligands, such as tumor necrosis factor-related apoptosis-inducing is a promising strategy of its tumor specificity.^[@bib4]^ TRAIL-based therapies such as recombinant TRAIL and death receptor agonists have been developed and have shown success in preclinical models.^[@bib5]^ Similarly, activating intrinsic apoptosis by inhibiting the antiapoptotic Bcl-2 Bcl-2 and Bcl-XL, with BH3 has success in preclinical tumor models.^[@bib6],\ [@bib7]^ However, major obstacle in proapoptotic therapies is innate or acquired resistance of tumor to the proapoptotic agents.^[@bib8]^ Aberrant regulation of the apoptosis pathway components could be responsible the failure of desired response to the proapoptotic agents.^[@bib3]^ One well-characterized misregulation is the silencing of proapoptotic genes, such as death receptor 4 (*DR4*).^[@bib9]^ However, epigenetic regulation of chemotherapy of cancer cells has been demonstrated to be dynamic and reversible.^[@bib10]^ Numerous studies have shown that overcoming TRAIL resistance is possible by using secondary agents, as histone deacetylase inhibitors,^[@bib11],\ [@bib12],\ [@bib13]^ corroborating idea that the resistance can be reversed by reprogramming. The epigenome cells are maintained by dynamic histone and DNA modifications throughout the chromatin by a group of enzymes (CMEs) called 'writers' such as histone acetyltransferases, histone methyltransferases (HMTs) and DNA methyltransferases (DNMTs).^[@bib14]^ These modifications can be removed by 'erasers' such as histone deacetylases (HDACs) and histone demethylases (HDMs), or can by 'readers' such as bromodomain-containing the molecular leading to aberrant cancer epigenomes are becoming better understood,^[@bib16],\ [@bib17],\ [@bib18]^ most studies in GBM merely focus on DNA hyper/hypomethylation.^[@bib19]^ an unbiased and comprehensive assessment roles of CMEs in apoptosis resistance is needed. In this study, we aimed to interrogate the function of CMEs regulating apoptotic response in and undertook a loss-of-function approach using short-hairpin (shRNAs) that designed against a select but diverse set of CMEs and associated proteins. We have shown of KDM2B -- a H3K36-specific histone demethylase -- primed GBM cells for apoptosis through the induction of proapoptotic genes and the suppression antiapoptotic genes. Our results suggest KDM2B as a novel central epigenetic regulator of cell apoptosis and it as a potential proapoptotic future of GBM treatment. Results ======= Interrogation of the CMEs reveals KDM2B as a modulator TRAIL-induced apoptosis in GBM cells ---------------------------------------------------------------------------------------------- To identify the CMEs regulating the apoptotic response of GBM cells, we applied a shRNA-based loss-of-function screen in U87MG cell line ([Figure 1a](#fig1){ref-type="fig"}). We a library of 60 shRNAs published before,^[@bib20]^ and expanded it to shRNAs targeting 48 different CME genes with two or three separate shRNAs. The targeted CMEs included DNMTs, HDMs, DNA-binding proteins, polycomb-group proteins (PRCs) and a few transcription factors ([Figure We first assessed the effects of shRNAs alone on U87MG cell viability, to identify shRNAs displaying intrinsic before TRAIL treatment. Two of the 125 shRNAs were eliminated from further analysis in the screen as they caused \~100% cell death puromycin selection, possibly problems with viral packaging. 6 out of 125 shRNAs reduced cell viability more than 2 S.D. compared with shControl ([Figure 1c](#fig1){ref-type="fig"}). These were shRNAs targeting KDM5C (68±1%), KDM4C (71±1%), KDM4A (75±1%), Set1A (77±1%) KDM3B (77±1%). Five out of shRNAs MeCP2 (115±1%), AHCY (117±5%), TCF3 and EZH1 caused increases in viability. However, majority the shRNAs did not significantly alter cell viability ([Figure 1c](#fig1){ref-type="fig"}). To identify shRNAs that modulated TRAIL sensitivity, we assessed the percent viability changes after TRAIL treatment. Accordingly, we categorized CMEs into two groups based on the observed upon their silencing compared with shControl cells: (1) suppressors of apoptosis, if their knockdown sensitized the cells to TRAIL, (2) enhancers of apoptosis, if their knockdown conferred cells more TRAIL resistant ([Figure 1d](#fig1){ref-type="fig"}). While TRAIL caused 25±4% in the viability of shControl cells, there were seven shRNAs sensitized cells to TRAIL. These were targeting five genes, namely *Suv39H2* (41±2%), *G9A* (42±2%), *NR2F2* (45±4%), *RING1A* (48±2 or 50±3%) and *KDM2B* (41±2 or 44±2%) ([Figure We then focused on the *RING1A* and *KDM2B*, whose knockdown led to a phenotype with two independent shRNAs ([Figure together, screen identified KDM2B RING1, an demethylase and E3 ubiquitin-protein ligase of H2AK119, respectively, as novel regulators of TRAIL response. Loss of KDM2B cooperates TRAIL reduce GBM cell viability ---------------------------------------------------------------- To assess the function of the novel apoptosis-modulating CMEs we identified in first checked the knockdown efficiency of the shRNAs and observed silencing down to 17--40% ([Supplementary Figure then focused on KDM2B, as its protumorigenic have been demonstrated in various and hematological malignancies;^[@bib21],\ however, its in GBMs was not defined. (qRT-PCR) analysis of *KDM2B* that both shRNAs *KDM2B* mRNA levels down to \~50% ([Figure 2a](#fig2){ref-type="fig"}). However, led to a more robust reduction at protein ([Figure To assess whether the targeting KDM2B is specific, we checked the levels of KDM family members upon KDM2B knockdown and no major alterations in their levels ([Supplementary Figure To further validate the screen results, we conducted ATP-based cell viability analysis of cells transduced with both shKDM2B vectors and verified that cells with reduced KDM2B exhibit cell death significantly more than the controls ([Figure 2c](#fig2){ref-type="fig"}). To examine these differences cell death further, we used an assay that measures cell growth in time, where cells' electrical in a well is measured transformed into a cell index. silencing of KDM2B not only augmented TRAIL response but also accelerated the process of cell death ([Figure 2d](#fig2){ref-type="fig"}). There was no observable difference between untreated cells, that KDM2B knockdown has no significant effects on short-term (24 h) proliferation dynamics. phenotype was validated by live-cell where the morphology of cells, as well as cell processes, was observed in real time ([Figure and [Supplementary Videos 1--4](#sup1){ref-type="supplementary-material"}). As shown by automated quantification of cellular blebs that were indicative of apoptotic bodies, the process of apoptosis was accelerated in shKDM2B cells compared with ([Figure 2f](#fig2){ref-type="fig"}). The number of apoptotic bodies per frame reached its maxima within h of TRAIL in shKDM2B cells, and significantly in higher numbers than shControl cells. To whether KDM2B effects can be in additional GBM cell lines, we used a more TRAIL-sensitive line, T98G in parallel. There, KDM2B | gLIOblaStoMa MUltiFoRme (Gbm) IS THe MoSt CoMmOn aNd AggRessiVE pRIMaRY Brain tuMOr WiTH A MEDian surViVAl oF 12 mONths. CoNVENTiONaL theRapEUtIc StrATegieS of rAdIOthERAPY AND chemothERApy ArE inAdeqUAtE TO ErAdicaTE GbMs becausE of THE DiFfusE naTUre OF gBm, acQUIRed oR iNNAtE ResIsTAnce To ThERAPy AnD the PRESEnCE of blooD--BRaIn--BArRier (bBB).^[@Bib1],\ [@bib2]^ To altER tHE *STatUs QuO* tHAT HAs REMaiNeD uNChAngeD oveR 25 yeArs, nOvEl taRGeTs aNd theraPEutIc sTRateGIES NeEd To Be dEVeLOpEd.
evASion oF ApOpToSIs is a Key hALLmarK OF cAnCeRs THat ExhIBIT reSisTAnCe AGaInst therapEuTiCs,^[@bIb3]^ maKing tHe ReActIVatiOn OF dormAnt aPOPtoTIc PROGrAMS A FAVoraBLE aPPrOaCH in TREAtMEnt. ACTIVaTinG exTriNsIc aPOPtOsiS usinG dEaTh LiGands, suCh as TuMor NeCROSis factOr-RElatEd APopTOsis-INdUcInG LIGand (TRAil), IS A PRomISING STrAteGY beCAUSE OF ITS TUMOr SpeCIfICIty.^[@Bib4]^ SeveraL trail-baSed therapIeS suCH AS RecoMbiNanT HumaN TRAIl aNd dEATH recEPTor agOnistS HAvE bEeN DEVelOpED aND hAve ShoWN sUccESS In PrEClInicaL moDELs.^[@bib5]^ sImilARlY, ACTIVatING iNtrinSic aPOPTOsIS BY INHiBItIng The AntIapoPTotIC Bcl-2 prOteinS, bcL-2 ANd BcL-XL, WIth Bh3 pEpTIdes haS SHOwN SUccESs in pRECLINICaL TuMor mODEls.^[@bIB6],\ [@Bib7]^ howeVeR, thE mAjor oBStACLE IN proAPoptotic therapIEs IS InNAte OR aCQuIRed reSISTANce OF tumOr Cells to tHe ProapOPTotIc agENtS.^[@bIb8]^
AberRAnT reGuLATIoN OF THE apOptoSis paTHway COMPoNENts cOulD Be REspoNsiBlE FOR ThE FaILUre oF desIREd rESPONsE tO tHE PrOAPOPtotiC AGents.^[@bib3]^ ONe WELL-CharacTerized MiSreGulaTiON IS tHE ePIgeneTIC SILENCIng Of pRoaPopTOtiC geNEs, sUCH As DeatH REcEpToR 4 (*DR4*).^[@biB9]^ HOwever, EpIGeneTIc reguLaTIoN OF cheMOTHErapY rEspoNSe OF caNceR cELLs has bEEn DEmonsTRAted tO be dYNaMic And ReveRSIbLE.^[@bIb10]^ NUMEroUs sTuDIes HaVE SHOWN tHaT oVERcOMIng TraiL RESistancE Is PoSsible bY USING seCondary aGEnts, such as tHE hISTOnE deacetYlASE inhIbItOrs,^[@Bib11],\ [@bIb12],\ [@Bib13]^ CORROBORAtInG THe IdEa thAt tHE reSIstaNCE caN be rEvErSEd BY EpIgenetiC REPRoGrAMMInG.
tHe EPiGeNOME OF ceLLS Are MaiNtAIneD By dYnaMic hisTONE and DNA MODIfiCAtIOnS ThrOUgHOUT The chromAtiN by A groUP oF chrOMATin-mOdIFYinG ENZYmES (cMes) cALlED 'WRiteRS' SuCH AS HiStoNE aCETylTRAnsFEraSES, hisToNe METhyLTranSfERaseS (HMTs) AND dNA meThYltRaNsFerASes (DnmTS).^[@bIB14]^ thESe MOdiFICAtions CaN Be RemOvEd by 'eRAseRS' suCh AS HisToNE DEaCETyLASes (HdAcs) AND hiSToNE dEMethylaSeS (hdmS), or can bE RECoGniZED bY 'rEadERS' such As bRomODOMAIn-COntaInIng pROTEinS.^[@BIB15]^ aLthOugH ThE mOLEculAr mEcHAnISms LeAdinG To abERrANt caNcer ePiGEnoMES Are BEcoMIng BeTTER UnDERsTOOD,^[@Bib16],\ [@bib17],\ [@BiB18]^ mOst studIEs iN gbM MErElY fOcus ON DNA hYpEr/hyPOmETHYLatiOn.^[@bIB19]^ THEREFOre, an UnbiAsed AnD comPrEhENsIVe AssesSMeNT Of ROlEs Of CMES IN APoptoSis reSiStANCE Is NeeDed.
iN tHiS sTudy, wE AiMEd tO INTERrOgAte the FUncTiON OF cmes REGulAtIng apOPtOTiC reSpONSE IN GBM aND uNdeRtOoK A LosS-Of-FuNcTION APpRoACH uSinG ShORT-haIRPIN rNas (shrnaS) tHaT werE DesIGnEd aGAINST A SeLEct BUt dIvErsE SEt oF CmES AnD asSOciated pROtEInS. WE HavE showN thaT LOSS OF kDM2b -- a h3k36-SpECiFIc HIstoNE demEthYLaSE -- pRIMed gBM Cells fOr aPoptoSIs THroUGh THE iNDUCtiON OF PROApOptOtIc GenEs and ThE suPprESSIoN of aNTiApOpTOTIC GENes. OUr ResULTS sUgGEST kdm2B as a NOvel cenTRal EPigENEtiC REgULAToR Of gbM celL ApOPtOSiS anD iDENTIFY It As A poteNtiAl PRoaPOPTOtIC taRGeT fOR fuTuRe Of gbm TREaTMENT.
rEsults
=======
InTErRogATiON of tHe cmes rEvEalS KDM2b as a moduLaToR Of Trail-INDuceD aPoPtoSIs IN gBm ceLLS
----------------------------------------------------------------------------------------------
tO IDEntIFY thE kEY CmEs ReGULATINg ThE aPoPTotIC rESpOnsE OF GBM CEllS, We apPLIeD a sHRNa-BASEd LOss-oF-FuNCTION screEN In u87MG CeLl lINE ([FiGURe 1a](#fIG1){rEF-tYPE="FiG"}). we UsED A lIBRaRY of 60 ShrNas PubLiSHEd beFORE,^[@BIb20]^ anD EXpAnDED iT to 125 ShrnaS tArgEtiNG 48 DiffEReNt cmE GeneS WiTH TWo Or THree sEpArATe sHRNAS. tHe targEteD cMES incluDed dNmTS, HmTs, hDmS, meTHylaTeD DNa-BiNDing prOtEiNS, poLYcOMB-gRouP PROTeiNs (PRCS) AnD a FEW transcRiptiON fACtORs ([fIGure 1b](#fIG1){Ref-TYpE="fIg"}). We firsT asSeSSED tHe EffeCTS of sHRnas aloNE ON U87mG ceLL ViABIlIty, TO IDENtIFy SHRNAs DISPlaYING INTRINSIC TOxICiTY beFORe tRAIl TrEAtment. twO of the 125 sHRNaS WErE elIMinATed From FURThEr AnalySis IN tHE scREEn aS tHeY caUSeD \~100% CELL DeAth AfteR PurOmyciN sEleCTiOn, POSsIblY DUe To prOBLems WITh vIrAL PacKaging. ACCORdinGlY, 6 oUt of 125 ShrNas RedUcEd Cell VIabILitY MoRe THaN 2 S.d. ComPaREd wiTH SHCOntrOl ([fIGurE 1c](#Fig1){rEF-tYpE="FiG"}). tHESe wEre shrnaS tARgetInG kdM5c (68±1%), KDm4C (71±1%), kDm4A (75±1%), Kdm4A (75±1%), SeT1A (77±1%) And Kdm3b (77±1%). fIve OuT oF 125 sHRNas TARGEting mEcP2 (115±1%), MBd1 (115±1%), ahCy (117±5%), tCf3 (117±1%) AnD ezH1 (121±2%) cAUsed MinOR iNCreaSES in VIabIlItY. HOweVER, tHe mAjoRity Of the sHrnAS Did NOT SignIfiCANtLY ALTER CEll ViabiLity ([figuRE 1c](#FIg1){Ref-TYpE="fiG"}). to IDEnTify shrnas THAt MOdulAteD TrAIL sEnsItiVitY, wE ASSessEd the pErCeNT vIAbILiTy CHaNGeS afTEr tRAIl trEaTMenT. ACCoRDinGly, we caTegorized cMES InTO two gROuPs bAseD oN ThE PheNOtyPE oBseRveD upOn tHeIR siLENcING CoMpArEd WITh ShCOntRoL cELlS: (1) sUPpREsSOrs OF apOpTOSis, iF THeIr KNOckdoWn sENSitIzed ThE CeLLS to tRaIl, (2) enHANCers Of apOPtOSis, if THeIr KNOckDoWN CoNFErRed cElLS moRE tRail ReSIStAnt ([figUrE 1D](#FiG1){REf-tYpe="FIG"}). WHile trAil cAUsed 25±4% rEDucTION in THe VIaBILItY oF sHCoNtrOl CELlS, THeRe WerE SeVeN ShrnaS ThAt SeNsitiZeD cElLS to trAIL. ThEsE WeRe TARGeTing five genes, NAmEly *suV39H2* (41±2%), *g9a* (42±2%), *nR2f2* (45±4%), *RING1a* (48±2 or 50±3%) aND *KDm2b* (41±2 Or 44±2%) ([fIgUrE 1E](#FIg1){rEF-tYpE="fIG"}). WE thEn FOCUSEd On thE gEnES *rING1a* aND *kdm2B*, WhoSe KNoCkdOWn LEd tO a pHEnOTyPe WITh two InDepENDEnT SHRNAS ([FigUrE 1e](#FIg1){ReF-TYpE="FIg"}). takeN tOGEthEr, OuR sCreEN iDENtifIEd kDM2b aNd rIng1, An H3K36-sPecIfIC DeMeTHYlasE and aN E3 UbiqUiTIN-pRoTeIn ligase Of H2aK119, rESPectiVELy, As nOVEL REguLatORS OF TRAIl ReSponse.
LOss of KDM2b COOpERATEs wITH TraiL tO rEdUCe gBm CELL ViabILiTY
----------------------------------------------------------------
TO asSEss THe fUNcTioN of thE NOvEL aPOptOSIS-MoDulATInG cmEs we IdENtiFIEd iN gBmS, WE FIRsT chECKED tHe knOCkDOwn EfFiCieNCY of ThE SHRNaS aNd OBseRveD silEnCinG down To 17--40% ([sUppLEMeNTARY FIGuRe 1](#SuP1){REf-tYpE="suPpLeMeNTAry-mATERIAl"}). WE ThEn FOcusEd on KDM2B, As iTs ProTUmOrIGEnic FUNCTIoNs HAve BeEn demONSTRATeD In variOUS SOlId AnD hemAToLogiCal mAliGnancIes;^[@Bib21],\ [@BIB22]^ hoWEVER, ITS fUnCtiON in gbms waS Not DeFiNED. quAntiTative Rt-Pcr (QRT-PcR) AnalYsis OF *Kdm2B* LEveLS reVEAled tHAT both ShRnAs REDuCeD *KDm2b* MRNA leveLS DoWn TO \~50% ([fIgUre 2a](#Fig2){rEf-Type="fIG"}). hoWEVEr, Shkdm2b-2 leD to a more RoBUst REduCTION aT pRoteIN leVels ([fIguRE 2b](#fIG2){rEf-Type="fig"}). TO ASSesS WhETheR the sHRNaS TaRgetiNG KDM2b Is spEcifIc, wE checkeD ThE LEvEls OF oTHer kdM faMIly meMbERS uPON kDm2B kNoCKdOwn aNd oBserveD No MAJoR ALtEraTionS iN tHEIR leVELs ([SUpPLeMeNtaRY FIGUrE 2](#sup1){ref-TYpe="supPlementArY-maTeRiaL"}).
TO fuRther vaLIDAtE The sCREEN REsULtS, WE ConDuCtEd aTP-BaSED CELl viabiliTy aNAlySIs oF cElLs tRAnSDUCeD wITh BOtH ShkDm2b VeCToRs ANd veriFIEd THaT cElLs WitH ReDuCEd KDm2B eXHIBit ceLl DEaTh siGNifIcantLY MoRe Than The COntrols ([figUre 2c](#Fig2){Ref-type="fIG"}). tO EXAmIne these dIfferEncEs in CEll DEAth FUrTHeR, we used an aSsAY thAT meaSUreS CELL GRoWTH in reAl TimE, wHERe CeLLs' ELECtriCaL IMpEdAnce IN a wELL is mEaSured And TRanSFOrMeD IntO a CelL IndEx. ACcoRdInglY, SilENciNg OF KDm2b nOT ONly AUgMenTeD TraiL response buT AlSO AcCeleRAtEd THE ProCess of ceLl DeAtH ([FIguRe 2D](#Fig2){reF-tYpE="fiG"}). ThERe WaS No ObSerVAblE dIfFeRence bEtWEEn UnTReATEd celLS, ShOwInG ThAt kdM2b KNocKdoWN HAS no sIGNIFicaNT EffEcTS ON SHorT-teRm (24 h) PRoLIfERATioN dyNAmiCS. This PHeNoTYPE WAS ALSo vaLIdAted by lIVE-Cell IMaGInG, WheRE ThE MOrpHology OF iNDIViDUal CElls, As WELl As ceLL deatH pRoCEsseS, Was OBsERveD IN REAl timE ([FIguRE 2e](#fIG2){REF-tYpE="fIg"} AnD [SUpPLEMentaRy viDEOs 1--4](#suP1){ref-TyPe="sUPpLeMEnTary-MateriaL"}). aS sHOWN BY auTOmATed QuanTifICaTiOn oF ceLlUlar BLEbs ThaT WEre INDicatIVE OF aPOPToTic bODieS, THE pRocEsS oF aPoPtosis wAs AcCEleraTeD in SHkDm2b CELLs CompARED WITh conTrOlS ([FIgure 2F](#fIG2){reF-TypE="FIg"}). thE numbEr Of APoPTOTic BODIes PeR fRAme REACHeD itS maxIma wiTHiN 6 H OF TraIl TREaTmeNt in shKDM2b cells, eArlIeR AnD sIgnIFiCAntLy in HIgheR NuMBERS tHAN SHCoNtRol CEllS. TO eXamiNe WHetHeR kdm2b eFfEcTS CaN BE ReCapiTUlated In AddItionAl GbM CElL LiNeS, We uSED a MOre tRaIl-SensiTIve LiNE, T98G IN ParaLLEl. tHEre, kdm2b | Glioblastomamultiforme (GBM) is the most common and aggressive primary brain tumor with a median survival of 12 months.Conventional therapeutic strategies ofradiotherapy and chemotherapy are inadequate toeradicate GBMs because of the diffuse nature of GBM, acquired or innate resistance to therapy and the presence of blood--brain--barrier (BBB).^[@bib1],\ [@bib2]^ Toalter the *status quo* that has remainedunchanged over 25 years, novel targets and therapeutic strategies need tobedeveloped. Evasionofapoptosis is a key hallmark of cancersthat exhibit resistance against therapeutics,^[@bib3]^ making the reactivationofdormant apoptotic programs a favorable approachin treatment. Activating extrinsicapoptosis using death ligands, suchas tumor necrosis factor-related apoptosis-inducingligand (TRAIL), is a promising strategy because of its tumor specificity.^[@bib4]^ Several TRAIL-based therapies suchas recombinant human TRAIL and death receptoragonists have been developed and have shown success in preclinical models.^[@bib5]^ Similarly, activating intrinsic apoptosis by inhibiting the antiapoptoticBcl-2 proteins, Bcl-2 and Bcl-XL, with BH3 peptides has shownsuccess inpreclinical tumor models.^[@bib6],\ [@bib7]^ However, the major obstacle in proapoptotic therapies is innate or acquiredresistance of tumor cells to the proapoptotic agents.^[@bib8]^ Aberrant regulation of the apoptosis pathway components could be responsible for the failure of desired response to the proapoptotic agents.^[@bib3]^ One well-characterized misregulation is theepigenetic silencing of proapoptoticgenes,such asdeathreceptor 4 (*DR4*).^[@bib9]^ However, epigenetic regulation of chemotherapy response of cancer cells hasbeen demonstrated tobe dynamic and reversible.^[@bib10]^ Numerous studieshaveshown that overcoming TRAILresistance is possible by using secondary agents, such as the histone deacetylase inhibitors,^[@bib11],\ [@bib12],\ [@bib13]^ corroborating theideathat the resistancecan be reversed by epigenetic reprogramming. The epigenomeof cells are maintained bydynamic histone and DNAmodifications throughout the chromatin by a group ofchromatin-modifying enzymes(CMEs) called 'writers' such ashistone acetyltransferases, histone methyltransferases (HMTs) and DNA methyltransferases (DNMTs).^[@bib14]^ These modifications can be removed by 'erasers' such as histone deacetylases (HDACs) and histone demethylases (HDMs), or can be recognized by 'readers' such as bromodomain-containingproteins.^[@bib15]^ Althoughthe molecular mechanisms leading to aberrant cancer epigenomesare becoming better understood,^[@bib16],\ [@bib17],\ [@bib18]^most studies in GBM merely focus on DNA hyper/hypomethylation.^[@bib19]^ Therefore, an unbiasedand comprehensive assessment ofroles of CMEs in apoptosis resistance is needed. In this study, we aimed to interrogate the function ofCMEs regulating apoptotic responsein GBM and undertook a loss-of-function approach using short-hairpin RNAs (shRNAs) that were designed against aselect but diverse set of CMEs and associated proteins. We have shownthat lossof KDM2B -- aH3K36-specific histone demethylase -- primed GBM cells for apoptosis throughthe induction of proapoptotic genes and the suppression of antiapoptoticgenes.Our results suggestKDM2B as a novel centralepigenetic regulator of GBM cell apoptosis and identify it as a potential proapoptotic target for future of GBM treatment. Results ======= Interrogation of the CMEsreveals KDM2B as a modulator of TRAIL-induced apoptosisin GBM cells ---------------------------------------------------------------------------------------------- To identify thekey CMEs regulatingthe apoptotic response of GBM cells, we applied a shRNA-based loss-of-function screen in U87MG cell line ([Figure1a](#fig1){ref-type="fig"}). We usedalibrary of 60shRNAs published before,^[@bib20]^ and expandedit to 125shRNAs targeting48 different CME genes withtwo or three separate shRNAs. Thetargeted CMEs included DNMTs, HMTs,HDMs, methylated DNA-binding proteins, polycomb-group proteins (PRCs) and a few transcription factors ([Figure 1b](#fig1){ref-type="fig"}). We first assessedthe effectsof shRNAs alone on U87MG cell viability, to identify shRNAs displaying intrinsic toxicity beforeTRAILtreatment. Two of the 125 shRNAs were eliminated from further analysis in the screen as they caused \~100% cell death after puromycin selection, possiblydue toproblems with viral packaging. Accordingly, 6 out of 125 shRNAsreduced cell viability more than 2 S.D. comparedwith shControl ([Figure 1c](#fig1){ref-type="fig"}). These wereshRNAs targeting KDM5C (68±1%), KDM4C (71±1%),KDM4A (75±1%), KDM4A (75±1%), Set1A (77±1%) and KDM3B (77±1%). Five out of 125 shRNAs targeting MeCP2 (115±1%), MBD1 (115±1%),AHCY (117±5%), TCF3 (117±1%) and EZH1 (121±2%) caused minor increases inviability. However, the majority of the shRNAs did notsignificantly alter cellviability ([Figure 1c](#fig1){ref-type="fig"}). To identify shRNAs that modulated TRAIL sensitivity, we assessedthe percent viability changes after TRAIL treatment. Accordingly, we categorized CMEs into two groupsbased on the phenotype observed upon their silencing compared with shControl cells: (1) suppressors of apoptosis,if their knockdown sensitizedthe cells to TRAIL, (2) enhancers of apoptosis, if theirknockdown conferred cells more TRAIL resistant ([Figure 1d](#fig1){ref-type="fig"}).While TRAIL caused 25±4%reductionin theviabilityofshControl cells, there wereseven shRNAs that sensitized cells to TRAIL. These were targetingfive genes, namely *Suv39H2* (41±2%), *G9A* (42±2%), *NR2F2*(45±4%), *RING1A*(48±2 or50±3%) and *KDM2B* (41±2 or 44±2%) ([Figure 1e](#fig1){ref-type="fig"}). Wethen focusedon the genes *RING1A* and *KDM2B*,whose knockdown led to a phenotype with two independent shRNAs ([Figure 1e](#fig1){ref-type="fig"}). Taken together, our screen identified KDM2B and RING1, an H3K36-specific demethylase and anE3 ubiquitin-protein ligase of H2AK119, respectively, as novel regulatorsof TRAIL response. Lossof KDM2B cooperateswithTRAIL to reduce GBM cell viability ---------------------------------------------------------------- To assess the function of the novel apoptosis-modulating CMEs we identified in GBMs, we first checkedthe knockdown efficiency of theshRNAsand observed silencing down to 17--40%([Supplementary Figure 1](#sup1){ref-type="supplementary-material"}). We then focused on KDM2B, as its protumorigenic functions have been demonstrated in varioussolid and hematological malignancies;^[@bib21],\ [@bib22]^ however, itsfunction inGBMs was notdefined. Quantitative RT-PCR (qRT-PCR) analysisof *KDM2B* levels revealed that both shRNAs reduced *KDM2B* mRNAlevels down to \~50% ([Figure 2a](#fig2){ref-type="fig"}). However, shKDM2B-2 led to a more robust reductionatprotein levels ([Figure 2b](#fig2){ref-type="fig"}). To assess whetherthe shRNAs targeting KDM2B is specific, we checkedthelevels of other KDM family members upon KDM2B knockdown and observed no major alterations in their levels([Supplementary Figure 2](#sup1){ref-type="supplementary-material"}). To further validate thescreen results, weconducted ATP-based cell viability analysis ofcells transduced with both shKDM2B vectors and verified that cells withreduced KDM2B exhibit cell death significantlymore thanthecontrols ([Figure 2c](#fig2){ref-type="fig"}). To examine these differences in cell death further, we used an assay that measures cell growth in real time, where cells' electrical impedancein a well ismeasured and transformedintoa cell index. Accordingly,silencing of KDM2Bnot only augmented TRAIL response but also accelerated the process of cell death ([Figure 2d](#fig2){ref-type="fig"}). There was no observable difference between untreated cells, showing that KDM2B knockdown has no significanteffects on short-term (24 h) proliferationdynamics. This phenotype wasalso validated by live-cell imaging, where the morphology ofindividual cells, aswell as cell death processes,was observed in real time ([Figure 2e](#fig2){ref-type="fig"} and[Supplementary Videos 1--4](#sup1){ref-type="supplementary-material"}). As shown by automatedquantification of cellular blebs that were indicative of apoptotic bodies, the processof apoptosis was accelerated in shKDM2B cells compared withcontrols ([Figure 2f](#fig2){ref-type="fig"}). The number of apoptotic bodies per frame reached its maxima within 6 hofTRAIL treatment in shKDM2B cells, earlier and significantly in higher numbers than shControl cells. To examine whether KDM2B effects can be recapitulated in additional GBM celllines, we used a more TRAIL-sensitive line, T98G in parallel. There, KDM2B | Glioblastoma multiforme (GBM) _is_ the most common _and_ aggressive primary brain tumor with a _median_ survival _of_ 12 months. _Conventional_ therapeutic strategies _of_ _radiotherapy_ and chemotherapy are inadequate to eradicate GBMs because of the diffuse nature of GBM, acquired _or_ innate resistance _to_ _therapy_ and _the_ presence of blood--brain--barrier (BBB).^[@bib1],\ [@bib2]^ To alter _the_ *status quo* _that_ has _remained_ unchanged over 25 years, novel _targets_ and therapeutic strategies need to be _developed._ _Evasion_ of _apoptosis_ is a key hallmark of _cancers_ that exhibit resistance against therapeutics,^[@bib3]^ making the _reactivation_ of dormant apoptotic programs a favorable _approach_ in _treatment._ Activating extrinsic apoptosis using death ligands, _such_ as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), is a promising strategy because of its tumor specificity.^[@bib4]^ _Several_ TRAIL-based _therapies_ such as recombinant human TRAIL and death receptor agonists _have_ been _developed_ _and_ have shown success in _preclinical_ models.^[@bib5]^ Similarly, activating intrinsic apoptosis by inhibiting the antiapoptotic Bcl-2 proteins, Bcl-2 and Bcl-XL, with _BH3_ peptides has shown success in preclinical tumor models.^[@bib6],\ [@bib7]^ However, _the_ _major_ obstacle in proapoptotic therapies is innate or acquired resistance of tumor cells to the proapoptotic agents.^[@bib8]^ Aberrant regulation of the apoptosis pathway components could be responsible for the failure of _desired_ response to the proapoptotic _agents.^[@bib3]^_ One _well-characterized_ misregulation is _the_ _epigenetic_ silencing of proapoptotic genes, such as death receptor 4 (*DR4*).^[@bib9]^ However, epigenetic regulation of chemotherapy response of cancer _cells_ _has_ been demonstrated to _be_ dynamic and reversible.^[@bib10]^ _Numerous_ studies have shown that overcoming TRAIL _resistance_ is possible by using secondary agents, _such_ as the histone deacetylase inhibitors,^[@bib11],\ [@bib12],\ [@bib13]^ corroborating the idea _that_ the _resistance_ can be reversed by epigenetic reprogramming. The epigenome _of_ cells are maintained by dynamic histone and DNA modifications throughout the chromatin by a _group_ of chromatin-modifying enzymes (CMEs) called 'writers' _such_ _as_ histone _acetyltransferases,_ _histone_ methyltransferases (HMTs) and _DNA_ methyltransferases _(DNMTs).^[@bib14]^_ These modifications can _be_ removed by 'erasers' _such_ as _histone_ deacetylases _(HDACs)_ and histone demethylases (HDMs), _or_ can be recognized by 'readers' such _as_ bromodomain-containing proteins.^[@bib15]^ Although the molecular mechanisms leading to aberrant cancer _epigenomes_ are becoming better understood,^[@bib16],\ [@bib17],\ _[@bib18]^_ _most_ studies in GBM merely focus on DNA hyper/hypomethylation.^[@bib19]^ Therefore, an unbiased and _comprehensive_ assessment of roles of CMEs in _apoptosis_ resistance _is_ _needed._ In this study, we aimed to interrogate _the_ _function_ of CMEs regulating apoptotic response in GBM and undertook a _loss-of-function_ approach _using_ short-hairpin RNAs (shRNAs) that were _designed_ against a select but diverse set _of_ CMEs and associated proteins. We have shown that loss of KDM2B -- a H3K36-specific histone demethylase -- primed GBM cells for _apoptosis_ through _the_ induction of proapoptotic genes and the suppression _of_ antiapoptotic _genes._ Our results suggest KDM2B as a novel _central_ epigenetic regulator of GBM _cell_ apoptosis _and_ _identify_ it as a _potential_ proapoptotic target _for_ future of GBM treatment. Results ======= _Interrogation_ of _the_ CMEs _reveals_ _KDM2B_ as a modulator of TRAIL-induced _apoptosis_ in GBM _cells_ ---------------------------------------------------------------------------------------------- To identify the key CMEs regulating _the_ apoptotic _response_ of GBM cells, we _applied_ _a_ shRNA-based loss-of-function screen in _U87MG_ cell line _([Figure_ _1a](#fig1){ref-type="fig"})._ We _used_ a _library_ of 60 _shRNAs_ published before,^[@bib20]^ and expanded it to 125 shRNAs targeting 48 different CME _genes_ with _two_ or three separate shRNAs. _The_ targeted _CMEs_ included _DNMTs,_ HMTs, HDMs, methylated DNA-binding proteins, _polycomb-group_ proteins (PRCs) _and_ a few transcription factors _([Figure_ 1b](#fig1){ref-type="fig"}). We _first_ assessed _the_ effects _of_ shRNAs alone _on_ U87MG _cell_ _viability,_ to identify _shRNAs_ displaying intrinsic toxicity _before_ TRAIL treatment. Two of the 125 shRNAs were _eliminated_ from further analysis _in_ the screen as they _caused_ \~100% cell death after puromycin selection, possibly _due_ to problems with viral packaging. Accordingly, 6 _out_ _of_ _125_ _shRNAs_ _reduced_ cell viability more than _2_ S.D. compared with shControl ([Figure 1c](#fig1){ref-type="fig"}). These were shRNAs _targeting_ KDM5C _(68±1%),_ KDM4C _(71±1%),_ KDM4A (75±1%), KDM4A (75±1%), Set1A _(77±1%)_ and KDM3B (77±1%). Five out _of_ 125 shRNAs targeting MeCP2 (115±1%), _MBD1_ (115±1%), AHCY _(117±5%),_ TCF3 (117±1%) and _EZH1_ (121±2%) caused minor increases in viability. However, the majority of the shRNAs did not significantly alter cell _viability_ ([Figure 1c](#fig1){ref-type="fig"}). _To_ identify shRNAs that modulated TRAIL sensitivity, we assessed the percent viability changes after TRAIL treatment. Accordingly, we categorized CMEs _into_ _two_ groups _based_ on the _phenotype_ _observed_ upon their _silencing_ compared with shControl cells: (1) _suppressors_ of apoptosis, if their knockdown sensitized the cells to TRAIL, (2) _enhancers_ of apoptosis, if their _knockdown_ conferred cells more _TRAIL_ resistant _([Figure_ _1d](#fig1){ref-type="fig"})._ While TRAIL _caused_ 25±4% reduction in the viability of shControl cells, there were _seven_ shRNAs that sensitized cells to TRAIL. These were targeting five genes, namely *Suv39H2* (41±2%), *G9A* (42±2%), *NR2F2* _(45±4%),_ _*RING1A*_ (48±2 or 50±3%) and *KDM2B* (41±2 or 44±2%) ([Figure 1e](#fig1){ref-type="fig"}). We then focused on _the_ _genes_ *RING1A* and *KDM2B*, whose knockdown led to a phenotype with two independent shRNAs ([Figure 1e](#fig1){ref-type="fig"}). Taken together, our screen identified KDM2B and RING1, an H3K36-specific demethylase _and_ an E3 ubiquitin-protein ligase of H2AK119, respectively, _as_ novel regulators of TRAIL response. _Loss_ of _KDM2B_ cooperates _with_ TRAIL to reduce GBM cell viability ---------------------------------------------------------------- To _assess_ the function of the novel apoptosis-modulating CMEs we identified _in_ _GBMs,_ _we_ first checked the _knockdown_ efficiency of _the_ shRNAs and observed _silencing_ _down_ to 17--40% ([Supplementary Figure 1](#sup1){ref-type="supplementary-material"}). _We_ then focused on KDM2B, as its protumorigenic functions have been demonstrated in various solid and hematological malignancies;^[@bib21],\ [@bib22]^ however, its function in GBMs was not defined. _Quantitative_ RT-PCR (qRT-PCR) analysis of *KDM2B* levels revealed that both shRNAs reduced *KDM2B* mRNA levels down _to_ _\~50%_ ([Figure 2a](#fig2){ref-type="fig"}). However, _shKDM2B-2_ led to a more robust reduction _at_ _protein_ levels ([Figure 2b](#fig2){ref-type="fig"}). _To_ _assess_ _whether_ _the_ _shRNAs_ targeting KDM2B is _specific,_ _we_ checked the levels of other KDM family members upon KDM2B knockdown _and_ _observed_ no major alterations in their levels ([Supplementary Figure 2](#sup1){ref-type="supplementary-material"}). To further validate _the_ _screen_ results, we conducted ATP-based cell viability analysis of _cells_ transduced with both shKDM2B vectors and verified that _cells_ with _reduced_ _KDM2B_ exhibit cell death significantly more than the controls _([Figure_ 2c](#fig2){ref-type="fig"}). To examine _these_ differences _in_ _cell_ _death_ _further,_ we used _an_ _assay_ _that_ measures cell growth _in_ real _time,_ where cells' electrical impedance in a well is measured _and_ transformed into a cell index. Accordingly, _silencing_ of KDM2B not _only_ augmented TRAIL _response_ _but_ also accelerated the process of _cell_ death ([Figure 2d](#fig2){ref-type="fig"}). There was no observable difference between _untreated_ cells, _showing_ that KDM2B knockdown has no significant effects on short-term (24 h) _proliferation_ dynamics. This phenotype was also validated by _live-cell_ imaging, where the morphology of individual cells, as well as cell death processes, _was_ observed in _real_ time ([Figure 2e](#fig2){ref-type="fig"} and [Supplementary Videos 1--4](#sup1){ref-type="supplementary-material"}). As shown by _automated_ quantification of _cellular_ _blebs_ _that_ were indicative _of_ apoptotic _bodies,_ the process of _apoptosis_ was accelerated _in_ shKDM2B cells compared _with_ controls ([Figure 2f](#fig2){ref-type="fig"}). The number of apoptotic bodies per frame _reached_ its _maxima_ within _6_ h of TRAIL treatment in shKDM2B _cells,_ earlier and significantly in _higher_ numbers than shControl cells. To _examine_ whether _KDM2B_ _effects_ _can_ be recapitulated in additional _GBM_ cell lines, we used a more TRAIL-sensitive line, T98G in parallel. _There,_ KDM2B |
1. Introduction {#sec1-ijerph-17-04838}
===============
Recent studies show a rise in overweight and obesity among children and adolescents to 18% worldwide in 2016 \[[@B1-ijerph-17-04838]\], with one in five school-aged children in Europe being overweight or obese \[[@B2-ijerph-17-04838],[@B3-ijerph-17-04838]\]. Also in The Netherlands, childhood obesity rates are high. In 2019, 12% of children aged 4 to 12 were overweight or obese \[[@B4-ijerph-17-04838]\]. Overweight or obesity in childhood is especially problematic, since this often continues into adolescence and adulthood and is related to an increased risk of negative health consequences, such as type II diabetes, hypertension, respiratory disease and various types of cancer \[[@B2-ijerph-17-04838],[@B3-ijerph-17-04838],[@B5-ijerph-17-04838]\]. Overweight and obesity are preventable and treatable \[[@B6-ijerph-17-04838]\], especially at a young age \[[@B5-ijerph-17-04838],[@B7-ijerph-17-04838]\], by improving healthy nutrition behaviours and increasing physical activity (PA) (also called energy balance-related behaviours (EBRBs)) \[[@B8-ijerph-17-04838],[@B9-ijerph-17-04838],[@B10-ijerph-17-04838]\]. To target children's EBRBs \[[@B8-ijerph-17-04838],[@B11-ijerph-17-04838]\], multiple settings \[[@B12-ijerph-17-04838]\], such as the school \[[@B13-ijerph-17-04838]\] and home environment should be involved in interventions \[[@B8-ijerph-17-04838],[@B13-ijerph-17-04838]\].
Since children spend a large proportion of their weekdays at school, and schools reach many children, schools have been a popular intervention setting for decades \[[@B13-ijerph-17-04838],[@B14-ijerph-17-04838],[@B15-ijerph-17-04838]\]. However, the influence of the home setting on children's EBRBs is profound, especially for young children, since parents determine the food availability at home, and influence children's nutrition and PA behaviour by practices like modelling and rule setting \[[@B16-ijerph-17-04838],[@B17-ijerph-17-04838],[@B18-ijerph-17-04838],[@B19-ijerph-17-04838]\]. Therefore it is important to also focus on the home setting when implementing school-based energy balance-related interventions \[[@B15-ijerph-17-04838],[@B17-ijerph-17-04838]\].
Most effective changes in the home setting are accomplished through interventions with direct parental involvement (e.g., parents attending educational sessions, or counselling sessions) \[[@B13-ijerph-17-04838],[@B16-ijerph-17-04838],[@B19-ijerph-17-04838],[@B20-ijerph-17-04838],[@B21-ijerph-17-04838]\]. However, directly involving parents is often resource- and labour-intensive, making these types of interventions less feasible \[[@B20-ijerph-17-04838]\]. Also, the recruitment \[[@B22-ijerph-17-04838]\] and prolonged engagement of parents in these types of interventions have proven to be challenging \[[@B3-ijerph-17-04838],[@B22-ijerph-17-04838],[@B23-ijerph-17-04838]\]. Only about one-third of invited families participate in any intervention activity \[[@B3-ijerph-17-04838],[@B24-ijerph-17-04838]\], 40 to 60% of whom drop out \[[@B24-ijerph-17-04838]\]. In addition, the participants of parental involvement interventions tend to be mostly high socioeconomic status (SES) parents \[[@B25-ijerph-17-04838]\], and it is particularly challenging to engage parents with a low SES \[[@B3-ijerph-17-04838],[@B25-ijerph-17-04838]\], a lower educational level, single parents and those of ethnic minority groups \[[@B24-ijerph-17-04838]\]. This is discouraging, since these parents/families are most in need of interventions. For example, research from a large Dutch cohort study has shown that there are socioeconomic and ethnic inequalities in child health \[[@B26-ijerph-17-04838]\]. At a young age, non-Western children were more likely to be overweight compared to Dutch children, with mother's educational level being one of the contributing factors in explaining this higher prevalence in overweight in these children \[[@B27-ijerph-17-04838]\]. Beyond socioeconomic health disparities, child characteristics and the supportiveness of the home environment to perform healthy behaviours are also important predictors of children's PA and nutrition behaviour. For instance, children's nutrition behaviour is determined by their preference for healthy and/or unhealthy foods, and their PA and sedentary behaviour is associated with their activity preferences \[[@B28-ijerph-17-04838]\]. Also, children raised in an environment in which healthy nutrition \[[@B29-ijerph-17-04838]\] and sufficient PA \[[@B29-ijerph-17-04838],[@B30-ijerph-17-04838]\] are less valued are more at risk for developing unhealthy EBRBs. Therefore, vulnerability of the population is not restricted to well-known socioeconomic differences, but also includes specific child preferences and the health climate regarding EBRBs in the home environment.
To involve more parents, and particularly those of vulnerable populations, the strategy of indirect parental involvement is an alternative. Even though indirect parental involvement is assumed to be less effective in changing health behaviours compared to direct parental involvement, it can lead to greater adoption and implementation rates \[[@B21-ijerph-17-04838]\]. In indirect parental involvement, parents are engaged in a way that the intervention implementers do not communicate or engage directly (i.e., face to face, or personally) with them. Instead, parents are informed via school media, or children function as the messenger. Examples of indirect parental involvement are the provision of information (newsletters, tip sheets), invitations to participate in or attend activities (events, educational sessions) and prompts or assignments directed at the child and/or parent with the aim of involving parents \[[@B19-ijerph-17-04838]\]. Previous research has shown that some of these strategies are more promising than others, for example prompting children to engage in intervention activities together with their parent(s) seems more promising than providing newsletters or invitations for optional intervention activities \[[@B17-ijerph-17-04838],[@B19-ijerph-17-04838]\]. The difference between these strategies could be described as passive (not requiring a specific action or response on the part of the parents) indirect involvement versus active (performing activities) indirect involvement.
To our knowledge, there is little to no research on the participation rates of the strategy of the latter type of indirect parental involvement in school-based interventions, while this information is crucial to be able to draw conclusions on potential strategies to involve parents in (school-based) interventions \[[@B21-ijerph-17-04838]\]. Given the fact that children of vulnerable populations are less active, more sedentary and have unhealthier diets \[[@B31-ijerph-17-04838],[@B32-ijerph-17-04838],[@B33-ijerph-17-04838]\], it is possible that these children and their parents are less interested in energy balance-related interventions. Therefore, gaining insight in participation rates of children and parents in interventions using an indirect parental involvement strategy is needed. Moreover, empirical evidence is lacking on who is engaged in this strategy and therefore, it is warranted to know whether vulnerable children and parents engage in these interventions using this strategy.
In the current study, we evaluated the potential of Challenge Me to engage children and parents, especially those of vulnerable populations. Challenge Me was a parental involvement intervention in which children are challenged to perform PA and nutrition-related activities by themselves and with their parents. The main aim of the current study is to examine whether conducting challenges is a feasible intervention strategy to engage children and parents in school-based energy balance-related interventions. With engagement, we refer to participation in the intervention, i.e., performing the intervention activities, and not enrolment. Additionally, a second aim is to gain insight in whether children in need for improvements, i.e., vulnerable populations, are engaged in the intervention using this indirect-involvement strategy. For this aim, we focussed on child demographics, child characteristics (preferences) and the climate towards health behaviours in the home environment.
2. Materials and Methods {#sec2-ijerph-17-04838}
========================
2.1. Design {#sec2dot1-ijerph-17-04838}
-----------
An exploratory cross-sectional study design was conducted. The Medical Ethics Committee of the Maastricht University Medical Centre and Maastricht University provided ethical approval for this study (METC163027, national number: NL58554.068.16).
2.2. Challenge Me Intervention {#sec2dot2-ijerph-17-04838}
------------------------------
### 2.2.1. Intervention Development {#sec2dot2dot1-ijerph-17-04838}
The intervention, called Challenge Me, challenged children to perform PA and nutrition-related challenges together with their parent(s) or guardian(s). Challenge Me has been developed as part of a larger evaluation \[[@B34-ijerph-17-04838]\], and is specifically focused on improving parental involvement. In the larger evaluation study, eight primary schools, located in low SES neighbourhoods (based on scoring of The Netherlands Institute for | 1. introduction { # sec1 - ijerph - 17 - 04838 } = = = = = = = = = = = = = = = recent studies show a rise in age and obesity among children and adolescents to 18 % worldwide in 2016 \ [ [ @ b1 - ijerph - 17 - 04838 ] \ ], with one in five school - aged children in europe under overweight or obese \ [ [ @ b2 - ijerph - 17 - 04838 ], [ @ b3 - ijerph - 17 - 04838 ] \ ]. also in the netherlands, childhood obesity rates are high. in 2019, 12 % of children aged 4 to 12 were overweight or obese \ [ [ @ b4 - ijerph - 17 - 04838 ] \ ]. overweight or obesity in africa is especially problematic, since this often continues into adolescence and adulthood it is related for an increased risk of negative health risks, such as type ii diabetes, hypertension, respiratory disease and various types of cancer \ [ [ @ b2 - ijerph - 17 - 04838 ], [ @ b3 - ijerph - 17 - 04838 ], [ @ b5 - ijerph - 17 - 04838 ] \ ]. overweight and obesity are preventable and treatable \ [ [ @ b6 - ijerph - 17 - 04838 ] \ ], especially at a young age \ [ [ @ b5 - ijerph - 17 - 04838 ], [ @ b7 - ijerph - 5 - 108 ] \ ], by promoting healthy nutrition behaviours and increasing physical activity ( pa ) ( also called energy balance - related behaviours ( ebrbs ) ) \ [ [ @ b8 - ijerph - 17 - 04838 ], [ @ b9 - ijerph - 17 - 04838 ], [ @ b10 - ijerph - 17 - 04838 ] \ ]. to target children ' s ebrbs \ [ [ @ b8 - ijerph - 17 - 04838 ], [ @ b11 - ijerph - 17 - 04838 ] \ ], multiple settings \ [ [ @ b12 - ijerph - 17 - 04838 ] \ ], such as the school \ [ [ @ c2 - ijerph - 17 - 04838 ] \ ] and home environment should be involved in interventions \ [ [ @ b8 - ijerph - 17 - 04838 ], [ @ b13 - ijerph - 17 - 04838 ] \ ]. since children spend a large proportion of their weekdays at school, and schools reach many children, schools have been a popular intervention setting for decades \ [ [ @ b13 - ijerph - 17 - 04838 ], [ @ b14 - ijerph - 17 - 04838 ], [ @ b15 - ijerph - 17 - 04838 ] \ ]. however, the influence of the home setting on children ' s ebrbs is profound, especially for young children, since parents determine the food availability at home, and influence children ' s nutrition and pa behaviour by practices like modelling and rule setting \ [ [ @ b16 - ijerph - 17 - 04838 ], [ @ b17 - ijerph - 17 - 04838 ], [ @ b18 - ijerph - 17 - 04838 ], [ @ b19 - ijerph - 17 - 04838 ] \ ]. therefore it is important to also focus on the home setting when implementing school - based energy balance - related interventions \ [ [ @ b15 - ijerph - 17 - 04838 ], [ @ b17 - ijerph - 17 - 04838 ] \ ]. most effective changes in the home setting are accomplished through interventions with direct parental involvement ( e. g., parents attending educational sessions, or counselling sessions ) \ [ [ @ b13 - ijerph - 17 - 04838 ], [ @ b16 - ijerph - 17 - 04838 ], [ @ b19 - ijerph - 17 - 04838 ], [ @ b20 - ijerph - 17 - 04838 ], [ @ b21 - ijerph - 17 - 04838 ] \ ]. however, directly involving parents is often resource - and labour - intensive, making these types of interventions less feasible \ [ [ @ b20 - ijerph - 17 - 04838 ] \ ]. also, the recruitment \ [ [ @ b22 - ijerph - 17 - 04838 ] \ ] and prolonged engagement of parents in these types of interventions have proven to be challenging \ [ [ @ b3 - ijerph - 17 - 04838 ], [ @ b22 - ijerph - 17 - 04838 ], [ @ b23 - ijerph - 17 - 04838 ] \ ]. only about one - third of invited families participate in any intervention activity \ [ [ @ b3 - ijerph - 17 - 04838 ], [ @ b24 - ijerph - 17 - 04838 ] \ ], 40 to 60 % of whom drop out \ [ [ @ b24 - ijerph - 17 - 04838 ] \ ]. in addition, the participants of parental involvement interventions tend to be mostly high socioeconomic status ( ses ) parents \ [ [ @ b25 - ijerph - 17 - 04838 ] \ ], and it is particularly challenging to engage parents with a low ses \ [ [ @ b3 - ijerph - 17 - 04838 ], [ @ b25 - ijerph - 17 - 04838 ] \ ], a lower educational level, single parents and those of ethnic minority groups \ [ [ @ b24 - ijerph - 17 - 04838 ] \ ]. this is discouraging, since these parents / families are most in need of interventions. for example, research from a large dutch cohort study has shown that there are socioeconomic and ethnic inequalities in child health \ [ [ @ b26 - ijerph - 17 - 04838 ] \ ]. at a young age, non - western children were more likely to be overweight compared to dutch children, with mother ' s educational level being one of the contributing factors in explaining this higher prevalence in overweight in these children \ [ [ @ b27 - ijerph - 17 - 04838 ] \ ]. beyond socioeconomic health disparities, child characteristics and the supportiveness of the home environment to perform healthy behaviours are also important predictors of children ' s pa and nutrition behaviour. for instance, children ' s nutrition behaviour is determined by their preference for healthy and / or unhealthy foods, and their pa and sedentary behaviour is associated with their activity preferences \ [ [ @ b28 - ijerph - 17 - 04838 ] \ ]. also, children raised in an environment in which healthy nutrition \ [ [ @ b29 - ijerph - 17 - 04838 ] \ ] and sufficient pa \ [ [ @ b29 - ijerph - 17 - 04838 ], [ @ b30 - ijerph - 17 - 04838 ] \ ] are less valued are more at risk for developing unhealthy ebrbs. therefore, vulnerability of the population is not restricted to well - known socioeconomic differences, but also includes specific child preferences and the health climate regarding ebrbs in the home environment. to involve more parents, and particularly those of vulnerable populations, the strategy of indirect parental involvement is an alternative. even though indirect parental involvement is assumed to be less effective in changing health behaviours compared to direct parental involvement, it can lead to greater adoption and implementation rates \ [ [ @ b21 - ijerph - 17 - 04838 ] \ ]. in indirect parental involvement, parents are engaged in a way that the intervention implementers do not communicate or engage directly ( i. e., face to face, or personally ) with them. instead, parents are informed via school media, or children function as the messenger. examples of indirect parental involvement are the provision of information ( newsletters, tip sheets ), invitations to participate in or attend activities ( events, educational sessions ) and prompts or assignments directed at the child and / or parent with the aim of involving parents \ [ [ @ b19 - ijerph - 17 - 04838 ] \ ]. previous research has shown that some of these strategies are more promising than others, for example prompting children to engage in intervention activities together with their parent ( s ) seems more promising than providing newsletters or invitations for optional intervention activities \ [ [ @ b17 - ijerph - 17 - 04838 ], [ @ b19 - ijerph - 17 - 04838 ] \ ]. the difference between these strategies could be described as passive ( not requiring a specific action or response on the part of the parents ) indirect involvement versus active ( performing activities ) indirect involvement. to our knowledge, there is little to no research on the participation rates of the strategy of the latter type of indirect parental involvement in school - based interventions, while this information is crucial to be able to draw conclusions on potential strategies to involve parents in ( school - based ) interventions \ [ [ @ b21 - ijerph - 17 - 04838 ] \ ]. given the fact that children of vulnerable populations are less active, more sedentary and have unhealthier diets \ [ [ @ b31 - ijerph - 17 - 04838 ], [ @ b32 - ijerph - 17 - 04838 ], [ @ b33 - ijerph - 17 - 04838 ] \ ], it is possible that these children and their parents are less interested in energy balance - related interventions. therefore, gaining insight in participation rates of children and parents in interventions using an indirect parental involvement strategy is needed. moreover, empirical evidence is lacking on who is engaged in this strategy and therefore, it is warranted to know whether vulnerable children and parents engage in these interventions using this strategy. in the current study, we evaluated the potential of challenge me to engage children and parents, especially those of vulnerable populations. challenge me was a parental involvement intervention in which children are challenged to perform pa and nutrition - related activities by themselves and with their parents. the main aim of the current study is to examine whether conducting challenges is a feasible intervention strategy to engage children and parents in school - based energy balance - related interventions. with engagement, we refer to participation in the intervention, i. e., performing the intervention activities, and not enrolment. additionally, a second aim is to gain insight in whether children in need for improvements, i. e., vulnerable populations, are engaged in the intervention using this indirect - involvement strategy. for this aim, we focussed on child demographics, child characteristics ( preferences ) and the climate towards health behaviours in the home environment. 2. materials and methods { # sec2 - ijerph - 17 - 04838 } = = = = = = = = = = = = = = = = = = = = = = = = 2. 1. design { # sec2dot1 - ijerph - 17 - 04838 } - - - - - - - - - - - an exploratory cross - sectional study design was conducted. the medical ethics committee of the maastricht university medical centre and maastricht university provided ethical approval for this study ( metc163027, national number : nl58554. 068. 16 ). 2. 2. challenge me intervention { # sec2dot2 - ijerph - 17 - 04838 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - # # # 2. 2. 1. intervention development { # sec2dot2dot1 - ijerph - 17 - 04838 } the intervention, called challenge me, challenged children to perform pa and nutrition - related challenges together with their parent ( s ) or guardian ( s ). challenge me has been developed as part of a larger evaluation \ [ [ @ b34 - ijerph - 17 - 04838 ] \ ], and is specifically focused on improving parental involvement. in the larger evaluation study, eight primary schools, located in low ses neighbourhoods ( based on scoring of the netherlands institute for | 1. Introduction {# sec1 - ijerph - 17 - 04838} = = = = = = = = = = = = = = = Recent studies show a rise in overweight and obesity among children and adolescents to 18% worldwide in 2016 \ [[ @ B1 - ijerph - 17 - 04838] \ ], with one in five school - Qg#d children in Europe being overweight or obese \ [[ @ B2 - ijerph - 17 - 04838 ], [@ B3 - ijerph - 17 - 04838] \ ]. Also in The Netherlands, childhood obesity rates are high. In 2019, 12% of children aged 4 to 12 were overweight or obese \ [[ @ B4 - ijerph - 17 - 04838] \ ]. Overweight or obesity in childhood is especially problematic, since this often continues into adolescence and adulthood and is related to an increased risk of negative health consequences, such as type II diabetes, hypertension, respiratory disease and various types of cancer \ [[ @ B2 - ijerph - 17 - 04838 ], [@ B3 - ijerph - 17 - 04838 ], [@ B5 - ijerph - 17 - 04838] \ ]. Overweight and obesity are preventable and treatable \ [[ @ B6 - ijerph - 17 - 04838] \ ], especially at a young age \ [[ @ B5 - ijerph - 17 - 04838 ], [@ B7 - ijerph - 17 - 04838] \ ], by improving healthy nutrition behaviours and increasing physical aftlvity (PA) (also called energy balance - related behaviours (EBRBs) ) \ [[ @ B8 - ijerph - 17 - 04838 ], [@ B9 - ijerph - 17 - 04838 ], [@ B10 - ijerph - 17 - 04838] \ ]. To target children ' s EBRBs \ [[ @ B8 - ijerph - 17 - 04838 ], [@ B11 - ijerph - 17 - 04838] \ ], multiple settings \ [[ @ B12 - ijerph - 17 - 04838] \ ], such as the school \ [[ @ B13 - ijerph - 17 - 04838] \] and home environment should be involved in interventions \ [[ @ B8 - ijerph - 17 - 04838 ], [@ B13 - ijerph - 17 - 04838] \ ]. Since children spend a large proportion of their weekdays at school, and schools reach many children, schools have been a popular intervention setting for decades \ [[ @ B13 - ijerph - 17 - 04838 ], [@ B14 - ijerph - 17 - 04838 ], [@ B15 - ijerph - 17 - 04838] \ ]. However, the influence of the home setting on children ' s EBRBs is profound, especially for young children, since parents determine the food availability at home, and influence children ' s nutrition and PA behaviour by practices like modelling and rule setting \ [[ @ B16 - ijerph - 17 - 04838 ], [@ B17 - ijerph - 17 - 04838 ], [@ B18 - ijerph - 17 - 04838 ], [@ B19 - ijerph - 17 - 04838] \ ]. Therefore it is important to also focus on the home setting when implementing school - based energy balance - related interventions \ [[ @ B15 - UjerpU - 17 - 04838 ], [@ B17 - ijerph - 17 - 04838] \ ]. Most effective changes in the home setting are accomplished through interventions with direct parental involvement (e. g. , parents attending educational sessions, or counselling sessions) \ [[ @ B13 - ijerph - 17 - 04838 ], [@ B16 - ijerph - 17 - 04838 ], [@ B19 - ijerph - 17 - 04838 ], [@ B20 - ijerph - 17 - 04838 ], [@ B21 - ijerph - 17 - 04838] \ ]. However, directly involving parents is often resource - and labour - intensive, making these types of interventions less feasible \ [[ @ B20 - ijerph - 17 - 04838] \ ]. Also, the recruitment \ [[ @ B22 - ijerph - 17 - 04838] \] and prolonged engagement of parents in these types of interventions have proven to be challenging \ [[ @ B3 - ijerph - 17 - 04838 ], [@ B22 - ijerph - 17 - 04838 ], [@ B23 - ijerph - 17 - 04I3O] \ ]. Only about one - third of invited families participate in any intervention activity \ [[ @ B3 - ijerph - 17 - 04838 ], [@ B24 - ijerph - 17 - 04838] \ ], 40 to 60% of whom drop out \ [[ @ B24 - ijerph - 17 - 04838] \ ]. In addition, the participants of parental involvement interventions tend to be mostly high socioeconomic status (SES) parents \ [[ @ B25 - ijerph - 17 - 04838] \ ], and it is particularly challenging to engage parents with a low SES \ [[ @ B3 - ijerph - 17 - 04838 ], [@ B25 - ijerph - 17 - 04838] \ ], a lower educational level, single parents and those of ethnic minority groups \ [[ @ B24 - ijerph - 17 - 04838] \ ]. This is discouraging, since these parents / families are most in need of interventions. For example, research from a large Dutch cohort study has shown that there are socioeconomic and ethnic inequalities in child health \ [[ @ B26 - ijerph - 17 - 04838] \ ]. At a young age, non - Western children were more likely to be overweight compared to Dutch children, with mother ' s 4ducat7onal level being one of the contributing factors in explaining this higher prevalence in overweight in these children \ [[ @ B27 - ijerph - 17 - 04838] \ ]. Beyond socioeconomic health disparities, child characteristics and the supportiveness of the home environment to perform healthy behaviours are also important predictors of children ' s PA and nutrition behaviour. For instance, children ' s nutrition behaviour is determined by their preference for healthy and / or unhealthy foods, and their PA and sedentary behaviour is associated with their activity preferences \ [[ @ B28 - ijerph - 17 - 04838] \ ]. Also, children raised in an environment in which healthy nutrition \ [[ @ B29 - ijerph - 17 - 04838] \] and sufficient PA \ [[ @ B29 - ijerph - 17 - 04838 ], [@ B30 - ijerph - 17 - 04838] \] are less valued are more at risk for developing unhealthy EBRBs. Therefore, vulnerability of the population is not restricted to well - known socioeconomic differences, but also includes specific child preferences and the health climate regarding EBRBs in the home environment. To involve more parents, and particularly those of vulnerable populations, the strategy of indirect parental involvement is an alternative. Even though indirect parental involvement is assumed to be less effective in changing health behaviours compared to direct parental involvement, it can lead to greater adoption and implementation rates \ [[ @ B21 - ijerph - 17 - 04838] \ ]. In indirect parental involvement, parents are engaged in a way that the intervention implementers do not communicate or engage directly (i. e. , face to face, or personally) with them. Instead, parents are informed via school media, or children function as the messenger. Examples of indirect parental involvement are the provision of information (newsletters, tip sheets ), invitations to participate in or attend activities (events, educational sessions) and prompts or assignments directed at the child and / or parent with the aim of involving parents \ [[ @ B19 - ijerph - 17 - 04838] \ ]. Previous research has shown that some of these strategies are more promising than others, for example prompting children to engage in intervention activities together with their parent (s) seems more promising than providing newsletters or invitations for optional intervention activities \ [[ @ B17 - ijerph - 17 - 04838 ], [@ B19 - ijerph - 17 - 04838] \ ]. The difference between these strategies could be described as passive (not requiring a specific action or response on the part of the parents) indirect involvement versus active (performing acHidities) indirect involvement. To our knowledge, there is little to no research on the participation rates of the strategy of the latter type of indirect parental involvement in school - based interventions, while this information is crucial to be able to deWw conclusions on potential strategies to involve parents in (school - based) interventions \ [[ @ B21 - ijerph - 17 - 04838] \ ]. Given the fact that children of vulnerable populations are less active, more sedentary and have unhealthier diets \ [[ @ B31 - ijerph - 17 - 04838 ], [@ B32 - ijerph - 17 - 04838 ], [@ B33 - ijerph - 17 - 04838] \ ], it is possible that these children and their parents are less interested in energy balance - related interventions. Therefore, gaining insight in participation rates of children and parents in interventions using an indirect parental involvement strategy is needed. Moreover, empirical evidence is lacking on who is engaged in this strategy and therefore, it is warranted to know whether vulnerable children and parents engage in these interventions using this strategy. In the current sHudJ, we evaluated the potential of Challenge Me to engage children and parents, especially those of vulnerable populations. Challenge Me was a parental involvement intervention in which children are challenged to perform PA and nutrition - related activities by themselves and with their parents. The main aim of the current study is to examine whether conducting challenges is a feasible intervention strategy to engage children and parents in school - based energy balance - related interventions. With engagement, we refer to participation in the intervention, i. e. , performing the intervention activities, and not enrolment. Additionally, a second aim is to gain insight in whether children in need for improvements, i. e. , vulnerable populations, are engaged in the intervention using this indirect - involvement strategy. For this aim, we focussed on child demographics, child characteristics (preferences) and the climate towards health behaviours in the home environment. 2. Materials and Methods {# sec2 - ijerph - 17 - 04838} = = = = = = = = = = = = = = = = = = = = = = = = 2. 1. Design {# sec2dot1 - ijerph - 17 - 04838} - - - - - - - - - - - An exploratory cross - sectional study design was conducted. The Medical Ethics Committee of the Maastricht University Medical Centre and Maastricht University provided ethical approval for this study (METC163027, national number: NL58554. 068. 16 ). 2. 2. Challenge Me Intervention {# sec2dot2 - ijerph - 17 - 04838} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - # # # 2. 2. 1. Intervention Development {# sec2dot2dot1 - ijerph - 17 - 04838} The intervention, called Challenge Me, challenged children to perform PA and nutrition - related challenges together with their parent (s) or guardian (s ). Challenge Me has been developed as part of a larger evaluation \ [[ @ B34 - ijerph - 17 - 04838] \ ], and is specifically focused on improving parental invpivement. In the larger evaluation study, eight primary schools, located in low SES neighbourhoods (based on sxorkng of The Netherlands Institute for | 1. Introduction {#sec1-ijerph-17-04838} =============== Recent show a in overweight and obesity children and adolescents to 18% worldwide in 2016 \[[@B1-ijerph-17-04838]\], with in five school-aged children in Europe being overweight or obese \[[@B2-ijerph-17-04838],[@B3-ijerph-17-04838]\]. Also in The Netherlands, childhood rates are high. In 2019, 12% of children aged 4 to 12 were overweight or obese \[[@B4-ijerph-17-04838]\]. Overweight or obesity in childhood is especially problematic, this often continues into adolescence and adulthood and is to an increased risk negative health consequences, such type II diabetes, hypertension, disease and various types cancer \[[@B2-ijerph-17-04838],[@B3-ijerph-17-04838],[@B5-ijerph-17-04838]\]. Overweight and obesity are preventable and treatable \[[@B6-ijerph-17-04838]\], especially at a young \[[@B5-ijerph-17-04838],[@B7-ijerph-17-04838]\], by improving healthy nutrition behaviours and increasing physical activity (PA) (also called energy balance-related behaviours (EBRBs)) \[[@B8-ijerph-17-04838],[@B9-ijerph-17-04838],[@B10-ijerph-17-04838]\]. To target children's EBRBs \[[@B8-ijerph-17-04838],[@B11-ijerph-17-04838]\], multiple \[[@B12-ijerph-17-04838]\], such as the school \[[@B13-ijerph-17-04838]\] and home environment should be involved in interventions \[[@B8-ijerph-17-04838],[@B13-ijerph-17-04838]\]. Since children spend a proportion of their weekdays at and schools reach many children, schools have been a popular intervention setting for decades \[[@B13-ijerph-17-04838],[@B14-ijerph-17-04838],[@B15-ijerph-17-04838]\]. However, the influence of the home on children's EBRBs is profound, especially for young children, since parents determine the food availability at home, and influence and PA by practices like modelling and rule setting \[[@B16-ijerph-17-04838],[@B17-ijerph-17-04838],[@B18-ijerph-17-04838],[@B19-ijerph-17-04838]\]. Therefore it is important to also focus on the home setting when implementing school-based energy balance-related interventions \[[@B15-ijerph-17-04838],[@B17-ijerph-17-04838]\]. Most effective in the setting through with parental involvement (e.g., parents attending educational sessions, or counselling sessions) \[[@B13-ijerph-17-04838],[@B16-ijerph-17-04838],[@B19-ijerph-17-04838],[@B20-ijerph-17-04838],[@B21-ijerph-17-04838]\]. However, involving parents is often resource- and labour-intensive, making these types of interventions less feasible \[[@B20-ijerph-17-04838]\]. Also, the recruitment \[[@B22-ijerph-17-04838]\] and prolonged engagement of parents in these types of interventions have proven be challenging \[[@B3-ijerph-17-04838],[@B22-ijerph-17-04838],[@B23-ijerph-17-04838]\]. Only one-third of invited participate in any intervention activity \[[@B3-ijerph-17-04838],[@B24-ijerph-17-04838]\], 40 to 60% of whom drop out In participants of parental involvement interventions tend to be mostly high socioeconomic status (SES) parents \[[@B25-ijerph-17-04838]\], and it is particularly challenging to engage parents with a low SES \[[@B3-ijerph-17-04838],[@B25-ijerph-17-04838]\], a lower level, single parents and those of ethnic minority groups \[[@B24-ijerph-17-04838]\]. This is since these parents/families are most in need of interventions. For example, research from a large Dutch cohort study has shown there are socioeconomic and ethnic inequalities in child health At a age, non-Western children were more likely to be compared to Dutch children, with mother's educational level being one of the contributing factors in explaining this higher prevalence in overweight in these children \[[@B27-ijerph-17-04838]\]. Beyond socioeconomic health disparities, characteristics and the supportiveness of the home environment to perform healthy behaviours are also important predictors of children's PA and nutrition behaviour. For instance, children's nutrition behaviour is determined by their preference for healthy and/or unhealthy and their PA and sedentary behaviour associated with their activity preferences \[[@B28-ijerph-17-04838]\]. children raised in an environment in which healthy nutrition \[[@B29-ijerph-17-04838]\] and sufficient PA are less valued more at risk for developing unhealthy EBRBs. Therefore, vulnerability of the population is not restricted to well-known differences, but also includes specific child preferences and the health climate regarding EBRBs in the To more parents, and those of vulnerable populations, the strategy of indirect parental is alternative. Even though parental involvement is assumed to be less effective in changing health behaviours compared to direct parental involvement, it can to greater adoption and implementation rates \[[@B21-ijerph-17-04838]\]. In indirect parental involvement, are engaged in a way that the intervention implementers do communicate or engage directly (i.e., face to face, or personally) with them. Instead, parents informed via school media, or children function as messenger. Examples of indirect parental involvement are the information (newsletters, tip sheets), invitations to participate or attend activities (events, educational sessions) and prompts or assignments directed at the child and/or parent with the aim of involving parents Previous research has shown that some of these strategies are more promising than others, for prompting children to engage in intervention activities together with their parent(s) seems more than providing invitations for optional intervention activities \[[@B17-ijerph-17-04838],[@B19-ijerph-17-04838]\]. The difference between these strategies could be described as (not requiring a specific action or response on the part of the involvement active activities) indirect involvement. To our knowledge, there is to no research on the participation rates of the of the latter type of indirect parental involvement in school-based while this information is crucial to be able to draw conclusions on potential strategies to involve parents in (school-based) interventions \[[@B21-ijerph-17-04838]\]. Given the fact that children of vulnerable populations are less active, more sedentary and have unhealthier diets \[[@B31-ijerph-17-04838],[@B32-ijerph-17-04838],[@B33-ijerph-17-04838]\], is possible that these children and their parents are less interested in energy balance-related interventions. Therefore, gaining insight in participation rates of children parents in interventions using an indirect involvement strategy is needed. Moreover, empirical evidence is lacking on who is engaged in this strategy and therefore, it is warranted know whether vulnerable children parents engage in these interventions using this strategy. In the current study, we evaluated the potential of Challenge Me children and parents, those of vulnerable Challenge Me was a parental involvement intervention which children are challenged to perform and nutrition-related activities by themselves and with their parents. main aim of the current study is examine whether conducting challenges a feasible intervention to engage children and parents in school-based energy interventions. With engagement, we refer to participation in the intervention, i.e., performing intervention activities, and enrolment. Additionally, a second aim is to gain in whether children in need for vulnerable populations, are engaged in the intervention using this indirect-involvement strategy. For this aim, we focussed child demographics, child characteristics and the climate towards health behaviours the home 2. Materials and Methods {#sec2-ijerph-17-04838} ======================== 2.1. Design {#sec2dot1-ijerph-17-04838} ----------- An exploratory cross-sectional study design Medical Ethics Committee of the Maastricht University Centre and Maastricht University provided ethical approval for this study (METC163027, national number: NL58554.068.16). 2.2. Challenge Me Intervention {#sec2dot2-ijerph-17-04838} ------------------------------ ### 2.2.1. Intervention Development {#sec2dot2dot1-ijerph-17-04838} The intervention, Challenge children to perform PA nutrition-related challenges with their parent(s) or guardian(s). Challenge Me been as part of a larger evaluation \[[@B34-ijerph-17-04838]\], is specifically focused on improving parental involvement. In the larger evaluation eight primary schools, located in SES neighbourhoods (based on scoring of The Netherlands Institute for | 1. intRoDUCTION {#sec1-IJeRph-17-04838}
===============
recENT stUdIeS show A RiSE In oVErweighT And ObeSiTy AMong cHiLDRen ANd aDoLeScenTs tO 18% WOrldwIDe in 2016 \[[@B1-IjeRpH-17-04838]\], wITH OnE iN FIvE sCHOoL-AgED ChILdREn IN eurOpe beInG overwEigHT Or oBese \[[@b2-ijerpH-17-04838],[@B3-ijERpH-17-04838]\]. alSo In thE nEThErlANds, ChiLdhooD oBESIty rAtES arE HigH. IN 2019, 12% of ChiLDREn AGED 4 TO 12 WeRe OveRWeIgHT Or obESE \[[@B4-IJERph-17-04838]\]. OVErWEIGHt Or obESitY iN CHiLdhOoD Is especIalLy proBLEMAtIc, sINCe this OFteN CoNTINUeS Into aDOlesCEnCE and aDulTHOod ANd iS reLAtEd tO aN increaSed RISk oF neGAtive HEAlth CoNseQUENces, sUch aS TYpE ii diABeTEs, HypertensioN, resPIRaToRy diSEASE aND VaRiouS TYpEs OF CAnCEr \[[@B2-ijERph-17-04838],[@b3-ijerph-17-04838],[@B5-IjerPH-17-04838]\]. ovErWEIGht AnD OBesITY Are pReVEntAble anD trEatAblE \[[@B6-IJeRPh-17-04838]\], ESpECIAllY at a YouNg AgE \[[@b5-ijERph-17-04838],[@b7-IjeRpH-17-04838]\], by imprOving HEALThy NUtRItIoN BEHAviOuRS And iNcreasinG pHySicaL ACTiVitY (pa) (ALso CALleD ENERgy bAlanCe-ReLAtEd BEHavIoUrs (Ebrbs)) \[[@B8-IJeRPH-17-04838],[@b9-Ijerph-17-04838],[@B10-iJErpH-17-04838]\]. TO taRGET childRen'S EbrBS \[[@b8-IJeRph-17-04838],[@b11-ijERPH-17-04838]\], MulTIPle seTTiNgS \[[@b12-IJeRPh-17-04838]\], sUch as tHe scHooL \[[@b13-IjErpH-17-04838]\] and homE enVIroNMEnT shouLd BE INVOlVED in iNtERVeNtIONS \[[@b8-iJErpH-17-04838],[@b13-IJerpH-17-04838]\].
sInce CHilDReN sPenD a laRge PROpORTIoN OF Their WEEKdAYS at SChooL, aNd SchoOlS ReAch maNy chIlDReN, SChOoLs Have BEEn A PoPUlAr inTERVeNTiON sEtTiNG for DECAdeS \[[@b13-ijERpH-17-04838],[@B14-Ijerph-17-04838],[@B15-IjeRPH-17-04838]\]. HoWevER, thE InFLUENCe oF tHe HoME seTTiNg oN CHIldReN'S eBRbs Is PROfOund, eSpeCIaLLy FoR yOUNG CHildrEN, siNce ParentS DeTeRmine tHe foOd aVaiLaBILItY aT hOME, and inFlueNCe cHildRen's nutrItion and PA behAViOUR By pRaCtIcES LiKE mODELLING And rUlE seTTing \[[@b16-IJerPh-17-04838],[@B17-ijeRpH-17-04838],[@b18-IjERPH-17-04838],[@B19-IJerPH-17-04838]\]. THERefOre iT IS IMPORTant To ALso focuS On The HOme SEttinG whEn IMplEMeNTiNg SCHoOl-BAsed EneRGy balANce-relATeD INTerveNtiONS \[[@b15-IjERPH-17-04838],[@b17-IJErPh-17-04838]\].
mOst effecTiVe changEs IN tHe hOMe SetTINg ARE ACCOMPliSheD ThRoUgh IntervEnTIonS WIth diReCt PARENTaL INvOLvEmeNt (e.g., PaRenTS atTEnDINg EducAtIONaL sESSiOns, Or coUnsElLINg SESsiOns) \[[@b13-ijeRph-17-04838],[@B16-IjERPh-17-04838],[@b19-IjerpH-17-04838],[@b20-iJErpH-17-04838],[@B21-IJErph-17-04838]\]. hOWeVER, DireCTLy iNvOlVInG pArENtS IS Often rESOuRcE- aND laBOUR-inTEnsIVE, makiNG TheSE tyPes oF iNTerveNTIoNS less FeaSible \[[@B20-IJeRph-17-04838]\]. ALso, tHe RECruItMeNt \[[@B22-IJErph-17-04838]\] anD PROLONgeD eNGaGement of PAREntS iN theSe types of iNTervEntiONs HAvE PrOvEN To BE ChAllEngInG \[[@b3-ijeRPh-17-04838],[@B22-IJERPh-17-04838],[@B23-ijErpH-17-04838]\]. OnLy AboUt one-THiRd of invIteD fAmILIEs PARtICipate IN ANY inTervENTioN ACtIvItY \[[@b3-IjERPH-17-04838],[@b24-IjErPH-17-04838]\], 40 To 60% of WHoM DRoP OUT \[[@b24-ijeRph-17-04838]\]. in addItIon, The ParTIcipANTS oF pareNtAl InvoLVeMenT iNteRVentIonS TENd To bE MoSTLY hIGh SOCioEcONOmIC STAtUS (SeS) PaRents \[[@b25-IJeRPh-17-04838]\], aNd iT Is particUlarLY ChaLLeNGIng tO EnGaGe parEnts wITh a loW SEs \[[@B3-iJErph-17-04838],[@B25-iJeRPh-17-04838]\], A lOweR EDUcaTiOnAL levEl, SingLE PArENTS aNd tHOSe OF eThNIc mINoRiTy groUPs \[[@b24-iJerPH-17-04838]\]. THiS IS disCOurAging, SINCE THeSe PArENTS/famILieS aRe mOST In nEEd Of InteRVENtiONs. FoR ExaMPLE, reSeARCh from a laRge DutcH coHOrt STuDy hAs showN THat THERe ARE sOciOeCoNOmIc AnD etHNiC IneQUALItIeS in CHILD HEAlth \[[@b26-IjeRpH-17-04838]\]. aT a young AgE, NoN-wEStern cHIlDreN weRE MOre LIkELy to BE ovErwEiGHt COmPAReD TO dutch chilDrEn, witH MOTHEr's educatIONAl leVeL BEing ONE oF THE coNTRIbuTInG faCtoRs In expLaINInG tHis hiGHEr PreVaLEnce In oVErWEighT iN tHESE CHiLDrEn \[[@B27-iJeRph-17-04838]\]. BeyoNd SoCIoecOnOmIC HEalth diSpARItIEs, chIlD CharAcTeRISTIcs AND ThE SupPORTiveNESs OF tHE hOmE ENvIRonMeNt to pERfORM HeAlthy behAvIouRs are AlSo imporTaNT PREDICtors of ChILDrEn's pa anD NuTRITIon BEHAVIour. fOr INsTAnCE, CHILDRen's NutriTION behAviour Is DETeRmIned By THEIr pReFErENce fOR hEAlthy AND/OR unhEALthy fOODs, aND ThEir PA AnD sEdeNtaRY behaviOuR iS aSsocIaTEd wItH THEIR ACtIViTY PreferENCeS \[[@b28-IJeRph-17-04838]\]. aLso, cHilDREN RAIsed IN AN eNVIrOnMenT iN WHIcH HEaLThy NuTRiTION \[[@b29-iJErPH-17-04838]\] ANd SuffIcIent Pa \[[@b29-IJerPH-17-04838],[@b30-IjerpH-17-04838]\] ArE LESs ValUeD Are more AT riSk fOr DEveLOPinG unHeALthY ebRBS. thErEforE, VuLNERAbIlity oF THe popULAtiON is Not REsTriCtED To WeLl-KNOWN SoCiOECONoMIC DIFfeRENCeS, buT alsO INCLudEs sPECiFiC child PREFeReNCes aND thE hEALTh CLImaTe regaRding ebrBS in tHE HoMe EnviROnMeNt.
TO INvOLVE moRe pAReNtS, AND PARTICUlaRLy THOsE OF vULneRable pOPUlatiOns, ThE StRAtEgY OF iNdIRecT pARenTal InvOlVeMeNT IS aN aLTErNaTIVE. eveN THOUGH iNDirECT pArENTAl iNVoLveMEnt is ASSumed TO bE lesS EFFEcTive iN cHanGINg HEalTH behAViOUrS ComPareD to DIRect ParenTAl invOlVemeNt, It CAN leAd To GrEAtEr ADopTION aND ImPlEMenTaTion ratEs \[[@B21-IJerPH-17-04838]\]. IN iNdIREct paReNtal inVolvemenT, PaRentS Are enGaged iN A WaY tHAT THe interVeNtIon ImplEMEnTers Do NOt COmmuniCAte OR ENGAgE DIrecTLy (I.E., faCE TO FacE, oR pERSonalLY) wiTH theM. iNSTead, PArenTs Are INFormEd VIa SChOOl mEdia, oR ChILdrEN FuNcTIoN AS tHE meSSEnGer. EXaMPLEs Of iNDIREct PareNtaL iNVOlVeMenT aRe The PRoViSiON OF iNFORMatioN (NewsleTtErs, tIp SHEETS), InVitATIoNS tO paRtICIPaTe IN Or ATTeND ACTiVitIeS (events, educaTIOnaL sessIOns) ANd PrOmpts or ASsigNMENts dIRECTED at tHE ChIlD aND/Or PAReNt WiTh tHe Aim Of INVolviNG ParENtS \[[@b19-ijeRpH-17-04838]\]. prEvIouS rESEArch HAS shOwN THAT SOme Of ThEse sTRaTEGieS aRE mORe pRoMiSinG thAn OTHErs, For exAmPle pROmptIng cHiLDREn tO ENgAGE in INTErventION ACTivITiES tOgetHEr WItH ThEir PARenT(S) SEeMs moRe PrOMisiNG than PrOVidiNG nEWSletterS OR INVItatIons For OpTIoNAl INTERVEntIoN aCTIvITiEs \[[@b17-ijErPH-17-04838],[@B19-iJeRPH-17-04838]\]. The DIFfereNce BETWEEn THesE sTrATEgIEs cOuLD bE dEScRiBed aS pASsIvE (NOt rEQuiRIng a sPECiFIC ActIoN or rEspoNSE on THE pART of THe PareNTS) iNDiReCT involveMeNT VERsus ACTive (pErfoRmInG actIVItieS) iNdIrEcT invOlVement.
TO oUR KNowLEDge, tHERe iS lITTle to no RESeARCH oN the PartIciPAtiON rATes Of thE stRATegy oF The LatTer tYPE Of IndIRECt PAreNTAl iNVOlveMenT IN ScHOol-BaSEd InTERVenTiOnS, WhIle ThIS INFOrmatIOn is cRUcIAl TO bE aBLe tO DraW ConcluSIonS On pOtenTiaL sTRAteGIEs to InvoLVe pAReNts iN (ScHOOL-bAsED) IntERVeNTIOnS \[[@B21-IJeRpH-17-04838]\]. GiveN tHE fACt THAt chiLdrEN of VULnerABle POPulatIOns ARE LeSs acTiVe, MoRE sEdeNtARy AnD Have UnHeAltHIer dietS \[[@b31-iJerPh-17-04838],[@b32-ijERph-17-04838],[@B33-iJErpH-17-04838]\], iT iS posSiBLE thAT ThESE ChIldrEn And ThEir PArenTs ARE leSS InTeresTED iN eNeRgy BalaNce-ReLaTED IntErVEntioNs. tHerefORE, GAININg iNSiGHt in pArtICiPATiOn raTes of CHildREN AnD pAReNtS IN iNTErVEnTIOnS USiNg an iNDIRect pARENTaL InvoLVEMent stRATEGy IS NeedED. MoREOVer, EMpIRiCAl EviDEnce IS LACking oN WhO is ENGaGed in This STRATEgY AnD tHerefoRE, it iS WaRranted tO KnOW WHEtHER VulNerAblE chIlDReN ANd pArENts ENGAGe in ThESE INtervenTIOnS USiNG THis stRAteGy.
in thE CUrrEnT STUDy, wE EValuaTED tHe POtENTial of chaLLenGE me tO eNgAge chIlDrEn anD pAReNtS, EspeCIAlly thOsE Of vulNErAbLE poPulatiOns. chAlLeNGE mE WAs A paReNTaL InvolveMEnT INtERVenTiON IN WHIch cHiLDrEn aRE cHALlenGed tO PeRFOrm Pa ANd nUtriTIon-RELATeD aCtIVItIeS By tHEMseLvES And wITH TheIr PARENTS. tHe mAin AIM Of tHe cURreNT STUDy Is TO EXamINE WHetHer coNDuCtinG CHAllENGes is A feaSIBLE interVEnTION StraTegY tO engaGE childreN aNd PaREntS IN scHooL-bAsED eNeRGy BalANCE-ReLated INtervENTiONS. wITH eNgageMeNT, WE REfeR TO PARtiCIpatiOn in tHe InTeRVENtiOn, I.e., PERForMiNg The INteRVEnTiOn ACTIVitIES, AND noT EnROLmENt. addiTioNALly, A sEcOnD AiM iS tO gAin INsIgHT in wheThER CHildrEn IN neeD FOr IMProvEmentS, I.e., VUlnERaBLE populaTIOns, arE EnGAGeD iN thE IntERVenTiON Using ThIs indIRECt-InvOlVEMeNt stratEgY. FOR thIS AIM, wE FocUssED on Child DemoGrApHics, ChILD chaRACTeRiStICS (PREFEReNCEs) AnD THe CLImaTe ToWArDS hEALth bEhAViours In THe HomE ENvIRoNmENT.
2. maTeRiALs ANd MEtHODs {#SEc2-ijErPh-17-04838}
========================
2.1. DesIgN {#Sec2DoT1-IjERpH-17-04838}
-----------
AN EXPLORAtOry CRoSs-sECtIONAl sTuDy DesIgN waS coNdUcted. ThE mEDicaL ETHICS cOMMittEE of the MAastRiCHt uNiveRsITy meDicaL cENTRe aND mAaStRiCHt UniVERSItY provIDED eTHiCal APPrOVAL FoR thIS StUdY (mEtC163027, naTIOnAL NUMbEr: nl58554.068.16).
2.2. CHaLlenge me InTErveNTION {#seC2Dot2-ijErPh-17-04838}
------------------------------
### 2.2.1. INTErVentioN DeVeloPMeNt {#SEc2DOt2dOT1-ijerPH-17-04838}
the InTERvENtION, cALlEd challEnGe Me, ChalLenGED chILDrEn To pErFORM PA AND NutRItIoN-relAtED chALLeNgeS tOGETher wItH theIr paReNt(S) or GuardIAN(S). cHALlEnGe mE has BEEn DEVelopEd AS PARt of a LaRgeR EVaLUATIon \[[@B34-IJerph-17-04838]\], AnD IS SpecIfiCALLY FocUsEd on IMPROViNG PaRENTAL INvoLVeMENT. IN THE LArGEr EVAlUAtION sTuDY, EIGHt prIMarY ScHOOLs, LoCATed in Low SeS NEiGhbouRhOoDs (BaSED on sCORiNg OF thE NEtHERlaNdS inSTITute FoR | 1. Introduction {#sec1-ijerph-17-04838} =============== Recent studies show arise in overweight and obesity among children and adolescentsto18%worldwide in 2016 \[[@B1-ijerph-17-04838]\], with onein five school-aged children in Europe being overweight or obese \[[@B2-ijerph-17-04838],[@B3-ijerph-17-04838]\]. Also in The Netherlands, childhood obesity rates are high. In 2019, 12% of children aged 4 to 12 were overweight or obese \[[@B4-ijerph-17-04838]\].Overweightor obesity inchildhood is especially problematic, sincethis often continues into adolescence and adulthood and is related to an increased riskof negative health consequences, such as type II diabetes, hypertension, respiratory disease and various types of cancer\[[@B2-ijerph-17-04838],[@B3-ijerph-17-04838],[@B5-ijerph-17-04838]\]. Overweight and obesityare preventableand treatable \[[@B6-ijerph-17-04838]\], especiallyat ayoung age \[[@B5-ijerph-17-04838],[@B7-ijerph-17-04838]\], by improving healthy nutrition behavioursand increasing physicalactivity (PA) (also called energy balance-relatedbehaviours (EBRBs)) \[[@B8-ijerph-17-04838],[@B9-ijerph-17-04838],[@B10-ijerph-17-04838]\]. To targetchildren's EBRBs \[[@B8-ijerph-17-04838],[@B11-ijerph-17-04838]\],multiple settings \[[@B12-ijerph-17-04838]\], such as the school \[[@B13-ijerph-17-04838]\] and home environment should be involved in interventions \[[@B8-ijerph-17-04838],[@B13-ijerph-17-04838]\]. Since children spend a largeproportion of their weekdays at school, and schools reach manychildren, schools have been a popular interventionsetting fordecades \[[@B13-ijerph-17-04838],[@B14-ijerph-17-04838],[@B15-ijerph-17-04838]\].However, the influence of the home setting onchildren's EBRBs isprofound, especially for young children, since parents determine the food availability at home, and influence children's nutrition and PA behaviour by practices like modelling and rule setting\[[@B16-ijerph-17-04838],[@B17-ijerph-17-04838],[@B18-ijerph-17-04838],[@B19-ijerph-17-04838]\]. Therefore it is important to also focus on the home setting when implementingschool-based energy balance-related interventions\[[@B15-ijerph-17-04838],[@B17-ijerph-17-04838]\]. Most effective changes inthe home setting are accomplishedthrough interventions with direct parental involvement (e.g.,parents attending educational sessions, or counselling sessions) \[[@B13-ijerph-17-04838],[@B16-ijerph-17-04838],[@B19-ijerph-17-04838],[@B20-ijerph-17-04838],[@B21-ijerph-17-04838]\]. However, directly involving parentsis often resource- and labour-intensive, making these types of interventionsless feasible \[[@B20-ijerph-17-04838]\]. Also, the recruitment \[[@B22-ijerph-17-04838]\] and prolonged engagement ofparentsin these types of interventions have proven tobe challenging \[[@B3-ijerph-17-04838],[@B22-ijerph-17-04838],[@B23-ijerph-17-04838]\]. Onlyabout one-thirdofinvited families participate in any intervention activity\[[@B3-ijerph-17-04838],[@B24-ijerph-17-04838]\], 40 to 60% of whom drop out \[[@B24-ijerph-17-04838]\]. In addition, the participants of parental involvement interventions tend to be mostly high socioeconomic status(SES) parents \[[@B25-ijerph-17-04838]\], and it is particularly challenging to engage parents with a low SES \[[@B3-ijerph-17-04838],[@B25-ijerph-17-04838]\], a lower educational level, singleparents and those of ethnic minority groups \[[@B24-ijerph-17-04838]\]. This is discouraging, since these parents/familiesare most in need of interventions. Forexample, research from alarge Dutchcohort study has shownthat there are socioeconomic and ethnic inequalities in child health \[[@B26-ijerph-17-04838]\].At a young age, non-Western children were more likely to beoverweight compared to Dutch children, with mother's educational level being one of the contributing factors in explaining thishigher prevalence inoverweight in these children \[[@B27-ijerph-17-04838]\].Beyond socioeconomic health disparities, child characteristics and the supportivenessof thehome environment to perform healthy behaviours are also important predictors ofchildren's PA and nutrition behaviour. Forinstance, children's nutrition behaviour is determined bytheir preference for healthy and/or unhealthy foods, and their PA and sedentarybehaviour is associated with their activitypreferences \[[@B28-ijerph-17-04838]\]. Also, children raised in an environment in which healthy nutrition \[[@B29-ijerph-17-04838]\] and sufficient PA\[[@B29-ijerph-17-04838],[@B30-ijerph-17-04838]\]are less valued are moreat risk for developingunhealthy EBRBs.Therefore, vulnerability of the population is not restricted to well-known socioeconomic differences, but also includes specific child preferencesand the health climate regarding EBRBs in thehome environment. To involve more parents, and particularly those ofvulnerable populations, the strategy of indirect parental involvement is an alternative. Even though indirect parental involvement is assumed to be less effective in changing health behaviours compared to direct parentalinvolvement, itcan lead to greater adoption and implementation rates \[[@B21-ijerph-17-04838]\]. In indirectparental involvement, parents are engaged ina way that the intervention implementers do not communicate or engage directly (i.e., face to face, or personally) with them. Instead, parents are informed via school media, or children function as the messenger. Examples of indirect parental involvement are the provision of information (newsletters, tip sheets), invitations to participate in or attend activities (events, educational sessions) and prompts or assignments directed atthe child and/or parent with the aim of involving parents \[[@B19-ijerph-17-04838]\]. Previous research has shown that some of these strategies are more promising than others, for example prompting children to engage in intervention activities together with their parent(s) seems more promising than providing newsletters or invitations for optionalintervention activities \[[@B17-ijerph-17-04838],[@B19-ijerph-17-04838]\].The difference between these strategies could be described as passive (not requiring a specific action or response onthepart of the parents) indirect involvement versus active (performing activities) indirect involvement.To our knowledge, there is little to no research on the participation rates of the strategy of the latter type of indirect parentalinvolvement in school-based interventions, while this information is crucial to be able to draw conclusions on potentialstrategies to involve parents in (school-based) interventions \[[@B21-ijerph-17-04838]\]. Giventhe factthat children ofvulnerable populations are less active, more sedentary and have unhealthier diets \[[@B31-ijerph-17-04838],[@B32-ijerph-17-04838],[@B33-ijerph-17-04838]\], it is possible that these children and their parents are less interested inenergy balance-related interventions. Therefore, gaininginsight in participation rates of children and parents in interventions using anindirectparentalinvolvement strategy is needed. Moreover, empirical evidence is lacking on who is engaged in this strategy and therefore,it is warrantedto know whether vulnerable children and parents engagein these interventions using this strategy. In the current study, we evaluated the potential of ChallengeMe to engage children and parents, especiallythose of vulnerable populations. Challenge Me was a parental involvement intervention in which children are challenged to perform PA and nutrition-related activities by themselves and with their parents. The main aim of the current study is toexamine whether conducting challenges is a feasible intervention strategy to engage childrenand parents in school-based energy balance-related interventions. With engagement, we refer to participation in theintervention, i.e., performing the intervention activities, and not enrolment. Additionally, a second aim is to gain insight in whether children in need for improvements, i.e., vulnerablepopulations, are engaged in the intervention using this indirect-involvement strategy. For this aim, wefocussed on child demographics,child characteristics (preferences)andthe climate towards health behaviours in the home environment. 2. Materials and Methods {#sec2-ijerph-17-04838} ======================== 2.1. Design {#sec2dot1-ijerph-17-04838} ----------- An exploratory cross-sectional study design was conducted. The Medical Ethics Committee of the Maastricht University Medical Centre and Maastricht University provided ethical approval for this study (METC163027, national number: NL58554.068.16). 2.2. Challenge Me Intervention {#sec2dot2-ijerph-17-04838} ------------------------------ ###2.2.1. Intervention Development {#sec2dot2dot1-ijerph-17-04838}The intervention, calledChallenge Me, challenged children to perform PA and nutrition-related challenges together with their parent(s) or guardian(s).Challenge Me has been developed as part of a larger evaluation \[[@B34-ijerph-17-04838]\], and is specifically focused on improving parentalinvolvement. Inthe larger evaluationstudy, eight primary schools, located inlow SES neighbourhoods (based on scoring of The Netherlands Institute for | 1. _Introduction_ _{#sec1-ijerph-17-04838}_ =============== Recent studies show a _rise_ in overweight and obesity among _children_ _and_ adolescents to _18%_ _worldwide_ in _2016_ \[[@B1-ijerph-17-04838]\], with one in five school-aged children in Europe _being_ overweight or obese _\[[@B2-ijerph-17-04838],[@B3-ijerph-17-04838]\]._ Also in _The_ Netherlands, childhood obesity rates are high. _In_ 2019, 12% of children _aged_ 4 to 12 _were_ overweight or obese \[[@B4-ijerph-17-04838]\]. Overweight or _obesity_ in childhood _is_ especially problematic, since this often _continues_ into adolescence and _adulthood_ and is related to an increased risk of _negative_ health consequences, such as type II diabetes, _hypertension,_ respiratory disease and various _types_ of cancer \[[@B2-ijerph-17-04838],[@B3-ijerph-17-04838],[@B5-ijerph-17-04838]\]. _Overweight_ and obesity are preventable and treatable \[[@B6-ijerph-17-04838]\], especially at a _young_ age \[[@B5-ijerph-17-04838],[@B7-ijerph-17-04838]\], by improving healthy nutrition behaviours and increasing physical activity (PA) _(also_ called energy _balance-related_ behaviours (EBRBs)) \[[@B8-ijerph-17-04838],[@B9-ijerph-17-04838],[@B10-ijerph-17-04838]\]. To target _children's_ EBRBs _\[[@B8-ijerph-17-04838],[@B11-ijerph-17-04838]\],_ multiple settings \[[@B12-ijerph-17-04838]\], _such_ as the school \[[@B13-ijerph-17-04838]\] and home _environment_ should be involved in interventions \[[@B8-ijerph-17-04838],[@B13-ijerph-17-04838]\]. Since _children_ spend a large proportion of their weekdays at school, and schools reach many children, schools have been a popular intervention setting _for_ _decades_ \[[@B13-ijerph-17-04838],[@B14-ijerph-17-04838],[@B15-ijerph-17-04838]\]. _However,_ the influence _of_ _the_ home _setting_ on children's EBRBs is _profound,_ especially for young _children,_ since parents determine the food availability at home, _and_ influence children's _nutrition_ and PA behaviour by practices like modelling _and_ rule setting \[[@B16-ijerph-17-04838],[@B17-ijerph-17-04838],[@B18-ijerph-17-04838],[@B19-ijerph-17-04838]\]. Therefore it _is_ important _to_ also focus on _the_ home setting _when_ implementing school-based energy balance-related interventions _\[[@B15-ijerph-17-04838],[@B17-ijerph-17-04838]\]._ Most effective changes in the home setting are accomplished _through_ interventions with direct parental _involvement_ (e.g., parents attending educational sessions, _or_ counselling sessions) _\[[@B13-ijerph-17-04838],[@B16-ijerph-17-04838],[@B19-ijerph-17-04838],[@B20-ijerph-17-04838],[@B21-ijerph-17-04838]\]._ However, directly involving parents is often resource- _and_ _labour-intensive,_ _making_ these _types_ of interventions less feasible _\[[@B20-ijerph-17-04838]\]._ Also, the recruitment \[[@B22-ijerph-17-04838]\] and prolonged _engagement_ of parents in these types of interventions have proven to be challenging \[[@B3-ijerph-17-04838],[@B22-ijerph-17-04838],[@B23-ijerph-17-04838]\]. _Only_ about one-third of invited _families_ participate in any intervention activity _\[[@B3-ijerph-17-04838],[@B24-ijerph-17-04838]\],_ _40_ to 60% of _whom_ drop out \[[@B24-ijerph-17-04838]\]. _In_ addition, the participants of parental involvement interventions tend to be mostly high _socioeconomic_ status (SES) parents \[[@B25-ijerph-17-04838]\], and _it_ is particularly challenging _to_ engage _parents_ _with_ a low SES \[[@B3-ijerph-17-04838],[@B25-ijerph-17-04838]\], _a_ lower _educational_ _level,_ single parents and those of ethnic minority groups \[[@B24-ijerph-17-04838]\]. This is discouraging, _since_ these parents/families are _most_ in need _of_ interventions. For example, research from a large Dutch cohort study has shown that there are socioeconomic and ethnic inequalities in child health \[[@B26-ijerph-17-04838]\]. At _a_ young age, non-Western children were _more_ _likely_ _to_ _be_ _overweight_ compared to Dutch children, _with_ mother's _educational_ level being one _of_ _the_ contributing factors in explaining this higher prevalence in overweight in these children _\[[@B27-ijerph-17-04838]\]._ Beyond socioeconomic health disparities, child characteristics and the supportiveness of the _home_ environment to perform healthy behaviours _are_ _also_ important predictors of children's PA _and_ nutrition _behaviour._ For instance, children's nutrition behaviour is determined by their preference _for_ healthy and/or unhealthy foods, and their PA and sedentary behaviour _is_ associated _with_ their _activity_ _preferences_ \[[@B28-ijerph-17-04838]\]. Also, children _raised_ in an environment in _which_ healthy nutrition \[[@B29-ijerph-17-04838]\] and sufficient _PA_ \[[@B29-ijerph-17-04838],[@B30-ijerph-17-04838]\] are less valued are more at risk for developing _unhealthy_ EBRBs. Therefore, vulnerability of the population is not restricted to well-known socioeconomic _differences,_ _but_ _also_ includes specific child _preferences_ and the health _climate_ regarding _EBRBs_ in the home _environment._ _To_ involve more parents, and particularly _those_ of vulnerable _populations,_ the strategy of indirect parental involvement is an alternative. _Even_ though indirect parental involvement _is_ assumed to be less effective in changing health behaviours _compared_ to direct parental _involvement,_ it can lead to greater adoption and _implementation_ _rates_ _\[[@B21-ijerph-17-04838]\]._ In indirect parental involvement, parents _are_ engaged in a _way_ that the intervention implementers do not communicate or engage directly (i.e., face to face, _or_ personally) with them. Instead, parents are informed via _school_ media, or children function as the messenger. Examples _of_ indirect parental involvement are the provision of information (newsletters, tip sheets), invitations to _participate_ in _or_ _attend_ activities (events, educational sessions) _and_ _prompts_ or assignments directed _at_ the child and/or parent with the aim of _involving_ _parents_ \[[@B19-ijerph-17-04838]\]. Previous _research_ has shown that some of these strategies are more _promising_ than others, for _example_ prompting children to engage in _intervention_ activities together with their parent(s) seems more promising than providing newsletters or _invitations_ for _optional_ intervention activities \[[@B17-ijerph-17-04838],[@B19-ijerph-17-04838]\]. The difference between these strategies could be described as passive (not requiring a specific action or response on the part of the parents) indirect involvement _versus_ active (performing activities) indirect involvement. _To_ our knowledge, there is little to no research on the participation rates of _the_ strategy of _the_ latter type of indirect _parental_ involvement in school-based _interventions,_ while this information _is_ crucial to _be_ able to draw conclusions on potential strategies to _involve_ parents in _(school-based)_ _interventions_ \[[@B21-ijerph-17-04838]\]. Given the fact that children of _vulnerable_ populations are less _active,_ more sedentary and have unhealthier diets \[[@B31-ijerph-17-04838],[@B32-ijerph-17-04838],[@B33-ijerph-17-04838]\], _it_ is possible that these children and their _parents_ are less interested in energy _balance-related_ _interventions._ _Therefore,_ gaining insight in participation _rates_ _of_ children and parents in interventions using an indirect parental involvement strategy is _needed._ _Moreover,_ empirical evidence is _lacking_ on who is engaged in this _strategy_ and therefore, it is _warranted_ to _know_ whether vulnerable _children_ and _parents_ engage in these interventions using this strategy. In the current _study,_ we evaluated the potential of Challenge Me _to_ engage children and parents, especially _those_ of vulnerable populations. Challenge Me was a _parental_ involvement intervention in which children are challenged to perform _PA_ and nutrition-related activities by themselves and with their _parents._ The main aim of the current study is to _examine_ whether conducting challenges _is_ a feasible intervention _strategy_ _to_ engage children _and_ parents in _school-based_ energy balance-related interventions. With engagement, we refer _to_ _participation_ in the intervention, i.e., performing the _intervention_ activities, _and_ _not_ _enrolment._ Additionally, a second aim is _to_ _gain_ insight _in_ whether children in _need_ for improvements, i.e., _vulnerable_ populations, are engaged in _the_ intervention _using_ _this_ indirect-involvement strategy. For this _aim,_ we focussed on child demographics, child _characteristics_ (preferences) and the climate towards health behaviours in the home environment. 2. Materials and _Methods_ {#sec2-ijerph-17-04838} ======================== 2.1. Design {#sec2dot1-ijerph-17-04838} ----------- An exploratory cross-sectional study design _was_ conducted. _The_ Medical Ethics Committee of the Maastricht University Medical Centre _and_ _Maastricht_ University provided ethical approval _for_ this study (METC163027, national number: _NL58554.068.16)._ 2.2. Challenge Me Intervention {#sec2dot2-ijerph-17-04838} ------------------------------ ### 2.2.1. _Intervention_ Development {#sec2dot2dot1-ijerph-17-04838} The intervention, called Challenge Me, challenged children _to_ perform _PA_ _and_ nutrition-related _challenges_ together with their _parent(s)_ or guardian(s). _Challenge_ Me has been developed as part _of_ _a_ larger evaluation \[[@B34-ijerph-17-04838]\], _and_ is specifically focused on improving parental involvement. In the larger _evaluation_ _study,_ eight primary schools, located in low SES neighbourhoods (based on _scoring_ of _The_ Netherlands _Institute_ _for_ |
All relevant data are within the paper and its Supporting Information files.
Introduction {#sec001}
============
Ecosystem services are defined as the benefits that nature provides to society \[[@pone.0235320.ref001]\]. Ecosystems such as forests, grasslands, croplands, coastal zones, and urban areas offer different services to society. These include provisioning services (food, water, wood, and fibers), regulation services (which affect climate, flooding, disease, waste, and water quantity and quality), cultural services (recreational opportunities, aesthetic, and spiritual values), and support services (soil formation, photosynthesis, and nutrient cycling) \[[@pone.0235320.ref001], [@pone.0235320.ref002]\]. Regulating services are obtained directly from ecosystems without any transformational process \[[@pone.0235320.ref001]\]. Water regulation is one of such services, which has great importance to society by providing adequate quality water and maintaining the water cycle \[[@pone.0235320.ref003], [@pone.0235320.ref004]\].
Water regulation in forest ecosystems involves the processes that take place after precipitation. These include interception, evapotranspiration, surface and subsurface flow, infiltration, soil erosion control, water quality, and groundwater replenishing, among others \[[@pone.0235320.ref001]\]. These water and soil movement-related processes are affected by various climatic and topographic factors, as well as by soil and vegetation cover types. In a forest system, a portion of the rain is intercepted by the top and other layers of the canopy. The intercepted rain evaporates and returns to the atmosphere, while the non-intercepted portion reaches the ground and deep parts of the soil \[[@pone.0235320.ref005]\]. Forest cover plays an important role in intercepting, capturing, and channeling rainfall. In addition, it is one factor that can be directly manipulated by resource managers using silvicultural practices that consist of different harvesting techniques of various intensities.
For the purpose of this study, we focused on the processes that regulate water movement, starting from the canopy, to the forest floor, and into the stream channels. This includes the processes of throughfall, stemflow, and surface runoff. Throughfall refers to the amount of water that passes directly through the forest canopy or drips from branches and leaves of trees \[[@pone.0235320.ref006]\]. It accounts for 60 to 90% of rainfall \[[@pone.0235320.ref007]\]. Stemflow is the fraction of the water that comes in contact with the forest canopy and runs down the trunks of trees and bushes, before being deposited on the ground \[[@pone.0235320.ref008], [@pone.0235320.ref009]\]. It is often ignored in rainfall studies because it is thought to be insignificant and expensive to measure, particularly when forests are composed by rough-barked trees \[[@pone.0235320.ref010]--[@pone.0235320.ref012]\]. Stemflow values represent between 1 to 4% of total rainfall, although some studies reported values up to 20% for certain forest types \[[@pone.0235320.ref007], [@pone.0235320.ref011], [@pone.0235320.ref012]\]. In many areas, particularly semi-arid ones, stemflow creates important islands of soil moisture and nutrients around the stem and contributes to streamflow and groundwater generation \[[@pone.0235320.ref007]\].
Surface runoff refers to the rainfall that flows over the surface of the soil directly into nearby channels and bodies of water \[[@pone.0235320.ref011]\]. It is often referred to as sheet flow, e.g., the water that resembles a braiding pattern of threads, without forming channels larger than rills and gullies \[[@pone.0235320.ref013]\]. In addition to vegetation and other surface obstructions, the rate of flow is dependent upon soil characteristics. There are numerous studies that describe the effect of soil infiltration capacity on surface runoff \[[@pone.0235320.ref014]--[@pone.0235320.ref017]\]. However, there are only a few studies that have addressed the impacts of vegetation cover on surface runoff in Mexico. Furthermore, there are only a limited number of studies in Mexico that have assessed the relationship between surface runoff and forest density, which, as we said above, can be manipulated through direct silvicultural treatments.
Silvicultural treatments affect hydrological fluxes. Intensive silvicultural treatments (e.g. stand thinning from above or clear-cutting) change forest density \[[@pone.0235320.ref018]\], eventually modifying throughfall and stemflow at both stand level and individual tree level \[[@pone.0235320.ref019]\], while increasing surface runoff \[[@pone.0235320.ref020]\]. The potential impact of the increased water from the surface flow may eventually affect site productivity and the provision or regulation of other ecosystem services (e.g., plant diversity, soil erosion control, carbon sequestration, etc.) \[[@pone.0235320.ref021], [@pone.0235320.ref022]\]. Varying levels of tree density affect water cycle components (namely interception, evapotranspiration, infiltration, and surface runoff), causing variations in water soil movement and groundwater reserves \[[@pone.0235320.ref021], [@pone.0235320.ref023]\]. For example, heavy rainfall occurrences, following highly intensive vegetation cover treatments (such as clear-cuts), result in increased surface runoff causing soil erosion, flooding, and water turbidity \[[@pone.0235320.ref023]\]. For short periods, large water and soil movements can modify the quality, quantity, and distribution of water resources \[[@pone.0235320.ref024]\].
This study used hydrological models to analyze throughfall, stemflow, and surface runoff in a managed pine-oak forest in northern Mexico. Hydrological models, which relate the flow of water to some stand variables, enable an evaluation of the impact of changes in forest cover on the water resources within a watershed \[[@pone.0235320.ref025]\]. The models can help determine the best forest management scenarios in places where regulation services are combined with provisioning ecosystem services. The objectives of this study were to evaluate the effects that forests, in terms of some forest tree and stand variables, have on throughfall, stemflow, and surface runoff in a temperate area of northern Mexico. The working hypotheses are that throughfall and stemflow are different depending on tree size and genus, and that stand density affects surface runoff.
Materials and methods {#sec002}
=====================
The study area is located in the mountainous region of the Sierra Madre Occidental, within the municipality of Durango, which lies in the southern part of the state of Durango. The experimental site is located in a private property known as Molinillos ([Fig 1](#pone.0235320.g001){ref-type="fig"}). The owners of this 2,866-hectare property have played a leading role in promoting a healthy silvicultural management, biodiversity conservation, and ecotourism in the region \[[@pone.0235320.ref026]\]. They allowed us to conduct research and field measurements on their property. The current management plan includes the application of non-intensive tree regeneration methods (selective harvesting) as well as intensive methods (seed tree retention or clear-cuts) in different parts of the property \[[@pone.0235320.ref018], [@pone.0235320.ref026]\]. Of the total area, about 2,050 ha are under timber management, with the following treatment distribution: clear-cuts 3.5%, tree retention 14%, thinning 27%, and individual selection 55.5% \[[@pone.0235320.ref026]\].
{#pone.0235320.g001}
The climate in the region is temperate and sub-humid, with moderate levels of rainfall in the summer and parts of December and January. In the coldest month (January), daily temperatures can reach anywhere between -3 °C and 18 °C. In the warmest month (June), daily temperatures vary between 15 °C and 35 °C. Historical records show that the mean annual temperature varies from 8 °C to 26 °C, while the annual average is 13.3 °C. The annual rainfall varies from 443 to 1450 mm, with an average of 917 mm \[[@pone.0235320.ref027]\].
Regional elevation ranges from 1,500 to 3,000 meters above sea level. However, the elevation in the plots is closer between 2,360 and 2,630 meters. The typical slope ranges between 20% and 60%. Runoff water flows toward the hydrographic system of the Acaponeta River basin and eventually into the Pacific Ocean. The natural, pine-oak forests include mixtures of *Pinus strobiformis*, *P*. *cooperii*, *P*. *durangensis*, *P*. *engelmannii*, *P*. *teocote*, *P*. *leiophylla*, *Quercus coccolobifolia*, *Q*. *ruogosa*, *Q*. *sideroxilla*, *Q*. *obtusata*, and *Arbutus* spp. The type of soils are Regosol, Litosol, Eutric Cambisol, and Luvisol cromico types \[[@pone.0235320.ref026]\].
Tree and stand variables {#sec003}
------------------------
The information of the tree and | all relevant data are within the paper and its supporting information files. introduction { # sec001 } = = = = = = = = = = = = ecosystem services are defined as the benefits that nature provides to society \ [ [ @ pone. 0235320. ref001 ] \ ]. ecosystems such as forests, grasslands, croplands, coastal zones, and urban areas offer different services to society. these include administrative services ( food, agriculture, wood, and fibers ), regulation services ( which affect climate, flooding, disease, waste, and water quantity and quality ), cultural services ( recreational opportunities, aesthetic, and ornamental values ), and support services ( soil formation, erosion, induced nutrient cycling ) \ [ [ @ pone. 0235320. ref001 ], [ @ pone. 0235320. ref002 ] \ ]. regulating services are obtained directly from ecosystems without any transformational process \ [ [ @ pone. 0235320. ref001 ] \ ]. water regulation is one of such services, which has great importance to society by providing adequate quality water and maintaining the water cycle \ [ [ @ pone. 0235320. ref003 ], [ @ pone. 0235320. ref004 ] \ ]. water regulation in forest ecosystems involves the processes that take place after precipitation. these include interception, evapotranspiration, surface and subsurface flow, infiltration, soil erosion control, water quality, and groundwater replenishing, by others \ [ [ @ pone. 0235320. ref001 ] \ ]. these water and soil movement - related processes are affected by various climatic and topographic factors, as well as by soil and vegetation cover types. in a forest system, a portion of the rain is intercepted by the rain and other layers of the canopy. the intercepted rain evaporates and returns to the atmosphere, while the non - intercepted portion reaches the ground and deep parts of the soil \ [ [ @ pone. 0235320. ref005 ] \ ]. forest cover plays an important role in intercepting, capturing, and channeling rainfall. in addition, it presents one factor that can be directly manipulated by resource managers using silvicultural practices that consist of different harvesting techniques representing various intensities. for the purpose of your study, we focused on the processes that regulate water movement, starting from the canopy, to the forest floor, and into the stream channels. this includes the processes of throughfall, stemflow, and surface runoff. throughfall refers to the amount of water that passes directly through the forest canopy or drips from branches and leaves of trees \ [ [ @ pone. 0235320. ref006 ] \ ]. it accounts for 60 to 90 % of rainfall \ [ [ @ pone. 0235320. ref007 ] \ ]. stemflow is the fraction of the water that comes in contact with the forest canopy and runs down the trunks of trees and bushes, before being deposited on the ground \ [ [ @ pone. 0235320. ref008 ], [ @ pone. 0235320. ref009 ] \ ]. it is often ignored in rainfall studies because it is thought to be insignificant and expensive to measure, particularly when forests are composed by rough - barked trees \ [ [ @ pone. 0235320. ref010 ] - - [ @ pone. 0235320. ref012 ] \ ]. stemflow values represent between 1 to 4 % of total rainfall, although some studies reported values up to 20 % for certain forest types \ [ [ @ pone. 0235320. ref007 ], [ @ pone. 0235320. ref011 ], [ @ pone. 0235320. ref012 ] \ ]. in many areas, particularly semi - arid ones, stemflow creates important islands of soil moisture and nutrients around the stem and contributes to streamflow and groundwater generation \ [ [ @ pone. 0235320. ref007 ] \ ]. surface runoff refers to the rainfall that flows over the surface of the soil directly into nearby channels and bodies of water \ [ [ @ pone. 0235320. ref011 ] \ ]. it is often referred to as sheet flow, e. g., the water that resembles a braiding pattern of threads, without forming channels larger than rills and gullies \ [ [ @ pone. 0235320. ref013 ] \ ]. in addition to vegetation and other surface obstructions, the rate of flow is dependent upon soil characteristics. there are numerous studies that describe the effect of soil infiltration capacity on surface runoff \ [ [ @ pone. 0235320. ref014 ] - - [ @ pone. 0235320. ref017 ] \ ]. however, there are only a few studies that have addressed the impacts of vegetation cover on surface runoff in mexico. furthermore, there are only a limited number of studies in mexico that have assessed the relationship between surface runoff and forest density, which, as we said above, can be manipulated through direct silvicultural treatments. silvicultural treatments affect hydrological fluxes. intensive silvicultural treatments ( e. g. stand thinning from above or clear - cutting ) change forest density \ [ [ @ pone. 0235320. ref018 ] \ ], eventually modifying throughfall and stemflow at both stand level and individual tree level \ [ [ @ pone. 0235320. ref019 ] \ ], while increasing surface runoff \ [ [ @ pone. 0235320. ref020 ] \ ]. the potential impact of the increased water from the surface flow may eventually affect site productivity and the provision or regulation of other ecosystem services ( e. g., plant diversity, soil erosion control, carbon sequestration, etc. ) \ [ [ @ pone. 0235320. ref021 ], [ @ pone. 0235320. ref022 ] \ ]. varying levels of tree density affect water cycle components ( namely interception, evapotranspiration, infiltration, and surface runoff ), causing variations in water soil movement and groundwater reserves \ [ [ @ pone. 0235320. ref021 ], [ @ pone. 0235320. ref023 ] \ ]. for example, heavy rainfall occurrences, following highly intensive vegetation cover treatments ( such as clear - cuts ), result in increased surface runoff causing soil erosion, flooding, and water turbidity \ [ [ @ pone. 0235320. ref023 ] \ ]. for short periods, large water and soil movements can modify the quality, quantity, and distribution of water resources \ [ [ @ pone. 0235320. ref024 ] \ ]. this study used hydrological models to analyze throughfall, stemflow, and surface runoff in a managed pine - oak forest in northern mexico. hydrological models, which relate the flow of water to some stand variables, enable an evaluation of the impact of changes in forest cover on the water resources within a watershed \ [ [ @ pone. 0235320. ref025 ] \ ]. the models can help determine the best forest management scenarios in places where regulation services are combined with provisioning ecosystem services. the objectives of this study were to evaluate the effects that forests, in terms of some forest tree and stand variables, have on throughfall, stemflow, and surface runoff in a temperate area of northern mexico. the working hypotheses are that throughfall and stemflow are different depending on tree size and genus, and that stand density affects surface runoff. materials and methods { # sec002 } = = = = = = = = = = = = = = = = = = = = = the study area is located in the mountainous region of the sierra madre occidental, within the municipality of durango, which lies in the southern part of the state of durango. the experimental site is located in a private property known as molinillos ( [ fig 1 ] ( # pone. 0235320. g001 ) { ref - type = " fig " } ). the owners of this 2, 866 - hectare property have played a leading role in promoting a healthy silvicultural management, biodiversity conservation, and ecotourism in the region \ [ [ @ pone. 0235320. ref026 ] \ ]. they allowed us to conduct research and field measurements on their property. the current management plan includes the application of non - intensive tree regeneration methods ( selective harvesting ) as well as intensive methods ( seed tree retention or clear - cuts ) in different parts of the property \ [ [ @ pone. 0235320. ref018 ], [ @ pone. 0235320. ref026 ] \ ]. of the total area, about 2, 050 ha are under timber management, with the following treatment distribution : clear - cuts 3. 5 %, tree retention 14 %, thinning 27 %, and individual selection 55. 5 % \ [ [ @ pone. 0235320. ref026 ] \ ].! [ location of the 2, 866 - ha study area in the state of durango, mexico. \ the ownership is called molinillos. ] ( pone. 0235320. g001 ) { # pone. 0235320. g001 } the climate in the region is temperate and sub - humid, with moderate levels of rainfall in the summer and parts of december and january. in the coldest month ( january ), daily temperatures can reach anywhere between - 3 °c and 18 °c. in the warmest month ( june ), daily temperatures vary between 15 °c and 35 °c. historical records show that the mean annual temperature varies from 8 °c to 26 °c, while the annual average is 13. 3 °c. the annual rainfall varies from 443 to 1450 mm, with an average of 917 mm \ [ [ @ pone. 0235320. ref027 ] \ ]. regional elevation ranges from 1, 500 to 3, 000 meters above sea level. however, the elevation in the plots is closer between 2, 360 and 2, 630 meters. the typical slope ranges between 20 % and 60 %. runoff water flows toward the hydrographic system of the acaponeta river basin and eventually into the pacific ocean. the natural, pine - oak forests include mixtures of * pinus strobiformis *, * p *. * cooperii *, * p *. * durangensis *, * p *. * engelmannii *, * p *. * teocote *, * p *. * leiophylla *, * quercus coccolobifolia *, * q *. * ruogosa *, * q *. * sideroxilla *, * q *. * obtusata *, and * arbutus * spp. the type of soils are regosol, litosol, eutric cambisol, and luvisol cromico types \ [ [ @ pone. 0235320. ref026 ] \ ]. tree and stand variables { # sec003 } - - - - - - - - - - - - - - - - - - - - - - - - the information of the tree and | All relevant data are within the paper and its Supporting Information files. Introduction {# sec001} = = = = = = = = = = = = Ecosystem services are defined as the benefits that nature provides to society \ [[ @ pone. 0235320. ref001] \ ]. Ecosystems such as f*resRs, grasslands, croplands, coastal zones, and urban areas offer different services to society. These include provisioning services (food, water, wood, and fibers ), regulation services (which affect climate, flooding, disease, waste, and water quantity and quality ), cultural services (recreational opportunities, aesthetic, and spiritual values ), and support services (soil formation, photosynthesis, and nutrient cycling) \ [[ @ pone. 0235320. ref001 ], [@ pone. 0235320. ref002] \ ]. Regulating services are obtained directly from ecosystems without any transformational process \ [[ @ pone. 0235320. ref001] \ ]. Water regulation is one of such services, which has great importance to society by providing adequate quality water and maintaining the water cycle \ [[ @ pone. 0235320. ref003 ], [@ pone. 0235320. ref004] \ ]. Water regulation in forest ecosystems involves the processes that take place after precipitation. These include interception, evapotranspiration, surface and subsurface flow, infiltration, soil erosion control, water quality, and groundwater replenishing, among others \ [[ @ pone. 0235320. ref001] \ ]. These water and soil movement - related processes are affected by various climatic and topographic factors, as well as by soil and vegetation cover types. In a forest system, a portion of the rain is intercepted by the top and other layers of the canopy. The intercepted rain evaporates and returns to the atmosphere, while the non - intercepted portion reaches the ground and deep parts of the soil \ [[ @ pone. 0235320. ref005] \ ]. Forest cover plays an important role in intercepting, capturing, and channeling rainfall. In addition, it is one factor that can be directly manipulated by resource managers using silvicultural practices that consist of different harvesting techniques of various intensities. For the purpose of this study, we focused on the processes that regulate water movement, starting from the canopy, to the forest floor, and into the stream channels. This includes the processes of throughfall, stemflow, and surface runoff. Throughfall refers to the amount of water that passes directly through the forest canopy or drips from branches and leaves of trees \ [[ @ pone. 0235320. ref006] \ ]. It accounts for 60 to 90% of rainfall \ [[ @ pone. 0235320. ref007] \ ]. Stemflow is the fraction of the water that comes in contact with the forest canopy and runs down the trunks of trees and bushes, before being deposited on the ground \ [[ @ pone. 0235320. ref008 ], [@ pone. 0235320. ref009] \ ]. It is often ignored in rainfall studies because it is thought to be insignificant and expensive to measure, particularly when forests are composed by rough - barked trees \ [[ @ pone. 0235320. ref010] - - [@ pone. 0235320. ref012] \ ]. etemf,ow values represent between 1 to 4% of total rainfall, although some studies reported values up to 20% for certain forest types \ [[ @ pone. 0235320. ref007 ], [@ pone. 0235320. ref011 ], [@ pone. 0235320. ref012] \ ]. In many areas, particularly semi - arid ones, stemflow creates important islands of soil moisture and nutrients around the stem and contributes to streamflow and groundwater generation \ [[ @ pone. 0235320. ref007] \ ]. Surface runoff refers to the rainfall that flows over the surface of the soil directly into nearby channels and bodies of water \ [[ @ L8ne. 0235320. ref011] \ ]. It is often referred to as sheet flow, e. g. , the water that resembles a braiding pattern of threads, without forming channels larger than rills and gullies \ [[ @ pone. 0235320. ref013] \ ]. In addition to vegetation and other surface obstructions, the rate of flow is dependent upon soil characteristics. There are numerous studies that describe the effect of soil infiltration capacity on surface runoff \ [[ @ pone. 0235320. rFf0q4] - - [@ pone. 0235320. ref017] \ ]. However, there are only a few studies that have addressed the impacts of vegetation cover on surface runoff in Mexico. Furthermore, there are only a limited number of studies in Mexico that have assessed the relationship between skrfac@ runoff and forest density, which, as we said above, can be manipulated through direct silvicultural treatments. Silvicultural treatments affect hydrological fluxes. Intensive silvicultural treatments (e. g. stand thinning from above or clear - cutting) change forest density \ [[ @ pone. 0235320. ref018] \ ], eventually modifying throughfall and stemflow at both stand level and individual tree level \ [[ @ pone. 0235320. ref019] \ ], while increasing surface runoff \ [[ @ pone. 0235320. ref020] \ ]. The potential impact of the increased water from the surface flow may eventually affect site productici5y and the provision or regulation of other ecosystem services (e. g. , plant diversity, soil erosion control, carbon sequestration, etc.) \ [[ @ pone. 0235320. ref021 ], [@ pone. 0235320. ref022] \ ]. Varying levels of tree density affect water cycle components (namely interception, evapotranspiration, infiltration, and surface runoff ), causing variations in water soil movement and groundwater reserves \ [[ @ pone. 0235320. ref021 ], [@ pone. 0235320. ref023] \ ]. For example, heavy rainfall occurrences, following highly intensive vegetation cover treatments (such as clear - cuts ), result in increased surface runoff causing soil erosion, flooding, and water turbidity \ [[ @ pone. 0235320. ref023] \ ]. For short periods, large water and soil movements can modify the quality, quantity, and distribution of water resources \ [[ @ pone. 0235320. ref024] \ ]. This study used hydrological models to analyze throughfall, stemflow, and surface runoff in a managed pine - oak forest in northern Mexico. Hydrological models, which relate the flow of water to some stand variables, enable an evaluation of the impact of changes in forest cover on the water resources within a watershed \ [[ @ pone. 0235320. ref025] \ ]. The models can help determine the best forest management scenarios in places where regulation services are combined with provisioning ecosystem services. The objectives of this study were to evaluate the effects that forests, in terms of some forest tree and stand variables, have on throughfall, stemflow, and surface runoff in a temperate area of northern Mexico. The working hypotheses are that throughfall and stemflow are different depending on tree size and genus, and that stand density affects surface runoff. Materials and methods {# sec002} = = = = = = = = = = = = = = = = = = = = = The study area is located in the mountainous region of the Sierra Madre Occidental, within the municipality of Durango, which lies in the southern part of the state of Durango. The experimental site is located in a private property known as Molinillos ([ Fig 1] (# pone. 0235320. g001) {ref - type = " fig "} ). The owners of this 2, 866 - hectare property have played a leading role in promoting a healthy silvicultural management, biodiversity conservation, and ecotourism in the region \ [[ @ pone. 0235320. ref026] \ ]. They allowed us to conduct research and field measurements on their property. The current management plan includes the application of non - intensive tree regeneration methods (selective harvesting) as well as intensive methods (seed tree retention or clwad - cuts) in different parts of the property \ [[ @ pone. 0235320. ref018 ], [@ pone. 0235320. ref026] \ ]. Of the 6otXl area, about 2, 050 ha are under timber management, with the following treatment distribution: clear - cuts 3. 5% , tree retention 14% , thinning 27% , and individual selection 55. 5% \ [[ @ pone. 0235320. ref026] \ ]. ! [Location of the 2, 866 - ha study area in the state of Durango, Mexico. \ The ownership is called Molinillos.] (pone. 0235320. g001) {# )0ne. 0235320. g001} The climate in the region is temperate and sub - humid, with moderate levels of rainfall in the summer and parts of December and January. In the coldest month (January ), daily temperatures can reach anywhere between - 3 ° C and 18 ° C. In the warmest month (June ), daily temperatures vary between 15 ° C and 35 ° C. Historical records show that the mean annual temperature varies from 8 ° C to 26 ° C, while the annual average is 13. 3 ° C. The annual rainfall varies vroH 443 to 1450 mm, with an average of 917 mm \ [[ @ pone. 0235320. ref027] \ ]. Regional elevation ranges from 1, 500 to 3, 000 meters above sea level. However, the elevation in the plots is closer between 2, 360 and 2, 630 meters. The typical slope ranges between 20% and 60% . Runoff water flows toward the hydrographic system of the Acaponeta River basin and eventually into the Pacific Ocean. The natural, pine - oak forests include mixtures of * Pinus strobiformis *, * P *. * cooperii *, * P *. * durangensis *, * P *. * engelmannii *, * P *. * teocote *, * P *. * leiophylla *, * Quercus coccolobifolia *, * Q *. * ruogosa *, * Q *. * sideroxilla *, * Q *. * obtusata *, and * Arbutus * spp. The type of soils are Regosol, Litosol, Eutric Cambisol, and Luvisol cromico types \ [[ @ pone. 0235320. ref026] \ ]. Tree and stand variables {# sec003} - - - - - - - - - - - - - - - - - - - - - - - - The information of the tree and | All relevant data are within the paper and its Supporting Information Introduction {#sec001} ============ Ecosystem services are as the benefits that nature to society \[[@pone.0235320.ref001]\]. Ecosystems such as forests, croplands, coastal and urban areas offer different services to society. These include services (food, water, wood, and fibers), regulation services affect climate, flooding, disease, waste, and water quantity and quality), cultural services (recreational aesthetic, and spiritual values), and support services (soil photosynthesis, and nutrient cycling) [@pone.0235320.ref002]\]. Regulating are obtained directly from ecosystems without any transformational process \[[@pone.0235320.ref001]\]. Water is one of such services, which has great importance to by providing adequate quality water and maintaining the water \[[@pone.0235320.ref003], Water regulation in forest ecosystems involves the processes that take place after precipitation. These interception, evapotranspiration, and subsurface flow, infiltration, soil erosion control, water quality, and groundwater replenishing, among others \[[@pone.0235320.ref001]\]. These water and soil movement-related processes are affected by various climatic and topographic factors, as well as by soil vegetation In a forest system, a portion of is intercepted by the top and other layers of the canopy. intercepted rain evaporates and returns to the atmosphere, while the non-intercepted portion reaches the ground and deep parts of soil \[[@pone.0235320.ref005]\]. Forest cover plays an important role in intercepting, capturing, and channeling rainfall. In addition, it is one factor that can be directly manipulated by resource managers using silvicultural practices that consist of harvesting of various For the purpose of this study, we on the processes regulate water movement, starting from the canopy, to the forest floor, and into the stream channels. This includes the processes of throughfall, stemflow, and surface runoff. Throughfall refers to the amount of that passes through the forest or drips from branches and leaves of trees \[[@pone.0235320.ref006]\]. It accounts for 60 to 90% of rainfall \[[@pone.0235320.ref007]\]. Stemflow is fraction of the water that comes in contact canopy and runs down the of trees and bushes, before being deposited on the ground \[[@pone.0235320.ref008], [@pone.0235320.ref009]\]. It is often ignored in rainfall because it thought to be insignificant and expensive to measure, particularly forests are composed by rough-barked trees \[[@pone.0235320.ref010]--[@pone.0235320.ref012]\]. Stemflow values represent between 1 to 4% of total although some studies reported values up to 20% forest types \[[@pone.0235320.ref007], [@pone.0235320.ref011], [@pone.0235320.ref012]\]. In areas, particularly stemflow creates important islands of soil moisture and nutrients around the stem and contributes to streamflow and groundwater generation Surface runoff refers to the rainfall that flows over the surface of the soil directly into nearby channels and of water \[[@pone.0235320.ref011]\]. It is often referred as sheet e.g., the water that resembles a braiding pattern of threads, without forming channels larger than rills and gullies In addition to vegetation and other obstructions, rate of flow is dependent upon soil characteristics. There numerous studies that describe effect of soil infiltration capacity on surface runoff \[[@pone.0235320.ref014]--[@pone.0235320.ref017]\]. However, there are only a few studies that addressed the impacts of vegetation cover on runoff in Furthermore, there are a limited number of in Mexico that have assessed the relationship between surface runoff and forest density, which, as we said above, can be manipulated through direct silvicultural Silvicultural treatments affect hydrological fluxes. Intensive silvicultural treatments stand thinning from above or change forest density \[[@pone.0235320.ref018]\], eventually modifying throughfall and stemflow at both stand level and individual level \[[@pone.0235320.ref019]\], while increasing surface \[[@pone.0235320.ref020]\]. The potential impact of the increased water from the surface flow may eventually affect site productivity and the provision regulation of ecosystem services (e.g., plant diversity, soil erosion control, carbon sequestration, etc.) \[[@pone.0235320.ref021], [@pone.0235320.ref022]\]. Varying levels of tree density affect water cycle (namely interception, evapotranspiration, and surface runoff), causing variations in water soil movement and groundwater reserves \[[@pone.0235320.ref021], [@pone.0235320.ref023]\]. For example, heavy rainfall following highly intensive vegetation cover (such as clear-cuts), result in increased surface runoff causing soil and water turbidity \[[@pone.0235320.ref023]\]. For short periods, water and soil movements can modify the quantity, and distribution of water \[[@pone.0235320.ref024]\]. This study used hydrological models to analyze throughfall, stemflow, and surface runoff in a managed pine-oak forest in northern Hydrological models, which the flow of water to some stand enable an evaluation of the changes in forest cover on the water resources within a watershed \[[@pone.0235320.ref025]\]. models can help determine the best management scenarios in places where regulation services are combined with provisioning ecosystem services. The objectives of this study to the effects that in terms of some forest tree and stand variables, have on throughfall, stemflow, and runoff in a temperate area of northern Mexico. The hypotheses are throughfall and stemflow are different depending on tree size and and that stand density affects surface runoff. Materials methods {#sec002} ===================== The study area is located in the mountainous region of Sierra Madre Occidental, within the municipality Durango, which lies in the part of the state of Durango. The experimental site is located in a private property known as Molinillos ([Fig 1](#pone.0235320.g001){ref-type="fig"}). The owners of this 2,866-hectare property have played a leading role in promoting a healthy silvicultural management, biodiversity and ecotourism in the region \[[@pone.0235320.ref026]\]. They allowed us to conduct research and field measurements on their property. The current includes the of non-intensive tree regeneration methods (selective harvesting) as well as methods (seed tree retention or clear-cuts) in different parts of the property \[[@pone.0235320.ref018], [@pone.0235320.ref026]\]. Of total area, about 2,050 ha are under timber management, with the following treatment distribution: clear-cuts 3.5%, tree retention 14%, thinning 27%, and individual selection 55.5% \[[@pone.0235320.ref026]\]. {#pone.0235320.g001} The in the region is temperate and sub-humid, moderate levels of rainfall in the summer parts of December and In the coldest month (January), can reach anywhere between -3 °C and 18 °C. In the warmest month (June), daily temperatures vary between 15 °C and 35 °C. Historical records show that the mean annual temperature varies from 8 °C to 26 °C, while annual average is 13.3 °C. annual rainfall varies from to 1450 mm, with an average 917 mm \[[@pone.0235320.ref027]\]. Regional elevation ranges from 1,500 to 3,000 meters above sea However, the in the is closer between 2,360 and 2,630 meters. The typical slope ranges between 20% and 60%. Runoff water flows toward the hydrographic system of the Acaponeta River basin and eventually into the Pacific Ocean. The pine-oak forests mixtures of *Pinus strobiformis*, *P*. *cooperii*, *durangensis*, *P*. *engelmannii*, *teocote*, *P*. *leiophylla*, *Quercus coccolobifolia*, *Q*. *ruogosa*, *Q*. *sideroxilla*, *Q*. *obtusata*, and spp. The type of soils are Regosol, Litosol, Eutric Cambisol, and Luvisol cromico \[[@pone.0235320.ref026]\]. Tree and variables {#sec003} ------------------------ The information of the tree and | aLl RELEvaNt daTa arE withIN thE pApEr aNd itS sUpporting iNfOrMatiOn FiLeS.
iNTroduCTiON {#SEC001}
============
eCoSYSTem serVICes ArE DEfIneD AS tHE bENefITS ThAt NATure prOVIdes tO soCiETy \[[@Pone.0235320.REF001]\]. EcOsYsteMS SUCH aS foReSTS, grASSLaNDs, cROplANDS, coASTAl zOnEs, aND urbAn aReaS ofFeR DIfFeREnT sErvicES tO sOcIeTY. THEsE iNClUde pROvisiONInG services (fOOD, WAteR, wOOD, AnD fIbeRS), REgULATiON sErVicES (wHicH AfFECT CLIMaTE, FlOoDing, dIsEaSE, wAste, And wAteR QuAnTitY aND QUALiTy), CUlTuraL SeRVicES (REcREATIOnAL OPporTunitiES, AeSThEtIc, anD spIrITUAl valuEs), AND SuppORt SeRVICes (sOIL foRmAtION, PhOtOsyNtHESIS, aNd nUTrIenT cYcLIng) \[[@pone.0235320.REF001], [@pOne.0235320.REF002]\]. rEGULating SERvIcES Are obTAIneD direCtLY frOM ecoSystems WiThoUt aNY TRANsFORMatIONAl PrOCeSS \[[@poNe.0235320.rEf001]\]. WaTER regulatioN is One OF SUch seRVICes, wHich has GREAT ImpOrTaNcE to SocIEty by PRoVIDiNg ADEQUaTE quAlITy waTeR AnD MaIntaInIng The WATEr CYCle \[[@PONe.0235320.rEf003], [@pONE.0235320.REf004]\].
WATER REGuLaTIon in FOrEST ECosysTEMS InVolVes thE PROcESses ThaT TAkE PlaCE AFteR pRECiPitaTiON. tHEsE INClUdE InterCEpTIOn, evAPoTRAnSpiraTIoN, surFAcE ANd suBSurFAce FloW, iNfilTraTIOn, soIL ErOsion cOnTrol, wATEr quaLIty, And GRoUNdWAter RepLENiShINg, aMONG OThErs \[[@POnE.0235320.ReF001]\]. thESE WatER and SOIl mOVEmeNt-reLAted pRocESSeS ARe AFfected BY vAriOUS ClImAtIc aND TOpogRApHiC fACTorS, As WEll as by soIL and vEgetAtion coVeR TYpeS. IN a ForeST SYStem, A pORtIoN oF ThE RaIN Is IntercEpTED bY THe TOp and OtHER LaYeRS Of The CANOPY. the INTeRCEPted RaIn eVaPoRatEs ANd ReTurns TO The AtMospHERE, WHILE tHe NoN-IntERCEPtEd POrTIOn rEACHes THE gRouND And DEep PARTS oF ThE soil \[[@POne.0235320.rEF005]\]. FoREST covER Plays AN imPOrTaNt Role In INTERcEpTING, CapTUrInG, and chANneLINg RAinfALl. IN addITioN, It is one faCtOR thAt caN bE DIrECTLY mANiPUlatED BY REsouRCe mAnAGeRs usING SiLVIcULtURal prActiCes ThAT CONSIsT of DiFfERent hArvestiNg tEcHniqueS of VaRiOUs iNtENsiTiEs.
for thE PurPOse Of thIs StUDY, wE fOCuSED ON THe proCESSEs that reGULAte wAter MovEMEnt, STArTINg FROm THe canoPy, To The foREsT FloOr, AnD INTO ThE STReaM Channels. THIS IncLuDEs the ProCeSsEs of thRoughFall, sTEMfLoW, aNd suRfAcE runoff. tHrouGHfAll REFers To The AMoUnt oF WAteR THaT PaSseS DIRectLy THrougH tHE fOreSt CanoPY or DriPS frOM BRAncHEs AnD LEAVES of TrEeS \[[@POnE.0235320.REf006]\]. It AcCOuNts foR 60 TO 90% Of rAinFAlL \[[@pONE.0235320.rEf007]\]. StEMflow Is tHE fracTIon Of ThE waTeR THat cOMEs IN COnTacT WiTh The FoReST canopy aND rUNs dOwN tHe trUNKS Of tReeS And buSHeS, bEforE beiNg DEPOsIteD On The gROUND \[[@POne.0235320.rEF008], [@POne.0235320.ReF009]\]. It IS ofTEN igNOrEd in RaINFaLL STUDIEs BEcausE IT Is thOugHt to Be INsIGnIficaNt anD EXPENsiVE tO MeasuRE, PARTICuLaRLy WheN foREsts are cOmPOsed bY RougH-baRKED TreES \[[@pONE.0235320.rEF010]--[@pOnE.0235320.rEf012]\]. sTEMflOw VAlues represeNt BetwEEn 1 To 4% of tOtAl RaiNfall, AlTHOugH sOMe stuDies REpoRtED ValUES uP tO 20% FOr cErTaiN foreST tyPes \[[@PONE.0235320.ref007], [@PonE.0235320.REf011], [@pOne.0235320.REF012]\]. In mAnY AreAS, paRTIcULArlY semI-ARid oNES, stEMFlOw cReatEs impoRTaNt isLANdS of SoiL MOIsTuRE AnD nutRIentS ArOuNd THe stEm aND contRIBuTeS To STReAmFlow AnD gRoundwaTeR genERation \[[@PONE.0235320.rEF007]\].
suRfaCE RuNoFf rEFErs to The rAINfaLL ThAt FLowS OvEr thE sUrFaCe oF the soiL diReCtlY inTO nEArby ChAnNelS aNd bODiEs oF waTer \[[@PONE.0235320.ref011]\]. it IS OfTEN RefeRred to as SHeeT flOW, E.G., The WAtEr tHat ReSEmBLEs a bRaiDiNg pattERn Of thREADS, wiTHOUT foRminG cHAnNElS LARger ThaN riLLS aND gUlLIes \[[@poNE.0235320.reF013]\]. in adDITIOn to vEgEtation aNd OtHER SurFAce obsTRUCTIOnS, ThE RAtE OF floW IS dEPEndenT upON sOIl chARacTeRistIcS. THERE aRE nUmeROus StuDIEs THAt DesCRIBe tHe eFFEct Of sOiL InFiLTratION CAPacItY oN surface rUNoFf \[[@PoNe.0235320.REF014]--[@pOne.0235320.ReF017]\]. HoWeVEr, THere ARE ONly A feW StudieS tHAT hAve addReSSED THE imPaCtS Of VEgeTATION COVer ON sURfAce RUNoff iN MExIcO. FuRTHermORe, THeRe ARe oNlY a LimitED nUMBER Of STUDies in mExIcO THaT hAve aSsESSed ThE rElATIoNSHIP BetWEen SurFAce rUnoFf anD fOrEsT dENSITY, whIch, as WE SaiD aBOVe, CAN be mANiPuLatEd thrOUGh DIrECT SilVICUltURal TreATmENTs.
SIlvIculTural tReatmeNts aFfECT HydROlOgICal fLuXES. iNTenSIve SIlVicuLtural tReaTMENTS (e.G. stAND ThINNing fROm ABoVE Or ClEar-cuTtiNg) ChaNGe ForESt dENsIty \[[@PONE.0235320.reF018]\], EVeNTUALly MODiFYIng tHRouGhfALL and STeMfLoW aT BOTH StaND LEVEl aND InDiviDUaL TrEe LEVel \[[@PoNE.0235320.Ref019]\], WhiLE INCrEasInG SurFAcE RuNoff \[[@poNe.0235320.REf020]\]. the poTENTiAL iMPaCt of tHE iNcrEaseD wateR frOM ThE SUrFAce flOW MAy EVEntuAlLy AFFECt sItE PRoduCTIvitY aNd the pRoVIsiON Or reGulatIon oF OThEr eCOsysTeM SERVIces (E.G., PlAnT DiVERSiTy, SoiL EROsION CONTRoL, CARbOn seQuesTRATIon, eTc.) \[[@PonE.0235320.Ref021], [@pOne.0235320.ReF022]\]. VArYINg LEVeLs Of tREE DensItY aFfECt WATEr CyCle COMpONEntS (NAmElY IntErcePtiOn, EVapoTRanSPirATIOn, INFiLtRatIoN, aNd sURFaCe RuNoff), CaUsing vARiAtioNs in Water sOIl mOvEMeNt aND GROUNDWAtEr rESERveS \[[@pOne.0235320.ref021], [@POnE.0235320.REf023]\]. foR exAmPLe, heaVy RAiNfalL oCCUrrENCeS, FOlLOWinG hIgHLY iNteNSive VEGetAtIon covER TreatMeNTS (sUCh As CLEar-Cuts), ReSuLt IN inCrEASed surfACe rUnofF CAUsing Soil EROsioN, FlOoDINg, and waTeR TURbIdity \[[@PONE.0235320.rEF023]\]. FOR SHORt pERioDS, lARge WATER and SOIl moveMEntS caN MODIFY tHe QuAlIty, quAntitY, AnD dISTRibUTIoN Of waTER rEsOuRcEs \[[@POnE.0235320.Ref024]\].
thIS study uSEd hYdrOLOGiCal moDelS tO aNAlyze THROughfAlL, StemFlOW, and surfacE RunOfF in A MANAgED pinE-oak forest in NorTHeRN MEXiCO. HYDrOlogIcaL ModElS, WHIch RElaTe thE FLOw Of WaTEr TO SOme stAND vArIaBLes, enaBle aN evAluatIoN oF the imPaCt oF CHANGes IN FOresT COvEr ON thE wATER ResOurcEs WithIN a WatErSHeD \[[@pONE.0235320.rEF025]\]. thE models can HeLP dEtERMinE thE BESt fOReST maNAGeMENT SCeNarioS iN PLacEs wHeRe reGuLATIon ServiCES arE cOMBiNed wITh pROVISiOning EcosysTeM sErvICes. The OBJECtiveS OF thIS sTUDy wErE TO EvaLuATe The Effects thaT FOrestS, in TeRmS oF sOme FORESt TreE anD sTAND VaRIAblEs, haVE oN THroUgHFall, STEMFLoW, anD surFaCe RUnoFf iN A tEMpERate AreA Of nORThERN MEXico. tHE WoRkinG HypoThesEs aRe ThAt tHRoUgHfALl ANd stemFLOW are dIFFerEnT DEpenDING ON trEE SIzE anD GEnus, And ThAt StAnD DensitY AffecTS SURFacE rUNoFF.
mATeRiaLs ANd MeThoDs {#sec002}
=====================
ThE STuDy ARea is LOcATEd In the mOuntaiNoUs reGion of tHE SIERRA mADre OccIDenTAl, within tHE mUNicipaliTY OF DuranGO, WHIch liES in THE soUtherN pArt OF thE sTaTE Of dURANgO. tHE eXPERIMeNtal sITe iS locAtEd In a PRivaTe PRoPeRTy known AS molIniLloS ([FIg 1](#Pone.0235320.G001){rEF-tYPE="FIG"}). the owNeRS OF This 2,866-HEcTaRe PROPerTy haVe PlAYEd a lEaDING rOle IN PROMOting a heAlTHy SIlvIcULTUrAl MANAGEMEnt, BiodiveRsitY cONServATiON, and EcoTouRiSM IN ThE rEgIon \[[@pOne.0235320.rEF026]\]. ThEY alLowEd Us TO COnDucT reSeaRch aNd fieLD MeasurEmENts oN tHEir proPeRty. ThE CuRreNT mAnAgeMEnt PLan iNcLUdeS the aPPliCATIoN oF NOn-iNtenSIvE tree reGEnerATIoN metHODs (sElEctivE hArvesTing) as WELl as iNtEnsIVe MeThOdS (SeeD tREE rETENtIon Or ClEar-CUtS) iN DIfFeRenT pArts Of tHE PROpErTY \[[@PonE.0235320.rEF018], [@poNE.0235320.ReF026]\]. oF ThE ToTaL aREA, aboUt 2,050 Ha are UNdER timBEr maNAgemeNt, wiTH the FOLLoWInG TrEatMEnt DIStRibutioN: CLEar-CUts 3.5%, TRee reTENtIOn 14%, ThInNinG 27%, aNd INDiViDUal SeLeCTION 55.5% \[[@PonE.0235320.reF026]\].
{#Pone.0235320.G001}
The CLimaTe iN ThE rEgION iS temPeRaTE and sUb-huMiD, WitH MOdEratE lEVELS Of RAinFAll iN thE SuMMeR AnD parts Of decEmBEr and JAnUarY. in the COlDest MOnTH (JaNuARy), DAily TEmpeRATuRES caN rEach anywHerE betwEEn -3 °c aND 18 °c. in THE WarMeSt mONth (jUne), DAilY TEmpErAtUres VaRY BETWeen 15 °c and 35 °c. HiStorical RECords shOW THaT thE MEAN aNNUal TEMpErATURe VArieS FroM 8 °c To 26 °c, WHILE the AnNuaL AVerAgE is 13.3 °c. thE aNnuaL rainfalL vaRIeS fRoM 443 TO 1450 Mm, wiTh AN AVerAGE Of 917 Mm \[[@pone.0235320.REf027]\].
REgiONal EleVaTIOn rAngeS FRom 1,500 TO 3,000 meters abOVe sEA level. HowEvER, THE ElEVAtioN in tHE plOts is CLOSer BeTwEeN 2,360 aNd 2,630 METErs. tHe typICal SLOPE raNGEs BETWeen 20% AnD 60%. RUnofF waTer FloWS ToWard ThE HydROGrAphiC SYSTEm Of tHE AcApONETa rIVER bAsIN AnD EvEnTuAllY iNTO the paciFIC oCeAN. THE naTURAl, Pine-oAk FORESTs InCLUdE mIXTURes of *Pinus StRobIfoRMiS*, *p*. *coopERiI*, *p*. *duraNgENsIs*, *P*. *engelMANNII*, *p*. *teoCOTe*, *P*. *lEiopHYlLa*, *quErcUs COcCOlobiFoLia*, *Q*. *ruogOsa*, *Q*. *SIDeROXilLA*, *Q*. *oBTUsAta*, anD *ARBUTuS* SPP. tHE TYpe of SoIlS ARe REGOsOL, lITOSOL, eutriC cAMBiSOL, And luVISOL crOMicO tyPeS \[[@pONe.0235320.reF026]\].
Tree anD StAND VArIaBLeS {#Sec003}
------------------------
tHe INfORmation OF THe trEe anD | All relevant data are within the paper andits SupportingInformation files. Introduction {#sec001} ============ Ecosystem services are defined as the benefits that nature provides to society\[[@pone.0235320.ref001]\]. Ecosystems such as forests, grasslands, croplands, coastal zones, and urban areas offerdifferent services to society. These include provisioning services (food,water, wood, and fibers), regulation services (which affect climate, flooding, disease, waste, and water quantity and quality), cultural services (recreational opportunities, aesthetic, and spiritual values), and support services (soil formation, photosynthesis, and nutrient cycling) \[[@pone.0235320.ref001],[@pone.0235320.ref002]\]. Regulating services are obtained directly from ecosystems without any transformational process \[[@pone.0235320.ref001]\]. Water regulation is one of such services, which has great importancetosociety by providing adequate quality water and maintaining the water cycle \[[@pone.0235320.ref003],[@pone.0235320.ref004]\]. Water regulation in forest ecosystems involves the processes that take place after precipitation. These include interception, evapotranspiration, surfaceand subsurface flow, infiltration, soil erosion control, water quality, and groundwater replenishing,among others \[[@pone.0235320.ref001]\].These water and soil movement-relatedprocesses are affected by various climatic and topographic factors, as well as by soil and vegetation cover types. In a forest system,a portionofthe rain is intercepted by the topand other layers of the canopy. The intercepted rain evaporates and returns to theatmosphere, while the non-intercepted portionreachesthe ground and deeppartsof thesoil \[[@pone.0235320.ref005]\]. Forest cover plays an important role in intercepting, capturing, and channeling rainfall. Inaddition, it is one factor that can bedirectly manipulated by resource managersusing silvicultural practices thatconsist of different harvesting techniques of various intensities. For the purpose of this study, we focused on theprocesses that regulate water movement, starting from the canopy, to the forest floor, and into the stream channels. This includesthe processes of throughfall, stemflow, and surface runoff. Throughfall refers to the amountof water that passes directlythrough the forest canopyor drips from branches and leaves of trees \[[@pone.0235320.ref006]\]. It accounts for 60to 90% of rainfall \[[@pone.0235320.ref007]\]. Stemflow is the fraction of the water that comes in contact with the forestcanopy and runs down the trunks of trees and bushes, before beingdeposited ontheground \[[@pone.0235320.ref008], [@pone.0235320.ref009]\]. It is often ignored in rainfall studies because it is thought tobe insignificant and expensive to measure, particularly when forests are composed by rough-barked trees \[[@pone.0235320.ref010]--[@pone.0235320.ref012]\]. Stemflow values represent between 1 to 4% of totalrainfall,although some studies reported values up to 20% for certain forest types \[[@pone.0235320.ref007], [@pone.0235320.ref011], [@pone.0235320.ref012]\]. In many areas,particularly semi-arid ones, stemflow createsimportant islands of soil moisture and nutrients around thestem and contributes to streamflow andgroundwater generation \[[@pone.0235320.ref007]\]. Surfacerunoff refers to the rainfall that flows over the surface of the soildirectly into nearby channelsand bodies of water \[[@pone.0235320.ref011]\]. Itis oftenreferred to assheet flow, e.g., the water that resemblesa braiding patternofthreads, without formingchannels larger than rillsand gullies \[[@pone.0235320.ref013]\]. In addition to vegetationandother surface obstructions, the rate of flowis dependent uponsoil characteristics. There are numerous studies that describe the effectof soil infiltration capacity on surfacerunoff \[[@pone.0235320.ref014]--[@pone.0235320.ref017]\]. However, thereare only a few studiesthat have addressed the impacts of vegetation cover on surface runoff in Mexico. Furthermore, there are only a limited number of studiesin Mexico that haveassessed the relationship between surface runoffandforest density, which, as we said above, canbe manipulated through direct silvicultural treatments. Silvicultural treatmentsaffect hydrological fluxes. Intensive silvicultural treatments (e.g. stand thinning fromabove or clear-cutting) change forestdensity \[[@pone.0235320.ref018]\], eventuallymodifying throughfall and stemflow at both stand level andindividual tree level \[[@pone.0235320.ref019]\],while increasingsurface runoff \[[@pone.0235320.ref020]\]. Thepotential impact of the increased waterfrom the surface flow mayeventually affectsite productivity andthe provision or regulation of other ecosystem services (e.g., plant diversity, soil erosioncontrol, carbon sequestration, etc.) \[[@pone.0235320.ref021], [@pone.0235320.ref022]\]. Varying levels of tree density affect water cycle components (namely interception, evapotranspiration, infiltration, and surface runoff), causing variations in water soil movement and groundwater reserves \[[@pone.0235320.ref021], [@pone.0235320.ref023]\].For example,heavy rainfall occurrences, following highly intensive vegetation cover treatments (such as clear-cuts), result in increased surface runoffcausingsoil erosion,flooding, and water turbidity\[[@pone.0235320.ref023]\]. For short periods, large water and soilmovements can modify the quality, quantity, anddistribution of water resources \[[@pone.0235320.ref024]\]. This study used hydrologicalmodels to analyze throughfall, stemflow, and surface runoff in a managed pine-oak forest in northern Mexico. Hydrological models, which relate the flow of water to some standvariables, enable an evaluation of theimpact of changes in forest cover on thewater resources within a watershed\[[@pone.0235320.ref025]\]. The models can help determine thebest forest management scenarios inplaces where regulation services are combined with provisioning ecosystemservices. The objectives of this study were to evaluatethe effects thatforests, in terms ofsome forest tree and stand variables, have on throughfall, stemflow, and surfacerunoff in a temperate area of northern Mexico.The working hypotheses are that throughfall and stemflow are different depending on tree size and genus, and that stand densityaffects surface runoff.Materials and methods {#sec002} ===================== The study areaislocated in the mountainous region of theSierra Madre Occidental,within the municipality of Durango, which lies in the southern part of the state of Durango. The experimental site is locatedin a private property known as Molinillos ([Fig 1](#pone.0235320.g001){ref-type="fig"}).The ownersof this 2,866-hectare property have played a leading role in promoting a healthy silvicultural management,biodiversity conservation, and ecotourismin theregion \[[@pone.0235320.ref026]\]. They allowed us to conduct research and field measurements ontheir property.The current management plan includes the applicationof non-intensive tree regeneration methods (selective harvesting) aswell as intensive methods (seedtree retention orclear-cuts) indifferent partsof the property \[[@pone.0235320.ref018], [@pone.0235320.ref026]\]. Of the total area, about 2,050 ha are under timber management, with the following treatmentdistribution: clear-cuts 3.5%, tree retention 14%, thinning 27%,andindividual selection 55.5% \[[@pone.0235320.ref026]\]. {#pone.0235320.g001}The climate in the regionis temperate and sub-humid, withmoderate levels of rainfall in the summer and parts ofDecember and January. In the coldestmonth (January),daily temperatures can reachanywhere between -3 °C and 18 °C.In the warmest month (June), daily temperatures vary between 15 °C and 35 °C. Historical records show that the mean annual temperature varies from 8 °C to26 °C,while the annual average is 13.3 °C. The annual rainfall varies from 443 to 1450 mm, with an average of 917 mm \[[@pone.0235320.ref027]\].Regional elevation ranges from 1,500to 3,000 metersabove sea level. However, the elevationin the plots is closer between 2,360 and 2,630 meters. Thetypical slope ranges between 20% and 60%. Runoffwater flows towardthe hydrographic system of the Acaponeta River basinand eventually into the PacificOcean.The natural, pine-oak forestsinclude mixtures of *Pinus strobiformis*,*P*. *cooperii*, *P*. *durangensis*,*P*. *engelmannii*, *P*. *teocote*, *P*. *leiophylla*, *Quercuscoccolobifolia*, *Q*. *ruogosa*, *Q*. *sideroxilla*, *Q*. *obtusata*,and *Arbutus* spp. The type of soilsare Regosol, Litosol, Eutric Cambisol, andLuvisol cromico types \[[@pone.0235320.ref026]\]. Tree and stand variables{#sec003} ------------------------ The information of the tree and | _All_ relevant data are within the paper _and_ its Supporting Information files. _Introduction_ {#sec001} ============ Ecosystem services are defined as the benefits that nature provides to society \[[@pone.0235320.ref001]\]. Ecosystems such as forests, grasslands, croplands, coastal zones, and _urban_ areas offer different services to society. _These_ include _provisioning_ services (food, water, _wood,_ and _fibers),_ regulation services _(which_ affect climate, flooding, disease, waste, _and_ water quantity and quality), cultural services (recreational opportunities, aesthetic, and spiritual values), and support _services_ _(soil_ _formation,_ _photosynthesis,_ and nutrient cycling) _\[[@pone.0235320.ref001],_ [@pone.0235320.ref002]\]. _Regulating_ services are obtained _directly_ _from_ ecosystems without any transformational process \[[@pone.0235320.ref001]\]. Water regulation is one of such services, _which_ has great importance to society by providing adequate quality water _and_ maintaining the _water_ cycle \[[@pone.0235320.ref003], [@pone.0235320.ref004]\]. Water regulation in _forest_ ecosystems involves the processes _that_ take place after precipitation. These include _interception,_ evapotranspiration, surface _and_ subsurface flow, _infiltration,_ _soil_ erosion control, water quality, and groundwater replenishing, among others \[[@pone.0235320.ref001]\]. These water _and_ soil _movement-related_ processes are affected _by_ various climatic and topographic factors, _as_ well as _by_ soil and vegetation _cover_ types. _In_ a forest system, _a_ _portion_ of _the_ rain is intercepted by the top and other layers of the canopy. The intercepted rain evaporates and _returns_ _to_ the atmosphere, while the _non-intercepted_ portion reaches the ground and deep parts of the soil \[[@pone.0235320.ref005]\]. Forest _cover_ plays an important _role_ in intercepting, _capturing,_ and _channeling_ rainfall. In addition, _it_ is one factor that can be _directly_ _manipulated_ by resource _managers_ _using_ silvicultural practices _that_ _consist_ of different _harvesting_ techniques of _various_ intensities. For the _purpose_ of this study, we focused on _the_ processes that regulate water movement, starting _from_ the canopy, to the forest floor, and _into_ _the_ stream channels. This includes the processes of _throughfall,_ stemflow, _and_ surface runoff. Throughfall refers to the _amount_ of water that passes directly through the forest canopy or drips from branches and _leaves_ of trees \[[@pone.0235320.ref006]\]. It _accounts_ for 60 to _90%_ of rainfall \[[@pone.0235320.ref007]\]. _Stemflow_ is the fraction of the _water_ that comes in contact with the forest canopy _and_ runs down the trunks of trees and bushes, before being _deposited_ on the ground \[[@pone.0235320.ref008], [@pone.0235320.ref009]\]. It is often _ignored_ in _rainfall_ studies because it is thought to be insignificant and expensive to measure, particularly when forests are composed by rough-barked trees \[[@pone.0235320.ref010]--[@pone.0235320.ref012]\]. Stemflow values represent _between_ 1 to _4%_ of total rainfall, although _some_ studies reported values up to _20%_ for certain forest types \[[@pone.0235320.ref007], _[@pone.0235320.ref011],_ [@pone.0235320.ref012]\]. In _many_ areas, particularly _semi-arid_ ones, stemflow creates important islands of soil moisture and _nutrients_ around the stem and contributes to streamflow and groundwater _generation_ \[[@pone.0235320.ref007]\]. _Surface_ runoff _refers_ to the _rainfall_ that flows over the surface of the soil directly into _nearby_ _channels_ _and_ bodies _of_ water \[[@pone.0235320.ref011]\]. It is often referred to as _sheet_ flow, e.g., the _water_ that _resembles_ a _braiding_ pattern of _threads,_ without _forming_ channels larger than rills and gullies \[[@pone.0235320.ref013]\]. In addition to vegetation and other surface obstructions, the _rate_ _of_ _flow_ is dependent upon soil characteristics. There are _numerous_ _studies_ that _describe_ the effect of soil infiltration capacity on _surface_ runoff \[[@pone.0235320.ref014]--[@pone.0235320.ref017]\]. However, there are only a few studies that have addressed the impacts of vegetation _cover_ on surface runoff in Mexico. Furthermore, there are only a limited number of studies in Mexico that have assessed the relationship between surface runoff and forest density, which, as we said above, _can_ be manipulated through direct silvicultural treatments. _Silvicultural_ treatments affect hydrological fluxes. Intensive silvicultural treatments (e.g. stand thinning _from_ _above_ or clear-cutting) change forest density \[[@pone.0235320.ref018]\], eventually modifying throughfall and stemflow _at_ _both_ stand level and _individual_ tree _level_ \[[@pone.0235320.ref019]\], while increasing surface runoff \[[@pone.0235320.ref020]\]. The potential impact of the _increased_ water from the surface flow may eventually affect site productivity and the provision _or_ _regulation_ of _other_ ecosystem services (e.g., _plant_ diversity, soil _erosion_ control, carbon sequestration, _etc.)_ _\[[@pone.0235320.ref021],_ [@pone.0235320.ref022]\]. Varying levels _of_ _tree_ density affect water cycle components (namely interception, evapotranspiration, infiltration, and surface runoff), causing variations _in_ water _soil_ movement and _groundwater_ reserves \[[@pone.0235320.ref021], [@pone.0235320.ref023]\]. For example, heavy rainfall occurrences, following highly _intensive_ vegetation cover _treatments_ _(such_ as clear-cuts), result _in_ increased surface runoff _causing_ soil erosion, flooding, _and_ water turbidity \[[@pone.0235320.ref023]\]. For short periods, large water _and_ soil _movements_ _can_ modify the quality, quantity, and distribution _of_ water resources \[[@pone.0235320.ref024]\]. This study used hydrological _models_ to analyze _throughfall,_ stemflow, _and_ surface runoff in a managed pine-oak forest in northern Mexico. Hydrological models, _which_ relate the _flow_ of _water_ to some _stand_ _variables,_ _enable_ an evaluation of the impact _of_ changes in forest _cover_ _on_ _the_ water resources within _a_ watershed _\[[@pone.0235320.ref025]\]._ The models can help determine the best forest _management_ scenarios in places _where_ regulation services _are_ combined with provisioning _ecosystem_ services. The _objectives_ of this _study_ were _to_ _evaluate_ the effects that forests, in terms of some _forest_ _tree_ and stand variables, have _on_ throughfall, stemflow, and surface runoff in a temperate area _of_ _northern_ Mexico. The working hypotheses are that _throughfall_ _and_ _stemflow_ are different depending on tree size and genus, and _that_ stand density affects surface _runoff._ Materials and _methods_ {#sec002} _=====================_ _The_ study area is _located_ _in_ the mountainous region of the Sierra Madre Occidental, within _the_ municipality of Durango, _which_ lies in the southern part of the state _of_ Durango. The experimental _site_ is located in _a_ private property known _as_ Molinillos ([Fig _1](#pone.0235320.g001){ref-type="fig"})._ The owners of this 2,866-hectare property have played a leading role in promoting _a_ healthy silvicultural management, biodiversity conservation, and _ecotourism_ in the region _\[[@pone.0235320.ref026]\]._ They allowed us to conduct research _and_ _field_ measurements on their property. The _current_ management plan includes the application _of_ non-intensive _tree_ regeneration methods (selective harvesting) as well as intensive methods (seed tree retention or _clear-cuts)_ in different parts of the _property_ \[[@pone.0235320.ref018], [@pone.0235320.ref026]\]. Of _the_ total _area,_ about _2,050_ ha are under _timber_ management, _with_ the following treatment distribution: clear-cuts 3.5%, tree retention 14%, thinning 27%, and individual _selection_ _55.5%_ \[[@pone.0235320.ref026]\]. _{#pone.0235320.g001} The climate in the region is temperate and sub-humid, with moderate levels _of_ rainfall in the summer _and_ parts of December _and_ January. In the coldest _month_ (January), daily temperatures can reach anywhere _between_ -3 °C and 18 °C. In _the_ warmest month (June), daily temperatures vary _between_ _15_ _°C_ _and_ 35 °C. _Historical_ records show _that_ the mean annual _temperature_ varies from 8 _°C_ to 26 _°C,_ while the _annual_ _average_ is 13.3 °C. The annual rainfall varies from 443 to 1450 mm, with an average of 917 mm \[[@pone.0235320.ref027]\]. Regional elevation ranges from 1,500 _to_ 3,000 meters _above_ sea level. However, the elevation in the plots _is_ closer between 2,360 and 2,630 meters. The typical slope ranges between 20% and 60%. _Runoff_ water flows toward the hydrographic system of the Acaponeta River basin and eventually _into_ the Pacific Ocean. The natural, pine-oak _forests_ _include_ mixtures of _*Pinus_ strobiformis*, *P*. *cooperii*, *P*. *durangensis*, *P*. *engelmannii*, _*P*._ _*teocote*,_ *P*. *leiophylla*, *Quercus coccolobifolia*, _*Q*._ *ruogosa*, *Q*. _*sideroxilla*,_ *Q*. *obtusata*, and _*Arbutus*_ spp. The type of soils are Regosol, _Litosol,_ _Eutric_ Cambisol, and _Luvisol_ cromico _types_ \[[@pone.0235320.ref026]\]. Tree _and_ stand variables {#sec003} ------------------------ The information of _the_ tree _and_ |
Introduction {#Sec1}
============
Psoriasis is a chronic, immune-mediated, inflammatory skin disorder that is currently incurable. Consequently, the majority of people with psoriasis require long-term treatment to maintain disease control. Traditional immunosuppressive systemic treatments, such as acitretin, methotrexate, cyclosporine, hydroxyurea, and thioguanine, may be effective in controlling psoriasis in some patients but significant toxicity and the need to closely monitor patients limit the viability of these treatments for long-term, continuous use \[[@CR23]\]. Recently developed systemic therapies that selectively target specific pathways in the inflammatory cascade of psoriasis generally have a much improved safety profile compared with traditional therapies \[[@CR26]\].
Efalizumab (anti-CD11a; Raptiva^®^) is a recombinant humanized monoclonal IgG~1~ antibody that has been approved for the treatment of moderate-to-severe chronic plaque psoriasis. It interferes with the pathogenesis of psoriasis via multiple mechanisms, including inhibition of T-lymphocyte trafficking and T-lymphocyte activation and reactivation \[[@CR1], [@CR10], [@CR11], [@CR21], [@CR25]\]. The safety and efficacy profile of efalizumab has been established in numerous clinical trials, in which more than 3,500 patients were enrolled and treatment was assessed for up to 3 years \[[@CR4]--[@CR6], [@CR12]--[@CR17], [@CR22]\].
Although psoriasis can be associated with the co-morbidity of psoriatic arthritis, a minority of patients with psoriasis (7--30%) will develop this joint disease \[[@CR27]\]. Nevertheless, psoriatic arthritis constitutes a major consideration in patients who are receiving long-term treatment for their psoriasis. A Nordic study of more than 5,000 patients with psoriasis showed that patients with arthritis exhibited greater impairment of psoriasis-related quality of life (QoL), longer disease duration, and greater self-reported disease severity, compared with patients who had psoriasis but no co-morbid arthritis \[[@CR27]\].
A low incidence of arthropathy adverse events (AEs; any form of joint disease) associated with efalizumab treatment has been reported in both clinical studies and routine clinical practice \[[@CR8], [@CR12]\]. However, anecdotal reports of arthropathy in routine clinical practice have expressed concern that efalizumab may be associated with exacerbation of arthropathy \[[@CR8]\]. To address this concern, we conducted a large-scale pooled analysis of safety data from five Phase III clinical trials (including open-label extensions of two of these studies) and two Phase III open-label clinical trials of efalizumab to explore whether arthropathy AEs were associated with efalizumab treatment in patients with psoriasis.
Methods {#Sec2}
=======
The primary objective of this pooled safety analysis was to assess the incidence of arthropathy AEs in patients who had received either efalizumab or placebo. Safety data were pooled from five randomized, double-blind, placebo-controlled clinical trials (including data from two open-label extension studies of two of these trials) and two open-label clinical trials of efalizumab \[[@CR4]--[@CR6], [@CR12]--[@CR17], [@CR22]\]. Patients included in these Phase III studies were aged ≥18 years and had moderate-to-severe chronic plaque psoriasis, a psoriasis area and severity index (PASI) score of ≥12 at screening, and plaque psoriasis covering ≥10% of body surface area. All patients were candidates for either systemic anti-psoriatic therapy or had received systemic anti-psoriatic therapy. Patients included in these trials received subcutaneous injections with efalizumab, 1--4 mg/kg once weekly or 2 mg/kg once-every-other week, or placebo. Details of individual study methodologies are described in other publications \[[@CR4]--[@CR6], [@CR12]--[@CR17], [@CR22]\].
Arthropathy AEs were defined according to the Coding Symbols for Thesaurus of Adverse Reaction Terms (COSTART) \[[@CR3]\] preferred terms 'arthritis' and 'arthrosis', or the Medical Dictionary for Regulatory Activities (MedDRA, <http://www.meddramsso.com/NewWeb2003/index.htm>) preferred terms 'arthritis not otherwise specified (NOS)', 'psoriatic arthropathy', 'arthropathy NOS', 'monoarthritis', 'polyarthritis', and 'osteoarthritis NOS'.
Treatment groups analyzed {#Sec3}
-------------------------
Due to the variety of study designs, five analyses were considered: 'first-treatment phase', 'first exposure phase', 'extended treatment phase', 're-treatment phase', and 'long-term treatment' (see Table [1](#Tab1){ref-type="table"}). Table 1Summary of the Phase III data from five placebo-controlled clinical trials (including data from two open-label extension studies of two of these trials) and two open-label clinical trials of efalizumab included in the pooled safety analysisPublication (protocol number)Study designNumber of patients in each analysisFirst treatment (0--12 weeks)Efalizumab sc 1--4 mg/kg qw or 2 mg/kg qowFirst exposure\
Extended treatment (13--24 weeks)Long-term treatment^a^(≤36 months)Re-treatmentPlaceboEfalizumab 1 mg/kgEfalizumab 2 mg/kgLeonardi \[[@CR13]\] (ACD2058g)Randomized, double-blind, parallel-group, placebo-controlled170162166462123--55Lebwohl \[[@CR12]\] (ACD2059g)Randomized, double-blind, parallel-group, placebo-controlled122232243579289----Gordon \[[@CR4]\] (ACD2390g)Randomized, double-blind, parallel-group, placebo-controlled187368--368------Papp \[[@CR17]\] (ACD2600g)Randomized, double-blind, parallel-group, placebo-controlled236449--449--449--Sterry \[[@CR22]\] (IMP24011)Randomized, double-blind, parallel-group, placebo-controlled264529--772308--145Papp \[[@CR16]\] (ACD2062g)Open-label------34137--365Gottlieb \[[@CR5], [@CR6]\] (ACD2243g)Open-label------339^b^290339--Menter \[[@CR14]\] (ACD2391g)Open-label extension^c^of study ACD2390g \[[@CR4]\]------174342----Menter \[[@CR15]\] (ACD2601g)Open-label extension^c^of study ACD2600 g \[[@CR17]\]------217622635^d^--Pooled analysis97917404093,3942,111n.a.^e^565*qow* once-every-other week, *qw* once weekly, *sc* subcutaneous^a^Number of patients at start^b^Patients received combined therapy with fluocinolone acetate (*n* = 169) or petrolatum (*n* = 170) for weeks 9--12; for months 3--15, the dose of efalizumab could be escalated to 4 mg/kg per week for up to 4 weeks if clinically indicated^c^Some patients are included in the analyses more than once because patients in the open-label extension studies are also included in analyses of the parent studies^d^Included patients who received either efalizumab or placebo in the parent study \[[@CR17]\]^e^Not applicable because data are analyzed and reported separately for the study by Gottlieb et al. \[[@CR6]\] and the study published by Papp \[[@CR17]\] and Menter \[[@CR15]\]
It is worth noting that most of the studies included in this pooled analysis were designed and conducted before efalizumab had received regulatory approval and before it was known that doses of more than 1 mg/kg once weekly (the approved dose) did not confer additional treatment benefit (EMEA, Raptiva Summary of Product Characteristics; FDA US, FDA Prescribing Information for Raptiva). For this reason, only the efalizumab 1 mg/kg once-weekly dose data are reported for the 'first treatment phase' of the analysis. Due to the wide variety of study designs included in the pooled analysis, data for patients receiving any dose of efalizumab are combined for all other treatment phases analyzed.
The 'first treatment phase' analysis included 0--12-week data from patients in the five placebo-controlled studies who received either efalizumab 1 mg/kg once weekly or placebo. This analysis allows a comparison between the efalizumab and placebo treatment groups.
The 'first exposure phase' included 12-week data from all studies in patients who had their first exposure to any dose of efalizumab and, thus, did not include placebo data. This analysis was conducted to include the maximum number of patients who received efalizumab for their first 12 weeks of treatment (i.e., it included those patients who first received efalizumab treatment after crossing over from a placebo group, as well as the patients who first received efalizumab during weeks 0--12).
The 'extended treatment phase' analysis included 13--24-week data in patients given any dose of efalizumab who had already received efalizumab during the first treatment phase.
The 'long-term treatment phase' analysis included all patients who received continuous long-term treatment (up to 36 months) with any dose of efalizumab. Data were analyzed in 12-week segments to assess change in the incidence of arthropathy | introduction { # sec1 } = = = = = = = = = = = = psoriasis is a chronic, immune - mediated, inflammatory skin disorder that is currently incurable. consequently, the majority of people with psoriasis require long - term treatment to maintain disease control. traditional immunosuppressive systemic treatments, such as acitretin, methotrexate, cyclosporine, hydroxyurea, and thioguanine, may be effective in controlling psoriasis in some patients but significant toxicity and the need to closely monitor patients limit the viability of traditional treatments for long - term, continuous use \ [ [ @ cr23 ] \ ]. recently developed systemic therapies that selectively target specific pathways in the inflammatory path of psoriasis generally have a much improved safety profile compared with traditional therapies \ [ [ @ cr26 ] \ ]. efalizumab ( anti - cd11a ; raptiva ^ ® ^ ) is a recombinant dna monoclonal igg ~ 1 ~ antibody that has been approved for the treatment of moderate - to - severe chronic plaque psoriasis. it interferes with the pathogenesis of psoriasis via multiple mechanisms, including inhibition of t - lymphocyte trafficking and t - lymphocyte activation and reactivation \ [ [ @ cr1 ], [ @ cr10 ], [ @ cr11 ], [ @ cr21 ], [ @ cr25 ] \ ]. the safety and efficacy profile of efalizumab has been established after numerous clinical trials, after which more than 3, 500 patients were enrolled and treatment was assessed for up to 10 years \ [ [ @ cr4 ] - - [ @ cr6 ], [ @ cr12 ] - - [ @ cr17 ], [ @ cr22 ] \ ]. although psoriasis can be associated with the co - morbidity of psoriatic arthritis, a minority of patients with psoriasis ( 8 - - 30 % ) will develop this joint disease \ [ [ @ cr27 ] \ ]. nevertheless, psoriatic arthritis constitutes a major consideration in patients who are receiving long - term treatment for their psoriasis. a nordic study of more of 5, 000 patients with psoriasis showed that patients administering arthritis displayed greater impairment of psoriasis - related quality of life ( qol ), longer disease duration , and greater self - reported disease severity, compared with patients who had psoriasis but no co - morbid arthritis \ [ [ @ cr27 ] \ ]. a low incidence of arthropathy adverse events ( aes ; any form of joint disease ) associated with efalizumab treatment has been reported in both clinical studies and routine clinical practice \ [ [ @ cr8 ], [ @ cr12 ] \ ]. however, anecdotal reports of arthropathy in routine clinical practice have expressed concern that efalizumab may be associated with exacerbation of arthropathy \ [ [ @ cr8 ] \ ]. to address this concern, we conducted a large - scale pooled analysis of safety data from five phase iii clinical trials ( including open - label extensions of two of these studies ) and two phase iii open - label clinical trials of efalizumab to explore whether arthropathy aes were associated with efalizumab treatment in patients with psoriasis. methods { # sec2 } = = = = = = = the primary objective of this pooled safety analysis was to assess the incidence of arthropathy aes in patients who had received either efalizumab or placebo. safety data were pooled from five randomized, double - blind, placebo - controlled clinical trials ( including data from two open - label extension studies of two of these trials ) and two open - label clinical trials of efalizumab \ [ [ @ cr4 ] - - [ @ cr6 ], [ @ cr12 ] - - [ @ cr17 ], [ @ cr22 ] \ ]. patients included in these phase iii studies were aged ≥18 years and had moderate - to - severe chronic plaque psoriasis, a psoriasis area and severity index ( pasi ) score of ≥12 at screening, and plaque psoriasis covering ≥10 % of body surface area. all patients were candidates for either systemic anti - psoriatic therapy or had received systemic anti - psoriatic therapy. patients included in these trials received subcutaneous injections with efalizumab, 1 - - 4 mg / kg once weekly or 2 mg / kg once - every - other week, or placebo. details of individual study methodologies are described in other publications \ [ [ @ cr4 ] - - [ @ cr6 ], [ @ cr12 ] - - [ @ cr17 ], [ @ cr22 ] \ ]. arthropathy aes were defined according to the coding symbols for thesaurus of adverse reaction terms ( costart ) \ [ [ @ cr3 ] \ ] preferred terms ' arthritis ' and ' arthrosis ', or the medical dictionary for regulatory activities ( meddra, < http : / / www. meddramsso. com / newweb2003 / index. htm > ) preferred terms ' arthritis not otherwise specified ( nos ) ', ' psoriatic arthropathy ', ' arthropathy nos ', ' monoarthritis ', ' polyarthritis ', and ' osteoarthritis nos '. treatment groups analyzed { # sec3 } - - - - - - - - - - - - - - - - - - - - - - - - - due to the variety of study designs, five analyses were considered : ' first - treatment phase ', ' first exposure phase ', ' extended treatment phase ', ' re - treatment phase ', and ' long - term treatment ' ( see table [ 1 ] ( # tab1 ) { ref - type = " table " } ). table 1summary of the phase iii data from five placebo - controlled clinical trials ( including data from two open - label extension studies of two of these trials ) and two open - label clinical trials of efalizumab included in the pooled safety analysispublication ( protocol number ) study designnumber of patients in each analysisfirst treatment ( 0 - - 12 weeks ) efalizumab sc 1 - - 4 mg / kg qw or 2 mg / kg qowfirst exposure \ extended treatment ( 13 - - 24 weeks ) long - term treatment ^ a ^ ( ≤36 months ) re - treatmentplaceboefalizumab 1 mg / kgefalizumab 2 mg / kgleonardi \ [ [ @ cr13 ] \ ] ( acd2058g ) randomized, double - blind, parallel - group, placebo - controlled170162166462123 - - 55lebwohl \ [ [ @ cr12 ] \ ] ( acd2059g ) randomized, double - blind, parallel - group, placebo - controlled122232243579289 - - - - gordon \ [ [ @ cr4 ] \ ] ( acd2390g ) randomized, double - blind, parallel - group, placebo - controlled187368 - - 368 - - - - - - papp \ [ [ @ cr17 ] \ ] ( acd2600g ) randomized, double - blind, parallel - group, placebo - controlled236449 - - 449 - - 449 - - sterry \ [ [ @ cr22 ] \ ] ( imp24011 ) randomized, double - blind, parallel - group, placebo - controlled264529 - - 772308 - - 145papp \ [ [ @ cr16 ] \ ] ( acd2062g ) open - label - - - - - - 34137 - - 365gottlieb \ [ [ @ cr5 ], [ @ cr6 ] \ ] ( acd2243g ) open - label - - - - - - 339 ^ b ^ 290339 - - menter \ [ [ @ cr14 ] \ ] ( acd2391g ) open - label extension ^ c ^ of study acd2390g \ [ [ @ cr4 ] \ ] - - - - - - 174342 - - - - menter \ [ [ @ cr15 ] \ ] ( acd2601g ) open - label extension ^ c ^ of study acd2600 g \ [ [ @ cr17 ] \ ] - - - - - - 217622635 ^ d ^ - - pooled analysis97917404093, 3942, 111n. a. ^ e ^ 565 * qow * once - every - other week, * qw * once weekly, * sc * subcutaneous ^ a ^ number of patients at start ^ b ^ patients received combined therapy with fluocinolone acetate ( * n * = 169 ) or petrolatum ( * n * = 170 ) for weeks 9 - - 12 ; for months 3 - - 15, the dose of efalizumab could be escalated to 4 mg / kg per week for up to 4 weeks if clinically indicated ^ c ^ some patients are included in the analyses more than once because patients in the open - label extension studies are also included in analyses of the parent studies ^ d ^ included patients who received either efalizumab or placebo in the parent study \ [ [ @ cr17 ] \ ] ^ e ^ not applicable because data are analyzed and reported separately for the study by gottlieb et al. \ [ [ @ cr6 ] \ ] and the study published by papp \ [ [ @ cr17 ] \ ] and menter \ [ [ @ cr15 ] \ ] it is worth noting that most of the studies included in this pooled analysis were designed and conducted before efalizumab had received regulatory approval and before it was known that doses of more than 1 mg / kg once weekly ( the approved dose ) did not confer additional treatment benefit ( emea, raptiva summary of product characteristics ; fda us, fda prescribing information for raptiva ). for this reason, only the efalizumab 1 mg / kg once - weekly dose data are reported for the ' first treatment phase ' of the analysis. due to the wide variety of study designs included in the pooled analysis, data for patients receiving any dose of efalizumab are combined for all other treatment phases analyzed. the ' first treatment phase ' analysis included 0 - - 12 - week data from patients in the five placebo - controlled studies who received either efalizumab 1 mg / kg once weekly or placebo. this analysis allows a comparison between the efalizumab and placebo treatment groups. the ' first exposure phase ' included 12 - week data from all studies in patients who had their first exposure to any dose of efalizumab and, thus, did not include placebo data. this analysis was conducted to include the maximum number of patients who received efalizumab for their first 12 weeks of treatment ( i. e., it included those patients who first received efalizumab treatment after crossing over from a placebo group, as well as the patients who first received efalizumab during weeks 0 - - 12 ). the ' extended treatment phase ' analysis included 13 - - 24 - week data in patients given any dose of efalizumab who had already received efalizumab during the first treatment phase. the ' long - term treatment phase ' analysis included all patients who received continuous long - term treatment ( up to 36 months ) with any dose of efalizumab. data were analyzed in 12 - week segments to assess change in the incidence of arthropathy | Introduction {# Sec1} = = = = = = = = = = = = Psoriasis is a chronic, immune - mediated, inflammatory skin disorder that is currently incurable. Consequently, the majority of people with psoriasis require long - term treatment to maintain disease control. Traditional immunosuppressive systemic treatments, such as acitretin, methotrexate, cyclosporine, hydroxyurea, and thioguanine, may be effective in controlling psoriasis in some patients but significant toxicity and the need to closely monitor patients limit the viability of these treatments for long - term, continuoHD use \ [[ @ CR23] \ ]. Recen6.y developed systemic therapies that selectively target specific pathways in the inflammatory cascade of psoriasis generally have a much improved safety profile compared with traditional therapies \ [[ @ CR26] \ ]. Efalizumab (anti - CD11a; Raptiva ^ ® ^) is a recombinant humanized monoclonal IgG ~ 1 ~ antibody that has been approved for the treatment of moderate - to - severe chronic plaque psoriasis. It interferes with the pathogenesis of psoriasis via multiple mechanisms, including inhibition of T - lymphocyte trafficking and T - lymphocyte activation and reactivation \ [[ @ CR1 ], [@ CR10 ], [@ CR11 ], [@ CR21 ], [@ CR25] \ ]. The safety and efficacy profile of efalizumab has been established in numerous clinical trials, in which more than 3, 500 patients were enrolled and tr@atJent was assessed for up to 3 years \ [[ @ CR4] - - [@ CR6 ], [@ CR12] - - [@ CR17 ], [@ CR22] \ ]. Although psoriasis can be associated with the co - morbidity of psoriatic arthritis, a minority of patients with psoriasis (7 - - 30%) will develop this joint disease \ [[ @ CR27] \ ]. Nevertheless, psoriatic arthritis constitutes a major consideration in patients who are receiving long - term treatment for their psoriasis. A Nordic study of more than 5, 000 patients with psoriasis showed that patients with arthritis exhibited greater impairment of psoriasis - related quality of life (QoL ), longer disease duration, and greater self - reported disease severity, compared with patients who had psoriasis but no co - morbid arthritis \ [[ @ CR27] \ ]. A low incidence of arthropathy adverse events (AEs; any form of joint disease) associated with efalizumab treatment has been reported in both clinical studies and routine clinical practice \ [[ @ CR8 ], [@ CR12] \ ]. However, anecdotal reports of arthropathy in routine clinical practice have expressed concern that efalizumab may be associated with exacerbation of arthropathy \ [[ @ CR8] \ ]. To address this concern, we conducted a large - scale pooled analysis of safety data from five Phase III clinical trials (including open - label extensions of two of these studies) and two Phase III open - label clinical trials of efalizumab to explore whether arthropathy AEs were associated with efalizumab treatment in patients with psoriasis. Methods {# Sec2} = = = = = = = The primary objective of this pooled safety analysis was to assess the incidence of arthropathy AEs in patients who had received either efalizumab or placebo. Safety data were pooled from five randomized, dougpe - blind, placebo - controlled clinical trials (including data from two open - label extension studies of two of these trials) and two open - label clinical trials of efalizumab \ [[ @ CR4] - - [@ CR6 ], [@ CR12] - - [@ CR17 ], [@ CR22] \ ]. Patients included in these Phase III studies were aged ≥ 18 years and had moderate - to - severe chronic plaque psoriasis, a psoriasis area and severity index (PASI) score of ≥ 12 at screening, and plaque psoriasis covering ≥ 10% of body surface area. All patients were candidates for either systemic anti - psoriatic therapy or had received systemic anti - psoriatic therapy. Patients included in these trials received subcutaneous injections with efalizumab, 1 - - 4 mg / kg once weekly or 2 mg / kg once - every - other week, or placebo. Details of individual study methodologies are described in other publications \ [[ @ CR4] - - [@ CR6 ], [@ CR12] - - [@ CR17 ], [@ CR22] \ ]. Arthropathy AEs were defined according to the Coding Symbols for Thesaurus of Adverse Reaction Terms (COSTART) \ [[ @ CR3] \] preferred terms ' arthritis ' and ' arthrosis ', or the Medical Dictionary for Regulatory Activities (MedDRA, <http: / / www. meddramsso. com / NewWeb2003 / index. htm> ) preferred terms ' arthritis not otherwise specified (NOS) ', ' psoriatic arthropathy ', ' arthropathy NOS ', ' monoarthritis ', ' polyarthritis ', and ' osteoarthritis NOS '. Treatment groups analyzed {# Sec3} - - - - - - - - - - - - - - - - - - - - - - - - - Due to the variety of study designs, five analyses were considered: ' first - treatment phase ', ' first exposure phase ', ' extended treatment phase ', ' re - treatment phase ', and ' long - term treatment ' (see Table [1] (# Tab1) {ref - type = " table "} ). Table 1Summary of the Phase III Ratx from five placebo - controlled clinical trials (including data from two open - label extension studies of two of these trials) and two open - label clinical trials of efalizumab included in the pooled safety analysisPublication (protocol number) Study designNumber of patients in each analysisFirst treatment (0 - - 12 weeks) Efalizumab sc 1 - - 4 mg / kg qw or 2 mg / kg qowFirst exposure \ Extended treatment (13 - - 24 weeks) Long - term treatment ^ a ^ (≤ 36 months) Re - treatmentPlaceboEfalizumab 1 mg / kgEfalizumab 2 mg / kgLeonardi \ [[ @ CR13] \] (ACD2058g) Randomized, double - blind, parallel - group, placebo - controlled170162166462123 - - 55Lebwohl \ [[ @ CR12] \] (ACD2059g) Randomized, double - blind, parallel - group, placebo - controlled122232243579289 - - - - Gordon \ [[ @ CR4] \] (ACD2390g) Randomized, double - blind, parallel - group, placebo - controlled187368 - - 368 - - - - - - Papp \ [[ @ CR17] \] (ACD2600g) Randomized, double - blind, parallel - group, placebo - controlled236449 - - 449 - - 449 - - Sterry \ [[ @ CR22] \] (IMP24011) Randomized, double - blind, parallel - group, placebo - controlled264529 - - 772308 - - 145Papp \ [[ @ CR16] \] (ACD2062g) Open - label - - - - - - 34137 - - 365Gottlieb \ [[ @ CR5 ], [@ CR6] \] (ACD2243g) Open - label - - - - - - 339 ^ b ^ 290339 - - Menter \ [[ @ CR14] \] (ACD2391g) Open - label extension ^ c ^ of study ACD2390g \ [[ @ CR4] \] - - - - - - 174342 - - - - Menter \ [[ @ CR15] \] (ACD2601g) Open - label extension ^ c ^ of study ACD2600 g \ [[ @ CR17] \] - - - - - - 217622635 ^ d ^ - - Pooled analysis97917404093, 3942, 111n. a. ^ e ^ 565 * qow * once - every - other week, * qw * once weekly, * sc * subcutaneous ^ a ^ Number of patients at start ^ b ^ Patients received combined therapy with fluocinolone acetate (* n * = 169) or petrolatum (* n * = 170) for weeks 9 - - 12; for months 3 - - 15, the dose of efalizumab could be escalated to 4 mg / kg per week for up to 4 weeks if clinically indicated ^ c ^ Some patients are included in the analyses more than once because patients in the open - label extension studies are also included in analyses of the parent sGudKes ^ d ^ Included patients who received either efalizumab or placebo in the parent study \ [[ @ CR17] \] ^ e ^ Not applicable because data are analyzed and reported separately for the study by Gottlieb et al. \ [[ @ CR6] \] and the study published by Papp \ [[ @ CR17] \] and Menter \ [[ @ CR15] \] It is worth noting that most of the studies included in this pooled analysis were designed and conducted before efalizumab had received regulatory approval and before it was known that doses of ,or$ than 1 mg / kg once weekly (the approved dose) did not confer additional treatment benefit (EMEA, Raptiva Summary of Product Characteristics; FDA US, FDA Prescribing Information for Raptiva ). For this reason, only the efalizumab 1 mg / kg once - weekly dose data are reported for the ' first treatment phase ' of the analysis. Due to the wide variety of study designs included in the pooled analysis, data for patients receiving any dose of efalizumab are combined for all other treatment phases analyzed. The ' first treatment phase ' analysis included 0 - - 12 - week data from patients in the five placebo - controlled studies who received either efalizumab 1 mg / kg once weekly or placebo. This analysis allows a comparison between the efalizumab and placebo treatment groups. The ' first exposure phase ' included 12 - week data from all studies in patients who had their first exposure to any dose of efalizumab and, thus, did not include placebo data. TJiX analysis was conducted to include the maximum number of patients who received efalizumab for their first 12 weeks of treatment (i. e. , it included those patients who first received efalizumab treatment after crossing over from a placebo group, as well as the patients who first received efalizumab during weeks 0 - - 12 ). The ' extended treatment phase ' analysis included 13 - - 24 - week data in patients given any dose of efalizumab who had already received efalizumab durJBg the first treatment phase. The ' lomR - term treatment phase ' analysis included all patients who received continuous long - term treatment (up to 36 months) with any dose of efalizumab. Data were analyzed in 12 - week segments to assess change in the incidence of arthropathy | Introduction {#Sec1} Psoriasis is chronic, inflammatory disorder that is incurable. Consequently, the majority of people with psoriasis require long-term treatment to maintain disease control. Traditional immunosuppressive systemic treatments, as acitretin, methotrexate, cyclosporine, hydroxyurea, and thioguanine, may be effective in controlling psoriasis in some patients but significant and the need to monitor patients limit the viability of these for long-term, use \[[@CR23]\]. Recently developed systemic therapies that selectively target pathways in the inflammatory cascade of psoriasis generally have a much improved safety profile compared with traditional \[[@CR26]\]. Efalizumab (anti-CD11a; Raptiva^®^) a humanized monoclonal IgG~1~ that has been approved for the treatment of moderate-to-severe plaque psoriasis. It interferes with the pathogenesis of psoriasis via multiple mechanisms, including inhibition of T-lymphocyte trafficking T-lymphocyte activation reactivation \[[@CR1], [@CR10], [@CR11], [@CR21], [@CR25]\]. The safety and efficacy profile of efalizumab has been established in numerous clinical trials, in which more than patients were and treatment was for up to 3 years \[[@CR4]--[@CR6], [@CR12]--[@CR17], [@CR22]\]. Although psoriasis can be associated with the co-morbidity of psoriatic a minority of patients with psoriasis (7--30%) will develop this joint disease \[[@CR27]\]. Nevertheless, psoriatic arthritis constitutes a major consideration in patients who are long-term treatment for their psoriasis. A Nordic study of more than patients with psoriasis showed that patients with arthritis exhibited greater of psoriasis-related quality of life (QoL), longer disease duration, and self-reported disease severity, compared with patients who had psoriasis but no co-morbid arthritis \[[@CR27]\]. A low incidence of arthropathy adverse events (AEs; any form of joint disease) associated with efalizumab treatment has been reported clinical studies and routine clinical practice \[[@CR8], [@CR12]\]. However, anecdotal of arthropathy in routine clinical practice have expressed concern efalizumab may be associated with of arthropathy \[[@CR8]\]. To address this concern, we conducted a large-scale pooled analysis of safety data from five Phase III trials (including open-label extensions of two of these studies) and two III clinical trials of efalizumab to explore whether arthropathy AEs were associated with efalizumab treatment in patients with psoriasis. Methods {#Sec2} ======= primary objective of this pooled safety analysis was to assess the incidence of arthropathy AEs patients who had received either efalizumab or Safety data were pooled from five randomized, double-blind, placebo-controlled clinical trials data two open-label extension studies of two of these trials) and two open-label clinical trials efalizumab \[[@CR4]--[@CR6], [@CR12]--[@CR17], Patients included in these Phase III studies were aged ≥18 years and had moderate-to-severe plaque psoriasis, a psoriasis area and severity index (PASI) score of ≥12 and plaque psoriasis covering of body surface area. All patients were for either systemic therapy or had received systemic anti-psoriatic therapy. Patients included in these trials received subcutaneous injections with 1--4 once weekly or 2 once-every-other week, or placebo. Details individual are described in other publications \[[@CR4]--[@CR6], [@CR22]\]. Arthropathy AEs were defined to the Coding Symbols for Thesaurus of Adverse Reaction Terms (COSTART) \[[@CR3]\] preferred terms 'arthritis' and 'arthrosis', or the Medical Dictionary for Activities (MedDRA, <http://www.meddramsso.com/NewWeb2003/index.htm>) preferred terms 'arthritis not otherwise specified (NOS)', 'psoriatic 'arthropathy NOS', 'monoarthritis', 'polyarthritis', and 'osteoarthritis NOS'. Treatment {#Sec3} Due to the variety of study designs, five analyses were considered: 'first-treatment phase', 'first exposure phase', treatment phase', 're-treatment phase', and 'long-term treatment' Table Table of the Phase III data five placebo-controlled clinical trials (including data from two open-label studies of two of these trials) and two open-label clinical trials efalizumab included in the pooled safety analysisPublication number)Study designNumber of patients in each analysisFirst treatment (0--12 weeks)Efalizumab sc 1--4 mg/kg qw or 2 mg/kg exposure\ Extended treatment (13--24 weeks)Long-term treatment^a^(≤36 months)Re-treatmentPlaceboEfalizumab 1 mg/kgEfalizumab 2 mg/kgLeonardi \[[@CR13]\] (ACD2058g)Randomized, double-blind, parallel-group, placebo-controlled170162166462123--55Lebwohl \[[@CR12]\] (ACD2059g)Randomized, double-blind, parallel-group, placebo-controlled122232243579289----Gordon \[[@CR4]\] (ACD2390g)Randomized, double-blind, placebo-controlled187368--368------Papp \[[@CR17]\] (ACD2600g)Randomized, parallel-group, placebo-controlled236449--449--449--Sterry \[[@CR22]\] double-blind, parallel-group, placebo-controlled264529--772308--145Papp \[[@CR16]\] (ACD2062g)Open-label------34137--365Gottlieb [@CR6]\] (ACD2243g)Open-label------339^b^290339--Menter \[[@CR14]\] (ACD2391g)Open-label extension^c^of study ACD2390g \[[@CR4]\]------174342----Menter \[[@CR15]\] extension^c^of study ACD2600 g \[[@CR17]\]------217622635^d^--Pooled analysis97917404093,3942,111n.a.^e^565*qow* once-every-other *qw* once *sc* subcutaneous^a^Number of patients at start^b^Patients received combined therapy with fluocinolone acetate (*n* = 169) or petrolatum (*n* = 170) for weeks 9--12; for months 3--15, the dose of efalizumab could be escalated to 4 mg/kg per week for up to 4 weeks if clinically indicated^c^Some patients included the analyses more than once because patients in extension studies are also included in analyses of the parent patients who received either efalizumab or placebo in the parent study \[[@CR17]\]^e^Not applicable because data are analyzed and reported separately for the study by et al. \[[@CR6]\] and the study published by Papp \[[@CR17]\] and Menter \[[@CR15]\] It is worth noting that most of the studies included in pooled designed and conducted before efalizumab had received regulatory approval before it was known that of more than 1 mg/kg once weekly (the approved dose) did not confer additional treatment benefit (EMEA, Raptiva Summary of Product Characteristics; FDA US, FDA Prescribing Information for Raptiva). For this reason, only the efalizumab 1 mg/kg once-weekly dose data are reported for the 'first treatment phase' of the analysis. Due the wide variety of study designs in the pooled analysis, data patients receiving any dose of are for all other treatment phases analyzed. The 'first treatment phase' analysis included 0--12-week data from in the five placebo-controlled studies who received either efalizumab mg/kg once weekly or placebo. This analysis allows a between the efalizumab placebo treatment groups. The 'first exposure phase' included 12-week data from all studies in patients who had their first exposure to any dose of efalizumab and, did not include placebo data. This analysis was conducted to include the maximum number of patients received efalizumab for their first 12 weeks it included those patients first received efalizumab treatment after crossing over a placebo group, as well as the patients who first received efalizumab during weeks 0--12). 'extended treatment phase' analysis included 13--24-week data patients given any dose of efalizumab who had already efalizumab during first phase. The 'long-term treatment phase' analysis included all patients who received continuous long-term treatment (up to 36 months) with any dose of efalizumab. analyzed in 12-week segments to assess change in the incidence of arthropathy | InTrodUCtion {#sec1}
============
pSoriaSIS Is a Chronic, ImmUne-MEdIATed, INFLAmMaTorY SkIN DisORDEr thAt Is cUrREnTly INCurABLe. CONsEqUENtLy, ThE MAjority OF pEoPlE wIth pSorIAsis rEqUirE LoNG-TeRM tREaTmeNt tO maiNTAiN diseaSE coNTRoL. trAdITIonal imMuNosuPpResSiVe sYsTEMiC tREAtmENTS, SuCh AS AciTreTIN, MetHotrExaTE, cYcLOSpORInE, HYdrOxYuReA, and THioguaNInE, maY be EFfEcTiVe IN CoNTRolLiNg psOriASiS in SOmE PaTIENTS But SIGNIficANT tOXiciTY anD The NEED to ClOsEly mONitOR pAtIENTs LiMiT the vIaBilItY of THese tREatmeNts FOr Long-Term, CoNTInUOUS usE \[[@CR23]\]. RECEntLy deVELoPED SySteMIc TherAPIES That SELeCTIVelY TaRgeT SpecIfic PAthWays IN the INfLamMAtory cAscade OF PsoriASis GEneraLLY HavE a Much ImProVeD SAFeTy PRofIle cOMPARED WiTh TRAdItiOnal THeRapiEs \[[@Cr26]\].
efAlIZumAB (anTI-CD11a; rAPtiVA^®^) is a REcomBiNAnt HumAnIZEd mONoclONAL IGg~1~ aNtIbODy ThAt hAs BeeN apProVED FOr The TreATMenT oF mODEraTe-to-severe CHrOniC PLAQUE PsOriAsIs. IT interFeRes wItH ThE PatHOGeNEsis OF PsoRiASIS vIA MuLtIpLE mechaniSmS, InCluDing iNHIBiTIoN Of t-lympHOcYTE tRaFfIcKiNG And t-LYmPHoCYtE ACtIVatiON AnD reAcTiVaTiOn \[[@Cr1], [@Cr10], [@cR11], [@CR21], [@CR25]\]. THE SAFetY anD EFfIcacy ProFIle oF efaLIzUMaB HAs BEEN EsTabliSheD IN nUmErOuS clINICaL trIalS, IN WHICH moRe THaN 3,500 pAtIEnTS weRe enrOlLEd anD TreatMENT wAs assESSEd fOR UP To 3 YeARs \[[@CR4]--[@cR6], [@CR12]--[@cR17], [@cR22]\].
ALTHoUGH PSOriAsIs Can BE AsSocIATED witH ThE Co-MoRBiDiTY of pSorIATic aRtHRItiS, a MINoRItY of PaTienTS witH psoriAsis (7--30%) wIlL DEVELOp thIs JoInT DiSeAsE \[[@Cr27]\]. NEvErTHeless, PSOriATIc ArthRItIS cONStItutes A Major CONSideRATioN IN pATiEntS WHO ARe rEcEivIng LoNg-term treATmENt for theIR PsoRiASiS. A NorDIC sTUdy of mORE tHan 5,000 pATieNtS wITh pSORIASIS sHOWeD THaT PatienTs wITH aRTHRiTiS exhiBItEd GREAtEr iMpAIRmeNT of PSOrIasIs-reLATeD quALity of LifE (qOl), lonGER DISEaSE duRAtioN, And GReATer SELf-repoRteD diSease SEveRiTy, COMPArEd wiTh paTIEnTS WHO hAd PSoriASiS but no CO-morBid aRthrItIS \[[@Cr27]\].
A low inCiDeNcE of aRthrOPAthY advErSE EVenTS (Aes; aNY FOrm OF jOiNt dISEasE) ASsOcIated wiTh efALizumAb tREaTMEnT haS bEEn reporTEd In BOTh clIniCAl studieS aNd RoUtiNE clINICal prActIcE \[[@cr8], [@CR12]\]. howEVer, aNeCDOTaL RePoRts oF ARtHRoPaThY In RoUTInE clinIcaL PrACTICe havE ExPrEsSED COnCERn THAT eFaLIzuMaB mAY be ASSocIAtED wIth ExacErBaTion OF ArTHROPAtHY \[[@CR8]\]. to ADdRESS tHIs CoNCerN, We CoNDuCTED a larGE-scale pOOlED aNAlYsIS of SaFEty dATa from FIVe phasE iIi clInIcAL tRIAlS (incLudiNg OpEN-LaBel eXtENsiONs OF TwO of tHEsE sTUdiES) aND TWo pHASe IiI open-lABeL ClINIcAL tRiAls Of EfalIZuMAB to ExpLoRE WHethEr aRtHRopaThy aes weRE aSSoCiAted WiTh eFalIZUMab TrEatmeNT IN paTiENtS with psoRIasiS.
meTHoDS {#sEc2}
=======
ThE PriMArY oBjECTivE oF ThIs pOOleD Safety ANALysIS WaS TO AsseSs THE InCideNCe of ARthrOpAThY AeS IN patiENTS who had ReCEivED eiThER EFAlizuMAb Or PLAcEbo. SafEtY DatA WERe pOolEd FroM Five ranDoMIZeD, DouBle-blINd, placEBo-COntrOlLeD CLinicaL TRIalS (InCluDING daTA from Two oPen-LABel EXtEnsiON STUDieS oF two oF THeSe triAlS) ANd TwO Open-lABeL cLiNiCAL trIaLs of eFalizuMAB \[[@cR4]--[@Cr6], [@cR12]--[@cR17], [@cr22]\]. paTIeNts InClUDed in TheSe PhaSe IIi StudIEs WeRe AGED ≥18 YEArs AND hAD ModeRATE-TO-sevErE ChRONIc PlAQUe PSoriaSIS, A psORIaSIs aREA aNd SEVeriTY iNDex (PAsI) scOre OF ≥12 aT sCrEEnING, aNd pLAQUE PSOrIAsIS cOveRinG ≥10% Of boDy sUrfACE aREA. alL PATIENtS wERE CANDidateS For EITHeR sYstEMiC anTi-PsOriatiC THErApY or Had ReceiVED SYsTEMic AnTI-PSoRIatIC tHeRapY. PaTIentS INcLudEd in THESe trials ReceiVed subCuTAneoUs iNjEctIons With efaliZUmab, 1--4 mg/kg onCe weEKLY oR 2 Mg/KG onCE-everY-oTHeR wEEK, or PlacEBo. dETAiLS of InDIviDuaL StUDy mETHOdoLogies are DESCrIbED In otHer PubLICATIONS \[[@cr4]--[@Cr6], [@Cr12]--[@cr17], [@cr22]\].
ArthrOPathy aeS werE DEFiNeD acCoRding To thE CodING syMboLS FOR thESauruS OF aDVERsE rEacTion TERMs (CostARt) \[[@cr3]\] prEfERrEd teRmS 'aRthriTis' anD 'ARthRoSiS', Or thE MEDICaL DICtionAry FOR RegUlaTory activItiES (MEddra, <HtTP://wWw.meDdRaMssO.coM/NEwWEB2003/InDEX.Htm>) pReFERRED TermS 'arTHRItIs noT OtHeRWiSE SPEciFieD (nOS)', 'PSOriaTic aRTHrOPAtHy', 'ARTHROPathY nOs', 'MOnOarTHrItIs', 'pOLyArthrITIS', AND 'OSTeoartHRItIS nos'.
tReAtMenT grOUps ANAlyzed {#sEC3}
-------------------------
DUe tO tHe variEtY Of StUdY deSiGns, FIve ANALYsEs WERE cONsidErEd: 'FIrSt-TrEatMENt PHaSe', 'first ExpOsUre PHASe', 'EXtEnDeD TREatmEnT PHasE', 'RE-trEATmENT pHasE', and 'lOng-TerM tREatmeNT' (SeE TAbLe [1](#TAB1){REF-tYpE="tabLE"}). Table 1SummaRy oF thE phAse IiI dATA FrOm FIVE PlaceBo-coNTRollED cLiNICal TrIALs (InCLudIng daTA FROM tWO opEN-lABEL eXTensIoN stUdieS Of Two OF THeSe trIaLS) aNd twO OPEn-LabEl clINIcaL tRIaLs Of eFaliZumAB InCLudED In THe PoOLEd saFEtY ANAlYSisPubLicAtiON (pRotOcol NumbEr)sTUdy DesignnumbER OF patIEnts IN EAch ANAlySISFirst trEatMEnT (0--12 weEKs)EFAlIZUmab sC 1--4 Mg/kG qW or 2 mg/kg qOwFirST EXPosuRE\
ExtENDeD TREATMENt (13--24 wEeKS)LOnG-TERM tReAtmENt^A^(≤36 monTHS)Re-TrEatmeNTpLACebOEFalizumaB 1 mG/kGeFAlIzUMAB 2 Mg/kGleonarDi \[[@Cr13]\] (aCD2058G)RANdomIZeD, DOuBLE-BliND, PARallel-GrOup, PlaCEBO-CoNtRolLED170162166462123--55leBwOhL \[[@CR12]\] (ACd2059G)rANDoMiZeD, DouBle-bliNd, pARAlLel-GrOUP, plaCEBo-COnTRollEd122232243579289----GORdOn \[[@cr4]\] (aCd2390G)RandoMIzeD, dOuble-BlINd, PARALLEl-GrOup, PLAcEbo-cONtrOllEd187368--368------pAPp \[[@CR17]\] (acd2600G)RAnDOMizED, DOUblE-BlINd, PaRallEL-grOup, PLACEBO-cOntrolLEd236449--449--449--SteRRy \[[@Cr22]\] (Imp24011)RaNdOmiZeD, DOubLE-bLiND, PArAlLel-GROup, placEBo-cONTROLLeD264529--772308--145pApP \[[@cR16]\] (aCD2062G)OPEn-lABEl------34137--365gOTtlieb \[[@Cr5], [@cR6]\] (Acd2243G)oPEn-lABel------339^B^290339--mEnTeR \[[@cr14]\] (acd2391G)OPeN-lAbEL exteNsioN^C^of sTudY ACd2390g \[[@Cr4]\]------174342----MenTeR \[[@Cr15]\] (acD2601g)opEn-lABeL extensiON^c^Of STUDY Acd2600 G \[[@Cr17]\]------217622635^D^--Pooled ANalYSIS97917404093,3942,111n.A.^e^565*qOW* oncE-EVERY-otHEr WeEK, *qw* OncE WEekly, *SC* SuBCuTAnEoUS^A^nUmBer Of PatIENTS At sTaRt^B^PATients RECEIvEd cOmbiNEd THERApy WITH fluOcINolOne aCEtAtE (*N* = 169) OR PetroLatuM (*n* = 170) foR wEEkS 9--12; FOR mONThs 3--15, ThE doSe Of eFalizuMAb cOuLD Be eSCaLaTEd to 4 mG/KG PER Week For UP To 4 WeEks IF cLInicalLy iNDiCAteD^c^SomE patiENtS are InCludeD In ThE AnAlYseS More ThAn OnCE becAuse paTIEnts iN THE OpEN-laBeL exTeNsioN sTudieS ArE ALso incluDeD IN anAlYsES Of tHE PaRENT sTUDIES^D^inCluDED paTieNTS wHO rEcEivEd eIThER EFAliZUMaB Or PLAceBo iN the pAReNt STudy \[[@CR17]\]^e^not apPLicABle bEcAUse daTa arE anaLyZED AnD ReporTEd SEpARatELy foR ThE sTUdy bY goTtliEB eT Al. \[[@CR6]\] and ThE sTudY puBLisheD BY PAPp \[[@CR17]\] aND MeNtER \[[@Cr15]\]
IT IS wORTH NOtIng THat MoSt Of thE StUdieS IncLUdEd In THIs pOOLED AnaLYsiS weRe DeSIgnEd ANd conDuCtED bEForE eFAliZumAb hAD RECeiveD reGUlaTory aPPRovAl aNd bEfORe It Was kNOWN thAT DoSes OF MoRE ThAN 1 MG/kG OncE weEKLY (tHE appRovED Dose) dId not cOnfer AdDiTioNaL TReaTmeNt bENefit (eMEA, RaPTIVa SumMaRy OF prODUct CHARaCTERISTIcS; fdA Us, Fda PrescRibing inFOrmaTiON FoR RapTivA). FOR This reaSOn, OnlY the EfAlizuMAb 1 MG/kg oNce-WEeklY dOSE DAta ARe RePOrTed fOr tHE 'fIrST TreAtmEnt phase' oF The AnAlYSiS. DUe to The WIdE VarieTY Of sTUDy DesignS INCluDeD in the pOOlED AnalySiS, dATA FoR patIENTS recEiving ANY DOSE oF eFaLiZumAB ARE COmbINED FOR alL oTHEr tReAtmenT PhASeS ANALyzED.
tHe 'first TreATmenT Phase' ANAlYsIS INcLuDEd 0--12-WeeK Data FrOm PATIentS in ThE FivE PlaCebo-CONTRoLLED STUDieS Who receIVEd eiThEr EFAliZUmAB 1 Mg/kG once weEKLy OR pLacEBO. tHis ANaLySIs AlLowS a cOMPARISON BetwEEN thE EfaLIZUmAb anD plACEbO trEAtment grouPS.
The 'fiRst EXPosUre pHasE' inCluDed 12-WEek dAtA FroM ALl StudIeS In patIENts Who had theiR FirsT expOSuRe To any dOSE Of efAlizuMab anD, ThUS, dId NOT iNcluDe pLAcebo daTa. tHiS anALYsIs waS ConDUCted tO iNcluDE THE maXIMUm nUmbeR oF PAtIenTs wHO REceIVeD eFalizumaB FOR tHeIr FirSt 12 WEEkS of tREAtmENt (i.e., It INClUdEd tHosE paTIenTS WhO FIrSt rECeIveD EFALiZumAb trEatMenT AFTER cROSSiNG oveR FROM A PlaCEBO GROuP, As WelL AS tHe paTiENTs who FiRst ReceIVEd efalIzuMaB DuriNG WeEKs 0--12).
tHe 'exTeNdeD TrEatMENt phAsE' ANAlYsis inclUDeD 13--24-week dATa IN paTIEnts gIVen anY dose of EFALizuMaB wHO HaD alrEadY reCeivEd eFALIzuMAb DuRING tHE FiRSt TrEatment PhASE.
tHe 'loNG-TERM TrEatMenT pHAsE' anaLySIS IncLUDeD alL PATiENTS who RECeived CONTINuoUs long-teRm tReAtMENT (Up tO 36 mONtHs) wiTH any DoSe Of efALizumaB. DATa wEre aNALYZEd IN 12-WeEK SegMeNTs to assesS chANGe iN tHe INciDEncE Of arThrOpathY | Introduction {#Sec1} ============ Psoriasis isa chronic,immune-mediated, inflammatory skin disorder that is currentlyincurable.Consequently, the majority of people with psoriasis require long-term treatment tomaintain diseasecontrol. Traditional immunosuppressivesystemic treatments, such as acitretin, methotrexate, cyclosporine, hydroxyurea,and thioguanine, may be effective in controllingpsoriasisin some patients butsignificant toxicity and the need toclosely monitor patients limit the viability of these treatmentsfor long-term, continuous use \[[@CR23]\]. Recently developed systemic therapiesthat selectively target specific pathwaysinthe inflammatory cascade of psoriasis generally have a much improved safetyprofilecompared with traditional therapies\[[@CR26]\]. Efalizumab (anti-CD11a; Raptiva^®^) is a recombinant humanized monoclonal IgG~1~ antibody that has been approvedfor the treatment of moderate-to-severe chronic plaquepsoriasis. It interfereswith the pathogenesis of psoriasis viamultiple mechanisms, including inhibition of T-lymphocytetrafficking and T-lymphocyte activation and reactivation \[[@CR1], [@CR10], [@CR11],[@CR21], [@CR25]\]. The safety andefficacy profile of efalizumab has been established innumerous clinical trials, in which more than 3,500 patients were enrolled and treatment was assessed forup to3 years \[[@CR4]--[@CR6], [@CR12]--[@CR17], [@CR22]\]. Although psoriasis can be associated with the co-morbidityof psoriatic arthritis, a minority of patients with psoriasis (7--30%) will develop thisjoint disease \[[@CR27]\]. Nevertheless, psoriatic arthritis constitutesa major consideration in patients who are receiving long-term treatment for their psoriasis. A Nordic study ofmore than 5,000 patients with psoriasis showed that patients with arthritis exhibited greater impairment of psoriasis-related quality of life (QoL), longer disease duration, and greater self-reporteddisease severity, compared withpatients who had psoriasis but no co-morbid arthritis \[[@CR27]\]. A low incidence of arthropathy adverse events (AEs; any form of joint disease) associated with efalizumab treatment has been reported in both clinical studies and routine clinical practice\[[@CR8], [@CR12]\].However, anecdotal reports of arthropathy in routine clinical practice have expressed concern that efalizumab maybe associated with exacerbationof arthropathy \[[@CR8]\]. To address this concern, we conducted alarge-scale pooled analysisof safety data fromfive PhaseIIIclinical trials (including open-label extensions of two of thesestudies) and two PhaseIII open-label clinical trials of efalizumab to explore whether arthropathy AEs were associated withefalizumabtreatment inpatients with psoriasis. Methods {#Sec2} ======= The primary objective of thispooled safety analysis was to assess the incidence of arthropathy AEsinpatients who had received eitherefalizumab orplacebo. Safety data were pooled from fiverandomized,double-blind, placebo-controlled clinical trials (including data from two open-label extension studies oftwo of these trials) and two open-label clinical trials ofefalizumab \[[@CR4]--[@CR6], [@CR12]--[@CR17], [@CR22]\]. Patients included inthese PhaseIII studies were aged ≥18 years andhad moderate-to-severe chronicplaque psoriasis, a psoriasis area andseverityindex (PASI) score of ≥12 at screening, and plaque psoriasis covering ≥10% of bodysurface area. All patients werecandidates for either systemic anti-psoriatic therapy or hadreceived systemic anti-psoriatic therapy. Patients included inthese trials received subcutaneousinjections with efalizumab,1--4 mg/kg once weekly or 2 mg/kg once-every-other week, or placebo. Details of individual study methodologies are described in otherpublications \[[@CR4]--[@CR6], [@CR12]--[@CR17], [@CR22]\]. Arthropathy AEs were definedaccording to the CodingSymbols for Thesaurus of AdverseReaction Terms (COSTART) \[[@CR3]\] preferred terms 'arthritis' and 'arthrosis',or the Medical Dictionary for Regulatory Activities (MedDRA, <http://www.meddramsso.com/NewWeb2003/index.htm>) preferred terms 'arthritis not otherwise specified (NOS)', 'psoriaticarthropathy', 'arthropathy NOS', 'monoarthritis', 'polyarthritis', and 'osteoarthritis NOS'. Treatment groupsanalyzed {#Sec3}-------------------------Duetothe variety of study designs, five analyses were considered: 'first-treatment phase', 'first exposure phase', 'extended treatment phase', 're-treatment phase', and 'long-term treatment' (see Table [1](#Tab1){ref-type="table"}). Table1Summary of the Phase III data fromfive placebo-controlled clinical trials (including data from twoopen-labelextension studies of two of these trials) and two open-label clinicaltrials of efalizumab included in the pooled safety analysisPublication(protocolnumber)Study designNumber of patients ineach analysisFirst treatment (0--12 weeks)Efalizumab sc 1--4mg/kg qw or2 mg/kg qowFirst exposure\ Extended treatment (13--24 weeks)Long-term treatment^a^(≤36 months)Re-treatmentPlaceboEfalizumab 1 mg/kgEfalizumab 2 mg/kgLeonardi \[[@CR13]\] (ACD2058g)Randomized, double-blind, parallel-group, placebo-controlled170162166462123--55Lebwohl \[[@CR12]\] (ACD2059g)Randomized, double-blind, parallel-group, placebo-controlled122232243579289----Gordon \[[@CR4]\] (ACD2390g)Randomized,double-blind, parallel-group, placebo-controlled187368--368------Papp\[[@CR17]\] (ACD2600g)Randomized, double-blind, parallel-group,placebo-controlled236449--449--449--Sterry \[[@CR22]\] (IMP24011)Randomized, double-blind, parallel-group, placebo-controlled264529--772308--145Papp \[[@CR16]\] (ACD2062g)Open-label------34137--365Gottlieb \[[@CR5], [@CR6]\] (ACD2243g)Open-label------339^b^290339--Menter \[[@CR14]\] (ACD2391g)Open-label extension^c^of studyACD2390g \[[@CR4]\]------174342----Menter\[[@CR15]\] (ACD2601g)Open-label extension^c^ofstudyACD2600 g \[[@CR17]\]------217622635^d^--Pooled analysis97917404093,3942,111n.a.^e^565*qow* once-every-other week, *qw* once weekly, *sc* subcutaneous^a^Number of patients at start^b^Patients received combined therapy with fluocinolone acetate (*n* = 169)or petrolatum (*n* = 170) for weeks 9--12; for months 3--15, the dose of efalizumab couldbeescalated to4 mg/kg per week for up to 4 weeks if clinically indicated^c^Some patients are included in theanalyses more than once becausepatients in the open-label extension studies are also included inanalyses of the parent studies^d^Included patients who receivedeither efalizumab or placebo in the parent study\[[@CR17]\]^e^Not applicablebecause data are analyzed andreported separately for the study by Gottlieb et al. \[[@CR6]\] andthe study published byPapp \[[@CR17]\] and Menter \[[@CR15]\] It is worth noting that most of the studies included in this pooled analysis weredesigned and conducted before efalizumab had receivedregulatory approval and before itwas known thatdoses of more than1 mg/kg once weekly (the approved dose) did not confer additional treatment benefit (EMEA, Raptiva Summary of ProductCharacteristics; FDAUS, FDA Prescribing Information for Raptiva).For this reason,only the efalizumab 1mg/kg once-weekly dose data arereportedfor the 'firsttreatment phase' ofthe analysis. Due to the wide varietyof study designs included in the pooled analysis, data for patients receiving any dose of efalizumab are combined for all other treatment phases analyzed. The 'first treatment phase' analysis included 0--12-week data from patients in the five placebo-controlled studies who received either efalizumab 1 mg/kg once weekly or placebo. Thisanalysis allows a comparison between the efalizumab andplacebo treatmentgroups. The 'firstexposure phase' included 12-weekdata from allstudies in patients who had their first exposure to any dose of efalizumab and, thus, did not include placebo data. This analysis wasconducted to include the maximum number of patients who received efalizumab for their first 12 weeks of treatment (i.e., it included those patientswhofirst receivedefalizumab treatment after crossing over from a placebo group, as well as the patients who first received efalizumab during weeks 0--12). The 'extended treatment phase' analysis included13--24-week data inpatients given any dose of efalizumab who hadalready receivedefalizumab during the first treatment phase. The 'long-term treatment phase' analysis included all patients who received continuous long-term treatment (up to 36months) with any dose of efalizumab. Datawere analyzed in 12-week segments toassesschange in the incidence of arthropathy | _Introduction_ {#Sec1} ============ Psoriasis is a chronic, _immune-mediated,_ _inflammatory_ skin disorder that is _currently_ incurable. Consequently, the majority of people with psoriasis require long-term treatment _to_ maintain disease control. Traditional immunosuppressive systemic treatments, _such_ as acitretin, methotrexate, cyclosporine, hydroxyurea, _and_ _thioguanine,_ may be effective in _controlling_ psoriasis in some patients _but_ significant toxicity and the need to _closely_ monitor patients limit the _viability_ of these treatments _for_ _long-term,_ continuous _use_ \[[@CR23]\]. _Recently_ developed systemic _therapies_ that selectively _target_ specific pathways in _the_ inflammatory _cascade_ of psoriasis generally have _a_ much improved _safety_ _profile_ compared with traditional therapies \[[@CR26]\]. _Efalizumab_ (anti-CD11a; Raptiva^®^) is a recombinant _humanized_ monoclonal _IgG~1~_ _antibody_ that has _been_ approved for the _treatment_ of moderate-to-severe chronic plaque _psoriasis._ It interferes with _the_ pathogenesis of psoriasis via multiple mechanisms, including inhibition of T-lymphocyte _trafficking_ and T-lymphocyte activation _and_ reactivation \[[@CR1], [@CR10], _[@CR11],_ [@CR21], [@CR25]\]. The _safety_ and _efficacy_ profile of efalizumab has been established in numerous _clinical_ trials, in _which_ more than 3,500 patients were enrolled and _treatment_ _was_ assessed for up to 3 years _\[[@CR4]--[@CR6],_ _[@CR12]--[@CR17],_ [@CR22]\]. Although psoriasis can be associated with the co-morbidity of _psoriatic_ arthritis, a minority _of_ patients with _psoriasis_ (7--30%) will develop _this_ joint disease \[[@CR27]\]. Nevertheless, psoriatic arthritis constitutes a major consideration in patients who are receiving long-term treatment for their psoriasis. A _Nordic_ study of more than 5,000 patients with psoriasis showed that patients with arthritis exhibited greater impairment of psoriasis-related quality of life (QoL), longer disease duration, and _greater_ self-reported disease severity, compared _with_ patients who had psoriasis but no co-morbid arthritis \[[@CR27]\]. A low incidence of _arthropathy_ adverse events _(AEs;_ any form of joint disease) associated with efalizumab treatment has been reported in _both_ clinical studies and _routine_ _clinical_ practice \[[@CR8], _[@CR12]\]._ However, anecdotal reports of arthropathy in routine clinical _practice_ have expressed concern that efalizumab may _be_ associated _with_ exacerbation _of_ arthropathy \[[@CR8]\]. To address this concern, we conducted a large-scale pooled analysis _of_ _safety_ data from five Phase III clinical trials (including open-label extensions of two of these studies) and _two_ _Phase_ III open-label _clinical_ trials _of_ efalizumab to explore whether _arthropathy_ _AEs_ were associated with efalizumab treatment in patients with _psoriasis._ Methods _{#Sec2}_ ======= The _primary_ objective of this pooled safety _analysis_ was to assess the _incidence_ of arthropathy AEs _in_ patients who _had_ received either efalizumab _or_ placebo. Safety _data_ _were_ pooled from five randomized, double-blind, placebo-controlled clinical trials _(including_ data _from_ two open-label extension studies _of_ two of these _trials)_ and _two_ open-label clinical trials of _efalizumab_ \[[@CR4]--[@CR6], [@CR12]--[@CR17], [@CR22]\]. _Patients_ included in these _Phase_ III studies were aged ≥18 years _and_ had moderate-to-severe _chronic_ plaque psoriasis, a psoriasis area and _severity_ index _(PASI)_ score of ≥12 at screening, and plaque psoriasis covering ≥10% of body surface _area._ _All_ patients were _candidates_ _for_ either systemic anti-psoriatic therapy _or_ had received systemic anti-psoriatic _therapy._ Patients included in these trials received subcutaneous injections with efalizumab, 1--4 _mg/kg_ once weekly or _2_ mg/kg once-every-other week, or placebo. Details of individual study methodologies are _described_ in _other_ publications _\[[@CR4]--[@CR6],_ [@CR12]--[@CR17], [@CR22]\]. Arthropathy _AEs_ were defined according _to_ the Coding _Symbols_ for Thesaurus of Adverse _Reaction_ _Terms_ (COSTART) \[[@CR3]\] preferred terms 'arthritis' and 'arthrosis', or _the_ Medical Dictionary for Regulatory Activities (MedDRA, <http://www.meddramsso.com/NewWeb2003/index.htm>) preferred terms 'arthritis not otherwise specified (NOS)', 'psoriatic _arthropathy',_ 'arthropathy NOS', 'monoarthritis', 'polyarthritis', and 'osteoarthritis NOS'. Treatment _groups_ analyzed _{#Sec3}_ ------------------------- Due to the variety of study designs, five analyses were _considered:_ _'first-treatment_ _phase',_ 'first exposure phase', _'extended_ treatment phase', 're-treatment phase', and 'long-term treatment' (see Table _[1](#Tab1){ref-type="table"})._ Table 1Summary of the Phase III data from five _placebo-controlled_ clinical trials (including data from two open-label extension studies of two of these trials) and two open-label clinical trials _of_ efalizumab _included_ in the _pooled_ _safety_ analysisPublication (protocol number)Study designNumber of patients in each analysisFirst treatment (0--12 weeks)Efalizumab sc 1--4 mg/kg _qw_ _or_ 2 _mg/kg_ qowFirst exposure\ Extended treatment (13--24 _weeks)Long-term_ treatment^a^(≤36 months)Re-treatmentPlaceboEfalizumab 1 mg/kgEfalizumab 2 mg/kgLeonardi \[[@CR13]\] (ACD2058g)Randomized, _double-blind,_ parallel-group, placebo-controlled170162166462123--55Lebwohl _\[[@CR12]\]_ (ACD2059g)Randomized, double-blind, parallel-group, placebo-controlled122232243579289----Gordon \[[@CR4]\] (ACD2390g)Randomized, double-blind, parallel-group, placebo-controlled187368--368------Papp _\[[@CR17]\]_ (ACD2600g)Randomized, double-blind, parallel-group, placebo-controlled236449--449--449--Sterry \[[@CR22]\] (IMP24011)Randomized, double-blind, parallel-group, placebo-controlled264529--772308--145Papp \[[@CR16]\] _(ACD2062g)Open-label------34137--365Gottlieb_ \[[@CR5], [@CR6]\] (ACD2243g)Open-label------339^b^290339--Menter \[[@CR14]\] (ACD2391g)Open-label extension^c^of study ACD2390g \[[@CR4]\]------174342----Menter \[[@CR15]\] (ACD2601g)Open-label _extension^c^of_ study ACD2600 g \[[@CR17]\]------217622635^d^--Pooled analysis97917404093,3942,111n.a.^e^565*qow* _once-every-other_ week, *qw* once weekly, _*sc*_ subcutaneous^a^Number _of_ patients at start^b^Patients _received_ combined therapy with fluocinolone acetate (*n* = _169)_ or _petrolatum_ (*n* = 170) for weeks _9--12;_ _for_ months 3--15, the dose _of_ efalizumab _could_ be escalated to 4 _mg/kg_ per week _for_ up to 4 weeks if clinically indicated^c^Some patients are included in the analyses more than _once_ because patients in the open-label extension studies are also included _in_ analyses of the parent studies^d^Included patients _who_ received either _efalizumab_ or placebo in _the_ parent study \[[@CR17]\]^e^Not applicable _because_ data are _analyzed_ and reported _separately_ for the study by Gottlieb et al. \[[@CR6]\] and the _study_ _published_ by _Papp_ _\[[@CR17]\]_ and Menter _\[[@CR15]\]_ It is _worth_ noting that most _of_ the _studies_ included in this pooled analysis were designed _and_ _conducted_ _before_ efalizumab had received regulatory approval and _before_ _it_ _was_ known that doses of more _than_ 1 _mg/kg_ _once_ weekly (the approved dose) did not _confer_ additional treatment benefit (EMEA, Raptiva Summary of Product Characteristics; FDA US, FDA Prescribing _Information_ for _Raptiva)._ For this _reason,_ only the efalizumab 1 mg/kg once-weekly dose _data_ are reported for _the_ 'first treatment phase' of the analysis. Due to the wide variety of study designs included in the pooled _analysis,_ data for patients receiving _any_ dose _of_ efalizumab are _combined_ _for_ all other _treatment_ phases analyzed. The _'first_ _treatment_ phase' analysis _included_ 0--12-week data from patients in the five placebo-controlled _studies_ who _received_ either efalizumab 1 _mg/kg_ once weekly or placebo. This analysis allows a comparison between the efalizumab and _placebo_ treatment groups. The 'first exposure phase' included 12-week data from all studies in patients who _had_ their first exposure to any _dose_ of efalizumab and, thus, _did_ not include placebo data. This analysis was conducted to include the maximum number of _patients_ who received efalizumab for their first 12 weeks of _treatment_ _(i.e.,_ it _included_ those patients who _first_ _received_ efalizumab treatment after _crossing_ _over_ from a _placebo_ group, as well as the patients who first received efalizumab during weeks 0--12). The _'extended_ treatment _phase'_ analysis included 13--24-week data in _patients_ given _any_ dose _of_ _efalizumab_ who had already _received_ efalizumab during the first _treatment_ phase. The _'long-term_ treatment phase' analysis included _all_ _patients_ who received continuous long-term _treatment_ (up to 36 months) with any _dose_ of _efalizumab._ Data were analyzed in _12-week_ segments to _assess_ change in the incidence of arthropathy |
Background {#Sec1}
==========
Hypoxia is common after stroke, and associated with poor outcomes. In this article, we have reviewed the physiology of oxygen transport, the cerebrovascular response to hypoxia and pathophysiology, incidence and aetiology behind hypoxia in stroke and its subsequent clinical consequences. We have then reviewed all randomised clinical trials looking at the use of supplemental oxygen therapy in acute stroke and made conclusions regarding current evidence and recommendations for clinical practice.
Oxygen physiology {#Sec2}
=================
The normal adult range of arterial oxygen pressure (PaO2) is 11.0--14.4 kPa and the normal range for arterial oxygen saturation (SaO2) is 95--98% \[[@CR1]\]. The term hypoxia refers to oxygen levels below normal. It includes both tissue (e.g. brain, myocardium) hypoxia and hypoxia in the blood (hypoxaemia). Tissue hypoxia is defined by the concentration of oxygen in blood and also tissue perfusion, whilst hypoxaemia is defined by the concentration of oxygen in inspired air and its transfer into the blood \[[@CR2]\].
Following inhalation oxygen is taken up in the lung capillaries via diffusion down an oxygen concentration gradient across the alveoli \[[@CR3], [@CR4]\]. Oxygen binds to the haemoglobin molecule, which can carry four oxygen molecules; each binding and changing the shape of the haemoglobin molecule and increasing its affinity for oxygen \[[@CR5]\]. A small amount of oxygen is also dissolved in plasma. This proportion increases in hyperoxia, when all haemoglobin is saturated \[[@CR6]\]. Oxygen dissociates from the haemoglobin molecule in the tissues owing to the relatively hypercapnic and acidic environment (the Bohr effect) \[[@CR3]\].
Oxygen is a vital substrate that supports virtually all metabolic processes. 90% of oxygen intake is engaged in the cytochrome C oxidase system in the mitochondria \[[@CR7]\] generating adenosine triphosphate (ATP), which acts as the main energy substrate within cells. A continuous supply of oxygen is required to secure a continuous supply of ATP maintaining sufficient energy for cerebral neuronal and cellular activity. This facilitates an efficient energy producing process making 38 molecules of ATP during aerobic respiration, equivalent to 1270 joules (J) energy, in comparison to two molecules of ATP (67 J of energy) during anaerobic respiration \[[@CR7], [@CR8]\].
The anoxic brain {#Sec3}
================
20% of all human oxygen consumption is utilised by the brain \[[@CR9]\]. The brain has no oxygen or glucose (the other important substrate in the ATP producing equation) stores. Thus complete disruption of cerebral blood flow very rapidly results in an anoxic, hypoglycaemic state, which via a variety of mechanisms ultimately leads to cell death. Excitatory neurotransmitters, such as glutamate, bind to a variety of receptors and allow for an influx of calcium ions that help formulate the chemical signal for depolarisation \[[@CR10], [@CR11]\]. Normally the re-uptake of glutamate is an active energy-driven process. In the absence of ATP this process fails, resulting in an extracellular accumulation of glutamate, which continually stimulates receptors leading to a persistent influx of calcium ions \[[@CR12]\]. Furthermore, the Na+/Ca2+ ATP driven pump normally used to eliminate calcium fails, also due to a lack of ATP \[[@CR13]\]. The resultant high intracellular calcium triggers multiple cascades that ultimately lead to mitochondrial dysfunction and cell death. Furthermore, instead of producing ATP, glial cells have been shown to release ATP extracellularly \[[@CR11]\]. Aside from rendering this unusable by mitochondria, ATP also stimulates the P2X7 receptor, which again leads to significant calcium influx and ultimately cell death \[[@CR13]\]. The other major mechanism of cellular demise is via the formation of free radicals facilitated by the reduction of iron from its ferric (Fe3+) to its ferrous (Fe2+) form and the initiation of inflammatory cascades \[[@CR12]\].
Cerebral blood flow in hypoxia {#Sec4}
==============================
In normoxic states, cerebral blood flow is very tightly controlled by the partial pressure of carbon dioxide (PaCO~2~). Any hypocapnic state will result in vasoconstriction and reduction in regional cerebral blood flow and a hypercapnic state leads to the reverse with vasodilatation and an increase in cerebral blood flow. Cerebral blood flow is somewhat less responsive to changes in PaO~2~, which has the opposite effect to carbon dioxide; a hypoxic state causing cerebral vasodilatation with the aim of improving oxygen delivery and a hyperoxic state causing vasoconstriction \[[@CR14], [@CR15]\].
In a hypoxic state, whilst the vasodilatory response improves flow, the detection of hypoxia by peripheral chemoreceptors will in turn lead to an increase in respiratory drive, increasing arterial oxygen content. However, the consequence of this is also an increase in the clearance of carbon dioxide, which would theoretically cause vasoconstriction and reduced cerebral blood flow \[[@CR9]\]. It appears there is a threshold to which the hypoxic response predominates (and the carbon dioxide one attenuated) at a PaO~2~ of around 50--60 mmHg \[[@CR9], [@CR14]\]. Whilst the carbon dioxide mediated vascular response is mediated via a direct change in vessel wall pH \[[@CR15]\], the oxygen response appears to be mediated by the deoxygenated erythrocyte via a number of mechanisms; which include release of ATP and the subsequent actions of endothelial nitric oxide synthase on the vessel wall, reduction of nitrite to nitric oxide and the activity of S-nitrosohaemoglobin \[[@CR9]\].
The cerebral vascular response to hypoxia is not uniform. A study found that in an induced isocapnic hypoxic state increases in cerebral blood flow were most prominent in basal ganglia nuclei, the putamen, thalamus, nucleus accumbens and pallidum \[[@CR16]\]. Studies of blood flow in individual vessels have found that flow in the internal carotid artery is maintained during hypoxia and that vertebral artery flow is increased \[[@CR17]\]. This had led to the hypothesis that blood flow is increased in this region to preserve vital brainstem structures, or that possibly the posterior circulation vasculature is less susceptible to the effects of carbon dioxide for similar reasons.
Neurological effects of hypoxia {#Sec5}
===============================
The neurological consequences of hypoxia are dependent upon the speed of onset, the severity of hypoxia, and the level of tissue perfusion. Rapid decreases in PaO~2~, as in a cardiorespiratory arrest, can lead to permanent neurological damage within minutes. However, lower, less abrupt changes, can be tolerated if the decrease in oxygen occurs in a gradual manner, such as ascending at altitude, where individuals can acclimatise and develop tolerance to lower oxygen partial pressures or, (to a lesser degree), in chronic smokers. Initial clinical features include altered judgement, difficulty in completing complex tasks, and impairment in short term memory \[[@CR18], [@CR19]\], but in the longer term deficits can be more widespread and span physical and neuropsychological domains. Seizures occur in up to a third of individuals within a day of exposure to hypoxia, and are commonly partial complex or myoclonic in nature. Intractable forms of either of these types of seizure are associated with a poor prognosis \[[@CR20]\]. Cognitive impairment domains include amnesia, visuospatial deficits, frontal lobe symptoms, impairment of executive function, and impairments in language \[[@CR21]\]. These are covered in more detailed reviews on the subject \[[@CR22], [@CR23]\]. Involvement of the basal ganglia, a region particularly susceptible to hypoxic injury, can result in delayed Parkinsonism in older subjects, dystonia mainly in younger people, choreo-athetosis, and tremors \[[@CR20]\]. Varying degrees of unilateral or bilateral motor impairment may be observed depending on both the anatomical level and extent of corticospinal tract involvement. In very rare cases, the syndrome of delayed post-hypoxic leukoencephalopathy may occur weeks after a seemingly rapid recovery from the original insult. This condition is characterised by rapid deterioration in cognition, emergence of extra-pyramidal signs, and loss of executive function as a results of severe demyelination \[[@CR24]\]. The severity of leukoencephalopathy can be assessed by magnetic resonance imaging (MRI) \[[@CR20]\]. Electroencephalography, somatosensory evoked potentials and MRI can provide valuable information about the severity of hypoxic injury, but also aid in prognostication together with overall clinical state \[[@CR25]\].
Hypoxia in the context of a stroke {#Sec6}
==================================
There is no specific definition as to what constitutes hypoxia in an acute stroke, and it is therefore reasonable to assume that normal values for the general population apply.
Sulter and colleagues \[[@CR26]\] monitored 49 consecutive patients who presented with an acute stroke within 12 h duration using pulse oximetry for 48 h. Patients were considered hypoxic and treated with supplemental oxygen if saturations were below 96% for more than 5 min. This occurred in 63% \[[@CR31]\] of patients, with 28 of those returning to 'normal' oxygen saturations following administration of up to 5 L/min of oxygen. The remaining three required much higher concentrations. Factors associated with hypoxia in this group were stroke severity, presence of dysphagia, and older age. Roffe et al. \[[@CR27]\] recruited 118 patients (100 of whom had adequate measurements by pulse oximetry) and found that the mean daytime awake SO~2~ was 94.5 ± 1.7% in stroke patients and 95.8 ± 1.7% in healthy controls. Nocturnal saturations were reduced to 93.5 ± 1.9% in the stroke group and 94.3 ± 1.9% in controls. In the stroke group the average 4% oxygen desaturation index (ODI) (number of times per | background { # sec1 } = = = = = = = = = = hypoxia is prevalent after stroke, and associated with poor outcomes. in this article, we have reviewed the physiology of oxygen transport, the cerebrovascular response to hypoxia and pathophysiology, incidence and aetiology behind hypoxia in stroke and its subsequent clinical consequences. we have then reviewed all randomised clinical trials looking at the use of supplemental oxygen therapy in acute stroke and made notes regarding current evidence and recommendations for clinical practice. oxygen physiology { # sec2 } = = = = = = = = = = = = = = = = = the normal normal range of arterial oxygen pressure ( pao2 ) is 11. 0 - - 14. 4 kpa and the normal range for arterial oxygen concentration ( sao2 ) is 17 - - 98 % \ [ [ @ cr1 ] \ ]. the term hypoxia refers to oxygen levels below normal. it includes both tissue ( e. g. brain, myocardium ) hypoxia from hypoxia in the blood ( hypoxaemia ). tissue hypoxia is defined by the concentration of oxygen in blood and also tissue perfusion, whilst hypoxaemia is defined by the concentration of oxygen in inspired air and its transfer into the blood \ [ [ @ cr2 ] \ ]. following inhalation oxygen is taken up in the lung capillaries via diffusion down an oxygen concentration gradient across the alveoli \ [ [ @ cr3 ], [ @ cr4 ] \ ]. oxygen binds to the haemoglobin molecule, which can carry four oxygen molecules ; each binding and changing the shape of the haemoglobin molecule and increasing its affinity of oxygen \ [ [ @ cr5 ] \ ]. a small amount of oxygen is also dissolved in plasma. this proportion increases in hyperoxia, when all haemoglobin is saturated \ [ [ @ cr6 ] \ ]. oxygen dissociates from the haemoglobin molecule in the heart owing to the relatively hypercapnic and acidic environment ( the bohr effect ) \ [ [ @ cr3 ] \ ]. oxygen is a vital substrate that supports virtually all bodily processes. 90 % of oxygen intake is engaged in the cytochrome c oxidase system in arterial mitochondria \ [ [ @ cr7 ] \ ] generating adenosine triphosphate ( atp ), which acts as the main energy substrate within cells. a continuous supply of oxygen is required to secure a continuous supply of atp maintaining sufficient energy for cerebral neuronal and cellular activity. this facilitates an efficient energy producing process making 38 molecules of atp during aerobic respiration, equivalent to 1270 joules ( j ) energy, in comparison to two molecules of atp ( 67 j of energy ) during anaerobic respiration \ [ [ @ cr7 ], [ @ cr8 ] \ ]. the anoxic brain { # sec3 } = = = = = = = = = = = = = = = = 20 % of all human oxygen consumption is utilised by the brain \ [ [ @ cr9 ] \ ]. the brain has no oxygen or glucose ( the other important substrate in the atp producing equation ) stores. thus complete disruption of cerebral blood flow very rapidly results in an anoxic, hypoglycaemic state, which via a variety of mechanisms ultimately leads to cell death. excitatory neurotransmitters, such as glutamate, bind to a variety of receptors and allow for an influx of calcium ions that help formulate the chemical signal for depolarisation \ [ [ @ cr10 ], [ @ cr11 ] \ ]. normally the re - uptake of glutamate is an active energy - driven process. in the absence of atp this process fails, resulting in an extracellular accumulation of glutamate, which continually stimulates receptors leading to a persistent influx of calcium ions \ [ [ @ cr12 ] \ ]. furthermore, the na + / ca2 + atp driven pump normally used to eliminate calcium fails, also due to a lack of atp \ [ [ @ cr13 ] \ ]. the resultant high intracellular calcium triggers multiple cascades that ultimately lead to mitochondrial dysfunction and cell death. furthermore, instead of producing atp, glial cells have been shown to release atp extracellularly \ [ [ @ cr11 ] \ ]. aside from rendering this unusable by mitochondria, atp also stimulates the p2x7 receptor, which again leads to significant calcium influx and ultimately cell death \ [ [ @ cr13 ] \ ]. the other major mechanism of cellular demise is via the formation of free radicals facilitated by the reduction of iron from its ferric ( fe3 + ) to its ferrous ( fe2 + ) form and the initiation of inflammatory cascades \ [ [ @ cr12 ] \ ]. cerebral blood flow in hypoxia { # sec4 } = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = in normoxic states, cerebral blood flow is very tightly controlled by the partial pressure of carbon dioxide ( paco ~ 2 ~ ). any hypocapnic state will result in vasoconstriction and reduction in regional cerebral blood flow and a hypercapnic state leads to the reverse with vasodilatation and an increase in cerebral blood flow. cerebral blood flow is somewhat less responsive to changes in pao ~ 2 ~, which has the opposite effect to carbon dioxide ; a hypoxic state causing cerebral vasodilatation with the aim of improving oxygen delivery and a hyperoxic state causing vasoconstriction \ [ [ @ cr14 ], [ @ cr15 ] \ ]. in a hypoxic state, whilst the vasodilatory response improves flow, the detection of hypoxia by peripheral chemoreceptors will in turn lead to an increase in respiratory drive, increasing arterial oxygen content. however, the consequence of this is also an increase in the clearance of carbon dioxide, which would theoretically cause vasoconstriction and reduced cerebral blood flow \ [ [ @ cr9 ] \ ]. it appears there is a threshold to which the hypoxic response predominates ( and the carbon dioxide one attenuated ) at a pao ~ 2 ~ of around 50 - - 60 mmhg \ [ [ @ cr9 ], [ @ cr14 ] \ ]. whilst the carbon dioxide mediated vascular response is mediated via a direct change in vessel wall ph \ [ [ @ cr15 ] \ ], the oxygen response appears to be mediated by the deoxygenated erythrocyte via a number of mechanisms ; which include release of atp and the subsequent actions of endothelial nitric oxide synthase on the vessel wall, reduction of nitrite to nitric oxide and the activity of s - nitrosohaemoglobin \ [ [ @ cr9 ] \ ]. the cerebral vascular response to hypoxia is not uniform. a study found that in an induced isocapnic hypoxic state increases in cerebral blood flow were most prominent in basal ganglia nuclei, the putamen, thalamus, nucleus accumbens and pallidum \ [ [ @ cr16 ] \ ]. studies of blood flow in individual vessels have found that flow in the internal carotid artery is maintained during hypoxia and that vertebral artery flow is increased \ [ [ @ cr17 ] \ ]. this had led to the hypothesis that blood flow is increased in this region to preserve vital brainstem structures, or that possibly the posterior circulation vasculature is less susceptible to the effects of carbon dioxide for similar reasons. neurological effects of hypoxia { # sec5 } = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = the neurological consequences of hypoxia are dependent upon the speed of onset, the severity of hypoxia, and the level of tissue perfusion. rapid decreases in pao ~ 2 ~, as in a cardiorespiratory arrest, can lead to permanent neurological damage within minutes. however, lower, less abrupt changes, can be tolerated if the decrease in oxygen occurs in a gradual manner, such as ascending at altitude, where individuals can acclimatise and develop tolerance to lower oxygen partial pressures or, ( to a lesser degree ), in chronic smokers. initial clinical features include altered judgement, difficulty in completing complex tasks, and impairment in short term memory \ [ [ @ cr18 ], [ @ cr19 ] \ ], but in the longer term deficits can be more widespread and span physical and neuropsychological domains. seizures occur in up to a third of individuals within a day of exposure to hypoxia, and are commonly partial complex or myoclonic in nature. intractable forms of either of these types of seizure are associated with a poor prognosis \ [ [ @ cr20 ] \ ]. cognitive impairment domains include amnesia, visuospatial deficits, frontal lobe symptoms, impairment of executive function, and impairments in language \ [ [ @ cr21 ] \ ]. these are covered in more detailed reviews on the subject \ [ [ @ cr22 ], [ @ cr23 ] \ ]. involvement of the basal ganglia, a region particularly susceptible to hypoxic injury, can result in delayed parkinsonism in older subjects, dystonia mainly in younger people, choreo - athetosis, and tremors \ [ [ @ cr20 ] \ ]. varying degrees of unilateral or bilateral motor impairment may be observed depending on both the anatomical level and extent of corticospinal tract involvement. in very rare cases, the syndrome of delayed post - hypoxic leukoencephalopathy may occur weeks after a seemingly rapid recovery from the original insult. this condition is characterised by rapid deterioration in cognition, emergence of extra - pyramidal signs, and loss of executive function as a results of severe demyelination \ [ [ @ cr24 ] \ ]. the severity of leukoencephalopathy can be assessed by magnetic resonance imaging ( mri ) \ [ [ @ cr20 ] \ ]. electroencephalography, somatosensory evoked potentials and mri can provide valuable information about the severity of hypoxic injury, but also aid in prognostication together with overall clinical state \ [ [ @ cr25 ] \ ]. hypoxia in the context of a stroke { # sec6 } = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = there is no specific definition as to what constitutes hypoxia in an acute stroke, and it is therefore reasonable to assume that normal values for the general population apply. sulter and colleagues \ [ [ @ cr26 ] \ ] monitored 49 consecutive patients who presented with an acute stroke within 12 h duration using pulse oximetry for 48 h. patients were considered hypoxic and treated with supplemental oxygen if saturations were below 96 % for more than 5 min. this occurred in 63 % \ [ [ @ cr31 ] \ ] of patients, with 28 of those returning to ' normal ' oxygen saturations following administration of up to 5 l / min of oxygen. the remaining three required much higher concentrations. factors associated with hypoxia in this group were stroke severity, presence of dysphagia, and older age. roffe et al. \ [ [ @ cr27 ] \ ] recruited 118 patients ( 100 of whom had adequate measurements by pulse oximetry ) and found that the mean daytime awake so ~ 2 ~ was 94. 5 ± 1. 7 % in stroke patients and 95. 8 ± 1. 7 % in healthy controls. nocturnal saturations were reduced to 93. 5 ± 1. 9 % in the stroke group and 94. 3 ± 1. 9 % in controls. in the stroke group the average 4 % oxygen desaturation index ( odi ) ( number of times per | Background {# Sec1} = = = = = = = = = = Hypoxia is common after stroke, and associated with poor outcomes. In this article, we have reviewed the physiology of oxygen transport, the cerebrovascular response to hypoxia and pathophysiology, incidence and aetiology behind hypoxia in stroke and its subsequent clinical consequences. We have then reviewed all randomised clinical trials looking at the use of supplemental oxygen therapy in acute stroke and made conclusions regarding current evidence and recommendations for clinical practice. Oxygen physiology {# Sec2} = = = = = = = = = = = = = = = = = The normal adult range of arterial oxygen pressure (PaO2) is 11. 0 - - 14. 4 kPa and the normal range for arterial oxygen saturation (SaO2) is 95 - - 98% \ [[ @ CR1] \ ]. The term hypoxia refers to oxygen levels below normal. It ijclKdes both tissue (e. g. brain, myocardium) hypoxia and hypoxia in the blood (hypoxaemia ). Tissue hypoxia is defined by the concentration of oxygen in blood and also tissue perfusion, whilst hypoxaemia is defined by the concentration of oxygen in inspired air and its transfer into the blood \ [[ @ CR2] \ ]. Following inhalation oxygen is taken up in the lung capillaries via diffusion down an oxygen concentration gradient across the alveoli \ [[ @ CR3 ], [@ CR4] \ ]. Oxygen binds to the haemoglobin molecule, which can carry four oxygen molecules; each binding and changing the shape of the haemoglobin molecule and increasing its affinity for oxygen \ [[ @ CR5] \ ]. A small amount of oxygen is also dissolved in plasma. This proportion increases in hyperoxia, when all haemoglobin is saturated \ [[ @ CR6] \ ]. Oxygen dissociates from the haemoglobin molecule in the tissues owing to the relatively hypercapnic and acidic environment (the Bohr effect) \ [[ @ CR3] \ ]. Oxygen is a vital substrate that supports virtually all metabolic processes. 90% of oxygen intake is engaged in the cytochrome C oxidase system in the mitochondria \ [[ @ CR7] \] generating adenosine triphosphate (ATP ), which acts as the main energy substrate within cells. A continuous supply of oxygen is required to secure a continuous supply of ATP maintaining sufficient energy for cerebral neuronal and cellular Qctidity. This facilitates an efficient energy producing process making 38 molecules of ATP during aerobic respiration, equivalent to 1270 joules (J) energy, in comparison to two molecules of ATP (67 J of energy) during anaerobic respiration \ [[ @ CR7 ], [@ CR8] \ ]. The anoxic brain {# Sec3} = = = = = = = = = = = = = = = = 20% of all human oxygen consumption is utilised by the brain \ [[ @ CR9] \ ]. The brain has no oxygen or glucose (the other important substrate in the ATP producing equation) stores. Thus complete disruption of cerebral blood flow very rapidly results in an anoxic, hypoglycaemic state, which via a variety of mechanisms ultimately leads to cell death. Excitatory neurotransmitters, such as glutamate, bind to a variety of receptors and allow for an influx of calcium ions that help formulate the chemical signal for depolarisation \ [[ @ CR10 ], [@ CR11] \ ]. Normally the re - uptake of glutamate is an active energy - driven process. In the absence of ATP this process fails, resulting in an extracellular accumulation of glutamate, which continually stimulates receptors leading to a persistent influx of calcium ions \ [[ @ CR12] \ ]. Furthermore, the Na + / Ca2 + ATP driven pump normally used to eliminate calcium fails, also due to a lack of ATP \ [[ @ CR13] \ ]. The resultant high intracellular calcium triggers multiple cascades that ultimately lead to mitochondrial dysfunction and cell death. Furthermore, instead of producing ATP, glial cells have been shown to release ATP extracellularly \ [[ @ CR11] \ ]. Aside from rendering this unusable by mitochondria, ATP also stimulates the P2X7 receptor, which again leads to significant calcium influx and ultimately cell death \ [[ @ CR13] \ ]. The other major mechanism of cellular demise is via the formation of free radicals facilitated by the reduction of iron from its ferric (Fe3 +) to its ferrous (Fe2 +) form and the initiation of inflammatory cascades \ [[ @ CR12] \ ]. Cerebral blood flow in hypoxia {# Sec4} = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = In normoxic states, cerebral blood flow is very tightly controlled by the partial pressure of carbon dioxide (PaCO ~ 2 ~ ). Any hypocapnic state will result in vasoconstriction and reduction in regional cerebral blood flow and a hypercapnic state leads to the reverse with vasodilatation and an increase in cerebral blood flow. Cerebral blood flow is somewhat less responsive to changes in PaO ~ 2 ~, which has the opposite effect to carbon dioxide; a hypoxic state causing cerebral vasodilatation with the aim of improving oxygen delivery and a hyperoxic state causing vasoconstriction \ [[ @ CR14 ], [@ CR15] \ ]. In a hypoxic state, whilst the vasodilatory response improves flow, the detection of hypoxia by peripheral chemoreceptors will in turn lead to an increase in respiratory drive, increasing arterial oxygen content. However, the consequence of this is also an increase in the clearance of carbon dioxide, which would theoretically cause vasoconstriction and reduced cerebral blood flow \ [[ @ CR9] \ ]. It appears there is a threshold to which the hypoxic response predominates (and the carbon dioxide one attenuated) at a PaO ~ 2 ~ of around 50 - - 60 mmHg \ [[ @ CR9 ], [@ CR14] \ ]. Whilst the carbon dioxide mediated vascular response is mediated via a direct change in vessel wall pH \ [[ @ CR15] \ ], the oxygen response appears to be mediated by the deoxygenated erythrocyte via a number of mechanisms; which include release of ATP and the subsequent actions of endothelial nitric oxide synthase on the vessel wall, reduction of nitrite to nitric oxide and the activity of S - nitrosohaemoglobin \ [[ @ CR9] \ ]. The cerebral vascular response to hypoxia is not uniform. A study found that in an induced isocapnic hypoxic X^ate increases in cerebral blood flow were most prominent in basal ganglia nuclei, the putamen, thalamus, nucleus accumbens and pallidum \ [[ @ CR16] \ ]. Studies of blood flow in individual vessels have found that flow in the internal carotid artery is maintained during hypoxia and that vetteNral artery flow is increased \ [[ @ CR17] \ ]. This had led to the hypothesis that blood flow is increased in this region to preserve vital brainstem structures, or that possibly the posterior circulation vasculature is less susceptible to the effects of carbon dioxide for similar reasIgs. Neurological effects of hypoxia {# Sec5} = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = The neurological consequences of hypoxia are dependent upon the speed of onset, the severity of hypoxia, and the level of tissue perfusion. Rapid decreases in PaO ~ 2 ~, as in a cardiorespiratory arrest, can lead to permanent neurological damage within minutes. However, lower, less abrupt changes, can be tolerated if the decrease in oxygen occurs in a gradual manner, such as ascending at altitude, where individuals can acclimatise and develop tolerance to lower oxygen partial pressures or, (to a lesser degree ), in chronic smokers. Initial clinical features include altered judgement, difficulty in completing complex tasks, and impairment in short term memory \ [[ @ CR18 ], [@ CR19] \ ], but in the longer term deficits can be more widespread and span physical and neuropsychological domains. Seizures occur in up to a third of indibidualA within a day of exposure to hypoxia, and are commonly partial complex or myoclonic in nature. Intractable forms of either of these types of seizure are associated with a poor prognosis \ [[ @ CR20] \ ]. Cognitive impairment domains include amnesia, visuospatial deficits, frontal >ofe symptoms, impairment of executive function, and impairments in language \ [[ @ CR21] \ ]. These are covered in more detailed reviews on the subject \ [[ @ CR22 ], [@ CR23] \ ]. Involvement of the basal ganglia, a region particularly susceptible to hypoxic injury, can result in delayed Parkinsonism in )ldeT subjects, dystonia mainly in younger people, choreo - athetosis, and tremors \ [[ @ CR20] \ ]. Varying degrees of unilateral or bilateral motor impairment may be observed depending on both the anatomical level and extent of corticospinal tract involvement. In very rare cases, the syndrome of delayed post - hypoxic leukoencephalopathy may occur weeks after a seemingly rapid recovery from the original insult. This condition is characterised by rapid deterioration in cognition, emergence of extra - pyramidal signs, and loss of executive function as a results of severe demyelination \ [[ @ CR24] \ ]. The severity of leukoencephalopathy can be assessed by magnetic resonance imaging (MRI) \ [[ @ CR20] \ ]. Electroencephalography, comatosenCory evoked potentials and MRI can provide valuable information about the severity of hypoxic inj8rj, but also aid in prognostication together with overall clinical state \ [[ @ CR25] \ ]. Hypoxia in the context of a stroke {# Sec6} = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = There is no specific definition as to what constitutes hypoxia in an acute stroke, and it is therefore reasonable to assume that normal values for the general population apply. Sulter and colleagues \ [[ @ CR26] \] monitored 49 consecutive patients who presented with an acute stroke within 12 h duration using pulse oximetry for 48 h. Patients were considered hypoxic and treated with supplemental oxygen if saturations were below 96% for more than 5 min. This occurred in 63% \ [[ @ CR31] \] of patients, with 28 of those returning to ' normal ' oxygen saturations following administration of up to 5 L / min of oxygen. The remaining three required much higher concentrations. Factors associated with hypoxia in this group were stroke severity, presence of dysphagia, and older age. Roffe et al. \ [[ @ CR27] \] recruited 118 patients (100 of whom had adequate measurements by pulse oximetry) and found that the mean daytime awake SO ~ 2 ~ was 94. 5 ± 1. 7% in stroke patients and 95. 8 ± 1. 7% in healthy controls. Nocturnal saturations were reduced to 93. 5 ± 1. 9% in the stroke group and 94. 3 ± 1. 9% in controls. In the stroke group the average 4% oxygen desaturation index (ODI) (number of times per | Background {#Sec1} ========== Hypoxia is common stroke, and associated with poor outcomes. In this article, we have reviewed the physiology of oxygen transport, the cerebrovascular response to and pathophysiology, incidence aetiology behind hypoxia in stroke and its subsequent clinical consequences. We have then reviewed all clinical trials looking at the use of supplemental oxygen therapy in stroke and made regarding current evidence and recommendations for clinical practice. Oxygen physiology {#Sec2} ================= normal adult range of arterial oxygen pressure (PaO2) is 11.0--14.4 kPa and the normal range for arterial oxygen saturation (SaO2) is 95--98% \[[@CR1]\]. The term hypoxia refers to oxygen levels below normal. It includes both tissue (e.g. brain, myocardium) hypoxia and hypoxia in the blood (hypoxaemia). Tissue hypoxia defined the concentration of oxygen in blood and also tissue perfusion, whilst hypoxaemia is defined by the concentration of oxygen in inspired air and transfer into the blood Following inhalation oxygen is taken up in the lung capillaries via diffusion down an oxygen concentration gradient across the alveoli \[[@CR3], [@CR4]\]. Oxygen binds to the molecule, which can carry oxygen each binding and changing the shape of the haemoglobin molecule and increasing its affinity for oxygen \[[@CR5]\]. A small amount of is also dissolved in plasma. This proportion increases in hyperoxia, when all is \[[@CR6]\]. Oxygen dissociates from the haemoglobin molecule the tissues owing to the relatively hypercapnic and acidic environment (the effect) \[[@CR3]\]. Oxygen is a vital substrate that supports virtually all metabolic oxygen intake is engaged in the cytochrome C oxidase system in the mitochondria \[[@CR7]\] generating adenosine (ATP), which acts as the main energy substrate within cells. A continuous supply of oxygen is required to secure a continuous supply of ATP maintaining sufficient energy for cerebral neuronal cellular activity. This facilitates an efficient energy producing process making 38 molecules of ATP during aerobic respiration, equivalent to 1270 joules (J) energy, in comparison to two molecules of ATP (67 J energy) during \[[@CR7], [@CR8]\]. The brain {#Sec3} ================ of all human oxygen consumption is utilised by the brain \[[@CR9]\]. The brain has no oxygen or glucose (the important substrate in the ATP producing equation) stores. Thus complete disruption of cerebral blood flow very rapidly results in an anoxic, hypoglycaemic state, which via a variety of mechanisms ultimately leads cell death. Excitatory neurotransmitters, such as glutamate, bind a variety of receptors allow for an influx of calcium ions that help formulate the chemical signal depolarisation \[[@CR10], [@CR11]\]. Normally the re-uptake of is an active energy-driven process. In the absence of ATP this process fails, resulting in an extracellular accumulation glutamate, continually stimulates receptors leading to a persistent influx of ions \[[@CR12]\]. Furthermore, the ATP driven pump normally used to eliminate calcium fails, also due to a lack ATP \[[@CR13]\]. The resultant high intracellular calcium triggers cascades that ultimately lead to mitochondrial dysfunction and cell death. Furthermore, instead of producing ATP, glial cells have been shown to release ATP extracellularly \[[@CR11]\]. Aside from rendering this unusable by mitochondria, ATP also stimulates the P2X7 receptor, which again leads to significant calcium influx and ultimately cell \[[@CR13]\]. The other major of demise is via the formation of free radicals facilitated by the reduction of from its ferric (Fe3+) to its ferrous (Fe2+) form and the initiation of inflammatory cascades \[[@CR12]\]. Cerebral blood flow in {#Sec4} ============================== In normoxic states, blood is very tightly controlled by the partial pressure of carbon dioxide (PaCO~2~). hypocapnic will result in vasoconstriction and reduction in cerebral blood flow and a hypercapnic state leads the with vasodilatation and an increase cerebral blood flow. Cerebral blood flow is less responsive to changes in PaO~2~, which has the opposite effect to carbon dioxide; a state causing cerebral vasodilatation with the aim of improving oxygen and a hyperoxic state vasoconstriction \[[@CR14], [@CR15]\]. a hypoxic state, whilst the response improves flow, the detection of hypoxia peripheral chemoreceptors will in turn lead to an increase in respiratory drive, arterial oxygen content. However, the consequence of this is also increase in clearance of carbon dioxide, which would theoretically cause and reduced cerebral blood \[[@CR9]\]. appears there a threshold to which hypoxic response predominates (and carbon one at a PaO~2~ of around 50--60 mmHg \[[@CR9], [@CR14]\]. Whilst the carbon dioxide mediated vascular response is mediated via a direct change in wall pH \[[@CR15]\], the oxygen response appears to be mediated by the deoxygenated erythrocyte via a number of mechanisms; which include release of ATP and the subsequent actions of endothelial nitric synthase on the vessel reduction of nitrite to nitric oxide and the activity of S-nitrosohaemoglobin \[[@CR9]\]. The cerebral vascular response to is not A study found that in an induced isocapnic hypoxic state increases in cerebral blood flow were most prominent in basal nuclei, putamen, thalamus, nucleus accumbens and pallidum \[[@CR16]\]. Studies blood flow in individual vessels have found that flow in the internal carotid artery is maintained during hypoxia and that vertebral artery is \[[@CR17]\]. This had led to the hypothesis blood flow is increased in this region to preserve vital brainstem structures, or that possibly posterior circulation vasculature is less susceptible to the effects of carbon dioxide for reasons. Neurological effects of hypoxia =============================== The neurological consequences of hypoxia are dependent upon the speed of onset, the severity of hypoxia, and the level of tissue perfusion. Rapid decreases in PaO~2~, in a cardiorespiratory arrest, can lead to permanent neurological damage within minutes. However, less abrupt changes, can be tolerated if decrease in oxygen in a gradual manner, such as ascending at altitude, where individuals can acclimatise and develop tolerance to lower oxygen partial or, (to a degree), in chronic smokers. Initial features include altered judgement, in completing complex tasks, and impairment in short term \[[@CR18], [@CR19]\], but in term deficits can be more widespread and span physical neuropsychological domains. Seizures occur in up to a third of within a day of to hypoxia, and are commonly partial complex or myoclonic in nature. Intractable forms of either of these types of seizure associated with a poor prognosis \[[@CR20]\]. Cognitive impairment domains include amnesia, visuospatial deficits, frontal lobe symptoms, impairment of executive function, impairments in language \[[@CR21]\]. These are covered in more detailed reviews on the subject \[[@CR22], [@CR23]\]. of the basal ganglia, a region particularly to hypoxic injury, can in delayed Parkinsonism older subjects, dystonia mainly in younger people, choreo-athetosis, and \[[@CR20]\]. Varying degrees of unilateral or bilateral motor impairment may be observed on both the level and extent of corticospinal tract involvement. In very cases, the syndrome of delayed post-hypoxic leukoencephalopathy may occur weeks after a seemingly rapid recovery from the original insult. This condition is characterised by rapid deterioration in cognition, emergence of extra-pyramidal signs, loss of executive function as results of severe demyelination \[[@CR24]\]. The of leukoencephalopathy can be by magnetic resonance (MRI) \[[@CR20]\]. Electroencephalography, somatosensory evoked potentials and MRI can provide valuable information about the of hypoxic injury, but also in prognostication with clinical state Hypoxia in the a stroke {#Sec6} ================================== no specific definition as to what constitutes hypoxia in an acute and is reasonable to assume that normal values for the population apply. Sulter and colleagues \[[@CR26]\] monitored 49 consecutive patients who presented with acute stroke 12 h duration pulse oximetry for 48 h. Patients were considered and treated with supplemental if saturations below 96% for more 5 min. This occurred in 63% \[[@CR31]\] of patients, with 28 of those returning to 'normal' oxygen saturations following administration of up to 5 L/min of oxygen. The remaining three required much higher concentrations. with hypoxia in this group were stroke severity, presence of and older Roffe et al. \[[@CR27]\] recruited 118 patients (100 of whom had adequate measurements by pulse oximetry) and that daytime awake SO~2~ 94.5 ± 1.7% in stroke patients and 95.8 ± 1.7% in healthy controls. Nocturnal saturations were reduced ± in the stroke group and 94.3 1.9% in controls. In the stroke group the average 4% oxygen desaturation index (ODI) (number of times per | BacKgrOuNd {#sEC1}
==========
hYpoXIA iS ComMON AfTER STrOkE, and aSsOCIated wiTh pOOR OuTcOMeS. iN ThIS ArTicLE, We have reviEWED ThE phySioloGy Of oXYGeN TRanspORt, the cErEbrOvAsculAr RESpoNSe to hypOXia AnD paTHOphySiOloGy, iNCIdenCe aNd aETIoloGy BehInD HyPOxIA In StrOkE aND ItS SUBSeQuenT cLinICal CoNSEquENcES. wE haVE tHEn RevieweD alL RAndoMIseD cLINIcal tRiAlS LOoKIng aT thE Use Of SuPpLemENtAL OxYgeN thErapy IN acute sTrOKE AnD madE CoNcluSIOns ReGarDiNG CURrent EViDEnCe And recOMMeNdations fOr cLiniCaL pRAcTIce.
OXygEN pHYsIoLogY {#sec2}
=================
THe norMAL aduLt rANge OF ARTErIal OxYgeN PrESsuRe (PAo2) is 11.0--14.4 KpA ANd the nORmal RanGe for ArTeRIAl oxYGen sAtURAtiON (sao2) iS 95--98% \[[@Cr1]\]. THE TErm HyPOXIA reFeRs To oxygen lEveLs BEloW NOrmAl. IT iNCLudES BoTH TisSUE (e.g. bRAiN, MYocaRdIUm) HyPOxIA And hypoXIA iN ThE BlOOd (hYPoXaeMIa). TisSUE hyPoXIa IS DefInED bY tHE ConcEntraTION of OXyGEN in blooD AnD also TISSue peRFUsion, WHiLST HyPOXaeMIA iS deFiNED bY tHE cOnceNtRATIOn Of OXygen IN inSPiREd AIr AND Its TransFER inTO thE blOOd \[[@CR2]\].
foLlOwing inHALAtion OXygen IS tAkEN up in tHE LUNG caPIlLARIEs Via DIFFusiOn DOwN aN oXygEn CONcenTRaTION GRadIeNt acrOsS tHe ALveoLI \[[@cr3], [@CR4]\]. oxygEN BINDs TO thE HaeMoGlObiN mOLEcuLe, whiCh cAn carry fOUr oxYgen moleCules; eACh bindING aNd chanGINg thE shApe of thE HaemoGLoBiN MOleCUle anD InCrEASiNg ITs affiniTy FoR oXygEn \[[@cr5]\]. a SmaLl AMoUNT of OxygEN is also dIsSOLveD IN PLasMA. THIs PrOportIOn iNcreaseS in HYPeRoxiA, WHeN aLl HAEMoGLObIn IS satUrATed \[[@Cr6]\]. oXyGeN dISsoCiATes FRom tHe HAemOGlObIn MOlEculE In THe tiSsuEs OwiNg to thE rELAtiVEly HYpERCApniC and ACIdIC EnViRonmeNT (THE BohR efFecT) \[[@Cr3]\].
OxygEN is a viTaL suBstrATe thaT SUPpoRts VirTUaLLY ALL metaBOLiC pROcEssES. 90% oF oXygeN InTakE IS eNgAgeD IN tHE CYTOChRome C OXIdaSe SySteM iN thE miTochOndrIa \[[@cR7]\] GeNErAtIng ADeNosINe trIphosPhate (ATP), whiCh aCts as thE MAin eNERGy SuBsTraTe WithIN CelLS. a CoNtiNUOUs sUPpLY of OxYGeN iS REqUIReD TO SEcuRe A CoNTINuouS SuppLy of atp MaiNTaininG SuFfICIENT ENergY fOr CeREbRAl NEUroNaL aND ceLlULAr ACtIViTy. thIS faCIliTAtes an EffiCIEnT eNerGY pROdUcing proCess MakING 38 moLeCULES Of ATp DUrInG AErOBIC REsPiRAtIon, eQUIVaLenT to 1270 JOULeS (j) eNErgy, IN coMparISON To twO mOLECUleS Of ATP (67 J OF ENErgy) DUrinG AnaerOBiC ReSpIrAtiOn \[[@CR7], [@cR8]\].
tHe ANOxiC BrAIN {#sEc3}
================
20% of ALl hUmaN OxygEN conSUmpTion Is utIliseD bY The bRaIN \[[@cR9]\]. thE brain haS nO oxYgen OR GLucosE (THE othEr iMpOrtANt sUbstrATE IN ThE Atp prOdUcINg EqUaTion) STOrEs. tHus cOMPLeTe diSRUpTiON oF cERebrAL BlOOd Flow VEry RApidLY ResULTS IN aN anOXIC, HYPogLycaemiC stATE, whICH vIa a vARiety of MecHaniSMS uLTImATeLY lEadS TO Cell DEATH. ExcitaTOrY NeURotRAnSmiTTErS, SUCH As GLuTAmATE, biND tO a vARiETY oF REcePTOrs ANd ALLow fOr AN inFlUX Of CAlCIUM ions THaT hElP fOrMUlATe thE chEmiCAL siGNAL FOr dEpOLarISaTIon \[[@Cr10], [@Cr11]\]. noRMallY thE rE-UpTake Of glUtamatE Is an acTiVe eNErgY-drIVEn PRocesS. in tHE ABSEnCe OF aTP this proCeSs FaIlS, rEsULTiNG In aN eXtRACElLuLaR AcCumUlAtiON OF gLUtamAtE, WHICh CoNtINUaLlY stIMuLATes RECePtORs LEaDING To a peRsIsTEnT INfLUX of CALcIuM iOns \[[@cr12]\]. fURtHErMoRE, ThE NA+/ca2+ ATP DRiVen pUMp nORMALlY uSeD to ElImINATe CAlCium FAiLS, alSO DuE TO a lack of Atp \[[@cr13]\]. The reSUlTant hIGh IntRacEllUlar CALCium TriggeRS MuLtIplE CAsCaDEs thAt ultimATeLy LeAd To MiToChonDrIAl DYSfUNCtIoN AND ceLL DeATh. FuRtHeRmoRe, iNSTEaD oF pRoDUcINg atP, gLIal CelLs HavE been shown tO reLease aTP ExtRacelluLaRly \[[@Cr11]\]. AsidE FRom reNdeRinG thIs uNUsAble By MiTOCHoNdrIa, Atp aLsO stimuLaTES tHE P2X7 REcEPToR, whiCh agAiN LEAdS To SIgnIfICanT CALcIuM InFlUX and UlTimaTElY CeLL DeAtH \[[@cR13]\]. THE OthEr MAJoR MecHaNism Of CeLluLar deMIsE is VIa tHe FOrMATIon of fREe RAdicaLs faCIlItated BY the ReduCTiOn Of Iron FrOM its fErric (fe3+) To its fERROus (fE2+) foRm ANd THE iniTiAtIoN oF INFLAMmAToRY CAsCADes \[[@cR12]\].
CeReBRal bLOod flOw In hYPoxia {#seC4}
==============================
iN nORMOxiC STatES, CErEBRaL blood FlOw iS vERy TiGhTly contrOLLED BY The ParTIaL PReSsUre oF CARboN dIoXide (PACo~2~). ANy HYpocapNIc StaTe wiLl rEsUlT IN VasOconstrICtIoN And REdUCtiON iN rEgIoNaL CERebraL bloOd FlOw and A HYpeRcApnIC STaTe lEAdS TO ThE REVErSE WiTh vasODilaTaTion aND AN InCREaSe in CEREbRal bLooD FLOW. cErEbRaL blOoD FLOw iS sOmewhaT LESS ReSPoNSIVe To ChANgES iN paO~2~, WHiCh hAS tHe opPoSITE efFECT TO carbon dioXiDe; A HYpOxIC STaTe cauSinG cErEBRaL vASODIlaTatiON wITH the aim OF imPRovInG oxYgEn DeliVERY aNd a HYPErOxIC stAte CAuSINg vaSoconsTrICTIon \[[@cr14], [@CR15]\].
iN A HYPOXIc StATe, WHILSt THE VASoDIlatorY REspoNSE IMPRoVeS flOw, THE DEtEctioN Of HypOXiA By pEripheraL CHemOreCepTOrs wiLL in Turn LEad TO an inCREASe in REspirAtoRY dRIVe, IncREasinG ArTErIAL oxygen CONTENT. HOWEVer, tHe CONSEQuENCE Of THIs is aLSo An INcreAse in ThE cLearanCe Of CArbOn DIoxIDe, WhiCh WOUld THEOretICALly cause VaSoconstriCtIon aND REDucED ceReBral BlOOD FLOw \[[@CR9]\]. It aPpeARS thERe Is a thresholD tO whIcH The HyPOxIC ReSPonSe PReDOmINAtes (aND thE cArboN diOxidE oNe aTtENUAted) At a pAo~2~ Of AROuNd 50--60 MMHg \[[@Cr9], [@CR14]\]. WhILST THe CARbOn diOXiDe mEdIaTEd VaSCulaR RESPonsE is meDIatEd VIA A dIReCT ChaNgE IN vesSEL wALl Ph \[[@cr15]\], THe oxYgen reSPonsE ApPeARs tO Be mEDIATEd By ThE dEoxYGenATed ErYThRocYTe ViA A NuMBEr Of mEChAniSms; wHiCH INclUde rELEAse Of Atp and thE sUBseQUENT aCTIOns oF endOtHelIaL nItRIc OXIDe SYNthase oN ThE vESSEL waLl, rEduCTION OF NITrite tO NitRiC oxide AnD THE ACTIvITY oF s-NitRosOHaEmOgLoBin \[[@CR9]\].
the CeReBrAl VasCulAr rESPonSe tO hyPOxIa iS NoT unIfORm. a sTUDy FOunD that In An INDucEd IsOcaPnIC hYpoxiC sTAte INcrEases IN CEReBRAL BlooD FLOW wERE MOsT PROMINENT iN basAl GANglIA Nuclei, tHE PUtamEN, tHAlaMUS, nUCLeus ACcuMBens and pallIdum \[[@cr16]\]. stUdieS Of BlooD flOw IN indIVIDUAL VEssELs haVE FOund tHAT fLOW iN ThE inTErnal CARoTiD aRTeRY Is mAiNTaInEd dUring HypoXiA aNd THAt VErteBRaL aRterY FlOw is InCrEaSed \[[@CR17]\]. ThIs hAD lED TO the hYpotHeSiS ThaT BLOOd FLOW iS inCrEAsEd IN THIS ReGioN to pREsErVE vitAl brAInStEM STRuCtUrEs, oR That PoSsIbLY thE POSTeRIOr CiRCULATion vAscUlaTUre Is lESS sUscepTIBlE to tHe efFEcTS oF caRbOn DiOxide fOR simILAr REaSonS.
nEUROLoGiCaL EfFectS of HYPoxIA {#SEc5}
===============================
tHE nEuROLOGicAl CONseqUEnCES OF HYPOXiA Are DePEndeNT UPOn THe Speed of onSET, THE sEVeRIty OF hYpOXIa, And THe leVeL OF tIssue pErFUsiON. RAPiD dEcREASES IN pao~2~, AS In A cArDioREsPIratORY aRreST, Can leaD to PerManEnT NeUROloGICaL damagE wIthIN MiNUTes. howeveR, lOweR, LESS ABRupT chANges, can bE ToLERaTED iF thE DEcrEaSE iN oxyGEN OcCUrS In A GraDuAl ManNER, SUCH AS ASCeNdINg AT altiTuDe, WHEre inDIviDuaLs CAn ACClImaTIsE aND DevELOp toleRANce To lowER oxygeN partIaL PResSURES Or, (tO A lesseR degREE), in chROniC SmOKErS. InItiaL ClINical fEatUReS INClUDe ALTeReD JuDgeMeNT, DIffIcuLTY iN coMPLETinG CompLex TAsKS, And iMpAiRMeNT IN shOrT teRM meMOry \[[@CR18], [@CR19]\], BUT iN The lOngEr TERm DefiCITs cAN BE mORE WiDesprEAD ANd SPAN phySICAL AND nEuRopsYchOLoGiCal DoMaINS. sEiZuRes OcCur IN uP tO A thIrd Of IndiviDuaLS WIThiN A DAY oF ExpOsUrE TO hyPoxiA, anD Are CommONly pARTIAl coMPLEX or mYocLoNIc IN naTuRE. InTRaCTabLe FormS oF EItHEr oF TheSe typEs of SEiZure aRE ASSoCIAtEd wITH A PoOr PRogNoSIS \[[@CR20]\]. COgnItIVe iMpaiRmenT DOmaiNs Include aMNeSiA, vISUospATiAl dEFiCITS, FrontAL lOBe SymptOMs, IMpaIRmENt oF eXECUTiVe fUnCtIOn, anD imPaiRMeNTs in laNGuAGe \[[@Cr21]\]. tHese Are cOvEred IN MoRE dETaILeD ReViEwS ON tHe suBJECt \[[@Cr22], [@cr23]\]. INvoLVEmeNt of THe BasAL ganGlia, a reGioN ParTicULARLY SUScEpTIBLE To HyPoxic InJurY, Can ReSULt in DelaYED pARKINsonISM iN oLdeR SUBJECts, DystoniA MAinLy iN yoUNGER PeoPLe, CHoreO-AtHETOSis, AND tReMoRS \[[@CR20]\]. vARyInG deGrEEs OF UNIlateRAL oR biLATErAL mOtoR impAirmENT may BE OBServeD dePEndiNg ON BOTH thE aNatoMical LeVEL AnD exteNT Of CoRticOspiNaL TRACt InvOlveMeNt. iN VErY RarE cASes, tHe sYnDromE of DElayed pOst-HyPoxiC lEUKOEncePhALopATHY mAY ocCUr weeKS AFTER A sEEMinGlY RApId RECoveRy FrOm The oRiginal InsuLt. tHIs Condition IS cHARacTERisED by rAPId DETERioRaTiON iN cOgNition, EMERgenCe oF EXTrA-pyRAmidAl sigNs, And lOSS of execUTiVe FUnCtIoN aS A ReSults Of SEvErE DeMyELiNATION \[[@cr24]\]. ThE seVERItY OF LEukOEnCEpHaLoPatHY can Be aSSESsED bY mAGNETic ResONANce iMaGiNG (mRI) \[[@cr20]\]. elECtrOEncephaLogRAPHy, sOMATosENsory evoKeD POtenTiALs aNd Mri can pRoviDE ValuablE inFOrMAtion about THE SeverITY oF hYPOxiC InjurY, bUT alSo aiD in ProgNOSTIcatIOn toGeTHeR With oVERaLL cLInIcal StATe \[[@Cr25]\].
HyPOXia iN thE conTExt Of a stRoke {#SEc6}
==================================
ThERE IS No SpecIFIC dEFInITIoN AS To WhaT consTItuTES HypoXIA In An acUtE strOkE, aND iT iS tHEreFore REasonaBle To AssumE tHat NorMAL vALUEs fOR THe gENEral populatIOn aPPLY.
sulTer aNd cOLLEaguEs \[[@cR26]\] MOnitOrED 49 COnseCutiVE paTiENtS wHO pReSENtEd wiTH an aCUTe StROke witHiN 12 H DuRatiON UsIng PuLSe OXiMetRy fOr 48 h. PatIEnTS were COnSIdErEd HypoXic aND TReAted WItH sUppLEMENtal oXyGen If SAtUraTioNs WEre bElOw 96% For mOrE thaN 5 MIN. tHIs oCcURreD in 63% \[[@CR31]\] of PatiEnTS, WItH 28 oF ThosE ReTurniNG To 'NoRMal' oXYgen SATuRaTIONs FOLlOWiNG aDmiNISTratioN oF up to 5 L/MIN Of oxYgen. The REmaining three ReqUired mUcH hIgheR ConceNTratIONS. FActORS aSsociaTed WITh HYpoxiA iN tHIS grouP weRe sTroke severITY, prEseNce oF dYsPhAGIA, ANd Older age. RoFFE et Al. \[[@CR27]\] rEcrUitED 118 PAtiEntS (100 oF wHom haD aDequaTE mEAsURemEnTs bY pULSE OxIMEtrY) And foUnd tHat tHe MEaN DayTIMe AWAke So~2~ was 94.5 ± 1.7% in StRoKe paTIenTS anD 95.8 ± 1.7% in heAlthy CONtRolS. NoctuRNAl SATUrATioNS WeRe reDucEd To 93.5 ± 1.9% In The sTrOKE GrOUp anD 94.3 ± 1.9% in CONTrols. in thE STroKE GRoUp THe averAGe 4% oXyGEN DesAturAtIoN INdEx (odI) (NuMBer OF TIMeS pER | Background {#Sec1} ========== Hypoxia is common afterstroke, andassociated with poor outcomes. In this article, we have reviewed the physiology of oxygen transport, the cerebrovascular response to hypoxia and pathophysiology, incidence and aetiology behind hypoxia in strokeand its subsequent clinical consequences. We have then reviewedall randomised clinical trials lookingat the use ofsupplemental oxygen therapyin acute stroke and made conclusions regarding current evidence andrecommendations for clinical practice.Oxygen physiology {#Sec2} ================= The normal adult rangeofarterial oxygenpressure(PaO2) is 11.0--14.4 kPa and the normalrange for arterial oxygensaturation (SaO2) is 95--98% \[[@CR1]\]. The termhypoxia refersto oxygen levels below normal. Itincludes both tissue (e.g. brain, myocardium) hypoxia and hypoxia in the blood (hypoxaemia). Tissue hypoxia isdefined by the concentration of oxygen in blood and also tissue perfusion, whilst hypoxaemia is defined by theconcentration of oxygen in inspiredair and its transfer into the blood \[[@CR2]\].Following inhalation oxygen is taken up in the lung capillaries viadiffusiondown anoxygen concentration gradient across thealveoli \[[@CR3], [@CR4]\]. Oxygenbinds to the haemoglobinmolecule,whichcan carryfour oxygenmolecules; each binding andchanging the shape of thehaemoglobin moleculeand increasing its affinity foroxygen \[[@CR5]\]. A small amount of oxygen is alsodissolved in plasma.This proportion increases in hyperoxia, when all haemoglobin is saturated\[[@CR6]\]. Oxygen dissociatesfrom the haemoglobin molecule in thetissues owing to therelatively hypercapnic and acidic environment(the Bohr effect) \[[@CR3]\]. Oxygen is a vital substrate that supports virtually all metabolic processes. 90% of oxygen intake is engaged in the cytochrome C oxidase system in the mitochondria \[[@CR7]\]generating adenosinetriphosphate (ATP), which acts as the main energy substrate within cells. Acontinuous supplyof oxygen is required to securea continuous supply of ATP maintaining sufficient energyfor cerebral neuronal and cellular activity. This facilitates an efficient energy producing process making 38 molecules of ATP during aerobic respiration, equivalent to 1270 joules (J) energy, incomparison to two molecules of ATP (67 J of energy) duringanaerobic respiration \[[@CR7], [@CR8]\]. The anoxic brain {#Sec3} ================ 20% of all human oxygen consumption is utilised by the brain \[[@CR9]\]. Thebrain has no oxygen orglucose (the other important substrate in the ATP producing equation) stores. Thus complete disruption of cerebral blood flow very rapidly results in ananoxic, hypoglycaemic state,which via a variety of mechanisms ultimately leads to cell death. Excitatory neurotransmitters, such as glutamate,bind to a variety of receptorsand allow foran influx of calcium ions that help formulate the chemical signal for depolarisation \[[@CR10], [@CR11]\]. Normally the re-uptake of glutamate isan active energy-driven process. In theabsence ofATP this process fails, resulting in an extracellularaccumulation of glutamate, which continually stimulates receptors leading to apersistent influx ofcalcium ions\[[@CR12]\]. Furthermore, the Na+/Ca2+ATPdriven pump normally used to eliminate calcium fails, alsodueto alackof ATP \[[@CR13]\]. The resultant high intracellularcalcium triggers multiple cascades thatultimately lead to mitochondrial dysfunction and cell death. Furthermore, instead of producing ATP, glial cellshave been shown to release ATP extracellularly \[[@CR11]\]. Aside from rendering thisunusable bymitochondria, ATP also stimulates the P2X7 receptor, which again leads to significantcalcium influx and ultimately cell death \[[@CR13]\]. The other major mechanism of cellular demise is viathe formation of free radicals facilitated by the reduction of iron from its ferric (Fe3+) to its ferrous (Fe2+) form and the initiation of inflammatory cascades \[[@CR12]\]. Cerebral blood flow inhypoxia {#Sec4} ============================== In normoxic states, cerebral blood flowis very tightly controlled by the partial pressure ofcarbon dioxide (PaCO~2~). Any hypocapnic state will result invasoconstriction and reduction in regional cerebral blood flow and a hypercapnic state leads tothe reverse with vasodilatationand an increasein cerebral blood flow. Cerebral blood flow is somewhat less responsive tochanges in PaO~2~, which has the opposite effect to carbon dioxide; a hypoxic state causing cerebralvasodilatation with the aim of improving oxygen delivery and ahyperoxic state causing vasoconstriction \[[@CR14], [@CR15]\]. In a hypoxic state, whilst the vasodilatory response improves flow, the detection ofhypoxia by peripheral chemoreceptorswillin turn lead to an increase in respiratory drive, increasing arterial oxygen content. However, theconsequence of this is also an increase in the clearanceof carbon dioxide, whichwould theoretically causevasoconstriction andreduced cerebral bloodflow\[[@CR9]\]. Itappears there is a threshold to which the hypoxic response predominates (and the carbondioxide one attenuated)at aPaO~2~ of around50--60 mmHg \[[@CR9], [@CR14]\]. Whilst thecarbon dioxidemediated vascular response is mediated via a direct change in vessel wall pH \[[@CR15]\], the oxygen response appears to be mediated by the deoxygenated erythrocyte via a number ofmechanisms; which include release of ATP and the subsequent actions of endothelial nitric oxide synthase on the vessel wall, reduction of nitrite tonitric oxideand the activity of S-nitrosohaemoglobin \[[@CR9]\]. Thecerebral vascular response to hypoxia is not uniform. A study found that in aninducedisocapnic hypoxic state increases in cerebral blood flow were mostprominentin basal ganglia nuclei, the putamen, thalamus, nucleus accumbens and pallidum \[[@CR16]\]. Studies of blood flow in individual vessels have found thatflow in the internal carotid artery is maintained during hypoxia andthat vertebral artery flow is increased \[[@CR17]\]. This had led to the hypothesis that bloodflow is increased in this region to preservevitalbrainstem structures, or that possibly the posterior circulation vasculatureis less susceptible to the effects of carbon dioxide forsimilar reasons.Neurological effects of hypoxia {#Sec5} =============================== The neurologicalconsequences of hypoxia are dependent upon the speed ofonset, the severity of hypoxia, and thelevel of tissue perfusion.Rapid decreases in PaO~2~, as ina cardiorespiratory arrest, can lead to permanent neurologicaldamagewithin minutes. However, lower, less abrupt changes, can be tolerated if the decrease in oxygen occurs in agradual manner, suchas ascending at altitude, where individuals can acclimatise and develop tolerancetoloweroxygen partial pressures or, (to a lesser degree), inchronic smokers.Initial clinical features include altered judgement, difficulty in completing complextasks, and impairment in short term memory \[[@CR18],[@CR19]\], but in thelongerterm deficits can bemore widespread and span physical and neuropsychological domains.Seizures occur in up to a third of individuals within aday of exposure to hypoxia, andare commonly partialcomplex or myoclonic in nature. Intractable forms of either of these types of seizure are associatedwith a poor prognosis\[[@CR20]\].Cognitive impairment domainsinclude amnesia, visuospatial deficits, frontal lobe symptoms, impairment of executivefunction, and impairments in language\[[@CR21]\]. These are covered inmoredetailedreviewson the subject \[[@CR22],[@CR23]\]. Involvement of the basal ganglia, a region particularly susceptible to hypoxic injury, can result in delayedParkinsonismin oldersubjects, dystonia mainly in younger people, choreo-athetosis, and tremors \[[@CR20]\]. Varying degrees of unilateral orbilateral motor impairment may beobserved dependingon both the anatomical level and extent of corticospinal tract involvement. In very rare cases, thesyndrome of delayed post-hypoxic leukoencephalopathy mayoccur weeks after aseemingly rapidrecovery from the original insult. This condition is characterisedby rapid deterioration in cognition, emergence of extra-pyramidal signs, andloss of executive functionas a results of severe demyelination \[[@CR24]\]. The severity of leukoencephalopathy can be assessedby magnetic resonance imaging (MRI) \[[@CR20]\]. Electroencephalography,somatosensory evoked potentials and MRI can provide valuable information about the severityof hypoxic injury, but also aid in prognosticationtogether with overall clinical state \[[@CR25]\]. Hypoxia in thecontext of a stroke {#Sec6} ==================================There is no specific definition asto what constitutes hypoxia in an acute stroke, and it is therefore reasonable toassume that normal values for the general population apply. Sulterand colleagues \[[@CR26]\] monitored 49 consecutive patients who presentedwith an acute stroke within12h durationusing pulse oximetryfor 48 h. Patients were considered hypoxic and treated with supplemental oxygen if saturations were below 96% for more than 5 min. This occurred in 63% \[[@CR31]\] of patients, with 28 of those returning to'normal' oxygen saturations following administration of up to 5 L/minof oxygen. The remaining threerequired much higher concentrations. Factors associated with hypoxiain this groupwere stroke severity, presence of dysphagia, and older age.Roffe et al. \[[@CR27]\]recruited 118 patients (100 of whom hadadequatemeasurementsby pulse oximetry) and found that the mean daytime awakeSO~2~was 94.5 ± 1.7% in stroke patients and 95.8 ± 1.7% in healthy controls. Nocturnal saturationswere reduced to 93.5 ±1.9% in the stroke group and 94.3 ± 1.9% in controls. Inthe stroke groupthe average 4% oxygen desaturation index (ODI) (number of times per | Background {#Sec1} ========== Hypoxia is common after stroke, and associated with _poor_ outcomes. In this article, we have reviewed _the_ physiology _of_ oxygen _transport,_ the cerebrovascular response to hypoxia and pathophysiology, incidence and aetiology behind hypoxia _in_ stroke _and_ its _subsequent_ clinical consequences. We have _then_ reviewed all randomised clinical _trials_ looking at the use of supplemental oxygen _therapy_ in _acute_ stroke and made _conclusions_ regarding current evidence and recommendations for clinical practice. Oxygen physiology {#Sec2} ================= _The_ normal _adult_ range of arterial oxygen pressure (PaO2) is 11.0--14.4 kPa and the normal range _for_ arterial oxygen saturation _(SaO2)_ is 95--98% \[[@CR1]\]. The term hypoxia refers to oxygen _levels_ below _normal._ _It_ includes _both_ tissue (e.g. brain, myocardium) hypoxia and hypoxia in _the_ blood (hypoxaemia). Tissue hypoxia is defined by the _concentration_ of oxygen _in_ _blood_ and also tissue perfusion, whilst hypoxaemia is defined by the concentration of _oxygen_ in inspired air and its transfer _into_ the blood \[[@CR2]\]. _Following_ inhalation oxygen _is_ taken up in the _lung_ capillaries via diffusion _down_ an oxygen concentration gradient across the alveoli _\[[@CR3],_ [@CR4]\]. Oxygen _binds_ to the haemoglobin molecule, which can _carry_ four oxygen molecules; _each_ _binding_ and changing the shape of the haemoglobin molecule and increasing _its_ affinity _for_ oxygen \[[@CR5]\]. A _small_ amount of oxygen is also dissolved in plasma. This proportion increases in hyperoxia, when all haemoglobin _is_ _saturated_ \[[@CR6]\]. _Oxygen_ dissociates from the haemoglobin _molecule_ _in_ _the_ tissues owing to _the_ relatively hypercapnic _and_ acidic environment (the Bohr effect) _\[[@CR3]\]._ Oxygen is _a_ vital substrate that supports virtually _all_ metabolic processes. _90%_ _of_ oxygen intake _is_ _engaged_ in the cytochrome C oxidase _system_ in the mitochondria \[[@CR7]\] generating adenosine _triphosphate_ (ATP), which acts as the main energy substrate within cells. A continuous supply of oxygen is required to secure _a_ continuous supply of ATP maintaining sufficient energy for cerebral neuronal and _cellular_ activity. This facilitates an efficient energy producing process making 38 molecules of _ATP_ during aerobic respiration, equivalent to 1270 joules (J) energy, in comparison to two _molecules_ of ATP (67 J of _energy)_ during _anaerobic_ respiration \[[@CR7], _[@CR8]\]._ The anoxic brain {#Sec3} _================_ 20% of all human oxygen consumption _is_ utilised by the brain \[[@CR9]\]. The brain has _no_ oxygen or glucose _(the_ other important substrate _in_ the _ATP_ _producing_ equation) stores. _Thus_ complete disruption of cerebral blood flow _very_ rapidly _results_ in _an_ anoxic, hypoglycaemic state, which via a variety of _mechanisms_ ultimately leads to cell death. Excitatory neurotransmitters, _such_ as glutamate, bind to a variety of receptors and _allow_ for an influx _of_ _calcium_ ions _that_ _help_ formulate the _chemical_ signal for _depolarisation_ \[[@CR10], _[@CR11]\]._ Normally _the_ _re-uptake_ _of_ glutamate is an active energy-driven process. In the absence of ATP this process fails, resulting in an extracellular accumulation of glutamate, _which_ _continually_ stimulates receptors leading to a persistent influx of calcium ions \[[@CR12]\]. _Furthermore,_ _the_ _Na+/Ca2+_ _ATP_ _driven_ pump _normally_ _used_ to _eliminate_ calcium fails, _also_ due to a lack of ATP \[[@CR13]\]. _The_ resultant high intracellular calcium triggers multiple _cascades_ that ultimately lead to mitochondrial dysfunction and cell death. _Furthermore,_ instead of producing ATP, _glial_ cells have been shown to release _ATP_ extracellularly \[[@CR11]\]. _Aside_ from rendering this unusable by mitochondria, ATP also stimulates the P2X7 _receptor,_ which again leads to significant calcium influx and ultimately _cell_ death \[[@CR13]\]. The other major mechanism of _cellular_ demise is via the formation of free radicals _facilitated_ by the reduction _of_ _iron_ from its _ferric_ (Fe3+) _to_ its _ferrous_ (Fe2+) form and the _initiation_ of inflammatory cascades _\[[@CR12]\]._ Cerebral blood flow in hypoxia {#Sec4} _==============================_ In _normoxic_ states, cerebral _blood_ _flow_ _is_ very _tightly_ controlled by the partial pressure of carbon dioxide (PaCO~2~). Any hypocapnic state will _result_ in vasoconstriction _and_ _reduction_ in regional _cerebral_ blood flow and a hypercapnic state _leads_ to _the_ reverse with _vasodilatation_ and an increase in cerebral blood _flow._ _Cerebral_ blood flow is somewhat _less_ _responsive_ to changes in PaO~2~, which has the opposite effect to carbon dioxide; a hypoxic state causing cerebral _vasodilatation_ with the aim of _improving_ _oxygen_ delivery and a hyperoxic state causing vasoconstriction \[[@CR14], [@CR15]\]. _In_ a hypoxic _state,_ whilst the vasodilatory response improves _flow,_ the _detection_ of hypoxia by peripheral chemoreceptors will _in_ turn _lead_ to an _increase_ in respiratory drive, _increasing_ arterial oxygen content. However, the consequence of this is also _an_ increase _in_ the clearance of _carbon_ dioxide, which would theoretically cause vasoconstriction _and_ reduced cerebral blood flow \[[@CR9]\]. It appears there is a threshold to which the hypoxic response predominates (and _the_ carbon dioxide one _attenuated)_ at _a_ PaO~2~ of _around_ _50--60_ _mmHg_ \[[@CR9], [@CR14]\]. Whilst the carbon dioxide mediated _vascular_ _response_ _is_ mediated via _a_ direct change in vessel wall pH \[[@CR15]\], the oxygen _response_ _appears_ to be mediated by the deoxygenated erythrocyte via a number _of_ _mechanisms;_ which include _release_ of ATP _and_ the subsequent actions _of_ endothelial _nitric_ oxide synthase on the vessel wall, reduction _of_ nitrite to nitric oxide and the activity of S-nitrosohaemoglobin \[[@CR9]\]. The cerebral vascular response _to_ hypoxia _is_ not uniform. _A_ study found that in _an_ induced isocapnic hypoxic state increases in cerebral blood flow were most prominent in basal ganglia _nuclei,_ the putamen, thalamus, nucleus _accumbens_ and pallidum \[[@CR16]\]. Studies of _blood_ flow in _individual_ vessels _have_ found that _flow_ in the _internal_ carotid artery is maintained during hypoxia and that vertebral artery flow is _increased_ _\[[@CR17]\]._ _This_ _had_ led to the hypothesis that blood flow is _increased_ in this region to preserve _vital_ _brainstem_ structures, or that possibly the _posterior_ circulation vasculature is less susceptible to the effects of carbon dioxide for similar reasons. Neurological effects _of_ hypoxia {#Sec5} =============================== The neurological consequences _of_ _hypoxia_ are dependent _upon_ the speed of _onset,_ the severity of hypoxia, and _the_ _level_ of _tissue_ perfusion. Rapid decreases in PaO~2~, as in _a_ cardiorespiratory arrest, can lead to permanent neurological damage _within_ minutes. However, lower, less abrupt _changes,_ can be tolerated if the _decrease_ in oxygen occurs _in_ a gradual manner, such as ascending _at_ altitude, where individuals can acclimatise and develop tolerance to lower oxygen partial _pressures_ or, (to _a_ _lesser_ degree), in chronic smokers. Initial clinical features include altered judgement, difficulty in completing complex tasks, _and_ impairment in _short_ term memory \[[@CR18], [@CR19]\], but in the longer term deficits can be more widespread _and_ span physical and neuropsychological domains. Seizures occur in up _to_ a _third_ of individuals within a day of _exposure_ to hypoxia, and are commonly partial complex or _myoclonic_ _in_ _nature._ Intractable _forms_ of _either_ of these types of seizure are _associated_ with a poor prognosis \[[@CR20]\]. Cognitive impairment domains include amnesia, visuospatial deficits, frontal lobe _symptoms,_ impairment of _executive_ _function,_ _and_ impairments in language \[[@CR21]\]. _These_ are covered _in_ more _detailed_ reviews on the subject \[[@CR22], _[@CR23]\]._ Involvement _of_ the basal ganglia, _a_ region particularly susceptible to hypoxic injury, can result _in_ _delayed_ Parkinsonism _in_ older subjects, dystonia mainly in younger people, choreo-athetosis, _and_ tremors \[[@CR20]\]. Varying _degrees_ of unilateral or bilateral motor _impairment_ may be observed depending on both the anatomical level and extent _of_ corticospinal _tract_ _involvement._ In _very_ rare cases, the syndrome of delayed post-hypoxic leukoencephalopathy may occur weeks after a _seemingly_ rapid recovery from _the_ original insult. This condition is characterised by rapid deterioration in cognition, emergence of extra-pyramidal _signs,_ and _loss_ of executive function as a results _of_ severe demyelination \[[@CR24]\]. The severity of leukoencephalopathy can be assessed _by_ magnetic _resonance_ imaging (MRI) \[[@CR20]\]. Electroencephalography, somatosensory evoked potentials _and_ MRI can provide _valuable_ information about the _severity_ of hypoxic injury, _but_ also aid in prognostication together with overall clinical state _\[[@CR25]\]._ Hypoxia in the context of a stroke {#Sec6} ================================== _There_ _is_ no specific _definition_ as to what _constitutes_ hypoxia in an acute _stroke,_ and it is therefore reasonable _to_ assume that normal _values_ for the _general_ population apply. Sulter and colleagues \[[@CR26]\] monitored 49 consecutive patients who presented with an _acute_ stroke within 12 h duration _using_ pulse _oximetry_ for 48 h. Patients _were_ _considered_ hypoxic and treated with supplemental _oxygen_ if saturations were below 96% for more than 5 min. This _occurred_ in 63% \[[@CR31]\] of patients, with 28 of those _returning_ to 'normal' oxygen saturations following administration of up to 5 L/min of oxygen. The remaining three required _much_ higher concentrations. Factors associated with _hypoxia_ in _this_ group were stroke _severity,_ presence of dysphagia, _and_ older age. Roffe et al. \[[@CR27]\] recruited _118_ patients (100 of whom had adequate measurements by pulse oximetry) and found that the mean _daytime_ awake SO~2~ was 94.5 ± 1.7% in _stroke_ patients _and_ 95.8 _±_ 1.7% in _healthy_ controls. _Nocturnal_ _saturations_ were reduced to 93.5 ± 1.9% in the _stroke_ group and 94.3 ± 1.9% _in_ _controls._ In the stroke group the average 4% oxygen desaturation index (ODI) (number of times per |
Raw deep sequencing cannot be provided because these data are derived from patient samples. Because of the Minimum Necessary Requirement of the HIPAA Privacy Rule these data may not be deposited into a public repository. A Supporting Information File accompanying the submission contains the de-identified results and interpretations of all clinical NGS tests included in our analysis next to the results of single-gene testing of the same specimen.
Introduction {#sec001}
============
The advance of next-generation sequencing (NGS) is a cornerstone of a recent development in molecular pathology, variably referred to as "personalized," "precision," or "individualized" medicine. Much of the focus of clinical NGS has been on oncology, as there are clear diagnostic, prognostic, and therapeutic implications for a multitude of genomic mutations in both solid and liquid malignancies. For example, in non-small cell lung cancers, activating mutations of the *EGFR* gene predict therapeutic response to tyrosine kinase inhibitors (TKI) such as erlotinib, gefitinib, and afatinib \[[@pone.0152851.ref001]--[@pone.0152851.ref003]\]. The KRAS protein acts downstream of EGFR, and thus mutations of the *KRAS* gene predict resistance to TKI \[[@pone.0152851.ref002],[@pone.0152851.ref004]--[@pone.0152851.ref006]\]. In metastatic melanoma, *BRAF* V600 mutations predict response to dabrafenib, vemurafenib, and trametinib \[[@pone.0152851.ref007]\]. Mutations of the *FLT3* and *NPM1* genes affect the prognosis of karyotypically normal acute myeloid leukemia and aid in the decision whether or not to pursue hematopoietic stem cell transplantation \[[@pone.0152851.ref008]\]. In addition, activating mutations of *JAK2*, which encodes a tyrosine kinase essential for cytokine and growth factor signaling, are found in a large proportion of patients with myeloproliferative neoplasms \[[@pone.0152851.ref009]\]. Ruxolitinib is a JAK1/JAK2 inhibitor approved for the treatment of myelofibrosis \[[@pone.0152851.ref010]\], and additional JAK2 inhibitors are in clinical development \[[@pone.0152851.ref011]\].
Detection of mutations in *EGFR*, *KRAS*, *BRAF*, *FLT3*, *NPM1*, and *JAK2* is most commonly accomplished by targeted tests that are designed to detect one or at most a small number of mutations in a single gene. However, NGS is gaining momentum as a complementary test for a number of reasons. Firstly, clinical trials for targeted cancer therapies rely on detection of mutations that are frequently not covered by existing targeted tests. In contrast to the laborious and lengthy process of validating and implementing a new molecular assay testing for one or a few mutations, NGS greatly simplifies the task of providing coverage of one of more additional mutations of interest. Secondly, targeted tests can provide misleading results. For example, the widely used FDA-approved cobas® *EGFR* Mutation Test only detects exon 19 deletion and L858R mutations, which together only comprise the mutations found in 85% of *EGFR*-mutated lung cancers \[[@pone.0152851.ref012]\]. In a significant proportion of cases, this test fails to identify therapeutically targetable mutations. Thirdly, targeted tests may fail to detect the very mutation they are designed to detect. Our group has recently reported a striking failure of two separate single-gene tests for the *BRAF* gene to detect a V600E mutation in a melanoma specimen \[[@pone.0152851.ref013]\]. The mutation was clearly demonstrated by concurrent NGS analysis. Finally, as has recently become apparent, tumors frequently harbor mutations that are therapeutically targetable but are not typically seen in that tumor type. Due to its massively parallel nature, NGS is very well-suited for detecting mutations in unexpected genes. One study reported a three-fold increased yield of clinically actionable mutations with NGS as compared to traditional molecular approaches targeting mutation hotspots \[[@pone.0152851.ref014]\].
Multiple recent studies have investigated the potential utility of NGS for detection of clinically actionable cancer mutations with encouraging results \[[@pone.0152851.ref014]--[@pone.0152851.ref025]\]. With the exception of one study \[[@pone.0152851.ref021]\], all demonstrated excellent performance of NGS on various platforms as measured by detection of point mutations and small insertions and deletions (indels). In many cases, additional potentially important variants were uncovered by NGS. All of the aforementioned studies were designed to validate clinical NGS pipelines that were not yet in clinical practice. As a consequence, they enrolled selected samples from previously examined specimens. With the exception of one commercially-sponsored study \[[@pone.0152851.ref014]\], in all cases a very limited number of samples was re-examined (range 13--61), often from only a single tissue type. While these important contributions confirm the potential usefulness of clinical NGS, they do not address the important question whether a well-validated NGS pipeline performs at an acceptable level in day-to-day clinical practice. Here we present a summative analysis of mutation results and quality control metrics obtained during the first year (March 2013 through March 2014) of clinical solid and liquid malignancy NGS carried out at the Center for Personalized Diagnostics at the University of Pennsylvania Health System. More than 900 specimens were submitted and processed during this time frame. We report that using our validated molecular and bioinformatics pipeline \[[@pone.0152851.ref026]\] with pre-determined tumor percentage and DNA quality cutoffs, we achieved excellent NGS data quality as determined by virtually perfect concordance between NGS and targeted single-gene tests for various genes in a large number of solid and liquid malignancy specimens.
Materials and Methods {#sec002}
=====================
Specimen Characteristics and Processing {#sec003}
---------------------------------------
Over the course of the study duration, 938 liquid and solid tumor specimens were submitted to the Center for Personalized Diagnostics ([Table 1](#pone.0152851.t001){ref-type="table"}). Specimens were eligible for NGS if they passed the tumor percentage, DNA quality, and DNA quantity thresholds that had been determined at the time of the validation of the NGS assay, which preceded the study period. Briefly, specimens with \<10% tumor were not eligible for NGS, because sequencing of samples with lower tumor percentages frequently yielded changes that were represented in fewer than five unique reads, making it difficult to distinguish true variants from sequencing artifacts. For similar reasons, DNA quality and quantity were judged to be insufficient, and the specimen was ineligible for NGS, if the DNA concentration was \<1 ng/μL; the DNA concentration was \<5 ng/μL with \>20% DNA degraded; the DNA concentration was \<50 ng/μL with \>45% DNA degraded; or DNA degradation was \>60%. Degraded DNA was defined as the proportion of DNA under 1000 bp in length.
10.1371/journal.pone.0152851.t001
###### Characteristics of Specimens by Tumor Site.
{#pone.0152851.t001g}
Tumor site Number of specimens submitted for NGS Specimens with DNA quality or quantity inadequate for NGS analysis Number of "shared" specimens (i.e., results are available from both NGS and targeted tests)
------------------ --------------------------------------- -------------------------------------------------------------------- ---------------------------------------------------------------------------------------------
Lung 196 13 (6.6%) 101 (51.5%)
Brain 174 9 (5.2%) 6 (3.4%)
Bone Marrow 161 0 95 (59.0%)
Lymph Node 70 8 (11.4%) 32 (45.7%)
Peripheral blood 56 0 29 (51.8%)
Liver 36 4 (11.1%) 5 (13.9%)
Skin 23 2 (8.7%) 5 (21.7%)
Other 222 24 (10.8%) 31 (14.0%)
Tumor site is not necessarily tissue of origin. Please note that in contrast to solid tumor specimens, all liquid specimens (bone marrow and peripheral blood) were adequate for NGS processing.
To determine the tumor percentage and volume of solid tumors, hematoxylin- and eosin-stained tissue specimens were evaluated by an anatomic pathologist, and the region with the highest tumor burden was marked. Genomic DNA was extracted from fresh bone marrow or peripheral blood using the Gentra Puregene Cell Kit (Qiagen, Netherlands). For formalin-fixed, paraffin-embedded (FFPE) specimens, tissues were macro-dissected from 5 μM or 10μM slides. Scrapings were dewaxed with Qiagen Deparaffinization Solution and purified with Gentra Puregene Tissue reagents following the manufacturer's protocol (Qiagen, Netherlands). DNA quantification was performed using the Qubit Broad Range assay following manufacturer's protocols (Life Technologies, CA). Agilent Genomic TapeScreens were used following manufacturer's protocols to assess the degree of DNA degradation (Agilent, CA).
Targeted Molecular Testing {#sec004}
--------------------------
### *EGFR*, *KRAS*, and *BRAF* Assays {#sec005}
The mutational status of *EGFR* exons 19 and 21 was determined using a laboratory-developed test (LDT) as previously described \[[@pone.0152851 | raw deep sequencing cannot be provided because these data are derived from patient samples. because of the minimum necessary requirement of the hipaa privacy rule these data may not be deposited into a public domain. a supporting information file accompanying the submission allows the de - identified results and interpretations of all clinical ngs tests included in our analysis next to the results of single - gene testing of the same specimen. introduction { # sec001 } = = = = = = = = = = = = immediate advance of next - generation sequencing ( ngs ) is a cornerstone of a recent development in molecular pathology, variably referred to as " personalized, " " precision, " or " individualized " medicine. much of the focus of clinical ngs also been on oncology, as there are clear diagnostic, prognostic, and therapeutic implications for a multitude of genomic mutations in both solid und liquid malignancies. for example, in non - small cell lung cancers, activating mutations of the * egfr * gene predict therapeutic response to tyrosine kinase inhibitors ( tki ) such as erlotinib, gefitinib, and afatinib \ [ [ @ pone. 0152851. ref001 ] - - [ @ pone. 0152851. ref003 ] \ ]. the kras protein acts downstream of egfr, and thus mutations of the * kras * gene predict resistance to tki \ [ [ @ pone. 0152851. ref002 ], [ @ pone. 0152851. ref004 ] - - [ @ pone. 0152851. ref006 ] \ ]. in metastatic melanoma, * braf * v600 mutations predict response to dabrafenib, vemurafenib, and ras \ [ [ @ pone. 0152851. ref007 ] \ ]. mutation of the * flt3 * genes * npm1 * genes affect the prognosis of severe normal acute myeloid leukemia and aid in the decision whether or not to pursue hematopoietic stem cell transplantation \ [ [ @ pone. 0152851. ref008 ] \ ]. in addition, activating mutations ‘ * jak2 *, which encodes a tyrosine kinase essential for cytokine and growth factor signaling, are found in a large proportion of patients with myeloproliferative neoplasms \ [ [ @ pone. 0152851. ref009 ] \ ]. ruxolitinib is a jak1 / jak2 inhibitor approved for the treatment of myelofibrosis \ [ [ @ pone. 0152851. ref010 ] \ ], and additional jak2 inhibitors are in clinical development \ [ [ @ pone. 0152851. ref011 ] \ ]. detection of mutations in * egfr *, * kras *, * braf *, * flt3 *, * npm1 *, and * jak2 * is most commonly accomplished by targeted tests that are designed to detect one or at most a small number of mutations in a single gene. however, ngs is gaining momentum as a complementary test for a number of reasons. firstly, clinical trials for targeted cancer therapies rely on detection of mutations that are frequently not covered by existing targeted tests. in contrast to the laborious and lengthy process of validating and implementing a new molecular assay testing for one or a few mutations, ngs greatly simplifies the task of providing coverage of one of more additional mutations of interest. secondly, targeted tests can provide misleading results. for example, the widely used fda - approved cobas® * egfr * mutation test only detects exon 19 deletion and l858r mutations, which together only comprise the mutations found in 85 % of * egfr * - mutated lung cancers \ [ [ @ pone. 0152851. ref012 ] \ ]. in a significant proportion of cases, this test fails to identify therapeutically targetable mutations. thirdly, targeted tests may fail to detect the very mutation they are designed to detect. our group has recently reported a striking failure of two separate single - gene tests for the * braf * gene to detect a v600e mutation in a melanoma specimen \ [ [ @ pone. 0152851. ref013 ] \ ]. the mutation was clearly demonstrated by concurrent ngs analysis. finally, as has recently become apparent, tumors frequently harbor mutations that are therapeutically targetable but are not typically seen in that tumor type. due to its massively parallel nature, ngs is very well - suited for detecting mutations in unexpected genes. one study reported a three - fold increased yield of clinically actionable mutations with ngs as compared to traditional molecular approaches targeting mutation hotspots \ [ [ @ pone. 0152851. ref014 ] \ ]. multiple recent studies have investigated the potential utility of ngs for detection of clinically actionable cancer mutations with encouraging results \ [ [ @ pone. 0152851. ref014 ] - - [ @ pone. 0152851. ref025 ] \ ]. with the exception of one study \ [ [ @ pone. 0152851. ref021 ] \ ], all demonstrated excellent performance of ngs on various platforms as measured by detection of point mutations and small insertions and deletions ( indels ). in many cases, additional potentially important variants were uncovered by ngs. all of the aforementioned studies were designed to validate clinical ngs pipelines that were not yet in clinical practice. as a consequence, they enrolled selected samples from previously examined specimens. with the exception of one commercially - sponsored study \ [ [ @ pone. 0152851. ref014 ] \ ], in all cases a very limited number of samples was re - examined ( range 13 - - 61 ), often from only a single tissue type. while these important contributions confirm the potential usefulness of clinical ngs, they do not address the important question whether a well - validated ngs pipeline performs at an acceptable level in day - to - day clinical practice. here we present a summative analysis of mutation results and quality control metrics obtained during the first year ( march 2013 through march 2014 ) of clinical solid and liquid malignancy ngs carried out at the center for personalized diagnostics at the university of pennsylvania health system. more than 900 specimens were submitted and processed during this time frame. we report that using our validated molecular and bioinformatics pipeline \ [ [ @ pone. 0152851. ref026 ] \ ] with pre - determined tumor percentage and dna quality cutoffs, we achieved excellent ngs data quality as determined by virtually perfect concordance between ngs and targeted single - gene tests for various genes in a large number of solid and liquid malignancy specimens. materials and methods { # sec002 } = = = = = = = = = = = = = = = = = = = = = specimen characteristics and processing { # sec003 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - over the course of the study duration, 938 liquid and solid tumor specimens were submitted to the center for personalized diagnostics ( [ table 1 ] ( # pone. 0152851. t001 ) { ref - type = " table " } ). specimens were eligible for ngs if they passed the tumor percentage, dna quality, and dna quantity thresholds that had been determined at the time of the validation of the ngs assay, which preceded the study period. briefly, specimens with \ < 10 % tumor were not eligible for ngs, because sequencing of samples with lower tumor percentages frequently yielded changes that were represented in fewer than five unique reads, making it difficult to distinguish true variants from sequencing artifacts. for similar reasons, dna quality and quantity were judged to be insufficient, and the specimen was ineligible for ngs, if the dna concentration was \ < 1 ng / μl ; the dna concentration was \ < 5 ng / μl with \ > 20 % dna degraded ; the dna concentration was \ < 50 ng / μl with \ > 45 % dna degraded ; or dna degradation was \ > 60 %. degraded dna was defined as the proportion of dna under 1000 bp in length. 10. 1371 / journal. pone. 0152851. t001 # # # # # # characteristics of specimens by tumor site.! [ ] ( pone. 0152851. t001 ) { # pone. 0152851. t001g } tumor site number of specimens submitted for ngs specimens with dna quality or quantity inadequate for ngs analysis number of " shared " specimens ( i. e., results are available from both ngs and targeted tests ) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - lung 196 13 ( 6. 6 % ) 101 ( 51. 5 % ) brain 174 9 ( 5. 2 % ) 6 ( 3. 4 % ) bone marrow 161 0 95 ( 59. 0 % ) lymph node 70 8 ( 11. 4 % ) 32 ( 45. 7 % ) peripheral blood 56 0 29 ( 51. 8 % ) liver 36 4 ( 11. 1 % ) 5 ( 13. 9 % ) skin 23 2 ( 8. 7 % ) 5 ( 21. 7 % ) other 222 24 ( 10. 8 % ) 31 ( 14. 0 % ) tumor site is not necessarily tissue of origin. please note that in contrast to solid tumor specimens, all liquid specimens ( bone marrow and peripheral blood ) were adequate for ngs processing. to determine the tumor percentage and volume of solid tumors, hematoxylin - and eosin - stained tissue specimens were evaluated by an anatomic pathologist, and the region with the highest tumor burden was marked. genomic dna was extracted from fresh bone marrow or peripheral blood using the gentra puregene cell kit ( qiagen, netherlands ). for formalin - fixed, paraffin - embedded ( ffpe ) specimens, tissues were macro - dissected from 5 μm or 10μm slides. scrapings were dewaxed with qiagen deparaffinization solution and purified with gentra puregene tissue reagents following the manufacturer ' s protocol ( qiagen, netherlands ). dna quantification was performed using the qubit broad range assay following manufacturer ' s protocols ( life technologies, ca ). agilent genomic tapescreens were used following manufacturer ' s protocols to assess the degree of dna degradation ( agilent, ca ). targeted molecular testing { # sec004 } - - - - - - - - - - - - - - - - - - - - - - - - - - # # # * egfr *, * kras *, and * braf * assays { # sec005 } the mutational status of * egfr * exons 19 and 21 was determined using a laboratory - developed test ( ldt ) as previously described \ [ [ @ pone. 0152851 | Raw deep sequencing cannot be provided because these data are derived from patient samples. Because of the Minimum Necessary Requirement of the HIPAA Privacy Rule these data may not be deposited into a public repository. A Supporting Information File accompanying the submission contains the de - identified results and interpretations of all clinical NGS tests included in our analysis next to the results of single - gene testing of the same specimen. Introduction {# sec001} = = = = = = = = = = = = The advance of next - generation sequencing (NGS) is a cornerstone of a recent development in molecular pathology, variably referred to as " personalized, " " precision, " or " individualized " medicine. Much of the focus of clinical NGS has been on oncology, as there are clear diagnostic, prognostic, and therapeutic implications for a multitude of genomic mutations in both solid and liquid malignancies. For example, in non - small cell lung cancers, activating mutations of the * EGFR * gene predict therapeutic response to tyrosine kinase iMhifitors (TKI) such as erlotinib, gefitinib, and afatinib \ [[ @ pone. 0152851. ref001] - - [@ pone. 0152851. ref003] \ ]. The KRAS protein acts downstream of EGFR, and thus mutations of the * KRAS * gene predict resistance to TKI \ [[ @ pone. 0152851. ref002 ], [@ pone. 0152851. ref004] - - [@ pone. 0152851. ref006] \ ]. In metastatic melanoma, * BRAF * V600 mutations predict response to dabrafenib, vemurafenib, and trametinib \ [[ @ pone. 0152851. ref007] \ ]. Mutations of the * FLT3 * and * NPM1 * genes affect the prognosis of karyotypically normal acute myeloid leukemia and aid in the decision whether or not to pursue hematopoietic stem cell transplantation \ [[ @ pone. 0152851. ref008] \ ]. In addition, activating mutations of * JAK2 *, which encodes a tyrosine kinase essential for cytokine and growth factor signaling, are found in a large proportion of patients with myeloproliferative neoplasms \ [[ @ pone. 0152851. ref009] \ ]. Ruxolitinib is a JAK1 / JAK2 inhibitor approved for the treatment of myelofibrosis \ [[ @ pone. 0152851. ref010] \ ], and additional JAK2 inhibitors are in clinical development \ [[ @ pone. 0152851. rrV011] \ ]. Detection of mutations in * EGFR *, * KRAS *, * BRAF *, * FLT3 *, * NPM1 *, and * JAK2 * is most commonly accomplished by targeted tests that are designed to detect one or at most a small number of mutations in a single gene. However, NGS is gaining momentum as a complementary test for a number of reasons. Firstly, clinical trials for targeted cancer therapies rely on detection of mutations that are frequently not covered by existing targeted tests. In contrast to the laborious and lengthy process of validating and implementing a new molecular assay testing for one or a few mutations, NGS greatly simplifies the task of providing coverage of one of more additional mutations of interest. Secondly, targeted tests can provide misleading results. For example, the widely used FDA - approved cobas ® * EGFR * Mutation Test only detects exon 19 deletion and L858R mutations, which together only comprise the mutations found in 85% of * EGFR * - mutated lung cancers \ [[ @ pone. 0152851. ref012] \ ]. In a significant proportion of cases, this test fails to identify therapeutically targetable mutations. Thirdly, targeted tests may fail to detect the very mutation they are designed to detect. Our group has recently reported a striking failure of two separate single - gene teaFs for the * BRAF * gene to detect a V600E mutation in a melanoma specimen \ [[ @ pone. 0152851. ref013] \ ]. The mutation was clearly demonstrated by concurrent NGS analysis. Finally, as has recently become apparent, tumors frequently harbor mutations that are therapeutically targetable but are not typically seen in that tumor type. Due to its massively parallel nature, NGS is very well - suited for detecting mutations in unexpected genes. One study reported a three - fold increased yield of clinically actionable mutations with NGS as compared to traditional molecular approaches targeting mutation hotspots \ [[ @ pone. 0152851. ref014] \ ]. Multiple recent studies have investigated the potential utility of NGS for detection of clinically actionable cancer mutations with encouraging results \ [[ @ pone. 01^2751. ref014] - - [@ pone. 0152851. ref025] \ ]. With the exception of one study \ [[ @ pone. 0152851. ref021] \ ], all demonstrated excellent performance of NGS on various platforms as measured by detection of point mutations and small insertions and deletions (indels ). In many cases, additional potentially important variants were uncovered by NGS. All of the aforementioned studies were designed to validate clinical NGS pipelines that were not yet in clinical practice. As a consequence, they enrolled selected samples from previously examined specimens. With the exception of one commercially - sponsored study \ [[ @ pone. 0152851. ref014] \ ], in all cases a very limited number of samples was re - examined (range 13 - - 61 ), often from only a single tissue type. While these important contributions confirm the potential usefulness of clinical NGS, they do not address the important question whether a well - validated NGS pipeline performs at an acceptable level in day - to - day clinical practice. Here we present a summative analysis of mutation results and quality control metrics obtained during the first year (March 2013 through March 2014) of clinical solid and liquid malignancy NGS carried out at the Center for Personalized Diagnostics at the University of Pennsylvania Health System. More than 900 specimens were submitted and processed during this time frame. We report that 7sinb our validated molecular and bioinformatics pipeline \ [[ @ pone. 0152851. ref026] \] with pre - determined tumor percentage and DNA quality cutoffs, we achieved excellent NGS data quality as determined by virtually perfect concordance between NGS and targeted single - gene tests for various genes in a large number of solid and liquid malignancy specimens. Materials and nethoSs {# sec002} = = = = = = = = = = = = = = = = = = = = = Specimen Characteristics and Processing {# sec003} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Over the course of the study duration, 938 liquid and solid tumor specimens were submitted to the Center for Personalized Diagnostics ([ Table 1] (# pone. 0152851. %00Q) {ref - type = " table "} ). Specimens were eligible for NGS if they passed the tumor percentage, DNA quality, and DNA quantity thresholds that had been determined at the time of the validation of the NGS assay, which preceded the study period. Briefly, specimens with \ <10% tumor were not eligible for NGS, because sequencing of samples with lower tumor percentages frequently yielded changes that were represented in fewer than five unique reads, making it difficult to distinguish true variants from sequencing artifacts. For similar reasons, DNA quality and quantity were judged to be insufficient, and the specimen was ineligible for NGS, if the DNA concentration was \ <1 ng / μL; the DNA concentration was \ <5 ng / μL with \> 20% DNA degraded; the DNA concentration was \ <50 ng / μL with \> 45% DNA degraded; or DNA degradation was \> 60% . Degraded DNA was defined as the proportion of DNA under 1000 bp in length. 10. 1371 / journal. pone. 0152851. t001 # # # # # # Characteristics of Specimens by Tumor Site. ! [] (pone. 0152851. t001) {# pone. 0152851. t001g} Tumor site Number of specimens submitted for NGS Specimens with DNA quality or quantity inadequate for NGS analysis Number of " shared " specimens (i. e. , results are available from both NGS and targeted tests) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Lung 196 13 (6. 6%) 101 (51. 5%) Brain 174 9 (5. 2%) 6 (3. 4%) Bone Marrow 161 0 95 (59. 0%) Lymph Node 70 8 (11. 4%) 32 (45. 7%) Peripheral blood 56 0 29 (51. 8%) Liver 36 4 (11. 1%) 5 (13. 9%) Skin 23 2 (8. 7%) 5 (21. 7%) Other 222 24 (10. 8%) 31 (14. 0%) Tumor site is not necessarily tissue of origin. Please note that in contrast to solid tuml% specimens, all liquid specimens (bone marrow and peripheral blood) were adequate for NGS processing. To determine the tumor percentage and volume of solid tumors, hematoxylin - and eosin - stained tissue specimens were evaluated by an anatomic pathologist, and the region with the highest tumor burden was marked. Genomic DNA was extracted from fresh bone marrow or peripheral blood using the Gentra Puregene Cell Kit (Qiagen, Netherlands ). For formalin - fixed, paraffin - embedded (FFPE) specimens, tissues were macro - dissected from 5 μM or 10μM slides. Scrapings were dewaxed with Qiagen Deparaffinization Solution and purified with Gentra Puregene Tissue reagents following the manufacturer ' s protocol (Qiagen, Netherlands ). DNA quantification was performed using the Qubit Broad Range aEsZy following manufacturer ' s protocols (Life Technologies, CA ). Agilent Genomic TapeScreens were used following manufacturer ' s protocols to assess the degree of DNA degradation (Agilent, CA ). Targeted Molecular Testing {# sec004} - - - - - - - - - - - - - - - - - - - - - - - - - - # # # * EGFR *, * KRAS *, and * BRAF * Assays {# sec005} The mutational status of * EGFR * exons 19 and 21 was determined using a laboratory - developed test (LDT) as pr4v&ously described \ [[ @ pone. 0152851 | Raw deep sequencing cannot be provided because these data are derived from patient samples. Because of the Minimum Necessary Requirement the HIPAA Privacy Rule these data may not be deposited into a public repository. A Information accompanying the submission contains the de-identified results and interpretations of clinical NGS tests included in our analysis next to the results of single-gene testing of the same specimen. Introduction {#sec001} ============ The advance of next-generation sequencing (NGS) is a cornerstone of a recent development molecular pathology, variably referred to as "personalized," "precision," or "individualized" medicine. Much of the focus of clinical NGS has been on oncology, as there are clear diagnostic, prognostic, and therapeutic implications for a multitude of genomic mutations in both solid and liquid malignancies. example, non-small cell lung activating mutations of the *EGFR* predict therapeutic response to inhibitors such as erlotinib, gefitinib, and \[[@pone.0152851.ref001]--[@pone.0152851.ref003]\]. The protein acts downstream of EGFR, and thus mutations the *KRAS* gene predict resistance to TKI \[[@pone.0152851.ref002],[@pone.0152851.ref004]--[@pone.0152851.ref006]\]. In metastatic melanoma, *BRAF* V600 mutations predict response to dabrafenib, vemurafenib, and trametinib \[[@pone.0152851.ref007]\]. Mutations of the *FLT3* *NPM1* genes affect the prognosis of karyotypically normal acute myeloid leukemia and aid in the decision or not to pursue hematopoietic stem cell transplantation \[[@pone.0152851.ref008]\]. In addition, activating mutations of *JAK2*, which encodes a tyrosine kinase essential for cytokine and growth factor signaling, found in a large proportion of patients with neoplasms \[[@pone.0152851.ref009]\]. Ruxolitinib is a JAK1/JAK2 inhibitor approved for treatment of myelofibrosis \[[@pone.0152851.ref010]\], and additional JAK2 inhibitors are in clinical development \[[@pone.0152851.ref011]\]. Detection of mutations in *EGFR*, *KRAS*, *BRAF*, *FLT3*, *NPM1*, *JAK2* is most commonly accomplished by targeted tests that are designed to detect one or at most a small number of mutations in a single gene. However, is momentum as a complementary test for a number of Firstly, clinical trials for targeted cancer on detection mutations that are frequently not covered by targeted tests. In contrast to the laborious and lengthy of validating and implementing a new molecular assay testing for one or a few mutations, NGS greatly simplifies task of providing of one more additional mutations of interest. Secondly, targeted tests can provide misleading For example, the widely used FDA-approved *EGFR* Mutation Test only detects exon 19 deletion and L858R mutations, which together only comprise the found in of *EGFR*-mutated lung cancers In a significant proportion of cases, this test fails to identify therapeutically targetable mutations. Thirdly, targeted tests may fail to detect very mutation are designed to Our group has recently reported a striking failure of two separate single-gene tests for the *BRAF* gene to detect a V600E mutation in a melanoma specimen \[[@pone.0152851.ref013]\]. The mutation was clearly demonstrated by NGS analysis. Finally, as has recently become apparent, tumors frequently harbor mutations that are therapeutically targetable but are not typically seen in that tumor type. to its massively parallel nature, NGS is very for detecting in unexpected genes. study reported a increased yield of actionable mutations NGS as compared traditional molecular approaches targeting mutation hotspots \[[@pone.0152851.ref014]\]. Multiple recent studies have investigated the utility of NGS for detection of clinically actionable cancer mutations with encouraging results \[[@pone.0152851.ref014]--[@pone.0152851.ref025]\]. the exception of one study \[[@pone.0152851.ref021]\], all demonstrated excellent of NGS on various platforms as measured by detection of point mutations small insertions and deletions (indels). cases, additional potentially important variants were uncovered by NGS. All of the aforementioned studies were to validate clinical pipelines that not in clinical practice. As a consequence, they enrolled selected samples from previously examined specimens. With exception of one commercially-sponsored study \[[@pone.0152851.ref014]\], in all cases a very limited number of was re-examined (range 13--61), often from only a single tissue type. While these important contributions confirm the potential usefulness clinical NGS, they do not address the important whether a well-validated NGS performs at acceptable level in day-to-day clinical practice. Here we present a analysis of results and quality control metrics obtained during the first 2013 through March 2014) of clinical solid and liquid malignancy carried out at the for Personalized Diagnostics at the University of Pennsylvania System. More 900 specimens were submitted and processed during this time frame. We that using validated molecular and bioinformatics pipeline with pre-determined tumor percentage and DNA quality cutoffs, we achieved excellent NGS data quality as determined by virtually perfect concordance between NGS and targeted single-gene for various genes in a number of solid and liquid malignancy specimens. Materials and {#sec002} Specimen Characteristics and Processing {#sec003} --------------------------------------- Over the course of study duration, 938 liquid and solid tumor specimens were submitted to the Center for Personalized ([Table 1](#pone.0152851.t001){ref-type="table"}). Specimens were NGS if they passed the tumor percentage, quality, and quantity thresholds that had been determined at the time of the validation of the NGS assay, which the study period. specimens with \<10% tumor were not eligible for NGS, sequencing of samples with lower tumor percentages frequently changes that were represented fewer than five unique reads, making it difficult to distinguish true variants from sequencing artifacts. similar reasons, DNA quality and quantity were judged to insufficient, and the specimen was for if the DNA concentration was \<1 ng/μL; the DNA concentration was \<5 ng/μL with \>20% DNA degraded; the DNA concentration was \<50 ng/μL with \>45% degraded; or DNA degradation was \>60%. Degraded DNA defined as the proportion of DNA under bp in 10.1371/journal.pone.0152851.t001 ###### Characteristics of Specimens by Tumor Site. {#pone.0152851.t001g} Tumor site Number of specimens submitted NGS Specimens with DNA quality or quantity inadequate for Number of "shared" specimens (i.e., results are available from both NGS and targeted tests) ------------------ --------------------------------------- -------------------------------------------------------------------- --------------------------------------------------------------------------------------------- Lung 196 13 (6.6%) 101 (51.5%) (5.2%) 6 (3.4%) Marrow 161 0 95 (59.0%) Lymph Node 70 8 (11.4%) 32 (45.7%) blood 56 29 (51.8%) 36 4 (11.1%) 5 (13.9%) 23 2 (8.7%) 5 (21.7%) Other 222 24 (10.8%) 31 (14.0%) site is not necessarily tissue of origin. note in contrast to solid tumor specimens, all liquid specimens (bone marrow and peripheral blood) were adequate for NGS processing. To determine the tumor percentage and volume of solid tumors, and eosin-stained tissue were evaluated by an pathologist, and the region with the highest tumor burden was marked. Genomic DNA was extracted from fresh bone marrow or peripheral blood using the Gentra Puregene Cell Kit (Qiagen, Netherlands). For formalin-fixed, paraffin-embedded (FFPE) specimens, tissues were macro-dissected 5 μM or 10μM slides. Scrapings dewaxed with Qiagen Deparaffinization Solution and purified with Gentra Puregene Tissue following the manufacturer's protocol (Qiagen, Netherlands). DNA quantification performed using the Qubit Broad Range assay following manufacturer's protocols (Life Technologies, Agilent Genomic TapeScreens were used manufacturer's to assess the degree of DNA degradation (Agilent, CA). Targeted Molecular Testing {#sec004} ### *EGFR*, *KRAS*, and *BRAF* Assays {#sec005} The mutational status *EGFR* exons 19 and 21 was determined using a laboratory-developed test (LDT) as previously described \[[@pone.0152851 | rAw deep SEquEnCiNg cANnoT Be PRovidEd BECAusE thEse DAtA aRe dERIvED FROm paTient sAMpLeS. beCausE of tHE miNImuM neCESsARy REquiREmenT OF THE hIPAa prIVACy Rule tHeSE DaTA MAy nOt BE DePOsited iNtO a PuBlIC rEposiTORy. A SupPortIng iNFoRmation FilE aCCOmpanyiNG ThE subMisSIon CoNTaiNS ThE dE-iDentiFIED ReSUlts aNd iNtERpretAtions of ALL clInical NGs TEsTS INcludeD in oUR ANALYSIS nEXt to The ResuLTs Of SINGle-gEnE tesTing OF The saMe specImen.
INTROdUCTIOn {#SEc001}
============
ThE aDVANcE of NEXT-gENERATIOn sEqUeNciNG (ngs) IS A cORnersTone oF a reCenT devElOpMenT IN moLecULAR PAtHoLOGY, VaRIablY refErReD To as "peRsOnAlIZed," "PrECIsiOn," OR "iNDIvIDUALIZeD" MEdiCiNe. muCh Of The fOCus OF cLINICAL nGS HaS bEeN On onCOlOgy, As tHeRE ArE CleaR DiAGnOsTiC, PRognOStic, anD THeRApeuTic ImPlICAtiOnS foR a mULtiTuDe of GenOmIC MUtATIONS In bOTH sOLid anD liQuiD maLIgNANCies. fOR eXAmpLe, in nON-sMAll Cell luNg CancErS, aCTIvaTiNg mutAtIoNS of tHe *egfR* geNe prEdict tHEraPeuTIC respoNsE to tYrosINE KiNASe inhiBItORs (tkI) sUCH AS eRLoTInIb, GEfItINib, AnD aFatiNib \[[@pone.0152851.ref001]--[@pOne.0152851.reF003]\]. ThE KRAs prOTEin Acts DowNstREam oF EGfR, anD ThUS MUTAtIoNs OF thE *krAs* Gene prEDicT rEsIsTAncE TO tki \[[@poNe.0152851.ref002],[@POnE.0152851.ReF004]--[@pONe.0152851.rEf006]\]. IN MetastAtIc mElANoMA, *BRAf* V600 mutaTions pREdIct resPOnSe To DabRAfEnIb, VemuraFEnIb, AnD traMETiNiB \[[@POne.0152851.Ref007]\]. MUTatIOns OF tHe *flt3* And *nPm1* GENes AfFEcT The PrognosIS Of kaRyoTYpICaLLy noRmAL ACUTE MyelOID leUKemIA And aID iN THe dEcISIoN whEtHEr or NOT tO PUrsUe HEMATopoIeTIc sTem CElL trAnSpLantaTIOn \[[@pOne.0152851.Ref008]\]. IN aDdItion, AcTIVATing mutaTIOns Of *JaK2*, whIcH enCOdES A TyrOsINE KINASe ESSENTIAL FOr cYtOKiNE aND groWth FaCtor SIGnaLInG, Are Found in a LArgE pROpoRtIOn OF pATieNTS WitH mYELoProliFeRATIVe neOplASmS \[[@pOnE.0152851.rEf009]\]. ruXOliTIniB Is A JAK1/Jak2 INhIbItOr approveD for The TREatmENT OF mYELoFiBrOSiS \[[@PoNe.0152851.ref010]\], ANd adDITioNal JaK2 InHIbItORS arE in ClINIcAL deveLopMEnt \[[@Pone.0152851.ReF011]\].
deTecTIoN oF MuTATioNs iN *eGFR*, *kras*, *BRaf*, *flT3*, *Npm1*, ANd *jak2* is mOsT COmMonLy AccompLisHeD bY tARGetEd tEsTS tHAt aRE DESIGNeD To dEtEct one oR At MOsT a sMaLl nuMBER oF mUTATioNS IN a SinGle GenE. HoWEveR, NGs IS GaINinG momENtum AS a complEMENTArY TesT fOr a NUMbER OF REAsons. FIrsTLy, ClinicaL TrIalS For TarGeted CanCER THERaPiEs rELY ON DEteCtIOn oF mUtaTIONS ThaT Are FReQUENtLY Not covERed by ExistINg TArgetED Tests. in CoNtrasT to tHE laboRIoUs aND lEngTHY prOCEss OF VaLidATING AND implEmEnTIng a NeW MoleCulAr aSSAY TestINg FOr ONe Or A Few MuTations, NgS GREAtlY SIMpLIfIES The task oF proVIDING COveRAge oF One oF more aDDiTIonAl MuTATIoNs Of INTEREST. SECONdlY, TaRgEted teSTs caN ProVIde mISleaDinG REsULTs. fOr exAmplE, tHe WIdElY usED fda-ApPRoVed cOBaS® *EGFr* mUTAtION TESt onLY deTECTs ExOn 19 DELEtIOn anD L858r muTations, Which toGEthER OnlY comPrise tHe mutATiONs FouNd IN 85% OF *EgfR*-muTAtEd lung CaNCers \[[@PONe.0152851.rEF012]\]. In a SIGnificANt PropoRTioN Of cAseS, thIs teST faILs To iDENTifY thERapeUtICALly tARgEtABlE MUtatIOns. ThiRdly, TArgetED TesTS mAY FAiL To DETECt THE vErY muTATION thEy are deSiGNED to detECt. Our GRoUP haS REcEntLY rEPOrtEd A STRiKInG failUrE Of TwO SeparAte SInGle-gENe tests for tHe *BRaF* geNe tO detect a V600E MUTation IN A MeLAnoMa sPEcimeN \[[@POne.0152851.rEf013]\]. tHE MUtaTION wAs CLeaRlY DemOnstratEd bY cONcurrENt ngs aNALYsis. finALlY, AS has reCeNtlY BecomE aPPArent, tUmoRS freQuENTLY hArbOr MuTATiONS THaT aRE THeRApEUtIcAllY TARgEtAble bUT are NoT TYpIcaLlY SEEN iN THAt Tumor tyPE. dUe tO iTs MAssiVElY PARalLel naTUre, Ngs IS very WELL-suIteD FOR DeTECTINg mUTaTiOnS iN UneXpected gENEs. one stuDY rEpORTeD A thrEE-folD iNcreASed YieLD of cLINiCally AcTIOnabLe muTatIOnS WITh Ngs as cOmpared to tRAdiTioNaL MOLEcuLaR aPpROAchES taRgEtinG MutatIon hotSPOtS \[[@poNE.0152851.rEF014]\].
MUlTiPlE rECeNT STUdieS have InveStIgAtED THe poTentiAl UTilITy OF NGs fOR dEtEctIoN OF cLINiCALLy aCTIONaBle cANcEr mUTATions WIth eNCouragiNg ReSuLTS \[[@pOne.0152851.Ref014]--[@ponE.0152851.REF025]\]. with tHe EXCeptiOn of oNE sTuDY \[[@PONe.0152851.REf021]\], ALl DEmoNSTrATEd exCElLeNT PerfOrManCE OF ngs on VARious PLATfOrMS as MEAsUreD bY dETEcTiOn oF POINT mUTAtiONs AnD sMAll INsertIONS AnD dELetionS (iNdELs). in MaNY caseS, aDdiTIOnal POtentiALly IMPoRtAnt VArIaNtS Were uncoVEreD By Ngs. AlL Of THE afOreMeNtiONED stuDiEs werE DeSIGneD To VALIdatE CliNICal ngS PipeLINes tHAT WeRe NOt yEt iN CLiNicaL pRaCtICe. as a conSequENCE, they ENROLlEd seLEcteD SAMPLeS frOM PrevIoUsly EXAmINEd sPeCIMeNS. wIth THe ExCEptIon of oNE comMeRciAlLy-spONSOrED sTUdy \[[@pone.0152851.REf014]\], in aLl caseS a vERY lIMited NUMber OF sAmPlES WAs re-eXAMIneD (RANGE 13--61), OFtEN fRom ONly a SiNGLe TIssUE tYpE. WHiLE tHESE impOrTAnt ContRiBuTIonS coNfIrm The POteNTial usEFulNess of ClIniCaL ngs, theY dO noT address tHE IMPorTaNT QuEStION whethEr a WEll-Validated nGS PIPELINe PeRfOrMs AT an accePtablE lEvEL in Day-tO-dAy CLInicAL PraCTICE. HERe we PResENT A sUMMatIvE AnALySIS Of Mutation RESUlts AnD qUALitY CONtroL MetrICs obTaINEd DUring ThE FIRSt YeAR (MaRCH 2013 thROUgH MarCH 2014) OF cLINiCal soLid And lIquID MAliGNaNcY Ngs carRiED ouT AT THE cENtEr fOR pERsoNAlIZEd dIagNOSTIcS aT tHe uniVerSity of PeNNSYLvaNIA heaLTH SYsteM. MorE Than 900 spECiMenS wEre SUBmItTED AnD pROcEsSEd DuRInG tHiS TiME FRAMe. wE RepoRT THaT usinG oUr VALidateD MoLecULar And BIoInFORmaTiCS PIPelinE \[[@PoNE.0152851.reF026]\] WitH pRE-detERmiNed tUMoR PeRCENTagE AND DnA qualitY cUToFfS, We acHieVed ExCelLEnT nGs DAtA QUaLItY AS dETeRMiNed By vIRTUAlLy pErFect cONcORDaNCe bETwEEN ngS anD tArGeTed SINGLe-Gene testS fOr VArIouS geNES In A lArGE NUmBer OF sOLID ANd Liquid maLigNANcy SPECiMeNs.
MATERialS aNd methODs {#SeC002}
=====================
SPEcimEn cHarAcTeristIcs anD prOcesSiNG {#Sec003}
---------------------------------------
OvER THe CoUrse oF tHE STUDY dUraTIoN, 938 LiQUId aNd SoLId TUmoR SpecIMeNs WeRe SubMITTED To THE CenTEr foR perSONalIZeD DiagNoStICS ([tAbLE 1](#pONe.0152851.T001){rEf-TYPE="TaBle"}). SPecIMeNS WeRe EliGiBlE FOr ngs IF thEy paSsed tHe TuMOR PErcentAGe, dNA QUalIty, aND DNA quAnTity thresHOLdS ThAT hAd BEen DetERmiNEd At THe tImE of tHe VAliDaTiON Of THe NGs aSsay, WhIcH PreceDeD The StUdY PEriOd. BRieFlY, sPeciMeNS wITh \<10% tUMOR WERE NOt eLiGIBlE FOR NGS, becAUSE SEquEncinG oF SaMpleS with lowER TuMOr PErcEntaGes FREqUeNTly YIEldED chAnges ThaT WerE RePrESEnTEd IN fEWeR ThAn fiVE uniQuE reAdS, mAKING it diFFIcUlt TO distiNguiSh trUe VariANTS fROM sEQUencING arTiFaCTS. FOR SiMILAR REASoNs, DNa qUAlIty aNd quAntITy WeRe jUdged to be INSUFfIcient, aNd THE sPeCImen was InelIgibLE foR ngs, if The dnA COnceNtrAtiOn wAS \<1 ng/ΜL; THE Dna conCENtrATiOn was \<5 Ng/Μl wITH \>20% DNa DegRaDEd; tHe dna COnCENTRatION wAs \<50 ng/ΜL witH \>45% dNA DegRADEd; Or DnA DeGrAdAtiOn waS \>60%. DegradED Dna Was defINed AS ThE proPOrTiOn OF DnA UNder 1000 bP In LeNGTh.
10.1371/JOuRnAl.poNE.0152851.t001
###### chARaCtERIStICS of SPecImeNS BY tuMor SIte.
{#PonE.0152851.t001G}
TumOR SIte NuMBer OF sPEciMEnS sUBMItteD FOR ngS SpEcImeNs WitH DNA Quality Or qUaNtitY InadEquATE fOr Ngs ANaLYsiS NUmBEr Of "SHaRED" SPeCiMens (I.E., REsULTs ArE aVaiLABlE fRom BOTh NGs aND TargeTEd tesTs)
------------------ --------------------------------------- -------------------------------------------------------------------- ---------------------------------------------------------------------------------------------
lUNG 196 13 (6.6%) 101 (51.5%)
BraiN 174 9 (5.2%) 6 (3.4%)
BonE MarROw 161 0 95 (59.0%)
Lymph NODe 70 8 (11.4%) 32 (45.7%)
periPHERal BlooD 56 0 29 (51.8%)
LIVer 36 4 (11.1%) 5 (13.9%)
SkIN 23 2 (8.7%) 5 (21.7%)
oTHEr 222 24 (10.8%) 31 (14.0%)
tUmOR siTe is nOt nECESSaRiLY tisSue oF oRIgIN. PLEAse nOtE THAt In ConTrAsT tO SoLID tUMor SpeCImenS, AlL LiQUid spEciMeNs (boNe mArrOW and peRIphERaL BlOOd) weRE AdequaTe FOr NgS ProCEssIng.
to DeTERMiNE the tUmOR peRcENtAge AnD vOlUMe Of sOlID TUmoRs, HEMaTOxYLin- and EoSIN-StAiNed tIssUE spEciMEns wERE eVaLUaTed By an AnATomIC pAtHOLogist, ANd thE regioN WIth tHe hIghESt tUmor bURDen Was maRkeD. gENoMic DNa wAS eXtraCTed FROM Fresh BOne maRROW or PeriPheraL blOOd uSINg THe gENTra puReGenE ceLl KIT (qiAgeN, NethErlANDs). For foRMaLin-FIXED, paraFFin-emBeDDEd (fFpE) SPeCimens, tiSsuES WeRe maCro-dissecTeD frOM 5 Μm or 10μm sliDES. sCrApINGS Were DEwaxed wItH qIaGEn dePaRAFFiniZatiON sOLUTion AnD PuriFIed wiTh GEntra PUregenE Tissue reAgENTs folloWIng The MaNuFactUrer'S proTOcoL (QIAgEN, NEtherLaNdS). DNa QUanTiFIcATiOn waS PerFOrMeD USinG THE QUbIT brOaD RAnGe AsSAY FollowINg MAnuFacturer'S PRoTOCOlS (LIfE tEcHnOlOgiES, cA). AGILeNT gENoMiC TApeSCrEens were used fOLLOwIng manUFACTuRER'S PROtocOlS to AsSeSs thE dEGREE oF dna DegRaDatION (AgIlENt, CA).
tARGeTeD molEcuLAR tESTIng {#SEC004}
--------------------------
### *egfR*, *KRAS*, ANd *BrAF* AsSaYs {#sEc005}
THE MUtaTionAL StAtUS oF *egfr* EXoNS 19 aND 21 was dETERMINeD UsiNg A LaBoratORY-DevEloPEd Test (lDT) AS pRevIoUSly DESCriBed \[[@PONE.0152851 | Raw deep sequencing cannot be provided because these data are derived from patient samples. Because of the Minimum Necessary Requirement of the HIPAA Privacy Rule these data may not bedeposited into a public repository. A Supporting Information File accompanying thesubmission contains the de-identified resultsand interpretations of all clinical NGStests included in our analysis next tothe results of single-gene testingof the samespecimen.Introduction {#sec001} ============The advance of next-generation sequencing (NGS) is a cornerstone of a recent development in molecular pathology, variably referred to as "personalized," "precision," or "individualized" medicine. Much ofthe focus of clinical NGS has beenon oncology, as there are clear diagnostic, prognostic, and therapeutic implicationsfor a multitude of genomic mutations in both solidand liquid malignancies. For example, in non-small cell lung cancers, activating mutations of the *EGFR* gene predict therapeutic response to tyrosine kinase inhibitors (TKI) such as erlotinib, gefitinib, and afatinib \[[@pone.0152851.ref001]--[@pone.0152851.ref003]\]. The KRAS protein acts downstream of EGFR, and thus mutations of the *KRAS* gene predict resistance toTKI \[[@pone.0152851.ref002],[@pone.0152851.ref004]--[@pone.0152851.ref006]\].In metastatic melanoma, *BRAF* V600 mutations predict response to dabrafenib, vemurafenib, and trametinib \[[@pone.0152851.ref007]\]. Mutations of the *FLT3* and *NPM1* genes affect the prognosis ofkaryotypically normal acute myeloid leukemia and aid in the decision whether or not to pursue hematopoietic stem celltransplantation\[[@pone.0152851.ref008]\]. In addition, activating mutations of *JAK2*, which encodesatyrosine kinase essential forcytokine andgrowth factor signaling, are found in a large proportion of patients with myeloproliferative neoplasms \[[@pone.0152851.ref009]\]. Ruxolitinib is a JAK1/JAK2inhibitor approved for the treatment of myelofibrosis \[[@pone.0152851.ref010]\], and additional JAK2 inhibitors are in clinicaldevelopment\[[@pone.0152851.ref011]\]. Detection ofmutations in*EGFR*, *KRAS*, *BRAF*, *FLT3*, *NPM1*, and *JAK2* ismost commonly accomplished by targeted tests that are designed todetect oneor at most a small number of mutations in a single gene. However,NGSis gaining momentum as a complementary test for a number of reasons. Firstly, clinical trials for targeted cancer therapies rely on detection of mutations thatarefrequently not covered by existing targeted tests. Incontrast to the laborious andlengthy process of validating andimplementinga new molecular assay testing for one or a few mutations, NGS greatlysimplifies the task of providing coverage of oneof more additional mutations of interest. Secondly, targeted tests can providemisleading results. For example, the widely used FDA-approved cobas® *EGFR* MutationTest only detects exon 19deletion and L858Rmutations,which together only comprise the mutations found in 85% of *EGFR*-mutated lung cancers \[[@pone.0152851.ref012]\]. In a significant proportionofcases, this test fails to identify therapeuticallytargetablemutations.Thirdly,targeted tests may fail to detect the very mutation theyaredesignedto detect. Our group has recently reported a striking failure of two separatesingle-gene tests for the*BRAF* gene todetect a V600E mutation in a melanoma specimen \[[@pone.0152851.ref013]\].The mutation was clearly demonstrated by concurrent NGS analysis. Finally, as hasrecentlybecome apparent, tumors frequently harbor mutations that are therapeutically targetable but arenot typically seen inthat tumor type. Due to its massively parallel nature, NGS isvery well-suited for detecting mutationsin unexpected genes. Onestudy reported a three-fold increased yield of clinicallyactionablemutations with NGS as compared to traditional molecular approaches targetingmutation hotspots\[[@pone.0152851.ref014]\]. Multiple recentstudies have investigated the potential utilityof NGS for detection ofclinically actionable cancer mutations with encouraging results \[[@pone.0152851.ref014]--[@pone.0152851.ref025]\]. With theexception of one study\[[@pone.0152851.ref021]\], all demonstrated excellent performance ofNGSon various platforms as measured by detection of point mutationsand small insertions and deletions (indels). In many cases, additional potentially important variants were uncovered by NGS. All of the aforementioned studies were designed tovalidate clinical NGS pipelines that were not yet in clinical practice. As a consequence, they enrolled selected samples from previously examined specimens.Withthe exception of one commercially-sponsored study\[[@pone.0152851.ref014]\], in all cases a very limited number of samples was re-examined (range 13--61), often from onlya single tissue type. While these important contributions confirm the potential usefulness of clinical NGS, theydo not address the important question whether a well-validated NGS pipeline performs atan acceptable level in day-to-day clinical practice. Herewe present a summative analysis of mutation resultsandquality control metrics obtained during the first year (March 2013 through March 2014) of clinical solid and liquid malignancy NGS carried out at theCenter for Personalized Diagnostics at the University of PennsylvaniaHealth System. More than 900 specimens were submitted and processed during thistimeframe. We reportthat using our validatedmolecularand bioinformaticspipeline \[[@pone.0152851.ref026]\] withpre-determined tumor percentage and DNA quality cutoffs, weachieved excellent NGSdata quality as determined byvirtually perfect concordance betweenNGS and targeted single-gene tests for various genes in a large number of solid and liquid malignancy specimens. Materials andMethods {#sec002} ===================== SpecimenCharacteristics and Processing {#sec003} --------------------------------------- Over the course of the study duration, 938 liquid and solid tumor specimens were submitted tothe Center for Personalized Diagnostics([Table 1](#pone.0152851.t001){ref-type="table"}). Specimens wereeligible for NGS if they passed the tumor percentage, DNA quality, and DNA quantity thresholds thathad been determined atthetime of thevalidation of the NGS assay, which preceded the studyperiod. Briefly,specimens with \<10%tumor were not eligible for NGS, because sequencing of samples with lower tumor percentages frequently yielded changes that wererepresented in fewer than five unique reads, making it difficult todistinguishtrue variants from sequencing artifacts. Forsimilar reasons, DNA qualityand quantity were judged to be insufficient, and the specimen was ineligible for NGS, if the DNA concentration was \<1 ng/μL; the DNA concentration was \<5 ng/μL with \>20% DNA degraded;the DNA concentrationwas\<50 ng/μL with \>45% DNA degraded; or DNA degradationwas \>60%.Degraded DNA was defined as the proportion of DNA under 1000 bp in length. 10.1371/journal.pone.0152851.t001 ###### Characteristics of Specimensby Tumor Site. {#pone.0152851.t001g} Tumor site Number of specimens submitted for NGS Specimens with DNA quality or quantity inadequate for NGS analysis Number of "shared" specimens (i.e., results are available from both NGS and targeted tests) ------------------ -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Lung 196 13 (6.6%) 101 (51.5%)Brain 174 9 (5.2%) 6 (3.4%)Bone Marrow 161 0 95 (59.0%) Lymph Node 708 (11.4%) 32 (45.7%) Peripheralblood 56 0 29 (51.8%) Liver 36 4 (11.1%) 5(13.9%) Skin 23 2 (8.7%) 5 (21.7%) Other 22224 (10.8%) 31 (14.0%) Tumor site is not necessarily tissue of origin. Please note that in contrast to solidtumor specimens, allliquid specimens (bone marrow and peripheral blood) were adequate forNGS processing.To determine the tumor percentage and volume ofsolid tumors, hematoxylin-and eosin-stained tissue specimens were evaluated by an anatomicpathologist, and the region with the highest tumor burden wasmarked.Genomic DNA wasextractedfrom fresh bone marrow or peripheral blood using theGentra Puregene Cell Kit (Qiagen, Netherlands).For formalin-fixed, paraffin-embedded(FFPE) specimens, tissues were macro-dissected from 5 μM or 10μM slides. Scrapings were dewaxed with Qiagen Deparaffinization Solution and purified withGentra Puregene Tissue reagents followingthe manufacturer's protocol (Qiagen, Netherlands). DNA quantification wasperformedusing the Qubit BroadRange assay following manufacturer's protocols(Life Technologies, CA). Agilent Genomic TapeScreens were used following manufacturer's protocols toassessthe degree of DNA degradation (Agilent, CA). Targeted Molecular Testing {#sec004} -------------------------- ### *EGFR*, *KRAS*, and *BRAF* Assays {#sec005} The mutational statusof *EGFR* exons 19 and 21 wasdetermined using a laboratory-developed test (LDT) as previously described \[[@pone.0152851 | _Raw_ deep sequencing cannot be provided _because_ these data are _derived_ from patient samples. Because of the Minimum _Necessary_ Requirement of the HIPAA Privacy Rule these data _may_ not be deposited into _a_ public repository. A _Supporting_ Information File accompanying the _submission_ contains the _de-identified_ results and interpretations of all _clinical_ NGS tests included in our analysis next _to_ the results of _single-gene_ testing of the same specimen. Introduction _{#sec001}_ ============ _The_ advance of _next-generation_ sequencing (NGS) is _a_ cornerstone of _a_ recent _development_ in molecular _pathology,_ variably referred to as _"personalized,"_ "precision," or "individualized" medicine. Much of _the_ focus of clinical NGS has been on oncology, as _there_ are clear diagnostic, prognostic, and therapeutic implications for a multitude of _genomic_ mutations in both solid and liquid malignancies. For example, in _non-small_ cell lung cancers, _activating_ mutations of the *EGFR* gene predict therapeutic response _to_ tyrosine kinase inhibitors (TKI) such as _erlotinib,_ gefitinib, and _afatinib_ \[[@pone.0152851.ref001]--[@pone.0152851.ref003]\]. _The_ _KRAS_ protein acts downstream of _EGFR,_ and thus mutations of _the_ *KRAS* gene predict resistance to TKI \[[@pone.0152851.ref002],[@pone.0152851.ref004]--[@pone.0152851.ref006]\]. In metastatic melanoma, *BRAF* V600 mutations predict response to dabrafenib, _vemurafenib,_ and trametinib \[[@pone.0152851.ref007]\]. Mutations of the _*FLT3*_ and *NPM1* genes affect _the_ prognosis of karyotypically normal acute myeloid leukemia and _aid_ in the decision whether or _not_ to _pursue_ hematopoietic stem cell _transplantation_ \[[@pone.0152851.ref008]\]. In addition, activating mutations _of_ *JAK2*, which encodes a _tyrosine_ kinase essential for cytokine and growth factor _signaling,_ _are_ _found_ in a large proportion of patients with myeloproliferative neoplasms \[[@pone.0152851.ref009]\]. _Ruxolitinib_ _is_ a _JAK1/JAK2_ _inhibitor_ _approved_ for the treatment of _myelofibrosis_ \[[@pone.0152851.ref010]\], and additional JAK2 inhibitors are in clinical development \[[@pone.0152851.ref011]\]. Detection _of_ _mutations_ _in_ *EGFR*, *KRAS*, *BRAF*, *FLT3*, _*NPM1*,_ and _*JAK2*_ is most commonly _accomplished_ by targeted tests that are _designed_ to detect _one_ or at most _a_ small _number_ _of_ mutations in _a_ single gene. However, NGS _is_ _gaining_ _momentum_ _as_ _a_ _complementary_ _test_ _for_ a number of reasons. Firstly, clinical trials for targeted cancer _therapies_ rely on _detection_ of mutations _that_ are frequently not covered by existing _targeted_ _tests._ _In_ contrast _to_ the _laborious_ _and_ lengthy process of validating and implementing a _new_ molecular assay testing for one or a few mutations, _NGS_ greatly simplifies the task of providing _coverage_ of one of _more_ _additional_ mutations of interest. Secondly, targeted _tests_ can provide misleading results. _For_ example, _the_ _widely_ used FDA-approved cobas® _*EGFR*_ Mutation Test only detects exon 19 deletion and L858R mutations, which together _only_ comprise the mutations found in 85% _of_ *EGFR*-mutated lung cancers \[[@pone.0152851.ref012]\]. In _a_ significant proportion of cases, this test fails to _identify_ _therapeutically_ targetable mutations. Thirdly, targeted _tests_ may fail to _detect_ _the_ very _mutation_ they are designed _to_ detect. Our group has _recently_ reported a _striking_ _failure_ _of_ two separate single-gene tests for the *BRAF* gene to _detect_ _a_ V600E _mutation_ in _a_ _melanoma_ specimen \[[@pone.0152851.ref013]\]. The mutation was clearly demonstrated by concurrent NGS analysis. _Finally,_ as has _recently_ _become_ apparent, tumors frequently harbor mutations that are therapeutically targetable but are not typically seen in that tumor _type._ Due to its _massively_ _parallel_ _nature,_ _NGS_ is very _well-suited_ for _detecting_ mutations in unexpected genes. One study reported a three-fold increased _yield_ of _clinically_ actionable mutations with NGS as compared to traditional molecular approaches targeting _mutation_ hotspots \[[@pone.0152851.ref014]\]. Multiple _recent_ _studies_ have investigated the _potential_ utility of NGS for detection of clinically actionable cancer mutations with encouraging results \[[@pone.0152851.ref014]--[@pone.0152851.ref025]\]. With _the_ exception _of_ one study \[[@pone.0152851.ref021]\], all demonstrated _excellent_ performance _of_ _NGS_ on various platforms as measured by _detection_ of point mutations and small insertions _and_ deletions (indels). In many cases, additional potentially important variants _were_ uncovered by NGS. All of the aforementioned studies were designed to _validate_ clinical NGS pipelines that _were_ not yet in clinical practice. As a consequence, _they_ enrolled selected samples from _previously_ _examined_ _specimens._ With the exception of one commercially-sponsored study \[[@pone.0152851.ref014]\], in all cases _a_ very limited number of samples was re-examined (range 13--61), often _from_ only a single _tissue_ _type._ While these important contributions confirm the _potential_ usefulness of clinical _NGS,_ they do not address the important _question_ whether a well-validated NGS _pipeline_ performs at an _acceptable_ level in _day-to-day_ clinical practice. Here we present a summative analysis of mutation results and quality control metrics obtained during the first year (March 2013 through _March_ 2014) of _clinical_ solid and liquid malignancy NGS _carried_ _out_ at the Center for _Personalized_ Diagnostics at _the_ _University_ _of_ Pennsylvania _Health_ System. More than _900_ _specimens_ were submitted _and_ processed _during_ this time frame. We _report_ _that_ using our validated molecular _and_ _bioinformatics_ pipeline _\[[@pone.0152851.ref026]\]_ with pre-determined tumor percentage and DNA quality _cutoffs,_ we achieved excellent NGS data quality as determined by virtually perfect concordance _between_ NGS and _targeted_ _single-gene_ tests for various _genes_ in _a_ large number of solid _and_ liquid _malignancy_ specimens. Materials _and_ _Methods_ {#sec002} ===================== Specimen Characteristics and Processing _{#sec003}_ _---------------------------------------_ Over _the_ course of the _study_ duration, 938 liquid and solid tumor specimens were submitted to _the_ Center for Personalized Diagnostics ([Table _1](#pone.0152851.t001){ref-type="table"})._ _Specimens_ _were_ eligible for NGS if they passed the tumor _percentage,_ DNA _quality,_ and DNA quantity thresholds that _had_ been determined at the time _of_ the _validation_ of the NGS assay, which preceded _the_ study _period._ Briefly, specimens with _\<10%_ tumor were not eligible for NGS, because sequencing of samples _with_ lower tumor percentages _frequently_ yielded changes _that_ were represented in fewer than _five_ unique reads, making it difficult to _distinguish_ true _variants_ from sequencing artifacts. For similar reasons, DNA quality and quantity were judged to be insufficient, and the specimen was ineligible for NGS, if the DNA concentration was \<1 _ng/μL;_ the DNA concentration was \<5 ng/μL with \>20% DNA _degraded;_ the _DNA_ concentration _was_ \<50 ng/μL with \>45% _DNA_ _degraded;_ or DNA degradation was \>60%. _Degraded_ DNA was defined _as_ the proportion of DNA under 1000 bp in length. 10.1371/journal.pone.0152851.t001 ###### _Characteristics_ of Specimens by Tumor Site. {#pone.0152851.t001g} Tumor site Number of specimens _submitted_ for NGS _Specimens_ with DNA quality or quantity inadequate _for_ NGS _analysis_ Number of "shared" specimens (i.e., results are available from both NGS and targeted tests) ------------------ _---------------------------------------_ -------------------------------------------------------------------- --------------------------------------------------------------------------------------------- Lung _196_ 13 (6.6%) 101 (51.5%) Brain 174 9 (5.2%) 6 (3.4%) Bone Marrow 161 _0_ _95_ (59.0%) Lymph Node _70_ 8 (11.4%) 32 (45.7%) Peripheral blood 56 0 29 (51.8%) Liver _36_ 4 (11.1%) 5 (13.9%) _Skin_ _23_ 2 (8.7%) 5 (21.7%) Other 222 24 _(10.8%)_ 31 _(14.0%)_ _Tumor_ _site_ is _not_ necessarily tissue of origin. Please note _that_ in _contrast_ to _solid_ _tumor_ specimens, _all_ liquid _specimens_ _(bone_ marrow and peripheral blood) were _adequate_ for NGS processing. To determine the _tumor_ percentage and _volume_ of solid _tumors,_ hematoxylin- and _eosin-stained_ _tissue_ specimens were _evaluated_ by an _anatomic_ pathologist, and the region with the _highest_ tumor burden was marked. Genomic DNA was _extracted_ from fresh bone marrow or peripheral blood _using_ the Gentra Puregene Cell Kit (Qiagen, _Netherlands)._ For formalin-fixed, paraffin-embedded _(FFPE)_ specimens, tissues were macro-dissected from 5 μM or 10μM slides. Scrapings were dewaxed _with_ _Qiagen_ _Deparaffinization_ _Solution_ and purified with Gentra Puregene Tissue reagents following the _manufacturer's_ protocol _(Qiagen,_ _Netherlands)._ DNA _quantification_ was performed _using_ _the_ Qubit _Broad_ Range assay following manufacturer's protocols (Life Technologies, CA). _Agilent_ Genomic TapeScreens were used _following_ manufacturer's protocols _to_ assess the degree _of_ DNA degradation (Agilent, _CA)._ Targeted Molecular Testing {#sec004} -------------------------- _###_ *EGFR*, *KRAS*, and *BRAF* Assays {#sec005} The mutational _status_ of _*EGFR*_ exons 19 and 21 was determined _using_ a laboratory-developed test (LDT) as _previously_ described \[[@pone.0152851 |
Introduction {#s1}
============
All organisms live in environments that vary through time and such environmental heterogeneity can impose highly variable selection pressures on populations. In this situation, an allele may be beneficial during one environmental regime and subsequently deleterious during another. Such an allele would be subject to short bursts of directional selection, alternately being favored and disfavored. When this situation occurs in diploids, the heterozygote can have a higher geometric mean fitness than either homozygote and allelic variation at this locus could be maintained for long periods despite being subject to directional selection at any given time [@pgen.1004775-Gillespie1]--[@pgen.1004775-Hedrick1]. This situation is referred to as marginal overdominance and is a form of balancing selection.
There is substantial evidence for the maintenance of phenotypic and genetic variation by temporally variable selection in a variety of organisms. For instance, evolutionary response to rapid changes in selection pressures has been demonstrated for morphological and life-history traits in mammals [@pgen.1004775-Gershenson1], [@pgen.1004775-Grant1], birds [@pgen.1004775-Tarwater1]--[@pgen.1004775-Wall1], plants [@pgen.1004775-Brakefield1], invertebrates [@pgen.1004775-Hairston1]--[@pgen.1004775-RodriguezTrelles1], and others (reviewed in [@pgen.1004775-Bell1], [@pgen.1004775-Siepielski1]). Chromosomal inversions and allozyme alleles in a variety of drosophilids vary among seasons [@pgen.1004775-Dobzhansky1]--[@pgen.1004775-Ananina1] suggesting that these polymorphisms confer differential fitness in alternating seasons. Further, in some species of drosophilids, life-history [@pgen.1004775-Bouletreaumerle1], [@pgen.1004775-Schmidt1], morphological [@pgen.1004775-Stalker1], [@pgen.1004775-Tantawy1] and stress tolerance traits [@pgen.1004775-Miyo1], [@pgen.1004775-Dev1] also fluctuate seasonally suggesting that these traits respond to seasonal shifts in selection pressures.
Although theoretical models suggest that temporal variation in selection pressures can maintain fitness-related genetic variation in populations [@pgen.1004775-Gillespie1]--[@pgen.1004775-Hedrick1] and empirical evidence from a variety of species [@pgen.1004775-Gershenson1]--[@pgen.1004775-Dev1] demonstrates that variation in selection pressures over short time periods does alter phenotypes and allele frequencies, we still lack a basic understanding of many fundamental questions about the genetics and evolutionary history of alleles that undergo rapid adaptation in response to temporal variation in selection pressures. Specifically, we do not know how many loci respond to temporally variable selection within a population, the strength of selection at each locus, nor the effects of such strong selection on neutral genetic differentiation through time. We do not know whether adaptation at loci that respond to temporally variable selection is predictable nor do we know the relationship between loci that respond to temporally variable selection and spatially varying selection. Finally, it is unclear whether rapid adaptation to temporally variable selection pressures is primarily fueled by young alleles that constantly enter the population but cannot be maintained for long periods of time or, rather, by old alleles that have possibly been maintained by variable selection associated with environmental heterogeneity despite short bursts of strong directional selection.
To address these questions, we estimated allele frequencies genome-wide from samples of *D. melanogaster* individuals collected along a broad latitudinal cline in North America and in the spring and fall over three consecutive years in a single temperate orchard. We demonstrate that samples of flies collected in a single Pennsylvania orchard over the course of several years are as differentiated as populations separated by 5--10° latitude. We identify hundreds of polymorphisms that are subject to strong, temporally varying selection and argue that genetic draft [@pgen.1004775-Gillespie3] in the wake of rapid, multilocus adaptation is sufficient to explain the high degree of genetic turnover that we observe in this population over several years. We examine the genome-wide relationship between spatial and temporal variation in allele frequencies and find that spatial genetic differentiation, but not clinality *per se*, in allele frequency is a good predictor of temporal variation in allele frequency. Moreover, at SNPs subject to seasonal fluctuations in selection pressures, northern populations are more similar to spring populations than southern ones are. Next, we show that allele frequencies at SNPs subject to seasonal fluctuations in selection pressures become more 'spring-like' (i.e., they move towards the average spring frequency) immediately following a hard frost event and that seasonally variably SNPs tend to be associated with two seasonally variable phenotypes, chill coma recovery time and starvation tolerance. Finally, we demonstrate that some of the loci that respond to temporal variation in selection pressures are likely ancient, balanced polymorphisms that predate the split of *D. melanogaster* from its sister species, *D. simulans*. Taken together, our results are consistent with a model in which temporally variable selection maintains fitness-related genetic variation at hundreds of loci throughout the genome for millions of generations if not millions of years.
Results/Discussion {#s2}
==================
Genomic differentiation through time and space {#s2a}
----------------------------------------------
To test for the genomic signatures of balancing selection caused by seasonal fluctuations in selection pressures, we performed whole genome, pooled resequencing of samples of male flies collected in the spring and fall over three consecutive years (2009--2011) in a temperate, Pennsylvanian orchard. We contrast changes in allele frequencies through time with estimates of allele frequencies we made from five additional populations spanning Florida to Maine along the east coast of North America over a number of years (2003--2010) largely during periods of peak abundance of *D. melanogaster* ([Fig. 1A](#pgen-1004775-g001){ref-type="fig"}, [Table S1](#pgen.1004775.s008){ref-type="supplementary-material"}). From each population and time point, we sampled approximately 50--100 flies and resequenced each sample to average read depth of 20--200× coverage ([Table S1](#pgen.1004775.s008){ref-type="supplementary-material"}, and see [Text S1](#pgen.1004775.s011){ref-type="supplementary-material"}). Estimates of allele frequency using this sampling design have been shown to be highly accurate [@pgen.1004775-Gillespie3].
![Experimental design and genomic turnover through time and space.\
(A) Map of sampling locations in North America used in this study. Grey boxes represent individual samples from each locale. Genome-wide differentiation among spatially (B) and temporally (C) separated samples, measured as genome-wide average *F~ST~* (y-axis). Lines represent the predicted value of *F~ST~* based on the linear (A; y = a+bx) and non-linear (B; y = ab^X^) regression. Note: Pennsylvanian samples are not represented in (B) and the negative *F~ST~* in (B) results from the conservative correction of heterozygosity [@pgen.1004775-Rashkovetsky1], [@pgen.1004775-Turelli1]. In addition, please note that there are four estimates of pairwise *F~ST~* between the two replicate Maine and Florida samples (corresponding to a difference in latitude of 20°) and that there are two estimates of *F~ST~* between each of the remaining clinal populations and each Maine and Florida replicate sample. Error bars represent 95% confidence intervals based on 500 blocked bootstrap samples of ∼2000 SNPs.](pgen.1004775.g001){#pgen-1004775-g001}
As a point of departure and to provide context for understanding the magnitude of genetic variation through the seasons, we first examined genetic differentiation along the cline ([Fig. 1B](#pgen-1004775-g001){ref-type="fig"}, [Fig. S1A](#pgen.1004775.s001){ref-type="supplementary-material"}). We calculated genome-wide average *F~ST~* among pairs of populations (excluding Pennsylvanian populations; hereafter 'spatial *F~ST~*') as well as the proportion of SNPs where average spatial *F~ST~* between a pair of populations is greater than expected by chance conditional on our sampling design and assuming panmixia using allele frequency estimates of 500,000 common polymorphisms ([Table S1](#pgen.1004775.s008){ref-type="supplementary-material"}). Genome-wide average spatial *F~ST~* ([Fig. 1B](#pgen-1004775-g001){ref-type="fig"}) as well as the proportion of SNPs where spatial *F~ST~* is greater than expected by chance ([Fig. S1A](#pgen.1004775.s001){ref-type="supplementary-material"}) is positively correlated with geographic distance (*r* = 0.75; *p* = 7e-5), a pattern consistent with isolation by distance [@pgen.1004775-Wright1]. Pooled resequencing did identify polymorphisms in or near genes previously shown to be clinal in North American populations (see [Text S1](#pgen.1004775.s011){ref-type="supplementary-material"}) demonstrating that clines are stable over multiple years. This suggests that populations sampled along the cline represent resident | introduction { # s1 } = = = = = = = = = = = = all organisms live in environments that vary through time and such environmental heterogeneity can impose highly variable selection pressures on populations. in this situation, an allele may be beneficial during one environmental regime while subsequently deleterious during another. such an allele would be subject to short bursts of directional selection, alternately being favored and disfavored. when this situation occurs alongside diploids, the heterozygote can have a higher geometric mean fitness than either homozygote and allelic variation at this locus could be maintained for long periods simultaneously being subject to directional selection at any given time [ @ pgen. 1004775 - gillespie1 ] - - [ @ pgen. 1004775 - hedrick1 ]. this situation is referred to as marginal overdominance and is a consequence of balancing selection. there is substantial evidence for the maintenance of phenotypic and genetic variation by temporally variable selection in a variety in organisms. for instance, evolutionary response to rapid changes in selection pressures has been demonstrated for morphological characteristics life - history traits in mammals [ @ pgen. 1004775 - gershenson1 ], [ @ pgen. 1004775 - grant1 ], birds [ @ pgen. 1004775 - tarwater1 ] - - [ @ pgen. 1004775 - wall1 ], plants [ @ pgen. 1004775 - brakefield1 ], yeast [ @ pgen. 1004775 - hairston1 ] - - [ @ sen. 1004775 - rodrigueztrelles1 ], and others ( reviewed in [ @ pgen. 1004775 - bell1 ], [ @ pgen. 1004775 - siepielski1 ] ). chromosomal inversions and allozyme alleles in a variety of drosophilids vary between seasons [ @ pgen. 1004775 - dobzhansky1 ] - - [ @ pgen. 1004775 - ananina1 ] proposes that these polymorphisms confer differential fitness in alternating seasons. further, in some species of drosophilids, life - history [ @ pgen. 1004775 - bouletreaumerle1 ], [ @ pgen. 1004775 - schmidt1 ], morphological [ @ pgen. 1004775 - stalker1 ] , [ @ pgen. 1004775 - tantawy1 ] and stress tolerance traits [ @ pgen. 1004775 - miyo1 ], [ @ pgen. 1004775 - dev1 ] also fluctuate seasonally suggesting that these traits respond to seasonal shifts in selection pressures. although theoretical models suggest that temporal variation in selection pressures can maintain fitness - related genetic variation in populations [ @ pgen. 1004775 - gillespie1 ] - - [ @ pgen. 1004775 - hedrick1 ] and empirical evidence from a variety of species [ @ pgen. 1004775 - gershenson1 ] - - [ @ pgen. 1004775 - dev1 ] demonstrates that variation in selection pressures over short time periods does alter phenotypes and allele frequencies, we still lack a basic understanding of many fundamental questions about the genetics and evolutionary history of alleles that undergo rapid adaptation in response to temporal variation in selection pressures. specifically, we do not know how many loci respond to temporally variable selection within a population, the strength of selection at each locus, nor the effects of such strong selection on neutral genetic differentiation through time. we do not know whether adaptation at loci that respond to temporally variable selection is predictable nor do we know the relationship between loci that respond to temporally variable selection and spatially varying selection. finally, it is unclear whether rapid adaptation to temporally variable selection pressures is primarily fueled by young alleles that constantly enter the population but cannot be maintained for long periods of time or, rather, by old alleles that have possibly been maintained by variable selection associated with environmental heterogeneity despite short bursts of strong directional selection. to address these questions, we estimated allele frequencies genome - wide from samples of * d. melanogaster * individuals collected along a broad latitudinal cline in north america and in the spring and fall over three consecutive years in a single temperate orchard. we demonstrate that samples of flies collected in a single pennsylvania orchard over the course of several years are as differentiated as populations separated by 5 - - 10° latitude. we identify hundreds of polymorphisms that are subject to strong, temporally varying selection and argue that genetic draft [ @ pgen. 1004775 - gillespie3 ] in the wake of rapid, multilocus adaptation is sufficient to explain the high degree of genetic turnover that we observe in this population over several years. we examine the genome - wide relationship between spatial and temporal variation in allele frequencies and find that spatial genetic differentiation, but not clinality * per se *, in allele frequency is a good predictor of temporal variation in allele frequency. moreover, at snps subject to seasonal fluctuations in selection pressures, northern populations are more similar to spring populations than southern ones are. next, we show that allele frequencies at snps subject to seasonal fluctuations in selection pressures become more ' spring - like ' ( i. e., they move towards the average spring frequency ) immediately following a hard frost event and that seasonally variably snps tend to be associated with two seasonally variable phenotypes, chill coma recovery time and starvation tolerance. finally, we demonstrate that some of the loci that respond to temporal variation in selection pressures are likely ancient, balanced polymorphisms that predate the split of * d. melanogaster * from its sister species, * d. simulans *. taken together, our results are consistent with a model in which temporally variable selection maintains fitness - related genetic variation at hundreds of loci throughout the genome for millions of generations if not millions of years. results / discussion { # s2 } = = = = = = = = = = = = = = = = = = genomic differentiation through time and space { # s2a } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - to test for the genomic signatures of balancing selection caused by seasonal fluctuations in selection pressures, we performed whole genome, pooled resequencing of samples of male flies collected in the spring and fall over three consecutive years ( 2009 - - 2011 ) in a temperate, pennsylvanian orchard. we contrast changes in allele frequencies through time with estimates of allele frequencies we made from five additional populations spanning florida to maine along the east coast of north america over a number of years ( 2003 - - 2010 ) largely during periods of peak abundance of * d. melanogaster * ( [ fig. 1a ] ( # pgen - 1004775 - g001 ) { ref - type = " fig " }, [ table s1 ] ( # pgen. 1004775. s008 ) { ref - type = " supplementary - material " } ). from each population and time point, we sampled approximately 50 - - 100 flies and resequenced each sample to average read depth of 20 - - 200× coverage ( [ table s1 ] ( # pgen. 1004775. s008 ) { ref - type = " supplementary - material " }, and see [ text s1 ] ( # pgen. 1004775. s011 ) { ref - type = " supplementary - material " } ). estimates of allele frequency using this sampling design have been shown to be highly accurate [ @ pgen. 1004775 - gillespie3 ].! [ experimental design and genomic turnover through time and space. \ ( a ) map of sampling locations in north america used in this study. grey boxes represent individual samples from each locale. genome - wide differentiation among spatially ( b ) and temporally ( c ) separated samples, measured as genome - wide average * f ~ st ~ * ( y - axis ). lines represent the predicted value of * f ~ st ~ * based on the linear ( a ; y = a + bx ) and non - linear ( b ; y = ab ^ x ^ ) regression. note : pennsylvanian samples are not represented in ( b ) and the negative * f ~ st ~ * in ( b ) results from the conservative correction of heterozygosity [ @ pgen. 1004775 - rashkovetsky1 ], [ @ pgen. 1004775 - turelli1 ]. in addition, please note that there are four estimates of pairwise * f ~ st ~ * between the two replicate maine and florida samples ( corresponding to a difference in latitude of 20° ) and that there are two estimates of * f ~ st ~ * between each of the remaining clinal populations and each maine and florida replicate sample. error bars represent 95 % confidence intervals based on 500 blocked bootstrap samples of [UNK] snps. ] ( pgen. 1004775. g001 ) { # pgen - 1004775 - g001 } as a point of departure and to provide context for understanding the magnitude of genetic variation through the seasons, we first examined genetic differentiation along the cline ( [ fig. 1b ] ( # pgen - 1004775 - g001 ) { ref - type = " fig " }, [ fig. s1a ] ( # pgen. 1004775. s001 ) { ref - type = " supplementary - material " } ). we calculated genome - wide average * f ~ st ~ * among pairs of populations ( excluding pennsylvanian populations ; hereafter ' spatial * f ~ st ~ * ' ) as well as the proportion of snps where average spatial * f ~ st ~ * between a pair of populations is greater than expected by chance conditional on our sampling design and assuming panmixia using allele frequency estimates of 500, 000 common polymorphisms ( [ table s1 ] ( # pgen. 1004775. s008 ) { ref - type = " supplementary - material " } ). genome - wide average spatial * f ~ st ~ * ( [ fig. 1b ] ( # pgen - 1004775 - g001 ) { ref - type = " fig " } ) as well as the proportion of snps where spatial * f ~ st ~ * is greater than expected by chance ( [ fig. s1a ] ( # pgen. 1004775. s001 ) { ref - type = " supplementary - material " } ) is positively correlated with geographic distance ( * r * = 0. 75 ; * p * = 7e - 5 ), a pattern consistent with isolation by distance [ @ pgen. 1004775 - wright1 ]. pooled resequencing did identify polymorphisms in or near genes previously shown to be clinal in north american populations ( see [ text s1 ] ( # pgen. 1004775. s011 ) { ref - type = " supplementary - material " } ) demonstrating that clines are stable over multiple years. this suggests that populations sampled along the cline represent resident | Introduction {# s1} = = = = = = = = = = = = All organisms live in environments that vary through time and such environmental heterogeneity can impose highly variable selection pressures on populations. In this situation, an allele may be beneficial during one environmental regime and subsequemt;y deleterious during another. Such an qlIele would be subject to short bursts of directional selection, alternately being favored and disfavored. When this situation occurs in diploids, the heterozygote can have a higher geometric mean fitness than either homozygote and allelic variation at this locus could be maintained for long periods despite being subject to directional selection at any given time [@ pgen. 1004775 - Gillespie1] - - [@ pgen. 1004775 - Hedrick1 ]. This situation is referred to as marginal overdominance and is a form of balancing selection. There is substantial evidence for the maintenance of phenotypic and genetic variation by temporally variable selection in a variety of organisms. For instance, evolutionary response to rapid changes in selection pressures has been demonstrated for morphological and life - history traits in mammals [@ pgen. 1004775 - Gershenson1 ], [@ pgen. 1004775 - Grant1 ], birds [@ pgen. 1004775 - Tarwater1] - - [@ pgen. 1004775 - Wall1 ], plants [@ pgen. 1004775 - Brakefield1 ], invertebrates [@ pgen. 1004775 - Hairston1] - - [@ pgen. 1004775 - RodriguezTrelles1 ], and others (reviewed in [@ pgen. 1004775 - Bell1 ], [@ pgen. 1004775 - Siepielski1] ). Chromosomal inversions and allozyme alleles in a variety of drosophilids vary among seasons [@ pgen. 1004775 - Dobzhansky1] - - [@ pgen. 1004775 - Ananina1] suggesting that these polymorphisms confer diffeDentiao fitness in alternating seasons. Further, in some species of drosophilids, life - history [@ pgen. 1004775 - Bouletreaumerle1 ], [@ pgen. 1004775 - Schmidt1 ], morphological [@ pgen. 1004775 - Stalker1 ], [@ pgen. 1004775 - Tantawy1] and stress tolerance traits [@ pgen. 1004775 - Miyo1 ], [@ pgen. 1004775 - Dev1] also fluctuate seasonally suggesting that these traits respond to seasonal shifts in selection pressures. Although theoretical models suggest that temporal variation in selection pressures can maintain fitness - related genetic variation in populations [@ pgen. 1004775 - Gillespie1] - - [@ pgen. 1004775 - Hedrick1] and ekplrical evidence from a variety of species [@ pgen. w00$775 - Gershenson1] - - [@ pgen. 1004775 - Dev1] demonstrates that variation in selection pressures over dho#t time periods does alter phenotypes and allele frequencies, we still lack a basic understanding of many fundamental questions about the genetics and evolutionary history of alleles that undergo rapid adaptation in response to temporal variation in selection pressures. Specifically, we do not know how many loci respond to temporally variable selection within a population, the strength of selection at each locus, nor the effects of such strong selection on neutral genetic differentiation through time. We do not know whether adaptation at loci that respond to temporally variable seles5ion is predictable nor do we know the relationship between loci that respond to temporally variable selection and spatially varying selection. Finally, it is unclear whether rapid adaptation to temporally variable selection pressures is primarily fueled by young alleles that constantly enter the population but cannot be maintained for long periods of time or, rather, by old alleles that have possibly been maintained by variable selection associated with environmental heterogeneity despite short bursts of strong directional selection. To address these questions, we estimated allele frequencies genome - wide from samples of * D. melanogaster * individuals collected along a broad latitudinal cline in North America and in the spring and fall over three consecutive years in a single temperate orchard. We demonstrate that samples of flies collected in a single Pennsylvania orchard over the course of several years are as differentiated as populations separated by 5 - - 10 ° latitude. We identify hundreds of polymorphisms that are subject to strong, temporally varying selection and argue that genetic draft [@ pgen. 1004775 - Gillespie3] in the wake of rapid, multilocus adaptation is sufficient to explain the high degree of genetic turnover that we observe in this population over several years. We examine the genome - wix4 relationship between spatial and temporal variation in allele frequencies and find that spatial genetic differentiation, but not clinality * per se *, in allele frequency is a good predictor of temporal variation in allele frequency. Moreover, at SNPs subject to seasonal fluctuations in selection pressures, northern populations are more similar to spring populations than southern ones are. Next, we show that allele frequencies at SNPs subject to seasonal fluctuations in selection pressures become more ' spring - like ' (i. e. , they move towards the average spring frequency) immediately following a hard frost event and that seasonally variably SNPs tend to be associated with two seasonally variable phenotypes, chill coma recovery time and starvation tolerance. Finally, we demonstrate that some of the loci that respond to temporal variation in selection pressures are likely ancient, balanced polymorphisms that predate the split of * D. melanogaster * from its sister species, * D. simulans *. Taken together, our results are consistent with a model in which temporally variable selection maintains fitness - related genetic variation at hundreds of loci throughout the genome for mklliohs of generations if not millions of years. Results / Discussion {# s2} = = = = = = = = = = = = = = = = = = Genomic differentiation through time and space {# s2a} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - To test for the genomic signatures of balancing selection caused by seasonal fluctuations in selection pressures, we performed whole genome, pooled resequencing of samples of male flies collected in the spring and fall over three consecutive years (2009 - - 2011) in a temperate, Pennsylvanian orchard. We contrast changes in allele frequencies through time with estimates of allele frequencies we made from five additional populations spanning Florida to Maine along the east coast of North America over a number of years (2003 - - 2010) largely during periods of peak abundance of * D. melanogaster * ([ Fig. 1A] (# pgen - 1004775 - g001) {ref - type = " fig " }, [Table S1] (# pgen. 1004775. s008) {ref - type = " supplementary - material "} ). From each population and time point, we sampled approximately 50 - - 100 flies and resequenced each sample to average read depth of 20 - - 200 × coverage ([ Table S1] (# pgen. 1004775. s008) {ref - type = " supplementary - material " }, and see [Text S1] (# pgen. 1004775. s011) {ref - type = " supplementary - material "} ). Estimates of allele frequency using this sampling design have been shown to be highly accurate [@ pgen. 1004775 - Gillespie3 ]. ! [Experimental design and genomic turnover through time and space. \ (A) Map of sampling locations in North America used in this study. Grey boxes represent individual samples from each locale. Genome - wide differentiation among spatially (B) and temporally (C) separated samples, measured as genome - wide average * F ~ ST ~ * (y - axis ). Lines represent the predicted value of * F ~ ST ~ * based on the linear (A; y = a + bx) and non - linear (B; y = ab ^ X ^) regression. Note: Pennsylvanian samples are not represented in (B) and the negative * F ~ ST ~ * in (B) results from the conservative correction of heterozygosity [@ pgen. 1004775 - Rashkovetsky1 ], [@ pgen. 1004775 - Turelli1 ]. In addition, please note that there are four estimates of pairwise * F ~ ST ~ * between the two replicate Maine and Florida samples (corresponding to a difference in latitude of 20 °) and that 5tere are two estimates of * F ~ ST ~ * between each of the remaining clinal populations and each Maine and Florida replicate sample. Error bars represent 95% confidence intervals based on 500 blocked bootstrap samples of ∼ 2000 SNPs.] (pgen. 1004775. g001) {# pgen - 1004775 - g001} As a point of departure and to provide context for understanding the magnitude of genetic variation through the seasons, we first examined genetic differentiation along the cline ([ Fig. 1B] (# pgen - 1004775 - g001) {ref - type = " fig " }, [Fig. S1A] (# pgen. 1004775. s001) {ref - type = " supplementary - material "} ). We calculated genome - wide average * F ~ ST ~ * among pairs of populations (excluding Pennsylvanian populations; hereafter ' spatial * F ~ ST ~ * ') as well as the proportion of SNPs where average spatial * F ~ ST ~ * between a pair of populations is greater than expected by chance conditional on our sampling design and assuming panmixia using allele frequency estimates of 500, 000 common polymorphisms ([ Table S1] (# pgen. 1004775. s008) {ref - type = " supplementary - material "} ). Genome - wide average spatial * F ~ ST ~ * ([ Fig. 1B] (# pgen - 1004775 - g001) {ref - type = " fig "} ) as well as the proportion of SNPs where spatial * F ~ ST ~ * is greater than expected by chance ([ Fig. S1A] (# pgen. 1004775. s001) {ref - type = " supplementary - material "} ) is positively correlated with geographic distance (* r * = 0. 75; * p * = 7e - 5 ), a pattern consistent with isolation by distance [@ pgen. 1004775 - Wright1 ]. Pooled resequencing did identify polymorphisms in or near genes previously shown to be clinal in North American populations (see [Text S1] (# pgen. 1004775. s011) {ref - type = " supplementary - material "} ) demonstrating that clines are stable over multiple years. This suggests that populations sampled along the cline represent resident | Introduction {#s1} ============ All organisms live in environments that vary through and such environmental heterogeneity can impose highly variable selection pressures on In this situation, allele may be beneficial during one environmental regime subsequently deleterious during Such an allele would be subject to bursts of directional alternately being favored and When this occurs in diploids, the heterozygote have a higher mean fitness than either homozygote and allelic variation at this could be maintained for long periods despite being subject to directional selection at any given time This situation is referred to as marginal overdominance and is a form of selection. There is substantial evidence for the maintenance of phenotypic and genetic variation by temporally variable selection in variety of organisms. For instance, evolutionary response to rapid changes in selection pressures has for morphological and life-history traits mammals [@pgen.1004775-Gershenson1], birds [@pgen.1004775-Tarwater1]--[@pgen.1004775-Wall1], plants [@pgen.1004775-Brakefield1], invertebrates [@pgen.1004775-Hairston1]--[@pgen.1004775-RodriguezTrelles1], and others (reviewed in [@pgen.1004775-Bell1], [@pgen.1004775-Siepielski1]). Chromosomal inversions and allozyme alleles in a variety of drosophilids vary among seasons [@pgen.1004775-Dobzhansky1]--[@pgen.1004775-Ananina1] suggesting that these polymorphisms confer differential fitness in seasons. Further, some species of drosophilids, life-history [@pgen.1004775-Bouletreaumerle1], [@pgen.1004775-Schmidt1], morphological [@pgen.1004775-Stalker1], [@pgen.1004775-Tantawy1] and stress tolerance traits [@pgen.1004775-Miyo1], [@pgen.1004775-Dev1] also fluctuate seasonally suggesting that these traits respond to seasonal shifts in selection pressures. Although models suggest that temporal variation in selection pressures maintain fitness-related genetic variation in populations [@pgen.1004775-Gillespie1]--[@pgen.1004775-Hedrick1] and empirical evidence from a variety of species [@pgen.1004775-Gershenson1]--[@pgen.1004775-Dev1] demonstrates that variation in selection pressures short time periods does alter phenotypes and allele frequencies, we still lack a basic understanding many fundamental questions about the genetics and evolutionary history of alleles that undergo rapid adaptation in response variation in pressures. Specifically, we do not know how many respond to temporally variable selection within a population, the strength selection at each locus, nor effects of such strong selection on neutral genetic differentiation time. We not know whether adaptation loci that respond to temporally variable selection is predictable nor know the relationship between loci that respond temporally variable selection and spatially varying selection. Finally, it is unclear whether rapid adaptation to temporally selection pressures is primarily fueled by young alleles that constantly enter the population but cannot be maintained for long periods of time rather, by alleles that possibly been maintained by selection associated with environmental heterogeneity despite short bursts of strong directional selection. To address these questions, we estimated allele frequencies from samples of *D. melanogaster* individuals collected along a broad latitudinal cline in North America and in the spring over three consecutive years in a single temperate orchard. We that samples of flies in a single Pennsylvania orchard over the course of several years are as differentiated as populations separated by 5--10° latitude. We identify hundreds of polymorphisms that are subject to strong, temporally varying selection and argue that genetic draft [@pgen.1004775-Gillespie3] in the wake of multilocus adaptation is sufficient to explain the high degree of genetic turnover we observe in this population over several We examine the genome-wide relationship between and temporal variation in allele frequencies and find that spatial genetic differentiation, but not clinality *per se*, in allele frequency is a good predictor of temporal variation allele frequency. Moreover, at SNPs to seasonal fluctuations in pressures, northern populations are more similar to spring populations than southern are. Next, we show that allele frequencies SNPs subject to seasonal fluctuations in selection pressures become more 'spring-like' (i.e., they move towards the average spring frequency) immediately following a hard frost event and that seasonally variably SNPs tend to be associated with two seasonally phenotypes, chill coma recovery time and tolerance. Finally, we demonstrate that some of loci that respond to temporal variation in selection pressures are likely ancient, balanced polymorphisms that the split of *D. melanogaster* from sister species, *D. simulans*. Taken our results are consistent with a in which temporally variable selection maintains fitness-related genetic variation at hundreds of loci throughout the genome for millions of if not millions years. Results/Discussion {#s2} ================== Genomic differentiation through time and space {#s2a} ---------------------------------------------- To test for the genomic signatures of balancing caused by seasonal fluctuations in selection pressures, we performed whole genome, pooled resequencing of samples of flies in the spring and over three consecutive years (2009--2011) in temperate, Pennsylvanian We contrast changes allele frequencies through time with of allele frequencies we made from five additional populations spanning Florida to Maine along the east of North America over a number years (2003--2010) largely during periods of peak abundance *D. melanogaster* ([Fig. 1A](#pgen-1004775-g001){ref-type="fig"}, [Table S1](#pgen.1004775.s008){ref-type="supplementary-material"}). each population and time point, we sampled approximately 50--100 flies resequenced each sample to average read of 20--200× coverage ([Table S1](#pgen.1004775.s008){ref-type="supplementary-material"}, and see [Text S1](#pgen.1004775.s011){ref-type="supplementary-material"}). Estimates of allele frequency using sampling design have been shown to be highly accurate [@pgen.1004775-Gillespie3]. ![Experimental and genomic turnover through time and space.\ (A) Map sampling locations in North America used this study. Grey boxes represent samples each locale. Genome-wide differentiation spatially (B) and temporally (C) separated samples, measured as genome-wide average *F~ST~* (y-axis). Lines represent the predicted value of *F~ST~* on the linear (A; y a+bx) and non-linear (B; y = ab^X^) regression. Note: Pennsylvanian samples are represented in (B) and the *F~ST~* in (B) results from the conservative correction of heterozygosity [@pgen.1004775-Rashkovetsky1], [@pgen.1004775-Turelli1]. In addition, note that there are four estimates of pairwise *F~ST~* between the replicate Maine and Florida samples to a difference in latitude of and that there are two estimates of *F~ST~* each of the remaining populations and each Maine and replicate sample. Error bars represent 95% confidence intervals based on 500 blocked bootstrap samples of ∼2000 SNPs.](pgen.1004775.g001){#pgen-1004775-g001} As a point of and to context for understanding the magnitude genetic variation through the seasons, we first examined genetic differentiation along the cline ([Fig. 1B](#pgen-1004775-g001){ref-type="fig"}, [Fig. S1A](#pgen.1004775.s001){ref-type="supplementary-material"}). We calculated genome-wide average *F~ST~* among pairs of populations Pennsylvanian populations; hereafter 'spatial *F~ST~*') as well the proportion of SNPs where average spatial *F~ST~* between a pair of populations is greater than expected chance conditional on our sampling design and assuming panmixia allele frequency estimates of 500,000 common polymorphisms ([Table S1](#pgen.1004775.s008){ref-type="supplementary-material"}). Genome-wide average spatial *F~ST~* ([Fig. 1B](#pgen-1004775-g001){ref-type="fig"}) as well the proportion of SNPs where spatial *F~ST~* is greater by chance ([Fig. S1A](#pgen.1004775.s001){ref-type="supplementary-material"}) is correlated with geographic distance (*r* 0.75; *p* = a pattern consistent with isolation [@pgen.1004775-Wright1]. Pooled resequencing identify polymorphisms in or near genes previously to be clinal in North American populations (see [Text demonstrating that are stable over multiple years. that populations sampled along the cline represent resident | iNtrOdUCTioN {#S1}
============
aLl OrgANISMs LIVe in EnVIRonmEnTS THAt VarY thROUgH tIME ANd sucH eNVIRoNmEntAL HeTErogeNeiTY cAn iMpOsE HiGhLy VaRIAbLe SELEctioN PREsSures On populATioNs. In THiS sITUaTioN, aN ALlElE may bE BEnEFiCIaL DUriNG One enVIrOnMENtAl ReGIMe aND SUbSEqUEnTly DELeterIOuS dURing anoTHer. suCh aN aLlele WoULD be subjEct to ShorT BURsTS of dIRectiONAL seLEcTION, aLtErNATely BEInG faVOrED ANd diSFavOReD. wHen tHiS SituaTION OccuRs IN DIPLOIdS, tHe HEtErozYGOTE cAn HAve A HighER geOMEtRic meaN FitnESS THaN eitHeR hoMOZygOte ANd AlLeLIC varIAtioN at tHIs LOCUs COuld Be MaINtAInED foR LONg peRiODS DeSPitE beInG suBjeCt TO DIRECTIOnal SELeCTion AT aNY giVEN TimE [@PgEN.1004775-gILlesPiE1]--[@pGEN.1004775-heDRicK1]. tHis sITUaTIon Is ReFeRREd TO As mArGiNal oVERDOMINaNCE aND IS a foRM Of baLaNCiNG SELecTioN.
THerE iS suBSTAntIAL evIDence for tHE MaInTEnanCE Of phENOtyPic aND GENeTIc VARiaTIon by TeMpoRALLy vaRIABLe sELeCtiOn IN A vaRietY oF orgAnismS. FoR INstANCe, evolUTIoNaRY ResPONSe TO rapid chAnGES iN seLEction PReSSuRES haS BEen DEmonsTraTEd for mOrpHOLogIcAl aND LiFE-hISToRY TrAItS In maMMaLs [@pgEn.1004775-GeRsHeNsOn1], [@PGEn.1004775-graNt1], BirdS [@PGEN.1004775-taRWateR1]--[@pgen.1004775-wALl1], PLAntS [@pGen.1004775-brAkEfIEld1], InVERtEBrateS [@pgen.1004775-HaIRSTon1]--[@PgEN.1004775-rODRIgUEZtrELlES1], ANd OthERS (REVIewEd iN [@pGen.1004775-belL1], [@pgeN.1004775-siEPIelski1]). chROMOsOMaL iNversioNs aNd allozyme aLLeLeS IN a VArieTy Of DRosOpHiLiDs Vary aMONg seAsoNS [@PgEn.1004775-dObZHAnSkY1]--[@PgeN.1004775-ananinA1] SuggeStiNG tHat tHesE POlyMOrPhISms CoNfER diFfERentIaL FITnESS IN AlTernATinG SeAsONS. fuRtHER, iN somE sPEcies OF drOSophiLIds, LiFe-hISToRy [@pgEn.1004775-boUlEtreAUMErLE1], [@PgeN.1004775-ScHMIdT1], mOrPHOLOGiCAl [@PGen.1004775-STaLKEr1], [@PGeN.1004775-TANtawY1] anD StreSs tolEraNcE tRaItS [@pGEn.1004775-MiYo1], [@PgeN.1004775-deV1] ALso FluctuATE SEasOnaLLY SuGgEStIng ThAt tHese trAITS REspOnD To SEasONaL SHiftS in sELecTIOn PReSSureS.
ALtHoUGH THEORETicAl mODeLS SuggeSt tHat teMpORaL VaRiATioN iN SeleCtiON pRESsures can MAiNtAIn FiTnEss-ReLaTED genETIC vARiaTIoN In poPuLATIoNs [@Pgen.1004775-GilLespIe1]--[@pgeN.1004775-hEdRiCk1] AnD empIriCal EViDEncE FrOM A vAriety of spEcIeS [@pGEN.1004775-geRShENSOn1]--[@Pgen.1004775-dev1] DEMonStRaTeS thAt VaRiatIoN iN seLecTION pResSures Over SHORT TImE PerIOdS Does AlTEr pHenOtYPES And allElE FrEqUeNCIES, WE STill LACk A basIC uNDERstanDIng OF maNY FundamentaL QueSTIOns aBouT THE GENeTiCS AND EVoLUTIOnAry hIstORY oF alleLes tHaT uNdERGo RapId ADApTAtIoN in RESPOnSE to TEmPOraL vaRIaTioN IN SeLECTIoN pREssurEs. sPecIFICALlY, wE DO noT knoW how manY locI REspoNd tO tEmPOraLLY VARiaBLE SEleCTIoN wiThIn A pOpuLAtion, tHE sTreNgTh OF sElEcTIOn At eAcH lOcUS, Nor THe EFFectS OF SucH sTroNg SELECtiOn oN NEUTraL genetIC DifFerEnTiAtIoN tHrOUGh TIme. WE dO noT knOw wHEtHEr AdaPtAtiON aT LOCI ThAt respOnD to TeMPoRaLlY varIABle SeLecTiOn Is prEdicTABLE nOR do wE KnOW the rELATIOnsHip BETWEen loCi thAT reSPOnD tO tEmPOrAllY vARIable sElEctIoN and spATiALLY vArYInG sElECTIOn. finaLly, It Is uncleAR wHEtheR RapiD adAptatIoN To tEmPORALlY VARiaBlE sEleCtioN prEssUrEs is prImaRily fuEleD by YOuNG AllELeS tHaT conStanTLy eNter THE pOPulAtiON buT CANNOt Be maINtaIneD FOR lONG pERIODs OF TIMe Or, rATHer, BY Old aLleleS ThAt HaVE POssIbly Been MAINtainED by vaRIaBle seleCtioN AsSOCiaTed wItH eNVIRONmEnTAl hEtErOgENeIty DespITE SHOrt bUrSTS OF sTrong DIRECtioNAL SelECTIOn.
TO AddrESS thESE qUEstiONS, WE EStImateD alLELE fREQueNcIeS genOmE-wIDe FrOM sAmPLES OF *D. MElaNoGaSTer* inDIViDualS COlLEcteD aLonG a BrOAd laTitUDINaL cliNE iN nORTh amERICA aND In thE sPrINg aND Fall over ThRee CoNsEcUtivE yeARs IN a sInGlE TeMpErATe OrcHArd. We DEMOnstrATE thaT SAMplES OF fLiES coLlEcTeD iN a SInGlE peNnsyLVANIA oRCHArd oVEr The couRSE Of SeveRAl YeARs arE AS dIfFerENtiaTed aS PopulaTiOns SEPaRAted BY 5--10° LATITUde. wE IdENtIfY hUnDRedS Of poLYMoRPHISMs thaT aRe suBJEcT TO sTrONG, teMporALlY Varying selEction AnD aRgue thaT geneTIc drAFt [@Pgen.1004775-GillEspIe3] In tHE WAke Of RAPiD, MUltIlOcus ADaptATION is SuFFiCIeNt tO expLaIN the HiGH degrEE Of GENEtic TurnoVER thAT We obSerVe in tHIs pOpULAtION OvEr SevErAL YeArs. we EXaminE tHe GenoME-widE ReLaTiOnsHIp bEtWEEN SpAtIAL aND temPORal VAriatIOn In ALleLE FReQuENCIeS AnD fiND ThAt spaTiAL gEneTiC DiFFEReNTiAtIoN, But noT ClinAlITY *peR Se*, In alLELe frEqUENCy is a GooD preDIcTOr OF teMpoRaL VariaTioN In alLEle FREQUenCY. morEoVeR, AT SnPs SuBjEct To SeASoNaL FLucTUAtioNs IN sELeCTioN presSUrES, nOrtHERn pOPULATioNS aRE mOre sIMilAR To sPRiNg POpULaTIONs ThAn soUtHeRN oNEs aRe. next, We shoW ThaT aLLEle FREQueNCieS At snPS sUBject TO SEASONaL FlUCTuatioNS In SElEcTioN PrEssUREs bEcOMe MOrE 'sPriNG-LIke' (i.e., theY MOvE ToWardS The aVerAgE SPriNg fREquENcY) iMmeDiAteLy foLlOWiNG A HArd fRost evEnT anD That SeAsONALly varIAblY sNPS tEnD TO bE AsSOCIATED WITh TWO SEaSoNAlLY VariAbLe pHENoTYPeS, cHiLl COMA rECoVerY Time and StARvatIoN toLERAnce. fInAlly, We DEMonsTRaTE tHAt some of thE LOci tHAT ResPOND tO tEmpoRal VaRIATion IN SelectIoN PrEssURES aRe LikelY aNcienT, BaLaNCeD pOLYMoRpHIsMs THaT prEdATE the Split Of *d. MelAnoGAstER* From its SiSter speciEs, *d. SiMuLans*. TAKeN ToGethER, OUR reSULTS ArE consISTEnt WIth A MoDEl IN WHicH TemPOraLLy VARiABle SelEcTion MAINTaINs fITneSs-RElAtED GenetIc vaRIAtiON AT hUNDReDS Of lOCi thRoUGhout tHe genOME foR MILLIoNS oF geNerATioNs If NoT MILLIONS oF YeaRS.
resUlts/diSCUssIoN {#s2}
==================
gEnOmic DifFEREnTIaTION thrOUgh TIme And space {#s2A}
----------------------------------------------
To tesT fOr THe GENomic SIgNatUReS Of BalaNCINg seLecTIoN cAusED bY seaSOnAL fLUCTUaTIoNS in seLectiON prEsSUres, WE PERfORmEd WHole GEnOME, poOlEd rEsequEncing Of sAMpLEs Of mALE FlIeS cOLLected In The sprINg AND FAll oVEr THree cONsECUTIve yEArs (2009--2011) In A TEMPErate, penNsYlVaNIan ORChard. WE cOnTRaSt chAngeS In alLeLe FrEqUENCIes ThrOUgh Time wITh EStIMaTEs OF ALlelE FrEQUenCIeS wE made fROM FivE AdDITIOnAL POPulAtIOns spaNNInG flORIDA To MaINe AloNG THE eAST COasT of NorTh AMerICa OVer A NumbeR OF yeaRS (2003--2010) LarGELy DuriNg PeRIODs oF peaK abUnDANCe Of *D. mElaNogASteR* ([fIG. 1A](#pgeN-1004775-g001){reF-tYpe="fIg"}, [table s1](#pGeN.1004775.S008){rEf-tyPe="sUpPLEmEntAry-mateRIAl"}). FrOM eAcH POPUlATIoN AND tIMe PoINT, we SaMPlED apPrOXIMAteLY 50--100 FliEs anD REseqUeNced EaCH saMplE To aveRAgE rEAD DePTh of 20--200× COveragE ([tAble S1](#PGen.1004775.S008){reF-TYpE="sUppleMeNtAry-MATErIaL"}, AnD sEE [Text s1](#pgEn.1004775.S011){REF-tYPE="SUPpLemeNTAry-maTeRIAl"}). esTiMAtES oF aLLele FrEqUENCY usInG This SAMpLINg DEsIgN HAVe been sHoWn To BE HIGHLy accurATe [@pGen.1004775-GiLlespie3].
![ExPErimentAL dEsIgN aND gENOmiC TUrnovER thROugh TImE and SpacE.\
(A) MAP OF saMPLinG locaTIoNs In NorTh AMeriCa USeD iN THiS StUDY. gReY boxEs rEPREsENT iNdivIdUAL sAMPlES From eACh lOcAle. gEnome-WIde difFErEnTiatIOn AmonG sPatiALlY (B) AND tEMPoRalLy (C) sEparated sampLEs, MeAsUReD as gENome-wide AVeRaGe *f~st~* (Y-AXIS). lines rEpReSeNt tHe pRediCted vALuE Of *F~ST~* BAseD ON The LiNEAr (a; Y = a+bX) And nON-linear (B; y = Ab^x^) regrEssion. nOte: pENnSylvaNIAN samPLeS are noT repReSented in (b) AND THE NEgaTIve *f~St~* iN (B) ResulTS from tHe CONSErvAtIve cOrrECTIOn OF HEtEroZYGOsITY [@Pgen.1004775-RashKOVEtSKy1], [@pgEN.1004775-tURELLI1]. In aDDItIon, pLeaSE NOtE thAt THERE ARe FOur eSTimAtES Of PAirwISe *f~st~* bEtWEen the tWo rEpLIcATe mAiNe anD floRIda SaMPlEs (CoRrESPONDINg TO a DIfFerEncE In LaTiTuDE of 20°) and THat TherE are TWo esTiMATEs Of *F~st~* BetwEeN eAch Of THE REMAiNiNg cLinAl PopULatIons AND eacH mAInE aND FlORIdA rePlicate sAMpLe. eRror bArS repreSeNt 95% CONfiDEncE iNterVaLS bAseD on 500 blOckEd boOTSTRAP SAmPlEs Of ∼2000 sNpS.](PGEN.1004775.g001){#pGEn-1004775-G001}
as A pOinT Of dEPaRtURe AND TO pRoVIDE coNTeXt fOr uNdeRStandInG the maGNITUDE of GEneTic varIaTIOn THrOugH tHE sEAsoNS, We fiRST eXaMINED GeneTIC DIffeREntiATION ALoNg tHe cliNE ([fIg. 1B](#pGEN-1004775-g001){ref-tYpE="fiG"}, [Fig. S1a](#pgEn.1004775.s001){rEf-TYPE="SUPpLEmENtaRY-maTeRial"}). We CAlCulAted GENoME-wIdE aVEraGE *f~sT~* aMong PAIRS oF POPUlATioNs (exclUDing pENnsyLvanIan poPULaTioNS; HeReAfTEr 'spAtIal *f~st~*') as WelL aS tHE PrOPorTIon OF SnPS Where AvERage sPatIAl *f~St~* bEtWeEN A paIR OF pOpUlAtIoNs iS GREatER THan exPeCted bY ChaNCe conDitIONal on OUr saMpLinG dEsigN and AsSUMiNg PaNMIXiA uSInG allele FrEquEnCy EsTimAtEs OF 500,000 cOmmoN poLymorPHisMs ([TaBLE s1](#Pgen.1004775.s008){Ref-TyPe="sUppleMeNTaRy-matErIAL"}). gEnOme-WIDe AveRAGe SPaTIaL *F~sT~* ([fIG. 1B](#PGEN-1004775-g001){rEF-TyPE="FIg"}) As Well as the pROPoRtiOn Of snPs whERe spAtial *F~St~* IS gREatEr than eXpEcTEd BY chanCe ([Fig. s1a](#pgEN.1004775.S001){Ref-Type="SuPPlemEntAry-materIal"}) iS POSItiVELY correlateD wIth geograPHIc DiStaNcE (*r* = 0.75; *p* = 7e-5), A PatteRn consIstEnT witH ISoLaTIon By DiStANCE [@Pgen.1004775-wrIght1]. poolEd resEquencING DID iDEntiFy POLYMoRphISMS in or Near GEnES PrevIOusly ShoWn tO be CLiNAL In nOrTh AmericAn pOPULAtIoNs (seE [teXT s1](#pgEn.1004775.s011){rEF-TyPe="SUPPlemeNTary-mATErIaL"}) dEMOnstraTIng ThAT ClINEs ARE StABLe OvER MULTIPlE YEARS. ThIs sUgGESts ThAt PoPUlAtIonS SamPLEd alONg THe cLIne RePRESent rEsidEnT | Introduction{#s1}============ All organisms live in environmentsthat varythrough time and such environmental heterogeneity can impose highly variable selection pressures on populations.In this situation, an allele may be beneficial during one environmental regime and subsequently deleterious during another. Such an allele would be subject to short bursts of directional selection, alternatelybeing favored and disfavored. When this situationoccurs in diploids, the heterozygote can haveahighergeometric mean fitnessthaneither homozygote and allelic variation at this locus could be maintainedfor long periodsdespite being subject to directional selection at any given time [@pgen.1004775-Gillespie1]--[@pgen.1004775-Hedrick1]. This situation is referred to as marginal overdominance and is a form of balancing selection. There is substantialevidencefor the maintenance ofphenotypic and genetic variation by temporally variable selection in a variety of organisms. For instance, evolutionary response to rapidchanges in selection pressureshas been demonstratedfor morphological and life-history traits in mammals [@pgen.1004775-Gershenson1], [@pgen.1004775-Grant1], birds[@pgen.1004775-Tarwater1]--[@pgen.1004775-Wall1],plants[@pgen.1004775-Brakefield1], invertebrates [@pgen.1004775-Hairston1]--[@pgen.1004775-RodriguezTrelles1], and others (reviewed in [@pgen.1004775-Bell1], [@pgen.1004775-Siepielski1]). Chromosomal inversions and allozyme alleles in avariety of drosophilids vary amongseasons [@pgen.1004775-Dobzhansky1]--[@pgen.1004775-Ananina1] suggesting that these polymorphisms confer differential fitness inalternating seasons. Further, insome species of drosophilids, life-history [@pgen.1004775-Bouletreaumerle1], [@pgen.1004775-Schmidt1], morphological [@pgen.1004775-Stalker1], [@pgen.1004775-Tantawy1] and stress tolerance traits [@pgen.1004775-Miyo1], [@pgen.1004775-Dev1] also fluctuate seasonally suggesting that thesetraitsrespond to seasonal shifts in selection pressures.Although theoretical modelssuggest that temporal variationin selection pressures can maintain fitness-related genetic variation in populations [@pgen.1004775-Gillespie1]--[@pgen.1004775-Hedrick1]and empirical evidencefrom a variety of species [@pgen.1004775-Gershenson1]--[@pgen.1004775-Dev1]demonstrates that variation in selection pressuresover short time periods doesalter phenotypes and allele frequencies, we still lack a basic understanding ofmany fundamental questions about the genetics and evolutionary historyof alleles that undergo rapid adaptation in response to temporal variationin selection pressures. Specifically, we do not know how many loci respond to temporally variable selection within apopulation, the strength of selection ateach locus, nor the effects of such strong selection on neutral genetic differentiation through time. We do notknowwhether adaptation at loci that respond to temporally variable selection is predictablenor dowe know the relationshipbetween loci that respond to temporally variable selection and spatially varying selection. Finally, it isunclear whether rapid adaptation to temporally variable selectionpressures is primarily fueled byyoung alleles that constantlyenter thepopulationbut cannot be maintained forlongperiods of time or, rather, by old alleles that have possiblybeen maintained by variable selection associatedwith environmental heterogeneity despite short bursts of strong directionalselection. To address these questions, we estimatedallele frequencies genome-wide from samples of *D. melanogaster* individuals collected along a broad latitudinal cline inNorth America and in the spring and fall overthree consecutive yearsin asingle temperate orchard. We demonstrate that samples of flies collected in a single Pennsylvania orchard over the course of several yearsare as differentiated as populationsseparated by5--10° latitude. We identify hundreds of polymorphisms that aresubject to strong, temporally varying selection and argue thatgenetic draft [@pgen.1004775-Gillespie3] in the wake ofrapid, multilocus adaptation is sufficient to explain the high degree of genetic turnover that we observein this population over several years.Weexamine the genome-wide relationship between spatial and temporal variation in allele frequencies and find that spatial genetic differentiation, but not clinality *perse*, in allele frequency is a goodpredictor of temporal variation in allelefrequency. Moreover, at SNPssubject to seasonal fluctuations in selection pressures, northern populations are more similar to spring populations than southern onesare. Next, we show that allele frequencies at SNPs subject to seasonal fluctuations in selection pressuresbecome more 'spring-like' (i.e., they move towards the average spring frequency) immediately following a hard frost event andthat seasonally variably SNPstend to beassociated with two seasonally variable phenotypes, chill coma recovery time and starvation tolerance. Finally, we demonstrate that some of the loci that respond totemporal variation in selection pressures are likely ancient, balanced polymorphisms that predate the splitof *D. melanogaster* from its sister species, *D. simulans*. Taken together, our results are consistent with a model in which temporally variable selection maintains fitness-related genetic variation at hundreds of locithroughout the genome for millionsofgenerations if not millions of years. Results/Discussion {#s2} ================== Genomic differentiation through time and space {#s2a} ---------------------------------------------- To test for the genomic signatures of balancing selection caused by seasonal fluctuations in selectionpressures, we performed wholegenome, pooled resequencingofsamples ofmale flies collected in the spring and fall overthreeconsecutive years (2009--2011) in a temperate,Pennsylvanian orchard. We contrast changes in allele frequenciesthrough time with estimates ofallelefrequencieswe made from five additionalpopulations spanning Floridato Mainealong the east coast of North America over a number of years (2003--2010) largely during periods ofpeak abundance of *D. melanogaster* ([Fig. 1A](#pgen-1004775-g001){ref-type="fig"}, [Table S1](#pgen.1004775.s008){ref-type="supplementary-material"}). From each population and time point, we sampled approximately 50--100 fliesandresequenced each sample to average read depth of 20--200× coverage ([TableS1](#pgen.1004775.s008){ref-type="supplementary-material"},andsee [Text S1](#pgen.1004775.s011){ref-type="supplementary-material"}). Estimates of allele frequency using this sampling design have beenshown to be highly accurate [@pgen.1004775-Gillespie3]. ![Experimental design and genomic turnover through time andspace.\ (A)Mapof sampling locations in NorthAmerica used in this study. Grey boxes represent individual samples from each locale. Genome-wide differentiation amongspatially(B) andtemporally (C) separated samples, measured as genome-wide average *F~ST~* (y-axis). Lines represent the predicted value of*F~ST~* based on the linear (A; y = a+bx) and non-linear (B; y = ab^X^) regression. Note: Pennsylvanian samples are not represented in (B)and the negative *F~ST~* in (B) results from theconservative correction of heterozygosity [@pgen.1004775-Rashkovetsky1], [@pgen.1004775-Turelli1]. In addition, please note that there are four estimates of pairwise*F~ST~* between the two replicate Maine and Florida samples (corresponding to a differencein latitude of20°) and that there are two estimates of *F~ST~*between each of the remaining clinal populations and each Maine and Florida replicate sample. Error bars represent 95% confidenceintervals based on500blocked bootstrapsamples of ∼2000 SNPs.](pgen.1004775.g001){#pgen-1004775-g001} As a point of departure and to provide context forunderstanding the magnitude of geneticvariation through the seasons, we first examined genetic differentiationalongthecline ([Fig. 1B](#pgen-1004775-g001){ref-type="fig"},[Fig.S1A](#pgen.1004775.s001){ref-type="supplementary-material"}). We calculated genome-wide average *F~ST~* among pairs of populations (excluding Pennsylvanian populations; hereafter 'spatial *F~ST~*') as well as the proportion of SNPs where averagespatial *F~ST~*between a pairof populationsis greater thanexpected bychance conditional on our sampling design and assuming panmixia using allele frequency estimates of 500,000 common polymorphisms ([Table S1](#pgen.1004775.s008){ref-type="supplementary-material"}). Genome-wideaverage spatial *F~ST~* ([Fig.1B](#pgen-1004775-g001){ref-type="fig"}) as well as theproportion of SNPs where spatial *F~ST~*is greater than expected bychance ([Fig. S1A](#pgen.1004775.s001){ref-type="supplementary-material"}) is positively correlated with geographic distance (*r* = 0.75;*p* = 7e-5), a pattern consistentwith isolation by distance [@pgen.1004775-Wright1]. Pooled resequencing did identify polymorphisms in ornear genes previously shown to beclinal in North American populations (see [Text S1](#pgen.1004775.s011){ref-type="supplementary-material"})demonstratingthat clines arestable over multiple years. Thissuggests that populations sampled along the clinerepresent resident | Introduction {#s1} ============ All organisms live in environments that vary _through_ time and such environmental heterogeneity can impose highly _variable_ selection pressures on populations. _In_ this situation, an allele may _be_ beneficial during one environmental regime and subsequently deleterious during another. Such an allele would be _subject_ to short bursts of _directional_ _selection,_ alternately being favored and disfavored. When this situation occurs in diploids, the heterozygote can have a _higher_ geometric mean fitness than either _homozygote_ and allelic variation at _this_ locus could be _maintained_ for long periods despite being subject to directional selection at _any_ given time [@pgen.1004775-Gillespie1]--[@pgen.1004775-Hedrick1]. This _situation_ is referred to as marginal overdominance and is a form of balancing _selection._ There _is_ _substantial_ evidence for the _maintenance_ of phenotypic and genetic variation _by_ _temporally_ variable selection in a variety of organisms. For instance, evolutionary response _to_ _rapid_ changes _in_ selection pressures _has_ been demonstrated for morphological and life-history traits in mammals [@pgen.1004775-Gershenson1], [@pgen.1004775-Grant1], _birds_ [@pgen.1004775-Tarwater1]--[@pgen.1004775-Wall1], plants [@pgen.1004775-Brakefield1], invertebrates [@pgen.1004775-Hairston1]--[@pgen.1004775-RodriguezTrelles1], and others (reviewed _in_ [@pgen.1004775-Bell1], _[@pgen.1004775-Siepielski1])._ _Chromosomal_ inversions and allozyme _alleles_ in a variety _of_ _drosophilids_ vary among seasons [@pgen.1004775-Dobzhansky1]--[@pgen.1004775-Ananina1] _suggesting_ that these polymorphisms confer differential _fitness_ _in_ alternating seasons. Further, _in_ _some_ species of drosophilids, life-history [@pgen.1004775-Bouletreaumerle1], [@pgen.1004775-Schmidt1], morphological [@pgen.1004775-Stalker1], [@pgen.1004775-Tantawy1] and stress _tolerance_ _traits_ [@pgen.1004775-Miyo1], _[@pgen.1004775-Dev1]_ also fluctuate _seasonally_ suggesting that _these_ traits respond to seasonal shifts in selection _pressures._ _Although_ theoretical _models_ suggest that _temporal_ variation in _selection_ pressures can maintain fitness-related genetic variation in populations [@pgen.1004775-Gillespie1]--[@pgen.1004775-Hedrick1] and empirical evidence from a variety of species [@pgen.1004775-Gershenson1]--[@pgen.1004775-Dev1] demonstrates that _variation_ in selection pressures over short _time_ periods does alter phenotypes _and_ _allele_ frequencies, we still lack a _basic_ understanding of _many_ fundamental questions about the genetics and evolutionary history of alleles that undergo rapid _adaptation_ in response _to_ temporal variation in selection pressures. Specifically, we do _not_ know how many _loci_ respond to temporally _variable_ selection within _a_ population, the strength of selection at _each_ locus, nor the effects of such strong selection on neutral _genetic_ differentiation through time. _We_ _do_ not know whether _adaptation_ at loci that respond _to_ temporally variable selection is _predictable_ nor do we know the relationship _between_ loci that respond to temporally variable _selection_ and spatially _varying_ selection. Finally, it is unclear whether rapid _adaptation_ to temporally variable selection pressures is primarily fueled by young alleles that constantly enter _the_ population but _cannot_ be maintained for long periods _of_ time or, rather, by old alleles that have possibly been _maintained_ by variable selection associated with environmental heterogeneity despite short bursts _of_ strong directional _selection._ _To_ address _these_ questions, we estimated _allele_ frequencies genome-wide from samples of *D. melanogaster* individuals collected _along_ a broad latitudinal cline in North America and in the spring and fall over three consecutive years in a single _temperate_ _orchard._ We _demonstrate_ that _samples_ of flies collected in a single _Pennsylvania_ orchard over the course of several years _are_ as differentiated _as_ populations separated by _5--10°_ latitude. We _identify_ _hundreds_ _of_ polymorphisms _that_ are subject _to_ strong, temporally varying _selection_ _and_ argue that genetic _draft_ [@pgen.1004775-Gillespie3] in _the_ wake of rapid, multilocus adaptation is sufficient to explain the high _degree_ of genetic turnover that we observe in this population _over_ _several_ years. We examine the genome-wide relationship between spatial and temporal variation _in_ _allele_ _frequencies_ and find that spatial genetic _differentiation,_ but not clinality *per se*, in allele frequency is a good predictor _of_ temporal _variation_ in allele _frequency._ Moreover, at SNPs _subject_ to seasonal fluctuations in _selection_ pressures, northern _populations_ are more similar to spring populations than southern _ones_ are. Next, we show _that_ allele _frequencies_ _at_ SNPs subject to seasonal _fluctuations_ _in_ selection pressures become more 'spring-like' (i.e., they move towards the average spring frequency) immediately following _a_ hard frost _event_ and that seasonally variably _SNPs_ tend to be associated with two seasonally variable _phenotypes,_ chill coma _recovery_ _time_ and starvation tolerance. Finally, we _demonstrate_ that some of the loci that _respond_ _to_ temporal variation in selection pressures _are_ likely _ancient,_ balanced polymorphisms that predate the split of _*D._ melanogaster* from its sister species, _*D._ simulans*. Taken together, our results are consistent with a model in which temporally variable selection _maintains_ _fitness-related_ _genetic_ variation at _hundreds_ of loci throughout the genome for millions of generations if not millions of years. Results/Discussion {#s2} ================== _Genomic_ differentiation _through_ time and space _{#s2a}_ ---------------------------------------------- To test for the _genomic_ signatures of balancing selection caused by seasonal fluctuations in selection pressures, we performed whole genome, _pooled_ _resequencing_ of samples of male flies collected in the spring and fall _over_ three _consecutive_ years (2009--2011) _in_ a temperate, Pennsylvanian orchard. We contrast changes in _allele_ frequencies through time with estimates of _allele_ frequencies we made _from_ five additional populations spanning Florida to _Maine_ along _the_ _east_ _coast_ of _North_ America over _a_ _number_ of years (2003--2010) largely during periods of peak abundance of *D. _melanogaster*_ ([Fig. 1A](#pgen-1004775-g001){ref-type="fig"}, [Table S1](#pgen.1004775.s008){ref-type="supplementary-material"}). From each population and time point, we sampled approximately 50--100 _flies_ _and_ _resequenced_ each sample to average read depth of 20--200× coverage ([Table S1](#pgen.1004775.s008){ref-type="supplementary-material"}, and see [Text S1](#pgen.1004775.s011){ref-type="supplementary-material"}). _Estimates_ of _allele_ frequency using this sampling design _have_ been shown _to_ be highly _accurate_ [@pgen.1004775-Gillespie3]. _![Experimental_ _design_ and genomic turnover _through_ _time_ and space.\ (A) Map _of_ sampling locations in North America used in this study. Grey _boxes_ represent individual samples from each _locale._ _Genome-wide_ _differentiation_ among spatially (B) and _temporally_ (C) separated samples, _measured_ _as_ genome-wide average *F~ST~* _(y-axis)._ Lines represent _the_ predicted value of *F~ST~* _based_ on the linear (A; y = _a+bx)_ and non-linear (B; y = _ab^X^)_ _regression._ Note: _Pennsylvanian_ _samples_ are not represented in (B) and the negative _*F~ST~*_ in (B) results from _the_ conservative correction of heterozygosity [@pgen.1004775-Rashkovetsky1], [@pgen.1004775-Turelli1]. In addition, please note that _there_ _are_ four _estimates_ of pairwise *F~ST~* _between_ the two replicate _Maine_ and _Florida_ samples _(corresponding_ to a _difference_ in latitude of _20°)_ and _that_ there are _two_ estimates of *F~ST~* between each of the remaining clinal _populations_ and each Maine and Florida replicate sample. Error bars represent 95% confidence _intervals_ based on 500 blocked bootstrap samples of _∼2000_ SNPs.](pgen.1004775.g001){#pgen-1004775-g001} As _a_ point _of_ departure and _to_ _provide_ context for understanding the magnitude of _genetic_ variation through the _seasons,_ we first examined genetic differentiation along the _cline_ ([Fig. 1B](#pgen-1004775-g001){ref-type="fig"}, [Fig. S1A](#pgen.1004775.s001){ref-type="supplementary-material"}). We calculated genome-wide average _*F~ST~*_ among pairs _of_ _populations_ (excluding Pennsylvanian _populations;_ _hereafter_ _'spatial_ _*F~ST~*')_ as well as the proportion _of_ _SNPs_ where average _spatial_ *F~ST~* between a pair of _populations_ is greater than expected by chance conditional _on_ _our_ sampling design and assuming panmixia using allele frequency estimates _of_ 500,000 common polymorphisms ([Table S1](#pgen.1004775.s008){ref-type="supplementary-material"}). Genome-wide average _spatial_ _*F~ST~*_ ([Fig. 1B](#pgen-1004775-g001){ref-type="fig"}) as well _as_ _the_ proportion of SNPs where spatial *F~ST~* is greater than expected by _chance_ ([Fig. S1A](#pgen.1004775.s001){ref-type="supplementary-material"}) is positively _correlated_ with geographic distance (*r* = 0.75; *p* = 7e-5), a _pattern_ _consistent_ with isolation by distance [@pgen.1004775-Wright1]. _Pooled_ resequencing did _identify_ polymorphisms in or _near_ genes previously _shown_ to be clinal in North American populations _(see_ [Text S1](#pgen.1004775.s011){ref-type="supplementary-material"}) demonstrating _that_ clines are stable over multiple years. This suggests that populations sampled along the cline _represent_ resident |
###### Strengths and limitations of this study
- The study data were obtained through a questionnaire carried out by face-to-face interviews. The youth were trained to obtain dietary diet from their parent and grandparent. They were accompanied by the research assistant during data collection.
- This study provides important insights of differences in food types consumed between the younger and older Pacific generation.
- Exploratory factor analyses was used to examine dietary data between groups, providing robust findings.
```{=html}
<!-- -->
```
- A major limitation is the small sample size of the study; however, this is a feasibility study, and the results are limited to the study participants.
- The use of the dietary diversity questionnaire is based on a limited sampling frame of food group diversity over a 7-day period; therefore, it may not be completely representative of all food items and food groups that may have been consumed by the different age generations.
Introduction {#s1}
============
Pacific peoples in New Zealand (NZ) are at greater risk of developing long-term conditions such as prediabetes, diabetes[@R1] and cardiovascular disease[@R4] due to obesity, defined as having a body mass index (BMI) \>30 kg/m^2^. The prevalence of obesity (67% in adults aged 15+ years) in Pacific peoples is twice that in the general population (33%).[@R5] Obesity has been viewed as a result of the changing westernised environmental pressures,[@R6] leading to an energy expenditure/energy storage mismatch that operates through dietary behaviours.[@R6] Often modernised dietary patterns, characterised by high energy dense and palatable food,[@R8] and eating habits, such as having more access to less healthy snacks and food,[@R9] explain the weight gain.[@R11] The findings from the NZ national nutritional survey on Pacific peoples reported similar patterns of energy, micronutrient intake, dietary supplement use and dietary habits (eg, consumption of breakfast and other food types, fruit and vegetable intake and salt intake), compared with the general population.[@R12]
Obesity rates continue to rise for all young people aged 15--24 years, with the highest increase among young Pacific peoples (up by 50% between 2007 and 2012).[@R13] Previous NZ research has documented the impact of family meals on the dietary quality of young people[@R14] and found that eating family meals together had an overall positive effect on the home food environment[@R15] and that it encouraged the availability of more healthy food.[@R10] Results from the Youth'12 Survey highlighted similar findings that shared family meals were associated with positive outcomes for young people, which were accentuated for those living in the lower deprivation neighbourhoods (66%), compared with those in the higher deprivation areas (58%).[@R16] However, the survey results do not provide any clues as to the obesity-related mechanisms to further understanding about why young people continue to eat unhealthily. It has also been shown that young people experiencing poverty, irrespective of living in either low deprivation or more affluent neighbourhoods, does not explain overweight and obesity issues for young people.[@R17] Little research has focused on understanding the Pacific concept of socialisation and food as inter-related activities, and there is increasing recognition of the importance of this if obesity prevention strategies are to effective.[@R18]
In 2014, we investigated obesity-related health issues among 30 Pacific youth from the Wellington and Auckland regions of NZ in the pilot study, '*Chewing the facts on fat! What does that say about me?*'. The methodology and scope of the study has been published elsewhere.[@R19] As part of that study, we also examined Pacific youths' diet and eating habits as it relates to obesity development. In particular, we explored dietary diversity as a form of investigating diet quality, which could be useful in identifying dietary component needs and provide insights to guide development of intervention strategies to improve diet quality and health outcomes for young Pacific peoples. The aim of the current paper is to examine the intergenerational dietary patterns among 30 young Pacific adults aged 15--24 years, and 34 parents and grandparents, from Wellington and Auckland regions.
Method {#s2}
======
From a pilot study of 30 young Pacific adults aged 15--24 years in Wellington and Auckland, NZ, we investigated the social cultural determinants of the obesogenic environment. The study was conducted in two phases, and the original study methodology has been previously published,[@R19] which described the recruitment, questionnaire data and the social demography of the participants, from phase 1. This paper presents data mainly from phase 2, obtained from young Pacific peoples who were trained to interview and obtain information from one Pacific parent and one Pacific grandparent to examine and compare older generation's dietary habits with those of the Pacific youth group (phase 1) using the Pasifika dietary diversity questionnaire (described below). As Pacific peoples are not a homogenous group, we will refer to them as Pasifika, defined as a collective group of people representing the different Pacific Island nations and their respective languages, social cultural realities and protocols.
Patient involvement {#s2a}
-------------------
Patients were not involved in the planning or design of the study.
Demography {#s2b}
----------
Basic descriptive data were obtained from the parent and grandparent as per the protocol (face to face) described in phase 1 of the study,[@R19] including measured weight and height, from which BMI was determined. We used the international standard cut-offs in defining obesity.[@R20] BMI was analysed as a continuous variable, with a BMI of ≥30 kg/m^2^ and 25--29.9 kg/m^2^ defined as being obese and overweight, respectively.[@R21] Waist-to-hip circumference was also measured, from which the waist--hip ratio (WHR) was determined to provide a measure of central adiposity to indicate associated risk of incident cardiovascular events.[@R22] The waist-to-height ratio (WtHR) was also calculated, as an adjunct measure of central obesity, which is less prone to measurement error than WHR.[@R23] Deprivation was assessed using the NZDep2013 measure,[@R25] a small area-based measure of deprivation derived from the 2013 Census, which uses nine variables (benefit income, employment, household income, communication, transport, support, qualifications, living space and home ownership) from the Census to place small area blocks on a deprivation scale from 1 to 10 with 10 representing the most deprived 10% of NZ areas, while 1 represents the 10% least deprived areas. For analyses, deprivation was categorised into quintiles combining deciles 1--2, 3--4, 5--6, 7--8 and 9--10. Ethnicity was defined according to standard NZ Census data[@R26] and self-identified by each participant; however, for participation in the project, parents and grandparents needed to be self-identifiable as being of Pacific ethnicity. Where possible, the participants were also invited to record any physician-diagnosed conditions to highlight the presence of morbidity.
Dietary diversity {#s2c}
-----------------
The Pacific dietary diversity questionnaire was compiled by the research team to assess the scope of individual foods and food groups in Pacific peoples' diets. The dietary diversity questionnaire aims to capture the 'range of food' that people consume over a 7-day period and not to measure quantity of food items like the 'food frequency questionnaire'. This type of assessment has been used among other indigenous groups successfully, and it has been proven to be effective than a quantitative assessment of describing the quality of the diet.[@R27] This questionnaire was pretested among an independent community group of Pasifika youth (n=30) from Wellington, and it was adapted to include common food that would be consumed by Pasifika people in NZ (eg, povi masima \[salted meat\]).
Data on different individual foods and food groups that were consumed over a 7-day period (reference period) were collected and recorded (dichotomised: yes/no) by the Pacific youth who were participants in phase 1 and were trained by the research team at a single day workshop. The training involved familiarising and understanding the questionnaire and prompts. Following the training day, each youth arranged and organised a face-to face interview (accompanied by a research assistant), with a parent and grandparent. The questionnaire included both nutritious and discretionary food and food groups to encapsulate the diversity of food groups and of food items. The questionnaire data produced a total of 26 food groups (15 nutritious and 11 discretionary) specifically consumed by the study participants. However, using exploratory factor analyses (EFA) (see below) in order to create meaningful summary patterns that describe types of diet, we had refined the food groups down to 13, as determined by the total percentage of items consumed within a food grouping, per person. The groupings are: group 1: meats, poultry and fish diversity; group 2: dairy products diversity; group 3: bread, cereals and starchy vegetable diversity; group 4: legumes and nut diversity; group 5: fruit diversity; group 6: vegetable diversity; group 7: oil and fat diversity; group 8: drinks diversity; group 9: alcohol diversity; group 10: sauces, spreads and flavouring diversity; group 11: sweets and sweet snacks diversity; group 12: savoury snacks diversity; and group 13: take way food diversity.
Eating habits and meal patterns, food choices and related cultural and social influences were also investigated, but the results are not presented here.
We also included a measure of acculturation using a tool developed by coauthor JK and his colleagues[@R33 | # # # # # # strengths and limitations of this study - the study data were extracted through a questionnaire carried out by face - to - face interviews. the youth were trained to obtain dietary diet from their parent and grandparent. they were accompanied by the research assistant during data collection. - this study provides important insights of differences in food types consumed between the younger and older pacific generation. - exploratory empirical analyses was used to examine the data between groups, providing robust findings. ` ` ` { = html } <! - - - - > ` ` ` - a major limitation is the small sample size of the study ; however, this is a feasibility study, and the results are limited to the study participants. - the use of the dietary diversity questionnaire is based on a limited sampling frame of food group diversity over a 120 - day period ; therefore, it may not be completely representative of all food items and food groups that may have been consumed by the different age generations. introduction { # s1 } = = = = = = = = = = = = pacific peoples in new zealand ( nz ) are at greater risk of developing long - term conditions such as prediabetes, diabetes [ @ r1 ] and cardiovascular disease [ @ r4 ] due to obesity, defined as having a body mass index ( bmi ) \ > 30 kg / m ^ 2 ^. the prevalence of obesity ( 67 % in adults aged 15 + years ) in pacific peoples is twice that in the general population ( 33 % ). [ @ r5 ] obesity has been viewed as a result of the changing westernised environmental pressures, [ @ r6 ] leading to an energy expenditure / energy storage mismatch that operates through dietary behaviours. [ @ r6 ] often modernised dietary patterns, characterised by high energy dense and palatable food, [ @ r8 ] and eating habits, such as having more access to less healthy snacks and food, [ @ r9 ] explain the weight gain. [ @ r11 ] the findings from the nz national nutritional survey on pacific peoples reported food patterns of energy, micronutrient intake, dietary supplement use and shopping habits ( eg, consumption of breakfast and other food types, fruit and vegetable intake versus salt intake ), compared with the general population. [ @ 5 ] obesity statistics continue to rise for all young people years 15 - - 24 years, with the highest increase among young pacific peoples ( up by 50 % between 2007 and 2012 ). [ @ r13 ] previous nz research has documented the impact of family meals on the dietary quality of young people [ @ r14 ] and found that eating family meals together had an overall positive effect on the home food environment [ @ r15 ] and that it encouraged the availability of more healthy food. [ @ r10 ] results from the youth ' 12 survey highlighted similar findings that shared family meals were associated with positive outcomes for young people, which were accentuated for those living in the lower deprivation neighbourhoods ( 66 % ), compared with those in the higher deprivation areas ( 58 % ). [ @ r16 ] however, the survey results do not provide any clues as to the obesity - related mechanisms to further understanding about why young people continue to eat unhealthily. it has also been shown that young people experiencing poverty, irrespective of living in either low deprivation or more affluent neighbourhoods, does not explain overweight and obesity issues for young people. [ @ r17 ] little research has focused on understanding the pacific concept of socialisation and food as inter - related activities, and there is increasing recognition of the importance of this if obesity prevention strategies are to effective. [ @ r18 ] in 2014, we investigated obesity - related health issues among 30 pacific youth from the wellington and auckland regions of nz in the pilot study, ' * chewing the facts on fat! what does that say about me? * '. the methodology and scope of the study has been published elsewhere. [ @ r19 ] as part of that study, we also examined pacific youths ' diet and eating habits as it relates to obesity development. in particular, we explored dietary diversity as a form of investigating diet quality, which could be useful in identifying dietary component needs and provide insights to guide development of intervention strategies to improve diet quality and health outcomes for young pacific peoples. the aim of the current paper is to examine the intergenerational dietary patterns among 30 young pacific adults aged 15 - - 24 years, and 34 parents and grandparents, from wellington and auckland regions. method { # s2 } = = = = = = from a pilot study of 30 young pacific adults aged 15 - - 24 years in wellington and auckland, nz, we investigated the social cultural determinants of the obesogenic environment. the study was conducted in two phases, and the original study methodology has been previously published, [ @ r19 ] which described the recruitment, questionnaire data and the social demography of the participants, from phase 1. this paper presents data mainly from phase 2, obtained from young pacific peoples who were trained to interview and obtain information from one pacific parent and one pacific grandparent to examine and compare older generation ' s dietary habits with those of the pacific youth group ( phase 1 ) using the pasifika dietary diversity questionnaire ( described below ). as pacific peoples are not a homogenous group, we will refer to them as pasifika, defined as a collective group of people representing the different pacific island nations and their respective languages, social cultural realities and protocols. patient involvement { # s2a } - - - - - - - - - - - - - - - - - - - patients were not involved in the planning or design of the study. demography { # s2b } - - - - - - - - - - basic descriptive data were obtained from the parent and grandparent as per the protocol ( face to face ) described in phase 1 of the study, [ @ r19 ] including measured weight and height, from which bmi was determined. we used the international standard cut - offs in defining obesity. [ @ r20 ] bmi was analysed as a continuous variable, with a bmi of ≥30 kg / m ^ 2 ^ and 25 - - 29. 9 kg / m ^ 2 ^ defined as being obese and overweight, respectively. [ @ r21 ] waist - to - hip circumference was also measured, from which the waist - - hip ratio ( whr ) was determined to provide a measure of central adiposity to indicate associated risk of incident cardiovascular events. [ @ r22 ] the waist - to - height ratio ( wthr ) was also calculated, as an adjunct measure of central obesity, which is less prone to measurement error than whr. [ @ r23 ] deprivation was assessed using the nzdep2013 measure, [ @ r25 ] a small area - based measure of deprivation derived from the 2013 census, which uses nine variables ( benefit income, employment, household income, communication, transport, support, qualifications, living space and home ownership ) from the census to place small area blocks on a deprivation scale from 1 to 10 with 10 representing the most deprived 10 % of nz areas, while 1 represents the 10 % least deprived areas. for analyses, deprivation was categorised into quintiles combining deciles 1 - - 2, 3 - - 4, 5 - - 6, 7 - - 8 and 9 - - 10. ethnicity was defined according to standard nz census data [ @ r26 ] and self - identified by each participant ; however, for participation in the project, parents and grandparents needed to be self - identifiable as being of pacific ethnicity. where possible, the participants were also invited to record any physician - diagnosed conditions to highlight the presence of morbidity. dietary diversity { # s2c } - - - - - - - - - - - - - - - - - the pacific dietary diversity questionnaire was compiled by the research team to assess the scope of individual foods and food groups in pacific peoples ' diets. the dietary diversity questionnaire aims to capture the ' range of food ' that people consume over a 7 - day period and not to measure quantity of food items like the ' food frequency questionnaire '. this type of assessment has been used among other indigenous groups successfully, and it has been proven to be effective than a quantitative assessment of describing the quality of the diet. [ @ r27 ] this questionnaire was pretested among an independent community group of pasifika youth ( n = 30 ) from wellington, and it was adapted to include common food that would be consumed by pasifika people in nz ( eg, povi masima \ [ salted meat \ ] ). data on different individual foods and food groups that were consumed over a 7 - day period ( reference period ) were collected and recorded ( dichotomised : yes / no ) by the pacific youth who were participants in phase 1 and were trained by the research team at a single day workshop. the training involved familiarising and understanding the questionnaire and prompts. following the training day, each youth arranged and organised a face - to face interview ( accompanied by a research assistant ), with a parent and grandparent. the questionnaire included both nutritious and discretionary food and food groups to encapsulate the diversity of food groups and of food items. the questionnaire data produced a total of 26 food groups ( 15 nutritious and 11 discretionary ) specifically consumed by the study participants. however, using exploratory factor analyses ( efa ) ( see below ) in order to create meaningful summary patterns that describe types of diet, we had refined the food groups down to 13, as determined by the total percentage of items consumed within a food grouping, per person. the groupings are : group 1 : meats, poultry and fish diversity ; group 2 : dairy products diversity ; group 3 : bread, cereals and starchy vegetable diversity ; group 4 : legumes and nut diversity ; group 5 : fruit diversity ; group 6 : vegetable diversity ; group 7 : oil and fat diversity ; group 8 : drinks diversity ; group 9 : alcohol diversity ; group 10 : sauces, spreads and flavouring diversity ; group 11 : sweets and sweet snacks diversity ; group 12 : savoury snacks diversity ; and group 13 : take way food diversity. eating habits and meal patterns, food choices and related cultural and social influences were also investigated, but the results are not presented here. we also included a measure of acculturation using a tool developed by coauthor jk and his colleagues [ @ r33 | # # # # # # Strengths and limitations of this study - The study data were obtained through a questionnaire carried out by face - to - face interviews. The youth were trained to obtain dietary diet from their parent and grandparent. They were accompanied by the research assistant duriHt data collection. - This study provides important insights of differences in food types consumed between the younger and older Pacific generation. - Exploratory factor analyses was used to examine dietary data between groups, providing robust findings. ` ` ` {= html} <! - - - -> ` ` ` - A major limitation is the small sample size of the study; however, this is a feasibility study, and the results are limited to the study participants. - The use of the dietary diversity questionnaire is based on a limited sampling frame of food group diversity over a 7 - day period; therefore, it may not be completely representative of all food items and food groups that may have been consumed by the different age generations. Introduction {# s1} = = = = = = = = = = = = Pacific peoples in New Zealand (NZ) are at greater risk of developing long - term conditions s&sh as prediabetes, diabetes [@ R1] and cardiovascular disease [@ R4] due to obesity, defined as having a body mass index (BMI) \> 30 kg / m ^ 2 ^. The prevalence of obesity (67% in adults aged 15 + years) in Pacific peoples is twice that in the general population (33% ). [@ R5] Obesity has been viewed as a result of the changing westernised environmental pressures, [@ R6] leading to an energy expenditure / energy storage mismatch that operates through dietary behaviours. [@ R6] Often modernised dietary patterns, characterised by high energy dense and palatable food, [@ R8] and eating habits, such as having more access to less healthy snacks and food, [@ R9] explain the weight gain. [@ R11] The findings from the NZ national nutritional survey on Pacific peoples reported similar patterns of energy, micronutrient intake, dietary supplement use and dietary habits (eg, consumption of breakfast and other food types, fruit and vegetable intake and salt intake ), compared with the general population. [@ R12] Obesity rates continue to rise for all young people aged 15 - - 24 years, with the highest increase among young Pacific peoples (up by 50% between 2007 and 2012 ). [@ R13] Previous NZ research has documented the impact of family meals on the dietary quality of young people [@ R14] and found that eating family meals together had an overall positive effect on the home food environment [@ R15] and that it encouraged the availability of more healthy food. [@ R10] Results from the Youth ' 12 Survey highlighted similar findings that shared family meals were associated with positive outcomes for young people, which were accentuated for those living in the lower deprivation neighbourhoods (66% ), compared with those in the higher deprivation areas (58% ). [@ R16] However, the survey results do not provide any clues as to the obesity - related mechanisms to further understanding about why young people continue to eat unhealthily. It has also been shown that young people experiencing poverty, irrespective of living in either low deprivation or more affluent neighbourhoods, does not explain overweight and obesity issues for young people. [@ R17] Little research has focused on understanding the Pacific concept of socialisation and food as inter - related activities, and there is increasing recognition of the importance of this if obesity prevention strategies are to effective. [@ R18] In 2014, we investigated obesity - related health issues among 30 Pacific yoKtB from the Wellington and Auckland regions of NZ in the pilot study, ' * Chewing the facts on fat! What does that say about me? * '. The methodology and scope of the study has been published elsewhere. [@ R19] As part of that study, we also examined Pacific youths ' diet and eating habits as it relates to obesity development. In particular, we explored dietary diversity as a form of investigating diet quality, which could be useful in identifying dietary component needs and provide insights to guide development of intervention strategies to iHprov$ diet quality and health outcomes for young Pacific peoples. The aim of the current paper is to examine the intergenerational dietary patterns among 30 young Pacific adults aged 15 - - 24 years, and 34 patenhs and grandparents, from Wellington and Auckland regions. Method {# s2} = = = = = = From a pilot study of 30 young Pacific adults aged 15 - - 24 years in Wellington and Auckland, NZ, we investigated the social cultural determinants of the obesogenic environment. The study was conducted in two phases, and the original study methodology has been previously published, [@ R19] which described the recruitment, questionnaire data and the social demography of the participants, from phase 1. This paper presents data mainly from phase 2, obtained from young Pacific peoples who were trained to interview and obtain information from one Pacific parent and one Pacific grandparent to examine and compare older generation ' s dietary habits with those of the Pacific youth group (phase 1) using the Pasifika dietary diversity questionnaire (described below ). As Pacific peoples are not a homogenous group, we will refer to them as Pasifika, defined as a collective group of people Tepres#nting the different PacLfiV Island nations and their respective languages, social cultural realities and protocols. Patient involvement {# s2a} - - - - - - - - - - - - - - - - - - - Patients were not involved in the planning or design of the study. Demography {# s2b} - - - - - - - - - - Basic descriptive data were obtained from the parent and grandparent as per the protocol (face to face) described in phase 1 of the study, [@ R19] including measured weight and height, from which BMI was determined. We used the international standard cut - offs in defining obesity. [@ R20] BMI was analysed as a continuous variable, with a BMI of ≥ 30 kg / m ^ 2 ^ and 25 - - 29. 9 kg / m ^ 2 ^ defined as being obese and overweight, respectively. [@ R21] Waist - to - hip circumference was also measured, from which the waist - - hip ratio (WHR) was determined to provide a measure of central adiposity to indicate associated risk of incident cardiovascular events. [@ R22] The wQizt - to - height ratio (WtHR) was also calculated, as an adjunct measure of central obesity, which is less prone to measurement error than WHR. [@ R23] Deprivation was assessed using the NZDep2013 measure, [@ R25] a small area - based measure of deprivation derived from the 2013 Census, which uses nine variables (benefit income, employment, household income, communication, transport, support, qualifications, living sLqce and home ownership) from the Census to place small area blocks on a deprivation scale from 1 to 10 with 10 representing the most deprived 10% of NZ areas, while 1 represents the 10% least deprived areas. For analyses, deprivation was categorised into quintiles combining deciles 1 - - 2, 3 - - 4, 5 - - 6, 7 - - 8 and 9 - - 10. Ethnicity was defined according to standard NZ Census data [@ R26] and self - identified by each participant; however, for participation in the project, parents and grandparents needed to be self - identifiable as being of Pacific ethnicity. Where possible, the participants were also invited to record any physician - diagnosed conditions to highlight the presence of morbidity. Dietary diversity {# s2c} - - - - - - - - - - - - - - - - - The Pacific dietary diversity questionnaire was compiled by the research team to assess the scope of individual foods and food groups in Pacific peoples ' diets. The dietary diversity questionnaire aims to capture the ' range of food ' that people consume over a 7 - day period and not to measure quantity of food items like the ' food frequency questionnaire '. This type of assessment has been used among other indigenous groups successfully, and it has been proven to be effective than a quantitative assessment of describing the quality of the diet. [@ R27] This questionnaire was pretested among an independent community group of Pasifika youth (n = 30) from Wellington, and it was adapted to include common food that would be consumed by Pasifika people in NZ (eg, povi masima \ [salted meat \] ). Data on different individual foods and food groups that were consumed over a 7 - day period (reference period) were collected and recorded (dichotomised: yes / no) by the Pacific youth who were participants in phase 1 and were trained by the research team at a single day workshop. The training involved familiarising and understanding the questionnaire and prompts. Following the training day, each youth arranged and organised a face - to face interview (accompanied by a research assistant ), with a parent and grandparent. The questionnaire included both nutritious and discretionary food and food groups to encapsulate the diversity of food groups and of food items. The questionnaire data produced a total of 26 food groups (15 nutritious and 11 discretionary) specifically sonsumew by the study participants. However, using exploratory factor analyses (EFA) (see below) in order to create meaningful summary patterns that describe types of diet, we had refined the food groups down to 13, as determined by the total percentage of items consumed within a food grouping, per person. The groupings are: group 1: meats, poultry and fish diversity; group 2: dairy products diversity; group 3: bread, cereals and starchy vegetable diversity; group 4: legumes and nut diversity; group 5: fruit diversity; group 6: vegetable diversity; group 7: oil and fat diversity; group 8: drinks diversity; group 9: alcohol diversity; group 10: sauces, spreads and flavouring diversity; group 11: sweets and sweet snacks diversity; group 12: savoury snacks diversity; and group 13: take way food diversity. Eating habits and meal patterns, food choices and related cultural and social influences were also investigated, but the results are not presented here. We also included a measure of acculturation using a tool developed by coauthor JK and his colleagues [@ R33 | ###### and limitations of this study - The study data were through a questionnaire carried out by face-to-face interviews. The youth to obtain dietary diet from their parent and grandparent. They were accompanied by the research assistant during data collection. - This study provides important insights of differences in types consumed between the younger older Pacific generation. - Exploratory factor analyses was used to dietary data between groups, providing robust findings. ```{=html} <!-- --> ``` - A major limitation is small sample size of the study; however, this is a study, and the results are limited to the study participants. - The of the dietary questionnaire is based on a limited sampling frame of food group diversity a 7-day period; therefore, it may not be completely representative of all food items and food groups that may have consumed by the different age generations. Introduction {#s1} ============ Pacific peoples in New Zealand (NZ) are at greater risk of developing conditions as prediabetes, and disease[@R4] to obesity, defined as having a body mass index (BMI) \>30 kg/m^2^. The prevalence of obesity (67% in adults aged 15+ years) Pacific peoples is twice that in the general population (33%).[@R5] Obesity has been a result of changing westernised environmental pressures,[@R6] leading an energy expenditure/energy storage mismatch that operates through dietary behaviours.[@R6] Often modernised dietary patterns, characterised by high energy dense and palatable food,[@R8] and eating habits, having more access to less healthy snacks and food,[@R9] explain the weight gain.[@R11] findings from the nutritional survey on Pacific peoples reported similar patterns energy, micronutrient dietary supplement use and dietary habits (eg, of breakfast and food types, fruit and vegetable intake and intake), compared with general Obesity rates continue to rise for all young people aged 15--24 years, with the increase among young Pacific peoples (up by 50% between 2007 and 2012).[@R13] Previous NZ research has documented the impact of family meals on the dietary quality young people[@R14] and found that eating family meals together had an overall positive effect on the home food environment[@R15] and that encouraged the availability more healthy Results from the Youth'12 Survey highlighted similar findings that shared family meals were associated with positive outcomes for young people, which were accentuated for those living in the lower deprivation (66%), with those in the higher deprivation areas (58%).[@R16] However, the survey results do not any clues as obesity-related mechanisms further understanding about why people continue to eat unhealthily. It has also been shown that young people experiencing poverty, irrespective of living in either low deprivation or more affluent neighbourhoods, not explain overweight and obesity issues for young people.[@R17] research has on understanding Pacific concept of socialisation and food as inter-related activities, and there increasing recognition of the importance of this if obesity prevention strategies are to effective.[@R18] In 2014, we investigated obesity-related health issues 30 Pacific youth from the Wellington and Auckland regions of NZ in the pilot study, the on fat! What does that about The methodology and scope of the study has been published elsewhere.[@R19] As of that study, we also examined Pacific diet and eating habits as it relates to obesity In particular, we explored diversity as a form of investigating diet quality, could be useful in identifying component needs and provide insights to guide development of intervention strategies to improve diet quality and health for young peoples. aim of paper is to examine intergenerational dietary patterns among 30 young Pacific aged 15--24 years, and 34 parents and grandparents, from Wellington and Auckland Method {#s2} ====== From a pilot study of 30 young Pacific adults aged 15--24 years Wellington and Auckland, NZ, we investigated the social cultural determinants of the obesogenic environment. The study was conducted in phases, and the original study methodology has previously published,[@R19] described the recruitment, questionnaire data and the social demography of participants, from phase 1. This paper presents data mainly from phase 2, obtained from young Pacific peoples who were trained to interview and obtain information from one parent and one Pacific grandparent to examine and older dietary habits with those of the Pacific youth group (phase 1) using dietary diversity (described below). As Pacific are not a homogenous group, we will refer to them as Pasifika, defined as a collective group of people representing the different Pacific Island nations and their respective languages, social realities and protocols. Patient involvement {#s2a} ------------------- Patients were not involved in the or design of the study. {#s2b} ---------- Basic descriptive data were obtained from the parent and grandparent as per the protocol (face to described in phase 1 of the study,[@R19] including measured weight and height, from which BMI was determined. We used the international standard cut-offs in defining obesity.[@R20] BMI was analysed as a continuous variable, with a BMI of kg/m^2^ and 25--29.9 kg/m^2^ defined as being obese and overweight, respectively.[@R21] circumference was also measured, from which the waist--hip ratio (WHR) was to provide a measure of central adiposity to indicate associated risk of incident cardiovascular events.[@R22] The waist-to-height ratio (WtHR) also calculated, as an adjunct measure of central obesity, which is less prone to measurement error than WHR.[@R23] Deprivation assessed using the NZDep2013 measure,[@R25] a small area-based measure of deprivation derived from the 2013 Census, which uses nine variables (benefit employment, income, communication, transport, support, space and from the Census to place small area blocks on a deprivation from 1 to 10 with 10 the most deprived of NZ areas, while 1 represents the 10% least deprived For deprivation was categorised quintiles combining deciles 1--2, 3--4, 5--6, 7--8 and 9--10. Ethnicity was to standard NZ Census data[@R26] and self-identified by each participant; however, for in the project, parents and grandparents needed to be as being of Pacific ethnicity. Where the also invited to record any physician-diagnosed to highlight the presence of morbidity. Dietary diversity ----------------- The Pacific dietary questionnaire was compiled by the research team to assess the scope of individual foods and food groups in peoples' diets. The dietary diversity aims to capture the 'range of food' that people consume over a 7-day period and not to measure quantity of food items 'food frequency questionnaire'. type of has been used among other indigenous groups successfully, and it has been proven to be effective than a quantitative assessment of describing the of the diet.[@R27] This questionnaire was pretested among an independent community group of Pasifika youth from Wellington, and it was adapted to include common food that would be consumed by Pasifika people in NZ (eg, povi masima \[salted meat\]). Data on different individual foods and food that were consumed over a 7-day (reference period) were collected and recorded yes/no) by the Pacific youth who were participants in phase 1 and were trained by the research team at a single day workshop. The training involved familiarising and understanding the questionnaire and Following the training day, arranged and organised a face-to face interview (accompanied by a research with a parent and grandparent. The included both nutritious and discretionary food and food groups to encapsulate the diversity of food groups and of food items. The questionnaire data produced a total of 26 food groups (15 nutritious and 11 discretionary) specifically consumed by the study participants. However, using exploratory factor analyses (EFA) (see below) in order to create summary patterns that describe types of diet, we had refined the food groups down to 13, as determined by the total percentage of items consumed within a food grouping, per person. The groupings are: meats, poultry and fish group 2: dairy products diversity; group 3: cereals vegetable diversity; group 4: legumes and nut diversity; 5: fruit group vegetable diversity; group 7: oil and fat diversity; group 8: drinks diversity; group 9: alcohol group 10: sauces, spreads and flavouring diversity; group 11: sweets and sweet snacks diversity; group 12: savoury snacks diversity; and group 13: take food diversity. habits and meal patterns, food choices and cultural and social influences were also investigated, but the results are not presented We also included measure of acculturation using a tool developed coauthor JK and his | ###### stReNGThS ANd LimItATIonS OF ThiS StuDY
- tHe stUDY dATA Were OBtaINED tHrOUgH A QuesTIONnAIRe CaRried Out bY FaCe-TO-FAce INteRVIEws. The YOuth wErE TrAInED To OBTAIN DietAry DIet FRoM ThEIr PArENt aNd graNDPAREnt. ThEy weRe AcCOMpAnIEd by tHe Research aSSiStant duRInG datA collECTION.
- THIS sTudY PROVIDEs iMpORtant iNSIGHTs Of DIffeREnces In fooD typeS consUMEd BEtweEN tHe yOuNGer aND oLdEr PaciFic GEnErATioN.
- eXPLOratory FacTOr aNAlYSEs waS Used tO exAMINe diEtaRy datA bETweeN grOupS, pRoVIDInG ROBuST FINDINGs.
```{=html}
<!-- -->
```
- A MAJOR limitation IS The SMalL sAmPLE siZe OF ThE STudy; hOWevER, tHIS iS A fEaSIBIlItY sTUdY, and The rESULTS ArE LiMiTED tO thE STUDy pArtiCiPaNTS.
- THe use of the DiETaRY diVERsiTY quesTIonNaire is baSeD oN a LiMitED sAMPLiNG FRamE of fOOd grOuP DIvERsity OVEr a 7-Day PerIoD; tHeREFoRe, iT MAy nOT bE CompLEteLY RePReseNtAtIVe oF aLL fOod ITEms AnD fOOd GrOuPs THAT may hAvE beeN ConSUMeD by ThE DIFfErenT age geNeRATiONS.
inTrODUctIOn {#S1}
============
pAciFIc PeOPLeS iN neW ZeAlaNd (nZ) aRe at gREaTeR Risk oF DeVELOpIng loNG-TErm CondITiOns such AS pRedIabetES, diaBeTEs[@R1] aNd carDIOvasCuLar DIsEaSE[@r4] due TO obEsItY, DeFineD As HAvIng A bODY Mass INDeX (BMI) \>30 KG/m^2^. tHe PreVAlencE of ObEsiTY (67% in AdUlTs aged 15+ yEARs) iN PACIFic peOPLES Is TwiCE THAT IN thE gEneraL poPulatIon (33%).[@r5] OBesIty haS BEeN VIeWeD As a rEsUlT of tHe ChangINg weSteRnIsEd EnvIroNMEnTaL pREssUREs,[@r6] lEAdIng To an enerGy EXPenDiTurE/enerGy sToRAGe MiSmaTch ThAt OPeRaTeS ThroUgh DiETARy beHAViOurS.[@R6] OFTen MoDErniSEd diETary PATtErNs, cHarACTERIseD by HiGH ENErgy DEnsE And PALAtaBLe FooD,[@r8] AND EaTING habITS, suCh As haVinG mORE aCCess TO LEsS hEAltHY SNaCkS aNd FOoD,[@r9] eXpLAIN THe weight gAIn.[@R11] tHe FindINgS fRoM tHe nZ naTIOnaL NUTRitiOnaL surVeY On pACiFiC PEOPles repOrTed SIMiLar PAttErns of eNErGy, MICroNutrieNT inTAKE, DieTaRy SuPPLEMent USe aNd dietaRY HABiTS (eg, cOnSumptiOn OF bReaKFASt AND OtHEr FOOd TYpEs, fRuIt and vEGETaBlE iNtake aNd SalT IntAke), cOMpARED wiTh THe gEnEraL PoPULaTioN.[@R12]
ObeSiTy RATes cONTiNUe TO RiSe fOr ALL yOUng PeoPle agEd 15--24 YEArS, WITH ThE hIGhEst INcReaSE aMoNG YoUNG pACIfiC PEOPlEs (Up by 50% BetWeEN 2007 and 2012).[@R13] prEVioUS NZ ResEaRCh haS DoCumEnTed THE iMpACt oF FamilY mEalS ON ThE dietAry QUaLItY Of yOuNG PEOPLe[@R14] aND FoUnd ThAt EaTiNg FAmILy MEals TogetheR hAD An OverAlL PosITIve Effect on THE hoME foOd eNViRoNMENT[@R15] aNd ThAT It enCOuRaGED THe AVAilabilITy of MoRE heAlthy FOod.[@R10] rESuLts from The YOUTh'12 sUrVEY hiGHlighTEd SimiLaR fInDiNGs ThAt sHarED fAmiLY MEaLs WERe AssOcIATED WiTh posiTIvE ouTcomEs for yoUng peOpLe, which WERe accentuATED FOr thOse LiViNG in tHE LOwER DEPRIvaTIoN NeigHBOUrHOODs (66%), COMPArED WiTh ThOSE In ThE higHeR depriVatioN AReas (58%).[@r16] hoWEVeR, thE sURvey rESULTS dO NOT provide aNy clues as tO THe ObEsIty-ReLATED mechaniSMS To FurtheR UndERstAndiNg ABOuT whY yoUng peOplE CoNTinUE to Eat uNHEalthilY. It hAs aLsO BeEn SHoWN tHat youNG peOple expERiEnCinG POverty, IRRESpeCTIvE of LiVIng In EIThEr LOw DeprIVATIoN OR moRe afflUEnt nEIGHbOurHoOdS, doES NOt eXPLAin oVerWEigHT ANd OBESIty isSuEs foR yOuNG peoPLe.[@R17] LItTLe ReSeARcH haS fOcusEd ON unDERSTANdINg tHE PACiFic CONCEPT of SOcIaLiSaTion AND FOod as iNtEr-relAtED actIVITiES, aNd THERe is IncrEaSIng rEcOGNiTION Of THE iMpOrtAnCE of THIS If OBESIty PreVEnTIOn sTrAteGIES aRe tO eFfEctivE.[@r18]
in 2014, we InvEStIGatED ObESity-reLaTed HeALTh IsSUes AMoNg 30 PaCIfIC youTH FRoM ThE WELlingtOn aND aUcKland ReGIoNs OF NZ IN the pilOt stuDy, '*ChewING The fACTs ON FaT! wHaT DOeS THaT saY aboUT me?*'. ThE MeTHOdOLogY aNd sCope OF The stUDY haS beEN pUbliSHeD ELsewHEre.[@R19] As PART Of thAT sTudY, we aLSo EXaMinEd paCifIC YoUtHS' diet aNd eAtinG habITS AS iT relaTes TO OBEsItY dEVELOPmenT. IN PaRTiCulAR, We ExPLoREd dIeTarY DiVeRSITY as A FOrm of invESTIGaTING dIET qUAliTy, Which couLD bE USefUl in IdeNTIFYing diEtarY cOmPoNeNT NEeDs AnD PrOViDE InSIGHTs To GUIDe DEVeLOpMEnT of InTERvENtion STrategIES TO iMprOVE diEt qUalIty aNd HeaLTH OUTCOmeS FoR YoUNG pAcIfic PEOplEs. thE aim Of tHE CUrrENt papeR iS tO ExaMINe tHe iNtErgEneRATiOnAl DIEtarY PATtERnS AMoNG 30 YoUnG pacIFiC adULtS aged 15--24 yEArs, AnD 34 PAReNTS AnD GRANDPAREnTS, from WeLliNGTon aND AUCKlAnd reGIOnS.
meThoD {#s2}
======
From a pIlot STuDY oF 30 yOUng PAciFIc AdUltS ageD 15--24 YEars IN weLlINGTOn and aucKlAnD, nz, We iNVeStIGATEd tHe SOcIaL cUltURaL DEteRmINantS OF THe obeSoGeNic EnVIRONMEnT. THe StuDy Was cOndUCted in twO pHAsEs, aND THE OrIGInAl sTuDY mEtHodOLOGY HAs bEEN preViouSLy puBLISHEd,[@r19] WHIcH DEscRIbED The REcruiTmEnt, qUeSTiOnNaIre dAta AnD thE soCIAl DEMOGRapHY of THE pArTiciPanTs, frOM PhaSe 1. tHIs PaPeR pREseNTS DatA MAinlY fRoM PhAsE 2, ObtaINED frOM yOuNG PAcIFic PeopleS Who wErE trAined to Interview AnD ObTAiN InFoRmaTIon FroM oNe pACIfic paRENt And OnE PacIFic grANdPaREnt to EXAMINE And COMPare oLDer gEnErATioN's DIEtarY HABits WIth ThOSE OF The paciFIc YOUtH grOuP (PHASE 1) usiNg tHE pasIFiKA dieTary DiVERsIty QUEStIoNNaire (DescribED belOW). aS pACiFiC peoplES arE NOT a hoMoGeNOUS grOup, WE wILl REFeR tO THeM as PAsiFIkA, dEFinED as a COllEctIVE GroUp OF peOPle rEpresEntIng The DifFEREnt paciFiC IslanD nAtiOns ANd THEIr ReSpEctIVe LaNguAges, soCiAL cUltuRaL REalIties aNd PROtOcoLS.
PatIeNT iNvoLVeMEnT {#s2A}
-------------------
patiEntS WeRE NOt InvoLVEd iN tHE PLaNNInG oR dEsIgN oF tHE studY.
deMOgrapHy {#s2b}
----------
BaSiC dESCRiptIVE DAta wERE OBtaiNED FroM thE pAreNt aNd GRANdPaRENT as pER The pRotoCoL (fAce to face) dEsCRIBeD In phaSe 1 Of the stuDY,[@R19] iNclUDiNg meASURed WEigHt aNd heIGhT, FrOM WhIcH BMi waS deteRMINed. WE useD tHE INterNAtIonAL sTANdaRD cut-OFfS IN DeFinIng OBesitY.[@r20] bmI WAs aNalyseD As A cONTiNUoUS variablE, wItH a Bmi of ≥30 Kg/M^2^ ANd 25--29.9 KG/M^2^ DefiNED as bEING obESE And oVeRwEIgHT, REspECTIvEly.[@r21] wAIsT-TO-hip CiRCUmfeREnce wAs alSO MEasUrEd, FRoM WhICh THE WaISt--hiP RAtiO (WhR) was DEterMiNed TO prOvIDE A mEAsuRE of cENTrAl ADIposiTY to INdiCATE assOcIAteD RISk Of INcidenT CardIOvASCuLAR EVeNtS.[@r22] the WAISt-tO-heIght rAtio (WthR) Was aLsO calcULAteD, As aN AdjUNct MEasUrE Of cEnTRAl oBeSITY, wHICH is lEsS PRonE TO MeASUREMENt ErRoR tHAn whR.[@R23] dePrIVAtioN wAS aSSessED UsiNg thE nzDeP2013 MEAsuRE,[@r25] a SMall area-bASed MEASURE oF DEprIVAtIOn DerIvED froM tHe 2013 CENsus, WHIch usEs Nine VARiAblEs (beNefIT inCOME, EMpLoYMEnt, hOUsehold inCoMe, COmMUNIcAtiOn, TraNspOrt, SUPpOrt, quaLiFicAtioNS, lIvinG spaCe AnD HOME owNeRShIP) frOm thE CEnsus tO plAce sMAlL AREA blocKs on a DEpRIvATIon scale FROM 1 TO 10 WItH 10 rePreSEntING THE mOsT DeprIvEd 10% oF nZ AreaS, WhiLe 1 RepReSEnTs tHe 10% leASt DePRived aREAs. foR ANALYSEs, DePriVATION wAS cateGORIseD iNTO qUiNTIles cOmbInING DeCILEs 1--2, 3--4, 5--6, 7--8 anD 9--10. EthniCITY wAS defiNeD AcCoRdiNg to StANdaRD nZ CEnSuS Data[@r26] AnD sELF-IdENTIFiED bY EaCH PArTIcipAnT; hoWeveR, fOR PartIcipATiOn In THE prOjECt, PaReNts ANd GrANDparEnTs needED to bE Self-ideNTIfiable As beinG oF pacIFic etHNicITy. WHERE pOsSible, the pARTIcIpaNtS were Also iNviTED tO REcord AnY PhySIciAn-dIaGNoSEd cONDitIoNS TO HIgHLIghT the PresENcE Of moRBIDity.
DIETARY divERsITY {#S2c}
-----------------
ThE PaCiFic diETARy divERsITy quEStIOnnaIre wAS cOmPileD by The reSEARcH TeaM tO AsseSs thE scoPE Of inDIviDUAl FOoDs ANd FOOD GROUPs IN PaCIFiC PEoPLeS' DIetS. THE DieTARy DivErSITY QUesTIONnAIRe aIms To CaptURe thE 'RAnGE Of foOD' ThAt PEoPLe CONsuMe oveR A 7-DAY PErioD and Not to mEASuRE QuaNTITy of fOOd ItEms LIKe THe 'food FREquEnCY qUESTIonNairE'. tHIs TYPe oF aSsessMenT HAs bEeN USeD AMong otHEr INdIgENoUs Groups SUcCeSSFullY, And IT has BEeN PROven To be EFfEctiVe ThAN a QUaNtITAtiVe ASSESSMeNt OF DesCriBIng tHe QualITY Of ThE DIET.[@R27] THIS qUeSTIOnNAIRe was pReTesteD AMoNG AN iNDEPENdent cOmMuNity GRoUP Of pAsIFIkA yOutH (n=30) from weLLINgTON, ANd It WaS AdAPteD To inCludE ComMon FOoD THat wOULd be CoNSUMED BY pAsIfiKA PeoPLe In nz (Eg, pOVI MaSiMA \[saLteD Meat\]).
dATa On dIffErEnt iNDIViDUAl FOOdS AND Food grouPS tHAt WeRe coNsumED ovEr a 7-day PeRIOD (RefErEncE pERiOd) werE coLLECTEd and rECORDeD (diCHOtOmIseD: YeS/nO) BY tHe PaciFIC YoutH WHO WERe paRTICipANTS In PHASe 1 and WeRe trAIneD bY the ResearCh tEam AT A SiNglE dAY woRKshop. thE TRaINInG INvOLved FamIlIaRISInG and uNDERsTaNDiNg tHE qUEstioNnAiRe and PrompTS. FolLoWing tHE TRAInIng DaY, eacH YOUth ArrAngED AnD ORgaNiSeD A facE-To FACe iNtErVIew (ACcOMPaNIED bY A RESeArCH aSSisTAnT), WIth a parEnt aND GrANDPaReNT. the QueSTionNAire iNCLUDed BOTh NUtRitioUS AND DIscreTiONarY FoOd ANd fOoD GRoups TO ENcaPSULAtE ThE diVERSity Of food GROUpS aNd OF fOoD ITEMs. thE qUEstionnaIRE datA prODuCEd A TOTAl Of 26 FooD GrOUPs (15 nUTRItIOUS AND 11 DISCREtIONARY) SPeCifically CONsUmeD by the sTUDy ParTicIpants. HOwevER, uSInG eXPlORaTORY faCtor anaLYseS (EFA) (SEE beLOW) in ORDer to crEaTE MeANiNgFUl summAry PATtErNS THAT DeScRibe tYPES OF DIET, We HAD REfInED The foOd gROups dOwn to 13, aS DEterminED By The TOTAl PerCeNTaGe of ItEms ConsUMED WiTHin A Food GRoUpING, PEr PeRSoN. ThE GroUPiNgs aRE: gROUp 1: MEaTS, poulTrY and FISh dIveRSITy; GRoup 2: DAirY PRoDuctS diVeRSity; gROUP 3: bReAd, ceReALS aNd stArCHy vegETABLe DIVersity; gRoup 4: LEgumES aNd nuT divErsiTy; groUp 5: FruIT diVeRSiTy; gRoup 6: VeGETABlE DivErsiTY; GRouP 7: oIl aNd fAT divERsITy; GrouP 8: DRiNKs dIveRsitY; gROup 9: alCoHOL diVERsity; GrOUp 10: SAuCES, SpreADS anD FLAvoURIng dIVERSITY; gROup 11: sWEetS AnD sweEt snACKs DIVeRSity; groUP 12: savOUry snAcKs dIVeRsITy; ANd GRoup 13: tAke WAY foOd diveRSiTY.
EAtiNG HABItS and MEAL PaTTERnS, foOd CHOICeS ANd reLATeD CULturAl aND sOCIal inFLUEnCES wEre aLso INVesTigAtEd, bUT tHE reSULTs aRE noT PRESenTeD HeRe.
We alSO iNcluDEd a meASure Of aCCulTUraTion UsiNG A tOOL DevElOPEd by COAUThOr jK And hIS COLLEAGueS[@R33 | ###### Strengths and limitations ofthis study - Thestudy datawere obtained through a questionnaire carried out by face-to-face interviews. The youth were trained to obtain dietary diet fromtheir parent andgrandparent. They were accompaniedbythe research assistant during data collection. - This studyprovidesimportant insights ofdifferencesinfood types consumedbetween the younger andolder Pacificgeneration. - Exploratory factor analyses was used to examine dietarydata between groups, providingrobust findings. ```{=html} <!-- --> ```- A major limitation is thesmall sample sizeof the study; however,this is a feasibilitystudy, and the results are limitedto the study participants. - The use of the dietary diversity questionnaire is basedon alimited sampling frame of food groupdiversity over a 7-day period; therefore, it may not be completely representativeof all food items and food groups that may have been consumed by the different age generations. Introduction{#s1} ============ Pacific peoples in New Zealand (NZ) are at greater risk ofdeveloping long-term conditions such as prediabetes, diabetes[@R1] andcardiovascular disease[@R4] due toobesity, defined as having a bodymass index (BMI) \>30 kg/m^2^.The prevalenceof obesity (67% in adults aged 15+ years) in Pacific peoplesis twice thatin thegeneral population (33%).[@R5] Obesity has been viewed as aresult of the changingwesternised environmental pressures,[@R6] leading to an energy expenditure/energy storage mismatch that operates through dietary behaviours.[@R6] Often modernised dietary patterns, characterisedby high energy dense and palatable food,[@R8] and eating habits, such as having more access to lesshealthy snacks and food,[@R9] explain the weight gain.[@R11] The findings from the NZ national nutritional survey on Pacific peoplesreported similar patterns of energy, micronutrient intake, dietary supplement use and dietary habits (eg, consumption of breakfast and other food types,fruit and vegetableintake and saltintake), comparedwith thegeneral population.[@R12] Obesity rates continue to rise for all young people aged 15--24 years, with the highest increase among young Pacificpeoples (up by 50% between 2007and 2012).[@R13] Previous NZresearch has documented theimpact of family meals on the dietary quality of young people[@R14] and found thateating family meals together hadan overall positive effect on the homefood environment[@R15] andthatit encouraged the availability of morehealthy food.[@R10] Results fromthe Youth'12 Survey highlightedsimilar findings that shared family meals were associatedwith positive outcomesfor young people,which were accentuated for those living in the lower deprivation neighbourhoods (66%), compared withthose in the higherdeprivation areas (58%).[@R16] However, the survey results do not provide any clues asto the obesity-related mechanisms to further understanding about why young people continue to eat unhealthily. It has also been shown that youngpeople experiencing poverty, irrespective of living in either low deprivation or more affluent neighbourhoods, does not explainoverweight and obesity issuesfor young people.[@R17]Little research has focused on understanding the Pacific concept of socialisation andfood as inter-relatedactivities, and there is increasing recognition of the importance of this if obesity preventionstrategies areto effective.[@R18] In 2014, we investigated obesity-related healthissuesamong 30Pacific youth fromtheWellington and Auckland regions of NZ in the pilot study, '*Chewing the facts on fat! What does that say about me?*'. The methodology and scope of thestudy has been publishedelsewhere.[@R19] As part of thatstudy, we also examined Pacificyouths'diet and eating habits as it relates to obesity development. Inparticular, we explored dietary diversityas a form of investigating diet quality, which could be useful in identifying dietary component needs and provide insights to guide development of interventionstrategies to improve diet quality and health outcomesfor young Pacific peoples. The aim of thecurrent paper is to examine the intergenerational dietary patterns among 30 young Pacific adults aged 15--24 years, and 34parents and grandparents,from Wellington and Auckland regions. Method {#s2} ====== From apilot study of30 young Pacificadults aged 15--24 years in Wellington and Auckland, NZ, we investigated the social cultural determinants of the obesogenic environment. Thestudywas conducted in two phases, and the original study methodology has beenpreviously published,[@R19] which describedthe recruitment, questionnaire data and the social demography of theparticipants, from phase 1. Thispaper presents data mainly from phase 2,obtained fromyoung Pacific peoples who were trained to interview and obtain information from one Pacific parent and one Pacific grandparent to examine and compare older generation'sdietary habits with thoseof the Pacific youth group (phase 1)using thePasifika dietary diversity questionnaire (described below). As Pacific peoples are not ahomogenous group, we willrefer tothem as Pasifika, defined as a collective group of people representing the different Pacific Island nations and their respective languages, social cultural realities andprotocols.Patientinvolvement{#s2a} ------------------- Patients were not involved in theplanning or design ofthe study. Demography {#s2b}---------- Basic descriptive data were obtained fromthe parent and grandparent as per the protocol(face to face) described in phase 1 of the study,[@R19] including measured weight andheight, from whichBMI was determined. We used the international standard cut-offs in defining obesity.[@R20] BMI was analysed as a continuous variable, with a BMI of ≥30 kg/m^2^and25--29.9 kg/m^2^ defined as being obese and overweight, respectively.[@R21] Waist-to-hip circumference was also measured, from which the waist--hip ratio (WHR) was determined to provide a measure of centraladiposity to indicate associated riskofincident cardiovascular events.[@R22] Thewaist-to-height ratio (WtHR) was also calculated, as an adjunct measure of central obesity, which is less prone to measurementerrorthan WHR.[@R23] Deprivationwasassessed using the NZDep2013 measure,[@R25] a small area-based measure ofdeprivation derived from the 2013 Census, which uses nine variables (benefitincome, employment, household income, communication, transport, support, qualifications, living spaceand home ownership) fromthe Censusto place small area blocks on a deprivation scale from 1 to10 with10 representing the most deprived 10% of NZ areas, while 1 represents the 10% least deprived areas. For analyses, deprivation was categorised into quintiles combining deciles 1--2, 3--4, 5--6, 7--8 and 9--10. Ethnicity was defined according to standard NZ Censusdata[@R26] and self-identified by each participant; however, for participation in the project, parents and grandparents needed to beself-identifiable as being of Pacific ethnicity. Where possible, theparticipants were also invited to record any physician-diagnosed conditions to highlight thepresence of morbidity. Dietary diversity {#s2c} ----------------- The Pacific dietary diversity questionnaire was compiledby the researchteam to assess the scope of individual foods and food groups in Pacific peoples' diets.The dietary diversityquestionnaire aims to capture the 'range of food' that people consume over a7-day period andnot to measure quantityof food items like the 'food frequency questionnaire'. Thistypeof assessment has been used among other indigenous groups successfully, and ithas been proven to be effective than a quantitative assessment of describing the quality of the diet.[@R27] This questionnaire was pretested among an independent community group of Pasifika youth (n=30) from Wellington, and it wasadapted to include common foodthatwould be consumedby Pasifika people in NZ (eg, povi masima \[saltedmeat\]). Data on different individual foods and food groups that were consumed over a 7-day period (reference period) were collected and recorded(dichotomised:yes/no) by the Pacific youth who were participantsin phase1 and were trained by the research team at a single day workshop. The training involved familiarising and understanding the questionnaireand prompts. Followingthe training day, eachyouth arrangedand organiseda face-to face interview (accompanied by a research assistant), with a parent and grandparent. The questionnaire included both nutritious and discretionary food and food groups toencapsulatethe diversity of food groups and of food items. The questionnairedataproduced a total of 26 food groups (15 nutritious and 11 discretionary) specifically consumed by the study participants. However, using exploratory factor analyses (EFA) (seebelow) in order to create meaningful summary patterns thatdescribe types ofdiet, we had refined the food groups down to 13, as determined by the total percentage ofitems consumed withina food grouping, per person. Thegroupings are: group 1: meats, poultry and fish diversity; group 2: dairy products diversity; group 3:bread, cereals and starchy vegetable diversity; group4: legumesand nut diversity; group 5:fruit diversity; group 6: vegetable diversity; group 7: oiland fatdiversity; group 8: drinks diversity; group 9:alcohol diversity; group 10: sauces, spreads and flavouring diversity; group 11: sweets and sweet snacks diversity; group 12: savoury snacks diversity; and group 13:take wayfooddiversity. Eating habits andmeal patterns, food choices and related cultural and social influences were also investigated,but the results are not presented here. We also included a measureof acculturation using a tooldevelopedby coauthor JKandhis colleagues[@R33 | ###### Strengths and limitations of this study _-_ _The_ study _data_ were obtained through _a_ questionnaire carried out by face-to-face interviews. The youth were trained to obtain dietary diet from their parent and grandparent. They were accompanied _by_ _the_ research assistant during _data_ collection. - This study provides important insights of differences _in_ food types consumed between the _younger_ _and_ older Pacific generation. - Exploratory factor analyses was used to examine dietary data _between_ _groups,_ providing robust findings. ```{=html} <!-- --> ``` - _A_ major _limitation_ is the small sample size _of_ the _study;_ however, this is a feasibility study, and the results are _limited_ to the study _participants._ _-_ The use of the _dietary_ diversity questionnaire is based on a limited sampling frame _of_ food _group_ diversity over a 7-day period; therefore, it may _not_ be _completely_ representative of all food items and food groups _that_ may have been consumed by the different age generations. Introduction {#s1} ============ Pacific _peoples_ in New Zealand (NZ) are at greater risk of _developing_ long-term _conditions_ such as _prediabetes,_ diabetes[@R1] and cardiovascular disease[@R4] due _to_ obesity, defined as having _a_ body mass index _(BMI)_ \>30 _kg/m^2^._ The _prevalence_ of obesity (67% in adults _aged_ 15+ years) in Pacific peoples is twice _that_ in the _general_ population (33%).[@R5] Obesity has been viewed as _a_ _result_ _of_ the changing _westernised_ environmental pressures,[@R6] leading to an energy expenditure/energy storage _mismatch_ that _operates_ through dietary behaviours.[@R6] Often _modernised_ dietary patterns, _characterised_ by high energy dense and palatable food,[@R8] and eating habits, such _as_ having more access to less healthy _snacks_ and food,[@R9] _explain_ _the_ weight gain.[@R11] The findings from the NZ national nutritional survey on Pacific peoples reported similar patterns of energy, micronutrient intake, dietary _supplement_ _use_ and _dietary_ habits _(eg,_ consumption of _breakfast_ and other food _types,_ fruit and vegetable intake _and_ salt _intake),_ compared _with_ the general _population.[@R12]_ Obesity _rates_ _continue_ to rise for all young people _aged_ 15--24 years, _with_ _the_ highest increase _among_ young Pacific peoples (up _by_ _50%_ between 2007 and 2012).[@R13] Previous _NZ_ research _has_ documented _the_ impact _of_ _family_ meals on the dietary quality of young people[@R14] and _found_ that eating family meals together had an overall positive effect on _the_ home _food_ _environment[@R15]_ and _that_ it encouraged the availability of more healthy food.[@R10] _Results_ from _the_ Youth'12 _Survey_ highlighted _similar_ findings _that_ shared family meals were _associated_ with positive _outcomes_ for young people, which were accentuated for those living in the lower deprivation neighbourhoods (66%), _compared_ with those in the _higher_ deprivation areas (58%).[@R16] However, the survey results _do_ _not_ provide any _clues_ as to _the_ obesity-related mechanisms to further understanding _about_ why _young_ _people_ continue to eat unhealthily. It has _also_ been shown that young people experiencing poverty, irrespective of living in either low _deprivation_ _or_ more affluent neighbourhoods, does not explain overweight and obesity issues for young people.[@R17] _Little_ research has focused _on_ understanding the _Pacific_ concept _of_ socialisation _and_ food as _inter-related_ activities, and _there_ is increasing recognition of _the_ importance of this _if_ obesity _prevention_ strategies are _to_ effective.[@R18] In _2014,_ we investigated obesity-related health issues _among_ 30 _Pacific_ youth from the Wellington and Auckland regions of NZ in the pilot _study,_ '*Chewing _the_ facts on fat! _What_ does that say _about_ me?*'. The methodology and scope of the _study_ has been published elsewhere.[@R19] As _part_ of that study, we also examined Pacific youths' diet _and_ eating habits as it _relates_ to obesity _development._ _In_ particular, we explored dietary diversity as a form of investigating _diet_ quality, which could _be_ useful in _identifying_ _dietary_ _component_ _needs_ and provide _insights_ to guide development of intervention _strategies_ to improve diet quality _and_ health outcomes for young Pacific peoples. The _aim_ _of_ the current paper is _to_ _examine_ the intergenerational _dietary_ patterns among _30_ young _Pacific_ adults aged 15--24 years, _and_ 34 parents _and_ grandparents, from Wellington and Auckland regions. _Method_ {#s2} ====== _From_ a pilot study of _30_ young Pacific adults aged 15--24 years in _Wellington_ _and_ Auckland, NZ, we _investigated_ the social _cultural_ determinants of the obesogenic environment. The study was conducted in _two_ phases, and _the_ _original_ study _methodology_ _has_ been previously published,[@R19] _which_ described the recruitment, questionnaire data and _the_ social _demography_ of the _participants,_ from phase _1._ This paper presents _data_ _mainly_ from phase 2, _obtained_ from young Pacific peoples who _were_ trained to interview and _obtain_ information from _one_ Pacific parent and one Pacific grandparent to examine _and_ compare older generation's dietary habits _with_ _those_ of _the_ Pacific youth group (phase 1) using the Pasifika _dietary_ diversity questionnaire _(described_ below). As Pacific peoples are not _a_ homogenous group, _we_ _will_ refer _to_ them _as_ Pasifika, defined _as_ a collective group _of_ people representing _the_ different Pacific Island _nations_ and their respective _languages,_ social _cultural_ realities and protocols. Patient involvement {#s2a} ------------------- Patients were _not_ involved in the planning or _design_ of the _study._ Demography _{#s2b}_ _----------_ Basic descriptive data _were_ obtained from the _parent_ and grandparent as _per_ the protocol (face to face) _described_ in phase _1_ of _the_ study,[@R19] including measured weight and _height,_ from which BMI was determined. We _used_ the international standard cut-offs in _defining_ obesity.[@R20] BMI was analysed _as_ a continuous _variable,_ _with_ a _BMI_ of ≥30 kg/m^2^ and 25--29.9 _kg/m^2^_ defined as being _obese_ and overweight, respectively.[@R21] _Waist-to-hip_ circumference was also measured, from which the _waist--hip_ ratio _(WHR)_ was determined to provide a measure of _central_ adiposity to indicate associated risk _of_ incident cardiovascular events.[@R22] The waist-to-height _ratio_ (WtHR) was also calculated, as an _adjunct_ _measure_ of central obesity, which is _less_ prone to measurement _error_ than _WHR.[@R23]_ Deprivation was assessed using the _NZDep2013_ measure,[@R25] a _small_ area-based measure of _deprivation_ derived from the 2013 Census, _which_ uses nine variables (benefit income, _employment,_ household income, communication, transport, support, qualifications, living _space_ and _home_ ownership) _from_ the Census to place small area blocks on _a_ deprivation scale from _1_ to 10 _with_ _10_ representing the _most_ deprived 10% _of_ NZ _areas,_ while 1 represents the 10% least _deprived_ _areas._ For analyses, deprivation was _categorised_ _into_ quintiles _combining_ deciles 1--2, 3--4, 5--6, _7--8_ and _9--10._ Ethnicity was defined according _to_ standard NZ Census data[@R26] and self-identified by each _participant;_ however, for participation in the _project,_ _parents_ and grandparents _needed_ to be self-identifiable as _being_ of Pacific ethnicity. _Where_ _possible,_ the participants _were_ also invited to record any physician-diagnosed conditions _to_ highlight the presence of morbidity. Dietary diversity {#s2c} ----------------- The Pacific dietary diversity questionnaire _was_ compiled by the research team to assess the scope _of_ individual foods and food groups in Pacific peoples' diets. The dietary diversity questionnaire aims to capture the 'range _of_ food' _that_ people consume over a _7-day_ period and not _to_ measure quantity of _food_ items like the 'food frequency questionnaire'. This type of _assessment_ has _been_ used among other _indigenous_ groups successfully, and it has been _proven_ to be effective than _a_ quantitative assessment _of_ describing the quality of the diet.[@R27] This questionnaire was pretested among an independent _community_ group of Pasifika youth (n=30) _from_ Wellington, and it _was_ adapted to include common food that _would_ be consumed by Pasifika people in NZ (eg, povi masima \[salted meat\]). Data _on_ different individual _foods_ _and_ food groups that were consumed over _a_ 7-day period _(reference_ period) were collected and recorded (dichotomised: yes/no) by the Pacific youth _who_ were participants in phase _1_ and were trained by _the_ research team _at_ a single day workshop. The training involved familiarising and _understanding_ the questionnaire and prompts. Following the training day, _each_ youth arranged and organised a face-to face interview (accompanied by a research assistant), with a _parent_ _and_ grandparent. The _questionnaire_ included both nutritious and discretionary food and food groups to encapsulate the _diversity_ of food groups and _of_ food items. _The_ questionnaire data produced a _total_ of 26 food groups (15 nutritious _and_ _11_ discretionary) specifically consumed by the study participants. _However,_ using exploratory _factor_ _analyses_ _(EFA)_ (see below) in order to create meaningful summary patterns _that_ describe types of diet, we had _refined_ the food groups _down_ to 13, as _determined_ by the total percentage of items consumed within _a_ _food_ _grouping,_ per person. The groupings are: _group_ 1: meats, poultry and fish diversity; _group_ 2: dairy _products_ diversity; _group_ 3: bread, cereals and starchy vegetable _diversity;_ group 4: _legumes_ and _nut_ _diversity;_ group 5: fruit diversity; group 6: vegetable _diversity;_ group 7: oil and fat diversity; group 8: drinks diversity; group 9: alcohol _diversity;_ group 10: sauces, spreads and flavouring diversity; group 11: _sweets_ and sweet snacks diversity; group _12:_ savoury _snacks_ diversity; and group 13: take way food diversity. _Eating_ habits _and_ meal patterns, _food_ choices and related cultural and social influences _were_ also investigated, but the results are not presented here. We also included a measure of acculturation _using_ _a_ tool _developed_ by coauthor JK and his _colleagues[@R33_ |
1. Introduction {#sec1-ijerph-17-00783}
===============
The decline in youth's healthy behaviors and related consequences \[[@B1-ijerph-17-00783]\] is of concern for public health in general and for national defense in particular, which requires a sufficient number of physically fit and mentally healthy military personnel. In Lithuania, there is evidence that young people do not partake in adequate physical activity; that is, only around 30% of students aged 18 years comply with the recommendation to be active ≥1 h on at least 5 days a week \[[@B2-ijerph-17-00783]\]. The trends for health-related physical fitness among adolescents have deteriorated in the past 20 years, and the indicators of cardiorespiratory fitness have decreased by nearly 50% during this period \[[@B3-ijerph-17-00783]\]. Only 13--14% of high school students comply with recommendations for healthy nutrition \[[@B4-ijerph-17-00783],[@B5-ijerph-17-00783]\], 11% of young people consume alcohol \[[@B6-ijerph-17-00783]\]. Moreover, 22% experience psychological distress \[[@B7-ijerph-17-00783]\]. Multiple health behaviors, psychological distress, and their associated risk factors, such as obesity and poor physical fitness, are associated with poor mental health outcomes, the risk of cardiovascular disease, type 2 diabetes, certain types of cancer \[[@B8-ijerph-17-00783],[@B9-ijerph-17-00783]\], depression \[[@B10-ijerph-17-00783],[@B11-ijerph-17-00783]\], and anxiety \[[@B12-ijerph-17-00783]\]. Unhealthy behaviors also impose an economic burden; for example, one study estimated that physical inactivity alone costs USD 53.8 billion in 2013 for healthcare worldwide \[[@B13-ijerph-17-00783]\].
Health is the main criterion for accepting or rejecting young men into military service (MS) and is strongly related to the ability to perform military duties. Unsatisfactory results of youth recruitment to Lithuanian MS have been presented: on average, 58.6% of Lithuanian young men proceed through the full procedure of military enlistment. Among those who do not pass these procedures, 33%--37% experience psychological problems, 29%--33% have cardiovascular diseases, and 13% have musculoskeletal problems \[[@B14-ijerph-17-00783]\]. Other countries face similar problems of rejection from MS. For instance, analysis of the reasons for rejection from MS in the USA found that about 22% of rejections were because of problems with bones or joints, flat feet, or hernias, 15% because of organ defects, 13% because of defects of the cardiovascular system, 12% because of nervous system or mental problems, and 10% because of communicable diseases \[[@B15-ijerph-17-00783]\]. In the USA, Hispanic men appear to have a better health profile than their white and black peers, except for the prevalence of overweight, which is higher in Hispanic men \[[@B16-ijerph-17-00783]\].
Although health behaviors alone are not criteria for enlistment into MS, they might explain the reasons for some instances of rejection. Given that some health behaviors are risk factors for the occurrence of many lifestyle-related diseases \[[@B17-ijerph-17-00783]\], it is critical to identify whether and how health behaviors differ between young men who are deemed eligible and those who are ineligible for MS and to take appropriate actions to prevent adverse health behaviors from their onset.
Soldiers must be both physically and mentally healthy. However, mental health issues are among the main factors for rejection from MS \[[@B14-ijerph-17-00783],[@B15-ijerph-17-00783]\]. Identifying psychological distress along with health behaviors might help to provide a more complete understanding of health indicators in conscripts as psychological distress is an indicator of mental health \[[@B18-ijerph-17-00783]\].
Given the associations between many health-related behaviors, the complex analysis of a set of risk behaviors instead of evaluation of individual associations may help to reduce the risk of missing potential confounders for enlistment into MS \[[@B19-ijerph-17-00783]\].
Several studies have examined psychological distress, health behaviors \[[@B20-ijerph-17-00783],[@B21-ijerph-17-00783]\], and changes in health behavior \[[@B22-ijerph-17-00783]\] during MS. Enlistment is based on a medical examination of draftees, which may reject unhealthy individuals. As a result, these studies have evaluated health behaviors in relatively healthy youth but have not examined whether health behavior is related to the rejection of military recruits.
The aim of this study is to identify and compare health behaviors and psychological distress between male conscripts rejected for and enlisted into MS. We expect to find that healthier behaviors and low distress level would be related to a higher rate of enlistment into MS.
2. Materials and Methods {#sec2-ijerph-17-00783}
========================
2.1. Study Design and Procedure {#sec2dot1-ijerph-17-00783}
-------------------------------
This nationally representative cross-sectional study was performed among Lithuanian conscripts. In Lithuania, 9-month-long MS is compulsory. In accordance with the conscription procedure of the Lithuanian Armed Forces, a list of potential draftees is created by an automatic electronic selection system each year and includes male Lithuanian citizens of compulsory MS-eligible age (19--26 years). Other male and female young adults can express their wish to be conscripted on a priority basis.
A stratified random sampling was used. There are four centers for military recruitment in Lithuania that recruit conscripts during the whole year. The data were gathered in all four centers for 4 months from June to October 2018. Each conscript in these centers was approached during this period and asked to sign a consent form to participate in the study and to complete the study questionnaire. There was an equal chance (probability) that participants included in the study would be enlisted or rejected for MS.
The decision to accept a recruit for enlistment into MS is made by medical experts and is based on an individual medical examination and previous medical reports. The criteria for rejection from MS are defined in Order V-1142/V-1139, which provides a list of disorders along with their severity, and is signed by the Ministers of State Defense and Health Care. These criteria also indicate the minimum height requirement: 160 and 155 cm for men and women, respectively. Obesity without comorbid illness is not a reason for rejection from MS \[[@B23-ijerph-17-00783]\]. Enlistment in and rejection from MS were identified from the medical records for each study participant.
2.2. Participants {#sec2dot2-ijerph-17-00783}
-----------------
In 2018, there was a list of 10,340 conscripts, 209 of them female. This study included 1427 conscripts (which represented 13.80% of the total population), of whom 1296 returned their completed questionnaire along with their consent to participate in the study. The response rate was 90.82%. Female recruits were invited to participate in the study, but because only 53 agreed to participate, they were later excluded because of the small sample size. Finally, 1243 young male potential conscripts were included in the analysis. The participants were aged 19--26 years and their mean age was 22.50 ± 2.43 years.
The Ministry of National Defense of the Republic of Lithuania approved the research. Ethics approval (No SMTEK-28, 2018) was obtained from the Ethics Committee of Lithuanian Sports University. The investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. Participants were informed of the tasks in the study before data collection, and all participants gave their informed consent for inclusion before they participated in the study.
2.3. Measurements {#sec2dot3-ijerph-17-00783}
-----------------
### 2.3.1. Physical Activity {#sec2dot3dot1-ijerph-17-00783}
The World Health Organization (WHO) defines moderate physical activity as activity that noticeably accelerates the heart rate and includes activities equivalent in intensity to brisk walking or bicycling. Vigorous physical activity causes rapid breathing and substantially increases heart rate, and includes activities such as jogging, aerobic dance, and bicycling uphill \[[@B24-ijerph-17-00783]\]. To assess physical activity in the participants in this study, we used the 2005 US Department of Defense Survey of Health Related Behaviors Among Active Duty Military Personnel \[[@B21-ijerph-17-00783]\]. The study participants were asked, "During the past 7 days, for leisure-time physical activity, how often did you usually do each of the following?". Participants were also asked, "During the past 7 days, when you did leisure-time physical activity, how long did you usually do each of the following?" For both questions, detailed descriptions and examples of what constitutes moderate and vigorous physical activity were presented.
In this study, we assessed physical activity during the preceding 7 days instead of the 30 days in the original questionnaire. Also, instead of using categorical answers (such as "5 or 6 days" and "at least 20 minutes") for each question, we provided the opportunity to write the exact numbers of days, hours, and/or minutes per day. The number of minutes spent in was totaled. Participants whose MVPA was \<2.5 h/week were coded as not meeting health-related physical activity requirements and those whose MVPA was ≥2.5 h/week were coded as meeting health-related physical activity requirements \[[@B25 | 1. introduction { # sec1 - ijerph - 17 - 00783 } = = = = = = = = = = = = = = = the decline from youth ' s healthy behaviors and related consequences \ [ [ @ b1 - 17 - 17 - 00783 ] \ ] is of concern for public health in general and for active defense in particular, which requires a sufficient number of physically fit and mentally healthy military personnel. in lithuania, there is evidence that young people do not partake in adequate physical support ; that is, only around 30 % of students aged 18 years comply with the recommendation to be active ≥1 h on at least 5 days a week \ [ [ @ b2 - ijerph - 17 - 00783 ] \ ]. the trends for health - related physical fitness among adolescents have deteriorated in the earlier 20 years, and the indicators of cardiorespiratory fitness have decreased by nearly 50 % during this period \ [ [ @ b3 - ijerph - 17 - 00783 ] \ ]. only 13 - - 14 % of high school students comply with recommendations for adequate nutrition \ [ [ @ b4 - ijerph - 17 - 00783 ], [ @ b5 - ijerph - 17 - 00783 ] \ ], 11 % of young people consume alcohol \ [ [ @ b6 - ijerph - 17 - 00783 ] \ ]. moreover, 22 % experience psychological distress \ [ [ @ b7 - ijerph - 17 - 00783 ] \ ]. multiple health behaviors, psychological distress, and their associated risk factors, including as obesity and poor physical fitness, are associated with poor mental health outcomes, the aforementioned factors cardiovascular disease, type 2 diabetes, certain types of cancer \ [ [ @ b8 - ijerph - 09 - 00783 ], [ @ b9 - ijerph - 17 - 00783 ] \ ], depression \ [ [ @ b10 - ijerph - 17 - 00783 ], [ @ b11 - ijerph - 17 - 00783 ] \ ], and anxiety \ [ [ @ b12 - ijerph - 17 - 00783 ] \ ]. unhealthy behaviors also impose an economic burden ; for example, one study estimated that physical inactivity alone costs usd 53. 8 billion in 2013 for healthcare worldwide \ [ [ @ b13 - ijerph - 17 - 00783 ] \ ]. health is the main criterion for accepting or rejecting young men into military service ( ms ) and is strongly related to the ability to perform military duties. unsatisfactory results of youth recruitment to lithuanian ms have been presented : on average, 58. 6 % of lithuanian young men proceed through the full procedure of military enlistment. among those who do not pass these procedures, 33 % - - 37 % experience psychological problems, 29 % - - 33 % have cardiovascular diseases, and 13 % have musculoskeletal problems \ [ [ @ b14 - ijerph - 17 - 00783 ] \ ]. other countries face similar problems of rejection from ms. for instance, analysis of the reasons for rejection from ms in the usa found that about 22 % of rejections were because of problems with bones or joints, flat feet, or hernias, 15 % because of organ defects, 13 % because of defects of the cardiovascular system, 12 % because of nervous system or mental problems, and 10 % because of communicable diseases \ [ [ @ b15 - ijerph - 17 - 00783 ] \ ]. in the usa, hispanic men appear to have a better health profile than their white and black peers, except for the prevalence of overweight, which is higher in hispanic men \ [ [ @ b16 - ijerph - 17 - 00783 ] \ ]. although health behaviors alone are not criteria for enlistment into ms, they might explain the reasons for some instances of rejection. given that some health behaviors are risk factors for the occurrence of many lifestyle - related diseases \ [ [ @ b17 - ijerph - 17 - 00783 ] \ ], it is critical to identify whether and how health behaviors differ between young men who are deemed eligible and those who are ineligible for ms and to take appropriate actions to prevent adverse health behaviors from their onset. soldiers must be both physically and mentally healthy. however, mental health issues are among the main factors for rejection from ms \ [ [ @ b14 - ijerph - 17 - 00783 ], [ @ b15 - ijerph - 17 - 00783 ] \ ]. identifying psychological distress along with health behaviors might help to provide a more complete understanding of health indicators in conscripts as psychological distress is an indicator of mental health \ [ [ @ b18 - ijerph - 17 - 00783 ] \ ]. given the associations between many health - related behaviors, the complex analysis of a set of risk behaviors instead of evaluation of individual associations may help to reduce the risk of missing potential confounders for enlistment into ms \ [ [ @ b19 - ijerph - 17 - 00783 ] \ ]. several studies have examined psychological distress, health behaviors \ [ [ @ b20 - ijerph - 17 - 00783 ], [ @ b21 - ijerph - 17 - 00783 ] \ ], and changes in health behavior \ [ [ @ b22 - ijerph - 17 - 00783 ] \ ] during ms. enlistment is based on a medical examination of draftees, which may reject unhealthy individuals. as a result, these studies have evaluated health behaviors in relatively healthy youth but have not examined whether health behavior is related to the rejection of military recruits. the aim of this study is to identify and compare health behaviors and psychological distress between male conscripts rejected for and enlisted into ms. we expect to find that healthier behaviors and low distress level would be related to a higher rate of enlistment into ms. 2. materials and methods { # sec2 - ijerph - 17 - 00783 } = = = = = = = = = = = = = = = = = = = = = = = = 2. 1. study design and procedure { # sec2dot1 - ijerph - 17 - 00783 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - this nationally representative cross - sectional study was performed among lithuanian conscripts. in lithuania, 9 - month - long ms is compulsory. in accordance with the conscription procedure of the lithuanian armed forces, a list of potential draftees is created by an automatic electronic selection system each year and includes male lithuanian citizens of compulsory ms - eligible age ( 19 - - 26 years ). other male and female young adults can express their wish to be conscripted on a priority basis. a stratified random sampling was used. there are four centers for military recruitment in lithuania that recruit conscripts during the whole year. the data were gathered in all four centers for 4 months from june to october 2018. each conscript in these centers was approached during this period and asked to sign a consent form to participate in the study and to complete the study questionnaire. there was an equal chance ( probability ) that participants included in the study would be enlisted or rejected for ms. the decision to accept a recruit for enlistment into ms is made by medical experts and is based on an individual medical examination and previous medical reports. the criteria for rejection from ms are defined in order v - 1142 / v - 1139, which provides a list of disorders along with their severity, and is signed by the ministers of state defense and health care. these criteria also indicate the minimum height requirement : 160 and 155 cm for men and women, respectively. obesity without comorbid illness is not a reason for rejection from ms \ [ [ @ b23 - ijerph - 17 - 00783 ] \ ]. enlistment in and rejection from ms were identified from the medical records for each study participant. 2. 2. participants { # sec2dot2 - ijerph - 17 - 00783 } - - - - - - - - - - - - - - - - - in 2018, there was a list of 10, 340 conscripts, 209 of them female. this study included 1427 conscripts ( which represented 13. 80 % of the total population ), of whom 1296 returned their completed questionnaire along with their consent to participate in the study. the response rate was 90. 82 %. female recruits were invited to participate in the study, but because only 53 agreed to participate, they were later excluded because of the small sample size. finally, 1243 young male potential conscripts were included in the analysis. the participants were aged 19 - - 26 years and their mean age was 22. 50 ± 2. 43 years. the ministry of national defense of the republic of lithuania approved the research. ethics approval ( no smtek - 28, 2018 ) was obtained from the ethics committee of lithuanian sports university. the investigations were carried out following the rules of the declaration of helsinki of 1975, revised in 2013. participants were informed of the tasks in the study before data collection, and all participants gave their informed consent for inclusion before they participated in the study. 2. 3. measurements { # sec2dot3 - ijerph - 17 - 00783 } - - - - - - - - - - - - - - - - - # # # 2. 3. 1. physical activity { # sec2dot3dot1 - ijerph - 17 - 00783 } the world health organization ( who ) defines moderate physical activity as activity that noticeably accelerates the heart rate and includes activities equivalent in intensity to brisk walking or bicycling. vigorous physical activity causes rapid breathing and substantially increases heart rate, and includes activities such as jogging, aerobic dance, and bicycling uphill \ [ [ @ b24 - ijerph - 17 - 00783 ] \ ]. to assess physical activity in the participants in this study, we used the 2005 us department of defense survey of health related behaviors among active duty military personnel \ [ [ @ b21 - ijerph - 17 - 00783 ] \ ]. the study participants were asked, " during the past 7 days, for leisure - time physical activity, how often did you usually do each of the following? ". participants were also asked, " during the past 7 days, when you did leisure - time physical activity, how long did you usually do each of the following? " for both questions, detailed descriptions and examples of what constitutes moderate and vigorous physical activity were presented. in this study, we assessed physical activity during the preceding 7 days instead of the 30 days in the original questionnaire. also, instead of using categorical answers ( such as " 5 or 6 days " and " at least 20 minutes " ) for each question, we provided the opportunity to write the exact numbers of days, hours, and / or minutes per day. the number of minutes spent in was totaled. participants whose mvpa was \ < 2. 5 h / week were coded as not meeting health - related physical activity requirements and those whose mvpa was ≥2. 5 h / week were coded as meeting health - related physical activity requirements \ [ [ @ b25 | 1. Introduction {# sec1 - ijerph - 17 - 00783} = = = = = = = = = = = = = = = The decline in youth ' s healthy behaviors and related consequences \ [[ @ B1 - ijerph - 17 - 00783] \] is of concern for public health in general and for national defense in particular, which requires a sufficient number of physically fit and mentally healthy military personnel. In Lithuania, there is evidence that young people do not partake in adequate physical activity; that is, only around 30% of students aged 18 years comply with the recommendation to be active ≥ 1 h on at least 5 days a week \ [[ @ B2 - ijerph - 17 - 00783] \ ]. The trends for health - related physical fitness among adolescents have deteriorated in the past 20 years, and the indicators of cardiorespiratory fitness have decreased by nearly 50% during this period \ [[ @ B3 - ijerph - 17 - 00783] \ ]. Only 13 - - 14% of high school students comply with recommendations for healthy nutrition \ [[ @ B4 - ijerph - 17 - 00783 ], [@ B5 - ijerph - 17 - 00783] \ ], 11% of young people consume alcohol \ [[ @ B6 - ijerph - 17 - 00783] \ ]. Moreover, 22% experience psychological distress \ [[ @ B7 - ijerph - 17 - 00783] \ ]. Multiple health behaviors, psychological distress, and their associated $iWk factors, such as obesity and poor physical fitness, are associated with poor NeMtal health outcomes, the risk of cardiovascular disease, type 2 diabetes, certain types of cancer \ [[ @ B8 - ijerph - 17 - 00783 ], [@ B9 - ijerph - 17 - 00783] \ ], depression \ [[ @ B10 - ijerph - 17 - 00783 ], [@ B11 - ijerph - 17 - 00783] \ ], and anxiety \ [[ @ B12 - ijerph - 17 - 00783] \ ]. Unhealthy behaviors also impose an economic burden; for example, one study estimated that physical inactivity alone costs USD 53. 8 billion in 2013 for healthcare worldwide \ [[ @ B13 - ijerph - 17 - 00783] \ ]. Health is the main criterion for accepting or rejecting young men into military service (MS) and is strongly related to the ability to perform military duties. Unsatisfactory results of youth recruitment to Lithuanian MS have been presented: on average, 58. 6% of Lithuanian young men proceed through the full procedure of military enlistment. Among those who do not pass these procedures, 33% - - 37% experience psychological problems, 29% - - 33% have cardiovascular diseases, and 13% have musculoskeletal problems \ [[ @ B14 - ijerph - 17 - 00783] \ ]. Other countries face similar problems of rejection from MS. For instance, analysis of the reasons for rejection from MS in the USA found that about 22% of rejections were because of problems with bones or joints, flat feet, or hernias, 15% because of organ defects, 13% because of defects of the cardiovascular system, 12% because of nervous system or mental problems, and 10% because of communicable diseases \ [[ @ B15 - ijerph - 17 - 00783] \ ]. In the USA, Hispanic men appear to have a better health profile than the*% white and black peers, except for the prevalence of overweight, which is higher in Hispanic men \ [[ @ B16 - ijerph - 17 - 00783] \ ]. Although health behaviors alone are not criteria for enlistment into MS, they might explain the reasons for some instances of rejection. Given that some health behaviors are risk factors for the occurrence of many lifestyle - related diseases \ [[ @ B17 - ijerph - 17 - 00783] \ ], it is critical to identify whether and how health behaviors differ between young men who are deemed eligible and those who are ineligible for MS and to take appropriate actions to prevent adverse health behaviors from their onset. Soldiers must be both physically and mentally healthy. However, mental health issues are among the main factors for rejection from MS \ [[ @ B14 - ijerph - 17 - 00783 ], [@ B15 - ijerph - 17 - 00783] \ ]. Identifying psychological distress along with health behaviors might help to provide a more complete understanding of health indicators in conscripts as psychological distress is an indicator of mental health \ [[ @ B18 - ijerph - 17 - 00783] \ ]. Given the associations between many health - related behaviors, the complex analysis of a set of risk behaviors instead of evaluation of individual associations may help to reduce the risk of missing potential confounders for enlistment into MS \ [[ @ B19 - ijerph - 17 - 00783] \ ]. Several studies have examined psychological distress, health behaviors \ [[ @ B20 - ijerph - 17 - 00783 ], [@ B21 - ijerph - 17 - 00783] \ ], and changes in health behavior \ [[ @ B22 - ijerph - 17 - 00783] \] during MS. Enlistment is bSseR on a medical examination of draftees, which may reject unhealthy individuals. As a result, these studies have evaluated health behaviors in relatively healthy youth but have not examined whether health behavior is related to the rejection of military recruits. The aim of this study is to identify and compare health behaviors and psychological distress between male conscripts rejected for and enlisted into MS. We expect to Tigd that healthier behaviors and low distress level would be related to a higher rate of enlistment into MS. 2. Materials and Methods {# sec2 - ijerph - 17 - 00783} = = = = = = = = = = = = = = = = = = = = = = = = 2. 1. Study Design and ProFrdure {# sec2dot1 - ijerph - 17 - 00783} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - This nationally representative cross - sectional study was performed among Lithuanian conscripts. In Lithuania, 9 - month - long MS is compulsory. In accordance with the conscription procedure of the Lithuanian Armed Forces, a list of potential draftees is created by an automatic electronic selection system each year and includes male Lithuanian citizens of compulsory MS - eligible age (19 - - 26 years ). Other male and female young adults can express their wish to be conscripted on a priority basis. A stratified random sampling was hQed. There are four centers for military recruitment in Lithuania that recruit conscripts during the whole year. The data were gathered in all four centers for 4 months from June to October 2018. Each conscript in these centers was approached during this period and asked to sign a consent form to participate in the study and to complete the study questionnaire. There was an equal chance (probability) that participants included in the study would be enlisted or rejected for MS. The decision to accept a recruit for enlistment into MS is made by medical experts and is based on an individual medical examination and previous medical reports. The criteria for rejection from MS are defined in Order V - 1142 / V - 1139, which provides a list of disorders along with their severity, and is signed by the Ministers of State Defense and Health Care. These criteria also indicate the minimum height requirement: 160 and 155 cm for men and women, respectively. Obesity without comorbid illness is not a reason for rejection from MS \ [[ @ B23 - ijerph - 17 - 00783] \ ]. Enlistment in and rejection from MS were identified from the medical records for each study participant. 2. 2. Participants {# sec2dot2 - ijerph - 17 - 00783} - - - - - - - - - - - - - - - - - In 2018, there was a list of 10, 340 conscripts, 209 of them female. This study included 1427 conscripts (which represented 13. 80% of the total population ), of whom 1296 returned their completed questionnaire along with their consent to participate in the study. The response rate was 90. 82% . Female recruits were invited to pa4ticiLate in the study, but because only 53 agreed to participate, they were later excluded because of the small sample size. Finally, 1243 young male potential conscripts were included in the analysis. The participants were aged 19 - - 26 years and their mean age was 22. 50 ± 2. 43 years. The Ministry of National Defense of the Republic of Lithuania approved the research. Ethics approval (No SMTEK - 28, 2018) was obtained from the Ethics Committee of Lithuanian Sports University. The investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. Participants were informed of the tasks in the study before data collection, and all participants gave their informed consent for inclusion before they participated in the study. 2. 3. Measurements {# sec2dot3 - ijerph - 17 - 00783} - - - - - - - - - - - - - - - - - # # # 2. 3. 1. Physical Activity {# sec2dot3dot1 - ijerph - 17 - 00783} The World Health Organization (WHO) defines moderate physical activity as activity that noticeably accelerates the heart rate and includes activities equivalent in intensity to brisk walking or bicycling. Vigorous physical activity causes rapid breathing and substantially increases heart rate, and includes activities such as jogging, aerobic dance, and bicycling uphill \ [[ @ B24 - ijerph - 17 - 00783] \ ]. To assess physical activity in the participants in this study, we used the 2005 US Department of Defense Survey of Health Related Behaviors Among Active Duty Military Personnel \ [[ @ B21 - ijerph - 17 - 00783] \ ]. The study participants were asked, " During the past 7 dzgs, for leisure - time physical activity, how often did you usually do each of the following? ". Participants were also asked, " During the past 7 days, when you did leisure - time physical activity, how long did you usually do each of the following? " For both questions, detailed descriptions and examples of what constitutes moderate and vigorous physical activity were presented. In this study, we assessed physical activity during the preceding 7 days instead of the 30 days in the original questionnaire. Also, instead of using catego58cal answers (such as " 5 or 6 days " and " at least 20 minutes ") for each question, we provided the opportunity to write the exact numbers of days, hours, and / or minutes per day. The number of minutes spent in was totaled. Participants whose MVPA was \ <2. 5 h / week were coded as not meeting health - related physical activity requirements and those whose MVPA was ≥ 2. 5 h / week were coded as meeting health - related physical activity requirements \ [[ @ B25 | 1. Introduction {#sec1-ijerph-17-00783} =============== decline in youth's healthy behaviors and related consequences \[[@B1-ijerph-17-00783]\] is of concern for public health in general and for national defense in particular, which requires a sufficient number of physically and healthy military personnel. In Lithuania, there is evidence that young people do not partake in adequate physical activity; that is, only around 30% of students aged 18 years comply with the to active ≥1 h at least 5 a week \[[@B2-ijerph-17-00783]\]. The trends for health-related physical fitness among adolescents have deteriorated in the past 20 and the indicators cardiorespiratory fitness have decreased by nearly 50% during this period \[[@B3-ijerph-17-00783]\]. Only 13--14% of high school students comply with recommendations nutrition 11% of young people consume alcohol \[[@B6-ijerph-17-00783]\]. Moreover, 22% experience psychological distress \[[@B7-ijerph-17-00783]\]. Multiple health behaviors, psychological and their associated factors, such as obesity and poor physical fitness, are associated with mental health outcomes, the cardiovascular disease, type 2 diabetes, certain types of cancer \[[@B8-ijerph-17-00783],[@B9-ijerph-17-00783]\], depression \[[@B10-ijerph-17-00783],[@B11-ijerph-17-00783]\], and anxiety \[[@B12-ijerph-17-00783]\]. Unhealthy behaviors impose an economic burden; for example, one study estimated that physical inactivity alone costs USD 53.8 billion in 2013 for healthcare worldwide \[[@B13-ijerph-17-00783]\]. is the main for accepting or rejecting young men into military service (MS) and is strongly related to to perform military duties. Unsatisfactory results of youth to Lithuanian have been presented: on average, 58.6% of Lithuanian young men proceed through the procedure of military enlistment. Among those who do not pass these procedures, 33%--37% experience psychological 29%--33% have cardiovascular diseases, and 13% have musculoskeletal problems Other countries face similar of rejection from MS. For instance, analysis of the reasons for rejection from MS in the USA found that about 22% of rejections were because of problems with bones or joints, flat feet, or hernias, 15% because of organ defects, 13% of defects of the cardiovascular 12% because of nervous system or mental problems, 10% because of communicable diseases \[[@B15-ijerph-17-00783]\]. In the USA, Hispanic men appear to have a health profile their white and black peers, except for the prevalence overweight, which is higher in Hispanic \[[@B16-ijerph-17-00783]\]. Although behaviors alone are not criteria for enlistment into MS, they might explain the reasons for instances of rejection. Given that some health behaviors are risk factors for the occurrence of many lifestyle-related diseases \[[@B17-ijerph-17-00783]\], is critical to identify whether and how health behaviors between young men deemed eligible and those who are ineligible for MS to take appropriate actions to prevent adverse health behaviors from their onset. Soldiers be physically and mentally healthy. However, mental health issues are among the main factors for rejection from MS \[[@B14-ijerph-17-00783],[@B15-ijerph-17-00783]\]. Identifying psychological distress along with health behaviors might help to a more complete understanding of health indicators in conscripts as psychological distress is an indicator of mental health \[[@B18-ijerph-17-00783]\]. Given the associations between many health-related behaviors, the complex analysis of a set of risk instead of evaluation of associations may help to reduce the risk of missing potential confounders enlistment into MS \[[@B19-ijerph-17-00783]\]. studies have examined psychological distress, health \[[@B20-ijerph-17-00783],[@B21-ijerph-17-00783]\], changes in health behavior \[[@B22-ijerph-17-00783]\] during MS. Enlistment is based on a medical examination of draftees, which may unhealthy As a result, these studies have health behaviors relatively healthy youth but have not examined whether health behavior is to the rejection of recruits. The aim of this study is to identify and health behaviors psychological distress between male conscripts for and into MS. We expect to find that healthier behaviors and distress level would be related to a higher rate of enlistment into MS. 2. Materials and Methods {#sec2-ijerph-17-00783} ======================== 2.1. Study Design and ------------------------------- This nationally representative cross-sectional study was performed among Lithuanian conscripts. In Lithuania, MS is compulsory. In accordance with the conscription procedure of the Armed Forces, list of potential is by an automatic electronic selection system each year and includes male Lithuanian citizens of compulsory MS-eligible age (19--26 years). Other male and female young adults can express their wish to be on basis. stratified random sampling was There are four centers for military recruitment in Lithuania that recruit conscripts during the whole The data were gathered in all four centers for 4 months from to October 2018. Each conscript in these centers was approached during this period and asked to sign consent form to participate the study and to complete the study questionnaire. There was an equal chance that participants included in the study would be enlisted or rejected for MS. The decision to accept a recruit for enlistment into MS is made by medical experts and is based on individual medical and previous medical reports. The rejection from MS are defined in Order V-1142/V-1139, which provides a list of disorders along their severity, and is by the Ministers of State and Health Care. These criteria indicate the minimum height requirement: and 155 cm for men and women, respectively. Obesity without comorbid illness is not a reason for rejection from MS \[[@B23-ijerph-17-00783]\]. Enlistment in and rejection from MS were identified from the medical records for each study participant. 2.2. Participants {#sec2dot2-ijerph-17-00783} ----------------- In 2018, there was list of 10,340 conscripts, 209 of them female. study included 1427 conscripts (which represented 13.80% of the total population), of whom 1296 returned their completed questionnaire along with their consent to participate in the study. The rate was 90.82%. Female recruits were invited participate the study, but only 53 agreed to participate, were excluded because of the small sample size. Finally, 1243 young potential conscripts were included in the analysis. The participants were aged 19--26 years and their mean age was 22.50 ± 2.43 years. The Ministry of of the Republic of approved the research. Ethics approval (No SMTEK-28, 2018) was obtained from the Ethics Committee of Lithuanian Sports University. The investigations were carried out following the rules of the Declaration of Helsinki 1975, revised in 2013. Participants were informed of the in before data collection, all participants gave their informed before they participated in the 2.3. Measurements ----------------- ### 2.3.1. {#sec2dot3dot1-ijerph-17-00783} The World Health Organization (WHO) defines moderate as activity that accelerates the heart rate and includes activities equivalent in intensity to brisk walking or bicycling. physical activity causes rapid breathing and substantially heart rate, includes activities such as jogging, aerobic dance, bicycling uphill \[[@B24-ijerph-17-00783]\]. To assess physical activity in in this study, we used the 2005 Department of Defense Survey of Health Related Behaviors Among Active Duty Military Personnel \[[@B21-ijerph-17-00783]\]. study participants were asked, "During the past 7 days, for leisure-time physical activity, how often did usually do each of following?". Participants were also asked, "During the past 7 days, when you did leisure-time activity, how long did you usually do each of the following?" For both questions, detailed descriptions and examples of what constitutes moderate and vigorous physical activity were presented. In this study, we assessed physical activity during the preceding 7 days instead of the 30 in the original questionnaire. Also, instead of using categorical answers (such "5 or 6 days" and "at least minutes") for each question, we provided the opportunity to write the exact numbers of days, hours, and/or minutes per The number of minutes spent in was totaled. Participants whose MVPA was \<2.5 h/week were coded as not meeting health-related physical requirements and those whose was ≥2.5 h/week were coded as health-related physical requirements \[[@B25 | 1. INTrODUctioN {#sec1-IjeRpH-17-00783}
===============
THe dEClIne IN yoUth's HealTHY behaviOrS And ReLaTed CONsEqUeNCes \[[@b1-iJeRPh-17-00783]\] iS Of ConcERN FoR PUBlIC Health in GENERAl AND FoR NaTiOnAl DEFenSE in paRticuLar, WhICh reqUIRes a SUfFicIeNt nuMbEr OF physiCaLly fit ANd meNtalLy heALthy miliTARY pERsONnEl. iN LIThuaNIA, THErE is eViDENcE ThAT YoUNG PEOplE do NOT parTakE in aDEqUATE PHYsicAL actiViTy; THaT iS, ONLY aroUND 30% OF STUdENts aGED 18 YeaRs cOMPlY wiTH THe rEcOmmENDaTIOn to Be aCtIVe ≥1 h on aT LeAST 5 DAyS A wEEK \[[@b2-ijerPh-17-00783]\]. tHe TRENdS fOr HEALth-rElatED pHySIcal FITNeSs aMONG ADOleScENtS hAVe dEtErIoRaTEd iN tHE PaSt 20 YeArS, aND ThE indICatorS oF carDiORESpiRaTOrY FiTNeSs HaVe dEcrEased by nearlY 50% DURiNG thIs PErIOD \[[@B3-IJeRpH-17-00783]\]. Only 13--14% oF High sCHool StuDEnTs cOmPLY wiTh recoMMENdations FOr HeALthy nutrItiON \[[@b4-IjerpH-17-00783],[@b5-IjeRpH-17-00783]\], 11% of yOuNG peoplE cONsume ALcoHoL \[[@b6-ijErpH-17-00783]\]. mOrEovER, 22% eXPErIenCE pSYCHOLoGIcal diStRess \[[@b7-ijERph-17-00783]\]. mulTIPlE HeALth BEHAviors, PSYChoLOGICal diSTResS, And thEiR ASsOciAtED risK fActOrs, SuCH as ObEsITy And POor phYSICAL fItnEss, ArE asSOciATed wITH pOOR MeNtal heAlTH ouTcOmes, tHe rISK oF cARdiOVasCulAR dIseaSe, Type 2 DiaBETEs, certAiN TypES Of caNCer \[[@b8-IJeRpH-17-00783],[@B9-iJErph-17-00783]\], dEPREsSION \[[@B10-IjErPH-17-00783],[@b11-IJERPH-17-00783]\], aNd anXiEty \[[@b12-iJerPH-17-00783]\]. UnhEALtHy beHAvIOrS ALsO IMPoSE aN EcoNomic BurDeN; foR eXamplE, One sTudY EStImateD thaT PhYsICAl iNacTiVItY AlOne costS uSd 53.8 bILliOn In 2013 fOR HealThcARe worlDwide \[[@b13-ijERPh-17-00783]\].
HEAlTH Is thE mAin CRitErIoN For accEpting oR reJeCTIng yoUnG mEN iNTO mIlITaRy ServicE (mS) ANd is strongLY RElAtEd to the AbIlItY to PeRFoRM MILiTARY DUTies. unsATisfACtoRy ReSULtS oF YoUtH rEcRUITmENt to LithUAnIAN mS HavE BEeN PreseNTed: ON avEragE, 58.6% OF lIthuaNIAN Young MEn proCEeD thRoUGH THE fUll pROCEduRE Of MiLiTary ENLIStmEnt. amOng thOse who DO noT PAsS tHese pRoCedUrEs, 33%--37% EXpeRIencE psychOlOgicAl pRObLEms, 29%--33% haVE cARdioVascULar DISEaSES, and 13% haVe MusCulosKELetal PRObLEms \[[@b14-IjErph-17-00783]\]. oTHEr coUNTRIeS faCE SimILAR probLEmS oF REJeCtIon from Ms. FOr InSTancE, ANalYSiS oF THE REASoNs fOr REJEcTION FrOM mS in ThE uSA FoUnD THaT aBout 22% Of ReJECTioNs wEre BecAUSe oF pRObLemS wiTh BONeS or joiNtS, FLaT FEET, Or hERNIAS, 15% bECauSE of OrgAn defECtS, 13% BECAuse OF dEfeCTS OF tHE cArDiOvascUlAR sySTEM, 12% BeCAusE OF nERvoUs systeM or mENTAL PRoBLEms, and 10% beCaUSE of COMmUnICaBLE dISeASEs \[[@b15-iJeRPH-17-00783]\]. in tHE usA, hIsPANiC Men apPEar To hAvE A BEtTeR hEalTH proFile THan ThEIr WhitE AnD BlACk PEers, eXCEpt for THE PReVaLence Of OveRWeiGHt, WHICH is HiGheR in HISpaNIc MeN \[[@B16-IJErph-17-00783]\].
ALThOugh hEALtH bEhAviOrS ALOnE ARe Not CriTErIa for EnLiSTMeNt into Ms, THEy MighT ExplaIN ThE rEASONS FOr SoMe instAnCes of rEjection. GIVEn THat SomE HEaltH BEHaviorS ARE RiSk FactORS for thE oCCUrrence OF manY LifestyLE-relatEd DiSEASeS \[[@B17-IjeRpH-17-00783]\], IT Is crItiCAL To identify WHEThER AND HoW heaLTh bEHAvIors DiFfEr bEtWEen yOung men WHo arE DEEmED eLigIblE AND THOse wHO aRe iNELiGiBlE for MS AND tO tAke apPROPriAtE acTionS TO prEVEnT advErSE heAltH beHAvIORs FROm thEIr oNseT.
sOlDIERS must BE BOTh PhYsICALlY And MeNTaLLy heaLtHY. HOWEVer, MeNtal HEAlth IsSuEs arE AmoNg thE MaIn factOrs fOr reJectIon From Ms \[[@B14-IjeRph-17-00783],[@B15-IJERph-17-00783]\]. iDeNtiFYiNg PsYchOlogicAL DIstress ALoNg WiTH hEaLTh BehaVIOrs MigHt hELp tO ProvIde A mORe coMPLEtE uNdeRsTAndINg OF hEAltH indiCatorS iN conSCRiPTs as PSYchOloGIcal DISTRESS Is aN INdICaTOR oF mEnTal HEaLTh \[[@b18-IjErPh-17-00783]\].
giVEN THe aSSociATIoNS BetweeN ManY healtH-RelAteD behAvIORs, The COmPLEx ANALySiS OF a sEt oF RIsK BeHaVIOrS INstEaD OF eVaLuAtIon of IndivIDual ASSOciATions mAy HELp to RedUCE The RISK of mIssing potENtIAL COnFoUNDErS FoR EnlISTmENT into mS \[[@B19-IJeRPh-17-00783]\].
SEVErAl stuDiEs hAvE eXamined PsYchoLogIcal DiStRESs, HeAlTh BehaViors \[[@b20-IjeRPH-17-00783],[@b21-ijerPh-17-00783]\], AND CHAngEs In hEALTH BEHaviOR \[[@b22-IJeRpH-17-00783]\] DURiNg MS. enLisTmenT is BAsed On a mediCaL EXAMINatioN OF dRAFteeS, whICh mAY rEJect UnheALTHy InDIvIdualS. As A rEsult, ThESe STUdIES have EvALuaTED HEaLTh bEhavIOrS in ReLaTiVeLy heAlThY YOUth BUt have nOT eXaMINeD whether HeAlTH bEHavIOr IS ReLatEd TO ThE rejEcTioN oF mILitaRy RECruITs.
thE aIM OF tHis STudy IS tO IDeNTiFY and CompaRE HeaLth beHAviORs AnD psYchOlOGicaL DISTresS BEtwEEn MalE cOnScrIpTs REJECTeD fOr aND eNListeD InTo Ms. wE EXpecT TO fIND THat heALThIER bEhaviORs aND LoW DiSTReSS lEvel Would BE rELATeD To a hIGheR RAte OF EnliStMeNT iNtO Ms.
2. mateRIAlS AnD metHoDs {#seC2-ijeRPH-17-00783}
========================
2.1. STUdY DEsigN ANd prOceDURe {#sEC2dot1-IjERPh-17-00783}
-------------------------------
THiS nAtionAlLY REprESEnTaTIVe CrosS-secTioNaL STUDY WAs PeRFOrMED amOng liTHuANiAn CONscripTs. In LItHuAnia, 9-moNtH-Long MS is CoMPUlsORY. in AccoRdANCe WIth THE conScrIPtIoN procEDuRE of tHe LIThUaNiAN arMED FOrceS, a liST OF PotENtiaL dRAFtEEs IS cReATED By aN AUtomATic ELEctRonIc SeLEctION SYsTEM each Year And iNClUdES Male liThUanIAN CitizeNs Of COMPULsOry Ms-ElIGIbLE agE (19--26 YEarS). OtHER male aND FeMALe YouNG aduLTS CAn ExPreSS THEiR wiSh tO be ConSCRIPTeD On A PrioRiTY bASIS.
a sTRatIfied RanDOM sAmPlING wAs used. tHEre ARE four CENtERs FOR miLItAry RECruITmENt IN lIthUania ThAT ReCrUIt conscRiPtS duRiNg The whOlE yEAR. tHE dAtA wERE gatHeRED in AlL FOUR CeNtERs fOr 4 montHs frOM juNe to OCtOber 2018. EACH COnSCRIpt In tHesE cenTeRS Was ApproaCHeD DUriNg thiS pERioD And ASked TO SIGn a CoNsenT foRM TO PARtICIPATE In THe StUDy ANd To complETE ThE StUdy qUEsTIOnnaiRE. tHere was an EQuaL chAnCE (PRobABilitY) tHat PArtiCIPANts iNCLUDeD IN THe stUDY WOULd Be enLISteD OR REJECteD foR ms.
ThE DecisIoN to aCcepT A rEcRuit fOr EnliStMeNT InTO MS Is MAde BY meDical expeRTs AND IS bAseD oN An IndividUAl MEdicAL examinAtiOn AND prEviOUs mEdiCAl RePORTs. THE criTEria FOr RejEcTIon fRoM mS Are DeFiNEd In OrdeR V-1142/V-1139, WhIch prOviDES a LiSt Of disoRdeRs alOng wiTh tHEIR sEvERiTY, anD is sIgNEd By THe MInIsTErs Of sTaTE dEFeNSe ANd hEAlTH CaRe. ThEsE CriTeria aLsO indiCaTe tHE mIniMUM HeiGht ReQUIReMENT: 160 aNd 155 cM fOr men And WOMen, RESpectIvELY. oBesItY WiTHouT cOMorbId iLLnEsS iS NoT A REASOn FOR RejEction frOm MS \[[@b23-IJERPH-17-00783]\]. ENlisTMENT IN and RejECTion FROm Ms WEre iDEntIFIed FROM THE mEdIcal reCoRDS FOR EacH study PaRTICIPaNT.
2.2. PARTIcIpants {#SEc2doT2-IJeRpH-17-00783}
-----------------
IN 2018, TheRe WAS A liSt OF 10,340 coNScriPTS, 209 of THEm FEmaLe. This sTUDy iNCLuDeD 1427 CONSCrIPTs (WHIch RepResenTed 13.80% OF thE TOTaL PopuLaTiON), Of WHOM 1296 ReTuRNEd thEiR CoMpleTed quEstiOnnaiRe ALoNG WitH THeIr consENT tO pARTICiPaTe in tHe STudy. tHe ReSpONsE RaTE Was 90.82%. femAle reCrUiTS WeRe INVitEd to PArTicIpaTE in tHE sTudY, but bEcAUSE Only 53 aGReeD tO PaRtiCipATE, THEy weRE LATeR ExCLUdED BECause OF thE sMALl sample SIZE. FiNALLy, 1243 yOung mAle POtentIaL ConscrIptS wErE iNCLUDEd IN ThE AnalysiS. THE ParTICiPaNts weRE Aged 19--26 yEARs AND thEIr meAn aGe wAS 22.50 ± 2.43 YeARS.
the minIstrY oF NATiOnaL DEFenSE OF thE REpublic of litHuania APPROVEd tHE ResearcH. ETHiCS APPrOVal (no SmTEK-28, 2018) WaS ObTaINed FroM The ethICs coMmITteE Of lITHuaNIan SPOrtS UnivErsIty. tHe inVEstIgaTiONs werE CArriED OUt fOLlOwING the ruleS Of the dECLArATiON of heLsiNki of 1975, RevIsEd in 2013. PArTICIpanTS WerE inforMed oF THE tAsKs in ThE StUDY BefOre datA CoLLecTion, and aLl parTiCipaNtS gAVE tHeIR INforMED cOnsEnt FOR IncluSIoN BeFOre ThEY PARticipATED in tHE sTuDY.
2.3. MEasUremeNts {#seC2DoT3-IjerPH-17-00783}
-----------------
### 2.3.1. PHysIcAl ACtiviTY {#sec2dOT3DOT1-IJERpH-17-00783}
thE woRLd HEALtH OrGANizATioN (WhO) DEfiNeS modeRaTe pHysICaL acTIVitY AS ACTiVIty tHaT NOTiCeaBLy ACCeLERAtes tHE hEaRT Rate AND incluDES actIVITIeS eQuIvALeNt In InTEnSItY tO brIsk wALKinG or BicYCLIng. viGOrouS PHYSical aCtIVIty Causes rApID bREaThINg AND sUbSTaNTially INcrEAseS HEaRt Rate, and IncluDES aCtiViTiES SuCh AS jOGgiNg, aerOBIc dANCe, aNd bICyclIng UpHilL \[[@b24-ijeRPH-17-00783]\]. tO assEss pHysiCAl aCTivIty In tHe pArtIcIpaNTS iN THiS sTUdY, WE UsEd The 2005 US DEpArTMeNt Of dEFEnSE sUrVEY Of health RElateD bEhAVIOrS amoNG AcTiVe DutY MilITARY peRSonnEl \[[@B21-Ijerph-17-00783]\]. thE sTuDY partiCipantS weRE AsKEd, "DURinG THe past 7 DaYs, fOr lEiSUre-TIMe phYsiCAl AcTIvItY, HOW ofTen DiD YOU UsuALlY dO EaCH oF thE folLOwiNG?". partIcIPanTS WeRE aLSO aSkeD, "DUriNG tHe PasT 7 Days, whEN YOu DID leiSUre-TIME PHYsiCal actiViTy, how lonG did yOu usUALLy do EAcH of thE FollOWINg?" FOR bOtH QUEstIoNs, DeTaIlEd descRIPTioNs AND exAMPLeS OF WhaT coNSTituTeS MODeRaTE And ViGOroUs phySICal AcTivitY weRe preseNTed.
In tHIS study, wE ASSesSeD pHySICaL AcTiVITY duRinG tHE PrEceDINg 7 DAYs insTeaD of the 30 dAYS iN ThE oRIginAL qUEsTIonnairE. AlSo, insTEad OF UsInG CaTEGOricAL answeRS (sUch As "5 or 6 dAyS" aND "AT lEast 20 mInUtes") FOr EACh queSTION, we pRoVIDed THE oPpoRTuniTY tO WritE THE exACT numBers of dayS, hoUrS, ANd/or MInutES PER DAY. THe NumBEr Of minUtes speNT in WAS TOtaleD. PaRTiCipants Whose MVPA Was \<2.5 h/WeEK WERe CODeD AS Not meeTINg HEalth-ReLaTed phYSicAl aCtivity reQuIreMents AND THoSE whoSe MvPa was ≥2.5 h/WeeK weRe cOdED as mEEtIng HEaLtH-reLATeD PhySiCAL ActIViTy ReQUIrEmenTs \[[@b25 | 1. Introduction {#sec1-ijerph-17-00783} =============== The decline in youth's healthy behaviorsand related consequences \[[@B1-ijerph-17-00783]\]isof concern forpublic health in generaland fornational defense in particular, which requires a sufficientnumber of physically fit and mentally healthymilitary personnel. In Lithuania, there is evidence that young people donot partakein adequate physical activity; that is, onlyaround 30% ofstudents aged 18 years comply with the recommendation to be active≥1h on at least 5 days a week \[[@B2-ijerph-17-00783]\]. The trendsfor health-related physical fitness among adolescents havedeteriorated in the past 20years, and the indicators of cardiorespiratory fitness have decreased by nearly 50% during this period \[[@B3-ijerph-17-00783]\]. Only 13--14% of high school students comply with recommendations for healthy nutrition \[[@B4-ijerph-17-00783],[@B5-ijerph-17-00783]\], 11% ofyoung people consume alcohol \[[@B6-ijerph-17-00783]\]. Moreover, 22% experience psychological distress \[[@B7-ijerph-17-00783]\].Multiple health behaviors, psychological distress,and their associated risk factors, such as obesity and poor physical fitness,are associated withpoor mental health outcomes,the riskof cardiovascular disease,type 2 diabetes, certaintypesofcancer \[[@B8-ijerph-17-00783],[@B9-ijerph-17-00783]\], depression \[[@B10-ijerph-17-00783],[@B11-ijerph-17-00783]\], and anxiety \[[@B12-ijerph-17-00783]\]. Unhealthy behaviorsalso impose an economic burden;for example, one study estimated that physical inactivity alone costsUSD 53.8 billion in 2013 for healthcare worldwide \[[@B13-ijerph-17-00783]\]. Health is themain criterion for accepting or rejecting young men into militaryservice (MS) and is stronglyrelated to theability to perform militaryduties. Unsatisfactory results of youthrecruitment to Lithuanian MS have been presented: on average, 58.6% of Lithuanian youngmenproceed through the full procedure of military enlistment.Among those who donotpasstheseprocedures,33%--37%experience psychological problems, 29%--33% have cardiovascular diseases, and 13% have musculoskeletal problems \[[@B14-ijerph-17-00783]\]. Other countries face similar problems ofrejection fromMS. For instance, analysis of thereasons for rejection from MS in the USAfound that about22% of rejections were because of problems withbones orjoints, flatfeet, or hernias, 15% because of organ defects, 13% because ofdefects of the cardiovascularsystem, 12%because of nervous system or mental problems, and 10% because ofcommunicable diseases \[[@B15-ijerph-17-00783]\]. In the USA,Hispanic men appear to have a better health profilethantheir white and black peers, except for the prevalence of overweight, which is higher in Hispanic men \[[@B16-ijerph-17-00783]\]. Although health behaviorsalone are not criteria for enlistment into MS, they might explain the reasons for some instances of rejection. Given that some health behaviors are risk factors for the occurrence ofmanylifestyle-related diseases \[[@B17-ijerph-17-00783]\],it is critical to identifywhether and howhealth behaviors differ between young men whoare deemed eligible andthosewho are ineligible for MS and to take appropriateactionsto prevent adverse health behaviors from their onset. Soldiers must be both physicallyand mentallyhealthy. However, mental healthissues are amongthe main factors for rejection from MS \[[@B14-ijerph-17-00783],[@B15-ijerph-17-00783]\]. Identifying psychological distress along with healthbehaviors might help to provide a morecomplete understanding of health indicatorsin conscripts as psychologicaldistress isan indicator of mental health\[[@B18-ijerph-17-00783]\].Giventhe associations between many health-related behaviors, the complex analysis of a set ofrisk behaviors instead ofevaluation of individualassociations may helpto reducethe risk of missing potential confounders for enlistmentinto MS \[[@B19-ijerph-17-00783]\]. Several studies haveexamined psychological distress, health behaviors\[[@B20-ijerph-17-00783],[@B21-ijerph-17-00783]\], and changes in health behavior \[[@B22-ijerph-17-00783]\] during MS. Enlistmentis basedon a medicalexamination of draftees, which may rejectunhealthy individuals. As a result, these studies have evaluated health behaviors in relativelyhealthy youth buthave not examined whether health behavior is related to the rejection of militaryrecruits. The aim of this study is to identify and compare healthbehaviors and psychological distress between male conscripts rejected for andenlisted into MS.Weexpect tofind that healthier behaviors and low distress level would be related to a higher rate of enlistment into MS. 2. Materials andMethods {#sec2-ijerph-17-00783} ======================== 2.1. Study Design and Procedure {#sec2dot1-ijerph-17-00783} ------------------------------- This nationally representative cross-sectional study was performed among Lithuanianconscripts. In Lithuania, 9-month-long MSis compulsory. In accordance with the conscription procedure of the Lithuanian Armed Forces,a list of potentialdraftees is created by an automatic electronic selectionsystem each year and includes male Lithuanian citizens of compulsoryMS-eligible age (19--26 years). Other male and female young adults can express their wishto be conscripted ona priority basis. A stratified random sampling wasused. There are four centers for military recruitment in Lithuania thatrecruit conscripts during the whole year. The datawere gathered in all four centers for4 months from June to October 2018. Each conscript inthese centers was approached during this period and asked to sign a consent form toparticipate in thestudy and to complete the studyquestionnaire. There was an equal chance (probability) thatparticipants included inthe study would be enlisted or rejected for MS. The decision to accept a recruit for enlistment intoMS is made by medical experts and is based on an individual medicalexamination and previous medical reports. The criteria for rejection from MSare definedin Order V-1142/V-1139,which provides alist of disorders along withtheir severity, and issigned by the Ministers of State Defense and Health Care. These criteria alsoindicate the minimumheight requirement: 160 and 155 cm for men andwomen, respectively. Obesity without comorbid illness is not a reason for rejection from MS \[[@B23-ijerph-17-00783]\].Enlistment in and rejection from MS were identified from themedical records foreach study participant. 2.2. Participants {#sec2dot2-ijerph-17-00783}----------------- In 2018, there was a list of 10,340conscripts, 209 of them female. This study included 1427 conscripts (which represented 13.80% of the total population), of whom 1296 returned their completed questionnaire along with their consent to participate in the study.Theresponse rate was90.82%. Female recruits wereinvited to participate in the study, but because only 53 agreed to participate, they were later excluded because of the small sample size.Finally, 1243 youngmale potential conscripts were includedin theanalysis. The participants were aged 19--26 years and theirmean age was 22.50 ± 2.43 years. The Ministry of National Defense of the Republic of Lithuania approved the research. Ethics approval (No SMTEK-28, 2018)was obtained from the Ethics Committee of Lithuanian SportsUniversity. The investigations werecarried out following the rulesofthe Declaration of Helsinki of 1975, revisedin 2013. Participants were informed of the tasks in thestudy before datacollection, and all participants gave their informed consentfor inclusion before they participated in the study.2.3. Measurements {#sec2dot3-ijerph-17-00783} ----------------- ### 2.3.1. Physical Activity{#sec2dot3dot1-ijerph-17-00783} The World Health Organization (WHO) defines moderate physical activity as activity that noticeably accelerates the heart rate and includes activities equivalent in intensity to brisk walking or bicycling. Vigorous physical activity causes rapid breathing and substantially increases heart rate, and includes activities suchas jogging, aerobicdance, and bicycling uphill\[[@B24-ijerph-17-00783]\]. To assess physical activityin the participants in this study,we used the 2005 USDepartment of Defense Survey ofHealth Related Behaviors Among Active DutyMilitary Personnel \[[@B21-ijerph-17-00783]\]. The study participantswere asked, "During the past 7 days, for leisure-timephysicalactivity,how often did you usually do each of the following?". Participantswere also asked, "Duringthe past 7 days,whenyou did leisure-time physical activity, how long did you usually do each of the following?" Forboth questions, detailed descriptionsand examples of what constitutes moderateand vigorous physical activity were presented.In this study,we assessedphysicalactivityduringthe preceding7 days instead of the 30 days in theoriginal questionnaire. Also, instead of using categorical answers (such as "5or 6 days" and "at least20 minutes") for each question, we provided the opportunity to write the exact numbersof days, hours, and/or minutes per day. The number of minutes spent in wastotaled. Participants whose MVPA was \<2.5 h/week were coded as not meetinghealth-related physical activity requirements and those whose MVPA was ≥2.5 h/week were coded as meeting health-related physical activity requirements \[[@B25 | 1. Introduction {#sec1-ijerph-17-00783} =============== The decline in _youth's_ healthy _behaviors_ _and_ related consequences \[[@B1-ijerph-17-00783]\] _is_ of concern for public health in general and _for_ _national_ defense in particular, _which_ requires a sufficient _number_ of physically fit and mentally _healthy_ military personnel. _In_ Lithuania, there is evidence that young people do _not_ partake in adequate physical activity; that is, only around 30% of students _aged_ 18 _years_ comply with _the_ _recommendation_ to _be_ active _≥1_ h on at least 5 days a week \[[@B2-ijerph-17-00783]\]. The _trends_ for health-related physical _fitness_ among adolescents have deteriorated in the past 20 _years,_ and the indicators _of_ cardiorespiratory _fitness_ _have_ decreased by nearly 50% _during_ this period _\[[@B3-ijerph-17-00783]\]._ Only _13--14%_ _of_ high school students comply with recommendations for healthy nutrition \[[@B4-ijerph-17-00783],[@B5-ijerph-17-00783]\], 11% of young people consume alcohol \[[@B6-ijerph-17-00783]\]. _Moreover,_ 22% experience psychological distress _\[[@B7-ijerph-17-00783]\]._ Multiple health behaviors, psychological distress, and their associated _risk_ factors, such as obesity and poor physical _fitness,_ _are_ associated with poor mental health outcomes, the risk of cardiovascular disease, type 2 diabetes, _certain_ types of cancer _\[[@B8-ijerph-17-00783],[@B9-ijerph-17-00783]\],_ depression \[[@B10-ijerph-17-00783],[@B11-ijerph-17-00783]\], and anxiety _\[[@B12-ijerph-17-00783]\]._ _Unhealthy_ behaviors also impose _an_ economic burden; for example, one study estimated that physical inactivity alone _costs_ USD 53.8 billion in 2013 for healthcare worldwide \[[@B13-ijerph-17-00783]\]. _Health_ _is_ the main criterion for accepting or rejecting young men into _military_ service (MS) and is strongly related _to_ the ability _to_ _perform_ military duties. Unsatisfactory results of youth recruitment to _Lithuanian_ MS have been presented: _on_ _average,_ 58.6% _of_ Lithuanian young men proceed through the full _procedure_ of military enlistment. Among those who do not _pass_ these procedures, _33%--37%_ experience psychological problems, 29%--33% have cardiovascular diseases, _and_ _13%_ have _musculoskeletal_ problems _\[[@B14-ijerph-17-00783]\]._ _Other_ _countries_ face similar problems of rejection from MS. For instance, analysis of the reasons for rejection from MS in the _USA_ found that about _22%_ of rejections were because of problems with bones or _joints,_ flat feet, or _hernias,_ 15% because _of_ organ _defects,_ 13% because of defects _of_ the _cardiovascular_ system, 12% because _of_ nervous system or _mental_ problems, and 10% _because_ of communicable _diseases_ \[[@B15-ijerph-17-00783]\]. In the USA, Hispanic _men_ appear to have a better health _profile_ _than_ _their_ _white_ and black peers, except for the prevalence of overweight, which is higher in _Hispanic_ men \[[@B16-ijerph-17-00783]\]. Although health behaviors alone _are_ not criteria for enlistment into MS, _they_ _might_ explain _the_ reasons for _some_ instances of rejection. _Given_ that some health behaviors are risk factors _for_ the occurrence of _many_ lifestyle-related diseases _\[[@B17-ijerph-17-00783]\],_ it is _critical_ to identify whether and how _health_ behaviors differ _between_ young men who are deemed eligible _and_ _those_ who are ineligible for MS and to take appropriate actions to prevent adverse health behaviors _from_ their onset. Soldiers must be both _physically_ and mentally healthy. _However,_ mental health issues _are_ among the _main_ _factors_ for rejection from MS \[[@B14-ijerph-17-00783],[@B15-ijerph-17-00783]\]. Identifying psychological distress along with health behaviors might help _to_ provide _a_ more _complete_ understanding of health _indicators_ in conscripts as psychological distress _is_ _an_ indicator of mental _health_ \[[@B18-ijerph-17-00783]\]. Given the associations between many _health-related_ _behaviors,_ the complex analysis _of_ a set of risk behaviors instead of evaluation of individual _associations_ may help to _reduce_ the risk of missing potential confounders for enlistment into MS \[[@B19-ijerph-17-00783]\]. _Several_ _studies_ have examined _psychological_ distress, _health_ _behaviors_ _\[[@B20-ijerph-17-00783],[@B21-ijerph-17-00783]\],_ and changes in _health_ behavior \[[@B22-ijerph-17-00783]\] _during_ MS. Enlistment is based on a _medical_ examination of draftees, which may reject unhealthy individuals. As a result, these studies have _evaluated_ _health_ behaviors in relatively _healthy_ _youth_ but have not examined whether health behavior _is_ related to _the_ rejection _of_ military recruits. _The_ _aim_ _of_ this study is to identify and compare health behaviors and psychological _distress_ _between_ male conscripts rejected for and enlisted into MS. We _expect_ _to_ find that _healthier_ behaviors and low distress level _would_ be related to a higher rate of enlistment _into_ MS. 2. Materials _and_ Methods {#sec2-ijerph-17-00783} ======================== 2.1. Study _Design_ and Procedure _{#sec2dot1-ijerph-17-00783}_ ------------------------------- _This_ nationally representative cross-sectional _study_ was performed _among_ _Lithuanian_ conscripts. In _Lithuania,_ 9-month-long MS _is_ compulsory. In accordance with _the_ _conscription_ procedure _of_ the Lithuanian Armed Forces, a list of _potential_ draftees is created by an automatic electronic selection system each _year_ and includes male Lithuanian citizens of _compulsory_ MS-eligible age _(19--26_ years). Other _male_ and female young adults can express their wish to be conscripted on a priority basis. A stratified random _sampling_ was used. There are four centers for _military_ recruitment in Lithuania that recruit conscripts during _the_ whole year. _The_ data were _gathered_ _in_ all _four_ centers for 4 months from June to October 2018. Each conscript in these _centers_ _was_ approached during this period and asked to _sign_ a consent form to participate in the study and to _complete_ the _study_ questionnaire. _There_ was an equal chance (probability) that participants _included_ in the study _would_ be enlisted _or_ rejected for MS. The decision to accept _a_ _recruit_ for enlistment into MS is made _by_ medical experts _and_ is _based_ on _an_ individual medical examination and previous _medical_ reports. The criteria for rejection from MS are _defined_ in Order _V-1142/V-1139,_ which provides a _list_ of _disorders_ along with _their_ severity, and is signed by the Ministers of State _Defense_ and Health Care. These criteria also indicate the minimum _height_ requirement: 160 and 155 cm for men _and_ women, respectively. _Obesity_ without comorbid illness is not _a_ reason for rejection _from_ MS \[[@B23-ijerph-17-00783]\]. Enlistment in and _rejection_ from _MS_ were identified _from_ the _medical_ records for _each_ study _participant._ 2.2. Participants {#sec2dot2-ijerph-17-00783} ----------------- In _2018,_ _there_ was a _list_ of 10,340 _conscripts,_ _209_ of them _female._ This _study_ included _1427_ _conscripts_ (which _represented_ _13.80%_ of the total population), of whom _1296_ _returned_ their completed questionnaire _along_ with their _consent_ to participate in the _study._ The response _rate_ was 90.82%. Female recruits were invited to participate in the study, _but_ because only 53 agreed to participate, they were later excluded because of the _small_ sample size. Finally, 1243 young male potential conscripts were included in the analysis. The participants were _aged_ 19--26 _years_ and _their_ _mean_ age was 22.50 ± 2.43 years. The Ministry of National Defense of the Republic of Lithuania approved the research. _Ethics_ _approval_ (No SMTEK-28, 2018) was _obtained_ _from_ the Ethics Committee of _Lithuanian_ _Sports_ University. The investigations were _carried_ out _following_ the rules of _the_ _Declaration_ of Helsinki of 1975, revised in 2013. Participants were informed of the tasks _in_ the study before _data_ collection, and _all_ participants gave their _informed_ _consent_ for inclusion before they participated _in_ the study. 2.3. Measurements {#sec2dot3-ijerph-17-00783} ----------------- _###_ _2.3.1._ Physical _Activity_ {#sec2dot3dot1-ijerph-17-00783} The World _Health_ Organization (WHO) _defines_ moderate physical activity as activity that noticeably accelerates the heart rate and includes _activities_ equivalent _in_ intensity to brisk walking _or_ bicycling. _Vigorous_ _physical_ activity causes rapid _breathing_ and substantially increases heart rate, and _includes_ _activities_ such as _jogging,_ _aerobic_ dance, and bicycling uphill _\[[@B24-ijerph-17-00783]\]._ To assess _physical_ activity in the participants _in_ this study, _we_ used the 2005 US Department of _Defense_ Survey of _Health_ Related Behaviors Among Active Duty _Military_ Personnel \[[@B21-ijerph-17-00783]\]. The study participants were asked, "During the past 7 days, for leisure-time physical activity, how often did you usually do each of the following?". Participants were also asked, "During the past 7 days, when you did _leisure-time_ physical activity, how long _did_ you usually do each of _the_ _following?"_ For both questions, detailed descriptions and examples of what constitutes moderate and vigorous physical activity were presented. In this study, we assessed physical _activity_ during the preceding 7 days instead of the _30_ days in the original _questionnaire._ _Also,_ instead _of_ using categorical answers (such as _"5_ or _6_ _days"_ and "at least 20 minutes") for each question, we provided _the_ _opportunity_ to _write_ the exact _numbers_ of days, hours, _and/or_ minutes per day. The number of minutes spent in was totaled. Participants whose MVPA was \<2.5 h/week _were_ coded as not _meeting_ health-related physical activity requirements and _those_ whose MVPA was ≥2.5 _h/week_ _were_ coded as meeting health-related physical activity requirements \[[@B25 |
Background {#Sec1}
==========
The prevalence of obesity in the general population has increased dramatically over the last 30 years and it seems likely that the environmental changes that have provoked these increases have also affected people with severe mental illness (SMI); in fact, the rates of overweight and obesity have increased even more rapidly in this cohort \[[@CR1]\]. Obesity adversely affects the physical health and psychological well-being of people with SMI and if weight gain is attributed to treatment, this can lead to non-adherence and risk of relapse.
Schizophrenia is a major psychiatric disorder that alters the individual's perception, thoughts, affect and behaviour and may involve a loss of insight and has a lifetime prevalence of approximately 1% \[[@CR2]\]. Schizoaffective disorder is recognised as a separate condition to schizophrenia and is more likely to occur in women at a later age. This disorder affects an individual's thoughts and emotions \[[@CR3]\]. Although individuals with first-episode psychosis do not fulfil the diagnostic criteria for schizophrenia or schizoaffective disorder, 90--95% of people presenting with a non-affective psychotic episode (i.e. not mania and not depressive psychosis) will still meet the criteria for a schizophrenia spectrum disorder 2 years later. Mortality rates are increased two to three fold in people with SMI and life expectancy is reduced by 10--20 years. Approximately 75% of all deaths in people with schizophrenia are caused by physical illness with cardiovascular disease being the commonest cause \[[@CR4]\]. Overweight and obesity contribute to this excess morbidity and mortality. Recent studies indicate that obesity is two to three times more common among people with SMI \[[@CR5]\]. Obesity occurs early in the natural history of schizophrenia with a significant proportion of people with first-episode psychosis being overweight prior to any treatment. Substantial weight gain (\> 7%) often occurs rapidly within 6--8 weeks after antipsychotic-treatment initiation \[[@CR6]\]. While most weight gain occurs early in treatment, longer-term observational studies suggest that weight gain continues for at least 4 years albeit at a slower rate \[[@CR7]\].
Individuals with schizophrenia are more likely to consume a diet that is rich in fat and refined carbohydrates while containing less fibre, fruit and vegetables than the general population \[[@CR8]\]. Although there are fewer studies, people with first-episode psychosis also have poor diets \[[@CR9]\]. Physical inactivity and the social and urban deprivation experienced by those with schizophrenia may contribute further to the increased obesity rates \[[@CR8], [@CR10]\]. There may be disease-specific effects of schizophrenia, such as genetic susceptibility, that have additive or synergistic actions to increase body weight further \[[@CR5]\]. However, the most important factor related to weight gain in people with SMI is the use of antipsychotic medications, which are among the most obesogenic drugs. Weight gain is the commonest side effect of second-generation antipsychotic medication, affecting between 15 and 72% of patients \[[@CR11]\]. Other psychotropic drugs are often prescribed to people with schizophrenia and include some antidepressants and mood-stabilising drugs, such as lithium and sodium valproate; these may also induce significant weight gain \[[@CR12]\].
Both lifestyle and pharmacological interventions lead to significant reductions in body weight in the general population. Not only are the interventions clinically effective, they are also cost-effective because of the benefits of long-term improved health outcomes, including decreased mortality \[[@CR13]\]. It is likely that similarly effective interventions for people with schizophrenia will also lead to improvements in health and would be a major step towards reducing the health inequalities experienced by people with schizophrenia. As the weight gain associated with antipsychotic medication result in some people discontinuing their medication, we hypothesis that effective weight-management strategies may also lead to improved adherence to antipsychotic medication and reduced relapse and hospitalisation rates.
Some studies have suggested that short-term lifestyle interventions could support weight reduction in people with SMI. A meta-analysis of non-pharmacological interventions in people with SMI \[[@CR14]\] reported a mean reduction in weight of 3.12 kg over a period of 8--24 weeks. However, the results of longer-term studies are more mixed. A recent meta-analysis found significant weight loss in only two of six studies with interventions lasting longer than a year \[[@CR15]\]. Most studies have included a mixed population of people with SMI and two large studies, which included only people with schizophrenia, found no effect of a lifestyle intervention on body weight \[[@CR16], [@CR17]\]. These latter studies suggest the weight management in people with schizophrenia may require a different approach from other SMIs such as bipolar disorder.
Given the challenges of implementing lifestyle change in people with schizophrenia and the lack of long-term effectiveness, alternative approaches are needed to manage overweight and obesity. A wide variety of treatments have been subject to clinical studies but currently no drug treatments are licensed for the treatment of antipsychotic-medication-associated weight gain or obesity in people with SMI with the exception of orlistat \[[@CR18]\]. The long-term use of the latter, however, is extremely limited by high discontinuation rates, making it of little value in routine clinical practice \[[@CR19]\].
To date, there have also been three completed trials of glucagon-like peptide 1 (GLP-1)-receptor agonists in people with SMI, two of which used exenatide and one used liraglutide (maximum dosage 1.8 mg) \[[@CR20]--[@CR22]\]. Liraglutide is a GLP-1-receptor agonist, with 97% homology to human GLP-1, which induces weight loss in humans mainly by reducing appetite and caloric intake, rather than increasing energy expenditure. There were contrasting results in the exenatide studies with one showing no difference between groups after 12 weeks of treatment \[[@CR20]\] but in the other the exenatide arm had greater mean weight loss (− 5.29 vs − 1.12 kg; *P* = 0.015), and reduced glycosylated haemoglobin (HbA~1c~) levels (− 0.21% vs 0.03%; *P* = 0.004) \[[@CR21]\]. In the liraglutide (maximum dosage 1.8 mg) study, glucose tolerance improved in the liraglutide group and body weight decreased compared with placebo (− 5.3 kg; 95% confidence interval (CI) − 7.0 to − 3.7 kg) \[[@CR22]\].
Liraglutide is approved for the management of obesity at a dosage of 3.0 mg daily, which is higher than the dosage used to treat diabetes \[[@CR23]\]. In a 56-week, double-blind trial involving 3731 participants without type-2 diabetes, 63.2% of the intervention arm compared with 27.1% in the placebo arm group lost at least 5% of their body weight, and 33.1% and 10.6%, respectively, lost more than 10% of their body weight \[[@CR23]\]. We have, therefore, chosen to use liraglutide (maximum dosage 3.0 mg) as we postulate that a higher dosage of liraglutide may offer even greater weight loss in people with SMI than the 1.8-mg dosage employed in the previous study or other currently available GLP-1-receptor agonists.
Aims and objectives {#Sec2}
-------------------
The aim of this pilot study is to undertake a double-blind, randomised controlled trial (RCT) of the use of liraglutide (maximum dosage 3.0 mg daily) in comparison to placebo in 60 obese or overweight people with schizophrenia, schizoaffective disorder or first-episode psychosis to assess the feasibility and acceptability of delivering a full-scale trial evaluating treatment with liraglutide in people with schizophrenia, schizoaffective disorder and first-episode psychosis.
Methods {#Sec3}
=======
Design {#Sec4}
------
This study is a double-blind, randomised pilot study of the use of liraglutide (maximum dosage 3.0 mg daily) in comparison to placebo (Fig. [1](#Fig1){ref-type="fig"}). It is important to include a double-blind placebo for two main reasons; there is evidence that people would be less likely to consent to a trial that includes a placebo arm because of the risk of not receiving an active treatment. As our ability to recruit to the trial was one of our key aims, it is important to assess whether the inclusion of a placebo prevented us from recruiting to the trial. Previous experience from weight-management trials that include pharmaceuticals have been plagued by high dropout rates in the placebo arm as the participants are able to assess the effectiveness of treatment. To adequately power a full RCT of liraglutide, we will need to know the likely dropout rate in the placebo group in this patient population. Fig. 1Consolidated Standards of Reporting Trials (CONSORT) study diagram
Setting {#Sec5}
-------
The study will be take place in a variety of community and inpatient mental health locations in the Southern Health NHS Foundation Trust. Public and Patient Involvement was actively included in the development of the trial and will continue throughout the trial.
Ethics approval and consent to participate {#Sec6}
------------------------------------------
South Central -- Hampshire B Research Ethics Committee (REC) approved the study on 17 April 2018 with REC reference: 18/SC/0085. Only those who agree to provide written informed consent will be included in the study. The study will be conducted in keeping with Good Clinical Practice (GCP) and the International Conference of Harmonisation (ICH) standards. The Trial Steering | background { # sec1 } = = = = = = = = = = the prevalence of obesity in the general population has increased dramatically over the last 30 years and it seems likely that the environmental changes that have provoked these increases have also affected people with severe mental illness ( smi ) ; in fact, the rates of overweight and obesity have increased even more rapidly in this situation \ [ [ @ cr1 ] \ ]. obesity adversely affects the physical health and psychological well - being of people with smi and if weight gain is brought to treatment, this can lead to non - adherence and risk of relapse. schizophrenia is a major psychiatric disorder that alters the individual ' s perception, thoughts, affect and behaviour and may involve a loss of insight and has a lifetime prevalence of approximately 1 % \ [ [ @ cr2 ] \ ]. schizoaffective disorder is recognised as a separate condition to schizophrenia and is more likely to occur in women at a specific age. this disorder affects an individual ' s thoughts and emotions \ [ [ @ cr3 ] \ ]. although individuals with first - episode psychosis do not fulfil the diagnostic criteria on schizophrenia or schizoaffective disorder, 90 - - 95 % of people presenting with a non - affective psychotic episode ( u. e. not mania and not depressive psychosis ) will still meet the criteria for a schizophrenia spectrum disorder 2 years later. mortality rates are increased two to three fold in people with smi and life expectancy is reduced by 10 - - 20 years. approximately 75 % of all deaths in people with schizophrenia being caused by physical illness with cardiovascular illnesses being the commonest cause \ [ [ @ cr4 ] \ ]. overweight and obesity contribute to this excess morbidity and mortality. recent studies estimate that obesity is two to three times more common among people with smi \ [ [ @ cr5 ] \ ]. obesity occurs early in the natural history of schizophrenia with a significant proportion of people with first - episode psychosis being overweight prior to any treatment. substantial weight gain ( \ > 7 % ) often occurs rapidly within 6 - - 8 weeks after antipsychotic - treatment initiation \ [ [ @ c ] \ ]. while most weight gain occurs early in treatment, longer - term observational studies suggest that weight gain continues for at least 4 months albeit at a slower rate \ [ [ @ cr7 ] \ ]. individuals with schizophrenia are more likely to consume a diet that is rich in fat and refined carbohydrates while containing less fibre, fruit and vegetables than the general population \ [ [ @ cr8 ] \ ]. although there are fewer studies, people with first - episode psychosis also have poor diets \ [ [ @ cr9 ] \ ]. physical inactivity and the social and urban deprivation experienced by those with schizophrenia may contribute further to the increased obesity rates \ [ [ @ cr8 ], [ @ cr10 ] \ ]. there may be disease - specific effects of schizophrenia, such as genetic susceptibility, that have additive or synergistic actions to increase body weight further \ [ [ @ cr5 ] \ ]. however, the most important factor related to weight gain in people with smi is the use of antipsychotic medications, which are among the most obesogenic drugs. weight gain is the commonest side effect of second - generation antipsychotic medication, affecting between 15 and 72 % of patients \ [ [ @ cr11 ] \ ]. other psychotropic drugs are often prescribed to people with schizophrenia and include some antidepressants and mood - stabilising drugs, such as lithium and sodium valproate ; these may also induce significant weight gain \ [ [ @ cr12 ] \ ]. both lifestyle and pharmacological interventions lead to significant reductions in body weight in the general population. not only are the interventions clinically effective, they are also cost - effective because of the benefits of long - term improved health outcomes, including decreased mortality \ [ [ @ cr13 ] \ ]. it is likely that similarly effective interventions for people with schizophrenia will also lead to improvements in health and would be a major step towards reducing the health inequalities experienced by people with schizophrenia. as the weight gain associated with antipsychotic medication result in some people discontinuing their medication, we hypothesis that effective weight - management strategies may also lead to improved adherence to antipsychotic medication and reduced relapse and hospitalisation rates. some studies have suggested that short - term lifestyle interventions could support weight reduction in people with smi. a meta - analysis of non - pharmacological interventions in people with smi \ [ [ @ cr14 ] \ ] reported a mean reduction in weight of 3. 12 kg over a period of 8 - - 24 weeks. however, the results of longer - term studies are more mixed. a recent meta - analysis found significant weight loss in only two of six studies with interventions lasting longer than a year \ [ [ @ cr15 ] \ ]. most studies have included a mixed population of people with smi and two large studies, which included only people with schizophrenia, found no effect of a lifestyle intervention on body weight \ [ [ @ cr16 ], [ @ cr17 ] \ ]. these latter studies suggest the weight management in people with schizophrenia may require a different approach from other smis such as bipolar disorder. given the challenges of implementing lifestyle change in people with schizophrenia and the lack of long - term effectiveness, alternative approaches are needed to manage overweight and obesity. a wide variety of treatments have been subject to clinical studies but currently no drug treatments are licensed for the treatment of antipsychotic - medication - associated weight gain or obesity in people with smi with the exception of orlistat \ [ [ @ cr18 ] \ ]. the long - term use of the latter, however, is extremely limited by high discontinuation rates, making it of little value in routine clinical practice \ [ [ @ cr19 ] \ ]. to date, there have also been three completed trials of glucagon - like peptide 1 ( glp - 1 ) - receptor agonists in people with smi, two of which used exenatide and one used liraglutide ( maximum dosage 1. 8 mg ) \ [ [ @ cr20 ] - - [ @ cr22 ] \ ]. liraglutide is a glp - 1 - receptor agonist, with 97 % homology to human glp - 1, which induces weight loss in humans mainly by reducing appetite and caloric intake, rather than increasing energy expenditure. there were contrasting results in the exenatide studies with one showing no difference between groups after 12 weeks of treatment \ [ [ @ cr20 ] \ ] but in the other the exenatide arm had greater mean weight loss ( − 5. 29 vs − 1. 12 kg ; * p * = 0. 015 ), and reduced glycosylated haemoglobin ( hba ~ 1c ~ ) levels ( − 0. 21 % vs 0. 03 % ; * p * = 0. 004 ) \ [ [ @ cr21 ] \ ]. in the liraglutide ( maximum dosage 1. 8 mg ) study, glucose tolerance improved in the liraglutide group and body weight decreased compared with placebo ( − 5. 3 kg ; 95 % confidence interval ( ci ) − 7. 0 to − 3. 7 kg ) \ [ [ @ cr22 ] \ ]. liraglutide is approved for the management of obesity at a dosage of 3. 0 mg daily, which is higher than the dosage used to treat diabetes \ [ [ @ cr23 ] \ ]. in a 56 - week, double - blind trial involving 3731 participants without type - 2 diabetes, 63. 2 % of the intervention arm compared with 27. 1 % in the placebo arm group lost at least 5 % of their body weight, and 33. 1 % and 10. 6 %, respectively, lost more than 10 % of their body weight \ [ [ @ cr23 ] \ ]. we have, therefore, chosen to use liraglutide ( maximum dosage 3. 0 mg ) as we postulate that a higher dosage of liraglutide may offer even greater weight loss in people with smi than the 1. 8 - mg dosage employed in the previous study or other currently available glp - 1 - receptor agonists. aims and objectives { # sec2 } - - - - - - - - - - - - - - - - - - - the aim of this pilot study is to undertake a double - blind, randomised controlled trial ( rct ) of the use of liraglutide ( maximum dosage 3. 0 mg daily ) in comparison to placebo in 60 obese or overweight people with schizophrenia, schizoaffective disorder or first - episode psychosis to assess the feasibility and acceptability of delivering a full - scale trial evaluating treatment with liraglutide in people with schizophrenia, schizoaffective disorder and first - episode psychosis. methods { # sec3 } = = = = = = = design { # sec4 } - - - - - - this study is a double - blind, randomised pilot study of the use of liraglutide ( maximum dosage 3. 0 mg daily ) in comparison to placebo ( fig. [ 1 ] ( # fig1 ) { ref - type = " fig " } ). it is important to include a double - blind placebo for two main reasons ; there is evidence that people would be less likely to consent to a trial that includes a placebo arm because of the risk of not receiving an active treatment. as our ability to recruit to the trial was one of our key aims, it is important to assess whether the inclusion of a placebo prevented us from recruiting to the trial. previous experience from weight - management trials that include pharmaceuticals have been plagued by high dropout rates in the placebo arm as the participants are able to assess the effectiveness of treatment. to adequately power a full rct of liraglutide, we will need to know the likely dropout rate in the placebo group in this patient population. fig. 1consolidated standards of reporting trials ( consort ) study diagram setting { # sec5 } - - - - - - - the study will be take place in a variety of community and inpatient mental health locations in the southern health nhs foundation trust. public and patient involvement was actively included in the development of the trial and will continue throughout the trial. ethics approval and consent to participate { # sec6 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - south central - - hampshire b research ethics committee ( rec ) approved the study on 17 april 2018 with rec reference : 18 / sc / 0085. only those who agree to provide written informed consent will be included in the study. the study will be conducted in keeping with good clinical practice ( gcp ) and the international conference of harmonisation ( ich ) standards. the trial steering | Background {# Sec1} = = = = = = = = = = The prevalence of obesity in the general population has increased dramatically over the last 30 years and it seems likely that the environmental changes that have provoked these increases have also affected people with severe mental illness (SMI ); in fact, the rates of overweight and obesity have increased even more rapidly in this cohort \ [[ @ CR1] \ ]. Obesity adversely affects the physical health and psychological well - being of people with SMI and if weight gain is attributed to treatment, this can lead to non - adherence and risk of relapse. Schizophrenia is a major psychiatric disorder that alters the individual ' s perception, thoughts, affect and behaviour and may involve a loss of insight and has a lifetime prevalence of approximately 1% \ [[ @ CR2] \ ]. Schizoaffective disorder is recognised as a separXtd condition to schizophrenia and is more lik#Iy to occur in women at a later age. This disorder affects an individual ' s thoughts and emotions \ [[ @ CR3] \ ]. Although individuals with first - episode psychosis do not fulfil the diagnostic criteria for schizophrenia or schizoaffective disorder, 90 - - 95% of people presenting with a non - affective psychotic episode (i. e. not mania and not depressive psychosis) will still meet the criteria for a schizophrenia spectrum disorder 2 years later. Mortality rates are increased two to tufee fold in people with SMI and life expectancy is reduced by 10 - - 20 years. Approximately 75% of all deaths in people with schizophrenia are caused by physical illness with cardiovascular disease being the commonest cause \ [[ @ CR4] \ ]. Overweight and obesity contribute to this excess morbidity and mortality. Recent studies indicate that obesity is two to three times more common among people with SMI \ [[ @ CR5] \ ]. Obesity occurs early in the natural history of schizophrenia with a significant proportion of people with first - episode psychosis being overweight prior to any treatment. Substantial weight gain (\> 7%) often occurs rapidly within 6 - - 8 weeks after antipsychotic - treatment initiation \ [[ @ CR6] \ ]. While most weight gain occurs eZ%ly in treatment, longer - term observational studies suggest that weight gain continues for at least 4 years albeit at a slower rate \ [[ @ CR7] \ ]. Individuals with schizophrenia are more likely to consume a diet that is rich in fat and refined carbohydrates while containing less fibre, fruit and vegetables than the general population \ [[ @ CR8] \ ]. Although there are fewer studies, people with first - episode psychosis also have poor diets \ [[ @ CR9] \ ]. Physical inactivity and the social and urban deprivation experienced by those with schizophrenia may contribute further to the increased obesity rates \ [[ @ CR8 ], [@ CR10] \ ]. There may be disease - specific effects of schizophrenia, such as genetic susceptibility, that have Zdvitive or synergistic actions to increase body weight further \ [[ @ CR5] \ ]. However, the most important factor related to weight gain in people with SMI is the use of antipsychotic medications, which are among the most obesogenic drugs. Weight gain is the commonest side effect of second - generation antipsychotic medication, affecting between 15 and 72% of patients \ [[ @ CR11] \ ]. Other psychotropic drugs are often prescribed to people with schizophrenia and include some antidepressants and mood - stabilising drugs, such as lithium and sodium valproate; these may also induce significant weight gain \ [[ @ CR12] \ ]. Both lifestyle and pharmacological interventions lead to significant reductions in body weight in the general population. Not only are the interventions clinically effective, they are also cost - effective because of the benefits of long - term improved health outcomes, including decreased mortality \ [[ @ CR13] \ ]. It is likely that similarly effective interventions for people with schizi9hrenia will also lead to improvements in health and would be a major step towards reducing the health inequalities experienced by people with schizophrenia. As the weight gain associated with antipsychotic medication result in some people discontinuing their medication, we hypothesis that effective weight - management strategies may also lead to improved adherence to antipsychotic medication and reduced relapse and hospitalisation rates. Some studies have suggested that short - term lifestyle interventions could support weight reduction in people with SMI. A meta - analysis of non - pharmacological interventions in people with SMI \ [[ @ CR14] \] reported a HeWn reduction in weight of 3. 12 kg over a period of 8 - - 24 weeks. However, the results of longer - term studies are more mixed. A recent meta - analysis found significant weight loss in only two of six studies with interventions lasting longer than a year \ [[ @ CR15] \ ]. Most studies have included a mixed population of people with SMI and two large studies, which included only people with schizophrenia, found no effect of a lifestyle intervention on body weight \ [[ @ CR16 ], [@ CR17] \ ]. These latter studies suggest the weight management in people with schizophrenia may require a different approach from other SMIs such as bipolar disorder. Given the challenges of implementing lifestyle change in people with schizophrenia and the lack of long - term effectiveness, alternative approaches are needed to manage overweight and obesity. A wide variety of treatments have been subject to clinical studies but currently no drug treatments are licensed for the treatment of antipsychotic - medication - associated weight gain or obesity in people with SMI with the exception of orlistat \ [[ @ CR18] \ ]. The long - term use of the latter, however, is extremely limited by high discontinuation rates, making it of little value in routine clinical practice \ [[ @ CR19] \ ]. To date, there have also been three completed trials of glucagon - like peptide 1 (GLP - 1) - receptor agonists in people with SMI, two of which used exenatide and one used liragoutiSe (maximum dosage 1. 8 mg) \ [[ @ CR20] - - [@ CR22] \ ]. Liraglutide is a GLP - 1 - receptor agonist, with 97% homology to human GLP - 1, which induces weight loss in humans mainly by reducing appetite and caloric intake, rather than increasing energy expenditure. There were contrasting results in the exenatide studies with one showing no difference between groups after 12 weeks of treatment \ [[ @ CR20] \] but in the other the exenatide arm had greater mean weight loss (− 5. 29 vs − 1. 12 kg; * P * = 0. 015 ), and reduced glycosylated jaemogloFin (HbA ~ 1c ~) levels (− 0. 21% vs 0. 03% ; * P * = 0. 004) \ [[ @ CR21] \ ]. In the liraglutide (maximum dosage 1. 8 mg) study, glucose tolerance improved in the liraglutide group and body weight decreased compared with placebo (− 5. 3 kg; 95% confidence interval (CI) − 7. 0 to − 3. 7 kg) \ [[ @ CR22] \ ]. Liraglutide is approved for the management of obesity at a dosage of 3. 0 mg daily, which is higher than the dosage used to treat diabetes \ [[ @ CR23] \ ]. In a 56 - week, double - blind trial involving 3731 participants without type - 2 diabetes, 63. 2% of the intervention arm compared with 27. 1% in the placebo arm group lost at least 5% of their body weight, and 33. 1% and 10. 6% , respectively, lost more than 10% of their body weight \ [[ @ CR23] \ ]. We have, therefore, chosen to use liraglutide (maximum dosage 3. 0 mg) as we postulate that a higher dosage of liraglutide may offer even greater weight loss in people with SMI than the 1. 8 - mg dosage employed in the previous study or other currently available GLP - 1 - receptor agonists. Aims and objectives {# Sec2} - - - - - - - - - - - - - - - - - - - The aim of this pilot study is to undertake a double - blind, randomised controlled trial (RCT) of the use of liraglutide (maximum dosage 3. 0 mg daily) in comparison to placebo in 60 obese or overweight people with schizophrenia, schizoaffective disorder or first - episode psychosis to assess the feasibility and acceptability of delivering a full - scale trial evaluating treatment with liraglutide in people with scBizophreniQ, schizoaffective disorder and first - episode psychosis. Methods {# Sec3} = = = = = = = Design {# Sec4} - - - - - - This study is a double - blind, randomised pilot study of the use of liraglutide (maximum dosage 3. 0 mg daily) in comparison to placebo (Fig. [1] (# Fig1) {ref - type = " fig "} ). It is important to include a double - blind placebo for two main reasons; there is evidence that people would be less likely to consent to a trial that includes a placebo arm because of the risk of not receiving an active treatment. As our ability to recruit to the trial was one of our key aims, it is important to assess whether the inclusion of a placebo prevented us from recruiting to the trial. Previous experience from weight - management trials that include pharmaceuticals have been plagued by high dropout rates in the placebo arm as the participants are able to assess the effectiveness of treatment. To adequately power a full RCT of liraglutide, we will need to know the likely dropout rate in the placebo group in this patient population. Fig. 1Consolidated Standards of Reporting Trials (CONSORT) study diagram Setting {# Sec5} - - - - - - - The study will be take place in a variety of community and inpatient mental health locations in the Southern Health NHS Foundation Trust. Public and Patient Involvement was actively included in the development of the trial and will continue throughout the trial. Ethics approval and consent to participate {# Sec6} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - South Central - - Hampshire B Research Ethics Committee (REC) approved the study on 17 April 2018 with REC reference: 18 / SC / 0085. Only those who agree to provide written informed consent will be included in the study. The study will be conducted in keeping with Good Clinical Practice (GCP) and the International Conference of Harmonisation (ICH) standards. The Trial Steering | Background ========== The prevalence of obesity in the general population has increased dramatically over the last 30 years and it seems likely that the environmental changes that have provoked these increases have also affected with severe mental illness (SMI); in fact, the rates of and obesity have increased even more rapidly in this cohort \[[@CR1]\]. Obesity adversely affects the physical health and psychological well-being of people with SMI and if gain is attributed to treatment, this lead non-adherence and risk relapse. is a psychiatric disorder that alters the individual's perception, thoughts, affect and behaviour and may involve a loss of insight and has a lifetime prevalence of approximately 1% \[[@CR2]\]. Schizoaffective disorder is recognised as a separate condition to schizophrenia and is more likely occur in at a later age. This disorder an individual's thoughts and emotions \[[@CR3]\]. individuals with first-episode do not fulfil the criteria for schizophrenia or schizoaffective disorder, 90--95% of presenting with a non-affective psychotic episode (i.e. not mania and not will still meet the criteria for a schizophrenia spectrum disorder 2 years later. Mortality rates are increased two to three fold in people with SMI and life expectancy is reduced 10--20 years. Approximately 75% deaths in people with schizophrenia are caused physical illness with cardiovascular disease being the commonest cause Overweight and obesity contribute to this excess and mortality. Recent studies indicate that obesity is two to three times more among people with SMI \[[@CR5]\]. Obesity occurs early in the natural history of schizophrenia with a significant proportion of people with first-episode psychosis being overweight prior to any treatment. Substantial weight gain (\> 7%) often occurs rapidly within 6--8 weeks after antipsychotic-treatment initiation While weight gain occurs early in treatment, longer-term studies that weight gain continues for at least 4 years albeit at a slower rate \[[@CR7]\]. Individuals schizophrenia more likely to consume a diet that is rich in fat and refined carbohydrates while containing less fibre, fruit and than the general population \[[@CR8]\]. Although there are fewer studies, people with first-episode psychosis also have poor diets \[[@CR9]\]. Physical inactivity and the and urban deprivation experienced by with may contribute further the increased obesity rates \[[@CR8], [@CR10]\]. There may be disease-specific of schizophrenia, such as genetic susceptibility, that have additive or synergistic actions to body weight further \[[@CR5]\]. However, the most important related to weight gain in people with SMI is the use of antipsychotic medications, which are among the most drugs. gain is commonest side effect of second-generation antipsychotic medication, affecting between 15 and 72% patients \[[@CR11]\]. Other psychotropic are often to people with schizophrenia and include some antidepressants and mood-stabilising drugs, such as lithium and sodium valproate; these may also induce significant weight gain \[[@CR12]\]. Both lifestyle and pharmacological interventions to significant reductions in body weight in the general population. only are the clinically they are cost-effective because of the benefits of long-term improved health outcomes, including decreased mortality \[[@CR13]\]. It is likely that similarly effective for people with schizophrenia will also lead to in health and would be a major step towards reducing the health inequalities experienced by people with schizophrenia. As the weight gain associated with antipsychotic medication result some discontinuing their medication, we hypothesis that effective weight-management strategies may also to improved adherence and reduced relapse and hospitalisation rates. Some studies have suggested that short-term lifestyle could support weight reduction in people with SMI. A meta-analysis non-pharmacological interventions in people \[[@CR14]\] reported a mean reduction in weight 3.12 kg over a period of 8--24 weeks. However, the results of longer-term studies are more mixed. A recent meta-analysis found significant weight in only two of six studies with interventions lasting longer than a year \[[@CR15]\]. Most studies have included a mixed of people with SMI and two large studies, which included only people with schizophrenia, found no effect of a lifestyle intervention body weight \[[@CR16], [@CR17]\]. These latter studies suggest the weight management in people with schizophrenia may require a different approach from other SMIs such bipolar disorder. Given the challenges of implementing lifestyle change in people with schizophrenia and the lack of long-term effectiveness, approaches are needed to manage overweight and obesity. A wide variety of treatments have been subject to clinical studies but currently no drug treatments are licensed for the of antipsychotic-medication-associated gain or obesity in people SMI with the of orlistat \[[@CR18]\]. The long-term use of the latter, however, is extremely limited high discontinuation making it of value in routine clinical practice \[[@CR19]\]. To date, there have also three completed trials of glucagon-like peptide 1 (GLP-1)-receptor agonists in people with of which used exenatide and one liraglutide (maximum dosage 1.8 mg) Liraglutide a GLP-1-receptor agonist, with 97% homology to human GLP-1, which induces weight loss in humans mainly by reducing appetite and caloric intake, rather than energy expenditure. were contrasting in the exenatide studies with no difference between groups after 12 weeks of treatment but in the other the exenatide arm had greater mean weight loss (− 5.29 − 1.12 kg; *P* = 0.015), reduced glycosylated haemoglobin (HbA~1c~) levels (− 0.21% vs 0.03%; = 0.004) \[[@CR21]\]. In the (maximum dosage 1.8 mg) study, tolerance improved in the liraglutide group and body weight decreased compared with placebo (− 5.3 kg; 95% interval (CI) − 7.0 to − kg) \[[@CR22]\]. Liraglutide is approved for the management of at a dosage of 3.0 daily, which higher than the dosage used to treat diabetes \[[@CR23]\]. In a 56-week, double-blind trial involving 3731 participants without type-2 diabetes, of the intervention arm compared with 27.1% the arm group lost at least 5% of their body weight, and 33.1% and 10.6%, respectively, lost more than 10% of their body weight \[[@CR23]\]. We have, therefore, to use liraglutide (maximum dosage 3.0 mg) as we postulate that a higher dosage of liraglutide may offer even greater weight loss in people with SMI than the 1.8-mg dosage employed in the previous study or other currently available GLP-1-receptor agonists. Aims objectives {#Sec2} ------------------- The of pilot study is to undertake double-blind, randomised controlled trial of the use of liraglutide (maximum dosage 3.0 mg daily) in comparison to placebo in 60 obese or overweight people with schizophrenia, schizoaffective or first-episode psychosis to assess the feasibility and acceptability of delivering full-scale trial evaluating with liraglutide in people with schizophrenia, schizoaffective disorder first-episode psychosis. Methods {#Sec3} ======= Design {#Sec4} This study is a double-blind, randomised pilot study of the use of liraglutide (maximum dosage 3.0 mg daily) in comparison placebo (Fig. [1](#Fig1){ref-type="fig"}). It is important to include a double-blind placebo for two main reasons; there is evidence people would be less likely consent to a that includes a placebo arm because of the risk not receiving an active treatment. As our ability to recruit to the was one of our key aims, it is important the inclusion of a placebo us recruiting to the trial. Previous experience from weight-management trials that include pharmaceuticals have plagued by high dropout rates in the placebo the are able to assess the effectiveness of treatment. To adequately power full RCT of liraglutide, we will need to know the likely dropout rate in the group in this patient population. Fig. 1Consolidated Standards Reporting Trials (CONSORT) study diagram Setting {#Sec5} ------- The study will be take place in a variety of community and inpatient mental health locations in Southern Health NHS Foundation Trust. and Patient Involvement was included in the development of the trial and continue throughout the trial. Ethics approval and consent {#Sec6} ------------------------------------------ South Central -- B Research Ethics Committee (REC) approved the study on April 2018 with REC 18/SC/0085. Only those who to provide written informed consent will be included in the study. The study will be conducted in keeping with Clinical Practice (GCP) and International Conference of Harmonisation Trial Steering | BAcKgrOUNd {#seC1}
==========
THE pRevALeNCE of oBeSITy in tHe GENeral poPulatION HAS IncreASED DRAMatICALLy Over The LAST 30 yeArS And iT sEeMS lIkElY ThAT ThE EnvirOnmenTAl CHanges THAt hAve PrOVOked tHEse INCREAseS HaVE ALSO aFFEcTEd pEOPle WITH seVerE mEntaL iLLNesS (SmI); iN fAct, THe raTeS Of OveRweighT ANd obeSItY HAVE InCreasED evEn moRE RApIdlY In THIS cOHoRt \[[@CR1]\]. ObeSItY AdVersELy afFecTs thE pHysicAl hEaltH aNd PSYCHOLOGiCal welL-BEING OF PEoPLe WITh sMi anD iF wEIgHt GaIn is aTtRIbUted TO TrEaTmENt, tHis CAn LEad To NON-adHErence AnD RISk of RElAPsE.
scHIZOPHREnIA Is a mAjOR psYchiAtric diSORDER tHAT alTers thE inDiviDuaL's perceptiOn, ThougHTs, afFeCt AnD BehavIOUR and may iNvOLvE A losS of insigHt aNd haS a LifEtiMe PreVaLENcE Of apProxiMATeLy 1% \[[@CR2]\]. SCHizoaFfECtive DIsoRDer is rEcOgNISEd As A SEPARATE CoNdiTIon To scHIZopHrENIA AND iS mOre LikELY tO OccUr iN Women AT a LateR AgE. THiS DISOrder aFfECts an INDIvIDUAL'S tHoUghts and eMOtIONS \[[@Cr3]\]. AlThouGh indIViDUAls wIth fIrSt-EPISodE pSychOSIs do Not fULfiL THe dIagNOSTic cRiterIa For ScHIzoPHREniA Or schizoAFfecTivE DisORdeR, 90--95% oF PEOPle PREsEntInG WiTH A non-AfFeCtive PSycHOTIc EPiSode (I.e. nOt maNIA AND nOt DePrESsIvE PsYcHOsis) wiLL stilL MeET tHe CriteRIA for A schizOphrenia sPECtRUM DIsOrder 2 YEArS LATeR. MorTALITy RatEs arE INCREasED tWO tO thReE foLD IN pEopLE WITh SMi ANd liFe EXpeCtANcy IS reDuced By 10--20 yearS. aPPRoXIMately 75% of all DEAtHS in pEOple wiTh ScHIZophRenia ArE causEd BY PHYsIcAl iLLnEss wiTH CARdIovAscuLAr dISEAse BEIng THE COMMoNeST CaUSe \[[@CR4]\]. oVeRwEight aND obeSITy cONTRIBuTe tO this excess MorbiDIty aNd MorTALiTy. ReceNT studiES inDicaTe thAT ObESItY Is twO TO tHrEE tIMES mORE cOMMoN AMOnG PeoPle wiTh smi \[[@cR5]\]. ObESITy ocCurs EaRLy In tHe NATUrAL HistoRy OF SChiZOpHreNIA wiTh a siGnifICanT ProporTION of pEOPle WiTh fIRSt-ePisOde psycHosiS bEINg oVErWEIGht pRIor tO Any TREatmeNT. suBStAnTiAL WEIGHT gaIN (\> 7%) oftEn OcCURS raPiDLY wIthIn 6--8 wEEKS AFtEr anTipsychOtiC-tREATMent INItIATiON \[[@cr6]\]. whiLe most wEIGhT GAIn OCcUrS earLY In treATMENT, LoNGEr-tErm ObsErVationAL stuDies SUggeST ThaT wEiGhT GAIN CoNTInues fOr aT leaST 4 yEarS ALBEIT aT a slower rATe \[[@cR7]\].
iNDIvIDUALS wiTH schIZOPhReniA arE MORE Likely tO CONSUme a DiET THAt Is rICh in fAT and refInEd CaRBoHydrAteS wHile CoNtAiNiNG LEsS FIbRe, FRuIT AnD VEGetABleS thAn ThE GeneRAl poPULATION \[[@CR8]\]. ALtHOUgH THERe aRe FEWeR STuDies, PeOPLe WitH fIRSt-EpiSOde psYcHOSis ALsO HavE Poor DIetS \[[@Cr9]\]. pHysicAl INActIviTY aND THe SOciAl AnD UrBaN DeprivATion ExPeRIeNcEd by those With scHizoPHReNiA mAy ConTRIbUTe FurtHeR to THe InCReASed oBESiTy RaTes \[[@CR8], [@Cr10]\]. THeRE MAY BE dIsEaSE-speCIfIc EffeCTS OF scHIzophRenIa, suCH AS GEnEtic suScEPtIbiLITy, THat HaVe AdDItIVe OR syNErGisTic ACTIOnS to iNcRease bODY WeIgHT fUrThEr \[[@cR5]\]. hOwevER, THe most iMPORTANt FACTOr rElated to wEIght GAIN IN peoPLe wiTh sMi iS tHe uSE OF ANtiPsYChOtIc meDiCAtIons, WhICh ArE AmOnG THE mOsT OBeSoGeNIC drugs. weighT gaIn Is THE CoMMoNesT SIde EfFeCt oF SecOnD-GENeRAtIon anTIpSYcHoTIC MedICaTiOn, AfFeCtINg BEtWEEn 15 anD 72% Of pATIEnts \[[@cr11]\]. otHEr pSychOtROpIC dRUGS ArE ofteN PrescRibED To peOPle wItH scHiZOPHrENiA ANd INclUDE sOME aNTiDEprEsSANTS aNd mOod-StabilisInG DRUgS, SUch As litHiuM AnD soDiuM valPROATE; THesE mAy ALSO INdUCE SiGnIfiCaNT weIghT gaiN \[[@cR12]\].
botH lIFeSTyle aNd phArmAcOLoGICAL iNtERVENTioNS lEAd tO sIGNifIcant RedUCTIons iN BOdy weIGht In THe geNERAl populATIon. nOt OnLy arE the InTeRVENTionS clIniCAlLy eFFEcTiVE, tHeY ArE alSO coSt-EFFECtIvE becAuSe of THe BeNeFIts Of LONG-TeRM imProvEd HEAlth outcoMeS, incLuDIng DECReaSeD mORTalitY \[[@Cr13]\]. iT IS LIkelY THAT SIMiLARLy EffeCtive INtERVeNtIonS fOr PeOple with SChIzOPHrEniA wILl aLSO LEad TO IMProVeMENTs IN HeALTH aNd WOuld bE A MaJOr step TOwaRdS REducIng the HEaLtH iNEQualIties EXPeriENceD BY pEOpLe WitH sChiZOpHRENiA. aS THe WEIgHt GAin associATEd wIth antIpsYChOtic MediCatiON Result in sOMe pEOplE disCONtinuING theIr mEdIcAtion, wE hyPoTHESIs tHat effectivE WeigHt-manAGEmENt strAtEGiES MAy Also LEAD To iMproveD aDhEreNCe TO anTIPsyChOtIc MedicatIon anD ReDuCeD rELAPsE anD HOsPItALISaTION RAtes.
soME StuDies HavE suggEStED thAt SHORT-TeRM lIfEStYLE iNTerVEnTIonS CouLD SuPPOrt weIGHt REdUctiOn iN peoplE WIth sMi. A mETA-ANaLYSIS OF nOn-PhArmacOlOgicAL iNTERvENTions In PEoplE wItH smi \[[@Cr14]\] REPorTEd a MeAN REDUCtion IN WeiGHT of 3.12 KG oVer A periOD Of 8--24 WEekS. hOWEVEr, tHe reSuLtS Of longER-TERM sTudIes ARE mOre mIxED. a rECeNT MetA-aNaLySiS fOuND sIgNIFICANt wEiGHt LOsS in oNlY tWo OF SIX sTudieS wiTh IntERVenTiOns laSTinG lonGeR tHAn a yeAr \[[@cr15]\]. MoST stUDIEs HAvE iNcLUDed a MIXEd pOpulatiOn OF pEOPLe wItH SMi aND tWO LARge stUDies, whicH InclUdEd oNLY PEOple with SCHizOphrEnIA, fOUnD nO EffEct Of A LIfeStyle iNtERVeNTIon on bODY wEIGHt \[[@CR16], [@cr17]\]. tHesE LaTTer StUdieS sUggEst THe wEiGht MaNagEmeNt in peoPLE WitH SChIzoPhrENia mAy REquIRE A DIfFeRent ApProach FRoM otheR sMis sucH aS BIpoLAR dIsorDEr.
GIven ThE CHAlLENGEs OF implEmENting lIFEStyLe ChANGe In PeOpLe With sCHizopHReniA and ThE LAck Of lonG-teRm EfFEctivEneSS, ALTErnAtIVE apProAches are NeeDED TO MAnage OVeRweight ANd obeSITY. a WiDE VArIETY oF tREatMenTs haVe BeEN SUBJeCt TO ClINicAL STudiES bUT CUrrently No dRug TReatMentS Are LiCEnSEd FOR tHE tReatmEnT OF ANtiPSycHotiC-MeDiCAtIoN-aSsoCIAted wEIGht GAIN or OBEsITy in PEOplE WITH smi WitH THE ExceptiOn OF OrlisTAt \[[@CR18]\]. THe LoNg-teRM uSE oF tHe LAtTEr, HOWevER, IS extrEMely LImiTEd bY High diScOnTINUATion ratEs, MaKInG iT oF LiTTLE vALUe In ROuTiNE ClinicAL pRacTice \[[@cr19]\].
tO daTe, THErE hAVe aLsO beeN threE cOMpLeteD triAlS OF GLUCAGON-lIkE PePtIDE 1 (gLP-1)-RECepTOR aGONIStS In pEoPLE WIth SMI, tWo OF WHiCH USeD eXENatIdE and one useD LIRAgluTide (mAXiMUm dosaGE 1.8 mG) \[[@cR20]--[@CR22]\]. LirAgLutide IS a glp-1-recEPToR agoNiST, WitH 97% hOMoloGY TO human gLP-1, WhICh iNduces WEIght LoSS In hUMANs mAInLY By REdUCIng AppETITE ANd calorIc InTaKe, ratHEr THAN INCreaSING EnERgY ExPENdItURE. tHErE WERe cOnTRAstiNG RESuLtS in THE EXENaTIdE sTudIes With one ShOWInG no DiFfEReNCe beTween gROUPs AftEr 12 wEeks Of treaTmEnT \[[@cR20]\] But in tHE other THe EXENaTIDe ARM Had grEATEr Mean WEIgHT LOSS (− 5.29 Vs − 1.12 kg; *p* = 0.015), And ReduCed gLycoSYLaTEd HaEmOgLObIn (hbA~1c~) LevEls (− 0.21% Vs 0.03%; *P* = 0.004) \[[@Cr21]\]. In ThE LIRAglutiDE (mAxIMuM DoSAGe 1.8 mG) studY, gLUCoSe TOLERANCE IMproVEd iN tHe LirAGlutide GRouP anD boDY wEiGHt dECrEasED COmParED WIth PLACebO (− 5.3 kG; 95% cONFiDEnce INterVaL (Ci) − 7.0 tO − 3.7 KG) \[[@cr22]\].
LiragluTidE IS APprovEd fOr tHE MANagEmEnt Of obeSITY at A DoSAgE of 3.0 Mg daiLY, wHiCH Is higHER THan thE DoSaGe usEd to TREat diabetEs \[[@cr23]\]. in a 56-Week, douBle-BLInd TRIal inVoLviNg 3731 pARTICIpaNTS withOut TyPE-2 diAbeTeS, 63.2% Of tHe interVenTION Arm coMPArEd wItH 27.1% In The pLAcEBo ArM gROuP LOST AT least 5% oF theIr BOdY wEiGHt, ANd 33.1% AnD 10.6%, rEspecTiveLY, LoST morE thAn 10% Of TheIR boDY weiGHT \[[@cR23]\]. WE HAVE, ThEREFOre, cHOsen To uSE liRAGlUTIde (MAximUM dOSAGe 3.0 Mg) aS We PosTUlaTe THAt A HiGher DOsaGe of lIRAGLuTiDe mAy OFFEr EvEn gREAteR WEIGHt LOss IN peOpLE With SMI thAN ThE 1.8-MG DOsage emPLoYed In THE PrEViOUS STUDY oR other CuRrentLY aVAILable GLP-1-REcEPTOr AgoNistS.
aIMs and obJEctIves {#seC2}
-------------------
ThE Aim Of tHiS PILOt sTUDy IS tO UNdeRTAKe a DOUbLE-BlInd, RAndOMiSed cONTRollED TRiaL (rCt) oF The use oF LIrAglUtiDE (maXImuM DosAGE 3.0 mg daIlY) IN coMparISoN To plACEBo in 60 obesE OR OveRWEighT PEoPle WITh sCHiZOPhReNia, SCHIZOAFFEctIve DiSORdEr OR FiRSt-epISOdE psyCHOSis tO aSsEsS thE FeAsIBILiTY AnD AccePtABilITy of DelIVERING A fuLL-ScAle TriAL EVAlUATING treaTmENT wItH lIRAglutiDe In PeOple WItH sCHiZOPhrENIa, SchIzoAfFeCTiVe DiSorDeR aNd fIRsT-ePisodE PSycHoSIS.
MeTHOdS {#SEc3}
=======
dEsign {#sEc4}
------
THis StUDy IS A doUble-bLInD, RANdOmiSed pilOt StudY oF THE USE Of LirAglutiDE (maxiMum DOsAge 3.0 Mg DaILy) IN ComPaRIsOn To PLaCeBo (FiG. [1](#FiG1){rEf-tYPE="FIg"}). It is iMporTANT to InclUdE A DoUBlE-blIND pLAceBO FOR TWO mAIN rEASonS; theRe iS evidENcE tHAT PEOPLE WOulD bE LesS LiKeLy to cONsEnt To A TrIAl THAt INclUdeS a pLacebo ARm BecaUse of THE RIsk oF noT rEceIvING an ActIve TReaTMEnt. AS OUR aBILity TO REcRuit tO thE TRiaL wAs one of oUR kEY Aims, it is iMPORTAnT to ASsESs wHEThEr ThE INClUSION of A PlACEBO PreVenTED uS frOm rEcrUiTINg To thE TrIal. pREvIOUS eXperIeNce frOm weIghT-MaNAGEMENT TrIaLS ThaT inCLuDe phArmAcEutICals HAVe BEEN pLaGUEd By higH DROpoUt RAteS in thE pLACeBo Arm aS the PartIciPanTS are AblE to ASSeSS THe effECTIveness OF tREaTment. To adEqUaTElY PoweR A FulL RCT Of lIrAGlutIDE, we wILl nEED TO knOw tHe LIkelY DrOPOut RatE iN the pLACEBo GrouP In THIS paTIENT pOPULATiOn. FIg. 1CoNSoliDAteD sTaNdArds oF rePOrTing TRiALs (ConSoRT) StuDY diaGRAM
SeTting {#SEc5}
-------
tHE sTUdy WILL bE tAke PLACe IN A VarIEty of cOmMuNIty And INpATIEnT mENtAl HeAlTH LocaTIoNs In The SoUTHerN HEAlTh nHs FoUnDatION tRUsT. PUbLIc anD PatienT InVoLVeMenT WAs ActIvely INcLUDed iN the dEVeLOpMENT of thE trIal aND wILl conTInUe tHRoughOUT tHe TriAl.
Ethics APPRoVal And conSENt To PArtiCipate {#sec6}
------------------------------------------
SoUTh CENtrAL -- haMpSHIrE b REsearch ETHICS coMMITtEe (rEC) appRoVEd the StUDy oN 17 aprIL 2018 WITh reC REfeREnCe: 18/SC/0085. only ThOSe WhO aGrEe To PrOViDE WritTEN INforMed conSenT WIlL Be inCLUDEd IN THe Study. ThE stUdy wILl BE CONDUCted iN KEepinG wITh GoOd cLiniCAl PRACTicE (GCP) anD thE InterNATIonal coNFeREncE of harMoNISaTion (ICH) stAnDarDs. ThE tRIal StEerIng | Background {#Sec1} ========== The prevalence ofobesity in the general populationhas increased dramatically overthe last 30 years and it seems likely that the environmental changes that have provoked theseincreases havealso affected people with severe mental illness (SMI); in fact, the ratesof overweight andobesity have increased even more rapidly in this cohort \[[@CR1]\]. Obesity adverselyaffectsthe physical health and psychological well-being of people withSMI and ifweight gain is attributed to treatment,thiscan lead to non-adherence andrisk of relapse. Schizophrenia is a majorpsychiatric disorder that alters the individual's perception, thoughts, affectand behaviour and may involve a loss of insightand has a lifetime prevalence of approximately 1% \[[@CR2]\]. Schizoaffectivedisorder is recognised asa separate condition to schizophrenia andis more likely to occur in womenata later age. Thisdisorder affects an individual's thoughts and emotions \[[@CR3]\]. Although individuals with first-episode psychosis do not fulfil the diagnostic criteria for schizophrenia or schizoaffective disorder, 90--95%of people presenting with anon-affective psychotic episode (i.e. notmania and not depressive psychosis) will stillmeet thecriteria for a schizophrenia spectrum disorder 2 years later. Mortality rates are increased two to three fold inpeople with SMI and life expectancy is reducedby 10--20 years. Approximately 75% of all deaths in people with schizophrenia are caused byphysical illness withcardiovascular disease being the commonest cause \[[@CR4]\]. Overweight andobesity contribute to this excess morbidity and mortality. Recent studies indicate that obesityis two to three times more commonamong people with SMI \[[@CR5]\]. Obesity occurs early in the natural history of schizophrenia with a significant proportion of people with first-episode psychosis being overweight prior to any treatment. Substantial weight gain (\> 7%) often occurs rapidly within 6--8 weeks after antipsychotic-treatmentinitiation\[[@CR6]\]. While mostweight gain occurs early in treatment, longer-termobservational studies suggest that weightgain continues forat least 4years albeit at a slower rate \[[@CR7]\].Individuals with schizophrenia are more likely toconsume a diet thatis rich in fat and refined carbohydrates while containing less fibre, fruit and vegetables than thegeneral population \[[@CR8]\]. Although there are fewer studies, peoplewith first-episode psychosis alsohave poor diets \[[@CR9]\]. Physical inactivity and the socialand urban deprivation experienced by those with schizophrenia may contribute further to the increased obesity rates \[[@CR8], [@CR10]\].There may be disease-specific effects of schizophrenia, such as genetic susceptibility, thathave additive or synergistic actionstoincrease body weight further \[[@CR5]\]. However, the most important factor related to weight gain in people with SMI is the use of antipsychotic medications, which areamong the mostobesogenicdrugs. Weight gainis the commonest side effect of second-generation antipsychotic medication, affecting between 15 and72% of patients \[[@CR11]\]. Other psychotropic drugs are often prescribed to people with schizophrenia and include some antidepressants and mood-stabilising drugs,such aslithium and sodium valproate; these may alsoinduce significant weight gain\[[@CR12]\]. Both lifestyle and pharmacologicalinterventions lead to significantreductions inbody weightin the generalpopulation. Not only are the interventions clinically effective, theyare also cost-effective because of the benefits of long-termimproved health outcomes, including decreased mortality \[[@CR13]\]. It is likely that similarly effective interventionsforpeople with schizophrenia will also lead to improvements inhealth and wouldbe a major step towards reducing the health inequalities experienced by people with schizophrenia. As the weight gain associatedwith antipsychotic medication result insome peoplediscontinuing their medication, we hypothesis that effective weight-management strategies mayalso leadto improved adherence to antipsychotic medication and reduced relapse andhospitalisation rates. Some studies havesuggested that short-term lifestyle interventions could support weight reduction in peoplewithSMI. A meta-analysis of non-pharmacological interventionsin people with SMI\[[@CR14]\] reporteda mean reduction in weight of 3.12kg over a period of 8--24 weeks.However, the resultsof longer-term studies are more mixed. A recent meta-analysis found significant weight loss in only two of six studies withinterventions lasting longer than a year \[[@CR15]\]. Most studies have included a mixed population of peoplewithSMI and two large studies, which included onlypeoplewith schizophrenia, found no effect of a lifestyle intervention on body weight \[[@CR16], [@CR17]\]. Theselatter studiessuggest the weight management inpeople withschizophrenia may require adifferent approach from other SMIssuch as bipolar disorder. Giventhe challenges of implementing lifestyle change in people withschizophrenia and thelack of long-term effectiveness, alternative approaches are needed to manage overweight and obesity. A widevariety of treatments havebeen subject to clinical studies butcurrently no drug treatments are licensed for the treatment of antipsychotic-medication-associated weightgain or obesity in peoplewithSMI with theexception of orlistat \[[@CR18]\]. The long-term use of the latter, however, isextremely limited byhigh discontinuation rates, making itof little value in routine clinical practice \[[@CR19]\].To date, there have alsobeenthree completed trials of glucagon-like peptide 1 (GLP-1)-receptor agonists in people with SMI,two of which used exenatide and one used liraglutide (maximumdosage 1.8 mg) \[[@CR20]--[@CR22]\]. Liraglutideis a GLP-1-receptor agonist, with 97% homology to human GLP-1, which induces weight lossinhumans mainly by reducing appetite and caloric intake, rather than increasing energy expenditure. There were contrasting results intheexenatidestudies with oneshowing no difference between groups after 12 weeks of treatment \[[@CR20]\]butin theotherthe exenatide armhadgreater mean weight loss (− 5.29 vs − 1.12 kg; *P*= 0.015), and reduced glycosylated haemoglobin (HbA~1c~) levels(− 0.21% vs 0.03%; *P* = 0.004) \[[@CR21]\].In the liraglutide(maximum dosage 1.8 mg) study, glucose toleranceimproved in theliraglutide group and body weight decreased compared withplacebo (− 5.3 kg; 95% confidence interval (CI) − 7.0 to − 3.7 kg) \[[@CR22]\]. Liraglutide is approved for the managementof obesityat a dosage of3.0 mg daily, which is higher than the dosage used to treat diabetes \[[@CR23]\]. In a 56-week, double-blind trial involving 3731 participants without type-2 diabetes,63.2%ofthe intervention arm comparedwith27.1% in the placebo armgrouplost at least 5% of their bodyweight, and 33.1% and 10.6%, respectively, lost more than 10% of their body weight\[[@CR23]\].We have, therefore, chosen to use liraglutide (maximumdosage 3.0 mg) as we postulate that a higher dosage of liraglutide may offer even greater weight loss inpeople with SMI than the1.8-mg dosage employed in theprevious study or other currently available GLP-1-receptor agonists.Aims and objectives {#Sec2} -------------------The aim of this pilot study is to undertake a double-blind, randomised controlled trial (RCT) of the use of liraglutide (maximum dosage3.0 mg daily) in comparison to placebo in 60 obese or overweight people withschizophrenia, schizoaffective disorder or first-episode psychosis to assess thefeasibility and acceptability ofdelivering a full-scale trial evaluating treatment with liraglutide in people with schizophrenia, schizoaffective disorder and first-episode psychosis. Methods {#Sec3} ======= Design {#Sec4}------ This study is a double-blind, randomised pilot study of the useofliraglutide (maximumdosage 3.0 mg daily) in comparison to placebo (Fig. [1](#Fig1){ref-type="fig"}). It is important to include a double-blind placebo for two mainreasons; there is evidencethat people would be less likely to consent to a trial that includes a placebo arm because of the risk of notreceiving an active treatment. As our ability torecruit to thetrial was one of our key aims, it isimportanttoassess whether the inclusionof a placebo prevented us from recruiting to the trial. Previousexperience from weight-management trialsthat include pharmaceuticals have been plagued by high dropout rates in the placebo arm as the participants are able toassessthe effectiveness of treatment. To adequately power a fullRCT of liraglutide,we willneed to knowthe likely dropout rate in the placebo group in thispatient population. Fig.1Consolidated Standards of ReportingTrials (CONSORT) study diagram Setting {#Sec5} ------- Thestudy will be take place in a variety ofcommunity and inpatient mental health locations in the Southern Health NHS Foundation Trust. Public and Patient Involvement was actively included in the development of the trial and will continue throughout thetrial. Ethics approval and consentto participate {#Sec6} ------------------------------------------ South Central-- Hampshire B Research Ethics Committee (REC) approved the study on17 April 2018 with REC reference: 18/SC/0085. Only those who agree to provide written informed consent will be included in thestudy.The study will be conducted in keeping withGood Clinical Practice (GCP) and the International Conference of Harmonisation (ICH)standards.TheTrial Steering | Background _{#Sec1}_ ========== _The_ prevalence _of_ obesity in the general population has _increased_ dramatically over _the_ last 30 years and it seems likely that the environmental changes that have provoked these increases have also affected people with severe _mental_ _illness_ (SMI); _in_ fact, the rates of overweight _and_ obesity have increased even more rapidly _in_ this cohort _\[[@CR1]\]._ Obesity adversely affects the physical health and psychological well-being _of_ people _with_ SMI and if weight gain is _attributed_ to treatment, this can lead to non-adherence _and_ risk of _relapse._ Schizophrenia is a major psychiatric disorder that alters the individual's perception, thoughts, affect and behaviour and may involve a _loss_ of insight and _has_ a _lifetime_ prevalence _of_ approximately 1% \[[@CR2]\]. Schizoaffective disorder _is_ recognised as a separate condition to _schizophrenia_ and is more likely to occur in women at _a_ _later_ _age._ This disorder _affects_ an individual's thoughts and emotions \[[@CR3]\]. _Although_ individuals with first-episode psychosis do _not_ fulfil the diagnostic _criteria_ for schizophrenia or schizoaffective disorder, 90--95% of people presenting with _a_ non-affective psychotic episode (i.e. not mania and not _depressive_ _psychosis)_ will _still_ meet the criteria for a schizophrenia spectrum _disorder_ 2 years later. Mortality rates are _increased_ _two_ to _three_ fold in people with SMI and _life_ expectancy is reduced by 10--20 years. Approximately 75% of all deaths in people _with_ schizophrenia are _caused_ by physical illness _with_ cardiovascular disease being _the_ commonest cause \[[@CR4]\]. Overweight and _obesity_ contribute to this excess morbidity _and_ mortality. _Recent_ _studies_ indicate _that_ obesity is two to _three_ times more common among _people_ with SMI \[[@CR5]\]. Obesity occurs early in the natural history of schizophrenia with _a_ significant proportion of people with first-episode psychosis being overweight _prior_ to any _treatment._ Substantial weight gain (\> 7%) often occurs _rapidly_ within 6--8 _weeks_ after antipsychotic-treatment initiation \[[@CR6]\]. While _most_ weight _gain_ occurs _early_ in treatment, _longer-term_ observational studies suggest that _weight_ gain continues for at least _4_ years _albeit_ at a slower rate \[[@CR7]\]. Individuals with schizophrenia are _more_ _likely_ _to_ consume _a_ diet that _is_ rich in fat and refined carbohydrates _while_ containing less fibre, fruit and vegetables than the general population \[[@CR8]\]. _Although_ there are fewer _studies,_ people _with_ first-episode psychosis _also_ _have_ poor diets _\[[@CR9]\]._ Physical inactivity and the social and urban deprivation _experienced_ by those with schizophrenia _may_ _contribute_ further _to_ the _increased_ obesity _rates_ \[[@CR8], _[@CR10]\]._ There may _be_ disease-specific effects _of_ schizophrenia, such as genetic susceptibility, that have additive or synergistic actions to increase body _weight_ further _\[[@CR5]\]._ _However,_ _the_ most important _factor_ related _to_ weight gain in people with SMI _is_ the use _of_ antipsychotic medications, which are among the most obesogenic drugs. Weight gain is the commonest side _effect_ of _second-generation_ _antipsychotic_ medication, affecting between 15 and 72% of patients \[[@CR11]\]. Other psychotropic drugs are _often_ _prescribed_ to people with schizophrenia and _include_ some _antidepressants_ and mood-stabilising _drugs,_ such as lithium and sodium valproate; these may also induce significant weight gain \[[@CR12]\]. Both lifestyle and pharmacological interventions _lead_ to significant reductions in body weight in _the_ general _population._ Not only are the interventions clinically effective, _they_ are also _cost-effective_ because of the benefits _of_ long-term improved health outcomes, including decreased _mortality_ \[[@CR13]\]. It is _likely_ that similarly effective interventions for _people_ with schizophrenia will also lead to improvements in health and would _be_ a major step _towards_ reducing the health inequalities _experienced_ by people with schizophrenia. As the weight gain associated with antipsychotic _medication_ result in some people discontinuing their medication, we hypothesis that effective weight-management strategies may _also_ lead _to_ improved adherence to antipsychotic medication and reduced relapse and hospitalisation _rates._ Some studies have _suggested_ that short-term lifestyle interventions could support weight reduction in people with SMI. A _meta-analysis_ _of_ non-pharmacological _interventions_ _in_ people with SMI _\[[@CR14]\]_ reported a mean reduction in weight of 3.12 kg over a _period_ of 8--24 weeks. However, the results of longer-term studies are more mixed. _A_ recent meta-analysis found significant weight loss _in_ only two of six studies with interventions lasting longer than _a_ _year_ \[[@CR15]\]. Most studies have _included_ a mixed population _of_ people with SMI and two large studies, which included only people with schizophrenia, found no effect of a lifestyle _intervention_ on _body_ _weight_ \[[@CR16], _[@CR17]\]._ These latter studies suggest the weight management in _people_ with schizophrenia may require _a_ different approach from other SMIs such as _bipolar_ disorder. Given the challenges of implementing lifestyle change in people with schizophrenia and the lack _of_ long-term effectiveness, alternative approaches are _needed_ to manage overweight _and_ obesity. A wide variety of treatments _have_ been subject _to_ _clinical_ _studies_ but _currently_ _no_ drug treatments are licensed for the treatment _of_ _antipsychotic-medication-associated_ _weight_ gain or _obesity_ in people with SMI _with_ the exception of orlistat \[[@CR18]\]. The long-term use of the latter, _however,_ is extremely limited by high discontinuation rates, making it _of_ little value in routine clinical practice \[[@CR19]\]. To date, _there_ have also _been_ three completed trials of glucagon-like peptide 1 (GLP-1)-receptor agonists in people with SMI, two _of_ which _used_ exenatide and _one_ used liraglutide (maximum _dosage_ _1.8_ mg) _\[[@CR20]--[@CR22]\]._ Liraglutide is _a_ GLP-1-receptor agonist, with 97% homology to human GLP-1, _which_ induces weight loss in _humans_ mainly by _reducing_ appetite and caloric intake, rather than increasing energy expenditure. There were contrasting results in the _exenatide_ studies with one showing _no_ difference between groups after 12 weeks of treatment \[[@CR20]\] but _in_ the other the _exenatide_ arm had _greater_ mean weight loss (− 5.29 vs − _1.12_ kg; *P* = 0.015), _and_ _reduced_ glycosylated haemoglobin (HbA~1c~) levels _(−_ 0.21% vs 0.03%; _*P*_ = 0.004) \[[@CR21]\]. In the liraglutide (maximum _dosage_ 1.8 mg) study, _glucose_ _tolerance_ improved _in_ the liraglutide group _and_ _body_ weight _decreased_ compared _with_ placebo (− 5.3 kg; 95% confidence _interval_ (CI) _−_ 7.0 to _−_ 3.7 _kg)_ \[[@CR22]\]. Liraglutide is approved for _the_ management of obesity at a _dosage_ of 3.0 mg _daily,_ which is _higher_ than the dosage used to treat diabetes \[[@CR23]\]. In a 56-week, double-blind trial involving 3731 participants without type-2 diabetes, 63.2% of the intervention arm compared _with_ 27.1% in _the_ _placebo_ arm group lost at least 5% of their _body_ weight, and _33.1%_ _and_ 10.6%, respectively, lost more than 10% of their body weight \[[@CR23]\]. We have, therefore, chosen to _use_ liraglutide (maximum dosage _3.0_ mg) as _we_ postulate that a higher dosage of liraglutide may offer even greater weight loss in people with SMI than _the_ 1.8-mg dosage employed _in_ _the_ previous study or other currently available GLP-1-receptor agonists. Aims and objectives {#Sec2} ------------------- The aim _of_ this pilot study is to _undertake_ _a_ double-blind, randomised _controlled_ trial (RCT) of the _use_ of _liraglutide_ (maximum dosage 3.0 mg daily) _in_ comparison to placebo in 60 _obese_ or _overweight_ people with schizophrenia, schizoaffective disorder or first-episode psychosis to _assess_ the feasibility and acceptability of _delivering_ a full-scale trial _evaluating_ treatment _with_ liraglutide in people with schizophrenia, schizoaffective disorder and first-episode _psychosis._ Methods {#Sec3} _=======_ Design {#Sec4} ------ This study is a double-blind, randomised pilot study of the use of liraglutide (maximum dosage 3.0 mg _daily)_ in comparison to placebo (Fig. [1](#Fig1){ref-type="fig"}). It is important to _include_ a double-blind placebo for two main reasons; there is evidence that people _would_ be less likely _to_ consent _to_ a _trial_ that includes a placebo arm because of the risk of not _receiving_ _an_ active treatment. As _our_ _ability_ to recruit to _the_ trial was one of _our_ key aims, it is important to assess whether _the_ inclusion _of_ a placebo prevented us from recruiting to the trial. Previous experience from weight-management _trials_ _that_ include pharmaceuticals have _been_ plagued _by_ high dropout rates in the placebo _arm_ as the participants _are_ able to assess the effectiveness of treatment. To adequately power a full RCT _of_ liraglutide, _we_ will need to know the _likely_ dropout rate _in_ the placebo group in this patient population. Fig. _1Consolidated_ Standards _of_ _Reporting_ Trials (CONSORT) study diagram Setting {#Sec5} ------- The study will be _take_ place in a _variety_ of community and inpatient mental _health_ locations in the Southern Health NHS Foundation Trust. Public and _Patient_ Involvement was actively included in the _development_ of the trial _and_ will continue throughout _the_ _trial._ Ethics approval and consent to participate {#Sec6} _------------------------------------------_ South Central _--_ Hampshire _B_ Research Ethics Committee (REC) approved the _study_ on 17 April 2018 with REC reference: 18/SC/0085. _Only_ those who _agree_ to provide _written_ informed _consent_ will be included in the _study._ The study will be conducted in keeping with Good Clinical Practice _(GCP)_ and the _International_ Conference of Harmonisation (ICH) standards. The _Trial_ _Steering_ |
Introduction {#s1}
============
Traditionally, ecological and eco-evolutionary epidemiological models describe the dynamics of infectious diseases by considering susceptible, infected and recovered hosts with host-to-host, or host-environment-host transmission [@pone.0071621-Kermack1]--[@pone.0071621-Hudson1]. A number of modifications--such as seasonality [@pone.0071621-Altizer1], or within-host dynamics [@pone.0071621-Mideo1]--have been introduced to the SI- and SIR-models in various attempts to explain recurrent outbreak disease dynamics. However, natural epidemics often show a variety of dynamics that do not correspond to the predictions made by the classical models. One reason for this is that the underlying assumptions on disease transmission are unrealistic for pathogens that spend a considerable amount, or even the most part of their life cycle, in the outside-host environment. A large proportion of opportunist pathogen species also grow actively in the outside-host environment. These environmental pathogens form an increasing problem for human health [@pone.0071621-Cangelosi1], and thus a better theoretical understanding of their epidemiology is required. Currently, most models for environmental transmission allow only decay of pathogens in the outside-host environment [@pone.0071621-Codeco1], [@pone.0071621-Day1], but see [@pone.0071621-Merikanto1]. In addition, the outside-host environment is riddled with other microbes which frequently interact with the pathogen. This means that while active growth as well as ecological interactions in the environment are likely to be profoundly important, they are yet poorly understood factors in disease dynamics.
The role of environmental transmission in disease dynamics and the evolution of virulence has attracted increasing interest [@pone.0071621-Day1], [@pone.0071621-Roche1], both due to human pathogen outbreaks such as cholera [@pone.0071621-Colwell1] and emergent animal diseases, e.g. columnaris disease [@pone.0071621-Pulkkinen1]. Worldwide, there is an ongoing battle against opportunistic infections, which are often persistent due to the pathogens' ability to grow outside hosts. Broad-spectrum antibiotics and disinfectants are used *en masse* to prevent environmental infections to humans and cultivated animals. This is likely to cause changes in the composition of environmental communities and have an impact on ecosystem functioning, health and disease [@pone.0071621-Ding1]. Theoretically, an environmentally transmitted pathogen can be highly lethal as the trade-offs between transmission and virulence associated with obligate pathogens are reduced [@pone.0071621-Walther1]. This is because by killing a host--which is not required to be alive for pathogen transmission--the pathogen gains access to an enormously rich resource for saprotrophic growth, which can lead to a positive transmission-virulence relationship [@pone.0071621-Kunttu1]. In most studies, however, the environment represents simply a reservoir into which the pathogen particles are shed from infected hosts and from which the surviving pathogen individuals may re-enter susceptible hosts, without explicit description of the outside-host dynamics other than the decay rate of the pathogen.
Interspecific interactions, such as competition, mutualism, predation, and parasitism constitute the core of ecological research. An important implication of these interactions is that the dynamics and stability of individual populations within ecological networks (e.g., communities or food webs) can strongly depend on the composition of these networks and the details of between-species interactions [@pone.0071621-DeRuiter1]--[@pone.0071621-Pimm1]. In general, similar co-occurring species compete for limiting resources [@pone.0071621-Hibbing1] and are attacked by parasites and predators [@pone.0071621-Abedon1]. All of these different ecological interactions can affect the density of pathogens and other interacting species in the community, thereby affecting the probabilities of infection outbreaks. Therefore, understanding the role of ecological interactions in the outside-host environment is likely to be of great importance for uncovering mechanisms behind the dynamics of many environmentally transmitted diseases such as *Vibrio cholera* [@pone.0071621-deMagny1], group A *streptococci* [@pone.0071621-Beres1], *Staphylococcus aureus* [@pone.0071621-Eveillard1], and *Flavobacterium columnare* [@pone.0071621-Pulkkinen1].
We explore the dynamics of a model that combines environmental opportunist pathogen--host dynamics to community dynamics outside the host. By the term environmental opportunist pathogen we mean an organism that is both (i) able to grow in the environment in the absence of hosts and (ii) infect susceptible hosts. Whether the pathogen can infect one or several host species is not important in this simple model in which we consider all (susceptible) hosts similar from the pathogens perspective. The model contains susceptible and infected hosts as in a classical SI-model and a competitive community in the outside-host environment. One of the competitors is a pathogen that can return from a dead host to the environment and thus the host does not represent an ecological or evolutionary dead end for the pathogen. This is opposite to the common assumption of the theory of "co-incidental virulence" [@pone.0071621-Brown1]. Further, we assume that there is a trade-off between virulence and environmental competitive ability. This assumption also differs from the expectation under co-incidental virulence theory [@pone.0071621-Brown1], where resource acquisition or fighting against natural enemies outside the host are positively linked to a pathogens ability to cause infections. Life-history trade-offs can reduce virulence because the machineries for resource acquisition and defence in the outside- vs. inside-host environments require specialisation [@pone.0071621-Gower1], [@pone.0071621-Mikonranta1]. The choice of the functional form of the infectivity response can crucially affect the model dynamics [@pone.0071621-Boldin1], [@pone.0071621-McCallum1]. We explore both linear and sigmoidal infection rate in response to pathogen density, and assume that the infected hosts can either recover back to the susceptible class or die from the disease. The sigmoidal infectivity response incorporates dose-dependence, i.e., exposure to pathogen densities below a certain level is unlikely to cause an infection, as observed in many laboratory experiments [@pone.0071621-Aaby1]--[@pone.0071621-McLean1]. The model behavior is explored in a parameter range that is likely to cover typical environmentally growing opportunist micro-parasites (e.g., bacteria, protozoa, or fungi) that infect multicellular hosts ranging from taxa with fast growth rates (e.g., nematodes and insects) to taxa with slow growth rates (e.g., vertebrates).
A striking feature of the model is that reducing competitor species richness in the outside-host environment can lead to an abrupt emergence of disease outbreaks. If the infectivity response of the pathogen is a sigmoidal function of pathogen density in the environment, epidemiological dynamics are sensitive to the intensity of competitive suppression by the outside-host community. With linear infectivity response and pathogen growth in the environment no disease outbreaks are observed, i.e. population dynamics remain stable, and pathogen and host densities are relatively insensitive to manipulation of diversity. Under sigmoidal infectivity, reduction of competitive pressure on the pathogen, either due to loss of diversity from the outside-host community (e.g., due to use of disinfectants), or increased loss rate of all species in the outside-host community (e.g., due to application of non-specific antibiotics) can lead to catastrophic disease outbreaks.
Methods {#s2}
=======
The dynamical model is a combination of the classical SI-disease dynamics [@pone.0071621-Kermack1] for hosts and the Lotka--Volterra competition model for an outside-host community. The susceptible and infected hosts at time *t* are denoted by *S*(*t*) and *I*(*t*), respectively. The pathogen, *P*(*t*), has *n* competitors, with densities denoted by *B~i~*(*t*), i.e., community size is *N* = *n* +1. The population densities vary according to the following differential equations:
In the absence of an infection, susceptible hosts follow a logistic growth model with a growth rate *r~h~* and a carrying capacity *K~h~* (eqn. 1.1). In the presence of an infective pathogen infected host individuals are formed. The infected individuals compete for resources with the susceptible individuals, but do not contribute to host reproduction (this assumption has no qualitative effect on the dynamics). Susceptible hosts are infected with a rate *βSf*(*P*) depending on the infectivity response *f*(*P*). Two alternative infectivity response functions were explored. Most previous theoretical work has assumed a linear infectivity response. However, we argue that a more realistic assumption for many circumstances is a sigmoidal dose-dependent response that is supported by empirical data [@pone.0071621-Aaby1]--[@pone.0071621-McLean1] as well as theoretical analysis [@pone.0071621-Pujol1]. The sigmoidal infectivity function has the following mechanistic interpretation. With low pathogen densities the immune system can effectively overcome most pathogen invasions and therefore the probability of an infection per unit time must increase nonlinearly at low densities. With high pathogen densities the effect of increasing pathogen density on the probability of infection per time unit must saturate since the | introduction { # s1 } = = = = = = = = = = = = traditionally, ecological and eco - evolutionary epidemiological models describe the dynamics of infectious diseases by considering susceptible, infected and recovered hosts with host - to - host, or host - environment - host transmission [ @ pone. 0071621 - kermack1 ] - - [ @ pone. 0071621 - hudson1 ]. a number of mechanisms - - such as seasonality [ @ pone. 0071621 - altizer1 ], or within - host dynamics [ @ pone. 0071621 - mideo1 ] - - have been introduced to the si - and sir - models in various attempts to explain recurrent outbreak disease dynamics. however, natural epidemics often show a variety of dynamics that do not correspond to the predictions made by the classical models. one reason for this is that the underlying assumptions on disease transmission are unrealistic for pathogens that spend a considerable amount, or even the most part of their life cycle, in the outside - host environment. a large proportion of opportunist pathogen species also grow actively in the outside - host environment. these environmental pathogens form an increasing problem for human health [ @ pone. 0071621 - cangelosi1 ], and thus a better theoretical understanding of their epidemiology is required. currently, most approaches for environmental transmission allow only propagation of pathogens in the outside - susceptible environment [ @ pone. 0071621 - codeco1 ], [ @ pone. 0071621 - day1 ], but see [ @ pone. 10 - merikanto1 ]. in addition, the outside - host environment is saturated with other microbes which frequently interact with the pathogen. this means that while active growth as high as ecological interactions from the environment are likely to be profoundly important, they are yet poorly understood factors in disease dynamics. the role of environmental transmission influences disease dynamics and the evolution of virulence has attracted increasing interest [ @ pone. 09 - day1 ], [ @ pone. 0071621 - roche1 ], both due to human pathogen outbreaks such as cholera [ @ pone. 0071621 - colwell1 ] and emergent animal diseases, e. g. columnaris disease [ @ pone. 0071621 - pulkkinen ##1 ]. worldwide, there is an ongoing battle against opportunistic infections, which are often persistent due to the pathogens ' ability to grow outside hosts. broad - spectrum antibiotics and disinfectants are used * en masse * to prevent environmental infections to humans and cultivated animals. this is likely to cause changes in the composition of environmental communities and have an impact on ecosystem functioning, health and disease [ @ pone. 0071621 - ding1 ]. theoretically, an environmentally transmitted pathogen can be highly lethal as the trade - offs between transmission and virulence associated with obligate pathogens are reduced [ @ pone. 0071621 - walther1 ]. this is because by killing a host - - which is not required to be alive for pathogen transmission - - the pathogen gains access to an enormously rich resource for saprotrophic growth, which can lead to a positive transmission - virulence relationship [ @ pone. 0071621 - kunttu1 ]. in most studies, however, the environment represents simply a reservoir into which the pathogen particles are shed from infected hosts and from which the surviving pathogen individuals may re - enter susceptible hosts, without explicit description of the outside - host dynamics other than the decay rate of the pathogen. interspecific interactions, such as competition, mutualism, predation, and parasitism constitute the core of ecological research. an important implication of these interactions is that the dynamics and stability of individual populations within ecological networks ( e. g., communities or food webs ) can strongly depend on the composition of these networks and the details of between - species interactions [ @ pone. 0071621 - deruiter1 ] - - [ @ pone. 0071621 - pimm1 ]. in general, similar co - occurring species compete for limiting resources [ @ pone. 0071621 - hibbing1 ] and are attacked by parasites and predators [ @ pone. 0071621 - abedon1 ]. all of these different ecological interactions can affect the density of pathogens and other interacting species in the community, thereby affecting the probabilities of infection outbreaks. therefore, understanding the role of ecological interactions in the outside - host environment is likely to be of great importance for uncovering mechanisms behind the dynamics of many environmentally transmitted diseases such as * vibrio cholera * [ @ pone. 0071621 - demagny1 ], group a * streptococci * [ @ pone. 0071621 - beres1 ], * staphylococcus aureus * [ @ pone. 0071621 - eveillard1 ], and * flavobacterium columnare * [ @ pone. 0071621 - pulkkinen1 ]. we explore the dynamics of a model that combines environmental opportunist pathogen - - host dynamics to community dynamics outside the host. by the term environmental opportunist pathogen we mean an organism that is both ( i ) able to grow in the environment in the absence of hosts and ( ii ) infect susceptible hosts. whether the pathogen can infect one or several host species is not important in this simple model in which we consider all ( susceptible ) hosts similar from the pathogens perspective. the model contains susceptible and infected hosts as in a classical si - model and a competitive community in the outside - host environment. one of the competitors is a pathogen that can return from a dead host to the environment and thus the host does not represent an ecological or evolutionary dead end for the pathogen. this is opposite to the common assumption of the theory of " co - incidental virulence " [ @ pone. 0071621 - brown1 ]. further, we assume that there is a trade - off between virulence and environmental competitive ability. this assumption also differs from the expectation under co - incidental virulence theory [ @ pone. 0071621 - brown1 ], where resource acquisition or fighting against natural enemies outside the host are positively linked to a pathogens ability to cause infections. life - history trade - offs can reduce virulence because the machineries for resource acquisition and defence in the outside - vs. inside - host environments require specialisation [ @ pone. 0071621 - gower1 ], [ @ pone. 0071621 - mikonranta1 ]. the choice of the functional form of the infectivity response can crucially affect the model dynamics [ @ pone. 0071621 - boldin1 ], [ @ pone. 0071621 - mccallum1 ]. we explore both linear and sigmoidal infection rate in response to pathogen density, and assume that the infected hosts can either recover back to the susceptible class or die from the disease. the sigmoidal infectivity response incorporates dose - dependence, i. e., exposure to pathogen densities below a certain level is unlikely to cause an infection, as observed in many laboratory experiments [ @ pone. 0071621 - aaby1 ] - - [ @ pone. 0071621 - mclean1 ]. the model behavior is explored in a parameter range that is likely to cover typical environmentally growing opportunist micro - parasites ( e. g., bacteria, protozoa, or fungi ) that infect multicellular hosts ranging from taxa with fast growth rates ( e. g., nematodes and insects ) to taxa with slow growth rates ( e. g., vertebrates ). a striking feature of the model is that reducing competitor species richness in the outside - host environment can lead to an abrupt emergence of disease outbreaks. if the infectivity response of the pathogen is a sigmoidal function of pathogen density in the environment, epidemiological dynamics are sensitive to the intensity of competitive suppression by the outside - host community. with linear infectivity response and pathogen growth in the environment no disease outbreaks are observed, i. e. population dynamics remain stable, and pathogen and host densities are relatively insensitive to manipulation of diversity. under sigmoidal infectivity, reduction of competitive pressure on the pathogen, either due to loss of diversity from the outside - host community ( e. g., due to use of disinfectants ), or increased loss rate of all species in the outside - host community ( e. g., due to application of non - specific antibiotics ) can lead to catastrophic disease outbreaks. methods { # s2 } = = = = = = = the dynamical model is a combination of the classical si - disease dynamics [ @ pone. 0071621 - kermack1 ] for hosts and the lotka - - volterra competition model for an outside - host community. the susceptible and infected hosts at time * t * are denoted by * s * ( * t * ) and * i * ( * t * ), respectively. the pathogen, * p * ( * t * ), has * n * competitors, with densities denoted by * b ~ i ~ * ( * t * ), i. e., community size is * n * = * n * + 1. the population densities vary according to the following differential equations : in the absence of an infection, susceptible hosts follow a logistic growth model with a growth rate * r ~ h ~ * and a carrying capacity * k ~ h ~ * ( eqn. 1. 1 ). in the presence of an infective pathogen infected host individuals are formed. the infected individuals compete for resources with the susceptible individuals, but do not contribute to host reproduction ( this assumption has no qualitative effect on the dynamics ). susceptible hosts are infected with a rate * βsf * ( * p * ) depending on the infectivity response * f * ( * p * ). two alternative infectivity response functions were explored. most previous theoretical work has assumed a linear infectivity response. however, we argue that a more realistic assumption for many circumstances is a sigmoidal dose - dependent response that is supported by empirical data [ @ pone. 0071621 - aaby1 ] - - [ @ pone. 0071621 - mclean1 ] as well as theoretical analysis [ @ pone. 0071621 - pujol1 ]. the sigmoidal infectivity function has the following mechanistic interpretation. with low pathogen densities the immune system can effectively overcome most pathogen invasions and therefore the probability of an infection per unit time must increase nonlinearly at low densities. with high pathogen densities the effect of increasing pathogen density on the probability of infection per time unit must saturate since the | Introduction {# s1} = = = = = = = = = = = = Traditionally, ecological and eco - evolutionary epidemiological models describe the dynamics of infectious diseases by considering susceptible, infected and recovered hosts with host - to - host, or host - environment - host transmission [@ pone. 0071621 - Kermack1] - - [@ pone. 0071621 - Hudson1 ]. A number of modifications - - such as seasonality [@ pone. 0071621 - Altizer1 ], or within - host dynamics [@ pone. 0071621 - Mideo1] - - have been introduced to the SI - and SIR - models in various attempts to explain recurrent outbreak disease dynamics. However, natural epidemics often show a variety of dynamics that do not correspond to the predictions made by the classical models. One reason for this is that the underlying assumptions on disease transmission are unrealistic for pathogens that spend a considerable amount, or even the most part of their life cycle, in the outside - host environment. A large proportion of opportunist pathogen species also grow actively in the outside - host environment. These environmental pathogens form an increasing problem for human health [@ pone. 0071621 - Cangelosi1 ], and thus a better theoretical understanding of their epidemiology is required. Currently, most models for environmental transmission allow only decay of pathogens in the outside - host environment [@ pone. 0071621 - Codeco1 ], [@ pone. 0071621 - Day1 ], but see [@ pone. 0071621 - Merikanto1 ]. In addition, the outside - host environment is riddled with other microbes which frequently interact with the pathogen. This meSgs that while active growth as well as ecological interactions in the environment are likely to be profoundly important, they are yet poorly understood factors in disease dynamics. The role of environmental transmission in disease dynamics and the evolution of virulence has attracted increasing interest [@ pone. 0071621 - Day1 ], [@ pone. 0071621 - Roche1 ], both due to human pathogen outbreaks such as cholera [@ pone. 0071621 - Colwell1] and emergent animal diseases, e. g. columnaris disease [@ pone. 0071621 - Pulkkinen1 ]. Worldwide, there is an ongoing battle against opportunistic infections, which are often persistent due to the pathogens ' ability to grow outside hosts. Broad - spectrum antibiotics and disinfectants are used * en masse * to prevent environmental infections to humans and cultivated animals. This is likely to cause changes in the composition of environmental communities and have an impact on ecosystem functioning, health and disease [@ pone. 0071621 - Ding1 ]. Theoretically, an environmentally transmitted pathogen can be highly lethal as the trade - offs between transmission and virulence aEWociated with obligate pathogens are reduced [@ pone. 0071621 - Walther1 ]. This is because by killing a host - - which is not required to be alive for pathogen transmission - - the pathogen gains access to an enormously rich resource for saprotrophic growth, which can lead to a positive transmission - virulence relationship [@ pone. 0071621 - Kunttu1 ]. In most studies, however, the environment represents simply a reservoir into which the pathogen particles are shed from infected hosts and from which the surviving pathogen individuals may re - enter susceptible hosts, without explicit description of the outside - host dynamics other than the decay rate of the pathogen. Interspecific interactions, such as competition, mutualism, predation, and parasitism constitute the core of ecological research. An important implication of these interactions is that the dynamics and stability of individual populations within ecological networks (e. g. , commJnitiSs or food webs) can strongly depend on the composition of these networks and the details of between - species interactions [@ pone. 0071621 - DeRuiter1] - - [@ pone. 0071621 - Pimm1 ]. In general, similar co - occurring species compete for limiting resources [@ pone. 0071621 - Hibbing1] and are attaFk4d by parasites and predators [@ pone. 0071621 - Abedon1 ]. All of these different ecological interactions can affect the density of pathogens and other interacting species in the community, thereby affecting the probabilities of infection outbreaks. Therefore, understanding the role of ecological interactions in the outside - host environment is likely to be of great importance for uncovering mechanisms behind the dynamics of many environmentally transmitted diseases such as * Vibrio cholera * [@ pone. 0071621 - deMagny1 ], group A * streptococci * [@ pone. 0071621 - Beres1 ], * Staphylococcus aureus * [@ pone. 0071621 - Eveillard1 ], and * Flavobacterium columnare * [@ pone. 0071621 - Pulkkinen1 ]. We explore the dynamics of a model that combines environmental opportunist pathogen - - host dynamics to community dynamics outside the host. By the term environmental opportunist pathogen we mean an organism that is both (i) able to grow in the environment in the absence of hosts and (ii) infect susceptible hosts. Whether the pathogen can infect one or several host species is not important in this simple model in which we consider all (susceptible) hosts similar from the pathogens perspective. The model contains susceptible and infected hosts as in a classical SI - model and a competitive community in the o8^side - host environment. One of the competitors is a pathogen that can return from a dead host to the environment and thus the host does not represent an ecological or evolutionary dead end for the pathogen. This is opposite to the common assumption of the theory of " co - incidental virulence " [@ pone. 0071621 - Brown1 ]. Further, we assume that there is a trade - off between virulence and environmental competitive ability. This assumption also differs from the expectation under co - incidental virulence theory [@ pone. 0071621 - Brown1 ], where resource acquisition or fighting against natural enemies outside the host are positively linked to a pathogens ability to cause infections. Life - history trade - offs can reduce virulence because the machineries for resource acquisition and defence in the outside - vs. inside - host environments require specialisation [@ pone. 0071621 - Gower1 ], [@ pone. 0071621 - Mikonranta1 ]. The choice of the functional form of the infectivity response can crucially affect the model dUnamJcs [@ pone. 0071621 - Boldin1 ], [@ pone. 0071621 - McCallum1 ]. We explore both linear and sjnmoidal infection rate in response to pathogen density, and assume that the infected hosts can either recover back to the susceptible class or die from the disease. The sigmoidal infectivity response incorporates dose - dependence, i. e. , exposure to pathogen densities below a certain level is unlikely to cause an infection, as observed in many laboratory experiments [@ pone. 0071621 - Aaby1] - - [@ pone. 0071621 - McLean1 ]. The model behavior is explored in a parameter range that is likely to cover typical environmentally growing opportunist micro - parasites (e. g. , bacteria, protozoa, or fungi) that infect multicellular hosts ranging from taxa with fast growth rates (e. g. , nematodes and insects) to taxa with slow growth rates (e. g. , vertebrates ). A striking feature of the model is that reducing competitor species richness in the outside - host environment can lead to an abrupt emergence of disease outbreaks. If the infectivity response of the pathogen is a sigmoidal function of pathogen density in the environment, epidemiological dynamics are sensitive to the intensity of competitive suppression by the outside - host community. With linear infectivity response and pathogen growth in the environment no disease outbreaks are observed, i. e. population dynamics remain stable, and pathogen and host densities are relatively insensitive to manipulation of diversity. Under sigmoidal infectivity, reduction of competitive pressure on the pathogen, either due to loss of diversity from the outside - host community (e. g. , due to use of disinfectants ), or increased loss rate of all species in the outside - host community (e. g. , due to application of non - specific antibiotics) can lead to catastrophic disease outbreaks. Methods {# s2} = = = = = = = The dynamical model is a combination of the classical SI - disease dynamics [@ pone. 0071621 - Kermack1] for hosts and the Lotka - - Volterra competition model for an outside - host community. The susceptible and infected hosts at time * t * are denoted by * S * (* t *) and * I * (* t * ), respectively. The pathogen, * P * (* t * ), has * n * competitors, with densities denoted by * B ~ i ~ * (* t * ), i. e. , community size is * N * = * n * + 1. The population densities vary according to the following differential equations: In the absence of an infection, susceptible hosts follow a logistic growth model with a growth rate * r ~ h ~ * and a carrying capacity * K ~ h ~ * (eqn. 1. 1 ). In the presence of an infective pathogen infected host individuals are formed. The infected individuals compete for resources with the susceptible individuals, but do not contribute to host reproduction (this assumption has no qualitative effect on the dynamics ). Susceptible hosts are infected with a rate * βSf * (* P *) depending on the infectivity response * f * (* P * ). Two alternative infectivity response functions were explored. Most previous theoretical work has assumed a linear infectivity response. However, we argue that a more realistic assumption for many circumstances is a sigmoidal dose - dependent response that is supportrV by empirical data [@ pone. 0071621 - Aaby1] - - [@ pone. 0071621 - McLean1] as well as theoretical analysis [@ pone. 0071621 - Pujol1 ]. The sigmoidal infectivity function has the following mechanistic interpretation. With low pathogen densities the immune system can effectively overcome most pathogen invasions and therefore the LrobabUlity of an infection per unit Gike must increase nonlinearly at low densities. With high pathogen densities the effect of increasing pathogen density on the probability of infection per time unit must saturate since the | Introduction {#s1} Traditionally, ecological and eco-evolutionary epidemiological models describe dynamics of infectious diseases considering susceptible, and recovered hosts with host-to-host, or transmission [@pone.0071621-Kermack1]--[@pone.0071621-Hudson1]. A number of modifications--such as seasonality [@pone.0071621-Altizer1], or within-host dynamics [@pone.0071621-Mideo1]--have been introduced to the SI- and SIR-models in attempts to explain recurrent disease However, natural epidemics often a variety of dynamics that do not correspond to the predictions made by the classical models. One reason for is that underlying assumptions on disease transmission are unrealistic for pathogens that spend a considerable amount, or even the most part of their cycle, in the outside-host environment. A large proportion of opportunist pathogen also actively in the outside-host environment. These environmental pathogens form increasing problem for human health [@pone.0071621-Cangelosi1], and thus a better theoretical understanding of their is required. Currently, most for environmental transmission allow only decay of pathogens the outside-host environment [@pone.0071621-Codeco1], [@pone.0071621-Day1], but see [@pone.0071621-Merikanto1]. In addition, the environment is riddled with microbes which frequently interact with the pathogen. This means that while growth as well as ecological interactions in the are likely be profoundly important, are yet poorly understood factors in disease dynamics. The of environmental transmission in dynamics and the evolution of virulence has attracted increasing interest [@pone.0071621-Day1], [@pone.0071621-Roche1], both due to human pathogen outbreaks such as cholera [@pone.0071621-Colwell1] and emergent animal diseases, e.g. columnaris disease [@pone.0071621-Pulkkinen1]. Worldwide, is an ongoing battle against opportunistic infections, which are often persistent due to the pathogens' ability to grow outside hosts. Broad-spectrum antibiotics are used *en masse* to environmental infections to humans and cultivated animals. This is to cause changes in composition of environmental communities have an impact on ecosystem functioning, health and disease [@pone.0071621-Ding1]. Theoretically, an environmentally pathogen can highly lethal as the trade-offs between transmission and virulence associated with obligate pathogens are reduced [@pone.0071621-Walther1]. This because by killing a not required to be alive for pathogen transmission--the pathogen access to an enormously rich for saprotrophic growth, which lead to a positive transmission-virulence relationship [@pone.0071621-Kunttu1]. In most studies, however, the environment represents simply a reservoir into which the pathogen particles are shed from infected hosts and from which the pathogen individuals re-enter susceptible hosts, without explicit description of the outside-host other than the decay rate of the pathogen. Interspecific such as mutualism, predation, and parasitism constitute the core of ecological research. An important implication of interactions is that the dynamics and stability of individual populations ecological networks (e.g., communities or food webs) can strongly depend on the composition of these networks and the details of between-species interactions [@pone.0071621-DeRuiter1]--[@pone.0071621-Pimm1]. In general, similar co-occurring species compete for limiting resources [@pone.0071621-Hibbing1] and attacked by parasites and predators [@pone.0071621-Abedon1]. All of these different ecological can affect density of pathogens and other interacting species in the community, thereby affecting the of infection outbreaks. understanding the role ecological interactions in the outside-host environment is likely to be of great importance uncovering mechanisms behind the dynamics of many environmentally transmitted diseases such as *Vibrio cholera* [@pone.0071621-deMagny1], group A *streptococci* [@pone.0071621-Beres1], *Staphylococcus aureus* [@pone.0071621-Eveillard1], and *Flavobacterium columnare* [@pone.0071621-Pulkkinen1]. We explore the dynamics of a model that environmental opportunist pathogen--host dynamics to community dynamics outside the host. By the environmental opportunist pathogen mean an organism that both (i) able to grow the environment in the absence hosts and (ii) infect hosts. Whether the pathogen infect one or several host species is not important in this simple model in which we consider all (susceptible) hosts similar from the pathogens perspective. The model susceptible and infected hosts as in classical and a competitive community in the outside-host environment. One of the competitors is pathogen can return from a dead host to the environment and thus the host does not represent an or evolutionary dead for the pathogen. This is opposite to the common assumption of the theory "co-incidental [@pone.0071621-Brown1]. Further, we assume that there is a trade-off between virulence and environmental competitive This assumption also differs from expectation under co-incidental virulence theory where acquisition fighting against natural enemies outside the host are positively linked to a pathogens ability to cause infections. Life-history trade-offs can reduce virulence because the machineries for resource acquisition and defence in the outside- vs. inside-host environments require [@pone.0071621-Mikonranta1]. The choice of the functional form of the response can crucially affect model [@pone.0071621-Boldin1], [@pone.0071621-McCallum1]. We linear and sigmoidal rate in response to pathogen density, and assume that the infected hosts can either recover back to the susceptible class or die from disease. The sigmoidal infectivity response incorporates dose-dependence, exposure to pathogen densities below a certain level unlikely to cause an infection, observed in many laboratory experiments [@pone.0071621-Aaby1]--[@pone.0071621-McLean1]. The model behavior is explored a parameter range that likely to cover typical environmentally growing opportunist micro-parasites (e.g., bacteria, protozoa, or fungi) that infect multicellular hosts ranging from taxa fast growth (e.g., nematodes and insects) to taxa with slow growth rates (e.g., vertebrates). A striking feature of the model is that reducing competitor species in the environment can lead to an abrupt of disease outbreaks. If the infectivity response of the pathogen a function pathogen density in the environment, epidemiological dynamics are sensitive to intensity of competitive suppression by the outside-host community. With linear infectivity response and pathogen growth in the environment disease outbreaks are observed, i.e. population dynamics remain stable, and pathogen host densities are relatively insensitive to manipulation of diversity. Under sigmoidal infectivity, reduction competitive pressure on the pathogen, either to loss of diversity from community due to of disinfectants), or loss rate of all species in the community (e.g., due application of non-specific antibiotics) can lead to catastrophic disease outbreaks. Methods {#s2} ======= The dynamical model is a combination of the classical dynamics [@pone.0071621-Kermack1] hosts the Lotka--Volterra competition model for an outside-host community. susceptible and infected hosts at time *t* are denoted *S*(*t*) and *I*(*t*), respectively. pathogen, *P*(*t*), has *n* competitors, with densities denoted by *B~i~*(*t*), i.e., community size is *N* = *n* +1. The population densities vary according to the following differential equations: the absence of an infection, susceptible hosts follow a logistic growth model with a growth rate *r~h~* and a carrying capacity (eqn. 1.1). the presence of an infective pathogen infected host individuals are formed. The infected individuals compete for resources with the susceptible but do not contribute to host assumption has no qualitative effect on the dynamics). Susceptible hosts are infected a rate *βSf*(*P*) depending on the infectivity response *f*(*P*). Two alternative infectivity response functions were explored. Most previous theoretical work assumed a linear infectivity response. However, we argue that a more realistic assumption for many circumstances is a sigmoidal dose-dependent response that is supported by empirical data [@pone.0071621-Aaby1]--[@pone.0071621-McLean1] as well theoretical analysis [@pone.0071621-Pujol1]. The sigmoidal infectivity function has the following mechanistic interpretation. With low pathogen densities the immune system can effectively overcome most pathogen invasions and therefore the probability of an infection per unit time must nonlinearly at low densities. With high pathogen densities the effect of increasing pathogen density on the probability of infection per time unit must saturate since | iNtRoDuCtIOn {#s1}
============
TRadiTioNAlLY, ECoLOgIcAL ANd EcO-EVoLUTiONARY ePidEMIoLogIcaL mOdELs dEscRiBE tHE DYnamicS of inFEcTIous DisEaSEs bY CoNSidERiNg SuScePtible, iNfectED aND rECovErEd hOsTS WitH HoST-To-HOST, OR HOsT-EnVIrOnmENt-HoST tRAnsmISSIOn [@PoNe.0071621-kERmacK1]--[@ponE.0071621-HudsON1]. a NuMBeR oF moDIfIcATioNs--sUch as sEasoNAliTY [@POnE.0071621-ALtIzEr1], oR wIthiN-HOst DYnaMICS [@PoNe.0071621-MIdeo1]--hAve BeEN InTRoDUced tO tHE SI- And siR-modELs In VArioUs ATTempts tO exPlain ReCURRENT oUtBreAk DIseASE dyNaMICS. HOWEVER, NAtuRal EpiDEmicS oFtEn shOw a variETY OF DynAMICs tHAt do NOT CORrEspoNd to the prEdICtIoNS MADe by The clasSIcAL mOdELs. ONE rEAsON FoR thIs is ThAt THE uNDerlyING ASSUMpTioNs ON dIseaSE TRANSmIsSIoN ARe UnrEaLIsTIc FoR PAThOgEnS ThaT sPeNd a CoNsideRable aMoUNT, or eVEN THE mOST pARt Of tHeir LiFE cycLE, In thE OutsiDe-hOSt eNviroNMent. a LARGE PrOPoRtioN of OPpOrtuNiST PAtHoGeN species aLso groW AcTIvEly IN thE OUTsIDE-hOst enviROnMent. thesE ENvIRonmenTaL pathogeNs fOrm AN INcREAsINg prObleM foR hUmAn HEalTH [@POnE.0071621-caNgElOSI1], aND THus A BettER theOREticAL undErSTaNDiNG oF THEiR ePiDeMIOLOGy Is rEQUIRED. cURRENTLY, MOST MOdeLS fOR enviRonmENtAL TRaNsmiSsiON aLlow onLy DecaY Of pAThogENs In The ouTsiDe-HosT ENvirONMEnT [@POne.0071621-cOdECO1], [@ponE.0071621-Day1], bUT SeE [@PONE.0071621-meRiKanTO1]. iN adDiTiON, THe oUTSIdE-HosT enVironMenT iS ridDleD WiTH otHEr miCroBeS which FREqUeNtlY INteRAct WiTh THe PaThOGEN. ThIs meANS ThAT wHIlE ActIve gRoWtH aS welL AS ecOLOgiCaL INTERaCTIoNs IN ThE EnViROnMENT aRE likeLy To bE PROFOuNDLy IMpOrTant, tHey aRe yet PooRlY UnDErsToOD FaCTORS iN DIseASE DyNAmiCS.
thE rOLe OF EnVIROnMeNTaL tRaNsMIsSIoN IN DiSeasE dYnAmICS and THE EVOLUtIOn of viRuLenCe hAS AtTRACteD IncReAsing iNTEREst [@Pone.0071621-DAy1], [@pone.0071621-RoChe1], bOTH DUe To HUmAN PaTHoGEn oUtBREAKS suCH as CHOLERA [@PonE.0071621-cOLWeLL1] And EmERGEnT AnImAl dIsEASES, e.G. COLuMnaRIs dIseAse [@POne.0071621-pUlkKINEN1]. WORLdWIde, thEre IS an OnGOIng bAtTle AGainST opPORTuniStIc InFectIons, whicH aRE oFtEn pERsIsTEnt Due TO ThE pAthOGens' aBIlItY tO grow oUTsIDE HOsts. bRoAd-SPecTrum ANTIBiOTics ANd diSInFeCtants are UsED *en maSSe* To pReVeNt eNvirONmEntaL iNfEctions to hUMaNS AnD cuLTiVatED anImals. tHIs is lIkely To CausE cHangES In THe coMPOSItion oF eNviRONmeNtAl cOmMuNitieS and have aN iMpaCt on ECoSYSTeM funCtiONinG, hEAltH AnD diSEaSe [@POnE.0071621-dIng1]. THeoRetICALlY, aN eNVIROnmEnTaLly TraNsMiTteD pAtHogEN Can be HigHlY LeTHAl AS tHe traDe-ofFs BetweEn traNsmisSIon aND viRuleNce asSOCIAtEd With ObLiGATe pATHogENs aRE REDUCED [@poNE.0071621-WALthEr1]. ThIS IS bEcAUSe By KilLINg a hOSt--whicH is NOT ReQuIrEd TO bE ALIVE foR paThoGeN TrANSMiSsION--tHE PAThogEn gaiNS aCcEss To An enorMOuSlY rIch REsourcE For sAprotRophIC gRowth, WHiCh caN LEad TO A pOsitIvE tRanSmiSsIOn-vIrUlENce rElAtIoNshIp [@pOnE.0071621-kUnTTU1]. in MoSt STudIeS, HOWeVer, tHE enVIRONmENt ReprESenTs sIMpLY a ResErVoiR InTO WHiCh THE patHOGeN pArTICLes Are SHeD FROM iNFeCTED hOsTs aND fRom whICH tHe survivInG pAthOGEn indiVIDuAls MAy Re-entEr suscEPTIBLe HOStS, WITHoUT ExPLIcIT deSCRIPTioN of ThE OuTsIde-HOSt dYNaMiCS other tHAN the DeCay rATE OF THe PAthoGeN.
INteRSPEcifiC InteRACtIons, SUCh as CoMpETitIOn, MutuaLIsm, PrEDatiOn, aND pARaSiTIsM CoNStitUTe THe cOre Of EcologIcAl rEsEARCh. An iMPORTaNt imPLICATION of tHESe inteRaCTiONs iS tHAt tHe DYNAMiCs anD StabIlITy OF iNDIVIdUaL POPULATIoNs wIthiN eColOgiCal nEtwORks (e.g., coMmUNiTIeS Or fOod WeBs) cAN sTROnGLy DepEnD on THe COMPOsiTiON Of theSe netWoRkS And the detAILs OF BeTWEen-SpeCies InteRAcTiONs [@pone.0071621-dERUiTER1]--[@pOnE.0071621-pimM1]. in GENERal, SiMiLAr cO-OccUrRiNg SpECIeS COMpetE FOR lImitiNG ResourCes [@PonE.0071621-HIBbiNg1] aNd ARe aTtAcked BY paraSItes AnD PRedatOrs [@PONE.0071621-ABedon1]. ALL Of thEsE DiFFErenT ecoLogIcaL INTErACtIoNs CAn aFFeCt THE densiTy OF paThOGEns AnD OThEr INterACTING speCIEs IN ThE CoMMUNitY, TheReBy AffEctiNg THE PRoBabIlitIeS Of iNFECTIon oUTbREAKS. tHEREfORE, UnDeRSTAndIng the role Of ECoLogicaL interACTIONS IN The OUTSiDe-HoSt eNviRONmeNT is LikElY tO be OF GreAT imPoRtANCe fOr UNCOVeriNG mechanisMS bEhINd tHE dYnaMiCS Of manY enVIRoNmenTalLY TrAnsmiTtED dIseasEs SUCH As *viBRiO Cholera* [@pOnE.0071621-DemagNY1], gRouP a *sTrePtOcOcci* [@poNe.0071621-bereS1], *stapHYlococCus aUReus* [@PoNE.0071621-EVEilLaRD1], AND *fLavobacTeRium CoLuMnarE* [@PONE.0071621-PuLKkiNen1].
We EXplOrE thE dYNamICS oF a MoDEl tHAT cOmbInes ENvirOnMenTAL oPportuNisT PAThOgeN--HoST dYNaMICs To CoMmunITy dYNamiCs oUTSIde tHe Host. By THE TerM envIroNmEnTaL oPPoRTUnIST pathOGen we MeAN aN orgaNISM tHAt is BoTh (i) ABlE To gROw IN tHE ENviROnMenT IN The AbsEnce Of hOSts ANd (II) InfecT sUscEpTIblE HoSTs. WHethEr THe pathogEN CaN InFecT one oR SeverAL hOst spEcieS Is not IMporTaNT in ThiS SIMPLe mODel in WHIcH wE CONsidER aLl (suscEptiBLE) hoSts similAr FRom THe PAtHOGeNs PersPecTIve. tHE ModEL CoNTaiNs SUScEptibLE AnD infEcTED HostS as in A classIcAl SI-mOdEL AND a cOMpeTITIVe COmmUnitY IN The OUTSiDe-HosT ENVIrOnMenT. OnE oF THE COMPEtItOrs Is A pAThOgen THAt cAN rETuRN fROM A DEad HOst tO the enVironMent AND tHUs ThE HOsT dOes NOT rEpresenT aN eCOlogicaL oR evOLuTIoNaRy DeAD END fOr tHe PATHOGEn. this iS OPpOsItE to thE coMMOn aSSUMpTIon of THE tHEOry OF "Co-inciDEntaL VIRULENCe" [@pONE.0071621-BROwN1]. FurTHeR, We ASSumE ThAT tHerE is A tradE-oFF BEtWEEn vIRUlEnce and EnvIRonMENtaL COmPEtitIVE abILITY. THiS AsSuMPtIon AlSO dIfFeRS FrOM THe exPECtATiOn unDeR Co-inciDEnTal VIRuLenCe THEORY [@ponE.0071621-BROwN1], wHerE rEsOurCE AcqUiSiTIoN or FIGhTiNg AgaiNst nAtUrAl EnEMIeS ouTSidE The HosT arE pOsITIVElY LiNKED tO a PaTHogEnS aBiliTY tO cauSe InfectIONs. liFe-HiSTORY tRadE-oFFS CaN reduCe VIrulence bECaUSE THe macHINERIeS FOr ReSoUrCe acqUISitiOn And deFEnce In thE OuTSiDe- vS. InSIDe-hOST envirOnMENtS rEQUIre spECIAlIsAtION [@pone.0071621-GoWeR1], [@PONE.0071621-MIkoNRAnTa1]. ThE cHoiCE of tHe FUNctIOnAl fOrm oF the InfECTIviTY RESponSe cAn CruciALLY AfFECt The moDEl DyNamiCs [@PoNE.0071621-bOlDIn1], [@pONe.0071621-MCcalLuM1]. wE eXPlORE boTh lInear AND SIGMoiDal iNfEcTIoN RATe in ReSpOnsE TO PAthOGEN DEnsiTy, AND ASsumE THaT ThE InfectEd hosTs cAN eItheR REcOver bAck to ThE SUScEptIbLe ClAss OR die fROM ThE diseAsE. tHe sigmoIdAL inFEctIVity respONse INCorpOrATeS dOSE-DePENDENCE, i.E., ExpoSURE tO PatHoGEN densiTIeS BELOW A Certain Level iS uNliKeLy tO CauSe An INFeCtion, aS ObSERVeD iN MaNy LaboratorY eXpEriments [@POne.0071621-AAby1]--[@pOne.0071621-mclean1]. tHE MOdEl bEHavIoR is EXPloRed iN A PAraMETeR rAngE thaT Is LiKElY tO coVer TypIcaL ENViRonMENTalLY GROwinG OpPORtunIsT MiCrO-parASiTes (E.G., BacteriA, pROtoZOA, oR fuNgI) thaT Infect muLTICeLLULar hoSts RAnGIng FROM TaXA WITh FAsT GRoWTh RAteS (e.G., NeMaTodes ANd InSects) tO TaXA wITH SLoW GroWTH rAtEs (e.g., verTebrates).
a sTRikiNg FEaTURe of tHE mODEl is THat ReduciNG CoMpeTitOR SPeCIes ricHNEsS in thE OUTsiDe-hoST enViRoNMEnt CaN lEad TO An aBRupt EMErgENcE OF dISEAsE oUtbrEaKs. If tHe iNFECtiViTY RespONsE Of tHe pathoGeN iS a SIgmoIDaL fuNCtIoN of pathOgeN DenSITy iN The EnViroNMEnT, ePideMiolOGIcAl DynAmiCs aRe SENSitIVE tO the iNtenSitY Of comPETiTiVe sUPprEssiOn By tHe OUtSIdE-hoSt CoMmUNITY. WiTh lIneAr INfECTIViTy REsPOnse and PAthOgEn GrOwth iN tHe envIRoNMEnt no DiseAse oUTBREakS are oBSERveD, I.e. popUlAtioN DYNAmIcs RemAin stable, aNd pATHoGen AND HosT DeNSITiES aRe rElATiVely INsENsItiVe to MAniPuLAtion Of diVERsiTy. UNDeR sIgMoIDal INFeCtIviTY, RedUcTion of cOmPetitive PrESSuRE oN The pAthoGen, eiTHeR dUe To LosS oF diVeRSITY FroM THe ouTsiDE-HOsT coMMunITy (e.G., due TO uSE Of DIsinfECtAnTs), OR INCReaSEd LOSs ratE of ALl SpeCieS in tHe OuTSiDe-Host cOmmUniTY (E.G., dUE to APPLicatiOn oF NOn-SpeCIFIC ANtibiOTicS) cAN LEaD to cATAStroPHiC DISEaSe OutbREakS.
MEthoDs {#s2}
=======
tHE dYnaMICal mOdEL is A COmBINaTION OF tHe ClAssicAL Si-dIsEAsE dYNaMIcs [@PONE.0071621-KeRMaCK1] foR hOSts ANd thE LOtKa--voLTeRRA compeTITion MODeL FOR An OutsIDe-hOST coMmuNItY. THE sUScepTIBle AnD InfECted hostS at time *T* Are DEnoTED by *S*(*t*) aND *i*(*T*), rEsPectIvEly. thE PaTHOGEn, *p*(*t*), HAs *N* COMpetIToRS, WiTh DensiTIeS deNoteD By *b~I~*(*T*), i.e., CoMMUniTy siZe iS *n* = *n* +1. tHE PoPuLATion DensItiEs VAry acCoRDiNg tO The FollOWInG DIfFEreNTiaL equAtIONs:
In thE abSeNCE Of an INfECTioN, suSCEpTIblE hOSTS FoLloW a lOGIstIc groWtH mOdel WitH a GRoWTH RATe *R~H~* anD a CaRRYInG CAPACITy *k~H~* (Eqn. 1.1). IN THe PrEseNcE oF aN infeCtive patHogEn INfECTEd HOSt InDIvIdUAls arE ForMEd. the INfEcTed IndIvIdUaLS COmPEte for REsOURces with ThE SUsCEptIBLe iNdiVidUALS, BuT DO NOT coNtriBUTE to host reProdUCtioN (thiS AsSUmPTioN hAS no QuAlItatIve EfFecT oN the dyNAMIcs). sUSCEPtIBLE hoSTs ArE INfectED wItH a RAte *βsF*(*P*) DePeNdiNg On The infECTIvITy resPonse *f*(*P*). tWO aLtERNaTIVE iNfeCtIViTY rEspoNSe FuNCTiONs WErE exPLOred. MoST preVIoUS thEOreTiCAl work has AsSUmeD A linEar infEctIviTy rEsPonse. hOwEvER, WE ARGUe thAt A More RealIsTIc AsSUMPtIoN for MANy circUmSTanCEs iS a sigMOidAL dOsE-dePENDenT RespOnSE tHaT Is sUPpoRtED BY eMpIRIcAL dATa [@PONe.0071621-aABY1]--[@PoNe.0071621-MclEAn1] AS well aS tHeoREtIcal anaLySiS [@POnE.0071621-pUJol1]. tHE SIGMOIDAl iNFeCTivITY FUNCTiOn haS thE FolLoWIng MECHAniSTic interPREtaTION. wItH lOW paThOgen DENSItieS thE iMmuNE SYstem can effecTiVeLy OvErcOme MOsT patHoGEn InVasIonS AnD TheReForE thE probAbIliTY of AN iNfEctiON PeR uNit tIMe muSt INcrEaSe noNliNEaRLY at LoW deNsITies. wITh HIgh PATHOgen dENsItieS the effecT Of iNCrEAsInG PaTHOGeN DeNsiTy oN THE PROBAbILiTY oF inFECtIoN PeR TiME unIT muST sAtUrate sincE THe | Introduction {#s1} ============ Traditionally, ecological and eco-evolutionary epidemiological models describethe dynamics of infectiousdiseases by considering susceptible, infected and recovered hosts with host-to-host, or host-environment-host transmission [@pone.0071621-Kermack1]--[@pone.0071621-Hudson1]. Anumber ofmodifications--such as seasonality [@pone.0071621-Altizer1], or within-host dynamics [@pone.0071621-Mideo1]--have been introduced to the SI- and SIR-models in variousattempts to explain recurrent outbreak disease dynamics.However, natural epidemics often show a variety of dynamics that do not correspondto the predictions made by theclassical models. One reason for this is that the underlying assumptions ondiseasetransmission are unrealistic for pathogens that spendaconsiderable amount,oreven the mostpart of their life cycle, in the outside-host environment. A large proportion of opportunist pathogen species also grow actively in the outside-host environment.These environmental pathogensform an increasing problem for human health [@pone.0071621-Cangelosi1], and thus a better theoretical understanding of their epidemiology is required. Currently, most models for environmental transmission allow only decay of pathogens in the outside-hostenvironment [@pone.0071621-Codeco1], [@pone.0071621-Day1], but see [@pone.0071621-Merikanto1]. In addition, the outside-hostenvironment isriddled withother microbes which frequently interact with the pathogen. This means that while active growth as well as ecological interactions in the environment are likely to be profoundly important, they are yet poorly understood factors in disease dynamics. The role of environmental transmission in diseasedynamics and the evolution of virulence has attracted increasing interest [@pone.0071621-Day1], [@pone.0071621-Roche1], both due to human pathogen outbreaks such as cholera [@pone.0071621-Colwell1] and emergent animal diseases, e.g. columnaris disease [@pone.0071621-Pulkkinen1]. Worldwide, there is an ongoing battleagainst opportunistic infections, which are often persistent due to the pathogens' ability to grow outside hosts. Broad-spectrum antibiotics and disinfectants areused *en masse* to prevent environmental infections to humans and cultivated animals. This is likely to cause changes in the composition ofenvironmental communities and have an impact on ecosystem functioning, health and disease [@pone.0071621-Ding1]. Theoretically, an environmentally transmitted pathogen can be highly lethal asthetrade-offs between transmission and virulence associated with obligate pathogens are reduced [@pone.0071621-Walther1]. This is because bykilling a host--which is not required to be alivefor pathogen transmission--the pathogen gains access to anenormously rich resource for saprotrophic growth, which can lead to a positive transmission-virulence relationship [@pone.0071621-Kunttu1]. In most studies, however, theenvironment represents simply a reservoir into which the pathogenparticles areshed from infected hosts and from which the survivingpathogen individualsmay re-enter susceptible hosts,withoutexplicit descriptionof theoutside-host dynamics otherthanthe decay rate of the pathogen. Interspecific interactions, suchas competition, mutualism, predation,and parasitism constitute the core of ecological research. Animportant implication of these interactions is that the dynamics and stability of individual populations within ecological networks (e.g., communities or food webs) can strongly depend on the compositionof these networks and thedetails of between-species interactions [@pone.0071621-DeRuiter1]--[@pone.0071621-Pimm1]. In general, similar co-occurringspecies compete for limiting resources [@pone.0071621-Hibbing1] and are attacked by parasites and predators [@pone.0071621-Abedon1]. All of these different ecologicalinteractions can affect thedensity of pathogens and other interacting species in thecommunity, thereby affecting the probabilities of infection outbreaks. Therefore, understanding therole of ecological interactions in the outside-host environment is likely tobe of greatimportance for uncovering mechanisms behindthe dynamics of many environmentally transmitted diseases such as *Vibrio cholera* [@pone.0071621-deMagny1], group A *streptococci* [@pone.0071621-Beres1], *Staphylococcus aureus* [@pone.0071621-Eveillard1], and *Flavobacterium columnare* [@pone.0071621-Pulkkinen1]. We explore the dynamics of amodel that combines environmental opportunist pathogen--host dynamics to community dynamics outside the host. Bythe term environmental opportunist pathogen we mean an organism that isboth (i) able to growin the environment in the absence of hosts and(ii) infect susceptible hosts.Whether the pathogen can infect oneor several host species is notimportant in this simplemodel in which we consider all (susceptible)hosts similar from the pathogens perspective. The model containssusceptible and infected hosts as in a classical SI-model and a competitive communityin theoutside-host environment. One of the competitors is apathogen thatcan return from a dead host to the environment and thus the host does not representan ecological or evolutionarydead end for the pathogen. This is opposite to thecommonassumption of the theory of"co-incidental virulence" [@pone.0071621-Brown1].Further, we assume that there isa trade-off between virulence and environmental competitive ability. This assumption also differs from the expectation under co-incidental virulence theory [@pone.0071621-Brown1], where resource acquisition or fighting against natural enemies outside the host are positively linked to a pathogens abilityto cause infections. Life-historytrade-offs can reduce virulence because the machineries forresource acquisitionand defence in the outside- vs. inside-host environments require specialisation [@pone.0071621-Gower1], [@pone.0071621-Mikonranta1]. The choice of the functional form of the infectivity response can crucially affectthe model dynamics[@pone.0071621-Boldin1], [@pone.0071621-McCallum1]. We explore both linear and sigmoidal infection rate in response topathogen density, and assume that the infected hosts can either recoverbackto the susceptible class or die from the disease. The sigmoidal infectivity response incorporates dose-dependence, i.e.,exposure to pathogen densities below a certain level is unlikelytocause an infection,as observed in many laboratory experiments [@pone.0071621-Aaby1]--[@pone.0071621-McLean1]. The model behavior isexplored in a parameterrange that is likely to cover typicalenvironmentally growing opportunistmicro-parasites (e.g., bacteria, protozoa, or fungi) thatinfect multicellular hosts ranging from taxa with fastgrowth rates (e.g., nematodesand insects) to taxa with slow growth rates (e.g., vertebrates).A striking feature of the model is that reducing competitor speciesrichness in the outside-host environment can lead toan abrupt emergence of diseaseoutbreaks. If the infectivity responseofthe pathogen isasigmoidal function of pathogen density in theenvironment, epidemiological dynamics are sensitive to the intensityof competitive suppression by the outside-host community. With linear infectivity response and pathogen growth in the environment no disease outbreaks are observed, i.e.population dynamics remain stable, and pathogen andhostdensities are relatively insensitive to manipulationof diversity. Under sigmoidal infectivity,reduction of competitivepressure on thepathogen, either due to loss of diversity from the outside-hostcommunity (e.g., due to use of disinfectants),or increased loss rateof all speciesin the outside-hostcommunity (e.g.,due to application of non-specific antibiotics)can lead to catastrophic disease outbreaks. Methods{#s2} ======= The dynamical modelisa combination of the classical SI-diseasedynamics [@pone.0071621-Kermack1] for hosts and the Lotka--Volterra competition model foran outside-host community. The susceptible and infectedhosts at time *t*are denoted by *S*(*t*) and *I*(*t*), respectively. The pathogen, *P*(*t*), has*n* competitors, with densities denoted by *B~i~*(*t*), i.e., communitysize is *N*= *n* +1. The population densities vary according to the following differential equations:Inthe absence of an infection, susceptible hosts follow a logistic growth model with agrowth rate *r~h~* and a carrying capacity *K~h~*(eqn. 1.1). In thepresence of an infective pathogen infected host individuals are formed. Theinfected individuals compete for resourceswith the susceptibleindividuals, but do not contribute to host reproduction (this assumption has no qualitative effect onthedynamics). Susceptible hostsareinfected with a rate*βSf*(*P*) depending on the infectivity response*f*(*P*). Twoalternative infectivity response functions were explored. Most previoustheoretical work has assumed a linear infectivityresponse. However, we argue that a more realisticassumption for manycircumstances is asigmoidaldose-dependent response that is supported by empirical data [@pone.0071621-Aaby1]--[@pone.0071621-McLean1] as well as theoretical analysis [@pone.0071621-Pujol1]. The sigmoidal infectivity function has the following mechanistic interpretation. With low pathogen densitiesthe immunesystemcan effectivelyovercome most pathogen invasions and therefore the probability of an infection per unit time must increase nonlinearly at lowdensities. With high pathogen densities the effectof increasing pathogen density onthe probability of infection per time unit must saturate since the | _Introduction_ {#s1} _============_ Traditionally, ecological and eco-evolutionary epidemiological _models_ _describe_ the dynamics of infectious diseases _by_ _considering_ susceptible, _infected_ and recovered hosts with host-to-host, or host-environment-host transmission [@pone.0071621-Kermack1]--[@pone.0071621-Hudson1]. A number of modifications--such as seasonality [@pone.0071621-Altizer1], or within-host dynamics [@pone.0071621-Mideo1]--have been introduced to the SI- _and_ SIR-models _in_ various attempts to explain recurrent outbreak _disease_ dynamics. _However,_ natural epidemics often show a variety of dynamics that _do_ not correspond _to_ the predictions made by the classical models. One reason for this is that _the_ underlying assumptions on disease transmission are unrealistic for pathogens that _spend_ a _considerable_ amount, _or_ even the most part of their _life_ cycle, _in_ _the_ _outside-host_ environment. A large _proportion_ of opportunist pathogen _species_ _also_ grow actively in the _outside-host_ environment. These environmental _pathogens_ form _an_ increasing problem for _human_ health [@pone.0071621-Cangelosi1], and thus a better theoretical understanding of _their_ _epidemiology_ is required. Currently, most models for environmental transmission allow only _decay_ of pathogens in the outside-host environment [@pone.0071621-Codeco1], [@pone.0071621-Day1], but see [@pone.0071621-Merikanto1]. In addition, the outside-host _environment_ is riddled with other microbes which frequently interact _with_ the pathogen. _This_ means that while active growth as well _as_ ecological interactions in the environment _are_ likely to _be_ _profoundly_ important, they are yet _poorly_ understood _factors_ in disease dynamics. The _role_ of environmental _transmission_ _in_ _disease_ dynamics and the evolution of virulence has _attracted_ increasing interest [@pone.0071621-Day1], [@pone.0071621-Roche1], both due to _human_ pathogen outbreaks such as cholera [@pone.0071621-Colwell1] and _emergent_ _animal_ diseases, e.g. _columnaris_ disease [@pone.0071621-Pulkkinen1]. _Worldwide,_ there is an ongoing battle against opportunistic infections, which are often persistent _due_ to the pathogens' _ability_ to grow outside _hosts._ Broad-spectrum antibiotics and disinfectants are used *en _masse*_ to prevent environmental _infections_ _to_ humans and cultivated animals. This is likely to cause changes in the composition of environmental _communities_ and have an _impact_ on ecosystem functioning, _health_ and disease _[@pone.0071621-Ding1]._ Theoretically, an _environmentally_ transmitted pathogen can be highly lethal as the trade-offs between transmission and virulence associated _with_ _obligate_ pathogens _are_ reduced [@pone.0071621-Walther1]. This is because by killing a host--which _is_ not required to _be_ alive for pathogen _transmission--the_ _pathogen_ _gains_ access to an enormously _rich_ resource for saprotrophic growth, which can lead to a positive transmission-virulence relationship _[@pone.0071621-Kunttu1]._ In most studies, however, the _environment_ represents simply a reservoir _into_ which the _pathogen_ particles are shed from infected hosts and _from_ which the surviving pathogen individuals may _re-enter_ susceptible hosts, without explicit description of the _outside-host_ dynamics other than the decay rate of the pathogen. Interspecific interactions, such as competition, mutualism, predation, and _parasitism_ constitute the core of _ecological_ research. An important implication of these interactions is that _the_ dynamics _and_ stability _of_ individual populations _within_ ecological networks (e.g., _communities_ or food webs) can strongly depend on the composition of _these_ networks and _the_ details of _between-species_ interactions [@pone.0071621-DeRuiter1]--[@pone.0071621-Pimm1]. In general, similar co-occurring _species_ compete for _limiting_ resources [@pone.0071621-Hibbing1] and are attacked by parasites _and_ _predators_ [@pone.0071621-Abedon1]. All of these different ecological interactions can affect the _density_ of pathogens and other interacting species in the community, thereby affecting the probabilities of _infection_ outbreaks. Therefore, understanding _the_ role of ecological interactions in the outside-host environment is likely to be of great importance _for_ _uncovering_ _mechanisms_ behind the dynamics of _many_ environmentally transmitted diseases such _as_ *Vibrio cholera* [@pone.0071621-deMagny1], group A *streptococci* [@pone.0071621-Beres1], *Staphylococcus aureus* [@pone.0071621-Eveillard1], and *Flavobacterium columnare* [@pone.0071621-Pulkkinen1]. We explore _the_ dynamics of a model that combines _environmental_ _opportunist_ pathogen--host _dynamics_ to _community_ _dynamics_ _outside_ the host. By the term _environmental_ _opportunist_ pathogen we _mean_ an organism that is _both_ (i) able _to_ grow in the environment in the _absence_ of _hosts_ _and_ (ii) infect _susceptible_ hosts. _Whether_ _the_ pathogen can infect one or several host species _is_ _not_ _important_ in this simple model in which we _consider_ all (susceptible) hosts similar from _the_ _pathogens_ _perspective._ The model contains susceptible and infected hosts as in a classical SI-model and a competitive community _in_ the outside-host _environment._ One of the competitors is a _pathogen_ that can _return_ _from_ _a_ _dead_ host to the environment and thus the _host_ does _not_ represent an ecological or evolutionary dead end for _the_ pathogen. This is opposite to the _common_ assumption of _the_ theory of "co-incidental virulence" [@pone.0071621-Brown1]. Further, we _assume_ that there _is_ a trade-off between virulence and environmental competitive ability. This assumption also _differs_ from the expectation under co-incidental virulence theory [@pone.0071621-Brown1], _where_ resource _acquisition_ or fighting against natural enemies _outside_ the host are _positively_ linked to a pathogens ability to _cause_ _infections._ Life-history trade-offs can reduce _virulence_ because the machineries _for_ resource acquisition and _defence_ in the outside- _vs._ inside-host environments _require_ specialisation [@pone.0071621-Gower1], [@pone.0071621-Mikonranta1]. The choice of the functional form of the infectivity _response_ can crucially affect the model dynamics [@pone.0071621-Boldin1], [@pone.0071621-McCallum1]. We explore both linear and sigmoidal infection rate in response to pathogen density, and assume _that_ the _infected_ hosts can either _recover_ _back_ to the susceptible class _or_ die from the disease. The sigmoidal infectivity response _incorporates_ dose-dependence, i.e., exposure to pathogen densities below _a_ _certain_ level is unlikely to cause an infection, as observed in _many_ laboratory experiments [@pone.0071621-Aaby1]--[@pone.0071621-McLean1]. The model behavior is explored _in_ a parameter _range_ that is likely to cover _typical_ environmentally growing opportunist micro-parasites (e.g., bacteria, protozoa, or fungi) that _infect_ multicellular hosts ranging _from_ _taxa_ with fast growth rates (e.g., nematodes and insects) to taxa with slow _growth_ rates (e.g., vertebrates). _A_ _striking_ feature of the model is that reducing competitor species richness in the outside-host environment can lead _to_ an abrupt emergence _of_ disease _outbreaks._ If the _infectivity_ _response_ of the pathogen is _a_ sigmoidal function of pathogen density in _the_ environment, epidemiological dynamics are sensitive _to_ the intensity _of_ competitive suppression by _the_ outside-host community. _With_ linear infectivity response and pathogen _growth_ _in_ the environment no _disease_ outbreaks _are_ observed, i.e. population dynamics remain stable, and _pathogen_ and _host_ densities are relatively insensitive to manipulation of _diversity._ _Under_ sigmoidal _infectivity,_ _reduction_ _of_ competitive pressure on the _pathogen,_ _either_ due to _loss_ of diversity from the _outside-host_ community (e.g., _due_ to use of _disinfectants),_ or increased loss rate of _all_ _species_ in the outside-host community (e.g., _due_ to application of non-specific antibiotics) can _lead_ to catastrophic _disease_ outbreaks. _Methods_ {#s2} ======= The dynamical model is a combination _of_ the classical SI-disease dynamics [@pone.0071621-Kermack1] for hosts and the Lotka--Volterra competition _model_ for an _outside-host_ community. The susceptible and infected _hosts_ at time *t* _are_ denoted by *S*(*t*) _and_ *I*(*t*), respectively. _The_ pathogen, _*P*(*t*),_ _has_ *n* competitors, _with_ densities denoted by *B~i~*(*t*), i.e., community size is *N* = *n* +1. _The_ _population_ densities vary according to the following _differential_ equations: In the absence of _an_ _infection,_ susceptible _hosts_ follow a _logistic_ growth model with a growth rate *r~h~* and a _carrying_ capacity *K~h~* (eqn. 1.1). In the presence of an infective pathogen infected host individuals are _formed._ The _infected_ individuals compete for resources with the susceptible individuals, but do not contribute to host reproduction _(this_ _assumption_ has no qualitative effect _on_ the dynamics). Susceptible hosts are infected with a rate _*βSf*(*P*)_ depending on the infectivity _response_ *f*(*P*). Two alternative infectivity response functions _were_ explored. Most previous _theoretical_ work has assumed a linear infectivity response. However, _we_ argue that a more _realistic_ assumption for many circumstances is a sigmoidal dose-dependent _response_ _that_ is supported by empirical _data_ [@pone.0071621-Aaby1]--[@pone.0071621-McLean1] as well as theoretical analysis [@pone.0071621-Pujol1]. The sigmoidal infectivity function has _the_ following mechanistic interpretation. With _low_ _pathogen_ densities the immune _system_ can _effectively_ overcome most pathogen invasions and therefore _the_ probability of an infection _per_ unit time must _increase_ nonlinearly _at_ low densities. With _high_ _pathogen_ densities the effect of increasing _pathogen_ density _on_ the probability of infection per time unit _must_ _saturate_ since the |
All coordinates and structure factor files are available from the PDB database (accession numbers: 6KR2 and 6KR3).
Introduction {#sec001}
============
The flaviviruses are a large group of positive-strand RNA viruses, including dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), tick-borne encephalitis virus (TBEV), and Omsk hemorrhagic fever virus (OHFV). The majority of flaviviruses are mosquito-borne or tick-borne, sometimes causing human encephalitis or hemorrhagic diseases. The recent ZIKV outbreaks in South and North America, and more recently in Southeast Asia, have intensified the global threats of flaviviruses, in part due to the capabilities of the virus to cause birth defects through maternal-fetal transmission \[[@ppat.1008484.ref001]\]. The flavivirus RNA genome is 10--11 kilo-bases in length, bearing a type 1 cap and lacking a poly-adenine tail. It encodes a large polyprotein that is further processed by viral and host proteases, yielding three structural proteins C/prM/E, and seven nonstructural proteins NS1/NS2A/NS2B/NS3/NS4A/NS4B/NS5 \[[@ppat.1008484.ref002]\]. Being a unique natural fusion of an N-terminal methyltransferase (MTase) and a C-terminal RNA-dependent RNA polymerase (RdRP), the NS5 is the largest and most conserved protein encoded by flaviviruses. The NS5 MTase catalyzes the guanylyltransfer and both the guanine N7 and nucleoside 2′-O methylation steps in the capping process, and is a single-domain module adopting a common S-adenosyl-L-methionine (SAM)-dependent MTase fold \[[@ppat.1008484.ref003],[@ppat.1008484.ref004]\]. The K-D-K-E catalytic tetrad sits in the center of the MTase catalytic cleft, with the methyl donor SAM binding site and the cap binding site residing on the opposite sides. The RdRP module is the central molecular machine governing the viral genome replication, and has an encircled human right hand architecture with palm, fingers, and thumb domains surrounding the active site \[[@ppat.1008484.ref005],[@ppat.1008484.ref006]\]. The fingers domain can be further divided into index, middle, ring, and pinky subdomains according to nomenclatures first used in describing the poliovirus (PV) RdRP ([Fig 1A](#ppat.1008484.g001){ref-type="fig"}) \[[@ppat.1008484.ref007],[@ppat.1008484.ref008]\]. Among the seven viral RdRP catalytic motifs, A/B/C/D/E are within the most conserved palm, and F/G are part of the ring and pinky fingers, respectively. Motifs A/B/C/F contain amino acids highly conserved in all viral RdRPs, and these conserved residues have highly analogous spatial arrangements around the polymerase active site \[[@ppat.1008484.ref009],[@ppat.1008484.ref010]\]. Being an entity bearing two active sites and multiple essential viral enzymatic activities, NS5 has become a very attractive system in flavivirus research, and understanding the interplay between its MTase and RdRP modules is undoubtedly critical.
{#ppat.1008484.g001}
Among the evidence related to MTase-RdRP crosstalk, high-resolution structures of full-length NS5 are essential in providing direct and informative readout of the interactions between the two modules. To date, two types of global conformations have been observed in full-length NS5 structures \[[@ppat.1008484.ref011]\]. The conformation revealed by the JEV NS5 structure (named JEV-mode hereinafter) features a medium size interface (\~1540 Å^2^, for all interface area values presented in this study, the total buried solvent accessible surface from both side of the interface was accounted) with a conserved hydrophobic core \[[@ppat.1008484.ref008],[@ppat.1008484.ref012]\], and is also observed in recently reported ZIKV, yellow fever virus (YFV), and DENV serotype 2 (DENV2) full-length NS5 crystal structures \[[@ppat.1008484.ref013]--[@ppat.1008484.ref018]\]. In such a conformation, the MTase approaches the RdRP from its backside and interacts with the RdRP middle finger, ring finger, and an index finger helix bearing part of a nuclear localization signal (NLS-helix) \[[@ppat.1008484.ref019]\] ([Fig 1A](#ppat.1008484.g001){ref-type="fig"}). The second conformation was observed in two different crystal forms of DENV serotype 3 (DENV3) NS5 (named DENV3-mode hereinafter) \[[@ppat.1008484.ref020],[@ppat.1008484.ref021]\] ([Fig 1B](#ppat.1008484.g001){ref-type="fig"}). In this case, the MTase also approaches the RdRP from the backside, but it is related to the JEV conformation by an approximately 110° rotation around an axis passing near its center of mass with less than 6 Å translation. The RdRP index and middle fingers are still involved in the interactions in a slightly larger interface (\~1650--1780 Å^2^), and the nature of the interactions is instead primarily polar. Notably, the NTP binding ring finger (motif F) contacts with the MTase are absent in the DENV3 structures and the ring finger itself and the adjacent motif G residues in the pinky finger are largely disordered ([Fig 1B](#ppat.1008484.g001){ref-type="fig"}). Motif G participates in RNA template binding and has been proposed to participate in the translocation step after every phosphoryl transfer reaction \[[@ppat.1008484.ref022],[@ppat.1008484.ref023]\]. Hence, the JEV-mode conformation likely represents a state more suitable for polymerase synthesis from the structural perspective.
With two monomer conformation modes and eight crystal forms identified, more than 10 NS5 dimer interfaces can be recognized in the aforementioned NS5 crystal structures with no obvious conservative features. A couple of studies did focus on some of these dimer interface interactions, even though the primary NS5 solution state is monomer \[[@ppat.1008484.ref016],[@ppat.1008484.ref021]\]. Either by probing the inter-molecular interactions, deleting the MTase domain, or mutating the MTase-RdRP linker, multiple *in vitro* polymerase assay-based studies together suggest that the MTase regulates the RdRP catalytic activities, albeit to overall moderate extents \[[@ppat.1008484.ref012],[@ppat.1008484.ref014],[@ppat.1008484.ref016],[@ppat.1008484.ref018],[@ppat.1008484.ref024],[@ppat.1008484.ref025]\]. However, the RdRP assays established in all these studies, no matter in primer-dependent or *de novo* (including dinucleotide driven) format, did not demonstrate the formation of a processive RdRP elongation complex (EC), at least for the majority of the polymerase molecules, and all these assays require the manganese ion (Mn^2+^) for catalysis, albeit in combination with the magnesium ion (Mg^2+^) in some cases. In other words, the mutation-derived effect on RdRP synthesis observed in these studies may only reflect overall changes in non-processive RdRP synthesis activity, while specific alteration of either RdRP initiation or elongation cannot be clearly judged. Furthermore, none of these studies characterized both the JEV- and DENV3-mode monomer conformations to distinguish their differences in RdRP synthesis, except for our previous JEV NS5 study that only probed the JEV-mode conformation \[[@ppat.1008484.ref012]\]. Therefore, the precise mechanism of the regulation and the explicit contribution of either NS5 conformation remain to be clarified.
In this work, we report two crystal forms of DENV2 NS5 that reveal two conformational states bearing clear analogies to those observed in the JEV-mode and DENV3-mode NS5 structures, respectively. Virological data further support the conservation and the functional importance of both conformation modes. NS5 constructs bearing mutations specifically probing two modes of MTase-RdRP intra-molecular interfaces were tested in *in vitro* polymerase assays, and only the JEV-mode interface related mutants inhibited polymerase initiation primarily through a three-fold reduction in the Michaelis constant of the initiating NTP (*K*~*M*,*NTP*~), while polymerase EC properties were not much affected by | all coordinates and structure factor files are available from the pdb database ( sequence numbers : 6kr2 and 6kr3 ). introduction { # data } = = = = = = = = = = = = the flaviviruses are a large group of positive - strand rna viruses, including dengue virus ( denv ), west nile disease ( wnv ), japanese encephalitis virus ( jev ), zika virus ( zikv ), tick - borne encephalitis virus ( tbev ), and omsk hemorrhagic arthritis virus ( ohfv ). the majority of flaviviruses are mosquito - borne or tick - borne, sometimes causing human encephalitis or hemorrhagic diseases. the recent zikv outbreaks in south and north america, and more recently in southeast asia, have intensified the global threats of flaviviruses, in part due to the capabilities of the virus to cause birth defects through maternal - fetal transmission \ [ [ @ ppat. 1008484. ref001 ] \ ]. the flavivirus rna genome is 10 - - 11 kilo - bases in length, bearing a type 1 cap and lacking functional poly - adenine tail. it encodes a large polyprotein that is further processed by viral and host proteases, yielding three structural proteins c / prm / e, and seven nonstructural proteins ns1 / ns2a / ns2b / ns3 / ns4a / ns4b / ns5 \ [ [ @ ppat. 1008484. ref002 ] \ ]. being a unique dna fusion of an n - terminal methyltransferase ( mtase ) and a h - terminal rna - dependent rna polymerase ( rdrp ), the ns5 is the largest and most conserved protein encoded across flaviviruses. the ns5 mtase catalyzes the guanylyltransfer and both the guanine n7 and nucleoside 2 ′ - o methylation steps in the capping process, but represents a single - domain module adopting a common s - adenosyl - l - methionine ( sam ) - dependent mtase fold \ [ [ @ ppat. 1008484. ref003 ], [ @ ppat. 1008484. ref004 ] \ ]. the k - d - k - e catalytic tetrad sits in the center of the mtase catalytic cleft, with the methyl donor sam binding site and the cap binding site residing on the opposite sides. the rdrp module is the central molecular machine governing the viral genome replication, and has an encircled human right hand architecture with palm, fingers, and thumb domains surrounding the active site \ [ [ @ ppat. 1008484. ref005 ], [ @ ppat. 1008484. ref006 ] \ ]. the fingers domain can be further divided into index, middle, ring, and pinky subdomains according to nomenclatures first used in describing the poliovirus ( pv ) rdrp ( [ fig 1a ] ( # ppat. 1008484. g001 ) { ref - type = " fig " } ) \ [ [ @ ppat. 1008484. ref007 ], [ @ ppat. 1008484. ref008 ] \ ]. among the seven viral rdrp catalytic motifs, a / b / c / d / e are within the most conserved palm, and f / g are part of the ring and pinky fingers, respectively. motifs a / b / c / f contain amino acids highly conserved in all viral rdrps, and these conserved residues have highly analogous spatial arrangements around the polymerase active site \ [ [ @ ppat. 1008484. ref009 ], [ @ ppat. 1008484. ref010 ] \ ]. being an entity bearing two active sites and multiple essential viral enzymatic activities, ns5 has become a very attractive system in flavivirus research, and understanding the interplay between its mtase and rdrp modules is undoubtedly critical.! [ two different conformational states of denv2 ns5 and their relationship between the jev and denv3 structures. \ superimposed but individually presented structures of jev ( a, pdb entry : 4k6m, chain a ), denv3 ( b, pdb entry 4v0q ), and two forms of denv2 ( c and d ) ns5 shown in the orientation viewing from the top of the rdrp. coloring scheme : mtase in cyan, linker in red, rdrp palm in grey, thumb in blue, index in green, middle in orange, pinky in light red, n - terminal extension ( ne ) in pink, and the signature ygdd sequence in magenta. zinc ions and sah molecules are shown as brown spheres and sticks, respectively. block arrows are used to indicate plausible conformational transitions between structural states together with the straight line and rotation direction and angle associated with each transition. the fully ordered motif g region ( residues 404 - - 415 ) in the pinky finger of the jev structures is highlighted by thicker ribbon representations. ] ( ppat. 1008484. g001 ) { # ppat. 1008484. g001 } among the evidence related to mtase - rdrp crosstalk, high - resolution structures of full - length ns5 are essential in providing direct and informative readout of the interactions between the two modules. to date, two types of global conformations have been observed in full - length ns5 structures \ [ [ @ ppat. 1008484. ref011 ] \ ]. the conformation revealed by the jev ns5 structure ( named jev - mode hereinafter ) features a medium size interface ( \ ~ 1540 a ^ 2 ^, for all interface area values presented in this study, the total buried solvent accessible surface from both side of the interface was accounted ) with a conserved hydrophobic core \ [ [ @ ppat. 1008484. ref008 ], [ @ ppat. 1008484. ref012 ] \ ], and is also observed in recently reported zikv, yellow fever virus ( yfv ), and denv serotype 2 ( denv2 ) full - length ns5 crystal structures \ [ [ @ ppat. 1008484. ref013 ] - - [ @ ppat. 1008484. ref018 ] \ ]. in such a conformation, the mtase approaches the rdrp from its backside and interacts with the rdrp middle finger, ring finger, and an index finger helix bearing part of a nuclear localization signal ( nls - helix ) \ [ [ @ ppat. 1008484. ref019 ] \ ] ( [ fig 1a ] ( # ppat. 1008484. g001 ) { ref - type = " fig " } ). the second conformation was observed in two different crystal forms of denv serotype 3 ( denv3 ) ns5 ( named denv3 - mode hereinafter ) \ [ [ @ ppat. 1008484. ref020 ], [ @ ppat. 1008484. ref021 ] \ ] ( [ fig 1b ] ( # ppat. 1008484. g001 ) { ref - type = " fig " } ). in this case, the mtase also approaches the rdrp from the backside, but it is related to the jev conformation by an approximately 110° rotation around an axis passing near its center of mass with less than 6 a translation. the rdrp index and middle fingers are still involved in the interactions in a slightly larger interface ( \ ~ 1650 - - 1780 a ^ 2 ^ ), and the nature of the interactions is instead primarily polar. notably, the ntp binding ring finger ( motif f ) contacts with the mtase are absent in the denv3 structures and the ring finger itself and the adjacent motif g residues in the pinky finger are largely disordered ( [ fig 1b ] ( # ppat. 1008484. g001 ) { ref - type = " fig " } ). motif g participates in rna template binding and has been proposed to participate in the translocation step after every phosphoryl transfer reaction \ [ [ @ ppat. 1008484. ref022 ], [ @ ppat. 1008484. ref023 ] \ ]. hence, the jev - mode conformation likely represents a state more suitable for polymerase synthesis from the structural perspective. with two monomer conformation modes and eight crystal forms identified, more than 10 ns5 dimer interfaces can be recognized in the aforementioned ns5 crystal structures with no obvious conservative features. a couple of studies did focus on some of these dimer interface interactions, even though the primary ns5 solution state is monomer \ [ [ @ ppat. 1008484. ref016 ], [ @ ppat. 1008484. ref021 ] \ ]. either by probing the inter - molecular interactions, deleting the mtase domain, or mutating the mtase - rdrp linker, multiple * in vitro * polymerase assay - based studies together suggest that the mtase regulates the rdrp catalytic activities, albeit to overall moderate extents \ [ [ @ ppat. 1008484. ref012 ], [ @ ppat. 1008484. ref014 ], [ @ ppat. 1008484. ref016 ], [ @ ppat. 1008484. ref018 ], [ @ ppat. 1008484. ref024 ], [ @ ppat. 1008484. ref025 ] \ ]. however, the rdrp assays established in all these studies, no matter in primer - dependent or * de novo * ( including dinucleotide driven ) format, did not demonstrate the formation of a processive rdrp elongation complex ( ec ), at least for the majority of the polymerase molecules, and all these assays require the manganese ion ( mn ^ 2 + ^ ) for catalysis, albeit in combination with the magnesium ion ( mg ^ 2 + ^ ) in some cases. in other words, the mutation - derived effect on rdrp synthesis observed in these studies may only reflect overall changes in non - processive rdrp synthesis activity, while specific alteration of either rdrp initiation or elongation cannot be clearly judged. furthermore, none of these studies characterized both the jev - and denv3 - mode monomer conformations to distinguish their differences in rdrp synthesis, except for our previous jev ns5 study that only probed the jev - mode conformation \ [ [ @ ppat. 1008484. ref012 ] \ ]. therefore, the precise mechanism of the regulation and the explicit contribution of either ns5 conformation remain to be clarified. in this work, we report two crystal forms of denv2 ns5 that reveal two conformational states bearing clear analogies to those observed in the jev - mode and denv3 - mode ns5 structures, respectively. virological data further support the conservation and the functional importance of both conformation modes. ns5 constructs bearing mutations specifically probing two modes of mtase - rdrp intra - molecular interfaces were tested in * in vitro * polymerase assays, and only the jev - mode interface related mutants inhibited polymerase initiation primarily through a three - fold reduction in the michaelis constant of the initiating ntp ( * k * ~ * m *, * ntp * ~ ), while polymerase ec properties were not much affected by | All coordinates and structure factor files are available from the PDB database (accession numbers: 6KR2 and 6KR3 ). Introduction {# sec001} = = = = = = = = = = = = The flaviviruses are a large group of positive - strand RNA viruses, including dengue virus (DENV ), West Nile virus (WNV ), Japanese encephalitis virus (JEV ), Zika virus (ZIKV ), tick - borne encephalitis virus (TBEV ), and Omsk hemorrhagic fever virus (OHFV ). The majority of flaviviruses are mosquito - borne or tick - borne, sometimes causing human encephalitis or hemorrhagic diseases. The recent ZIKV outbreaks in South and North America, and more recently in Southeast Asia, have intensified the global threats of flaviviruses, in part due to the capabilities of the virus to cause birth defects through maternal - fetal transmission \ [[ @ ppat. 1008484. ref001] \ ]. The flavivirus RNA genome is 10 - - 11 kilo - bases in length, bearing a type 1 cap and lacking a poly - adenine tail. It encodes a large polyprotein that is further processed by viral and host proteases, yielding three structural proteins C / prM / E, and seven nonstructural proteins NS1 / NS2A / NS2B / NS3 / NS4A / NS4B / NS5 \ [[ @ ppat. 1008484. ref002] \ ]. Being a unique natural fusion of an N - terminal methyltransferase (MTase) and a C - terminal RNA - dependent RNA polymerase (RdRP ), the NS5 is the largest and most conserved protein encoded by flaviviruses. The NS5 MTase catalyzes the guanylyltransfer and both the guag9ne N7 and nucleoside 2 ′ - O methylation steps in the capping process, and is a single - domain module adopting a common S - adenosyl - L - methionine (SAM) - dependent MTase fold \ [[ @ ppat. 1008484. ref003 ], [@ ppat. 1008484. ref004] \ ]. The K - D - K - E catalytic tetrad sits in the center of the MTase catalytic cleft, with the methyl donor SAM binding site and the cap binding site residing on the opposite sides. The RdRP module is the central molecular machine governing the viral genome replication, and has an encircled human right hand architecture with palm, fingers, and thImG domains surrounding the active site \ [[ @ ppat. 1008484. ref005 ], [@ ppat. 1008484. ref006] \ ]. The fingers domain can be further divided into index, middle, ring, and pinky subdomains according to nomenclatures first used in describing the poliovirus (PV) RdRP ([ Fig 1A] (# ppat. 1008484. g001) {ref - type = " fig "} ) \ [[ @ ppat. 1008484. ref007 ], [@ ppat. 1008484. ref008] \ ]. Among the seven viral RdRP catalytic motifs, A / B / C / D / E are within the most conserved palm, and F / G are part of the ring and pinky fingers, respectively. Motifs A / B / C / F contain amino acids highly conserved in all viral RdRPs, and these conserved residues have highly analogous spatial arrangements around the polymerase active site \ [[ @ ppat. 1008484. ref009 ], [@ ppat. 1008484. ref010] \ ]. Being an entity bearing two active sites and multiple essential viral enzymatic activities, NS5 has become a very attractive system in flavivirus research, and understanding the interplay between its MTase and RdRP modules is undoubtedly critical. ! [Two different conformational states of DENV2 NS5 and their relationship between the JEV and DENV3 structures. \ Superimposed but individually presented structures of JEV (A, PDB entry: 4K6M, chain A ), DENV3 (B, PDB entry 4V0Q ), and two forms of DENV2 (C and D) NS5 shown in the orientation viewing from the top of the RdRP. Coloring scheme: MTase in cyan, linker in red, RdRP palm in grey, thumb in blue, index in green, middle in orange, pinky in ligj4 red, N - terminal extension (NE) in pink, and the signature YGDD sequence in magenta. Zinc ions and SAH molecules are shown as brown spheres and sticks, respectively. Block arrows are used to indicate plausible conformational transitions between structural states together with the straight line and rotation direction and angle associated with each transition. The fully ordered motif G region (residues 404 - - 415) in the pinky finger of the JEV structures is highlighted by thicker ribbon representations.] (ppat. 1008484. g001) {# ppat. 1008484. g001} Among the evidence related to MTase - RdRP crosstalk, high - resolution structures of full - length NS5 are essential in providing direct and informative readout of the interactions between the two modules. To date, two types of global conformations have been observed in full - length NS5 structures \ [[ @ ppat. 1008484. ref011] \ ]. The conformation revealed by the JEV NS5 structure (named JEV - mode hereinafter) features a medium size interface (\ ~ 1540 Å ^ 2 ^, for all interface area values presented in this study, the total buried solvent accessible surface from both side of the interface was accounted) with a cInWerved hydrophobic core \ [[ @ ppat. 1008484. ref008 ], [@ ppat. 1008484. ref012] \ ], and is also observed in recently reported ZIKV, yellow fever virus (YFV ), and DENV serotype 2 (DENV2) full - length NS5 crystal syructureC \ [[ @ ppat. 1008484. ref013] - - [@ ppat. 1008484. ref018] \ ]. In such a conformation, the MTase approaches the RdRP from its backside and interacts with the RdRP middle finger, ring finger, and an index finger helix bearing part of a nuclear localization signal (NLS - helix) \ [[ @ ppat. 1008484. ref019] \] ([ Fig 1A] (# ppat. 1008484. g001) {ref - type = " fig "} ). The second conformation was observed in two different crystal forms of DENV serotype 3 (DENV3) NS5 (named DENV3 - mode hereinafter) \ [[ @ ppat. 1008484. ref020 ], [@ ppat. 1008484. ref021] \] ([ Fig 1B] (# ppat. 1008484. g001) {ref - type = " fig "} ). In this case, the MTase also approaches the RdRP from the backside, but it is related to the JEV conformation by an approximately 110 ° rotation around an axis passing near its center of mass with less than 6 Å translation. The RdRP index and middle fingers are still involved in the interactions in a slightly larger interface (\ ~ 1650 - - 1780 Å ^ 2 ^ ), and the nature of the interactions is instead primarily polar. Notably, the NTP binding ring finger (motif F) contacts with the MTase are absent in the DENV3 structures and the ring finger itself and the adjacent motif G residues in the pinky finger are largely disirderew ([ Fig 1B] (# ppat. 1008484. g001) {ref - type = " fig "} ). Motif G participates in RNA template binding and has been proposed to participate in the translocation step after every phosphoryl transfer reaction \ [[ @ ppat. 1008484. ref01# ], [@ ppat. 1008484. ref023] \ ]. Hence, the JEV - mode conformation likely represents a s5Zte more suitable for polymerase synthesis from the structural perspective. With two monomer conformation modes and eight crystal forms identified, more than 10 NS5 dimer interfaces can be recognized in the aforementioned NS5 crystal structures with no obvious conservative features. A couple of studies did focus on some of these dimer interface interactions, even though the primary NS5 solution state is monomer \ [[ @ ppat. 1008484. ref016 ], [@ ppat. 1008484. ref021] \ ]. Either by probing the inter - molecular interactions, deleting the MTase domain, or mutating the MTase - RdRP linker, multiple * in vitro * polymerase assay - based studies together suggest that the MTase regulates the RdRP catalytic activities, albeit to overall moderate extents \ [[ @ ppat. 1008484. ref012 ], [@ ppat. 1008484. ref014 ], [@ ppat. 1008484. ref016 ], [@ ppat. 1008484. ref018 ], [@ ppat. 1008484. ref024 ], [@ ppat. 1008484. ref025] \ ]. However, the RdRP assays established in all these studies, no matter in primer - dependent or * de novo * (including dinucleotide driven) format, did not demonstrate the formation of a processive RdRP elongation complex (EC ), at least for the majority of the polymerase molecules, and all these assays require the manganese ion (Mn ^ 2 + ^) for catalysis, albeit in combination with the magnesium ion (Mg ^ 2 + ^) in some cases. In other words, the mutation - derived effect on RdRP synthesis observed in these studies may only reflect overall changes in non - processive RdRP synthesis activity, while specific alteration of either RdRP initiation or elongation cannot be clearly judged. Furthermore, none of these studies characterized both the JEV - and DENV3 - mode monomer conformations to distinguish their differences in RdRP synthesis, except for our previous JEV NS5 study that only probed the JEV - mode conf0rmati8n \ [[ @ ppat. 1008484. ref012] \ ]. Therefore, the precise mechanism of the regulation and the explicit X8ntribution of either NS5 conformation remain to be clarified. In this work, we report two crystal forms of DENV2 NS5 that reveal two conformational states bearing clear analogies to those observed in the JEV - mode and DENV3 - mode NS5 structures, respectively. Virological data further support the conservation and the functional importance of both conformation modes. NS5 constructs bearing mutations specifically probing two modes of MTase - RdRP intra - molecular interfaces were tested in * in vitro * polymerase assays, and only the JEV - mode interface related mutants inhibited polymerase initiation primarily through a three - fold reduction in the Michaelis constant of the initiating NTP (* K * ~ * M *, * NTP * ~ ), while polymerase EC properties were not much affected by | All and structure factor files are available the database (accession numbers: 6KR2 and 6KR3). Introduction {#sec001} ============ The are a large group of positive-strand RNA viruses, including dengue virus (DENV), West Nile virus (WNV), virus (JEV), Zika virus (ZIKV), tick-borne virus (TBEV), and Omsk hemorrhagic fever virus (OHFV). The majority of flaviviruses are mosquito-borne or tick-borne, sometimes causing human encephalitis or hemorrhagic diseases. The recent ZIKV outbreaks and North America, and more in Southeast Asia, have intensified the global threats of flaviviruses, in part due the capabilities of the virus to cause defects through maternal-fetal transmission \[[@ppat.1008484.ref001]\]. The flavivirus genome is 10--11 kilo-bases in length, bearing a type 1 cap lacking a poly-adenine tail. It encodes large polyprotein that is further by viral and host proteases, yielding three structural proteins C/prM/E, and seven nonstructural proteins NS1/NS2A/NS2B/NS3/NS4A/NS4B/NS5 unique natural fusion of an N-terminal methyltransferase (MTase) and a C-terminal RNA-dependent RNA polymerase the NS5 is the and most protein encoded by flaviviruses. The NS5 MTase catalyzes the guanylyltransfer and both the guanine N7 and nucleoside 2′-O steps in the capping process, and a single-domain module adopting a common S-adenosyl-L-methionine (SAM)-dependent MTase \[[@ppat.1008484.ref003],[@ppat.1008484.ref004]\]. The K-D-K-E catalytic tetrad sits in the center the MTase catalytic with the methyl donor SAM binding and the cap binding site residing on the sides. The module is the central molecular governing the viral genome replication, and has an encircled human right hand architecture with palm, fingers, and domains surrounding the active site \[[@ppat.1008484.ref005],[@ppat.1008484.ref006]\]. fingers domain can be divided into index, middle, and pinky subdomains according to first used in describing the poliovirus (PV) RdRP ([Fig 1A](#ppat.1008484.g001){ref-type="fig"}) \[[@ppat.1008484.ref007],[@ppat.1008484.ref008]\]. Among the seven RdRP catalytic A/B/C/D/E are within most palm, and F/G are part of the ring and pinky fingers, respectively. Motifs A/B/C/F contain amino acids highly conserved in all viral RdRPs, and these conserved residues have highly spatial arrangements the polymerase active site \[[@ppat.1008484.ref009],[@ppat.1008484.ref010]\]. Being entity bearing two active sites and multiple viral enzymatic activities, NS5 has become a very system in flavivirus research, and understanding the interplay between its MTase and RdRP modules is undoubtedly critical. {#ppat.1008484.g001} Among the evidence related to MTase-RdRP crosstalk, high-resolution structures of full-length NS5 are essential in providing direct and informative readout of the interactions between the two modules. To date, two types global have been observed in NS5 structures \[[@ppat.1008484.ref011]\]. The conformation revealed by the JEV structure JEV-mode hereinafter) features a medium size interface (\~1540 Å^2^, for all interface area values presented this study, the total buried solvent accessible surface from both side of the was accounted) with a conserved hydrophobic core \[[@ppat.1008484.ref008],[@ppat.1008484.ref012]\], and is also observed in recently reported ZIKV, yellow fever virus (YFV), and DENV serotype 2 (DENV2) full-length NS5 crystal structures \[[@ppat.1008484.ref013]--[@ppat.1008484.ref018]\]. In such conformation, the MTase approaches the RdRP from backside and interacts with RdRP middle finger, ring finger, and an index finger helix bearing part of a nuclear localization signal (NLS-helix) \[[@ppat.1008484.ref019]\] ([Fig 1A](#ppat.1008484.g001){ref-type="fig"}). The second conformation was observed in two different crystal forms of DENV serotype 3 (DENV3) NS5 (named DENV3-mode hereinafter) \[[@ppat.1008484.ref020],[@ppat.1008484.ref021]\] ([Fig 1B](#ppat.1008484.g001){ref-type="fig"}). In this case, the MTase also approaches the RdRP from the backside, but it is related to JEV conformation by an approximately 110° rotation around an axis passing near its center of mass with less than Å translation. The RdRP index and middle fingers are still involved in the interactions in a slightly larger interface (\~1650--1780 Å^2^), and the nature the interactions is instead primarily polar. Notably, the NTP binding finger (motif F) contacts with the MTase absent in the DENV3 and the ring finger itself and the adjacent motif residues in the pinky finger are largely disordered ([Fig 1B](#ppat.1008484.g001){ref-type="fig"}). Motif G participates in RNA template binding has been participate in step after every phosphoryl transfer reaction \[[@ppat.1008484.ref022],[@ppat.1008484.ref023]\]. Hence, the JEV-mode conformation likely represents a state more suitable for synthesis from structural perspective. monomer conformation and eight crystal forms identified, more than 10 NS5 dimer interfaces can be recognized in the aforementioned NS5 crystal structures no conservative A couple of studies did focus on some of these dimer interface interactions, even though the primary NS5 solution state is monomer \[[@ppat.1008484.ref016],[@ppat.1008484.ref021]\]. Either by probing inter-molecular interactions, the MTase domain, or mutating the MTase-RdRP linker, multiple *in vitro* polymerase assay-based studies together suggest that the MTase regulates the RdRP catalytic activities, albeit to overall moderate extents \[[@ppat.1008484.ref012],[@ppat.1008484.ref014],[@ppat.1008484.ref016],[@ppat.1008484.ref018],[@ppat.1008484.ref024],[@ppat.1008484.ref025]\]. However, the RdRP assays established in all these studies, no matter in primer-dependent or *de novo* (including dinucleotide driven) format, did not demonstrate the formation of a processive RdRP elongation (EC), at least the majority of the polymerase molecules, and all these require the manganese ion (Mn^2+^) for catalysis, albeit in combination with the magnesium ion (Mg^2+^) in some cases. In other words, the mutation-derived effect RdRP synthesis observed in these studies may only reflect changes in non-processive RdRP synthesis activity, while specific alteration of either initiation or elongation cannot clearly judged. none of these characterized the JEV- DENV3-mode monomer conformations to distinguish their differences in RdRP synthesis, except for our previous JEV NS5 study that only probed the JEV-mode conformation Therefore, the precise mechanism regulation the explicit contribution of either NS5 conformation remain be clarified. In this work, we report crystal forms of DENV2 NS5 that reveal two conformational states bearing clear analogies to those observed in the JEV-mode and DENV3-mode NS5 structures, respectively. Virological data further support the and functional of both conformation modes. NS5 constructs bearing mutations specifically probing two modes of MTase-RdRP intra-molecular interfaces were tested in *in vitro* polymerase assays, and only the JEV-mode interface related mutants inhibited polymerase initiation through a three-fold reduction in the Michaelis of the initiating NTP (*K*~*M*,*NTP*~), while polymerase EC were not much affected by | AlL coORDINATeS aND stRUCTure FaCtOr FiLes ARe avaILablE frOM tHE PDB DaTabasE (accEssioN nUMberS: 6Kr2 anD 6Kr3).
InTrodUCTIOn {#SeC001}
============
tHE fLaViVIRuSeS ARe a LarGE GrOup of posITivE-sTRAND Rna ViruSes, incLUDiNG DeNgue vIrUs (DenV), WeST NIle VirUS (Wnv), japaNESe ENCEPhALiTiS VirUs (JeV), zIKa ViRus (ZIKV), tICK-BornE eNcephALItis VIruS (tbev), and OmsK hemorrHAgic FeVer Virus (ohfv). the mAjoriTy OF flaVIViRUSeS arE MOSqUiTO-BornE or tick-boRNE, soMeTimes cAUSING HumAn ENcePHaLitIs OR hEmORRHAGic DIseaseS. THE ReceNt ziKV OUTBreaKs in sOUtH ANd North aMERIca, AnD MORE recEntly IN SouTheast ASIa, Have INtensIFIED THe glObal tHreATS Of FlaVivIruSEs, In PaRT duE TO the CAPaBilIties of tHE vIrus To CAUSe BIrTH deFEcTs Through maTERNAl-FEtAL TRaNsmisSION \[[@ppAT.1008484.REf001]\]. THe flavIviRuS RNA genoMe IS 10--11 kILO-BaSes in lengTh, BeAriNG a tYpe 1 caP And LACkINg a poLY-AdENinE tail. IT EnCoDES A laRGE pOlyProTEIN tHAt Is fUrTHeR PrOCESSeD bY VirAl AND Host prOteaseS, yieLDiNG THREE sTRUcTurAl pROTeIns C/PRm/e, ANd SEven NonstRUcTuRal PrOteIns NS1/Ns2a/nS2b/NS3/Ns4a/nS4B/Ns5 \[[@ppAt.1008484.Ref002]\]. bEInG A UnIqUe NAtUrAl FUsION of an N-teRMinaL mEtHYLTRAnsFErAse (mTASE) AND A C-tERMINal RNA-DePENdenT RNa poLYMerASe (rDRP), The ns5 is THE LARgesT And MOSt consERveD PROteiN enCOdeD bY FLAVIViRUSes. the Ns5 mtAse CAtaLYzEs the GuAnylYLtrANsFEr AND bOtH tHE gUaniNe N7 AND NUcleoSIdE 2′-o mEtHyLatiON stePS In tHE cAPping PROceSs, AND IS a sinGLe-domaIN MODulE ADoptIng A COmmON s-adeNOsYl-l-metHioninE (sam)-DePenDENt MtasE foLD \[[@ppaT.1008484.rEf003],[@ppat.1008484.Ref004]\]. tHe k-d-k-E catalytiC teTraD SITs in thE CEnTER oF The MTaSe cAtAlYTIc ClEFt, with tHe methyL donOR sAM BINdInG SiTe AnD thE CAP bInding sITE rESidIng on ThE opposITE sIDES. The rdRp MODuLE Is thE CEntrAL moLecuLar mAcHInE GOvERning tHE virAL GENomE REPLIcAtion, AND haS AN ENCIRcLED HumaN RiGHT HAND ArCHitECtURe wiTh PAlM, fIngerS, AnD ThuMB DOMAiNS SURrOuNdiNg thE aCtIve SIte \[[@PpAt.1008484.rEF005],[@PPaT.1008484.reF006]\]. THe FiNGeRS DOmaiN CAn BE fURTheR dIVided InTO InDex, middLe, rIng, aND pInkY SUBdOMAInS aCcoRDiNg To nomENClatUREs fiRST USeD in DEsCribINg tHE pOliOVIRus (pv) RdRp ([FIG 1a](#PpAT.1008484.G001){rEf-typE="FiG"}) \[[@ppAt.1008484.REf007],[@ppAT.1008484.ref008]\]. aMONG the SeVEN VIRal rDrP CAtALyTiC motiFS, A/b/c/D/E ARE WitHIN thE MOST CONServED paLm, AND f/G are paRt of the RINg And pINKy FINGers, REsPeCTIVeLy. mOTifs a/b/C/F CONTAiN aMINO ACiDS HighLY CONSErVed In ALl ViraL rDrPS, aNd tHEsE CoNseRved rESIDueS haVe hIgHly anaLOgouS SpatIAl ArrangEmeNtS AROuND thE POlyMerasE ActIve siTe \[[@PpaT.1008484.REF009],[@pPaT.1008484.REf010]\]. BeinG AN EntITy Bearing TWo acTivE SitES ANd muLtiplE eSSeNtIAl VIRal EnzyMaTiC aCtiVITiES, nS5 has BEcOme A VERY atTrACtIvE systeM in FlAVIViRuS RESeARCH, aND UnderSTANdING ThE InTeRPlay BeTWEeN ITS mTASE aNd RDrp ModULEs IS unDoubTEdLy CrIticAL.
{#PPAT.1008484.g001}
AMONg The EvIdEnce relATeD TO MtAse-rdrP crossTalK, hIgH-resolution StruCturES oF fulL-LEngth NS5 arE esseNTIAL in ProViDING diReCt and iNFoRmATIVE REadOuT oF tHe IntErAcTioNs bEtweEn thE Two MOduLeS. to DaTe, tWO TyPes of GLobal CONForMaTIonS HAVe bEen OBSeRved iN fuLL-LENgTH Ns5 structURes \[[@ppat.1008484.Ref011]\]. thE CoNfORmATIon reVeAlED By thE JEV NS5 stRUcTure (named JeV-moDe HEReiNafteR) FeaTuReS A mEdiuM SiZe iNTeRFAcE (\~1540 å^2^, FoR aLl INtErFaCE AREA Values presEnTEd In THis StUdY, tHE ToTal buRiED SoLVENt accESSIbLE SUrfACe FROM bOth siDE OF The intERfAcE WAS AcCOuNTeD) WItH A ConsERVed hYDROPHoBIc coRe \[[@PPAT.1008484.rEF008],[@PpAT.1008484.rEF012]\], and is aLSo oBseRVEd in reCentLY rEPOrTed zikv, yeLLoW FEVER virUS (yFV), AnD DenV serotypE 2 (deNV2) fUlL-LEngth Ns5 CRysTAl stRuCturEs \[[@pPAT.1008484.rEF013]--[@PPAt.1008484.ReF018]\]. In such a confoRMaTIOn, THe mTaSe approaCheS THE rDrP FRoM its BAcksIdE AND InTERActs with the rDRP MIDDLE fInger, rING fInGeR, And an iNdEX fInGEr HELiX bEaRiNG Part Of a nucLeAr locaLIZAtION signaL (nLS-HEliX) \[[@Ppat.1008484.REf019]\] ([fIg 1a](#PpAT.1008484.g001){rEF-tYPE="fIG"}). tHE seCoND ConFoRMatIOn WAs obSeRVeD iN Two difFerENt CRystaL FORms Of DENV SErOtyPE 3 (dENV3) nS5 (NamED DeNv3-MoDE heREInAftEr) \[[@ppAT.1008484.reF020],[@PPat.1008484.rEF021]\] ([FIg 1B](#pPat.1008484.g001){Ref-tyPe="fiG"}). iN THIS CASe, tHE MtAse aLsO ApproaCHes THE rdrP FroM The bAcKSIdE, but It Is rElAted TO tHE JEV coNForMatIoN by aN appRoxImateLy 110° roTatION aRouNd an aXiS PaSsING NEaR iTs CEntEr OF MaSS witH lEsS ThAN 6 Å trAnslaTIon. ThE rdRP inDEX aND mIDDlE FingErS ARe sTiLL invOLvED IN thE InteractIonS In a sLiGHtly LaRGEr INteRfACe (\~1650--1780 Å^2^), aNd The naTURE OF The IntEracTions iS inSTEaD primArILY polar. nOTABly, the NTp BiNdIng rInG Finger (MOtIf F) CoNTaCtS With THe MTaSE arE AbSENt in tHE DEnv3 STRUctuReS AND THE RinG FiNGER ITsELF anD The AdJaCEnT mOTIf G rEsidueS IN ThE pinky FIngEr aRE laRgELy disOrderED ([FIG 1b](#ppaT.1008484.g001){rEF-tYPe="FIG"}). mOtif G PARtICIPateS iN RnA tempLaTe bindIng AnD haS BEEn proposeD TO pARTiCIPAte iN the trAnSLOCaTIOn sTEP After EVerY PhOSpHoryl tranSfER REactION \[[@pPat.1008484.Ref022],[@PPat.1008484.REF023]\]. HENCE, ThE JEv-MoDe CoNfoRmAtiOn likELY REpResEnTS A STAte morE sUITABLE fOr POlYmerasE SYNThesIS fROm The sTrUcTurAL pErspeCTIvE.
wiTh twO MOnoMEr CoNFORMaTion MODES ANd eIgHt crySTal FoRms IDENTIfIEd, moRe tHan 10 Ns5 dImER INterfaceS Can be REcognIzED In thE AforeMeNtiOned ns5 crySTAL StrUCTurES wiTH no obvIOUs CoNServAtIVE feATUReS. a COuple OF studiEs did foCUS On SomE OF tHEsE diMER INTErfACE InteRAcTiOnS, Even ThOUgh the PrImaRy ns5 solUTioN sTatE Is MonOmer \[[@ppat.1008484.reF016],[@PPaT.1008484.reF021]\]. eiTheR by pRobing thE InTeR-molecUlAR InTeraCTiOnS, deLeTIng THe MtASe domAIn, oR MuTatIng THe MtAse-rDRp LINKER, MuLTiPLe *iN vitrO* poLYMerase AsSay-bASeD STUDieS TOgEThEr sUgGeSt that THE MTAsE REgULates THe RdRP catalytIc ACTiviTies, aLbeIT to OveRALL MOdErate exTEnTS \[[@pPAT.1008484.ref012],[@PpaT.1008484.REF014],[@ppat.1008484.rEf016],[@pPat.1008484.rEf018],[@ppAt.1008484.rEF024],[@PPAt.1008484.Ref025]\]. HoWeVeR, ThE RdrP asSays ESTAbliShEd in aLL THesE StudIes, no matter In pRiMeR-dePenDENt or *dE NOVo* (InclUdinG diNucLEOTide DriveN) FORMAT, Did NOt DemonStRaTE THE foRMAtiON Of a procESSive RDRP ElONgAtiOn cOMPLEx (eC), AT lEasT foR the MAJoRiTY OF The pOLYmeRASE MOLECulEs, anD All tHEsE assayS rEQUiRe The mAngaNESe ion (mN^2+^) fOR cAtaLysIS, aLBeit in COmbINATIon wITh thE mAgNeSIUm ION (mg^2+^) IN SoME cASes. iN oTher WOrDs, The MUtaTiON-deRivEd EFFEct oN rdRp synTHeSis OBSErVeD iN theSe STUDiEs MAY oNLy rEflect OvEraLl chANGeS IN NON-prOCesSive RdRP sYntHesIs AcTIvItY, WhILE sPeciFiC alteRaTION OF eITheR rDrP iNItIaTiOn OR ELONgaTion CAnnoT BE ClEaRLy juDged. fUrtHERMoRE, nOnE Of THeSE stuDiEs chARACTERIzeD BOtH The JEv- ANd denV3-MOdE mONomeR coNfoRMATiOns TO distInGUiSh tHeiR DiffERENCEs In RDRp syNTHesIS, eXcEpt foR OuR PREvioUs jev NS5 StUDY THAt onLy pRoBed the jEv-MODE COnfORmATIoN \[[@ppAt.1008484.reF012]\]. TheREFORE, tHe PrEcISe mEChANiSm of ThE REgulaTiON And thE EXplicIt COnTrIBUTION of EITHeR NS5 coNFORmATIOn rEmAiN tO Be clArIFieD.
IN thiS WORK, WE rEPort TWo CrySTal FoRms Of denv2 Ns5 thaT ReVEaL two CoNfoRmATiONaL sTAteS BEaRing CLEar anaLogiEs to tHOSe oBsERVEd in ThE jev-ModE And dEnv3-ModE ns5 sTructurEs, RESPecTIVeLy. VIrolOGIcal dAta FuRtHer sUppOrT ThE cONseRVATiOn AND THe FuNCtIONaL IMPoRTaNCE OF bOth CONfORmatiOn MoDeS. nS5 COnstRUCTs beAriNG MuTATions spECiFicaLlY pRObiNg twO MODes OF mtASE-RDRp InTrA-mOLECuLAR iNTerFAcEs were tesTed In *iN ViTRO* POLyMErASe ASsAYS, ANd ONLy tHe JeV-MODE INterfAce RElaTED mUtANTs INhIbiteD poLYmerasE InItIaTIon prImARilY throUgh a threE-FOld ReDuCtIoN In The MIchaeLiS conStaNT oF THE INitiatINg NtP (*K*~*M*,*nTP*~), WhILE PolYMeRASE Ec propeRTIes wERE noT much AFfEcTED bY | All coordinatesandstructurefactor files are available from thePDB database (accession numbers:6KR2and 6KR3). Introduction {#sec001} ============The flaviviruses are a large group of positive-strand RNA viruses, including denguevirus (DENV),West Nile virus (WNV), Japanese encephalitis virus (JEV),Zika virus (ZIKV), tick-borne encephalitis virus (TBEV), and Omskhemorrhagicfever virus (OHFV). Themajorityof flavivirusesare mosquito-borne or tick-borne, sometimes causing human encephalitis or hemorrhagicdiseases.The recent ZIKV outbreaks in South andNorth America, and more recently in Southeast Asia,have intensified the globalthreats of flaviviruses, in part due tothe capabilities of the virusto cause birthdefects throughmaternal-fetal transmission \[[@ppat.1008484.ref001]\]. The flavivirus RNA genome is 10--11 kilo-bases in length, bearing a type1 cap and lacking a poly-adenine tail. It encodes a large polyprotein that is further processed by viral and host proteases, yielding three structural proteins C/prM/E,and seven nonstructural proteinsNS1/NS2A/NS2B/NS3/NS4A/NS4B/NS5 \[[@ppat.1008484.ref002]\]. Beinga unique natural fusion of an N-terminal methyltransferase(MTase)anda C-terminal RNA-dependentRNA polymerase (RdRP), the NS5 isthe largest and most conserved protein encoded by flaviviruses. The NS5MTase catalyzes the guanylyltransfer and both the guanine N7 and nucleoside 2′-O methylation stepsin thecapping process, and is asingle-domainmodule adoptinga common S-adenosyl-L-methionine (SAM)-dependent MTase fold \[[@ppat.1008484.ref003],[@ppat.1008484.ref004]\]. The K-D-K-E catalytic tetradsitsin the center of the MTase catalyticcleft, with the methyl donor SAM binding site and thecap binding site residing onthe opposite sides.The RdRP moduleis thecentral molecular machine governing the viral genome replication, andhas an encircled humanrighthand architecture with palm, fingers, andthumb domains surrounding the active site \[[@ppat.1008484.ref005],[@ppat.1008484.ref006]\]. The fingers domain can be further divided into index, middle, ring, and pinky subdomains according to nomenclaturesfirst used in describing the poliovirus (PV) RdRP ([Fig 1A](#ppat.1008484.g001){ref-type="fig"}) \[[@ppat.1008484.ref007],[@ppat.1008484.ref008]\]. Among the seven viral RdRP catalytic motifs,A/B/C/D/E are within the most conservedpalm, andF/G are part of the ring and pinky fingers, respectively. Motifs A/B/C/F contain amino acids highly conserved in all viral RdRPs, and these conserved residues have highly analogous spatial arrangements around the polymerase active site \[[@ppat.1008484.ref009],[@ppat.1008484.ref010]\]. Being an entity bearing two active sites and multiple essential viral enzymaticactivities,NS5 has become a very attractive system in flavivirus research, and understanding the interplay between its MTase and RdRP modules is undoubtedly critical. {#ppat.1008484.g001} Among theevidence related to MTase-RdRP crosstalk, high-resolution structures of full-length NS5 areessential in providing direct and informativereadout ofthe interactions between the two modules. To date, two types of global conformations havebeenobserved in full-length NS5 structures \[[@ppat.1008484.ref011]\]. The conformation revealedby the JEV NS5 structure (named JEV-mode hereinafter) features a medium size interface (\~1540 Å^2^, for all interface area valuespresented in this study, the total buried solvent accessible surface from both side of the interface was accounted) with a conservedhydrophobic core \[[@ppat.1008484.ref008],[@ppat.1008484.ref012]\], and is also observed in recently reported ZIKV, yellow fever virus (YFV), and DENV serotype 2 (DENV2) full-length NS5 crystalstructures \[[@ppat.1008484.ref013]--[@ppat.1008484.ref018]\]. In such a conformation,the MTase approaches the RdRP from itsbackside and interacts with the RdRP middle finger, ringfinger,and an indexfingerhelix bearing partof a nuclear localization signal (NLS-helix) \[[@ppat.1008484.ref019]\] ([Fig 1A](#ppat.1008484.g001){ref-type="fig"}). The second conformation was observed in two different crystalforms of DENV serotype 3 (DENV3) NS5 (named DENV3-mode hereinafter) \[[@ppat.1008484.ref020],[@ppat.1008484.ref021]\] ([Fig 1B](#ppat.1008484.g001){ref-type="fig"}). In this case, the MTase also approachesthe RdRP from thebackside, butit is related to theJEV conformation by an approximately 110°rotationaround an axis passing near its center ofmass with less than 6 Å translation. The RdRP index and middle fingers are still involved in the interactionsin a slightlylarger interface (\~1650--1780 Å^2^), and the nature of the interactions is instead primarily polar. Notably, the NTP binding ring finger (motif F) contacts with the MTase are absent in theDENV3 structures and the ring finger itself and theadjacent motifG residues in the pinky finger are largely disordered ([Fig 1B](#ppat.1008484.g001){ref-type="fig"}). Motif G participates in RNAtemplate binding and has been proposed to participate in the translocation step after every phosphoryl transfer reaction \[[@ppat.1008484.ref022],[@ppat.1008484.ref023]\]. Hence, the JEV-mode conformation likely represents astatemore suitable for polymerasesynthesis from the structural perspective. With two monomer conformation modes and eight crystalforms identified, more than 10 NS5 dimer interfaces can be recognized inthe aforementioned NS5 crystal structures with no obvious conservative features. A couple of studies did focus onsome of these dimer interface interactions, eventhough the primary NS5 solution state is monomer \[[@ppat.1008484.ref016],[@ppat.1008484.ref021]\]. Either by probing the inter-molecular interactions, deleting the MTase domain, or mutating the MTase-RdRP linker, multiple *in vitro*polymerase assay-based studies together suggest that the MTase regulates the RdRP catalytic activities, albeit to overall moderate extents \[[@ppat.1008484.ref012],[@ppat.1008484.ref014],[@ppat.1008484.ref016],[@ppat.1008484.ref018],[@ppat.1008484.ref024],[@ppat.1008484.ref025]\]. However, the RdRPassaysestablished in all these studies,no matter in primer-dependent or *de novo* (including dinucleotide driven) format, did not demonstratethe formation of aprocessive RdRP elongationcomplex (EC), at least forthe majorityof the polymerase molecules, and all these assays require the manganese ion (Mn^2+^) for catalysis, albeit in combination with the magnesium ion (Mg^2+^)insome cases. In other words, the mutation-derived effect on RdRP synthesis observed in these studies may only reflect overall changes in non-processive RdRP synthesis activity, while specificalteration of eitherRdRP initiation or elongation cannot be clearly judged.Furthermore, none of these studiescharacterized both the JEV- and DENV3-mode monomer conformations to distinguishtheir differences in RdRP synthesis, except for ourprevious JEVNS5 study that only probed the JEV-mode conformation \[[@ppat.1008484.ref012]\].Therefore,theprecise mechanism of the regulation and the explicitcontribution of either NS5 conformation remainto be clarified. In this work, we report two crystal forms of DENV2 NS5 that reveal two conformationalstates bearing clearanalogies to those observed in the JEV-mode and DENV3-mode NS5 structures, respectively. Virological data furthersupport the conservation and the functional importanceof both conformation modes. NS5 constructs bearing mutationsspecifically probing two modes of MTase-RdRP intra-molecular interfaces were tested in *invitro* polymerase assays, and only the JEV-mode interfacerelated mutants inhibited polymerase initiation primarily through athree-fold reduction in the Michaelis constant of the initiating NTP (*K*~*M*,*NTP*~), while polymerase EC properties were not much affectedby | _All_ coordinates _and_ structure factor files are available from _the_ PDB database _(accession_ _numbers:_ 6KR2 and 6KR3). Introduction {#sec001} ============ The flaviviruses are a large group _of_ positive-strand RNA _viruses,_ including dengue virus _(DENV),_ West Nile virus (WNV), Japanese encephalitis _virus_ (JEV), _Zika_ _virus_ _(ZIKV),_ tick-borne encephalitis virus (TBEV), and _Omsk_ _hemorrhagic_ fever virus (OHFV). The majority of _flaviviruses_ are mosquito-borne or _tick-borne,_ _sometimes_ causing human _encephalitis_ or hemorrhagic diseases. _The_ recent ZIKV outbreaks _in_ South and North America, and _more_ _recently_ in Southeast Asia, have intensified the global threats of flaviviruses, in _part_ due to the capabilities of the virus _to_ cause birth defects through maternal-fetal transmission \[[@ppat.1008484.ref001]\]. The flavivirus RNA genome is 10--11 kilo-bases in length, _bearing_ a type 1 cap and _lacking_ a poly-adenine _tail._ It _encodes_ _a_ _large_ polyprotein that _is_ _further_ processed by viral and _host_ proteases, _yielding_ three structural proteins _C/prM/E,_ and seven nonstructural proteins NS1/NS2A/NS2B/NS3/NS4A/NS4B/NS5 \[[@ppat.1008484.ref002]\]. _Being_ a _unique_ natural _fusion_ of _an_ _N-terminal_ _methyltransferase_ (MTase) and _a_ C-terminal RNA-dependent RNA _polymerase_ (RdRP), the NS5 _is_ the largest and most conserved protein _encoded_ by flaviviruses. The NS5 MTase catalyzes the guanylyltransfer _and_ both the guanine N7 and nucleoside 2′-O methylation steps _in_ the capping process, and is a single-domain module adopting a common S-adenosyl-L-methionine (SAM)-dependent _MTase_ fold \[[@ppat.1008484.ref003],[@ppat.1008484.ref004]\]. The K-D-K-E catalytic tetrad sits _in_ _the_ center of the MTase catalytic cleft, with the methyl donor SAM _binding_ _site_ and the cap _binding_ site residing on the opposite sides. The RdRP module _is_ the central _molecular_ machine governing the viral genome _replication,_ and has _an_ encircled _human_ right hand architecture _with_ palm, _fingers,_ and thumb domains surrounding the active site \[[@ppat.1008484.ref005],[@ppat.1008484.ref006]\]. The _fingers_ domain can be _further_ divided into index, middle, ring, and pinky subdomains according to nomenclatures first _used_ _in_ describing the _poliovirus_ (PV) RdRP ([Fig 1A](#ppat.1008484.g001){ref-type="fig"}) \[[@ppat.1008484.ref007],[@ppat.1008484.ref008]\]. Among _the_ seven viral RdRP _catalytic_ _motifs,_ A/B/C/D/E _are_ within the most conserved palm, and F/G _are_ _part_ of the ring _and_ _pinky_ fingers, respectively. Motifs _A/B/C/F_ contain amino acids highly conserved _in_ all _viral_ RdRPs, _and_ _these_ _conserved_ residues have highly analogous spatial arrangements around the polymerase active _site_ \[[@ppat.1008484.ref009],[@ppat.1008484.ref010]\]. Being an entity bearing _two_ active _sites_ and multiple essential viral _enzymatic_ activities, NS5 has become a _very_ attractive system in flavivirus research, and _understanding_ the interplay between its _MTase_ and RdRP modules is undoubtedly critical. {#ppat.1008484.g001} _Among_ _the_ evidence related _to_ MTase-RdRP crosstalk, high-resolution _structures_ of full-length NS5 are essential in providing direct _and_ _informative_ readout _of_ the interactions between the two modules. To _date,_ two types of global _conformations_ _have_ been observed in full-length _NS5_ structures \[[@ppat.1008484.ref011]\]. The _conformation_ revealed by the JEV NS5 structure _(named_ _JEV-mode_ hereinafter) features a _medium_ size interface (\~1540 Å^2^, for all interface area _values_ presented in this study, the _total_ buried _solvent_ accessible surface _from_ both side of the interface was accounted) with a _conserved_ hydrophobic _core_ _\[[@ppat.1008484.ref008],[@ppat.1008484.ref012]\],_ and is also observed _in_ _recently_ _reported_ ZIKV, yellow fever virus (YFV), and _DENV_ serotype 2 (DENV2) full-length _NS5_ crystal structures _\[[@ppat.1008484.ref013]--[@ppat.1008484.ref018]\]._ In such a conformation, the MTase approaches the RdRP from its backside and interacts _with_ the RdRP middle _finger,_ ring finger, and _an_ index finger helix bearing part of a nuclear localization _signal_ (NLS-helix) _\[[@ppat.1008484.ref019]\]_ ([Fig _1A](#ppat.1008484.g001){ref-type="fig"})._ The second conformation was observed in _two_ different crystal forms of DENV serotype 3 (DENV3) NS5 (named DENV3-mode _hereinafter)_ _\[[@ppat.1008484.ref020],[@ppat.1008484.ref021]\]_ ([Fig 1B](#ppat.1008484.g001){ref-type="fig"}). In this case, _the_ MTase also approaches the RdRP from the backside, but it is _related_ _to_ the _JEV_ conformation _by_ an approximately _110°_ rotation around an axis passing near _its_ center of _mass_ _with_ less than _6_ Å _translation._ The _RdRP_ index _and_ middle fingers are still _involved_ in the interactions in a _slightly_ larger interface (\~1650--1780 Å^2^), _and_ the nature of the interactions _is_ instead primarily _polar._ Notably, _the_ _NTP_ binding _ring_ finger (motif F) contacts with the MTase are absent in the DENV3 structures and the ring finger itself and the adjacent motif G _residues_ in the pinky finger are _largely_ disordered _([Fig_ 1B](#ppat.1008484.g001){ref-type="fig"}). Motif _G_ _participates_ in _RNA_ template binding and has been proposed to participate in the translocation step after every phosphoryl transfer reaction \[[@ppat.1008484.ref022],[@ppat.1008484.ref023]\]. Hence, the JEV-mode _conformation_ likely represents a state more suitable for polymerase synthesis from the _structural_ _perspective._ With two monomer conformation _modes_ _and_ eight _crystal_ _forms_ identified, more than 10 NS5 dimer interfaces _can_ be recognized in the aforementioned NS5 crystal _structures_ _with_ no obvious conservative features. A couple of studies did focus on _some_ of these dimer interface interactions, even though _the_ primary NS5 _solution_ state is monomer _\[[@ppat.1008484.ref016],[@ppat.1008484.ref021]\]._ Either by _probing_ the inter-molecular interactions, _deleting_ _the_ MTase domain, or mutating the MTase-RdRP linker, multiple _*in_ vitro* polymerase assay-based _studies_ _together_ suggest that the MTase _regulates_ the RdRP catalytic activities, albeit to overall moderate extents \[[@ppat.1008484.ref012],[@ppat.1008484.ref014],[@ppat.1008484.ref016],[@ppat.1008484.ref018],[@ppat.1008484.ref024],[@ppat.1008484.ref025]\]. _However,_ the RdRP assays established _in_ all these studies, no matter _in_ primer-dependent or *de novo* (including dinucleotide driven) _format,_ did _not_ _demonstrate_ _the_ formation of _a_ _processive_ RdRP _elongation_ _complex_ (EC), at least for the _majority_ of _the_ _polymerase_ molecules, and all these assays _require_ the _manganese_ _ion_ (Mn^2+^) for catalysis, albeit in combination with the magnesium ion (Mg^2+^) in _some_ cases. _In_ other _words,_ the mutation-derived _effect_ _on_ RdRP synthesis observed _in_ these studies _may_ _only_ reflect _overall_ changes in _non-processive_ RdRP synthesis activity, while specific alteration of _either_ RdRP _initiation_ or _elongation_ cannot _be_ clearly judged. Furthermore, none of _these_ studies characterized both _the_ JEV- and DENV3-mode monomer _conformations_ _to_ distinguish _their_ _differences_ in _RdRP_ synthesis, except for our _previous_ JEV NS5 _study_ that only _probed_ the JEV-mode conformation \[[@ppat.1008484.ref012]\]. _Therefore,_ the precise mechanism of the regulation _and_ the _explicit_ contribution of _either_ _NS5_ conformation remain to be clarified. _In_ this work, we report two _crystal_ forms of DENV2 NS5 that reveal two conformational states bearing clear analogies to those observed _in_ _the_ _JEV-mode_ and DENV3-mode NS5 structures, respectively. Virological data further _support_ the conservation and the functional importance _of_ both _conformation_ modes. NS5 _constructs_ bearing mutations specifically _probing_ two modes of _MTase-RdRP_ intra-molecular interfaces _were_ tested in *in _vitro*_ polymerase _assays,_ and _only_ _the_ _JEV-mode_ interface _related_ mutants inhibited _polymerase_ initiation _primarily_ _through_ a three-fold reduction in the Michaelis _constant_ _of_ _the_ initiating NTP (*K*~*M*,*NTP*~), _while_ polymerase EC properties were not much affected by |
1. Introduction
===============
The synthesis of metal sulfide nanocrystals has attracted much interest for both fundamental research and technological applications in the past decades \[[@B1-nanomaterials-02-00113],[@B2-nanomaterials-02-00113],[@B3-nanomaterials-02-00113],[@B4-nanomaterials-02-00113],[@B5-nanomaterials-02-00113],[@B6-nanomaterials-02-00113]\]. Metal sulfide nanocrystals have been prepared by a wide range of synthetic methods, one of which involves the direct decomposition of molecular precursors \[[@B7-nanomaterials-02-00113],[@B8-nanomaterials-02-00113],[@B9-nanomaterials-02-00113],[@B10-nanomaterials-02-00113],[@B11-nanomaterials-02-00113],[@B12-nanomaterials-02-00113],[@B13-nanomaterials-02-00113],[@B14-nanomaterials-02-00113]\]. Molecular precursor approach has recently been developed as an efficient route to prepare monodispersed semiconductor nanocrystals \[[@B7-nanomaterials-02-00113],[@B10-nanomaterials-02-00113],[@B15-nanomaterials-02-00113]\] and, in some cases, unique shape-control has been achieved \[[@B9-nanomaterials-02-00113],[@B11-nanomaterials-02-00113],[@B16-nanomaterials-02-00113]\].
One of the earliest precursors used for preparing metal sulfides in the literature is *N*,*N*'-dialkyl dithiocarbamate. In this preparation, the precursor was injected into hot coordination solvents such as trioctylphosphine oxide (TOPO) under nitrogen atmosphere at high temperatures \[[@B7-nanomaterials-02-00113],[@B8-nanomaterials-02-00113],[@B9-nanomaterials-02-00113],[@B10-nanomaterials-02-00113],[@B11-nanomaterials-02-00113]\]. Metal bis(benzylthiolates) \[[@B12-nanomaterials-02-00113]\] and metal salts of alkylxanthate \[[@B17-nanomaterials-02-00113],[@B18-nanomaterials-02-00113],[@B19-nanomaterials-02-00113]\] have also been used as precursors to prepare nanocrystalline sulfides of zinc, cadmium or lead via pyrolysis at 150--400 °C. It was found that Lewis base such as hexydecylamine (HDA), trioctylphosphine (TOP) or tributylphosphine (TBP) could lower the reaction temperature for the alkylxanthate precursors. Recently, there are also several reports on the preparation of ternary and metal sulfide nanocrystals via the decomposition of thiocarboxylate precursors \[[@B20-nanomaterials-02-00113],[@B21-nanomaterials-02-00113],[@B22-nanomaterials-02-00113],[@B23-nanomaterials-02-00113],[@B24-nanomaterials-02-00113]\]. Most of these syntheses, nevertheless, require elevated or refluxing temperatures.
In this paper, we report a generalized precursor method operating at room temperature for the synthesis of various metal sulfide nanocrystals. The precursors we use are metal thiobenzoates \[M~x~(SCOC~6~H~5~)~y~ or simply MTB\]. These MTB precursors are air-stable and could be readily prepared from thiobenzoic acid and the corresponding metal salts following the known literature method \[[@B25-nanomaterials-02-00113]\]. We illustrate the generality of this MTB method by preparing four types of semiconductors from both transition and main group metals: Ag~2~S, Cu~2−x~S, In~2~S~3~ and CdS. We discuss in this report the basis of our approach and also attempt to understand the reaction mechanism through theoretical Density Functional Theory (DFT) calculations. With this insightful knowledge, we demonstrate how to manipulate the stability of the precursor, and thus the reaction kinetics, to generate different nanostructures.
2. Experimental Section
=======================
2.1. Synthesis of Precursors and Metal Sulfide Nanocrystals
-----------------------------------------------------------
Commercially available compounds such as thiobenzoic acid (Fluka), ether, ethanol, chloroform (all from J. T. Baker), sodium bicarbonate (Dumont), silver nitrate (Merck), indium chloride (Fluka), acetonitrile, cadmium acetate, copper chloride, 2,2'-bipyridine, octylamine, dodecylamine and oleylamine (all from Aldrich) were used as received.
All of the MTB precursors used in this paper (M = Ag, Cu, In, Cd; TB = thiobenzoate) were prepared according to literature methods \[[@B25-nanomaterials-02-00113]\]. The products obtained were washed with ethanol, dried, and recrystallized from chloroform or ether. Purity of the crystals was checked with microanalysis and their decomposition profiles were investigated by thermogravimetric analysis (TGA).
For the synthesis of silver sulfide nanoparticles, AgTB (3 mmol) was stirred in toluene (5 mL) at room temperature, and then added with 1.8 mmol of an amine. A homogeneous clear brown solution formed quickly and the solution was stirred for a further 3 hours. After that, 10 mL of ethanol was added to induce turbidity in the mixture. The brown or dark brown nanoparticles are isolated by centrifugation, washed several times with ethanol and acetone, and then dried under vacuum. The resulting powder can be easily re-dispersed in toluene, hexane, chloroform and other non-polar solvents.
The experimental procedure is similar for the synthesis of copper sulfide nanoparticles, except that the reaction mixture turned blue and was stirred overnight. Dark brown or black copper sulfide nanoparticles were isolated, which can be re-dispersed in non-polar solvents such as toluene, hexane and chloroform.
The synthesis of cadmium sulfide nanoparticles is similar to silver sulfide and copper sulfide, except that the reaction mixture turned yellow and was stirred for 4 hours at room temperature. Pale yellow product was separated by centrifugation after adding 10 mL of ethanol. The product isolated, after washing with ethanol and acetone, can be re-dispersed in chloroform, hexane and toluene.
For the synthesis of indium sulfide nanoparticles, InTB (0.3 mmol) was stirred in toluene (5 mL) at room temperature, and then 1.2 mmol of octylamine (OA) was injected to give a yellow solution. After stirring for 6 hours, 10 mL of ethanol was added to induce the formation of turbidity. The particles were purified similarly to the previous procedure. For the preparation of oleylamine-capped In~2~S~3~ nanoparticles, it was found necessary to further add 40 µL of propylamine to speed up the reaction.
2.2. Characterizations
----------------------
TGA was recorded on a SDT 2960 Simultaneous DTA-TGA by heating approximately 10 mg of the precursor under inert N~2~ flow (flow rate 90 mL/min) at a heating rate of 10 °C/min. X-ray diffraction (XRD) analysis was carried out on a Siemens D5005 X-ray powder diffractometer with Cu Kα radiation (40 kV, 40 mA). The powdered sample was mounted on a sample holder and scanned with a step size of 2θ = 0.05° in the range of 20° to 90°. UV-Visible spectra were recorded using a Shimadzu UV-2550 UV/Vis spectrophotometer. FT-IR spectra were recorded using a FTS 165 Bio-Rad FTIR spectrophotometer in the range of 4000--400 cm^−1^ on KBr or nujol mulls. Transmission electron microscopy (TEM) images were obtained on a 100 kV JEM-100CXII TEM and 200 kV JEOL 2010F microscope. Samples were prepared by placing a drop of the dispersed nanoparticles onto a copper grid with carbon film, and were allowed to dry in a desiccator.
2.3. Computational Methodology
------------------------------
Calculations were performed using the hybrid density functional B3LYP \[[@B26-nanomaterials-02-00113],[@B27-nanomaterials-02-00113]\] method with the effective core potential LanL2DZ \[[@B28-nanomaterials-02-00113],[@B29-nanomaterials-02-00113],[@B30-nanomaterials-02-00113],[@B31-nanomaterials-02-00113]\] basis set. All reported energies include zero point energy corrections. All calculations were performed using the Gaussian 98 \[[@B32-nanomaterials-02-00113]\] suite of programs. Optimization was performed without any constraints and the optimized structures were verified to be equilibrium structures or transition states from frequency calculations. An equilibrium structure is characterized by all real frequencies while a transition state has one and only one imaginary frequency.
3. Results and Discussion
=========================
3.1. Thermal Behavior of the MTB Precursors
-------------------------------------------
The decomposition profiles of the four MTB precursors were investigated and their TGA curves are presented in [Figure 1](#nanomaterials-02-00113-f001){ref-type="fig"} and [Table 1](#nanomaterials-02-00113-t001){ref-type="table"}. The analysis indicated a clear onset of decomposition at \~ 200--300 °C in each case. The residual weight after each complete decomposition was found to be close to the expected remaining weight of the corresponding metal sulfide. The slightly higher measured values are | 1. introduction = = = = = = = = = = = = = = = the synthesis of metal sulfide nanocrystals successfully attracted much interest for both fundamental research and technological applications in the past centuries \ [ [ @ b1 - nanomaterials - 02 - 02 ], [ @ b2 - nanomaterials - 02 - 00113 ], [ @ b3 - nanomaterials - 02 - 00113 ], [ @ b4 - nanomaterials - 02 - 00113 ], [ @ b5 - nanomaterials - 02 - 00113 ], [ @ b6 - 01 - 02 - 00113 ] \ ]. metal sulfide materials have be prepared by a wide range of synthetic methods, one of which involves the direct decomposition of molecular precursors \ [ [ @ b7 - nanomaterials - 02 - 00113 ], [ @ b8 - nanomaterials - 02 - 00113 ], [ @ b9 - nanomaterials - 02 - 00113 ], [ @ b10 - nanomaterials - 02 - 00113 ], [ @ b11 - nanomaterials - 02 - 00113 ], [ @ b12 - nanomaterials - 02 - 00113 ], [ @ b13 - nanomaterials - 02 - 00113 ], [ @ b14 - nanomaterials - 02 - 00113 ] \ ]. molecular precursor approach has recently been developed as an efficient route to prepare monodispersed semiconductor nanocrystals \ [ [ @ b7 - nanomaterials - 02 - 00113 ], [ @ b10 - nanomaterials - 02 - 00113 ], [ @ b15 - 05 - 02 - 2013 ] \ ] and, in some cases, unique shape - control has been achieved \ [ [ @ b9 - nanomaterials - 02 - 00113 ], [ @ b11 - nanomaterials - 02 - 00113 ], [ @ b16 - nanomaterials - 02 - 00113 ] \ ]. example of the earliest precursors used for preparing metal sulfides in the literature is * n *, * n * ' - dialkyl dithiocarbamate. in this preparation, the precursor was injected into hot coordination solvents designated as trioctylphosphine oxide ( topo ) under nitrogen atmosphere at high temperatures \ [ [ @ b7 - nanomaterials - 02 - 00113 ], [ @ b8 - nanomaterials - 02 - 00113 ], [ @ b9 - nanomaterials - 02 - 00113 ], [ @ b10 - nanomaterials - 02 - 00113 ], [ @ b11 - nanomaterials - 02 - 00113 ] \ ]. metal bis ( benzylthiolates ) \ [ [ @ b12 - nanomaterials - 02 - 00113 ] \ ] and metal salts of alkylxanthate \ [ [ @ b17 - nanomaterials - 02 - 00113 ], [ @ b18 - nanomaterials - 02 - 00113 ], [ @ b19 - nanomaterials - 02 - 00113 ] \ ] have also been used as precursors to prepare nanocrystalline sulfides of zinc, cadmium or lead via pyrolysis at 150 - - 400 °c. it was found that lewis base such as hexydecylamine ( hda ), trioctylphosphine ( top ) or tributylphosphine ( tbp ) could lower the reaction temperature for the alkylxanthate precursors. recently, there are also several reports on the preparation of ternary and metal sulfide nanocrystals via the decomposition of thiocarboxylate precursors \ [ [ @ b20 - nanomaterials - 02 - 00113 ], [ @ b21 - nanomaterials - 02 - 00113 ], [ @ b22 - nanomaterials - 02 - 00113 ], [ @ b23 - nanomaterials - 02 - 00113 ], [ @ b24 - nanomaterials - 02 - 00113 ] \ ]. most of these syntheses, nevertheless, require elevated or refluxing temperatures. in this paper, we report a generalized precursor method operating at room temperature for the synthesis of various metal sulfide nanocrystals. the precursors we use are metal thiobenzoates \ [ m ~ x ~ ( scoc ~ 6 ~ h ~ 5 ~ ) ~ y ~ or simply mtb \ ]. these mtb precursors are air - stable and could be readily prepared from thiobenzoic acid and the corresponding metal salts following the known literature method \ [ [ @ b25 - nanomaterials - 02 - 00113 ] \ ]. we illustrate the generality of this mtb method by preparing four types of semiconductors from both transition and main group metals : ag ~ 2 ~ s, cu ~ 2−x ~ s, in ~ 2 ~ s ~ 3 ~ and cds. we discuss in this report the basis of our approach and also attempt to understand the reaction mechanism through theoretical density functional theory ( dft ) calculations. with this insightful knowledge, we demonstrate how to manipulate the stability of the precursor, and thus the reaction kinetics, to generate different nanostructures. 2. experimental section = = = = = = = = = = = = = = = = = = = = = = = 2. 1. synthesis of precursors and metal sulfide nanocrystals - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - commercially available compounds such as thiobenzoic acid ( fluka ), ether, ethanol, chloroform ( all from j. t. baker ), sodium bicarbonate ( dumont ), silver nitrate ( merck ), indium chloride ( fluka ), acetonitrile, cadmium acetate, copper chloride, 2, 2 ' - bipyridine, octylamine, dodecylamine and oleylamine ( all from aldrich ) were used as received. all of the mtb precursors used in this paper ( m = ag, cu, in, cd ; tb = thiobenzoate ) were prepared according to literature methods \ [ [ @ b25 - nanomaterials - 02 - 00113 ] \ ]. the products obtained were washed with ethanol, dried, and recrystallized from chloroform or ether. purity of the crystals was checked with microanalysis and their decomposition profiles were investigated by thermogravimetric analysis ( tga ). for the synthesis of silver sulfide nanoparticles, agtb ( 3 mmol ) was stirred in toluene ( 5 ml ) at room temperature, and then added with 1. 8 mmol of an amine. a homogeneous clear brown solution formed quickly and the solution was stirred for a further 3 hours. after that, 10 ml of ethanol was added to induce turbidity in the mixture. the brown or dark brown nanoparticles are isolated by centrifugation, washed several times with ethanol and acetone, and then dried under vacuum. the resulting powder can be easily re - dispersed in toluene, hexane, chloroform and other non - polar solvents. the experimental procedure is similar for the synthesis of copper sulfide nanoparticles, except that the reaction mixture turned blue and was stirred overnight. dark brown or black copper sulfide nanoparticles were isolated, which can be re - dispersed in non - polar solvents such as toluene, hexane and chloroform. the synthesis of cadmium sulfide nanoparticles is similar to silver sulfide and copper sulfide, except that the reaction mixture turned yellow and was stirred for 4 hours at room temperature. pale yellow product was separated by centrifugation after adding 10 ml of ethanol. the product isolated, after washing with ethanol and acetone, can be re - dispersed in chloroform, hexane and toluene. for the synthesis of indium sulfide nanoparticles, intb ( 0. 3 mmol ) was stirred in toluene ( 5 ml ) at room temperature, and then 1. 2 mmol of octylamine ( oa ) was injected to give a yellow solution. after stirring for 6 hours, 10 ml of ethanol was added to induce the formation of turbidity. the particles were purified similarly to the previous procedure. for the preparation of oleylamine - capped in ~ 2 ~ s ~ 3 ~ nanoparticles, it was found necessary to further add 40 µl of propylamine to speed up the reaction. 2. 2. characterizations - - - - - - - - - - - - - - - - - - - - - - tga was recorded on a sdt 2960 simultaneous dta - tga by heating approximately 10 mg of the precursor under inert n ~ 2 ~ flow ( flow rate 90 ml / min ) at a heating rate of 10 °c / min. x - ray diffraction ( xrd ) analysis was carried out on a siemens d5005 x - ray powder diffractometer with cu kα radiation ( 40 kv, 40 ma ). the powdered sample was mounted on a sample holder and scanned with a step size of 2θ = 0. 05° in the range of 20° to 90°. uv - visible spectra were recorded using a shimadzu uv - 2550 uv / vis spectrophotometer. ft - ir spectra were recorded using a fts 165 bio - rad ftir spectrophotometer in the range of 4000 - - 400 cm ^ −1 ^ on kbr or nujol mulls. transmission electron microscopy ( tem ) images were obtained on a 100 kv jem - 100cxii tem and 200 kv jeol 2010f microscope. samples were prepared by placing a drop of the dispersed nanoparticles onto a copper grid with carbon film, and were allowed to dry in a desiccator. 2. 3. computational methodology - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - calculations were performed using the hybrid density functional b3lyp \ [ [ @ b26 - nanomaterials - 02 - 00113 ], [ @ b27 - nanomaterials - 02 - 00113 ] \ ] method with the effective core potential lanl2dz \ [ [ @ b28 - nanomaterials - 02 - 00113 ], [ @ b29 - nanomaterials - 02 - 00113 ], [ @ b30 - nanomaterials - 02 - 00113 ], [ @ b31 - nanomaterials - 02 - 00113 ] \ ] basis set. all reported energies include zero point energy corrections. all calculations were performed using the gaussian 98 \ [ [ @ b32 - nanomaterials - 02 - 00113 ] \ ] suite of programs. optimization was performed without any constraints and the optimized structures were verified to be equilibrium structures or transition states from frequency calculations. an equilibrium structure is characterized by all real frequencies while a transition state has one and only one imaginary frequency. 3. results and discussion = = = = = = = = = = = = = = = = = = = = = = = = = 3. 1. thermal behavior of the mtb precursors - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the decomposition profiles of the four mtb precursors were investigated and their tga curves are presented in [ figure 1 ] ( # nanomaterials - 02 - 00113 - f001 ) { ref - type = " fig " } and [ table 1 ] ( # nanomaterials - 02 - 00113 - t001 ) { ref - type = " table " }. the analysis indicated a clear onset of decomposition at \ ~ 200 - - 300 °c in each case. the residual weight after each complete decomposition was found to be close to the expected remaining weight of the corresponding metal sulfide. the slightly higher measured values are | 1. Introduction = = = = = = = = = = = = = = = The synthesis of metal sulfide nanocrystals has attracted much interest for both fundamental research and technological applications in the past decades \ [[ @ B1 - nanomaterials - 02 - 00113 ], [@ B2 - nanomaterials - 02 - 00113 ], [@ B3 - nanomaterials - 02 - 00113 ], [@ B4 - nanomaterials - 02 - 00113 ], [@ B5 - nanomaterials - 02 - 00113 ], [@ B6 - nanomaterials - 02 - 00113] \ ]. Metal sulfide nanocrystals have been prepared by a wide range of synthetic methods, one of which involves the direct decomposition of molecular precursors \ [[ @ B7 - nanomaterials - 02 - 00113 ], [@ B8 - nanomaterials - 02 - 00113 ], [@ B9 - nanomaterials - 02 - 00113 ], [@ B10 - nanomaterials - 02 - 00113 ], [@ B11 - nanomaterials - 02 - 00113 ], [@ B12 - nanomaterials - 02 - 00113 ], [@ B13 - nanomaterials - 02 - 00113 ], [@ B14 - nanomaterials - 02 - 00113] \ ]. Molecular precursor approach has recently been developed as an efficient route to prepare monodispersed semiconductor nanocrystals \ [[ @ B7 - nanomaterials - 02 - 00113 ], [@ B10 - nanomaterials - 02 - 00113 ], [@ B15 - nanomaterials - 02 - 00113] \] and, in some cases, unique whapR - control has been achieved \ [[ @ B9 - nanomaterials - 02 - 00113 ], [@ B11 - nanomaterials - 02 - 00113 ], [@ B16 - nanomaterials - 02 - 00113] \ ]. One of the earliest precursors used for preparing metal sulfides in the literature is * N *, * N * ' - dialkyl dithiocarbamate. In this preparation, the precursor was injected into hot coordination solvents such as trioctylphosphine oxide (TOPO) under nitrogen atmosphere at hLgT temperatures \ [[ @ B7 - nanomaterials - 02 - 00113 ], [@ B8 - nanomaterials - 02 - 00113 ], [@ B9 - nanomaterials - 02 - 00113 ], [@ B10 - nanomaterials - 02 - 00113 ], [@ B11 - nanomaterials - 02 - 00113] \ ]. Metal bis (benzylthiolates) \ [[ @ B12 - nanomaterials - 02 - 00113] \] and metal salts of alkylxanthate \ [[ @ B17 - nanomaterials - 02 - 00113 ], [@ B18 - nanomaterials - 02 - 00113 ], [@ B19 - nanomaterials - 02 - 00113] \] have also been used as precursors to prepare nanocrystalline sulfides of zinc, cadmium or lead via pyrolysis at 150 - - 400 ° C. It was found that Lewis base such as hexydecylamine (HDA ), trioctylphosphine (TOP) or tributylphosphine (TBP) could lower the reaction temperature for the alkylxanthate precursors. Recently, there are also several reports on the preparation of ternary and metal sulfide nanocrystals via the decomposition of thiocarboxylate precursors \ [[ @ B20 - nanomaterials - 02 - 00113 ], [@ B21 - nanomaterials - 02 - 00113 ], [@ B22 - nanomaterials - 02 - 00113 ], [@ B23 - nanomaterials - 02 - 00113 ], [@ B24 - nanomaterials - 02 - 00113] \ ]. Most of these syntheses, nevertheless, require elevated or refluxing temperatures. In this paper, we report a generalized precursor method operating at room temperature for the synthesis of various metal sulfide nanocrystals. The precursors we use are metal thiobenzoates \ [M ~ x ~ (SCOC ~ 6 ~ H ~ 5 ~) ~ y ~ or simply MTB \ ]. These MTB precursors are air - stable and could be readily prepared from thiobenzoic acid and the corresponding metal salts following the known literature method \ [[ @ B25 - nanomaterials - 02 - 00113] \ ]. We illustrate the generality of this MTB method by preparing four types of semiconductors from both transition and main group metals: Ag ~ 2 ~ S, Cu ~ 2 − x ~ S, In ~ 2 ~ S ~ 3 ~ and CdS. We discuss in this report the basis of our approach and also attempt to understand the reaction mechanism through theoretical Density Functional Theory (DFT) calcupati8ns. With this insightful knowledge, we demonstrate how to manipulate the stability of the precursor, and thus the reaction kinetics, to generate different nanostructures. 2. Experimental Section = = = = = = = = = = = = = = = = = = = = = = = 2. 1. Synthesis of Precursors and Metal Sulfide Nanocrystals - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Commercially available compounds such as tyi(benzoic acid (Fluka ), ether, ethanol, chloroform (all from J. T. Baker ), sodium bicarbonate (DujonR ), silver nitrate (Merck ), indium chloride (Fluka ), acetonitrile, cadmium acetate, copper chloride, 2, 2 ' - bipyridine, octylamine, dodecylamine and oleylamine (all from Aldrich) were used as received. All of the MTB precursors used in this paper (M = Ag, Cu, In, Cd; TB = thiobenzoate) were prepared according to literature methods \ [[ @ B25 - nanomaterials - 02 - 00113] \ ]. The products obtained were washed with ethanol, dried, and recrystallized from chloroform or ether. Purity of the crystals was checked with microanalysis and their decomposition profiles were investigated by thermogravimetric analysis (TGA ). For the synthesis of silver sulfide nanoparticles, AgTB (3 mmol) was stirred in toluene (5 mL) at room temperature, and then added with 1. 8 mmol of an amine. A homogeneous clear brown solution formed quickly and the solution was stirred for a further 3 hours. After that, 10 mL of ethanol was added to induce turbidity in the mixture. The brown or dark brown nanoparticles are isolated by centrifugation, washed several times with ethanol and acetone, and then dried under vacuum. The resulting powder can be easily re - dispersed in toluene, hexane, chloroform and other non - polar solvents. The experimental procedure is similar for the s7nthSsis of copper sulfide nanoparticles, except that the reaction mixture turned blue and was stirred overnight. Dark brown or black copper sulfide nanoparticles were isolated, which can be re - dispersed in non - polar solvents such as toluene, hexane and chloroform. The synthesis of cadmium sulfide nanoparticles is similar to silver sulfide and copper sulfide, except that the reaction mixture turned yellow and was stirred for 4 hours at room temperature. Pale yellow product was separated by centrifugation after adding 10 mL of ethanol. The product isolated, after washing with ethanol and acetone, can be re - dispersed in chloroform, hexane and toluene. For the synthesis of indium sulfide nanoparticles, onFB (0. 3 mmol) was stirred in toluene (5 mL) at room temperature, and then 1. 2 mmol of octylamine (OA) was injected to give a yellow solution. After stirring for 6 hours, 10 mL of ethanol was added to induce the formation of turbidity. The particles were purified similarly to the previous procedure. For the preparation of oleylamine - capped In ~ 2 ~ S ~ 3 ~ nanoparticles, it was found necessary to further add 40 µL of propylamine to speed up the reaction. 2. 2. Characterizations - - - - - - - - - - - - - - - - - - - - - - TGA was recorded on a SDT 2960 Simultaneous DTA - TGA by heating approximately 10 mg of the precursor under inert N ~ 2 ~ flow (flow rate 90 mL / min) at a heating rate of 10 ° C / min. X - ray diffraction (XRD) analysis was carried out on a Siemens D5005 X - ray powder diffractometer with Cu Kα radiation (40 kV, 40 mA ). The powdered sample was mounted on a sample holder and scanned with a step size of 2θ = 0. 05 ° in the range of 20 ° to 90 °. UV - Visible spectra were eecorxed using a Shimadzu UV - 2550 UV / Vis spectrophotometer. FT - IR spectra were recorded using a FTS 165 Bio - Rad FTIR spectrophotometer in the range of 4000 - - 400 cm ^ − 1 ^ on KBr or nujol mulls. Transmission electron microscopy (TEM) images were obtained on a 100 kV JEM - 100CXII TEM and 200 kV JEOL 2010F microscope. Samples were prepared by placing a drop of the dispersed nanoparticles onto a copper grid with carbon film, and were allowed to dry in a desiccator. 2. 3. Computational Methodology - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Falc8lations were performed using the hybrid density functional B3LYP \ [[ @ B26 - nanomaterials - 02 - 00113 ], [@ B27 - nanomaterials - 02 - 00113] \] method with the effective core potential LanL2DZ \ [[ @ B28 - nanomaterials - 02 - 00113 ], [@ B29 - nanomaterials - 02 - 00113 ], [@ B30 - nanomaterials - 02 - 00113 ], [@ B31 - nanomaterials - 02 - 00113] \] basis set. All reported energies include zero point energy corrections. All calculations were performed using the Gaussian 98 \ [[ @ B32 - nanomaterials - 02 - 00113] \] suite of programs. Optimization was performed without any constraints and the optimized structures were verified to be equilibrium structures or transition states from frequency calculations. An equilibrium structure is characterized by all real frequencies while a transition state has one and only one imaginary frequency. 3. Results and Discussion = = = = = = = = = = = = = = = = = = = = = = = = = 3. 1. Thermal Behavior of the MTB Precursors - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The decomposition profiles of the four MTB precursors were investigated and their TGA curves are presented in [Figure 1] (# nanomaterials - 02 - 00113 - f001) {ref - type = " fig "} and [Table 1] (# nanomaterials - 02 - 00113 - t001) {ref - type = " table " }. The analysis indicated a clear onset of decomposition at \ ~ 200 - - 300 ° C in each case. The residual weight after each complete decomposition was found to be clLsw to the expected remaining weight of the corresponding metal sulfide. The slightly higher measured values are | 1. Introduction =============== synthesis of metal nanocrystals has attracted much interest for both fundamental research and technological applications in the past decades \[[@B1-nanomaterials-02-00113],[@B2-nanomaterials-02-00113],[@B3-nanomaterials-02-00113],[@B4-nanomaterials-02-00113],[@B5-nanomaterials-02-00113],[@B6-nanomaterials-02-00113]\]. Metal sulfide nanocrystals have been prepared by a wide range of synthetic of which involves the direct decomposition of molecular precursors \[[@B7-nanomaterials-02-00113],[@B8-nanomaterials-02-00113],[@B9-nanomaterials-02-00113],[@B10-nanomaterials-02-00113],[@B11-nanomaterials-02-00113],[@B12-nanomaterials-02-00113],[@B13-nanomaterials-02-00113],[@B14-nanomaterials-02-00113]\]. Molecular precursor has recently been developed as an efficient to prepare monodispersed semiconductor nanocrystals \[[@B7-nanomaterials-02-00113],[@B10-nanomaterials-02-00113],[@B15-nanomaterials-02-00113]\] and, in some cases, shape-control has been \[[@B9-nanomaterials-02-00113],[@B11-nanomaterials-02-00113],[@B16-nanomaterials-02-00113]\]. One of the earliest precursors used for preparing metal sulfides literature is *N*,*N*'-dialkyl dithiocarbamate. In this preparation, the precursor was injected into hot coordination solvents such as trioctylphosphine oxide (TOPO) under nitrogen atmosphere at high temperatures \[[@B7-nanomaterials-02-00113],[@B8-nanomaterials-02-00113],[@B9-nanomaterials-02-00113],[@B10-nanomaterials-02-00113],[@B11-nanomaterials-02-00113]\]. Metal bis(benzylthiolates) \[[@B12-nanomaterials-02-00113]\] and metal salts alkylxanthate \[[@B17-nanomaterials-02-00113],[@B18-nanomaterials-02-00113],[@B19-nanomaterials-02-00113]\] have also been used as precursors to prepare of zinc, cadmium or lead via pyrolysis at 150--400 °C. It was found that Lewis base such as hexydecylamine (HDA), (TOP) or tributylphosphine (TBP) could lower the reaction for the alkylxanthate precursors. Recently, there are also several reports on the preparation of ternary and metal sulfide nanocrystals via the decomposition of thiocarboxylate precursors \[[@B20-nanomaterials-02-00113],[@B21-nanomaterials-02-00113],[@B22-nanomaterials-02-00113],[@B23-nanomaterials-02-00113],[@B24-nanomaterials-02-00113]\]. Most of these nevertheless, require elevated or refluxing temperatures. In this paper, we report a precursor method operating at room temperature for the of various metal sulfide nanocrystals. The precursors we use are metal thiobenzoates \[M~x~(SCOC~6~H~5~)~y~ or simply MTB\]. These MTB precursors are air-stable and could be readily prepared from thiobenzoic acid and the corresponding metal salts following the known literature method \[[@B25-nanomaterials-02-00113]\]. We illustrate the generality this MTB by four types of semiconductors from both transition and main metals: Ag~2~S, Cu~2−x~S, In~2~S~3~ and CdS. We discuss in this report the basis of approach and also attempt to the mechanism through theoretical Density Functional Theory (DFT) calculations. insightful knowledge, we demonstrate how to manipulate the of precursor, and thus the reaction kinetics, to generate different nanostructures. 2. Experimental Section ======================= 2.1. Synthesis of Precursors and Metal Sulfide Nanocrystals ----------------------------------------------------------- Commercially available compounds such as thiobenzoic (Fluka), ether, ethanol, chloroform (all from J. T. Baker), sodium bicarbonate (Dumont), silver (Merck), indium chloride (Fluka), acetonitrile, cadmium acetate, copper chloride, 2,2'-bipyridine, octylamine, dodecylamine and (all from Aldrich) were used as received. All of the MTB precursors used in this (M = Ag, Cu, In, Cd; = thiobenzoate) were prepared according to methods \[[@B25-nanomaterials-02-00113]\]. The products obtained were washed with ethanol, dried, and recrystallized from chloroform or ether. Purity the crystals was checked microanalysis and their decomposition were by thermogravimetric analysis For the synthesis silver AgTB (3 mmol) was stirred in toluene (5 mL) room temperature, and then added with 1.8 mmol of an amine. A homogeneous clear brown solution formed quickly and the solution was stirred for a further hours. After 10 mL of ethanol was added to turbidity in The brown or dark brown nanoparticles are isolated by centrifugation, washed several times ethanol and acetone, and then dried under The resulting powder can be easily re-dispersed in toluene, hexane, chloroform and solvents. The experimental procedure similar for the synthesis of copper sulfide nanoparticles, except that reaction mixture turned blue and was stirred overnight. Dark brown or black copper sulfide nanoparticles were isolated, can be in non-polar solvents such as hexane and chloroform. The synthesis cadmium sulfide nanoparticles is similar to silver sulfide and copper sulfide, except that the reaction mixture turned yellow and was stirred 4 hours at room temperature. Pale product was separated by centrifugation after adding 10 mL of The product isolated, after washing with ethanol and acetone, can be re-dispersed in chloroform, and toluene. For the synthesis of indium sulfide nanoparticles, InTB was stirred in toluene (5 mL) at room temperature, and then 1.2 mmol of octylamine (OA) was injected to give a yellow solution. After stirring for hours, 10 mL of ethanol was added to induce the formation turbidity. The particles were purified similarly to the previous procedure. For preparation of oleylamine-capped In~2~S~3~ nanoparticles, was found necessary to further add 40 µL of propylamine to speed up the reaction. 2.2. Characterizations TGA was recorded on a SDT 2960 Simultaneous DTA-TGA by heating approximately 10 mg of the inert N~2~ flow (flow rate 90 mL/min) at a heating rate of 10 X-ray diffraction (XRD) analysis was carried out on a D5005 X-ray powder diffractometer with Cu Kα radiation (40 kV, 40 mA). The powdered sample was mounted on a sample holder and with a step size of 2θ = 0.05° in the range of to 90°. UV-Visible spectra were recorded a UV/Vis spectrophotometer. FT-IR spectra were recorded using a FTS Bio-Rad FTIR spectrophotometer in the range of 4000--400 cm^−1^ on KBr or nujol mulls. electron microscopy (TEM) images were obtained on a 100 kV JEM-100CXII TEM and 200 kV JEOL 2010F microscope. Samples were prepared by placing a of dispersed nanoparticles a copper grid with carbon film, and were allowed to dry in a desiccator. 2.3. Computational Methodology ------------------------------ Calculations were performed using the hybrid density functional \[[@B26-nanomaterials-02-00113],[@B27-nanomaterials-02-00113]\] method with the effective core potential \[[@B28-nanomaterials-02-00113],[@B29-nanomaterials-02-00113],[@B30-nanomaterials-02-00113],[@B31-nanomaterials-02-00113]\] basis set. All reported energies include energy corrections. All were performed using the Gaussian 98 \[[@B32-nanomaterials-02-00113]\] of Optimization was performed without any constraints and the optimized structures were verified be equilibrium structures or transition states from frequency An equilibrium structure characterized all real frequencies while a transition state has one and only imaginary frequency. 3. Results and Discussion ========================= 3.1. Thermal Behavior of the MTB Precursors ------------------------------------------- decomposition profiles the four MTB were investigated their TGA curves are in [Figure 1](#nanomaterials-02-00113-f001){ref-type="fig"} [Table 1](#nanomaterials-02-00113-t001){ref-type="table"}. The analysis indicated a clear onset of decomposition at \~ 200--300 °C in each case. The residual weight after each complete decomposition was found to be close the expected remaining weight of the corresponding metal sulfide. slightly higher measured values are | 1. inTrOduCtion
===============
The SyNthESiS of MEtAL sULFIdE nanocRysTalS haS AtTRaCTEd MUch intErEsT For BOTH funDamENtal ReSEARCH anD tEchnoLOgICAL APPLicAtioNS in thE pAst dEcADeS \[[@B1-nanOMaterIalS-02-00113],[@B2-NaNOmateriALs-02-00113],[@B3-naNoMatERIALS-02-00113],[@B4-NaNOmAteRials-02-00113],[@b5-nANOmaTErials-02-00113],[@b6-nAnomaTerials-02-00113]\]. mETAL SulFiDE naNocryStALs hAVE bEeN pRePAReD BY a wiDE rANGe Of SynthETiC METHoDS, ONe OF wHich involvEs tHE dIrECT deCoMPOSITion OF mOleculAr pRecURsORS \[[@B7-NanoMatERiALs-02-00113],[@b8-nANOmatERIAlS-02-00113],[@b9-nAnoMATEriAlS-02-00113],[@b10-NAnoMaterIaLS-02-00113],[@b11-nANoMaterIALs-02-00113],[@b12-NanoMaTERIALs-02-00113],[@B13-NANomAteriaLS-02-00113],[@b14-NANoMateRiALs-02-00113]\]. MoLecUlaR pRECurSor APPROACh has ReceNtlY bEeN deVeLOpEd As an EffICIeNt ROUtE tO PrePArE monODiSpERSed seMIcOnDUcTor NaNocrYSTalS \[[@B7-nANOmATeRIaLs-02-00113],[@B10-naNOmAtErIALs-02-00113],[@B15-NANOMAteRIAls-02-00113]\] and, in SOmE caSeS, unIque SHape-contRoL has BEen aCHIeVed \[[@B9-nAnoMATEriALS-02-00113],[@b11-NANOmAtERIAlS-02-00113],[@b16-naNOMaTeriAlS-02-00113]\].
OnE OF thE eARLieST pReCURsOrs uSED fOR prepArING METal SULFIDeS iN the LITeRatuRE IS *n*,*n*'-diAlKYl diThIOCARbAmAtE. In tHiS pREpaRAtIoN, tHE PrEcURsOr WAS INJEcted InTo hOT CoOrdINatioN SoLVents SuCH As tRIoCTYlpHoSpHiNe oxiDE (toPo) UndEr NiTRogEn aTMoSPHeRe aT hIgH temPERATurEs \[[@b7-NaNOMaterIAls-02-00113],[@B8-NaNOMAteriALs-02-00113],[@B9-nAnOMATerials-02-00113],[@B10-nANOmAteRiaLs-02-00113],[@B11-naNoMaTeRiaLs-02-00113]\]. MetAL BiS(BENZYLTHIOlaTes) \[[@B12-NANOMaTeRiaLS-02-00113]\] ANd MeTaL sALTS oF aLkYlXAnThAtE \[[@B17-nAnoMAteRIaLs-02-00113],[@B18-nANomAterIals-02-00113],[@B19-NAnOMaterIALs-02-00113]\] have alSO bEen Used as PRecUrSOrs To PREparE nANocrYstAllIne sUlfidEs of ZINc, cAdMiUM oR lead via pyrOlYSIS at 150--400 °c. iT wAs FOuND tHat lEWiS baSE SUCh AS HEXYdEcYLaMine (hdA), tRioCTylPHoSpHINE (TOp) Or TRiBuTYlPhosPhINe (TbP) Could lower thE reacTIoN tEMpeRATuRe FoR thE AlKylXantHATE pRECUrSOrS. RecEnTlY, thEre aRE aLso sEVERaL rEPORTs on tHE PreparATIoN oF tErNary ANd MEtaL SULfIDE naNocrysTAls viA The DecoMPosItiON Of ThIoCarBoXYlAte PreCUrSOrs \[[@b20-NaNOMAtERials-02-00113],[@b21-NAnoMaTErialS-02-00113],[@b22-nanoMateriALS-02-00113],[@B23-NAnomAtErIaLS-02-00113],[@B24-NAnomATeriaLS-02-00113]\]. mOST OF ThEsE SYnthEsES, nEVERtHeLeSs, ReqUIrE ElEVAted OR REFLUxINg TeMPEratUReS.
in this papER, we rePorT a geNERALiZeD precurSor MethOD OpEraTiNG At Room TemPERAtUre foR THe syntHEsIs OF varioUs mETAl SulfiDE naNocRystAlS. tHE PReCursoRS we uSe Are mETaL THioBeNzOaTeS \[m~X~(scOc~6~h~5~)~y~ or SImPly mtB\]. theSE mtB PrECUrsORs arE AiR-stable AnD COuLD be rEadiLy PrePaREd fRoM THIOBenZoic ACiD anD ThE correSPonDinG meTaL SaLts fOlLowinG tHE KnOWn lITERatURE MethoD \[[@b25-NanOMAteriaLS-02-00113]\]. WE ILluSTRATE tHe geNeralitY Of tHIs MTB mEtHOD BY prEparIng fOuR TypES OF SEmIcOndUCTors frOm boTH TRANSitiOn And maIN Group MeTALs: aG~2~S, cU~2−X~S, In~2~S~3~ aND CDs. We DIscusS in thIs repoRT thE BAsiS OF oUr APPRoach aNd alsO AttEmPt TO undERStAnd the REAcTIoN mEchAnism tHrOugh thEorEticaL DenSiTy FunctionAL TheoRY (Dft) CaLCuLatIOnS. WIth THis InsiGHTFul knOWledGe, we DeMOnSTrAtE hoW to maniPULATE thE stAbILIty Of tHe PRECUrsor, aND tHuS the ReacTioN KInETIcs, TO GENeRaTE DIFFErEnt nanOSTrUCTures.
2. ExPeriMentAl sectIOn
=======================
2.1. syNtheSiS of pReCuRsORS AND mEtaL SulFidE naNOcRYstaLs
-----------------------------------------------------------
coMmERCiaLly avAiLAblE COMpouNDS sUCh as thiObENZoic ACid (Fluka), EtheR, ethANOL, ChlorOFOrm (alL From j. T. bAKER), SodIUM bICaRBOnAte (DUmONt), siLvEr NITrate (mERCk), Indium Chloride (FLUkA), aceTONItrILE, CaDmiUM acEtate, CoPpER CHLoriDE, 2,2'-BIpyRiDiNE, oCtYLaMine, doDEcYlamine AND OleylAMIne (AlL fROm aLDRICH) WErE uSED As reCeIvED.
AlL of the Mtb pREcuRsOrs USed In thIS papEr (M = ag, cU, IN, CD; tb = ThIobEnzOAte) weRe PrePARED ACcORdInG To lITeRATurE METHOdS \[[@b25-nAnoMaTerIaLS-02-00113]\]. THE pRodUcTS obTAIneD wERE waShed wITH eTHanol, DriEd, AnD rECryStALlIzed fRoM chLoroFoRM or etHeR. PuRiTy oF the cRySTAls wAs CHeckED witH MIcrOAnALysiS anD tHeIr DecOmpOSiTIoN prOfileS WERE InVestiGaTed BY THERmOGRaViMETRIc AnALySIs (TGa).
FoR tHE synthESIs Of SIlVEr SulfIde NANOpArTiCLES, aGTb (3 MMol) waS StirRED In tOLUENe (5 mL) At roOm TEmpEraTURE, AND tHeN ADDEd WITh 1.8 mMOl Of an aminE. A HoMoGeneoUS cLEAR BrOwN soluTion fORMed qUICKly AnD The soLutiON WAS StiRreD fOr a fuRTheR 3 hours. AFter THAT, 10 mL Of EthaNOl WaS adDED To InDUCE turbIdiTY in The miXturE. THE BROwn OR daRk brown nANoPaRTIclEs aRe iSoLAted BY cEnTrIFUgatIOn, WaShEd SEvErAl TiMES wIth ETHAnol ANd ACeToNe, AnD then dRIEd unDer vAcUum. tHe REsUltIng PoWdER CAN BE eAsILY RE-dISPErSeD In TOlueNe, heXane, cHLoRoform aNd OThEr nOn-POlar SOLvEnts.
ThE exPerIMEnTAL ProcedURe iS simIlar fOR tHE SynTHEsIS Of COppER suLFIde NaNOpARTicLes, exCEpT that tHE reACTIon mIXTurE TUrnEd BluE ANd wAS STIrReD OVErnIgHT. DarK bRoWN or BLack coPPER sULfiDE naNoPartIClEs werE ISolATeD, Which caN BE Re-DISperSeD iN nOn-pOLAR solVEnTs suCH As tolUENe, heXaNE AnD CHlOrOFOrm.
The synTHeSis Of cadmIuM SuLfIDE NAnoPARtICles is siMILAR tO silVeR SULFIDe and cOpPer SUlFiDE, eXCePt ThAt thE ReACTION MiXTURE turneD yeLlOW AnD Was STiRRed FOR 4 hOurs AT rooM TemperaTuRe. pALe YElLoW pRoDuct Was SeparATeD BY CeNtRiFUgatIOn aftER ADDing 10 ML OF EThanOL. tHe product isolAtED, afTER WAsHING with eThANOl anD ACeTOne, cAn bE re-DISPeRSed In CHlorOforM, hexAnE anD tolUeNE.
for THe SyNtHESIS OF iNDIum SUlFIDe NanOpARticlEs, inTb (0.3 Mmol) wAS stIrrED iN TOLUenE (5 Ml) at RoOM teMpErAtUrE, AND THEn 1.2 MMoL oF OcTyLamine (oa) waS inJeCted to giVe A yEllOw SOlUTIoN. AFTer stIrrING FOr 6 HOuRS, 10 ML Of ETHanOl was aDDED To iNDUCE the fOrmATIon oF turBIDItY. The PaRtiCLES Were purIFIeD SImilarlY TO thE PREVIOuS Procedure. fOr thE PRepArATiOn oF oLEyLAmIne-cApPED iN~2~s~3~ nANoPartIclEs, iT waS fouNd neceSsARY TO FuRtHER aDd 40 ΜL OF pRopYlaMiNE to sPEed uP THe REaCtiOn.
2.2. cHaractERIZaTioNs
----------------------
tgA Was REcORDEd oN a sDt 2960 siMULtAneOUS dtA-tga By HeATIng APProxImaTELY 10 MG Of THe pREcUrSOr UndEr iNERT n~2~ FLOW (FLOW raTe 90 Ml/min) at a Heating RATe oF 10 °c/mIn. x-Ray DiFFrAcTIoN (XRD) AnalYsis waS carRiEd out On A SIEMEnS D5005 X-RAY POwDEr difFRACTOmEtEr WiTH Cu KΑ rAdiAtIoN (40 kV, 40 Ma). THE POwDereD sAMPLe WAS mounTED ON a SAMPLe HoLdER AnD scANnED WITh a STEp SIzE oF 2Θ = 0.05° iN ThE rAnGe oF 20° To 90°. UV-visIblE SPecTra WEre recORDEd usINg a SHIMADZU Uv-2550 UV/ViS speCtROPhOtometeR. fT-ir SpEctRa wERe recOrDeD USInG A fTs 165 BiO-rAd fTIr sPecTROphOToMETeR IN The rangE OF 4000--400 CM^−1^ ON kbR Or NujOl MullS. trAnsmIssion EleCtron MIcRosCOPy (Tem) IMAGeS WErE ObtAiNed oN a 100 KV Jem-100CXii TeM and 200 KV jeol 2010f micRosCOpE. SAMpLes WerE PREPARED bY PLaciNG a drOp OF tHE DIsPeRsed NaNoPaRTicleS oNto A coppEr gRid WITh cArBoN Film, anD WeRE aLlowEd To DRY In a desIcCAtoR.
2.3. CoMpUTAtionAl MEtHOdOLOgy
------------------------------
CalcuLAtIOnS WeRE PeRFOrmED USiNg the HYbRid dEnSitY funCtiONAL B3LYp \[[@B26-nAnOMaterials-02-00113],[@b27-nAnomaterIAls-02-00113]\] meTHOD WItH THe efFeCtivE CORe pOTeNtiAl lanL2DZ \[[@B28-NAnomaTErIAls-02-00113],[@B29-NANOMAterialS-02-00113],[@b30-naNOMaTERiAlS-02-00113],[@B31-NANomateRials-02-00113]\] basis Set. All repoRTEd ENerGiES INCLUDE ZeRO pOINT enerGy CoRreCTIOnS. ALl CaLCuLatIOnS wERE PErFOrMed USInG THE gAusSIAn 98 \[[@b32-NaNOMateriaLS-02-00113]\] SuiTE of PrOgRamS. oPTIMiZation wAS PeRFoRmED wITHOuT aNY ConStRAinTS And the oPTIMized StRuctURES werE vErIFieD tO BE eqUiliBriUm sTrucTUres OR tRaNSitION stAtes fROM fREQueNCY cALcULATIoNs. aN EqUILIbRIUm structURe Is CHARACterIZed By aLl ReAl fReqUenciEs WHILE A trAnsiTION sTate hAs oNe and ONly oNE iMAgiNaRY FrEqueNCy.
3. rEsULtS AnD dIscUssION
=========================
3.1. TheRMAL BEHaViOr oF tHE mTb pRecurSors
-------------------------------------------
ThE dECOMpOsition prOfILeS OF THe FOUR mTb PrECuRsOrs wERE INvEStIGaTEd anD thEIr TgA cURveS are preSenteD in [fIguRe 1](#nAnoMAtEriALS-02-00113-F001){ref-TYpe="fIg"} and [TABLe 1](#nANoMAterials-02-00113-T001){reF-tYpE="TAblE"}. tHe AnalYsis INdIcaTed a CleaR onSEt OF decomposITioN aT \~ 200--300 °c in eAch CASe. thE RESiduaL wEight AfteR EacH CompLETe DECOmpOsItiOn WAS foUNd To bE cLose tO The ExPectEd rEmAiNING weighT OF thE coRResPONding MeTal suLfIdE. tHE sLIGhtly hiGHEr MeasUrEd vaLueS ArE | 1.Introduction =============== The synthesis of metalsulfide nanocrystals has attracted much interest for both fundamentalresearch and technological applications in the past decades \[[@B1-nanomaterials-02-00113],[@B2-nanomaterials-02-00113],[@B3-nanomaterials-02-00113],[@B4-nanomaterials-02-00113],[@B5-nanomaterials-02-00113],[@B6-nanomaterials-02-00113]\]. Metalsulfide nanocrystals have been prepared by a wide range ofsyntheticmethods, one of whichinvolvesthedirect decomposition ofmolecular precursors \[[@B7-nanomaterials-02-00113],[@B8-nanomaterials-02-00113],[@B9-nanomaterials-02-00113],[@B10-nanomaterials-02-00113],[@B11-nanomaterials-02-00113],[@B12-nanomaterials-02-00113],[@B13-nanomaterials-02-00113],[@B14-nanomaterials-02-00113]\]. Molecularprecursor approach has recently been developed as anefficient route to preparemonodispersedsemiconductor nanocrystals \[[@B7-nanomaterials-02-00113],[@B10-nanomaterials-02-00113],[@B15-nanomaterials-02-00113]\] and, insome cases, unique shape-control has been achieved \[[@B9-nanomaterials-02-00113],[@B11-nanomaterials-02-00113],[@B16-nanomaterials-02-00113]\]. One of the earliestprecursorsused for preparingmetal sulfides in the literatureis *N*,*N*'-dialkyl dithiocarbamate. Inthispreparation, the precursor was injected intohot coordination solventssuchastrioctylphosphine oxide (TOPO) under nitrogen atmosphere at high temperatures \[[@B7-nanomaterials-02-00113],[@B8-nanomaterials-02-00113],[@B9-nanomaterials-02-00113],[@B10-nanomaterials-02-00113],[@B11-nanomaterials-02-00113]\].Metal bis(benzylthiolates) \[[@B12-nanomaterials-02-00113]\] and metal salts of alkylxanthate\[[@B17-nanomaterials-02-00113],[@B18-nanomaterials-02-00113],[@B19-nanomaterials-02-00113]\] have also been used as precursors to prepare nanocrystalline sulfides of zinc,cadmium or lead viapyrolysisat 150--400 °C.It was found that Lewis base such as hexydecylamine (HDA), trioctylphosphine (TOP) or tributylphosphine (TBP) could lowerthereaction temperature for the alkylxanthate precursors. Recently, there are also severalreports onthepreparation of ternary and metalsulfide nanocrystals via the decompositionof thiocarboxylate precursors \[[@B20-nanomaterials-02-00113],[@B21-nanomaterials-02-00113],[@B22-nanomaterials-02-00113],[@B23-nanomaterials-02-00113],[@B24-nanomaterials-02-00113]\]. Most of thesesyntheses, nevertheless,requireelevated or refluxing temperatures. In this paper, we report a generalizedprecursor method operating atroom temperaturefor the synthesis of various metal sulfide nanocrystals. The precursorsweuse are metal thiobenzoates \[M~x~(SCOC~6~H~5~)~y~ or simply MTB\].These MTBprecursors are air-stable and could be readily prepared from thiobenzoic acid and the corresponding metal salts following the known literature method \[[@B25-nanomaterials-02-00113]\]. We illustrate the generality ofthis MTBmethod by preparing four types of semiconductorsfromboth transition and main groupmetals: Ag~2~S,Cu~2−x~S, In~2~S~3~ andCdS. We discuss in this report the basisof our approach and also attempt to understand the reaction mechanism through theoretical Density Functional Theory (DFT)calculations.With thisinsightful knowledge, we demonstrate how to manipulate the stability of the precursor, and thus the reaction kinetics, to generate different nanostructures. 2. Experimental Section ======================= 2.1. Synthesis of Precursors and Metal Sulfide Nanocrystals ----------------------------------------------------------- Commercially available compounds such as thiobenzoic acid (Fluka), ether, ethanol,chloroform (all from J.T. Baker), sodium bicarbonate (Dumont), silver nitrate (Merck), indium chloride(Fluka),acetonitrile, cadmium acetate, copperchloride, 2,2'-bipyridine,octylamine, dodecylamine and oleylamine (all fromAldrich) were used as received. All of theMTB precursors used in this paper (M =Ag, Cu, In,Cd; TB = thiobenzoate) were prepared according toliterature methods \[[@B25-nanomaterials-02-00113]\]. The products obtained werewashed with ethanol,dried, and recrystallized from chloroform or ether. Purityof the crystalswaschecked with microanalysis and their decomposition profiles were investigated by thermogravimetric analysis (TGA). For the synthesis of silver sulfide nanoparticles, AgTB (3 mmol)wasstirredin toluene (5 mL) atroom temperature, and then added with 1.8 mmol of an amine. A homogeneous clear brown solution formedquicklyand thesolution was stirred for afurther3 hours. Afterthat, 10mL of ethanol was added to induce turbidity in the mixture. The brown or darkbrown nanoparticles are isolated by centrifugation, washed several times with ethanol and acetone, and then dried under vacuum. The resulting powder can beeasily re-dispersed intoluene, hexane, chloroform and other non-polar solvents. The experimental procedure is similar for the synthesisof copper sulfide nanoparticles,except that the reaction mixture turned blue and was stirred overnight. Dark brown or black coppersulfide nanoparticles were isolated, which can bere-dispersedin non-polar solvents such as toluene, hexane and chloroform. The synthesis of cadmium sulfide nanoparticles is similar tosilver sulfide and copper sulfide, except thatthereaction mixture turned yellow and was stirred for 4 hours at room temperature. Pale yellow product was separated by centrifugation after adding 10 mL of ethanol. The productisolated, after washing with ethanol andacetone,can be re-dispersed in chloroform, hexane and toluene. For the synthesis of indium sulfide nanoparticles, InTB (0.3 mmol)was stirred in toluene (5 mL) at room temperature, and then 1.2 mmol of octylamine (OA) wasinjected to give ayellow solution. After stirring for 6 hours, 10mL of ethanolwas added to induce the formation of turbidity. The particleswere purified similarly to the previous procedure. For the preparation of oleylamine-capped In~2~S~3~ nanoparticles, it was found necessary to further add 40 µL of propylamine to speed up the reaction. 2.2. Characterizations ----------------------TGA was recorded on aSDT 2960 Simultaneous DTA-TGA byheating approximately 10 mg of the precursor under inert N~2~ flow(flow rate 90 mL/min) at a heating rate of 10 °C/min. X-ray diffraction (XRD) analysiswas carried out on a Siemens D5005 X-ray powderdiffractometer with Cu Kαradiation (40 kV, 40 mA). Thepowdered sample was mounted on a sample holder and scanned witha step size of 2θ = 0.05° inthe range of20° to 90°. UV-Visible spectra were recorded using a Shimadzu UV-2550 UV/Vis spectrophotometer. FT-IR spectra were recorded using a FTS165 Bio-Rad FTIR spectrophotometer in the rangeof 4000--400 cm^−1^ on KBr or nujol mulls. Transmission electron microscopy (TEM) images were obtained on a 100 kV JEM-100CXII TEM and 200 kV JEOL 2010Fmicroscope. Samples were prepared by placing a dropof the dispersed nanoparticlesonto acopper grid with carbon film, and were allowed to dryin a desiccator. 2.3.Computational Methodology------------------------------ Calculations wereperformed using the hybriddensity functional B3LYP \[[@B26-nanomaterials-02-00113],[@B27-nanomaterials-02-00113]\] method with the effective core potential LanL2DZ \[[@B28-nanomaterials-02-00113],[@B29-nanomaterials-02-00113],[@B30-nanomaterials-02-00113],[@B31-nanomaterials-02-00113]\] basis set. All reported energies include zero point energycorrections. All calculations were performed using the Gaussian 98 \[[@B32-nanomaterials-02-00113]\] suite of programs. Optimization was performed without any constraints and the optimized structures were verified to be equilibrium structures or transition states from frequency calculations. An equilibrium structureis characterizedby all real frequencieswhile atransitionstate has one andonly one imaginary frequency. 3. Results and Discussion========================= 3.1. Thermal Behavior of the MTB Precursors -------------------------------------------The decomposition profiles of the fourMTB precursorswere investigated and their TGA curves are presented in [Figure 1](#nanomaterials-02-00113-f001){ref-type="fig"} and [Table 1](#nanomaterials-02-00113-t001){ref-type="table"}. The analysis indicateda clear onset ofdecomposition at\~ 200--300 °Cin each case. The residual weight aftereach complete decomposition was found to be close to the expected remaining weight of the corresponding metal sulfide. The slightly higher measuredvalues are | 1. _Introduction_ =============== The synthesis of _metal_ sulfide nanocrystals has attracted much interest for both fundamental _research_ and technological applications in the past decades \[[@B1-nanomaterials-02-00113],[@B2-nanomaterials-02-00113],[@B3-nanomaterials-02-00113],[@B4-nanomaterials-02-00113],[@B5-nanomaterials-02-00113],[@B6-nanomaterials-02-00113]\]. Metal _sulfide_ nanocrystals have _been_ prepared by a wide range of _synthetic_ _methods,_ one of which _involves_ the direct decomposition of _molecular_ precursors _\[[@B7-nanomaterials-02-00113],[@B8-nanomaterials-02-00113],[@B9-nanomaterials-02-00113],[@B10-nanomaterials-02-00113],[@B11-nanomaterials-02-00113],[@B12-nanomaterials-02-00113],[@B13-nanomaterials-02-00113],[@B14-nanomaterials-02-00113]\]._ Molecular precursor approach has recently been _developed_ as an efficient route to prepare monodispersed semiconductor nanocrystals \[[@B7-nanomaterials-02-00113],[@B10-nanomaterials-02-00113],[@B15-nanomaterials-02-00113]\] _and,_ in _some_ _cases,_ unique _shape-control_ _has_ _been_ achieved \[[@B9-nanomaterials-02-00113],[@B11-nanomaterials-02-00113],[@B16-nanomaterials-02-00113]\]. One of the earliest precursors used for preparing _metal_ sulfides in _the_ _literature_ is _*N*,*N*'-dialkyl_ _dithiocarbamate._ In this preparation, the precursor was injected into hot _coordination_ solvents such _as_ trioctylphosphine oxide (TOPO) under nitrogen _atmosphere_ _at_ high temperatures \[[@B7-nanomaterials-02-00113],[@B8-nanomaterials-02-00113],[@B9-nanomaterials-02-00113],[@B10-nanomaterials-02-00113],[@B11-nanomaterials-02-00113]\]. Metal bis(benzylthiolates) \[[@B12-nanomaterials-02-00113]\] and metal salts _of_ alkylxanthate \[[@B17-nanomaterials-02-00113],[@B18-nanomaterials-02-00113],[@B19-nanomaterials-02-00113]\] _have_ also been _used_ _as_ precursors to prepare _nanocrystalline_ sulfides of zinc, cadmium or lead via pyrolysis at 150--400 °C. It was found that Lewis _base_ _such_ _as_ _hexydecylamine_ (HDA), trioctylphosphine (TOP) _or_ tributylphosphine _(TBP)_ could lower the reaction _temperature_ for _the_ alkylxanthate precursors. _Recently,_ there are also several reports on _the_ preparation of ternary _and_ metal sulfide _nanocrystals_ via the decomposition of thiocarboxylate precursors \[[@B20-nanomaterials-02-00113],[@B21-nanomaterials-02-00113],[@B22-nanomaterials-02-00113],[@B23-nanomaterials-02-00113],[@B24-nanomaterials-02-00113]\]. Most of these syntheses, nevertheless, _require_ _elevated_ _or_ refluxing temperatures. In _this_ _paper,_ we report a generalized precursor _method_ operating at _room_ _temperature_ for the synthesis of various metal sulfide nanocrystals. The precursors we use are metal _thiobenzoates_ _\[M~x~(SCOC~6~H~5~)~y~_ or simply MTB\]. These MTB precursors are air-stable and _could_ be _readily_ prepared from thiobenzoic acid and the corresponding _metal_ salts following the _known_ literature _method_ \[[@B25-nanomaterials-02-00113]\]. _We_ _illustrate_ the generality of this MTB method _by_ preparing four types of semiconductors _from_ both transition _and_ main group metals: Ag~2~S, Cu~2−x~S, In~2~S~3~ and CdS. _We_ discuss in this report the basis of our approach and _also_ attempt to understand the reaction _mechanism_ through theoretical Density _Functional_ Theory (DFT) calculations. With this insightful _knowledge,_ we demonstrate how to _manipulate_ the stability of the precursor, _and_ thus the _reaction_ kinetics, _to_ _generate_ different nanostructures. 2. _Experimental_ Section ======================= 2.1. Synthesis of Precursors and Metal Sulfide Nanocrystals _-----------------------------------------------------------_ _Commercially_ available compounds such as _thiobenzoic_ acid (Fluka), ether, ethanol, chloroform (all _from_ J. T. Baker), sodium bicarbonate (Dumont), silver nitrate (Merck), indium _chloride_ (Fluka), acetonitrile, cadmium acetate, copper chloride, 2,2'-bipyridine, octylamine, _dodecylamine_ _and_ oleylamine _(all_ from _Aldrich)_ were used as received. All of the MTB precursors used in _this_ paper (M = Ag, Cu, In, _Cd;_ TB = thiobenzoate) were prepared according to literature methods \[[@B25-nanomaterials-02-00113]\]. _The_ products obtained were washed with ethanol, dried, and recrystallized from chloroform or ether. _Purity_ of the crystals was checked with _microanalysis_ and their decomposition profiles _were_ _investigated_ by _thermogravimetric_ analysis (TGA). For the synthesis _of_ silver sulfide _nanoparticles,_ _AgTB_ (3 mmol) was stirred in toluene _(5_ mL) _at_ room temperature, and then added with 1.8 mmol _of_ an _amine._ A homogeneous clear _brown_ solution formed quickly and the solution was stirred for a further 3 hours. After that, 10 mL of ethanol _was_ added to induce turbidity in the _mixture._ _The_ brown _or_ dark _brown_ nanoparticles are isolated by _centrifugation,_ washed several times with ethanol and acetone, _and_ then dried under vacuum. The resulting powder can be easily _re-dispersed_ in toluene, hexane, chloroform and other non-polar _solvents._ The _experimental_ procedure is similar for the synthesis _of_ copper sulfide nanoparticles, except that the reaction _mixture_ _turned_ blue and _was_ stirred _overnight._ Dark brown or _black_ copper sulfide nanoparticles were isolated, which _can_ be re-dispersed in _non-polar_ solvents such _as_ toluene, hexane and chloroform. The synthesis of cadmium sulfide nanoparticles _is_ similar to _silver_ sulfide and copper _sulfide,_ except that _the_ _reaction_ mixture turned _yellow_ and was _stirred_ for _4_ hours at room temperature. Pale _yellow_ product was separated by centrifugation after adding 10 mL of _ethanol._ The product isolated, after _washing_ with ethanol and _acetone,_ can be re-dispersed _in_ chloroform, hexane and _toluene._ For the _synthesis_ of indium sulfide nanoparticles, InTB (0.3 _mmol)_ was stirred in _toluene_ _(5_ mL) at room temperature, and then 1.2 _mmol_ of octylamine (OA) was _injected_ to give a yellow _solution._ After _stirring_ for _6_ hours, 10 mL _of_ ethanol was added _to_ induce _the_ formation of turbidity. The _particles_ were purified similarly to the previous _procedure._ _For_ _the_ preparation of oleylamine-capped _In~2~S~3~_ nanoparticles, it was found necessary _to_ further add 40 µL of propylamine _to_ _speed_ _up_ the _reaction._ 2.2. Characterizations ---------------------- _TGA_ was recorded on _a_ _SDT_ 2960 Simultaneous DTA-TGA by heating approximately _10_ mg _of_ _the_ precursor under _inert_ N~2~ flow (flow rate 90 mL/min) _at_ a heating rate of 10 _°C/min._ X-ray _diffraction_ (XRD) analysis _was_ carried out _on_ a Siemens D5005 X-ray powder diffractometer _with_ Cu _Kα_ radiation (40 _kV,_ 40 mA). The powdered sample _was_ mounted _on_ a sample holder and scanned with a _step_ size of 2θ = 0.05° in the range _of_ 20° to 90°. UV-Visible spectra were recorded using a Shimadzu UV-2550 _UV/Vis_ spectrophotometer. FT-IR spectra were recorded using a FTS 165 Bio-Rad FTIR spectrophotometer in the _range_ of 4000--400 _cm^−1^_ on KBr or nujol mulls. Transmission electron _microscopy_ (TEM) _images_ were obtained on a 100 kV JEM-100CXII TEM and 200 kV JEOL 2010F microscope. Samples _were_ prepared _by_ placing a drop of the dispersed _nanoparticles_ _onto_ _a_ copper grid with _carbon_ film, and _were_ allowed to dry in a desiccator. 2.3. Computational Methodology ------------------------------ Calculations _were_ performed using _the_ hybrid density functional _B3LYP_ \[[@B26-nanomaterials-02-00113],[@B27-nanomaterials-02-00113]\] _method_ _with_ the effective _core_ potential LanL2DZ \[[@B28-nanomaterials-02-00113],[@B29-nanomaterials-02-00113],[@B30-nanomaterials-02-00113],[@B31-nanomaterials-02-00113]\] basis _set._ _All_ reported energies include zero point energy corrections. _All_ calculations were _performed_ using the Gaussian 98 \[[@B32-nanomaterials-02-00113]\] suite of programs. Optimization was performed without any constraints and the _optimized_ structures _were_ verified to be equilibrium structures or _transition_ states _from_ frequency calculations. An _equilibrium_ structure is characterized by all real frequencies while a _transition_ state _has_ one and only one _imaginary_ frequency. 3. _Results_ and Discussion ========================= 3.1. Thermal Behavior of the MTB Precursors ------------------------------------------- The decomposition profiles of the four _MTB_ precursors were investigated and _their_ TGA curves are presented in [Figure _1](#nanomaterials-02-00113-f001){ref-type="fig"}_ and _[Table_ 1](#nanomaterials-02-00113-t001){ref-type="table"}. _The_ analysis _indicated_ a clear onset of decomposition at \~ 200--300 _°C_ in _each_ case. _The_ _residual_ _weight_ after _each_ complete decomposition _was_ found to be close _to_ _the_ expected _remaining_ weight of the corresponding _metal_ _sulfide._ _The_ slightly higher _measured_ values _are_ |
Introduction
============
Porcine reproductive and respiratory syndrome (PRRS), first reported in 1987 in the USA, is a global infectious disease that has resulted in widespread economic loss in the swine industry. PRRS is characterized by reproductive failure in pregnant sows (*e.g.* abortions and stillbirths), respiratory distress in pigs of different ages, and severe immune suppression \[[@B13],[@B16]\]. The PRRS virus (PRRSV) is the recognized causative agent of this syndrome. Two different genotypes of PRRSV have been described: European or type 1, and North American or type 2 \[[@B6],[@B21]\]. In China, most isolated strains are of type 2 \[[@B3]\].
PRRSV is an enveloped, positive, single-stranded RNA virus in the genus *Arterivirus*, which belongs to the family Arteriviridae within the order Nidovirales \[14\].The PRRSV genome is 15 kb in length and contains at least 10 open reading frames (ORFs). Of these, ORF1a and ORF1b represent nearly 75% of the viral genome and encode two large polyproteins (pp), pp1a and pp1ab, respectively. These pps can be hydrolyzed into at least 16 small nonstructural proteins (Nsps). Of these 16 Nsps, at least 14 are involved in viral genome replication and transcription \[[@B10]\]. The Nsp1, Nsp2, and Nsp7 proteins elicit a strong immune response in pigs. Fourteen days after pigs are infected with PRRSV, anti-PRRSV Nsp7 antibodies can be detected, and high levels of antibodies in pigs can last for up to 202 days \[[@B2]\].
A diagnosis of PRRSV is important for its prevention and control. The laboratory diagnostic tests commonly used at present include reverse transcriptase polymerase chain reaction (PCR), quantitative real-time PCR \[[@B19],[@B22]\], and four serological detection methods \[[@B1],[@B4],[@B5],[@B20]\]: the indirect fluorescence assay (IFA), the immunoperoxidase monolayer assay (IPMA), the serum neutralization test (SN), and the enzyme-linked immunosorbent assay (ELISA). In recent years, by using the PRRSV N protein as an antigen, the commercial IDEXX ELISA kit (IDEXX Laboratories, USA) has been widely used for the detection of PRRSV antibodies. Recently, an ELISA kit that uses recombinant Nsp7 protein as the antigen has been introduced, and the concurrence rate between that kit and the IDEXX ELISA assay has been reported to be up to 97.6% \[[@B2]\]. Although traditional laboratory tests offer good sensitivity and specificity, these detection methods require professional and technical personnel and specialized equipment. In addition, there are many methodological aspects that must be constantly improved and complemented.
In developing countries, vaccination is an important strategy for preventing and controlling PRRSV. To monitor the titer of anti-PRRSV antibodies after vaccination in order to confirm the effectiveness of the vaccination, quick and easy techniques suitable for field testing are needed. Compared with the traditional detection methods, an immunochromatographic strip method has the advantages of being easy to use, providing an answer rapidly and at a low cost, and not requiring specialized equipment or technical personnel, thus such a method is suitable for field testing for antigens or antibodies. In this study, we developed an immunochromatographic test strip for detecting anti-PRRSV antibodies and conducted a preliminary field study of the strip.
Materials and Methods
=====================
Serum samples
-------------
Pigs were experimentally inoculated with either of two strains of PRRSV having differing levels of virulence, HN07-1 and BJ-4, in order to provide positive test serum samples. HN07-1 strain was isolated during an atypical PRRSV outbreak in Henan Province, China in 2007 \[[@B17]\]. The BJ-4 strain was a typical North American (VR2332)-like PRRSV isolated in 1996 in China \[[@B17]\]. The field sera for antibody testing were collected from several swine herds (all sampled pigs were more than 2-weeks-old) within Henan Province, China. Positive serum samples for porcine circovirus-2 (PCV-2), pseudorabies virus (PRV), classical swine fever virus (CSFV), porcine parvovirus (PPV), and foot-and-mouth disease virus (FMDV) were collected and stored in the Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences.
Preparation of recombinant Nsp7 protein of PRRSV
------------------------------------------------
For the expression of a recombinant Nsp7 proteins in *Escherichia* (*E.*) *coli*, reverse transcriptase PCR was performed on viral genomic RNA from PRRSV isolate BJ-4, obtained from Dr. Hanchun Yang of China Agricultural University. To amplify the Nsp7 gene the following oligonucleotide primers were used: forward primer, 5′-CGC**GGATCC**TCTCTGACTGGTGCCCTCGCTATG-3′ and reverse primer, 5′-CCG**CTCGAG**TTCCCATTGAACTCTTCCAT-3′ (restriction sites in bold font). The amplified gene was then ligated into the pET-28a vector. The recombinant plasmid was transformed to *E. coli* BL21-competent cells. Single colonies were obtained and tested by PCR and sequencing, and a positive clone was grown at 37℃ in LB broth supplemented with 100 µg/mL ampicillin to an optical density of 0.8 at 600 nm. Expression of the recombinant protein was induced by 100 mM isopropyl-β-D-thiogalactopyranoside (IPTG, TAKARA Bio, China) for 8 h at 37℃. Cells were then harvested by centrifugation (7000 × g for 30 min).
Purification of recombinant Nsp7 protein
----------------------------------------
The recombinant Nsp7 protein was purified by immobilized-metal affinity chromatography (IMAC) using a polyhistidine tag and further purified by a gel filtration column Superdex200 (GE Healthcare, Sweden). The cell pellet was suspended and lysed by sonication on ice. The lysate was centrifuged at 16,000 × g for 30 min, and the supernatant was collected and transferred to a Ni-NTA His Band Resin column pre-equilibrated with binding buffer (500 mM NaCl, 20 mM Tris, 5 mM imidazole). More than five column-volumes of washing buffer (500 mM NaCl, 20 mM Tris, 20 mM imidazole) was added to remove the nonspecific binding proteins. The target protein was eluted with elution buffer (500 mM NaCl, 20 mM Tris, 400 mM imidazole). The purity and relative concentration of the recombinant Nsp7 was determined by SDS-PAGE. The protein was further fractionated by gel filtration on a column of Superdex200 in a buffer of 50 mM Tris, 150 mM NaCl by using the Bio-Rad BioLogic system (Bio-Rad Laboratories, USA). The protein of interest was collected in different fractions according to its different states of aggregation. The final protein products were examined by SDS-PAGE before storing at −80℃.
Western blot
------------
For western blot analysis, 4 µg purified recombinant Nsp7 protein were subjected to 15% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. The membrane was washed with phosphate-buffered saline-Tween20 (PBST) and blocked with 5% skimmed milk. After washing three times with PBST, the membranes were reacted with PRRSV-positive sera; a PRRSV-negative serum were used as a negative control. After incubating at 37℃ for 1 h, the resulting blot was treated with secondary antibody horseradish peroxidase-conjugated rabbit-anti-pig IgG (Abbkine; WuHan AmyJet Scientific, China) for 1 h. As the substrate for color development, 3-amino-9-ethylcarbazole (AEC) was used.
The antigenicity of the separated protein fractions compared by ELISA
---------------------------------------------------------------------
The antibody binding capability of the monomer, dimer, and larger aggregate of the recombinant Nsp7, which were separated by Superdex200 gel filtration column, were compared by indirect ELISA assay. The separated proteins were diluted to the appropriate concentration in 50 mM sodium carbonate bicarbonate buffer (pH 9.6). After incubation for 14 h at 4℃, antigen-coated plates were washed five times with phosphate-buffered saline (PBS) containing 0.05% Tween 80 then blocked with 5% skimmed milk powder dissolved by PBST for 1 h at 37℃. Then, appropriate dilutions in PBST of PRRSV-positive HN07-1, PRRSV-positive BJ-4, and PRRSV-negative pig sera were incubated in the antigen-coated wells at 37℃ for 30 min. Secondary antibody horseradish peroxidase-conjugated rabbit-anti-pig IgG was added at a final dilution of 1:2,000, and the mixture incubated for a further 30 min at 37℃. Finally, 3′,3′,5′,5′-tetramethylbenzidine was added as a substrate. Color development was stopped with 2 M H~2~SO~4~, and the OD value at 450 nm was read on a spectrophotometer.
Conjugation of antigen with colloidal gold
------------------------------------------
Colloidal gold with an average particle diameter of approximately 20 to 25 nm was obtained by reduction of a HAuCl4 solution with 1% trisodium citrate. Three milliliters of 1% trisodium citrate (w/v) was added to 100 mL of 0.01% HAuCl~4~·3H~2 | introduction = = = = = = = = = = = = porcine reproductive and respiratory syndrome ( prrs ), first reported in 1987 in the usa, is a global infectious disease that has resulted in widespread economic loss in the swine industry. prrs is explained by reproductive failure in pregnant sows ( * e. g. * abortions and stillbirths ), respiratory distress in pigs of different ages, and severe immune suppression \ [ [ @ b13 ], [ @ c2 ] \ ]. the prrs virus ( prrsv ) is the recognized causative agent of this syndrome. two different kinds of prrsv have been described : european or type 1, and north american or type 2 \ [ [ @ b6 ], [ @ b21 ] \ ]. in china, most isolated strains are of type 2 \ [ [ @ b3 ] \ ]. prrsv is an enveloped, positive, single - stranded rna virus in the genus * arterivirus *, which belongs to the family arteriviridae containing the order nidovirales \ [ 14 \ ]. the prrsv genome is 15 kb in length and contains at least 10 open reading frames ( orfs ). of these, orf1a and orf1b represent nearly 75 % of the viral genome and encode two large polyproteins ( pp ), pp1a and pp1ab, respectively. these pps can be hydrolyzed into at least 16 small nonstructural proteins ( nsps ). of these 16 nsps, at least 14 are involved in viral genome replication and transcription \ [ [ @ b10 ] \ ]. the nsp1, nsp2, and nsp7 proteins elicit a strong immune response in pigs. 150 days after pigs are infected with prrsv, anti - prrsv nsp7 antibodies can be detected, and high levels of antibodies in pigs can last for up to 202 days \ [ [ @ b2 ] \ ]. correct diagnosis of infection is important for its prevention and control. the laboratory diagnostic procedures commonly used are present include reverse transcriptase polymerase chain reaction ( pcr ), four real - time pcr \ [ [ @ b19 ], [ @ b22 ] \ ], and four serological detection methods \ [ [ @ b1 ], [ @ b4 ], [ @ b5 ], [ @ b ##20 ] \ ] : the indirect fluorescence assay ( ifa ), the immunoperoxidase monolayer assay ( ipma ), the serum neutralization test ( sn ), and the enzyme - linked immunosorbent assay ( elisa ). in recent years, by using the prrsv n protein as an antigen, the commercial idexx elisa kit ( idexx laboratories, usa ) has been widely used for the detection of prrsv antibodies. recently, an elisa kit that uses recombinant nsp7 protein as the antigen has been introduced, and the concurrence rate between that kit and the idexx elisa assay has been reported to be up to 97. 6 % \ [ [ @ b2 ] \ ]. although traditional laboratory tests offer good sensitivity and specificity, these detection methods require professional and technical personnel and specialized equipment. in addition, there are many methodological aspects that must be constantly improved and complemented. in developing countries, vaccination is an important strategy for preventing and controlling prrsv. to monitor the titer of anti - prrsv antibodies after vaccination in order to confirm the effectiveness of the vaccination, quick and easy techniques suitable for field testing are needed. compared with the traditional detection methods, an immunochromatographic strip method has the advantages of being easy to use, providing an answer rapidly and at a low cost, and not requiring specialized equipment or technical personnel, thus such a method is suitable for field testing for antigens or antibodies. in this study, we developed an immunochromatographic test strip for detecting anti - prrsv antibodies and conducted a preliminary field study of the strip. materials and methods = = = = = = = = = = = = = = = = = = = = = serum samples - - - - - - - - - - - - - pigs were experimentally inoculated with either of two strains of prrsv having differing levels of virulence, hn07 - 1 and bj - 4, in order to provide positive test serum samples. hn07 - 1 strain was isolated during an atypical prrsv outbreak in henan province, china in 2007 \ [ [ @ b17 ] \ ]. the bj - 4 strain was a typical north american ( vr2332 ) - like prrsv isolated in 1996 in china \ [ [ @ b17 ] \ ]. the field sera for antibody testing were collected from several swine herds ( all sampled pigs were more than 2 - weeks - old ) within henan province, china. positive serum samples for porcine circovirus - 2 ( pcv - 2 ), pseudorabies virus ( prv ), classical swine fever virus ( csfv ), porcine parvovirus ( ppv ), and foot - and - mouth disease virus ( fmdv ) were collected and stored in the henan provincial key laboratory of animal immunology, henan academy of agricultural sciences. preparation of recombinant nsp7 protein of prrsv - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - for the expression of a recombinant nsp7 proteins in * escherichia * ( * e. * ) * coli *, reverse transcriptase pcr was performed on viral genomic rna from prrsv isolate bj - 4, obtained from dr. hanchun yang of china agricultural university. to amplify the nsp7 gene the following oligonucleotide primers were used : forward primer, 5 ′ - cgc * * ggatcc * * tctctgactggtgccctcgctatg - 3 ′ and reverse primer, 5 ′ - ccg * * ctcgag * * ttcccattgaactcttccat - 3 ′ ( restriction sites in bold font ). the amplified gene was then ligated into the pet - 28a vector. the recombinant plasmid was transformed to * e. coli * bl21 - competent cells. single colonies were obtained and tested by pcr and sequencing, and a positive clone was grown at [UNK] in lb broth supplemented with 100 µg / ml ampicillin to an optical density of 0. 8 at 600 nm. expression of the recombinant protein was induced by 100 mm isopropyl - β - d - thiogalactopyranoside ( iptg, takara bio, china ) for 8 h at [UNK]. cells were then harvested by centrifugation ( 7000 × g for 30 min ). purification of recombinant nsp7 protein - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the recombinant nsp7 protein was purified by immobilized - metal affinity chromatography ( imac ) using a polyhistidine tag and further purified by a gel filtration column superdex200 ( ge healthcare, sweden ). the cell pellet was suspended and lysed by sonication on ice. the lysate was centrifuged at 16, 000 × g for 30 min, and the supernatant was collected and transferred to a ni - nta his band resin column pre - equilibrated with binding buffer ( 500 mm nacl, 20 mm tris, 5 mm imidazole ). more than five column - volumes of washing buffer ( 500 mm nacl, 20 mm tris, 20 mm imidazole ) was added to remove the nonspecific binding proteins. the target protein was eluted with elution buffer ( 500 mm nacl, 20 mm tris, 400 mm imidazole ). the purity and relative concentration of the recombinant nsp7 was determined by sds - page. the protein was further fractionated by gel filtration on a column of superdex200 in a buffer of 50 mm tris, 150 mm nacl by using the bio - rad biologic system ( bio - rad laboratories, usa ). the protein of interest was collected in different fractions according to its different states of aggregation. the final protein products were examined by sds - page before storing at [UNK]. western blot - - - - - - - - - - - - for western blot analysis, 4 µg purified recombinant nsp7 protein were subjected to 15 % sds - page gel and transferred to polyvinylidene difluoride membranes. the membrane was washed with phosphate - buffered saline - tween20 ( pbst ) and blocked with 5 % skimmed milk. after washing three times with pbst, the membranes were reacted with prrsv - positive sera ; a prrsv - negative serum were used as a negative control. after incubating at [UNK] for 1 h, the resulting blot was treated with secondary antibody horseradish peroxidase - conjugated rabbit - anti - pig igg ( abbkine ; wuhan amyjet scientific, china ) for 1 h. as the substrate for color development, 3 - amino - 9 - ethylcarbazole ( aec ) was used. the antigenicity of the separated protein fractions compared by elisa - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the antibody binding capability of the monomer, dimer, and larger aggregate of the recombinant nsp7, which were separated by superdex200 gel filtration column, were compared by indirect elisa assay. the separated proteins were diluted to the appropriate concentration in 50 mm sodium carbonate bicarbonate buffer ( ph 9. 6 ). after incubation for 14 h at [UNK], antigen - coated plates were washed five times with phosphate - buffered saline ( pbs ) containing 0. 05 % tween 80 then blocked with 5 % skimmed milk powder dissolved by pbst for 1 h at [UNK]. then, appropriate dilutions in pbst of prrsv - positive hn07 - 1, prrsv - positive bj - 4, and prrsv - negative pig sera were incubated in the antigen - coated wells at [UNK] for 30 min. secondary antibody horseradish peroxidase - conjugated rabbit - anti - pig igg was added at a final dilution of 1 : 2, 000, and the mixture incubated for a further 30 min at [UNK]. finally, 3 ′, 3 ′, 5 ′, 5 ′ - tetramethylbenzidine was added as a substrate. color development was stopped with 2 m h ~ 2 ~ so ~ 4 ~, and the od value at 450 nm was read on a spectrophotometer. conjugation of antigen with colloidal gold - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - colloidal gold with an average particle diameter of approximately 20 to 25 nm was obtained by reduction of a haucl4 solution with 1 % trisodium citrate. three milliliters of 1 % trisodium citrate ( w / v ) was added to 100 ml of 0. 01 % haucl ~ 4 ~ · 3h ~ 2 | Introduction = = = = = = = = = = = = Porcine reproductive and respiratory syndrome (PRRS ), first reported in 1987 in the USA, is a global infectious disease that has resulted in widespread economic loss in the swine industry. PRRS is characterized by reproductive failure in pregnant sows (* e. g. * abortions and stillbirths ), respiratory distress in pigs of different ages, and severe immune suppression \ [[ @ B13 ], [@ B16] \ ]. The PRRS virus (PRRSV) is the recognized causative agent of this syndrome. Two different genotypes of PRRSV have been described: European or type 1, and North American or type 2 \ [[ @ B6 ], [@ B21] \ ]. In China, most isolated strains are of type 2 \ [[ @ B3] \ ]. PRRSV is an enveloped, positive, single - stranded RNA virus in the genus * Arterivirus *, which belongs to the family Arteriviridae within the order Nidovirales \ [14 \ ]. The PRRSV genome is 15 kb in length and contains at least 10 open reading frames (ORFs ). Of these, ORF1a and ORF1b represent nearly 75% of the viral genome and encode two large polyproteins (pp ), pp1a and pp1ab, respectively. These pps can be hydrolyzed into at least 16 small nonstructural proteins (Nsps ). Of these 16 Nsps, at least 14 are involved in viral genome replication and transcription \ [[ @ B10] \ ]. The Nsp1, Nsp2, and Nsp7 proteins elicit a strong immune response in pigs. Fourteen days after pigs are infected with PRRSV, anti - PRRSV Nsp7 antibodies can be detected, and high levels of antibodies in pigs can last for up to 202 days \ [[ @ B2] \ ]. A diagnosis of PRRSV is important for its prevention and control. The laboratory diagnostic tests commonly used at present include reverse transcriptase polymerase chain reaction (PCR ), quantitative real - time PCR \ [[ @ B19 ], [@ B22] \ ], and four serological detection methods \ [[ @ B1 ], [@ B4 ], [@ B5 ], [@ B20] \ ]: the indirect fluorescence assay (IFA ), the immunoperoxidase monolayer assay (IPMA ), the serum neutralization test (SN ), and the enzyme - linked immunosorbent assay (ELISA ). In recent years, by using the PRRSV N protein as an antigen, the commercial IDEXX ELISA kit (IDEXX Laboratories, USA) has been widely used for the detection of PRRSV antibodies. Recently, an ELISA kit that uses recombinant Nsp7 protein as the antigen has been introduced, and the concurrence rate between that kit and the IDEXX ELISA assay has feem reported to be up to 97. 6% \ [[ @ B2] \ ]. Although traditional laboratory tests offer good sensitivity and specificity, these detection methods require professional and technical personnel and specialized equipment. In addition, there are many methodological aspects that must be constantly improved and complemented. In developing countries, gaccinxtion is an important strategy for preventing and controlling PRRSV. To monitor the titer of anti - PRRSV antibodies after vaccination in order to confirm the effectiveness of the vaccination, quick and easy techniques suitable for field testing are needed. Compared with the traditional detection methods, an immunochromatographic strip method has the advantages of being easy to use, providing an answer rapidly and at a low cost, and not requiring specialized equipment or technical personnel, thus such a method is suitable for field testing for antigens or antibodies. In this study, we developed an immunochromatographic test strip for detecting anti - PRRSV antibodies and conducted a preliminary field study of the strip. Materials and Methods = = = = = = = = = = = = = = = = = = = = = Serum ESmples - - - - - - - - - - - - - Pigs were experimentally inoculated with either of two strains of PRRSV having differing levels of virulence, HN07 - 1 and BJ - 4, in order to provide positive test serum samples. Hh0i - 1 strain was isolated during an atypical PRRSV outbreak in Henan Province, China in 2007 \ [[ @ B17] \ ]. The BJ - 4 strain was a typical North Amegixan (VR2332) - like PRRSV isolated in 1996 in China \ [[ @ B17] \ ]. The field sera for antibody testing were collected from several swine herds (all sampled pigs were more than 2 - weeks - old) within Henan Province, China. Positive serum samples for porcine circovirus - 2 (PCV - 2 ), pseudorabies virus (PRV ), classical swine fever virus (CSFV ), porcine parvovirus (PPV ), and foot - and - mouth disease virus (FMDV) were cpllested and stored in the HWnXn Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences. Preparation of recombinant Nsp7 protein of PRRSV - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - For the expression of a recombinant Nsp7 proteins in * Escherichia * (* E. *) * coli *, reverse transcriptase PCR was performed on viral genomic RNA from PRRSV isolate BJ - 4, obtained from Dr. Hanchun Yang of China Agricultural University. To amplify the Nsp7 gene the following oligonucleotide primers were used: forward primer, 5 ′ - CGC * * GGATCC * * TCTCTGACTGGTGCCCTCGCTATG - 3 ′ and reverse primer, 5 ′ - CCG * * CTCGAG * * TTCCCATTGAACTCTTCCAT - 3 ′ (restriction sites in bold font ). The amplified gene was then ligated into the pET - 28a vector. The recombinant plasmid was transformed to * E. coli * BL21 - competent cells. Single colonies were obtained and tested by PCR and sequencing, and a positive clone was grown at 37 ℃ in LB broth supplemented with 100 µg / mL ampicillin to an optical density of 0. 8 at 600 nm. Expression of the recombinant protein was induced by 100 mM isopropyl - β - D - thiogalactopyranoside (IPTG, TAKARA Bio, China) for 8 h at 37 ℃. Cells were then harvested by centrifugation (7000 × g for 30 min ). Purification of recombinant Nsp7 protein - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The recombinant Nsp7 protein was purified by immobilized - metal affinity chromatography (IMAC) using a polyhistidine tag and further purified by a gel fultratiPn column Superdex200 (GE Healthcare, Sweden ). The cell pellet was suspended and lysed by sonication on ice. The lysate was centrifuged at 16, 000 × g for 30 min, and the supernatant was collected and transferred to a Ni - NTA His Band Resin column pre - equilibrated with binding buffer (500 mM NaCl, 20 mM Tris, 5 mM imidazole ). More than five column - volumes of washing buffer (500 mM NaCl, 20 mM Tris, 20 mM imidazole) was added to remove the nonspecific binding proteins. The target protein was eluted with elution buffer (500 mM NaCl, 20 mM Tris, 400 mM imidazole ). The purity and relative concentration of the recombinant Nsp7 was determined by SDS - PAGE. The protein was further fractionated by gel filtration on a column of Superdex200 in a buffer of 50 mM Tris, 150 mM NaCl by using the Bio - Rad BioLogic system (Bio - Rad Laboratories, USA ). The protein of interest was collected in different fractions according to its different states of aggregation. The final protein products were examined by SDS - PAGE before storing at − 80 ℃. Western blot - - - - - - - - - - - - For western blot analysis, 4 µg purified recombinant Nsp7 protein were subjected to 15% SDS - PAGE gel and transferred to polyvinylidene difluoride membranes. The membrane was washed with phosphate - buffered saline - Tween20 (PBST) and blocked with 5% skimmed milk. After washing three times with PBST, the membranes were reacted with PRRSV - positive sera; a PRRSV - negative serum were used as a negative control. After incubating at 37 ℃ for 1 h, the resulting blot was treated with secondary antibody horseradish peroxidase - conjugated rabbit - anti - pig IgG (Abbkine; WuHan AmyJet Scientific, China) for 1 h. As the substrate for color development, 3 - amino - 9 - ethylcarbazole (AEC) was used. The antigenicity of the separated protein fractions compared by ELISA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The antibody binding capability of the monomer, dimer, and larger aggregate of the recombinant Nsp7, which were separated by Superdex200 gel filtration column, were compared by indirect ELISA assay. The separated proteins were diluted to the appropriate concentration in 50 mM sodium carbonate bicarbonate buffer (pH 9. 6 ). After incubation for 14 h at 4 ℃, antigen - coated plates were washed five times with phosphate - buffered saline (PBS) containing 0. 05% Tween 80 then blocked with 5% skimmed milk powder dissolved by PBST for 1 h at 37 ℃. Then, appropriate dilutions in PBST of PRRSV - positive HN07 - 1, PRRSV - positive BJ - 4, and PRRSV - negative pig sera were incubated in the antigen - coated wells at 37 ℃ for 30 min. Secondary antibody horseradish peroxidase - conjugated rabbit - anti - pig IgG was added at a final dilution of 1: 2, 000, and the mixture incubated for a further 30 min at 37 ℃. Finally, 3 ′, 3 ′, 5 ′, 5 ′ - tetramethylbenzidine was added as a substrate. Color development was stopped with 2 M H ~ 2 ~ SO ~ 4 ~, and the OD value at 450 nm was 5eaf on a spectrophotlme^er. Conjugation of antigen with colloidal gold - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Colloidal gold with an average particle diameter of approximately 20 to 25 nm was obtained by reduction of a HAuCl4 solution with 1% trisodium citrate. Three milliliters of 1% trisodium citrate (w / v) was added to 100 mL of 0. 01% HAuCl ~ 4 ~ · 3H ~ 2 | Introduction ============ Porcine reproductive and respiratory syndrome (PRRS), reported in 1987 in the USA, is a global infectious disease that has in widespread economic loss in the swine industry. PRRS is characterized by reproductive failure in pregnant sows (*e.g.* abortions and stillbirths), respiratory distress of different and immune suppression \[[@B13],[@B16]\]. PRRS virus (PRRSV) is the recognized causative agent of this syndrome. Two different genotypes of PRRSV have been described: European type 1, and North American or type 2 \[[@B6],[@B21]\]. In China, most isolated strains are of type 2 \[[@B3]\]. PRRSV is an enveloped, positive, single-stranded RNA virus in genus *Arterivirus*, which belongs to the family within the Nidovirales \[14\].The PRRSV genome is 15 kb in length and at least 10 frames (ORFs). Of these, ORF1a and ORF1b represent nearly 75% of the and encode two large polyproteins (pp), pp1a and pp1ab, respectively. These pps be at least 16 small nonstructural proteins (Nsps). Of these 16 Nsps, at least 14 are involved in viral genome replication and transcription \[[@B10]\]. The Nsp1, Nsp2, and Nsp7 proteins elicit strong response in pigs. Fourteen days after pigs are infected with PRRSV, anti-PRRSV Nsp7 antibodies can be detected, and high levels of antibodies in pigs can last for up to 202 days A diagnosis of PRRSV is important its prevention and control. The laboratory diagnostic tests commonly used at present include reverse transcriptase polymerase chain reaction (PCR), quantitative real-time PCR \[[@B19],[@B22]\], and four detection \[[@B1],[@B4],[@B5],[@B20]\]: the indirect fluorescence assay (IFA), the immunoperoxidase assay (IPMA), the serum neutralization test (SN), and enzyme-linked immunosorbent assay In recent years, by using the N protein as an antigen, the commercial IDEXX ELISA kit (IDEXX Laboratories, USA) been widely used for the detection PRRSV antibodies. Recently, an ELISA kit uses as the antigen been and the concurrence rate between that kit and the IDEXX ELISA assay has been reported to be up to 97.6% \[[@B2]\]. Although traditional laboratory offer sensitivity and specificity, these detection methods require professional and technical personnel and equipment. In addition, there are methodological aspects that must be constantly improved and complemented. developing countries, vaccination is an strategy for preventing and controlling PRRSV. To monitor titer of anti-PRRSV antibodies after vaccination in order to confirm the effectiveness of the vaccination, quick and easy techniques suitable for field testing are needed. Compared the traditional detection methods, immunochromatographic strip method has the advantages of being easy to use, providing an answer rapidly at low cost, and not requiring specialized equipment or technical personnel, thus such a is suitable for field testing for antigens or antibodies. In this study, we developed an immunochromatographic test strip for detecting antibodies and conducted preliminary field study of Materials and Methods ===================== Serum samples Pigs were experimentally inoculated with either of two strains of PRRSV having differing levels of HN07-1 and BJ-4, in order positive test serum samples. HN07-1 strain was isolated during an atypical PRRSV outbreak in Henan China in 2007 \[[@B17]\]. The BJ-4 strain was a typical American (VR2332)-like PRRSV isolated in in China \[[@B17]\]. The field sera for antibody testing were collected from several swine herds sampled pigs were more than 2-weeks-old) within Henan Province, China. Positive serum samples for porcine circovirus-2 pseudorabies virus (PRV), swine fever virus (CSFV), porcine parvovirus (PPV), and disease virus (FMDV) were and in the Henan Provincial Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences. Preparation of recombinant Nsp7 protein of PRRSV ------------------------------------------------ For the expression of recombinant Nsp7 proteins in *Escherichia* (*E.*) *coli*, transcriptase PCR was performed on viral genomic RNA from PRRSV isolate BJ-4, from Dr. of China Agricultural University. To amplify the Nsp7 gene the following oligonucleotide primers were used: forward primer, 5′-CGC**GGATCC**TCTCTGACTGGTGCCCTCGCTATG-3′ and reverse primer, 5′-CCG**CTCGAG**TTCCCATTGAACTCTTCCAT-3′ (restriction sites in bold font). The amplified gene was then ligated into pET-28a vector. The recombinant plasmid was transformed to *E. coli* BL21-competent cells. Single colonies were obtained and PCR and sequencing, and a positive clone was grown at 37℃ in LB broth supplemented with 100 µg/mL ampicillin to an optical 0.8 at 600 nm. Expression of the recombinant protein was induced 100 mM isopropyl-β-D-thiogalactopyranoside (IPTG, TAKARA Bio, for 8 h at 37℃. Cells were then harvested by centrifugation (7000 × g for 30 min). Purification of recombinant Nsp7 protein ---------------------------------------- The recombinant Nsp7 protein purified by immobilized-metal affinity (IMAC) using a polyhistidine tag and further purified by a gel filtration column Superdex200 (GE Sweden). The cell pellet was suspended and lysed by sonication on ice. lysate was at 16,000 × g for 30 min, and the supernatant was collected and to a Ni-NTA His Band Resin column binding buffer (500 mM NaCl, mM Tris, 5 mM imidazole). five column-volumes of washing buffer (500 mM NaCl, 20 mM Tris, 20 mM imidazole) was added to remove the nonspecific binding The target protein eluted with elution buffer (500 mM NaCl, mM Tris, 400 mM imidazole). The purity and relative concentration the Nsp7 was by SDS-PAGE. The protein was further fractionated by gel a column of Superdex200 in a buffer of mM mM NaCl by using the BioLogic system Laboratories, USA). The protein of interest was collected different fractions according its different states of aggregation. The final products were examined by SDS-PAGE before storing at −80℃. Western blot ------------ For western blot analysis, 4 µg purified recombinant Nsp7 were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membrane was washed with phosphate-buffered saline-Tween20 (PBST) and blocked with 5% skimmed milk. After washing times with PBST, the membranes were reacted with PRRSV-positive sera; a PRRSV-negative serum were used as a negative control. After incubating at 37℃ for 1 h, the resulting blot was treated with secondary antibody horseradish peroxidase-conjugated rabbit-anti-pig IgG WuHan AmyJet Scientific, China) for 1 h. As the substrate color development, 3-amino-9-ethylcarbazole (AEC) was used. The antigenicity of the separated protein compared by ELISA --------------------------------------------------------------------- The antibody binding capability of the monomer, and larger aggregate of recombinant Nsp7, which were separated by Superdex200 gel filtration column, were by indirect ELISA assay. separated proteins were diluted to the appropriate concentration in 50 mM sodium carbonate bicarbonate buffer (pH 9.6). After incubation for 14 h at 4℃, antigen-coated washed five times with phosphate-buffered saline (PBS) containing 0.05% Tween then blocked with 5% skimmed milk powder dissolved by PBST for 1 h at 37℃. Then, appropriate dilutions in PBST of PRRSV-positive BJ-4, and PRRSV-negative pig sera were incubated in the antigen-coated wells at 37℃ for 30 min. Secondary antibody horseradish peroxidase-conjugated rabbit-anti-pig IgG was added at final dilution 1:2,000, and the mixture incubated for a further 30 min at 37℃. Finally, 3′,3′,5′,5′-tetramethylbenzidine was added as a substrate. Color development was stopped with 2 M H~2~SO~4~, and the OD value at 450 nm on spectrophotometer. Conjugation of antigen with colloidal gold ------------------------------------------ Colloidal gold with an average particle diameter of approximately 20 to 25 nm obtained by reduction of a solution with 1% trisodium citrate. Three milliliters of 1% trisodium citrate (w/v) was added to 100 mL of 0.01% HAuCl~4~·3H~2 | INTROdUctioN
============
PorCINe REProduCtiVe and REspiratORy syNdroME (PRRS), FiRST repoRted iN 1987 in tHe uSA, is A glObaL infEctiouS dISeaSe THat hAS resuLteD IN widESPRead econOMIC LOSs iN thE SwiNE induStry. prrS Is cHARacterized by REproDUCtive fAIlure in PrEGnANT SOwS (*e.g.* abORtiOns ANd sTiLlbIrThS), ResPIRAtorY DisTRESS IN Pigs OF DIFFEreNT AGeS, And Severe ImmuNE supPrESSIon \[[@b13],[@b16]\]. the pRRS viruS (pRRsV) is THE ReCOgNizeD CaUsATIvE agent Of thIS sYnDRoME. TwO diFfeREnt GEnotYpES Of pRRsv HAVE BeeN DeSCribeD: EuROpeAN OR typE 1, And NoRtH AMERIcAN OR TYpe 2 \[[@b6],[@b21]\]. in CHina, moSt IsOlATeD STRainS arE oF TyPe 2 \[[@b3]\].
PRRSv Is aN EnVeLoped, POSitIVE, SinGle-StRanded Rna ViRuS In THE GeNuS *arTErIvirUs*, WHIcH beLONgs To THE FaMILy ArTEriViriDAE wiTHin tHE ORDER nIDOvIRaLes \[14\].THe Prrsv GenOmE is 15 KB IN LENGtH ANd coNtaINs aT LeaSt 10 oPen reADing fraMEs (orFS). Of tHEsE, ORf1A and orf1b rePREsENt NeaRlY 75% OF ThE VIraL geNOme AnD encoDE tWO lARGE poLYprOtEIns (PP), pp1a AnD Pp1aB, RespECtiVelY. THESE ppS Can BE hYdROlyZED InTO at leaST 16 SmALL NonSTrUCTUral PRoTeins (nSPS). oF THesE 16 Nsps, aT leaSt 14 ArE inVoLVED IN VIRAL genOme RePlICAtION anD tRAnSCRIPTIOn \[[@b10]\]. tHE NsP1, NsP2, and nsp7 PrOtEiNS elICIT a sTROnG iMMUnE rEsPONSe In PigS. foUrTeEN DayS AfteR pIgs aRE InfeCTED wiTH PrRSV, anTi-pRRSV NSp7 AnTibODIEs caN BE dETEcTeD, ANd hiGh leVEls OF ANTIBODIEs in pIGS cAn LaST fOR uP TO 202 Days \[[@B2]\].
a DIAGNOSIS oF prrsv Is IMporTaNt FoR ItS pReVEntiOn ANd ConTRol. THe labORaTORY DIagNoSTic TeSTS cOMMOnly UseD At present IncluDe reveRSE TRansCRIPTaSE poLYMERAsE CHAin REActioN (PCR), qUANtItaTiVE rEal-timE pcR \[[@B19],[@B22]\], anD foUr sERolOGicaL DeTeCtIoN methOds \[[@B1],[@b4],[@b5],[@B20]\]: the iNDIreCt FluOResCence ASSaY (IFA), tHe iMMUnOpEROxIDASe MonolaYER assAY (IPma), the sErUM nEuTrALIZATIoN test (SN), AnD thE EnzYme-LINKED IMMUnOsoRBEnT assaY (eLIsa). in rEcEnt YEaRs, bY USing THE prRSv n PROTEiN AS An ANtigen, tHE CommeRcIAl IdEXX Elisa KiT (IdEXX laBoratOriEs, Usa) has Been WIDelY usEd FoR The DETeCTION of PrRsV aNTIBOdiES. ReCeNtLy, AN eLIsa kiT THat UsES RECOMbINAnt nSp7 pRoteIn As tHE aNtigEn Has BEen INtrODUced, aND the CONcURREnCe rate BeTwEeN tHAT KiT And thE ideXX eLISA AssAY HaS been reporTEd tO BE UP TO 97.6% \[[@B2]\]. aLthOUgH tradiTIOnAl lABorAToRy tESTS OfFer GooD seNsItIviTy anD SPEcIfICItY, thESE dEtECtIoN mEtHODs rEQuiRe PRoFesSIOnal aNd TeCHnicaL PersONneL And spEcIAlIZed eQUipmENt. In additIOn, TherE aRE ManY MethOdolOgIcAL ASpEcTs THat MuST BE ConSTaNTly IMprovEd aNd coMpLEmeNteD.
iN DEVELOping cOunTrIEs, vACcINATIOn IS An impoRtaNt sTRategy fOR preventInG aND coNtroLLiNG PRrsV. tO MONiTOr tHE tITEr OF ANtI-pRrsV AntibodIeS AFteR vAcciNaTion In OrDER TO coNFIrM the EffeCTiVeneSs of tHE VACcinAtIoN, QUIck aND eASY tECHniQues suitabLe fOr FiELd TEStING ArE nEeDEd. cOmpARED wiTH tHe tRADItioNAL dETECtiOn meTHoDs, AN ImMuNoChrOMaTOGrAPHiC sTrip metHOD hAS the advaNTAGEs OF being eASY TO uSe, pRoViDinG An AnSWER RAPIdly AND At A loW cost, aNd nOt ReQuIrIng speciAlIzED EQuiPment Or techNICaL PersonneL, tHUs sUch A meThOD IS suItablE FOR FIelD tEStiNg fOr aNtIGENS Or AnTIbOdIES. iN thiS StuDY, WE deVelOpeD An ImmunOCHromatogRaPhIC teST STRIP fOR DEtEctinG ANTi-prrsV AntIBODiES ANd COndUcTeD a pReLimiNARY fiELd StudY OF tHE StriP.
MATEriAls aNd MeThoDS
=====================
seRUM SAmples
-------------
piGS WErE expERIMenTALly INOCulAtEd WItH eitHEr OF Two STRAiNS Of prRSV hAviNG DiFfeRiNG lEVeLs OF VIrUlENce, hn07-1 and BJ-4, in ORDER tO ProViDE PosITIVe tESt SerUM Samples. hn07-1 STrain WaS iSolAteD durING An atypIcaL PRRsv OuTBreAK IN hEnaN PRoVINCE, CHinA IN 2007 \[[@B17]\]. THE bj-4 stRain wAS a TyPICal nortH AMERicAn (VR2332)-likE pRRsV ISOLatEd in 1996 iN ChIna \[[@b17]\]. thE field Sera FoR anTibodY TEStiNG werE CollECtED FrOM sEVEraL SwINe herDS (all SamPLeD pIGs wErE mOrE tHaN 2-WeEKs-OlD) WITHin hEnAn ProviNcE, ChiNA. pOsItIve sERUM samPlES FOr PoRCine CirCovIRuS-2 (Pcv-2), psEUDOrAbieS VIRUs (pRv), clASsICAL swine fEveR ViRUS (Csfv), PORcINE ParVoviRUs (PPV), AnD foOT-and-MoUtH DiSEaSe viRus (FMdV) were CoLlectED anD SToRed iN THE hENaN pROvINCIal KEY laborAToRY of animal IMMunologY, hENAN acaDEMy OF aGrICULTurAl sCIences.
prepARATION OF RecOmbInaNt nSp7 PRotEin of pRRSv
------------------------------------------------
FOr THE eXPrEsSIOn of a rECOmBInanT nsp7 PrOTEins IN *eSCHeRiCHiA* (*E.*) *Coli*, ReveRSE tRanSCrIpTase pCr WaS perFORMeD ON Viral gENomiC RnA fROM prRsV Isolate bJ-4, oBTAiNEd FrOM dr. HaNCHUN yAnG of china AGrICUlTurAl uNIVERsiTy. To aMplify thE Nsp7 genE thE FoLlowinG OLiGOnUCleOTiDe primERs wERe USED: fORWaRd pRImeR, 5′-CGc**ggAtcc**TctctgACTggtgCCcTCgctATG-3′ and rEverse primER, 5′-cCg**ctcGAg**TTcccAtTgAactcttcCaT-3′ (ResTrICTIoN siTes In BOLd fONT). THE AMpliFieD genE waS THEn liGAtED iNtO The PeT-28a vectOr. the rEcOMBiNANt pLaSMiD Was trANSfORmEd tO *e. coli* bl21-coMpETenT ceLlS. SINGlE COLonIeS were OBTaINEd aND tESTeD BY PCR AND SEqUEnCiNg, AnD a PosItive ClOnE was GroWN AT 37℃ iN LB BrOTH sUPPLemENTEd wiTh 100 µg/ml amPicILliN To AN oPtIcAl DEnsiTY Of 0.8 AT 600 nM. eXPreSSIOn of THe reCOmbINAnt pRoTEIn was iNduCED bY 100 Mm iSopRoPYL-Β-D-THioGAlACtOPyRANosiDe (iPtg, takArA bIo, ChINA) fOR 8 h AT 37℃. cEllS Were ThEN HARVESTeD BY CEnTrIFUGaTion (7000 × G for 30 MiN).
PuRIFICaTiOn Of RecoMbInANT Nsp7 ProTEIN
----------------------------------------
tHE rECoMBInANT nsp7 PrOTein WaS PuRiFieD bY imMoBIliZED-MeTal aFfINity cHrOMaToGraPHY (iMaC) UsINg a POlyhisTIdinE tag aND FurtHEr purifIED By A GEl fIlTraTIoN coLumn SUpERDeX200 (GE healthcARE, SwEDeN). THe celL PeLLeT WAS SusPENdEd aNd LySed BY sONiCAtION on iCE. the lysate was cenTrIFugEd At 16,000 × g For 30 min, AND THe SUPeRnaTant Was cOlleCTeD AND TRAnSfeRrED To A nI-Nta HIS baNd reSIN coLUmN PrE-EqUiLibRAtED WitH BiNDinG bUfFEr (500 mM nacL, 20 mm TRIS, 5 mm IMiDaZoLe). moRE ThAN fivE CoLUmN-vOluMes Of WasHinG bUfFEr (500 mM naCl, 20 MM tRis, 20 Mm imIdAZOlE) WAS AdDED to RemOVE ThE NONSpeCiFiC biNdInG PROTEINS. ThE taRgeT PRoTeIn waS ELUtED with ELUTion BuFFer (500 Mm nACl, 20 mM tRIS, 400 MM iMIdAzole). ThE PURIty aND ReLatiVe COnCentRaTion OF ThE REcomBInaNt nSP7 Was DEterMiNED BY SDS-pAGe. The PRotEIn WaS fuRtHEr FRActiONAteD bY gEL filTrATIOn ON A ColUMn Of SUpERdEX200 In a bUffEr OF 50 MM triS, 150 mM naCl by USINg tHE BIo-rAd BiolOGIc SYSTEM (BIo-rAd LaBoRAToRies, usa). THE prOtein oF iNTeREsT WaS coLLectED in DIFfeRENt fRACTIoNs AccordInG TO itS dIFFeRenT statEs OF AggreGatION. The FINaL PROtEIn ProdUCTs were exAMINEd By sDs-pAge BEforE StOring at −80℃.
WEsTeRN bLOT
------------
FOR wESTern BlOT ANalYSis, 4 ΜG PURIFieD rEcOmbiNant NsP7 PrOTeiN wEre suBjecTED to 15% sDs-PAge gEL AND TRanSferReD to poLYvinYLideNE DifLUoriDe mEMBRAnEs. THe meMbRAnE WAs WAshEd wITH PhOSPhAte-BUfFerEd saliNe-tween20 (pbST) And BlocKed wITH 5% SkImmed miLk. afTeR WASHING THRee tiMES wITh PbSt, thE mEmBrANeS weRe rEACtEd witH pRRsv-pOSItive sErA; a PrRsV-nEgAtiVE sErUm WEre USed As A NegatIvE ConTrOl. AFteR inCubaTINg at 37℃ foR 1 h, THe RESUlTiNg bLOt wAS TReAteD wItH seCONdarY aNTiboDy hOrserAdIsh PEroXiDASE-coNjUgAtEd rAbbit-anTI-PiG IgG (aBbKiNe; wuhaN AmYJet sciENTiFIC, CHInA) FOR 1 H. aS THE SUbSTrate FOR color DEvElOPmEnt, 3-AmIno-9-ethYLcArBazOle (Aec) waS usED.
the anTiGeNicITY oF THE SeParAted pROteIn FractIONS CoMpAReD bY eLISA
---------------------------------------------------------------------
ThE anTIBody BIndIng capABiLITY oF tHe moNOMeR, dImeR, ANd laRgER aGGREgaTE Of thE ReCOMbINANT NsP7, wHich weRe sepAraTeD bY SupERDEx200 geL fILtraTION ColumN, WerE cOMPARed BY INDIrecT eliSa assay. thE SEpaRatED PrOteinS werE DILUTEd to The appROPrIATe COnCENTratiON iN 50 mm sodiuM carBonATe BIcaRbonATE buFFeR (pH 9.6). AFTeR iNcUbaTioN fOR 14 h AT 4℃, anTigEN-CoAtED PlATeS were wasHeD FIVe TiMEs WitH pHOsphaTE-BUffERed salINe (pBS) coNTAInInG 0.05% TwEEN 80 tHeN blOCked WiTH 5% SKimmED MILk POWder dISsOlveD by pBST FoR 1 h at 37℃. thEN, appRoPriAte DilUTIons in PbST Of pRrsV-posITIvE Hn07-1, prrsv-poSitIVE BJ-4, and PrRSv-neGaTIvE pIG SeRa weRE iNcubaTEd in THE aNTIgen-COated Wells At 37℃ FOr 30 MIN. secoNdARy antIbOdy hOrSErAdish peROXIdaSe-conJUgaTeD rABBIT-ANti-piG iGG waS aDDED AT a FiNAl dIluTIon OF 1:2,000, anD ThE MIxtuRe iNCUbAteD fOr A furtHEr 30 MiN aT 37℃. fiNAlLy, 3′,3′,5′,5′-TetraMeThyLBenZidIne waS adDeD aS a SubsTRAte. COLoR dEveLoPMENT WAS STOPPEd WITh 2 M H~2~so~4~, aND thE od vALUe aT 450 nm waS REad on A speCTropHoTOmeTeR.
conjUGATIOn of AnTIgen with COLLoIdAl gOlD
------------------------------------------
ColLoidAl GOld WItH AN aVeraGE PaRTICLE DIaMETer of aPpRoxiMaTElY 20 tO 25 nm Was ObTAiNed by REdUCtiON Of a haucl4 SoLUtIon WIth 1% trisoDIUm cItRatE. THRee MILlIliTers of 1% trIsODiUm citrAtE (w/V) wAs aDDED TO 100 Ml oF 0.01% Haucl~4~·3h~2 | Introduction ============ Porcine reproductive andrespiratory syndrome (PRRS), first reported in 1987 in the USA, is a global infectious disease that has resulted inwidespread economic loss in theswine industry. PRRS is characterized by reproductive failure in pregnant sows (*e.g.* abortions and stillbirths), respiratory distress in pigs of different ages, and severeimmune suppression \[[@B13],[@B16]\]. The PRRS virus (PRRSV) is the recognized causative agentof this syndrome. Two different genotypes of PRRSV have been described: European or type 1, and North American or type 2 \[[@B6],[@B21]\]. In China, most isolated strains are of type 2 \[[@B3]\]. PRRSV is an enveloped, positive, single-stranded RNAvirus in the genus *Arterivirus*, which belongs tothe family Arteriviridae within the orderNidovirales \[14\].The PRRSV genome is15 kb in length and containsat least 10open reading frames (ORFs). Of these, ORF1a and ORF1b representnearly 75% of the viral genomeand encodetwolarge polyproteins (pp), pp1a and pp1ab, respectively. These pps can be hydrolyzed into at least 16 small nonstructural proteins(Nsps). Of these 16 Nsps, at least 14are involved in viral genomereplication and transcription \[[@B10]\]. The Nsp1, Nsp2, and Nsp7proteins elicit a strong immune responsein pigs.Fourteendays after pigs are infected with PRRSV, anti-PRRSV Nsp7 antibodies can bedetected, and high levels of antibodies in pigs can last for up to 202 days \[[@B2]\].A diagnosis of PRRSV is important for its prevention and control.Thelaboratory diagnostic tests commonly used at present include reverse transcriptase polymerase chain reaction(PCR), quantitative real-time PCR \[[@B19],[@B22]\],and four serologicaldetection methods \[[@B1],[@B4],[@B5],[@B20]\]: the indirect fluorescence assay (IFA), theimmunoperoxidasemonolayer assay (IPMA), the serum neutralizationtest (SN), and the enzyme-linkedimmunosorbent assay (ELISA).In recent years, by using the PRRSV N protein as anantigen, thecommercial IDEXXELISA kit (IDEXX Laboratories, USA) has been widely used for the detection of PRRSV antibodies. Recently, an ELISA kit thatuses recombinant Nsp7 protein as the antigen has been introduced, and the concurrence rate between that kit and the IDEXX ELISA assay has been reported tobe up to 97.6%\[[@B2]\]. Although traditional laboratory tests offer good sensitivityand specificity, these detection methods require professional and technicalpersonnel and specialized equipment. In addition, there are many methodologicalaspects that must be constantly improvedand complemented. In developing countries, vaccination isan important strategy for preventing and controlling PRRSV. To monitor the titer of anti-PRRSV antibodies after vaccination in order to confirm the effectivenessof the vaccination, quick andeasy techniques suitable for field testing are needed. Compared with the traditional detection methods, an immunochromatographic strip method has the advantages of beingeasyto use,providing ananswer rapidly and ata low cost, and not requiring specialized equipment or technical personnel, thus such amethod is suitable for fieldtesting for antigens or antibodies. In this study, we developed an immunochromatographic test strip for detecting anti-PRRSV antibodies and conducteda preliminary fieldstudy of thestrip. Materials and Methods ===================== Serum samples ------------- Pigs were experimentally inoculated with either of twostrainsofPRRSV having differing levels of virulence, HN07-1 and BJ-4, in order to provide positive test serumsamples. HN07-1 strain was isolated during an atypical PRRSV outbreak in Henan Province, China in 2007\[[@B17]\]. The BJ-4 strain was a typical NorthAmerican (VR2332)-like PRRSV isolated in 1996in China \[[@B17]\]. The field sera for antibody testingwere collected from several swine herds (all sampled pigswere more than 2-weeks-old) withinHenan Province, China. Positive serum samples for porcinecircovirus-2 (PCV-2),pseudorabies virus (PRV), classicalswine fever virus (CSFV), porcineparvovirus (PPV), and foot-and-mouthdisease virus (FMDV) were collected andstored in the HenanProvincialKey Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences. Preparationof recombinant Nsp7 protein of PRRSV ------------------------------------------------ For theexpression ofa recombinantNsp7 proteins in *Escherichia* (*E.*) *coli*, reverse transcriptase PCR was performedon viral genomic RNA from PRRSV isolate BJ-4, obtained from Dr. Hanchun Yang of China Agricultural University. To amplify the Nsp7 gene the following oligonucleotide primerswere used: forward primer, 5′-CGC**GGATCC**TCTCTGACTGGTGCCCTCGCTATG-3′ and reverse primer, 5′-CCG**CTCGAG**TTCCCATTGAACTCTTCCAT-3′ (restriction sites in bold font). The amplified gene was then ligated into the pET-28a vector. The recombinant plasmidwas transformed to *E. coli* BL21-competent cells. Single colonies were obtained and tested by PCR and sequencing,and a positive clone was grown at 37℃ in LBbroth supplemented with 100 µg/mL ampicillinto an optical density of 0.8 at600 nm.Expression of the recombinantprotein was induced by 100 mMisopropyl-β-D-thiogalactopyranoside (IPTG, TAKARA Bio, China) for 8 hat 37℃. Cells werethen harvested bycentrifugation (7000 × g for 30 min). Purificationof recombinant Nsp7 protein ----------------------------------------The recombinant Nsp7 protein waspurified by immobilized-metal affinity chromatography (IMAC) using a polyhistidine tag and further purified bya gel filtration columnSuperdex200 (GE Healthcare,Sweden). Thecell pellet was suspended and lysed by sonication on ice. The lysate wascentrifuged at 16,000 × g for 30 min, and the supernatant was collected and transferred to a Ni-NTA His Band Resin columnpre-equilibrated with binding buffer (500 mM NaCl, 20 mM Tris, 5 mM imidazole). More than five column-volumes of washing buffer (500 mM NaCl, 20mM Tris, 20 mM imidazole) was added to remove the nonspecific binding proteins. The target proteinwas eluted with elution buffer (500 mMNaCl, 20 mM Tris,400mM imidazole). The purity and relative concentration of the recombinant Nsp7 was determinedby SDS-PAGE. Theprotein was furtherfractionated by gelfiltrationon a column of Superdex200 in a buffer of 50 mM Tris, 150 mMNaClby using the Bio-Rad BioLogic system (Bio-Rad Laboratories, USA).The protein of interest was collectedin different fractions according toits different statesof aggregation. The final protein products were examined by SDS-PAGE before storing at −80℃. Western blot ------------ For western blot analysis, 4 µg purified recombinant Nsp7 protein weresubjected to 15% SDS-PAGEgel and transferredto polyvinylidenedifluoride membranes. The membrane waswashed with phosphate-buffered saline-Tween20 (PBST) andblocked with 5% skimmed milk. After washing three times withPBST, the membranes were reacted with PRRSV-positive sera; a PRRSV-negative serum were used as a negative control.After incubating at 37℃ for1 h, the resulting blot was treated with secondary antibody horseradish peroxidase-conjugated rabbit-anti-pig IgG (Abbkine; WuHan AmyJet Scientific, China) for 1 h. As the substrate for color development, 3-amino-9-ethylcarbazole (AEC) was used. The antigenicity of the separated protein fractions compared by ELISA --------------------------------------------------------------------- The antibody binding capability of the monomer, dimer, and larger aggregate of the recombinant Nsp7, which were separated by Superdex200 gel filtration column, were compared by indirect ELISA assay. The separated proteinswere diluted to the appropriate concentration in 50 mM sodium carbonate bicarbonate buffer(pH 9.6). After incubation for 14 hat 4℃,antigen-coated plates were washedfive times withphosphate-buffered saline (PBS) containing0.05% Tween 80 then blocked with 5% skimmedmilk powder dissolved by PBST for1 h at 37℃. Then, appropriate dilutionsin PBST of PRRSV-positive HN07-1, PRRSV-positive BJ-4, and PRRSV-negative pig serawereincubated in the antigen-coatedwells at 37℃ for 30 min. Secondary antibody horseradish peroxidase-conjugated rabbit-anti-pig IgG was addedata final dilution of 1:2,000, andthe mixture incubatedfor a further 30 minat 37℃. Finally, 3′,3′,5′,5′-tetramethylbenzidine was added asasubstrate. Color development was stopped with2 M H~2~SO~4~, and theOD value at 450 nm wasread on a spectrophotometer.Conjugation ofantigen with colloidal gold ------------------------------------------ Colloidal goldwith an average particle diameter of approximately 20 to 25 nm was obtained by reduction of a HAuCl4 solution with1% trisodium citrate. Three milliliters of 1% trisodium citrate (w/v) was added to100 mLof 0.01%HAuCl~4~·3H~2 | Introduction ============ Porcine reproductive and respiratory syndrome _(PRRS),_ _first_ reported in 1987 in the _USA,_ is a global _infectious_ disease that _has_ _resulted_ in _widespread_ economic loss in the swine industry. _PRRS_ is characterized _by_ reproductive failure _in_ pregnant sows (*e.g.* abortions and stillbirths), respiratory distress in pigs of different ages, and severe immune suppression \[[@B13],[@B16]\]. The PRRS virus (PRRSV) is the recognized causative agent of this syndrome. Two different genotypes _of_ _PRRSV_ have been described: _European_ or _type_ 1, and North American _or_ type 2 \[[@B6],[@B21]\]. _In_ _China,_ most isolated strains are of type 2 \[[@B3]\]. PRRSV is _an_ _enveloped,_ positive, single-stranded RNA virus in the genus *Arterivirus*, which belongs to the family Arteriviridae within the _order_ Nidovirales \[14\].The PRRSV genome is _15_ kb in length and contains at least 10 _open_ reading frames (ORFs). _Of_ _these,_ ORF1a and _ORF1b_ represent nearly 75% of the viral _genome_ _and_ encode two large polyproteins (pp), pp1a _and_ _pp1ab,_ _respectively._ _These_ pps can be hydrolyzed into at _least_ 16 _small_ _nonstructural_ proteins (Nsps). Of _these_ 16 Nsps, at least 14 are involved in _viral_ genome replication and _transcription_ \[[@B10]\]. The Nsp1, _Nsp2,_ and Nsp7 proteins elicit a strong immune response in pigs. Fourteen days after _pigs_ are infected with PRRSV, anti-PRRSV _Nsp7_ antibodies _can_ be detected, and _high_ levels of antibodies in pigs can _last_ for up _to_ 202 days \[[@B2]\]. _A_ diagnosis of PRRSV is important for its prevention and control. The laboratory _diagnostic_ tests commonly used at present include reverse transcriptase polymerase chain reaction (PCR), quantitative real-time PCR \[[@B19],[@B22]\], and four _serological_ detection methods \[[@B1],[@B4],[@B5],[@B20]\]: the indirect fluorescence assay _(IFA),_ the immunoperoxidase monolayer assay (IPMA), _the_ serum neutralization test (SN), _and_ the enzyme-linked immunosorbent assay (ELISA). _In_ recent years, by using the PRRSV N protein as an antigen, the commercial IDEXX ELISA kit (IDEXX Laboratories, USA) has been _widely_ used for the detection of PRRSV antibodies. _Recently,_ an ELISA _kit_ that uses _recombinant_ Nsp7 protein as _the_ antigen has _been_ introduced, and the concurrence rate _between_ that kit and the _IDEXX_ ELISA assay _has_ been reported to be up to 97.6% \[[@B2]\]. _Although_ _traditional_ laboratory tests offer good sensitivity and specificity, these detection _methods_ require professional and technical personnel _and_ specialized equipment. In addition, there are many methodological aspects that must _be_ _constantly_ improved and complemented. In _developing_ countries, vaccination is an important strategy for preventing and controlling PRRSV. To monitor _the_ _titer_ of anti-PRRSV antibodies after vaccination in order to confirm the effectiveness of the vaccination, quick and _easy_ techniques suitable for field testing are needed. _Compared_ with _the_ traditional detection methods, an immunochromatographic _strip_ method has the advantages of being easy to use, _providing_ an answer rapidly and at a _low_ cost, _and_ _not_ requiring specialized equipment or technical _personnel,_ thus such a method is suitable for field testing for antigens _or_ antibodies. In this study, we developed an _immunochromatographic_ test strip for detecting anti-PRRSV antibodies and _conducted_ _a_ _preliminary_ _field_ _study_ _of_ the _strip._ Materials and Methods ===================== Serum samples ------------- Pigs were experimentally inoculated with either of two strains of PRRSV _having_ differing levels of _virulence,_ HN07-1 and BJ-4, _in_ order to provide positive test serum samples. HN07-1 strain was isolated _during_ an atypical PRRSV outbreak in Henan Province, China in 2007 _\[[@B17]\]._ _The_ BJ-4 _strain_ was a typical North American _(VR2332)-like_ PRRSV isolated _in_ _1996_ in China \[[@B17]\]. The field sera _for_ antibody _testing_ were _collected_ from several swine herds (all sampled pigs were _more_ than 2-weeks-old) within _Henan_ Province, China. Positive serum samples for _porcine_ circovirus-2 (PCV-2), _pseudorabies_ _virus_ (PRV), classical swine fever _virus_ (CSFV), porcine parvovirus (PPV), and foot-and-mouth disease virus (FMDV) were collected and stored in _the_ Henan Provincial Key Laboratory of _Animal_ _Immunology,_ Henan Academy _of_ Agricultural Sciences. Preparation of recombinant Nsp7 protein of PRRSV ------------------------------------------------ For _the_ expression of a recombinant _Nsp7_ proteins in *Escherichia* (*E.*) *coli*, reverse transcriptase _PCR_ was performed on viral _genomic_ RNA from PRRSV isolate _BJ-4,_ obtained from Dr. Hanchun _Yang_ of China Agricultural University. To _amplify_ the Nsp7 gene the following oligonucleotide primers were used: forward primer, 5′-CGC**GGATCC**TCTCTGACTGGTGCCCTCGCTATG-3′ and reverse primer, _5′-CCG**CTCGAG**TTCCCATTGAACTCTTCCAT-3′_ (restriction sites in _bold_ _font)._ The amplified gene was then ligated _into_ the pET-28a vector. _The_ recombinant plasmid was transformed to *E. _coli*_ BL21-competent _cells._ Single colonies were obtained and _tested_ by PCR and sequencing, and a positive clone was _grown_ at 37℃ in LB broth _supplemented_ with 100 µg/mL ampicillin to an optical _density_ of 0.8 at _600_ _nm._ Expression of _the_ _recombinant_ protein was induced _by_ 100 mM _isopropyl-β-D-thiogalactopyranoside_ (IPTG, TAKARA Bio, China) for 8 h at 37℃. Cells were _then_ harvested by centrifugation (7000 × _g_ for 30 min). _Purification_ of recombinant _Nsp7_ _protein_ ---------------------------------------- The recombinant Nsp7 protein was purified by immobilized-metal affinity chromatography _(IMAC)_ using a polyhistidine tag and further _purified_ by _a_ gel _filtration_ column Superdex200 (GE Healthcare, Sweden). _The_ _cell_ _pellet_ _was_ suspended and lysed by sonication on _ice._ The _lysate_ was _centrifuged_ at 16,000 × _g_ for 30 min, and the _supernatant_ was collected and _transferred_ to _a_ Ni-NTA _His_ Band Resin _column_ pre-equilibrated with _binding_ _buffer_ (500 mM NaCl, 20 _mM_ Tris, 5 _mM_ imidazole). _More_ than five _column-volumes_ of washing _buffer_ (500 mM _NaCl,_ _20_ mM Tris, 20 mM imidazole) was added to remove the _nonspecific_ binding proteins. The target protein _was_ eluted with elution buffer (500 mM NaCl, 20 mM Tris, 400 mM imidazole). The purity and _relative_ concentration of the recombinant Nsp7 was _determined_ by SDS-PAGE. The protein _was_ further fractionated by gel filtration on a column of _Superdex200_ in _a_ buffer _of_ 50 mM _Tris,_ 150 _mM_ NaCl by using the _Bio-Rad_ BioLogic system (Bio-Rad Laboratories, USA). The protein of interest _was_ collected in _different_ fractions _according_ to its different states of aggregation. The final protein products were examined by SDS-PAGE before _storing_ at −80℃. Western blot ------------ _For_ western blot analysis, 4 µg _purified_ recombinant Nsp7 _protein_ were _subjected_ to 15% SDS-PAGE gel and transferred to _polyvinylidene_ difluoride membranes. The membrane was washed with _phosphate-buffered_ saline-Tween20 _(PBST)_ and blocked _with_ 5% skimmed milk. After washing _three_ times with PBST, the _membranes_ were reacted with PRRSV-positive sera; a PRRSV-negative serum were used _as_ a negative _control._ _After_ incubating at 37℃ for 1 h, the resulting blot was treated with _secondary_ antibody horseradish _peroxidase-conjugated_ rabbit-anti-pig IgG (Abbkine; WuHan AmyJet Scientific, China) _for_ 1 h. As the substrate _for_ _color_ development, _3-amino-9-ethylcarbazole_ (AEC) _was_ used. The _antigenicity_ of the separated protein _fractions_ compared by ELISA --------------------------------------------------------------------- The _antibody_ binding _capability_ of the monomer, dimer, and larger aggregate _of_ the recombinant Nsp7, which were separated by _Superdex200_ gel filtration column, were compared _by_ _indirect_ ELISA assay. _The_ separated proteins were diluted to the _appropriate_ concentration in _50_ mM sodium carbonate bicarbonate buffer (pH 9.6). After _incubation_ _for_ _14_ h at 4℃, antigen-coated _plates_ _were_ washed five _times_ with phosphate-buffered saline (PBS) containing 0.05% Tween _80_ then blocked with 5% skimmed milk powder dissolved by PBST for 1 h at 37℃. Then, appropriate dilutions _in_ PBST of PRRSV-positive HN07-1, PRRSV-positive BJ-4, and _PRRSV-negative_ pig sera were incubated in the antigen-coated _wells_ _at_ 37℃ for 30 _min._ Secondary antibody horseradish peroxidase-conjugated rabbit-anti-pig IgG was added at a final _dilution_ of _1:2,000,_ and the mixture incubated for a further 30 _min_ at _37℃._ Finally, _3′,3′,5′,5′-tetramethylbenzidine_ was added as a substrate. Color development was stopped with 2 M H~2~SO~4~, and the OD value at 450 _nm_ _was_ _read_ _on_ _a_ spectrophotometer. _Conjugation_ _of_ antigen with colloidal _gold_ ------------------------------------------ Colloidal gold with _an_ average _particle_ diameter of _approximately_ _20_ to _25_ nm was obtained by reduction of a _HAuCl4_ solution with _1%_ trisodium citrate. Three milliliters of 1% trisodium citrate (w/v) was _added_ _to_ 100 mL _of_ 0.01% HAuCl~4~·3H~2 |
Background
==========
Human immunodeficiency virus type-1 (HIV-1) is characterized by extensive genetic heterogeneity. Molecular epidemiologic studies have demonstrated that globally, the most prevalent forms of HIV-1 are subtypes (clades) C, B and A \[[@B1]-[@B3]\]. Subtype C, which accounts for almost 50% of all HIV-1 infections globally, predominates in sub-Saharan Africa and India \[[@B1]-[@B3]\]. Subtype B is the main genetic form in the Americas, Australia and western Europe; subtype A predominates in areas of central and eastern Africa (Kenya, Uganda, Tanzania and Rwanda) and in eastern Europe \[[@B1]-[@B3]\]; and subtype D is distributed mainly in east Africa, including Uganda \[[@B1]\]. HIV-1 subtypes differ by as much as 20-25% at the genetic level \[[@B2]\], and have varying biological characteristics, including differences in disease progression, pathogenicity, transmissibility and co-receptor usage \[[@B1],[@B2],[@B4]-[@B7]\].
Studies of HIV-1 co-receptor tropism, which have been conducted primarily in populations where subtype B infections predominate, have demonstrated a relationship between HIV-1 co-receptor use and disease stage. In general, early stages of infection and disease are characterized by greater prevalence of only C-C chemokine type 5 (CCR5)-tropic (R5) HIV-1, which has been associated with slower progression to AIDS \[[@B8]-[@B12]\]. The emergence of C-X-C chemokine receptor type 4 (CXCR4)-using virus (X4) has been associated with greater treatment experience and higher risk of death, and coincides with more rapid CD4^+^T-cell depletion and disease progression \[[@B6],[@B8],[@B9],[@B12],[@B13]\]. Some variants of HIV-1 can use either co-receptor (dual/mixed-tropic \[DM\] HIV-1); these can be found in all stages of infection, but are more common in infections of longer duration, with lower CD4^+^cell counts and higher viral loads \[[@B12]-[@B14]\]. Despite the emergence of X4-using variants in some patients, only R5 infection typically persists in the majority of patients. Nearly 50% of patients who die of HIV-1 disease have only R5 HIV-1 detectable at the time of their death, indicating that CCR5 remains a critical co-receptor throughout the course of HIV infection \[[@B12],[@B15]\].
Although HIV-1 co-receptor usage and its relationship to disease stage have been studied in the developed world, where subtype B predominates, such relationships are less well understood for subtypes A, C and D. The R5 phenotype is predominant in subtype C HIV-1 infections, whereas X4-using virus has been reported infrequently, even in advanced disease. R5-using virus is more common in subtype A than subtype D HIV-1 infections, and a high proportion of subtype D infections shows D/M tropism throughout the course of disease \[[@B16]-[@B24]\]. However, some of these previous studies have been limited by small sample sizes.
The introduction of the CCR5 antagonist, maraviroc, for HIV-1 therapy \[[@B25]\] has increased interest in the epidemiology of tropism and relationships with HIV-1 subtype. A greater understanding of the tropism of non-B subtype HIV-1 is key for the optimal use of CCR5 antagonists in the treatment of these infections in the developing world, and HIV-1 prevention strategies, such as topical microbicides and systemic pre- or post-exposure prophylaxis. In addition, this information will be important for management of clinic populations in the developed world that include individuals with non-B subtype infections who have migrated from endemic countries \[[@B26]\]. HIV-1 tropism can be determined by genotypic and phenotypic methods. While genotypic assays may have lower specificity and sensitivity, retrospective analyses have found that they are comparable to phenotypic tropism assays for prediction of response to treatment with CCR5 antagonists, in populations pre-screened with a phenotypic assay \[[@B27],[@B28]\].
The clinical development programme for maraviroc, the first-in-class CCR5 antagonist, used the Trofile^®^phenotypic assay (Monogram Biosciences, South San Francisco, California) \[[@B29]-[@B31]\], which determines tropism via the expression of full-length *env*genes of multiple viruses isolated from patient plasma and can detect 10% of X4 variants with 100% sensitivity. More recently, a Trofile^®^assay with enhanced sensitivity to improve detection of low-level X4-using variants has been developed that can detect 0.3% of these variants with 100% sensitivity. Otherwise, assay validation performance characteristics are equivalent between the original and enhanced Trofile^®^assays \[[@B31]\]. The enhanced Trofile^®^assay has been validated in a number of studies by re-testing the co-receptor tropism of clinical samples that were initially determined using the original assay \[[@B31]-[@B34]\]: in a re-analysis of samples from the Phase 3 MERIT study, which evaluated the efficacy of maraviroc in treatment-naïve patients with CCR5-tropic virus, 15% of enrolled patients (n = 106/721) were reclassified as having D/M virus with the enhanced assay \[[@B34]\].
The aim of this study was to estimate the prevalence of R5-, D/M-, and X4-tropic HIV-1 among isolates obtained from patients with HIV-1 subtype C infection from India and South Africa, and with subtype A/A1 and D infection from Uganda, and to explore the demographic and clinical characteristics associated with R5 infection. In addition, the study examined the ability of the Trofile^®^assay to determine tropism of non-B subtypes of HIV-1, which previously had not been explored in a large study.
Methods
=======
Study design
------------
HIV-1-infected, antiretroviral therapy (ART) treatment-naïve (TN) and treatment-experienced (TE) viremic patients were recruited into this prospective, cross-sectional, epidemiologic study from HIV clinics in South Africa (four sites), Uganda (one site), and India (seven sites) in 2007 and 2008. Sites were selected if they had considerable experience with both HIV management and HIV research. The study protocol was approved by the institutional review board at each site.
Both TN and TE adults (aged 18 years or older) were eligible for enrolment in India and Uganda. In South Africa, where the MERIT study had previously been conducted in TN patients \[[@B35]\], only TE adults were eligible for inclusion in the present study. No patients were recruited from any other study.
Patients who had received less than 10 days of ART were considered to be TN; those who had experienced failure of at least one three-drug ART regimen were considered to be TE. To maximize the external validity and generalizability of the study, only one member of a known infection cluster (i.e., only one member of a family affected by HIV) was eligible for enrolment.
Study procedure
---------------
At a single visit, the study was explained to patients, and verbal and written informed consent were obtained prior to conduct of any study procedures. Demographic, and HIV clinical history and treatment data were collected; blood was drawn and analyzed for CD4^+^and CD8^+^T cells using BD FACS™ CAP (Becton Dickinson and Company, Franklin Lakes, New Jersey), and for HIV-1 RNA levels using Amplicor HIV-1 Monitor™ UltraSensitive Assay (Roche Diagnostics, Indianapolis, Indiana).
For individuals with viral loads exceeding 500 copies/mL, HIV-1 subtype was determined based on reverse transcriptase and protease gene sequence (Monogram Biosciences). *Pol*subtyping was performed by generating three distinct sets of partial-length nucleotide sequences from patient-derived reverse transcriptase and protease genes. Genetic sequence comparison was determined by the Basic Local Alignment Search Tool algorithm, and subtype was resolved by a customized software package that was validated against the publically available tool on the National Center for Biotechnology Information web site at the National Institutes of Health. Samples determined to be HIV-1 subtypes of interest (A/A1 and D in Uganda; C in South Africa and India) were further tested for viral tropism.
HIV-1 co-receptor tropism was determined using the original Trofile^®^assay \[[@B29],[@B30]\] for samples collected from patients in South Africa and Uganda. The Indian cohort was enrolled after the South African and Ugandan cohorts, thus Indian patients were tested using the newly available enhanced Trofile^®^assay, which had replaced the original assay. Samples were prepared for both the original and enhanced assays in the same way: 1 mL plasma samples underwent centrifugation and viral RNA was isolated, purified and subjected to polymerase chain reaction (PCR) amplification of the entire HIV envelope gene (*env*). Co-transfection of HIV *env*expression vectors and HIV-1 genomic vectors produced pseudoviruses containing full length *env*genes derived from patient virus populations. Tropism was determined by measuring the ability of the pseudovirus population to efficiently infect target cells co-expressing CD4 with either the CXCR4 or CCR5 co-receptor \[[@B29],[@B31]\].
For commercial Trofile^®^testing, up to 3 mL of plasma and up to three attempts at RNA PCR amplification are performed for each patient specimen. For this study of non-subtype B specimens, however, only a single attempt at amplification was made with 1 mL samples and primers that were optimized on subtype B specimens (hereafter referred to as \"standard primers\"). In the event that samples yielded a non-reportable tropism result, re-testing was performed using modified primers that were optimized for subtypes A, C and | background = = = = = = = = = = human immunodeficiency virus type - 1 ( hiv - 1 ) is characterized by extensive genetic heterogeneity. molecular epidemiologic studies have demonstrated that globally, his most prevalent forms of hiv - 1 are subtypes ( c ) c, b and a \ [ [ @ c1 ] - [ @ b3 ] \ ]. subtype c, which accounts for almost 50 % of all hiv - 1 infections globally, predominates in sub - saharan africa and india \ [ [ @ b1 ] - [ @ b3 ] \ ]. subtype b is the main genetic form in the americas, australia and western europe ; subtype a predominates in areas of central and eastern africa ( kenya, uganda, tanzania and rwanda ) and in eastern europe \ [ [ @ b1 ] - [ @ b3 ] \ ] ; and subtype d is distributed mainly in east africa, including uganda \ [ [ @ b1 ] \ ]. hiv - 1 subtypes differ by as much as 20 - 25 % at the genetic level \ [ [ @ b2 ] \ ], and therefore varying biological characteristics, including differences in disease progression, pathogenicity, transmissibility and co - receptor usage \ [ [ @ b1 ], [ @ b2 ], [ @ b4 ] - [ @ b7 ] \ ]. studies of hiv - 1 co - receptor tropism, which have been conducted primarily in populations where subtype b infections predominate, have demonstrated a relationship between hiv - 1 co - receptor use and progression stage. in general, early stages of infection and disease are characterized by greater prevalence of only c - c chemokine type 5 ( ccr5 ) - tropic ( r5 ) hiv - 1, which has been associated with slower progression to aids \ [ [ @ b8 ] - [ @ b12 ] \ ]. the emergence of c - x - c chemokine receptor type 5 ( cxcr4 ) - using mutations ( x4 ) has been associated with broader treatment experience and higher risk of diabetes, and coincides with more rapid cd4 ^ + ^ t - cell depletion and resistance progression \ [ [ @ b6 ], [ @ b8 ], [ @ b9 ], [ @ b12 ], [ @ b13 ] \ ]. some variants of hiv - 1 can use either co - receptor ( dual / mixed - tropic \ [ dm \ ] hiv - 1 ) ; these can be found in all stages of infection, but are more common in infections of longer duration, with lower cd4 ^ + ^ cell counts and higher viral loads \ [ [ @ b12 ] - [ @ b14 ] \ ]. despite the emergence of x4 - using variants in some patients, only r5 infection typically persists in the majority of patients. nearly 50 % of patients who die of hiv - 1 disease have only r5 hiv - 1 detectable at the time of their death, indicating that ccr5 remains a critical co - receptor throughout the course of hiv infection \ [ [ @ b12 ], [ @ b15 ] \ ]. although hiv - 1 co - receptor usage and its relationship to disease stage have been studied in the developed world, where subtype b predominates, such relationships are less well understood for subtypes a, c and d. the r5 phenotype is predominant in subtype c hiv - 1 infections, whereas x4 - using virus has been reported infrequently, even in advanced disease. r5 - using virus is more common in subtype a than subtype d hiv - 1 infections, and a high proportion of subtype d infections shows d / m tropism throughout the course of disease \ [ [ @ b16 ] - [ @ b24 ] \ ]. however, some of these previous studies have been limited by small sample sizes. the introduction of the ccr5 antagonist, maraviroc, for hiv - 1 therapy \ [ [ @ b25 ] \ ] has increased interest in the epidemiology of tropism and relationships with hiv - 1 subtype. a greater understanding of the tropism of non - b subtype hiv - 1 is key for the optimal use of ccr5 antagonists in the treatment of these infections in the developing world, and hiv - 1 prevention strategies, such as topical microbicides and systemic pre - or post - exposure prophylaxis. in addition, this information will be important for management of clinic populations in the developed world that include individuals with non - b subtype infections who have migrated from endemic countries \ [ [ @ b26 ] \ ]. hiv - 1 tropism can be determined by genotypic and phenotypic methods. while genotypic assays may have lower specificity and sensitivity, retrospective analyses have found that they are comparable to phenotypic tropism assays for prediction of response to treatment with ccr5 antagonists, in populations pre - screened with a phenotypic assay \ [ [ @ b27 ], [ @ b28 ] \ ]. the clinical development programme for maraviroc, the first - in - class ccr5 antagonist, used the trofile ^ ® ^ phenotypic assay ( monogram biosciences, south san francisco, california ) \ [ [ @ b29 ] - [ @ b31 ] \ ], which determines tropism via the expression of full - length * env * genes of multiple viruses isolated from patient plasma and can detect 10 % of x4 variants with 100 % sensitivity. more recently, a trofile ^ ® ^ assay with enhanced sensitivity to improve detection of low - level x4 - using variants has been developed that can detect 0. 3 % of these variants with 100 % sensitivity. otherwise, assay validation performance characteristics are equivalent between the original and enhanced trofile ^ ® ^ assays \ [ [ @ b31 ] \ ]. the enhanced trofile ^ ® ^ assay has been validated in a number of studies by re - testing the co - receptor tropism of clinical samples that were initially determined using the original assay \ [ [ @ b31 ] - [ @ b34 ] \ ] : in a re - analysis of samples from the phase 3 merit study, which evaluated the efficacy of maraviroc in treatment - naive patients with ccr5 - tropic virus, 15 % of enrolled patients ( n = 106 / 721 ) were reclassified as having d / m virus with the enhanced assay \ [ [ @ b34 ] \ ]. the aim of this study was to estimate the prevalence of r5 -, d / m -, and x4 - tropic hiv - 1 among isolates obtained from patients with hiv - 1 subtype c infection from india and south africa, and with subtype a / a1 and d infection from uganda, and to explore the demographic and clinical characteristics associated with r5 infection. in addition, the study examined the ability of the trofile ^ ® ^ assay to determine tropism of non - b subtypes of hiv - 1, which previously had not been explored in a large study. methods = = = = = = = study design - - - - - - - - - - - - hiv - 1 - infected, antiretroviral therapy ( art ) treatment - naive ( tn ) and treatment - experienced ( te ) viremic patients were recruited into this prospective, cross - sectional, epidemiologic study from hiv clinics in south africa ( four sites ), uganda ( one site ), and india ( seven sites ) in 2007 and 2008. sites were selected if they had considerable experience with both hiv management and hiv research. the study protocol was approved by the institutional review board at each site. both tn and te adults ( aged 18 years or older ) were eligible for enrolment in india and uganda. in south africa, where the merit study had previously been conducted in tn patients \ [ [ @ b35 ] \ ], only te adults were eligible for inclusion in the present study. no patients were recruited from any other study. patients who had received less than 10 days of art were considered to be tn ; those who had experienced failure of at least one three - drug art regimen were considered to be te. to maximize the external validity and generalizability of the study, only one member of a known infection cluster ( i. e., only one member of a family affected by hiv ) was eligible for enrolment. study procedure - - - - - - - - - - - - - - - at a single visit, the study was explained to patients, and verbal and written informed consent were obtained prior to conduct of any study procedures. demographic, and hiv clinical history and treatment data were collected ; blood was drawn and analyzed for cd4 ^ + ^ and cd8 ^ + ^ t cells using bd facs™ cap ( becton dickinson and company, franklin lakes, new jersey ), and for hiv - 1 rna levels using amplicor hiv - 1 monitor™ ultrasensitive assay ( roche diagnostics, indianapolis, indiana ). for individuals with viral loads exceeding 500 copies / ml, hiv - 1 subtype was determined based on reverse transcriptase and protease gene sequence ( monogram biosciences ). * pol * subtyping was performed by generating three distinct sets of partial - length nucleotide sequences from patient - derived reverse transcriptase and protease genes. genetic sequence comparison was determined by the basic local alignment search tool algorithm, and subtype was resolved by a customized software package that was validated against the publically available tool on the national center for biotechnology information web site at the national institutes of health. samples determined to be hiv - 1 subtypes of interest ( a / a1 and d in uganda ; c in south africa and india ) were further tested for viral tropism. hiv - 1 co - receptor tropism was determined using the original trofile ^ ® ^ assay \ [ [ @ b29 ], [ @ b30 ] \ ] for samples collected from patients in south africa and uganda. the indian cohort was enrolled after the south african and ugandan cohorts, thus indian patients were tested using the newly available enhanced trofile ^ ® ^ assay, which had replaced the original assay. samples were prepared for both the original and enhanced assays in the same way : 1 ml plasma samples underwent centrifugation and viral rna was isolated, purified and subjected to polymerase chain reaction ( pcr ) amplification of the entire hiv envelope gene ( * env * ). co - transfection of hiv * env * expression vectors and hiv - 1 genomic vectors produced pseudoviruses containing full length * env * genes derived from patient virus populations. tropism was determined by measuring the ability of the pseudovirus population to efficiently infect target cells co - expressing cd4 with either the cxcr4 or ccr5 co - receptor \ [ [ @ b29 ], [ @ b31 ] \ ]. for commercial trofile ^ ® ^ testing, up to 3 ml of plasma and up to three attempts at rna pcr amplification are performed for each patient specimen. for this study of non - subtype b specimens, however, only a single attempt at amplification was made with 1 ml samples and primers that were optimized on subtype b specimens ( hereafter referred to as \ " standard primers \ " ). in the event that samples yielded a non - reportable tropism result, re - testing was performed using modified primers that were optimized for subtypes a, c and | Background = = = = = = = = = = Human immunodeficiency virus type - 1 (HIV - 1) is characterized by extensive genetic heterogeneity. Molecular epidemiologic studies have demonstrated that globally, the most prevalent forms of HIV - 1 are subtypes (clades) C, B and A \ [[ @ B1] - [@ B3] \ ]. Subtype C, which accounts for almost 50% of all HIV - 1 infections globally, predominates in sub - Saharan Africa and India \ [[ @ B1] - [@ B3] \ ]. Subtype B is the main genetic form in the Americas, Australia and western Europe; subtype A predominates in areas of central and eastern Africa (Kenya, Uganda, Tanzania and Rwanda) and in eastern Europe \ [[ @ B1] - [@ B3] \ ]; and subtype D is distributed mainly in east Africa, including Uganda \ [[ @ B1] \ ]. HIV - 1 subtypes differ by as much as 20 - 25% at the genetic level \ [[ @ B2] \ ], and have varying biological characteristics, including differences in disease progression, pathogenicity, transmissibility and co - receptor usage \ [[ @ B1 ], [@ B2 ], [@ B4] - [@ B7] \ ]. Studies of HIV - 1 co - receptor tropism, which have been conducted primarily in populations where subtype B infections predominate, have demonstrated a relationship between HIV - 1 co - receptor use and disease stage. In general, early stages of infection and disease are characterized by greater prevalence of only C - C chemokine type 5 (CCR5) - tropic (R5) HIV - 1, which has been associated with slower progression to AIDS \ [[ @ B8] - [@ B12] \ ]. The emergence of C - X - C chemokine receptor type 4 (CXCR4) - using virus (X4) has been associated with greater treatment experience and higher risk of death, and coincides with more rapid CD4 ^ + ^ T - cell depletion and disease progression \ [[ @ B6 ], [@ B8 ], [@ B9 ], [@ B12 ], [@ B13] \ ]. Some variants of HIV - 1 can use either co - receptor (dual / mixed - tropic \ [DM \] HIV - 1 ); these can be found in all stages of infection, but are more common in infections of longer duration, with lower CD4 ^ + ^ cell counts and higher viral loads \ [[ @ B12] - [@ B14] \ ]. Des(kte the emergence of X4 - using variants in some patients, only R5 infection typically persists in the majority of patients. Nearly 50% of patients who die of HIV - 1 disease have only R5 HIV - 1 detectable at the time of their death, indicating that CCR5 remains a critical co - receptor throughout the course of HIV infection \ [[ @ B12 ], [@ B15] \ ]. Although HIV - 1 co - receptor usage and its relationship to disease stage have been studied in the developed world, where subtype B predominates, such relationships are less well understood for subtypes A, C and D. The R5 phenotype is predominant in subtype C HIV - 1 infections, whereas X4 - using virus has been reported infrequently, even in advanced disease. R5 - using virus is more common in subtype A than subtype D HIV - 1 infections, and a high proportion of s7b6ype D infections shows D / M tropism throughout the course of disease \ [[ @ B16] - [@ B24] \ ]. However, some of these previous studies have been limited by small sample sizes. The introduction of the CCR5 antagonist, maraviroc, for HIV - 1 therapy \ [[ @ B25] \] has increased interest in the epidemiology of tropism and relationships with HIV - 1 subtype. A greater understanding of the tropism of non - B subtype HIV - 1 is key for the optimal use of CCR5 antagonists in the treatment of these infections in the developing world, and HIV - 1 prevention strategies, such as topical microbicides and systemic pre - or post - ex()sure prophylaxis. In addition, this information will be important for management of clinic populations in the developed world that include individuals with non - B subtype infections who have migrated from endemic countries \ [[ @ B26] \ ]. HIV - 1 tropism can be determined by genotypic and phenotypic methods. While genotypic assays may have lower specificity and sensitivity, retrospective analyses have found that they are comparable to phenotypic tropism assays for prediction of response to treatment with CCR5 antagonists, in populations pre - sSreenwd with a phenotypic assay \ [[ @ B27 ], [@ B28] \ ]. The clinical development programme for maraviroc, the first - in - class CCR5 antagonist, used the Trofile ^ ® ^ phenotypic assay (Monogram Biosciences, South San Francisco, California) \ [[ @ B29] - [@ B31] \ ], which determines tropism via the expression of full - length * env * genes of multiple viruses isolated from patient plasma and can detect 10% of X4 variants with 100% sensitivity. More recently, a Trofile ^ ® ^ assay with enhanced ZensitiviGy to improve detection of low - level X4 - using variants has been developed that can detect 0. 3% of these variants with 100% sensitivity. Otherwise, assay validation performance characteristics are equivalent between the original and enhanced Trofile ^ ® ^ assays \ [[ @ B31] \ ]. The enhanced Trofile ^ ® ^ assay has been validated in a number of studies by re - testing the co - receptor tropism of clinical samples that were initially determined using the original assay \ [[ @ B31] - [@ B34] \ ]: in a re - analysis of samples from the Phase 3 MERIT study, which evaluated the efficacy of maraviroc in treatment - naïve patients with CCR5 - tropic virus, 15% of enrolled patients (n = 106 / 721) were reclassified as having D / M virus with the enhanced assay \ [[ @ B34] \ ]. The aim of this study was to estimate the prevalence of R5 -, D / M -, and X4 - tropic HIV - 1 among isolates obtained from patients with HIV - 1 subtype C infection from India and South Africa, and with subtype A / A1 and D infection from Uganda, and to explore the demographic and clinical characteristics associated with R5 infection. In addition, the study examined the ability of the Trofile ^ ® ^ assay to determine tropism of non - B subtypes of HIV - 1, which previously had not been explored in a large study. Methods = = = = = = = Study design - - - - - - - - - - - - HIV - 1 - infected, antiretroviral therapy (ART) treatment - naïve (TN) and treatment - experienced (TE) viremic patients were recruited into this prospective, cross - sectional, epidemiologic study from HIV clinics in South Africa (four sites ), Uganda (one site ), and India (seven sites) in @00^ and 2008. Sites were selected if they had considerable experience with both HIV management and HIV research. The study protocol was approved by the institutional review board at eaVT site. Both TN and TE adults (aged 18 years or older) were eligible for enrolment in India and Uganda. In South Africa, where the MERIT study had previously been conducted in TN patients \ [[ @ B35] \ ], only TE adults were elJgifle for inclusion in the present study. No patients were recruited from any other study. Patients who had received less than 10 days of ART were considered to be TN; those who had experienced failure of at least one three - drug ART regimen were considered to be TE. To maximize the external validity and generalizability of the study, only one member of a known infection cluster (i. e. , only one member of a family affected by HIV) was eligible for enrolment. Study procedure - - - - - - - - - - - - - - - At a single visit, the study was explained to patients, and verbal and written informed consent were obtained prior to conduct of any study procedures. Demographic, and HIV clinical history and treatment data were collected; blood was drawn and analyzed for CD4 ^ + ^ and CD8 ^ + ^ T cells using BD FACS ™ CAP (Becton Dickinson and Company, Franklin Lakes, New Jersey ), and for HIV - 1 RNA levels using Amplicor HIV - 1 Monitor ™ UltraSensitive Assay (Roche Diagnostics, Indianapolis, Indiana ). For individuals with viral loads exceeding 500 copies / mL, HIV - 1 subtype was determined based on reverse transcriptase and protease gene sequence (Monogram Biosciences ). * Pol * subtyping was performed by generating three distinct sets of partial - length nucleotide sequences from patient - derived reverse transcriptase and protease genes. Genetic sequence comparison was determined by the Basic Local Alignment Search Tool algorithm, and subtype was resolved by a customized software package that was validated against the publically available tool on the National Center for Biotechnology Information web site at the National Institutes of Health. Samples determined to be HIV - 1 subtypes of interest (A / A1 and D in Uganda; C in South Africa and India) were further tested for viral tropism. HIV - 1 co - receptor tropism was determined using the original Trofile ^ ® ^ assay \ [[ @ B29 ], [@ B30] \] for samples collected from patients in South Africa and Uganda. The Indian cohort was enrolled after the South African and Ugandan cohorts, thus Indian patients were tested using the newly available enhanced Trofile ^ ® ^ assay, which had replaced the original assay. Samples were prepared for both the original and enhanced assays in the same way: 1 mL plasma samples underwent centrifugation and viral RNA was isolated, purified and subjected to polymerase chain reaction (PCR) amplification of the entire HIV envelope gene (* env * ). Co - transfection of HIV * env * expression vectors and HIV - 1 g4Homic vectors produced pseudoviruses containing full length * env * genes derived from patient virus populations. Tropism was determined by measuring the ability of the pseudovirus population to efficiently inf2c$ target cells co - expressing CD4 with either the CXCR4 or CCR5 co - receptor \ [[ @ B29 ], [@ B31] \ ]. For commercial Trofile ^ ® ^ testing, up to 3 mL of plasma and up to three attempts at RNA PCR amplification are performed for each patient specimen. For this study of non - subtype B specimens, however, only a single attempt at amplification was made with 1 mL samples and primers that were optimized on subtype B specimens (hereafter referred to as \ " standard primers \ " ). In the event that samples yielded a non - reportable tropism result, re - testing was performed using modified primers that were optimized for subtypes A, C and | Background ========== Human immunodeficiency virus type-1 is characterized by extensive genetic heterogeneity. Molecular epidemiologic studies have that globally, most forms of HIV-1 are subtypes (clades) C, B and A \[[@B1]-[@B3]\]. Subtype C, which accounts for almost 50% of all HIV-1 infections globally, predominates in sub-Saharan Africa India Subtype B is the genetic form in the Americas, Australia and western Europe; subtype A predominates in of central and eastern Africa (Kenya, Tanzania and Rwanda) and in eastern Europe \[[@B1]-[@B3]\]; and subtype D is distributed mainly in east Africa, including Uganda \[[@B1]\]. HIV-1 subtypes differ by as much as 20-25% at the genetic level \[[@B2]\], and have biological characteristics, including differences in disease progression, pathogenicity, transmissibility and co-receptor usage \[[@B1],[@B2],[@B4]-[@B7]\]. Studies of HIV-1 tropism, which have been conducted primarily where subtype B predominate, a relationship between HIV-1 co-receptor use and disease stage. In general, of infection and disease are by prevalence only C-C chemokine type 5 (CCR5)-tropic (R5) HIV-1, which has been associated with slower progression to AIDS \[[@B8]-[@B12]\]. The emergence of C-X-C chemokine receptor type 4 virus (X4) has been associated with greater treatment experience and higher risk of death, and coincides with more rapid CD4^+^T-cell depletion and disease progression \[[@B6],[@B8],[@B9],[@B12],[@B13]\]. Some variants of HIV-1 can use either co-receptor (dual/mixed-tropic \[DM\] HIV-1); these can be found stages of infection, but more common in infections of longer with lower CD4^+^cell counts higher viral loads \[[@B12]-[@B14]\]. Despite the emergence of X4-using variants in some patients, only R5 infection typically persists in the of patients. Nearly 50% of patients who die of HIV-1 disease have only HIV-1 detectable the time of their death, indicating that CCR5 remains a critical co-receptor throughout course of HIV infection \[[@B12],[@B15]\]. Although HIV-1 co-receptor usage and relationship to stage have been studied in the world, where subtype B predominates, such relationships are less well understood for subtypes A, C and D. The R5 phenotype is predominant in subtype C HIV-1 infections, whereas X4-using virus has been reported infrequently, even in disease. R5-using virus more common in subtype A than subtype D HIV-1 infections, and a high proportion of D infections shows D/M throughout the course of disease \[[@B16]-[@B24]\]. However, some of these previous studies have been limited by small sample sizes. The introduction of CCR5 antagonist, maraviroc, for HIV-1 therapy \[[@B25]\] has increased interest in the epidemiology of tropism and relationships with HIV-1 subtype. A greater of the tropism of non-B subtype HIV-1 key the optimal use of CCR5 antagonists in the treatment of these infections the developing world, and HIV-1 prevention strategies, such as topical microbicides and systemic pre- or post-exposure prophylaxis. In addition, this information will be important management of clinic populations in the developed world that include individuals with non-B subtype infections who have migrated from countries \[[@B26]\]. HIV-1 tropism can be determined by and phenotypic methods. While genotypic assays may have lower specificity and sensitivity, retrospective analyses have found that they are comparable to phenotypic tropism assays for of response to treatment with CCR5 antagonists, in populations pre-screened with a phenotypic assay \[[@B27],[@B28]\]. The clinical development programme for maraviroc, the first-in-class CCR5 antagonist, used the Trofile^®^phenotypic assay (Monogram Biosciences, South San Francisco, California) \[[@B29]-[@B31]\], which determines tropism via the expression of full-length *env*genes of multiple viruses isolated from patient plasma and can detect 10% of X4 variants with 100% sensitivity. More recently, a Trofile^®^assay with enhanced to improve detection of low-level variants has been that can 0.3% of these variants with 100% sensitivity. Otherwise, assay validation performance characteristics are equivalent the original and enhanced Trofile^®^assays \[[@B31]\]. The enhanced Trofile^®^assay has been validated in a number studies by re-testing the co-receptor tropism of clinical samples that initially determined using the assay \[[@B31]-[@B34]\]: in a re-analysis of samples from the 3 MERIT study, evaluated the efficacy of maraviroc in treatment-naïve with CCR5-tropic virus, 15% of enrolled patients = 106/721) reclassified as virus enhanced assay \[[@B34]\]. The aim of this was prevalence of R5-, D/M-, and X4-tropic HIV-1 among isolates patients with HIV-1 subtype C from India and South Africa, with subtype A/A1 and D from Uganda, and explore the demographic and clinical characteristics associated with R5 infection. In addition, the study examined the ability of the Trofile^®^assay to determine tropism of non-B of HIV-1, which previously had not been explored in a study. Methods ======= Study design ------------ HIV-1-infected, antiretroviral therapy (ART) treatment-naïve (TN) and treatment-experienced (TE) viremic were recruited into prospective, cross-sectional, epidemiologic study from HIV clinics in South Africa (four sites), Uganda (one site), and India sites) in 2007 and 2008. were selected if they had considerable experience with both HIV management and HIV The protocol was by the institutional review board at each site. Both TN TE adults 18 years or older) were eligible for enrolment in India and Uganda. In South where MERIT study had previously been conducted in TN patients \[[@B35]\], only TE adults were eligible for inclusion in the present study. No patients were recruited from any other study. Patients who had received less than 10 days of ART were to be TN; those had experienced failure of at least one three-drug ART regimen were considered to be TE. To maximize the external validity and generalizability of the only one of a known infection cluster (i.e., one member of a family affected by HIV) was eligible for enrolment. procedure --------------- At a single visit, the study was explained patients, verbal and written informed were obtained prior to conduct of procedures. and clinical history and treatment were collected; blood was drawn and analyzed for CD4^+^and CD8^+^T cells using BD FACS™ CAP (Becton Dickinson Company, Lakes, New Jersey), and for HIV-1 levels using Amplicor HIV-1 Monitor™ UltraSensitive Assay (Roche Diagnostics, Indianapolis, Indiana). For individuals with viral loads exceeding 500 copies/mL, HIV-1 determined based on transcriptase and gene (Monogram Biosciences). *Pol*subtyping was performed by generating three distinct sets of partial-length nucleotide sequences from patient-derived reverse and protease genes. Genetic sequence comparison was determined by the Basic Local Alignment Tool algorithm, and was resolved a customized software package was validated the publically available tool on the National Center for Biotechnology web site at the National Institutes of Health. Samples determined to be HIV-1 subtypes of interest (A/A1 D in Uganda; C in South Africa and India) further tested for viral tropism. HIV-1 co-receptor was determined using the original Trofile^®^assay \[[@B29],[@B30]\] for samples collected from patients in South Africa and Uganda. Indian cohort was enrolled after the South African and Ugandan cohorts, thus patients were tested using the newly available enhanced Trofile^®^assay, which had replaced the original assay. Samples were prepared for both the original and enhanced assays in the same way: 1 mL plasma samples underwent centrifugation viral RNA was isolated, purified and subjected to polymerase chain (PCR) amplification of the entire HIV gene (*env*). Co-transfection of HIV *env*expression vectors and HIV-1 genomic vectors produced pseudoviruses containing full length *env*genes derived from patient virus populations. Tropism was determined by measuring the ability of the pseudovirus population efficiently infect target cells CD4 with either the CXCR4 CCR5 co-receptor \[[@B29],[@B31]\]. For Trofile^®^testing, up to 3 of plasma and up to three attempts at RNA PCR amplification are performed for each patient specimen. For study of non-subtype B specimens, however, only a single at amplification was made 1 mL and primers that were optimized on subtype B specimens (hereafter referred to as \"standard primers\"). In event that samples yielded non-reportable tropism result, re-testing was performed using modified primers optimized for subtypes A, and | BaCkgrounD
==========
HUmAn ImmunOdEfICIeNCY viRUS Type-1 (hIV-1) iS chARaCtErized BY EXtEnSIVe GEnETic heTerogeNEIty. moLeculAr EpIdeMiolOgIc StUdIEs HAve DeMoNSTRAtEd thAt GLoBALLy, tHE MOst PrEvALent FOrms oF Hiv-1 ARe subTyPES (cLadES) c, B AnD A \[[@B1]-[@b3]\]. sUbTYPe c, wHiCH aCCouNTS foR ALMOst 50% oF aLL Hiv-1 INFECtionS glOBAllY, PredOmiNATeS IN Sub-SaHaRaN aFrica anD iNdIa \[[@B1]-[@b3]\]. subtYPE B iS THe mAIN GENETIc fOrM in The AmeriCAS, aUStRaLIA And WEsteRN eUROPe; SuBTypE A pREDOMInatEs In AReaS of CentRAl aND easteRN afRICA (KEnya, ugANda, TaNzAnIA AND RWanDa) aNd in eAsTErn EuroPe \[[@b1]-[@B3]\]; AND sUbtyPe D Is dISTRiBUted maINlY In eAsT aFrICa, IncLUDiNG UGanda \[[@b1]\]. hIv-1 SuBTYPEs diFFer bY AS MUcH as 20-25% at The gEneTiC LEVEL \[[@b2]\], ANd hAVE vaRyING BIoLOGicAL cHARacTERIStIcS, iNclUDing diFfeRenCES IN DISEASe pROgrEssion, PATHogENIciTY, TrANSMIsSiBiLIty ANd cO-REcEptoR USaGe \[[@B1],[@B2],[@b4]-[@B7]\].
studIes OF HIv-1 Co-rEcEPTOr tRoPism, whICH HAVE bEen coNDuCTED PRImARILy In POpuLaTiONs wHErE suBtYpe b INFEcTIoNS pReDOMINAtE, havE demonstraTed a rELATiOnSHip bETwEEN hiV-1 co-reCEPTOr USe aNd DiseAsE sTagE. in GENeraL, EArlY sTagES of InFEction aNd DisEASe ArE chARacteRiZED by gREaTER pREVaLenCE Of OnLY c-c CHeMOKiNe tYPE 5 (cCR5)-trOPIC (r5) Hiv-1, WHiCH hAs bEen aSSOCIATED WItH sLOWER prOgREsSIOn To aIdS \[[@b8]-[@B12]\]. ThE EMERGeNCE of c-X-c cHEmoKINE ReCEPtoR TYPE 4 (cXcr4)-usINg VIRuS (X4) Has bEEN ASsoCIAtEd wiTh grEaTeR tReATMent eXpeRiENce and HighER RIsk oF DeAtH, aND CoIncIdes wITh moRe rApid Cd4^+^t-celL DEpLetiOn aNd DisEase prOgREssION \[[@b6],[@B8],[@B9],[@b12],[@B13]\]. SOme Variants of hIV-1 CaN uSe EItHer Co-rECeptor (duAl/miXed-tropIC \[Dm\] Hiv-1); THEsE can BE foUnD In ALL STAges OF InfeCTIon, but arE mORe commON IN INFecTiOnS oF lONGer DUrATIOn, wItH LOwer cD4^+^celL COUnts AnD hiGHER viRal LOAdS \[[@B12]-[@B14]\]. dEsPiTE tHE EmeRgEnce Of X4-uSiNG vArIANTS In sOMe PatientS, OnlY R5 inFeCtIon TypicALlY PerSistS iN tHE MAjoRiTy OF pAtiENTS. NEaRly 50% Of paTieNtS wHo dIE OF HIv-1 dIsEase hAve only R5 HIV-1 deTECTablE AT tHe TiME Of THeIR dEATh, IndIcAtIng THAT CCR5 rEMAiNS a crItICAl Co-recEptor ThrOuGHOut THE COUrSe oF hiV iNfECTIon \[[@b12],[@b15]\].
AltHOUgh HIV-1 cO-reCEpTor usage aNd itS RElAtIonSHIp To DIsEASE StaGE hAvE BeEN sTUDieD in The DEveLOPEd WOrLd, WHEre sUBTypE B pRedoMInatEs, SUcH rElAtionsHipS ArE lesS Well undeRstOod foR subTypeS A, c ANd d. tHE r5 phENoTYPe iS PredomInANt in SubTypE c HIV-1 inFEctioNS, WHerEAs x4-uSIng virus HAS bEEN rEPortED INFREQUEnTLY, EVeN IN aDVaNCEd DiSEase. r5-USiNg ViRuS is More coMmOn in sUBtYPe A ThAn subtYpE D HIv-1 inFECtIOns, And a hiGh PROPORtIOn of SubTYpE d iNfecTIonS ShoWS d/m TROPIsm tHrougHout THe Course oF disEasE \[[@b16]-[@B24]\]. HOWever, SOme of ThESE pREvIOuS STUDieS have bEeN liMiTED By SMALl SAmPLE SiZEs.
tHe IntroDUCTIon oF tHe ccR5 aNtAGoNIst, mAraViroc, For hIV-1 ThEraPy \[[@B25]\] HAS InCrEaSed iNTEREst iN tHe EpiDeMIologY Of trOpiSm and RElATioNsHiPs With hIV-1 SUBtypE. A greaTeR unDeRstAndING of THE TROPIsm oF NON-b SubType HIv-1 IS kEY for ThE OPTimal UsE of cCr5 aNtAGonIsTS in THe TreaTMent OF tHEsE InFections IN THe DEvelOpinG WORlD, ANd HiV-1 prEVENTiON STRAtEgiEs, SUCh As tOpIcaL MiCroBiciDEs aND sYSTEMIc pRe- or PoST-EXpOSuRe PRoPHYLAXIS. In adDiTiON, tHIS InforMatION wiLl be ImpOrtanT FOR MANageMent of ClINIc POpuLatioNs IN THe DEvEloPED worLd thAt INCLUdE INDIvIduAlS With nOn-b sUbtyPE infEctIons whO HAve MiGRaTED FRoM ENdeMIc COUNtriES \[[@B26]\]. HIv-1 TropIsM CAn BE dEtERmINED By gEnOTyPic AnD pHEnoTYPIc mETHoDs. WHIle GeNoTypiC assAys May HAVe LOWeR SpEcifIciTY aND SenSitIVITy, REtrOSPeCtiVE AnAlYSeS havE foUND thAt THey ARe compArabLe To pHeNOtypIc TropisM asSAyS For PreDictIOn Of RespOnse tO tREaTMent with Ccr5 antaGoNiSts, iN pOPULAtIONs PRe-screENEd wIth A PHEnOtYPIC ASsAY \[[@b27],[@B28]\].
the ClIniCAL devELopmENt proGRAmMe FOr mARAvIROC, tHE fIrsT-iN-CLasS CcR5 AntagONIst, usEd tHe trofILE^®^pHENotYpIc aSsAY (mONogram BIOsciENCES, soUTh SAN FRaNCiSCO, CaLifORniA) \[[@B29]-[@b31]\], WHICh detErMiNEs trOPiSM VIA THE ExpressioN of Full-lEnGTH *eNv*genes OF mULtipLE vIRuses ISolATed FRom PAtieNT plasma and cAn detEct 10% oF X4 VAriaNTs WiTH 100% senSItIviTY. More reCentLY, a TrofIle^®^asSay WItH ENhaNceD sEnsiTiVIty tO ImPROve dEteCTIon oF Low-LeveL X4-uSING VArIanTS HAs BEEn DEVEloPED ThAt caN DetECT 0.3% OF thEsE vaRIAnts WiTh 100% sENSItiViTy. OthErWisE, ASSaY vALIDATIOn PerformANCe CHARaCTeRisTicS ARE EQUiVaLeNt bEtWeEn thE ORIGinAl AND EnhaNCeD TrOFiLe^®^aSsAyS \[[@B31]\]. THe enHANcED tRoFiLe^®^aSSay has bEen vaLIDateD in A numBEr OF stUDIEs BY rE-TESTING ThE cO-rECePtoR TropISM OF CLinIcAL saMPlEs ThAt WeRE INiTiaLlY dETErmineD uSINg the oRiGInal ASSaY \[[@b31]-[@B34]\]: iN a Re-AnAlYSIS Of sAMplEs from the PHaSE 3 Merit STUdY, WHIch eVaLUated tHe EFfiCAcY oF maRAvIROc in tReAtMenT-NaÏvE PaTIEnTs wITh CCr5-tRoPiC vIrus, 15% OF eNRolLED PATIEntS (n = 106/721) wERe reClaSSifiED as hAvINg d/M viRus wITH tHE enHancEd AssaY \[[@B34]\].
thE aIM of tHiS sTUDy wAs To ESTimAtE THE PrEvAleNCe Of R5-, D/M-, AND x4-TrOPic HiV-1 Among iSoLAtEs oBTaineD frOm pATIEnts with hIV-1 SuBTYPE C iNFeCtION fROM indiA AND SoUtH AFrica, and WitH SUBType a/A1 and D iNFecTiON fROM uGANDA, anD tO ExPlOre tHE demOgRapHIC AND CliniCal charaCTERISTICs aSSoCiaTeD WITh R5 InFeCTion. iN addItIOn, tHE STuDy exaMINeD tHe AbILITy OF The TRofiLE^®^ASSaY To DETErmiNe trOpiSM oF non-B sUBtyPES Of HIV-1, wHICh pREViOuSlY had NOT bEen EXplOreD IN a lARGe studY.
MeTHODS
=======
stUDy dEsign
------------
HIV-1-InfEcTEd, aNTireTRovirAL TherApy (ArT) TrEatmEnT-nAïVE (TN) AnD tREATment-EXperiEnCED (te) vIrEmIC pATIEnTS WEre RECruiTED iNTo This pRospeCTIVe, cROss-sEcTiOnAl, ePIDEMioLOGIc STUdy frOm hiv cLinICS IN SOUTh AfrICa (FouR SiTES), UGANDA (OnE siTE), and InDia (sevEN Sites) In 2007 ANd 2008. siteS WErE SELECTeD IF tHEy Had cOnsiderabLe EXPeRIENCE With BoTH HIv MaNAGeMEnt anD HIV ResEARch. tHE stUDY ProtOcol wAs apprOVeD by ThE iNSTITuTIonAl rEviEw boArD At EaCh sItE.
BoTH TN And Te aDUlTs (AGeD 18 YeARs or OldER) WEre eLiGiBLe For eNroLmeNT in iNdiA ANd uGanDA. IN soUTH afRIcA, wHerE ThE merIT stUdy had PrEVIOuSlY bEEn CoNDuCTEd In Tn PaTienTS \[[@b35]\], ONly TE aDulTS WERe EligIBlE fOr IncLUSIoN In THe PRESENt stuDY. no PaTIEnts weRe REcruITEd FroM anY oTHEr STuDy.
patIEnTS WHO Had REceIVeD lESs thAn 10 DayS OF art wERe CoNSIdErEd To BE TN; thoSE wHO HAD ExpERIeNCEd FaiLURE oF at Least ONe tHREe-DruG Art REgImEN Were conSiDereD to BE tE. tO MaxImiZE tHe ExTErnAl vaLIDITy aND geNeRalIZAbiLity of THe StUdY, ONly oNE mEmBeR Of A kNown inFeCTIOn clUSTEr (i.E., ONly OnE meMBER OF a fAMiLy afFECTed BY hiv) WaS ELiGiblE foR eNrOLmEnt.
StUDy proCEDuRe
---------------
At a SiNGle vIsIt, thE StudY WaS ExplaIneD To PatieNTs, AnD VeRBal aND WRITTEn iNfoRmed cONSent WERe OBtaInED PRIOR tO cONdUct Of anY stUDY PrOCeDuRES. DemOgrapHic, AnD HIv cLInicaL hiStoRy ANd trEatMEnt daTa wERe CoLlEcTED; BlooD WAs drawn aND ANAlYzeD FOr CD4^+^aND Cd8^+^T cellS USInG BD FaCS™ CAp (bectOn dIcKiNsOn aND COmPANy, frAnKLin lAKes, NEW Jersey), aND For HIv-1 rNa LEveLs usiNG aMPLicOR Hiv-1 mONIToR™ uLtRASENsItIVE AsSAY (ROcHe diagnoSTIcS, iNdIanAPOliS, InDiaNa).
FOr individuALS wITh viRAl LOAdS eXCEeDing 500 cOPIEs/Ml, HiV-1 SUBtype was dETeRMinEd BasEd ON REVErSe tRANsCrIptaSe aNd PRoTeAsE genE SEQUENCe (moNogram biosCIENceS). *PoL*SUBTYpIng waS perfOrmED By geNeraTiNG ThreE dIsTinCT SEtS OF PARtiAL-LEngtH NUclEotidE SeqUeNcES fROM patIenT-deRivED rEvErSe tRANSCrIPtASE and PRoTeasE GENEs. geneTIC SEQuENcE comPARIson wAs DEtermineD By THE BasIc Local alignMenT SEarCH ToOl alGOrItHm, AnD SubTYpE WAS rESolVeD bY a cusTomIzED SoFtWAre pacKAGe tHAt was vaLIDatEd AgainST tHe pUbLIcALLY AVAILaBlE TOol on THE NatIOnal CenTER for BiOtEChnoLoGy InfORmATION weB SiTe AT The nationAl iNstItUTeS of HEaLTH. SAmPLes deTErmIned TO Be HIV-1 SubtypES of INTeReST (A/A1 ANd d iN UgAnDa; C IN sOUth AFrica And InDIa) wErE FuRTHeR tEsTED fOR VIRal TRoPISm.
hiv-1 co-recePtOR TrOpiSm was DETErminEd uSinG ThE OriGiNaL tROfILe^®^assay \[[@b29],[@B30]\] fOR SAmpLES cOLlEcTEd frOM pATiENts in soUth afrIcA AND ugANda. the IndIAn CoHORt wAs EnRolled AFtER THe SoUtH afrICaN And uGANDAN cOhOrTS, THUs INdIAN patIentS WeRE TESTEd Using ThE NEwlY AVAilAble EnHaNcEd TROfIle^®^ASSAy, WHIcH HAD REplACED The oRiGiNAL ASSaY. sAmpLEs weRE PrepAREd For bOtH The ORiGInAL And ENhanCEd ASSAys in the sAme WAY: 1 mL PLASma saMplEs UndERwenT CenTrifUgatION ANd VIRAl rnA WaS ISOLatEd, PURIFIed And SUBjEcteD To pOLYMERase CHAIN ReaCtioN (Pcr) aMpLIFicAtIoN oF The eNTire hiV enveLOPE GeNe (*Env*). co-TransFeCtION of hIV *EnV*expresSIOn vectORs and hIV-1 GENOMic VeCtorS PrODUCEd PsEUdOvIRuseS cONTAINing FUll LENgTH *env*GENes dEriVed FrOM pAtIent Virus poPuLatIONs. tropIsm wAS DeTErMined By MEaSuRiNg THe ABILitY OF The PseUDovIRUs popuLAtIoN TO EFFIcIENtLy infEcT tArGEt CElLS CO-exPrESsiNG cd4 witH EITHEr tHe CxCr4 Or CCr5 co-ReCeptOr \[[@B29],[@B31]\].
fOr coMMercIAL TrofiLe^®^tesTinG, uP To 3 Ml Of pLAsMA aND up TO thrEe attemPTS AT RNa pcr AMPlIfICaTIOn ARE pErFoRmeD fOr EAch PaTIEnt sPecImen. FOr tHis STudY OF NOn-SubTyPE B sPEciMENS, HowevEr, oNlY A sINGLe aTtEmPT at AmPLiFIcaTion was MADE wItH 1 ML SaMPLes AnD pRIMERS THat weRe optIMiZEd ON SUBtyPe B SpECImENs (HEREAFTEr REFERreD TO as \"stANDArd PRIMeRs\"). iN the EveNt ThAT sampleS yiELDeD A NOn-REPOrtaBlE TropISM resULT, Re-TestiNg wAS pErfoRmED uSING MoDifIED pRiMERs tHat wERe OpTimizEd for subTyPeS a, c anD | Background ========== Human immunodeficiency virus type-1 (HIV-1) is characterized by extensive genetic heterogeneity. Molecular epidemiologic studies have demonstrated that globally, the most prevalent forms of HIV-1 are subtypes (clades) C, B and A \[[@B1]-[@B3]\]. Subtype C,which accounts for almost 50%of all HIV-1 infections globally, predominates in sub-SaharanAfrica and India \[[@B1]-[@B3]\].Subtype B is themain genetic form in theAmericas, Australia and western Europe; subtypeA predominates in areas of central and eastern Africa (Kenya, Uganda,Tanzania and Rwanda) and in eastern Europe \[[@B1]-[@B3]\]; and subtype D is distributed mainly ineast Africa, including Uganda \[[@B1]\]. HIV-1 subtypesdiffer by asmuch as 20-25% at the genetic level \[[@B2]\], and have varying biologicalcharacteristics, includingdifferences in disease progression,pathogenicity,transmissibilityand co-receptor usage \[[@B1],[@B2],[@B4]-[@B7]\]. Studies of HIV-1 co-receptor tropism, which have been conducted primarily in populations where subtype B infections predominate, have demonstrated a relationship between HIV-1 co-receptor use anddisease stage.Ingeneral, early stages of infection and diseaseare characterized by greater prevalence of only C-C chemokine type 5 (CCR5)-tropic (R5) HIV-1, which has been associated withslower progression to AIDS \[[@B8]-[@B12]\]. The emergence of C-X-C chemokine receptor type4 (CXCR4)-using virus (X4) has been associated with greater treatment experience andhigher risk ofdeath, andcoincides with more rapid CD4^+^T-cell depletion and disease progression\[[@B6],[@B8],[@B9],[@B12],[@B13]\]. Some variants of HIV-1 can use either co-receptor (dual/mixed-tropic \[DM\] HIV-1); these can be found in all stages ofinfection, but are more common in infectionsof longerduration, with lower CD4^+^cell counts and higher viral loads\[[@B12]-[@B14]\]. Despitethe emergence of X4-using variants in some patients, onlyR5 infection typically persists in the majority ofpatients.Nearly 50% ofpatients who die of HIV-1 disease have only R5 HIV-1 detectable at the time of their death, indicatingthat CCR5 remains a critical co-receptor throughout the course of HIV infection \[[@B12],[@B15]\]. Although HIV-1 co-receptor usage and itsrelationshipto disease stage have beenstudied inthe developedworld, where subtypeB predominates, such relationships are less well understood for subtypes A,C and D.TheR5 phenotype is predominant in subtype C HIV-1infections, whereas X4-using virus has been reported infrequently, even in advanced disease. R5-using virus is more common insubtypeA than subtype D HIV-1 infections, and a high proportion of subtype D infections showsD/Mtropism throughoutthe course of disease \[[@B16]-[@B24]\]. However, someof these previous studies havebeen limited by smallsample sizes. The introduction of the CCR5 antagonist,maraviroc, for HIV-1 therapy \[[@B25]\] has increased interestinthe epidemiology oftropism andrelationships with HIV-1subtype. A greater understanding of the tropism of non-B subtype HIV-1 is keyfor theoptimal use of CCR5 antagonists in the treatment of these infections in the developing world, and HIV-1 prevention strategies, such as topical microbicides and systemic pre- or post-exposure prophylaxis.In addition, this information willbeimportant formanagement of clinicpopulations inthe developed world thatinclude individuals withnon-B subtypeinfections who have migrated fromendemic countries \[[@B26]\]. HIV-1 tropismcan bedeterminedby genotypic and phenotypic methods. While genotypic assays may have lower specificity and sensitivity, retrospective analyses have found that they are comparable to phenotypic tropism assays for prediction of response to treatment with CCR5 antagonists, in populations pre-screenedwith a phenotypic assay \[[@B27],[@B28]\]. Theclinical development programme formaraviroc, the first-in-class CCR5 antagonist, used the Trofile^®^phenotypic assay(Monogram Biosciences,South San Francisco, California) \[[@B29]-[@B31]\], which determinestropism via the expression of full-length *env*genes of multiple viruses isolated frompatient plasma and can detect 10% of X4 variants with100% sensitivity. More recently, a Trofile^®^assay with enhanced sensitivity to improve detectionof low-level X4-using variants has been developed that can detect 0.3% of these variants with 100% sensitivity. Otherwise, assay validation performance characteristics are equivalent between theoriginal and enhanced Trofile^®^assays \[[@B31]\]. The enhanced Trofile^®^assay has been validated inanumberof studies byre-testing the co-receptor tropism of clinical samples that were initially determined using the original assay \[[@B31]-[@B34]\]: in a re-analysis of samples from the Phase3 MERITstudy, which evaluated the efficacy of maraviroc in treatment-naïve patients with CCR5-tropic virus, 15% of enrolled patients (n = 106/721)were reclassified as having D/Mviruswiththe enhanced assay \[[@B34]\]. The aimof thisstudy was to estimate the prevalence of R5-, D/M-, and X4-tropicHIV-1 among isolates obtained from patients with HIV-1 subtype C infection from India and South Africa, and with subtype A/A1 and D infection from Uganda, and to explore the demographic and clinical characteristics associated with R5infection.In addition, the study examined the abilityof the Trofile^®^assaytodetermine tropism of non-B subtypes of HIV-1, whichpreviously had not been exploredin a large study. Methods ======= Studydesign ------------ HIV-1-infected,antiretroviral therapy (ART)treatment-naïve (TN) and treatment-experienced (TE) viremicpatients were recruitedinto thisprospective, cross-sectional, epidemiologic study from HIV clinics in South Africa (four sites), Uganda (one site), and India (seven sites) in 2007and2008. Sites wereselected if they had considerable experience with both HIV managementand HIVresearch. The study protocol was approved by the institutional review board at each site. Both TN and TE adults(aged 18 years or older) were eligiblefor enrolment in India andUganda. In South Africa, wherethe MERIT studyhad previouslybeen conducted in TN patients \[[@B35]\], onlyTE adults were eligible for inclusion in the present study. No patients were recruited from any other study. Patientswho had received less than 10 days of ART were considered to beTN; thosewho had experiencedfailure of at least one three-drug ART regimen were considered to be TE.To maximize the external validity and generalizability of thestudy, only one member of a known infectioncluster (i.e., only one member of a family affected by HIV) was eligible for enrolment. Study procedure --------------- At a single visit, thestudy was explained to patients, and verbal and written informed consent were obtained prior to conductof any study procedures. Demographic, and HIVclinicalhistory and treatment data werecollected;blood was drawn and analyzed for CD4^+^and CD8^+^T cells using BD FACS™ CAP (Becton Dickinson and Company, FranklinLakes,New Jersey), andfor HIV-1 RNA levels using AmplicorHIV-1 Monitor™ UltraSensitive Assay (Roche Diagnostics, Indianapolis, Indiana).For individuals withviral loads exceeding 500 copies/mL, HIV-1 subtype was determined based on reverse transcriptase and protease gene sequence (MonogramBiosciences). *Pol*subtyping was performed by generating three distinct sets of partial-length nucleotide sequences frompatient-derived reverse transcriptase and protease genes. Genetic sequencecomparison was determined by the Basic Local Alignment Search Tool algorithm, and subtype was resolved by a customizedsoftware package that was validated against thepublicallyavailable tool on the National Center forBiotechnologyInformation web siteat the National Institutes of Health.Samples determinedto beHIV-1subtypes of interest (A/A1 and DinUganda; C in South Africa and India) were further tested for viral tropism. HIV-1 co-receptor tropismwas determined usingthe original Trofile^®^assay \[[@B29],[@B30]\] for samples collected from patients in South Africa and Uganda. TheIndian cohort was enrolledafterthe South African and Ugandan cohorts, thus Indian patients weretested using the newlyavailableenhancedTrofile^®^assay, which had replaced the original assay. Samples were prepared for boththe original and enhanced assays in the same way: 1 mL plasma samples underwent centrifugation and viral RNA was isolated, purified and subjected to polymerase chain reaction (PCR) amplification of the entire HIV envelope gene (*env*). Co-transfection of HIV *env*expression vectors and HIV-1 genomic vectors produced pseudoviruses containing full length *env*genes derived frompatient virus populations. Tropism was determined bymeasuringthe ability of the pseudovirus population to efficiently infect target cells co-expressing CD4 with eitherthe CXCR4 or CCR5 co-receptor \[[@B29],[@B31]\]. For commercial Trofile^®^testing,up to 3 mL of plasma and up to three attempts at RNAPCR amplification are performed for each patient specimen. Forthis study of non-subtype Bspecimens, however, onlya singleattempt at amplification was made with 1 mL samples and primers that were optimized onsubtype B specimens (hereafter referred to as \"standard primers\"). In the event that samples yielded a non-reportable tropism result, re-testing was performed using modified primers that wereoptimized for subtypes A, C and | Background ========== Human _immunodeficiency_ virus type-1 (HIV-1) _is_ characterized by extensive _genetic_ heterogeneity. Molecular epidemiologic studies have demonstrated _that_ globally, _the_ most prevalent forms of HIV-1 are subtypes (clades) C, B and A \[[@B1]-[@B3]\]. Subtype C, which accounts for almost 50% of all HIV-1 _infections_ globally, predominates _in_ _sub-Saharan_ Africa _and_ India \[[@B1]-[@B3]\]. Subtype B is the _main_ _genetic_ form _in_ the Americas, _Australia_ and _western_ Europe; subtype A predominates in areas of central and eastern Africa _(Kenya,_ _Uganda,_ Tanzania and Rwanda) and in eastern Europe \[[@B1]-[@B3]\]; _and_ _subtype_ D is _distributed_ mainly _in_ _east_ Africa, _including_ Uganda \[[@B1]\]. HIV-1 subtypes differ by as much as 20-25% at the genetic level \[[@B2]\], and have varying biological characteristics, including _differences_ _in_ disease progression, _pathogenicity,_ transmissibility and co-receptor usage \[[@B1],[@B2],[@B4]-[@B7]\]. Studies of HIV-1 co-receptor tropism, which have been conducted primarily _in_ populations where subtype B _infections_ predominate, _have_ demonstrated _a_ relationship _between_ HIV-1 co-receptor use and _disease_ _stage._ In general, early _stages_ of _infection_ and disease are characterized by greater prevalence of _only_ C-C _chemokine_ type 5 (CCR5)-tropic (R5) HIV-1, which _has_ been _associated_ with slower progression to AIDS _\[[@B8]-[@B12]\]._ _The_ _emergence_ _of_ C-X-C chemokine receptor _type_ _4_ (CXCR4)-using virus (X4) has been _associated_ with greater _treatment_ experience and higher risk _of_ death, and coincides with more _rapid_ _CD4^+^T-cell_ depletion and disease progression \[[@B6],[@B8],[@B9],[@B12],[@B13]\]. _Some_ _variants_ _of_ HIV-1 can _use_ either co-receptor (dual/mixed-tropic \[DM\] HIV-1); these can be found in all stages of infection, but are more common _in_ infections of longer duration, _with_ _lower_ CD4^+^cell counts and higher _viral_ loads _\[[@B12]-[@B14]\]._ Despite the emergence of X4-using variants in _some_ patients, only _R5_ infection typically persists in _the_ _majority_ _of_ patients. Nearly 50% of patients who die of _HIV-1_ disease have only R5 HIV-1 _detectable_ at the time _of_ their death, indicating that CCR5 remains a critical co-receptor throughout the course of HIV infection \[[@B12],[@B15]\]. Although HIV-1 co-receptor usage and _its_ relationship to _disease_ stage have been studied in the developed world, where subtype _B_ predominates, such relationships are less well _understood_ for subtypes A, _C_ and D. The R5 phenotype is predominant in subtype _C_ HIV-1 infections, whereas X4-using virus has been _reported_ infrequently, even in advanced _disease._ R5-using _virus_ is more common in _subtype_ A than subtype _D_ HIV-1 infections, and a high _proportion_ of subtype _D_ infections shows D/M tropism _throughout_ the _course_ of _disease_ \[[@B16]-[@B24]\]. However, some _of_ these previous studies have been limited by small sample sizes. The introduction _of_ _the_ CCR5 antagonist, maraviroc, for HIV-1 therapy \[[@B25]\] has _increased_ interest in _the_ _epidemiology_ of tropism _and_ relationships with _HIV-1_ subtype. A greater understanding of the _tropism_ of non-B subtype HIV-1 is _key_ for the _optimal_ use of CCR5 _antagonists_ in the treatment of these infections _in_ the developing world, _and_ HIV-1 _prevention_ strategies, such as topical microbicides _and_ _systemic_ _pre-_ _or_ post-exposure prophylaxis. In addition, this _information_ will be important _for_ _management_ of clinic populations _in_ the developed _world_ that _include_ individuals with non-B _subtype_ infections who have migrated from _endemic_ countries \[[@B26]\]. HIV-1 tropism _can_ be _determined_ by genotypic and phenotypic methods. _While_ genotypic assays may have lower _specificity_ and sensitivity, retrospective analyses have _found_ that _they_ _are_ comparable _to_ phenotypic tropism _assays_ for _prediction_ of _response_ _to_ treatment _with_ CCR5 _antagonists,_ in _populations_ pre-screened _with_ a phenotypic assay \[[@B27],[@B28]\]. The clinical development programme for maraviroc, the first-in-class CCR5 _antagonist,_ used the Trofile^®^phenotypic _assay_ _(Monogram_ Biosciences, South San Francisco, California) \[[@B29]-[@B31]\], which _determines_ _tropism_ via the expression of full-length *env*genes _of_ multiple viruses isolated from patient _plasma_ and can detect 10% of X4 variants _with_ 100% sensitivity. More recently, a Trofile^®^assay with enhanced sensitivity _to_ improve detection of low-level X4-using _variants_ has been developed that can detect _0.3%_ of these variants with 100% sensitivity. Otherwise, assay _validation_ performance characteristics are equivalent _between_ the original _and_ enhanced Trofile^®^assays _\[[@B31]\]._ The enhanced Trofile^®^assay _has_ _been_ validated in a number _of_ studies by re-testing the _co-receptor_ tropism _of_ _clinical_ samples that were _initially_ _determined_ using the _original_ _assay_ \[[@B31]-[@B34]\]: in a re-analysis of samples from the Phase 3 MERIT study, _which_ evaluated the efficacy _of_ maraviroc in treatment-naïve patients with CCR5-tropic virus, 15% of _enrolled_ patients _(n_ = 106/721) were reclassified _as_ having D/M virus with the enhanced assay \[[@B34]\]. The aim of _this_ _study_ was to estimate the prevalence of R5-, D/M-, and X4-tropic HIV-1 among isolates obtained from patients with HIV-1 subtype C infection from India _and_ South Africa, and with subtype A/A1 and D infection from Uganda, and _to_ explore _the_ demographic and clinical _characteristics_ associated _with_ _R5_ infection. In addition, the study examined the _ability_ of the Trofile^®^assay to determine tropism of non-B subtypes _of_ HIV-1, which previously _had_ _not_ been _explored_ in _a_ large study. Methods ======= Study design _------------_ HIV-1-infected, antiretroviral _therapy_ (ART) treatment-naïve (TN) and treatment-experienced _(TE)_ viremic patients were _recruited_ into this prospective, cross-sectional, epidemiologic _study_ from _HIV_ clinics _in_ South Africa (four sites), Uganda (one site), and _India_ (seven sites) in _2007_ and 2008. _Sites_ _were_ selected if they had considerable experience _with_ both HIV management _and_ HIV research. The study protocol was approved by the institutional review _board_ at each site. Both TN and _TE_ adults _(aged_ 18 years or older) were eligible for enrolment in India _and_ Uganda. In South _Africa,_ _where_ the MERIT study had previously been _conducted_ in TN patients \[[@B35]\], only TE adults were eligible for _inclusion_ in the present _study._ No _patients_ were recruited from _any_ other _study._ Patients who had received less than _10_ days of ART were considered _to_ be TN; those who had experienced failure of at least _one_ _three-drug_ ART _regimen_ _were_ _considered_ to be _TE._ To maximize the external validity _and_ generalizability of the study, only one member _of_ a known infection _cluster_ _(i.e.,_ only _one_ member _of_ a family affected by _HIV)_ _was_ eligible for enrolment. Study procedure --------------- At a single visit, the _study_ was explained to _patients,_ and verbal and written _informed_ _consent_ were obtained prior to conduct of any study procedures. Demographic, and HIV clinical _history_ and treatment data were collected; blood was drawn and analyzed for _CD4^+^and_ _CD8^+^T_ cells using BD FACS™ CAP (Becton Dickinson and Company, _Franklin_ Lakes, New Jersey), and for _HIV-1_ RNA levels using Amplicor _HIV-1_ _Monitor™_ UltraSensitive Assay _(Roche_ Diagnostics, Indianapolis, Indiana). For individuals with _viral_ loads exceeding _500_ _copies/mL,_ HIV-1 subtype was _determined_ based _on_ reverse _transcriptase_ and protease gene sequence (Monogram Biosciences). *Pol*subtyping was performed by generating three _distinct_ sets of _partial-length_ nucleotide sequences from patient-derived reverse _transcriptase_ and _protease_ genes. Genetic sequence comparison was determined by the Basic Local _Alignment_ Search Tool algorithm, and subtype _was_ _resolved_ _by_ a customized software package that was validated against _the_ publically available _tool_ on the National Center for Biotechnology Information web site at the National _Institutes_ of Health. Samples determined to be _HIV-1_ _subtypes_ of _interest_ (A/A1 and D in Uganda; C _in_ South Africa and India) were further tested for _viral_ tropism. HIV-1 co-receptor tropism was _determined_ using the original Trofile^®^assay _\[[@B29],[@B30]\]_ _for_ _samples_ _collected_ from _patients_ in South Africa and Uganda. The Indian cohort was _enrolled_ after the South African _and_ Ugandan cohorts, thus Indian _patients_ were tested _using_ the newly available enhanced Trofile^®^assay, which had replaced the _original_ _assay._ _Samples_ were _prepared_ _for_ both the original and enhanced assays in _the_ same way: 1 _mL_ plasma _samples_ underwent centrifugation and viral RNA was isolated, _purified_ and _subjected_ to polymerase chain reaction (PCR) _amplification_ _of_ the _entire_ _HIV_ envelope gene _(*env*)._ Co-transfection of HIV *env*expression vectors and HIV-1 _genomic_ vectors produced _pseudoviruses_ containing full length _*env*genes_ derived from patient virus populations. Tropism was determined by _measuring_ the ability of the pseudovirus population to efficiently _infect_ target _cells_ co-expressing CD4 _with_ _either_ the CXCR4 _or_ CCR5 co-receptor \[[@B29],[@B31]\]. For commercial Trofile^®^testing, up to _3_ _mL_ _of_ plasma and _up_ to three attempts _at_ RNA _PCR_ _amplification_ are _performed_ for each patient specimen. _For_ this study of non-subtype _B_ specimens, _however,_ _only_ a _single_ _attempt_ at amplification was made with 1 _mL_ _samples_ and primers _that_ were optimized on _subtype_ B specimens (hereafter referred _to_ as \"standard primers\"). In the event that samples yielded a non-reportable tropism result, re-testing was performed _using_ modified primers that were optimized _for_ subtypes A, C and |
Introduction
============
Myopia is the main cause of preventive blindness worldwide, especially in adolescents ([@B1], [@B2]). Thus, it is one of the main priorities among the five projects under the 'Vision 2020 Action' launched by WHO ([@B3]). In recent years, the incidence of myopia has increased rapidly worldwide ([@B4]), especially among adolescents in East and Southeast Asia ([@B5], [@B6]). The prevalence of myopia among adolescents is at 96.5% ([@B7]) in South Korea, 81.6% ([@B8]) in Singapore and 95.5% ([@B9]) in Shanghai.
Myopia not only affects adolescents' school performance and future career choice ([@B10]) but also causes glaucoma, cataract and other serious complications ([@B11]). Thus, many researchers have devoted themselves to gaining a more in-depth understanding of the prevention and control of adolescent myopia ([@B12], [@B13]).
Collaborative research networks can help other researchers expand their field of research or join groups conducting related studies. Bibliometric studies of scientific collaboration have been conducted in various fields ([@B14], [@B15]), providing different levels of cooperation frequency in research practice. One of the methods used to study such collaboration is the co-authorship network analysis, which focuses on finding patterns of contacts or interactions between social actors. Author, country, and institution are the subjects of cooccurrence relationship; thus, analyzing their cooccurrence relationship can better reflect the truth of scientific research and academic communication, because the cooperation of authors, institutions and countries can measure the cooperation at different levels ([@B15]).
However, to date, no bibliometric analysis of scientific literature in myopia prevention and control had been carried out and published. As such, this study aimed to describe the diversity of cooperation among authors, institutions, and countries in the study of adolescent myopia prevention and control. Specifically, for adolescent myopia prevention and control research, our main goal is to explore the following content: firstly, analysing the overall status of collaborative research among authors, institutions and countries; secondly, determining the institutions and authors at the core of the cooperative research network; and thirdly, identifying countries that have a strong cooperative relationship.
Materials and Methods
=====================
The search for papers to be included in the analysis was conducted in one day (Sep 25, 2017) to avoid bias resulting from daily updating in the database. The Web of Science Core Collection annually collects a large number of journals and records each publication, including bibliographic information (i.e., author, institution and country or region), which we used to locate publications. All papers published within the period of 1997--2016 were evaluated. Search terms included combinations of terms, such as 'adolescent', 'children', 'student', 'myopia', 'myopic', 'prevention', 'control' and 'management'. Literature types, such as meeting abstracts, letters, correction, news item, book chapter, retracted publication, editorial material, non-English literature and repeated articles, were excluded. To ensure reliability, profile information of each included article was extracted by two independent reviewers, resulting in a reliability check of 100% of the selected abstracts. A search query that was used for data extraction from Web of Science database looked like this: TS= ((adolescent myopia OR children myopia OR student myopia OR adolescent myopic OR children myopic OR student myopic) AND (prevention OR control OR management)).
Social network analysis (SNA) is a method of structural analysis applied in many research fields. It focuses on relationship research and is mainly used to describe and measure relationships and information between individuals ([@B16]). SNA has been proven to be effective in studies on scientific collaboration network ([@B17], [@B18]). The same method is used in the current study.
To analyze and identify critical issues, we used SATI (Statistical Analysis Toolkit for Informetrics) (ver. 3.2) to build the co-occurrence matrix ([@B19]) and transformed the data format with Ucinet 6.0 ([@B20]) to finally obtain co-occurrence mapping. VOS viewer (Visualisation of Similarities viewer) software (ver. 1.6.6) was employed to draw the co-country (region) maps by using literature title packets ([@B21]). Excel 2016 (Microsoft, Redmond, DC, USA) and Netdraw (ver. 2.118) were also used in the research. In addition, some measures of our network, including degree centrality, betweenness centrality, closeness centrality, density, and diameter, were evaluated ([@B22]). Degree centrality refers to the number of neighbors to a node in the network ([@B15]). In this case, the greater its connection to other nodes in the network, the more important is the node.
Betweenness centrality refers to the number of the shortest paths passing through a given node ([@B23]). The higher the betweenness centrality of the node, the greater the ability to control the information passed between the other nodes. The closeness centrality is used to measure the distance of one node to other nodes in a network. Nodes with high closeness centrality obtain information better than other nodes or tend to have a more direct influence on other nodes ([@B16]). Density is calculated through the actually observed ties divided by all possible ties whose value is between 0 and 1 ([@B24]). Density values tend to reach 0 in sparse networks, and close to one in tightly connected networks ([@B24]). The diameter represents the longest measuring distance in a connected network; it shows the number of steps required from one side of the network to the other ([@B16]).
Ethical considerations
----------------------
This study did not require any ethical consideration as it does not include any human or animal to be the object of study.
Results
=======
A systematic search for publications on adolescent myopia prevention and control retrieved 624 articles in Web of Science Core Collection, excluding one duplicate. After further screening of titles and abstracts, 9 editorial materials, 4 letters and a meeting abstract were removed, leaving 610 eligible papers.
The scale and overall trend of collaborative research
-----------------------------------------------------
[Figure 1](#F1){ref-type="fig"} shows the number of publications issued annually and the number of papers published through collaboration with authors, institutional cooperation and country (region) cooperation. The number of papers, co-authors, co-institutions and country (region) cooperative papers has increased significantly from 1997 to 2016, particularly after 2011. In general, the total number of published articles since 1997 has increased more than six-fold, from 11 in 1997 to 79 in 2016; the institutional cooperation increased more than five-fold, the author cooperation increased by twelve-fold and the country (region) cooperation increased by fifteen-fold.
{#F1}
[Fig. 2](#F2){ref-type="fig"} reveals the average number of authors, institutions and countries per article from 1997 to 2016. The average number shows a gradually increasing trend.
{#F2}
The increase in number of authors was from 3.91 to 4.34, from 1.36 to 2.51 for institutions and from 0.82 to 1.25 for countries per paper. Overall, the rates of cooperation among authors, institutions and countries were 93%, 57.9% and 21.5%, respectively.
In general, the number of SCI journal papers produced by institutional cooperation is the largest (accounting for 56.6%), followed by papers generated through intra-institutional collaboration (accounting for 36.1%) and papers produced without collaboration (accounting for only 7.4%). [Figure 3](#F3){ref-type="fig"} shows the percentage of papers studied in each of the different institution collaboration types and their changes over time. The percentages of single-author papers have decreased by 26.7% from 1997 to 2016, whereas that of institution-collaborated papers increased by 24.4%. The percentage of papers produced through single authorship has always been higher than that of institutional collaboration from 1997 to 2000 but decreased after 2006.
{#F3}
Authors' collaborative research
-------------------------------
Results of scientific research are published in the form of papers, and the status of co-authorship in papers reflects the collaboration among authors. Researchers who study the growth of co-authorship articles produced by multiple authors regard co-authorship of papers as a significant scientometric indicator of researching on cooperation among authors ([@B25]).
More important researchers were expected to have published more articles, thus scholars who published more than four articles were included in the co-authorship networks. Overall, 75 researchers with 371 co-authored experiments meet this condition. Five authors not cooperated with other authors were excluded. The research collaboration network between authors is shown in [Fig. 4](#F4){ref-type="fig"}.
{#F4}
Each node of the figure represents an author, and the connections among the nodes represent the collaboration relationships among authors. The weight of a link indicates the number of publications co-authored by two scholars. In this author's collaboration network, the highest degree centrality of Allen, Peter M. and O\'Leary, Daniel J. was 5.83, indicating that they had 5.83 collaborators and that they played a pivotal role in the co | introduction = = = = = = = = = = = = myopia is the main cause of preventive blindness worldwide, especially in adolescents ( [ @ b1 ], [ @ b2 ] ). thus, it remains one of the main priorities among the five projects under the ' vision 2020 action ' launched by schools ( [ @ b3 ] ). in recent years, the incidence of myopia has increased rapidly worldwide ( [ @ b4 ] ), especially among adolescents in east and southeast asia ( [ @ b5 ], [ @ b6 ] ). the prevalence by myopia among adolescents is at 96. 5 % ( [ @ b7 ] ) in south korea, 81. 6 % ( [ @ b8 ] ) in singapore and 95. 5 % ( [ @ b9 ] ) in germany. myopia not only affects adolescents ' school performance and future career development ( [ @ b10 ] ) but also causes glaucoma, cataract and other serious complications ( [ @ b11 ] ). thus, many researchers have devoted themselves to gaining a more in - depth understanding of the prevention and control of adolescent myopia ( [ @ b12 ], [ @ b13 ] ). collaborative research networks can help other researchers expand their field of research or join groups conducting related studies. bibliometric studies of scientific collaboration have been conducted in various fields ( [ @ b14 ], [ @ b15 ] ), providing different levels of cooperation frequency in research practice. one of the methods used to study such collaboration is the co - authorship network analysis, which focuses on finding patterns of contacts or interactions between social actors. author, country, and institution are the subjects of cooccurrence relationship ; thus, analyzing their cooccurrence relationship can better reflect the truth of scientific opinion and academic communication, because the cooperation of authors, institutions and countries can measure the cooperation at different levels ( [ @ b15 ] ). therefore, to date, no bibliometric analysis of scientific literature in myopia prevention and control had been carried through and published. as such, this study aimed to describe the diversity of cooperation among authors, institutions, and countries in the study of youth myopia prevention and control. specifically, for adolescent myopia prevention and control research, our main goal is to explore the following content : firstly, analysing the legal status of collaborative research among authors, institutions and countries ; secondly, determining the institutions and authors at the core of the cooperative research network ; and thirdly, identifying countries that have a strong cooperative relationship. materials and methods = = = = = = = = = = = = = = = = = = = = = the search for papers to be included in the analysis was conducted in one day ( sep 25, 2017 ) to avoid bias resulting from daily updating in the database. the web of science core collection annually collects a large number of journals and records each publication, including bibliographic information ( i. e., author, institution and country or region ), which we used to locate publications. all papers published within the period of 1997 - - 2016 were evaluated. search terms included combinations of terms, such as ' adolescent ', ' children ', ' student ', ' myopia ', ' myopic ', ' prevention ', ' control ' and ' management '. literature types, such as meeting abstracts, letters, correction, news item, book chapter, retracted publication, editorial material, non - english literature and repeated articles, were excluded. to ensure reliability, profile information of each included article was extracted by two independent reviewers, resulting in a reliability check of 100 % of the selected abstracts. a search query that was used for data extraction from web of science database looked like this : ts = ( ( adolescent myopia or children myopia or student myopia or adolescent myopic or children myopic or student myopic ) and ( prevention or control or management ) ). social network analysis ( sna ) is a method of structural analysis applied in many research fields. it focuses on relationship research and is mainly used to describe and measure relationships and information between individuals ( [ @ b16 ] ). sna has been proven to be effective in studies on scientific collaboration network ( [ @ b17 ], [ @ b18 ] ). the same method is used in the current study. to analyze and identify critical issues, we used sati ( statistical analysis toolkit for informetrics ) ( ver. 3. 2 ) to build the co - occurrence matrix ( [ @ b19 ] ) and transformed the data format with ucinet 6. 0 ( [ @ b20 ] ) to finally obtain co - occurrence mapping. vos viewer ( visualisation of similarities viewer ) software ( ver. 1. 6. 6 ) was employed to draw the co - country ( region ) maps by using literature title packets ( [ @ b21 ] ). excel 2016 ( microsoft, redmond, dc, usa ) and netdraw ( ver. 2. 118 ) were also used in the research. in addition, some measures of our network, including degree centrality, betweenness centrality, closeness centrality, density, and diameter, were evaluated ( [ @ b22 ] ). degree centrality refers to the number of neighbors to a node in the network ( [ @ b15 ] ). in this case, the greater its connection to other nodes in the network, the more important is the node. betweenness centrality refers to the number of the shortest paths passing through a given node ( [ @ b23 ] ). the higher the betweenness centrality of the node, the greater the ability to control the information passed between the other nodes. the closeness centrality is used to measure the distance of one node to other nodes in a network. nodes with high closeness centrality obtain information better than other nodes or tend to have a more direct influence on other nodes ( [ @ b16 ] ). density is calculated through the actually observed ties divided by all possible ties whose value is between 0 and 1 ( [ @ b24 ] ). density values tend to reach 0 in sparse networks, and close to one in tightly connected networks ( [ @ b24 ] ). the diameter represents the longest measuring distance in a connected network ; it shows the number of steps required from one side of the network to the other ( [ @ b16 ] ). ethical considerations - - - - - - - - - - - - - - - - - - - - - - this study did not require any ethical consideration as it does not include any human or animal to be the object of study. results = = = = = = = a systematic search for publications on adolescent myopia prevention and control retrieved 624 articles in web of science core collection, excluding one duplicate. after further screening of titles and abstracts, 9 editorial materials, 4 letters and a meeting abstract were removed, leaving 610 eligible papers. the scale and overall trend of collaborative research - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - [ figure 1 ] ( # f1 ) { ref - type = " fig " } shows the number of publications issued annually and the number of papers published through collaboration with authors, institutional cooperation and country ( region ) cooperation. the number of papers, co - authors, co - institutions and country ( region ) cooperative papers has increased significantly from 1997 to 2016, particularly after 2011. in general, the total number of published articles since 1997 has increased more than six - fold, from 11 in 1997 to 79 in 2016 ; the institutional cooperation increased more than five - fold, the author cooperation increased by twelve - fold and the country ( region ) cooperation increased by fifteen - fold.! [ numbers of papers on adolescent myopia prevention and control by collaboration type between 1997 and 2016 ] ( ijph - 48 - 621 - g001 ) { # f1 } [ fig. 2 ] ( # f2 ) { ref - type = " fig " } reveals the average number of authors, institutions and countries per article from 1997 to 2016. the average number shows a gradually increasing trend.! [ average number of different entities per paper ] ( ijph - 48 - 621 - g002 ) { # f2 } the increase in number of authors was from 3. 91 to 4. 34, from 1. 36 to 2. 51 for institutions and from 0. 82 to 1. 25 for countries per paper. overall, the rates of cooperation among authors, institutions and countries were 93 %, 57. 9 % and 21. 5 %, respectively. in general, the number of sci journal papers produced by institutional cooperation is the largest ( accounting for 56. 6 % ), followed by papers generated through intra - institutional collaboration ( accounting for 36. 1 % ) and papers produced without collaboration ( accounting for only 7. 4 % ). [ figure 3 ] ( # f3 ) { ref - type = " fig " } shows the percentage of papers studied in each of the different institution collaboration types and their changes over time. the percentages of single - author papers have decreased by 26. 7 % from 1997 to 2016, whereas that of institution - collaborated papers increased by 24. 4 %. the percentage of papers produced through single authorship has always been higher than that of institutional collaboration from 1997 to 2000 but decreased after 2006.! [ percentage of different collaboration types ] ( ijph - 48 - 621 - g003 ) { # f3 } authors ' collaborative research - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - results of scientific research are published in the form of papers, and the status of co - authorship in papers reflects the collaboration among authors. researchers who study the growth of co - authorship articles produced by multiple authors regard co - authorship of papers as a significant scientometric indicator of researching on cooperation among authors ( [ @ b25 ] ). more important researchers were expected to have published more articles, thus scholars who published more than four articles were included in the co - authorship networks. overall, 75 researchers with 371 co - authored experiments meet this condition. five authors not cooperated with other authors were excluded. the research collaboration network between authors is shown in [ fig. 4 ] ( # f4 ) { ref - type = " fig " }.! [ the structure map of the author collaboration network ] ( ijph - 48 - 621 - g004 ) { # f4 } each node of the figure represents an author, and the connections among the nodes represent the collaboration relationships among authors. the weight of a link indicates the number of publications co - authored by two scholars. in this author ' s collaboration network, the highest degree centrality of allen, peter m. and o \ ' leary, daniel j. was 5. 83, indicating that they had 5. 83 collaborators and that they played a pivotal role in the co | Introduction = = = = = = = = = = = = Myopia is the main cause of preventive blindness worldwide, especially in adolescents ([ @ B1 ], [@ B2] ). Thus, it is one of the main priorities aJpng the five projects under the ' Vision Q0E0 Action ' launched by WHO ([ @ B3] ). In recent years, the incidence of myopia has increased rapidly worldwide ([ @ B4] ), eZpeciwlly among adolescents in East and S8utJeast Asia ([ @ B5 ], [@ B6] ). The prevalence of myopia among adolescents is at 96. 5% ([ @ B7] ) in South Korea, 81. 6% ([ @ B8] ) in Singapore and 95. 5% ([ @ B9] ) in Shanghai. Myopia not only affects adolescents ' school performance and future career choice ([ @ B10] ) but also causes glaucoma, cataract and other serious complications ([ @ B11] ). Thus, many researchers have devoted themselves to gaining a more in - depth understanding of the prevention and control of adolescent myopia ([ @ B12 ], [@ B13] ). Collaborative research networks can help other researchers expand their C7eld of research or join groups conducting related s^udiWs. Bibliometric studies of scientific collaboration have been conducted in various fields ([ @ B14 ], [@ B15] ), providing different levels of cooperation frequency in research practice. One of the methods used to study such collaboration is the co - authorship network analysis, which focuses on finding patterns of contacts or interactions between social actors. Author, country, and institution are the subjects of cooccurrence relationship; thus, analyzing their cooccurrence relationship can better reflect the truth of scientific research and academic communication, because the cooperation of authors, institutions and countries can measure the cooperation at different levels ([ @ B15] ). However, to date, no bibliometric analysis of scientific literature in myopia prevention and control had been carried out and published. As such, this study aimed to describe the diversity of cooperation among authors, institutions, and countries in the study of adolescent myopia prevention and control. Specifically, for adolescent myopia prevention and control research, our main goal is to explore the following content: firstly, analysing the overall status of collaborative research among authors, institutions and countries; secondly, determining the institutions and authors at the core of the cooperative research network; and thirdly, identifying countries that have a strong cooperative relationship. Materials and Methods = = = = = = = = = = = = = = = = = = = = = The search for papers to be included in the analysis was conducted in one day (Sep 25, 2017) to avoid bias resulting from daily updating in the database. The Web of Science Core Collection annually collects a large number of journals and records each publication, including bibliographic information (i. e. , author, institution and country or region ), which we used to locate publications. All papers published within the period of 1997 - - 2016 were evaluated. Search terms included combinations of terms, such as ' adolescent ', ' children ', ' student ', ' myopia ', ' myopic ', ' prevention ', ' control ' and ' management '. Literature types, such as meeting abstracts, letters, correction, news item, book chapter, retracted publication, editorial material, non - English literature and repeated articles, were excluded. To ensure reliability, profile information of each included article was extracted by two independent reviewers, resulting in a reliability check of 100% of the selected abstracts. A search query that was used for data extraction from Web of Science database looked like this: TS = (( adolescent myopia OR children myopia OR student myopia OR adolescent myopic OR children myopic OR student myopic) AND (prevention OR control OR management) ). Social network analysis (SNA) is a method of structural analysis applied in many research fields. It focuses on relationship research and is mainly used to describe and measure relationships and information between individuals ([ @ B16] ). SNA has been proven to be effective in studies on scientific collaboration network ([ @ B17 ], [@ B18] ). The same method is used in the current study. To analyze and identify critical issues, we used SATI (Statistical Analysis Toolkit for Informetrics) (ver. 3. 2) to build the co - occurrence matrix ([ @ B19] ) and transformed the data format with Ucinet 6. 0 ([ @ B20] ) to finally obtain co - occurrence mapping. VOS viewer (Visualisation of Similarities viewer) software (ver. 1. 6. 6) was employed to draw the co - country (region) maps by using literature title packets ([ @ B21] ). Excel 2016 (Microsoft, Redmond, DC, USA) and Netdraw (ver. 2. 118) were also used in the research. In addition, some measures of our network, including degree centrality, betweenness centrality, closeness centrality, density, and diameter, were evaluated ([ @ B22] ). DegrW2 centrality refers to the number of neighbors to a node in the network ([ @ B15] ). In this case, the greater its connection to other nodes in the network, the more important is the node. Betweenness centrality refers to the number of the shortest paths passing through a given node ([ @ B23] ). The higher the betweenness centrality of the node, the greater the ability to control the information passed between the other nodes. The closeness centrality is used to measure the distance of one node to other nodes in a network. Nodes with high closeness centrality obtain information better than other nodes or tend to have a more direct influence on other nodes ([ @ B16] ). Density is calculated through the actually observed ties divided by all possible ties whose value is between 0 and 1 ([ @ B24] ). Density values tend to reach 0 in sparse networks, and close to one in tightly connected networks ([ @ B24] ). The diameter represents the longest measuring distance in a connected network; it shows the number of steps required from one side of the network to the other ([ @ B16] ). Ethical considerations - - - - - - - - - - - - - - - - - - - - - - This study did not require any ethical consideration as it does not include any human or animal to be the object of study. Results = = = = = = = A systematic search for publications on adolescent myopia prevention and control retrieved 624 articles in Web of Science Core Collection, excluding one duplicate. After further screening of titles and abstracts, 9 editorial materials, 4 letters and a meeting abstract were removed, leaving 610 eligible papers. The scale and overall trend of collaborative research - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - [Figure 1] (# F1) {ref - type = " fig "} shows the number of publications issued annually and the nuKb4r of papers published through collaboration with authors, institutional cooperation and country (region) cooperation. The number of papers, co - authors, co - institutions and country (region) cooperative papers has increased significantly from 1997 to 2016, particularly after 2011. In general, the total number of published articles since 1997 has increased more than six - foiX, from 11 in 1997 to 79 in 2016; the institutional cooperation increased more than five - fold, the author cooperation increased by twelve - fold and the country (region) cooperation increased by fifteen - fold. ! [Numbers of papers on adolescent myopia prevention and control by collaboration type between 1997 and 2016] (IJPH - 48 - 621 - g001) {# F1} [Fig. 2] (# F2) {ref - type = " fig "} reveals the average number of authors, institutions and countries per article from 1997 to 2016. The average number shows a gradually increasing trend. ! [Average number of different entities per paper] (IJPH - 48 - 621 - g002) {# F2} The increase in number of authors was from 3. 91 to 4. 34, from 1. 36 to 2. 51 for institutions and from 0. 82 to 1. 25 for countries per paper. Overall, the rates of cooperation among authors, institutions and countries were 93% , 57. 9% and 21. 5% , respectively. In general, the number of SCI journal papers produced by institutional cooperation is the largest (accounting for 56. 6% ), followed by papers generated through ibtrX - institutional collaboration (accounting for 36. 1%) and papers produced without collaboration (accounting for only 7. 4% ). [Figure 3] (# F3) {ref - type = " fig "} shows the percentage of papers studied in each of the different institution collaboration types and their changes over time. The percentages of single - author papers have decreased by 26. 7% from 1997 to 2016, whereas that of institution - collaborated papers increased by 24. 4% . The percentage of papers produced through single authorship has always been higher than that of institutional collaboration from 1997 to 2000 but decreased after 2006. ! [Percentage of different collaboration types] (IJPH - 48 - 621 - g003) {# F3} Authors ' collaborative research - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Results of scientific research are published in the form of papers, and the status of co - authorship in papers reflects the collaboration among authors. Researchers who study the growth of co - authorship articles produced by multiple authors regard co - authorship of papers as a significant scientometric indicator of researching on cooperation among authors ([ @ B25] ). More important researchers were expected to have published more articles, thus scholars who published more than four articles were included in the co - authorship networks. Overall, 75 researchers with 371 co - authored experiments meet this condition. Five authors not cooperated with other authors were excluded. The research collaboration network between authors is shown in [Fig. 4] (# F4) {ref - type = " fig " }. ! [The structure map of the author collaboration network] (IJPH - 48 - 621 - g004) {# F4} Each node of the figure represents an author, and the connections among the nodes represent the collaboration relationships among authors. The weight of a link indicates the number of publications co - authored by two scholars. In this author ' s collaboration network, the highest degree centrality of Allen, Peter M. and O \ ' Leary, Daniel J. was 5. 83, indicating that they had 5. 83 collaborators and that they played a pivotal role in the co | Introduction is the main cause of preventive blindness especially in adolescents ([@B1], [@B2]). Thus, it is the priorities among the five projects under 'Vision 2020 Action' launched by WHO ([@B3]). In recent years, the incidence of myopia has increased rapidly worldwide ([@B4]), especially among adolescents in East and Southeast Asia ([@B5], [@B6]). The prevalence of myopia among adolescents is at 96.5% ([@B7]) South 81.6% ([@B8]) in Singapore and 95.5% ([@B9]) in Shanghai. Myopia not affects adolescents' school performance future career choice ([@B10]) but causes glaucoma, cataract and other serious complications Thus, many researchers have devoted themselves to gaining a more in-depth understanding of the and control of adolescent myopia ([@B12], [@B13]). Collaborative research networks can help other researchers expand their field of research or join groups conducting related studies. Bibliometric studies of scientific collaboration have been conducted in various ([@B14], [@B15]), providing different levels of frequency research practice. One of methods used study such collaboration is the co-authorship network analysis, which focuses on finding patterns of contacts or interactions between social actors. Author, country, and institution are the subjects of cooccurrence thus, analyzing their cooccurrence relationship can better reflect the truth of scientific research and academic communication, because the cooperation authors, institutions and can measure the cooperation at different levels ([@B15]). However, to date, bibliometric analysis of scientific literature in myopia prevention and control had been carried out published. As such, this study aimed to describe the diversity of cooperation among authors, institutions, countries in the study of adolescent myopia prevention and for adolescent myopia prevention control research, main goal is to explore the content: firstly, analysing the overall status of collaborative research among authors, institutions and secondly, determining the institutions and authors at the core of the cooperative research network; and thirdly, identifying countries that have a strong cooperative relationship. Materials and Methods ===================== The search papers to be included in the analysis was conducted in one day (Sep 25, 2017) to avoid bias resulting from daily updating in database. The Web of Science Core Collection annually collects a large number of journals and records each publication, including bibliographic information (i.e., institution and country or region), which we used to publications. All papers published the period of 1997--2016 were Search terms included combinations of terms, such as 'adolescent', 'children', 'student', 'myopia', 'prevention', 'control' and 'management'. Literature such as abstracts, letters, news item, book chapter, retracted publication, editorial material, non-English and repeated were excluded. To ensure reliability, profile information of included article was extracted by two independent reviewers, resulting in a check of the selected abstracts. A search query that was used for data extraction from Web of Science database looked like this: TS= ((adolescent myopia OR children myopia OR myopia OR adolescent myopic OR children myopic OR student myopic) AND (prevention OR control OR management)). network analysis (SNA) is a method of analysis applied many research fields. It focuses on relationship research and is mainly used to describe and measure relationships and information between individuals ([@B16]). SNA has proven to be effective in studies on scientific collaboration network ([@B17], [@B18]). same method is used in the study. To analyze and identify critical issues, we used SATI (Statistical Analysis Toolkit for Informetrics) 3.2) to build co-occurrence matrix ([@B19]) and transformed the data format with Ucinet 6.0 ([@B20]) finally obtain co-occurrence mapping. VOS viewer (Visualisation of Similarities viewer) software (ver. 1.6.6) was employed to draw the co-country (region) maps by using literature title packets ([@B21]). Excel 2016 (Microsoft, Redmond, DC, USA) and Netdraw (ver. 2.118) were also used the research. In addition, some measures of our network, including degree centrality, betweenness centrality, closeness centrality, density, and were evaluated ([@B22]). Degree centrality refers to the number of neighbors to a node in the network ([@B15]). In this case, greater its connection to other nodes in the network, the more important is the node. Betweenness centrality refers to the number of shortest paths passing through a given node ([@B23]). The higher the betweenness centrality of the greater the ability to control the information passed between the other nodes. The closeness centrality is used to measure the distance of one node to other nodes in a network. Nodes with high closeness centrality obtain information better than other or tend have a more direct on other nodes ([@B16]). Density is calculated through the actually observed ties divided by possible ties whose value is between 0 and ([@B24]). Density values to reach 0 in sparse networks, and close to in connected networks ([@B24]). diameter represents the longest measuring distance in a connected network; shows the number of steps required from one side of the network the other ([@B16]). Ethical considerations ---------------------- This study did not require any ethical consideration it does not include any human or animal to be the study. Results ======= A systematic search for publications on adolescent prevention and control retrieved 624 articles in of Science Collection, excluding one duplicate. After further screening of titles and abstracts, 9 editorial materials, 4 letters and a meeting abstract were removed, leaving 610 eligible papers. The and overall of collaborative research 1](#F1){ref-type="fig"} the number of publications issued annually and the number of papers published through authors, institutional and country (region) cooperation. The of co-authors, and country (region) cooperative papers has increased significantly from 1997 to 2016, particularly after 2011. In general, the total number of published articles since 1997 has increased than six-fold, 11 1997 to 79 in 2016; the institutional cooperation more five-fold, the author cooperation increased by twelve-fold and the cooperation increased by fifteen-fold. {#F1} [Fig. 2](#F2){ref-type="fig"} reveals the average number of authors, institutions and countries per article from 1997 to 2016. The number shows gradually increasing trend. {#F2} The increase in number of authors was from 3.91 to 4.34, from 1.36 to 2.51 for institutions and from to for countries per paper. the of cooperation among authors, institutions were 93%, 57.9% and 21.5%, respectively. In general, of SCI journal papers produced by institutional is the largest for 56.6%), followed papers generated through intra-institutional collaboration (accounting for 36.1%) and papers produced without for only 7.4%). [Figure 3](#F3){ref-type="fig"} shows the percentage of studied in each of the different collaboration types and their changes over time. The percentages of single-author papers decreased by 26.7% from 1997 to 2016, whereas that of institution-collaborated papers increased by 24.4%. The papers through authorship has always been higher than that of institutional collaboration 1997 to 2000 but after 2006. {#F3} Authors' collaborative research ------------------------------- Results of scientific research are in the form papers, and the status in papers reflects the collaboration among authors. Researchers who study the growth of co-authorship articles produced by multiple authors regard co-authorship of papers as a significant scientometric researching on cooperation among ([@B25]). More important researchers were expected to have published more articles, thus scholars who published more than four articles were included in the co-authorship networks. Overall, 75 researchers with 371 co-authored experiments meet condition. Five authors not cooperated other authors were excluded. The research collaboration network between authors is shown 4](#F4){ref-type="fig"}. {#F4} Each node of the figure an author, and the connections among the nodes represent the collaboration relationships among authors. The weight of a link indicates the of publications co-authored by two scholars. this author's collaboration the highest degree centrality of Allen, Peter M. and O\'Leary, Daniel J. was 5.83, indicating that they had 5.83 collaborators and that they played pivotal role in the co | iNtroDUCtIOn
============
MyoPiA IS thE maIN CaUsE OF PreVeNtive BLInDnESS woRLDWidE, esPECIalLy IN AdOlesCEnTs ([@b1], [@B2]). thuS, It iS OnE OF tHE MAin pRiOrIties aMoNG thE fIVe pROjEcts UnDeR tHE 'ViSIon 2020 aCTioN' LAUnCHed bY whO ([@b3]). iN reCeNt yEArs, ThE incideNCE OF mYOPIa HaS INCReAsEd rAPidlY wORlDWIde ([@B4]), espEcIALLY AMong aDoLEscenTS In EaST aNd soUtHeASt ASiA ([@B5], [@b6]). thE prevALENCe oF myOPIa Among aDolescEntS is At 96.5% ([@b7]) IN soUth kOREA, 81.6% ([@b8]) IN sINGaporE ANd 95.5% ([@b9]) In shANGHaI.
mYOpia nOt oNLY AffECTs aDolEsCENTs' SCHoOL pERFoRMaNcE anD fUtUre CAReer cHoicE ([@B10]) but ALso CAUseS gLauCOmA, CATARact ANd OTher sErIOus ComPLicatIons ([@b11]). thUs, mAny rESEArCHERS HAvE dEVoTeD THemsElVEs TO GAiNINg A mOrE in-DeptH UNdeRStAnDiNG oF the PreVenTiON And conTRoL of adOlescENt MyOpia ([@B12], [@b13]).
cOlLAbORaTIVE RESeARCh NETwOrKs can HeLp OthEr RESEArChERS eXpand tHEiR fIelD of resEarcH Or jOin gROUPs cONduCTiNG RElAteD StUdiEs. BibLIOMetRIc STuDiES OF sCIEntiFiC cOlLaBoRAtioN HAVE BeEn COnDUcTEd IN VaRiouS fiElDs ([@b14], [@b15]), pRovIDing difFEReNT lEVEls of CoOpeRAtiOn frEQUeNcy IN ReSEARch prAcTIce. OnE of The MeThodS useD To stuDY suCh coLlabOrATiOn IS thE CO-aUthORshiP NetWORK aNAlYSIS, WHIch FocUsEs on finding pAtterns oF conTaCTS Or InteRACTioNs beTWeEn SOCial ACTorS. auTHOr, cOunTrY, aND institUTiOn arE the SuBJEcTs Of CoOcCURrencE relaTionship; tHus, AnALYZing theiR COOccUrrenCe ReLATIoNshiP CAn BETtER ReFlECT the truTH OF scientiFIc reSeARCh and aCadeMIc CoMmUnICAtioN, becauSe the cooPERATIoN OF AuTHoRS, iNsTITUTiONS aNd cOuntRIEs can mEAsURE The CoopEratioN at DIFFERENt lEVeLS ([@b15]).
HOWEvEr, To DatE, NO BIbLiOmEtRIc ANaLysIs Of sCiEntIFic LiTErAtUrE in myoPIA PReVENtiON AnD cOntROL HaD been cArRiEd OUt aNd PuBlIShED. AS SUCH, THIS StuDY Aimed TO DEScrIbE The dIVERSiTy Of COOPeRatIOn amOnG AuthORs, iNSTiTUtIons, aND cOuNtrIES iN tHe study Of adOlEScENT myopiA PrEvENtion AND CoNtrol. sPEcIfIcAlLY, foR AdolEsCent mYOpIA pReVEnTion AND COntrOL reSearcH, OUr mAiN goAl IS To ExPLOrE thE FOlLOwInG conTEnT: fIrstLy, anAlYSInG THe OvERaLl staTUs Of COllABOrative rEseArch Among AUtHORS, INstItutIons AnD COUntRIEs; secoNDLY, DEterMiniNg the InsTItUtiONs anD aUThoRS At tHE Core Of The COOpERATiVe rESEaRCh neTWoRK; AnD ThiRDly, iDenTIfYINg CoUnTRieS THAT HAve A StroNg cOopErAtIVe relatIONSHIP.
materiaLS ANd MethODs
=====================
tHE SEarCh fOR PaPeRs To be inClUDed IN thE ANaLYsiS wAS cOnDucTED in one dAY (SEp 25, 2017) TO AvOID bIAs RESULTIng FRoM dailY UpdaTing iN THe DATABAsE. tHe weB oF ScIenCE CorE coLLeCTiOn aNNUALly COlLECts a LarGe numBer OF JOUrNals And REcorDs eaCH pUbLICAtiOn, INCLuDINg BIBlIOgraphIC InfOrMaTIoN (I.E., aUTHOr, iNsTitutioN AnD couNTry Or REgIOn), WhiCh WE usEd to loCATe PUBLIcAtions. all PApERs pUBliSHEd wITHIn ThE peRiod Of 1997--2016 weRe EvalUATED. SEARcH TERmS InCLUDeD coMBinatIONS of terMs, sUch As 'AdOLeSceNT', 'CHILdRen', 'sTUDenT', 'MYopIA', 'MYopIC', 'PREVENTION', 'cONtROl' and 'manaGEmeNT'. lItErATURE tYPes, Such AS MeETING abSTRaCTs, LetTERS, CORREcTiOn, NEWS ITem, bOok CHapteR, RETRacTED PUBlication, EdiTOrIal MATeRiAl, noN-ENgLiSH lITEraTURe aND RePEAted arTiClEs, WERE ExcLUdeD. to EnSure rELIaBilITy, pROFIlE INFormatIOn OF EaCH INclUdED ARTIClE Was EXTRAcTeD BY TwO INDEpeNDenT ReviEwErS, REsulTIng In A rELIaBilITY cHecK OF 100% OF ThE sElECTEd absTractS. A SEarch qUEry thAt wAs UsED foR dATA eXTracTIoN FRom WEb oF sCienCe daTaBASe LOoKED lIke ThIS: Ts= ((ADOlESceNT mYopiA or ChIldrEn mYOpiA Or stUdent MyOpia oR ADOlEsCenT MyOpIc OR chIlDReN MyopIc oR stuDeNt myoPIc) AnD (pREVEnTION OR coNtroL oR MANAGeMeNt)).
socIaL NEtwOrk aNaLySiS (SnA) iS A mEthOD OF StrUcTUrAL AnaLYsiS APPlieD IN mANy RESEaRCh fIeLds. iT focUSes ON RElaTIONship ResEARcH anD IS MAinlY usEd TO DEsCRIbE aNd measuRe rElAtiONshIPs anD INFORMATiOn BeTweEN inDiViDUALS ([@b16]). sNA HaS bEeN PROvEn to BE effecTIVE IN stUDies oN SCienTIFic CollAboRATiON NETWORK ([@b17], [@b18]). The SAme MEthOd is usEd In the cUrrent STUDy.
tO aNALYZe anD idEntIfy CRITiCal issuES, WE UseD SaTI (StAtisTiCAL ANalysIS TooLkiT FOr INformeTrics) (ver. 3.2) To BuiLD The CO-OCcURrENce matRIx ([@B19]) AND TRaNsFoRmed the DAtA fOrMAT wiTH UCiNEt 6.0 ([@b20]) tO fiNaLLY ObTaiN Co-occUrRENce MappinG. VoS VIEwER (VisuaLISatIOn Of SImILArItieS vIEweR) SoftwARE (ver. 1.6.6) wAs EMpLOYed TO DrAW The CO-cOUNtRy (reGiON) MAps by UsING LitERAtuRE titLE PAcKeTs ([@B21]). ExCel 2016 (MiCrOSoft, reDMOnd, DC, USa) anD NETDraW (VEr. 2.118) WeRe AlSo UsEd iN thE resEarcH. In aDDitiON, SomE measureS oF ouR network, INcLudINg DEgree CEnTRALItY, bEtweENneSs cEnTrALitY, cLoSeneSS cEntRaLitY, densiTy, AND DiaMETeR, wErE evaLUAted ([@B22]). Degree cENTRALitY referS tO ThE nuMbeR OF NeIgHbOrs To A noDE in The NEtWorK ([@B15]). in THIS CaSe, tHe GreAteR ITs cONnection To oTHeR nodeS IN thE nETwoRk, The MoRE iMpORTant IS tHE NoDE.
BetWEenNeSs cENTrAlItY ReFERs TO the nUMBer oF The shORtest PatHS PasSINg ThROUgH a gIVen nODe ([@b23]). thE higHER tHe BetweENNESs CeNtraLItY oF tHE node, tHe greaTEr tHe ABIlITy To coNtrOL ThE InforMaTIOn PaSseD BETween the OTHeR nODes. tHE clOSenEss cENTRaLITY is used to mEASURe tHE DiSTAnCe OF onE NoDE tO otHER noDEs IN a nEtwORk. nOdEs WITH HiGh CLOSEneSS CEnTrAliTy oBTAin inFOrMAtIoN BeTTEr thaN oTHer NoDeS Or TenD To hAVe a mOre direct InFLuenCE ON OtHEr NOdes ([@B16]). deNSiTY is CaLcuLated ThROUgH thE AcTuallY OBSERVeD TIES DIViDeD BY ALL POsSIbLE TiES WHosE valUe Is BeTWeeN 0 aND 1 ([@B24]). DenSity vAlues Tend to rEAcH 0 In sPARSe NETworKs, AnD Close to oNe In tiGHTly CoNneCted nETwOrks ([@b24]). tHe diAMetEr rEPrESENtS ThE LoNGesT meASuriNg diStaNcE In a cOnNECteD netWorK; IT ShOWS THe NumBER OF StePS rEQuiReD fRom onE sidE Of thE NEtWOrk tO thE OThEr ([@B16]).
etHIcal coNSIdEraTiONs
----------------------
ThIs Study DiD nOT REqUirE ANY EThIcaL CONsIDEratioN AS it DoES NoT iNcLUde anY human or AnIMaL tO BE tHe ObJeCt Of sTuDy.
reSuLtS
=======
A sySTEMaTiC SeaRcH For pUblIcatiOns ON AdoLeScENt mYoPIa pREVENTiOn and coNtrOl retrieVED 624 ArticlES IN Web oF scIeNCE COrE COlLEctIon, ExCLUDIng OnE dUpLICaTE. aFTer fUrThEr ScreEnINg oF titLEs AnD ABsTrACTS, 9 EDItoRIAL mATeRiAls, 4 LETteRs and a mEetiNG ABsTraCT wErE removeD, leaviNg 610 ELigiBLe PApers.
ThE SCaLe And OVeRAlL tREnd of CollABOrATIve ReSEArch
-----------------------------------------------------
[FiGurE 1](#f1){reF-tYPE="fIG"} sHoWS ThE numbeR oF puBliCaTiOnS iSSued anNUally AND THE nUMBEr OF PAPeRs pubLiSHEd THrOuGh cOllaBoRaTION WitH authors, iNStituTiONaL CoOpeRatiOn aND COUntRY (rEGIoN) cOOPeratIon. thE NumbER OF pAPERs, cO-auThOrs, CO-iNsTiTUtIoNS anD COUNtrY (REGion) coopeRaTIVE PApERs Has iNcReASed SIgNiFiCantlY FRoM 1997 to 2016, PaRTICuLArLY AFteR 2011. In geNEral, THE Total nUmBEr of pUbLIShEd ARTicles SinCe 1997 hAS INCReaSED MoRE THAN Six-FoLD, FroM 11 In 1997 TO 79 iN 2016; The InSTiTUtIonAL CoOperAtIOn inCREaSEd MoRe THAn FiVE-foLd, the aUThoR COOPErATioN INcreaSED by tWelve-foLD AnD The COuNtrY (Region) cOoPeRatIoN iNcrEasEd By fIFTEEN-foLd.
{#f1}
[Fig. 2](#F2){rEf-tYPe="FiG"} REveAls The AvEraGE nUmBEr OF authoRS, INsTiTuTIONs aNd CoUNtRIEs PeR ArTiCLE fROM 1997 to 2016. thE AVerAGe nUmBer SHowS A GRAdualLY INCrEASINg treND.
{#f2}
tHE INcrEAse in NumBeR of AUtHoRS WAS FRoM 3.91 to 4.34, fROM 1.36 TO 2.51 FOR INstItutIoNS and FroM 0.82 to 1.25 FOr cOUnTRieS peR pAper. oVErALL, THE RatES OF COopeRAtIon aMONg autHors, InStItUtIONS AND coUntRIES were 93%, 57.9% and 21.5%, reSPeCTIvely.
iN GENERal, the nUmBEr Of sCI JoUrNal papers pRoDuCEd BY iNsTItutIOnAL coOPEraTIoN iS thE LArgeST (AccOUNtiNg For 56.6%), FolLOwed BY PApeRS genErAteD THrOugh INTra-iNstItUTional cOllAbORaTion (acCoUnting FOr 36.1%) and pApErs ProducED withoUT cOLLAboRATION (ACcOuNtINg FoR only 7.4%). [FIGURe 3](#f3){rEf-tYpE="fIG"} shOws tHe PeRcentAGE OF PaperS StuDieD iN EAcH oF tHE DiffEREnt INSTiTuTiON coLlABORATion typeS aNd tHeiR chaNGes OvEr Time. thE PeRceNtAges of singLE-aUtHor paPErs HaVE DecreaSED bY 26.7% FrOM 1997 TO 2016, wHEreAs tHAT of InsTITUTiON-CoLLAboRAtEd PAPErs IncREaSEd By 24.4%. The pERCEnTagE oF papErs prODuCEd thrOUGh SingLe AutHORSHip haS aLWAYS BeEn HIgHEr THAN THat Of INstITUTIonal COLlAboRatiOn FROm 1997 TO 2000 bUT decReaSEd afTer 2006.
{#f3}
aUThOrS' coLLaBoRATIvE ResEarCH
-------------------------------
rEsults oF scIEnTiFiC ResearCH ArE PUBLIsheD IN ThE fORM Of pAPErs, AnD ThE sTaTuS of CO-auTHorsHiP In paPERs reFlEcTs ThE coLLAbORAtIOn aMOng aUthORS. rEsEarcHERs who studY thE GrOwTH oF CO-AuThoRsHip arTiclES Produced bY mULtiplE authORs rEGard co-aUTHoRShIp Of papErs as a signIfiCanT sCIEnTomeTric iNDiCAtor Of ReseaRchING On cooperATiOn AMOng AUThors ([@B25]).
more iMPortant REsEArCHERS Were ExpeCtEd TO HAvE PUblisHeD MOrE artICles, thUS sCholarS wHO pUblisHed mOre ThaN four aRticlES weRE iNCLudEd iN ThE cO-AUTHorshiP NETwOrKs. overaLl, 75 ResearcheRS wiTh 371 Co-AuTHORED eXPERimenTS MEEt this cOnDItiOn. FIve autHoRs not cOoperatED wiTH OThEr aUthoRs were ExCLudEd. ThE ReseARcH colLABOrAtION nEtWOrK BEtWeEN auTHoRS Is shoWn in [FIG. 4](#F4){rEF-tyPE="fIG"}.
{#f4}
EAcH NOdE of tHE FiGUre repRESEnts an aUthoR, AnD tHe ConnECtIoNS aMOng ThE nODeS rEpRESeNt tHe CoLlAborAtion reLaTIoNsHiPs amonG aUTHOrS. tHe WEIGhT of A link indiCaTEs THE nUmbeR Of pubLIcAtiOnS Co-aUTHorEd bY Two SCHolARS. iN THIS autHoR's ColLAbORAtIoN nETwoRk, THE hiGHESt deGRee ceNtrAlIty Of Allen, pEtEr m. aNd O\'LEaRy, daNiEL j. WAS 5.83, inDicATInG thAT They had 5.83 ColLAboRAtORS And ThaT thEY PLAYed a pIvotAl RoLe in tHE cO | Introduction ============ Myopia is themain cause of preventiveblindnessworldwide, especially in adolescents ([@B1], [@B2]). Thus, it is one of the main priorities among the five projects underthe 'Vision 2020Action'launchedby WHO([@B3]). In recentyears,the incidence of myopia has increased rapidly worldwide ([@B4]), especially amongadolescents in East and Southeast Asia ([@B5], [@B6]). The prevalence of myopia amongadolescents isat 96.5% ([@B7]) in South Korea, 81.6%([@B8]) in Singapore and 95.5% ([@B9]) in Shanghai. Myopia not only affects adolescents' school performance and futurecareerchoice ([@B10]) but also causes glaucoma, cataract and other serious complications ([@B11]). Thus, many researchers have devotedthemselves to gainingamore in-depth understanding of the prevention and control of adolescent myopia ([@B12], [@B13]). Collaborative research networks can help other researchers expand their field of research orjoingroups conducting related studies. Bibliometric studies of scientificcollaboration havebeen conducted in various fields ([@B14], [@B15]), providing differentlevels of cooperation frequency in research practice. One of the methods used to study such collaboration is the co-authorship network analysis, which focuses onfindingpatterns of contacts or interactions between social actors. Author, country, and institution arethe subjects of cooccurrence relationship; thus,analyzing theircooccurrence relationship can better reflect the truth of scientific research and academic communication, because the cooperation ofauthors,institutions andcountriescan measure the cooperation at different levels ([@B15]). However, to date, no bibliometric analysis of scientific literature in myopia prevention and control had been carriedout andpublished. As such, this study aimed to describe the diversity of cooperation among authors, institutions, and countries in the study of adolescent myopia prevention and control. Specifically, for adolescent myopia preventionandcontrol research, our main goalis to explorethefollowing content: firstly, analysingthe overall status of collaborative research among authors, institutionsand countries; secondly, determining the institutions and authors at the core of the cooperative research network;andthirdly, identifying countries that have astrongcooperative relationship. Materialsand Methods===================== The search for papers to be includedin the analysis wasconducted in one day (Sep 25,2017)to avoid bias resulting from daily updating in the database. The Web of Science Core Collection annually collects a large number of journals and records eachpublication, including bibliographic information (i.e., author, institution and country or region), which weused to locate publications. All papers published within the period of 1997--2016 wereevaluated. Search terms includedcombinations ofterms,such as 'adolescent', 'children','student', 'myopia', 'myopic', 'prevention', 'control' and 'management'. Literature types, such as meeting abstracts, letters, correction, news item,book chapter, retractedpublication, editorial material, non-English literature and repeatedarticles, were excluded. Toensure reliability, profileinformation of each included article was extracted by two independent reviewers, resulting in a reliability check of 100% of theselected abstracts. A search query that wasused for data extraction from Web of Science database lookedlike this: TS= ((adolescent myopia OR children myopia OR student myopia OR adolescent myopic OR children myopic ORstudentmyopic) AND (prevention OR control OR management)). Social network analysis (SNA) isa method of structural analysis appliedin many research fields. It focuses on relationship research and is mainly used to describeand measure relationshipsand information between individuals([@B16]). SNA has been proven tobe effective in studies on scientificcollaboration network ([@B17],[@B18]).Thesame method is used in thecurrent study.To analyze and identify critical issues, we used SATI (Statistical Analysis Toolkit for Informetrics) (ver. 3.2) to build the co-occurrencematrix([@B19]) and transformed the data format withUcinet 6.0 ([@B20])to finally obtainco-occurrence mapping.VOS viewer (Visualisation of Similarities viewer) software (ver. 1.6.6) was employed to draw the co-country (region) maps by using literature title packets ([@B21]). Excel 2016 (Microsoft, Redmond, DC, USA) and Netdraw (ver. 2.118) were also used in the research. In addition, some measures of our network, including degree centrality, betweenness centrality,closeness centrality, density,anddiameter, were evaluated ([@B22]). Degree centrality refers to the number of neighbors to a nodein the network ([@B15]). In this case, the greater its connectiontoother nodes inthe network,the moreimportant is the node. Betweenness centrality refers to the number of the shortest paths passing through a given node ([@B23]).The higher the betweenness centrality of the node,the greater the ability to control the informationpassed between the othernodes. The closeness centrality is used to measurethe distance of onenode to othernodes in a network. Nodes with high closeness centralityobtain information better than other nodes or tend to have a more direct influence onother nodes([@B16]). Density is calculated throughthe actually observed ties divided by allpossible ties whose value is between 0and 1([@B24]).Density values tend to reach 0 in sparse networks, and closeto onein tightly connected networks ([@B24]). The diameter represents the longest measuring distance in a connectednetwork; it showsthe number of steps required from one side of the networkto the other([@B16]). Ethical considerations ---------------------- This study did not require anyethical considerationas it does not include any human or animal to be the object ofstudy. Results ======= A systematic search forpublications on adolescent myopia prevention and control retrieved624 articlesin Web of Science Core Collection,excluding one duplicate. After furtherscreening of titles and abstracts,9 editorial materials, 4 letters and a meeting abstract were removed,leaving 610 eligible papers. The scale and overall trend of collaborative research ----------------------------------------------------- [Figure 1](#F1){ref-type="fig"}showsthe number of publications issued annually and the number of paperspublished through collaboration withauthors, institutionalcooperation and country (region) cooperation. The number of papers, co-authors, co-institutions and country(region) cooperative papers has increased significantly from 1997 to 2016, particularly after 2011. In general,the total number of published articles since 1997 has increased more thansix-fold,from 11 in1997 to 79 in 2016;the institutional cooperation increased more than five-fold, the author cooperation increased by twelve-fold and the country (region) cooperation increasedby fifteen-fold. {#F1} [Fig. 2](#F2){ref-type="fig"} reveals the average numberof authors, institutions and countries per article from 1997 to 2016. The average number shows a gradually increasing trend. {#F2} The increase in number ofauthors was from 3.91 to 4.34, from 1.36 to 2.51 for institutions and from 0.82to 1.25 for countries per paper. Overall, the rates of cooperation among authors, institutions and countrieswere 93%, 57.9% and 21.5%, respectively. In general, the number of SCI journalpapers produced by institutional cooperation is thelargest (accounting for56.6%), followedby papers generated through intra-institutional collaboration (accounting for 36.1%) and papers produced without collaboration (accounting foronly7.4%). [Figure 3](#F3){ref-type="fig"} shows the percentage of papers studied ineach of the different institution collaboration types and theirchanges over time. The percentages of single-author papers have decreased by 26.7% from 1997 to 2016, whereas that of institution-collaborated papers increased by 24.4%. The percentageof papers producedthrough single authorship has always been higher than that of institutional collaboration from 1997 to 2000 but decreasedafter 2006.{#F3} Authors' collaborative research ------------------------------- Results of scientific research are published in theform of papers, and the status of co-authorship in papers reflects the collaborationamong authors. Researchers who study the growth of co-authorship articles produced by multipleauthorsregard co-authorship ofpapers as a significant scientometric indicatorofresearching on cooperation among authors ([@B25]). More importantresearchers were expected to havepublished more articles, thusscholars whopublished more than fourarticles were included inthe co-authorship networks. Overall, 75 researcherswith 371 co-authored experiments meet this condition. Five authors not cooperated with otherauthors were excluded. The research collaboration network betweenauthors is shown in [Fig. 4](#F4){ref-type="fig"}. {#F4} Each node ofthe figure represents an author, and the connections among the nodes represent the collaboration relationships among authors.The weight of a linkindicates the number ofpublications co-authored by two scholars. In this author's collaboration network, the highest degree centralityof Allen, Peter M. and O\'Leary, DanielJ. was 5.83, indicatingthat they had 5.83 collaboratorsand that they played a pivotal role in the co | Introduction ============ _Myopia_ _is_ _the_ main cause of preventive blindness worldwide, especially in adolescents _([@B1],_ [@B2]). _Thus,_ it _is_ one _of_ the main _priorities_ among the five _projects_ under _the_ 'Vision 2020 Action' launched by WHO ([@B3]). In _recent_ years, the incidence of myopia has increased rapidly _worldwide_ ([@B4]), especially _among_ _adolescents_ _in_ East and Southeast Asia ([@B5], [@B6]). The _prevalence_ of myopia among _adolescents_ is at 96.5% ([@B7]) in South _Korea,_ 81.6% ([@B8]) in Singapore and _95.5%_ ([@B9]) in Shanghai. _Myopia_ not _only_ _affects_ adolescents' school performance and future career choice ([@B10]) but also _causes_ glaucoma, cataract and other serious complications ([@B11]). Thus, many researchers have _devoted_ themselves to gaining a more in-depth understanding of the prevention and control of adolescent _myopia_ ([@B12], [@B13]). Collaborative research networks _can_ help other _researchers_ _expand_ their _field_ of _research_ or join groups conducting related studies. Bibliometric _studies_ of _scientific_ collaboration have been conducted in _various_ fields ([@B14], _[@B15]),_ providing different levels of cooperation frequency in research practice. One of the methods used to _study_ such collaboration is the co-authorship network analysis, which focuses on _finding_ _patterns_ of contacts or interactions between _social_ actors. _Author,_ country, and institution are the subjects of cooccurrence relationship; thus, _analyzing_ their cooccurrence relationship can better reflect the truth _of_ scientific research and academic communication, because _the_ _cooperation_ of authors, institutions _and_ _countries_ can _measure_ the cooperation at different levels ([@B15]). However, to _date,_ _no_ bibliometric analysis of scientific literature in myopia prevention _and_ control had _been_ carried out and published. As such, this _study_ _aimed_ to _describe_ the diversity of cooperation among authors, institutions, and countries in _the_ study of adolescent myopia prevention and _control._ Specifically, _for_ _adolescent_ myopia prevention and control research, our _main_ goal _is_ to explore the following content: firstly, analysing the overall status of _collaborative_ research among authors, institutions and countries; secondly, determining the institutions _and_ authors at the _core_ of the cooperative _research_ _network;_ and _thirdly,_ identifying _countries_ that have _a_ strong cooperative relationship. Materials and Methods ===================== The _search_ for papers to be _included_ in the analysis was _conducted_ _in_ one _day_ (Sep 25, 2017) _to_ _avoid_ bias resulting from daily updating in the database. The _Web_ of Science Core Collection annually collects a large number of journals and _records_ each _publication,_ including _bibliographic_ information (i.e., author, _institution_ and country _or_ region), which _we_ used to locate publications. All papers published _within_ the period _of_ 1997--2016 were _evaluated._ Search terms _included_ combinations of terms, such as _'adolescent',_ 'children', 'student', 'myopia', 'myopic', 'prevention', 'control' _and_ 'management'. Literature _types,_ _such_ as meeting abstracts, letters, _correction,_ news item, book chapter, retracted _publication,_ editorial material, non-English _literature_ and repeated articles, were excluded. To _ensure_ reliability, _profile_ information of each included _article_ was _extracted_ by two independent _reviewers,_ resulting in a reliability check of 100% of the selected abstracts. A search query that _was_ used for _data_ extraction from Web _of_ Science database looked like this: TS= ((adolescent myopia OR children myopia OR student myopia OR _adolescent_ myopic OR children _myopic_ OR student _myopic)_ AND _(prevention_ OR control OR _management))._ _Social_ network analysis (SNA) _is_ a method of structural analysis applied in many research fields. It _focuses_ on relationship research _and_ _is_ mainly used to describe and _measure_ relationships and information between individuals ([@B16]). SNA _has_ _been_ _proven_ to be effective in _studies_ on scientific collaboration _network_ ([@B17], [@B18]). The same method is _used_ in _the_ current study. To analyze and identify critical issues, we used SATI (Statistical Analysis Toolkit for Informetrics) (ver. 3.2) to build the _co-occurrence_ _matrix_ _([@B19])_ and transformed _the_ data format _with_ _Ucinet_ 6.0 _([@B20])_ to finally obtain _co-occurrence_ _mapping._ VOS _viewer_ (Visualisation of Similarities viewer) software (ver. _1.6.6)_ was _employed_ to draw the co-country (region) maps _by_ using literature title packets ([@B21]). Excel 2016 (Microsoft, Redmond, DC, USA) and _Netdraw_ (ver. 2.118) were _also_ used in the research. In _addition,_ _some_ _measures_ _of_ our network, including _degree_ centrality, betweenness centrality, closeness centrality, density, and diameter, were _evaluated_ ([@B22]). Degree _centrality_ refers _to_ the number of neighbors to a node in _the_ _network_ _([@B15])._ _In_ _this_ case, the greater its connection to other _nodes_ in _the_ network, the _more_ _important_ is the _node._ Betweenness centrality refers to the _number_ of the _shortest_ paths passing through a given node ([@B23]). _The_ higher _the_ betweenness centrality of the node, the greater _the_ _ability_ to control the information passed _between_ the other nodes. _The_ _closeness_ centrality is used to measure the distance of _one_ node to other nodes _in_ a network. Nodes with high _closeness_ centrality obtain information better _than_ other _nodes_ or tend to have a more direct influence on other nodes _([@B16])._ _Density_ is calculated through the actually observed ties divided by all possible ties whose _value_ is between 0 _and_ _1_ ([@B24]). Density values tend to reach 0 in _sparse_ _networks,_ and _close_ _to_ one in tightly connected _networks_ ([@B24]). The diameter represents _the_ longest measuring distance in _a_ _connected_ network; it shows the number _of_ steps required from one side of _the_ network to the _other_ ([@B16]). Ethical considerations ---------------------- This study did not require _any_ ethical consideration _as_ it does _not_ include any human or animal to be the _object_ of study. _Results_ ======= A systematic search for publications on _adolescent_ myopia _prevention_ and control retrieved 624 articles in Web of _Science_ _Core_ _Collection,_ excluding one duplicate. After further screening of _titles_ _and_ abstracts, 9 _editorial_ materials, 4 letters and _a_ meeting _abstract_ were _removed,_ leaving 610 _eligible_ papers. The scale and overall trend _of_ collaborative research ----------------------------------------------------- [Figure 1](#F1){ref-type="fig"} _shows_ the number of publications issued annually and the number of papers _published_ _through_ collaboration with authors, institutional cooperation and country (region) cooperation. _The_ _number_ of papers, co-authors, co-institutions and _country_ (region) cooperative _papers_ has increased significantly from 1997 to 2016, particularly after 2011. In general, the _total_ number of _published_ _articles_ since 1997 has increased more _than_ six-fold, _from_ 11 in _1997_ to 79 _in_ _2016;_ the institutional cooperation increased more than _five-fold,_ _the_ author cooperation increased by twelve-fold _and_ the country (region) cooperation increased by fifteen-fold. _{#F1} [Fig. 2](#F2){ref-type="fig"} reveals the average number of authors, institutions and countries per article from 1997 _to_ 2016. The _average_ _number_ shows a gradually increasing trend. _{#F2} The increase in number of authors was from 3.91 to 4.34, from 1.36 _to_ 2.51 for institutions and from _0.82_ to 1.25 _for_ countries _per_ paper. Overall, _the_ _rates_ of _cooperation_ _among_ authors, institutions and countries were 93%, _57.9%_ _and_ 21.5%, respectively. In general, the number of SCI journal papers produced by institutional cooperation is the largest (accounting for 56.6%), followed by papers generated _through_ _intra-institutional_ collaboration (accounting _for_ _36.1%)_ and papers produced without _collaboration_ _(accounting_ for _only_ 7.4%). [Figure _3](#F3){ref-type="fig"}_ shows the _percentage_ of papers studied in each _of_ the _different_ institution collaboration types and their changes over _time._ _The_ _percentages_ of single-author papers have decreased by 26.7% from 1997 to _2016,_ whereas that of _institution-collaborated_ papers increased by _24.4%._ The percentage _of_ papers _produced_ through single _authorship_ has always been higher _than_ that of institutional _collaboration_ from 1997 to 2000 _but_ decreased after 2006. {#F3} Authors' collaborative research ------------------------------- _Results_ _of_ _scientific_ research are published in the _form_ of papers, and the _status_ of co-authorship in papers reflects the collaboration among authors. Researchers who study the growth of co-authorship articles produced _by_ multiple authors regard co-authorship of papers as a _significant_ scientometric indicator of researching on _cooperation_ _among_ authors ([@B25]). More important researchers were expected to _have_ _published_ more articles, _thus_ scholars who published more than _four_ articles were included in the _co-authorship_ networks. Overall, 75 researchers with 371 _co-authored_ experiments meet this condition. Five authors _not_ cooperated _with_ other authors _were_ excluded. The research collaboration network between _authors_ is shown in [Fig. 4](#F4){ref-type="fig"}. {#F4} Each node of the figure represents an author, and the connections _among_ the nodes represent the collaboration relationships among authors. The _weight_ of a link indicates the number of _publications_ co-authored by two _scholars._ In this author's collaboration network, the _highest_ degree centrality _of_ Allen, Peter M. and O\'Leary, Daniel J. was 5.83, indicating that they had 5.83 collaborators _and_ _that_ they played a _pivotal_ role _in_ the co |
Introduction
============
Resistin is a cysteine-rich protein, which is mainly secreted from monocytes and macrophages in humans ([@b1-or-40-06-3392]--[@b3-or-40-06-3392]). It is associated with inflammation and malignant neoplasms ([@b4-or-40-06-3392]--[@b5-or-40-06-3392]). Blood resistin levels are demonstrated to be increased in certain cancer patients compared with healthy controls, including esophageal squamous cancer, gastric, colorectal, breast and endometrial cancer, and malignant lymphoma ([@b6-or-40-06-3392]--[@b12-or-40-06-3392]). Resistin is considered to be a risk factor for breast cancer ([@b9-or-40-06-3392],[@b13-or-40-06-3392]) and biomarker of disease progression of esophageal squamous cancer, gastric and colorectal cancer ([@b6-or-40-06-3392]--[@b8-or-40-06-3392]). It is an independent prognostic factor of pancreatic ductal adenocarcinoma ([@b5-or-40-06-3392]). Resistin can promote prostate cancer cell proliferation through the phosphatidylinositol 3 kinase (PI3K)/protein kinase B signaling pathway in human prostate cancer cell lines PC-3 and DU-145 ([@b14-or-40-06-3392]).
However, most of the reported studies only demonstrated the association between serum or plasma resistin and malignancy. Few reports measured the level of resistin expression in cancer tissues, even though it is less well studied and controversial in lung cancer. Certain reports demonstrated a higher concentration of resistin in the blood was demonstrated in non-small cell lung cancer (NSCLC) patients compared with the controls ([@b15-or-40-06-3392]--[@b17-or-40-06-3392]). One of the reports assessed resistin expression in the marginal area of lung cancer tissue and non-cancer region by immunofluorescence staining in 10 cases ([@b17-or-40-06-3392]). Another revealed that blood resistin levels were similar between cancer group and non-cancer group ([@b18-or-40-06-3392]). Furthermore, the clinical significance and biological function remain largely unknown.
However, lung cancer is one of the most common malignancies worldwide, with higher morbidity and poorer prognosis ([@b19-or-40-06-3392]--[@b20-or-40-06-3392]). The most common form of lung cancer is NSCLC, which includes lung adenocarcinoma and squamous carcinoma. At present, lung adenocarcinoma replaces squamous carcinoma as the dominating type of NSCLC. The aim of the present study is to determine the resistin expression in lung adenocarcinoma tissues, clinical significance and biological function *in vitro* and *in vivo*.
Materials and methods
=====================
### Patients and tissue samples
A total of 70 consecutive cases of newly diagnosed lung adenocarcinoma patients at Tianjin Medical University Cancer Institute and Hospital (Tianjin, China) from January to December 2008, with complete clinical and pathological data, were selected retrospectively in the present study and followed up for at least five years. Paired cancer and adjacent non-cancerous tissue samples, which were located more than 1 cm away from the tumor, were obtained through open surgeries. The paraffin-embedded tissue samples were stained with hematoxylin-eosin and confirmed lung as adenocarcinoma again.
The clinicopathological characteristics of the patients were recorded. The tumor staging of NSCLC was defined according to the tumor, node and metastasis system. The study was comprised of 70 cases of lung adenocarcinoma (38 male cases, 32 female cases), with an average age of 61 years old (36--77 years old). All patients received treatments (including operation, chemotherapy or radiotherapy), which conformed to the guidelines of NSCLC.
### Immunohistochemistry
For immunohistochemical staining, 5-µm paraffin-embedded tissue sections were heated for 1 h at 70°C, deparaffinized with a xylene soak, followed by rehydration via the addition of alcohol at decreasing concentrations (100, 95, 85 and 75%) for 5 min/step. A 96°C water-bath was used for antigen retrieval in 0.01 mol/l sodium citrate buffer (10 mM, pH 6.0) for 20 min. Next, endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide for 15 min at room temperature. Subsequently, slides were blocked with goat serum (cat. no. ZLI-9022; OriGene Technologies, Inc., Beijing, China; 1:1) at 37°C in a wet box for 30 min and then incubated by the primary antibody (rabbit polyclonal antibody against human resistin; cat. no. BS7730; Bioworld Technology, Inc., St. Louis Park, MN, USA; 1:100) overnight at 4°C in moist chambers. Following washing with 0.01 mol/L PBS (pH 7.2) three times, slides were incubated with a biotinylated secondary rabbit anti-mouse antibody (cat. no. PV-6000; OriGene Technologies, Inc.; 1:1) for 25 min at 37°C. Following incubating in horseradish peroxidase marked streptomycin avidin working fluid at 37°C for 30 min, slides were treated with avidin biotin-peroxidase complex using 3,3′-diaminobenzidine as a chromogen, and then counterstained with hematoxylin for 30 sec at room temperature and examined by light microscopy (Olympus Corporation, Tokyo, Japan). PBS was used as a negative control instead of a primary antibody.
### Evaluation of immunohistochemical staining
Existing tan or brown particles in the nucleus or cytoplasm indicated positive cells, which must conform to the following conditions: i) The cellular structure was clear; ii) the location of positive granules was accurate; iii) staining was significantly increased compared with the background.
In a 400× high power filed, randomly selected from 10 different cancer cell fields of view, the percentage of (a) positively stained cells was calculated as follows: 0--5% positive cells, score 0; 6--25% positive cells, score 1; 26--50% positive cells, score 2; 51--75% positive cells, score 3; 76--100% positive cells, score 4. Then, the (b) staining intensity was evaluated: Colorless, score 0; light yellow, score 1; deep yellow and tan, score 2; brown, score 3.
The expression of resistin was based on the product of (a) × (b): Score 0, negative (−); score 1--4, weakly positive (+); score 5--8, positive (++); score 9--12, strongly positive (+++). (−) and (+) were regarded as low expression group, (++) and (+++) were categorized as the high expression group ([Fig. 1A](#f1-or-40-06-3392){ref-type="fig"}). The result of each specimen was independently evaluated by two qualified and expert pathologists, blinded to the patients\' clinical data. The few cases with discordant results were reevaluated and final scores were consensual.
### Cell culture
Human lung adenocarcinoma cell lines A549 and H1975 were obtained from the Committee of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere of 95% air and 5% CO~2~. Cells were divided according to transfection of overexpression resistin plasmid for the resistin group and empty vector for the control group.
### Transfection and isolation of stable transfectants
Lipofectamine™ 2000 Reagent (Invitrogen; Thermo Fisher Scientific, Inc.), endo-free maxiplasmid kit (Tiangen Biotech Co., Ltd., Beijing, China); pcDNA3.1-(+)/resistin plasmids were established by OriGene Technologies, Inc. (cat. no. RC210942) and the primers were as follows: Forward primer, 5′-CCCACCGAGAGGGATGAAAG-3′ and reverse primer, 5′-CAGTGACATGTGGTCTCGGC-3′; forward primer, 5′-CAGCTCACCATGGATGATGATATC-3′ and reverse primer, 5′-AAGCCGGCCTTGCACAT-3′ (β-actin).
A fragment of the rat resistin cDNA fragment (285 bp) was inserted at the unique *Eco*RI site in the anti-sense orientation as determined by sequencing. The final concentration of resistin was 100 nM. A total of 2×10^6^ A549 and H1975 cells grown in 60 mm Petri dishes were transfected with 10 µg of the recombinant plasmid using lipofectamine, as described by the supplier (Gibco; Thermo Fisher Scientific, Inc.). A total of 24 h later, fresh RPMI-1640 media containing 10% FBS was added and replaced 48 h later. Monoclonal cells were selected with NeoR. Then the cells were cultured for 24 h following transfection.
### Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from the cells by TRIzol (cat. no. 15596026; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer\'s protocol. Prime Script RT reagent kit (DRR037A; Takara Bio, Inc., Otsu, Japan) were used for cDNA generation (42°C for 30--60 min, 70°C for 15 min). RT-qPCR was performed | introduction = = = = = = = = = = = = resistin is a cysteine - rich protein, which is mainly secreted from monocytes and macrophages in humans ( [ @ b1 - or - 40 - 06 - 3392 ] - - [ @ b3 - or - 40 - 09 - 3392 ] ). it is associated with inflammation and malignant neoplasms ( [ @ b4 - or - 40 - 06 - 3392 ] - - [ @ b5 - or - 40 - 06 - 3392 ] ). blood resistin levels have demonstrated to be increased in certain cancer patients compared with healthy controls, including esophageal squamous cancer, gastric, colorectal, breast and endometrial cancer, and malignant lymphoma ( [ @ b6 - or - 40 - 06 - 3392 ] - - [ @ b12 - or - 40 - 06 - 3392 ] ). resistin is considered may be a risk factor for breast cancer ( [ @ b9 - or - 40 - 06 - 540 ], [ @ b13 - ur - 40 - 06 - 3392 ] ) and biomarker of disease progression of esophageal squamous cancer, gastric and colorectal cancer ( [ @ b6 - or - 40 - 06 - 3392 ] - - [ @ b8 - or - 40 - 06 - 3392 ] ). it is an independent prognostic factor of pancreatic ductal adenocarcinoma ( [ @ b5 - or - 40 - 06 - 3392 ] ). resistin can promote prostate cancer cell proliferation through the phosphatidylinositol 3 kinase ( cdc ) / protein kinase b signaling pathway in human prostate cancer cell lines pc - 3 and du - 145 ( [ @ b14 - or - 40 - 06 - 3392 ] ). however, most in the reported studies only demonstrated the association between serum or plasma proliferation and malignancy. few reports demonstrate the level of resistin expression in cancer tissues, although though it is less well studied and controversial in lung cancer. certain reports demonstrated a higher concentration of resistin in the blood was demonstrated in non - small cell lung cancer ( nsclc ) patients compared with the controls ( [ @ b15 - or - 40 - 06 - 3392 ] - - [ @ b17 - or - 40 - 06 - 3392 ] ). one of the reports assessed resistin expression in the marginal area of lung cancer tissue and non - cancer region by immunofluorescence staining in 10 cases ( [ @ b17 - or - 40 - 06 - 3392 ] ). another revealed that blood resistin levels were similar between cancer group and non - cancer group ( [ @ b18 - or - 40 - 06 - 3392 ] ). furthermore, the clinical significance and biological function remain largely unknown. however, lung cancer is one of the most common malignancies worldwide, with higher morbidity and poorer prognosis ( [ @ b19 - or - 40 - 06 - 3392 ] - - [ @ b20 - or - 40 - 06 - 3392 ] ). the most common form of lung cancer is nsclc, which includes lung adenocarcinoma and squamous carcinoma. at present, lung adenocarcinoma replaces squamous carcinoma as the dominating type of nsclc. the aim of the present study is to determine the resistin expression in lung adenocarcinoma tissues, clinical significance and biological function * in vitro * and * in vivo *. materials and methods = = = = = = = = = = = = = = = = = = = = = # # # patients and tissue samples a total of 70 consecutive cases of newly diagnosed lung adenocarcinoma patients at tianjin medical university cancer institute and hospital ( tianjin, china ) from january to december 2008, with complete clinical and pathological data, were selected retrospectively in the present study and followed up for at least five years. paired cancer and adjacent non - cancerous tissue samples, which were located more than 1 cm away from the tumor, were obtained through open surgeries. the paraffin - embedded tissue samples were stained with hematoxylin - eosin and confirmed lung as adenocarcinoma again. the clinicopathological characteristics of the patients were recorded. the tumor staging of nsclc was defined according to the tumor, node and metastasis system. the study was comprised of 70 cases of lung adenocarcinoma ( 38 male cases, 32 female cases ), with an average age of 61 years old ( 36 - - 77 years old ). all patients received treatments ( including operation, chemotherapy or radiotherapy ), which conformed to the guidelines of nsclc. # # # immunohistochemistry for immunohistochemical staining, 5 - µm paraffin - embedded tissue sections were heated for 1 h at 70°c, deparaffinized with a xylene soak, followed by rehydration via the addition of alcohol at decreasing concentrations ( 100, 95, 85 and 75 % ) for 5 min / step. a 96°c water - bath was used for antigen retrieval in 0. 01 mol / l sodium citrate buffer ( 10 mm, ph 6. 0 ) for 20 min. next, endogenous peroxidase activity was quenched by incubation in 3 % hydrogen peroxide for 15 min at room temperature. subsequently, slides were blocked with goat serum ( cat. no. zli - 9022 ; origene technologies, inc., beijing, china ; 1 : 1 ) at 37°c in a wet box for 30 min and then incubated by the primary antibody ( rabbit polyclonal antibody against human resistin ; cat. no. bs7730 ; bioworld technology, inc., st. louis park, mn, usa ; 1 : 100 ) overnight at 4°c in moist chambers. following washing with 0. 01 mol / l pbs ( ph 7. 2 ) three times, slides were incubated with a biotinylated secondary rabbit anti - mouse antibody ( cat. no. pv - 6000 ; origene technologies, inc. ; 1 : 1 ) for 25 min at 37°c. following incubating in horseradish peroxidase marked streptomycin avidin working fluid at 37°c for 30 min, slides were treated with avidin biotin - peroxidase complex using 3, 3 ′ - diaminobenzidine as a chromogen, and then counterstained with hematoxylin for 30 sec at room temperature and examined by light microscopy ( olympus corporation, tokyo, japan ). pbs was used as a negative control instead of a primary antibody. # # # evaluation of immunohistochemical staining existing tan or brown particles in the nucleus or cytoplasm indicated positive cells, which must conform to the following conditions : i ) the cellular structure was clear ; ii ) the location of positive granules was accurate ; iii ) staining was significantly increased compared with the background. in a 400× high power filed, randomly selected from 10 different cancer cell fields of view, the percentage of ( a ) positively stained cells was calculated as follows : 0 - - 5 % positive cells, score 0 ; 6 - - 25 % positive cells, score 1 ; 26 - - 50 % positive cells, score 2 ; 51 - - 75 % positive cells, score 3 ; 76 - - 100 % positive cells, score 4. then, the ( b ) staining intensity was evaluated : colorless, score 0 ; light yellow, score 1 ; deep yellow and tan, score 2 ; brown, score 3. the expression of resistin was based on the product of ( a ) × ( b ) : score 0, negative ( − ) ; score 1 - - 4, weakly positive ( + ) ; score 5 - - 8, positive ( + + ) ; score 9 - - 12, strongly positive ( + + + ). ( − ) and ( + ) were regarded as low expression group, ( + + ) and ( + + + ) were categorized as the high expression group ( [ fig. 1a ] ( # f1 - or - 40 - 06 - 3392 ) { ref - type = " fig " } ). the result of each specimen was independently evaluated by two qualified and expert pathologists, blinded to the patients \ ' clinical data. the few cases with discordant results were reevaluated and final scores were consensual. # # # cell culture human lung adenocarcinoma cell lines a549 and h1975 were obtained from the committee of type culture collection of the chinese academy of sciences ( shanghai, china ). the cells were maintained in rpmi - 1640 ( gibco ; thermo fisher scientific, inc., waltham, ma, usa ) supplemented with 10 % fetal bovine serum ( fbs ; gibco ; thermo fisher scientific, inc. ) at 37°c in a humidified atmosphere of 95 % air and 5 % co ~ 2 ~. cells were divided according to transfection of overexpression resistin plasmid for the resistin group and empty vector for the control group. # # # transfection and isolation of stable transfectants lipofectamine™ 2000 reagent ( invitrogen ; thermo fisher scientific, inc. ), endo - free maxiplasmid kit ( tiangen biotech co., ltd., beijing, china ) ; pcdna3. 1 - ( + ) / resistin plasmids were established by origene technologies, inc. ( cat. no. rc210942 ) and the primers were as follows : forward primer, 5 ′ - cccaccgagagggatgaaag - 3 ′ and reverse primer, 5 ′ - cagtgacatgtggtctcggc - 3 ′ ; forward primer, 5 ′ - cagctcaccatggatgatgatatc - 3 ′ and reverse primer, 5 ′ - aagccggccttgcacat - 3 ′ ( β - actin ). a fragment of the rat resistin cdna fragment ( 285 bp ) was inserted at the unique * eco * ri site in the anti - sense orientation as determined by sequencing. the final concentration of resistin was 100 nm. a total of 2×10 ^ 6 ^ a549 and h1975 cells grown in 60 mm petri dishes were transfected with 10 µg of the recombinant plasmid using lipofectamine, as described by the supplier ( gibco ; thermo fisher scientific, inc. ). a total of 24 h later, fresh rpmi - 1640 media containing 10 % fbs was added and replaced 48 h later. monoclonal cells were selected with neor. then the cells were cultured for 24 h following transfection. # # # reverse transcription - quantitative polymerase chain reaction ( rt - qpcr ) total rna was extracted from the cells by trizol ( cat. no. 15596026 ; invitrogen ; thermo fisher scientific, inc. ) according to the manufacturer \ ' s protocol. prime script rt reagent kit ( drr037a ; takara bio, inc., otsu, japan ) were used for cdna generation ( 42°c for 30 - - 60 min, 70°c for 15 min ). rt - qpcr was performed | Introduction = = = = = = = = = = = = Resistin is a cysRejne - rich protein, which is mainly secreted from monocytes and macrophages in humans ([ @ b1 - or - 40 - 06 - 3392] - - [@ b3 - or - 40 - 06 - 3392] ). It is aswoviated with inflammation and malignant neoplasms ([ @ b4 - or - 40 - 06 - 3392] - - [@ b5 - or - 40 - 06 - 3392] ). Blood resistin levels are demonstrated to be increased in certain cancer patients compared with healthy controls, including esophageal squamous cancer, gastric, colorectal, breAqt and endometrial cancer, and malignant lymphoma ([ @ b6 - or - 40 - 06 - 3392] - - [@ b12 - or - 40 - 06 - 3392] ). Resistin is considered to be a risk factor for breast cancer ([ @ b9 - or - 40 - 06 - 3392 ], [@ b13 - or - 40 - 06 - 3392] ) and biomarker of disease progression of esophageal squamous cancer, gastric and colorectal cancer ([ @ b6 - or - 40 - 06 - 3392] - - [@ b8 - or - 40 - 06 - 3392] ). It is an independent prognostic factor of pancreatic ductal adenocarcinoma ([ @ b5 - or - 40 - 06 - 3392] ). Resistin can promote prostate cancer cell proliferation through the phosphatidylinositol 3 kinase (PI3K) / protein kinase B signaling pathway in human prostate cancer cell lines PC - 3 and DU - 145 ([ @ b14 - or - 40 - 06 - 3392] ). However, most of the reported studies only demonstrated the association between serum or plasma resistin and malignancy. Few reports measured the level of resistin expression in cancer tissues, even though it is less well studied and controversial in lung cancer. Certain reports demonstrated a higher concentration of resistin in the blood was demonstrated in non - small cell lung cancer (NSCLC) patients compared with the controls ([ @ b15 - or - 40 - 06 - 3392] - - [@ b17 - or - 40 - 06 - 3392] ). One of the reports assessed resistin expression in the marginal area of lung cancer tissue and non - cancer region by immunofluorescence staining in 10 cases ([ @ b17 - or - 40 - 06 - 3392] ). Another revealed that blood resistin levels were similar between cancer group and non - cancer group ([ @ b18 - or - 40 - 06 - 3392] ). Furthermore, the clinical significance and biological function reNaib largely unknown. However, lung cancer is one of the most common malignancies worldwide, with higher morbidity and poorer prognosis ([ @ b19 - or - 40 - 06 - 3392] - - [@ b20 - or - 40 - 06 - 3392] ). The most common form of lung cancer is NSCLC, which includes lung adenocarcinoma and squamous carcinoma. At present, lung adenocarcinoma replaces squamous carcinoma as the dominating type of NSCLC. The aim of the present study is to determine the resistin expression in lung adenocarcinoma tissues, xlinicSl significance and biological function * in vitro * and * in vivo *. Materials and methods = = = = = = = = = = = = = = = = = = = = = # # # Patients and tissue samples A total of 70 consecutive cases of newly diagnosed lung adenocarcinoma patients at Tianjin Medical University Cancer Institute and Hospital (Tianjin, China) from January to December 2008, with complete clinical and pathological data, were selected retrospectively in the present study and followed up for at least five years. Paired cancer and adjacent non - cancerous tissue samples, which were located more than 1 cm away from the tumor, were obtained through open surgeries. The paraffin - embedded tissue samples were stained with hematoxylin - eosin and confirmed lung as adenocarcinoma again. The clinicopathological characteristics of the patients were recorded. The tumor staging of NSCLC was defined according to the tumor, node and metastasis system. The study was comprised of 70 cases of lung adenocarcinoma (38 male cases, 32 female cases ), with an average age of 61 years old (36 - - 77 years old ). All patients received treatments (including operation, chemotherapy or radiotherapy ), which conformed to the guidelines of NSCLC. # # # Immunohistochemistry For immunohistochemical staining, 5 - µm paraffin - embedded tissue sections wegF heated for 1 h at 70 ° C, deparaffinized with a xylene soak, followed by rehydration via the addition of alcohol at decreasing concentrations (100, 95, 85 and 75%) for 5 min / step. A 96 ° C water - bath was used for antigen retrieval in 0. 01 mol / l sodium citrate buffer (10 mM, pH 6. 0) for 20 min. Next, endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide for 15 min at room temperature. Subsequently, slides were blocked with goat serum (cat. no. ZLI - 9022; OriGene Technologies, Inc. , Beijing, China; 1: 1) at 37 ° C in a wet box for 30 min and then incubated by the primary antibody (rabbit polyclonal antibody against human resistin; cat. no. BS7730; Bioworld Technology, Inc. , St. Louis Park, MN, USA; 1: 100) overnight at 4 ° C in moist chambers. Following washing with 0. 01 mol / L PBS (pH 7. 2) three times, slides were incubated with a biotinylated secondary rabbit anti - mouse antibody (cat. no. PV - 6000; OriGene Technologies, Inc. ; 1: 1) for 25 min at 37 ° C. Following incubating in horseradish peroxidase marked streptomycin avidin working fluid at 37 ° C for 30 min, slides were treated with avidin biotin - peroxidase complex using 3, 3 ′ - diaminobenzidine as a chromogen, and then counterstained with hematoxylin for 30 sec at room temperature and examined by light microscopy (Olympus Corporation, Tokyo, Japan ). PBS was used as a negative control instead of a primary antibody. # # # Evaluation of immunohistochemical staining Existing tan or brown particles in the nucleus or cytoplasm indicated positive cells, which must conform to the following conditions: i) The cellular structure was clear; ii) the location of positive granules was accurate; iii) staining was significantly increased compared with the background. In a 400 × high power filed, randomly selected from 10 different cancer cell fields of view, the percentage of (a) positively stained cells was calculated as follows: 0 - - 5% positive cells, score 0; 6 - - 25% positive cells, score 1; 26 - - 50% positive cells, score 2; 51 - - 75% positive cells, score 3; 76 - - 100% positive cells, score 4. Then, the (b) staining intensity was evaluated: Colorless, score 0; light yellow, score 1; deep yellow and tan, score 2; brown, score 3. The expression of resistin was based on the product of (a) × (b ): Score 0, negative (− ); score 1 - - 4, weakly positive (+ ); score 5 - - 8, positive (+ + ); score 9 - - 12, strongly positive (+ + + ). (−) and (+) were regarded as low expression group, (+ +) and (+ + +) were categorized as the high expression group ([ Fig. 1A] (# f1 - or - 40 - 06 - 3392) {ref - type = " fig "} ). The result of each specimen was independently evaluated by two qualified and expert pathologists, blinded to the patients \ ' clinical data. The few cases with discordant results were reevaluated and final scores were consensual. # # # Cell culture bumxn lung adenocarcinoma cell lines A549 and H1975 were obtained from the Committee of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China ). The cells were maintained in RPMI - 1640 (Gibco; Thermo Fisher Scientific, Inc. , Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37 ° C in a humidified atmosphere of 95% air and 5% CO ~ 2 ~. Cells were divided according to transfection of overexpression resistin plasmid for the resistin group and empty vector for the control group. # # # Transfection and isolation of stable transfectants Lipofectamine ™ 2000 Reagent (Invitrogen; Thermo Fisher Scientific, Inc. ), endo - free maxiplasmid kit (Tiangen Biotech Co. , Ltd. , Beijing, China ); pcDNA3. 1 - (+) / resistin plasmids were established by OriGene Technologies, Inc. (cat. no. RC210942) and the primers were as follows: Forward primer, 5 ′ - CCCACCGAGAGGGATGAAAG - 3 ′ and reverse primer, 5 ′ - CAGTGACATGTGGTCTCGGC - 3 ′; forward primer, 5 ′ - CAGCTCACCATGGATGATGATATC - 3 ′ and reverse primer, 5 ′ - AAGCCGGCCTTGCACAT - 3 ′ (β - actin ). A fragment of the rat resistin cDNA fragment (285 bp) was inserted at the unique * Eco * RI site in the anti - sense orientation as determined by sequencing. The final concentration of resistin was 100 nM. A total of 2 × 10 ^ 6 ^ A549 and H1975 cells grown in 60 mm Petri dishes were transfected with 10 µg of the revombinan% plasmid using lipofectamine, as described by the supplier (Gibco; Thermo TisTer Scientific, Inc. ). A total of 24 h later, fresh RPMI - 1640 media containing 10% FBS was added and replaced 48 h later. Monoclonal cells were selected with NeoR. Then the cells were cultured for 24 h following transfection. # # # Reverse transcription - quantitative polymerase chain reaction (RT - qPCR) Total RNA was extracted from the cells by TRIzol (cat. no. 15596026; 9nvktrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer \ ' s protocol. Prime Script RT reagent kit (DRR037A; Takara Bio, Inc. , Otsu, Japan) were used for cDNA generation (42 ° C for 30 - - 60 min, 70 ° C for 15 min ). RT - qPCR was performed | ============ Resistin is cysteine-rich protein, is mainly secreted from monocytes and macrophages in humans It is associated with inflammation and malignant neoplasms ([@b4-or-40-06-3392]--[@b5-or-40-06-3392]). Blood resistin levels are demonstrated be increased in certain cancer patients compared with healthy controls, including esophageal squamous cancer, gastric, colorectal, breast and endometrial cancer, and malignant lymphoma ([@b6-or-40-06-3392]--[@b12-or-40-06-3392]). Resistin is considered to be a risk factor for breast ([@b9-or-40-06-3392],[@b13-or-40-06-3392]) biomarker of disease progression esophageal squamous cancer, and cancer ([@b6-or-40-06-3392]--[@b8-or-40-06-3392]). is an independent prognostic factor of pancreatic ductal adenocarcinoma ([@b5-or-40-06-3392]). Resistin can promote prostate cancer cell proliferation through the phosphatidylinositol 3 kinase (PI3K)/protein B signaling pathway in human prostate cancer cell lines PC-3 ([@b14-or-40-06-3392]). However, most of the reported studies only demonstrated the association between serum or plasma resistin and malignancy. Few reports measured the level of expression in cancer tissues, though is less well studied and controversial in lung cancer. Certain reports demonstrated a higher concentration of resistin in the blood was demonstrated in non-small cell lung cancer (NSCLC) patients compared with the controls ([@b15-or-40-06-3392]--[@b17-or-40-06-3392]). One of the reports assessed resistin expression in the marginal area of lung cancer tissue and non-cancer region by immunofluorescence staining in 10 ([@b17-or-40-06-3392]). Another revealed that blood resistin levels were between cancer group and group ([@b18-or-40-06-3392]). Furthermore, the clinical significance and biological function remain largely unknown. However, lung one of the common malignancies worldwide, with higher morbidity and poorer prognosis ([@b19-or-40-06-3392]--[@b20-or-40-06-3392]). The most common form of lung cancer is NSCLC, which includes lung adenocarcinoma and squamous carcinoma. At present, lung adenocarcinoma replaces squamous carcinoma as the dominating of NSCLC. The of present study is to determine the resistin expression in lung adenocarcinoma tissues, clinical significance and biological function *in vitro* and *in vivo*. Materials and methods ===================== ### Patients and samples A total of 70 consecutive of newly diagnosed lung adenocarcinoma patients at Tianjin Medical University Cancer Institute and Hospital (Tianjin, China) from January to December 2008, with clinical and pathological data, were selected retrospectively the present study and followed up for at least five years. Paired cancer adjacent non-cancerous which were located more than 1 cm away from the were through open surgeries. The paraffin-embedded tissue samples were stained with and confirmed lung as adenocarcinoma The characteristics of the patients were recorded. The tumor staging of NSCLC was defined according to the tumor, node and metastasis system. The study was comprised of 70 cases of lung adenocarcinoma (38 male cases, 32 female cases), with an age of years old (36--77 years old). All patients received treatments operation, chemotherapy or radiotherapy), which to the guidelines of NSCLC. ### For immunohistochemical staining, 5-µm paraffin-embedded tissue sections were heated for h at 70°C, deparaffinized with a xylene soak, followed by rehydration via the addition of decreasing concentrations (100, 95, 85 and 75%) for 5 min/step. A 96°C water-bath was used for antigen retrieval in 0.01 mol/l sodium citrate buffer (10 mM, 6.0) for 20 min. Next, endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide for 15 min at room temperature. Subsequently, slides were blocked with goat serum (cat. no. ZLI-9022; OriGene Inc., Beijing, China; 1:1) at 37°C in a box for and then incubated the primary antibody (rabbit polyclonal antibody human resistin; cat. no. BS7730; Bioworld Technology, Inc., Louis Park, MN, USA; 1:100) overnight at 4°C moist chambers. Following washing with 0.01 mol/L PBS 7.2) three times, slides were incubated with a biotinylated secondary rabbit anti-mouse antibody (cat. no. PV-6000; OriGene Inc.; 1:1) for 25 37°C. Following incubating in horseradish peroxidase marked streptomycin avidin working fluid at for 30 min, slides were treated with avidin biotin-peroxidase complex as a chromogen, and then counterstained with 30 sec at room temperature and examined by light (Olympus Corporation, Tokyo, Japan). PBS was used as a negative control instead of a primary antibody. ### Evaluation of immunohistochemical staining Existing or brown particles in the nucleus or cytoplasm indicated cells, which must conform to the following conditions: i) The cellular structure was clear; ii) the location positive granules was accurate; iii) staining was significantly increased compared with the background. In a 400× high filed, randomly selected from 10 different cancer cell fields of view, the percentage of (a) positively stained cells calculated as follows: 0--5% positive cells, score 0; 6--25% positive positive cells, score 2; 51--75% positive cells, score 3; 76--100% positive cells, score 4. the (b) staining intensity was evaluated: Colorless, score 0; light yellow, score 1; deep yellow and score 2; brown, score The expression of resistin was based on the product of (a) × (b): Score 0, negative (−); 1--4, positive (+); 5--8, positive score strongly positive (+++). (−) and (+) were regarded as low expression group, (++) and (+++) were categorized as the high expression group ([Fig. The result of each specimen was independently by two qualified and expert pathologists, blinded to the patients\' clinical The few cases with discordant results were reevaluated and final scores were ### Cell culture Human lung adenocarcinoma cell lines A549 H1975 were obtained from Committee of Type Collection of the Chinese Academy of (Shanghai, China). The cells maintained in RPMI-1640 (Gibco; Thermo Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere of 95% air and CO~2~. Cells were divided according to transfection of resistin plasmid for resistin group and empty vector for the control group. ### Transfection and isolation of stable transfectants Lipofectamine™ 2000 Reagent (Invitrogen; Thermo Fisher Scientific, Inc.), endo-free maxiplasmid kit (Tiangen Biotech Co., Ltd., Beijing, China); pcDNA3.1-(+)/resistin were established OriGene Inc. (cat. no. RC210942) and the primers were as follows: Forward primer, 5′-CCCACCGAGAGGGATGAAAG-3′ and reverse primer, 5′-CAGTGACATGTGGTCTCGGC-3′; forward primer, 5′-CAGCTCACCATGGATGATGATATC-3′ and reverse primer, 5′-AAGCCGGCCTTGCACAT-3′ (β-actin). A fragment the rat resistin fragment (285 bp) was inserted the unique *Eco*RI site in the anti-sense as determined by sequencing. final concentration of resistin was 100 nM. A total of 2×10^6^ A549 and H1975 cells grown in 60 mm dishes were transfected with µg of recombinant plasmid using lipofectamine, as described by supplier (Gibco; Fisher Scientific, Inc.). A total of 24 h fresh RPMI-1640 media 10% FBS was and replaced 48 h later. Monoclonal cells were selected with NeoR. Then the cells were cultured for 24 h following ### Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Total RNA was extracted from the cells by TRIzol (cat. no. 15596026; Fisher Scientific, according to the manufacturer\'s protocol. Prime Script RT reagent kit Takara Bio, Inc., Otsu, Japan) were used for cDNA generation 30--60 70°C for 15 min). RT-qPCR was performed | iNTROduCtIon
============
ReSiStiN Is a cYsTeINE-ricH PrOteIN, WhicH IS maiNlY sECReTED FrOm MONoCyteS And MaCrophAges In HUmANs ([@B1-or-40-06-3392]--[@B3-or-40-06-3392]). IT iS AsSoCIaTED WItH iNFlaMmATIoN aNd mALIgNAnt nEOPLASmS ([@b4-Or-40-06-3392]--[@b5-Or-40-06-3392]). BLOoD rEsIsTin LEvElS arE deMonsTrATED to bE iNcReAsED iN CERTAIN caNcEr PAtients cOmParED With heAlTHy cONtrolS, iNcludINg eSOPHaGeAl SquAMOuS CaNCER, GASTRic, ColOrECTaL, BrEAst anD enDOMETRIaL CaNceR, ANd MALiGnAnt LyMPhOmA ([@b6-oR-40-06-3392]--[@B12-or-40-06-3392]). RESiStiN Is coNsIDEred tO BE A RiSK FACTOr foR BReAsT CanCer ([@B9-OR-40-06-3392],[@b13-OR-40-06-3392]) AnD BiomaRKEr oF DisEASe PRogresSion oF EsOPhAgeal SquAmOUS cANCeR, gaSTRIC And COLOrEcTal CAnCeR ([@b6-or-40-06-3392]--[@B8-OR-40-06-3392]). it Is an iNDependEnt PRognOsTiC fAcToR oF pAncreATIc DuCTal AdeNoCARCinoMa ([@b5-oR-40-06-3392]). rESisTin CAN promoTe PROState CanceR CeLl pRoLIfeRatiOn THRouGh ThE PHOSpHAtidyLInosiToL 3 KinasE (Pi3k)/PRoteIn kInAse B SIGnaLing PAtHWAY In hUmAn pRoStatE CanCER cell lINES pC-3 ANd dU-145 ([@B14-OR-40-06-3392]).
howEVeR, MOSt OF thE rePoRTed sTudIes onLY dEMONsTRAteD thE AsSoCiATiON BEtWeeN SErUm OR PlasmA REsIStin AND MAlignaNcY. FEW RepoRts mEAsUrED the LeVEL OF RESiSTiN eXprEsSIOn iN CaNcER TISSues, evEN ThOUgH it IS lEsS welL studIED ANd CONTRoversiaL in LuNg CancEr. CErtAiN RePorTs DeMOnstrATeD A HIGHER concentRATiON Of ResiSTin in ThE blOOD WAs DEmonstrated In NoN-sMAlL CELl Lung canCer (NsCLC) PAtiENTS cOmPaReD with tHe CONTROLS ([@B15-or-40-06-3392]--[@B17-oR-40-06-3392]). one of tHe rePortS asSeSsEd rEsISTiN eXPrEsSion In tHe maRgiNaL ArEA Of luNG CaNCer tISsue ANd Non-caNCeR reGioN By imMuNOfLuorEscEnCE StAIninG in 10 caSEs ([@B17-Or-40-06-3392]). AnOtheR rEveaLeD ThAT BLOOD ResisTIn leVELS WerE SIMilAr betwEEN cANceR GRoup aND non-caNcEr gRouP ([@b18-oR-40-06-3392]). fuRtHeRmORE, the cLIniCAl sIgNiFicanCe ANd BIOlogiCal fUNcTION reMain largEly UnknowN.
howeVER, LUnG CAnCer IS One Of tHE MOST commoN MalignaNcIES wORlDWIde, wITH highEr moRBIdiTY anD Poorer PRoGnosis ([@b19-OR-40-06-3392]--[@b20-oR-40-06-3392]). thE mosT cOmmon forM of LUnG CanceR is NSClC, WhIcH InclUDes luNg aDenocARCiNomA AnD SqUaMoUS CarCinomA. At preSenT, LuNG adeNoCaRCInOma ReplACes sQUAmoUs CarCiNoma as The doMInAtIng tYPE Of NSClc. thE AiM oF tHe PrEseNT sTUDy Is tO DETeRMiNE the ReSIStIn ExPResSIon IN LUnG ADenoCArcINoMa tissues, cLiNiCal sIgNIFIcAnCe AND BIolOGIcAl fUNcTiOn *In vitRo* and *iN VIVo*.
MAtErIALS AND MEthODs
=====================
### PaTiENTS AND TIssUE SAMpleS
A TOtaL Of 70 consecuTIVE CASEs of newLy DiaGNOSed lUNg AdenoCArCInoMA PAtIenTS aT tIANjIN MeDiCal univERSitY CanCeR InsTitUte aNd HosPiTAl (TianjiN, chINa) FROM JanuARy TO dECemBER 2008, wItH COmPLETe ClINICAl anD PAthOLoGiCaL daTA, WERE SELECTED REtrOSPecTIveLY IN THE PRESEnT stUDY and FoLLOWeD uP FOR at LEaST fIVE YEArS. pAireD CaNcer ANd adJacENT NON-CaNCerOuS TISSuE sAMPlES, wHich wEre LOCateD mOre ThaN 1 cM awaY frOM THe tumoR, WeRE obtAInED thrOUGH opeN suRgeRies. ThE paRafFIn-embEDdeD TISsuE SAmpLES weRe sTained wItH HEMAToxYLIN-EoSIn and cOnfIRmed LUng aS AdEnoCaRCiNoMA aGaIn.
THE clInicopatHOloGICal chaRActErISticS Of THe paTIEntS weRE rEcOrDed. thE TumOR staginG OF NsCLC wAS dEfINeD aCcorDinG TO the TUmor, NoDE anD meTaSTasiS SYStEM. THE sTUdy wAs comPrIsED Of 70 caSes of LUnG AdENoCARcinomA (38 MaLe CAsEs, 32 FEMale cASEs), With AN aVerAGE Age Of 61 yEARS Old (36--77 yeArS OLd). alL paTiEnTs receiVed trEaTMentS (IncLudINg oPeRAtioN, chEMOtheRapY oR rAdiOthERaPy), wHicH ConfoRMED to The GuIDEliNEs OF nSClc.
### iMMUnohiStOChEMistRY
foR iMMUnOHISToChemIcAl StAinING, 5-ΜM PaRAffIn-EMbeDded tISsue SeCTIONS werE HeatED FOR 1 H At 70°C, dEpaRaFFiNIZEd wITh A XYleNe soAK, FollowED By REHYdRATion VIA THe ADDition Of alcoHOl aT decrEAsiNg conCEntrAtioNS (100, 95, 85 aND 75%) FoR 5 mIn/SteP. a 96°C waTeR-baTH WAs USED FOr aNTigen RetRiEVAl In 0.01 MoL/l soDIUM cITrAte bUfFER (10 mm, ph 6.0) For 20 mIN. nexT, eNDOGenOUS PEROxIdaSE acTivITy waS QuENcHed BY INCubATIoN In 3% hydrogEN peRoxidE FOR 15 MIn At rOOm TeMPERATure. sUbsEQuEntLY, SlIDES WerE BLoCKED WiTH gOaT SErUm (caT. No. ZlI-9022; orIgEne tEChnOlOGieS, InC., beIJiNg, chinA; 1:1) aT 37°c iN A WEt Box FOR 30 MIn ANd tHen incUBateD BY tHE priMARy AnTIBoDY (RaBbIT polycLOnaL ANTiBoDy aGaiNST huMAN REsIsTIn; cAt. No. BS7730; BIOwOrLd tEChnOLogY, InC., ST. LOuis pArK, mN, Usa; 1:100) ovERnIGHt aT 4°C IN MOiSt cHAMBERS. FoLLOWiNg WasHinG wIth 0.01 MoL/L pBs (Ph 7.2) ThrEe TIMEs, SliDes werE INcuBAtED wiTh A bIOTiNyLAtED SeconDaRy RABbiT aNti-mOuse aNTIboDY (CAt. no. pv-6000; orIGenE TeCHNOLogIEs, inc.; 1:1) FOr 25 MiN at 37°c. fOLLOwIng inCubaTING In hORseRadiSh PeRoXidAse maRkED strEptomYcIn aViDIN WORkING FlUid aT 37°C For 30 miN, SliDEs wERe trEAted WItH avidIN bioTIn-pErOXidASE cOMpLEx USINg 3,3′-dIAMinObeNziDInE as A chroMOgen, aND tHEN CounTERstAIneD WITh hEMAtoxyLIn fOr 30 SEC AT rOoM TeMPERATuRe anD ExAMiNeD bY LIGHt MIcRoScOPy (oLYmPUs coRpoRATiOn, tokYo, jaPAn). PBs was useD as A NeGaTive contRoL INSTead of A prImARy anTibodY.
### eValuaTION Of IMMuNoHIsTOChemicAL STaInInG
eXiSTING Tan or BROWn particLeS IN THE NuClEus or cYTOPlasm INDICaTED posiTIvE ceLLs, WhICH must COnfORM tO ThE FolLOwing COndITions: I) tHe cELlulaR STrUCTURe WAS Clear; II) tHE LOCAtION OF POsItIVe grAnULES Was aCCuRATe; IIi) stAinINg wAS SIgniFICAntly INCReased coMPared WitH THe bAckGROUNd.
in A 400× hIGH POWeR FILEd, RAndomlY seLeCTED FROm 10 dIFfErENT cAnCER CEll fieLds oF ViEW, THE PercentAGE OF (a) pOsitiVELY sTaInEd cEllS wAs CalcuLAted as folLowS: 0--5% POsItIVe ceLls, SCORE 0; 6--25% poSITIVe CElLs, scORe 1; 26--50% POsitiVe CeLLs, sCORE 2; 51--75% POsiTivE CElLs, SCoRe 3; 76--100% POSITiVE CElls, SCore 4. then, the (b) sTAiniNG INtEnsITy WaS EVaLuaTED: ColorlEss, sCORE 0; lIght yelLow, SCore 1; dEeP YELLow aNd tAn, SCoRE 2; BrOWn, scOre 3.
thE EXPRESSIon oF rEsIStiN WaS BAsed ON thE pRoDUcT OF (A) × (b): SCOre 0, NEgATivE (−); sCoRE 1--4, WeaKLy pOSITiVe (+); SCORE 5--8, POsITIVE (++); scOrE 9--12, strOngly pOSitive (+++). (−) aNd (+) WeRe REgARDED as LOw ExPrESsioN gROup, (++) and (+++) WerE CAteGORIZeD AS thE higH expReSsiON GROuP ([fIg. 1A](#f1-or-40-06-3392){REf-TyPe="fIg"}). the REsULt Of EaCh SPEcIMeN wAS InDEpEndENtLy EValUaTed by tWO QuAliFIED aND experT PAThOLoGIsts, blindED to tHE PaTIeNTS\' clINICAL dAtA. The FEW Cases WiTH DISCORDAnt rEsULts weRE rEEValuateD AnD finaL SCoreS wERE CONSeNSUAl.
### CElL culTuRE
HuMan LuNG aDENOcarciNoMA ceLL LINeS a549 and H1975 WEre obtaiNEd FRoM the Committee OF tyPe CuLtuRE CoLlEctiON of thE ChineSE ACADEMY oF sciEnCes (shaNghai, cHiNa). The CELlS wERE mAintaiNED in RPMi-1640 (GIBCo; theRMO fiSHer sCIENTiFIC, INC., walthAM, Ma, uSa) SUpPLEMEnTeD WItH 10% FetAL boVINe seRUM (FBs; GIBcO; THermO FisHER sciEnTiFIC, Inc.) At 37°c In A hUMidified AtMOSphErE OF 95% AiR AND 5% Co~2~. CElLS wERE DIVIdED aCcOrdInG to TRansfECTioN oF OVerexPrESsion REsIStin PlaSmid for THE rESiSTin GRoup AnD Empty vECtOR FOR THE CONTRoL group.
### tRANsfeCTion and ISoLatIon Of sTabLe trANSfEctanTS
lipOfECTamiNE™ 2000 rEAgEnT (invITRogeN; THerMo fIshEr ScieNTIfic, Inc.), enDO-FReE MaxIpLaSMid KiT (tiANGen BIOTech cO., ltD., bEIjiNG, cHiNa); PCdnA3.1-(+)/resistIN PLASmIDs WERe establISheD bY oriGene tEcHNOloGIes, INc. (cAt. NO. rc210942) And THE PRiMerS wERe AS FoLLOWs: foRwARD PrImEr, 5′-CccAcCgaGaGGGAtGaaAG-3′ AND reVeRsE pRimer, 5′-cAgTGaCAtgtgGtCTcggc-3′; foRWaRD PRImeR, 5′-CAGctCACCATGGaTgATgATaTC-3′ aNd ReVersE pRimER, 5′-AAgCcgGccTtGcACaT-3′ (β-actIn).
a fRaGmEnt OF the rat REsIsTIN CDnA fragmenT (285 Bp) WaS inSErTED at the unIqUe *ECO*RI site In tHE AnTi-Sense oRIeNtATIOn As DETeRMineD bY SeQuencing. The fINaL ConcENTrAtion of ReSisTIn wAS 100 nM. a toTAl oF 2×10^6^ A549 ANd h1975 CElls GrOwn in 60 Mm pEtRi DiSheS WeRe tRaNsfectEd WItH 10 µG of ThE recoMBiNANT pLasmid uSIng lipoFECTAmine, AS desCRIbED BY THe SupPLiEr (gIBCO; tHermO fISHER ScientiFIc, INC.). A TOTAL Of 24 h LaTER, FrESh rpmI-1640 mEdiA ContaiNIng 10% FBs was AddeD aND RepLACED 48 h laTER. MonoClonAL ceLLS wEre seLECTED WIth NEoR. TheN tHE CeLLS werE cULTured FOR 24 h followINg TRAnsfeCtiON.
### reveRSE trANsCriPTiON-QUaNTitaTIVe PoLymErASE ChAIn reacTiOn (Rt-QPCR)
tOTAl rNa wAs extRACTeD frOm ThE ceLlS By TRIzoL (CAt. no. 15596026; INVITRogEN; theRmo FisHER ScIeNTIFiC, inc.) accoRdING to tHE MaNuFaCTUrER\'S ProTOCOl. priMe SCrIpT Rt rEagent kiT (DRR037a; taKARa Bio, INC., OtsU, jAPaN) werE uSeD FOr CDnA GENERaTIOn (42°C For 30--60 Min, 70°C FOR 15 MIN). rT-QpCR wAS pErFOrMED | Introduction ============ Resistin isacysteine-rich protein, which is mainly secreted from monocytesand macrophages in humans ([@b1-or-40-06-3392]--[@b3-or-40-06-3392]). It is associated with inflammation and malignant neoplasms([@b4-or-40-06-3392]--[@b5-or-40-06-3392]). Blood resistin levelsare demonstrated to be increased in certain cancer patients compared with healthy controls, including esophageal squamouscancer, gastric,colorectal, breast and endometrial cancer, and malignant lymphoma ([@b6-or-40-06-3392]--[@b12-or-40-06-3392]). Resistin is considered to be a risk factor for breast cancer ([@b9-or-40-06-3392],[@b13-or-40-06-3392]) and biomarker of disease progression of esophageal squamous cancer, gastricand colorectal cancer ([@b6-or-40-06-3392]--[@b8-or-40-06-3392]). It is an independent prognosticfactor of pancreatic ductal adenocarcinoma ([@b5-or-40-06-3392]). Resistin can promote prostate cancer cellproliferation throughthe phosphatidylinositol 3 kinase (PI3K)/protein kinase B signaling pathway in human prostatecancercell lines PC-3 and DU-145 ([@b14-or-40-06-3392]). However, most of the reported studiesonly demonstrated theassociation between serum orplasma resistin andmalignancy. Few reports measured the level of resistin expressionin cancer tissues, even though itis less well studiedand controversial in lungcancer. Certainreports demonstrateda higher concentrationofresistin in the blood was demonstrated in non-small cell lungcancer (NSCLC)patients compared withthe controls ([@b15-or-40-06-3392]--[@b17-or-40-06-3392]). One of the reports assessed resistin expression in the marginal area oflung cancer tissue and non-cancer region by immunofluorescence staining in 10 cases([@b17-or-40-06-3392]). Another revealed that blood resistin levels were similar between cancer group and non-cancer group ([@b18-or-40-06-3392]). Furthermore, the clinicalsignificanceand biological function remain largely unknown. However, lung cancer is one ofthe most commonmalignancies worldwide,with highermorbidity and poorer prognosis ([@b19-or-40-06-3392]--[@b20-or-40-06-3392]). The most common form of lung cancer is NSCLC, which includes lung adenocarcinoma and squamouscarcinoma. At present, lung adenocarcinoma replaces squamous carcinoma as the dominating type of NSCLC. The aim of the presentstudy is to determine the resistin expression in lung adenocarcinoma tissues, clinical significance and biological function *invitro* and*in vivo*. Materials and methods===================== ### Patients and tissue samples A totalof 70 consecutive cases of newly diagnosed lungadenocarcinoma patients at Tianjin Medical University Cancer Institute andHospital (Tianjin, China) from January to December 2008, with complete clinical and pathological data, were selected retrospectively in the present study and followed up for at least five years. Paired cancer and adjacent non-cancerous tissue samples, which were located morethan 1 cm away from the tumor, were obtained through open surgeries. The paraffin-embedded tissue samples werestained with hematoxylin-eosin and confirmed lung as adenocarcinoma again. The clinicopathological characteristics of the patients were recorded. The tumor staging of NSCLCwas defined according to the tumor, node and metastasis system. Thestudy was comprisedof 70 cases oflung adenocarcinoma(38 male cases, 32 female cases), with an average age of 61 yearsold (36--77 years old). Allpatients receivedtreatments (including operation, chemotherapyor radiotherapy), which conformed to the guidelines of NSCLC. ### Immunohistochemistry For immunohistochemical staining, 5-µm paraffin-embedded tissue sections were heated for 1 h at70°C,deparaffinized with a xylene soak, followed byrehydrationvia the addition of alcoholat decreasing concentrations (100, 95, 85 and 75%) for 5 min/step. A 96°C water-bath was usedfor antigen retrievalin 0.01 mol/lsodium citrate buffer (10 mM, pH 6.0) for 20 min. Next, endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide for 15min at room temperature. Subsequently, slides were blockedwith goat serum (cat. no. ZLI-9022; OriGeneTechnologies, Inc., Beijing, China; 1:1) at37°C in a wet box for 30 min and then incubated by theprimary antibody (rabbit polyclonal antibody against human resistin; cat.no. BS7730; Bioworld Technology, Inc., St. Louis Park,MN, USA; 1:100) overnight at 4°C in moistchambers. Followingwashing with0.01mol/LPBS (pH 7.2)three times, slides were incubated with a biotinylated secondary rabbitanti-mouse antibody (cat. no. PV-6000; OriGene Technologies, Inc.; 1:1)for 25 min at 37°C. Following incubatingin horseradish peroxidase marked streptomycin avidin working fluid at37°C for 30 min, slides were treated with avidin biotin-peroxidase complexusing 3,3′-diaminobenzidine as a chromogen, andthencounterstainedwith hematoxylin for 30 sec at room temperature and examined by light microscopy (Olympus Corporation, Tokyo, Japan). PBS was used asa negativecontrol instead of aprimary antibody. ### Evaluationof immunohistochemical staining Existing tanor brownparticles in thenucleus orcytoplasm indicated positive cells, which must conform to the following conditions: i) The cellular structure was clear; ii) the location of positive granules was accurate; iii)staining was significantly increased compared with the background. In a 400× high power filed, randomlyselectedfrom 10 different cancer cell fields of view,the percentage of (a) positively stained cells was calculated as follows: 0--5% positive cells, score 0; 6--25% positive cells, score 1;26--50%positive cells,score 2; 51--75% positivecells, score 3; 76--100% positive cells, score 4. Then, the (b)stainingintensity was evaluated: Colorless, score 0; light yellow, score 1; deep yellow and tan, score 2; brown, score 3. The expression ofresistin was based on the product of (a) × (b): Score 0, negative (−); score 1--4,weakly positive (+); score 5--8, positive (++); score 9--12, strongly positive (+++). (−) and (+)were regarded as low expression group, (++) and (+++) were categorized as the high expression group ([Fig. 1A](#f1-or-40-06-3392){ref-type="fig"}). Theresult of each specimen wasindependently evaluated by two qualified and expert pathologists, blinded to thepatients\' clinical data. The few cases withdiscordant results were reevaluated and final scores were consensual. ### Cell culture Human lungadenocarcinoma cell lines A549 and H1975 were obtained fromthe Committee of Type Culture Collection ofthe Chinese Academy ofSciences (Shanghai, China).The cells were maintained inRPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°Cin a humidified atmosphere of 95% air and 5% CO~2~. Cells were divided according totransfection of overexpressionresistinplasmid fortheresistin group and empty vector for the control group.### Transfection and isolation of stable transfectants Lipofectamine™ 2000 Reagent (Invitrogen; Thermo FisherScientific, Inc.), endo-free maxiplasmid kit (Tiangen Biotech Co., Ltd.,Beijing, China); pcDNA3.1-(+)/resistin plasmids were established by OriGene Technologies, Inc. (cat.no. RC210942) and the primers were as follows: Forward primer, 5′-CCCACCGAGAGGGATGAAAG-3′and reverse primer, 5′-CAGTGACATGTGGTCTCGGC-3′; forward primer, 5′-CAGCTCACCATGGATGATGATATC-3′ and reverse primer, 5′-AAGCCGGCCTTGCACAT-3′ (β-actin). A fragment of therat resistin cDNA fragment (285 bp) was inserted at the unique*Eco*RI sitein the anti-sense orientation as determined by sequencing. Thefinal concentration of resistin was 100 nM. A total of 2×10^6^ A549 and H1975 cellsgrown in 60mm Petridishes were transfectedwith 10 µg of the recombinant plasmid using lipofectamine, as described by the supplier (Gibco; Thermo FisherScientific, Inc.). Atotal of 24 h later, fresh RPMI-1640 media containing 10% FBS wasadded and replaced 48 h later.Monoclonal cells were selected with NeoR. Then the cells were culturedfor 24 h following transfection. ### Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Total RNA wasextracted from the cells by TRIzol(cat. no. 15596026; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer\'s protocol. Prime Script RT reagentkit (DRR037A; Takara Bio, Inc., Otsu,Japan) were used for cDNA generation (42°C for 30--60 min, 70°Cfor 15 min). RT-qPCR wasperformed | Introduction ============ Resistin is a cysteine-rich protein, which is mainly secreted from monocytes and macrophages in humans ([@b1-or-40-06-3392]--[@b3-or-40-06-3392]). _It_ is associated with inflammation and malignant neoplasms ([@b4-or-40-06-3392]--[@b5-or-40-06-3392]). Blood _resistin_ levels are demonstrated to be increased in _certain_ cancer patients compared with healthy controls, including _esophageal_ squamous _cancer,_ _gastric,_ _colorectal,_ breast _and_ endometrial _cancer,_ and malignant lymphoma ([@b6-or-40-06-3392]--[@b12-or-40-06-3392]). _Resistin_ is _considered_ to be a _risk_ factor for breast cancer ([@b9-or-40-06-3392],[@b13-or-40-06-3392]) and biomarker of disease _progression_ of esophageal _squamous_ cancer, gastric _and_ colorectal cancer _([@b6-or-40-06-3392]--[@b8-or-40-06-3392])._ It is an independent _prognostic_ _factor_ of pancreatic ductal _adenocarcinoma_ ([@b5-or-40-06-3392]). Resistin can promote prostate _cancer_ cell _proliferation_ through _the_ phosphatidylinositol 3 kinase (PI3K)/protein kinase B _signaling_ _pathway_ in _human_ prostate _cancer_ cell lines PC-3 and DU-145 ([@b14-or-40-06-3392]). However, most of the reported studies only demonstrated the association between serum or plasma resistin and malignancy. Few reports measured _the_ level of resistin expression in cancer tissues, even though it is less well studied and controversial in _lung_ cancer. _Certain_ reports demonstrated a _higher_ _concentration_ of _resistin_ in the blood was _demonstrated_ _in_ non-small _cell_ lung cancer (NSCLC) patients compared with _the_ controls ([@b15-or-40-06-3392]--[@b17-or-40-06-3392]). One of the _reports_ _assessed_ resistin expression in _the_ marginal _area_ of lung cancer tissue _and_ non-cancer region by immunofluorescence staining in 10 _cases_ _([@b17-or-40-06-3392])._ Another revealed that blood _resistin_ levels were similar between cancer _group_ and non-cancer group ([@b18-or-40-06-3392]). _Furthermore,_ _the_ _clinical_ significance and biological function _remain_ largely unknown. However, lung _cancer_ is one of the _most_ _common_ malignancies worldwide, with _higher_ _morbidity_ _and_ poorer prognosis ([@b19-or-40-06-3392]--[@b20-or-40-06-3392]). The most common _form_ of lung cancer is NSCLC, which includes _lung_ adenocarcinoma _and_ _squamous_ carcinoma. At present, _lung_ adenocarcinoma replaces squamous _carcinoma_ as _the_ dominating type of NSCLC. The aim of the _present_ _study_ is _to_ determine the resistin expression _in_ lung adenocarcinoma tissues, _clinical_ significance and _biological_ _function_ _*in_ vitro* _and_ *in vivo*. Materials _and_ methods ===================== ### Patients _and_ _tissue_ samples _A_ total of _70_ consecutive cases of _newly_ diagnosed _lung_ adenocarcinoma patients _at_ _Tianjin_ Medical University Cancer _Institute_ and _Hospital_ (Tianjin, China) from January to December 2008, with complete clinical and _pathological_ data, were _selected_ retrospectively in the present study and followed up for _at_ least five _years._ Paired cancer and adjacent non-cancerous tissue samples, which were located more than _1_ cm away from the tumor, were obtained through _open_ surgeries. The paraffin-embedded _tissue_ _samples_ _were_ stained _with_ hematoxylin-eosin _and_ confirmed lung as adenocarcinoma again. The _clinicopathological_ characteristics _of_ the patients were recorded. The tumor _staging_ of NSCLC _was_ defined according to the tumor, node _and_ metastasis system. The study was _comprised_ of 70 cases of lung adenocarcinoma (38 _male_ cases, 32 female cases), with an _average_ age of 61 years old (36--77 _years_ old). All _patients_ received treatments (including operation, chemotherapy or radiotherapy), which conformed to the guidelines _of_ _NSCLC._ _###_ _Immunohistochemistry_ For immunohistochemical staining, 5-µm paraffin-embedded tissue sections _were_ heated _for_ 1 h at _70°C,_ deparaffinized _with_ _a_ xylene soak, followed by _rehydration_ via _the_ addition of alcohol at decreasing concentrations (100, _95,_ 85 and 75%) _for_ _5_ min/step. A 96°C _water-bath_ was used for antigen _retrieval_ in _0.01_ mol/l sodium citrate _buffer_ (10 mM, pH 6.0) for 20 min. _Next,_ endogenous peroxidase _activity_ was quenched by _incubation_ in _3%_ hydrogen peroxide for 15 min _at_ room temperature. Subsequently, _slides_ were blocked with goat _serum_ (cat. _no._ ZLI-9022; OriGene Technologies, Inc., Beijing, China; _1:1)_ at _37°C_ in a _wet_ box for _30_ min and then incubated by the primary antibody (rabbit polyclonal antibody against human resistin; cat. no. _BS7730;_ Bioworld Technology, _Inc.,_ _St._ Louis _Park,_ _MN,_ USA; _1:100)_ overnight at 4°C in moist chambers. _Following_ washing with 0.01 mol/L PBS (pH 7.2) three _times,_ slides were incubated with a biotinylated secondary rabbit anti-mouse antibody (cat. no. _PV-6000;_ _OriGene_ Technologies, Inc.; 1:1) for 25 min _at_ 37°C. Following incubating in horseradish peroxidase marked streptomycin _avidin_ working fluid at 37°C for 30 min, slides were _treated_ _with_ avidin _biotin-peroxidase_ _complex_ using _3,3′-diaminobenzidine_ as a chromogen, and then counterstained with hematoxylin for _30_ sec at room temperature and examined _by_ light microscopy (Olympus Corporation, Tokyo, Japan). PBS was _used_ _as_ a negative control instead of a _primary_ antibody. ### Evaluation _of_ _immunohistochemical_ staining Existing tan _or_ brown _particles_ in the nucleus or _cytoplasm_ indicated positive cells, which must conform _to_ the following _conditions:_ _i)_ The cellular structure was _clear;_ ii) the _location_ of positive granules _was_ accurate; iii) _staining_ _was_ significantly _increased_ compared _with_ the background. In _a_ _400×_ _high_ power filed, randomly selected _from_ 10 different cancer _cell_ fields of view, the _percentage_ of (a) positively stained cells was calculated as follows: 0--5% positive cells, score 0; 6--25% positive cells, score 1; _26--50%_ positive cells, score 2; 51--75% positive cells, score _3;_ 76--100% positive cells, score 4. Then, the (b) staining intensity was evaluated: Colorless, score 0; light yellow, score 1; deep yellow _and_ tan, score 2; brown, score 3. The expression _of_ resistin was based on _the_ product of (a) × _(b):_ Score 0, _negative_ (−); score 1--4, weakly positive (+); score 5--8, positive _(++);_ _score_ 9--12, _strongly_ positive (+++). (−) and (+) were regarded as _low_ expression _group,_ (++) and _(+++)_ were categorized as the high expression group ([Fig. _1A](#f1-or-40-06-3392){ref-type="fig"})._ The result of each specimen was independently evaluated by two qualified and expert pathologists, blinded to the patients\' clinical _data._ The few _cases_ with discordant results were _reevaluated_ and final scores were consensual. _###_ Cell culture Human lung adenocarcinoma cell lines A549 and H1975 were obtained from _the_ Committee of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were _maintained_ in RPMI-1640 (Gibco; Thermo _Fisher_ Scientific, _Inc.,_ _Waltham,_ MA, USA) _supplemented_ with 10% fetal bovine serum (FBS; _Gibco;_ Thermo Fisher Scientific, _Inc.)_ at _37°C_ _in_ a humidified atmosphere of 95% air and 5% CO~2~. Cells _were_ _divided_ according to transfection of overexpression resistin plasmid for the resistin group and empty vector for the control group. ### _Transfection_ and isolation _of_ stable _transfectants_ _Lipofectamine™_ 2000 Reagent (Invitrogen; Thermo Fisher _Scientific,_ Inc.), endo-free maxiplasmid kit (Tiangen Biotech Co., Ltd., Beijing, China); pcDNA3.1-(+)/resistin plasmids were established _by_ OriGene Technologies, _Inc._ (cat. no. RC210942) and the primers were as follows: Forward primer, 5′-CCCACCGAGAGGGATGAAAG-3′ and reverse primer, 5′-CAGTGACATGTGGTCTCGGC-3′; forward _primer,_ _5′-CAGCTCACCATGGATGATGATATC-3′_ and reverse primer, 5′-AAGCCGGCCTTGCACAT-3′ (β-actin). _A_ fragment _of_ the rat resistin cDNA fragment (285 bp) was _inserted_ at the _unique_ *Eco*RI _site_ _in_ _the_ anti-sense orientation as determined by sequencing. The final concentration of resistin was 100 nM. _A_ total of _2×10^6^_ A549 and H1975 _cells_ grown _in_ 60 mm Petri dishes were transfected with 10 µg of the recombinant _plasmid_ _using_ lipofectamine, _as_ described _by_ the supplier (Gibco; Thermo Fisher Scientific, Inc.). A _total_ of 24 h later, fresh RPMI-1640 media _containing_ 10% _FBS_ _was_ added _and_ replaced 48 _h_ later. Monoclonal cells were selected _with_ NeoR. Then the cells _were_ cultured for _24_ h following transfection. _###_ _Reverse_ transcription-quantitative polymerase chain reaction (RT-qPCR) Total _RNA_ _was_ extracted _from_ the cells _by_ TRIzol (cat. no. 15596026; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer\'s protocol. Prime _Script_ _RT_ reagent _kit_ (DRR037A; _Takara_ Bio, Inc., Otsu, Japan) _were_ used for cDNA generation (42°C for 30--60 min, 70°C for 15 min). RT-qPCR was performed |
INTRODUCTION {#sec1-1}
============
The main goal of periodontal treatment is to control the inflammation in periodontal tissues and to regenerate the lost tissues predictably. To meet this goal it is critical to guide the tissues capable of regeneration.\[[@ref1][@ref2][@ref3]\] Guided tissue regeneration is an accepted method for enhancement of lost periodontal tissue. In this technique a barrier membrane is used to prevent epithelial cell migration and stabilization of the clot into the defect. This prevention results in the migration of periodontal ligament cells and osteoblasts into defect and these cells are known to be responsible for tissue regeneration.\[[@ref4]\] Different types of barrier membranes are introduced that had shown favorable results due to different studies.\[[@ref5]\] These membranes are different in composition and structure, but all of them prevent the migration of epithelial and gingival connective tissue cells into the defect and ideally, a barrier membrane should enhance the cell attachment and migration of the progenitor cells.\[[@ref5][@ref6][@ref7][@ref8][@ref9][@ref10]\] Wound healing is a complex process which includes cell migration, cell attachment to various extracellular matrix components, and cell proliferation.\[[@ref11][@ref12]\] Cell attachment process is a four-step sequence which includes adsorption of glycoproteins to the substrate surface, cell contact, attachment, and spreading.\[[@ref9][@ref10]\] Cell proliferation begins after these events.\[[@ref5]\] Tissue integration property ensures the stabilization of the wound and inhibits the migration of epithelial cells, which results in better gain of clinical attachment levels.\[[@ref13][@ref14][@ref15]\]
According to their degradation characteristics, barrier membranes are divided into two groups of resorbable and non-resorbable membranes. Collagen is the most common material used as resorbable membranes.\[[@ref5]\] It facilitates hemostasis and wound stability by promotion of platelet aggregation along with fibroblast migration which accelerates wound closure,\[[@ref16][@ref17]\] but collagenous membranes are not stiff enough to resist soft tissue pressure during healing.\[[@ref16][@ref18]\]
Polytetrafluroethylene (PTFE) is the main composition of non-resorbable membranes.\[[@ref19]\] Although their biocompatibility and positive effect on bone regeneration was shown, but a second surgery is required for their removal which may traumatize the newly formed immature periodontal tissue and causes patient discomfort and increases the treatment time and cost.\[[@ref20]\] Also, the membrane stiffness may result in tissue dehiscence which is the main reason of treatment failure 3 weeks after membrane placement and exposes the membrane which leads to bacterial infection and decrease in the levels of gained clinical attachment.\[[@ref21][@ref22][@ref23][@ref24]\] An alternative to an expanded PTFE membrane is a high-density polytetrafluroethylene (d-PTFE) membrane which is commercially available as TXT-200 and GBR-200. High-density polytetrafluroethylene membranes have small porosities, so bacterial contamination is eliminated and therefore there is no need of primary closure when they are being used and they can be left exposed to the oral cavity.\[[@ref25][@ref26][@ref27][@ref28]\]
The acellular dermal matrix (Alloderm) was originally introduced in medicine for reconstructive plastic surgeries but is also used in dentistry in various periodontal procedures like root coverage and keratinized tissue augmentation around teeth and implants.\[[@ref29][@ref30][@ref31]\] It has many advantages, but the absence of cells and vessels makes tissue incorporation slower, therefore, attempts of culturing fibroblasts on Alloderm were performed to achieve early wound healing and decrease wound contraction in periodontium.\[[@ref32][@ref33][@ref34][@ref35]\]
Fibroblasts play an important role in the healing process. It has been shown that the key factor in the success of regenerative treatment is the recruitment or delivery of cells to the defect site and the production of suitable extracellular matrix along with the periodontal tissues.\[[@ref36][@ref37]\]
Introduction of specific cell adhesion molecules to the membrane surfaces may lead to specific tissue responses. Different growth factors and proteins have been introduced and one of them is enamel matrix derivatives. A commercially available product of enamel matrix derivatives is called Emdogain^®^ (EMD). It is an acidic extract of low molecular weight procine enamel proteins mainly amelogenin and a propylene glycol alginate vehicle.\[[@ref38][@ref39]\] Different studies showed that EMD enhances the adhesion, proliferation, and matrix production of periodontal ligament fibroblasts, stimulates cell growth, and production of insulin growth factor-1 and transforming growth factor-β1 in periodontal ligament cells although it has no appreciable effect on osteoblastic differentiation and has no effect on epithelial cells.\[[@ref37][@ref38]\] All of the described characteristics of EMD make it a suitable functional material for regenerative treatments. Therefore, its effects on cell adhesion to different materials were investigated in the present study.
There was also no available study that had compared the fibroblast adhesion among TXT-200, GBR-200, Alloderm, and collagenous membrane (RTM Collagen, Cytoplast^®^) or the effect of EMD on fibroblast attachment to these common barrier membranes. The present study was performed to compare cell adhesion among the prementioned membranes and also to investigate the effect of EMD on gingival fibroblast attachment.
MATERIALS AND METHODS {#sec1-2}
=====================
For this experimental *in vitro* study, gingival fibroblast cells (NCBI Codece C165) were provided by Pasteur Institute of Iran. Cells were cultured in a culture flask and cultured in the presence of Dulbecco\'s modified Eagle medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) containing 10% Fetal Calf Serum and 100 μg/ml of penicillin, streptomycin, and amphotericin B. The flask was kept in 37°C in a 5% CO~2~ atmosphere in an incubator with humidity. The medium was changed twice a week. Cells were cultured for 3 weeks and passaged for five times.
Four different barrier membranes were used in this study. Two non-resorbable dense polytetrafluoroethylene membranes GBR-200 (GBR1224, LOT: 2541) (Cytoplast®, Osteogenic Biomedical, Lubbock, TX, USA), TXT-200 (TXT1224, LOT: 3688) (Cytoplast®), RTM Collagen (RTM2030, LOT:C2030263) (Cytoplast®) and acellular dermal matrix (ADM, 302111, LOT: B42234) (Alloderm, Biohorizons, Birmingham, AL, USA).
Each membrane was cut into two 6×6-mm pieces and washed with sterile saline solution according to the supplier\'s instructions. In RTM Collagen and ADM groups, membranes were washed with sterile saline solution until the protect paper was floating. A 48 wells culture plate was used in this experiment. Five groups of four close wells were selected. Four groups were used for membranes (each group containing four wells for each membrane). All of the membranes were adapted at the bottom of the selected group of wells. No membrane was added to the fifth group and it served as a control group to check the growth of seeded cells. 10 μg/mL of EMD (LOT: C2822, Emdogain®, Straumann, Malmö, Sweden) was added to two wells of each group (EMD+) and two wells were left without any EMD (EMD-). Cells were seeded at a density of 100,000 cell/well on the membranes. Plate was placed in a 37°C incubator with humidity and 5% CO~2~ atmosphere for 24 hours. The growth of seeded cells in the fifth group was evaluated by means of a light microscope.
Then cells were washed four times with phosphate buffer saline (PBS) to remove non-adherent cells. The membranes were fixed in 2.5% glutaraldehyde for 2 hours, washed five times with distilled water for 20 minutes, treated with 1% osmium tetroxide for 1 hour, washed again five times with distilled water for 20 minutes and finally dehydrated through a series of graded ethanol solutions and left for 24 hours in room temperature to dry. To finish the process, they were coated with gold and analyzed with Field Emission Scanning Electron Microscope (Hittachi s4160, Stanford, CA, USA). An operator not aware of the experimental set up analyzed the membranes with SEM. Each membrane was divided into four intellectual parts under SEM with ×300 magnifications and one image was taken from each part. Another two observers totally unaware of the experiment counted the cells on each image and if there was a difference, the least cell count was recorded.
Data was analyzed by independent t-test, one-way ANOVA, two-way ANOVA, and *post hoc* LSD test with SPSS18 (version 18;SPSS Inc, Chicago, IL, USA). *P* \< 0.05 in independent t-test analysis and *P* \< 0.001 in one-way ANOVA, two-way ANOVA, and *post hoc* LSD analysis was considered statistically significant.
RESULTS {#sec1-3}
=======
Figures [1](#F1){ref-type="fig"}--[4](#F4){ref-type="fig"} illustrates the membranes in EMD- and EMD+ groups under SEM with ×300 magnifications and [Table 1](#T1){ref-type="table"} shows the gained data after cell counting process by two observes.
{#F1}
![SEM illustration of TXT-200 membrane, a- EMD- group | introduction { # sec1 - 1 } = = = = = = = = = = = = the main goal of periodontal treatment is to control the inflammation in periodontal tissues and to regenerate the lost tissues predictably. to meet this goal it is critical to guide the tissues capable of regeneration. \ [ [ @ ref1 ] [ @ ref2 ] [ @ ref3 ] \ ] guided tissue regeneration is an accepted method for enhancement of lost periodontal tissue. in this technique a barrier membrane is used to prevent epithelial cell migration after stabilization of the clot into the defect. this prevention results in the migration of periodontal ligament cells and osteoblasts into defect and these cells are known to be responsible for tissue regeneration. \ [ [ @ ref4 ] \ ] different types of barrier membranes are introduced that had shown favorable results due to different environments. \ [ [ @ ref5 ] \ ] these membranes are different in composition and structure, thus all of them prevent the migration of epithelial and gingival connective tissue cells into the defect and ideally, a barrier membrane should facilitate the cell attachment and migration of the progenitor cells. \ [ [ @ ref5 ] [ @ ref6 ] [ @ ref7 ] [ @ ref8 ] [ @ ref9 ] [ @ ref10 ] \ ] wound healing is a complex process which includes cell migration, cell attachment to various extracellular matrix components, and cell proliferation. \ [ [ @ ref11 ] [ @ ref12 ] \ ] cell attachment behavior is a four - step sequence which includes adsorption of glycoproteins to the substrate surface, cell contact, attachment, and spreading. \ [ [ @ ref9 ] [ @ ref10 ] \ ] cell proliferation begins after these events. \ [ [ @ ref5 ] \ ] tissue integration property ensures the stabilization of the wound and inhibits the migration of epithelial cells, which results in better evaluation of clinical attachment levels. \ [ [ @ ref13 ] [ @ ref14 ] [ @ ref15 ] \ ] according to their degradation characteristics, barrier membranes are divided into two groups of resorbable and non - resorbable agents. below contains currently most common material used as resorbable membranes. \ [ [ @ ref5 ] \ ] it facilitates hemostasis and wound stability by promotion of platelet aggregation along with fibroblast migration which accelerate ##s wound closure, \ [ [ @ ref16 ] [ @ ref17 ] \ ] but collagenous membranes are not stiff enough to resist soft tissue pressure during healing. \ [ [ @ ref16 ] [ @ ref18 ] \ ] polytetrafluroethylene ( ptfe ) is the main composition of non - resorbable membranes. \ [ [ @ ref19 ] \ ] although their biocompatibility and positive effect on bone regeneration was shown, but a second surgery is required for their removal which may traumatize the newly formed immature periodontal tissue and causes patient discomfort and increases the treatment time and cost. \ [ [ @ ref20 ] \ ] also, the membrane stiffness may result in tissue dehiscence which is the main reason of treatment failure 3 weeks after membrane placement and exposes the membrane which leads to bacterial infection and decrease in the levels of gained clinical attachment. \ [ [ @ ref21 ] [ @ ref22 ] [ @ ref23 ] [ @ ref24 ] \ ] an alternative to an expanded ptfe membrane is a high - density polytetrafluroethylene ( d - ptfe ) membrane which is commercially available as txt - 200 and gbr - 200. high - density polytetrafluroethylene membranes have small porosities, so bacterial contamination is eliminated and therefore there is no need of primary closure when they are being used and they can be left exposed to the oral cavity. \ [ [ @ ref25 ] [ @ ref26 ] [ @ ref27 ] [ @ ref28 ] \ ] the acellular dermal matrix ( alloderm ) was originally introduced in medicine for reconstructive plastic surgeries but is also used in dentistry in various periodontal procedures like root coverage and keratinized tissue augmentation around teeth and implants. \ [ [ @ ref29 ] [ @ ref30 ] [ @ ref31 ] \ ] it has many advantages, but the absence of cells and vessels makes tissue incorporation slower, therefore, attempts of culturing fibroblasts on alloderm were performed to achieve early wound healing and decrease wound contraction in periodontium. \ [ [ @ ref32 ] [ @ ref33 ] [ @ ref34 ] [ @ ref35 ] \ ] fibroblasts play an important role in the healing process. it has been shown that the key factor in the success of regenerative treatment is the recruitment or delivery of cells to the defect site and the production of suitable extracellular matrix along with the periodontal tissues. \ [ [ @ ref36 ] [ @ ref37 ] \ ] introduction of specific cell adhesion molecules to the membrane surfaces may lead to specific tissue responses. different growth factors and proteins have been introduced and one of them is enamel matrix derivatives. a commercially available product of enamel matrix derivatives is called emdogain ^ ® ^ ( emd ). it is an acidic extract of low molecular weight procine enamel proteins mainly amelogenin and a propylene glycol alginate vehicle. \ [ [ @ ref38 ] [ @ ref39 ] \ ] different studies showed that emd enhances the adhesion, proliferation, and matrix production of periodontal ligament fibroblasts, stimulates cell growth, and production of insulin growth factor - 1 and transforming growth factor - β1 in periodontal ligament cells although it has no appreciable effect on osteoblastic differentiation and has no effect on epithelial cells. \ [ [ @ ref37 ] [ @ ref38 ] \ ] all of the described characteristics of emd make it a suitable functional material for regenerative treatments. therefore, its effects on cell adhesion to different materials were investigated in the present study. there was also no available study that had compared the fibroblast adhesion among txt - 200, gbr - 200, alloderm, and collagenous membrane ( rtm collagen, cytoplast ^ ® ^ ) or the effect of emd on fibroblast attachment to these common barrier membranes. the present study was performed to compare cell adhesion among the prementioned membranes and also to investigate the effect of emd on gingival fibroblast attachment. materials and methods { # sec1 - 2 } = = = = = = = = = = = = = = = = = = = = = for this experimental * in vitro * study, gingival fibroblast cells ( ncbi codece c165 ) were provided by pasteur institute of iran. cells were cultured in a culture flask and cultured in the presence of dulbecco \ ' s modified eagle medium ( dmem, sigma - aldrich, st. louis, mo, usa ) containing 10 % fetal calf serum and 100 μg / ml of penicillin, streptomycin, and amphotericin b. the flask was kept in 37°c in a 5 % co ~ 2 ~ atmosphere in an incubator with humidity. the medium was changed twice a week. cells were cultured for 3 weeks and passaged for five times. four different barrier membranes were used in this study. two non - resorbable dense polytetrafluoroethylene membranes gbr - 200 ( gbr1224, lot : 2541 ) ( cytoplast®, osteogenic biomedical, lubbock, tx, usa ), txt - 200 ( txt1224, lot : 3688 ) ( cytoplast® ), rtm collagen ( rtm2030, lot : c2030263 ) ( cytoplast® ) and acellular dermal matrix ( adm, 302111, lot : b42234 ) ( alloderm, biohorizons, birmingham, al, usa ). each membrane was cut into two 6×6 - mm pieces and washed with sterile saline solution according to the supplier \ ' s instructions. in rtm collagen and adm groups, membranes were washed with sterile saline solution until the protect paper was floating. a 48 wells culture plate was used in this experiment. five groups of four close wells were selected. four groups were used for membranes ( each group containing four wells for each membrane ). all of the membranes were adapted at the bottom of the selected group of wells. no membrane was added to the fifth group and it served as a control group to check the growth of seeded cells. 10 μg / ml of emd ( lot : c2822, emdogain®, straumann, malmo, sweden ) was added to two wells of each group ( emd + ) and two wells were left without any emd ( emd - ). cells were seeded at a density of 100, 000 cell / well on the membranes. plate was placed in a 37°c incubator with humidity and 5 % co ~ 2 ~ atmosphere for 24 hours. the growth of seeded cells in the fifth group was evaluated by means of a light microscope. then cells were washed four times with phosphate buffer saline ( pbs ) to remove non - adherent cells. the membranes were fixed in 2. 5 % glutaraldehyde for 2 hours, washed five times with distilled water for 20 minutes, treated with 1 % osmium tetroxide for 1 hour, washed again five times with distilled water for 20 minutes and finally dehydrated through a series of graded ethanol solutions and left for 24 hours in room temperature to dry. to finish the process, they were coated with gold and analyzed with field emission scanning electron microscope ( hittachi s4160, stanford, ca, usa ). an operator not aware of the experimental set up analyzed the membranes with sem. each membrane was divided into four intellectual parts under sem with ×300 magnifications and one image was taken from each part. another two observers totally unaware of the experiment counted the cells on each image and if there was a difference, the least cell count was recorded. data was analyzed by independent t - test, one - way anova, two - way anova, and * post hoc * lsd test with spss18 ( version 18 ; spss inc, chicago, il, usa ). * p * \ < 0. 05 in independent t - test analysis and * p * \ < 0. 001 in one - way anova, two - way anova, and * post hoc * lsd analysis was considered statistically significant. results { # sec1 - 3 } = = = = = = = figures [ 1 ] ( # f1 ) { ref - type = " fig " } - - [ 4 ] ( # f4 ) { ref - type = " fig " } illustrates the membranes in emd - and emd + groups under sem with ×300 magnifications and [ table 1 ] ( # t1 ) { ref - type = " table " } shows the gained data after cell counting process by two observes.! [ sem illustration of gbr - 200 membrane, a - emd - group, b - emd + group ] ( drj - 11 - 429 - g001 ) { # f1 }! [ sem illustration of txt - 200 membrane, a - emd - group | INTRODUCTION {# sec1 - 1} = = = = = = = = = = = = The main goal of periodontal treatment is to control the inflammation in periodontal tissues and to regenerate the lost tissues predictably. To meet this goal it is critical to guide the tissues capable of regeneration. \ [[ @ ref1] [@ ref2] [@ ref3] \] Guided tissue regeneration is an accepted method for enhancement of lost periodontal tissue. In this technique a barrier membrane is used to prevent epithelial cell migration and stabilization of the clot into the defect. This prevention results in the migration of periodontal ligament cells and osteoblasts into defect and these cells are known to be responsible for tissue regeneration. \ [[ @ ref4] \] Different types of barrier membranes are introduced that had shown favorable results due to different studies. \ [[ @ ref5] \] These membranes are different in composition and structure, but all of them prevent the migration of epithelial and gingival connective tissue cells into the defect and iXezlly, a barrier membrane should enhance the cell attachment and migration of the progenitor cells. \ [[ @ ref5] [@ ref6] [@ ref7] [@ ref8] [@ ref9] [@ ref10] \] Wound healing is a complex process which includes cell migration, cell attachment to various extracellular matrix components, and cell proliferation. \ [[ @ ref11] [@ ref12] \] Cell attachment process is a four - step sequence which includes adsorption of gl^Voproteins to the substrate surface, cell contact, attachment, and qprezding. \ [[ @ ref9] [@ ref10] \] Cell proliferation begins after these events. \ [[ @ ref5] \] Tissue integration property ensures the stabilization of the wound and inhibits the migration of epithelial cells, which results in better gain of clinical attachment levels. \ [[ @ ref13] [@ ref14] [@ ref15] \] According to their degradation characteristics, barrier membranes are divided into two groups of resorbable and non - resorbable membrqmes. Collagen is the most common material used as resorbable membranes. \ [[ @ ref5] \] It facilitates hemostasis and wound stability by promotion of platelet aggregation along with fibroblast migration which accelerates wound closure, \ [[ @ ref16] [@ ref17] \] but collagenous membranes are not stiff enough to resist soft tissue pressure during healing. \ [[ @ ref16] [@ ref18] \] Polytetrafluroethylene (PTFE) is the main composition of non - resorbable membranes. \ [[ @ ref19] \] Although their biocompatibility and positive effect on bone regeneration was shown, but a second surgery is required for their removal which may traumatize the newly formed immature periodontal tissue and causes patient discomfort and increases the treatment time and cost. \ [[ @ ref20] \] Also, the membrane stiffness may result in tissue dehiscence which is the main reason of treatment failure 3 weeks after membrane placement and exposes the membrane which leads to bacterial infection and decrease in the levels of gained clinical attachment. \ [[ @ ref21] [@ ref22] [@ ref23] [@ ref24] \] An alternative to an expanded PTFE membrane is a high - density polytetrafluroethylene (d - PTFE) membrane which is commercially available as TXT - 200 and GBR - 200. High - density polytetrafluroethylene membranes have small porosities, so bacterial contamination is eliminated and therefore there is no need of primary closure when they are being used and they can be left exposed to the oral cavity. \ [[ @ ref25] [@ ref26] [@ ref27] [@ ref28] \] The acellular dermal matrix (Alloderm) was originally introduced in medicine for reconstrKct7ve plastic surgeries but is Qoso used in dentistry in various periodontal procedures /iMe root coverage and keratinized tissue augmentation around teeth and implants. \ [[ @ ref29] [@ ref30] [@ ref31] \] It has many advantages, but the absence of cells and vessels makes tissue incorporation slo!eE, therefore, attempts of culturing fibroblasts on Alloderm were performed to achieve early wound healing and decrease wound contraction in periodontium. \ [[ @ ref32] [@ ref33] [@ ref34] [@ ref35] \] Fibroblasts play an important role in the healing process. It has been shown that the key factor in the success of regenerative treatment is the recruitment or delivery of cells to the defect site and the production of suitable extracellular matrix along with the periodontal tissues. \ [[ @ ref36] [@ ref37] \] Introduction of specific cell adhesion molecules to the membrane surfaces may lead to specific tissue responses. Different growth factors and proteins have been introduced and one of them is enamel matrix derivatives. A commercially available product of enamel matrix derivatives is called Emdogain ^ ® ^ (EMD ). It is an acidic extract of low molecular weight procine enamel proteins mainly amelogenin and a propylene glycol alginate vehicle. \ [[ @ ref38] [@ ref39] \] Different studies showed that EMD enhances the adhesion, proliferation, and matrix production of periodontal ligament fibroblasts, stimulates cell growth, and production of insulin growth factor - 1 and transforming growth factor - β1 in periodontal ligament cells although it has no appreciable effect on osteoblastic differentiation and has no effect on epithelial cells. \ [[ @ ref37] [@ ref38] \] All of the described characteristics of EMD make it a suitable functional material for regenerative treatments. Therefore, its effects on cell adhesion to different materials were investigated in the present study. There was also no available study that had compared the fibroblast adhesion among TXT - 200, GBR - 200, Alloderm, and collagenous membrane (RTM Collagen, Cytoplast ^ ® ^) or the effect of EMD on fibroblast attachment to these common barrier membranes. The present study was performed to compare cell adhesion among the prementioned membranes and also to investigate the effect of EMD on gingival fibroblast attachment. MATERIALS AND METHODS {# sec1 - 2} = = = = = = = = = = = = = = = = = = = = = For this experimental * in vitro * study, gingival fibroblast cells (NCBI Codece C165) were provided by Pasteur Institute of Iran. Cells were cultured in a culture flask and cultured in the presence of Dulbecco \ ' s modified Eagle medium (DMEM, Sigma - Aldrich, St. Louis, MO, USA) containing 10% Fetal Calf Serum and 100 μg / ml of penicillin, streptomycin, and amphotericin B. The flask was kept in 37 ° C in a 5% CO ~ 2 ~ atmosphere in an incubator with humidity. The medium was changed twice a week. Cells were cultured for 3 weeks and passaged for five times. Four different barrier membranes were used in this study. Two non - resorbable dense polytetrafluoroethylene membranes GBR - 200 (GBR1224, LOT: 2541) (Cytoplast ®, Osteogenic Biomedical, Lubbock, TX, USA ), TXT - 200 (TXT1224, LOT: 3688) (Cytoplast ® ), RTM Collagen (RTM2030, LOT: C2030263) (Cytoplast ®) and acellular dermal matrix (ADM, 302111, LOT: B42234) (Alloderm, Biohorizons, Birmingham, AL, USA ). Each membrane was cut into two 6 × 6 - mm pieces and washed with stFriOe saline solution according to the supplier \ ' s instructions. In RTM Collagen and ADM groups, membranes were washed with sterile saline solution until the protect paper was floating. A 48 wells culture plate was used in this experiment. Five groups of four close wells were selected. Four groups were used for membranes (each group containing four wells for each membrane ). All of the membranes were adapted at the bottom of the selected group of wells. No membrane was added to the fifth group and it served as a control group to check the growth of seeded cells. 10 μg / mL of EMD (LOT: C2822, Emdogain ®, Straumann, Malmö, Sweden) was added to two wells of each group (EMD +) and two wells were left without any EMD (EMD - ). Cells were seeded at a density of 100, 000 cell / well on the membranes. Plate was placed in a 37 ° C incubator with humidity and 5% CO ~ 2 ~ atmosphere for 24 hours. The growth of seeded cells in the fifth group was evaluated by means of a light microscope. Then cells were washed four times with phosphate buffer saline (PBS) to remove non - adherent cells. The membranes were fixed in 2. 5% glutaraldehyde for 2 hours, washed five times with distilled water for 20 minutes, treated with 1% osmium tetroxide for 1 hour, washed again five times with distilled water for 20 minutes and finally dehydrated through a series of graded ethanol solutions and left for 24 hours in room temperature to dry. To finish the process, they were coated with gold and analyzed with Field Emission Scanning Electron Microscope (Hittachi s4160, Stanford, CA, USA ). An operator not aware of the experimental set up analyzed the membranes with SEM. Each membrane was divided into fo&E intellectual parts under SEM with × 300 magnifications and one image was taken from each part. Another two observers totally unaware of the experiment counted the cells on each image and if there was a difference, the least cell count was recorded. Data was analyzed by independent t - test, one - way ANOVA, two - way ANOVA, and * post hoc * LSD test with SPSS18 (version 18; SPSS Inc, Chicago, IL, USA ). * P * \ <0. 05 in independent t - test analysis and * P * \ <0. 001 in one - way ANOVA, two - way ANOVA, and * post hoc * LSD analysis was considered statistically significant. RESULTS {# sec1 - 3} = = = = = = = Figures [1] (# F1) {ref - type = " fig "} - - [4] (# F4) {ref - type = " fig "} illustrates the membranes in EMD - and EMD + groups under SEM with × 300 magnifications and [Table 1] (# T1) {ref - type = " table "} shows the gained data after cell counting process by two observes. ! [SEM illustration of GBR - 200 membrane, a - EMD - group, b - EMD + group] (DRJ - 11 - 429 - g001) {# F1 }! [SEM illustration of TXT - 200 membrane, a - EMD - group | INTRODUCTION The main goal of periodontal treatment is control the inflammation in periodontal tissues and to regenerate lost predictably. To meet this goal it is critical to guide the tissues capable of regeneration.\[[@ref1][@ref2][@ref3]\] tissue regeneration is an accepted method for enhancement of lost periodontal tissue. In this technique a barrier membrane is used prevent epithelial cell migration and stabilization of the clot into the defect. This prevention results in the migration of periodontal ligament cells and osteoblasts into defect and these cells are to responsible for tissue Different types of barrier membranes are introduced that had shown favorable results due to studies.\[[@ref5]\] These membranes are different in composition and structure, all of them prevent the migration of epithelial and connective tissue cells into the defect and ideally, a barrier membrane should enhance cell and migration of the progenitor Wound healing is a process which includes cell migration, cell attachment to various extracellular matrix components, cell proliferation.\[[@ref11][@ref12]\] Cell attachment process is a four-step which includes adsorption of to the surface, cell contact, attachment, and spreading.\[[@ref9][@ref10]\] Cell begins after these events.\[[@ref5]\] Tissue integration property ensures stabilization of the wound and inhibits migration of epithelial cells, which results in better gain of clinical attachment levels.\[[@ref13][@ref14][@ref15]\] According to their degradation characteristics, barrier membranes are into two groups of resorbable and non-resorbable membranes. Collagen is the most common material used as resorbable membranes.\[[@ref5]\] It facilitates hemostasis wound stability by promotion of platelet aggregation along with fibroblast migration which wound closure,\[[@ref16][@ref17]\] but collagenous membranes are stiff enough to resist soft pressure during Polytetrafluroethylene (PTFE) is composition of non-resorbable Although their biocompatibility and positive effect bone regeneration was shown, but a second surgery is their which may traumatize the newly formed immature periodontal tissue and causes patient discomfort and increases the time and cost.\[[@ref20]\] Also, the membrane stiffness may in tissue dehiscence which is the main reason of treatment failure 3 weeks after membrane placement and exposes the which leads to infection and decrease in levels gained attachment.\[[@ref21][@ref22][@ref23][@ref24]\] An alternative to an expanded PTFE membrane is a high-density polytetrafluroethylene (d-PTFE) membrane which commercially available as TXT-200 and GBR-200. High-density polytetrafluroethylene membranes have porosities, so bacterial contamination is eliminated and therefore is no need of primary closure when they are being used and they can be exposed to the cavity.\[[@ref25][@ref26][@ref27][@ref28]\] The acellular dermal (Alloderm) was originally introduced medicine for reconstructive surgeries but is also used in dentistry in various periodontal procedures like coverage and keratinized tissue augmentation around teeth and implants.\[[@ref29][@ref30][@ref31]\] It has many advantages, the absence of cells and vessels makes tissue incorporation slower, therefore, attempts of culturing fibroblasts on Alloderm were performed achieve early wound and decrease wound contraction in periodontium.\[[@ref32][@ref33][@ref34][@ref35]\] Fibroblasts play an important role in the healing process. It has shown that key in the success of regenerative treatment is the recruitment or delivery of to the defect site and the production of suitable extracellular matrix along with periodontal tissues.\[[@ref36][@ref37]\] Introduction of specific cell adhesion molecules to the membrane surfaces may lead to specific tissue responses. Different growth factors and proteins have been introduced and one of them is enamel matrix derivatives. A commercially available product of enamel matrix derivatives is called Emdogain^®^ (EMD). It an acidic extract of low molecular weight enamel proteins mainly amelogenin and propylene alginate vehicle.\[[@ref38][@ref39]\] Different studies showed that EMD enhances the adhesion, proliferation, and production of periodontal ligament fibroblasts, stimulates cell growth, and of insulin growth factor-1 transforming growth factor-β1 in periodontal ligament cells although it has no appreciable effect on differentiation and has no on epithelial cells.\[[@ref37][@ref38]\] All of the described EMD make it a suitable functional for regenerative Therefore, its effects on cell adhesion to different materials were investigated in the present study. There was also no available study that had compared the fibroblast adhesion GBR-200, Alloderm, and collagenous membrane (RTM Collagen, Cytoplast^®^) or effect of EMD on fibroblast attachment to these common barrier membranes. The present was performed to compare cell adhesion the prementioned membranes and also to investigate the effect of on gingival fibroblast attachment. MATERIALS AND METHODS {#sec1-2} ===================== For this experimental *in vitro* study, gingival fibroblast cells (NCBI Codece were provided by Pasteur Institute of Iran. Cells were cultured in culture flask and cultured in the presence Dulbecco\'s modified Eagle medium Sigma-Aldrich, St. Louis, MO, containing 10% Fetal Calf Serum and 100 μg/ml of streptomycin, and amphotericin B. The flask was kept in 37°C in a 5% CO~2~ atmosphere in an incubator with humidity. The medium was changed twice a Cells were cultured for 3 weeks and passaged for five times. Four different barrier membranes were used in this study. Two non-resorbable polytetrafluoroethylene membranes GBR-200 (GBR1224, LOT: 2541) (Cytoplast®, Osteogenic Biomedical, Lubbock, USA), TXT-200 (TXT1224, LOT: (Cytoplast®), RTM Collagen (RTM2030, LOT:C2030263) (Cytoplast®) and acellular dermal matrix (ADM, 302111, LOT: B42234) (Alloderm, Biohorizons, Birmingham, AL, USA). Each membrane was cut into two 6×6-mm pieces and washed with sterile saline solution according to the supplier\'s instructions. In RTM Collagen and ADM groups, membranes were washed with sterile saline solution until the protect paper was A 48 wells culture plate was used in this experiment. Five groups of four close wells were selected. Four groups were used for (each group containing four wells for each membrane). All of the membranes adapted the bottom of the selected group of wells. No membrane was added to the group and it served as a control group to check growth of seeded cells. 10 μg/mL of EMD (LOT: C2822, Straumann, Malmö, Sweden) was added to two wells of each group (EMD+) and two were left without any EMD were at a density of 100,000 cell/well on membranes. Plate placed in a 37°C incubator with humidity and 5% CO~2~ atmosphere for 24 hours. The of seeded cells in the fifth group was evaluated by means of a light microscope. Then cells were washed four times with phosphate buffer saline (PBS) to remove non-adherent cells. membranes were fixed in glutaraldehyde for 2 hours, washed five times with distilled water for 20 minutes, treated with 1% osmium tetroxide 1 hour, washed five times with distilled water for 20 minutes and finally dehydrated through a series of graded ethanol solutions and left for 24 hours in room temperature To finish the process, they were coated with gold and analyzed with Field Emission Electron Microscope (Hittachi s4160, Stanford, USA). An operator not aware of the experimental set up analyzed the with SEM. Each membrane was divided into four intellectual parts SEM magnifications one image was from each part. Another two observers totally unaware of the experiment counted the cells on each image and if there was a difference, the least cell count was recorded. Data analyzed by independent t-test, one-way ANOVA, two-way ANOVA, and *post hoc* LSD test with (version 18;SPSS Inc, Chicago, IL, USA). *P* \< 0.05 in independent t-test analysis *P* \< 0.001 in one-way ANOVA, two-way ANOVA, and *post hoc* analysis was statistically significant. RESULTS {#sec1-3} ======= Figures [1](#F1){ref-type="fig"}--[4](#F4){ref-type="fig"} illustrates the membranes in EMD- and EMD+ under SEM with ×300 magnifications and [Table 1](#T1){ref-type="table"} shows the gained after counting by two {#F1} ![SEM illustration of TXT-200 membrane, a- EMD- | inTRoDuctiOn {#sec1-1}
============
ThE MaiN GoAl oF PerIoDOnTAL treaTmEnt Is to COntrOl thE iNFlamMAtiOn in pErIODoNtAL tIssues aNd to rEgenERaTE the LOST tiSsUES prEDIcTaBLY. TO MEet thIS GOaL it IS cRiTIcAl tO GuidE thE TISSues capAbLe Of RegeneRATiON.\[[@ReF1][@rEf2][@ReF3]\] GUIDEd tissue rEgENERatiOn iS AN aCCEPTEd metHOD FOR eNHaNCeMeNt oF lost pERIodONtAL tISSUE. IN tHIS TECHniQUe A bArRIER meMBRANe Is uSED to pREVENT EpithElial CElL MIGRaTiOn ANd sTaBiliZAtiOn OF tHe cLoT InTO THE defEcT. ThIs prEveNtIon rESULts In ThE mIGraTIoN OF PeRIoDoNTAl LiGamenT CElLs aNd OSteoBLASTS Into DefEct and THese cElLS ARE kNowN TO Be REsPOnSIbLe For tISsuE reGEneRaTIoN.\[[@rEf4]\] dIFFeReNt TYPeS of baRRiEr mEMBRanes Are INTRoDuCEd thAt HaD shOWN fAvOrABLe rEsultS Due tO DiffeReNt STuDies.\[[@reF5]\] tHese MEMbraneS ARE dIFfErENT iN coMPOSiTiOn and STrucTure, but aLL OF THeM preVent THE migRaTIoN of epITheliaL aNd GINGIvAL coNNecTiVe tIsSuE CeLLS INTO THe DefecT aND IDEallY, a barRIeR MembRAne shouLD EnHAnce thE CEll AttACHMENt And mIgrAtIOn of ThE prOgenITOr cElls.\[[@rEF5][@ref6][@REF7][@rEF8][@rEf9][@reF10]\] WounD HeALIng is a ComPleX prOCess wHicH Includes CELL mIGratIoN, cEll aTTacHMENt tO vArIOuS EXTrACELlUlaR mATRIx cOMpOnEntS, aNd cELl pRoLIFerATIoN.\[[@reF11][@reF12]\] CELl aTTacHMEnt pRoCEss Is A FoUR-STep SEquENcE whiCh INClUdes adsOrPTiOn OF GlYcOpROTeins to THE SUbsTRATe SUrfaCe, cEll COnTaCT, attachmEnT, anD spReAding.\[[@reF9][@REf10]\] CelL pROliferATIon BEgINs AftER ThESe EVEnTS.\[[@REF5]\] TiSSuE INTEGRatIOn prOPErty ENSures tHE STAbiLIZaTioN oF thE WounD AND iNHiBITS THE MigRATion oF EPITheliAl cells, wHich RESUlts In bETTEr GaIn oF CLINIcAL atTachMENt leVElS.\[[@rEf13][@ref14][@REf15]\]
AccORDING To TheIr DegradAtiON ChARaCTErIsTIcs, bARriEr MEMbRaNes Are dIViDEd INtO Two GROupS OF RESOrBABLe anD nON-ReSorBAbLe MembRANEs. CollAGEn iS The mOST cOMmON materIAl uSeD as rEsoRBAbLe mEmbRaNes.\[[@Ref5]\] IT fACilItatES HEmOstaSiS AnD Wound STaBilItY by PROMoTiON Of plAtEleT AggReGATION alOnG WiTH FIBRObLAsT miGRatiOn whiCh acCelErAtEs WouND ClosuRE,\[[@ref16][@ref17]\] But COllAgeNOUs meMbRANEs Are noT sTIFf ENOuGH to ReSiSt soft tISSuE pRESSUrE duRing HEaLING.\[[@Ref16][@reF18]\]
poLYtEtraflUROeThylEne (ptfE) IS ThE mAIn COMPOsitIon of noN-rEsOrbable MEmBRAnES.\[[@ref19]\] alTHough ThEIR biOcoMpATibiLitY ANd pOsitIVe eFfect oN bONe regenErAtIOn was SHOwn, buT A second SUrGerY Is rEQuiREd fOr tHeir rEMOVal WHicH mAy traUmATizE tHE neWly FOrMED iMMAtURe perIODONtAL tIsSue AND cAUSEs pATiEnt DIsCoMfoRT AnD iNcREASES tHe tREAtmenT TiME and coSt.\[[@REF20]\] AlSo, thE membrAnE stifFnesS mAy RESUlt iN TIssuE DEhISCEnce WHIch is tHE Main REAson of treatMenT faILure 3 weEks aftER MemBRanE PlaceMENt And ExPoSEs ThE MeMbRaNE WhIch LEAdS tO BAcTERiAl INfeCtIoN aND DEcreASE in tHe lEVeLs oF GAiNed cLiniCaL AttachMENt.\[[@rEf21][@rEf22][@rEF23][@rEF24]\] An aLterNAtIVE tO aN eXPANdeD PTfe MemBrAnE Is a High-denSiTy PoLytETrafluROEthyleNE (d-PTfe) mEMBraNE whIcH is coMMErCiaLly aVAILaBLE AS TXT-200 And gbr-200. HigH-dEnSitY POlyteTrAflurOetHYleNE MEMbrANES hAve smAlL PORosITIEs, so bACTErIAL coNtAmiNatIOn is elImINAted AnD tHEREFOre ThEre IS NO nEED of PrIMarY ClosuRE wheN thEy ArE BeinG USed And tHeY cAN bE LefT eXpOseD To the Oral cAvIty.\[[@rEF25][@reF26][@Ref27][@reF28]\]
The acelluLar dERMaL matrix (alLoderM) wAs origINaLlY INtRodUCeD In MedicIne for reConsTrUctIVe plASTIc SuRGERIes BUt is AlSO UsED IN DENtIsTRY in vaRiOuS pEriodONtAL procEDUREs lIKE rOoT COVeragE AnD keRaTinIzed tISSUE AUGmenTatioN AROUND tEETH anD implANts.\[[@REF29][@ReF30][@REF31]\] It hAS MAny aDVanTAgEs, but tHe AbSeNCe Of cELLS anD VeSseLs mAkes tiSSUE iNCOrPOrAtION SloWeR, tHEReFORE, aTTEmPtS OF cULTuRiNg fIbRObLASts on ALLodERM WeRE pERfORmeD to acHIEvE EARLy WoUnd hEaLing aNd dECREAse WoUNd coNtrAction In peRIODontiuM.\[[@REf32][@ref33][@REf34][@ref35]\]
fibrObLaSts pLaY aN impOrtAnt RolE IN THe HEALing PrOcess. It hAS BeEN SHoWN thaT tHe kEy FAcTOR IN THe succeSs of REgenEraTiVE tReATmENt Is THE reCRUItMent Or deliVeRy oF CELLS To tHE defECt siTE anD The pRodUCtion oF sUITAble eXTRAcELLuLaR MAtrIX ALonG wiTH tHe PErIODoNtal TIssuEs.\[[@reF36][@REf37]\]
intRODUctIon OF SPEciFIC CelL ADhEsiOn mOlEcules TO The membRANe sURfAcES may leAD to speCific tISSUE REsPOnSes. DIfFEreNT GrOwth FAcTOrS and proteInS havE bEeN iNTroducED anD one of TheM iS ENAmEl mATRiX DERiVatIVes. a COmmErCiaLly AVAILaBlE prodUct Of enAmel MAtRIx dEriVaTiveS is CaLLeD eMdOGAIN^®^ (Emd). IT iS An AciDiC EXTrACt of low MolECuLAR wEIGht prOcIne EnaMeL PROTEIns MaiNLy AMELogEnIn And A propYlEnE GLyCOL ALGInate veHicle.\[[@ReF38][@rEf39]\] DifFerenT STuDiEs SHOWeD THAt eMD enHaNCeS thE ADheSIOn, proLIfEraTiOn, aND mATRix prODUCtioN Of pERioDoNtal liGaMenT FIBROBlaStS, stIMULATes cELL grOWTh, ANd ProDUction Of insuLiN GRowtH factOR-1 AND tRaNsformING gRowTH FaCtoR-β1 IN PeRIOdonTAL LIgAmEnT cELlS AltHOuGh IT Has no APPrECIAblE EFfECt On oSTeOblastic DIffErENtiaTion ANd Has No EFFect on epiTHeLIAL CelLs.\[[@REf37][@rEf38]\] alL oF tHe dEScribeD ChaRACteRIsticS OF eMD maKE IT A sUitABLe fUncTIOnaL MAteriaL FOR REgEnErAtivE TREatmENTS. ThEreFORE, iTS EffeCTS ON Cell adhesioN TO DIFfEReNT mAtEriALs weRe InvestiGATED IN ThE PReSeNt StUDY.
tHere WaS ALSo no AVaIlABLe sTudy tHAt HAD CoMpareD thE FibRobLaSt aDHEsion AmONg txT-200, gbR-200, AlloDERm, AnD colLAGENOus meMBrANE (Rtm COLLAgeN, cYtOplaST^®^) Or The EffECt of EMD On FIbROBlasT ATtachmEnT TO THESE COmMon barRiER MEmbRanEs. THE PrESEnt STudY WaS PERFormed to COMpare cELl ADhesion amONg tHE PremENtIOnEd mEmbRaNEs And also TO INVestigATe The EFfeCT Of eMD on GinGIVal FiBRoblAST aTtACHmENt.
mAterIaLS AND MeThoDS {#Sec1-2}
=====================
FOr thiS eXPErimENtal *iN vITRO* sTUDY, GiNgIvAL fibroBLAst celLS (nCbI CodeCe c165) Were PRovIDed bY pAstEur InstITUtE oF iraN. cELLs werE CultUreD IN a culTure fLasK AND culTURed In the prEseNce of DULbECCo\'S MoDIfIEd Eagle medIuM (dMem, SigMA-alDRiCH, St. LoUIS, Mo, usA) CONTAIning 10% FETAl Calf serUm aND 100 μg/mL oF peNIciLlIn, stRePtOMycin, anD AmphOTerICin B. the FlAsK WAS kEpT IN 37°C iN A 5% CO~2~ ATMOSpHERE in AN InCUbaTOr WITh HuMIDiTY. The MediUm wAS ChANgED TWICE A wEEK. cEllS WERe cULTUrEd For 3 WeEks AnD PassagEd fOR FivE TIMEs.
FoUR DIFfERENT BaRrIEr mEMbraNes weRe USED iN ThIs Study. Two nOn-ResoRbABlE DENSE pOlYTETraflUOrOEthYLEne membRANES gbr-200 (gBR1224, lOt: 2541) (cYToPLast®, oSteogeniC BiomediCal, LubboCK, tX, USa), txT-200 (Txt1224, LOt: 3688) (cytoplASt®), RtM CollAgEN (RtM2030, lOt:c2030263) (CyTOPlasT®) AnD aCelLUlAR dermAl matrix (aDM, 302111, Lot: B42234) (AlLODErM, BIOHOrIZONS, biRmIngham, Al, USa).
eAcH mEmBRAne Was CUt intO tWo 6×6-Mm PIeceS And wAsHED wiTh sTeRIlE SalIne sOLuTiON AccoRdinG To thE SUppLier\'S InsTRUctiOns. IN RtM cOllAgEN ANd ADm groUpS, MEmbRANeS WeRE wasHEd witH sTErile sALiNE SOLuTION untIl tHe PROTEct pAPeR WAS fLoATIng. A 48 weLlS CulTuRE pLATe was uSED IN tHIs expERimeNT. fIVE Groups oF FOUR ClOsE wELLs wEre SeLecTEd. Four gROUps WERE uSED For MemBraNES (EAcH GrOUp cOnTaiNiNG fOuR welLs for eACH MemBrane). ALl Of thE mEmBrANEs WERE adApTEd At the BoTtOm oF tHe SeLeCTed groUp oF WELLS. NO MembRANE was aDDEd TO The fiFTh GROUp anD iT SErved as a conTrOl GROuP tO CHECk thE gRowtH Of sEeDeD cELLs. 10 Μg/Ml OF emD (LOt: C2822, EmdOgAin®, sTrAUmaNn, malmö, SWEdEN) waS ADDed To two weLLs oF EacH gROUp (eMD+) And TWO Wells wEre Left WiTHOut anY EMd (eMD-). cElLs wERE seEDeD aT A DENsiTY OF 100,000 CEll/welL On THe mEMBrANEs. PLatE WAs PLAceD IN A 37°c INCUbATOR wItH hUMIDiTy anD 5% co~2~ aTMosPHERe FOR 24 HoURs. the GROWTH Of seEded CElLs in tHe fifTH GROUP WaS EvaLUATeD bY meaNs of A liGHt MIcrOscoPe.
then cELLS wEre WaSHeD four Times WiTH pHOSPhAte buFFER SaliNe (pbs) TO rEMoVE nOn-adHerent celLs. thE MeMbranEs wErE FiXED iN 2.5% gLUtArAlDehyDE For 2 houRs, wASHed FIve tIMES with DiStillED WAtEr fOR 20 miNUTes, trEATed WiTh 1% OSMIuM tETRoXIdE For 1 HOUr, wAShEd agAin FIve TiMes WItH diSTiLLEd waTEr foR 20 MinUTeS AND FinALLY deHYDrAteD tHroUgH A sERiEs oF GRadEd eTHaNOL sOLUtIoNS AnD lEFt fOR 24 hourS in room TEmPERatuRe to DrY. TO fInISH thE PrOCEsS, THEy wERE CoATed wiTH gOlD aNd AnAlyZeD wiTH fiEld EMIssion scanNInG ElecTroN mICroScOpe (hITtaChi s4160, StAnFOrd, CA, UsA). An OPEratoR NOt AWaRe oF tHE exPeRimENtAl SeT Up aNALyzEd The MemBraNEs WItH seM. eACh MeMBRANe wAS DiVIDed iNTO FouR InTElLECTUal PARTs under sem WITH ×300 MagniFiCAtIonS aND onE IMaGE WAS TAkeN fROm EaCH part. anoTHeR two oBSErVerS tOtAllY uNaWArE Of The ExPERiMEnT cOunted thE cEllS oN eAcH ImAgE aND if THeRE WAs a dIffEREncE, The LEASt CElL cOunt WAs RECordED.
DaTa was anAlYzEd bY indePeNDEnT T-TESt, One-WaY ANovA, two-waY ANOVA, aNd *pOst HOC* lSD TeST WIth spsS18 (vERSIon 18;SPss InC, ChiCaGO, IL, USa). *p* \< 0.05 IN InDepeNdent T-tEst aNalYSis AnD *p* \< 0.001 iN ONe-WaY ANOva, two-WaY ANovA, aND *PoSt HoC* lSD aNALySiS WAs conSIdeReD StAtiStIcalLy sIgnIfiCant.
rESUlTs {#sEc1-3}
=======
FiGUres [1](#F1){reF-TYpe="fig"}--[4](#F4){Ref-TYpe="fiG"} illUSTRATES thE MembRAnES In emD- AnD EmD+ grouPs UNDER SeM wIth ×300 MagNIFICATiONs anD [TAblE 1](#T1){Ref-tYPE="TAble"} SHOWS tHE GAiNed DaTA afTeR ceLl CoUNtIng PrOcEsS By two OBserVes.
{#f1}
![Sem IlLuStratiON OF TxT-200 mEMBRanE, a- eMD- GRoUP | INTRODUCTION {#sec1-1} ============ The maingoal of periodontal treatment is to control the inflammation in periodontal tissues andto regenerate the losttissues predictably. Tomeet this goal it is critical to guide the tissues capable of regeneration.\[[@ref1][@ref2][@ref3]\] Guided tissue regenerationisan accepted method for enhancement oflost periodontal tissue. In this technique a barrier membrane is used to prevent epithelial cell migration and stabilization ofthe clot into the defect. This preventionresults in the migrationof periodontal ligament cells and osteoblasts into defect and these cells are known to beresponsible for tissueregeneration.\[[@ref4]\] Differenttypes of barriermembranesareintroduced thathad shown favorableresults due to different studies.\[[@ref5]\]These membranesare different in composition and structure, butall ofthem prevent the migration of epithelial andgingival connective tissue cells into the defect and ideally,a barrier membrane should enhance the cell attachmentand migration ofthe progenitor cells.\[[@ref5][@ref6][@ref7][@ref8][@ref9][@ref10]\] Wound healing is a complex process which includes cell migration, cell attachment to various extracellular matrixcomponents, and cellproliferation.\[[@ref11][@ref12]\] Cell attachment process is afour-step sequence which includes adsorption of glycoproteins to the substrate surface, cellcontact, attachment,and spreading.\[[@ref9][@ref10]\] Cellproliferation begins after these events.\[[@ref5]\] Tissue integration propertyensures the stabilization of the wound and inhibits the migration of epithelial cells, which results in better gain of clinical attachment levels.\[[@ref13][@ref14][@ref15]\]According to their degradationcharacteristics, barrier membranes are dividedinto two groups of resorbable and non-resorbable membranes. Collagen is the most common material used as resorbable membranes.\[[@ref5]\] It facilitates hemostasis and woundstability by promotion of platelet aggregation along with fibroblast migration which accelerates wound closure,\[[@ref16][@ref17]\] but collagenous membranes arenot stiff enough toresist softtissue pressure during healing.\[[@ref16][@ref18]\] Polytetrafluroethylene (PTFE) is the maincomposition of non-resorbable membranes.\[[@ref19]\] Although their biocompatibility and positive effect on boneregeneration was shown, but a secondsurgery is required for their removal which may traumatize thenewly formedimmature periodontal tissueand causes patient discomfort andincreases thetreatment time andcost.\[[@ref20]\] Also, the membrane stiffness may result in tissue dehiscence which is themain reason of treatment failure 3 weeks after membraneplacement and exposes the membranewhich leads to bacterial infection and decrease in the levels of gainedclinical attachment.\[[@ref21][@ref22][@ref23][@ref24]\]An alternative toan expanded PTFEmembrane is a high-density polytetrafluroethylene (d-PTFE)membrane which iscommercially availableas TXT-200and GBR-200. High-density polytetrafluroethylene membranes havesmall porosities, so bacterialcontamination is eliminated and therefore there is no need of primary closure when they are being used and they can be leftexposed to theoral cavity.\[[@ref25][@ref26][@ref27][@ref28]\] The acellular dermal matrix (Alloderm) was originally introduced in medicine for reconstructive plastic surgeries but isalsoused in dentistryin various periodontal procedureslike root coverageandkeratinized tissue augmentation around teethand implants.\[[@ref29][@ref30][@ref31]\]It has many advantages,but the absence of cells and vessels makes tissue incorporation slower, therefore, attempts of culturing fibroblasts onAlloderm were performed to achieve early wound healing anddecrease wound contraction in periodontium.\[[@ref32][@ref33][@ref34][@ref35]\] Fibroblasts play an important role in the healing process. It has been shownthatthe key factor inthe success of regenerative treatment is the recruitment or delivery of cells to thedefect site and the production of suitableextracellular matrix alongwiththe periodontal tissues.\[[@ref36][@ref37]\] Introduction of specific cell adhesion molecules to the membrane surfaces may lead to specific tissue responses. Different growth factorsandproteins have been introduced and one of them is enamel matrix derivatives. A commercially available product ofenamel matrix derivativesis called Emdogain^®^ (EMD). It is an acidic extract of low molecular weightprocineenamelproteinsmainlyamelogenin and apropylene glycol alginate vehicle.\[[@ref38][@ref39]\] Different studies showed that EMD enhances the adhesion, proliferation, and matrix production of periodontal ligament fibroblasts, stimulates cell growth, and production of insulin growth factor-1 andtransforminggrowth factor-β1 in periodontal ligament cells although it has no appreciable effect on osteoblastic differentiation and has no effect on epithelial cells.\[[@ref37][@ref38]\] All of the described characteristics of EMD make it a suitable functional material for regenerativetreatments. Therefore, its effects on cell adhesion to different materials were investigated in the present study. There was also no available study that had compared the fibroblast adhesionamong TXT-200, GBR-200, Alloderm, and collagenousmembrane (RTM Collagen,Cytoplast^®^) or the effect of EMD on fibroblast attachment to these common barrier membranes. The present study was performed to compare celladhesion amongthe prementionedmembranes and also to investigate the effect ofEMD on gingival fibroblast attachment. MATERIALS AND METHODS{#sec1-2} ===================== For thisexperimental *in vitro* study, gingival fibroblast cells (NCBI Codece C165) were provided by Pasteur Institute of Iran. Cells were culturedin a culture flask and cultured in the presence of Dulbecco\'s modified Eagle medium (DMEM, Sigma-Aldrich, St.Louis, MO, USA) containing 10% Fetal Calf Serum and100 μg/ml of penicillin, streptomycin,and amphotericin B. The flask was keptin 37°C ina 5% CO~2~ atmosphere in an incubator with humidity. The medium was changed twice a week. Cells were cultured for 3 weeks and passaged for five times. Four different barrier membranes were used in this study. Two non-resorbabledense polytetrafluoroethylene membranes GBR-200(GBR1224,LOT: 2541) (Cytoplast®, OsteogenicBiomedical, Lubbock, TX, USA), TXT-200 (TXT1224, LOT: 3688) (Cytoplast®), RTMCollagen (RTM2030, LOT:C2030263) (Cytoplast®) and acellulardermal matrix (ADM, 302111, LOT: B42234) (Alloderm, Biohorizons, Birmingham,AL, USA). Each membrane wascut into two 6×6-mm pieces and washed with sterile saline solution accordingto the supplier\'s instructions.In RTM Collagen and ADM groups, membranes were washed with sterile saline solution until the protect paper was floating. A 48 wells culture plate was used in thisexperiment. Five groups of four close wells were selected. Four groups were usedfor membranes (each group containing four wells for each membrane). All of the membranes were adapted at thebottomof the selected group of wells. No membrane was added to the fifth group and it served asa control groupto check the growth of seeded cells. 10 μg/mL of EMD (LOT: C2822, Emdogain®, Straumann,Malmö, Sweden) was added to two wells ofeach group (EMD+) and two wells were left without any EMD (EMD-). Cells were seeded at a density of 100,000 cell/well on the membranes. Plate was placed in a 37°C incubator with humidity and 5% CO~2~ atmosphere for 24 hours. The growth of seeded cells inthe fifth group was evaluated by means of a light microscope. Then cells were washed four times with phosphate buffersaline (PBS) toremove non-adherent cells. The membraneswere fixed in2.5% glutaraldehyde for2 hours, washed five times with distilledwater for 20 minutes, treatedwith 1% osmium tetroxide for 1 hour, washed again five times with distilled water for 20 minutesandfinally dehydrated through aseriesofgraded ethanol solutions and left for24 hours in room temperature to dry. To finish the process, they were coated with gold and analyzed withField Emission ScanningElectron Microscope (Hittachi s4160, Stanford, CA,USA). An operatornot aware of the experimental set up analyzed the membraneswith SEM. Each membrane was dividedinto fourintellectual partsunderSEM with ×300magnifications andone image was taken from each part. Another two observers totally unaware of the experiment counted the cells on each image and if there was adifference, theleast cellcount was recorded. Data was analyzed by independent t-test, one-wayANOVA, two-way ANOVA, and *post hoc* LSD test with SPSS18 (version 18;SPSS Inc, Chicago,IL, USA). *P* \< 0.05 in independent t-test analysis and *P* \<0.001 in one-way ANOVA, two-way ANOVA, and *post hoc* LSD analysis was considered statistically significant. RESULTS {#sec1-3} ======= Figures [1](#F1){ref-type="fig"}--[4](#F4){ref-type="fig"} illustrates the membranes in EMD- and EMD+ groups under SEM with ×300 magnifications and [Table 1](#T1){ref-type="table"} shows the gained data after cellcountingprocess by two observes.{#F1}![SEMillustration ofTXT-200 membrane, a- EMD- group | INTRODUCTION {#sec1-1} ============ The main goal of _periodontal_ treatment is to control the inflammation in _periodontal_ tissues and to regenerate the lost tissues predictably. _To_ meet _this_ _goal_ it is _critical_ to _guide_ the tissues capable of _regeneration.\[[@ref1][@ref2][@ref3]\]_ Guided _tissue_ _regeneration_ is an accepted method for enhancement of lost _periodontal_ tissue. _In_ this technique a barrier membrane _is_ _used_ to _prevent_ epithelial cell migration _and_ stabilization of the clot into _the_ _defect._ This prevention results in the migration _of_ _periodontal_ ligament cells and osteoblasts _into_ _defect_ and _these_ cells are known to _be_ responsible for tissue regeneration.\[[@ref4]\] Different types of barrier membranes are introduced that _had_ shown _favorable_ results due to different _studies.\[[@ref5]\]_ These membranes _are_ different _in_ composition _and_ _structure,_ but all of them _prevent_ the migration _of_ epithelial and gingival connective tissue cells into the _defect_ and ideally, _a_ barrier membrane should enhance the cell attachment _and_ _migration_ of the _progenitor_ cells.\[[@ref5][@ref6][@ref7][@ref8][@ref9][@ref10]\] _Wound_ healing is a complex process which includes cell migration, cell attachment to various extracellular matrix components, and cell proliferation.\[[@ref11][@ref12]\] Cell attachment process is a four-step sequence which includes _adsorption_ of glycoproteins to the substrate surface, _cell_ _contact,_ attachment, and spreading.\[[@ref9][@ref10]\] Cell proliferation begins after these _events.\[[@ref5]\]_ _Tissue_ integration property ensures the stabilization of the wound _and_ inhibits the _migration_ _of_ epithelial _cells,_ which results in better gain of clinical attachment levels.\[[@ref13][@ref14][@ref15]\] According _to_ their _degradation_ characteristics, _barrier_ membranes are divided into two _groups_ of resorbable _and_ non-resorbable membranes. Collagen _is_ the most common material _used_ as resorbable membranes.\[[@ref5]\] It facilitates hemostasis and _wound_ stability by promotion of platelet aggregation _along_ with fibroblast _migration_ which accelerates wound closure,\[[@ref16][@ref17]\] _but_ collagenous membranes are not stiff enough _to_ resist soft tissue pressure during _healing.\[[@ref16][@ref18]\]_ Polytetrafluroethylene (PTFE) is _the_ main composition of _non-resorbable_ membranes.\[[@ref19]\] Although their biocompatibility and positive effect on _bone_ _regeneration_ was shown, _but_ a second _surgery_ is required for _their_ removal _which_ may traumatize the newly _formed_ immature _periodontal_ tissue and causes patient _discomfort_ and increases _the_ treatment time and _cost.\[[@ref20]\]_ Also, _the_ membrane _stiffness_ may result in _tissue_ dehiscence which is the _main_ reason of _treatment_ _failure_ 3 weeks after membrane _placement_ and exposes the membrane which leads _to_ bacterial _infection_ _and_ decrease _in_ the levels of _gained_ clinical attachment.\[[@ref21][@ref22][@ref23][@ref24]\] An _alternative_ to _an_ _expanded_ PTFE _membrane_ is a high-density polytetrafluroethylene (d-PTFE) membrane which is _commercially_ available as _TXT-200_ and GBR-200. _High-density_ polytetrafluroethylene _membranes_ have small porosities, _so_ bacterial contamination is eliminated and therefore there is no need of primary closure when they are being _used_ and they can be left exposed to the oral cavity.\[[@ref25][@ref26][@ref27][@ref28]\] The acellular dermal matrix (Alloderm) _was_ originally _introduced_ in _medicine_ for reconstructive plastic surgeries but is also used in dentistry in various periodontal procedures _like_ _root_ coverage _and_ keratinized tissue augmentation around teeth and implants.\[[@ref29][@ref30][@ref31]\] It has many _advantages,_ but the _absence_ of cells and vessels _makes_ _tissue_ incorporation slower, therefore, attempts _of_ culturing fibroblasts on Alloderm were performed to achieve early wound _healing_ and decrease _wound_ contraction in periodontium.\[[@ref32][@ref33][@ref34][@ref35]\] Fibroblasts _play_ an important role in the healing process. It has been shown that the _key_ factor in the success of regenerative treatment _is_ the recruitment _or_ delivery _of_ cells to the defect site and the production of suitable extracellular matrix along with the _periodontal_ tissues.\[[@ref36][@ref37]\] _Introduction_ of specific cell _adhesion_ _molecules_ to _the_ membrane surfaces may lead to _specific_ tissue _responses._ Different growth _factors_ and proteins have been _introduced_ _and_ one of them is enamel matrix derivatives. _A_ _commercially_ available product of enamel matrix derivatives is _called_ Emdogain^®^ _(EMD)._ It is an acidic extract of low molecular _weight_ procine enamel _proteins_ _mainly_ amelogenin _and_ a propylene glycol alginate vehicle.\[[@ref38][@ref39]\] _Different_ studies showed that EMD enhances the adhesion, _proliferation,_ and matrix production of periodontal ligament fibroblasts, stimulates _cell_ _growth,_ _and_ production of insulin growth factor-1 and _transforming_ growth _factor-β1_ in periodontal ligament cells although it has _no_ appreciable effect on osteoblastic differentiation and _has_ no _effect_ on epithelial _cells.\[[@ref37][@ref38]\]_ All of the described characteristics of _EMD_ make it _a_ _suitable_ functional material for regenerative treatments. Therefore, _its_ _effects_ _on_ cell adhesion _to_ different materials were investigated in the _present_ study. There was also _no_ available _study_ _that_ had compared the fibroblast _adhesion_ _among_ TXT-200, _GBR-200,_ Alloderm, and collagenous membrane _(RTM_ Collagen, _Cytoplast^®^)_ or the effect of EMD on _fibroblast_ attachment _to_ these common barrier _membranes._ The _present_ study was _performed_ to compare _cell_ adhesion among the prementioned _membranes_ _and_ also to investigate the effect _of_ _EMD_ _on_ _gingival_ fibroblast attachment. MATERIALS AND METHODS {#sec1-2} ===================== For this experimental *in vitro* study, gingival _fibroblast_ cells (NCBI Codece C165) were provided by _Pasteur_ _Institute_ of Iran. Cells were cultured in _a_ culture _flask_ and cultured in _the_ presence of Dulbecco\'s modified Eagle medium (DMEM, Sigma-Aldrich, St. _Louis,_ MO, USA) containing 10% Fetal Calf Serum and 100 μg/ml of penicillin, streptomycin, and amphotericin B. The flask was kept in 37°C in _a_ 5% CO~2~ _atmosphere_ in _an_ incubator with humidity. The medium was changed twice a week. Cells _were_ cultured for 3 weeks and passaged for _five_ times. Four different barrier membranes were used in _this_ study. Two non-resorbable dense polytetrafluoroethylene membranes GBR-200 _(GBR1224,_ LOT: 2541) (Cytoplast®, Osteogenic Biomedical, Lubbock, TX, USA), TXT-200 (TXT1224, LOT: 3688) (Cytoplast®), _RTM_ _Collagen_ _(RTM2030,_ LOT:C2030263) (Cytoplast®) and _acellular_ dermal matrix (ADM, 302111, LOT: B42234) (Alloderm, Biohorizons, Birmingham, AL, USA). _Each_ membrane _was_ cut into two 6×6-mm pieces and washed with sterile saline _solution_ according to the supplier\'s instructions. _In_ RTM Collagen and ADM groups, membranes were washed with _sterile_ saline solution until _the_ protect paper was floating. _A_ 48 wells culture plate was used in this experiment. Five groups of four close wells _were_ selected. Four groups were used for membranes (each _group_ containing _four_ wells for each membrane). All of the membranes were adapted at the bottom _of_ the _selected_ group _of_ wells. No membrane was added to the fifth _group_ and it served as a control group to check the growth of seeded _cells._ 10 _μg/mL_ of _EMD_ (LOT: C2822, Emdogain®, Straumann, _Malmö,_ Sweden) was added to two wells of _each_ group (EMD+) and two _wells_ were left without _any_ EMD (EMD-). Cells were _seeded_ at a density of 100,000 _cell/well_ on the _membranes._ Plate was placed in a _37°C_ incubator with humidity and 5% _CO~2~_ atmosphere _for_ _24_ hours. The growth _of_ _seeded_ cells in the fifth group _was_ _evaluated_ by means of _a_ _light_ microscope. Then cells were washed _four_ _times_ with phosphate buffer saline (PBS) _to_ remove non-adherent cells. The membranes were fixed in 2.5% glutaraldehyde for 2 hours, _washed_ five times with distilled water _for_ 20 minutes, treated _with_ 1% osmium tetroxide for 1 hour, washed again five times with _distilled_ water for 20 minutes and finally _dehydrated_ through a series of graded _ethanol_ solutions and left for 24 hours _in_ _room_ temperature _to_ dry. To finish the process, they were coated with gold and analyzed with Field Emission Scanning Electron Microscope (Hittachi s4160, _Stanford,_ CA, USA). An operator not aware of the experimental _set_ up analyzed the membranes with SEM. Each membrane was _divided_ _into_ _four_ intellectual parts under SEM _with_ ×300 magnifications and one image was taken from each part. Another two _observers_ totally _unaware_ of the _experiment_ counted the cells on each _image_ and if there was a difference, the least cell count was recorded. Data _was_ _analyzed_ by independent _t-test,_ one-way ANOVA, two-way ANOVA, and *post _hoc*_ LSD test _with_ SPSS18 _(version_ 18;SPSS Inc, Chicago, IL, USA). *P* \< 0.05 in independent t-test _analysis_ and _*P*_ \< 0.001 in one-way ANOVA, two-way ANOVA, and *post hoc* LSD analysis was considered statistically _significant._ RESULTS {#sec1-3} _=======_ Figures [1](#F1){ref-type="fig"}--[4](#F4){ref-type="fig"} illustrates the membranes in EMD- and EMD+ groups under SEM with ×300 _magnifications_ and [Table 1](#T1){ref-type="table"} shows the gained data _after_ _cell_ counting process by two observes. {#F1} ![SEM illustration _of_ TXT-200 membrane, a- EMD- group |
Background {#Sec1}
==========
Bronchopleural fistula (BPF) is a relatively infrequent but potentially fatal complication of pulmonary resection. BPF can be divided into peripheral or central, based on the location of the leakage, and BPF occurs in about 1.5 to 28 % of pneumonectomy cases, and is associated with high death rate \[[@CR9], [@CR30]\]. It is estimated that incidence of BPF after pneumonectomy and lobectomy for lung cancer is 4.5--20 % and 0.5 %, respectively, and the incidence of BPF is highest after right pulmonary resection and right lower lobectomy \[[@CR31]\]. The etiology of BPF includes incomplete tumor resection, use of steroids, intraoperative infection and prolonged postoperative mechanical ventilation as major risk factors of BPF \[[@CR31]\]. The clinical manifestations of BPF can be frequently classified as acute, subacute, and chronic. An acute BPF presents as tension pneumothorax, with pleural cavity communicating abnormally with the airways, and is associated with purulent sputum expectoration, dyspnea, and reduction in established pleural effusion \[[@CR22]\]. The presentations of subacute and chronic BPF are commonly related to a pleural space with infection, manifesting as a more invisible form with fever, dry cough, and malaise with different levels of respiratory disorder \[[@CR33]\]. Traditional treatments of BPF include thoracotomy after drainage and primary repair, which is based on vascularized muscular flaps and omental grafts tissues \[[@CR20]\]. Amplatzer vascular plug, which was originally designed for the transcatheter closure of vascular structures, has also been reported as a safe and effective method to treat small postoperative BPF \[[@CR9]\]. Fruchter et al. also found that the technique of Amplatzer double-disk occluder implantation may be suitable for both large and small BPFs which originate from the main bronchi and lobar bronchi, respectively \[[@CR8]\]. Additionally, endoscopic approaches and bronchoscopy are common methods of treating BPF to avoid thoracotomy \[[@CR27], [@CR36]\].
Bronchofiberscope (BFS) is a precision instrument employed to diagnose bronchial diseases using of the light guide composed by the fine fibers formed by tens of thousands of high transmittance glass or acrylic resin \[[@CR12], [@CR16]\]. BFS is designed to offer advantageous features such as easy operation method, clear vision, mild trauma, tolerance of surgery by patients, and high safety profile, which reduces or avoids complications associated with tracheotomy and prevents local infection \[[@CR25], [@CR32]\]. Clinically, BFS has multiple uses, including removing foreign bodies, eliminating secretions, treating nasopharyngeal carcinoma, central lung cancer, alveolar cell carcinoma, esophageal fistula, hemoptysis, obstruction, assisting endotracheal intubation treatment and placing gastric tube \[[@CR1], [@CR11], [@CR21]\]. Previous studies have revealed that BFS is also an excellent diagnostic tool for early detection of various intrabronchial injuries, and the attached biopsy sampling feature is helpful in the identification of early lesions, and to carry out poly excision surgery for the studies on bronchus and lung diseases \[[@CR15], [@CR18], [@CR19]\]. Previous studies reported various treatment methods for BPF using BFS, and the methods include gelfoam, shot put plugs, and tissue adhesives. However, these methods have significant deficiencies, evident from the fact that treatment fistula under 3 mm was efficient using these methods, but they show poor efficacy in treatment of BPF beyond 3 mm, particularly those beyond 10 mm \[[@CR6], [@CR28], [@CR35], [@CR37]\]. Phenol, also named carbolic acid, is a sweet-smelling colorless liquid used to prepare resins, preservatives, fungicides, drugs (e.g., aspirin), and also is used to disinfect surgical instruments \[[@CR4], [@CR24], [@CR38]\]. 88 % carbolic acid was found to be efficacious with all alopecia areata patients and can be considered as a treatment of choice for stable alopecia areata \[[@CR3]\]. Moreover, spot peel with 88 % phenol can be a cost-effective procedure for idiopathic guttate hypomelanosis, which can be combined with other medical therapies \[[@CR26]\].
There are no studies using carbolic acid to treat BPF with the help of BFS at present. Therefore, we investigated the efficiency of carbolic acid treatment of BPF in post-pulmonectomy patients, by instilled 100 % carbolic acid with the aid of BFS.
Methods {#Sec2}
=======
Ethics statement {#Sec3}
----------------
This study was conducted with the approval of the Institutional Review Board of Liaoning Tumor Hospital, Shenyang. The informed written consent was collected from each eligible patient and the whole study was performed based on the Declaration of Helsinki \[[@CR14]\].
Study population {#Sec4}
----------------
A total of 12 patients with post-pulmonectomy BPF were enrolled at the Department of Thoracic Surgery, Liaoning Tumor Hospital, Shenyang between February 2009 and March 2012. Orificium fistulae were confirmed by bronchoscope and the average diameter was 4.5 mm. The eligible patients included eight males and three females, with an average age of 56 years (range, 45 \~ 71 years). Three patients had BPF after the right pneumonectomy, six after the left pneumonectomy, one after the right middle and low lobectomy and two after left upper lobectomy.
Preoperotive preparation {#Sec5}
------------------------
Electrocardiogram, routine blood tests and biochemical examination were performed in all the patients. Patients were fasted for 4 \~ 6 h in preparation for surgery and received 10 mg diazepam and 1 mg atropine via intramuscular injection about 30 min before operation. In addition, 1 % lidocaine was used for nasopharyngeal anesthesia by nebulizer.
Intraoperative methods {#Sec6}
----------------------
All patients were instructed to take supine position except 2 patients with short breath in sitting position. The BFS (Olympus BF1T40) was inserted into the trachea through nasal cavity. Heart rate, blood pressure and SpO2 was monitored. Patients received local nasopharyngeal anesthesia with 2 % lidocaine to alleviate irritant reaction. The bronchus around the suture was bubbling when the patient breathed deeply. The fistula was observed via BFS. After the drainage of secretion, hematocele or pus around the BPF, a bronchoscopy biopsy forceps was used for removing necrotic tissues and a 1.8 mm flexible tube was guided through the biopsy hole. The distal end of BFS was brought out and fixed 0.3 cm above the fistula. With breath holding, 100 % carbolic acid solution (0.5--1.0 ml) was instilled to bronchial mucosa through the BFS. The bronchial mucosa became pale after treatment and finally the flexible tube and bronchoscope were removed.
Postoperative and histological observation {#Sec7}
------------------------------------------
After the surgery, the patients were treated with closed drainage of thoracic cavity, anti-inflammatory, symptomatic and supportive treatments. Gas discharge in thoracic drainage tube was observed, and fistula healing were measured via BFS. The treatments were repeated if there was gas discharge from thoracic drainage tube, or further observations were made. Patients could leave hospital after blood routine test showing no evidence of dyspnea, fever, positive culture of fluid drainage (3 times). Paraffin sections (4 \~ 6 μm) of bronchial stump were stained by hematoxylin and eosin (HE) to observe the irritation of bronchial stump after instilled with carbolic acid solution.
Results {#Sec8}
=======
Outcome characteristics of BPF {#Sec9}
------------------------------
In the 12 patients with BPF, the median diameter of the BPF orifice was 4.5 mm, according to the intraoperative observation. Specifically, 3 patients showed a fistula diameter of 3 mm or smaller, 6 patients showed a fistula diameter of 3 \~ 5 mm, and 3 patients exhibited a fistula diameter of 5 mm or larger, 1 of whom had a fistula diameter of 7 mm (Table [1](#Tab1){ref-type="table"}). Serious complications, such as haemorrhage, severe dyspnea and SpO2 declines, did not occur in all the 12 patients during bronchoscopic therapy. Of note, BPF orifices in 5 patients closed after 5 treatments with carbolic acid, 1 patient through 2 treatments, 1 patient through 3 treatments, 2 patients through 4 treatments and 3 patients through 7 treatments (Fig. [1](#Fig1){ref-type="fig"}). Follow-up was conducted for six months after bronchoscopy. Based on the data collected, the average treatment time of the 12 patients was calculated as 20 min and the average time of fistula closure was 30 days. Importantly, the cure rate was 100 %.Table 1Characteristics and outcomes of BPF patientsAge\
(y)GenderInitial symptomSurgical methodBronchopleural fistulaeSize (mm)Treatment timesCure time (d)Follow up148maleLow feverRight PNY3535alive256maleHigh feverRight PNY3.5535alive350maleBlood sputumLeft PNY1321alive471maleIrritating coughLeft upper LBY1.5428alive557femaleLow feverRight upper LBY4535alive665maleFever/air bubbleLeft PNY5749unknown764maleCough/feverLeft PNY7749alive859male | background { # sec1 } = = = = = = = = = = bronchopleural fistula ( bpf ) is a relatively infrequent but potentially fatal complication of pulmonary resection. bpf can be divided into peripheral or respiratory, based on the location of the leakage, and bpf occurs in about 1. 5 to 28 % of pneumonectomy cases, which is associated with high death rate \ [ [ @ cr9 ], [ @ cr30 ] \ ]. it is estimated that incidence of bpf after insertion and lobectomy for lung cancer is 4. 5 - - 20 % and 0. 5 %, respectively, and the incidence of bpf is highest after right pulmonary resection and right lower lobectomy \ [ [ @ cr31 ] \ ]. the etiology of bpf includes incomplete tumor resection, use of steroids, intraoperative infection and prolonged postoperative mechanical ventilation as major risk factors of bpf \ [ [ @ cr31 ] \ ]. the clinical manifestations of bpf can be frequently classified as acute, diffuse, and chronic. an acute bpf presentations as tension pneumothorax, with pleural cavity communicating abnormally with the airways, and is associated with purulent sputum expectoration, dyspnea, and reduction in established pleural effusion \ [ [ @ cr22 ] \ ]. the presentations of subacute and chronic bpf are commonly related to a pleural space capsule infection, presenting as a more invisible form with fever, dry cough, and malaise with different levels of respiratory disorder \ [ [ @ cr33 ] \ ]. traditional treatments of bpf include thoracotomy after drainage and primary repair, which is based on vascularized muscular flaps and omental grafts tissues \ [ [ @ cr20 ] \ ]. amplatzer vascular plug, which was specially designed for the transcatheter closure of vascular structures, has also been reported offering a safe and effective method to treat small postoperative bpf \ [ [ @ cr9 ] \ ]. fruchter et al. also found that the technique of amplatzer double - disk occluder implantation may be suitable for both large and intermediate bpfs which originate from the main bronchi and lobar bronchi, respectively \ [ [ @ cr8 ] \ ]. additionally, endoscopic approaches and bronchoscopy are common methods of treating bpf to avoid thoracotomy \ [ [ @ cr27 ], [ @ cr36 ] \ ]. bronchofiberscope ( bfs ) is a precision instrument employed to diagnose bronchial diseases using of the light guide composed by the fine fibers formed by tens of thousands of high transmittance glass or acrylic resin \ [ [ @ cr12 ], [ @ cr16 ] \ ]. bfs is designed to offer advantageous features such as easy operation method, clear vision, mild trauma, tolerance of surgery by patients, and high safety profile, which reduces or avoids complications associated with tracheotomy and prevents local infection \ [ [ @ cr25 ], [ @ cr32 ] \ ]. clinically, bfs has multiple uses, including removing foreign bodies, eliminating secretions, treating nasopharyngeal carcinoma, central lung cancer, alveolar cell carcinoma, esophageal fistula, hemoptysis, obstruction, assisting endotracheal intubation treatment and placing gastric tube \ [ [ @ cr1 ], [ @ cr11 ], [ @ cr21 ] \ ]. previous studies have revealed that bfs is also an excellent diagnostic tool for early detection of various intrabronchial injuries, and the attached biopsy sampling feature is helpful in the identification of early lesions, and to carry out poly excision surgery for the studies on bronchus and lung diseases \ [ [ @ cr15 ], [ @ cr18 ], [ @ cr19 ] \ ]. previous studies reported various treatment methods for bpf using bfs, and the methods include gelfoam, shot put plugs, and tissue adhesives. however, these methods have significant deficiencies, evident from the fact that treatment fistula under 3 mm was efficient using these methods, but they show poor efficacy in treatment of bpf beyond 3 mm, particularly those beyond 10 mm \ [ [ @ cr6 ], [ @ cr28 ], [ @ cr35 ], [ @ cr37 ] \ ]. phenol, also named carbolic acid, is a sweet - smelling colorless liquid used to prepare resins, preservatives, fungicides, drugs ( e. g., aspirin ), and also is used to disinfect surgical instruments \ [ [ @ cr4 ], [ @ cr24 ], [ @ cr38 ] \ ]. 88 % carbolic acid was found to be efficacious with all alopecia areata patients and can be considered as a treatment of choice for stable alopecia areata \ [ [ @ cr3 ] \ ]. moreover, spot peel with 88 % phenol can be a cost - effective procedure for idiopathic guttate hypomelanosis, which can be combined with other medical therapies \ [ [ @ cr26 ] \ ]. there are no studies using carbolic acid to treat bpf with the help of bfs at present. therefore, we investigated the efficiency of carbolic acid treatment of bpf in post - pulmonectomy patients, by instilled 100 % carbolic acid with the aid of bfs. methods { # sec2 } = = = = = = = ethics statement { # sec3 } - - - - - - - - - - - - - - - - this study was conducted with the approval of the institutional review board of liaoning tumor hospital, shenyang. the informed written consent was collected from each eligible patient and the whole study was performed based on the declaration of helsinki \ [ [ @ cr14 ] \ ]. study population { # sec4 } - - - - - - - - - - - - - - - - a total of 12 patients with post - pulmonectomy bpf were enrolled at the department of thoracic surgery, liaoning tumor hospital, shenyang between february 2009 and march 2012. orificium fistulae were confirmed by bronchoscope and the average diameter was 4. 5 mm. the eligible patients included eight males and three females, with an average age of 56 years ( range, 45 \ ~ 71 years ). three patients had bpf after the right pneumonectomy, six after the left pneumonectomy, one after the right middle and low lobectomy and two after left upper lobectomy. preoperotive preparation { # sec5 } - - - - - - - - - - - - - - - - - - - - - - - - electrocardiogram, routine blood tests and biochemical examination were performed in all the patients. patients were fasted for 4 \ ~ 6 h in preparation for surgery and received 10 mg diazepam and 1 mg atropine via intramuscular injection about 30 min before operation. in addition, 1 % lidocaine was used for nasopharyngeal anesthesia by nebulizer. intraoperative methods { # sec6 } - - - - - - - - - - - - - - - - - - - - - - all patients were instructed to take supine position except 2 patients with short breath in sitting position. the bfs ( olympus bf1t40 ) was inserted into the trachea through nasal cavity. heart rate, blood pressure and spo2 was monitored. patients received local nasopharyngeal anesthesia with 2 % lidocaine to alleviate irritant reaction. the bronchus around the suture was bubbling when the patient breathed deeply. the fistula was observed via bfs. after the drainage of secretion, hematocele or pus around the bpf, a bronchoscopy biopsy forceps was used for removing necrotic tissues and a 1. 8 mm flexible tube was guided through the biopsy hole. the distal end of bfs was brought out and fixed 0. 3 cm above the fistula. with breath holding, 100 % carbolic acid solution ( 0. 5 - - 1. 0 ml ) was instilled to bronchial mucosa through the bfs. the bronchial mucosa became pale after treatment and finally the flexible tube and bronchoscope were removed. postoperative and histological observation { # sec7 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - after the surgery, the patients were treated with closed drainage of thoracic cavity, anti - inflammatory, symptomatic and supportive treatments. gas discharge in thoracic drainage tube was observed, and fistula healing were measured via bfs. the treatments were repeated if there was gas discharge from thoracic drainage tube, or further observations were made. patients could leave hospital after blood routine test showing no evidence of dyspnea, fever, positive culture of fluid drainage ( 3 times ). paraffin sections ( 4 \ ~ 6 μm ) of bronchial stump were stained by hematoxylin and eosin ( he ) to observe the irritation of bronchial stump after instilled with carbolic acid solution. results { # sec8 } = = = = = = = outcome characteristics of bpf { # sec9 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - in the 12 patients with bpf, the median diameter of the bpf orifice was 4. 5 mm, according to the intraoperative observation. specifically, 3 patients showed a fistula diameter of 3 mm or smaller, 6 patients showed a fistula diameter of 3 \ ~ 5 mm, and 3 patients exhibited a fistula diameter of 5 mm or larger, 1 of whom had a fistula diameter of 7 mm ( table [ 1 ] ( # tab1 ) { ref - type = " table " } ). serious complications, such as haemorrhage, severe dyspnea and spo2 declines, did not occur in all the 12 patients during bronchoscopic therapy. of note, bpf orifices in 5 patients closed after 5 treatments with carbolic acid, 1 patient through 2 treatments, 1 patient through 3 treatments, 2 patients through 4 treatments and 3 patients through 7 treatments ( fig. [ 1 ] ( # fig1 ) { ref - type = " fig " } ). follow - up was conducted for six months after bronchoscopy. based on the data collected, the average treatment time of the 12 patients was calculated as 20 min and the average time of fistula closure was 30 days. importantly, the cure rate was 100 %. table 1characteristics and outcomes of bpf patientsage \ ( y ) genderinitial symptomsurgical methodbronchopleural fistulaesize ( mm ) treatment timescure time ( d ) follow up148malelow feverright pny3535alive256malehigh feverright pny3. 5535alive350maleblood sputumleft pny1321alive471maleirritating coughleft upper lby1. 5428alive557femalelow feverright upper lby4535alive665malefever / air bubbleleft pny5749unknown764malecough / feverleft pny7749alive859male | Background {# Sec1} = = = = = = = = = = Bronchopleural fistula (BPF) is a relatively infrequent but potentially fatal complication of pulmonary resection. BPF can be divided into peripheral or central, based on the location of the leakage, and BPF occurs in about 1. 5 to 28% of pneumonectomy cases, and is associated with high death rate \ [[ @ CR9 ], [@ CR30] \ ]. It is estimated that incidence of BPF after pneumonectomy and lobectomy for lung cancer is 4. 5 - - 20% and 0. 5% , respectively, and the incidence of BPF is highest after right pulmonary resection and right lower lobectomy \ [[ @ CR31] \ ]. The etiology of BPF includes incomplete tumor resection, use of steroids, intraoperative infection and prolonged postoperative mechanical ventilation as major risk factors of BPF \ [[ @ CR31] \ ]. The clinical manifestations of BPF can be frequently classified as acute, subacute, and chronic. An acute BPF presents as tension pneumothorax, with pleural cavity communicating abnormally with the airways, and is associated with purulent sputum expectoration, dyspnea, and reduction in established pleural effusion \ [[ @ CR22] \ ]. The presentations of subacute and chronic BPF are commonly related to a pleural space with infection, manifesting as a more invisible form with fever, dry cough, and malaise with different levels of respiratory disorder \ [[ @ CR33] \ ]. Traditional treatments of BPF include thoracotomy after drainage and primary repair, which is based on vascularized muscular flaps and omental grafts tissues \ [[ @ CR20] \ ]. Amplatzer vascular plug, which was originally designed for the transcatheter closure of vascular structures, has also been reported as a safe and effective method to treat small postoperative BPF \ [[ @ CR9] \ ]. Fruchter et al. also found that the technique of Amplatzer double - disk occluder implantation may be suitable for both large and small BPFs which originate from the main bronchi and lobar bronchi, respectively \ [[ @ CR8] \ ]. Additionally, endoscopic approaches and bronchoscopy are common methods of treating BPF to avoid thoracotomy \ [[ @ CR27 ], [@ CR36] \ ]. Bronchofiberscope (BFS) is a precision instrument employed to diagnose vrobchial diseases using of the light guide composed by the fine fibers formed by tens of thousands of high transmittance glass or acrylic resin \ [[ @ CR12 ], [@ CR16] \ ]. BFS is sesKgned to offer advantageous features such as easy operation method, clear vision, mild trauma, tolerance of surgery by patients, and high safety profile, which reduces or avoids complications associated with tracheotomy and prevents local infection \ [[ @ CR25 ], [@ CR32] \ ]. Clinically, BFS has multiple uses, including removing foreign bodies, eliminating secretions, treating nasopharyngeal carcinoma, central lung cancer, alveolar cell carcinoma, esophageal fistula, hemoptysis, obstruction, assisting endotracheal intubation treatment and placing gastric tube \ [[ @ CR1 ], [@ CR11 ], [@ CR21] \ ]. Previous studies have revealed that BFS is also an excellent diagnostic tool for early detection of various intrabronchial injuries, and the attached biopsy sampling feature is helpful in the identification of early lesions, and to carry out poly excision surgery for the studies on bronchus and lung diseases \ [[ @ CR15 ], [@ CR18 ], [@ CR19] \ ]. Previous studies reported various treatment methods for BPF using BFS, and the methods include gelfoam, shot put plugs, and tissue adhesives. However, these methods have significant deficiencies, evident from the fact that treatment fistula under 3 mm was efficient using these methods, but they show poor efficacy in treatment of BPF beyond 3 mm, particularly those beyond 10 mm \ [[ @ CR6 ], [@ CR28 ], [@ CR35 ], [@ CR37] \ ]. Phenol, also named carbolic acid, is a sweet - smelling colorless liquid used to prepare resins, preservatives, fungicides, drugs (e. g. , aspirin ), and also is used to disinfect surgical instruments \ [[ @ CR4 ], [@ CR24 ], [@ CR38] \ ]. 88% carbolic acid was found to be efficacious with all alopecia areata patients and can be considered as a treatment of choice for stable alopecia areata \ [[ @ CR3] \ ]. Moreover, spot peel with 88% phenol can be a cost - effective procedure for idiopathic guttate hypomelanosis, which can be combined with other medical therapies \ [[ @ CR26] \ ]. There are no studies using carbolic acid to treat BPF with the help of BFS at present. Therefore, we investigated the efficiency of carbolic acid treatment of BPF in post - pulmonectomy patients, by instilled 100% carbolic acid with the aid of BFS. Methods {# Sec2} = = = = = = = Ethics statement {# Sec3} - - - - - - - - - - - - - - - - This study was conducted with the approval of the Institutional Review Board of Liaoning Tumor Hospital, Shenyang. The informed written consent was collected from each eligible patient and the whole study was performed based on the Declaration of Helsinki \ [[ @ CR14] \ ]. Study population {# Sec4} - - - - - - - - - - - - - - - - A total of 12 patients with post - pulmonectomy BPF were enrolled at the Department of Thoracic Surgery, Liaoning Tumor Hospital, Shenyang between February 2009 and March 2012. Orificium fistulae were confirmed by bronchoscope and the average Xiaheter was 4. 5 mm. The eligible patients included eight males and three females, with an average age of 56 years (range, 45 \ ~ 71 years ). Three patients had BPF after the right pneumonectomy, six afGe4 the left pneumonectomy, one after the right middle and low lobectomy and two after left upper lobectomy. Preoperotive )reparati*n {# Sec5} - - - - - - - - - - - - - - - - - - - - - - - - Electrocardiogram, routine blood tests and biochemical examination were performed in all the patients. Patients were fasted for 4 \ ~ 6 h in preparation for surgery and received 10 mg diazepam and 1 mg atropine via intramuscular injection about 30 min before operation. In addition, 1% lidocaine was used for nasopharyngeal anesthesia by nebulizer. Intraoperative methods {# Sec6} - - - - - - - - - - - - - - - - - - - - - - All patients were instructed to take supine position except 2 patients with short breath in sitting position. The BFS (Olympus BF1T40) was inserted into the trachea through nasal cavity. Heart rate, blood pressure and SpO2 was monitored. Patients received locZo nasopharyngeal anesthesia with 2% lidocaine to alleviate irritant reaction. The bronchus around the suture was bubbling when the patient breathed deeply. The fistula was observed via BFS. After the drainage of secretion, hematocele or pus around the BPF, a bronchoscopy biopsy forceps was used for removing necrotic tissues and a 1. 8 mm flexible tube was guided through the biopsy hole. The distal end of BFS was brought out and fixed 0. 3 cm above the fistula. With breath holding, 100% carbolic acid solution (0. 5 - - 1. 0 ml) was instilled to bronchial mucosa through the BFS. The bronchial mucosa became pale after treatment and finally the flexible tube and bronchoscope were removed. Oost9perative and histological observation {# Sec7} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - After the surgery, the patients Dege treated with closed drainage of thoracic cavity, anti - inflakmato$y, symptomatic and supportive treatments. Gas discharge in thoracic drainage tube was observed, and fistula healing were measured via BFS. The treatments were repeated if there was gas discharge from thoracic drainage tube, or further observations were made. Patients could leave hospital after blood routine test showing no evidence of dyspnea, fever, positive culture of fluid drainage (3 times ). Paraffin sections (4 \ ~ 6 μm) of bronchial stump were stained by hematoxylin and eosin (HE) to observe the irritation of bronchial stump after instilled with carbolic acid solution. Results {# Sec8} = = = = = = = Outcome characteristics of BPF {# Sec9} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - In the 12 patients with BPF, the median diameter of the BPF orifice was 4. 5 mm, according to the intraoperative observation. Specifically, 3 patients showed a fistula wiameyer of 3 mm or smaller, 6 patients showed a fistula diameter of 3 \ ~ 5 mm, and 3 patients exhibited a fistula diameter of 5 mm or larger, 1 of whom had a fistula diameter of 7 mm (Table [1] (# Tab1) {ref - type = " table "} ). Serious complications, such as haemorrhage, severe dyspnea and SpO2 declines, did not occur in all the 12 patients during bronchoscopic therapy. Of note, BPF orifices in 5 patients closed after 5 treatments with carbolic acid, 1 patient through 2 treatments, 1 patient through 3 treatments, 2 patients through 4 treatments and 3 patients through 7 treatments (Fig. [1] (# Fig1) {ref - type = " fig "} ). Follow - up was conducted for six months after bronchoscopy. Based on the data collected, the average treatment time of the 12 patients was calculated as 20 min and the average time of fistula closure was 30 days. Importantly, the cure rate was 100% . Table 1Characteristics and outcomes of BPF patientsAge \ (y) GenderInitial symptomSurgical methodBronchopleural fistulaeSize (mm) Treatment timesCure time (d) Follow up148maleLow feverRight PNY3535alive256maleHigh feverRight PNY3. 5535alive350maleBlood sputumLeft PNY1321alive471maleIrritating coughLeft upper LBY1. 5428alive557femaleLow feverRight upper LBY4535alive665maleFever / air bubbleLeft PNY5749unknown764maleCough / feverLeft PNY7749alive859male | Background {#Sec1} ========== Bronchopleural fistula (BPF) is a relatively infrequent but potentially fatal of pulmonary resection. BPF can be divided into peripheral or central, based on the location of the leakage, BPF occurs in about 1.5 to 28 % of pneumonectomy cases, and associated with high rate \[[@CR9], [@CR30]\]. is estimated that incidence of BPF after pneumonectomy and lobectomy for lung cancer is 4.5--20 % and 0.5 %, respectively, and the incidence of BPF is highest after right pulmonary resection right lower lobectomy \[[@CR31]\]. etiology of BPF includes incomplete tumor resection, use of steroids, intraoperative infection prolonged postoperative mechanical ventilation as major risk of BPF \[[@CR31]\]. The clinical of BPF can be frequently classified as acute, subacute, and chronic. An acute BPF presents as tension with pleural cavity communicating abnormally the airways, and associated with purulent sputum dyspnea, and reduction in established pleural effusion \[[@CR22]\]. The presentations of subacute and chronic BPF are commonly related to a pleural space with infection, manifesting as a more invisible form with dry cough, and malaise with levels of respiratory disorder \[[@CR33]\]. treatments of include thoracotomy after drainage and primary repair, which is based on muscular flaps and omental grafts tissues \[[@CR20]\]. Amplatzer plug, which originally designed for transcatheter closure of vascular structures, has also been reported as a safe and effective method to treat small postoperative BPF \[[@CR9]\]. Fruchter et al. found that the technique of Amplatzer double-disk occluder may be suitable for both large and small BPFs which originate from the main bronchi and lobar bronchi, respectively \[[@CR8]\]. Additionally, endoscopic approaches and bronchoscopy are common methods of BPF to avoid thoracotomy [@CR36]\]. Bronchofiberscope (BFS) is a precision instrument employed to diagnose bronchial diseases the light composed by the fine fibers formed tens of thousands of high transmittance glass or acrylic resin [@CR16]\]. BFS is designed to offer advantageous such as easy operation method, clear vision, mild trauma, tolerance of surgery by patients, and high safety reduces or avoids complications associated with tracheotomy and prevents \[[@CR25], [@CR32]\]. Clinically, BFS has multiple uses, including removing foreign bodies, eliminating secretions, treating nasopharyngeal carcinoma, central lung cancer, alveolar cell carcinoma, esophageal fistula, obstruction, assisting endotracheal intubation and gastric \[[@CR1], [@CR11], [@CR21]\]. studies have revealed that BFS is also an excellent diagnostic tool for early detection various intrabronchial injuries, and the biopsy sampling feature is helpful in the identification early lesions, and to carry out poly excision surgery for the studies bronchus and lung diseases \[[@CR15], [@CR18], [@CR19]\]. Previous studies reported various treatment methods for BPF using BFS, and the methods include gelfoam, shot put plugs, and tissue adhesives. However, these have significant deficiencies, evident from the fact that treatment fistula under 3 mm was efficient using these methods, but they show poor efficacy in treatment of BPF beyond 3 mm, those beyond 10 mm \[[@CR6], [@CR28], [@CR35], [@CR37]\]. Phenol, also named carbolic acid, is sweet-smelling colorless liquid used to prepare resins, fungicides, drugs (e.g., aspirin), and is used to disinfect surgical instruments \[[@CR4], [@CR24], [@CR38]\]. % acid was found be efficacious with all alopecia areata patients and can be considered as a of for stable alopecia areata \[[@CR3]\]. Moreover, spot peel with 88 % phenol can a cost-effective procedure for idiopathic guttate hypomelanosis, which can be combined with other medical therapies \[[@CR26]\]. There are no studies using acid to treat BPF with the help of BFS at present. Therefore, we investigated the efficiency of acid treatment of BPF post-pulmonectomy patients, by instilled 100 % acid with the aid of BFS. Methods {#Sec2} Ethics statement {#Sec3} ---------------- This study was with the approval of the Institutional Review Board of Liaoning Hospital, The informed written consent was collected each eligible and the whole study was performed based on the Declaration of Helsinki \[[@CR14]\]. Study population {#Sec4} ---------------- A total of 12 patients with post-pulmonectomy BPF were enrolled the Department of Thoracic Surgery, Liaoning Tumor Hospital, Shenyang between February 2009 and March 2012. Orificium were confirmed by and the average diameter was 4.5 mm. The patients included males and three females, with an average age of 56 years (range, 45 years). patients had BPF after the right pneumonectomy, six after the left one after the middle and low lobectomy and two after left upper lobectomy. Preoperotive preparation {#Sec5} ------------------------ Electrocardiogram, routine blood tests and biochemical examination were performed all the patients. Patients were fasted for \~ 6 h in preparation for surgery and received 10 mg diazepam and mg atropine via intramuscular injection about 30 min before operation. In addition, % was used for nasopharyngeal by nebulizer. ---------------------- All patients were instructed to take supine position except 2 patients with short breath sitting position. The BFS (Olympus BF1T40) was inserted into the trachea nasal Heart rate, blood pressure and SpO2 was monitored. Patients received local nasopharyngeal anesthesia 2 % lidocaine irritant reaction. The bronchus around the suture was bubbling the patient breathed deeply. The fistula was observed via BFS. the of secretion, hematocele or pus around the BPF, a bronchoscopy biopsy forceps was used for removing tissues and a 1.8 mm flexible tube was guided through the biopsy The distal end of BFS was brought out and fixed 0.3 cm the fistula. breath holding, 100 % carbolic acid solution (0.5--1.0 ml) was instilled to bronchial mucosa through the The mucosa became after treatment and finally the flexible tube and bronchoscope were removed. Postoperative and histological observation {#Sec7} ------------------------------------------ After the surgery, the were treated with closed drainage of thoracic cavity, anti-inflammatory, symptomatic and supportive treatments. Gas discharge in thoracic drainage tube was observed, and fistula healing were measured via BFS. The treatments were repeated if there was gas discharge from thoracic drainage tube, further observations made. Patients could leave hospital blood routine test showing no evidence of dyspnea, fever, positive culture of fluid (3 Paraffin sections (4 \~ 6 μm) of bronchial were stained by hematoxylin and eosin (HE) to observe the irritation of bronchial stump after instilled with carbolic acid solution. Results {#Sec8} ======= Outcome characteristics of {#Sec9} ------------------------------ In the 12 patients with BPF, the median diameter of the BPF orifice was 4.5 mm, to the intraoperative observation. patients showed fistula diameter of 3 mm or 6 patients showed a fistula diameter of 3 5 mm, and 3 patients exhibited a fistula diameter of 5 mm or larger, 1 of whom had a fistula of 7 mm (Table [1](#Tab1){ref-type="table"}). Serious complications, such as haemorrhage, severe dyspnea and SpO2 declines, did not occur in the 12 patients during bronchoscopic therapy. Of note, BPF orifices in 5 patients after 5 treatments with acid, 1 patient through 2 treatments, patient through 3 treatments, 2 patients 4 treatments and 3 patients through 7 treatments (Fig. [1](#Fig1){ref-type="fig"}). Follow-up was conducted for six months bronchoscopy. on the collected, the average treatment time of the 12 patients was as min and the average time of fistula closure was 30 days. Importantly, the cure rate was 100 1Characteristics and outcomes BPF patientsAge\ (y)GenderInitial symptomSurgical methodBronchopleural fistulaeSize (mm)Treatment timesCure time (d)Follow up148maleLow feverRight PNY3535alive256maleHigh feverRight PNY3.5535alive350maleBlood sputumLeft PNY1321alive471maleIrritating coughLeft upper LBY1.5428alive557femaleLow feverRight bubbleLeft PNY5749unknown764maleCough/feverLeft PNY7749alive859male | BAcKGRound {#SEc1}
==========
bRONCHoPleural FistuLA (BPf) IS a rELaTIVeLy iNfReQUEnt BUt pOTEnTiAllY Fatal COMPLiCatiOn OF PulmOnaRY rESEction. bPF cAN be dIVIDEd iNto peRIPHeRAl Or CentRal, BaSEd ON THE loCation OF THE LEAkaGe, aND BPF OcCUrS in ABOut 1.5 to 28 % OF pneUMONectOMY caseS, ANd IS AssocIated with HIGH DEATh rate \[[@cR9], [@cR30]\]. It Is eSTIMAtEd THat iNcIdENCE of bPf after PNEUMOneCTomy and lObeCtoMy for lUng cANCeR Is 4.5--20 % and 0.5 %, ReSPEctIVELy, ANd ThE iNcIdEnCe OF BPf is hiGhest afteR righT puLmONarY ReSEctioN AND RigHT LoWEr lobECToMY \[[@Cr31]\]. ThE eTIology Of BpF inCluDes incOMpLETE tumor RESection, UsE oF SteroiDs, iNTrAoPERaTIve iNfeCTiON aND pROlongEd pOstOpERATIve mEcHanicAL vEntILaTiOn AS maJor Risk FactORS OF BPF \[[@CR31]\]. THE clIniCaL manIFEStationS of bPf CaN BE FreQuenTLy CLASSIfIED aS ACUTE, suBaCuTE, AND ChrOnIC. aN Acute bpF presENtS aS tENsIon PnEumoThOrAX, WIth pLEUraL CAvitY ComMUnIcatINg abnoRMALly WITH ThE AIRways, AnD is AsSOCIAteD With PURUlEnt sPUTUm eXPecTORATioN, dYsPneA, anD reDuctIon in EsTAbLIsheD PlEuRal efFuSioN \[[@CR22]\]. tHe pResEntATIONS of sUbaCUtE And chRONiC BPf arE coMmONly ReLaTED tO A PLeuraL SPaCe WitH iNFECtioN, MaNifeSTInG as A MorE iNVisIbLe forM WiTh fEvEr, dry cOuGH, And mALaIse wITH difFEreNT leVeLs oF ReSpIRatORy dISOrDER \[[@cr33]\]. TRADITional TREAtmeNts oF Bpf INclUDe THorACOTOmY aFter DrAinage AnD primarY repAiR, WhIch is BAsED on vaScUlAriZEd mUscuLAr FLApS aNd omeNTAl GRAFTs tiSsuEs \[[@CR20]\]. AmPLatzER VASCULaR PlUg, WhicH wAS oRiGiNALlY dEsIgnED fOr the TranSCAtheTEr cLosURe OF vasCuLAr STRuCTuRes, has aLSo BeEN RePORTED aS a sAFe anD EFfECTIve meTHoD tO TreAt SMaLL pOstoPeRATIVe BpF \[[@cR9]\]. FrUcHTER ET al. also foUnD THAt THe teChNIque OF amPlATzer dOuBlE-DISK OccLUder iMplaNtatiON mAY bE suitaBLe fOr BOTh laRGe anD sMALl bpfS wHiCH oRIGiNatE FRoM THe MAIn BroncHI ANd lobaR bRoNchI, REspecTIVElY \[[@CR8]\]. ADdITiOnally, eNdOSCOpiC APPROaCHES aND broNchoscoPy are CoMMon mEthoDs oF TreAtInG BPF tO aVoid thoRaCotoMY \[[@cR27], [@Cr36]\].
BRoNcHOFibErSCoPE (bFS) Is A pRECiSIoN INStRument EmPLOyeD TO diaGnoSe BROnChiAl DiSEaSEs uSing of ThE LigHt guidE COmPosED bY tHe FinE FIBers FoRmeD by teNs OF tHOuSaNdS Of HiGh tRanSmITTANCe GlasS OR AcRylIC reSin \[[@CR12], [@cr16]\]. bfS Is deSigNeD to OFFEr ADvaNtagEous feaTUReS SucH As eAsy operaTioN mEtHOd, clear VIsioN, mILD TRauMa, ToLEraNce of surgERY bY PaTIeNTS, aND HigH SaFETY proFile, WHICH rEDUcES oR AVoiDS cOmplIcATIOns AssOcIaTED witH TRACheOToMY AND PreVENts loCal InFeCTiOn \[[@cR25], [@Cr32]\]. cLiNiCALLy, bfs has MUltIpLE USes, IncLUdiNG rEmOVING foreIGN bODiEs, ELimInaTIng SECReTioNs, TReATIng NasOphaRYnGEaL CaRcInomA, cEnTRAL lUng CAncer, aLveOLAr celL carCInOMA, esOphAGeAl fIstUlA, HEmOPtySIS, ObsTRuCtion, asSiStING EndOTrAChEAl iNTUBAtIOn trEATmENT AnD PLacinG gAStRiC TuBe \[[@CR1], [@Cr11], [@CR21]\]. prevIous sTuDieS havE ReVeALed tHaT BFS Is ALSO AN ExcelLent dIAgNOStiC tOoL fOR eArly deTECTiON oF VariouS iNTRaBRONchIAL INJUrIEs, aND thE attacHEd biOPSY SamPlING FeATurE Is hELpFul in THe iDenTifiCatIOn OF EarLy LEsIoNS, anD to CARRy out POly excisIOn surgEry FOr The studies On BronChUs anD LuNG DiSEAseS \[[@Cr15], [@CR18], [@cR19]\]. PrEvIoUs STuDIes rePOrTED VaRIOuS TREatmEnT mETHoDS fOr BpF USinG bFs, anD tHE MetHodS INclUdE gElfOam, shOT pUt plugS, aND tISsUE adHeSives. hoWeVER, thesE methodS have SIgNIfICAnt DEFicIENCIEs, EvIdeNT FrOm thE FAct thaT TreatmENt fISTuLa UnDer 3 mM WAS efFIcIenT usING TheSe mEthOdS, buT tHEy ShoW PoOr effIcaCY In treAtMEnT Of BPf BeYond 3 mM, paRTICULarlY THOse BEyOnD 10 mM \[[@CR6], [@Cr28], [@cR35], [@CR37]\]. phENOl, also NAmEd CarBoliC ACiD, is A sWeET-SMellInG ColoRlESS lIqUid USED To PREpare rEsiNS, pREservaTivES, fungicIdes, dRUgs (E.g., aSpIrIN), AnD AlSo iS USED to DISinfECt suRGiCAl INsTRuMents \[[@CR4], [@cr24], [@Cr38]\]. 88 % cARBOlIc ACId WAs fOUND tO Be EFfIcAciOus WItH All ALoPeCIa AReATA paTieNTS And CAN Be cOnsiDEred aS A TReatMenT of ChOIce fOR staBle aloPECIA aREAtA \[[@CR3]\]. moREOvER, sPoT PEEl WIth 88 % pHeNol can be A Cost-effecTive proceDUrE for idiopATHic GUtTAtE hYpOmELaNoSIs, which can Be cOMBINEd wiTH OthEr MedIcAl ThErapiEs \[[@CR26]\].
there ARE NO stUdiEs USing cArboLiC Acid to TreaT bpf wIth tHE Help of bFS at preSEnT. tHereFore, We inVESTiGaTed thE eFFiCienCy Of cARbOlIC AcID tREaTMEnT oF BPF in PosT-PUlmONECtOMY PATients, bY INsTillEd 100 % cArbOLIc aciD WitH THE Aid OF Bfs.
MeThODs {#seC2}
=======
etHics sTATemeNT {#sec3}
----------------
ThiS STUdy Was CONDUCTed wITh thE APPrOvAL of ThE InStiTUTIoNal rEView bOArd Of liAonING tuMOR HOspiTal, ShENYaNG. THe INfoRmED wRITtEn cONseNt was COlLected FROm EACH ELiGIBlE PATIENt aNd The wholE StuDY was pERFoRmEd BAseD ON THe DeClarAtiOn OF HElSiNkI \[[@Cr14]\].
sTuDy PoPULATiOn {#SEc4}
----------------
A total oF 12 pAtIeNTs WiTH POsT-pUlmonecTOmY BpF WerE ENROLled aT tHE dEpArtMeNT Of ThORACiC SurGeRy, LiaOniNG tuMOr HOSpitaL, shEnYaNG BETWeEN febRUARy 2009 and maRch 2012. ORifIciuM FIStulae WeRE COnfIRMED bY broNChoscOPe aNd the Average DIameteR wAs 4.5 Mm. the eLigIbLE pAtiENTs IncludEd eight MaLes and ThrEE FEmALES, With an aveRaGE age oF 56 yeArs (RANge, 45 \~ 71 yEARS). thrEe PAtIeNtS HAD BPf AFtER ThE rigHt PNeUMOnECToMy, SIX AFteR ThE left PnEUmOnecTOmy, ONE after tHe RIghT MIddle AnD lOw lOBEcTOmY ANd tWo AfTER LEft UPpeR loBeCtoMy.
preOPErOTivE pReparATION {#SeC5}
------------------------
eLEcTROCARdiOgrAm, ROUTiNE BlOOD tEstS And bIocheMICal exAmiNaTIOn weRE pERforMed IN aLL ThE pAtIEntS. paTIEnTS WEre fASTED FoR 4 \~ 6 H iN prEPARATion FOr SurGeRY anD ReCEiVeD 10 MG diaZEPAM anD 1 mg atropINe vIA inTramuScUlAr InjectiON aBOUt 30 MIn BeFore OPeRatIon. iN ADDItion, 1 % lIDOcAIne WaS USED FOr nASOphaRynGeal AnestHesiA BY nEBulIzER.
InTRAOPERATiVe METhoDS {#SEC6}
----------------------
AlL PaTieNts weRE iNstRUcted tO TakE suPinE PositiON ExCEpt 2 patiENts WIth short bREaTH in SITting pOsItION. tHE Bfs (oLYmPUS BF1T40) waS insERteD INTo thE trAChEa ThrOugH nasAL CaVITY. hEart RAtE, bLOOD pRESSuRe ANd SPo2 wAS moNiToRed. paTIENTs rECEived LoCAl naSOPhaRYNGEAl anesTHeSiA WitH 2 % LiDoCaIne tO AlLeviATE IrRITAnT ReaCTiON. ThE brOncHuS aroUNd THE SutuRE Was bubBling wHeN The PatIeNT BREatHeD deeply. thE fIstUla wAS oBseRVED vIa bFs. AFTEr tHe DrAINAGE of secretiOn, HemAtOCeLe oR PuS AroUND tHE BPF, a bRONCHosCOPy BIOpsY FOrcEPS wAS Used For RemOVINg neCROtic TISSUeS AND A 1.8 mM FLExiblE tubE Was GUiDeD THROugh tHE biOpSy Hole. thE distAl EnD Of BfS WaS bROUghT OUt ANd fIXED 0.3 CM aboVE THE FIstuLA. wiTh brEAtH hoLDIng, 100 % cArbOlic AciD SoLUtiON (0.5--1.0 mL) Was iNStILled tO broNcHIAL mUcoSA ThrOuGh The BfS. the BrONCHIaL muCOsA beCamE PaLE AftEr tREAtmenT aND fiNallY THe fLexIBle tuBE AND brOnCHOscopE Were REMOVED.
PosToPErAtIVE ANd hIsToLoGicaL oBSERvATIOn {#Sec7}
------------------------------------------
AftEr The SuRgErY, thE PATienTS WeRe tReatEd wiTH CLOsEd drAInAge of THorACIc cAviTy, ANti-InFLaMmATORy, syMpTomaTic ANd SUPpoRtIVE treATmentS. gAs DiscHArgE In tHoracIc DraiNaGe Tube wAs ObsERved, And fIStULa HEAlinG wERe MEASURED VIA BFS. The TReAtMents werE REpEatED if THere waS gAs dIscHarGE frOM ThoRacic draINAGE TuBe, OR furTHER obSErvATions WerE MAdE. PAtiEntS CoulD leAvE hosPITAl AFTEr BLOOD rOutINE TesT shoWING NO EViDEnCE oF DysPneA, fEVER, pOsitiVe CuLtuRE oF FluiD DRAinaGe (3 TIMes). pARAFfIN sEctIOns (4 \~ 6 μM) of bRoNcHial sTUMP were STaINED bY heMAtoXylIn and eosIn (he) to ObSERVe tHe iRRiTaTIOn OF bRONcHIaL sTumP AFTeR INStIlLEd wITh CarboliC AcID SolutioN.
results {#Sec8}
=======
oUTcome chARactEriSticS OF Bpf {#sEC9}
------------------------------
In The 12 PAtiENTs WiTh BPf, THE meDiAN DIaMeter of tHE bPf ORificE was 4.5 MM, aCcORdINg to tHE intrAopEratIVe obsERVATION. speCificALlY, 3 patienTs SHOWED a FiSTUla DIametEr of 3 mM OR SMALLer, 6 PAtiEnTS showEd A FistULa DIAmETER OF 3 \~ 5 mM, AND 3 paTiENts EXhibited A FISTula diametER Of 5 MM or LarGEr, 1 OF wHom hAd a fIStULA diametER Of 7 Mm (tABLe [1](#tAb1){REF-tyPE="TabLe"}). seRIoUs coMpLICATiONS, SUcH as haeMorrHAgE, SEVERE DySpnEA aNd spo2 DeCLiNES, DId nOT OCcuR in aLl ThE 12 PAtiEnts DurInG BronchOScOPIc tHERaPY. of nOTE, bPF ORIfICES in 5 paTIents Closed aFTer 5 TREaTmEnTS WitH cARbOLIC AcID, 1 pATieNT thRoUgH 2 tREAtMeNtS, 1 PaTIENT tHrOuGH 3 trEAtmENTS, 2 PAtieNts THRoUgh 4 tReATMENTs aNd 3 pAtiENtS THRouGH 7 trEAtmenTS (fIg. [1](#FiG1){ref-TYPe="Fig"}). FOlLOW-UP Was cOnDuCteD foR SiX MoNthS afTER BrOncHOscoPY. bASed oN the daTa cOlLeCteD, THe AverAGe trEAtment timE Of ThE 12 PaTieNtS Was CalculATed As 20 miN aND ThE aVeRAGE tIme OF FIsTulA CLOSUrE waS 30 DAYS. imPortANTly, THE CURE RatE was 100 %.tablE 1charaCTERistiCS AND oUtCoMEs oF bPF PatIeNtSaGe\
(Y)gEndeRiNitIAl symptOmSURGiCAl mEtHodBronChOPleuRaL FISTulaESizE (mm)TREaTMent TiMescUrE timE (D)foLLOW Up148mAlEloW FEvErrIgHT pNy3535AliVe256maLEhiGH feveRrigHT PNy3.5535Alive350MaLeblOOd sPUtUMLEft PNy1321ALIvE471MALEirRiTaTINg couGhLeft UPper lBY1.5428aLIvE557FEmALELOW feVerrIgHT UPPer lBY4535AliVe665mALefever/aIR bUBBLELeFt pNy5749UnKnoWN764mALecOUgH/fevERLEFt PNY7749ALiVe859MALe | Background {#Sec1} ========== Bronchopleuralfistula (BPF) is a relativelyinfrequentbutpotentially fatal complication of pulmonary resection. BPF can be dividedinto peripheral or central, based on the location of the leakage, and BPF occurs in about 1.5 to 28 % of pneumonectomycases,and is associated withhigh death rate \[[@CR9], [@CR30]\]. It isestimated that incidence of BPF after pneumonectomy and lobectomyfor lung cancer is 4.5--20 % and 0.5%, respectively, and the incidence of BPF is highestafter right pulmonaryresection and right lower lobectomy \[[@CR31]\]. Theetiology of BPF includes incomplete tumorresection, use of steroids, intraoperative infectionand prolonged postoperative mechanicalventilationas majorrisk factors of BPF \[[@CR31]\]. The clinical manifestations ofBPF can befrequently classified asacute, subacute,and chronic. An acute BPF presents as tension pneumothorax,with pleural cavity communicating abnormally with the airways, and is associated with purulent sputum expectoration, dyspnea, and reduction in established pleural effusion \[[@CR22]\]. The presentations of subacute andchronic BPF are commonly related to a pleural space with infection, manifestingas a more invisible form with fever,drycough,and malaise with different levelsof respiratorydisorder \[[@CR33]\]. Traditional treatments of BPFinclude thoracotomy after drainage and primary repair, which is based on vascularized muscular flaps and omentalgraftstissues \[[@CR20]\]. Amplatzer vascular plug, which was originally designed forthe transcatheter closure of vascular structures, has also been reported as a safe and effective method to treat small postoperative BPF \[[@CR9]\]. Fruchteret al. also found that the technique ofAmplatzer double-disk occluder implantation may be suitablefor bothlarge and small BPFs which originate from the main bronchi and lobar bronchi, respectively \[[@CR8]\]. Additionally,endoscopic approaches and bronchoscopy are common methods of treating BPF toavoid thoracotomy \[[@CR27], [@CR36]\]. Bronchofiberscope(BFS) isa precision instrument employed to diagnose bronchialdiseasesusing of the light guide composed by the fine fibersformed by tens of thousands of high transmittanceglass or acrylicresin\[[@CR12], [@CR16]\]. BFS is designed to offer advantageous features such as easy operation method, clear vision, mild trauma, toleranceofsurgerybypatients,and high safety profile, which reduces or avoids complications associatedwith tracheotomy and prevents local infection \[[@CR25],[@CR32]\]. Clinically, BFS has multipleuses, including removing foreign bodies, eliminatingsecretions, treating nasopharyngeal carcinoma, central lung cancer, alveolar cell carcinoma, esophageal fistula, hemoptysis, obstruction, assisting endotrachealintubation treatment and placinggastric tube \[[@CR1],[@CR11], [@CR21]\]. Previous studies have revealedthat BFS is alsoan excellent diagnostic tool for early detection of various intrabronchial injuries, and the attached biopsy sampling feature is helpful in the identification of early lesions, and to carry out poly excision surgery forthe studieson bronchus and lung diseases \[[@CR15], [@CR18], [@CR19]\]. Previous studiesreported various treatment methods for BPF using BFS, and the methods include gelfoam, shot put plugs, and tissue adhesives. However,these methods have significant deficiencies, evident from the fact that treatment fistula under 3 mm was efficient using these methods, buttheyshow poor efficacy in treatmentof BPFbeyond 3 mm, particularly thosebeyond 10 mm \[[@CR6], [@CR28], [@CR35], [@CR37]\]. Phenol, alsonamedcarbolic acid, is a sweet-smelling colorlessliquid used to prepare resins, preservatives, fungicides, drugs (e.g., aspirin), and also is used to disinfect surgical instruments \[[@CR4], [@CR24], [@CR38]\]. 88 % carbolic acid was found to beefficacious with all alopecia areata patients andcan be considered as a treatment of choice for stable alopecia areata \[[@CR3]\]. Moreover, spot peel with 88 % phenol can be a cost-effective procedure for idiopathic guttate hypomelanosis, which can be combinedwith other medical therapies \[[@CR26]\]. There are no studies using carbolic acid to treat BPF with the help of BFS atpresent. Therefore, we investigated theefficiency of carbolic acid treatment of BPF inpost-pulmonectomy patients, by instilled 100 %carbolic acid with the aidof BFS.Methods {#Sec2} ======= Ethicsstatement{#Sec3} ---------------- This study was conducted with the approvalof the Institutional Review Board of LiaoningTumor Hospital, Shenyang. The informed written consent was collectedfrom each eligible patient and thewhole study was performed based on the Declaration of Helsinki \[[@CR14]\]. Study population {#Sec4} ---------------- A total of 12patientswithpost-pulmonectomyBPF were enrolled at theDepartment of Thoracic Surgery, Liaoning Tumor Hospital, Shenyang between February 2009 and March 2012. Orificiumfistulae were confirmed by bronchoscopeandthe average diameter was 4.5 mm. The eligiblepatients included eight malesand three females, with an average age of 56years (range, 45 \~ 71years). Three patients had BPF after the right pneumonectomy, six after the left pneumonectomy, one after the right middle and low lobectomy and two after left upper lobectomy. Preoperotive preparation {#Sec5} ------------------------ Electrocardiogram, routinebloodtests and biochemical examination were performed inallthe patients. Patients were fasted for 4 \~6 h inpreparation for surgery and received10mg diazepam and 1 mg atropine via intramuscular injection about 30 minbefore operation.In addition,1 % lidocaine was used for nasopharyngeal anesthesia by nebulizer. Intraoperative methods {#Sec6} ---------------------- All patients were instructed to take supineposition except 2 patients with short breath in sitting position.TheBFS (Olympus BF1T40) was inserted into the trachea through nasal cavity.Heartrate, blood pressure and SpO2 was monitored. Patients received local nasopharyngeal anesthesia with2% lidocaine toalleviate irritant reaction. The bronchus around the suture was bubbling when the patient breathed deeply. The fistula was observed via BFS. After the drainage of secretion, hematocele or pus around the BPF,a bronchoscopy biopsy forceps was used for removing necrotic tissues and a 1.8 mm flexible tube was guided through the biopsy hole. The distal end of BFSwasbrought out and fixed 0.3 cm above the fistula. Withbreath holding,100 % carbolic acid solution (0.5--1.0 ml)was instilledto bronchial mucosa through the BFS. The bronchial mucosa became pale after treatment and finally the flexible tube and bronchoscope wereremoved. Postoperative and histological observation {#Sec7} ------------------------------------------ After thesurgery, the patients were treated with closed drainage of thoracic cavity, anti-inflammatory,symptomatic and supportive treatments. Gasdischarge in thoracic drainage tube was observed, and fistula healingweremeasured via BFS. The treatments were repeated ifthere was gas dischargefromthoracic drainage tube, or further observations were made. Patients could leave hospital after blood routine test showing no evidence ofdyspnea, fever, positiveculture of fluid drainage (3 times). Paraffin sections (4 \~ 6 μm) of bronchial stump were stained by hematoxylin and eosin (HE) to observe the irritation ofbronchial stump after instilled with carbolic acid solution. Results {#Sec8} ======= Outcome characteristics of BPF {#Sec9} ------------------------------ In the 12 patients with BPF, the median diameter of the BPForifice was 4.5 mm, according to the intraoperative observation. Specifically, 3 patients showed a fistula diameter of 3mm or smaller, 6 patients showed afistula diameter of 3 \~ 5 mm, and 3 patients exhibited a fistula diameterof 5mmorlarger, 1 of whom had a fistula diameter of 7 mm(Table [1](#Tab1){ref-type="table"}). Serious complications, such as haemorrhage,severe dyspnea and SpO2 declines,did notoccur in all the 12 patients duringbronchoscopic therapy. Of note, BPF orificesin 5 patients closed after5 treatmentswith carbolic acid, 1 patient through 2 treatments, 1 patient through3 treatments, 2 patients through 4 treatments and3 patientsthrough 7 treatments (Fig. [1](#Fig1){ref-type="fig"}). Follow-up was conducted for six months after bronchoscopy. Based on the data collected, the average treatment time of the12 patients was calculated as 20min andthe average time of fistula closure was 30 days. Importantly, the cure rate was100 %.Table 1Characteristics and outcomes of BPFpatientsAge\ (y)GenderInitial symptomSurgicalmethodBronchopleuralfistulaeSize (mm)TreatmenttimesCure time(d)Followup148maleLow feverRight PNY3535alive256maleHigh feverRight PNY3.5535alive350maleBloodsputumLeft PNY1321alive471maleIrritating coughLeft upper LBY1.5428alive557femaleLow feverRight upper LBY4535alive665maleFever/air bubbleLeft PNY5749unknown764maleCough/feverLeft PNY7749alive859male | Background {#Sec1} ========== Bronchopleural fistula (BPF) _is_ a _relatively_ infrequent but _potentially_ fatal complication of pulmonary _resection._ BPF can be _divided_ into peripheral or central, based on _the_ _location_ of the leakage, and _BPF_ _occurs_ in about 1.5 to _28_ % of pneumonectomy _cases,_ and _is_ associated with high _death_ rate \[[@CR9], [@CR30]\]. It is estimated that incidence of _BPF_ after pneumonectomy and lobectomy _for_ lung cancer is _4.5--20_ % and 0.5 %, respectively, _and_ _the_ _incidence_ of BPF _is_ _highest_ after right pulmonary resection _and_ right lower lobectomy \[[@CR31]\]. The etiology of BPF includes incomplete tumor resection, _use_ of steroids, intraoperative infection and _prolonged_ postoperative mechanical ventilation _as_ major risk _factors_ of _BPF_ \[[@CR31]\]. The clinical manifestations of BPF can be frequently classified _as_ acute, subacute, and _chronic._ An acute _BPF_ presents as tension pneumothorax, with pleural _cavity_ _communicating_ abnormally with _the_ airways, and is associated with purulent sputum expectoration, dyspnea, and reduction in established _pleural_ effusion \[[@CR22]\]. The presentations _of_ subacute and chronic BPF are _commonly_ related to a pleural space _with_ infection, _manifesting_ _as_ a more _invisible_ form with fever, _dry_ cough, and malaise with different levels of respiratory disorder \[[@CR33]\]. Traditional treatments of BPF include thoracotomy after drainage and primary repair, which is based _on_ vascularized muscular flaps and omental grafts tissues \[[@CR20]\]. Amplatzer _vascular_ plug, which _was_ _originally_ designed for the _transcatheter_ closure of vascular structures, has _also_ been reported as _a_ safe and effective method to treat small _postoperative_ _BPF_ \[[@CR9]\]. _Fruchter_ et al. also found that _the_ technique _of_ Amplatzer _double-disk_ occluder _implantation_ may be suitable for _both_ _large_ and small _BPFs_ which originate from the _main_ bronchi and lobar _bronchi,_ respectively _\[[@CR8]\]._ Additionally, endoscopic approaches and bronchoscopy _are_ common methods of treating BPF to _avoid_ thoracotomy \[[@CR27], [@CR36]\]. Bronchofiberscope (BFS) is _a_ precision _instrument_ employed to diagnose _bronchial_ diseases using of the light guide _composed_ by _the_ _fine_ fibers formed _by_ _tens_ of thousands of high transmittance glass or acrylic resin \[[@CR12], [@CR16]\]. BFS is designed to offer advantageous features such _as_ _easy_ operation method, clear vision, _mild_ trauma, tolerance _of_ surgery by patients, and _high_ safety profile, which _reduces_ or avoids complications associated with tracheotomy and prevents local infection \[[@CR25], [@CR32]\]. Clinically, BFS has _multiple_ uses, _including_ removing _foreign_ bodies, eliminating secretions, treating nasopharyngeal carcinoma, central lung cancer, alveolar cell carcinoma, esophageal fistula, _hemoptysis,_ obstruction, assisting endotracheal intubation _treatment_ and _placing_ gastric tube \[[@CR1], [@CR11], [@CR21]\]. _Previous_ studies have revealed that _BFS_ _is_ also an excellent _diagnostic_ _tool_ for early detection of various _intrabronchial_ injuries, and the attached biopsy _sampling_ _feature_ is helpful in the identification of early lesions, _and_ _to_ _carry_ out poly excision surgery for the studies on bronchus and lung diseases \[[@CR15], _[@CR18],_ [@CR19]\]. Previous studies reported various treatment methods for _BPF_ using _BFS,_ and the methods _include_ gelfoam, shot put _plugs,_ and _tissue_ adhesives. _However,_ these methods _have_ significant deficiencies, evident from the fact that treatment _fistula_ under _3_ mm was efficient using these methods, but _they_ _show_ poor _efficacy_ in treatment of BPF beyond 3 mm, particularly those _beyond_ 10 _mm_ \[[@CR6], [@CR28], [@CR35], [@CR37]\]. Phenol, also named carbolic acid, _is_ _a_ sweet-smelling colorless liquid used to prepare resins, preservatives, fungicides, drugs _(e.g.,_ _aspirin),_ _and_ also is used to disinfect _surgical_ instruments _\[[@CR4],_ [@CR24], [@CR38]\]. 88 % carbolic acid _was_ found to _be_ efficacious with all alopecia _areata_ patients and _can_ _be_ _considered_ as a treatment of choice _for_ stable alopecia areata \[[@CR3]\]. Moreover, spot peel with 88 % phenol _can_ be a cost-effective procedure for idiopathic guttate hypomelanosis, which can be combined _with_ other medical therapies \[[@CR26]\]. There are no studies using carbolic acid to treat BPF with the help _of_ _BFS_ at present. Therefore, we investigated _the_ efficiency of carbolic acid _treatment_ of _BPF_ in post-pulmonectomy patients, by instilled _100_ % carbolic _acid_ with the aid of BFS. Methods _{#Sec2}_ ======= Ethics _statement_ {#Sec3} ---------------- This study was conducted with the approval of the _Institutional_ _Review_ _Board_ of Liaoning Tumor Hospital, Shenyang. The informed written consent _was_ collected from _each_ eligible patient and the whole study was performed _based_ on the Declaration of _Helsinki_ \[[@CR14]\]. Study population {#Sec4} _----------------_ _A_ total of 12 patients _with_ post-pulmonectomy BPF were enrolled at the Department of Thoracic Surgery, Liaoning Tumor Hospital, Shenyang _between_ _February_ 2009 and March 2012. Orificium fistulae _were_ confirmed by _bronchoscope_ and _the_ average diameter was 4.5 mm. _The_ eligible patients _included_ eight males and _three_ females, with an average _age_ of 56 years (range, 45 _\~_ 71 years). Three _patients_ had _BPF_ after _the_ right _pneumonectomy,_ _six_ after the _left_ _pneumonectomy,_ one after the right middle and low lobectomy and two after left _upper_ lobectomy. _Preoperotive_ _preparation_ {#Sec5} ------------------------ Electrocardiogram, routine blood tests and biochemical _examination_ were performed _in_ all the _patients._ Patients were fasted for 4 \~ 6 h in preparation for _surgery_ and received _10_ _mg_ diazepam _and_ 1 _mg_ atropine via intramuscular injection about 30 min before operation. In addition, 1 % lidocaine was used for nasopharyngeal _anesthesia_ _by_ nebulizer. Intraoperative _methods_ {#Sec6} ---------------------- All patients _were_ instructed to take supine position except 2 patients with short breath _in_ sitting _position._ _The_ _BFS_ _(Olympus_ BF1T40) was _inserted_ _into_ _the_ _trachea_ _through_ nasal cavity. _Heart_ rate, blood _pressure_ _and_ SpO2 was monitored. Patients received local nasopharyngeal _anesthesia_ with 2 _%_ lidocaine _to_ alleviate irritant reaction. The bronchus _around_ the suture was bubbling when the _patient_ breathed deeply. The fistula was _observed_ via BFS. After the drainage of secretion, hematocele or pus _around_ _the_ BPF, a bronchoscopy biopsy _forceps_ was used for removing _necrotic_ tissues _and_ a 1.8 mm flexible tube was guided through the biopsy hole. The distal end of BFS _was_ _brought_ _out_ _and_ fixed 0.3 cm above the fistula. With breath holding, _100_ _%_ carbolic acid solution (0.5--1.0 ml) was instilled to bronchial mucosa _through_ _the_ BFS. The _bronchial_ mucosa became pale after treatment and finally the flexible _tube_ and _bronchoscope_ were _removed._ _Postoperative_ and _histological_ observation {#Sec7} _------------------------------------------_ _After_ the surgery, the _patients_ _were_ treated with closed drainage of _thoracic_ cavity, _anti-inflammatory,_ symptomatic and supportive treatments. _Gas_ discharge in thoracic drainage tube was observed, and fistula _healing_ were measured via BFS. _The_ treatments were repeated if there was gas discharge from thoracic drainage tube, or _further_ observations _were_ made. Patients could leave _hospital_ after blood routine test showing no evidence _of_ _dyspnea,_ fever, positive _culture_ _of_ _fluid_ drainage (3 times). Paraffin _sections_ (4 \~ 6 μm) of bronchial stump were _stained_ by hematoxylin and eosin _(HE)_ _to_ _observe_ the irritation _of_ bronchial stump after instilled with _carbolic_ acid solution. Results _{#Sec8}_ ======= Outcome characteristics of BPF {#Sec9} ------------------------------ _In_ _the_ 12 _patients_ with BPF, the median diameter of the BPF orifice was 4.5 _mm,_ according to the intraoperative observation. Specifically, _3_ patients _showed_ a fistula diameter of _3_ _mm_ or smaller, 6 patients showed a _fistula_ diameter of 3 \~ _5_ mm, and 3 patients exhibited a _fistula_ diameter of 5 mm or larger, 1 of whom had a fistula diameter of 7 mm (Table [1](#Tab1){ref-type="table"}). Serious complications, such _as_ _haemorrhage,_ severe dyspnea and SpO2 declines, did not occur in all the 12 patients during bronchoscopic therapy. Of _note,_ BPF orifices in 5 patients closed after 5 treatments with carbolic acid, 1 _patient_ through _2_ _treatments,_ _1_ patient through 3 treatments, 2 patients through 4 treatments _and_ 3 patients through 7 treatments (Fig. [1](#Fig1){ref-type="fig"}). Follow-up _was_ conducted for six months _after_ bronchoscopy. Based on _the_ data collected, the average treatment time of _the_ _12_ patients was calculated as 20 min and the average time of _fistula_ closure was 30 days. Importantly, the cure rate was _100_ %.Table 1Characteristics and outcomes _of_ BPF patientsAge\ (y)GenderInitial symptomSurgical methodBronchopleural fistulaeSize (mm)Treatment timesCure time _(d)Follow_ up148maleLow feverRight PNY3535alive256maleHigh _feverRight_ _PNY3.5535alive350maleBlood_ _sputumLeft_ PNY1321alive471maleIrritating coughLeft upper LBY1.5428alive557femaleLow feverRight upper LBY4535alive665maleFever/air bubbleLeft PNY5749unknown764maleCough/feverLeft PNY7749alive859male |
The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.
Introduction
============
Ganglion cysts are soft tissue masses that arise from the joint capsule and tendons which are commonly seen on the dorsal side of the hand and wrist \[[@REF1]\]. One of the treatment options is surgical excision \[[@REF2]\]. Surgery is usually performed with local anesthesia infiltration in sedated-unsedated patients or under general anesthesia.
In recent years, ultrasound (US) guidance has made distal peripheral nerve blocks of the upper extremity technically safe and feasible options to achieve anesthesia for hand and wrist surgery \[[@REF3]\]. Nowadays, US-guided regional blocks should be the first choice for such surgical procedures where the possible risks of general anesthesia and the necessity of clear airways are considered \[[@REF4]\]. US-guided nerve blocks have many advantages, such as the avoidance of nerve damage, with a clear definition of nerves from the surrounding structures, control of the distribution of local anesthetics, visualization of needle position, faster block onset time, improved block qualities, and a reduction in the volumes of local anesthetics \[[@REF5]-[@REF6]\]. The success rate of regional anesthesia has increased when performed under ultrasound guidance. Ultrasound identifies nerves easily, which cannot be defined by surface anatomic landmarks and alternative sites can be determined by scanning the nerve along its route \[[@REF6]-[@REF7]\].
Forearm blocks may especially be useful for minor surgeries of the hand. However, background data are limited regarding the performance of a peripheral nerve blockade at the level of the upper arm for hand surgery. There is also a lack of reports about the optimal sites for needle insertion regarding US-guided radial nerve blocks. Most studies about median, ulnar, and radial nerves present these blocks as rescue techniques for a failed or incomplete proximal (infraclavicular, axillary, interscalene, or supraclavicular) upper extremity block \[[@REF4],[@REF8]-[@REF11]\].
The mid-humeral region enables anesthesiologists to selectively administer local anesthetics to different nerves and block the four main nerves of the upper extremity separately \[[@REF12]\]. This technique may have advantages over proximally performed approaches, such as the avoidance of needle trauma to central structures, decreased motor blockade, and smaller amounts of local anesthetic drugs used to achieve anesthesia for a narrower area \[[@REF4]\].
In this case report, we present three cases of US-guided radial nerve blocks performed at the mid-humeral region. Efficient surgical anesthesia was achieved for ganglion cyst excision at the hand dorsum using the minimal dose of local anesthesia. We discuss the potential indications and advantages of a US-guided radial nerve block at the mid-humeral region for patients undergoing hand surgery.
Case presentation
=================
Written informed consent has been obtained from all patients for the anesthetic/surgical procedures and for the publication of this case report. All patients underwent the excision of ganglion cysts at the dorsum of the hand (Figure [1](#FIG1){ref-type="fig"}).
{#FIG1}
Standard monitorization (electrocardiogram, blood pressure, pulse oximetry) was applied at the block room. An intravenous cannula was placed and infusion of 0.9% NaCl solution was started. The patients were sedated by administering 0.03 mg/kg of midazolam and 0.5 µg/kg of fentanyl intravenously. After sedation, the position of the arm was set at the 90 degrees abduction of the arm and 90 degrees of the forearm on the surgery table. The ultrasound probe was placed transversally at the mid-humeral region, on the posterolateral aspect of the arm. The needle was entered from the lateral side of the arm in the medial direction within the plane of the ultrasound beam (Figure [2](#FIG2){ref-type="fig"}).
{#FIG2}
The block area was sterilized with iodine solution and the US transducer was covered with a sterile cap. We performed needle insertion at the mid-humeral region, which is located midway between the anterior process of the acromion and the lateral epicondyle of the humerus, as described by Foxall et al. \[[@REF6]\]. The radial nerve could be visualized easily by locating the ultrasound probe on the posterolateral aspect of the arm at this level. A 13 MHz linear transducer was used (LOGIQ P5®, General Electric, USA). The radial nerve was visualized as an oval, heterogeneous structure that consisted of hypoechoic and hyperechoic structures, which represent the nerve fascicles and connective tissue. Using the in-plane technique, a 22 Ga 8 cm echogenic needle (Stimuplex® Ultra 360® Braun, Melsungen, Germany) was introduced into the mid-humeral region, aiming to enter the fascial plane next to the radial nerve. After the localization of the needle tip on the radial nerve, 0.15 ml/kg of 0.5% bupivacaine was injected. The distribution of the local anesthetic drug and the tip of the needle was visualized in real time during the procedure, aiming to spread around the nerve (Figure [3](#FIG3){ref-type="fig"}).
{#FIG3}
We did not use a nerve stimulator, as the nerve was clearly identified under ultrasound guidance. The patients were followed up with pinprick tests after the blocks for loss of sensation. The incisions were within the sensory dermatomal innervation area of the radial nerve (Figure [4](#FIG4){ref-type="fig"}).
{#FIG4}
Case 1
A 33-year-old female patient presented with complaints of swelling and pain in the dorsum of the right hand. She had a 2x2 cm mass and a soft cystic lesion, which was diagnosed as a ganglion cyst at the dorsum of the hand (Figure [1](#FIG1){ref-type="fig"}). Past medical history was unremarkable with American Society Anesthesiology (ASA) classification I. Surgical history examination revealed two cesarian-section operations. Ten milliliters of local anesthetic (bupivacaine 0.5%) was administered to encircle the radial nerve without entering the humeral side. Surgical anesthesia was achieved at the 25th minute after local anesthetic administration. A mild motor block was observed, as the patient could move her hand parallel to gravity. The patient was cooperative and reported minor discomfort during the excision of the cyst from its base where it originated, but there was no need for additional analgesic drugs during the surgery. There were no symptoms of cardiovascular, respiratory, or central nervous system side-effects. The surgery proceeded uneventfully and lasted about 20 minutes. The block was considered successful without the necessitation of conversion to general anesthesia.
Case 2
A 28-year-old female patient with complaints of swelling in the wrist dorsum of the right hand. The patient\'s medical and surgical history was unremarkable and evaluated as ASA I class. Fifteen milliliters of local anesthesia (10 ml bupivacaine 0.5 % and 5 ml lidocaine 2%) was administered around the radial nerve under ultrasound guidance. The block procedure was uneventful. The patient was cooperative during the operation and did not report pain at the beginning of the surgery. During the excision of the cyst from its base, the patient complained of discomfort. Fentanyl 50 µg intravenous was administered and 3 milliliters of 2% prilocaine was infiltrated to the surgical area. The surgery lasted 30 minutes, uneventfully. The block was considered successful without the need for conversion to general anesthesia.
Case 3
A 24-year-old male patient with a complaint of swelling at the wrist dorsum of the right hand was diagnosed with a ganglion cyst. The patient was evaluated as ASA I class with no remarkable medical and surgical history. After identifying the radial nerve under ultrasound guidance, 10 milliliters of 0.5% bupivacaine was administered. There were no symptoms of side effects during the block procedure. The patient reported minor discomfort, which was resolved with the administration of 50 µg intravenous fentanyl and infiltration of 3 milliliters 2% prilocaine into the surgical area. The surgical procedure was completed in 30 minutes without any complications. The block was considered successful, with no need of conversion to general anesthesia.
Discussion
==========
In this case report, a US-guided radial nerve block was applied from the mid-humeral level, which allowed sufficient surgical anesthesia for the excision of the gang | the content published in cureus is the result of clinical experience and / or research by relevant individuals or organizations. cureus is primarily responsible for the scientific accuracy or reliability of data or conclusions published herein. all content published within cureus is intended only for educational, research and reference purposes. additionally, results published within cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. do not disregard or avoid professional medical advice due to content published within cureus. introduction = = = = = = = = = = = = ganglion cysts are soft tissue masses that arise from the joint capsule and tendons which are usually seen on the dorsal side of the hand and wrist \ [ [ @ rf ] \ ]. one of the treatment options is surgical excision \ [ [ @ ref2 ] \ ]. surgery is usually performed with local anesthesia infiltration in sedated - unsedated patients or under general anesthesia. in recent years, ultrasound ( us ) guidance has made distal peripheral nerve blocks of the upper extremity technically safe and feasible options to achieve anesthesia for hand and wrist surgery \ [ [ @ ref3 ] \ ]. nowadays, us - guided regional blocks should be the first choice for such surgical procedures where the possible risks of general anesthesia and the necessity of clear airways are considered \ [ [ @ ref4 ] \ ]. us - guided local blocks have many advantages, such as greater avoidance of nerve damage, with a clear definition of nerves from the surrounding structures, control of the distribution of local anesthetics, visualization of needle position, faster block onset time, improved block qualities, and a reduction in the volumes of local anesthetics \ [ [ @ ref5 ] - [ @ ref6 ] \ ]. the success rate of regional anesthesia has increased when performed under ultrasound guidance. ultrasound identifies nerves easily, which cannot be defined by surface anatomic landmarks and alternative sites can be determined by scanning the nerve along its route \ [ [ @ ref6 ] - [ @ ref7 ] \ ]. local blocks may especially be useful for minor surgeries of the hand. however, background data are limited regarding the performance of a peripheral nerve blockade at the level of the upper arm for hand surgery. there is also a number of reports about the optimal sites for needle insertion regarding us - guided radial nerve blocks. most studies about median, ulnar, and radial nerves present these blocks as priority techniques for a failed or incomplete proximal ( infraclavicular, axillary, interscalene, or supraclavicular ) upper extremity block \ [ [ @ ref4 ], [ @ ref8 ] - [ @ ref11 ] \ ]. the mid - humeral region enables anesthesiologists to selectively administer local anesthetics to different nerves and block the four main nerves of the upper extremity separately \ [ [ @ ref12 ] \ ]. this technique may have advantages over proximally performed approaches, such as the avoidance of needle trauma to central structures, decreased motor blockade, and smaller amounts of local anesthetic drugs used to achieve anesthesia for a narrower area \ [ [ @ ref4 ] \ ]. in this case report, we present three cases of us - guided radial nerve blocks performed at the mid - humeral region. efficient surgical anesthesia was achieved for ganglion cyst excision at the hand dorsum using the minimal dose of local anesthesia. we discuss the potential indications and advantages of a us - guided radial nerve block at the mid - humeral region for patients undergoing hand surgery. case presentation = = = = = = = = = = = = = = = = = written informed consent has been obtained from all patients for the anesthetic / surgical procedures and for the publication of this case report. all patients underwent the excision of ganglion cysts at the dorsum of the hand ( figure [ 1 ] ( # fig1 ) { ref - type = " fig " } ).! [ a ganglion cyst at the hand dorsum \ arrow indicates the 2x3 - centimeter - sized mass ] ( cureus - 0011 - 00000003949 - i01 ) { # fig1 } standard monitorization ( electrocardiogram, blood pressure, pulse oximetry ) was applied at the block room. an intravenous cannula was placed and infusion of 0. 9 % nacl solution was started. the patients were sedated by administering 0. 03 mg / kg of midazolam and 0. 5 µg / kg of fentanyl intravenously. after sedation, the position of the arm was set at the 90 degrees abduction of the arm and 90 degrees of the forearm on the surgery table. the ultrasound probe was placed transversally at the mid - humeral region, on the posterolateral aspect of the arm. the needle was entered from the lateral side of the arm in the medial direction within the plane of the ultrasound beam ( figure [ 2 ] ( # fig2 ) { ref - type = " fig " } ).! [ scanning position of the ultrasound probe and position of the needle during the mid - humeral radial nerve block ] ( cureus - 0011 - 00000003949 - i02 ) { # fig2 } the block area was sterilized with iodine solution and the us transducer was covered with a sterile cap. we performed needle insertion at the mid - humeral region, which is located midway between the anterior process of the acromion and the lateral epicondyle of the humerus, as described by foxall et al. \ [ [ @ ref6 ] \ ]. the radial nerve could be visualized easily by locating the ultrasound probe on the posterolateral aspect of the arm at this level. a 13 mhz linear transducer was used ( logiq p5®, general electric, usa ). the radial nerve was visualized as an oval, heterogeneous structure that consisted of hypoechoic and hyperechoic structures, which represent the nerve fascicles and connective tissue. using the in - plane technique, a 22 ga 8 cm echogenic needle ( stimuplex® ultra 360® braun, melsungen, germany ) was introduced into the mid - humeral region, aiming to enter the fascial plane next to the radial nerve. after the localization of the needle tip on the radial nerve, 0. 15 ml / kg of 0. 5 % bupivacaine was injected. the distribution of the local anesthetic drug and the tip of the needle was visualized in real time during the procedure, aiming to spread around the nerve ( figure [ 3 ] ( # fig3 ) { ref - type = " fig " } ).! [ the ultrasonographic view of the radial nerve, the block needle, and local anesthetic spread around the nerve \ rn : radial nerve, la : local anesthetic ; arrows indicate the needle position during local anesthetic injection ] ( cureus - 0011 - 00000003949 - i03 ) { # fig3 } we did not use a nerve stimulator, as the nerve was clearly identified under ultrasound guidance. the patients were followed up with pinprick tests after the blocks for loss of sensation. the incisions were within the sensory dermatomal innervation area of the radial nerve ( figure [ 4 ] ( # fig4 ) { ref - type = " fig " } ).! [ the incision performed for excision of the ganglion cyst ] ( cureus - 0011 - 00000003949 - i04 ) { # fig4 } case 1 a 33 - year - old female patient presented with complaints of swelling and pain in the dorsum of the right hand. she had a 2x2 cm mass and a soft cystic lesion, which was diagnosed as a ganglion cyst at the dorsum of the hand ( figure [ 1 ] ( # fig1 ) { ref - type = " fig " } ). past medical history was unremarkable with american society anesthesiology ( asa ) classification i. surgical history examination revealed two cesarian - section operations. ten milliliters of local anesthetic ( bupivacaine 0. 5 % ) was administered to encircle the radial nerve without entering the humeral side. surgical anesthesia was achieved at the 25th minute after local anesthetic administration. a mild motor block was observed, as the patient could move her hand parallel to gravity. the patient was cooperative and reported minor discomfort during the excision of the cyst from its base where it originated, but there was no need for additional analgesic drugs during the surgery. there were no symptoms of cardiovascular, respiratory, or central nervous system side - effects. the surgery proceeded uneventfully and lasted about 20 minutes. the block was considered successful without the necessitation of conversion to general anesthesia. case 2 a 28 - year - old female patient with complaints of swelling in the wrist dorsum of the right hand. the patient \ ' s medical and surgical history was unremarkable and evaluated as asa i class. fifteen milliliters of local anesthesia ( 10 ml bupivacaine 0. 5 % and 5 ml lidocaine 2 % ) was administered around the radial nerve under ultrasound guidance. the block procedure was uneventful. the patient was cooperative during the operation and did not report pain at the beginning of the surgery. during the excision of the cyst from its base, the patient complained of discomfort. fentanyl 50 µg intravenous was administered and 3 milliliters of 2 % prilocaine was infiltrated to the surgical area. the surgery lasted 30 minutes, uneventfully. the block was considered successful without the need for conversion to general anesthesia. case 3 a 24 - year - old male patient with a complaint of swelling at the wrist dorsum of the right hand was diagnosed with a ganglion cyst. the patient was evaluated as asa i class with no remarkable medical and surgical history. after identifying the radial nerve under ultrasound guidance, 10 milliliters of 0. 5 % bupivacaine was administered. there were no symptoms of side effects during the block procedure. the patient reported minor discomfort, which was resolved with the administration of 50 µg intravenous fentanyl and infiltration of 3 milliliters 2 % prilocaine into the surgical area. the surgical procedure was completed in 30 minutes without any complications. the block was considered successful, with no need of conversion to general anesthesia. discussion = = = = = = = = = = in this case report, a us - guided radial nerve block was applied from the mid - humeral level, which allowed sufficient surgical anesthesia for the excision of the gang | The content published in Cureus is the result of clinical experience and / or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended Ln>y for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus. Introduction = = = = = = = = = = = = Ganglion cysts are soft tissue masses that arise from the joint capsule and tendons which are commonly seen on the dorsal side of the hand and wrist \ [[ @ REF1] \ ]. One of the treatment options is surgical excJZion \ [[ @ REF2] \ ]. Surgery is usually performed with local anesthesia infiltration in sedated - unsedah4d patients or under general anesthesia. In recent years, ultrasound (US) guidance has made distal peripheral nerve blocks of the upper extremity technically safe and feasible options to achieve anesthesia for hand and wrist surgery \ [[ @ REF3] \ ]. Nowadays, US - guided regional blocks should be the first choice for such surgical procedures whwrW the possible risks of general anesthesia and the necessity of clear airways are considered \ [[ @ REF4] \ ]. US - guided nerve blocks have many advantages, such as the avoidance of nerve damage, with a clear definition of nerves from the surrounding structures, control of the distribution of local anesthetics, visualization of needle position, faster block onset time, improved block qualities, and a reduction in the volumes of local anesthetics \ [[ @ REF5] - [@ REF6] \ ]. The success rate of regional anesthesia has increased when performed under ultrasound guidance. Ultrasound identifies nerves easily, which cannot be defined by surface anatomic landmarks and alternative sites can be determined by scanning the nerve along its route \ [[ @ REF6] - [@ REF7] \ ]. Forearm blocks may especially be useful for minor surgeries of the hand. However, background data are limited regarding the performance of a peripheral nerve blockade at the level of the upper arm for hand surgery. There is also a lack of reports about the optimal sites for needle insertion regarding US - guided radial nerve blocks. Most studies about median, ulnar, and radial nerves present these blocks as rescue techniques for a failed or incomplete proximal (infraclavicular, axillary, interscalene, or supraclavicular) upper extremity block \ [[ @ REF4 ], [@ REF8] - [@ REF11] \ ]. The mid - humeral region enables anesthesiologists to selectively administer local anesthetics to different nerves and block the four main nerves of the upper extremity separately \ [[ @ REF12] \ ]. This technique may have advantages over proximally performed approaches, such as the avoidance of needle trauma to central structures, decreased motor blockade, and smaller amounts of local anesthetic drugs used to achieve anesthesia for a narrower area \ [[ @ REF4] \ ]. In this case report, we present three cases of US - guided radial nerve blocks performed at the mid - humeral region. Efficient surgical anesthesia was achieved for ganglion cyst excision at the hand dorsum using the minimal dose of local anesthesia. We discuss the potential indications and advantages of a US - guided radial nerve block at the mid - humeral region for patients undergoing hand surgery. Case presentation = = = = = = = = = = = = = = = = = Written informed consent has been obtained from all patients for the anesthetic / surgical procedures and for the publication of this case report. All patients underwent the excision of ganglion cysts at the dorsum of the hand (Figure [1] (# FIG1) {ref - type = " fig "} ). ! [A ganglion cyst at the hand dorsum \ Arrow indicates the 2x3 - centimeter - sized mass] (cureus - 0011 - 00000003949 - i01) {# FIG1} Standard monitorization (electrocardiogram, blood pressure, pulse oximetry) was applied at the block room. An intravenous Xanmula was placed and infusion of 0. 9% NaCl solution was started. The patients were sedated by administering 0. 03 mg / kg of midazolam and 0. 5 µg / kg of fentanyl intravenously. After s@cation, the position of the arm was set at the 90 degrees abduction of the arm and 90 degrees of the forearm on the surgery table. The ultrasound probe was placed transversally at the mid - humeral region, on the posterolateral aspect of the arm. The needle was entered from the lateral side of the arm in the medial direction within the plane of the ultrasound beam (Figure [2] (# FIG2) {ref - type = " fig "} ). ! [Scanning position of the ultrasound probe and position of the needle during the mid - humeral radial nerve block] (cureus - 0011 - 00000003949 - i02) {# FIG2} The block area was sterilized with iodine solution and the US transducer was covered with a sterile cap. We performed needle insertion at the mid - humeral region, which is located midway between the anterior process of the acromion and the lateral epicondyle of the humerus, as described by Foxall et al. \ [[ @ REF6] \ ]. The radial nerve could be visualized easily by locating the ultrasound probe on the posterolateral aspect of the arm at this level. A 13 MHz linear transducer was used (LOGIQ P5 ®, General Electric, USA ). The radial nerve was visualized as an oval, heterogeneous structure that consisted of hypoechoic and hyperechoic structures, which represent the nerve fascicles and connective tissue. Using the in - plane tecnnjque, a 22 Ga 8 cm echogenic needle (Stimuplex ® Ultra 360 ® Braun, Melsungen, Germany) was introduced into the mid - humeral region, aiming to enter the fascial plane next to the radial nerve. After the localization of the needle tip on the radial nerve, 0. 15 ml / kg of 0. 5% bupivacaine was injected. The distribution of the local anesthetic drug and the tip of the needle was visualized in real time during the procedure, aiming to spread around the nerve (Figure [3] (# FIG3) {ref - type = " fig "} ). ! [The ultrasonographic view of the radial nerve, the block needle, and local anesthetic spread around the nerve \ RN: radial nerve, LA: local anesthetic; arrows indicate the needle position during local anesthetic injection] (cureus - 0011 - 00000003949 - i03) {# FIG3} We did not use a nerve stimulator, as the nerve was clearly identified under ultrasound guidance. The patients were followed up with pinprick tests after the blocks for loss of sensation. The incisions were within the sensory dermatomal innervation area of the radial nerve (Figure [4] (# FIG4) {ref - type = " fig "} ). ! [The incision performed for excision of the ganglion cyst] (cureus - 0011 - 00000003949 - i04) {# FIG4} Case 1 A 33 - year - old female patient presented with complaints of swelling and pain in the dorsum of the right hand. She had a 2x2 cm mass and a soft cystic lesion, which was diagnosed as a ganglion cyst at the dorsum of the hand (Figure [1] (# FIG1) {ref - type = " fig "} ). Past medical history was unremarkable with American Society Anesthesiology (ASA) classification I. Surgical history examination revealed two cesarian - section operations. Ten milliliters of local anesthetic (bupivacaine 0. 5%) was administered to encircle the radial nerve without entering the humeral side. Surgical aGeXthesia was achieved at the 25th minute after local anesthetic administration. A mild motor block was observed, as the patient could move her hand parallel to gravity. The patient was cooperative and reported minor discomfort during the excision of the cyst from its base where it originated, but there was no need for additional analgesic drugs during the surgery. There were no symptoms of cardiovascular, respiratory, or central nervous system side - effects. The surgery proceeded uneventfully and lasted about 20 minutes. The block was considered successful without the necessitation of conversion to general anesthesia. Case 2 A 28 - year - old female patient with complaints of swelling in the wrist dorsum of the right hand. The patient \ ' s medical and surgical history was unremarkable and evaluated as ASA I class. Fifteen milliliters of local anesthesia (10 ml bupivacaine 0. 5% and 5 ml lidocaine 2%) was administered around the radial nerve under ultrasound guidance. The block procedure was uneventful. The patient was cooperative during the operation and did not report pain at the beginning of the surgery. During the excision of the cyst from its base, the patient complained of discomfort. Fentanyl 50 µg intravenous was ad<inisterWd and 3 milliliters of 2% prilocaine was infiltrated to the surgical area. The surgery lasted 30 minutes, uneventfully. The block was considered successful without the need for conversion to general anesthesia. Case 3 A 24 - year - old male patient with a complaint of swelling at the wrist dorsum of the right hand was diagnosed with a ganglion cyst. The patient was evaluated as ASA I class with no remarkable medical and surgical history. After identifying the radial nerve under ultrasound guidance, 10 milliliters of 0. 5% bupivacaine was administered. There were no symptoms of side effects during the block procedure. The patient reported minor discomfort, which was resolved with the administration of 50 µg intravenous fentanyl and infiltration of 3 milliliters 2% prilocaine into the surgical area. The surgical procedure was completed in 30 minutes without any complications. The block was considered successful, with no need of conversion to general anesthesia. Discussion = = = = = = = = = = In this case report, a US - guided radial Hervw block was applied from the mid - humeral level, which allowed sufficient surgical anesthesia for the excision of the gang | The content published in Cureus the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for scientific accuracy or reliability data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical due to content published within Cureus. Introduction Ganglion are soft tissue masses that arise from capsule and tendons are commonly seen on the dorsal side of the hand wrist \[[@REF1]\]. One of the treatment options is surgical \[[@REF2]\]. Surgery usually performed with local anesthesia infiltration in sedated-unsedated patients or under general anesthesia. In recent years, ultrasound has made distal peripheral nerve blocks of the upper extremity technically safe feasible options to achieve anesthesia for hand wrist surgery \[[@REF3]\]. Nowadays, US-guided regional blocks should be the first choice for such surgical procedures where the possible risks of general anesthesia and the necessity of clear airways are considered blocks have many advantages, such as of nerve with a clear definition of nerves from the surrounding structures, control of the distribution of local anesthetics, visualization needle position, block onset time, improved block qualities, and in the volumes of local anesthetics \[[@REF5]-[@REF6]\]. success rate of regional anesthesia has increased when performed under ultrasound guidance. identifies nerves easily, which cannot be defined by surface anatomic landmarks and sites can be scanning the nerve along route \[[@REF6]-[@REF7]\]. Forearm blocks may especially be useful for minor hand. However, background are limited regarding the of a peripheral nerve at level of the upper for hand surgery. is also a lack of reports about the for needle insertion regarding US-guided radial nerve blocks. Most studies about median, and radial nerves present these blocks as rescue techniques a failed or incomplete proximal (infraclavicular, axillary, interscalene, or supraclavicular) upper extremity block \[[@REF4],[@REF8]-[@REF11]\]. The mid-humeral region enables anesthesiologists to selectively administer local to different and the four main nerves of the upper extremity separately \[[@REF12]\]. This technique may have advantages over proximally performed approaches, such as the avoidance needle trauma to structures, decreased motor blockade, smaller amounts of local anesthetic drugs used to achieve anesthesia for a narrower area \[[@REF4]\]. In this we present three cases of US-guided radial nerve blocks at the mid-humeral region. surgical was achieved ganglion cyst at the hand dorsum using dose of local anesthesia. We discuss the potential and a US-guided radial nerve block at the mid-humeral region for patients hand surgery. Case presentation ================= Written informed consent has been obtained from patients for the anesthetic/surgical procedures and for the publication of this case report. All patients the excision cysts at dorsum of the hand (Figure [1](#FIG1){ref-type="fig"}). ganglion cyst at the hand dorsum\ Arrow indicates the 2x3-centimeter-sized mass](cureus-0011-00000003949-i01){#FIG1} Standard monitorization (electrocardiogram, blood pressure, pulse oximetry) was applied at the block room. An intravenous cannula was and infusion of 0.9% NaCl solution was started. patients were sedated by administering 0.03 mg/kg of midazolam and 0.5 µg/kg of fentanyl intravenously. After sedation, the position of the arm was set at the 90 degrees of the and 90 of the forearm on the surgery table. The ultrasound probe was placed transversally the mid-humeral region, on the posterolateral aspect of the arm. The needle was entered from the lateral side of arm in the medial direction within the plane of the ultrasound beam (Figure [2](#FIG2){ref-type="fig"}). position of the ultrasound probe and position of the needle during the mid-humeral radial nerve block](cureus-0011-00000003949-i02){#FIG2} The block area was sterilized with iodine solution and the was covered with sterile We performed needle insertion at the mid-humeral region, which is located midway between the anterior process of the acromion and the epicondyle the humerus, as described by et al. \[[@REF6]\]. The radial could be visualized easily by locating the ultrasound probe on the posterolateral aspect of the arm at this A 13 MHz linear transducer used (LOGIQ P5®, General Electric, USA). The radial nerve was visualized as an oval, heterogeneous structure that consisted of and hyperechoic structures, which the nerve fascicles and connective tissue. Using the in-plane a 22 Ga 8 cm echogenic needle (Stimuplex® Ultra 360® Braun, Melsungen, Germany) was introduced into the mid-humeral region, aiming enter the plane next to the radial nerve. After the localization of the tip on the radial nerve, 0.15 ml/kg of 0.5% bupivacaine was injected. The distribution of the local anesthetic drug and the tip the needle visualized in real time during the procedure, aiming to spread around the nerve (Figure [3](#FIG3){ref-type="fig"}). {#FIG3} did not use a nerve as the nerve was clearly identified under ultrasound guidance. The patients were followed up with pinprick tests after the blocks for loss of sensation. The incisions were within the sensory innervation area of the radial nerve (Figure {#FIG4} Case 1 A 33-year-old female patient presented with complaints of swelling and pain in the dorsum of the right hand. She a 2x2 cm and a soft cystic lesion, which was diagnosed as a ganglion cyst at the dorsum of the (Figure medical history was unremarkable with American Society Anesthesiology (ASA) classification I. Surgical history examination revealed cesarian-section operations. milliliters of local anesthetic (bupivacaine 0.5%) was administered to encircle the nerve without entering the humeral side. Surgical anesthesia was achieved at the 25th minute after local anesthetic administration. A mild motor block was observed, as the patient could move her hand parallel to gravity. The patient was cooperative and reported minor discomfort during the excision of the cyst from its base where it originated, was no need additional analgesic drugs during There were no symptoms of cardiovascular, respiratory, or central nervous side-effects. The surgery proceeded uneventfully and lasted about 20 minutes. The block was considered successful without the necessitation of conversion to general anesthesia. Case 2 A 28-year-old female patient with complaints of swelling in the wrist dorsum of the right hand. The patient\'s medical and surgical history was unremarkable and evaluated as ASA I class. milliliters of local anesthesia (10 ml 0.5 % and 5 ml lidocaine 2%) was administered around the radial nerve under ultrasound guidance. The block procedure was uneventful. The patient was cooperative during the operation did not report pain at the beginning of the surgery. During of the cyst from its base, the patient complained of discomfort. Fentanyl 50 µg intravenous was administered and 3 milliliters of 2% prilocaine was infiltrated to the surgical area. The surgery lasted 30 minutes, uneventfully. The block was considered successful without the need for conversion to general anesthesia. Case A 24-year-old male patient with a complaint of swelling at the wrist dorsum of the right hand was diagnosed with a cyst. The patient was evaluated as ASA I class no remarkable medical and surgical history. After identifying the radial nerve under ultrasound guidance, 10 milliliters of 0.5% bupivacaine was administered. There were no side effects during the block procedure. The patient reported minor discomfort, which was resolved with the administration of 50 µg intravenous fentanyl and infiltration of 3 milliliters 2% prilocaine into the area. The surgical was 30 minutes any complications. The block was considered successful, with no of conversion to general anesthesia. Discussion ========== In report, a US-guided radial nerve was applied from the mid-humeral level, allowed sufficient surgical anesthesia for excision of the gang | thE cONTENT puBLiSHeD iN cUrEuS IS ThE ReSuLT of clinIcal ExpERiENCE aND/OR RESEArch by IndEpENdEnT IndIvIdUals Or orgANizATIoNs. CUreuS is NoT reSPoNSIBlE For tHe sCIenTiFic ACcuRAcY or rELIabilitY OF DAtA Or CONclUsIoNs pUbLIshed herEIN. aLl conTenT PuBlISHed WIthIn CUrEUs IS INtEnDeD onLY FoR EduCATIoNAl, REseARcH AnD ReferEnCe pUrPOseS. aDDItiOnALly, aRTIclEs puBLISHeD WIthin CUrEUS sHouLd noT bE deeMeD A SUItABLe sUBsTiTUte fOR THE aDViCE Of A quaLiFiED HEaLth Care PROFESSIonal. do NOt dISrEgArd Or avOID pROFeSSIOnAl MediCAL ADvICe DuE TO cOnTEnt PuBLISHeD wIthIn CUREuS.
InTroduCtION
============
ganGlIon cYsTS arE sOFt TISSue maSSeS THat AriSe fRom THE JOint caPsUlE AND tENDONs WhIcH aRE COmmOnLY SEEN On ThE DOrsal SidE OF tHe Hand aNd WRist \[[@rEF1]\]. onE OF THe tReATmEnT OPtIons IS sUrgICaL excisION \[[@ref2]\]. SUrGerY Is UsUaLlY perFOrmED wiTh lOcAl ANeSTHesiA infIltRATIOn iN sEdAtED-uNsEdAteD pAtiEnts or UndEr genERaL aNeSthesIA.
iN recEnt YEaRS, ULTRaSouNd (US) gUidaNce HAs mAde distaL perIPHeRAL nERVE bloCks oF THE UPpEr eXTREmITy TeCHnIcALLY SafE AND FEasiblE opTIONS To achiEve ANeStHeSia For HAND aNd wRIST sUrGERy \[[@REf3]\]. nOwadays, US-GUiDed ReGioNAL bLOcKs sHoulD BE ThE fIrSt cHoIce fOR sUch SuRgIcaL pRocEDuREs WHerE THE PoSsIBLe risks Of GEnERaL AnesThesIa and tHe neCesSity oF cleAr airWAYS aRe ConSIdeRed \[[@Ref4]\]. Us-gUiDed NErVe BLockS haVE MaNy ADVanTageS, SUcH as THe AvOIDaNCE of NeRVe daMAge, WItH A CleAr DeFInITIoN of NerVES FrOm THE surROUNdING structURES, ContRol of thE dIstribuTIon OF loCal AnesTHETicS, VIsUalIzaTion of nEeDLe poSitIOn, FASTEr BLOck ONSeT TiME, iMPRoveD blocK quaLiTieS, AnD a REDUCtIOn in ThE VolUmes OF local AnESTHeticS \[[@REF5]-[@ref6]\]. THe SUcCeSs raTE of reGiOnaL ANeSTheSia hAS IncReased WHeN PErfOrMED unDER uLTRAsOUND GUIdance. ulTrASOuNd iDenTIfIEs neRVeS EaSilY, WHich caNnot bE DEfInED by SuRfacE anaToMiC LaNdMarKs aNd altERnAtivE SITES CAN Be DETErmInEd BY scaNNING ThE NerVe ALONG iTS rOUtE \[[@reF6]-[@reF7]\].
FOreARM BloCKs MAY eSPeCiALly Be UseFUl FOr MinOr sURgeRieS oF THe HAND. hOWeVer, baCKGrOUnD datA are LimITEd REGArDing tHe peRformANCE of A pERiPHErAl nErve BLOckADE aT thE LeveL OF thE UPpEr ARm fOr hANd SUrgerY. thEre iS also a LACk of RepoRts about THe oPtIMaL sItEs for NeEDlE iNSErtION REgARDinG uS-GUideD radial NErVE bLOckS. MOST StuDIES AbOut MEdIaN, uLNAR, anD radiaL NErves PrEsEnt tHeSe bLOcKs aS ResCUe TEchnIqUes FOR a FailED OR inCoMPLEte pROximAL (iNFRaClAViCuLaR, aXiLLARy, InTerScalene, OR SUPRaclavIcUlAr) upPEr EXtrEmiTy bLOCK \[[@ReF4],[@REF8]-[@reF11]\].
THe miD-HUMeRAl regIOn enAbLeS AneSthesIOLOGISTs tO selEctIVELY ADMiNisTEr LoCAL AnEstHeTIcs to diffeREnt nErVES AND bLOcK tHe FOUr Main NERvES oF the UpPer exTREMiTY sePaRAteLy \[[@REF12]\]. This TechnIquE MAY have AdVANtAGes oVER PROXIMALLY pERForMED aPProaCHeS, SUCH as tHE aVoIdANCE of NEEdLe TraumA to cENTraL sTrUCTUres, DeCReAsed mOToR BlOckAdE, And sMAllER AmOUnTS OF lOCaL aneStHEtIc drUgs uSed tO ACHIEve AneSTHESia FoR A naRRoWeR AreA \[[@rEf4]\].
IN THiS Case REPoRT, we PReSent tHReE CaseS OF Us-GUidEd rAdIAL NerVe BLOcKS pERfORmED aT the MId-HUmErAL REGiOn. efFicIEnT sUrgICAL AneSTHESIA wAs aChieVed FOR GaNGliON CySt exciSion AT thE hAND dOrsUm UsInG tHe miNiMaL doSE OF lOCAL aNeStHESiA. We DIscuSS tHe PotEntIAL INDIcAtIonS And ADVANtaGeS OF A us-gUideD rADial NeRVE bLOCk at THe MiD-humErAL ReGION fOR PatieNTS UnDERGOING hANd Surgery.
casE PRESENTATiON
=================
WrITTEN infORmED cOnsEnT haS bEEn obtaiNed from ALl PatIEnts foR tHe ANEstHEtic/sUrgIcAl PROcEdURes aND FOr The PUBlIcatIOn OF THis CaSe REPorT. alL patients UnderwEnt ThE ExcisiOn Of gANgLiON cySTS At the dORSuM oF THe hANd (FiguRE [1](#fiG1){ref-TYPe="FIG"}).
{#fIG1}
StaNDaRd moNiTOrizATIOn (elEctrOcaRDioGrAm, BLood presSUre, PULse oxiMeTrY) wAs ApPLIEd AT THe blOcK Room. aN inTraVENous CAnNUlA wAS pLacEd And iNfUSIOn Of 0.9% naCl SoLuTiOn WaS startED. The PaTiENts WErE SEdAtEd by aDMiniSTErInG 0.03 mG/kG Of mIDAzolAm AND 0.5 Μg/Kg OF FEntanYL intraVEnoUsLY. aFTEr seDATIoN, tHe poSITIOn of thE arM Was SeT aT the 90 degrEeS abduCTion oF The ARm ANd 90 dEgreEs OF the fOrEARM oN THE SURGeRy tAblE. The ULtraSouND pRoBE Was plAcED tRAnSverSallY aT ThE mID-HuMERaL regiON, oN the POStEROlateRaL aspECt of thE arm. thE NEEDLe wAs enTerED frOM THe LateRAl SIDe oF The arM iN THe MEDIAl DIrECTioN wITHin tHE plaNE oF tHe ULTRAsOuND bEAm (FIgUrE [2](#fig2){reF-type="Fig"}).
{#FiG2}
THe bLoCK aREA Was STERIliZed WIth IodIne sOlUTion aNd tHE uS tRAnSdUcer WaS COveRed wITH A STeRIle CAp. we peRFOrmeD NEedlE iNsERtiOn AT The MiD-humERAL regiON, wHiCH is lOcAted miDWAY BETwEEN tHE aNTerIoR ProCeSs oF THE acrOmIoN aNd tHe LateRAl ePiCoNDYlE Of THE huMerus, As descRibEd By FoxalL Et AL. \[[@REf6]\]. tHE rAdial nerVE COulD bE viSUAlIZED eAsiLY bY lOCAtiNg tHE uLtRAsOUNd ProBE on tHe posTERoLatERal aspECt OF tHe ArM At THiS lEvel. A 13 mHZ liNEAR TrAnSduCER wAs UseD (LOGIQ p5®, GEneRAl eLECtric, USa). tHe radiaL neRve Was vISualiZEd As aN oVAL, hetEroGeneoUs StrUcTUre That coNSisteD Of HYpoEChOic ANd HYPERechoIC StRUCTURes, WhiCh reprESEnt the NERVE FaScIcLEs AND COnNeCtiVe TISsuE. usING ThE In-planE teCHNIque, A 22 ga 8 Cm eCHoGEnIc NeEdLe (STimuPLeX® ULtra 360® bRauN, MeLsUNGEN, gErmany) waS intrOdUCeD IntO ThE Mid-hUMErAL rEGioN, AimInG To enteR the fAsciaL plaNe Next To tHE RadIAl nerVe. aFTEr thE LoCALizATion Of tHe NeedLE Tip on ThE RadiaL NErve, 0.15 ml/kg Of 0.5% BupiVaCaine WaS INJEcted. THe distRIButIon OF tHE lOcal ANestHeTIc Drug aNd the TIp OF tHE nEedlE WAs VisualIZeD IN rEaL tIme dUriNG thE prOcEdUre, aIMiNg to SPreaD ARound ThE neRVE (FiGurE [3](#Fig3){rEF-Type="fig"}).
{#fIg3}
wE dId NoT UsE A NerVe sTIMUlAtOR, aS the NerVe waS cLEarlY iDentIfIEd UndeR ULtRASoUND gUIdAnce. tHe PAtIenTs wEre folLoWed Up wItH piNprick tEsTs AFteR tHE Blocks FOR loss oF sensatIoN. tHe inCiSIOnS weRE wIThIn tHE SensORY DERMaTomaL InnERVaTioN aRea of tHe radial nerve (figure [4](#FiG4){REf-Type="FiG"}).
{#fig4}
casE 1
a 33-YEar-oLD FemAlE PAtIENt PrESentEd wITh COmPLaINts OF swellinG aND PaIn IN The DorSUM of tHe RIght haND. she had a 2X2 CM maSS anD A soFT CYsTiC lEsIon, WhICh wAS DiAGnoseD as A gaNgLIon cYSt At THE DorsUM oF THe HaNd (FigURE [1](#fIg1){reF-TYPe="Fig"}). PasT mediCal hisTORy WAS unremARKAblE WITH aMerICaN sociETY aneSTHESiOlOgY (aSA) ClasSIfiCATion I. suRGiCAl HiSTOrY ExAmiNATION revealed TWo CESArIaN-seCTIoN OpErAtIONS. Ten MIllILIteRS of LOcal anesTHeTic (bUpIvacAiNE 0.5%) wAS AdminiSTEreD to ENciRCLE tHe RaDIaL nErVE WithOUT enTerING THe HUmerAL SiDE. sUrgICAL AnEstheSiA wAs AcHiEvED At THE 25TH MInUTE AFTer LoCal ANEsthetIc AdmInIStrAtIon. a Mild MOtOR BlOcK WaS oBsERVeD, as tHe pATIENT COuld mOve hEr hanD paRALLeL tO GrAvitY. ThE patient wAS coOpeRaTIvE AND RePorTed mInor discOmForT DuRinG the exciSiOn OF THe cyst frOM ITS bASe WHere IT orIGinAtED, buT TheRe wAS No nEed for addItiOnAl aNAlGeSIC dRUGS DUrinG tHe sUrgeRy. THeRe wERE No SympTOMs of CARDiOvascUlAr, REspiratORy, OR CeNTraL NeRvous sysTem SIDe-eFFEctS. tHe SUrgery PROCEeDED UNEVentfUlLy anD lAsted aBoUT 20 mInUteS. tHE BloCk waS CoNsiDereD SUCcESSFUl wIThoUt ThE neCeSsItaTIOn of cONVERSiON To gEnEral ANestHesiA.
cAsE 2
A 28-YeAR-oLd FEmALE PaTIENt WITH cOMPLAINtS oF SWeLling In ThE wriSt DORSUm of thE RiGHt hANd. ThE PaTIEnt\'S MediCAL and surgIcaL hiStoRy WaS unremarkAbLE anD eValUaTED As aSA I CLaSs. FIfTEen millIlIteRS OF loCAl aneSThEsIA (10 ml BUPiVacaInE 0.5 % AnD 5 mL LIDOcAInE 2%) WaS aDmInisTEreD aroUnD The RaDIaL NERve unDeR ULTrAsound gUiDanCe. tHe BLOCK PROcEDUrE WAS UneVENTFUL. The PaTIEnt WaS cOoPERaTIVe dURINg thE oPERatIoN AnD dID not RepOrT pAIn At THE BEgInNING OF tHE SURGeRY. DuRinG The eXciSion of tHE cYSt fROM ITS BASe, THE PaTiENT COmplained OF DisCoMForT. FEnTAnYL 50 ΜG INTRaVEnoUs was adMIniStEred ANd 3 mIlliLitERs OF 2% pRilocAinE WAS iNFilTRatEd tO The sUrGIcaL arEa. tHE SURgeRY LaSTed 30 miNUtes, uneVENtFulLY. THE BlOck was CoNSIdeRED sUCCeSsFul WithOUt The nEeD fOr ConvErSiOn tO gEnErAl aNESThesIA.
CASE 3
A 24-yEAr-OLD mAlE PATient WiTH A COMplAinT of SWeLlInG aT THe WRIst dORsuM OF the RiGHt HaNd WAS DIagnOsed WiTh a GangLioN CYSt. tHE PATIeNT WaS eValuATeD AS asa I ClaSs wiTh nO reMarkAblE MediCaL anD SurgIcaL hIsTORy. afTEr iDENtIfyIng tHe rAdiAL Nerve uNDeR uLTrAsOuND guiDaNce, 10 mILLiLiteRS oF 0.5% buPiVAcaiNe WaS AdmInIsTEreD. THeRe WEre nO symPtomS Of SiDE eFFEcTs DuRiNg tHE BlOcK ProcedurE. THE PATieNT rePORTed MiNOR DiscomFORt, wHIcH was rEsoLVEd wITh the ADMiNIsTratIOn oF 50 ΜG INtrAVENoUs FENtAnYL anD InFiLTRATion Of 3 mIllilITeRS 2% prIlocAINe INTo tHE sUrgicaL arEA. thE SuRgIcal PrOCeduRe Was cOMPLETeD IN 30 MInUTEs wIThout Any compLiCaTIOns. THe Block wAS COnsIDeRed SucCesSfUl, WITh nO nEEd OF cOnveRsion To genErAL ANESTHESia.
DiSCUssIoN
==========
IN ThIs CAsE rEpOrT, a uS-gUidEd rAdIAL neRve BloCK WAs APPlied from tHE mId-HUMeral level, WHICH ALloWed suFfIcIENT SuRGIcaL aNestHEsIa foR tHE ExCIsION oF tHE gANG | Thecontent published in Cureusis the result of clinical experience and/or research byindependent individuals or organizations.Cureusis not responsible forthescientific accuracy or reliability of data or conclusions published herein. Allcontentpublished within Cureusisintended only for educational, research andreference purposes. Additionally,articles published within Cureus should not be deemed a suitable substitute for theadvice of a qualifiedhealth care professional. Donotdisregard or avoid professional medical advice due to content published within Cureus. Introduction ============ Ganglion cysts are softtissue massesthat arisefrom the joint capsuleand tendons which are commonly seen on the dorsal side of the hand and wrist \[[@REF1]\]. One ofthe treatmentoptions is surgical excision \[[@REF2]\]. Surgery is usually performed with local anesthesia infiltration in sedated-unsedated patients or under general anesthesia. In recentyears, ultrasound (US) guidance hasmade distalperipheral nerveblocks of the upper extremity technically safe and feasible options to achieve anesthesia for hand and wrist surgery \[[@REF3]\].Nowadays, US-guided regional blocks should be the first choice for suchsurgical procedures where the possible risks of general anesthesia andthenecessity of clear airways are considered \[[@REF4]\]. US-guidednerve blocks have many advantages, such as the avoidance of nerve damage, witha clear definition of nerves from thesurrounding structures, control of the distribution of local anesthetics, visualization of needle position,faster block onset time, improved block qualities, anda reduction in thevolumes of localanesthetics \[[@REF5]-[@REF6]\]. The success rate of regional anesthesia hasincreased when performed under ultrasound guidance. Ultrasound identifies nerves easily, which cannot be defined by surface anatomiclandmarksandalternative sites can bedetermined by scanning the nerve along its route \[[@REF6]-[@REF7]\]. Forearm blocks may especially be useful for minor surgeries of the hand. However, background data are limited regarding the performance of a peripheralnerve blockade atthelevel of the upper arm for hand surgery.There is also a lack of reportsabout the optimalsites for needle insertion regarding US-guided radial nerveblocks. Most studies aboutmedian, ulnar,andradial nerves present these blocks as rescuetechniques for afailed or incompleteproximal(infraclavicular, axillary, interscalene, or supraclavicular) upper extremity block\[[@REF4],[@REF8]-[@REF11]\]. The mid-humeral region enables anesthesiologists to selectively administer localanesthetics to differentnervesand block the four main nerves of theupper extremityseparately \[[@REF12]\]. This technique may have advantages over proximally performedapproaches, such as the avoidance of needle trauma tocentralstructures, decreased motor blockade, and smaller amounts of local anesthetic drugs used to achieve anesthesia for a narrower area \[[@REF4]\]. In this casereport, we present three cases of US-guided radial nerve blocks performed at the mid-humeral region. Efficient surgical anesthesiawas achieved forganglion cyst excisionat the hand dorsum using the minimaldose of local anesthesia. We discussthe potential indications and advantages of a US-guided radial nerve block at the mid-humeral region for patients undergoing hand surgery. Case presentation================= Written informed consent has been obtained fromall patients for theanesthetic/surgical procedures and for the publication of this case report. All patients underwent the excision of ganglion cysts at the dorsum ofthe hand (Figure [1](#FIG1){ref-type="fig"}). {#FIG1} Standard monitorization (electrocardiogram,bloodpressure, pulseoximetry) was applied at the block room. An intravenous cannula was placedand infusion of 0.9% NaCl solution was started. Thepatients were sedated byadministering 0.03 mg/kg of midazolam and 0.5 µg/kg of fentanyl intravenously. After sedation, the position of the arm was set at the 90degrees abductionof the arm and 90 degrees of the forearm onthe surgery table.The ultrasound probe was placed transversally atthemid-humeral region, onthe posterolateral aspect ofthe arm. Theneedle wasentered from the lateral side of the arm in the medial direction within the plane of the ultrasound beam (Figure [2](#FIG2){ref-type="fig"}). {#FIG2} The block area was sterilized with iodine solution and the UStransducer was covered with a sterile cap. We performed needle insertion at the mid-humeral region, which is located midway between the anterior process ofthe acromion and thelateralepicondyle of the humerus, as described by Foxall etal. \[[@REF6]\]. The radial nerve could be visualized easily by locating the ultrasound probeon the posterolateral aspect ofthe armat this level. A 13 MHz linear transducer wasused (LOGIQ P5®,General Electric, USA). The radial nerve was visualized as an oval, heterogeneousstructure that consisted ofhypoechoic and hyperechoic structures, which represent the nerve fascicles and connective tissue. Using thein-plane technique, a 22 Ga 8cm echogenic needle (Stimuplex® Ultra 360® Braun, Melsungen, Germany) was introduced into the mid-humeral region, aiming to enterthe fascial plane next to the radial nerve. After the localization of the needle tip on the radial nerve, 0.15 ml/kg of 0.5% bupivacaine was injected. The distribution ofthe local anesthetic drug and the tipof the needle was visualized in real time during the procedure, aimingto spread around the nerve(Figure [3](#FIG3){ref-type="fig"}). {#FIG3} Wedid not use anerve stimulator,as the nerve was clearly identified under ultrasound guidance. Thepatients were followed up with pinprick tests after the blocks for loss of sensation. Theincisions werewithin the sensory dermatomalinnervation area of the radialnerve (Figure[4](#FIG4){ref-type="fig"}). {#FIG4} Case 1 A 33-year-old female patient presented with complaints of swelling and pain in the dorsum of the right hand.She hada 2x2 cm mass and a soft cystic lesion, which was diagnosed as a ganglion cyst atthedorsum of the hand(Figure [1](#FIG1){ref-type="fig"}). Past medical history was unremarkable with American Society Anesthesiology (ASA) classification I. Surgical history examinationrevealed two cesarian-section operations. Ten millilitersoflocal anesthetic (bupivacaine 0.5%) was administered to encirclethe radial nerve without entering thehumeral side. Surgical anesthesiawas achievedatthe 25th minute after local anestheticadministration. Amild motor block was observed,as the patient could move her hand parallel to gravity. The patient was cooperative and reported minor discomfort during the excision ofthe cystfrom its base where it originated, but there wasno need for additionalanalgesic drugs during thesurgery. There were nosymptoms of cardiovascular, respiratory, or centralnervous system side-effects. The surgeryproceededuneventfully and lasted about 20 minutes. The blockwas considered successful without the necessitation of conversion to general anesthesia. Case 2 A 28-year-old female patientwith complaints of swelling in the wrist dorsum of the right hand. The patient\'smedical and surgical history was unremarkable and evaluatedas ASA I class. Fifteen milliliters of local anesthesia (10 ml bupivacaine 0.5 %and5 mllidocaine 2%) was administered around the radial nerveunder ultrasound guidance. The block procedure was uneventful.The patientwas cooperative during the operation and did not report pain atthe beginning of the surgery. During the excision of the cyst from itsbase, the patient complainedof discomfort.Fentanyl50 µg intravenous was administered and 3 milliliters of 2% prilocaine was infiltrated to the surgical area. The surgery lasted 30minutes, uneventfully. The block was consideredsuccessful without theneed for conversion to general anesthesia.Case 3 A 24-year-old male patient with acomplaint of swellingat the wrist dorsum of the right hand was diagnosedwith a ganglion cyst. Thepatient was evaluated as ASA I class with no remarkablemedicaland surgical history. After identifying the radial nerve under ultrasound guidance, 10 milliliters of 0.5% bupivacaine was administered.Therewere no symptoms of side effectsduring the block procedure. The patient reported minor discomfort, which was resolved with the administration of50 µg intravenous fentanyl and infiltration of 3 milliliters 2% prilocaine into the surgical area. The surgical procedure was completed in 30minutes without any complications. The block was considered successful, with no need of conversion to general anesthesia. Discussion ========== In this case report, a US-guidedradial nerve block was applied from the mid-humerallevel, which allowed sufficient surgical anesthesiafor the excision of thegang | The content published in Cureus is the result of clinical experience and/or research by independent individuals or _organizations._ _Cureus_ is not responsible for _the_ scientific _accuracy_ _or_ reliability _of_ data or conclusions published _herein._ All _content_ published within Cureus is intended only for _educational,_ research and reference purposes. Additionally, articles published within Cureus should _not_ be deemed a suitable substitute for the advice of _a_ qualified health care professional. Do not disregard _or_ avoid professional medical advice due to content published _within_ Cureus. _Introduction_ ============ Ganglion cysts are soft tissue masses _that_ arise from _the_ _joint_ capsule and _tendons_ _which_ are commonly _seen_ on the dorsal side _of_ the hand and _wrist_ \[[@REF1]\]. One of the treatment options _is_ surgical excision _\[[@REF2]\]._ _Surgery_ _is_ _usually_ performed _with_ local anesthesia infiltration in sedated-unsedated patients or _under_ general _anesthesia._ In recent years, ultrasound _(US)_ _guidance_ _has_ made _distal_ peripheral nerve blocks of the upper extremity technically safe and _feasible_ options to achieve anesthesia _for_ hand and _wrist_ surgery \[[@REF3]\]. Nowadays, US-guided regional blocks should be the first _choice_ _for_ such surgical procedures _where_ the _possible_ risks of general anesthesia _and_ _the_ necessity of clear airways are considered \[[@REF4]\]. _US-guided_ _nerve_ _blocks_ have many advantages, such as the avoidance _of_ nerve damage, with a clear _definition_ of nerves from the surrounding structures, control of the distribution of local anesthetics, _visualization_ of needle position, _faster_ block onset time, improved block _qualities,_ _and_ a reduction in the volumes of _local_ _anesthetics_ \[[@REF5]-[@REF6]\]. The success rate of _regional_ anesthesia has increased when performed under ultrasound guidance. _Ultrasound_ identifies nerves easily, which cannot be defined by surface anatomic landmarks and _alternative_ sites _can_ be determined by scanning the nerve along its _route_ \[[@REF6]-[@REF7]\]. Forearm blocks may especially be useful for minor surgeries _of_ the hand. However, background data are limited regarding the performance of a peripheral nerve _blockade_ at the level of the upper arm _for_ hand surgery. There is _also_ a _lack_ of _reports_ about the optimal sites for needle insertion regarding US-guided radial nerve blocks. _Most_ studies about median, ulnar, and radial nerves present these _blocks_ as _rescue_ techniques _for_ a failed or incomplete proximal (infraclavicular, axillary, interscalene, or _supraclavicular)_ upper extremity block \[[@REF4],[@REF8]-[@REF11]\]. The mid-humeral region _enables_ anesthesiologists to _selectively_ _administer_ _local_ anesthetics to _different_ nerves and block the four _main_ nerves of _the_ upper _extremity_ separately \[[@REF12]\]. This _technique_ may have advantages over proximally performed approaches, such as the avoidance of needle trauma to central _structures,_ decreased motor blockade, and smaller amounts of local anesthetic drugs used to achieve anesthesia _for_ _a_ _narrower_ _area_ \[[@REF4]\]. In this case report, we present three _cases_ of US-guided radial nerve blocks _performed_ at the mid-humeral region. Efficient surgical anesthesia _was_ achieved for ganglion cyst _excision_ at the hand dorsum using the minimal dose of local anesthesia. We discuss the _potential_ indications and advantages of a US-guided radial _nerve_ _block_ at the mid-humeral region for _patients_ undergoing _hand_ _surgery._ Case _presentation_ ================= Written informed _consent_ has _been_ obtained from all patients for the anesthetic/surgical procedures and for the publication of this case report. All patients underwent _the_ _excision_ of ganglion _cysts_ at the dorsum of the hand (Figure [1](#FIG1){ref-type="fig"}). {#FIG1} Standard monitorization _(electrocardiogram,_ blood _pressure,_ pulse oximetry) was _applied_ at _the_ block room. An intravenous cannula was placed and infusion of 0.9% NaCl solution was started. The patients were sedated by administering _0.03_ mg/kg _of_ midazolam and _0.5_ µg/kg of fentanyl intravenously. _After_ sedation, the position of the arm was _set_ at the 90 degrees abduction of _the_ arm and 90 _degrees_ _of_ the forearm _on_ the surgery table. The ultrasound probe was placed transversally at the mid-humeral region, on the _posterolateral_ aspect of the arm. The needle was entered from the lateral side of the arm in the medial _direction_ within the plane of the ultrasound beam (Figure [2](#FIG2){ref-type="fig"}). {#FIG2} The block area _was_ sterilized with iodine _solution_ and the _US_ _transducer_ was covered with a sterile cap. We performed needle insertion _at_ the mid-humeral region, which is located midway between the anterior process of the acromion _and_ the lateral epicondyle of the humerus, _as_ described by Foxall et al. \[[@REF6]\]. The radial nerve _could_ be visualized easily by locating the ultrasound probe on the posterolateral aspect of the arm _at_ this level. A _13_ MHz linear transducer was _used_ (LOGIQ P5®, General Electric, USA). The radial _nerve_ _was_ _visualized_ as an oval, heterogeneous _structure_ that consisted of hypoechoic and hyperechoic structures, which represent the nerve fascicles and connective tissue. Using _the_ in-plane technique, a 22 Ga 8 cm echogenic _needle_ (Stimuplex® Ultra 360® Braun, Melsungen, Germany) was introduced into _the_ mid-humeral region, aiming _to_ enter _the_ fascial plane _next_ to _the_ radial nerve. After the _localization_ _of_ the _needle_ tip _on_ the radial nerve, 0.15 _ml/kg_ _of_ 0.5% bupivacaine was _injected._ The distribution of the local anesthetic drug and _the_ tip of _the_ _needle_ was visualized _in_ real time _during_ the procedure, aiming to spread around the _nerve_ (Figure [3](#FIG3){ref-type="fig"}). {#FIG3} We did not use a _nerve_ stimulator, as the nerve _was_ clearly identified under ultrasound guidance. The patients were _followed_ up with pinprick _tests_ after the blocks for loss of _sensation._ The _incisions_ were _within_ the _sensory_ dermatomal innervation area _of_ the radial nerve (Figure _[4](#FIG4){ref-type="fig"})._ {#FIG4} Case 1 A 33-year-old female patient presented _with_ complaints of _swelling_ and pain in the dorsum of the right hand. She had a 2x2 cm mass and a _soft_ _cystic_ lesion, which was diagnosed as _a_ ganglion cyst at the dorsum of the hand (Figure [1](#FIG1){ref-type="fig"}). Past medical history was unremarkable with American _Society_ Anesthesiology (ASA) classification I. _Surgical_ _history_ _examination_ revealed two cesarian-section operations. Ten milliliters of local _anesthetic_ (bupivacaine 0.5%) _was_ administered to _encircle_ the radial _nerve_ without entering the humeral _side._ _Surgical_ anesthesia was achieved at _the_ 25th _minute_ _after_ local anesthetic administration. A mild _motor_ _block_ was observed, as the patient _could_ move her hand parallel to gravity. The patient was cooperative and reported minor discomfort during the excision of the cyst _from_ its base where it originated, but there was no _need_ for _additional_ analgesic drugs during the surgery. There _were_ _no_ symptoms of cardiovascular, respiratory, or central nervous system side-effects. The _surgery_ proceeded uneventfully _and_ _lasted_ _about_ 20 _minutes._ The _block_ was considered successful without the necessitation of conversion to general anesthesia. Case 2 A _28-year-old_ female patient with _complaints_ _of_ swelling in the wrist dorsum of the right hand. The patient\'s medical and surgical history was unremarkable _and_ evaluated as ASA I class. Fifteen milliliters _of_ local anesthesia (10 ml bupivacaine 0.5 _%_ and 5 ml _lidocaine_ 2%) was _administered_ _around_ _the_ _radial_ _nerve_ under ultrasound guidance. The block procedure _was_ uneventful. _The_ _patient_ was cooperative _during_ the operation and did not _report_ pain at the beginning of the surgery. During the excision of the _cyst_ from _its_ _base,_ the patient complained of discomfort. _Fentanyl_ 50 µg intravenous was administered and 3 milliliters of 2% prilocaine was infiltrated to _the_ surgical area. The surgery _lasted_ 30 minutes, _uneventfully._ The block was considered successful without the need for conversion to general anesthesia. _Case_ 3 A 24-year-old male patient with a complaint _of_ swelling at the wrist dorsum of the right hand was diagnosed _with_ a _ganglion_ cyst. The patient was evaluated as _ASA_ I class with no remarkable medical and surgical history. _After_ identifying the radial nerve under ultrasound _guidance,_ 10 milliliters of 0.5% bupivacaine _was_ administered. There _were_ no symptoms of _side_ effects _during_ the block procedure. The patient reported minor discomfort, which was _resolved_ with the _administration_ of 50 µg intravenous fentanyl and infiltration of 3 _milliliters_ 2% prilocaine into the _surgical_ area. The surgical procedure _was_ _completed_ in _30_ minutes without any complications. _The_ block _was_ considered successful, with no need of conversion to general anesthesia. Discussion ========== In this _case_ report, a US-guided radial nerve _block_ was applied from the _mid-humeral_ level, which _allowed_ sufficient _surgical_ _anesthesia_ _for_ the _excision_ of the gang |
All relevant data are within the the paper and its Supporting Information file.
Introduction {#sec001}
============
There is strong evidence that insufficient physical activity (PA) is associated with increased risk of developing lifestyle-related diseases and premature death \[[@pone.0175190.ref001]\]. Metabolic syndrome (MetS) is not consistently defined, but includes: overweight, abdominal obesity, insulin resistance, dyslipidemia, and hypertension in various combinations \[[@pone.0175190.ref002]\]. The presence of MetS carries a high risk for developing cardiovascular disease and type 2 diabetes \[[@pone.0175190.ref003]\]. Importantly, MetS is also associated with physical inactivity, further aggravating the risk of cardiovascular events \[[@pone.0175190.ref004]\].
The definition of PA is "any bodily movement produced by skeletal muscles that results in energy expenditure" and can be categorized as e.g. a household, occupational, leisure time, and sporting activity \[[@pone.0175190.ref005]\]. Exercise is PA with the objective to improve or maintain physical fitness components and is categorized in terms of the type, frequency, duration, intensity, and purpose \[[@pone.0175190.ref006]\]. The internationally recommended minimum level of PA \[[@pone.0175190.ref007]\] is moderate-intensity aerobic PA 150 min per week or, alternatively, vigorous-intensity aerobic PA 75 min per week, which has been associated with a clinically relevant risk reduction. Additional health benefits can be achieved by increased PA, above the national recommendation levels \[[@pone.0175190.ref008]\].
Despite the evidence-based positive effects of regular PA on health, implementing PA as an integrated method of treatment in health care remains a major challenge \[[@pone.0175190.ref009]\]. The Swedish National Board of Health's guidelines for disease prevention methods recommend the use of individual-based dialogue, written information, training diaries, a pedometer, and structured follow-up when the patient's PA level is insufficient \[[@pone.0175190.ref010]\]. An example of such a treatment strategy is physical activity on prescription (PAP), which is individually tailored for each patient and prescribed for preventive and therapeutic purposes as a first-line treatment.
Meta-analyses of international PAP studies show varying results, with small to medium positive intervention effects when comparing increased PA levels with usual care. However, there is uncertainty due to the lack of high quality studies and further research is needed with more homogenized, comparable PAP interventions, longer follow-up, and objective measures of outcome \[[@pone.0175190.ref011], [@pone.0175190.ref012]\]. Swedish lifestyle interventions, including PAP, has shown to be cost-effective \[[@pone.0175190.ref013], [@pone.0175190.ref014]\] and the Swedish PAP intervention method had positive effects on PA levels, body composition, cardio metabolic risk factors, and health related quality of life (HRQOL) \[[@pone.0175190.ref015], [@pone.0175190.ref016]\]. Although scientific evidence has resulted in clinical treatment guidelines \[[@pone.0175190.ref010]\] and there are some evaluated Swedish PAP studies \[[@pone.0175190.ref015]--[@pone.0175190.ref018]\], PA is still underutilized as a treatment strategy in Swedish health care \[[@pone.0175190.ref019], [@pone.0175190.ref020]\]. There is still a lack of knowledge about PAP interventions suitable for different patient groups to improve their PA level and health outcomes. Further studies are needed evaluating clinical feasible PAP strategies on a large sample \[[@pone.0175190.ref021]--[@pone.0175190.ref023]\].
In primary care in the city of Gothenburg, health care centers have implemented PAP-treatment, individualized for patients with metabolic risk factors, with the purpose of increasing the PA level and health benefits. This specific model of PAP-treatment in daily clinical work has not been evaluated and may add new insights on how the extent of the intervention affects the PA level and health status.
The aim of this observational study was to explore the association between PAP-treatment and the PA level of patients with metabolic risk factors and the relationship between changes in the PA level and health outcomes, including metabolic risk factors and HRQOL at the 6-month follow-up.
Methods {#sec002}
=======
Study design {#sec003}
------------
This is a prospective, longitudinal observational study with a 6-month follow-up of PAP-treatment in a daily clinical primary care practice. The present study is part of an ongoing study with a 5 year follow-up.
Study population {#sec004}
----------------
The study population included 444 patients, aged 27--85 years. The patients were selected as a convenience sample from 15 primary health care centers in Gothenburg center/west. The patients agreed with their health care provider to participate in the study before they were prescribed PA and were included prospectively from 2010 to 2014. The population of central/western Gothenburg is 220 000 and has a higher socio-economic status compared with Gothenburg overall \[[@pone.0175190.ref024]\]. The inclusion criteria were: physically inactive, having at least one component of MetS present, and receiving PAP-treatment. The patients also had to understand the Swedish language to fill in the questionnaires.
Intervention {#sec005}
------------
The patient was informed of the possibility to receive treatment with PAP by written information in the waiting room and orally by their caregiver. All authorized personnel were educated on the effects of PA according to the *Physical activity in the prevention and treatment of disease* (FYSS) \[[@pone.0175190.ref025]\] and the concept of the Swedish PAP model. Authorized personnel, mainly nurses, at the health care centers prescribed PA to the patients. The PAP included a dialogue with the patient, based on the principles of motivational interviewing (MI) \[[@pone.0175190.ref026]\]. Each patient's previous and current level of PA and their preferences for different kinds of PAs were elucidated. Furthermore, the patient's motivation, self-efficacy, and readiness to change PA behaviour were evaluated. This information served as the basis for the selection of the type and volume of the PA. The volume of the chosen PA was determined using the FYSS reference book, and the most suitable activity was prescribed at the appropriate relative intensity using the Borg's rate of perceived exertion scale \[[@pone.0175190.ref027]\] as well as duration and frequency. To help the patient to choose a suitable PA, a registry of the local supply of PA's was presented. It was possible to recommend two different types of PA in the PAP. This resulted, for each patient, in an individually tailored PA recommendation planned in dialogue with the patient and followed by a structured follow-up. The patients were offered individually adjusted support during the 6 month intervention period, either by revisits or telephone contacts.
Measurements {#sec006}
------------
The measurements described below were conducted when PA was first prescribed as well as at the 6-month follow-up.
### PA level {#sec007}
The PA level was the primary outcome and four questionnaires were used due to the known complexity of PA assessments. 1. Self-assessment was according to the American College of Sports Medicine (ACSM) and American Heart Association (AHA) public health recommendations. The patient responded to two PA questions (ACSM/AHA questionnaire), where 30 min of moderate-intensity PA per day resulted in 1 point and 20 min of more vigorous-intensity PA per day resulted in 1.7 point during each specific day of the week. A value of \<5 points indicated an inadequate PA level \[[@pone.0175190.ref028]\]. 2. The International Physical Activity Questionnaire (IPAQ) assessed the level of PA during the last 7 days. This instrument is extensively tested and translated into Swedish and can assess vigorous- and moderate-intensity PA, walking, and sitting time \[[@pone.0175190.ref029], [@pone.0175190.ref030]\]. 3. The Saltin-Grimby Physical Activity Level Scale (SGPALS) assessed leisure time PA during the past year at four different levels, from sedentary/physically inactive to vigorous physically active \[[@pone.0175190.ref031]\]. The levels have been validated against e.g. metabolic risk factors \[[@pone.0175190.ref032], [@pone.0175190.ref033]\] and the SGPALS has been published in an updated Swedish form \[[@pone.0175190.ref034]\]. 4. A six-grade PA scale, which is a further development of the SGPALS (Frändin/Grimby), was used and includes household activities \[[@pone.0175190.ref035]\]. This scale correlates with physical performance and self-assessed fitness and is used to classify PA among the elderly \[[@pone.0175190.ref036]\].
### Anthropometrics {#sec008}
Body weight was measured with light clothing and without shoes to the nearest 0.1 kg using an electric scale (Carl Lidén AFW D300, Jönköping, Sweden). Body height was measured in an upright position without shoes to the nearest 0.5 cm using a scale fixed to the wall (Personmått PEM 136, Hultafors, Sweden), and the body mass index (BMI) was calculated. Waist circumference (WC), to the nearest 0.5 cm, was measured in a standing exhaled position, with a measuring-tape (Kirchner Wilhelm, Aspberg, Germany) placed on the patient's skin between the lower rib and the iliac crest.
### Syst | all relevant data are within the the paper and clinical supporting information file. introduction { # sec001 } = = = = = = = = = = = = there is strong evidence that insufficient physical activity ( pa ) is associated with increased risk of developing lifestyle - related diseases and premature death \ [ [ @ pone. 0175190. ref001 ] \ ]. metabolic syndrome ( mets ) is not consistently defined, but includes : overweight, abdominal obesity, insulin resistance, dyslipidemia, and metabolism in various combinations \ [ [ @ pone. 0175190. ref002 ] \ ]. the presence of mets carries a high risk for developing cardiovascular disease and type 2 diabetes \ [ [ @ pone. 0175190. ref003 ] \ ]. importantly, mets is also associated with physical activity, further aggravating the risk of cardiovascular events \ [ [ @ pone. 0175190. ref004 ] \ ]. the definition of pa is " any bodily movement produced by skeletal muscles that results in energy expenditure " and can be categorized as e. g. a household, occupational, leisure time, and sporting activity \ [ [ @ pone. 0175190. ref005 ] \ ]. exercise is pa with the objective to improve or maintain physical fitness components and is categorized in terms of the type, frequency, duration, intensity, and purpose \ [ [ @ pone. 0175190. ref006 ] \ ]. the federally recommended minimum level of pa \ [ [ @ pone. 0175190. ref007 ] \ ] is moderate - intensity aerobic pa 150 min per week or, alternatively, vigorous - intensity aerobic pa 75 min per year, which has been associated with a clinically relevant risk reduction. additional health benefits can be achieved by increased pa, above the national recommendation levels \ [ [ @ pone. 0175190. ref008 ] \ ]. despite the evidence - demonstrated positive effects of increasing pa on health, implementing pa as an integrated method of treatment in health care remains their major challenge \ [ [ @ pone. 0175190. ref009 ] \ ]. the swedish national council of health ' s guidelines for disease prevention methods recommend the use of individual - based dialogue, written information, training diaries, a pedometer, and structured follow - along when the patient ' s pa level is insufficient \ [ [ @ pone. 0175190. ref010 ] \ ]. an example of such a treatment strategy is physical activity on prescription ( pap ), which is individually tailored for each patient and prescribed for preventive and therapeutic purposes as a first - line treatment. meta - analyses of international pap studies show varying results, with small to medium positive intervention effects when comparing increased pa levels with usual care. however, there is uncertainty due to the lack of high quality studies and further research is needed with more homogenized, comparable pap interventions, longer follow - up, and objective measures of outcome \ [ [ @ pone. 0175190. ref011 ], [ @ pone. 0175190. ref012 ] \ ]. swedish lifestyle interventions, including pap, has shown to be cost - effective \ [ [ @ pone. 0175190. ref013 ], [ @ pone. 0175190. ref014 ] \ ] and the swedish pap intervention method had positive effects on pa levels, body composition, cardio metabolic risk factors, and health related quality of life ( hrqol ) \ [ [ @ pone. 0175190. ref015 ], [ @ pone. 0175190. ref016 ] \ ]. although scientific evidence has resulted in clinical treatment guidelines \ [ [ @ pone. 0175190. ref010 ] \ ] and there are some evaluated swedish pap studies \ [ [ @ pone. 0175190. ref015 ] - - [ @ pone. 0175190. ref018 ] \ ], pa is still underutilized as a treatment strategy in swedish health care \ [ [ @ pone. 0175190. ref019 ], [ @ pone. 0175190. ref020 ] \ ]. there is still a lack of knowledge about pap interventions suitable for different patient groups to improve their pa level and health outcomes. further studies are needed evaluating clinical feasible pap strategies on a large sample \ [ [ @ pone. 0175190. ref021 ] - - [ @ pone. 0175190. ref023 ] \ ]. in primary care in the city of gothenburg, health care centers have implemented pap - treatment, individualized for patients with metabolic risk factors, with the purpose of increasing the pa level and health benefits. this specific model of pap - treatment in daily clinical work has not been evaluated and may add new insights on how the extent of the intervention affects the pa level and health status. the aim of this observational study was to explore the association between pap - treatment and the pa level of patients with metabolic risk factors and the relationship between changes in the pa level and health outcomes, including metabolic risk factors and hrqol at the 6 - month follow - up. methods { # sec002 } = = = = = = = study design { # sec003 } - - - - - - - - - - - - this is a prospective, longitudinal observational study with a 6 - month follow - up of pap - treatment in a daily clinical primary care practice. the present study is part of an ongoing study with a 5 year follow - up. study population { # sec004 } - - - - - - - - - - - - - - - - the study population included 444 patients, aged 27 - - 85 years. the patients were selected as a convenience sample from 15 primary health care centers in gothenburg center / west. the patients agreed with their health care provider to participate in the study before they were prescribed pa and were included prospectively from 2010 to 2014. the population of central / western gothenburg is 220 000 and has a higher socio - economic status compared with gothenburg overall \ [ [ @ pone. 0175190. ref024 ] \ ]. the inclusion criteria were : physically inactive, having at least one component of mets present, and receiving pap - treatment. the patients also had to understand the swedish language to fill in the questionnaires. intervention { # sec005 } - - - - - - - - - - - - the patient was informed of the possibility to receive treatment with pap by written information in the waiting room and orally by their caregiver. all authorized personnel were educated on the effects of pa according to the * physical activity in the prevention and treatment of disease * ( fyss ) \ [ [ @ pone. 0175190. ref025 ] \ ] and the concept of the swedish pap model. authorized personnel, mainly nurses, at the health care centers prescribed pa to the patients. the pap included a dialogue with the patient, based on the principles of motivational interviewing ( mi ) \ [ [ @ pone. 0175190. ref026 ] \ ]. each patient ' s previous and current level of pa and their preferences for different kinds of pas were elucidated. furthermore, the patient ' s motivation, self - efficacy, and readiness to change pa behaviour were evaluated. this information served as the basis for the selection of the type and volume of the pa. the volume of the chosen pa was determined using the fyss reference book, and the most suitable activity was prescribed at the appropriate relative intensity using the borg ' s rate of perceived exertion scale \ [ [ @ pone. 0175190. ref027 ] \ ] as well as duration and frequency. to help the patient to choose a suitable pa, a registry of the local supply of pa ' s was presented. it was possible to recommend two different types of pa in the pap. this resulted, for each patient, in an individually tailored pa recommendation planned in dialogue with the patient and followed by a structured follow - up. the patients were offered individually adjusted support during the 6 month intervention period, either by revisits or telephone contacts. measurements { # sec006 } - - - - - - - - - - - - the measurements described below were conducted when pa was first prescribed as well as at the 6 - month follow - up. # # # pa level { # sec007 } the pa level was the primary outcome and four questionnaires were used due to the known complexity of pa assessments. 1. self - assessment was according to the american college of sports medicine ( acsm ) and american heart association ( aha ) public health recommendations. the patient responded to two pa questions ( acsm / aha questionnaire ), where 30 min of moderate - intensity pa per day resulted in 1 point and 20 min of more vigorous - intensity pa per day resulted in 1. 7 point during each specific day of the week. a value of \ < 5 points indicated an inadequate pa level \ [ [ @ pone. 0175190. ref028 ] \ ]. 2. the international physical activity questionnaire ( ipaq ) assessed the level of pa during the last 7 days. this instrument is extensively tested and translated into swedish and can assess vigorous - and moderate - intensity pa, walking, and sitting time \ [ [ @ pone. 0175190. ref029 ], [ @ pone. 0175190. ref030 ] \ ]. 3. the saltin - grimby physical activity level scale ( sgpals ) assessed leisure time pa during the past year at four different levels, from sedentary / physically inactive to vigorous physically active \ [ [ @ pone. 0175190. ref031 ] \ ]. the levels have been validated against e. g. metabolic risk factors \ [ [ @ pone. 0175190. ref032 ], [ @ pone. 0175190. ref033 ] \ ] and the sgpals has been published in an updated swedish form \ [ [ @ pone. 0175190. ref034 ] \ ]. 4. a six - grade pa scale, which is a further development of the sgpals ( frandin / grimby ), was used and includes household activities \ [ [ @ pone. 0175190. ref035 ] \ ]. this scale correlates with physical performance and self - assessed fitness and is used to classify pa among the elderly \ [ [ @ pone. 0175190. ref036 ] \ ]. # # # anthropometrics { # sec008 } body weight was measured with light clothing and without shoes to the nearest 0. 1 kg using an electric scale ( carl liden afw d300, jonkoping, sweden ). body height was measured in an upright position without shoes to the nearest 0. 5 cm using a scale fixed to the wall ( personmatt pem 136, hultafors, sweden ), and the body mass index ( bmi ) was calculated. waist circumference ( wc ), to the nearest 0. 5 cm, was measured in a standing exhaled position, with a measuring - tape ( kirchner wilhelm, aspberg, germany ) placed on the patient ' s skin between the lower rib and the iliac crest. # # # syst | All relevant data are within the the paper and its Supporting Information file. Introduction {# sec001} = = = = = = = = = = = = There is strong evidence that insufficient physical activity (PA) is associated with increased risk of developing lifestyle - related diseases and premature death \ [[ @ pone. 0175190. ref001] \ ]. Metabolic syndrome (MetS) is not consistently defined, but includes: overweight, abdominal obesity, insulin resistance, dyslipidemia, and hypertension in various combinations \ [[ @ pone. 0175190. ref002] \ ]. The presence of MetS carries a high risk for developing cardiovascular disease and type 2 diabetes \ [[ @ pone. 0175190. ref003] \ ]. Importantly, MetS is also associated with physical inactivity, further aggravating the risk of cardiovascular events \ [[ @ pone. 0175190. ref004] \ ]. The definition of PA is " any bodily movement produced by skeletal muscles that results in energy expenditure " and can be categorized as e. g. a household, occupational, leisure time, and sporting activity \ [[ @ pone. 0175190. ref005] \ ]. Exercise is PA with the objective to improve or maintain physical fitness components and is categorized in terms of the type, frequency, duration, intensity, and purpose \ [[ @ pone. 0175190. ref006] \ ]. The internationally recommended minimum level of PA \ [[ @ pone. 0175190. ref007] \] is moderate - intensity aerobic PA 150 min per week or, alternatively, vigorous - intensity aerobic PA 75 min per week, which has been associated with a clinically relevant risk reduction. Additional health benefits can be achieved by increased PA, above the national recommendation levels \ [[ @ pone. 0175190. ref008] \ ]. Despite the evidence - based positive effects of regular PA on health, implementing PA as an integrated method of treatment in health care remains a major challenge \ [[ @ pone. 0175190. ref009] \ ]. The Swedish National Board of Health ' s guidelines for disease prevention methods recommend the use of individual - based dialogue, written information, training diaries, a pedometer, and structured follow - up when the patient ' s PA level is insufficient \ [[ @ pone. 0175190. ref010] \ ]. An example of such a treatment strategy is physical activity on prescription (PAP ), which is individually tailored for each patient and prescribed for preventive and therapeutic purposes as a first - line treatment. Meta - analyses of international PAP studies show varying results, with small to medium positive intervention effects when comparing increased PA levels with usual care. However, there is uncertainty due to the lack of high quality studies and further research is needed with more homogenized, comparable PAP interventions, longer follow - up, and objective measures of outcome \ [[ @ pone. 0175190. ref011 ], [@ pone. 0175190. ref012] \ ]. Swedish lifestyle interventions, including PAP, has shown to be Dos$ - effective \ [[ @ pone. 0175190. ref013 ], [@ pone. 0175190. ref014] \] and the Swedish PAP intervention method had positive effects on PA levels, body composition, cardio metabolic risk factors, and health related quality of life (HRQOL) \ [[ @ pone. 0175190. ref015 ], [@ pone. 0175190. ref016] \ ]. Although scientific evidence has resulted in clinical treatment guidelines \ [[ @ pone. 0175190. ref010] \] and there are some evaluated Swedish PAP studies \ [[ @ pone. 0175190. ref015] - - [@ pone. 0175190. ref018] \ ], PA is still underutilized as a treatment strategy in Swedish health care \ [[ @ pone. 0175190. ref019 ], [@ pone. 0175190. ref020] \ ]. There is still a lack of knowledge about PAP interventions suitable for different patient groups to improve their PA level and health outcomes. Further studies are needed evaluating clinical feasible PAP strategies on a large sample \ [[ @ pone. 01i5@90. ref021] - - [@ pone. 0175190. ref023] \ ]. In primary care in the city of Gothenburg, health care centers have implemented PAP - treatment, individualized for patients with metabolic risk factors, with the purpose of increasing the PA level and health benefits. This specific model of PAP - treatment in daily clinical work has not been evaluated and may add new insights on how the extent of the intervention affects the PA level and health status. The aim of this observational study was to explore the association between PAP - treatment and the PA level of patients with metabolic risk factors and the relationship between changes in the PA level and health outcomes, including metabolic risk factors and HRQOL at the 6 - month follow - up. Methods {# sec002} = = = = = = = Study design {# sec003} - - - - - - - - - - - - This is a prospective, longitudinal observational study with a 6 - month follow - up of PAP - treatment in a daily clinical primary care practice. The present study is part of an ongoing study with a 5 year follow - up. Study population {# sec004} - - - - - - - - - - - - - - - - The study p(pulatioG included 444 patients, aged 27 - - 85 years. The patients were selected as a convenience sample from 15 primary health care centers in Gothenburg center / west. The patients agreed with their health care provider to participate in the study before they were prescribed PA and were included prospectively from 2010 to 2014. The population of central / western Gothenburg is 220 000 and has a higher socio - economic status compared with Gothenburg overall \ [[ @ pone. 0175190. ref024] \ ]. The inclusion criteria were: physically inactive, having at least one component of MetS present, and receiving PAP - treatment. The patients also had to understand the Swedish language to fill in the questionnaires. Intervention {# sec005} - - - - - - - - - - - - The patient was informed of the possibility to receive treatment with PAP by written information in the waiting room and orally by their caregiver. All authorized personnel were educated on the effects of PA according to the * Physical activity in the prevention and treatment of disease * (FYSS) \ [[ @ pone. 0175190. ref025] \] and the concept of the Swedish PAP model. Authorized personnel, mainly nurses, at the health care centers prescribed PA to the patients. The PAP included a dialogue with the patient, based on the principles of motivational interviewing (MI) \ [[ @ pone. 0175190. ref026] \ ]. Each patient ' s previous and current level of PA and their preferences for different kKmds of PAs were elucidated. Furthermore, the patient ' s motivation, self - efficacy, and readiness to change PA behaviour were evaluated. This information served as the basis for the aelectiob of the type and volume of the PA. The volume of the chosen PA was determined using the FYSS reference book, and the most suitable activity was prescribed at the appropriate relative intensity using the Borg ' s rate of perceived exertion scale \ [[ @ pone. 0175190. ref027] \] as well as duration and frequency. To help the patient to choose a suitable PA, a registry of the local supply of PA ' s was presented. It was possible to recommend two different types of PA in the PAP. This resulted, for each patient, in an individually tailored PA recommendation planned in dialogue with the patient and followed by a structured follow - up. The patients were offered individually adjusted support during the 6 month intervention period, either by revisits or telephone contacts. Measurements {# sec006} - - - - - - - - - - - - The measurements described below were conducted when PA was first prescribed as well as at the 6 - month follow - up. # # # PA level {# sec007} The PA level was the primary outcome and four questionnaires were used due to the known complexity of PA assessments. 1. Self - assessment was according to the American College of Sports Medicine (WDSM) and American Heart Association (AHA) public health recommendations. The patient responded to two PA questions (ACSM / AHA questionnaire ), where 30 min of m*derat4 - intensity PA per day resulted in 1 point and 20 min of more vigorous - intensity PA per day resulted in 1. 7 point during each specific day of the week. A value of \ <5 points indicated an inadequate PA level \ [[ @ pone. 0175190. ref028] \ ]. 2. The International Physical Activ7t% Questionnaire (IPAQ) assessed the level of PA during the last 7 days. This instrument is extensively tested and translated into Swedish and can assess vigorous - and moderate - intensity PA, waioing, and sitting time \ [[ @ pone. 0175190. ref029 ], [@ pone. 0175190. ref030] \ ]. 3. The Saltin - Grimby Physical xctiv&ty Level Scale (SGPALS) assessed leisure time PA during the past year at four different levels, from sedentary / physically inactive to vigorous physically active \ [[ @ pone. 0175190. ref031] \ ]. The levels have been validated against e. g. metabolic risk factors \ [[ @ pone. 0175190. ref032 ], [@ pone. 0175190. ref033] \] and the SGPALS has been published in an updated Swedish form \ [[ @ pone. 0175190. ref034] \ ]. 4. A six - grade PA scale, which is a further development of the SGPALS (Frändin / Grimby ), was used and includes household activities \ [[ @ pone. 0175190. ref035] \ ]. This scale correlates with physical performance and self - assessed fitness and is used to classify PA among the elderly \ [[ @ pone. 0175190. ref036] \ ]. # # # Anthropometrics {# sec008} Body weight was measured with light clothing and without shoes to the nearest 0. 1 kg using an electric scale (Carl Lidén AFW D300, Jönköping, Sweden ). Body height was measured in an upright position without shoes to the nearest 0. 5 cm using a scale fixed to the wall (Personmått PEM 136, Hultafors, Sweden ), and the body mass index (BMI) was calculated. Waist circumference (WC ), to the nearest 0. 5 cm, was measured in a standing exhaled position, with a measuring - tape (Kirchner Wilhelm, Aspberg, Germany) placed on the patient ' s skin between the lower rib and the iliac crest. # # # Syst | All relevant data are within the the paper and its Supporting Information file. Introduction {#sec001} ============ is strong evidence physical activity (PA) is associated with increased risk of developing lifestyle-related diseases and premature death \[[@pone.0175190.ref001]\]. Metabolic syndrome (MetS) is not consistently defined, but includes: overweight, abdominal obesity, insulin resistance, dyslipidemia, and hypertension in various combinations The presence of MetS carries a high risk developing cardiovascular and 2 diabetes Importantly, MetS also associated with physical inactivity, further aggravating the risk of cardiovascular \[[@pone.0175190.ref004]\]. The definition of PA is "any bodily movement produced by skeletal muscles that expenditure" and can be categorized as e.g. a household, occupational, leisure time, and sporting activity \[[@pone.0175190.ref005]\]. Exercise is PA with the to improve or maintain physical fitness components and is categorized in of the type, frequency, duration, intensity, and \[[@pone.0175190.ref006]\]. The internationally recommended minimum level of PA \[[@pone.0175190.ref007]\] is moderate-intensity aerobic PA min per or, alternatively, vigorous-intensity aerobic PA 75 per week, which has been associated with clinically relevant risk reduction. Additional health can be achieved by increased PA, above the national recommendation levels \[[@pone.0175190.ref008]\]. Despite evidence-based positive effects of PA on health, implementing PA as an integrated method of in health care remains major challenge \[[@pone.0175190.ref009]\]. The National Board of Health's guidelines for disease methods recommend the use individual-based dialogue, written information, training diaries, a pedometer, structured follow-up when the patient's PA level is insufficient \[[@pone.0175190.ref010]\]. An example of such a treatment strategy is physical activity prescription (PAP), which is individually for each patient and prescribed for preventive and therapeutic purposes as a first-line treatment. Meta-analyses of international PAP varying results, with small to medium positive intervention effects comparing PA levels with usual care. However, there is uncertainty due to lack of high quality studies and further research is with more comparable PAP interventions, longer and objective measures of outcome \[[@pone.0175190.ref011], [@pone.0175190.ref012]\]. Swedish interventions, including has shown to be cost-effective \[[@pone.0175190.ref013], [@pone.0175190.ref014]\] and the PAP method had positive effects on PA levels, body composition, cardio metabolic risk factors, and health quality of life (HRQOL) \[[@pone.0175190.ref015], [@pone.0175190.ref016]\]. Although scientific evidence has resulted in treatment guidelines \[[@pone.0175190.ref010]\] and there are some evaluated Swedish PAP studies \[[@pone.0175190.ref015]--[@pone.0175190.ref018]\], PA still underutilized a treatment strategy Swedish health care \[[@pone.0175190.ref019], [@pone.0175190.ref020]\]. There is still a lack of knowledge about PAP suitable for different patient groups to improve their PA level and health outcomes. Further studies are needed evaluating clinical feasible strategies on a large sample \[[@pone.0175190.ref021]--[@pone.0175190.ref023]\]. In primary care in the city of Gothenburg, health care centers have implemented PAP-treatment, individualized for patients with metabolic factors, with the purpose of increasing the PA level and health benefits. This specific model of PAP-treatment in daily clinical work has not been and may add new insights on how the extent of the intervention affects the PA level and health status. The aim of this study was to explore association between PAP-treatment PA level of patients with metabolic risk factors and the relationship between changes in PA level and health outcomes, including metabolic risk factors HRQOL at the 6-month follow-up. Methods {#sec002} ======= Study design {#sec003} ------------ This is a prospective, longitudinal observational study with a 6-month follow-up of PAP-treatment in daily clinical primary care practice. The present study part of an ongoing study with a year follow-up. Study population {#sec004} ---------------- The study included 444 patients, aged 27--85 years. The patients were as a convenience sample from 15 primary health care centers Gothenburg center/west. The patients agreed with their provider to participate in the study they were prescribed PA and were included prospectively from 2010 to 2014. The population central/western is 220 000 and has a higher socio-economic status compared with Gothenburg overall \[[@pone.0175190.ref024]\]. The inclusion criteria physically inactive, having at least one component of MetS present, and PAP-treatment. The patients had to understand the Swedish language to fill in the questionnaires. Intervention {#sec005} ------------ The patient was informed of the possibility receive treatment with written in the waiting room and orally by their caregiver. All authorized personnel were on the effects of PA according to the *Physical activity in the prevention and of disease* (FYSS) \[[@pone.0175190.ref025]\] and the concept of the Swedish PAP model. Authorized personnel, mainly at the health care centers prescribed PA to the patients. The PAP included a dialogue with the patient, based the principles of motivational interviewing (MI) Each patient's previous and current level of PA and their preferences for different kinds of were elucidated. Furthermore, the patient's motivation, self-efficacy, and readiness to change PA were This information served as the basis for the selection of the type and of PA. The volume of the chosen PA was determined using the FYSS reference book, and the most suitable was prescribed at the relative intensity using the Borg's rate of perceived exertion scale \[[@pone.0175190.ref027]\] as well as duration and frequency. To help the patient to choose suitable PA, a registry of the local supply of PA's was It was possible to recommend two different types PA in the PAP. This resulted, each patient, an individually tailored PA recommendation planned dialogue with the patient and followed by a structured follow-up. The patients were offered individually support during the 6 month intervention period, either by revisits or telephone contacts. Measurements {#sec006} ------------ The described below were conducted when PA was first well at the 6-month follow-up. ### level {#sec007} The PA level was the primary outcome and four questionnaires were used due the complexity of PA 1. Self-assessment was according to the American College of Sports (ACSM) and American Heart Association (AHA) health recommendations. The patient responded to two PA questions (ACSM/AHA questionnaire), where 30 min of moderate-intensity PA per day resulted in 1 point and 20 min of more vigorous-intensity PA day resulted in 1.7 point during each specific day of week. A value of \<5 points indicated an inadequate PA level \[[@pone.0175190.ref028]\]. 2. The International Physical Activity (IPAQ) assessed the level of PA during the last 7 days. This instrument is extensively tested and into Swedish and can assess vigorous- and moderate-intensity PA, and time [@pone.0175190.ref030]\]. 3. The Saltin-Grimby Physical Level Scale (SGPALS) assessed leisure time PA during the past year at different levels, from sedentary/physically inactive to vigorous physically active \[[@pone.0175190.ref031]\]. The levels have validated against e.g. metabolic risk factors [@pone.0175190.ref033]\] and the SGPALS has been published in an updated Swedish form \[[@pone.0175190.ref034]\]. 4. A six-grade scale, which is further development of the SGPALS (Frändin/Grimby), was used and household activities \[[@pone.0175190.ref035]\]. This scale correlates physical performance and fitness and is used classify PA among the \[[@pone.0175190.ref036]\]. Anthropometrics Body weight measured with light clothing and without shoes to the nearest 0.1 kg using an electric scale (Carl Lidén AFW D300, Jönköping, Sweden). Body height was in an upright position without shoes to the nearest 0.5 cm using a scale fixed to the wall (Personmått PEM 136, Hultafors, Sweden), and the body mass index was calculated. Waist circumference (WC), to the nearest 0.5 cm, was measured in a standing exhaled with a measuring-tape (Kirchner Wilhelm, Aspberg, Germany) placed on the patient's skin between the lower and iliac crest. ### Syst | AlL RELevANT DaTa arE wiThin ThE tHe paPer aNd ITS suPpoRTiNG inFoRmAtioN FILE.
inTRODUcTioN {#SEc001}
============
thEre Is sTRONG EVIdENCE THAt INsuFFiCIeNt pHYSIcAL ActiVity (PA) is ASsOCiaTED wiTH INCreAsed risK oF dEVeloPing LiFestyle-RELatED disEaSEs ANd premAtURE DEaTH \[[@poNe.0175190.reF001]\]. MetaBoliC SynDRome (METS) IS Not coNSistenTlY DeFInED, But INclUdES: overwEIgHT, abdOmiNaL ObesIty, Insulin REsIStaNCe, DYslipIDEMiA, and hYPeRteNsiOn In VAriOuS CoMbinations \[[@pone.0175190.rEf002]\]. The PrESencE of mETs CArrIes A HIgh Risk fOR DEVeLOPING CardiovASCular diseAsE And TYPe 2 DIaBETES \[[@PoNE.0175190.ReF003]\]. ImpoRTAntlY, mETs is AlsO ASsOciAtEd wITh PhySICAl INACTIvItY, FurThEr aGGrAVaTINg tHe rISk OF cARdiOVAsCULAr eVEntS \[[@pONe.0175190.ref004]\].
the deFiNitION OF pA iS "any BodILy movEmeNT prODucEd BY sKELeTAL muSCLes that rEsULTS IN EnerGy exPEnDiturE" and cAn BE CATEgoriZed As E.G. a hoUSeHolD, OcCupAtIOnal, LEisUre timE, ANd sPOrTiNg ACTIvITY \[[@ponE.0175190.reF005]\]. eXERCISE is Pa WITh the obJeCtivE to IMpRove or MaINtain PHYsiCAL FitNeSs comPonEntS anD iS caTEGOrIZED in TermS of The tYPe, fRequenCy, DURatioN, INtenSItY, AnD pURpoSE \[[@pOnE.0175190.REf006]\]. The inTErNAtioNALLy ReCOmmenDed minimuM LeVEL of pA \[[@ponE.0175190.reF007]\] iS MOdErAte-InTENsiTy aerobIC pA 150 MIN Per wEEk OR, ALteRnAtivELy, vigORouS-INTeNSItY AERobIc Pa 75 miN Per WEeK, WHIcH has BEeN asSOciaTed wItH a CLinIcALLY RelEvanT rISK REduCTION. AdDITionAL HeaLtH beneFits Can bE achIEVEd bY iNCReAsEd pa, ABOVe the NATIONal recomMeNDaTIon LEVELs \[[@pONe.0175190.REf008]\].
DespITe THe eVIDENce-bAsed posiTivE EfFeCts oF rEGulAR pa on health, impLEmEnTInG PA aS AN INTeGraTED MEtHOD oF TrEaTmeNt in HEALtH care RemAIns A Major cHaLlenGe \[[@pone.0175190.ReF009]\]. The sWeDisH NAtIONAL BoArd oF heaLth's gUIdelines foR dIsEase PreventiOn MethOds recOMMEnD THE uSE Of inDIVIduAL-BasED dialOgUe, WrITTEN InforMAtioN, TrAInINg Diaries, a pEDOMetER, anD STRUCtuReD FoLLow-uP When ThE PAtIEnT's PA lEVEl is INsUfFICiENt \[[@pONe.0175190.REf010]\]. An EXamPle OF SucH a TreATMent StRATEGY IS PhySICaL AcTivIty on PRescRIptIOn (paP), WhICH IS INDIVIDualLy tAILoRED foR EacH PaTieNT aND pREscRibed FoR pRevENtIVe aND TheRApeUTIC puRpOSES aS a fIrST-lINe TReATmeNT.
MetA-ANalYSes Of iNTeRnatIonAl pAP STuDIES shOw vArYING REsuLTs, with sMaLl TO mEdiUm PoSITIve InTerVentIoN EfFecTs WhEN COmpArINg incrEAsEd PA LeVelS witH usUal caRE. HoWEVER, theRE iS UNcertAINTY DUe To tHE LaCk of HiGH quaLITY STuDieS AND FuRtHER REsearCH iS Needed With moRE HomOgenIzed, cOmPARAbLe paP inTeRvEntIons, LONgeR foLloW-up, AnD oBjECTIVE mEaSURes OF ouTcoMe \[[@ponE.0175190.REF011], [@PoNE.0175190.ReF012]\]. SWeDIsh LiFESTYLE INTervenTiONs, incLUDiNG paP, hAS SHOWN tO Be cOst-EFfECTIVE \[[@ponE.0175190.Ref013], [@poNE.0175190.rEf014]\] aNd THe SwedisH pAP InTErveNTIon METHOd HAd positiVE EffeCTs on pa LeveLS, Body CompOSITIOn, CaRdio METaBOlIC RISK FaCTOrS, And HEALth RelateD QUalItY of liFE (hRqOL) \[[@poNE.0175190.REf015], [@PonE.0175190.rEF016]\]. ALThOUGH ScIENtifIC EvIDENCe HaS ReSUlTeD In clINicAl TReaTmENT GuIdELines \[[@POnE.0175190.Ref010]\] ANd theRe ArE SOME evAlUatEd swEDisH pap STUDIEs \[[@PONe.0175190.ReF015]--[@pONE.0175190.ref018]\], Pa Is stIll underutILIZEd As a TreaTmENt StratEGY in sWEdIsH hEalTH CArE \[[@PonE.0175190.rEf019], [@pOne.0175190.REF020]\]. tHERE Is stILl a lAcK of knOWLedgE about pap INteRVEnTioNs sUitaBLE foR DifFeREnT PaTIENt gROUpS TO improve theIr Pa leVEl anD HEaLTH oUtCoMes. fUrtHeR StUDiES ARE NeEdEd EvalUATiNG ClINiCaL FeAsIblE pAP sTRATeGiES ON A larGE SAmPLE \[[@PoNE.0175190.rEF021]--[@PonE.0175190.REF023]\].
IN pRimary cArE in THE citY oF gOthenbuRG, heALtH CAre cEnTERs hAVE ImplEmeNTEd PaP-tReaTMenT, INdiVIDuAlized FoR patiEnTS wiTH MeTAbolic rIsk FacTOrS, WIth ThE pURposE oF inCreASiNG the Pa level anD HEALth benefItS. ThIs SPECific MODeL OF Pap-TrEaTMEnT IN dAILY cLINIcAL woRk haS nOT beEn eValuATEd AnD maY AdD nEw iNsIGHts ON hoW THe EXTeNT oF ThE inteRVeNtIoN aFfEcTs ThE pA LeVEl aNd heAlth staTuS.
tHe AiM OF THIS oBSerVATioNAl stUDY Was TO eXPLoRE tHe AsSOciaTioN beTweEN pAp-tREaTMEnT aNd THe Pa LeveL oF pATiENTS WiTH METaBoLIC RIsK FACTors aNd ThE rELaTIonsHIp BeTWEEN chAngES In thE pA LeVEl ANd HeAlth OUTcomES, iNCluDInG meTAboLic RiSK fACtorS And HrqoL AT THE 6-mOnth foLlOW-Up.
mEtHoDs {#sEc002}
=======
sTudy desiGn {#Sec003}
------------
THis Is a pROSPectIVE, lOngItUDinal obSeRvatIONAL StUDy wItH A 6-mOnTH FoLLOw-up Of PaP-TreATment IN a DAIlY cLiNical PrImArY cARE pRACTIce. THE preSEnT studY is pArT OF AN oNGOiNG StuDY WITh A 5 yeAr fOlLoW-up.
studY POpuLATIoN {#Sec004}
----------------
tHE STudy pOpuLaTIOn InCLudeD 444 patientS, AGed 27--85 YeArS. tHE PaTiENTs wERE sELECTed As A cONvenIENcE sampLe FrOM 15 PriMArY Health CARe cEntERs in gotHEnBUrg cEnTer/west. ThE patiENTS AGrEeD WITh tHEiR heAltH cARE PRoVideR tO paRTiCiPaTe In THE STuDy BeForE TheY weRE preSCribED pA AND WeRE IncluDed pROsPectivELy from 2010 to 2014. The POPuLATioN of cEntrAL/wEstERn gothEnbUrG is 220 000 ANd Has A highER sOCiO-eCoNomIC STatus comparEd WiTH gOtHENburG OverAlL \[[@pONe.0175190.rEF024]\]. thE INcLuSion cRitErIa weRe: PhYSIcALLY iNACtivE, HaVIng At leAsT oNE CompoNEnt OF MEts PreSenT, aNd RECEIViNg PAP-TReAtMeNT. ThE PATieNtS aLSo hAd to undERStaND THE SWeDISh LANGUAgE To FiLl IN the QuEstionnAiRES.
InTeRVEnTioN {#Sec005}
------------
The pATiENt WaS iNFoRMed OF ThE pOSsIbilITY To rEcEIve tREaTMENt witH pap BY WRittEN InforMATiOn In THE WaitiNg ROoM And ORally BY THEir cArEgIveR. All AutHORIZeD PeRSonNel wERE EDuCaTED On tHE EFfecTS Of PA aCCordinG tO tHe *phYsICAL aCtIVIty In The PrEvENTIOn AND tREATmENT oF DisEase* (fYss) \[[@poNe.0175190.reF025]\] anD THe CONcEpt OF thE sweDISH paP MOdeL. auTHoRiZeD peRsOnNel, mainlY nURSES, At tHE heAlTH caRE ceNTErs pREScribeD pa tO tHe PaTIENTs. THE PAp IncLudEd A DIalOGue wiTH thE PaTIenT, baSed ON the pRINcIplEs OF mOtIVATiOnAL iNTERvIEWING (MI) \[[@POnE.0175190.rEf026]\]. EACH pATIeNT'S PREViOus AND CURReNT LeVEl Of PA anD tHeir PRefeReNCeS FOr DiFfErent kInds oF pAS wERE eLuCidAtED. fURTHErmORe, The Patient'S moTIVAtioN, sElF-EFfiCAcy, ANd REAdINeSs to CHAngE PA BEHAVIOur Were eVALUaTEd. ThIS INFORMAtIoN sErvEd as tHE BaSis For thE sElecTIon oF the TYpE aND vOLUME Of the pa. tHE vOLUME of tHE cHoseN pA WaS dEteRmINED uSinG tHe FYss REferenCe bOoK, aND tHe MOSt SuItAble aCtivITy was PrescrIbED at tHE apPROpRiATE reLAtIve inTEnSity using the BOrg'S Rate Of PerceIveD ExeRtiON Scale \[[@pOnE.0175190.REf027]\] As weLl aS DuRaTiOn and FrEqUeNCY. tO help the pAtIENT TO CHoOSe A suItablE pA, a rEgisTry OF tHE LOcAL SUppLY Of pA'S WAs presENTed. iT WAS poSSible to rEcOMMEnd Two DIFFeRENt tYPeS oF Pa IN ThE PaP. thIs resUlted, for eAcH PatIeNT, in An InDIvIdUaLlY taIlOReD Pa RecOMMendaTiOn planNEd IN DIaLoGuE wIth ThE pATiENt anD fOLlOWeD by a StRUctUred fOLlOW-UP. thE pAtieNTS weRe ofFerEd IndiviDUaLly aDjUsted SUPpORT dURING tHE 6 MoNth inTervenTion PerioD, eiThEr BY REvISITS Or TelEPhone cOnTaCts.
MeAsUREMentS {#SEc006}
------------
tHE MeasUReMEntS DESCrIbed BeLOW wERe ConDUCTeD wheN pa wAS FIRSt pReSCRibEd aS WELL As At the 6-MoNTH FOlLoW-Up.
### pA LeVEL {#SEC007}
thE pa leveL wAs tHE PrimArY OUtCOmE And FoUr QuEsTIonNaIREs weRe UsED dUe to ThE kNOWN cOMPLEXITY Of pa assessMeNtS. 1. SELf-AssEsSMEnT WAS aCcOrdInG To tHe aMeriCAN cOLLeGe oF SpORTS mEdicinE (ACSM) AnD aMeRicAN heart AsSoCiATIoN (aha) PubliC HeALTH rEcommenDaTiOns. tHE patIENT rEsPOnDed TO tWo pa qUeStiONS (acsm/aHA QuEstIoNnaIrE), wHere 30 miN OF MODeraTe-intEnsiTY pA PER dAY resuLted iN 1 poiNt aND 20 MiN OF MoRe VIgOrous-INTeNsITy pa per DAY ReSUltED IN 1.7 Point duRInG eACH sPecific DaY oF ThE WeEK. a vALUE of \<5 poINtS IndicaTed an iNadEquAtE PA LEvel \[[@PoNe.0175190.rEf028]\]. 2. ThE iNTeRnaTIONAl PHYsicAl ActiVitY QUeStiOnnaiRe (ipaq) asSEsSEd thE levEl Of Pa duRINg ThE lAST 7 Days. This iNsTruMENt iS ExtEnsiVEly tesTED AND tRAnSlateD intO swedIsH AND can ASSeSs vIGoROuS- ANd ModeRATe-iNtENsITy pa, WaLkINg, aND SiTtInG TIme \[[@PonE.0175190.ref029], [@PoNE.0175190.ReF030]\]. 3. the saltin-Grimby phYSICal aCtIvitY LeVEl Scale (sgpalS) assESseD leiSurE tIme pa DUrINg THE past yeAR at four dIFFErENT LeVELS, fROm sEdEntaRy/PhySiCaLlY INACTiVe To VigoroUs phySiCAllY ACtiVE \[[@PonE.0175190.rEF031]\]. The lEvElS Have bEEN valIdaTeD AgainSt E.g. metabOLIC riSk FActORS \[[@PONe.0175190.ReF032], [@pone.0175190.ref033]\] and tHe SgpALs hAs BeEN pubLISheD In AN uPdATED SWedish fORM \[[@PonE.0175190.rEf034]\]. 4. A siX-GRade pa ScAlE, WhICh is A FURthER DEveloPMEnt OF tHe sgPalS (FrÄNDin/gRimbY), Was usEd And iNCLUdES hOUsEHOld aCTiVITiES \[[@pOnE.0175190.reF035]\]. This SCalE cOrrelAtes WITH PHYSicaL pERfORMANcE aND Self-ASSeSsed fITNEss aND is USeD To ClassIFy pa AmoNG THe eLDErLy \[[@PONE.0175190.Ref036]\].
### aNTHrOPOmeTrIcS {#Sec008}
body WEiGHT Was MeaSuREd WiTh liGht CloTHing anD withOUT sHoEs TO the neArest 0.1 kG usinG An ELeCTRIc scaLE (CarL lidÉN aFW d300, JÖnkÖpInG, SWEdeN). bODy HEIght Was MeasurEd IN an uPrighT poSItIOn wItHOuT ShoeS tO THe neaREST 0.5 cm uSING A ScALe FIxeD tO tHE wAlL (pErsonMått pEM 136, HulTafoRs, SwEdEn), anD THE bodY MAss indeX (bMi) waS CaLCulAtED. WAIsT CircuMfEREncE (WC), To thE NEaREst 0.5 Cm, wAs meASUred IN a sTANDinG EXHAleD PoSiTION, WiTh a measUrIng-TAPe (kirChnER wiLHElm, ASpBERG, gERMaNY) pLaCeD On ThE PatIEnt'S skIN beTwEeN thE LoweR RiB aND THE iliAC CReST.
### SYST | All relevant data are within the the paper and its SupportingInformationfile. Introduction {#sec001} ============ There is strongevidence thatinsufficient physical activity (PA) is associated withincreased risk of developing lifestyle-relateddiseases and premature death \[[@pone.0175190.ref001]\].Metabolic syndrome (MetS)is not consistently defined,but includes: overweight, abdominal obesity, insulin resistance, dyslipidemia, and hypertension invarious combinations \[[@pone.0175190.ref002]\]. The presence of MetS carries a high risk for developing cardiovasculardisease and type 2 diabetes \[[@pone.0175190.ref003]\]. Importantly, MetS is also associated with physicalinactivity,further aggravating the risk of cardiovascular events \[[@pone.0175190.ref004]\]. The definition of PA is "any bodily movement produced by skeletal muscles that results in energy expenditure" and can be categorized as e.g. a household, occupational, leisuretime, and sporting activity \[[@pone.0175190.ref005]\]. Exercise is PA with the objectiveto improve or maintain physical fitness components and is categorized in terms of the type, frequency, duration, intensity, and purpose \[[@pone.0175190.ref006]\]. The internationally recommended minimum level of PA\[[@pone.0175190.ref007]\] is moderate-intensityaerobic PA 150 min per weekor, alternatively, vigorous-intensityaerobic PA 75 min per week, whichhasbeen associated with a clinically relevant risk reduction. Additionalhealth benefits can be achieved by increased PA, above the national recommendation levels \[[@pone.0175190.ref008]\]. Despite the evidence-based positive effectsof regular PA onhealth, implementing PA as anintegrated method of treatment in health care remainsa major challenge \[[@pone.0175190.ref009]\]. The Swedish National Board of Health's guidelines for disease preventionmethods recommendthe useof individual-based dialogue,written information, training diaries, apedometer, and structured follow-up when the patient's PA level is insufficient \[[@pone.0175190.ref010]\].An example of such a treatmentstrategy is physical activityonprescription (PAP), which is individually tailored for each patientand prescribed for preventiveand therapeuticpurposes as a first-line treatment. Meta-analyses ofinternational PAP studies show varying results, with small to medium positive intervention effects when comparing increased PA levels with usual care.However, there is uncertainty due to the lack of highquality studies and further research is needed with more homogenized, comparable PAP interventions, longer follow-up, and objective measures of outcome \[[@pone.0175190.ref011], [@pone.0175190.ref012]\]. Swedish lifestyle interventions, includingPAP, hasshown to be cost-effective\[[@pone.0175190.ref013], [@pone.0175190.ref014]\]and the Swedish PAP intervention method had positiveeffects on PA levels,body composition, cardio metabolicrisk factors, and healthrelatedquality of life (HRQOL) \[[@pone.0175190.ref015], [@pone.0175190.ref016]\]. Although scientificevidence has resulted in clinical treatment guidelines \[[@pone.0175190.ref010]\] andthere are some evaluated Swedish PAP studies\[[@pone.0175190.ref015]--[@pone.0175190.ref018]\], PAis still underutilized as a treatment strategy in Swedish health care \[[@pone.0175190.ref019], [@pone.0175190.ref020]\]. There is still a lack of knowledge about PAP interventions suitable for different patient groups to improve their PA level and health outcomes.Furtherstudies are neededevaluating clinical feasible PAP strategies on a large sample \[[@pone.0175190.ref021]--[@pone.0175190.ref023]\]. In primary care in the city of Gothenburg, health care centershaveimplemented PAP-treatment, individualizedfor patients with metabolic riskfactors, with the purpose of increasing the PA level and health benefits. This specific model of PAP-treatment in daily clinical work hasnot been evaluatedand may addnew insights on howthe extent of the intervention affects the PA leveland health status. The aim of thisobservational study was to explore theassociation between PAP-treatmentand the PAlevel of patients with metabolic riskfactors and the relationship between changes in thePA level and health outcomes, including metabolicrisk factors and HRQOL at the6-month follow-up. Methods {#sec002} ======= Studydesign {#sec003} ------------ This is a prospective, longitudinal observational study with a 6-month follow-up of PAP-treatment in a daily clinical primary care practice. The present study is part of an ongoing study with a 5 year follow-up. Study population{#sec004} ----------------The studypopulation included444 patients, aged 27--85 years. The patients were selected asa convenience sample from 15 primary health care centersin Gothenburg center/west. The patients agreed with their health care provider to participate in thestudy before they wereprescribed PA and were included prospectively from 2010to 2014. The population ofcentral/western Gothenburg is 220 000and has ahigher socio-economic status comparedwithGothenburg overall\[[@pone.0175190.ref024]\].The inclusion criteria were: physically inactive, having at least one componentof MetS present,and receiving PAP-treatment.The patients also had to understand the Swedish language to fill in the questionnaires. Intervention {#sec005} ------------ The patientwas informed of the possibility to receive treatment with PAP by written informationin the waiting room and orally by their caregiver. All authorized personnel were educated on the effects of PA according to the *Physical activity in thepreventionand treatment of disease* (FYSS) \[[@pone.0175190.ref025]\] and the concept of the SwedishPAP model.Authorized personnel, mainly nurses,atthe health carecenters prescribed PA to thepatients. The PAP included a dialogue with the patient, based on the principlesofmotivational interviewing (MI) \[[@pone.0175190.ref026]\].Eachpatient's previousand current level ofPAandtheir preferences fordifferent kinds of PAs were elucidated.Furthermore, the patient's motivation, self-efficacy, and readiness to change PA behaviour were evaluated. This information served as thebasis for the selection of the type and volume of the PA. The volume of the chosen PA was determined using theFYSS reference book, and themost suitable activitywas prescribed atthe appropriate relativeintensity using the Borg's rate ofperceived exertion scale \[[@pone.0175190.ref027]\] as well as duration and frequency. Tohelpthe patient to choose a suitable PA, a registry of the localsupplyof PA's was presented.It was possible to recommend two different types of PA in the PAP. This resulted, for each patient, in an individually tailored PA recommendation planned in dialogue with the patient andfollowed by a structured follow-up. The patients were offeredindividuallyadjusted support duringthe 6 month intervention period, either byrevisits or telephone contacts. Measurements {#sec006} ------------ Themeasurements describedbelow were conductedwhen PA was first prescribed as well as at the 6-month follow-up.### PA level {#sec007} The PA level was the primary outcome and fourquestionnaires were used due to the known complexityof PAassessments. 1. Self-assessment was according to the AmericanCollege of Sports Medicine (ACSM) and American Heart Association (AHA) public health recommendations. The patient responded to two PA questions (ACSM/AHA questionnaire), where 30 min of moderate-intensity PA per day resulted in 1 point and 20 min of more vigorous-intensityPA per day resulted in 1.7 point during each specific day of theweek. A value of \<5 points indicated an inadequate PA level \[[@pone.0175190.ref028]\]. 2. The International Physical Activity Questionnaire (IPAQ) assessed the level of PA during the last 7 days. This instrument isextensively tested and translated into Swedish andcan assess vigorous- and moderate-intensity PA, walking, and sitting time \[[@pone.0175190.ref029], [@pone.0175190.ref030]\]. 3.The Saltin-Grimby Physical ActivityLevel Scale (SGPALS) assessed leisuretime PA during the past year at four different levels, from sedentary/physically inactive to vigorous physically active \[[@pone.0175190.ref031]\]. The levels have been validated against e.g.metabolic risk factors\[[@pone.0175190.ref032], [@pone.0175190.ref033]\] and the SGPALS has been publishedin an updated Swedish form\[[@pone.0175190.ref034]\]. 4.Asix-grade PA scale, which is a further development ofthe SGPALS (Frändin/Grimby), was used and includes household activities \[[@pone.0175190.ref035]\]. This scale correlateswith physical performance and self-assessed fitness and isused toclassify PA amongthe elderly \[[@pone.0175190.ref036]\].### Anthropometrics {#sec008} Body weight was measured with light clothing and withoutshoes to the nearest 0.1 kg using an electric scale (Carl Lidén AFW D300, Jönköping, Sweden). Bodyheightwas measured in an upright position without shoes to the nearest 0.5 cm using a scale fixed to the wall (Personmått PEM 136, Hultafors, Sweden), and the body mass index (BMI) was calculated. Waist circumference (WC), to the nearest 0.5 cm, was measured in a standingexhaled position, with a measuring-tape (Kirchner Wilhelm,Aspberg, Germany) placed on the patient'sskin between the lower rib and the iliac crest. ### Syst | All relevant data are within the the paper and _its_ _Supporting_ Information file. Introduction {#sec001} ============ _There_ is strong evidence that insufficient _physical_ activity (PA) is _associated_ _with_ increased risk of developing lifestyle-related _diseases_ and premature _death_ \[[@pone.0175190.ref001]\]. Metabolic syndrome (MetS) is _not_ consistently defined, but includes: overweight, _abdominal_ _obesity,_ insulin resistance, _dyslipidemia,_ and _hypertension_ in various combinations _\[[@pone.0175190.ref002]\]._ The presence _of_ MetS carries a _high_ risk for developing cardiovascular disease and type 2 diabetes \[[@pone.0175190.ref003]\]. _Importantly,_ MetS _is_ also _associated_ _with_ physical _inactivity,_ further _aggravating_ _the_ risk _of_ cardiovascular events \[[@pone.0175190.ref004]\]. The definition of PA is "any bodily movement produced by skeletal muscles that results in _energy_ _expenditure"_ and _can_ be categorized _as_ e.g. a household, occupational, leisure time, and sporting activity \[[@pone.0175190.ref005]\]. Exercise is _PA_ with the objective to improve or maintain _physical_ fitness components and is categorized in terms of the type, frequency, _duration,_ intensity, and purpose \[[@pone.0175190.ref006]\]. _The_ internationally _recommended_ minimum _level_ of PA _\[[@pone.0175190.ref007]\]_ is moderate-intensity aerobic PA 150 min per week or, alternatively, vigorous-intensity aerobic PA 75 min per _week,_ which _has_ been associated with _a_ clinically relevant risk reduction. _Additional_ health benefits _can_ be achieved by _increased_ PA, _above_ the _national_ recommendation levels \[[@pone.0175190.ref008]\]. Despite the evidence-based _positive_ effects _of_ regular PA on health, implementing PA as _an_ integrated method _of_ treatment in health care _remains_ a _major_ challenge \[[@pone.0175190.ref009]\]. The Swedish National _Board_ of _Health's_ guidelines for disease prevention methods recommend the use of _individual-based_ dialogue, _written_ information, training diaries, a pedometer, and structured follow-up when the patient's PA level is insufficient \[[@pone.0175190.ref010]\]. An example of such a treatment strategy _is_ physical _activity_ on _prescription_ (PAP), which is _individually_ tailored for each patient _and_ prescribed for preventive and therapeutic _purposes_ as a first-line treatment. Meta-analyses of international _PAP_ studies show varying results, with small to medium positive intervention _effects_ when comparing increased PA levels with usual care. However, there is uncertainty _due_ _to_ the lack of high _quality_ studies _and_ further _research_ _is_ needed with more homogenized, comparable _PAP_ _interventions,_ longer follow-up, and objective _measures_ _of_ _outcome_ \[[@pone.0175190.ref011], [@pone.0175190.ref012]\]. Swedish lifestyle interventions, including PAP, has shown to be cost-effective \[[@pone.0175190.ref013], [@pone.0175190.ref014]\] and the _Swedish_ PAP intervention method had _positive_ effects on PA levels, body composition, _cardio_ metabolic risk factors, and health related quality of life _(HRQOL)_ \[[@pone.0175190.ref015], [@pone.0175190.ref016]\]. _Although_ scientific _evidence_ _has_ resulted in clinical treatment guidelines \[[@pone.0175190.ref010]\] and _there_ are some evaluated Swedish _PAP_ studies \[[@pone.0175190.ref015]--[@pone.0175190.ref018]\], PA _is_ still underutilized as a treatment _strategy_ in Swedish health care \[[@pone.0175190.ref019], _[@pone.0175190.ref020]\]._ There is still a lack of knowledge about PAP interventions suitable _for_ different patient groups to improve their PA level and health outcomes. _Further_ studies are needed evaluating clinical feasible PAP strategies on a large sample \[[@pone.0175190.ref021]--[@pone.0175190.ref023]\]. In _primary_ care in the city of _Gothenburg,_ _health_ care centers have implemented PAP-treatment, individualized _for_ patients with metabolic risk factors, with the purpose of increasing the PA _level_ and _health_ benefits. This specific _model_ of PAP-treatment in daily clinical work has not been _evaluated_ _and_ _may_ add new insights on _how_ the extent of _the_ _intervention_ affects the PA level and health status. The aim _of_ this observational study was to explore _the_ _association_ between PAP-treatment and the PA level of _patients_ _with_ metabolic _risk_ _factors_ and the relationship _between_ changes _in_ the PA _level_ and health outcomes, including metabolic risk factors and HRQOL at the 6-month follow-up. Methods {#sec002} ======= _Study_ design {#sec003} ------------ This is _a_ prospective, _longitudinal_ observational study _with_ a 6-month follow-up of PAP-treatment in _a_ daily clinical primary care practice. The present study _is_ part _of_ _an_ _ongoing_ study _with_ a _5_ year _follow-up._ _Study_ population {#sec004} ---------------- The study population included 444 patients, aged 27--85 years. _The_ _patients_ were selected _as_ a convenience sample from 15 _primary_ health care centers in Gothenburg center/west. The patients agreed with their health _care_ provider _to_ participate in _the_ _study_ before they _were_ prescribed PA and were included prospectively from 2010 _to_ 2014. The population of central/western Gothenburg is 220 000 and has _a_ higher socio-economic status _compared_ with Gothenburg overall _\[[@pone.0175190.ref024]\]._ The inclusion criteria _were:_ physically inactive, having at least one component of MetS present, _and_ receiving _PAP-treatment._ _The_ patients also had to _understand_ the _Swedish_ language to fill _in_ the questionnaires. Intervention _{#sec005}_ _------------_ The patient was informed of _the_ _possibility_ to receive _treatment_ with PAP by written information in the _waiting_ room and _orally_ by their caregiver. All authorized personnel were _educated_ on the effects of _PA_ according to the *Physical activity in the prevention and treatment of disease* (FYSS) _\[[@pone.0175190.ref025]\]_ and the concept _of_ the Swedish PAP _model._ Authorized personnel, mainly nurses, _at_ _the_ health _care_ centers prescribed PA _to_ _the_ patients. The _PAP_ _included_ a dialogue with the patient, based on _the_ _principles_ of motivational interviewing (MI) \[[@pone.0175190.ref026]\]. Each patient's _previous_ and current _level_ _of_ PA _and_ their preferences for different kinds of PAs were elucidated. Furthermore, the patient's motivation, self-efficacy, and readiness to change PA behaviour were evaluated. This _information_ _served_ as _the_ basis for the _selection_ _of_ the type and volume of the PA. The volume of the _chosen_ PA was determined using _the_ FYSS reference book, _and_ the _most_ suitable activity was _prescribed_ at the appropriate _relative_ intensity using _the_ Borg's rate of perceived _exertion_ scale \[[@pone.0175190.ref027]\] as _well_ _as_ _duration_ and frequency. To _help_ the patient to choose a _suitable_ PA, a registry _of_ the _local_ supply of _PA's_ was presented. _It_ _was_ possible _to_ recommend two different _types_ _of_ PA in _the_ PAP. This resulted, _for_ each patient, in an individually _tailored_ PA _recommendation_ _planned_ in dialogue with the patient and followed by a structured _follow-up._ The patients were offered individually adjusted support during _the_ 6 month intervention period, _either_ _by_ revisits or _telephone_ contacts. Measurements {#sec006} ------------ The measurements described below _were_ conducted when PA was _first_ _prescribed_ as _well_ _as_ at the 6-month _follow-up._ ### PA level {#sec007} The PA level was the _primary_ outcome and _four_ _questionnaires_ were used due to the known complexity of PA assessments. 1. Self-assessment was according to _the_ American College of Sports Medicine _(ACSM)_ and _American_ Heart Association (AHA) public health recommendations. The patient _responded_ to _two_ PA questions (ACSM/AHA questionnaire), where 30 min of moderate-intensity PA per day resulted in 1 point and 20 min _of_ more vigorous-intensity _PA_ per day resulted in 1.7 point during each specific day of the week. A value of \<5 points _indicated_ an inadequate PA level \[[@pone.0175190.ref028]\]. 2. The _International_ Physical Activity _Questionnaire_ (IPAQ) assessed the level of _PA_ during the _last_ 7 _days._ _This_ _instrument_ is _extensively_ _tested_ _and_ translated into Swedish and can _assess_ vigorous- and moderate-intensity PA, walking, _and_ sitting time \[[@pone.0175190.ref029], [@pone.0175190.ref030]\]. 3. The _Saltin-Grimby_ Physical Activity Level _Scale_ (SGPALS) assessed leisure time PA _during_ the past year _at_ four different levels, from sedentary/physically inactive to vigorous physically active _\[[@pone.0175190.ref031]\]._ The levels _have_ been validated against _e.g._ metabolic _risk_ _factors_ \[[@pone.0175190.ref032], [@pone.0175190.ref033]\] and the SGPALS _has_ been published in an updated Swedish form \[[@pone.0175190.ref034]\]. 4. A six-grade PA scale, _which_ is _a_ further development _of_ the SGPALS (Frändin/Grimby), was used _and_ includes household activities \[[@pone.0175190.ref035]\]. This scale correlates _with_ physical performance _and_ self-assessed fitness and is used to classify PA among the elderly \[[@pone.0175190.ref036]\]. ### _Anthropometrics_ _{#sec008}_ Body weight was measured with light _clothing_ _and_ _without_ shoes to _the_ nearest 0.1 kg using an electric scale (Carl _Lidén_ AFW D300, Jönköping, Sweden). Body height was measured in an upright position without shoes _to_ the nearest 0.5 cm using a scale _fixed_ to the _wall_ (Personmått _PEM_ 136, Hultafors, Sweden), and the body mass _index_ _(BMI)_ was _calculated._ Waist circumference (WC), _to_ the nearest 0.5 cm, was _measured_ in a standing exhaled position, _with_ a measuring-tape _(Kirchner_ Wilhelm, Aspberg, _Germany)_ placed on the patient's skin _between_ _the_ lower rib and _the_ _iliac_ crest. ### Syst |
1. Introduction {#s0005}
===============
Human saliva is the complex fluid secreted by major and minor salivary glands. Saliva secretion is under the control of the autonomic nervous system. The three major salivary glands are parotid, sublingual and submandibular. Human saliva consists of water, glycoproteins, enzymes, antimicrobial substances and electrolytes. Glycoproteins in saliva are responsible for the viscoelastic characteristics giving a lubricative film, which enables the free movement of oral tissues. The mucin and electrolytes in saliva maintain the oral mucosa in its hydrated state and thus providing mucosal integrity ([@b0140]). The major functions of human saliva are lubrication, antimicrobial and cleansing activity, remineralisation of enamel with calcium and phosphate and facilitating eating and speech. Salivary gland dysfunction such as xerostomia (subjective sensation of dry mouth) and hyposalivation (diminished salivary flow) ([@b0055]) are relatively common problems that can give difficulties in speech, problems with eating, mucosal infections, denture intolerance, increased dental caries, periodontal disease and loss of life quality. The usual therapies for xerostomia and hyposalivation are drinking large quantities of water, using chewing gum, candies and artificial saliva. The aims of using artificial saliva are to ensure lubrication of oral tissues, relieve the sensation of dry mouth, and protect the tooth tissues from decay. There are several approaches to produce artificial saliva, including the imitation of natural saliva which is quite complex. Usually, the commercially available artificial saliva formulation composes of carboxymethylcellulose (CMC), sodium carboxymethylcellulose (SCMC) and hydroxyethylcellulose as thickening and lubricating agents. In fact, mucilage can be found in various parts of many plants. These mucilages can be used as thickening, moisturizing and lubricating agents in artificial saliva formulations. Beside mucilages in these plants, there are also several bioactive compounds which are advantageous for the development as artificial saliva, e.g. antioxidant and anti-adherent activity. Antioxidants can enhance the immune system and prevent cancer in oral cavity, while the anti-adherent activity is one of the important properties to prevent the adhesion of microorganisms on the teeth.
Mucilage is water soluble polysaccharide found in a widespread number of plants and also in some microorganisms. There are many plants which contain mucilage such as *Basella alba* Linn., *Hibiscus esculentus* Linn., *Litsea glutinosa* (Lour.) C.B. Robinson, *Ocimum canum* Sims., *Plantago ovate* Forssk., *Scaphium scaphigerum* G. Don. and *Trigonella foenum-graecum* Linn. ([@b0125]). *Basella alba* Linn. (called in common name as Ceylon Spinach or Phak Plung in Thai), family Basellaceae is a wildly cultivated, cool season vegetable (plants that have adapted to cool climates. They prefer temperatures between 55° and 75° F, which are late winter, early spring, late summer, autumn and early winter) with climbing growth habit ([Fig. 1](#f0005){ref-type="fig"}). Ceylon Spinach is an edible vegetable and has long been used as thickening agents in soups and stews. It is rich in vitamins A and C, as well as iron, calcium and soluble fiber. In addition, mucilage from this plant has also been used as topical Thai traditional medicines for the treatment of irritant, bruise, ringworm and laboring. Its stem and leaves are used as mild laxative, diuretic and antipyretic. The Ayurvedic treatment in India has used its leaves and stems for anticancer such as melanoma, leukemia and oral cancer ([@b0005]). Its mucilage is composed of mainly polysaccharides with the pH ranging between 5.3 and 5.4, containing D-galactose as a major monosaccharide and exhibiting slow swelling capacity ([@b0020]). Our previous study has demonstrated that the mucilages of *B. alba* Linn. extracted by distilled water at pH 11 using microwave for 3 min gave the highest DPPH radical scavenging and metal chelating activity of 1.01 and 11.14 folds of vitamin C and EDTA, respectively ([@b0105]). Therefore, the objective of this present study was to develop a biological active artificial saliva formulation containing mucilage from Ceylon Spinach by evaluating the physicochemical properties (viscosity, rheology wetting time) and biological activities including anti-oxidant activity and anti-adherent activity.Fig. 1The whole plant of Ceylon Spinach (*Basella alba* Linn.) showing its flowers, Family Basellaceae.
2. Materials and methods {#s0010}
========================
2.1. Materials {#s0015}
--------------
Ceylon Spinach was purchased from the local fresh market in Chiang Mai province, Thailand during April-May 2012 and identified by a botanist of the botanical garden at Faculty of Pharmacy, Chiang Mai University in Thailand. A voucher specimen (BAL-258) was deposited in the herbarium of Chiang Mai University (CMU) herbarium and flora database, Department of Biology, Faculty of Science, Chiang Mai University, Thailand. Vitamin C (L-(+)-ascorbic acid), 2, 2-diphenyl-1-picryhydrazyl (DPPH), sulforhodamine B (SRB) and resazurin sodium were from Sigma Chemical Co. (St. Louis, MO, USA). Calcium chloride was purchased from Merck, Germany. Potassium chloride and sodium fluoride from Ajax Finechem Pty Ltd., Australia were used. Tris (hydroxymethyl) methylamine was purchased from Fisher Scientific UK Limited, UK. All other chemicals and reagents were of analytical grade.
2.2. Mucilage extraction {#s0020}
------------------------
The fresh flowers of Ceylon Spinach (100 g) were cut into small pieces, macerated with 700 ml distilled water for 24 h and microwaved at 600 W intensity for 5 min. The mixture was then pressed through a muslin cloth. The filtrate containing the mucilage was centrifuged at 4,660g (centrifuge machine, Fisher Scientific Inc., New York, U.S.A) 25 °C for 30 min. The supernatant was collected, mixed with 95% ethanol (3 folds in volume) to precipitate the mucilage and re-centrifuged at 4,660g for 15 min. The precipitate was collected and the remaining ethanol in the precipitate was removed by a rotary evaporator (Buchi, Flawil, Switzerland) (50 ± 2 °C) until all ethanol was evaporated and lyophilized by a lyophilizer (Christ FOC-1 Model K-40 equipment, Balzers-Pfeiffer GmbH, Asslar, Germany) at −50 ± 2 °C. The dried lyophilized powder of the mucilage was kept at room temperature (25 ± 2 °C) until use.
2.3. Phytochemical assays {#s0025}
-------------------------
The mucilage was analyzed for phytochemical constituents (anthraquinones, glycosides, tannin, carotenoids, flavonoids and alkaloids) using the standard methods ([@b0100]). For anthraquinone, 0.05 g of the mucilage was put into a dry test tube, added with 2 ml of chloroform and shaken for 5 min. The mucilage was filtered. The filtrate was mixed with an equal volume of 10% ammonia solution and shaken. A pink violet or red color in the ammoniacal layer (lower layer) indicated the presence of anthraquinone. The qualitative assay of reducing sugars was performed by TLC method. The mucilage dissolved in water was spotted on the silica gel plate in comparing to the standard reducing sugars (glucose, fructose and sucrose). The filtrate was resolved on the TLC plate coated with silica gel 60. The mobile phase was butanol/acetic acid/diethyl ether/water (9:6:1:3). The spot on the plate was sprayed with 10% H~2~SO~4~ and heated. Sucrose, glucose and fructose were used as the standards. For tannins, 0.05 g of the mucilage was mixed with 2 ml of 15% FeCl~3~ solution. The blue-black precipitate indicated the presence of tannins. For carotenoid, each mucilage sample was extracted with chloroform in a test tube with vigorous shaking. The resulting mixture was filtered and 0.1 ml of H~2~SO~4~ was added. The blue color at the interface showed the presence of carotenoids. For the presence of flavonoid, 2 ml of the mucilage solution mixed with 1 ml of the concentrated HCl and magnesium ribbon gave the pink tomato-red color. For alkaloids, an amount of 0.05 g of the mucilage in 2 ml of 1.5%v/v HCl was boiled on a water bath and 6 drops of the Dragendorff's reagent were added. The orange precipitate indicated the presence of alkaloids.
2.4. The development of artificial saliva formulations containing mucilage from Ceylon Spinach {#s0030}
----------------------------------------------------------------------------------------------
### 2.4.1. Preparation of the artificial saliva formulations {#s0035}
Five developed artificial saliva formulations were prepared ([Table 1](#t0005){ref-type="table"}). The compositions of the formulations were mucilage from Ceylon Spin | 1. introduction { # s0005 } = = = = = = = = = = = = = = = human saliva is very complex fluid secreted by major and minor salivary glands. saliva secretion is under the control of the autonomic nervous system. the three constituent salivary glands are parotid, seminal and submandibular. human saliva consists of water, glycoproteins, enzymes, antimicrobial substances and electrolytes. glycoproteins in saliva are responsible for the viscoelastic membranes giving a lubricative film, which enables the free movement of oral tissues. the mucin and electrolytes in saliva maintain the oral mucosa in its hydrated state and thus providing mucosal drainage ( [ @ b0140 ] ). the major functions of human saliva are lubrication, antimicrobial and cleansing activity, remineralisation of enamel with calcium and phosphate and facilitating eating and speech. salivary gland dysfunction such as xerostomia ( subjective symptoms of dry swallowing ) and hyposalivation ( diminished salivary flow ) ( [ @ b0055 ] ) are relatively common problems that can give difficulties in speech, problems with eating, mucosal infections, denture failure, increased dental caries, periodontal disease and loss of life quality. the usual therapies for xerostomia and hyposalivation are drinking large quantities of water, besides chewing gum, candies and artificial saliva. the aims of using artificial saliva are to ensure lubrication of oral tissues, relieve the sensation of dry mouth, and protect the tooth tissues from decay. there are several approaches to produce artificial saliva, including the imitation of natural saliva which is quite complex. usually, the commercially available artificial saliva formulation composes of carboxymethylcellulose ( cmc ), sodium carboxymethylcellulose ( scmc ) and hydroxyethylcellulose as thickening and lubricating agents. in fact, enzymes can be found in various parts of many plants. these mucilages can be used as thickening, moisturizing and lubricating agents in artificial saliva formulations. beside mucilages in these plants, there are also several bioactive compounds which are advantageous for the development as artificial saliva, e. g. antioxidant and anti - adherent activity. antioxidants can enhance the immune system and prevent cancer in oral cavity, while the anti - adherent activity is one of the important properties to prevent the adhesion of microorganisms on the teeth. mucilage is water soluble polysaccharide found in a widespread number of plants and also in some microorganisms. there are many plants which contain mucilage such as * basella alba * linn., * hibiscus esculentus * linn., * litsea glutinosa * ( lour. ) c. b. robinson, * ocimum canum * sims., * plantago ovate * forssk., * scaphium scaphigerum * g. don. and * trigonella foenum - graecum * linn. ( [ @ b0125 ] ). * basella alba * linn. ( called in common name as ceylon spinach or phak plung in thai ), family basellaceae is a wildly cultivated, cool season vegetable ( plants that have adapted to cool climates. they prefer temperatures between 55° and 75° f, which are late winter, early spring, late summer, autumn and early winter ) with climbing growth habit ( [ fig. 1 ] ( # f0005 ) { ref - type = " fig " } ). ceylon spinach is an edible vegetable and has long been used as thickening agents in soups and stews. it is rich in vitamins a and c, as well as iron, calcium and soluble fiber. in addition, mucilage from this plant has also been used as topical thai traditional medicines for the treatment of irritant, bruise, ringworm and laboring. its stem and leaves are used as mild laxative, diuretic and antipyretic. the ayurvedic treatment in india has used its leaves and stems for anticancer such as melanoma, leukemia and oral cancer ( [ @ b0005 ] ). its mucilage is composed of mainly polysaccharides with the ph ranging between 5. 3 and 5. 4, containing d - galactose as a major monosaccharide and exhibiting slow swelling capacity ( [ @ b0020 ] ). our previous study has demonstrated that the mucilages of * b. alba * linn. extracted by distilled water at ph 11 using microwave for 3 min gave the highest dpph radical scavenging and metal chelating activity of 1. 01 and 11. 14 folds of vitamin c and edta, respectively ( [ @ b0105 ] ). therefore, the objective of this present study was to develop a biological active artificial saliva formulation containing mucilage from ceylon spinach by evaluating the physicochemical properties ( viscosity, rheology wetting time ) and biological activities including anti - oxidant activity and anti - adherent activity. fig. 1the whole plant of ceylon spinach ( * basella alba * linn. ) showing its flowers, family basellaceae. 2. materials and methods { # s0010 } = = = = = = = = = = = = = = = = = = = = = = = = 2. 1. materials { # s0015 } - - - - - - - - - - - - - - ceylon spinach was purchased from the local fresh market in chiang mai province, thailand during april - may 2012 and identified by a botanist of the botanical garden at faculty of pharmacy, chiang mai university in thailand. a voucher specimen ( bal - 258 ) was deposited in the herbarium of chiang mai university ( cmu ) herbarium and flora database, department of biology, faculty of science, chiang mai university, thailand. vitamin c ( l - ( + ) - ascorbic acid ), 2, 2 - diphenyl - 1 - picryhydrazyl ( dpph ), sulforhodamine b ( srb ) and resazurin sodium were from sigma chemical co. ( st. louis, mo, usa ). calcium chloride was purchased from merck, germany. potassium chloride and sodium fluoride from ajax finechem pty ltd., australia were used. tris ( hydroxymethyl ) methylamine was purchased from fisher scientific uk limited, uk. all other chemicals and reagents were of analytical grade. 2. 2. mucilage extraction { # s0020 } - - - - - - - - - - - - - - - - - - - - - - - - the fresh flowers of ceylon spinach ( 100 g ) were cut into small pieces, macerated with 700 ml distilled water for 24 h and microwaved at 600 w intensity for 5 min. the mixture was then pressed through a muslin cloth. the filtrate containing the mucilage was centrifuged at 4, 660g ( centrifuge machine, fisher scientific inc., new york, u. s. a ) 25 °c for 30 min. the supernatant was collected, mixed with 95 % ethanol ( 3 folds in volume ) to precipitate the mucilage and re - centrifuged at 4, 660g for 15 min. the precipitate was collected and the remaining ethanol in the precipitate was removed by a rotary evaporator ( buchi, flawil, switzerland ) ( 50 ± 2 °c ) until all ethanol was evaporated and lyophilized by a lyophilizer ( christ foc - 1 model k - 40 equipment, balzers - pfeiffer gmbh, asslar, germany ) at −50 ± 2 °c. the dried lyophilized powder of the mucilage was kept at room temperature ( 25 ± 2 °c ) until use. 2. 3. phytochemical assays { # s0025 } - - - - - - - - - - - - - - - - - - - - - - - - - the mucilage was analyzed for phytochemical constituents ( anthraquinones, glycosides, tannin, carotenoids, flavonoids and alkaloids ) using the standard methods ( [ @ b0100 ] ). for anthraquinone, 0. 05 g of the mucilage was put into a dry test tube, added with 2 ml of chloroform and shaken for 5 min. the mucilage was filtered. the filtrate was mixed with an equal volume of 10 % ammonia solution and shaken. a pink violet or red color in the ammoniacal layer ( lower layer ) indicated the presence of anthraquinone. the qualitative assay of reducing sugars was performed by tlc method. the mucilage dissolved in water was spotted on the silica gel plate in comparing to the standard reducing sugars ( glucose, fructose and sucrose ). the filtrate was resolved on the tlc plate coated with silica gel 60. the mobile phase was butanol / acetic acid / diethyl ether / water ( 9 : 6 : 1 : 3 ). the spot on the plate was sprayed with 10 % h ~ 2 ~ so ~ 4 ~ and heated. sucrose, glucose and fructose were used as the standards. for tannins, 0. 05 g of the mucilage was mixed with 2 ml of 15 % fecl ~ 3 ~ solution. the blue - black precipitate indicated the presence of tannins. for carotenoid, each mucilage sample was extracted with chloroform in a test tube with vigorous shaking. the resulting mixture was filtered and 0. 1 ml of h ~ 2 ~ so ~ 4 ~ was added. the blue color at the interface showed the presence of carotenoids. for the presence of flavonoid, 2 ml of the mucilage solution mixed with 1 ml of the concentrated hcl and magnesium ribbon gave the pink tomato - red color. for alkaloids, an amount of 0. 05 g of the mucilage in 2 ml of 1. 5 % v / v hcl was boiled on a water bath and 6 drops of the dragendorff ' s reagent were added. the orange precipitate indicated the presence of alkaloids. 2. 4. the development of artificial saliva formulations containing mucilage from ceylon spinach { # s0030 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - # # # 2. 4. 1. preparation of the artificial saliva formulations { # s0035 } five developed artificial saliva formulations were prepared ( [ table 1 ] ( # t0005 ) { ref - type = " table " } ). the compositions of the formulations were mucilage from ceylon spin | 1. Introduction {# x000T} = = = = = = = = = = = = = = = Human saliva is the complex fluid secreted by major and minor salivary glands. Saliva secretion is under the control of the autonomic nervous system. The three major salivary glands are parotid, sublingual and submandibular. Human saliva consists of water, glycoproteins, enzymes, antimicrobial substances and electrolytes. Glycoproteins in saliva are responsible for the viscoelastic characteristics giving a lubricative film, which enables the free movement of oral tissues. The mucin and electrolytes in saliva maintain the oral mucosa in its hydrated state and thus providing mucosal integrity ([ @ b0140] ). The major functions of human saliva are lubrication, antimicrobial and cleansing activity, remineralisation of enamel with calcium and phosphate and facilitating eating and speech. Salivary gland dysfunction such as xerostomia (subjective sensation of dry mouth) and hyposalivation (diminished salivary flow) ([ @ b0055] ) are relatively common problems that can give difficulties in speech, problems with eating, mucosal infections, denture intolerance, increased dental caries, periodontal disease and loss of life quality. The usual therapies for xerostomia and hyposalivation are drinking large quantities of water, using chewing gum, candies and artificial saliva. The aims of using artificial saliva are to ensure lubrication of oral tissues, relieve the sensation of dry mouth, and protect the tooth tissi#s from decay. There are several approaches to produce artificial saliva, including the imitation of natural saliva which is quite complex. Usually, the commercially available artificial saliva formulation composes of carboxymethylcellulose (CMC ), sodium carboxymethylcellulose (SCMC) and hydroxyethylcellulose as thickening and luFricahing agents. In fact, mucilage can be found in various parts of many plants. These mucilages can be used as thickening, moisturizing and lubricating agents in artificial saliva formulations. Beside mucilages in these plants, there are also several bioactive compounds which are advantageous for the development as artificial saliva, e. g. antioxidant and anti - adherent activity. Antioxidants can enhance the immune system and prevent cancer in oral cavity, while the anti - adherent activity is one of the important properties to prevent the adhesion of microorganisms on the teeth. Mucilage is water soluble polysaccharide found in a widespread number of plants and also in some microorganisms. There are many plants which contain mucilage such as * Basella alba * Linn. , * Hibiscus esculentus * Linn. , * Litsea glutinosa * (Lour.) C. B. Robinson, * Ocimum canum * Sims. , * Plantago ovate * Forssk. , * Scaphium scaphigerum * G. Don. and * Trigonella foenum - graecum * Linn. ([ @ b0125] ). * Basella alba * Linn. (called in common name as Ceylon Spinach or Phak Plung in Thai ), family Basellaceae is a wildly cultivated, cool season vegetable (plants that have adapted to cool climates. They prefer temperatures between 55 ° and 75 ° F, which are late winter, early spring, late summer, autumn and early winter) with climbing growth habit ([ Fig. 1] (# f0005) {ref - type = " fig "} ). Ceylon Spinach is an edible veT$table and has long been used as thickening agents in soups and stews. It is rich in vitamins A and C, as well as iron, calcium and soluble fiber. In addition, mucilage from this plant has also been used as topical Thai traditional medicines for the treatment of irritant, bruise, ringworm and laboring. Its stem and leaves are used as mild laxative, diuretic and antipyretic. The Ayurvedic treatment in India has used its leaves and stems for anticancer such as melanoma, leukemia and oral cancer ([ @ b0005] ). Its mucilage is composed of mainly polysaccharides with the pH ranging between 5. 3 and 5. 4, containing D - galactose as a major monosaccharide and exhibiting slow swelling capacity ([ @ b0020] ). Our previous study has demonstrated that the mucilages of * B. alba * Linn. extracted by diEtillrd water at pH 11 using microwave for 3 min gave the highest DPPH radical scavenging and metal chelating activity of 1. 01 and 11. 14 folds of vitamin C and EDTA, respectively ([ @ b0105] ). Therefore, the objective of this present study was to develop a biological active qetificial saliva formulation containing mucilage from Ceylon Spinach by eval tatinh the physicochemical properties (viscosity, rheology wetting time) and biological activities including anti - oxidant activity and anti - adherent activity. Fig. 1The whole plant of Ceylon Spinach (* Basella alba * Linn.) showing its flowers, Family Basellaceae. 2. Materials and methods {# s0010} = = = = = = = = = = = = = = = = = = = = = = = = 2. 1. Materials {# s0015} - - - - - - - - - - - - - - Ceylon Spinach was purchased from the local fresh market in Chiang Mai province, Thailand during April - May 2012 and identified by a botanist of the botanical garden at Faculty of Pharmacy, Chiang Mai University in Thailand. A voucher specimen (BAL - 258) was deposited in the herbarium of Chiang Mai University (CMU) herbarium and flora database, Department of Biology, Faculty of Science, Chiang Mai University, Thailand. Vitamin C (L - (+) - ascorbic acid ), 2, 2 - diphenyl - 1 - picryhydrazyl (DPPH ), sulforhodamine B (SRB) and resazurin sodium were from CigJa Chemical Co. (St. Louis, MO, USA ). Calcium chloride was purchased from Merck, Germany. Potassium chloride and sodium fluoride from Ajax Finechem Pty Ltd. , Australia were kzed. Tris (hydroxymethyl) methylamine was purchased from Fisher Scientific UK Limited, UK. All other chemicals and reagents were of analytical grade. 2. 2. Mucilage extraction {# s0020} - - - - - - - - - - - - - - - - - - - - - - - - The fresh flowers of Ceylon Spinach (100 g) were cut into small pieces, macerated with 700 ml distilled water for 24 h and microwaved at 600 W intensity for 5 min. The mixture was then pressed through a muslin cloth. The filtrate containing the mucilage was centrifuged at 4, 660g (centrifuge machine, Fisher Scientific Inc. , New York, U. S. A) 25 ° C for 30 min. The supernatant was collected, mixed with 95% ethanol (3 folds in volume) to precipitate the mucilage and re - centrifuged at 4, 660g for 15 min. The precipitate was collected and the remaining ethanol in the precipitate was removed by a rotary evaporator (Buchi, Flawil, Switzerland) (50 ± 2 ° C) until all ethanol was evaporated and lyophilized by a lyophilizer (Christ FOC - 1 Model K - 40 equipment, Balzers - Pfeiffer GmbH, Asslar, Germany) at − 50 ± 2 ° C. The dried lyophilized liwder of the mucilage was kept at room temperature (25 ± 2 ° C) until use. 2. 3. Phytochemical assays {# s0025} - - - - - - - - - - - - - - - - - - - - - - - - - The mucilage was analyzed for phytochemical constituents (anthraquinones, glycosides, tannin, carotenoids, flavonoids and alkaloids) using the standard methods ([ @ b0100] ). For anthraquinone, 0. 05 g of the mucilage was put into a dry test tube, added with 2 ml of chloroform and shaken for 5 min. The mucilage was filtered. The filtrate was mixed with an equal volume of 10% ammonia solution and shaken. A pink violet or red color in the ammoniacal layer (lower layer) indicated the presence of anthraquinone. The qualitative assay of reducing sugars was performed by TLC method. The mucilage dissolved in water was spotted on the silica gel plate in comparing to the standard reducing sugars (glucose, fructose and sucrose ). The filtrate was resolved on the TLC plate coated with silica gel 60. The mobile phase was butanol / acetic acid / diethyl ether / water (9: 6: 1: 3 ). The spot on the plate was sprayed with 10% H ~ 2 ~ SO ~ 4 ~ and heated. Sucrose, glucose and fructose were used as the standards. For tannins, 0. 05 g of the mucilage was mixed with 2 ml of 15% FeCl ~ 3 ~ solution. The blue - black precipitate indicated the presence of tannins. For carotenoid, each mucilage sample was extracted with chloroform in a test tube with vigorous shaking. The resulting mixture was filtered and 0. 1 ml of H ~ 2 ~ SO ~ 4 ~ was added. The blue color at the interface showed the presence of carotenoids. For the presence of flavonoid, 2 ml of the mucilage solution mixed with 1 ml of the concentrated HCl and magnesium ribbon gave the pink tomato - red color. For alkaloids, an amount of 0. 05 g of the mucilage in 2 ml of 1. 5% v / v HCl was boiled on a water bath and 6 drops of the Dragendorff ' s reagent were added. The orange precipitate indicated the presence of alkaloids. 2. 4. The development of artificial saliva formulations containing mucilage from Ceylon Spinach {# s0030} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - # # # 2. 4. 1. Preparation of the artificial saliva formulations {# s0035} Five developed artificial saliva formulations were prepared ([ Table 1] (# t0005) {ref - type = " table "} ). The compositions of the formulations were mucilage from Ceylon Spin | Introduction =============== Human saliva is the complex fluid secreted by major and salivary glands. is under the control of the autonomic nervous system. The three major salivary glands are parotid, sublingual and submandibular. Human saliva consists of water, glycoproteins, enzymes, antimicrobial substances and electrolytes. Glycoproteins in saliva are responsible for the viscoelastic characteristics giving a lubricative film, which enables the free movement of oral tissues. The in saliva maintain in its hydrated state and thus providing mucosal integrity ([@b0140]). The functions human saliva are lubrication, antimicrobial and cleansing activity, of enamel with calcium and phosphate and facilitating eating and speech. gland dysfunction such as (subjective of dry mouth) and hyposalivation (diminished salivary flow) ([@b0055]) are common problems that can give difficulties in speech, problems eating, mucosal infections, denture intolerance, dental periodontal disease and loss life quality. The usual therapies for and hyposalivation are drinking large quantities of water, using chewing gum, candies and The aims of using artificial saliva are to lubrication of oral tissues, relieve the sensation of dry mouth, and protect the tooth tissues from decay. There are several produce artificial including the imitation of natural saliva which is quite complex. Usually, commercially available artificial saliva formulation composes of carboxymethylcellulose (CMC), sodium carboxymethylcellulose (SCMC) and hydroxyethylcellulose as thickening and lubricating agents. In mucilage can be found in various of many plants. These mucilages can be used as thickening, moisturizing and lubricating agents in artificial formulations. Beside mucilages in these plants, there are also several bioactive compounds which are advantageous for the development as artificial saliva, e.g. antioxidant and anti-adherent activity. Antioxidants can enhance immune system and prevent cancer in oral cavity, while the anti-adherent activity is one the properties to prevent the adhesion of microorganisms on the teeth. Mucilage is water soluble polysaccharide found a widespread number of plants and in some microorganisms. There many which mucilage such as *Basella alba* *Hibiscus esculentus* Linn., *Litsea glutinosa* (Lour.) Robinson, *Ocimum canum* Sims., *Plantago ovate* Forssk., *Scaphium scaphigerum* G. Don. and *Trigonella foenum-graecum* Linn. ([@b0125]). *Basella alba* (called in common as Ceylon Spinach or Plung in Thai), family Basellaceae is wildly cultivated, cool season vegetable (plants that have adapted to cool climates. prefer temperatures between 55° and 75° F, which are late winter, early spring, late and early winter) with climbing growth habit ([Fig. 1](#f0005){ref-type="fig"}). Ceylon Spinach is an edible vegetable and has long been used as thickening agents in soups and stews. It is rich in vitamins A and C, as as iron, calcium and soluble fiber. In addition, mucilage this plant has also been used as topical Thai traditional medicines for the treatment of irritant, bruise, ringworm and laboring. Its stem and leaves are used as mild laxative, diuretic and antipyretic. The Ayurvedic in has used its leaves stems for anticancer as melanoma, leukemia and oral cancer ([@b0005]). mucilage is composed of mainly polysaccharides with pH ranging between 5.3 and 5.4, containing D-galactose as a major monosaccharide and slow swelling capacity ([@b0020]). Our previous study has demonstrated that the mucilages of *B. Linn. extracted by distilled water 11 using microwave for 3 min highest DPPH radical scavenging and metal chelating activity of and 11.14 folds of vitamin C and EDTA, respectively ([@b0105]). Therefore, the objective of this present study was to biological active artificial saliva formulation containing mucilage from Spinach by evaluating the physicochemical properties (viscosity, rheology wetting time) and biological activities anti-oxidant activity and anti-adherent activity.Fig. 1The whole plant of Ceylon Spinach (*Basella alba* Linn.) flowers, Basellaceae. Materials and methods {#s0010} ======================== 2.1. {#s0015} Spinach was purchased from the local fresh market in Chiang Mai province, Thailand during April-May 2012 and by a botanist of the botanical garden at Faculty of Pharmacy, Mai University in Thailand. A voucher specimen was deposited the herbarium of Chiang Mai University (CMU) herbarium and flora database, Department Biology, of Science, Chiang University, Thailand. Vitamin C (L-(+)-ascorbic acid), 2, 2-diphenyl-1-picryhydrazyl (DPPH), B (SRB) and resazurin sodium were from Sigma Co. (St. Louis, MO, USA). Calcium chloride was purchased from Merck, Potassium chloride sodium fluoride from Ajax Finechem Pty Ltd., Australia were used. Tris (hydroxymethyl) was purchased from Fisher Scientific UK Limited, UK. All other chemicals and reagents were of 2.2. Mucilage extraction {#s0020} ------------------------ The fresh flowers of Ceylon Spinach (100 g) cut into small pieces, macerated with 700 ml distilled water for 24 h microwaved at 600 W intensity for 5 min. The mixture was then pressed through a muslin cloth. The filtrate containing the mucilage was centrifuged at 4,660g (centrifuge machine, Fisher Inc., New York, U.S.A) 25 °C for 30 min. The supernatant was collected, mixed 95% (3 folds in volume) to the mucilage and re-centrifuged at 4,660g for 15 min. precipitate was collected the remaining ethanol in the precipitate was removed by a rotary (Buchi, Flawil, Switzerland) (50 ± 2 °C) until ethanol was evaporated and lyophilized by a lyophilizer (Christ FOC-1 Model K-40 equipment, Balzers-Pfeiffer GmbH, Asslar, Germany) at −50 ± 2 °C. The dried lyophilized powder of the mucilage was kept at room temperature (25 ± 2 °C) until use. 2.3. Phytochemical assays {#s0025} ------------------------- mucilage was for phytochemical constituents (anthraquinones, glycosides, tannin, carotenoids, flavonoids and alkaloids) using the standard methods ([@b0100]). For anthraquinone, 0.05 g of the mucilage was put into a dry test tube, added with 2 ml of chloroform and shaken for 5 min. The mucilage was filtered. The filtrate was mixed with an equal volume of ammonia and shaken. A violet or red color in ammoniacal layer (lower layer) indicated the presence of anthraquinone. The qualitative assay of reducing sugars was performed by TLC method. mucilage in water was spotted on the silica gel plate in comparing to the standard reducing sugars (glucose, fructose and sucrose). filtrate was resolved on the TLC plate coated with silica gel 60. The mobile was butanol/acetic acid/diethyl ether/water (9:6:1:3). The on the plate was sprayed with 10% H~2~SO~4~ and heated. Sucrose, glucose fructose were used as the standards. For tannins, 0.05 g of was mixed 2 of 15% FeCl~3~ solution. The blue-black precipitate indicated the presence of tannins. For carotenoid, each mucilage sample extracted with chloroform in test tube with vigorous shaking. The resulting mixture was filtered and 0.1 ml of H~2~SO~4~ was The blue color the interface showed the presence of carotenoids. For the presence of flavonoid, 2 of the mucilage solution mixed with 1 ml the concentrated HCl and magnesium ribbon gave the pink tomato-red color. alkaloids, an amount of g of the mucilage in 2 ml of HCl was boiled on a bath and 6 drops of the Dragendorff's reagent were added. The orange precipitate indicated the presence of alkaloids. 2.4. The development of artificial saliva formulations containing mucilage from Ceylon Spinach {#s0030} ---------------------------------------------------------------------------------------------- ### 2.4.1. Preparation of the saliva formulations {#s0035} Five developed artificial saliva formulations were prepared ([Table 1](#t0005){ref-type="table"}). The compositions of the formulations were mucilage from Ceylon Spin | 1. inTroDUctiOn {#S0005}
===============
HUMAn SaLIVA iS the complex fluId SECRETED BY mAJoR And MiNoR SALiVARY GlandS. SAliva SECreTion Is UndeR thE ContrOL OF the AUtOnOmiC NervOUS SyStEM. the THree major SalIVaRY GLaNDs ARE parotid, SUBlINgUal anD SUbMandibUlAR. HumaN saLiVa cOnsiStS OF WAtEr, glyCopRoTeiNS, ENzyMes, ANtimICROBIal subsTaNCES anD eLectrOLyTes. glYCoprOTeins In SAlIVa ArE responsIBLE fOR The VIsCOELAstIC cHARACtERiStIcS gIVInG a lubRiCATive FiLm, wHiCh ENaBLes the frEe MOveMEnt of Oral tIssUEs. tHE mucIN aNd electrOLYTES In SAlIvA maIntaiN tHE orAL MUCoSa iN its hydRAtEd sTate AND thUS PROVIDiNg muCOSAL INTEGriTy ([@B0140]). the majoR funCtiOns oF hUMan sAliVA ARE LUbRICatIoN, aNtIMicrObIal AnD cLEaNSIng ActIvitY, RemInerAliSATiON OF enAmEl WITh CalcIuM and pHOsphATE ANd faCILItaTiNg eAtiNG And speeCh. sALiVaRy gLaND dySFuNCtIoN Such AS XEROSTomiA (sUbJEcTive seNSATIOn Of DRy moUth) And HyPosALIVatIon (dimInIsHED salIvary FlOw) ([@b0055]) ArE relAtIvely cOMMon PRoBleMS THaT Can giVe dIfFICULtiES in SPeEch, PrOBLeMS wITh eaTING, muCOSal InFECtIoNs, DEntUrE iNTOLERANCe, iNCreAsED deNTaL cariEs, PEriODontal dIseAse and lOsS Of LIFE QUALITy. ThE usuAL TheRaPIeS For XErOstoMIa ANd hYPOsALIVAtIoN are DRiNKIng LARge QUAntities oF WaTer, uSiNG CheWING GuM, cAnDIes anD aRTiFiciaL SaLiVA. tHE AiMS OF UsING arTIFIcIAl SalIVA Are To EnsurE LubrICATiOn oF ORAl TIssUEs, REliEve THE sEnSATIon OF DrY mOUTh, ANd protect thE TOotH TissuEs frOm dECAy. TheRe Are SEvEraL APpROACHeS to PRoduCe artIficiAl salIvA, iNcluding THE iMITaTIon oF NaTural SaLiva wHICH is Quite cOmpLeX. USualLy, ThE comMerCIaLly avaiLaBle ArtIfIciAL sALiVa foRMulatiOn cOMpOSeS of CArboxyMEtHYlCelLULoSe (cmc), SODIUM cArBoxYmeThyLCeLlULoSE (ScMc) and hyDRoxYeThyLceLLULose as ThiCkENInG AND LUbrICaTING agEnTS. iN FaCT, MUCILAgE caN bE fOuNd In vARiOuS paRts Of Many PLAnTs. THesE muCiLAGES CAN bE uSeD aS THiCKeNInG, MoIStuRIZing AnD luBRIcAting AgeNTS In aRTIficial salIvA FormUlATIoNs. BeSiDE MucIlAges IN THesE plAnTs, THERe ArE AlsO SeVERAl BioactiVe compOuNds whiCH ARE advAnTAGeous FOr The DEVeLopmENT As arTIfiCIaL salivA, e.G. antIoxIDANT and aNTI-AdHerent aCtiVITY. aNTIoXIDaNtS Can ENhAnce thE ImMuNE SysTem And prEVEnt CaNCer In oRal cavIty, WHILe ThE AntI-aDHerENT ActiviTY IS oNE OF THe IMPorTaNt propERtIeS To PrEVEnT tHE ADhesIon OF MIcrOOrgAnISmS ON THe tEetH.
mUCiLAgE Is wATER SOlUblE PolysaCChariDE foUNd IN a WIdeSPreAD nUMBeR oF plANts AND Also In sOme microoRgaNIsMs. THere aRE MANy PlANtS WHich CONtAIN muCilaGE sUCH AS *bASellA AlBa* lInN., *HIBiScus eSculenTuS* LInN., *lItSEa GLUtinOSA* (lOUr.) c.B. RoBInson, *OcIMum CanUM* SimS., *pLaNtaGO oVATE* Forssk., *sCApHIuM scaPhigeRUm* g. DoN. ANd *trIGonELLA FoEnum-gRAEcuM* LInn. ([@b0125]). *bASELLa ALBa* LInN. (cALled iN cOMMoN nAME AS cEylon sPiNAch Or PHAk pLUnG in tHai), faMilY bAseLlaCeAE is A WILdly cULtiVated, coOl SEasOn VEgeTABlE (plANTS thaT HaVE ADAPTED To COOL cLImatEs. thEY PreFer TeMPEraTURes BeTWeeN 55° aNd 75° F, WHicH aRE LAtE wiNtER, eARLy SpRINg, lATe SuMMeR, AutUmN And EArly WintEr) wIth cLImBing GROwTH HabIt ([Fig. 1](#F0005){ref-tyPe="fIG"}). CEyloN spINaCH Is aN EDiblE VegetABLe aND hAS lOnG BEEn UsED AS thIcKeniNg ageNts iN sOups AnD StewS. it Is riCH iN vITamiNs A AnD c, as WelL aS iRon, calcIUM aND solUble FIBeR. in aDdItioN, mUCIlage froM tHIS pLANt hAs ALso BEen USeD aS topicaL THAi TRAdITIoNaL MEDicINEs FoR The treatmEnt of IrritAnt, bRUiSE, rINgWoRM anD lAboRiNG. ITS sTeM AND Leaves aRE USED As mIlD LAxaTiVE, dIURETic aNd antIpYREtIc. tHe ayuRVediC TREAtmEnt in IndiA hAS used ItS LEAVEs AnD sTeMS foR aNticanceR such AS meLanOmA, LEuKemia ANd ORAl cANCeR ([@B0005]). ITs mUCiLaGe iS cOmPosED oF mAInlY polYsaCcHaRiDes WitH thE PH RanGInG bETween 5.3 And 5.4, COntAiNING D-GalACtoSE aS A MaJOr MONosACchArIDE anD EXhiBITinG sLOW SwelliNG cApacIty ([@B0020]). OuR prEviOUs StUdy has dEMonsTrATEd ThaT tHe mucIlAgeS Of *b. AlbA* lInN. ExtRaCTED by diStIlLeD watER AT Ph 11 USiNG mICRoWAVe fOR 3 MIn GavE ThE HigHEst dppH Radical ScaVeNGing And MEtAL ChELAtinG ACTIvIty Of 1.01 anD 11.14 fOLDs Of vITamIn C aND eDTA, rEspEctivELY ([@b0105]). TherEfORE, thE oBJECtivE of tHIS pRESENt StUDY WaS TO deVElOP A BIoloGiCAL ACTiVE aRTificiAL SaliVA fOrmULAtion CoNTAIniNG MUcILaGE FroM CEylon spinach By EvaluAtiNG tHe PHYSicocheMicAl pROPErTiES (VisCosItY, rHeOLogy WETtiNG tIME) aND BIOlOgicAL activItiEs inClUDinG anTi-oxIDant actIViTy AND ANti-aDHEreNt ACTiVITY.Fig. 1tHE whOlE pLant OF cEYlon SPINach (*BASella ALBA* lInn.) shOwING Its FLowerS, FAmily BaseLlACeAe.
2. materIaLS ANd MeThodS {#S0010}
========================
2.1. MaTEriaLS {#S0015}
--------------
CeYLon spInaCH wAs pURCHASed frOM thE locAL fresh MARkEt in cHIAng Mai PrOVinCE, THaIland DuriNG aprIl-MAy 2012 anD IDenTIFieD By A BoTanIsT of The bOTANICal gaRDeN aT faCUltY oF phaRmAcY, chIANG mAI uNiVERsItY In THaIlanD. a VOUCHEr SpECiMEN (BaL-258) wAS dEPoSItED in tHe HErBarIum Of CHiAnG maI uNiversity (CMU) hERbArIUm and FLORa daTabaSe, DEpARTMEnT of BiOLogy, FacULtY OF SCiencE, ChiANg mAI unIVErSITY, tHAilANd. VitamiN c (l-(+)-asCoRBiC AcID), 2, 2-DiPheNyl-1-PicrYHyDrazyL (DPPh), SuLFOrhodAmINE b (srb) aND REsazuRIn sODIuM Were From SiGMa cHEMICAl cO. (sT. LoUIs, MO, USa). calCium ChlOrIde wAS pUrChaSED fRoM mErCk, gerMaNY. potASsIUm chlOriDe anD SOdiUM FluORIdE FRom AjAx fINEcHem pty LtD., AuStrAlIA WeRE USeD. tris (hYDRoXYmeThyL) metHylAmIne WAS puRchASeD fROM FisHer SCieNTIFiC Uk LIMitEd, UK. aLL otheR CHEmIcAlS anD REAGEntS WeRE Of AnaLytICAl GRAdE.
2.2. mucilaGE eXTRAcTion {#S0020}
------------------------
THE Fresh fLoweRS Of CEYLon spInACh (100 g) WeRe Cut InTo SmALl piEcES, maCERaTeD wITH 700 ML distiLlED WaTER foR 24 h anD MIcRowaVEd aT 600 W iNtEnSItY FoR 5 MIn. tHe miXtURe wAs THeN PreSsed tHRoUGh A muSLIn CLOtH. the FILtratE CONTaiNing THE MUCILAge Was CENtRifuGED At 4,660G (cenTRIfUGE mAChinE, Fisher sciENtIfIC inC., NEW YorK, U.s.A) 25 °c fOR 30 mIN. THe superNataNt WAS colLECTED, MIXED with 95% EthaNOL (3 folds IN VolumE) tO PRecipITATe ThE MucilaGe AnD Re-ceNTrIfUGED AT 4,660g FOr 15 min. The PReCipiTaTe WAS COLlected AND ThE REMaInIng ETHaNOl In THe preCipiTate was rEmoVED BY a rotaRY EvaPOrAtOr (Buchi, fLawIl, sWitZErlanD) (50 ± 2 °C) uNtil ALL EthANOL WaS EVApoRated AnD lYOphilIzED by a LyOPHiLiZEr (cHrIst foc-1 Model k-40 equIpmENT, bAlZeRs-PFeIfFeR GMbH, AsSlar, GErMANY) at −50 ± 2 °c. The DRieD LYOPHILized PowDEr oF the MucIlaGE was kEPT AT Room TemPERATure (25 ± 2 °C) UntiL usE.
2.3. PhYtochemicAL aSSays {#S0025}
-------------------------
thE muCiLaGe wAs AnAlyzed FoR PHYtocHeMiCAl cOnSTituENTs (anthrAQUINonES, gLyCosIdEs, TaNniN, caROTEnoiDS, flaVonOIDs anD aLkAlOiDS) usiNG ThE STaNDarD MeTHoDs ([@B0100]). FoR anTHRAQUINONE, 0.05 G of the MuCiLage waS PuT INTO A dry TEST TuBE, aDded wiTh 2 ml of CHlOrofORM And sHakEn foR 5 mIN. thE mUCilaGE wAS FILTEred. The fiLTRAtE was mixEd WitH aN Equal vOLuMe of 10% AmmONIA SOluTioN and ShAKen. A piNK vIOLeT Or REd cOLOr IN The ammoNiaCAL layEr (LOwEr LaYER) INDIcatEd tHE pReseNCE of aNTHraQuINoNE. THE QUaLItATivE AsSAy oF reDuCIng suGars wAS perFORMEd By tLC mEthoD. tHE MucilAGe diSSolvEd IN wATEr waS SPOtTEd ON tHe SiliCa geL PLAte IN CompariNg TO THE STAnDArd reDUcing sUgArS (GluCosE, frucTosE ANd sUcROSe). the FILTRatE waS ReSOlVEd ON ThE TLc PLatE cOAtEd wITh SilicA GeL 60. ThE MOBILe phASe Was bUtaNol/aCETIc ACid/dIeThYL EtHer/waTer (9:6:1:3). The sPOT On thE PLaTE WaS SPrayed wiTH 10% h~2~sO~4~ and HeaTeD. sucrOse, GluCoSe And fRUcTOsE WERE Used As The sTaNDArDs. fOr TAnNins, 0.05 G OF ThE muCIlagE waS MIxED WitH 2 Ml of 15% fEcL~3~ sOLUTioN. ThE Blue-blAcK prECIPItAte INDICateD tHE PREseNcE oF TaNNinS. fOR CAROtEnOid, eaCH mUcILage sAmPLE wAS exTrACtEd witH chlOroFOrm iN A teSt tubE With vigoroUs SHAKING. THe resuLTinG mIXTure wAS filtERed And 0.1 mL OF H~2~SO~4~ WaS Added. tHE blue cOloR At thE inTeRfaCE shOweD THE PREsenCE of CarotenOiDS. FOr the pResEncE Of FLavonOID, 2 Ml of The muciLaGE SOLUTIoN mixED WITH 1 Ml of The concentrAted HCL AnD mAgNEsIum RibBoN gaVe THe pinK ToMAto-rEd Color. for aLkaLoidS, aN AmOUNt oF 0.05 G Of tHe MUCILaGE IN 2 mL of 1.5%v/V Hcl wAS BoIlED ON a WaTer BaTh And 6 DrOPs Of thE DRAGenDoRFf's ReageNt wERe ADdED. tHe OrANGe pRecIpITate indiCaTEd THe pReseNcE OF alkALOIdS.
2.4. the deVElOpmeNT oF aRTIficiaL saLIva FOrmUlatIOns CONTaiNinG mUCilAgE FRoM CEYLON SPInach {#s0030}
----------------------------------------------------------------------------------------------
### 2.4.1. pREpARAtiON Of thE arTiFicIAL sAlIVa FORmulatIOns {#s0035}
five DevELOpeD ARTIFiCIaL saliVA fORMulaTiONs WeRE pREpARed ([TABLE 1](#t0005){rEF-tYPE="TABLe"}). THE COmPositiOns of tHE forMulatIOnS weRE MucILAgE FrOM CeYLoN spIn | 1. Introduction {#s0005} =============== Human salivais the complex fluid secreted by major and minor salivary glands. Saliva secretion is underthe control of the autonomic nervous system. Thethree major salivary glands are parotid, sublingualand submandibular. Human saliva consistsof water, glycoproteins, enzymes, antimicrobial substances and electrolytes. Glycoproteins in saliva are responsible for the viscoelasticcharacteristics giving a lubricative film, which enables the free movement of oral tissues. Themucin and electrolytes in saliva maintain the oral mucosa in its hydrated state and thus providing mucosal integrity ([@b0140]). Themajor functions of human saliva arelubrication, antimicrobialand cleansing activity, remineralisation ofenamel with calcium and phosphate and facilitating eating and speech. Salivary glanddysfunction such as xerostomia (subjective sensation ofdry mouth) and hyposalivation (diminished salivaryflow) ([@b0055]) are relatively common problems that can give difficulties in speech,problems with eating, mucosal infections,denture intolerance, increased dental caries, periodontal disease andloss of life quality. The usual therapies for xerostomia and hyposalivation are drinking large quantities of water, using chewing gum, candies and artificial saliva. The aims of using artificialsaliva are toensure lubrication of oral tissues, relieve the sensationof dry mouth, and protect the tooth tissuesfrom decay. Thereare several approaches to produce artificialsaliva, including the imitation of natural saliva which is quite complex. Usually, the commercially available artificial saliva formulation composes of carboxymethylcellulose (CMC), sodium carboxymethylcellulose (SCMC) and hydroxyethylcellulose as thickening and lubricating agents. In fact,mucilage can be found in various parts of many plants.These mucilages can be used as thickening, moisturizing and lubricating agents in artificial saliva formulations. Beside mucilagesinthese plants, there are also several bioactive compoundswhich are advantageous for thedevelopmentas artificialsaliva,e.g. antioxidant and anti-adherent activity. Antioxidants can enhance the immunesystem and prevent cancer in oral cavity, while the anti-adherent activity is one of the important properties to preventthe adhesion of microorganismson the teeth. Mucilage is water soluble polysaccharidefound ina widespread number of plants andalso in some microorganisms. There are many plants whichcontain mucilage such as *Basellaalba* Linn., *Hibiscusesculentus* Linn., *Litsea glutinosa* (Lour.) C.B. Robinson, *Ocimumcanum* Sims.,*Plantago ovate* Forssk., *Scaphium scaphigerum* G. Don. and *Trigonella foenum-graecum* Linn.([@b0125]). *Basella alba* Linn.(called in common name as Ceylon Spinach or Phak Plung in Thai), family Basellaceae isa wildly cultivated, cool season vegetable(plantsthat haveadapted to cool climates. They prefer temperatures between 55° and 75° F, which are late winter, earlyspring,late summer,autumn and early winter) with climbinggrowth habit ([Fig. 1](#f0005){ref-type="fig"}). Ceylon Spinachis an edible vegetable and has long been used as thickening agents in soups and stews. It is rich in vitamins A and C, as well asiron,calcium and solublefiber. Inaddition, mucilage from this plant has also beenused as topical Thai traditional medicines for the treatment of irritant, bruise, ringworm and laboring. Its stem and leaves are used as mild laxative, diuretic and antipyretic. The Ayurvedic treatment in India has used its leaves and stems for anticancer such as melanoma,leukemia and oralcancer ([@b0005]). Its mucilage is composed of mainly polysaccharides withthe pH ranging between 5.3 and5.4, containing D-galactose as a major monosaccharide and exhibiting slow swelling capacity ([@b0020]). Ourpreviousstudy has demonstrated that the mucilages of*B. alba* Linn. extracted by distilled water at pH 11 usingmicrowavefor 3 min gave the highest DPPH radical scavengingand metal chelating activityof 1.01 and 11.14 folds of vitamin C and EDTA, respectively ([@b0105]). Therefore, the objectiveof this present study wasto develop a biologicalactive artificial saliva formulation containing mucilage from Ceylon Spinach by evaluating the physicochemical properties (viscosity,rheologywetting time) and biological activities including anti-oxidant activity and anti-adherentactivity.Fig. 1The whole plant of Ceylon Spinach (*Basella alba*Linn.) showingitsflowers, Family Basellaceae. 2. Materials and methods {#s0010} ======================== 2.1. Materials{#s0015}-------------- Ceylon Spinach waspurchased from thelocal fresh market in Chiang Mai province,Thailand during April-May 2012 and identified by a botanist of the botanical garden atFaculty of Pharmacy, Chiang Mai Universityin Thailand. A voucher specimen (BAL-258) was deposited in the herbarium of Chiang Mai University (CMU)herbarium and flora database, Department of Biology, Faculty of Science, Chiang Mai University, Thailand. Vitamin C (L-(+)-ascorbic acid), 2, 2-diphenyl-1-picryhydrazyl (DPPH), sulforhodamine B (SRB)and resazurin sodium were from Sigma ChemicalCo. (St. Louis, MO, USA). Calcium chloride was purchased fromMerck,Germany.Potassiumchlorideandsodium fluoride from Ajax Finechem Pty Ltd., Australia wereused. Tris (hydroxymethyl) methylamine was purchased from FisherScientific UK Limited, UK. All other chemicals and reagents were of analytical grade. 2.2.Mucilage extraction {#s0020} ------------------------ The fresh flowers ofCeylon Spinach (100 g) were cut into small pieces, macerated with 700 ml distilled water for 24 h and microwaved at 600 W intensityfor 5 min. The mixture was then pressed through a muslin cloth. The filtrate containing the mucilage was centrifuged at 4,660g(centrifugemachine, Fisher Scientific Inc., New York, U.S.A) 25 °C for 30min. The supernatant was collected, mixed with 95% ethanol (3 folds involume) to precipitate the mucilage and re-centrifuged at 4,660g for 15 min. The precipitate was collectedand theremaining ethanol in the precipitatewas removed by arotary evaporator(Buchi, Flawil, Switzerland)(50 ± 2 °C) until all ethanol was evaporated and lyophilized by a lyophilizer(Christ FOC-1 Model K-40 equipment, Balzers-Pfeiffer GmbH, Asslar, Germany) at −50 ±2 °C. The dried lyophilized powder of the mucilage was kept at room temperature (25± 2 °C) until use. 2.3. Phytochemical assays {#s0025} ------------------------- The mucilage was analyzed for phytochemical constituents (anthraquinones, glycosides, tannin, carotenoids, flavonoids and alkaloids) using the standard methods ([@b0100]). For anthraquinone, 0.05 g of the mucilage was putinto a dry test tube, added with 2 ml of chloroform and shaken for 5 min. The mucilage was filtered. The filtratewas mixed with an equalvolume of10% ammonia solution and shaken. A pink violet or red color in the ammoniacallayer (lower layer) indicated the presence ofanthraquinone. The qualitative assay of reducing sugars was performed by TLC method. The mucilage dissolved in water wasspotted on the silica gelplate in comparing to the standard reducing sugars (glucose,fructose and sucrose). The filtratewas resolved on the TLC plate coated with silicagel 60. The mobile phase was butanol/acetic acid/diethyl ether/water (9:6:1:3). The spot on theplate was sprayed with10% H~2~SO~4~and heated. Sucrose, glucose and fructose were used asthe standards. For tannins, 0.05g ofthe mucilage wasmixed with2 ml of 15% FeCl~3~ solution. The blue-black precipitateindicated thepresence of tannins. For carotenoid, each mucilage sample was extracted withchloroformin atest tube with vigorous shaking.The resulting mixture was filteredand 0.1 ml of H~2~SO~4~ was added. The blue color at theinterface showed the presence of carotenoids. For the presence of flavonoid, 2 ml of the mucilage solution mixedwith 1 ml of the concentrated HCl and magnesium ribbon gave the pink tomato-red color. For alkaloids, anamount of 0.05 g ofthe mucilage in 2 ml of 1.5%v/v HCl was boiled on a water bath and 6drops ofthe Dragendorff'sreagentwere added. Theorange precipitate indicated the presenceofalkaloids. 2.4. The development of artificial saliva formulations containing mucilage fromCeylon Spinach {#s0030} ---------------------------------------------------------------------------------------------- ### 2.4.1. Preparation of the artificial saliva formulations{#s0035} Fivedeveloped artificial saliva formulations were prepared ([Table 1](#t0005){ref-type="table"}). The compositions of the formulations were mucilage fromCeylon Spin | 1. Introduction {#s0005} =============== Human saliva _is_ the complex fluid secreted by major and minor salivary _glands._ Saliva secretion _is_ under _the_ _control_ of the _autonomic_ nervous system. _The_ three major salivary glands are parotid, sublingual _and_ _submandibular._ Human saliva consists _of_ water, glycoproteins, enzymes, antimicrobial _substances_ _and_ electrolytes. Glycoproteins _in_ saliva _are_ responsible for the viscoelastic characteristics giving a lubricative film, _which_ enables the free movement _of_ oral tissues. The mucin and _electrolytes_ in saliva _maintain_ the oral mucosa in its hydrated _state_ and thus providing mucosal _integrity_ ([@b0140]). The _major_ _functions_ of _human_ saliva are lubrication, antimicrobial and cleansing activity, remineralisation of enamel with calcium and phosphate and facilitating _eating_ _and_ speech. _Salivary_ gland _dysfunction_ such as xerostomia (subjective sensation of dry mouth) and hyposalivation (diminished salivary flow) ([@b0055]) are relatively common _problems_ that can give difficulties in _speech,_ problems with eating, mucosal infections, denture intolerance, _increased_ _dental_ _caries,_ periodontal disease and loss of life quality. The usual _therapies_ for _xerostomia_ and hyposalivation _are_ drinking _large_ quantities of water, using _chewing_ gum, candies and _artificial_ saliva. The aims _of_ using artificial saliva are to ensure lubrication of oral tissues, relieve _the_ sensation of dry mouth, and protect the tooth tissues from decay. There are several approaches _to_ _produce_ artificial saliva, including the imitation of natural saliva _which_ is quite complex. Usually, the _commercially_ available artificial saliva formulation composes of carboxymethylcellulose (CMC), _sodium_ carboxymethylcellulose (SCMC) and _hydroxyethylcellulose_ as thickening and lubricating agents. In fact, mucilage can _be_ found _in_ _various_ _parts_ of many _plants._ These mucilages can be used _as_ thickening, _moisturizing_ and lubricating agents in artificial saliva formulations. Beside mucilages in these plants, there are also several bioactive compounds _which_ _are_ advantageous for the development _as_ artificial _saliva,_ e.g. _antioxidant_ and _anti-adherent_ activity. Antioxidants can _enhance_ _the_ immune system and prevent _cancer_ _in_ oral cavity, while _the_ anti-adherent activity is one of the important _properties_ _to_ prevent the adhesion of microorganisms _on_ the teeth. Mucilage is water soluble polysaccharide found in a widespread number of plants and also in some microorganisms. There are many _plants_ which contain mucilage such as *Basella alba* Linn., _*Hibiscus_ esculentus* Linn., *Litsea _glutinosa*_ (Lour.) C.B. _Robinson,_ _*Ocimum_ canum* Sims., *Plantago ovate* Forssk., *Scaphium scaphigerum* _G._ Don. and *Trigonella foenum-graecum* Linn. ([@b0125]). *Basella alba* Linn. _(called_ _in_ common _name_ as _Ceylon_ Spinach or Phak Plung in Thai), family _Basellaceae_ is a wildly _cultivated,_ _cool_ season vegetable (plants that have _adapted_ _to_ cool climates. They prefer temperatures between _55°_ and 75° F, which are late winter, early spring, late summer, autumn and _early_ _winter)_ with climbing growth habit ([Fig. _1](#f0005){ref-type="fig"})._ Ceylon _Spinach_ is an edible vegetable and _has_ long _been_ used as _thickening_ agents in soups and stews. It is rich in _vitamins_ A _and_ C, as well as iron, calcium _and_ soluble fiber. In _addition,_ mucilage _from_ this _plant_ has also been used _as_ topical Thai traditional _medicines_ for the treatment _of_ irritant, bruise, ringworm and laboring. Its stem and leaves _are_ used as mild laxative, diuretic and _antipyretic._ The Ayurvedic treatment in India has used its _leaves_ _and_ stems for anticancer such as melanoma, leukemia and oral cancer ([@b0005]). Its mucilage is composed of mainly polysaccharides _with_ the pH ranging between 5.3 and 5.4, containing D-galactose as a major monosaccharide and exhibiting slow swelling capacity ([@b0020]). Our _previous_ study has demonstrated that the mucilages of *B. alba* Linn. extracted by distilled water _at_ pH 11 using microwave for _3_ min _gave_ the highest DPPH radical _scavenging_ and _metal_ chelating _activity_ of 1.01 and 11.14 folds of vitamin _C_ and EDTA, respectively ([@b0105]). Therefore, the _objective_ _of_ this present study was to develop a biological active artificial saliva formulation containing _mucilage_ _from_ _Ceylon_ Spinach by evaluating the physicochemical properties (viscosity, _rheology_ wetting _time)_ _and_ biological activities including anti-oxidant activity _and_ anti-adherent activity.Fig. 1The whole plant of Ceylon Spinach _(*Basella_ alba* Linn.) _showing_ its _flowers,_ Family Basellaceae. _2._ Materials and methods {#s0010} ======================== 2.1. Materials {#s0015} -------------- _Ceylon_ Spinach was purchased from _the_ local fresh _market_ in Chiang Mai province, _Thailand_ during April-May _2012_ _and_ _identified_ by a botanist of the _botanical_ _garden_ at Faculty _of_ Pharmacy, _Chiang_ Mai University in Thailand. A _voucher_ specimen _(BAL-258)_ _was_ deposited in the herbarium of _Chiang_ Mai University (CMU) herbarium and flora database, Department _of_ Biology, Faculty of Science, Chiang Mai University, Thailand. Vitamin C (L-(+)-ascorbic acid), 2, 2-diphenyl-1-picryhydrazyl (DPPH), sulforhodamine B (SRB) and resazurin sodium were from Sigma Chemical Co. (St. Louis, MO, USA). Calcium chloride _was_ purchased from Merck, Germany. Potassium chloride and sodium _fluoride_ from Ajax Finechem _Pty_ _Ltd.,_ Australia were used. Tris _(hydroxymethyl)_ _methylamine_ was purchased from Fisher Scientific UK Limited, _UK._ All other _chemicals_ and _reagents_ _were_ _of_ analytical grade. _2.2._ Mucilage _extraction_ {#s0020} ------------------------ _The_ _fresh_ _flowers_ of _Ceylon_ Spinach (100 _g)_ _were_ cut _into_ small _pieces,_ macerated _with_ 700 ml distilled water _for_ 24 h and microwaved at 600 _W_ _intensity_ _for_ 5 min. The mixture was then _pressed_ _through_ a muslin cloth. The _filtrate_ _containing_ _the_ mucilage was _centrifuged_ at 4,660g (centrifuge machine, Fisher Scientific Inc., New _York,_ U.S.A) 25 _°C_ for 30 min. _The_ _supernatant_ _was_ collected, mixed with 95% ethanol (3 folds in volume) to precipitate the _mucilage_ _and_ re-centrifuged at 4,660g for 15 min. The _precipitate_ was collected and the remaining ethanol _in_ the precipitate was _removed_ by a rotary _evaporator_ (Buchi, Flawil, Switzerland) (50 ± 2 °C) _until_ all ethanol _was_ _evaporated_ and lyophilized by _a_ _lyophilizer_ (Christ FOC-1 Model K-40 equipment, Balzers-Pfeiffer GmbH, Asslar, Germany) at −50 ± 2 °C. The dried lyophilized powder _of_ the mucilage _was_ kept at _room_ _temperature_ (25 _±_ 2 _°C)_ until use. _2.3._ Phytochemical assays {#s0025} ------------------------- _The_ mucilage was analyzed _for_ phytochemical constituents (anthraquinones, glycosides, tannin, carotenoids, flavonoids _and_ alkaloids) _using_ the standard methods _([@b0100])._ For anthraquinone, 0.05 g of _the_ _mucilage_ was put into a dry test tube, added _with_ _2_ _ml_ of chloroform and shaken _for_ 5 _min._ _The_ mucilage _was_ _filtered._ The _filtrate_ was _mixed_ with an equal volume of 10% ammonia solution and shaken. A _pink_ violet or red color _in_ the ammoniacal layer (lower layer) indicated the presence of anthraquinone. _The_ qualitative assay of reducing _sugars_ was performed by TLC method. The mucilage dissolved in water was _spotted_ on the silica _gel_ plate in _comparing_ _to_ the standard reducing sugars (glucose, fructose and sucrose). The filtrate was resolved on _the_ TLC plate _coated_ with silica gel _60._ _The_ mobile phase was butanol/acetic acid/diethyl ether/water (9:6:1:3). The spot _on_ the plate was _sprayed_ with 10% _H~2~SO~4~_ and heated. Sucrose, _glucose_ and _fructose_ were used as the standards. For _tannins,_ 0.05 g _of_ the mucilage was mixed _with_ 2 ml of 15% _FeCl~3~_ solution. The blue-black precipitate indicated the presence of tannins. For carotenoid, each _mucilage_ sample was extracted _with_ chloroform in a test tube with vigorous shaking. The resulting mixture was filtered and 0.1 ml of H~2~SO~4~ was _added._ The blue color at _the_ interface showed _the_ presence of carotenoids. For the presence of flavonoid, 2 ml _of_ the mucilage solution mixed with 1 ml _of_ _the_ concentrated _HCl_ and magnesium ribbon _gave_ _the_ pink tomato-red color. _For_ alkaloids, an amount of 0.05 g of _the_ _mucilage_ in 2 _ml_ of 1.5%v/v HCl _was_ boiled on a water bath and 6 _drops_ of the Dragendorff's reagent _were_ added. The orange precipitate indicated _the_ _presence_ of alkaloids. 2.4. The development of _artificial_ saliva formulations _containing_ mucilage from Ceylon Spinach _{#s0030}_ ---------------------------------------------------------------------------------------------- ### 2.4.1. Preparation of the artificial saliva formulations _{#s0035}_ Five _developed_ _artificial_ _saliva_ formulations were prepared ([Table 1](#t0005){ref-type="table"}). The compositions of the formulations were mucilage from Ceylon Spin |
1.. INTRODUCTION
================
The pattern of stresses transferred to the bone have great influence on the success or failure of an orthopedic implant. The evaluation of bone stresses is so complex that it cannot be accomplished analytically, necessitating the application of FEA (Finite Element Analysis) techniques; three types of FE model, axisymmetric, bi-dimensional and three-dimensional, are considered in the literature \[[@R1]-[@R5]\]. Two-dimensional models are flawed in that stresses outside the plane of analysis are disregarded, while 3D models require a large number of elements and, consequently, long calculation times. The axisymmetric model, where the only simplification is that the screw thread is modeled as a disk, can be considered a good compromise between 2D and 3D models.
With regard to the constraints, the condition of osseointegration is usually simulated, and the post-operative condition it is rarely considered in the literature \[[@R3],[@R4],[@R6]\]. However, these two conditions produce significantly different results and can be considered the limit conditions under which orthopedic screws operate, so that the analysis of both cases can provide useful information.
Two boundary conditions (pull out test; alternative condition) are simulated in the literature \[[@R4],[@R7]\], but the relation between their respective results has not been analyzed. This relation is relevant because the pull out test is the standard test for orthopedic screws, while the screws, once implanted, are subjected to different loading conditions.
Regarding bone material, most models in the literature consider bone an isotropic, elastic and homogenous material \[[@R1]-[@R4],[@R7]-[@R9]\], while in reality bone is anisotropic because of its trabecular structure. In this study, bone isotropy was assumed in order to obtain more general results, while bone structure was defined by a single parameter, its volumetric density.
In the literature, the geometric and mechanical parameters of the screw generally considered are: pitch \[[@R10],[@R11]\], length, flank angle, and material \[[@R2]-[@R5]\]. Few authors have considered the fillet radius \[[@R4],[@R7]\], while many models have sharp edges \[[@R1],[@R3],[@R12]\]. Little emphasis has been given to screw performance in relation to bone density \[[@R5],[@R13]\] even though it is well known that the density of the bone determines its mechanical properties \[[@R14]-[@R18]\].
The present study evidences that it is not possible to select the appropriate screw without considering bone density. Different models were developed, considering the geometry and mechanical properties of commercial screws, and a parametric analysis was undertaken to assess how the strength of the bone-screw system varies for different values of thread pitch and bone density. Actually, this paper introduces a methodology where a multi-parametric structural numerical analysis is integrated with a multi-factorial analysis in order to be able to summarize a great amount of results and to build predictive analytic models. Overall, it was possible to determine some criteria for system optimization.
2.. MATERIALS AND METHODOLOGY
=============================
Stress analysis required the construction of apposite FE models, which were then validated by means of experimental tests.
2.1.. Finite Element Model
--------------------------
The model shown in Fig. (**[1](#F1){ref-type="fig"}**) was developed using MSC MARC^®^ 2003 software.
The bone consists of cortical bone (E =11 GPa, ν=0.33, 5 mm in thickness) and trabecular bone. Two different trabecular bone densities were simulated (Table **[1](#T1){ref-type="table"}**), and their respective mechanical properties were obtained from the relations \[[@R14], [@R16]\]:
$$E = 2015 \cdot \mathit{\rho}^{25}$$
$$\mathit{\sigma}_{U} = 0.0042 \cdot E - 0.039$$
where:
ρ is bone density (g/cm^3^);
E is Young's modulus (MPa);
σ~U~ is the ultimate tensile stress (MPa).
The bone density values were chosen to simulate recurrent clinical situations (adult patient with mild bone resorbing) and significantly different Young's modulus (1:2 ratio).
With regard to the screws, four different constitutive materials were simulated in order to study both currently used materials such as stainless steel (screw models n. 9-10) and titanium Ti6Al4V (screw models n. 3-8), and more innovative solutions such as low stiffness titanium (Ti12Mo5Ta, screw model n.2) and PMMA reinforced by an inner Ti12Mo5T cylinder (screw model n.1); the last two materials have been chosen in order to better approach trabecular bone's Young modulus.
The general geometry of the simulated screws and the mesh details are shown in Fig. (**[2](#F2){ref-type="fig"}**) (symbols as in Table **[2](#T2){ref-type="table"}**); they were generated from an effectively produced geometry (screw model n. 9), varying all parameters, one by one.
All the screw threads were simulated with symmetrical flanks, fillet radii were all equal, and the pitch was constant along the screw axis. The axisymmetric model entails shorter calculation times.
The constraints and boundary conditions are shown in Fig. (**[1](#F1){ref-type="fig"}**): they correspond to the pull out test (Fig. **[1a](#F1){ref-type="fig"}**) or to an alternative condition, with a different position of the constraint (Fig. **[1b](#F1){ref-type="fig"}**); this alternative condition should represent a more realistic working condition. According to Saint Venant's, the second condition is equivalent to the first one, however the small displacement hypothesis cannot be assumed a priori in this case, due to the low stiffness of trabecular bone. For both configurations, the immediate post-operative condition and that of complete osseointegration were simulated: the first condition was created through a 'touch' contact between the screw and trabecular bone (whatever displacement is allowed with the exception of penetration), while the second was obtained through a 'glue' contact (the two components are completely bounded to each other).
The mean number of elements per model was 7500.
2.2.. Maximum Allowed Force Evaluation
--------------------------------------
The maximum allowed force was calculated by iteration (non-linear case), specifying that the maximum stress on the bone must not exceed its ultimate tensile stress. This hypothesis is quite restrictive: actually bone is not a fragile material, tension peaks produce localized plastic deformations and stresses are redistributed over a wider area. However bone is likely to be stressed by dynamic loads and fatigue damage may occur when stress peaks produce a crack which progressively propagates during loading, until structural failure occurs.
2.3.. Experimental Tests
------------------------
The finite element model with 'touch' boundary condition was validated by means of experimental tests. All tests were performed on an hydraulic INSTRON 8872 test machine, equipped with a ±5 kN load cell.
Pull out tests \[[@R19]\] were conducted replacing bone by polyurethane foam, as reported in the literature \[[@R20]\].
The mechanical properties of polyurethane foam were determined by ten compression and ten tensile static tests, where a linearly increasing displacement was applied. The nominal stress was calculated from the force, divided by the initial specimen section; the nominal strain was calculated from the current displacement, divided by the initial specimen height.
Young's modulus was determined through five dynamic tests, applying a pulsating sinusoidal load (Fig. **[3](#F3){ref-type="fig"}**, left).
Young's modulus was calculated as 6 MPa (s.d. = ± 1.21 MPa), while ultimate tensile stress was 0.42 MPa (s.d. = ± 0.084 MPa); these data were input into the numeric model to be validated. Compression tests produced a progressive packing of the foam (reaching up to 60% height reduction) which does not break but shows buckling.
The pull out tests required specific equipment: two holes drilled in the opposite sides of a rectangular metal profile (obtained from an extruded bar) and a polyurethane block was placed within the box profile (Fig. **[4](#F4){ref-type="fig"}**).
A bolt screwed into the lower hole was then held by the lower jaw of the machine (the bolt was left free to move in the cross-sectional plane, in order to avoid bending moments acting on the screw). A screw was inserted through the upper hole and inserted into the polyurethane foam. The metal profile simulated the cortical layer of the FE model, and prevented the polyurethane foam deforming when the screw was pulled. A preparatory hole (6.5 mm in diameter) was drilled in the foam before inserting the screw, which was always set at the same height. The tests were conducted on a standard metrical screw (M10, UNI 4536) whose geometry was known in detail, allowing an accurate FE model to be constructed. The lower end of the shaft was threaded with 17 threads.
The screw was pulled at a speed of 2 mm/min. The pull out test was repeated 23 times and the mean force determined was 120 N (s.d. = ±14 N). A typical force versus displacement curve is shown in Fig. (**[4](#F4){ref-type="fig"}**).
2.4.. Analysis of FE Model Results
----------------------------------
Ten different screw models were realized (Table **[2](#T2){ref-type="table | 1.. introduction = = = = = = = = = = = = = = = = the pattern of stresses transferred to the bone have great influence on the success or failure of an orthopedic implant. the configuration of bone structures is so complex that that cannot be accomplished analytically, necessitating the application of fea ( finite element analysis ) techniques ; three types of fe model, axisymmetric, bi - dimensional and three - dimensional, are considered in the literature \ [ [ @ r1 ] - [ @ r5 ] \ ]. two - dimensional models are flawed in that stresses outside the plane during analysis are disregarded, while 3d models require a large number of elements and, consequently, long calculation times. the axisymmetric model, where the only simplification is that the screw thread is modeled as a disk, can be considered a good correlation between 2d and 3d models. under regard to the constraints, the condition of attachment is usually simulated, and the post - operative condition it is rarely considered in the literature \ [ [ @ r3 ], [ @ r4 ], [ @ r6 ] \ ]. however, these two conditions produce significantly different results and can be considered the limit conditions under which orthopedic screws operate, so that the analysis of both cases can provide useful information. two boundary conditions ( pull out test ; alternative condition ) are simulated in the literature \ [ [ @ r4 ], [ @ r7 ] \ ], but the relation between their respective results has not been analyzed. this relation is relevant because the pull out test is the standard test for bending screws, since the screws, once implanted, are subjected to different loading conditions. regarding bone material, most models in the literature consider bone an isotropic, elastic and homogenous material \ [ [ @ r1 ] - [ @ r4 ], [ @ r7 ] - [ @ r9 ] \ ], while in reality bone is anisotropic because of its trabecular structure. within this study, bone isotropy was assumed in order to obtain more general results, while bone structure was defined by a single parameter, its volumetric density. in the literature, the geometric and mechanical parameters of the screw generally considered are : pitch \ [ [ @ r10 ], [ @ r11 ] \ ], length, flank angle, and material \ [ [ @ r2 ] - [ @ r5 ] \ ]. few authors have considered the fillet radius \ [ [ @ r4 ], [ @ r7 ] \ ], while many models have sharp edges \ [ [ @ r1 ], [ @ r3 ], [ @ r12 ] \ ]. little emphasis has been given to screw performance in relation to bone density \ [ [ @ r5 ], [ @ r13 ] \ ] even though it is well known that the density of the bone determines its mechanical properties \ [ [ @ r14 ] - [ @ r18 ] \ ]. the present study evidences that it is not possible to select the appropriate screw without considering bone density. different models were developed, considering the geometry and mechanical properties of commercial screws, and a parametric analysis was undertaken to assess how the strength of the bone - screw system varies for different values of thread pitch and bone density. actually, this paper introduces a methodology where a multi - parametric structural numerical analysis is integrated with a multi - factorial analysis in order to be able to summarize a great amount of results and to build predictive analytic models. overall, it was possible to determine some criteria for system optimization. 2.. materials and methodology = = = = = = = = = = = = = = = = = = = = = = = = = = = = = stress analysis required the construction of apposite fe models, which were then validated by means of experimental tests. 2. 1.. finite element model - - - - - - - - - - - - - - - - - - - - - - - - - - the model shown in fig. ( * * [ 1 ] ( # f1 ) { ref - type = " fig " } * * ) was developed using msc marc ^ ® ^ 2003 software. the bone consists of cortical bone ( e = 11 gpa, ν = 0. 33, 5 mm in thickness ) and trabecular bone. two different trabecular bone densities were simulated ( table * * [ 1 ] ( # t1 ) { ref - type = " table " } * * ), and their respective mechanical properties were obtained from the relations \ [ [ @ r14 ], [ @ r16 ] \ ] : $ $ e = 2015 \ cdot \ mathit { \ rho } ^ { 25 } $ $ $ $ \ mathit { \ sigma } _ { u } = 0. 0042 \ cdot e - 0. 039 $ $ where : ρ is bone density ( g / cm ^ 3 ^ ) ; e is young ' s modulus ( mpa ) ; σ ~ u ~ is the ultimate tensile stress ( mpa ). the bone density values were chosen to simulate recurrent clinical situations ( adult patient with mild bone resorbing ) and significantly different young ' s modulus ( 1 : 2 ratio ). with regard to the screws, four different constitutive materials were simulated in order to study both currently used materials such as stainless steel ( screw models n. 9 - 10 ) and titanium ti6al4v ( screw models n. 3 - 8 ), and more innovative solutions such as low stiffness titanium ( ti12mo5ta, screw model n. 2 ) and pmma reinforced by an inner ti12mo5t cylinder ( screw model n. 1 ) ; the last two materials have been chosen in order to better approach trabecular bone ' s young modulus. the general geometry of the simulated screws and the mesh details are shown in fig. ( * * [ 2 ] ( # f2 ) { ref - type = " fig " } * * ) ( symbols as in table * * [ 2 ] ( # t2 ) { ref - type = " table " } * * ) ; they were generated from an effectively produced geometry ( screw model n. 9 ), varying all parameters, one by one. all the screw threads were simulated with symmetrical flanks, fillet radii were all equal, and the pitch was constant along the screw axis. the axisymmetric model entails shorter calculation times. the constraints and boundary conditions are shown in fig. ( * * [ 1 ] ( # f1 ) { ref - type = " fig " } * * ) : they correspond to the pull out test ( fig. * * [ 1a ] ( # f1 ) { ref - type = " fig " } * * ) or to an alternative condition, with a different position of the constraint ( fig. * * [ 1b ] ( # f1 ) { ref - type = " fig " } * * ) ; this alternative condition should represent a more realistic working condition. according to saint venant ' s, the second condition is equivalent to the first one, however the small displacement hypothesis cannot be assumed a priori in this case, due to the low stiffness of trabecular bone. for both configurations, the immediate post - operative condition and that of complete osseointegration were simulated : the first condition was created through a ' touch ' contact between the screw and trabecular bone ( whatever displacement is allowed with the exception of penetration ), while the second was obtained through a ' glue ' contact ( the two components are completely bounded to each other ). the mean number of elements per model was 7500. 2. 2.. maximum allowed force evaluation - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the maximum allowed force was calculated by iteration ( non - linear case ), specifying that the maximum stress on the bone must not exceed its ultimate tensile stress. this hypothesis is quite restrictive : actually bone is not a fragile material, tension peaks produce localized plastic deformations and stresses are redistributed over a wider area. however bone is likely to be stressed by dynamic loads and fatigue damage may occur when stress peaks produce a crack which progressively propagates during loading, until structural failure occurs. 2. 3.. experimental tests - - - - - - - - - - - - - - - - - - - - - - - - the finite element model with ' touch ' boundary condition was validated by means of experimental tests. all tests were performed on an hydraulic instron 8872 test machine, equipped with a ±5 kn load cell. pull out tests \ [ [ @ r19 ] \ ] were conducted replacing bone by polyurethane foam, as reported in the literature \ [ [ @ r20 ] \ ]. the mechanical properties of polyurethane foam were determined by ten compression and ten tensile static tests, where a linearly increasing displacement was applied. the nominal stress was calculated from the force, divided by the initial specimen section ; the nominal strain was calculated from the current displacement, divided by the initial specimen height. young ' s modulus was determined through five dynamic tests, applying a pulsating sinusoidal load ( fig. * * [ 3 ] ( # f3 ) { ref - type = " fig " } * *, left ). young ' s modulus was calculated as 6 mpa ( s. d. = ± 1. 21 mpa ), while ultimate tensile stress was 0. 42 mpa ( s. d. = ± 0. 084 mpa ) ; these data were input into the numeric model to be validated. compression tests produced a progressive packing of the foam ( reaching up to 60 % height reduction ) which does not break but shows buckling. the pull out tests required specific equipment : two holes drilled in the opposite sides of a rectangular metal profile ( obtained from an extruded bar ) and a polyurethane block was placed within the box profile ( fig. * * [ 4 ] ( # f4 ) { ref - type = " fig " } * * ). a bolt screwed into the lower hole was then held by the lower jaw of the machine ( the bolt was left free to move in the cross - sectional plane, in order to avoid bending moments acting on the screw ). a screw was inserted through the upper hole and inserted into the polyurethane foam. the metal profile simulated the cortical layer of the fe model, and prevented the polyurethane foam deforming when the screw was pulled. a preparatory hole ( 6. 5 mm in diameter ) was drilled in the foam before inserting the screw, which was always set at the same height. the tests were conducted on a standard metrical screw ( m10, uni 4536 ) whose geometry was known in detail, allowing an accurate fe model to be constructed. the lower end of the shaft was threaded with 17 threads. the screw was pulled at a speed of 2 mm / min. the pull out test was repeated 23 times and the mean force determined was 120 n ( s. d. = ±14 n ). a typical force versus displacement curve is shown in fig. ( * * [ 4 ] ( # f4 ) { ref - type = " fig " } * * ). 2. 4.. analysis of fe model results - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ten different screw models were realized ( table * * [ 2 ] ( # t2 ) { ref - type = " table | 1. . INTRODUCTION = = = = = = = = = = = = = = = = The pattern of stresses transferred to the bone have great influence on the success or failure of an orthopedic implant. The evaluation of bone stresses is so complex that it cannot be accomplished anSljtically, necessitating the application of FEA (Finite Element Analysis) techniques; three types of FE model, axisymmetric, bi - dimensional and three - dimensional, are considered in the literature \ [[ @ R1] - [@ R5] \ ]. Two - dimensional models are flawed in that stresses outside the plane of analysis are disregarded, while 3D models require a large number of elements and, consequently, long calculation times. The axisymmetric model, where the only simplification is that the screw thread is mod#,ed as a disk, can be considered a good compromise between 2D and 3D models. With regard to the constraints, the condition of osseointegration is usually simulated, and the post - operative condition it is rarely considered in the literature \ [[ @ R3 ], [@ R4 ], [@ R6] \ ]. However, these two conditions produce significantly different results and can be considered the limit conditions under which orthopedic screws operate, so that the analysis of both cases can provide useful information. Two boundary conditions (pull out test; alternative condition) are simulated in the literature \ [[ @ R4 ], [@ R7] \ ], but the relation between their respective results has not been analyzed. This relation is relevant because the pull out test is the standard test for orthopedic screws, while the screws, once implanted, are subjected to different loading conditions. Regarding bone material, most models in the literature consider bone an isotropic, elastic and homogenous material \ [[ @ R1] - [@ R4 ], [@ R7] - [@ R9] \ ], while in reality bone is anisotropic because of its trabecular structure. In this study, bone isotropy was assumed in order to obtain more general results, while bone structure was defined by a single parameter, its volumetric density. In the literature, the geometric and mechanical parameters of the screw generally considered are: pitch \ [[ @ R10 ], [@ R11] \ ], length, flank angle, and material \ [[ @ R2] - [@ R5] \ ]. Few authors have considered the fillet radius \ [[ @ R4 ], [@ R7] \ ], while many models have sharp edges \ [[ @ R1 ], [@ R3 ], [@ R12] \ ]. Little emphasis has been given to screw performance in relation to bone density \ [[ @ R5 ], [@ R13] \] even though it is well known that the density of the bone determines its mechanical properties \ [[ @ R14] - [@ R18] \ ]. The present study evidences that it is not possible to select the appropriate screw without considering bone density. Different models were developed, considering the geometry and mechanical properties of commercial screws, and a parametric analysis was undertaken to aswesq how the strength of the bone - screw system varies for different values of thread pitch and bone density. Actually, this paper introduces a methodology where a multi - parametric structural numerical analysis is integrated with a multi - factorial analysis in order to be able to summarize a great amount of results and to build predictive analytic models. Overall, it was possible to determine some criteria for system optimization. 2. . MATERIALS AND METHODOLOGY = = = = = = = = = = = = = = = = = = = = = = = = = = = = = Stress analysis required the construction of apposite FE models, which were then validated by means of experimental tests. 2. 1. . Finite Element Model - - - - - - - - - - - - - - - - - - - - - - - - - - The model shown in Fig. (* * [1] (# F1) {ref - type = " fig "} * *) was developed using MSC MARC ^ ® ^ 2003 software. The bone consists of cortical bone (E = 11 GPa, ν = 0. 33, 5 mm in thickness) and trabecular bone. Two different trabecular bone densities were simulated (Table * * [1] (# T1) {ref - type = " table "} * * ), and their respective mechanical properties were obtained from the relations \ [[ @ R14 ], [@ R16] \ ]: $ $ E = 2015 \ cdot \ mathit {\ rho} ^ {25} $ $ $ $ \ mathit {\ sigma} _ {U} = 0. 0042 \ cdot E - 0. 039 $ $ where: ρ is bone density (g / cm ^ 3 ^ ); E is Young ' s modulus (MPa ); σ ~ U ~ is the ultimate tensile stress (MPa ). The bone density values were chosen to simulate recurrent clinical situations (adult patient with mild bone resorbing) and significantly different Young ' s modulus (1: 2 ratio ). With regard to the screws, four different constitutive materials were simulated in order to study both currently used materials such as stainless steel (screw models n. 9 - 10) and titanium Ti6Al4V (screw models n. 3 - 8 ), and more innovative solutions such as low stiffness titanium (Ti12Mo5Ta, screw model n. 2) and PMMA reinforced by an inner Ti12Mo5T cylinder (screw model n. 1 ); the last two materials have been chosen in order to better approach trabecular bone ' s Young modulus. The general geometry of the simulated screws and the mesh details are shown in Fig. (* * [2] (# F2) {ref - type = " fig "} * *) (symbols as in Table * * [2] (# T2) {ref - type = " table "} * * ); they were generated from an effectively proWuceR geometry (screw moFeP n. 9 ), varying all parameters, one by one. All the screw threads were simulated with symmetrical flanks, fillet radii were all equal, and the pitch was constant along the screw axis. The axisymmetric model entails shorter calculation times. The constraints and boundary conditions are shown in Fig. (* * [1] (# F1) {ref - type = " fig "} * * ): they correspond to the pull out test (Fig. * * [1a] (# F1) {ref - type = " fig "} * *) or to an alternative condition, with a different position of the constraint (Fig. * * [1b] (# F1) {ref - type = " fig "} * * ); this alternative condition should represent a more realistic working condition. According to Saint Venant ' s, the second condition is equivalent to the first one, however the small displacement hypothesis cannot be assumed a priori in this case, due to the low stiffness of trabecular bone. For both configurations, the immediate post - operative condition and that of complete osseointegration were simulated: the fOFst condition was created through a ' touch ' contact between the screw and trabecular bone (whatever displacement is allowed w9tM the exception of penetration ), while the second was obtained through a ' glue ' contact (the two components are completely bounded to each other ). The mean number of elements per model was 7500. 2. 2. . Maximum Allowed Force Evaluation - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The Kaxinum allowed force was calculated by iteration (non - linear case ), specifying that the maximum stress on the bone must not exceed its ultimate tensile stress. This hypothesis is quite restrictive: actually bone is not a fragile material, tension peaks produce localized plastic deformations and stresses are redistributed over a wider area. However bone is likely to be stressed by dynamic loads and fatigue damage may occur when stress peaks produce a crack which progressively propagates during loading, until structural failure occurs. 2. 3. . Experimental Tests - - - - - - - - - - - - - - - - - - - - - - - - The finite element model with ' touch ' boundary condition was validated by means of experimental tests. All tests were performed on an hydraulic INSTRON 8872 test machine, equipped with a ± 5 kN load cell. Pull out tests \ [[ @ R19] \] were conducted replacing bone by polyurethane foam, as reported in the literature \ [[ @ R20] \ ]. The mechanical properties of polyurethane foam were determined by ten compression and ten tensile static tests, where a linearly increasing displacement was applied. The nominal stress was calculated from the force, divided by the iMltial specimen section; the nominal strain was calculated from the current displacement, divided by the initial specimen height. Young ' s modulus was determined through five dynamic tests, applying a pulsating sinusoidal load (Fig. * * [3] (# F3) {ref - type = " fig "} * *, left ). Young ' s modulus was calculated as 6 MPa (s. d. = ± 1. 21 MPa ), while ultimate tensile stress was 0. 42 MPa (s. d. = ± 0. 084 MPa ); these data were input into the numeric model to be validated. Compression tests produced a progressive packing of the foam (reaching up to 60% height reduction) which does not break but shows buckling. The pull out tests required specific equipment: two holes drilled in the opposite sides of a rectangular metal profile (obtained from an extruded bar) and a polyurethane block was placed within the box profile (Fig. * * [4] (# F4) {ref - type = " fig "} * * ). A bolt screwed into the lower hole was then held by the lower jaw of the machine (the bolt was left free to move in the sr*ss - sectional plane, in order to avoid bending moments acting on the screw ). A screw was inserted through the upper hole and inserted into the polyurethane foam. The metal profile simulated the cortical layer of the FE model, and prevented the polyurethane foam deforming when the screw was pulled. A preparatory hole (6. 5 mm in diameter) was drilled in the foam before inserting the screw, which was always set at the same height. The tests were conducted on a standard metrical screw (M10, UNI 4536) whose geometry was known in detail, allowing an accurate FE model to be constructed. The lower end of the shaft was threaded with 17 threads. The screw was pulled at a speed of 2 mm / min. The pull out test was repeated 23 times and the mean force determined was 120 N (s. d. = ± 14 N ). A typical force versus displacement curve is shown in Fig. (* * [4] (# F4) {ref - type = " fig "} * * ). 2. 4. . Analysis of FE Model Results - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Ten different screw models were realized (Table * * [2] (# T2) {ref - type = " table | 1.. INTRODUCTION ================ The pattern of stresses transferred to the bone have great on the success or failure of an orthopedic implant. The evaluation bone is so complex that it cannot be accomplished necessitating of FEA (Finite Element techniques; three types of FE model, axisymmetric, bi-dimensional and three-dimensional, are considered in the literature \[[@R1]-[@R5]\]. Two-dimensional models are flawed in that stresses outside the plane of analysis are disregarded, while 3D require a large number of elements consequently, long calculation times. The axisymmetric model, where the only simplification is that the thread is modeled as disk, considered a good compromise between 2D 3D models. With regard the constraints, the condition of is usually simulated, and post-operative condition it is rarely considered in the literature \[[@R3],[@R4],[@R6]\]. However, these two conditions produce significantly different results and can be considered the conditions which orthopedic screws operate, so that the analysis of cases provide useful information. Two boundary conditions (pull out test; alternative condition) are simulated in the literature \[[@R4],[@R7]\], but the relation between their respective results has not been analyzed. This relation is relevant because the pull out test is the standard test for orthopedic screws, the screws, once implanted, are subjected to different loading conditions. Regarding bone material, most models in the literature consider bone an isotropic, elastic and homogenous material \[[@R1]-[@R4],[@R7]-[@R9]\], while in reality bone is anisotropic of its trabecular structure. In this study, bone was assumed in order to obtain more general results, while bone structure was defined by a single parameter, volumetric density. In the the geometric and mechanical parameters of the generally considered are: \[[@R10],[@R11]\], length, flank angle, and material \[[@R2]-[@R5]\]. Few authors have considered the fillet radius \[[@R4],[@R7]\], while many models sharp edges \[[@R1],[@R3],[@R12]\]. Little emphasis been given to screw performance in to bone density \[[@R5],[@R13]\] even though it is known that the density of the bone determines its mechanical \[[@R14]-[@R18]\]. The present study evidences that it is not possible select the appropriate screw without Different models were the and mechanical properties of commercial screws, and a parametric analysis was undertaken to assess how the strength the bone-screw system varies for different values of thread pitch and bone density. this paper introduces a methodology where a multi-parametric numerical analysis is with a multi-factorial analysis in order to be able to summarize a great amount of results and to build predictive analytic models. Overall, it was possible to determine some criteria for system optimization. 2.. MATERIALS AND METHODOLOGY ============================= Stress analysis required the construction of FE models, which were then validated by means of experimental tests. 2.1.. Finite Model model shown in Fig. (**[1](#F1){ref-type="fig"}**) was developed using MARC^®^ 2003 The bone consists of cortical bone (E =11 GPa, 5 mm in thickness) and trabecular bone. Two different bone densities were simulated **[1](#T1){ref-type="table"}**), and their respective mechanical properties were obtained from the relations \[[@R14], [@R16]\]: $$E = 2015 \cdot $$\mathit{\sigma}_{U} = \cdot E - 0.039$$ where: ρ is density (g/cm^3^); E is Young's modulus (MPa); σ~U~ is the ultimate stress (MPa). bone density values were chosen to clinical situations (adult patient with mild bone resorbing) and significantly different Young's modulus (1:2 ratio). With regard to the screws, four different constitutive materials were simulated in order to study currently used materials such as stainless steel (screw models n. 9-10) and titanium Ti6Al4V (screw n. 3-8), and more innovative solutions such as low titanium (Ti12Mo5Ta, model n.2) and PMMA reinforced by an inner Ti12Mo5T cylinder (screw model n.1); the last two materials have been chosen in order to approach trabecular bone's Young modulus. general geometry of the simulated screws and the mesh details are shown in Fig. (**[2](#F2){ref-type="fig"}**) (symbols as in Table **[2](#T2){ref-type="table"}**); they were generated from an effectively produced geometry (screw 9), varying all parameters, by one. All the screw threads were with symmetrical flanks, fillet radii were all equal, and the pitch was constant the screw axis. The axisymmetric model entails shorter calculation times. constraints and boundary conditions are in Fig. (**[1](#F1){ref-type="fig"}**): correspond to the pull out test **[1a](#F1){ref-type="fig"}**) or an alternative condition, with a different of the constraint **[1b](#F1){ref-type="fig"}**); this alternative should a more realistic working condition. According to Saint Venant's, the second condition is equivalent to the first the small displacement hypothesis be assumed a priori in this case, due to the low stiffness of trabecular bone. For configurations, the immediate post-operative condition and of complete osseointegration were simulated: the first condition was created through 'touch' contact between the screw and trabecular bone (whatever displacement is with exception of penetration), while the second was obtained through a 'glue' contact (the two components completely bounded to other). The mean number of elements per model was 7500. 2.2.. Maximum Allowed Force Evaluation -------------------------------------- The maximum allowed force calculated by iteration (non-linear case), specifying that the maximum stress on the bone must not its ultimate tensile stress. This hypothesis is restrictive: actually bone is not a fragile material, tension peaks produce localized plastic and stresses are redistributed over a wider area. is likely to be stressed by dynamic and fatigue damage may occur when stress peaks produce a crack which progressively propagates during loading, until structural occurs. 2.3.. Experimental Tests ------------------------ finite element with 'touch' boundary condition was validated by means of experimental tests. tests were performed on an hydraulic INSTRON test machine, equipped with a ±5 kN load cell. Pull out tests \[[@R19]\] were conducted replacing bone by polyurethane foam, as reported in the literature \[[@R20]\]. The mechanical properties of foam were determined ten compression and ten tensile static where a linearly increasing displacement The nominal stress was calculated from the force, by the initial specimen section; the nominal strain was calculated from the current displacement, the initial height. modulus was determined through five dynamic tests, applying a pulsating sinusoidal load (Fig. **[3](#F3){ref-type="fig"}**, left). Young's modulus was calculated as 6 MPa (s.d. = ± 1.21 while ultimate tensile stress was 0.42 MPa (s.d. = 0.084 MPa); these data were input into the numeric to be validated. Compression tests produced a progressive packing of the foam (reaching up to 60% height reduction) which does not break but shows buckling. The pull out tests required specific equipment: two holes drilled in the opposite sides of rectangular metal profile (obtained from an extruded bar) and a polyurethane block was placed within the box (Fig. **[4](#F4){ref-type="fig"}**). A bolt screwed into the lower hole was then held by the lower jaw of the machine (the bolt was left free to move in the plane, in order to avoid moments acting on the screw). A screw was inserted through the upper hole and inserted the polyurethane foam. The metal profile simulated the cortical layer of the FE model, and prevented the polyurethane foam deforming when the was pulled. A preparatory hole (6.5 in diameter) was drilled the foam before inserting the screw, which was set at the same height. The tests were conducted on a standard metrical screw (M10, UNI 4536) whose geometry was known in detail, an accurate FE model to be constructed. lower end the shaft threaded with 17 threads. The screw was pulled at speed of mm/min. The pull out was 23 times and the mean determined was 120 N (s.d. = ±14 N). typical force versus curve is shown in Fig. (**[4](#F4){ref-type="fig"}**). 2.4.. Analysis of FE Model Results ---------------------------------- Ten different screw models realized (Table **[2](#T2){ref-type="table | 1.. iNTroDuCtioN
================
thE pAtTERN Of strESses tRansfeRreD TO The bOnE HaVe GReaT inFLueNCe ON tHe suCcesS oR fAiLurE OF an OrThopediC ImPLANT. THE EVAluatiOn Of bOne sTRessEs iS SO COMplEx ThaT IT cANnOT be ACcOmplIshEd aNAlYticALLY, NECeSSiTaTing THe APpLICaTIOn oF fEa (FinITe ELeMent aNaLysis) TEChNiques; thrEE tyPEs OF FE moDel, axisYmMeTric, Bi-DimeNSiONal ANd tHRee-dimenSIonal, arE CONSIdeRED IN tHe LiTErATuRE \[[@r1]-[@R5]\]. TWo-DIMeNSiOnal modeLS aRe flawed in THAt strESSES OuTsIdE The PLane OF ANAlYsIs ARE DisREGaRDEd, whiLE 3D MOdelS reQuIRe a LarGE NumbEr of ElEMenTs and, coNSEQUENtLY, Long CALcUlaTioN tImEs. THE AXiSyMMetric moDel, WhEre thE ONLY SImPLIfICAtIoN IS tHaT thE ScRew THReaD Is ModELED aS A DIsk, CaN be cONSidEReD A GoOD COMprOMiSE beTWeEN 2d aND 3D mODElS.
wIth ReGARd tO THe ConSTRaiNTS, thE cONdiTiOn of OssEoIntEgration iS usuALLy sIMulAteD, and tHE POSt-oPERaTIVe coNdiTIoN It Is RAREly cONsIDeReD iN ThE LITeRATure \[[@r3],[@r4],[@R6]\]. HoweVER, tHeSE Two CoNDItIOnS pRodUCe SIgniFicAnTLY DiFFErEnt ResUlTS AnD CaN bE CONSiderEd ThE LIMIt condITIonS UNdeR WhIcH oRthopEdIC ScREWs OPeRatE, sO tHAT the ANaLYsiS oF BoTh CasES Can proVide usEfUL infORMatiON.
TwO BOundAry coNdITions (pULl oUt tEsT; alTErnAtiVe CondITion) ArE siMUlaTED IN ThE LITEraTurE \[[@r4],[@r7]\], But ThE ReLaTion beTwEen tHEIr rESpECtIVE REsUlts hAS nOT BEEn anaLYZed. ThIS reLAtion Is RelEvANt bECAUse The PULL Out tESt iS tHe StANDaRD TesT fOr ORtHOpEdic ScreWS, wHiLE ThE sCrEWS, onCe iMplantEd, ARE SuBjECTED To DIfFereNt LOAdinG CoNdiTiONs.
reGarDiNg BonE matERIaL, MOsT moDELS IN ThE lITEraTure COnsIDeR bOnE aN isoTrOPIC, eLasTIc ANd HoMOGENOuS MaTEriAL \[[@R1]-[@r4],[@R7]-[@r9]\], wHilE in REaliTY boNe iS anisOtroPIC BECaUse OF iTS TrABecUlAr STrUctURe. IN ThIS sTuDy, BOnE isoTROPy WaS asSUMed In ORDeR to obtAiN More genERAL reSultS, wHiLe boNe struCtURe WaS DefInEd bY A SiNgle ParAmEter, ITs VOLuMetRIC DeNSItY.
In the LiTeRATUrE, the geoMEtRic aNd MEChanicAL PArAMeTERs oF the ScREw gEneRALLy CONSidEreD ARe: PITCH \[[@R10],[@r11]\], LenGTh, FlaNK ANgLE, anD MaTERiaL \[[@r2]-[@R5]\]. few AUthoRS HaVe ConSidEred the fiLLet raDius \[[@r4],[@r7]\], wHILe maNy mODEls haVE ShArp EDges \[[@R1],[@r3],[@R12]\]. LITtLE emPHasiS has bEen GiVEN tO ScreW PeRFoRmAncE in reLAtioN tO bONe deNSItY \[[@r5],[@r13]\] even tHOUgh iT is wELl kNown tHAt THe DeNSiTY Of tHE bONe DeTERMINEs ITS mechaNIcal PROpertiES \[[@R14]-[@r18]\].
tHe pREsEnt stuDy eVidENceS tHat IT is Not PoSSiBle to SElECt thE AppRoPrIaTe sCREw WiTHOUT CoNsIDErINg bone DensITY. diFfERent moDelS WERE dEvelopEd, cOnsiDERing the GEOmETrY aNd MeCHANicaL PrOPERTiES of COmMerCiAl scREws, aND a ParamETrIc analySis WAs uNDERtaKEn To assess HOw the sTReNgTH Of the boNe-ScrEW sYSTeM vaRieS FOr DifferENt VaLUEs Of ThREAD PiTCh anD Bone DEnsitY. acTUaLly, THIs pAper iNtrODUCeS A MethoDoLOgy WHeRe a MUlti-pARamEtRiC strUcTUrAL NuMeRIcAl ANalysis Is integratED WiTh a muLtI-FACTORIAl aNalySiS iN ORDer tO be ABLE To suMmarize A gReat AmOuNT Of RESuLts aNd to buIlD pREDIctivE AnALYtIC modELS. oVeralL, it wAS poSSIBle To deteRmInE soMe CrItEria FoR SYStem OPtIMiZAtion.
2.. materiaLS AnD meTHODoLogY
=============================
Stress ANAlySIs ReQuIReD tHe coNsTrUCtIon of AppoSiTe fe ModElS, wHIch werE THEN vaLIdaTed By mEaNs of EXpEriMeNTaL tEsts.
2.1.. fiNIte ELeMEnt moDEl
--------------------------
tHE ModEL sHOwN in Fig. (**[1](#F1){Ref-tyPe="FIG"}**) wAS DEveLopEd usING MSc marc^®^ 2003 sOfTWaRE.
tHe Bone coNsisTS OF CORtiCAL BOne (E =11 gpa, Ν=0.33, 5 mM iN ThIcKnESS) AND tRABECulAr boNE. Two differenT tRabecuLar boNE densItIES WeRe siMULaTed (table **[1](#t1){Ref-tyPE="taBlE"}**), aNd THEiR rEspEctiVe mECHaNiCaL pROPeRTIes wERE obTAINEd from THE RElaTioNs \[[@r14], [@r16]\]:
$$e = 2015 \cdot \matHiT{\rho}^{25}$$
$$\mAthIT{\SIgmA}_{u} = 0.0042 \cDot e - 0.039$$
WheRe:
ρ is BoNE deNsiTY (g/CM^3^);
e iS youNg's mOduluS (mPa);
Σ~U~ IS tHE UlTImatE TensilE STRESS (MPA).
The BONE DensitY valUeS were chOSEN To siMULATe RECUrreNt CliNiCal sITUATIONs (aDuLT PAtieNT WIth mIlD boNe REsoRBInG) and sIgniFIcaNtLY diFFeRENT YOuNG's mOdUlus (1:2 RATiO).
wITH rEgArd to tHe ScrewS, fOur DIffeRENT cONSTITuTiVe MaTErIals WEre sImUlATeD In ORder TO sTUDy BotH cURrEnTLy USEd MAtEriAlS SUCH aS StAInlesS STEEL (SCrEw MODELs N. 9-10) and TitaNiUM tI6al4v (Screw mODels n. 3-8), And MORE iNNOvAtIve SoLUtIoNs such aS lOw StiffNeSS TitaNiuM (tI12mO5tA, scrEW ModEL n.2) And PMma ReINForCED bY An iNnEr Ti12Mo5t CYlInder (scRew MoDEL n.1); ThE lAsT two mAtEriAlS haVE BeEN CHOsEN iN Order to bEttEr APPrOacH TrabECulAR boNE's yOUng modulUs.
thE General GeOMetRY OF THe simuLATEd ScREWs AND THe mESh DeTailS ARe ShOwN iN fIg. (**[2](#F2){reF-tYpe="fig"}**) (SYmbOLS As IN tablE **[2](#T2){reF-TyPe="taBlE"}**); they WEre geNerateD froM An efFeCTiVElY pRoduCed GeOMETRy (sCRew MOdEl N. 9), VAryinG aLL paramEtErs, onE By oNE.
ALl THe scrEw THreAds WeRE SimuLAteD With symmeTRICAL FLAnks, filleT raDiI WERE aLl eqUaL, AnD tHe pItcH was ConstANt AlONg The sCReW aXIS. THE AxisYmmetriC mOdeL entaIls ShOrTeR cALCulaTIOn TImEs.
THe coNsTrAints AnD bouNDARy ConDiTIOnS are sHOwn In fiG. (**[1](#F1){rEF-tyPE="Fig"}**): tHEY CoRresPoND tO THe puLl Out tEst (FIg. **[1a](#F1){REF-TYpE="fig"}**) Or to an AlteRNAtiVe cOnDItion, WITH a DIfFEReNt pOsItiON of the coNStRaiNT (FIg. **[1B](#f1){ReF-TYPe="fIG"}**); tHiS AltERnAtIVe COnDItiOn SHoUlD RepRESENT a mORe ReaLisTiC working CoNDITIoN. ACCorDINg tO saiNT veNAnt'S, tHe secOnd CoNdITiOn IS eQuIvALENT To ThE FiRSt One, howEvEr The SmalL DIsPLACEmENt HYPOtheSiS canNOT BE ASsUmed a PRIoRi in thIs CaSE, DUe tO THe loW StiFFnESs OF tRAbECULaR boNE. fOr BoTH confIgUrAtIONs, ThE ImmEDIAtE pOst-OpeRAtIVe coNditIoN And THaT OF COmPLEtE OSseOiNTEGrAtIon weRe sIMuLaTeD: tHE FIRsT conditIoN WaS CReatEd THrOugh A 'TOUCH' cOnTaCt beTwEEn the scrEW And TraBEculAR BOnE (WhaTeVeR dIsPlacEMEnT is ALLOWed WitH THE ExCePtioN OF pENEtRaTIOn), WhiLe tHE SeconD wAS obtaiNEd ThROuGH A 'GLUE' conTaCt (THE TwO coMpOnENTS ARe CompleTELY BoUNdEd tO EACH OTHer).
tHE MEan nuMBEr oF EleMenTS PEr Model wAS 7500.
2.2.. maxIMUM alloweD FoRCE eVAlUatiOn
--------------------------------------
THe mAxiMUM ALLoweD foRCE Was CaLCulATeD by iTERATioN (non-LiNeaR casE), SPECiFyING that The MaxiMUm STrESs oN The Bone mUST not exceed ITS uLTimATe tENsile sTRess. tHIs HYPOthEsis Is QuiTE reSTrICtIvE: actuaLLy BoNe iS NOT a FrAgiLe mATeriAl, teNsion pEAKS PRodUce locALiZed PLAStIc dEfoRmaTiONS ANd streSseS aRe REdistRIbUTEd OvER A WIDer ARea. hOwEVeR BONE Is LIKeLY tO be STREssEd BY DynAMIC loaDS anD FATIguE daMaGe MAy OCCUr when stRess Peaks prODUce A crAck whIcH PROGresSiVEly PROPAgATEs dUrINg loaDING, until StRUCTural FaILuRE OCcURS.
2.3.. EXPerIMenTAL tEStS
------------------------
THE fINIte ElemeNT MOdel WiTh 'TOuCh' BouNdary cOndITIOn was VAlIDaTed bY MeaNs of eXpeRimEntaL tests. alL tESTS weRE PErfoRMEd oN an hydRauliC InsTrOn 8872 TEst MACHINe, eQuiPPED wITh a ±5 Kn loAd Cell.
PuLl oUT TEsTS \[[@R19]\] WERE ConDucTed REpLACiNG BOne By pOlYURethanE FOAM, as rEportEd in the LITEraTurE \[[@r20]\].
The mECHAnicAL PropeRtiES OF poLYuretHanE foAm weRe dETErmiNeD bY teN CoMprEssiOn ANd teN TEnSilE StatIc tests, WHerE a LInEaRlY iNCReASinG DIsPLAcEMEnT was APPLIEd. the NOMinaL STrESs wAs cAlculaTED FroM The FORcE, DIvIDed BY tHe inITIAL SpeciMeN sectioN; the NOMInAl strAIn WAS CaLcULaTED from The cUrRENT DiSpLAcemEnt, DIvidED BY THe INITiAL SPecImen HEighT.
yoUnG's mOdUlUS WaS dEterminEd thrOuGh fivE dyNaMic TesTs, ApplyinG A PulsAtINg siNUsoIDAL LOAD (fig. **[3](#f3){ref-TyPE="FIg"}**, lEFT).
yOuNG'S mOdULus WAS CAlCuLAtEd aS 6 MpA (S.d. = ± 1.21 MPa), while uLtImaTe TeNSIlE streSS WaS 0.42 mpA (S.D. = ± 0.084 MPA); THese dATa wErE inPUT intO THE numeriC moDeL To Be vALIdAted. CoMpREsSiON tesTs ProDuceD A PRogReSsiVE PACKIng OF tHe FoaM (rEACHiNg up to 60% HEiGHT rEductIOn) Which does Not brEaK BuT shOWs BUCKling.
tHE pULL OUT tEStS rEqUirED sPECIFIc EQuipMeNt: Two holes drILlED In the oPpOSITe sIdes of A REctAngUlaR metAl prOfIlE (ObTainEd FroM AN exTruDED BAr) AnD A POLyurEthANe blOcK WAS plAcEd wiTHiN The BOX PROFile (fIg. **[4](#F4){ReF-type="FIg"}**).
A BoLT scREWed INtO the LowER HoLE Was tHEN heLd by thE lower jAw Of tHe MAChIne (The boLt waS lEft free tO MOVE IN tHE crOsS-secTIONAl PlaNE, IN ORDeR To AvoiD BeNDiNG moMEnTs aCtiNG oN The sCrEW). A ScREw wAS inserTed THRoUgh THe UPPEr hOLE AND iNSERteD INTO THE polyuRETHANe FOAM. thE meTAl ProfilE SimulatED THe cOrtiCaL LAyEr Of tHe fE mODEl, aNd PREVentEd ThE PolyuRETHaNE fOAM dEFOrMIng When the screw was PUllEd. A PrEPARaToRy HOlE (6.5 mM in diaMEtER) waS DrIlLed In The FoAm beFoRe InSERTiNg THe SCRew, WhiCH was AlWAYS sEt At the samE hEiGHt. the TeSts wEre condUCTED on a StANDaRD metrICaL ScREw (m10, unI 4536) WhoSE geomeTRy was KnoWN In deTaIL, ALloWinG an ACcURaTE Fe mODeL TO bE CONstRUCTEd. ThE LOWeR end of THe shAFT WAS tHrEaDEd wIth 17 tHReaDs.
the SCrEW WAs PULLeD At A SpeED OF 2 Mm/MiN. The Pull oUt teST Was REPEatED 23 TimES anD tHe meaN FOrCe deTERMineD Was 120 n (s.d. = ±14 n). a TyPIcal fORcE VErsUS dIspLacemEnT curVE iS shOWN in FiG. (**[4](#F4){ReF-TyPe="FIg"}**).
2.4.. ANALYSis oF fe model rESULtS
----------------------------------
tEn DIFFeRent ScrEw MoDELs were rEALiZED (taBle **[2](#T2){rEf-typE="tAbLe | 1.. INTRODUCTION ================ The pattern of stresses transferred to the bone have great influence onthe success or failure of an orthopedic implant.The evaluation of bone stresses is socomplex that it cannot be accomplished analytically,necessitating the application of FEA(FiniteElement Analysis) techniques; three types of FE model,axisymmetric, bi-dimensional and three-dimensional, are consideredin the literature \[[@R1]-[@R5]\]. Two-dimensional models areflawed in that stresses outsidetheplane of analysis are disregarded, while 3D models requirea large number of elements and, consequently,long calculation times.The axisymmetric model, where the only simplification is that the screwthreadis modeled as a disk, can be considered a good compromisebetween 2D and3D models. With regard to the constraints, thecondition of osseointegration is usually simulated, and the post-operative condition itis rarely consideredin the literature \[[@R3],[@R4],[@R6]\]. However, these two conditions produce significantly different results and can be considered the limitconditions under whichorthopedic screws operate, sothat the analysis of both cases can provide useful information.Two boundary conditions(pull out test; alternative condition) are simulated in the literature\[[@R4],[@R7]\], but the relation between their respective results has not been analyzed. This relation is relevant becausethe pull out test is the standard test fororthopedic screws, while the screws, onceimplanted, are subjectedto different loading conditions. Regarding bone material, most models in the literature consider bone an isotropic, elasticand homogenous material\[[@R1]-[@R4],[@R7]-[@R9]\], while in reality boneis anisotropicbecause of itstrabecular structure. In thisstudy, bone isotropy wasassumed in ordertoobtain more general results, while bone structure was defined by a single parameter, its volumetric density.In the literature, the geometric and mechanical parameters of the screw generally considered are: pitch \[[@R10],[@R11]\], length, flank angle, and material \[[@R2]-[@R5]\]. Few authors have considered thefillet radius \[[@R4],[@R7]\], whilemany modelshave sharp edges \[[@R1],[@R3],[@R12]\].Littleemphasis has been given to screw performance in relation to bone density \[[@R5],[@R13]\] even though it iswellknown that the densityof the bone determines its mechanical properties \[[@R14]-[@R18]\]. The present studyevidences that it isnot possible to select the appropriate screw without considering bone density.Differentmodels were developed,considering the geometry and mechanicalproperties of commercial screws, and a parametric analysis was undertaken to assess how thestrength of the bone-screwsystem varies for different valuesof thread pitch and bone density. Actually, this paper introducesa methodology where a multi-parametric structural numericalanalysis isintegrated with amulti-factorial analysisin order to be able to summarize a great amount of results and to build predictive analytic models.Overall,it was possible todeterminesome criteria forsystem optimization. 2..MATERIALS AND METHODOLOGY ============================= Stressanalysis required the construction of apposite FE models, which were then validatedby meansof experimental tests. 2.1.. Finite Element Model -------------------------- Themodel shown in Fig. (**[1](#F1){ref-type="fig"}**) was developedusingMSC MARC^®^ 2003 software. The boneconsists ofcorticalbone (E =11 GPa, ν=0.33,5 mm in thickness) and trabecularbone. Two different trabecular bone densities were simulated (Table **[1](#T1){ref-type="table"}**), and their respective mechanical properties were obtained fromthe relations\[[@R14], [@R16]\]:$$E = 2015 \cdot \mathit{\rho}^{25}$$ $$\mathit{\sigma}_{U} = 0.0042\cdot E - 0.039$$ where: ρ isbone density (g/cm^3^);E is Young's modulus (MPa); σ~U~ is the ultimate tensile stress (MPa). The bone density values were chosen to simulate recurrentclinical situations(adultpatient with mild bone resorbing) and significantly different Young's modulus (1:2 ratio). With regardto the screws, four differentconstitutive materials were simulated in orderto study both currently used materials such as stainless steel (screw modelsn. 9-10)and titanium Ti6Al4V (screw models n.3-8), and moreinnovative solutions such aslow stiffnesstitanium (Ti12Mo5Ta, screw model n.2)and PMMA reinforced by aninner Ti12Mo5Tcylinder (screw model n.1); the last twomaterials have been chosen in order tobetter approachtrabecular bone's Young modulus. Thegeneral geometry of the simulated screws andthe mesh details are shown in Fig. (**[2](#F2){ref-type="fig"}**) (symbols as in Table **[2](#T2){ref-type="table"}**); they were generated from an effectively produced geometry (screw model n. 9), varying all parameters, one byone. All thescrew threads were simulatedwith symmetrical flanks, fillet radii wereall equal, and the pitch was constant along the screw axis. The axisymmetric model entails shorter calculation times.The constraints and boundary conditions are shown in Fig. (**[1](#F1){ref-type="fig"}**): theycorrespond to the pull out test(Fig. **[1a](#F1){ref-type="fig"}**) or to analternative condition, with adifferent position of the constraint (Fig. **[1b](#F1){ref-type="fig"}**); this alternative condition should represent a more realistic working condition. According to Saint Venant's, the second condition is equivalent to the first one, however the small displacement hypothesiscannot be assumed a priori in this case, due to the low stiffness of trabecular bone. For both configurations, the immediate post-operative condition and that of complete osseointegrationwere simulated: the first condition wascreated through a 'touch' contact betweenthe screw and trabecular bone(whatever displacement is allowedwith the exceptionof penetration), while the second was obtained through a 'glue' contact(the two components are completely bounded toeach other). The mean number of elements per model was 7500. 2.2.. Maximum Allowed Force Evaluation -------------------------------------- The maximum allowed force was calculatedby iteration (non-linear case),specifying that the maximum stress on the bone must notexceed its ultimatetensile stress. Thishypothesis is quite restrictive: actually bone is nota fragile material, tension peaks produce localized plastic deformations andstresses are redistributed over a wider area. However bone is likely tobe stressedby dynamic loads and fatiguedamagemay occurwhen stress peaks produce a crackwhichprogressively propagates during loading, until structuralfailure occurs. 2.3.. Experimental Tests ------------------------ Thefinite element model with 'touch' boundary condition was validated bymeans of experimental tests. All testswere performed onan hydraulicINSTRON 8872 test machine, equipped with a ±5kN loadcell. Pull outtests \[[@R19]\] were conducted replacingbone by polyurethane foam, as reported in the literature \[[@R20]\]. The mechanical properties of polyurethane foamwere determinedby ten compressionand tentensile static tests, where a linearly increasing displacement was applied. The nominal stress was calculated from theforce, divided bythe initial specimen section; the nominal strain was calculated from the currentdisplacement, divided by the initial specimen height. Young's modulus was determined through five dynamic tests, applying a pulsatingsinusoidal load (Fig. **[3](#F3){ref-type="fig"}**, left).Young's modulus was calculated as6 MPa(s.d. = ± 1.21 MPa), while ultimate tensile stress was 0.42 MPa (s.d. = ± 0.084 MPa); these datawere input into the numeric model to be validated. Compression tests produced a progressive packing ofthe foam (reachingup to 60%height reduction) which does not break but shows buckling. The pull out tests required specific equipment: two holes drilled in theoppositesides of a rectangular metal profile (obtained from an extruded bar) and a polyurethane blockwas placed within the box profile (Fig. **[4](#F4){ref-type="fig"}**). Abolt screwed into the lower hole wasthen held by the lowerjaw of themachine (the bolt was left free to move in the cross-sectional plane, in order to avoid bending moments acting onthescrew). A screw was inserted through the upper hole andinserted into the polyurethanefoam.The metal profile simulated the cortical layer of the FE model, and prevented the polyurethane foamdeforming when the screwwas pulled. A preparatoryhole (6.5mm in diameter) wasdrilled in the foam before inserting the screw, which was always set at the same height.The tests were conducted on astandard metrical screw(M10, UNI 4536) whose geometry was known in detail, allowing an accurate FEmodel to be constructed. The lowerend of the shaft was threaded with 17 threads. The screw was pulled at a speed of 2 mm/min. The pull out test wasrepeated 23 times and the mean forcedetermined was 120 N (s.d. = ±14 N). A typical force versus displacement curve is shown in Fig. (**[4](#F4){ref-type="fig"}**).2.4..Analysis of FE Model Results ----------------------------------Ten different screw models wererealized (Table **[2](#T2){ref-type="table | 1.. INTRODUCTION ================ The pattern of stresses transferred to the bone have great _influence_ on _the_ _success_ or _failure_ of _an_ orthopedic implant. The evaluation of bone stresses _is_ so complex that it cannot _be_ _accomplished_ analytically, _necessitating_ the application of FEA (Finite Element Analysis) techniques; three types of FE model, _axisymmetric,_ bi-dimensional _and_ three-dimensional, are considered in the _literature_ _\[[@R1]-[@R5]\]._ _Two-dimensional_ models are flawed in that stresses outside the plane of analysis are disregarded, while 3D _models_ require _a_ large number of elements and, consequently, _long_ calculation times. The axisymmetric model, where the only simplification is _that_ the screw thread is modeled as _a_ disk, can be considered a good compromise between 2D and 3D _models._ With regard to the constraints, the condition of osseointegration is _usually_ simulated, and the post-operative _condition_ _it_ is rarely considered in the literature \[[@R3],[@R4],[@R6]\]. However, _these_ _two_ conditions produce _significantly_ different results and can _be_ considered _the_ limit conditions under which orthopedic screws operate, so that the analysis of both _cases_ can provide useful information. _Two_ boundary conditions _(pull_ out test; alternative condition) are _simulated_ in _the_ literature \[[@R4],[@R7]\], but _the_ relation between their respective results has not been analyzed. This relation is _relevant_ _because_ the pull out test is the standard test for orthopedic _screws,_ _while_ the screws, once implanted, are subjected to different loading _conditions._ Regarding bone _material,_ _most_ _models_ in the literature consider bone an isotropic, elastic and homogenous material _\[[@R1]-[@R4],[@R7]-[@R9]\],_ _while_ in reality bone is anisotropic _because_ of _its_ trabecular structure. In this study, _bone_ _isotropy_ was assumed in order to obtain more _general_ results, while bone structure was _defined_ by _a_ single parameter, its volumetric density. In the literature, the geometric and mechanical parameters _of_ _the_ screw _generally_ _considered_ are: pitch \[[@R10],[@R11]\], length, flank angle, and material \[[@R2]-[@R5]\]. Few authors have considered the fillet radius \[[@R4],[@R7]\], while many _models_ have sharp edges \[[@R1],[@R3],[@R12]\]. Little emphasis has _been_ given to screw _performance_ in relation to _bone_ density _\[[@R5],[@R13]\]_ even though it is well known that the density of the bone determines its mechanical properties _\[[@R14]-[@R18]\]._ The _present_ _study_ evidences that it is _not_ possible _to_ _select_ the appropriate screw without considering bone _density._ Different models were developed, considering the geometry and mechanical properties of _commercial_ _screws,_ _and_ a parametric analysis was undertaken to assess how the strength _of_ the bone-screw system varies _for_ different values of thread pitch _and_ bone _density._ _Actually,_ this paper introduces a methodology where a multi-parametric _structural_ numerical analysis is _integrated_ _with_ a multi-factorial _analysis_ _in_ order to be _able_ to summarize _a_ great amount _of_ results _and_ to build _predictive_ analytic models. Overall, it was possible to determine some criteria for system optimization. _2.._ _MATERIALS_ AND METHODOLOGY ============================= Stress analysis required the construction _of_ _apposite_ FE models, _which_ were then validated by means of experimental _tests._ _2.1.._ Finite Element Model -------------------------- The model shown in _Fig._ (**[1](#F1){ref-type="fig"}**) was developed using _MSC_ MARC^®^ 2003 software. _The_ _bone_ _consists_ of cortical bone _(E_ _=11_ GPa, ν=0.33, 5 _mm_ in _thickness)_ and trabecular bone. Two different trabecular _bone_ densities were simulated _(Table_ _**[1](#T1){ref-type="table"}**),_ and their _respective_ mechanical _properties_ were obtained from the relations \[[@R14], [@R16]\]: $$E = 2015 _\cdot_ _\mathit{\rho}^{25}$$_ $$\mathit{\sigma}_{U} = 0.0042 \cdot E - _0.039$$_ where: ρ is bone density (g/cm^3^); E _is_ Young's modulus (MPa); σ~U~ _is_ the _ultimate_ _tensile_ stress (MPa). The bone density _values_ were chosen _to_ simulate recurrent clinical _situations_ (adult patient _with_ mild bone resorbing) and significantly _different_ Young's _modulus_ (1:2 ratio). With regard to _the_ screws, four different constitutive materials were simulated in order to study both currently used materials _such_ as stainless _steel_ (screw models n. 9-10) and titanium Ti6Al4V _(screw_ _models_ _n._ _3-8),_ and more innovative solutions such as low stiffness titanium (Ti12Mo5Ta, screw model n.2) and PMMA reinforced by an inner _Ti12Mo5T_ cylinder (screw model n.1); the last two materials have been chosen in order to better _approach_ trabecular bone's Young modulus. _The_ general geometry of the _simulated_ _screws_ and the mesh details are shown in Fig. (**[2](#F2){ref-type="fig"}**) (symbols _as_ in Table **[2](#T2){ref-type="table"}**); they were generated from an effectively _produced_ geometry (screw _model_ n. 9), varying all _parameters,_ _one_ by one. All the _screw_ threads were simulated _with_ symmetrical flanks, fillet radii were all equal, and the pitch _was_ constant along the screw axis. The axisymmetric model entails shorter _calculation_ times. The constraints and boundary conditions are _shown_ _in_ _Fig._ (**[1](#F1){ref-type="fig"}**): _they_ correspond to the _pull_ out test _(Fig._ **[1a](#F1){ref-type="fig"}**) or to an alternative condition, with a different position of _the_ _constraint_ (Fig. _**[1b](#F1){ref-type="fig"}**);_ this alternative condition _should_ _represent_ a _more_ realistic working condition. According _to_ Saint _Venant's,_ the second condition is equivalent to the first one, however the _small_ _displacement_ hypothesis cannot be assumed a priori in this case, due to the low stiffness _of_ trabecular bone. For both _configurations,_ the immediate post-operative condition and that of _complete_ osseointegration were simulated: the first condition was created through a 'touch' contact between the screw _and_ _trabecular_ bone (whatever displacement is allowed _with_ the exception of penetration), while the _second_ was _obtained_ through a 'glue' contact (the _two_ components are completely _bounded_ to each other). The mean number _of_ elements per model _was_ 7500. 2.2.. Maximum _Allowed_ _Force_ Evaluation _--------------------------------------_ The maximum _allowed_ _force_ was calculated by iteration _(non-linear_ _case),_ specifying that the maximum stress on the _bone_ must not exceed its _ultimate_ tensile _stress._ This hypothesis is quite restrictive: actually _bone_ is not _a_ _fragile_ material, tension peaks produce localized plastic deformations and stresses are _redistributed_ over _a_ _wider_ area. However bone is likely to _be_ stressed by dynamic loads and fatigue damage may _occur_ when stress peaks produce a _crack_ which progressively propagates during _loading,_ until structural failure occurs. _2.3.._ Experimental Tests ------------------------ The finite element _model_ with 'touch' boundary condition _was_ validated by means of experimental tests. All tests were _performed_ on an _hydraulic_ INSTRON 8872 test _machine,_ equipped with a _±5_ _kN_ load cell. _Pull_ out _tests_ \[[@R19]\] were conducted replacing bone by polyurethane foam, as reported in the literature \[[@R20]\]. The mechanical properties of polyurethane foam were determined by ten compression _and_ ten tensile static tests, where _a_ linearly increasing displacement was applied. The _nominal_ _stress_ was calculated from the force, divided by the initial specimen section; the nominal strain _was_ calculated from the current displacement, _divided_ by the initial specimen height. Young's modulus was determined through five dynamic tests, applying a pulsating _sinusoidal_ load (Fig. **[3](#F3){ref-type="fig"}**, left). Young's modulus _was_ calculated as 6 MPa (s.d. = ± _1.21_ MPa), while ultimate tensile _stress_ was _0.42_ MPa (s.d. _=_ ± 0.084 MPa); these data were input into the numeric _model_ _to_ be validated. _Compression_ _tests_ produced a _progressive_ packing _of_ _the_ _foam_ (reaching _up_ _to_ _60%_ _height_ reduction) _which_ does not break _but_ shows buckling. The pull out tests required specific equipment: two holes drilled in the opposite sides of a rectangular _metal_ profile (obtained from an extruded bar) and a polyurethane block was _placed_ within the box profile _(Fig._ **[4](#F4){ref-type="fig"}**). A bolt screwed _into_ the _lower_ hole was then held _by_ _the_ lower _jaw_ of the machine (the bolt was _left_ free to _move_ in the cross-sectional plane, in order to _avoid_ bending moments acting on the screw). A screw _was_ inserted through the upper hole and inserted into _the_ polyurethane foam. _The_ _metal_ profile simulated the cortical layer of the FE _model,_ _and_ prevented the polyurethane foam deforming when the screw was pulled. A preparatory hole (6.5 mm in diameter) _was_ _drilled_ in the foam before inserting _the_ screw, _which_ _was_ always set _at_ the same _height._ The tests _were_ conducted on a standard metrical _screw_ (M10, UNI 4536) whose geometry was _known_ in detail, allowing an _accurate_ FE model _to_ be _constructed._ The _lower_ end of the shaft was threaded _with_ _17_ _threads._ The screw _was_ pulled at a speed of 2 _mm/min._ The _pull_ out test _was_ repeated 23 _times_ and the mean force determined was 120 N (s.d. = ±14 N). A typical force versus displacement _curve_ is shown in Fig. (**[4](#F4){ref-type="fig"}**). _2.4.._ Analysis of FE Model Results ---------------------------------- Ten _different_ _screw_ models were _realized_ (Table **[2](#T2){ref-type="table |
Introduction {#s1}
============
Iron (Fe) deficiency is among the most prevalent micronutrient deficiencies in humans. Since plants constitute the primary source of nutrients for a large part of the world's population, the improvement of plants in terms of nutrient bioavailability is considered a priority [@pone.0099234-deBenoist1]. Micronutrients like Fe are often present in an un-soluble form in the soil. Plants are able to mobilize such nutrients for uptake into the roots. Plants can also mobilize Fe from internal stores. Understanding the regulation of Fe acquisition and internal Fe utilization is of high importance for precision breeding of crops that are improved to either tolerate growth on alkaline and calcareous soils with poor Fe bio-availability or to accumulate a higher content of this micronutrient in bio-available form in the edible plant parts.
Genetic traits have been associated with micronutrient content and usage in plants, for example [@pone.0099234-Uauy1], [@pone.0099234-Baxter1]. Another trait was found in soybean as being linked to transcription factor genes encoding the soybean homologs of *BHLH38* and *BHLH39* [@pone.0099234-Peiffer1]. The potential importance of these two transcription factor genes for Fe mobilization had previously been uncovered in studies on the plant model *Arabidopsis thaliana*. *BHLH38* and *BHLH39* belong to the so-called subgroup Ib(2) *BHLH* genes [@pone.0099234-Pires1] and they are functionally redundant [@pone.0099234-Wang1]--[@pone.0099234-Wang2]. In fact, *BHLH38* and *BHLH39* are tandem duplicates on the chromosome, and they share similarity with two other *BHLH* genes, namely *BHLH100* and *BHLH101* [@pone.0099234-Wang1], [@pone.0099234-Heim1]--[@pone.0099234-ToledoOrtiz1]. All these four subgroup Ib(2) *BHLH* genes are highly induced by low Fe supply in roots and leaves while they are not usually found expressed under sufficient Fe supply [@pone.0099234-Wang1]. Expression of *BHLH39* and *BHLH101* in response to iron can be followed using the public microarray data in Arabidopsis [@pone.0099234-Bauer1]--[@pone.0099234-Yang1] and it was found that they occur in a co-expression network along with several Fe homeostasis genes like *FERRIC REDUCTASE OXIDASE3* (*FRO3*), *NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN4* (*NRAMP4*) and *NICOTIANAMINE SYNTHASE4* (*NAS4*) [@pone.0099234-Ivanov1]. From the co-expression with Fe homeostasis genes it can be concluded that the subgroup Ib(2) *BHLH* transcription factor genes likely perform regulatory functions in the context of Fe homeostasis and internal Fe mobilization. The bHLH protein POPEYE (PYE, belonging to another bHLH subgroup) is also induced by Fe deficiency within this co-expression network and it acts as a negative regulator of *FRO3*, *NRAMP4* and *NAS4,* presumably to avoid over-activation of Fe mobilization [@pone.0099234-Long1]. PYE is regulated by BRUTUS (BTS) that is also found in this co-expression network [@pone.0099234-Ivanov1], [@pone.0099234-Long1]. bHLH subgroup Ib(2) can physically interact with the bHLH FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) [@pone.0099234-Wang2], [@pone.0099234-Yuan1]. FIT is expressed specifically in roots and has been shown to be essential for Fe uptake [@pone.0099234-Bauer2]--[@pone.0099234-Yuan2] by regulating the expression of the genes encoding ARABIDOPSIS H^+^-ATPASE2 (AHA2) [@pone.0099234-Ivanov1], Fe reductase FERRIC OXIDASE2 (FRO2) [@pone.0099234-Jakoby1], [@pone.0099234-Robinson1] and the IRON-REGULATED TRANSPORTER1 (IRT1) [@pone.0099234-Jakoby1], [@pone.0099234-Eide1]. From ectopic FIT expression experiments along with yeast promoter activation assays and inducible FIT activation in plants, it can be concluded that FIT targets *FRO2* and *IRT1* gene promoters [@pone.0099234-Jakoby1], [@pone.0099234-Yuan2], [@pone.0099234-Meiser1], [@pone.0099234-Sivitz2]. However, FIT induces *IRT1* and *FRO2* only upon Fe deficiency even when overexpressed [@pone.0099234-Jakoby1], [@pone.0099234-Meiser1]. The activation of FIT at low Fe can be explained with the presence of bHLH subgroup Ib(2) factors. Indeed, the double overexpression of FIT together with either bHLH subgroup Ib(2) protein leads to an increase of Fe acquisition responses under sufficient Fe supply conditions, and it was therefore proposed that the function of bHLH subgroup Ib(2) might be to induce Fe deficiency responses in conjunction with FIT [@pone.0099234-Wang2], [@pone.0099234-Yuan1]. However, the occurrence of *BHLH* subgroup Ib(2) genes in the *PYE* coexpression network, their non-expression upon sufficient Fe (where *FIT* and *IRT1* are active although at low level) and their high induction upon Fe deficiency not only in roots but also in leaves (in contrast to Fe acquisition genes) renders this hypothesis questionable. Moreover, contradictory results have been published with regard to the function of bHLH subgroup Ib(2) proteins. In one report, double *bhlh100 bhlh101* knockout mutants were demonstrated to develop a more severe leaf chlorosis than the wild type upon Fe deficiency, while no phenotype was apparent upon Fe sufficiency. Although some Fe homeostasis genes appeared mis-expressed, the gene knockouts did not affect the plants' abilities for Fe uptake and the regulation of *FRO2* and *IRT1* upon sufficient or deficient Fe supply [@pone.0099234-Sivitz1]. In contrast to that, in another report, bHLH subgroup Ib(2) knockouts including *bhlh100 bhlh101* and a triple knockout *bhlh39 bhlh100 bhlh101* were demonstrated to affect Fe acquisition responses and to have low *FRO2* and *IRT1* expression upon sufficient or deficient Fe supply [@pone.0099234-Wang3]. This latter finding was rather puzzling, and it was not further explained how this finding fits to the observation that the *BHLH* genes are not normally expressed upon sufficient Fe supply, when Fe also needs to be acquired via FRO2 and IRT1 [@pone.0099234-Jakoby1], [@pone.0099234-Vert1]. Thus, the function of the bHLH subgroup Ib(2) transcription factors in Fe uptake is still open for debate.
Very interestingly, it has been shown that *BHLH38* and *BHLH39* were induced after application of salicylic acid ( = SA) by the SA-inducible Dof ( = DNA binding with one finger) transcription factor OBF BINDING PROTEIN3 (OBP3) [@pone.0099234-Kang1]. Binding of OBP3 to promoter elements in *BHLH38* and *BHLH39* genes and their subsequent activation was demonstrated (in these studies *BHLH38* and *BHLH39* were named *OBP3 RESPONSIVE GENE2*, *ORG2*, and *OBP3 RESPONSIVE GENE3*, *ORG3*) [@pone.0099234-Kang1]. Jasmonic acid negatively affects the onset of Fe mobilization and the induction of *FRO2* and *IRT1* [@pone.0099234-Maurer1], while ethylene enhances the responses [@pone.0099234-Garca1]--[@pone.0099234-Lingam1]. Since SA, jasmonic acid and ethylene act in stress response networks, the possibility exists that perhaps, there is a link between SA and the up-regulation of Fe deficiency responses.
Here, we made use of the triple knockout mutant *bhlh39 bhlh100 bhlh101* (*3xbhlh*) that we constructed to investigate the functions of these *BHLH* genes in the Fe deficiency response and to further shed light on the question whether SA is involved in mediating the onset of Fe uptake via the induction of *BHLH* subgroup Ib(2) genes. We discuss that *BHLH39*, *BHLH100* and *BHLH101* are essential for a subset of Fe deficiency responses but not including up-regulation of *IRT1* and *FRO2*. We suggest that these transcription | introduction { # → } = = = = = = = = = = = = iron ( fe ) deficiency is among the most prevalent micronutrient deficiencies in humans. since plants constitute the primary supply of phosphorus for a large part of the world ' s population, the improvement of plants in terms of nutrient bioavailability is considered a priority [ @ pone. 0099234 - debenoist1 ]. micronutrients like fe are often present in an enzyme - soluble form in the soil. plants are able to mobilize such nutrients for uptake into the roots. plants may also mobilize fe from internal stores. understanding the regulation of fe acquisition and internal fe utilization is of high importance for precision breeding of crops that are impossible to either tolerate growth on alkaline and calcareous soils with poor fe bio - supply or to accumulate a higher content of this micronutrient in bio - available form in the edible plant parts. genetic traits have been discovered with micronutrient content and usage in plants, for example [ @ pone. 0099234 - uauy1 ], [ @ pone. 0099234 - baxter1 ]. another trait was found in soybean as being linked to transcription factor transcription encoding the soybean homologs of * bhlh38 * and * bhlh39 * [ @ pone. 0099234 - peiffer1 ]. the potential importance of these two transcription factor genes for fe mobilization had previously been uncovered in studies on the plant model * arabidopsis thaliana *. * bhlh38 * and * bhlh39 * belong to the so - called subgroup ib ( 2 ) * bhlh * genes [ @ pone. 0099234 - pires1 ] and they are functionally redundant [ @ pone. 0099234 - wang1 ] - - [ @ pone. 0099234 - wang2 ]. in fact, * bhlh38 * and * bhlh39 * are tandem duplicates on the chromosome, and they share similarity with two other * bhlh * families, namely * bhlh100 * and * bhlh101 * [ @ pone. 0099234 - wang1 ], [ @ pone. 0099234 - heim1 ] - - [ @ pone. 0099234 - toledoortiz1 ]. all these four subgroup ib ( 2 ) * bhlh * genes are highly induced by low fe supply in roots and leaves while they are not usually found expressed under sufficient fe supply [ @ pone. 0099234 - wang1 ]. expression of * bhlh39 * and * bhlh101 * in response to iron can be followed using the public microarray data in arabidopsis [ @ pone. 0099234 - bauer1 ] - - [ @ pone. 0099234 - yang1 ] and it was found that they occur in a co - expression network along with several fe homeostasis genes like * ferric reductase oxidase3 * ( * fro3 * ), * natural resistance - associated macrophage protein4 * ( * nramp4 * ) and * nicotianamine synthase4 * ( * nas4 * ) [ @ pone. 0099234 - ivanov1 ]. from the co - expression with fe homeostasis genes it can be concluded that the subgroup ib ( 2 ) * bhlh * transcription factor genes likely perform regulatory functions in the context of fe homeostasis and internal fe mobilization. the bhlh protein popeye ( pye, belonging to another bhlh subgroup ) is also induced by fe deficiency within this co - expression network and it acts as a negative regulator of * fro3 *, * nramp4 * and * nas4, * presumably to avoid over - activation of fe mobilization [ @ pone. 0099234 - long1 ]. pye is regulated by brutus ( bts ) that is also found in this co - expression network [ @ pone. 0099234 - ivanov1 ], [ @ pone. 0099234 - long1 ]. bhlh subgroup ib ( 2 ) can physically interact with the bhlh fer - like iron deficiency - induced transcription factor ( fit ) [ @ pone. 0099234 - wang2 ], [ @ pone. 0099234 - yuan1 ]. fit is expressed specifically in roots and has been shown to be essential for fe uptake [ @ pone. 0099234 - bauer2 ] - - [ @ pone. 0099234 - yuan2 ] by regulating the expression of the genes encoding arabidopsis h ^ + ^ - atpase2 ( aha2 ) [ @ pone. 0099234 - ivanov1 ], fe reductase ferric oxidase2 ( fro2 ) [ @ pone. 0099234 - jakoby1 ], [ @ pone. 0099234 - robinson1 ] and the iron - regulated transporter1 ( irt1 ) [ @ pone. 0099234 - jakoby1 ], [ @ pone. 0099234 - eide1 ]. from ectopic fit expression experiments along with yeast promoter activation assays and inducible fit activation in plants, it can be concluded that fit targets * fro2 * and * irt1 * gene promoters [ @ pone. 0099234 - jakoby1 ], [ @ pone. 0099234 - yuan2 ], [ @ pone. 0099234 - meiser1 ], [ @ pone. 0099234 - sivitz2 ]. however, fit induces * irt1 * and * fro2 * only upon fe deficiency even when overexpressed [ @ pone. 0099234 - jakoby1 ], [ @ pone. 0099234 - meiser1 ]. the activation of fit at low fe can be explained with the presence of bhlh subgroup ib ( 2 ) factors. indeed, the double overexpression of fit together with either bhlh subgroup ib ( 2 ) protein leads to an increase of fe acquisition responses under sufficient fe supply conditions, and it was therefore proposed that the function of bhlh subgroup ib ( 2 ) might be to induce fe deficiency responses in conjunction with fit [ @ pone. 0099234 - wang2 ], [ @ pone. 0099234 - yuan1 ]. however, the occurrence of * bhlh * subgroup ib ( 2 ) genes in the * pye * coexpression network, their non - expression upon sufficient fe ( where * fit * and * irt1 * are active although at low level ) and their high induction upon fe deficiency not only in roots but also in leaves ( in contrast to fe acquisition genes ) renders this hypothesis questionable. moreover, contradictory results have been published with regard to the function of bhlh subgroup ib ( 2 ) proteins. in one report, double * bhlh100 bhlh101 * knockout mutants were demonstrated to develop a more severe leaf chlorosis than the wild type upon fe deficiency, while no phenotype was apparent upon fe sufficiency. although some fe homeostasis genes appeared mis - expressed, the gene knockouts did not affect the plants ' abilities for fe uptake and the regulation of * fro2 * and * irt1 * upon sufficient or deficient fe supply [ @ pone. 0099234 - sivitz1 ]. in contrast to that, in another report, bhlh subgroup ib ( 2 ) knockouts including * bhlh100 bhlh101 * and a triple knockout * bhlh39 bhlh100 bhlh101 * were demonstrated to affect fe acquisition responses and to have low * fro2 * and * irt1 * expression upon sufficient or deficient fe supply [ @ pone. 0099234 - wang3 ]. this latter finding was rather puzzling, and it was not further explained how this finding fits to the observation that the * bhlh * genes are not normally expressed upon sufficient fe supply, when fe also needs to be acquired via fro2 and irt1 [ @ pone. 0099234 - jakoby1 ], [ @ pone. 0099234 - vert1 ]. thus, the function of the bhlh subgroup ib ( 2 ) transcription factors in fe uptake is still open for debate. very interestingly, it has been shown that * bhlh38 * and * bhlh39 * were induced after application of salicylic acid ( = sa ) by the sa - inducible dof ( = dna binding with one finger ) transcription factor obf binding protein3 ( obp3 ) [ @ pone. 0099234 - kang1 ]. binding of obp3 to promoter elements in * bhlh38 * and * bhlh39 * genes and their subsequent activation was demonstrated ( in these studies * bhlh38 * and * bhlh39 * were named * obp3 responsive gene2 *, * org2 *, and * obp3 responsive gene3 *, * org3 * ) [ @ pone. 0099234 - kang1 ]. jasmonic acid negatively affects the onset of fe mobilization and the induction of * fro2 * and * irt1 * [ @ pone. 0099234 - maurer1 ], while ethylene enhances the responses [ @ pone. 0099234 - garca1 ] - - [ @ pone. 0099234 - lingam1 ]. since sa, jasmonic acid and ethylene act in stress response networks, the possibility exists that perhaps, there is a link between sa and the up - regulation of fe deficiency responses. here, we made use of the triple knockout mutant * bhlh39 bhlh100 bhlh101 * ( * 3xbhlh * ) that we constructed to investigate the functions of these * bhlh * genes in the fe deficiency response and to further shed light on the question whether sa is involved in mediating the onset of fe uptake via the induction of * bhlh * subgroup ib ( 2 ) genes. we discuss that * bhlh39 *, * bhlh100 * and * bhlh101 * are essential for a subset of fe deficiency responses but not including up - regulation of * irt1 * and * fro2 *. we suggest that these transcription | Introduction {# s1} = = = = = = = = = = = = Iron (Fe) deficiency is among the most prevalent micronutrient deficiencies in humans. Since plants constitute the primary source of nutrients for a large part of the world ' s population, the improvement of plants in terms of nutrient bioavailability is considered a priority [@ pone. 0099234 - deBenoist1 ]. Micronutrients like Fe are often present in an un - soluble form in the soil. Plants are able to mobilize such nutrients for uptake into the roots. Plants can also mobilize Fe from internal stores. Understanding the regulation of Fe acquisition and internal Fe utilization is of high omportanc# for precision breeding of crops that are improved to either tolerate growth on alkaline and calcareous soils with poor Fe bio - availability or to accumulate a higher content of this micronutrient in bio - available form in the edible plant parts. Genetic traits have been associated with micronutrient content and usage in plants, for example [@ pone. 0099234 - Uauy1 ], [@ pone. 0099234 - Baxter1 ]. Another trait was found in soybean as being linked to transcription factor genes encoding the soybean homologs of * BHLH38 * and * BHLH39 * [@ pone. 0099234 - Peiffer1 ]. The potential importance of these two transcription factor genes for Fe mobilization had previously been uncovered in studies on the plant model * Arabidopsis thaliana *. * BHLH38 * and * BHLH39 * belong to the so - called subgroup Ib (2) * BHLH * genes [@ pone. 0099234 - Pires1] and they are functionally redundant [@ pone. 0099234 - Wang1] - - [@ pone. 0099234 - Wang2 ]. In daSt, * BHLH38 * and * BHLH39 * are tandem duplicates on the chromosome, and they share similarity with two other * BHLH * genes, namely * BHLH100 * and * BHLH101 * [@ pone. 0099234 - Wang1 ], [@ pone. 0099234 - Heim1] - - [@ pone. 0099234 - ToledoOrtiz1 ]. All these four subgroup Ib (2) * BHLH * genes are highly induced by low Fe supply in roots and leaves while they are not usually found expressed under sufficient Fe supply [@ pone. 0099234 - Wang1 ]. Expression of * BHLH39 * and * BHLH101 * in response to iron can be followed using the public microarray data in Arabidopsis [@ pone. 0099234 - Bauer1] - - [@ pone. 0099234 - Yang1] and it was found HMat they occur in a co - expression network along with several Fe homeostasis genes like * FERRIC REDUCTASE OXIDASE3 * (* FRO3 * ), * NATURAL RESISTANCE - ASSOCIATED MACROPHAGE PROTEIN4 * (* NRAMP4 *) and * NICOTIANAMINE SYNTHASE4 * (* NAS4 *) [@ pone. 0099234 - Ivanov1 ]. From the co - expression with Fe homeostasis genes it can be concluded that the subgroup Ib (2) * BHLH * transcription factor genes likely perform regulatory functions in the context of Fe homeostasis and intF3nal Fe mobilization. The bHLH protein POPEYE (PYE, belonging to another bHLH subgroup) is also induced by Fe deficiency within this co - expression network and it acts as a negative regulator of * FRO3 *, * NRAMP4 * and * NAS4, * presumably to avoid over - activation of Fe mobilization [@ pone. 0099234 - Long1 ]. PYE is regulated by BRUTUS (BTS) that is also found in this co - expression network [@ pone. 0099234 - Ivanov1 ], [@ pone. 0099234 - Long1 ]. bHLH subgroup Ib (2) can physically interact with the bHLH FER - LIKE IRON DEFICIENCY - INDUCED TRANSCRIPTION FACTOR (FIT) [@ pone. 0099234 - Wang2 ], [@ pone. 0099234 - Yuan1 ]. FIT is expressed specifically in roots and has been shown to be essential for Fe uptake [@ pone. 00p92e4 - Bauer2] - - [@ pone. 0099234 - Yuan2] by regulating the expression of the genes encoding ARABIDOPSIS H ^ + ^ - ATPASE2 (AHA2) [@ pone. 0099234 - Ivanov1 ], Fe reductase FERRIC OXIDASE2 (FRO2) [@ pone. 0099234 - Jakoby1 ], [@ pone. 0099234 - Robinson1] and the IRON - REGULATED TRANSPORTER1 (IRT1) [@ pone. 0099234 - Jakoby1 ], [@ pone. 0099234 - Eide1 ]. From ectopic FIT expression experiments along with yeast promoter activation assays and inducible FIT activation in plants, it can be concluded that FIT targets * FRO2 * and * IRT1 * gene promoters [@ pone. 0099234 - Jakoby1 ], [@ pone. 0099234 - Yuan2 ], [@ pone. 0099234 - Meiser1 ], [@ pone. 0099234 - Sivitz2 ]. However, FIT induces * IRT1 * and * FRO2 * only upon Fe deficiency even when overexpressed [@ pone. 0099234 - Jakoby1 ], [@ pone. 0099234 - Meiser1 ]. The activation of FIT at low Fe can be explained with the presence of bHLH subgroup Ib (2) factors. Indeed, the double overexpression of FIT together with either bHLH subgroup Ib (2) protein leads to an increase of Fe acquisition responses under sufficient Fe supply conditions, and it was therefore proposed that the function of HHLG subgroup Ib (2) might be to induce Fe deficiency responses in conjunction with FIT [@ pone. 0099234 - Wang2 ], [@ pone. 0099234 - Yuan1 ]. However, the occurrence of * BHLH * subgroup Ib (2) genes in the * PYE * coexpression network, their non - expression upon sufficient Fe (where * FIT * and * IRT1 * are active although at low level) and their high induction upon Fe deficiency not only in roots but also in leaves (in contrast to Fe acquisition genes) renders this hypothesis questionable. Moreover, contradictory results have been published with regard to the function of bHLH subgroup Ib (2) proteins. In one report, double * bhlh100 bhlh101 * knockout mutants were demonstrated to develop a more severe leaf chlorosis than the wild type upon Fe deficiency, while no phenotype was apparent upon Fe sufficiency. Although some Fe homeostasis genes appeared mis - expressed, the gene knockouts did not affect the plants ' abilities for Fe uptake and the regulation of * FRO2 * and * IRT1 * upon sufficient or deficient Fe supply [@ pone. 0099234 - Sivitz1 ]. In contrast to that, in another report, bHLH subgroup Ib (2) knockouts including * bhlh100 bhlh101 * and a triple knockout * bhlh39 bhlh100 bhlh101 * were demonstrated to affect Fe acquisition responses and to have low * FRO2 * and * IRT1 * expression upon sufficient or deficient Fe eup9ly [@ pone. 0099234 - Wang3 ]. This latter finding was rather puzzling, and it was not further explained how this finding fits to the observation that the * BHLH * genes are not normally expressed upon sufficient Fe supply, when Fe also needs to be acquired via FRO2 and IRT1 [@ pone. 0099234 - Jakoby1 ], [@ pone. 0099234 - Vert1 ]. Thus, the function of the bHLH subgroup Ib (2) transcription factors in Fe uptake is still open for debate. Very interestingly, it has been shown that * BHLH38 * and * BHLH39 * were induced after application of salicylic acid (= SA) by the SA - inducible Dof (= DNA binding with one finger) transcription factor OBF BINDING PROTEIN3 (OBP3) [@ pone. 0099234 - Kang1 ]. Binding of OBP3 to promoter elements in * BHLH38 * and * BHLH39 * genes and their subsequent activation was demonstrated (in these studies * BHLH38 * and * BHLH39 * were named * OBP3 RESPONSIVE GENE2 *, * ORG2 *, and * OHP$ RESPONSIVE GENE3 *, * ORG3 *) [@ pone. 0099234 - Kang1 ]. Jasmonic acid negatively affects the onset of Fe mobilization and the induction of * FRO2 * and * IRT1 * [@ pone. 00iI234 - kwurer1 ], while ethylene enhances the responses [@ pone. 0099234 - Garca1] - - [@ pone. 0099234 - Lingam1 ]. Since SA, jasmonic acid and ethylene act in stress response networks, the possibility exists that perhaps, there is a link between SA and the up - regulation of Fe deficiency responses. Here, we made use of the triple knockout mutant * bhlh39 bhlh100 bhlh101 * (* 3xbhlh *) that we constructed to investigate the functions of these * BHLH * genes in the Fe deficiency response and to further shed light on the question whether SA is involved in mediating the onset of Fe uptake via the induction of * BHLH * subgroup Ib (2) genes. We discuss that * BHLH39 *, * BHLH100 * and * BHLH101 * are essential for a subset of Fe deficiency responses but not including up - regulation of * IRT1 * and * FRO2 *. We suggest that these transcription | Introduction {#s1} ============ Iron (Fe) deficiency is among the most micronutrient deficiencies in humans. Since plants constitute primary of nutrients a large part of the world's population, the of plants in terms of nutrient is considered a [@pone.0099234-deBenoist1]. Micronutrients like Fe are often present in an un-soluble form in the Plants able to mobilize such nutrients for uptake into the roots. Plants can also mobilize Fe from internal stores. Understanding the regulation Fe acquisition and internal Fe utilization is of high importance for precision breeding of that are improved to either tolerate growth on alkaline and calcareous soils with poor Fe bio-availability or to accumulate a higher content of this micronutrient in bio-available form in plant parts. Genetic traits have been associated with micronutrient content and usage in plants, for example [@pone.0099234-Uauy1], [@pone.0099234-Baxter1]. Another trait was found in soybean as being linked to transcription genes the soybean homologs of *BHLH38* [@pone.0099234-Peiffer1]. The potential importance of two transcription factor genes for Fe mobilization had previously uncovered in on the plant model *Arabidopsis thaliana*. *BHLH38* and *BHLH39* belong to the subgroup Ib(2) *BHLH* genes [@pone.0099234-Pires1] and they are functionally redundant In fact, *BHLH38* and *BHLH39* are tandem duplicates on the chromosome, and share similarity with two other *BHLH* genes, namely *BHLH100* [@pone.0099234-Heim1]--[@pone.0099234-ToledoOrtiz1]. All these four subgroup Ib(2) *BHLH* genes are highly induced by low Fe supply in roots and leaves while they are not usually expressed under sufficient Fe supply [@pone.0099234-Wang1]. Expression of *BHLH39* and *BHLH101* in to iron can be followed using the public microarray data in Arabidopsis [@pone.0099234-Bauer1]--[@pone.0099234-Yang1] it was found that occur in a co-expression network along with several Fe homeostasis genes like REDUCTASE OXIDASE3* (*FRO3*), *NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN4* (*NRAMP4*) and *NICOTIANAMINE SYNTHASE4* (*NAS4*) From the co-expression with Fe homeostasis it can be concluded the subgroup Ib(2) *BHLH* transcription factor genes likely perform functions in context of Fe homeostasis and internal Fe mobilization. The bHLH protein (PYE, belonging to bHLH subgroup) is also induced by Fe deficiency within this co-expression network and acts as a regulator of *FRO3*, *NRAMP4* and *NAS4,* presumably to avoid over-activation of Fe mobilization [@pone.0099234-Long1]. PYE is regulated (BTS) is also found in this co-expression [@pone.0099234-Ivanov1], [@pone.0099234-Long1]. bHLH subgroup Ib(2) can physically interact with the bHLH FER-LIKE IRON TRANSCRIPTION FACTOR (FIT) [@pone.0099234-Yuan1]. FIT is expressed and has been shown to be essential for Fe uptake [@pone.0099234-Bauer2]--[@pone.0099234-Yuan2] by regulating the expression of the genes encoding ARABIDOPSIS H^+^-ATPASE2 (AHA2) [@pone.0099234-Ivanov1], Fe reductase OXIDASE2 (FRO2) [@pone.0099234-Jakoby1], [@pone.0099234-Robinson1] and the IRON-REGULATED TRANSPORTER1 (IRT1) [@pone.0099234-Jakoby1], [@pone.0099234-Eide1]. From ectopic FIT expression experiments along with yeast promoter activation assays and FIT activation in plants, it can be concluded FIT targets *FRO2* and *IRT1* gene promoters [@pone.0099234-Jakoby1], [@pone.0099234-Yuan2], [@pone.0099234-Meiser1], [@pone.0099234-Sivitz2]. However, FIT induces *FRO2* only upon Fe deficiency even when overexpressed [@pone.0099234-Jakoby1], [@pone.0099234-Meiser1]. The activation of FIT at low Fe can be explained with presence of bHLH factors. the double of FIT together with either bHLH subgroup Ib(2) leads to an increase Fe acquisition responses under supply conditions, and it was therefore proposed that the function of subgroup Ib(2) might to induce Fe deficiency responses in conjunction with FIT [@pone.0099234-Wang2], [@pone.0099234-Yuan1]. However, the occurrence of *BHLH* subgroup Ib(2) in the *PYE* coexpression network, their non-expression upon sufficient Fe *FIT* and *IRT1* are active although at low level) and their high induction Fe deficiency not only in roots but also leaves (in contrast to Fe acquisition genes) renders this hypothesis questionable. Moreover, contradictory results have been published with regard to the of bHLH subgroup Ib(2) proteins. In one report, double *bhlh100 bhlh101* knockout mutants were demonstrated to develop a more severe leaf chlorosis than the wild type upon Fe while no phenotype was apparent upon Fe sufficiency. Although some Fe homeostasis genes appeared mis-expressed, the gene did not affect the plants' abilities for Fe uptake and the regulation of *FRO2* and *IRT1* upon sufficient or deficient Fe supply [@pone.0099234-Sivitz1]. In contrast to that, in another report, bHLH subgroup Ib(2) knockouts including *bhlh100 bhlh101* a triple knockout *bhlh39 bhlh100 bhlh101* were demonstrated to affect Fe acquisition responses to have *FRO2* *IRT1* expression upon sufficient or deficient Fe supply [@pone.0099234-Wang3]. This latter finding was rather puzzling, was not further explained how this finding fits to the observation that the *BHLH* genes not normally expressed upon sufficient Fe supply, when Fe needs to be acquired FRO2 IRT1 [@pone.0099234-Jakoby1], [@pone.0099234-Vert1]. Thus, the function of the bHLH subgroup Ib(2) transcription factors in Fe uptake is open for Very interestingly, it has been shown that *BHLH38* and *BHLH39* induced after application of salicylic acid ( SA) by the SA-inducible Dof ( = DNA binding with one finger) transcription factor OBF BINDING PROTEIN3 [@pone.0099234-Kang1]. Binding of OBP3 to elements *BHLH38* and *BHLH39* genes their activation was demonstrated (in these studies *BHLH38* and *BHLH39* were named *OBP3 RESPONSIVE *ORG2*, and *OBP3 RESPONSIVE GENE3*, *ORG3*) [@pone.0099234-Kang1]. Jasmonic acid negatively affects the onset of Fe mobilization and the induction of *FRO2* and [@pone.0099234-Maurer1], while ethylene enhances the responses [@pone.0099234-Garca1]--[@pone.0099234-Lingam1]. Since SA, jasmonic acid and ethylene act in stress response networks, the possibility exists that perhaps, there is a link between SA and the up-regulation of Fe deficiency responses. we made use of the triple mutant *bhlh39 bhlh100 bhlh101* (*3xbhlh*) that we constructed to the functions of these *BHLH* genes in the Fe deficiency response and to further shed light on the whether SA is involved in mediating the onset of Fe uptake via the induction of *BHLH* subgroup Ib(2) genes. We discuss that *BHLH39*, and *BHLH101* essential for a subset Fe responses but up-regulation of *IRT1* and *FRO2*. We suggest that these transcription | iNTRODucTiOn {#s1}
============
iroN (fE) DEFICiEncY iS AmOng ThE MosT pReValENt MiCRonuTriENT DEfIcieNCiES IN HUmANS. SincE PLanTS ConsTItuTE The PrIMARy sOuRce OF nuTRiENTs FoR A laRge pArT OF tHE woRlD's PopuLATiOn, tHe improvEMEnT oF PLANTs IN tErmS OF nuTRIeNT biOavaIlabILITY is cONsidEREd a pRioRitY [@poNe.0099234-debEnOiSt1]. micrONuTrienTs LiKe FE ARE OFTen pReSENT in an Un-SOlublE fORM IN The soIL. PLANTS arE aBLE to mobiLIze such NUTriEnTS FoR UpTaKe INTo ThE RoOts. pLANTs CAn AlSO MobILiZe FE FRom iNtERnAl stOREs. uNDerstAnDinG tHe regULAtION of FE ACQuiSitIon And InTeRnal fe UTilIzAtioN IS Of hiGH IMpOrTaNcE for pRecISiON BREeDing oF CroPs tHat aRe IMpRoVed tO EITHER toLeraTe gRoWth on aLKALinE And caLCaREoUS soIls WItH POoR FE BiO-aVAIlabIlIty Or TO aCCUMulaTE a HIGHER cOnTENT oF THIs miCroNUtrIeNt iN bio-AvaILabLe foRm In The EdiBle PlanT pARtS.
GeneTiC TRaiTs HavE beEn aSsoCIaTEd wITH mICRoNuTrIenT cONteNt aND uSaGE IN pLaNTs, fOR EXaMPle [@pone.0099234-uauy1], [@poNE.0099234-BaXtER1]. anothER TrAIT WaS fouNd IN SOYbEaN aS BEing LiNKeD tO TRaNSCRiPTiOn FACtOR GEneS ENCOding tHE soYBEan homoLogs OF *bhLh38* aND *BHLh39* [@POne.0099234-peifFER1]. tHe poteNtIAl importanCe oF TheSE twO TRAnscRIPtiON FAcToR GeneS foR FE MoBiLizatioN Had preVIOusly bEeN UncoVErEd IN sTUDiES on The PLaNT mOdel *ARabIdopsis tHaliaNA*. *bhlh38* AnD *bhLH39* BELong tO thE so-cALlEd sUBgRoUP IB(2) *bhLH* GeNEs [@pONe.0099234-PIRES1] And They are funCtioNALLY REdUNdaNt [@poNE.0099234-WAng1]--[@PoNE.0099234-Wang2]. iN fAct, *BHlH38* ANd *bHlh39* aRE tANDEM dupliCateS ON tHE cHrOmOSOme, And tHEY ShArE siMilARITy WITH TwO OTHER *bhlH* geNeS, nameLy *bHLH100* aNd *BHlH101* [@PONE.0099234-WaNg1], [@PONe.0099234-hEIm1]--[@Pone.0099234-tOLEdOORtiz1]. AlL these fOUr sUBGROuP iB(2) *bhlH* GEnes Are hIGHLY iNduCed BY lOW FE Supply in RoOTs and LeAVes wHilE They Are not UsUaLly foUnd eXPrEsSed UndER suFfIciENT fE SUppLY [@ponE.0099234-wang1]. ExPrESsion OF *BhlH39* and *bHlH101* iN reSPonSe To irOn CAN Be FOLLOWeD USing the pUbLIC MIcRoARraY DAta IN arABidopsIs [@PonE.0099234-BaueR1]--[@PONE.0099234-yaNG1] AND IT WAs foUnD ThAt theY oCCUR In A co-eXPrEsSIon nEtWork along WIth SEVerAL fe homeOstAsIS genEs LiKE *ferRIc rEDuCTaSE OxidaSe3* (*FRO3*), *NAturaL rEsiStaNCe-asSOciaTEd MacROPhAGE ProtEIn4* (*NraMP4*) And *nICotIanAMIne SYNthAsE4* (*NaS4*) [@POnE.0099234-Ivanov1]. frOm ThE CO-ExpRESsIoN WitH Fe hOmEostAsiS geneS It CaN be CoNCluDED ThAt The SUbgROup Ib(2) *bHLh* tRanscRIpTioN FacTOr gENeS lIKELy PErFORm rEgULAtORY FuNCTiOns In thE CoNTexT oF fE HomEOsTAsIS AND InTERNAL Fe mobILIzaTIoN. tHE bHLH PRoTEIn PopeYe (PYe, BelonGIng TO anoTHer BHLh sUbGroUP) iS aLso iNduCeD bY fE dEFIcIeNcy WithIn THiS Co-exPression nETwoRk AND It aCts AS A nEGatIVE regulAtoR OF *fRO3*, *nrAMP4* And *NAs4,* pReSuMabLY tO AVOiD oVER-acTiVATION of fE MObilizATIoN [@pone.0099234-long1]. PYe IS REGulAted by BRUTUS (Bts) THaT Is also fOUND iN THIS Co-exPrESsiON NeTwOrk [@PONe.0099234-Ivanov1], [@PONe.0099234-LonG1]. bhlh sUbGROUp IB(2) can pHySically iNTERaCT with thE bhlh Fer-LiKE irOn dEfIcIEncY-iNDUCED TrANScRIpTIOn faCtOr (fIT) [@PonE.0099234-wAnG2], [@pOnE.0099234-YuAN1]. FIT Is eXPResSED SpECIFiCALLy IN rooTS And HAs been sHOwN tO bE EssENtIAl FOr Fe uPTAKe [@PONE.0099234-BAuer2]--[@pONE.0099234-yUAN2] bY rEGulatIng the ExPRESSION of the GENeS EnCODIng arABiDOPSIS H^+^-AtpAsE2 (aHa2) [@pone.0099234-iVaNOv1], Fe reduCtASE ferRic oXIDAsE2 (fRO2) [@poNE.0099234-JAkoBy1], [@pone.0099234-RObInSOn1] aND THE IrOn-REGUlaTed trAnSPORtER1 (iRt1) [@pONe.0099234-jAkoBY1], [@PonE.0099234-eide1]. FRom eCToPiC fit exPRessION ExperimEntS aloNG wITH yEaST PRoMoteR acTiVAtiON ASsays AND INduCiBlE Fit ACTIVatIOn IN PlaNts, It CaN bE CoNClUDeD tHAt FIT TaRGeTS *FrO2* and *irT1* geNE PrOMoteRs [@pone.0099234-JakoBy1], [@ponE.0099234-YUAn2], [@PoNe.0099234-meiseR1], [@POnE.0099234-SIVitZ2]. HowEVER, fIT INDUCeS *iRt1* ANd *fRo2* onlY UPon FE deFicIEnCY eVEn whEN oVeREXPrEsSEd [@pOne.0099234-JAkoBY1], [@poNe.0099234-mEiSER1]. thE aCTivatION Of fIt At loW fe CAN bE EXPlaINED wITh THe pReSenCe Of bhLh sUbgRoUp iB(2) FaCTOrs. indeed, thE DoUbLE oVERexprESSioN Of fiT TOgEtHer WiTH EItHER BHlH SuBgRoUp Ib(2) prOTeIN Leads TO An IncreASE oF Fe aCqUIsITiOn reSPonses uNdER SUFFicIEnT FE SuppLy coNdItiOnS, aNd iT waS ThErEfOre PRopOSed THAT THE FUNCtiOn Of bhLH SUBgroUP IB(2) mighT be TO INdUCe FE DefIcIeNCY RESponSes iN cOnjUnCTIon WIth FiT [@POne.0099234-waNG2], [@POne.0099234-YUaN1]. HOweveR, The OCcurrENCe oF *BHLh* sUbGroUP ib(2) GENes in ThE *Pye* CoExpreSsION NETwOrk, THEir non-eXprESSIon uPoN sUFficienT fE (WhErE *Fit* aNd *Irt1* aRe ACtiVe ALThOugH at lOW LeVEl) AnD tHeiR higH INdUCTioN Upon Fe DefICIENCY NoT onLy in Roots bUT ALsO iN lEAveS (iN cONTRasT To Fe AcqUIsItioN Genes) RENDeRs thIS HYPOthEsIS qUEstIOnable. moreOveR, cONtRadiCtoRy ReSULtS Have BEen pUblIsHed wIth Regard To thE fuNction Of BHLH SubGroUp ib(2) pRoteins. in oNe RePoRt, douBLe *bhlH100 BHLh101* kNOckoUT mUTaNTS WerE demonstrAtED tO DevELOP A MOre seVere lEaf CHLOrosIS tHan the wild TyPe UPoN fE DeFIciEnCy, WhiLE NO pHEnoTyPE wAs appARENt upon fE sufFiciEnCy. ALtHouGH SOMe fE hOMEostAsIs gEnes aPpEared mis-eXpRESSed, The GeNE KnOckouts diD NOt afFECt THe planTs' aBIlitiEs for Fe UPtAke AnD the rEGUlatioN Of *Fro2* ANd *Irt1* uPON sUfFIcIeNt oR dEFiCient fE supPLy [@POnE.0099234-SiVITz1]. in cONTRaSt TO That, IN anotHeR Report, bHlh SubGrOUp ib(2) KnOCkOutS iNCLudIng *bhlh100 bHLh101* anD a triPlE knOCKOUt *bHLh39 BhlH100 BHlh101* wERE dEMonstraTEd To AFFEcT FE ACQUIsITiOn rESPonseS aND To haVE loW *FrO2* anD *iRt1* ExpReSsion UpON sufFIcIEnt OR DeFIcIEnt Fe SUpply [@PONe.0099234-WAnG3]. THis LATtER fInDinG waS RaThEr pUzzLING, anD it WAS NoT FuRTHeR ExpLAineD How This FINDInG FiTs tO THE ObSERvATIOn THat thE *BhlH* gEnES aRe not NOrmAllY expREsSEd UpOn sUFFicIeNt fe SUPplY, WhEN FE ALsO NeEDs To BE aCQUIrEd vIa fRo2 anD Irt1 [@PONe.0099234-JAKoBY1], [@PoNE.0099234-vERt1]. Thus, tHE FunCTion Of THE bhLH suBgroUp iB(2) TranSCRIptIoN factors iN Fe UPTaKe IS StiLl oPeN for DEbatE.
VerY INTEReStInGly, it HAs bEEn Shown tHAt *bHLH38* aNd *bHLh39* wEre inDuCeD AFter appLiCAtIoN OF SAlIcYLIc ACiD ( = Sa) By the sa-InduCIbLe dOf ( = dNA BINDING wiTh OnE FinGEr) TRaNscRIPTioN facTOr oBF bIndiNG pRoteiN3 (oBp3) [@ponE.0099234-KANG1]. biNdIng OF OBp3 tO pRoMoTEr eLEmENTs In *bhLh38* And *bhLH39* GENeS and Their SuBSEqUent ACTivaTioN WAs DemOnsTrATeD (IN tHEsE sTuDies *BhLh38* AnD *bHLH39* wErE NAMeD *oBp3 responsIvE GENe2*, *oRg2*, and *obP3 reSpoNSivE geNe3*, *ORG3*) [@poNe.0099234-kAng1]. JasMOnIc Acid NegATivELy aFfECTS ThE ONset of fE MobilIzatIoN anD the iNDuctiOn of *fRo2* aND *irt1* [@poNE.0099234-maUReR1], WHile ethylENE enHAncES The REspoNSes [@PONE.0099234-gARCa1]--[@pone.0099234-liNgAm1]. SInce Sa, JaSmoNic aCid And eTHyLeNe acT IN StREsS ReSPOnse nEtworks, ThE PoSSIBIlity eXiSTS that PerHapS, ThERe iS A lINK bEtwEen sa AnD thE up-regULATioN of fe DefICIENcY rESpOnsES.
hEre, WE Made use oF the tRiPLe knockoUT MutaNT *bHLh39 BHLh100 bhlh101* (*3XbHLh*) thAt we cOnStrUCTEd TO inVESTIGaTe The FUnctIonS of THEse *Bhlh* gEnes IN THe Fe DefICIencY RESponsE aNd to fUrThEr sHed lIGht oN THe QUestIoN WhEtheR sa is INVOLveD IN MEDiAtING THE onSET of Fe UPTakE ViA tHe INDUcTioN of *bhlh* SUbGroup iB(2) geNES. WE DiScUSS ThaT *BhLH39*, *bHLH100* AnD *BhLH101* arE ESSeNtIal fOr A subset oF FE dEfiCIenCy rESpONSES bUT noT InCluDing Up-RegUlaTIoN oF *irt1* aNd *FRo2*. wE suggEST thaT tHeSe trANscRiPTion | Introduction {#s1} ============Iron (Fe) deficiency is amongthe most prevalentmicronutrient deficiencies in humans. Sinceplants constitute the primary sourceof nutrients for a large partof the world's population, the improvement of plants in terms of nutrient bioavailability is considered a priority [@pone.0099234-deBenoist1]. Micronutrients likeFe are often present in an un-soluble formin the soil. Plants are ableto mobilizesuch nutrients for uptake into the roots.Plants can also mobilizeFe from internal stores. Understanding theregulationof Fe acquisition and internalFe utilizationis of high importance for precision breeding of crops that are improved to either tolerate growth on alkaline and calcareous soilswith poorFe bio-availability or toaccumulate a higher contentofthis micronutrient in bio-availableformin the edible plant parts. Genetic traits have been associated withmicronutrient content and usage in plants, forexample [@pone.0099234-Uauy1],[@pone.0099234-Baxter1]. Another trait was found in soybean as being linked to transcription factor genes encoding the soybean homologs of *BHLH38* and *BHLH39* [@pone.0099234-Peiffer1]. The potentialimportance ofthese two transcription factor genes forFe mobilization had previously been uncovered in studies on the plant model *Arabidopsis thaliana*. *BHLH38* and *BHLH39* belong to the so-called subgroupIb(2) *BHLH* genes [@pone.0099234-Pires1] and they are functionally redundant [@pone.0099234-Wang1]--[@pone.0099234-Wang2]. In fact, *BHLH38* and *BHLH39* are tandemduplicates on the chromosome,and they share similarity with twoother *BHLH* genes, namely *BHLH100* and *BHLH101* [@pone.0099234-Wang1], [@pone.0099234-Heim1]--[@pone.0099234-ToledoOrtiz1]. All these four subgroupIb(2) *BHLH* genes are highly induced by low Fe supply in roots and leaves while they are not usually found expressed under sufficient Fe supply [@pone.0099234-Wang1]. Expression of *BHLH39* and *BHLH101* in response to iron can be followed using thepublic microarray datain Arabidopsis [@pone.0099234-Bauer1]--[@pone.0099234-Yang1] and it was found that they occur in a co-expressionnetwork along with several Fe homeostasis geneslike *FERRIC REDUCTASE OXIDASE3* (*FRO3*), *NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN4* (*NRAMP4*) and *NICOTIANAMINE SYNTHASE4* (*NAS4*) [@pone.0099234-Ivanov1]. From theco-expression with Fe homeostasis genesit can be concluded that the subgroup Ib(2) *BHLH* transcription factorgenes likely perform regulatory functions in the context ofFe homeostasis and internalFe mobilization. The bHLH protein POPEYE (PYE, belonging to another bHLH subgroup) is also induced by Fe deficiency within this co-expression network and itacts as a negative regulatorof *FRO3*, *NRAMP4* and *NAS4,* presumablyto avoid over-activation of Fe mobilization [@pone.0099234-Long1]. PYE is regulated by BRUTUS (BTS) that isalso found inthis co-expression network[@pone.0099234-Ivanov1], [@pone.0099234-Long1]. bHLH subgroup Ib(2)can physically interact with the bHLHFER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) [@pone.0099234-Wang2], [@pone.0099234-Yuan1]. FIT is expressed specifically in roots and hasbeen shown to be essential for Feuptake[@pone.0099234-Bauer2]--[@pone.0099234-Yuan2] by regulating the expressionof the genes encoding ARABIDOPSIS H^+^-ATPASE2 (AHA2)[@pone.0099234-Ivanov1], Fe reductase FERRIC OXIDASE2 (FRO2) [@pone.0099234-Jakoby1], [@pone.0099234-Robinson1] and the IRON-REGULATED TRANSPORTER1 (IRT1) [@pone.0099234-Jakoby1], [@pone.0099234-Eide1]. From ectopic FIT expression experiments along with yeast promoteractivation assays and inducible FIT activationin plants,it can be concluded that FIT targets *FRO2* and *IRT1* gene promoters [@pone.0099234-Jakoby1],[@pone.0099234-Yuan2], [@pone.0099234-Meiser1], [@pone.0099234-Sivitz2].However, FIT induces *IRT1*and *FRO2* only upon Fe deficiency even whenoverexpressed[@pone.0099234-Jakoby1], [@pone.0099234-Meiser1]. The activation of FIT at lowFe can be explained with the presence of bHLH subgroupIb(2) factors. Indeed, thedouble overexpression of FIT together with either bHLHsubgroup Ib(2) proteinleads to an increase of Fe acquisition responsesunder sufficient Fe supply conditions, and it was therefore proposed that the functionof bHLH subgroup Ib(2)might be to induceFedeficiencyresponses in conjunction with FIT [@pone.0099234-Wang2], [@pone.0099234-Yuan1].However, theoccurrence of *BHLH*subgroup Ib(2)genes in the *PYE* coexpression network,their non-expression uponsufficient Fe (where *FIT* and *IRT1* are active althoughat lowlevel)andtheir high induction upon Fe deficiency not only in roots but also in leaves (in contrast to Fe acquisition genes)renders this hypothesis questionable.Moreover, contradictoryresultshave been published with regard to the function of bHLH subgroup Ib(2) proteins. In one report,double*bhlh100 bhlh101* knockout mutants were demonstrated to develop a more severeleafchlorosis than the wild type upon Fe deficiency, while no phenotype was apparent upon Fe sufficiency. Although some Fe homeostasisgenesappearedmis-expressed, thegene knockoutsdid not affect the plants' abilitiesfor Fe uptake and theregulation of *FRO2* and *IRT1* upon sufficient ordeficient Fe supply [@pone.0099234-Sivitz1]. In contrast to that, in anotherreport,bHLHsubgroup Ib(2) knockoutsincluding *bhlh100 bhlh101*and a triple knockout *bhlh39bhlh100 bhlh101* were demonstrated to affect Fe acquisition responsesand to have low *FRO2*and *IRT1* expression upon sufficient or deficientFe supply [@pone.0099234-Wang3]. This latterfinding wasrather puzzling, and it was not furtherexplained how this findingfits to the observation thatthe *BHLH* genes are not normally expressedupon sufficient Fe supply, when Fe alsoneeds to beacquired via FRO2 and IRT1 [@pone.0099234-Jakoby1], [@pone.0099234-Vert1].Thus, the function of the bHLH subgroup Ib(2) transcriptionfactorsin Fe uptake is stillopen for debate. Very interestingly, it has beenshown that *BHLH38* and*BHLH39* wereinduced after application of salicylicacid ( = SA) by the SA-inducible Dof ( = DNA binding with one finger) transcription factor OBF BINDING PROTEIN3 (OBP3) [@pone.0099234-Kang1]. Binding ofOBP3 to promoter elementsin *BHLH38* and *BHLH39* genes and their subsequent activation was demonstrated (inthese studies *BHLH38*and *BHLH39* were named*OBP3 RESPONSIVE GENE2*, *ORG2*, and *OBP3 RESPONSIVE GENE3*,*ORG3*) [@pone.0099234-Kang1]. Jasmonic acid negatively affects the onset of Fe mobilization and the induction of *FRO2* and *IRT1* [@pone.0099234-Maurer1], while ethylene enhances the responses [@pone.0099234-Garca1]--[@pone.0099234-Lingam1]. Since SA,jasmonic acid and ethylene act in stress responsenetworks, the possibility exists that perhaps, there is a linkbetween SA and the up-regulationof Fedeficiency responses. Here, we made use of the triple knockout mutant*bhlh39 bhlh100 bhlh101*(*3xbhlh*) that we constructedto investigate thefunctions ofthese *BHLH* genes in the Fe deficiencyresponse and to further shed light on the questionwhether SA is involved in mediating the onset ofFe uptake via the induction of*BHLH* subgroupIb(2) genes. Wediscuss that *BHLH39*, *BHLH100* and *BHLH101* are essential for a subset of Fe deficiencyresponses but not including up-regulation of *IRT1*and *FRO2*.We suggest that these transcription | Introduction {#s1} ============ Iron _(Fe)_ deficiency is among _the_ most prevalent micronutrient deficiencies in humans. Since plants constitute the _primary_ source of _nutrients_ _for_ _a_ _large_ part of the world's population, _the_ _improvement_ of plants in terms of nutrient bioavailability is considered a _priority_ [@pone.0099234-deBenoist1]. Micronutrients like _Fe_ are _often_ present in an _un-soluble_ _form_ in the soil. Plants are able to mobilize such nutrients for uptake _into_ the roots. _Plants_ _can_ also _mobilize_ Fe _from_ internal stores. Understanding the regulation of Fe _acquisition_ and internal Fe utilization _is_ of high importance for precision breeding of crops that are improved to either tolerate growth on alkaline and _calcareous_ soils with poor _Fe_ bio-availability _or_ to accumulate _a_ _higher_ content of _this_ micronutrient in bio-available form in the edible plant parts. Genetic traits have _been_ associated with micronutrient content and usage _in_ plants, for _example_ [@pone.0099234-Uauy1], [@pone.0099234-Baxter1]. Another _trait_ _was_ found in soybean as being linked _to_ _transcription_ factor genes encoding the _soybean_ homologs of *BHLH38* _and_ _*BHLH39*_ _[@pone.0099234-Peiffer1]._ The potential importance of these two transcription _factor_ genes for Fe mobilization had _previously_ been _uncovered_ _in_ studies on the plant model *Arabidopsis _thaliana*._ *BHLH38* and *BHLH39* belong to the so-called subgroup Ib(2) *BHLH* genes _[@pone.0099234-Pires1]_ and they _are_ functionally _redundant_ [@pone.0099234-Wang1]--[@pone.0099234-Wang2]. _In_ fact, *BHLH38* and *BHLH39* are tandem duplicates on the chromosome, and they share similarity with two other *BHLH* genes, namely *BHLH100* and *BHLH101* [@pone.0099234-Wang1], [@pone.0099234-Heim1]--[@pone.0099234-ToledoOrtiz1]. All these four _subgroup_ Ib(2) _*BHLH*_ genes are highly induced by _low_ Fe supply in roots and leaves while _they_ are _not_ usually found expressed under sufficient Fe _supply_ _[@pone.0099234-Wang1]._ Expression of *BHLH39* and _*BHLH101*_ _in_ response to iron can be followed using the public microarray _data_ in Arabidopsis [@pone.0099234-Bauer1]--[@pone.0099234-Yang1] and it was _found_ _that_ _they_ occur in _a_ co-expression network along _with_ several Fe homeostasis _genes_ like *FERRIC REDUCTASE _OXIDASE3*_ _(*FRO3*),_ *NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN4* (*NRAMP4*) and *NICOTIANAMINE SYNTHASE4* (*NAS4*) [@pone.0099234-Ivanov1]. From the co-expression with Fe homeostasis genes it can be concluded that the subgroup _Ib(2)_ *BHLH* transcription factor genes likely perform regulatory functions in the context _of_ _Fe_ homeostasis _and_ internal Fe mobilization. The bHLH _protein_ POPEYE _(PYE,_ belonging to another _bHLH_ subgroup) is also induced _by_ Fe deficiency within this co-expression _network_ and it acts as _a_ negative regulator of *FRO3*, *NRAMP4* and *NAS4,* _presumably_ _to_ avoid over-activation _of_ Fe mobilization [@pone.0099234-Long1]. PYE is regulated by _BRUTUS_ (BTS) that is also found in this co-expression network [@pone.0099234-Ivanov1], [@pone.0099234-Long1]. bHLH subgroup Ib(2) _can_ physically interact with the bHLH _FER-LIKE_ _IRON_ DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) _[@pone.0099234-Wang2],_ [@pone.0099234-Yuan1]. FIT is expressed specifically in roots _and_ has been _shown_ to be essential for Fe uptake [@pone.0099234-Bauer2]--[@pone.0099234-Yuan2] by regulating the expression of the genes encoding ARABIDOPSIS H^+^-ATPASE2 (AHA2) [@pone.0099234-Ivanov1], Fe reductase FERRIC OXIDASE2 (FRO2) [@pone.0099234-Jakoby1], _[@pone.0099234-Robinson1]_ and _the_ IRON-REGULATED TRANSPORTER1 (IRT1) [@pone.0099234-Jakoby1], [@pone.0099234-Eide1]. From ectopic FIT expression experiments _along_ with yeast _promoter_ activation _assays_ and inducible FIT activation _in_ plants, _it_ can be concluded that FIT targets *FRO2* and _*IRT1*_ gene promoters [@pone.0099234-Jakoby1], _[@pone.0099234-Yuan2],_ [@pone.0099234-Meiser1], [@pone.0099234-Sivitz2]. However, _FIT_ induces *IRT1* _and_ *FRO2* only upon Fe deficiency even when overexpressed [@pone.0099234-Jakoby1], _[@pone.0099234-Meiser1]._ _The_ _activation_ of FIT at _low_ Fe can _be_ explained with the presence _of_ bHLH subgroup Ib(2) factors. Indeed, the double overexpression of FIT _together_ _with_ _either_ bHLH subgroup Ib(2) protein leads to _an_ increase _of_ Fe acquisition _responses_ _under_ _sufficient_ Fe supply _conditions,_ and _it_ was therefore proposed that the _function_ _of_ bHLH subgroup Ib(2) _might_ _be_ to induce Fe deficiency responses in conjunction with _FIT_ _[@pone.0099234-Wang2],_ [@pone.0099234-Yuan1]. _However,_ the occurrence of *BHLH* _subgroup_ _Ib(2)_ _genes_ in the _*PYE*_ coexpression network, their non-expression upon sufficient Fe _(where_ *FIT* and _*IRT1*_ are active although _at_ low level) _and_ their high induction upon Fe _deficiency_ not only _in_ roots _but_ also in leaves (in contrast to Fe acquisition genes) renders this hypothesis _questionable._ Moreover, contradictory _results_ have _been_ published _with_ regard to _the_ function of bHLH _subgroup_ Ib(2) proteins. In one report, double *bhlh100 bhlh101* knockout mutants were demonstrated to develop a more severe leaf chlorosis _than_ the wild type upon Fe deficiency, while _no_ phenotype was _apparent_ upon Fe _sufficiency._ _Although_ some Fe homeostasis genes appeared mis-expressed, the gene _knockouts_ did not affect the _plants'_ abilities for Fe uptake and the regulation of *FRO2* and *IRT1* upon sufficient _or_ deficient _Fe_ supply [@pone.0099234-Sivitz1]. _In_ _contrast_ to that, _in_ another report, _bHLH_ _subgroup_ Ib(2) knockouts including *bhlh100 bhlh101* and a triple knockout _*bhlh39_ bhlh100 bhlh101* were _demonstrated_ to _affect_ _Fe_ acquisition responses and to have low *FRO2* and *IRT1* expression upon sufficient or deficient Fe supply [@pone.0099234-Wang3]. _This_ latter finding _was_ rather puzzling, _and_ it was not _further_ explained how this finding fits _to_ the observation that the _*BHLH*_ genes are not normally expressed _upon_ sufficient _Fe_ supply, when Fe also _needs_ to be acquired _via_ FRO2 and IRT1 [@pone.0099234-Jakoby1], [@pone.0099234-Vert1]. Thus, _the_ function of the bHLH _subgroup_ Ib(2) transcription _factors_ in Fe uptake _is_ still _open_ for _debate._ Very interestingly, it has been shown that *BHLH38* and *BHLH39* were induced after application of salicylic acid ( = SA) by the SA-inducible Dof ( = DNA _binding_ _with_ one _finger)_ transcription _factor_ OBF BINDING PROTEIN3 (OBP3) [@pone.0099234-Kang1]. Binding of _OBP3_ to promoter elements _in_ *BHLH38* and *BHLH39* _genes_ and their subsequent activation _was_ demonstrated _(in_ _these_ _studies_ *BHLH38* _and_ *BHLH39* were named *OBP3 RESPONSIVE GENE2*, *ORG2*, and *OBP3 RESPONSIVE GENE3*, _*ORG3*)_ _[@pone.0099234-Kang1]._ Jasmonic acid negatively affects the onset of _Fe_ mobilization and the induction of _*FRO2*_ and *IRT1* [@pone.0099234-Maurer1], _while_ ethylene enhances the responses [@pone.0099234-Garca1]--[@pone.0099234-Lingam1]. _Since_ SA, jasmonic acid and _ethylene_ act in _stress_ response networks, the possibility exists that perhaps, there _is_ a link between _SA_ and the up-regulation _of_ Fe _deficiency_ responses. Here, we made use _of_ _the_ triple _knockout_ mutant *bhlh39 bhlh100 bhlh101* _(*3xbhlh*)_ that we constructed to investigate _the_ functions of these *BHLH* genes in the Fe _deficiency_ _response_ and to further shed light on the question whether _SA_ is involved in mediating the onset of _Fe_ uptake via the induction _of_ *BHLH* subgroup _Ib(2)_ genes. We discuss that *BHLH39*, *BHLH100* and *BHLH101* are essential for a subset of Fe deficiency responses _but_ not including up-regulation of *IRT1* _and_ _*FRO2*._ We suggest that these transcription |
Introduction
============
RNA interference (RNAi) is a highly efficient gene-silencing mechanism in which a small interfering RNA (siRNA) binds a target mRNA, guiding mRNA cleavage via an RNA-induced silencing complex (RISC).^[@bib1],[@bib2]^ This biological phenomenon is widely used as a genetic tool in biomedical research. Advances in RNA chemistry have expanded siRNA applications toward therapeutic development, with robust efficacy seen in phase 2 clinical trials for liver diseases (*e.g.*, transthyretin amyloidosis).^[@bib3],[@bib4],[@bib5]^
Despite its prevalence in biomedical research, the use of RNAi in neurodegenerative research has been limited.^[@bib6]^ There is a significant unmet need for simple, effective, and nontoxic siRNA delivery methods to modulate gene expression in primary neurons and brain. A range of approaches has been evaluated,^[@bib7]^ including AAV viruses,^[@bib8],[@bib9]^ peptide conjugates,^[@bib10]^ oligonucleotide formulations,^[@bib11]^ infusion of naked or slightly modified siRNAs,^[@bib12],[@bib13]^ ultrasound,^[@bib14]^ and convection-enhanced based delivery.^[@bib15]^ None of these approaches has received wide acceptance due to toxicity, a requirement for extensive repetitive dosing, and/or limited spatial distribution. Lipofection and electroporation of siRNAs are challenging in primary neurons due to low transfection efficiencies and their extreme sensitivity to external manipulation.^[@bib16]^ Delivery of siRNA precursors (Lentiviruses and AAV) has been used successfully, but viral transduction cannot readily be turned off and requires extensive formulation and experimental optimization to achieve reproducible, nontoxic silencing in neuronal cells.^[@bib17],[@bib18],[@bib19],[@bib20],[@bib21],[@bib22]^
In this study, we describe the delivery, distribution, and silencing capacity of hydrophobically modified siRNAs (hsiRNAs) in primary neurons and in mouse brain. hsiRNAs are siRNA-antisense hybrids containing numerous chemical modifications (see **[Figure 1](#fig1){ref-type="fig"}** and **Supplementary Table S1** for exact chemical composition of compounds used) designed to promote biodistribution and stability while minimizing immunogenicity. As a model for our studies, we silenced the huntingtin (*Htt*) gene, the causative gene in Huntington\'s disease (HD). HD is an autosomal-dominant neurodegenerative disorder caused by a toxic expansion in the CAG repeat region of the huntingtin gene leading to a variety of molecular and cellular consequences. Tetrabenazine, the only FDA-approved therapy for HD, seeks to alleviate disease symptoms but does not treat the actual problem: the gain of toxic function caused by mutant *Htt*. Recent studies suggest that transient neuronal knockdown of *Htt* mRNA can reverse disease progression without compromising normal cellular function *in vivo*.^[@bib23]^ At present, RNA interference via siRNA or antisense oligonucleotide is one of the most promising therapeutic approaches for transient *Htt* mRNA silencing.
We performed a screen of hsiRNAs targeting *Htt* mRNA and identified multiple functional compounds. We showed that primary neurons internalize hsiRNA added directly to the culture medium, with membrane saturation occurring by 1 hour. Direct uptake in neurons induces potent and long-lasting silencing of *Htt* mRNA for up to 3 weeks *in vitro* without major detectable effects on neuronal viability. Additionally, a single injection of unformulated (without cationic lipid or AAV formulation) *Htt* hsiRNA into mouse brain silences *Htt* mRNA with minimal neuronal toxicity.
Efficient gene silencing in primary neurons and *in vivo* upon direct administration of unformulated hsiRNA represents a significant technical advance in the application of RNAi to neuroscience research, enabling technically achievable genetic manipulation in a native, biological context.
Results
=======
hsiRNAs are efficiently internalized by primary neurons
-------------------------------------------------------
hsiRNA is an asymmetric compound composed of a 15-nucleotide modified RNA duplex with a single-stranded 3′ extension on the guide strand (**[Figure 1a](#fig1){ref-type="fig"}** and **Supplementary Table S1**).^[@bib24],[@bib25]^ Pyrimidines in the hsiRNA are modified with 2′-O-methyl (passenger strand) or 2′-fluoro (guide strand) to promote stability, and the 3′ end of the passenger strand is conjugated to a hydrophobic teg-Chol (tetraethylene glycol cholesterol) to promote membrane binding and association.^[@bib26]^ The single-stranded tail contains hydrophobic phosphorothioate linkages and promotes cellular uptake by a mechanism similar to that of antisense oligonucleotides.^[@bib27]^ The presence of phosphorothioates, ribose modifications, and a cholesterol conjugate contribute to overall hydrophobicity and are essential for compound stabilization and efficient cellular internalization.
Previous studies have shown that hydrophobically modified siRNAs bind to a wide range of cells and is readily internalized without the requirement for a transfection reagent.^[@bib26],[@bib28],[@bib29]^ Here, we evaluated whether asymmetric hydrophobically modified siRNAs are efficiently internalized by primary neurons. We found that, when added to the culture medium, Cy3-labeled hsiRNAs rapidly associated with primary cortical neurons (**[Figure 1b](#fig1){ref-type="fig"}**). These Cy3-labeled hsiRNAs were observed in every cell in the culture, demonstrating efficient and uniform uptake. Initially, hsiRNAs mainly associate with neurites and, over time, accumulate in the cell bodies. Treatment of primary neurons with a previously identified hsiRNA targeting *Ppib*^[@bib26],[@bib28]^ (encodes cyclophilin B) reduced target mRNA levels by 90%, further supporting that the observed compound internalization results in potent gene silencing (**[Figure 1c](#fig1){ref-type="fig"}**).
Identification of hsiRNAs that silence huntingtin mRNA
------------------------------------------------------
Robust uptake and efficacy observed with hsiRNAs in primary cortical neurons encouraged us to identify functional compounds that target *Htt* mRNA, the single gene responsible for the development of Huntington\'s disease. The hsiRNA\'s extensive chemical scaffold^[@bib26],[@bib28]^ is essential for stability, minimization of innate immune response,^[@bib30],[@bib31]^ and cellular internalization but imposes significant restrictions on sequence space by potentially interfering with the compound\'s RISC-entering ability. To maximize the likelihood of identifying functional *Htt* hsiRNAs and to evaluate the hit rate for this type of chemistry, we designed (using conventional criteria described in Materials and Methods) and synthesized hsiRNAs targeting 94 sites across the human *Htt* mRNA (**Supplementary Table S1**). The panel of hsiRNAs was initially screened for efficacy in HeLa cells by adding hsiRNA directly to the culture medium (without lipofection or electroporation) to a final concentration of 1.5 µmol/l and evaluating impact on levels of *Htt* and housekeeping (*Ppib*) gene mRNA expression using the QuantiGene (Affymetrix, Santa Clara, CA) assay. At this concentration, 24 hsiRNAs reduced *Htt* mRNA levels to less than 50% of control levels, including 7 hsiRNAs that reduced *Htt* mRNA levels below 30% of control (**[Figure 2a](#fig2){ref-type="fig"}**). Unlike unmodified siRNA libraries, creating a library with extensive 2\'-O-methyl and 2\'-fluoro modifications introduces additional constraints on sequence selection. As a result, hit rates for modified siRNA screens are lower than that seen for conventional unmodified siRNA.^[@bib32],[@bib33],[@bib34],[@bib35]^ Functional hsiRNAs targeted sites distributed throughout the mRNA, except the distal end of the 3′ UTR, which later was shown to be part of the alternative *Htt* gene isoform^[@bib36]^ not expressed in HeLa cells (data not shown). Discounting the \~32 hsiRNAs targeting long 3′ UTR sites absent from the *Htt* isoform in HeLa cells, almost 40% of hsiRNAs showed some level of activity at 1.5 µmol/l, demonstrating that the evaluated chemical scaffold is well tolerated by the RNAi machinery and a functional compound can be easily identified against a wide range of targets.
Half-maximal inhibitory concentrations (IC~50~) for passive uptake of hsiRNAs ranged from 82 to 766 nmol/l (**Supplementary Table S1 and Figure S1**). In lipid-mediated delivery, eight of the most active hsiRNAs had IC~50~ values ranging from 4 to 91 pmol/l (**Supplementary Table S1**). The best clinically active siRNAs are usually characterized by IC~50~ values in the low pmol/l range.^[@bib37]^ An ability to identify highly potent compounds with low picomolar IC~50~ values suggests that the hsiRNA chemical scaffold does not interfere with siRNA biological activity in selected compounds. The most potent hsiRNA targeting position, 10150 (HTT10150), and an unmodified conventional siRNA version of HTT10150 showed similar IC~50~ values in lipid-mediated delivery (4 and 13 pmol/l respectively, **[Figure 2c](#fig2){ref-type="fig"}**), further confirming that the hsiRNA chemical scaffold does not interfere with RISC loading or function. Only the fully modified hsiRNA, and not the unmodified version, silenced *Htt* mRNA by passive uptake (**[Figure 2b](#fig2){ref-type="fig"}**). Thus, the chemical scaffold described here does not interfere with RISC assembly and is sufficient to support unformulated compound uptake and efficacy. HTT10150 was used for subsequent studies.
Potent and specific silencing with unformulated hsiRNAs in primary neurons
--------------------------------------------------------------------------
HTT10150 induced a concentration-dependent silencing at 72 hours and | introduction = = = = = = = = = = = = genetic interference ( rnai ) — one highly efficient gene - silencing mechanism in which a small interfering rna ( sirna ) binds a target genes, guiding mrna cleavage via an rna - induced silencing complex ( risc ). ^ [ @ bib1 ], [ @ bib2 ] ^ this biological phenomenon is widely used as a genetic tool in biomedical research. advances in rna chemistry have expanded sirna applications toward therapeutic development, with robust efficacy seen in phase 2 clinical trials for liver diseases ( * e. coli. *, transthyretin amyloidosis ). ^ [ @ bib3 ], [ @ bib4 ], [ @ bib5 ] ^ despite its prevalence in biomedical research, the use of rnai in neurodegenerative research has been limited. ^ [ @ bib6 ] ^ there is a significant unmet need for simple, effective, and nontoxic sirna delivery methods to modulate gene expression in primary neurons and brain. a range of approaches has been evaluated, ^ [ @ bib7 ] ^ including aav viruses, ^ [ @ bib8 ], [ @ bib9 ] ^ peptide conjugates, ^ [ @ bib10 ] ^ diverse formulations, ^ [ @ bib11 ] ^ infusion of naked or slightly modified sirnas, ^ [ @ bib12 ], [ @ bib13 ] ^ ultrasound, ^ [ @ bib14 ] ^ and convection - enhanced based delivery. ^ [ @ bib15 ] ^ none of these approaches has received wide acceptance due to toxicity, a requirement for extensive repetitive dosing, and / or limited spatial distribution. lipofection and electroporation of cells are challenging in primary neurons due by low transfection efficiencies and their extreme risks to external manipulation. ^ [ @ bib16 ] ^ delivery of sirna precursors ( lentiviruses and aav ) has been used successfully, but viral transduction cannot readily be turned off and requires extensive formulation and experimental optimization to achieve reproducible, viral silencing in neuronal cells. ^ [ @ bib17 ], [ @ bib18 ], [ @ bib19 ], [ @ bib20 ], [ @ bib21 ], [ @ bib22 ] ^ in this study , we describe the delivery, distribution, and silencing capacity of hydrophobically modified sirnas ( hsirnas ) in primary neurons and in mouse brain. hsirnas are sirna - antisense hybrids containing numerous chemical modifications ( see * * [ figure 1 ] ( # fig1 ) { ref - type = " fig " } * * and * * supplementary table s1 * * for exact chemical composition of compounds used ) designed to promote biodistribution and stability while minimizing immunogenicity. as a model for our studies, we silenced the huntingtin ( * htt * ) gene, the causative gene in huntington \ ' s disease ( hd ). hd is an autosomal - dominant neurodegenerative disorder caused by a toxic expansion in the cag repeat region of the huntingtin gene leading to a variety of molecular and cellular consequences. tetrabenazine, the only fda - approved therapy for hd, seeks to alleviate disease symptoms but does not treat the actual problem : the gain of toxic function caused by mutant * htt *. recent studies suggest that transient neuronal knockdown of * htt * mrna can reverse disease progression without compromising normal cellular function * in vivo *. ^ [ @ bib23 ] ^ at present, rna interference via sirna or antisense oligonucleotide is one of the most promising therapeutic approaches for transient * htt * mrna silencing. we performed a screen of hsirnas targeting * htt * mrna and identified multiple functional compounds. we showed that primary neurons internalize hsirna added directly to the culture medium, with membrane saturation occurring by 1 hour. direct uptake in neurons induces potent and long - lasting silencing of * htt * mrna for up to 3 weeks * in vitro * without major detectable effects on neuronal viability. additionally, a single injection of unformulated ( without cationic lipid or aav formulation ) * htt * hsirna into mouse brain silences * htt * mrna with minimal neuronal toxicity. efficient gene silencing in primary neurons and * in vivo * upon direct administration of unformulated hsirna represents a significant technical advance in the application of rnai to neuroscience research, enabling technically achievable genetic manipulation in a native, biological context. results = = = = = = = hsirnas are efficiently internalized by primary neurons - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - hsirna is an asymmetric compound composed of a 15 - nucleotide modified rna duplex with a single - stranded 3 ′ extension on the guide strand ( * * [ figure 1a ] ( # fig1 ) { ref - type = " fig " } * * and * * supplementary table s1 * * ). ^ [ @ bib24 ], [ @ bib25 ] ^ pyrimidines in the hsirna are modified with 2 ′ - o - methyl ( passenger strand ) or 2 ′ - fluoro ( guide strand ) to promote stability, and the 3 ′ end of the passenger strand is conjugated to a hydrophobic teg - chol ( tetraethylene glycol cholesterol ) to promote membrane binding and association. ^ [ @ bib26 ] ^ the single - stranded tail contains hydrophobic phosphorothioate linkages and promotes cellular uptake by a mechanism similar to that of antisense oligonucleotides. ^ [ @ bib27 ] ^ the presence of phosphorothioates, ribose modifications, and a cholesterol conjugate contribute to overall hydrophobicity and are essential for compound stabilization and efficient cellular internalization. previous studies have shown that hydrophobically modified sirnas bind to a wide range of cells and is readily internalized without the requirement for a transfection reagent. ^ [ @ bib26 ], [ @ bib28 ], [ @ bib29 ] ^ here, we evaluated whether asymmetric hydrophobically modified sirnas are efficiently internalized by primary neurons. we found that, when added to the culture medium, cy3 - labeled hsirnas rapidly associated with primary cortical neurons ( * * [ figure 1b ] ( # fig1 ) { ref - type = " fig " } * * ). these cy3 - labeled hsirnas were observed in every cell in the culture, demonstrating efficient and uniform uptake. initially, hsirnas mainly associate with neurites and, over time, accumulate in the cell bodies. treatment of primary neurons with a previously identified hsirna targeting * ppib * ^ [ @ bib26 ], [ @ bib28 ] ^ ( encodes cyclophilin b ) reduced target mrna levels by 90 %, further supporting that the observed compound internalization results in potent gene silencing ( * * [ figure 1c ] ( # fig1 ) { ref - type = " fig " } * * ). identification of hsirnas that silence huntingtin mrna - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - robust uptake and efficacy observed with hsirnas in primary cortical neurons encouraged us to identify functional compounds that target * htt * mrna, the single gene responsible for the development of huntington \ ' s disease. the hsirna \ ' s extensive chemical scaffold ^ [ @ bib26 ], [ @ bib28 ] ^ is essential for stability, minimization of innate immune response, ^ [ @ bib30 ], [ @ bib31 ] ^ and cellular internalization but imposes significant restrictions on sequence space by potentially interfering with the compound \ ' s risc - entering ability. to maximize the likelihood of identifying functional * htt * hsirnas and to evaluate the hit rate for this type of chemistry, we designed ( using conventional criteria described in materials and methods ) and synthesized hsirnas targeting 94 sites across the human * htt * mrna ( * * supplementary table s1 * * ). the panel of hsirnas was initially screened for efficacy in hela cells by adding hsirna directly to the culture medium ( without lipofection or electroporation ) to a final concentration of 1. 5 µmol / l and evaluating impact on levels of * htt * and housekeeping ( * ppib * ) gene mrna expression using the quantigene ( affymetrix, santa clara, ca ) assay. at this concentration, 24 hsirnas reduced * htt * mrna levels to less than 50 % of control levels, including 7 hsirnas that reduced * htt * mrna levels below 30 % of control ( * * [ figure 2a ] ( # fig2 ) { ref - type = " fig " } * * ). unlike unmodified sirna libraries, creating a library with extensive 2 \ ' - o - methyl and 2 \ ' - fluoro modifications introduces additional constraints on sequence selection. as a result, hit rates for modified sirna screens are lower than that seen for conventional unmodified sirna. ^ [ @ bib32 ], [ @ bib33 ], [ @ bib34 ], [ @ bib35 ] ^ functional hsirnas targeted sites distributed throughout the mrna, except the distal end of the 3 ′ utr, which later was shown to be part of the alternative * htt * gene isoform ^ [ @ bib36 ] ^ not expressed in hela cells ( data not shown ). discounting the \ ~ 32 hsirnas targeting long 3 ′ utr sites absent from the * htt * isoform in hela cells, almost 40 % of hsirnas showed some level of activity at 1. 5 µmol / l, demonstrating that the evaluated chemical scaffold is well tolerated by the rnai machinery and a functional compound can be easily identified against a wide range of targets. half - maximal inhibitory concentrations ( ic ~ 50 ~ ) for passive uptake of hsirnas ranged from 82 to 766 nmol / l ( * * supplementary table s1 and figure s1 * * ). in lipid - mediated delivery, eight of the most active hsirnas had ic ~ 50 ~ values ranging from 4 to 91 pmol / l ( * * supplementary table s1 * * ). the best clinically active sirnas are usually characterized by ic ~ 50 ~ values in the low pmol / l range. ^ [ @ bib37 ] ^ an ability to identify highly potent compounds with low picomolar ic ~ 50 ~ values suggests that the hsirna chemical scaffold does not interfere with sirna biological activity in selected compounds. the most potent hsirna targeting position, 10150 ( htt10150 ), and an unmodified conventional sirna version of htt10150 showed similar ic ~ 50 ~ values in lipid - mediated delivery ( 4 and 13 pmol / l respectively, * * [ figure 2c ] ( # fig2 ) { ref - type = " fig " } * * ), further confirming that the hsirna chemical scaffold does not interfere with risc loading or function. only the fully modified hsirna, and not the unmodified version, silenced * htt * mrna by passive uptake ( * * [ figure 2b ] ( # fig2 ) { ref - type = " fig " } * * ). thus, the chemical scaffold described here does not interfere with risc assembly and is sufficient to support unformulated compound uptake and efficacy. htt10150 was used for subsequent studies. potent and specific silencing with unformulated hsirnas in primary neurons - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - htt10150 induced a concentration - dependent silencing at 72 hours and | Introduction = = = = = = = = = = = = RNA interference (RNAi) is a highly efficient gene - silencing mechanism in which a small interfering RNA (siRNA) binds a target mRNA, guiding mRNA cleavage via an RNA - induced silencing complex (RISC ). ^ [@ bib1 ], [@ bib2] ^ This biological phenomenon is widely used as a genetic tool in biomedical research. Advances in RNA chemist#& have expanded siRNA applications toward therapeutic development, with robust efficacy seen in phase 2 clinical trials for liver diseases (* e. g. *, transthyretin amyloidosis ). ^ [@ bib3 ], [@ bib4 ], [@ bib5] ^ Despite its prevalence in biomedical research, the use of RNAi in neurodegenerative research has been limited. ^ [@ bib6] ^ There is a significant unmet need for simple, effective, and nontoxic siRNA delivery methods to modulate gene expression in primary neurons and brain. A range of approaches has been evaluated, ^ [@ bib7] ^ including AAV viruses, ^ [@ bib8 ], [@ bib9] ^ peptide conjugates, ^ [@ bib10] ^ oligonucleotide formulations, ^ [@ bib11] ^ infusion of naked or slightly modified siRNAs, ^ [@ bib12 ], [@ bib13] ^ ultrasound, ^ [@ bib14] ^ and convection - enhanced based delivery. ^ [@ bib15] ^ None of these approaches has received wide acceptance due to toxicity, a requirement for extensive repetitUvS dosing, and / or limited spatial distribution. Lipofection and electroporation of siRNAs are challenging in primary neurons due to low transfection efficiencies and their extreme sensitivity to external manipulation. ^ [@ bib16] ^ Delivery of siRNA precursors (Lentiviruses and AAV) has been used successfully, but viral transduction cannot readily be turned off and requires extensive formulation and experimental optimization to achieve reproducible, nontoxic silencing in neuronal cells. ^ [@ bib17 ], [@ bib18 ], [@ bib19 ], [@ bib20 ], [@ bib21 ], [@ bib22] ^ In this study, we describe the delivery, distribution, and silencing capacity of hydrophobically modified siRNAs (hsiRNAs) in primary neurons and in mouse brain. hsiRNAs are siRNA - antisense hybrids containing numerous chemical modifications (see * * [Figure 1] (# fig1) {ref - type = " fig "} * * and * * Supplementary Table S1 * * for exact chemical composition of compounds used) designed to promote biodistribution and stability while minimizing immunogenicity. As a model for our studies, we silenced the huntingtin (* Htt *) gene, the causative gene in Huntington \ ' s disease (HD ). HD is an autosomal - dominant neurodegenerative disorder caused by a toxic expansion in the CAG repeat region of the huntingtin gene leading to a variety of molecular and cellular consequences. Tetrabenazine, the only FDA - approved thwraoy for HD, seeks to alleviate disease symptoms but does not treat the actual problem: the gain of toxic function caused by mutant * Htt *. Recent studies suggest that transient neuronal knockdown of * Htt * mRNA can reverse disease progression without compromising normal cellular function * in vivo *. ^ [@ bib23] ^ At present, RNA interference via siRNA or antisense oligonucleotide is one of the most promising therapeutic approaches for transient * Htt * mRNA silencing. We performed a screen of hsiRNAs targeting * Htt * mRNA and identified multiple functional compounds. We showed that primary neurons internalize hsiRNA added directly to the culture medium, wK%h membrane saturation occurring by 1 hour. Direct uptake in neurons induces potent and long - lasting silencing of * Htt * mRNA for up to 3 weeks * in vitro * without major detectable effects on neuronal viability. Additionally, a single injection of unformulated (without cationic lipid or AAV formulation) * Htt * hsiRNA into mouse brain silences * Htt * mRNA with minimal neuronal toxicity. Efficient gene silencing in primary neurons and * in vivo * upon direct administration of unformulated hsiRNA represents a significant technical advance in the application of RNAi to neuroscience research, enabling technically achievable genetic manipulation in a native, biological context. Results = = = = = = = hsiRNAs are efficiently internalized by primary neurons - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - hsiRNA is an asymmetric compound composed of a 15 - nucleotide modified RNA duplex with a single - stranded 3 ′ extension on the guide strand (* * [Figure 1a] (# fig1) {ref - type = " fig "} * * and * * Supplementary Table S1 * * ). ^ [@ bib24 ], [@ bib25] ^ Pyrimidines in the hsiRNA are modified with 2 ′ - O - methyl (passenger strand) or 2 ′ - fluoro (guide strand) to prKmPte stability, and the 3 ′ end of the passenger strand is conjugated to a hydrophobic teg - Chol (tetraethylene glycol cholesterol) to promote membrane binding and association. ^ [@ bib26] ^ The single - stranded tail contains hydrophobic phosphorothioate linkages and promotes cellular uptake by a mechanism similar to that of antisense oligonucleotides. ^ [@ bib27] ^ The presence of phosphorothioates, ribose modifications, and a cholesterol conjugate contribute to overall hydrophobicity and are essential for compound stabilization and efficient cellular internalization. Previous studies have shown that hydrophobically modified siRNAs bind to a wUds range of cells and is readily internalized without the requirement for a transfection reagent. ^ [@ bib26 ], [@ bib28 ], [@ bib29] ^ Here, we evaluated whether asymmetric hydrophobically modified siRNAs are efficiently internalized by primary neurons. We found that, when added to the culture medium, Cy3 - labeled hsiRNAs rapidly associated with primary cortical neurons (* * [Figure 1b] (# fig1) {ref - type = " fig "} * * ). These Cy3 - labeled hsiRNAs were observed in every cell in the culture, demonstrating efficient and uniform uptake. Initially, hsiRNAs mainly associate with neurites and, over time, accumulate in the cell bodies. Treatment of primary neurons with a previously identified hsiRNA targeting * Ppib * ^ [@ bib26 ], [@ bib28] ^ (encodes cyclophilin B) reduced target mRNA levels by 90% , further supporting that the observed compound internalization results in potent gene silencing (* * [Figure 1c] (# fig1) {ref - type = " fig "} * * ). Identification of hsiRNAs that silence huntingtin mRNA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Robust uptake and efficacy observed with hsiRNAs in primary cortical neurons encouraged us to identify functional compounds that target * Htt * mRNA, the single gene responsible for the development of Huntington \ ' s disease. The hsiRNA \ ' s extensive chemical scaffold ^ [@ bib26 ], [@ bib28] ^ is essential for stability, minimization of innate immune response, ^ [@ bib30 ], [@ bib31] ^ and cellular internalization but imposes significant restrictions on sequence space by potentially interfering with the compound \ ' s RISC - entering ability. To maximize the likelihood of identifying functional * Htt * hsiRNAs and to evaluate the hit rate for this type of chemistry, we designed (using conventional criteria described in Materials and Methods) and synthesized hsiRNAs targeting 94 sites across the human * Htt * mRNA (* * Supplementary Table S1 * * ). The panel of hsiRNAs was initially screened for efficacy in HeLa cells by adding hsiRNA directly to the culture medium (without lipofection or elfvtroporation) to a final concentration of 1. 5 µmol / l and evaluating impact on levels of * Htt * and housekeeping (* Ppib *) gene mRNA expression using the QuantiGene (Affymetrix, Santa Clara, CA) assay. At this concentration, 24 hsiRNAs reduced * Htt * mRNA levels to less than 50% of control levels, including 7 hsiRNAs that reduced * Htt * mRNA levels below 30% of control (* * [Figure 2a] (# fig2) {ref - type = " fig "} * * ). Unlike unmodified siRNA libraries, creating a library with extensive 2 \ ' - O - methyl and 2 \ ' - fluoro modifications introduces additional constraints on sequence selection. As a result, hit rates for modified siRNA screens are lower than that seen for conventional unmodified siRNA. ^ [@ bib32 ], [@ bib33 ], [@ bib34 ], [@ bib35] ^ Functional hsiRNAs targeted sites distributed throughout the mRNA, except the distal end of the 3 ′ UTR, which later was shown to be part of the alternative * Htt * gene isoform ^ [@ bib36] ^ not expressed in HeLa cells (data not shown ). Discounting the \ ~ 32 hsiRNAs targeting long 3 ′ UTR sites absent from the * Htt * isoform in HeLa cells, almost 40% of hsiRNAs showed some level of activity at 1. 5 µmol / l, demonstrating that the evaluated chemical scaffold is well tolerated by the RNAi machinery and a functional compound can be easily identified against a wide range of targets. Half - maximal inhibitory concentrations (IC ~ 50 ~) for passive uptake of hsiRNAs ranged from 82 to 766 nmol / l (* * Supplementary Table S1 and Figure S1 * * ). In lipid - mediated delivery, eight of the most active hsiRNAs had IC ~ 50 ~ values ranging from 4 to 91 pmol / l (* * Supplementary Table S1 * * ). The best clinically active siRNAs are usually characterized by IC ~ 50 ~ values in the low pmol / l range. ^ [@ bib37] ^ An ability to identify highly potent compounds with low picomolar IC ~ 50 ~ values suggests that the hsiRNA chemical scaffold does not interfere with siRNA biological activity in selected compounds. The most potent hsiRNA targeting position, 10150 (HTT10150 ), and an unmodified conventional siRNA version of HTT10150 showed similar IC ~ 50 ~ values in lipid - mediated delivery (4 and 13 pmol / l respectively, * * [Figure 2c] (# fig2) {ref - R^pe = " fig "} * * ), further confirming that the hsiRNA chemical scaffold does not interfere with RISC loading or function. Only the fully modified hsiRNA, and not the unmodified version, silenced * Htt * mRNA by passive uptake (* * [Figure 2b] (# fig2) {ref - type = " fig "} * * ). Thus, the chemical scaffold described here does not interfere with RISC assembly and is Wkfficient to support unformulated compound uptake and efficacy. HTT10150 was ieed for subsequent studies. Potent and specific silencing with unformulated hsiRNAs in primary neurons - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - HTT10150 induced a concentration - dependent silencing at 72 hours and | Introduction ============ RNA interference (RNAi) is a efficient mechanism in which a small interfering RNA binds a target mRNA, guiding cleavage via an RNA-induced complex (RISC).^[@bib1],[@bib2]^ This biological phenomenon is widely used as a genetic biomedical research. Advances in RNA chemistry have expanded siRNA applications toward therapeutic development, with robust efficacy seen in phase 2 clinical trials for liver diseases (*e.g.*, amyloidosis).^[@bib3],[@bib4],[@bib5]^ Despite prevalence in biomedical research, the of in neurodegenerative research has been limited.^[@bib6]^ There is a significant unmet need simple, effective, and nontoxic siRNA delivery methods to modulate gene expression in primary neurons and brain. A of approaches has been evaluated,^[@bib7]^ including AAV viruses,^[@bib8],[@bib9]^ peptide conjugates,^[@bib10]^ formulations,^[@bib11]^ infusion of naked or slightly modified siRNAs,^[@bib12],[@bib13]^ and convection-enhanced based delivery.^[@bib15]^ None of these approaches has received wide acceptance due to toxicity, a requirement for repetitive dosing, and/or limited distribution. Lipofection and electroporation of siRNAs are challenging in primary neurons due to low transfection efficiencies their extreme sensitivity to external manipulation.^[@bib16]^ Delivery siRNA precursors (Lentiviruses and AAV) has been used successfully, but viral transduction cannot readily be turned off and extensive formulation and experimental optimization to achieve reproducible, nontoxic in neuronal cells.^[@bib17],[@bib18],[@bib19],[@bib20],[@bib21],[@bib22]^ In this study, describe the delivery, distribution, and silencing capacity of hydrophobically modified siRNAs (hsiRNAs) in primary neurons and in brain. hsiRNAs are siRNA-antisense hybrids numerous chemical modifications (see **[Figure 1](#fig1){ref-type="fig"}** and S1** for exact chemical composition of compounds used) designed to promote biodistribution and stability minimizing immunogenicity. As a model for our studies, we silenced the huntingtin (*Htt*) gene, the causative gene in Huntington\'s disease (HD). HD is an autosomal-dominant neurodegenerative disorder by a toxic expansion in the CAG region of the huntingtin gene to a variety of molecular and cellular consequences. Tetrabenazine, the only FDA-approved for HD, seeks to alleviate disease symptoms but does not the actual problem: the gain of toxic function caused by mutant *Htt*. Recent studies suggest that transient neuronal knockdown of *Htt* mRNA can reverse disease progression without compromising normal function *in vivo*.^[@bib23]^ At present, RNA interference via siRNA or antisense oligonucleotide is one the most promising therapeutic approaches transient *Htt* mRNA silencing. We performed a screen of hsiRNAs *Htt* and identified multiple functional compounds. showed that neurons internalize hsiRNA added directly to the culture medium, with membrane saturation occurring by 1 hour. Direct uptake in neurons induces potent and long-lasting *Htt* mRNA for up to weeks *in without major detectable on neuronal Additionally, a single injection of unformulated cationic lipid or AAV formulation) *Htt* hsiRNA into mouse brain silences *Htt* mRNA with minimal neuronal toxicity. Efficient gene silencing in primary neurons and *in upon direct administration of unformulated hsiRNA represents a significant in application of RNAi to neuroscience research, enabling achievable genetic manipulation in native, biological context. Results ======= are efficiently internalized by neurons ------------------------------------------------------- hsiRNA is an asymmetric compound composed of a 15-nucleotide modified RNA duplex with a single-stranded 3′ extension on the strand (**[Figure 1a](#fig1){ref-type="fig"}** and **Supplementary Table S1**).^[@bib24],[@bib25]^ Pyrimidines in the are with 2′-O-methyl (passenger or 2′-fluoro (guide strand) to promote and the 3′ end of the passenger strand is conjugated to a teg-Chol (tetraethylene glycol cholesterol) to promote membrane and association.^[@bib26]^ The single-stranded tail contains hydrophobic linkages and promotes cellular uptake by a mechanism similar that of antisense oligonucleotides.^[@bib27]^ The presence of phosphorothioates, ribose modifications, and a cholesterol contribute to overall hydrophobicity and are essential for compound stabilization and efficient cellular internalization. Previous have shown that hydrophobically modified siRNAs bind a wide range of cells and is readily internalized the requirement for a transfection reagent.^[@bib26],[@bib28],[@bib29]^ Here, we whether asymmetric hydrophobically modified siRNAs are efficiently internalized primary neurons. We found that, when added to the culture hsiRNAs rapidly associated with primary cortical neurons (**[Figure 1b](#fig1){ref-type="fig"}**). These hsiRNAs were observed in every cell in the culture, demonstrating efficient and uniform uptake. Initially, hsiRNAs associate with neurites and, over time, accumulate in the cell bodies. Treatment of primary neurons a previously identified hsiRNA targeting *Ppib*^[@bib26],[@bib28]^ (encodes cyclophilin B) reduced target mRNA levels by 90%, supporting that the observed compound internalization results in potent gene silencing (**[Figure 1c](#fig1){ref-type="fig"}**). Identification of hsiRNAs that mRNA ------------------------------------------------------ Robust uptake and observed with hsiRNAs in primary cortical neurons encouraged us to identify functional that target *Htt* the single gene responsible for the development of Huntington\'s disease. The hsiRNA\'s extensive chemical is essential for stability, minimization of response,^[@bib30],[@bib31]^ and cellular imposes significant restrictions space by potentially with the compound\'s RISC-entering ability. To maximize the likelihood of identifying functional *Htt* and to evaluate the hit rate for this type of chemistry, we (using conventional criteria described in Materials and Methods) and synthesized hsiRNAs targeting 94 sites across the human *Htt* (**Supplementary S1**). panel of hsiRNAs was initially screened for efficacy in HeLa cells by adding hsiRNA directly to the culture medium (without lipofection or electroporation) to a final concentration of 1.5 µmol/l and evaluating impact on levels of *Htt* and housekeeping (*Ppib*) gene mRNA expression using the QuantiGene (Affymetrix, Santa Clara, CA) assay. this concentration, 24 hsiRNAs reduced *Htt* mRNA levels to than 50% of control levels, including 7 hsiRNAs that *Htt* mRNA levels below 30% control (**[Figure 2a](#fig2){ref-type="fig"}**). Unlike unmodified siRNA libraries, creating a library extensive 2\'-O-methyl and modifications introduces additional constraints on sequence As result, hit rates for modified siRNA screens are lower than that seen for conventional unmodified siRNA.^[@bib32],[@bib33],[@bib34],[@bib35]^ Functional targeted sites distributed throughout the mRNA, except the distal end of the 3′ UTR, which later was shown to be part of the alternative *Htt* gene isoform^[@bib36]^ expressed in HeLa cells (data not shown). Discounting the \~32 hsiRNAs long 3′ UTR sites absent from the *Htt* isoform in HeLa cells, almost 40% of hsiRNAs showed some level of activity at 1.5 µmol/l, demonstrating that the evaluated chemical scaffold is well tolerated the machinery and a functional compound can be easily identified against a wide range of targets. Half-maximal inhibitory concentrations (IC~50~) for passive uptake of hsiRNAs from to 766 nmol/l (**Supplementary Table S1 and Figure S1**). In lipid-mediated delivery, eight of the most active hsiRNAs had IC~50~ values ranging from 4 91 pmol/l (**Supplementary Table S1**). The best active siRNAs are characterized by IC~50~ in the low range.^[@bib37]^ An ability identify highly potent compounds with low picomolar IC~50~ values suggests that the hsiRNA chemical scaffold does not interfere with siRNA biological activity selected compounds. The most potent hsiRNA targeting position, 10150 (HTT10150), and an unmodified conventional siRNA version of showed similar IC~50~ values in lipid-mediated delivery (4 and 13 pmol/l respectively, **[Figure 2c](#fig2){ref-type="fig"}**), further confirming chemical scaffold does not interfere with loading or function. the fully modified hsiRNA, and the unmodified version, silenced *Htt* mRNA uptake (**[Figure 2b](#fig2){ref-type="fig"}**). the scaffold described here does not interfere with RISC assembly and is sufficient to support unformulated compound uptake and efficacy. HTT10150 was for subsequent studies. Potent and specific silencing with unformulated hsiRNAs in primary neurons -------------------------------------------------------------------------- HTT10150 induced a concentration-dependent silencing at 72 hours and | IntRoduCTiON
============
RNA InTErfEreNcE (rnAI) Is A hIgHlY EffiCIENt genE-SIleNCInG MEChANisM IN WhIch a SmalL iNTErFERinG RNA (SiRNa) BiNDS a TaRGet Mrna, GUIDING mrna cLeAVAgE VIa AN rNa-inDUCeD SiLencINg cOMPLEx (RISc).^[@bib1],[@BIB2]^ tHIs bIOlOGiCal PHeNomenoN IS WIDeLY USeD As a geNEtiC tOoL In bIOMeDicAL reSeARCh. AdvaNCEs In RNa cHEmiSTRy HavE ExpanDED SirNA aPPliCationS TowaRd thERapEuTIc DEVeLopmenT, WIth rOBuSt effICaCY SEEN IN PHAsE 2 CLiNiCAl tRIALs fOr lIveR dIsEASes (*e.G.*, tranSthYrETiN aMYLoIdOSIS).^[@Bib3],[@bIb4],[@bib5]^
DESPiTe ITS PreVaLEnCe IN bIoMEdicAl resEArcH, the USe oF RnAi IN NeuRoDegEnErAtive reSeARcH Has bEEn LIMitED.^[@bib6]^ tHEre IS a SigniFiCANt unmeT NeED FOr siMPle, eFFECtIVe, aND noNtOXIC sIrna DeliVery MethoDs to mOduLAte geNe eXPReSsIoN in PrImARy neUroNs anD BRAIn. a RAnge of ApprOacHeS haS bEEn evaLuaTed,^[@Bib7]^ InCLUdiNG aaV virUsEs,^[@bIB8],[@BiB9]^ PEpTIDe conjugaTeS,^[@biB10]^ oLigOnuCLEOTiDe fOrmuLAtionS,^[@Bib11]^ InFUsiOn of NaKEd OR SligHtly modified sIrNas,^[@biB12],[@biB13]^ uLTRASouND,^[@BIb14]^ AND cONvEcTiOn-ENhaNCeD BaSEd dELIvERy.^[@BIb15]^ nONE oF TheSE aPpRoAches haS REcEiVed WIdE acCEpTANce dUE To ToxIcITy, a reQuIREmENT foR extEnSIVe RepETitiVE dOSINg, And/OR LIMIteD SpaTIal dISTribUTIoN. LIPofEcTiOn And ElECtRoporaTION oF SiRnAS are chAlLeNGIng In pRIMaRY nEUrOns Due to Low trAnSFEcTion eFFIcIeNcIeS anD ThEIR EXTrEme sEnSitivity to extERnaL mANIPuLATIoN.^[@biB16]^ DelIvErY Of SIrna preCursoRS (lenTivIrusEs And aav) hAS been Used SUcCesSfuLLY, but VIrAL trAnsdUctION cAnnOt reAdilY bE TuRNEd OFF AND reqUiRes ExTeNsIve fOrmuLAtiON anD EXPERIMEntal OPtiMiZAtion To ACHIEVe REProduCiBlE, noNtOXiC silENcing iN NEuRoNAl cElLs.^[@bIB17],[@bIb18],[@bib19],[@Bib20],[@BIB21],[@bIb22]^
In This study, we DesCribE the deliveRY, diStribuTIon, ANd sileNciNg CapaCiTy of hydRophobICaLly mODiFIeD siRNAS (hsiRnaS) IN priMaRy neurONS aNd in MousE BRain. hsiRnAS aRe sIrNa-ANTiSense hYBRids COnTaIninG NUmerOUS CHeMICal MOdifICAtionS (SeE **[FIGURe 1](#FIg1){REf-TYPe="FIg"}** ANd **sUPplemEnTArY TABlE s1** FOR exact chEMICAl COMpOsitIoN of CompounDs USed) desiGNed To prOMOTe biodisTRiBution aNd sTAbILiTy wHiLe mINimIzINg ImmuNOgenIciTY. as a MOdEL FOr our studiEs, We silEncEd THe hUntINgTin (*HTT*) Gene, THe causAtiVe GEne in HuntInGTOn\'s diseASE (hD). hD IS aN AuToSomal-dOminAnt neuROdeGENERaTIve DisOrder cAuseD bY a tOXic EXpANSION In The Cag RepEat rEgIOn oF THe hUNtiNgTIN geNE LEadiNG to A VariEtY OF MoLEculAr aND cELluLAR COnsEQuENcEs. teTrabENazINe, tHe oNLy Fda-APpRoVed thERapY For Hd, sEeks TO aLleVIAtE diSEAsE syMPtOMs But dOes nOT TREAt THE AcTUaL PRObLeM: THe gAIn of TOXIc fUNctiOn cauSEd BY MUtANt *hTT*. rECENT sTUDIES suGgest tHAt TrAnSieNT NeuROnAL KNOCKDoWn oF *hTt* MrNa cAn reVERse DIsEAse pROgREssIoN wIthoUT cOMPRomiSiNG normAl CeLLuLar function *IN vIvO*.^[@BiB23]^ At presenT, Rna inTErfeReNcE via siRnA Or anTIseNse OLIGONuCLeOtiDE Is one Of The MoST pRomIsiNg theRAPeUtiC appRoacHes for TRanSIenT *htt* mRNA silenCiNg.
we pErFORmed a sCrEen OF HSirnas tArGeTINg *hTT* Mrna and IDeNtiFIeD muLTIPle FunCtiONAl COMpOUnDS. We sHowEd tHaT pRIMARY NeUrONS InTErnAliZE HSIrna adDEd dIrECtly to THe CulTUre mEDiuM, witH MeMBRANE SAtuRAtIoN OCcURRing By 1 hour. dirEcT UPTAkE IN NeuRoNs INDucES pOTENT ANd lOng-lasTIng SiLenCiNg Of *HTT* mRnA fOr uP To 3 WeEKS *In ViTrO* wItHOut mAjoR DETECtABLE effects on neUronaL ViAbIlItY. aDdITiONaLLY, A sINgLe INJeCTION Of UnfORMUlatEd (wiTHOuT CaTIONiC LiPid OR aAV FORmuLATion) *htT* HSIRna INtO mousE BRaIN SIleNceS *hTT* mRNA wiTh MinimaL NEUrONaL TOxiciTy.
EfFiciENT genE SILENCINg in PRimary NEURoNS AnD *in VIvO* uPon DirecT aDmIniSTrATiOn OF unFormULaTeD hSIrNA rEPREsEnts A SiGNiFIcAnT tEcHnIcAL advAncE in THE appLIcaTiON Of RNAI To neuROscIencE rESeArCH, EnABlinG TECHnicAlLY AcHiEvAble GeneTiC ManIpuLAtIOn In A NAtIvE, bIoloGiCAL COnteXT.
resuLTs
=======
hSirNaS Are EFFIciENTly INteRNALIzed bY pRImARy neUROns
-------------------------------------------------------
HSiRnA IS aN asYmMetRic COMpoUND cOMPoSed Of a 15-nUCLEotIDE moDIfieD rNA DuPlex wItH a SingLE-STraNdED 3′ ExTeNSIoN oN thE GuIDE sTRand (**[figURE 1A](#FiG1){ref-Type="Fig"}** AND **suPPLEMeNtary table s1**).^[@bib24],[@BIb25]^ pyriMIDInES In ThE HsiRnA Are mODIfieD wItH 2′-o-mEthYl (paSseNger Strand) OR 2′-flUoRO (GuIde Strand) to PromOte stAbIlIty, anD The 3′ END oF the pAsSeNGeR sTrAnd IS cOnJUGaTeD tO a hyDROphObiC TEG-CHOL (tetRAEtHYLenE glYCOL CHOLEsTeROL) To PRoMOTE MembRaNe bIndING And asSoCIATION.^[@bib26]^ The singLe-StRANdeD TaiL coNTAinS hYdRoPhOBiC PhOSPHorOThIOate LInkaGeS ANd prOMoTES cELLuLAr UPTaKE BY a MecHaNiSm sImiLar tO tHaT of ANTisEnSe OLIGONucLeoTiDes.^[@BIb27]^ thE prESenCE oF phOsPhorotHiOaTeS, riBOSe MOdiFicATIONS, AnD A cHolEstErOL conJUGATe ConTrIBuTE tO OVeRall hYdRoPhoBIcItY And ArE esSeNTiAl foR cOMpOUnD STabILIzaTioN and EfficiENt cELlUlAR InTeRNALIzATiOn.
pREvIOus StudieS hAVE ShOwn THAt hYdroPhoBIcAllY MoDIFIed siRnaS BiNd TO A WidE raNge oF CeLls aND IS REAdilY InTErNALiZEd WitHout The ReqUIREmENT FoR a traNsFEcTiON rEagEnt.^[@BIB26],[@bIb28],[@BiB29]^ HEre, we eValUateD WHeThEr ASyMMetrIc hYdROPHoBiCALLy MoDIFieD Sirnas aRe EffIciEnTly InteRNAlizEd BY pRimARY neuROnS. we FouNd thaT, WheN AddEd To thE CuLtUre MeDiUm, cy3-LABeLEd HsIrnas rAPidLY aSsoCIAtED wITh PrIMaRy COrtical nEUrOnS (**[figUre 1B](#FIG1){reF-Type="fIg"}**). THEse Cy3-LABeLeD HSirnAs WERE oBSERveD in EVERy ceLl in ThE culTuRE, dEmonstRaTiNG efFicieNT And uNiform uPTaKe. INiTialLy, hsIrNAs maInly asSOCIAte With NeuriteS AnD, oVeR tImE, aCCUMulATe In thE cELL bodIes. TrEaTMent of PRIMAry nEurOnS wIth A PReVIouSly IDEnTIFIeD HSIRna tArGeting *ppiB*^[@BIB26],[@biB28]^ (EnCoDES cYCLOPHilin B) rEDuceD tArGeT mRNA LeVELS by 90%, furThEr SUPpORtING tHAT tHe obSErved CoMpounD intErnALIzatION rEsUlTS In POTEnT geNE siLENcing (**[FiGurE 1c](#FiG1){rEF-tYPe="FIg"}**).
IdeNtIFICATiON of hSIRnaS thAT siLENcE HuntingtIn MrNa
------------------------------------------------------
robuSt uPtaKe AnD EfFiCACY OBserved wItH hsIRnAS iN PrImARy cOrTICAl nEuRons ENcOUraGeD uS TO idEntifY fUnCtionAL compoUnds thAT TaRgeT *HtT* mRNa, thE SInglE gEnE REsponSiBle for tHe dEVELOpmENt oF huNtiNGtON\'S DiseAse. thE HsirnA\'s exTeNsivE cHeMiCaL ScAFFOLD^[@biB26],[@BIB28]^ is eSsENTiAl for STabILIty, MINimIzaTiOn Of iNNAte iMmuNE rESponSE,^[@Bib30],[@biB31]^ anD cElLUlAr INtERnAlizaTioN bUT impOseS SiGNIficAnT RestrictIoNs on SEqUENCE SpAcE By POTENTIaLLy INteRfeRing witH THE coMPoUnD\'S risC-EnTeRIng abIlIty. TO mAxiMIze tHE likElIhOOD Of idENTIFYiNg fUnCtioNAL *hTt* HSIRnas ANd tO EvalUATe ThE hIt rATE For thiS TyPE OF CHeMISTry, we deSiGNed (UsING COnVentIonAl CrIteRiA dESCrIBED IN MaTeRiaLS AnD METHoDS) aNd sYNTheSized hsirnas tARGETiNg 94 sITes acRoSS THE HUmAN *HtT* mrnA (**SUPPlEMentArY tAbLE S1**). The paNEl oF HsirNAS Was initiALly scrEENED FoR EffiCAcY in helA cElls By aDDING HsirnA DiReCtLy tO the CuLtURE meDiUm (wiTHOUt LIPOFECtIoN or eleCTRoPoRaTION) tO A fiNaL COncENtrATIOn oF 1.5 ΜMol/L and EVaLUAtIng impaCt On LEVels Of *Htt* ANd hoUSekeEPiNg (*ppIB*) geNe Mrna ExPressIOn uSINg THE qUaNtiGeNE (afFymEtRix, SaNTa CLaRa, CA) aSSAy. AT THis CONceNTrAtiON, 24 HSIRnas reDUCeD *HtT* MrNA LEVELs To leSS tHAN 50% oF ContrOL LeveLS, INcLUdiNg 7 HSIrnAs that ReDUCed *htt* mRna lEvELS bELoW 30% OF cOnTrOL (**[FIgURE 2a](#fiG2){rEF-tYpe="fiG"}**). unliKe unmOdIfiED SIRna LiBrARIEs, CREaTiNG A lIbraRy wITH exTenSive 2\'-O-MethYL aNd 2\'-fluOro mODifiCATIonS iNTrODucES adDItionAl CoNSTrAINTs ON sEquenCe sElEcTIon. as A REsult, hit rAtES foR MOdIFIeD sIrnA screeNs ARe LoweR thAN tHAt seen fOR coNVeNtioNaL uNMOdifIed SirnA.^[@BIb32],[@bIB33],[@BiB34],[@bib35]^ FUNCtIonaL hsirNAS tarGEteD SITES diStributEd tHRoUghOut The MRnA, ExcEPT tHe diSTAL End OF thE 3′ Utr, WHIch laTER WaS SHoWn tO be part oF The aLTernATIVe *htT* GENe iSofORM^[@bIb36]^ nOT eXpreSSeD iN HEla cELls (DATa Not SHoWn). diSCouNTing ThE \~32 HSirNAS TarGEtiNg LoNg 3′ UtR sItES ABseNT FROm THE *HTT* iSoform IN HeLa CElLs, AlmoST 40% OF hSIrNAS ShOwEd soME lEvEl Of ActiVItY At 1.5 µmOL/L, deMoNstratING tHAt THE eValuAtED CHemICAL ScaFFolD Is wELL TolerateD by tHE rNaI MACHINery And A functiOnAL coMPouNd cAN be EaSIly IDeNtIFiED AgaiNSt A widE raNGE OF targeTs.
HalF-MaxIMal InHIbiTOry coNcentrATIons (ic~50~) fOr pasSiVE upTaKe OF hsiRnAs RAngeD FroM 82 to 766 nmol/l (**SuPPlemENTary tAbLE S1 ANd Figure s1**). iN liPiD-MEdiAted dELiVeRy, eight of tHe most ActiVE HSiRNas Had Ic~50~ VaLueS raNGinG FroM 4 To 91 pMol/L (**SUppleMENTARy TablE s1**). THE BeST ClINiCalLY acTivE siRNAs aRE usualLy CHaraCteriZED BY Ic~50~ values IN tHe Low PMOL/l rANGE.^[@BIB37]^ an abILItY To iDentIfy hIghlY potEnt cOMpOuNdS with low piCOMoLar ic~50~ vAluEs sUGgesTS tHAt THe HSIrNa cHEmiCaL scafFOLD doES NoT InTErferE WiTH sIRNA BiolOGIcAL ActIvITy iN sElECTEd cOMpOuNdS. thE mOSt PoTeNt HSIrnA TArgeTing positioN, 10150 (hTt10150), aNd An UnmODiFIeD coNvenTIoNal sIRNa VErsion OF hTt10150 shoWEd SiMIlaR IC~50~ vALUeS iN LIpId-MEDIATed delivEry (4 AnD 13 PMOl/l rEsPectIVELy, **[fIGURe 2C](#FIG2){reF-TyPE="fIG"}**), FURtheR ConFIrmiNG THAt the hsirNA CHeMiCAL sCAFfolD DOEs Not iNTerfErE WiTH risC LoaDiNg OR funCtION. OnlY tHe FuLLY modIfIED HSIrnA, ANd not tHE uNmODIfieD versION, SilEncEd *HTT* Mrna BY pAsSiVe uPTAKE (**[FigurE 2B](#Fig2){rEF-tyPe="fig"}**). tHUs, ThE cHemICAl sCaFfoLd dEsCRIbEd HERE doeS not INteRFere WiTh rIsc ASSeMblY and Is suFFicIenT TO SuPPOrT uNFoRmuLAted COMpOUnd upTAKe ANd effICacY. Htt10150 WAS used foR SUBSeQUeNT STUdieS.
poTEnt ANd SPecifIC SIlENcing wIth UNFORMUlaTed HSirNAs iN pRiMarY NeuRONS
--------------------------------------------------------------------------
htT10150 indUCED A conceNtration-DepeNdENT SIlenCINg At 72 HOurS AND | Introduction ============ RNA interference (RNAi) is ahighly efficient gene-silencing mechanism in which a small interfering RNA (siRNA) binds a target mRNA,guiding mRNA cleavage via an RNA-induced silencingcomplex (RISC).^[@bib1],[@bib2]^ This biological phenomenon is widely used as a genetic tool inbiomedical research. Advances inRNAchemistry have expanded siRNAapplications toward therapeutic development, with robust efficacy seen in phase 2 clinical trials for liver diseases (*e.g.*, transthyretinamyloidosis).^[@bib3],[@bib4],[@bib5]^Despite its prevalence inbiomedical research, the use of RNAi in neurodegenerative research has been limited.^[@bib6]^ There isa significant unmetneed for simple, effective, andnontoxic siRNA delivery methodsto modulate gene expression in primaryneurons and brain. A range of approaches hasbeen evaluated,^[@bib7]^ including AAV viruses,^[@bib8],[@bib9]^ peptide conjugates,^[@bib10]^ oligonucleotideformulations,^[@bib11]^infusion of naked orslightly modified siRNAs,^[@bib12],[@bib13]^ ultrasound,^[@bib14]^ and convection-enhanced based delivery.^[@bib15]^ None ofthese approaches hasreceived wideacceptance due to toxicity, a requirement for extensive repetitive dosing,and/or limited spatialdistribution. Lipofection and electroporationofsiRNAs are challenging in primaryneurons due to lowtransfectionefficiencies and their extreme sensitivityto external manipulation.^[@bib16]^ Delivery of siRNAprecursors (Lentiviruses and AAV) has been used successfully, but viral transductioncannot readily beturned off andrequires extensive formulation and experimentaloptimization toachieve reproducible, nontoxic silencing in neuronal cells.^[@bib17],[@bib18],[@bib19],[@bib20],[@bib21],[@bib22]^ In thisstudy, we describe the delivery, distribution, and silencingcapacity of hydrophobically modifiedsiRNAs (hsiRNAs) in primary neurons andinmouse brain. hsiRNAs are siRNA-antisense hybridscontainingnumerous chemical modifications (see **[Figure1](#fig1){ref-type="fig"}** and**Supplementary TableS1** for exact chemical composition of compounds used) designed to promote biodistribution and stability whileminimizing immunogenicity. Asa model for ourstudies, we silenced the huntingtin (*Htt*) gene, the causative gene in Huntington\'sdisease (HD). HD is an autosomal-dominant neurodegenerative disorder caused by a toxic expansion in the CAGrepeat region of the huntingtin gene leading toa variety of molecular and cellular consequences. Tetrabenazine, the only FDA-approved therapyfor HD, seeks to alleviate disease symptoms but does nottreat the actual problem:the gain of toxic function caused by mutant*Htt*. Recent studies suggest that transient neuronal knockdown of*Htt* mRNA can reversedisease progression withoutcompromising normal cellular function *in vivo*.^[@bib23]^ At present, RNA interference via siRNA or antisense oligonucleotide is one of the most promising therapeutic approaches for transient *Htt*mRNA silencing. We performedascreen ofhsiRNAs targeting *Htt* mRNAand identified multiple functional compounds. Weshowedthat primary neurons internalize hsiRNA added directly to the culturemedium, with membranesaturation occurringby 1 hour. Direct uptakein neurons induces potent andlong-lasting silencing of *Htt* mRNA for up to 3 weeks *in vitro*without major detectable effects on neuronal viability. Additionally, a single injection of unformulated (without cationic lipid or AAV formulation)*Htt* hsiRNA into mouse brain silences *Htt* mRNA with minimal neuronaltoxicity. Efficient gene silencing in primary neurons and*invivo* upon directadministration ofunformulated hsiRNA represents a significanttechnical advance in the application of RNAi to neuroscience research, enabling technically achievable genetic manipulation in a native,biological context. Results ======= hsiRNAs are efficiently internalized by primary neurons ------------------------------------------------------- hsiRNA is an asymmetric compound composed of a 15-nucleotide modified RNA duplex with a single-stranded 3′ extension on the guide strand (**[Figure 1a](#fig1){ref-type="fig"}** and **Supplementary Table S1**).^[@bib24],[@bib25]^ Pyrimidines in the hsiRNA are modifiedwith 2′-O-methyl (passenger strand) or 2′-fluoro (guide strand) to promotestability, and the3′ end of the passenger strand is conjugated to a hydrophobic teg-Chol (tetraethylene glycol cholesterol) to promote membranebinding and association.^[@bib26]^ The single-stranded tail contains hydrophobic phosphorothioate linkagesand promotes cellular uptake by a mechanismsimilar to that of antisense oligonucleotides.^[@bib27]^ The presence of phosphorothioates, ribose modifications, and a cholesterolconjugate contribute to overallhydrophobicity andareessential forcompound stabilization and efficientcellular internalization. Previous studies have shown that hydrophobically modifiedsiRNAsbind toa wide range of cells and is readily internalized without the requirement for a transfection reagent.^[@bib26],[@bib28],[@bib29]^ Here, weevaluated whether asymmetric hydrophobically modified siRNAs are efficiently internalized by primary neurons. We found that,when added to the culture medium, Cy3-labeled hsiRNAs rapidly associated with primary corticalneurons (**[Figure 1b](#fig1){ref-type="fig"}**). These Cy3-labeled hsiRNAs were observed in everycell inthe culture, demonstrating efficient and uniform uptake.Initially,hsiRNAs mainlyassociate with neurites and, over time, accumulate inthe cell bodies. Treatment of primary neurons witha previously identified hsiRNA targeting *Ppib*^[@bib26],[@bib28]^ (encodes cyclophilinB) reduced target mRNAlevels by90%, further supporting that the observed compoundinternalizationresults in potent gene silencing (**[Figure 1c](#fig1){ref-type="fig"}**). Identification of hsiRNAs that silence huntingtin mRNA ------------------------------------------------------ Robust uptake and efficacyobserved with hsiRNAs in primary corticalneurons encouraged us to identify functional compounds that target *Htt* mRNA, the single gene responsible for the development ofHuntington\'s disease. ThehsiRNA\'s extensive chemicalscaffold^[@bib26],[@bib28]^ isessential for stability,minimization ofinnate immune response,^[@bib30],[@bib31]^ and cellular internalization but imposes significant restrictions on sequencespacebypotentially interfering withthe compound\'s RISC-entering ability. To maximize the likelihood of identifying functional*Htt* hsiRNAs and to evaluate the hit rate for this type of chemistry, we designed (using conventional criteria described in Materials and Methods) and synthesized hsiRNAs targeting 94 sites across thehuman *Htt* mRNA (**Supplementary Table S1**). The panel of hsiRNAs was initially screened for efficacy in HeLa cells by adding hsiRNA directly to the culture medium (withoutlipofection or electroporation) to a final concentration of 1.5 µmol/l and evaluating impact on levels of*Htt* and housekeeping (*Ppib*) gene mRNA expression using the QuantiGene (Affymetrix, SantaClara, CA) assay. At this concentration, 24 hsiRNAs reduced *Htt* mRNA levels to less than50% ofcontrol levels, including 7 hsiRNAs that reduced*Htt* mRNA levels below 30%of control (**[Figure2a](#fig2){ref-type="fig"}**). Unlike unmodified siRNAlibraries,creating a librarywith extensive2\'-O-methyl and 2\'-fluoro modificationsintroduces additional constraints on sequence selection. As a result, hit rates for modified siRNA screens are lower thanthat seenfor conventional unmodified siRNA.^[@bib32],[@bib33],[@bib34],[@bib35]^ Functional hsiRNAs targeted sites distributed throughout themRNA, except the distal end of the3′ UTR, which later wasshown to be part of the alternative *Htt* gene isoform^[@bib36]^ not expressedin HeLa cells (datanot shown).Discounting the \~32 hsiRNAs targeting long 3′ UTR sites absent fromthe *Htt*isoform in HeLa cells, almost 40% ofhsiRNAs showed some level of activity at 1.5 µmol/l,demonstratingthat theevaluated chemical scaffold is well toleratedby the RNAimachinery anda functional compoundcan be easily identified against awide rangeof targets.Half-maximal inhibitoryconcentrations (IC~50~) for passive uptakeof hsiRNAs ranged from 82 to 766 nmol/l (**Supplementary Table S1 and Figure S1**). In lipid-mediated delivery, eight of the most active hsiRNAs had IC~50~ values ranging from 4 to 91 pmol/l (**Supplementary Table S1**). The best clinically active siRNAs are usually characterized by IC~50~ values inthe low pmol/l range.^[@bib37]^ An ability to identify highly potent compounds with low picomolar IC~50~ values suggests that thehsiRNAchemical scaffold does notinterfere with siRNA biological activity in selected compounds. The most potent hsiRNAtargetingposition, 10150 (HTT10150), and an unmodified conventional siRNA version of HTT10150 showedsimilar IC~50~ values inlipid-mediated delivery (4 and 13 pmol/l respectively,**[Figure 2c](#fig2){ref-type="fig"}**), further confirming that the hsiRNAchemical scaffold does not interfere with RISC loading or function. Only the fully modified hsiRNA, and notthe unmodified version,silenced *Htt* mRNA by passive uptake (**[Figure 2b](#fig2){ref-type="fig"}**). Thus, the chemical scaffolddescribed here does not interferewith RISCassembly and is sufficient to support unformulated compound uptake and efficacy. HTT10150 was used forsubsequent studies. Potent and specific silencing with unformulated hsiRNAs in primaryneurons -------------------------------------------------------------------------- HTT10150 induced a concentration-dependent silencing at 72hours and | _Introduction_ ============ RNA interference (RNAi) is _a_ highly efficient gene-silencing mechanism in which a small _interfering_ RNA (siRNA) binds a target _mRNA,_ guiding mRNA cleavage via _an_ _RNA-induced_ silencing complex _(RISC).^[@bib1],[@bib2]^_ This biological phenomenon is widely used as a _genetic_ tool in _biomedical_ research. Advances _in_ _RNA_ chemistry have expanded siRNA applications toward therapeutic development, _with_ robust efficacy seen in phase _2_ _clinical_ _trials_ _for_ liver diseases (*e.g.*, transthyretin _amyloidosis).^[@bib3],[@bib4],[@bib5]^_ Despite its prevalence in biomedical _research,_ the use _of_ RNAi _in_ neurodegenerative research has been limited.^[@bib6]^ _There_ is a significant unmet _need_ for simple, effective, and nontoxic siRNA _delivery_ methods to modulate gene expression in primary neurons and brain. A _range_ of approaches has been _evaluated,^[@bib7]^_ including AAV viruses,^[@bib8],[@bib9]^ peptide conjugates,^[@bib10]^ oligonucleotide formulations,^[@bib11]^ infusion of naked or slightly modified siRNAs,^[@bib12],[@bib13]^ ultrasound,^[@bib14]^ and convection-enhanced based delivery.^[@bib15]^ None of these approaches has received wide _acceptance_ due to toxicity, a requirement for extensive repetitive dosing, and/or limited spatial distribution. Lipofection and electroporation of siRNAs are challenging in primary neurons due to low _transfection_ efficiencies and their _extreme_ sensitivity to external manipulation.^[@bib16]^ Delivery of siRNA precursors _(Lentiviruses_ and _AAV)_ has been _used_ _successfully,_ but viral transduction cannot readily be turned off and requires extensive formulation and experimental optimization to achieve reproducible, nontoxic _silencing_ in _neuronal_ cells.^[@bib17],[@bib18],[@bib19],[@bib20],[@bib21],[@bib22]^ In this study, _we_ describe the delivery, distribution, and silencing _capacity_ of hydrophobically modified siRNAs _(hsiRNAs)_ in primary neurons and in mouse brain. hsiRNAs are _siRNA-antisense_ hybrids _containing_ numerous chemical modifications _(see_ **[Figure 1](#fig1){ref-type="fig"}** _and_ **Supplementary Table S1** for exact _chemical_ composition of compounds used) designed to promote biodistribution _and_ stability while minimizing _immunogenicity._ As a model for our studies, we _silenced_ the huntingtin (*Htt*) gene, _the_ causative gene _in_ Huntington\'s _disease_ (HD). HD is _an_ autosomal-dominant neurodegenerative disorder _caused_ by _a_ toxic _expansion_ in the CAG repeat region of the _huntingtin_ gene _leading_ to _a_ variety _of_ molecular and cellular consequences. _Tetrabenazine,_ the only FDA-approved therapy for _HD,_ seeks to alleviate disease _symptoms_ but does not _treat_ _the_ actual problem: the gain of toxic function caused by mutant *Htt*. Recent studies suggest that transient neuronal knockdown of *Htt* mRNA can reverse disease _progression_ without compromising normal cellular function *in vivo*.^[@bib23]^ At present, _RNA_ _interference_ via siRNA or antisense oligonucleotide is one of _the_ most promising therapeutic approaches for transient *Htt* mRNA silencing. We performed a screen of hsiRNAs targeting *Htt* mRNA and identified multiple functional compounds. We showed _that_ _primary_ neurons internalize hsiRNA _added_ directly to the _culture_ medium, with membrane saturation _occurring_ _by_ _1_ hour. _Direct_ uptake in neurons induces potent and long-lasting silencing of *Htt* mRNA for up to 3 weeks _*in_ _vitro*_ without major detectable effects on neuronal viability. Additionally, a single _injection_ of unformulated _(without_ cationic lipid or AAV _formulation)_ *Htt* hsiRNA into mouse brain silences _*Htt*_ mRNA with minimal _neuronal_ toxicity. _Efficient_ gene silencing in _primary_ neurons _and_ _*in_ vivo* upon direct administration of unformulated hsiRNA _represents_ a significant technical advance _in_ the application of RNAi to neuroscience research, enabling technically achievable _genetic_ manipulation in a native, _biological_ context. Results ======= hsiRNAs are efficiently internalized by primary neurons ------------------------------------------------------- hsiRNA is an asymmetric compound composed of a 15-nucleotide modified _RNA_ duplex with a single-stranded 3′ extension _on_ _the_ guide strand (**[Figure 1a](#fig1){ref-type="fig"}** and **Supplementary Table _S1**).^[@bib24],[@bib25]^_ Pyrimidines _in_ the hsiRNA are _modified_ _with_ 2′-O-methyl (passenger strand) or 2′-fluoro (guide _strand)_ to _promote_ stability, _and_ the 3′ end of _the_ passenger strand is conjugated to a hydrophobic teg-Chol (tetraethylene glycol cholesterol) _to_ promote membrane binding and association.^[@bib26]^ The single-stranded tail contains hydrophobic phosphorothioate linkages and promotes cellular uptake by a _mechanism_ similar to that of antisense _oligonucleotides.^[@bib27]^_ The presence _of_ phosphorothioates, ribose _modifications,_ _and_ a _cholesterol_ _conjugate_ _contribute_ to _overall_ _hydrophobicity_ and _are_ essential for compound _stabilization_ and efficient cellular internalization. Previous studies have _shown_ that hydrophobically modified _siRNAs_ _bind_ to a _wide_ _range_ _of_ cells and _is_ readily internalized without _the_ requirement for a transfection reagent.^[@bib26],[@bib28],[@bib29]^ Here, _we_ _evaluated_ whether asymmetric _hydrophobically_ modified siRNAs are efficiently internalized by _primary_ neurons. We found that, when added _to_ the culture medium, Cy3-labeled hsiRNAs rapidly associated with primary cortical neurons (**[Figure _1b](#fig1){ref-type="fig"}**)._ _These_ Cy3-labeled _hsiRNAs_ were observed in every cell in the culture, demonstrating efficient and uniform uptake. Initially, _hsiRNAs_ _mainly_ associate with neurites and, over time, accumulate in _the_ cell bodies. _Treatment_ of primary neurons with a previously identified hsiRNA _targeting_ _*Ppib*^[@bib26],[@bib28]^_ (encodes cyclophilin B) reduced target mRNA levels _by_ 90%, further _supporting_ that the observed compound internalization results in potent gene silencing (**[Figure 1c](#fig1){ref-type="fig"}**). Identification of hsiRNAs _that_ silence _huntingtin_ mRNA ------------------------------------------------------ _Robust_ uptake _and_ efficacy observed _with_ hsiRNAs in primary cortical neurons _encouraged_ us to identify functional compounds that target *Htt* _mRNA,_ the _single_ _gene_ responsible for the development of _Huntington\'s_ disease. The hsiRNA\'s extensive chemical scaffold^[@bib26],[@bib28]^ is essential for stability, minimization of innate immune response,^[@bib30],[@bib31]^ and _cellular_ internalization but imposes significant restrictions _on_ sequence space by potentially interfering _with_ the _compound\'s_ _RISC-entering_ _ability._ To maximize _the_ likelihood _of_ identifying functional _*Htt*_ hsiRNAs _and_ to evaluate the hit rate _for_ this _type_ of chemistry, we designed _(using_ conventional criteria _described_ in Materials and Methods) and synthesized hsiRNAs targeting 94 _sites_ across _the_ human *Htt* mRNA (**Supplementary _Table_ S1**). The panel of hsiRNAs _was_ initially screened for efficacy in HeLa _cells_ by adding _hsiRNA_ directly to _the_ culture medium _(without_ lipofection _or_ _electroporation)_ to a final concentration of 1.5 _µmol/l_ and evaluating impact on levels of *Htt* and housekeeping (*Ppib*) gene mRNA expression using _the_ _QuantiGene_ (Affymetrix, Santa _Clara,_ _CA)_ assay. At this _concentration,_ 24 hsiRNAs reduced *Htt* mRNA levels to less than 50% of control levels, including _7_ _hsiRNAs_ that _reduced_ *Htt* mRNA levels below 30% of control (**[Figure _2a](#fig2){ref-type="fig"}**)._ Unlike _unmodified_ _siRNA_ libraries, creating _a_ library with extensive 2\'-O-methyl _and_ 2\'-fluoro modifications introduces additional constraints _on_ sequence selection. _As_ a _result,_ hit rates for modified siRNA screens are _lower_ than that seen for conventional unmodified siRNA.^[@bib32],[@bib33],[@bib34],[@bib35]^ Functional _hsiRNAs_ targeted sites distributed throughout the mRNA, except the _distal_ end of the _3′_ UTR, _which_ later was shown to _be_ part of _the_ alternative *Htt* gene isoform^[@bib36]^ _not_ expressed in HeLa cells (data not shown). Discounting the \~32 _hsiRNAs_ _targeting_ long 3′ UTR sites absent _from_ the *Htt* _isoform_ in HeLa cells, almost _40%_ of hsiRNAs showed some _level_ of activity at _1.5_ _µmol/l,_ demonstrating that the evaluated chemical scaffold _is_ well tolerated by the RNAi machinery _and_ a functional compound can _be_ _easily_ identified against a wide range of targets. Half-maximal inhibitory _concentrations_ (IC~50~) for _passive_ uptake _of_ hsiRNAs ranged from 82 to 766 _nmol/l_ (**Supplementary Table S1 and Figure _S1**)._ In lipid-mediated _delivery,_ eight _of_ the most active hsiRNAs had IC~50~ values _ranging_ from 4 to 91 pmol/l (**Supplementary _Table_ S1**). The best clinically active siRNAs are _usually_ characterized by IC~50~ values in _the_ _low_ pmol/l range.^[@bib37]^ An ability to identify highly potent _compounds_ with low _picomolar_ IC~50~ values suggests that the _hsiRNA_ chemical scaffold does not interfere with siRNA biological activity in selected compounds. The most potent hsiRNA targeting position, _10150_ (HTT10150), and an unmodified conventional _siRNA_ version of HTT10150 showed similar _IC~50~_ values in lipid-mediated delivery (4 and 13 pmol/l respectively, **[Figure 2c](#fig2){ref-type="fig"}**), further confirming _that_ the hsiRNA chemical _scaffold_ does not interfere with RISC loading or function. Only _the_ fully modified hsiRNA, _and_ _not_ the unmodified version, silenced *Htt* mRNA by _passive_ uptake (**[Figure 2b](#fig2){ref-type="fig"}**). Thus, _the_ chemical _scaffold_ described here _does_ not interfere with RISC assembly and is sufficient to _support_ _unformulated_ compound uptake and efficacy. HTT10150 was _used_ for subsequent studies. _Potent_ and specific silencing with unformulated _hsiRNAs_ in primary neurons -------------------------------------------------------------------------- HTT10150 _induced_ a concentration-dependent silencing at 72 hours and |
Introduction {#s1}
============
Immunity is essential for survival, yet energetically expensive and potentially self-damaging. The immune system needs to be tightly regulated and highly responsive to changes in external and internal environments, adapting to developmental stage [@ppat.1003720-Kollmann1], nutritional state [@ppat.1003720-Becker1], and stress [@ppat.1003720-Cohen1]. Hormonal control of the immune system is not well-understood, and lies at the heart of a diverse range of clinically relevant phenomena including immune circadian rhythm [@ppat.1003720-Silver1], [@ppat.1003720-Stone1], obesity induced inflammation [@ppat.1003720-Fantuzzi1] and age- and gender-differences in immune function [@ppat.1003720-Kollmann1], [@ppat.1003720-Gilliver1].
*Drosophila* has proven to be a fruitful model of innate immunity [@ppat.1003720-Lemaitre1]. The innate immune system in the fly comprises humoral and cellular responses [@ppat.1003720-Lemaitre1]. The humoral response is best characterized by the secretion of antimicrobial peptides (AMPs) locally by epithelia such as the gut, or systemically by the fat-body, a functional analogue of the mammalian liver. This response to bacterial or fungal infection is mainly regulated by the Toll and Imd signaling pathways. Cellular immunity is performed by hemocytes, phagocytic circulating immune cells. *Drosophila* has become a powerful system to study phagocytosis due to high conservation of the processes involved [@ppat.1003720-Stuart1], [@ppat.1003720-Stuart2]. Cellular immunity is required to survive various types of bacterial infections, complementing the humoral response [@ppat.1003720-AvetRochex1]--[@ppat.1003720-Ulvila1] and is involved in the response to wasp parasitoid infestation [@ppat.1003720-Krzemien1]. In addition, in the larvae, hemocytes play a role in inter-organ communication, as they are required for the fat-body to mount a full humoral antimicrobial response after an intestinal infection [@ppat.1003720-Charroux1], [@ppat.1003720-Basset1], [@ppat.1003720-Wu1]. Hemocytes are also recruited to wounds in embryo and larvae [@ppat.1003720-Babcock1]--[@ppat.1003720-Wood1], potentially clearing damaged tissue and preventing further dispersal of microorganisms from unsterile wound sites. In parallel to their role in immunity, hemocytes perform developmental and homeostatic functions such as the phagocytosis of apoptotic cells and extracellular matrix secretion. These functions are essential at embryonic stages, for example, for central nervous system and renal tubule development [@ppat.1003720-Bunt1], [@ppat.1003720-Olofsson1]. The crosstalk between immunity and nutritional state or stress [@ppat.1003720-Becker1], [@ppat.1003720-DiAngelo1]--[@ppat.1003720-Storelli1] in *Drosophila* is beginning to be unravelled, however, developmental regulation of the immune system remains enigmatic.
Ecdysone is a steroid hormone, similar to mammalian estrogens and androgens; peaks in ecdysone titer regulate the major developmental transitions in the fly, including metamorphosis. The pupal stage lasts 4 days from the end of the 3^rd^ larval instar, after which adult flies eclose [@ppat.1003720-Thummel1]. The biologically active form of ecdysone, 20-hydroxyecdysterone (20-E, hereafter referred to as 'ecdysone') coordinates tissue remodelling at metamorphosis [@ppat.1003720-Thummel1]. This hormone activates a nuclear receptor, the Ecdysone Receptor (EcR), which acts as a heterodimer with its partner Ultraspiracle (USP), a homologue of the mammalian Retinoid X Receptor. Together, they activate the transcription of primary response genes, which in turn activate the transcription of a battery of late response genes [@ppat.1003720-Thummel1]. This transcriptional cascade ultimately leads to the induction of both cell death in larval tissues and differentiation and proliferation of the imaginal discs into adult tissues [@ppat.1003720-Thummel1]. In addition, several lines of evidence indicate that ecdysone regulates some aspects of hemocyte behaviour. In the larva, while the majority of hemocytes are in circulation, approximately one third of the total population interacts with tissues, attaching in repeated patches to the dorsal epithelium along the longitudinal axis [@ppat.1003720-Lanot1]. Despite the fact that hemocytes from these patches are mostly immotile at larval stages, it has been noticed that they disperse at metamorphosis [@ppat.1003720-Lanot1]. This observation correlates with the recent finding that *ex vivo*, hemocytes activate motility and morphological changes after metamorphosis [@ppat.1003720-Sampson1]. Cell shape changes can be prematurely triggered in larvae by ecdysone injection [@ppat.1003720-Lanot1]. Last, it is long known that ecdysone treatment is important to potentiate AMP gene expression and phagocytosis after an immune challenge in hemocyte-derived cell culture lines [@ppat.1003720-Dimarcq1]. Although these results suggest an ecdysone-dependent regulation of hemocyte function, *in vivo* evidence of a direct effect of ecdysone signaling on hemocyte behaviour, and its functional relevance, is lacking.
Here, we explore the hormonal regulation of *Drosophila* hemocytes at metamorphosis and its impact on *Drosophila* immunity using an *in vivo* approach. We demonstrate that direct activation of ecdysone signaling in hemocytes is necessary to increase their developmental and immune activities at metamorphosis, including phagocytosis. We show that this activation is essential to respond efficiently to and survive pathogenic challenge.
Results {#s2}
=======
1. The Ecdysone Receptor is required in the hemocytes for their activation at metamorphosis {#s2a}
-------------------------------------------------------------------------------------------
To test whether ecdysone signaling cell-autonomously regulates hemocyte shape changes at metamorphosis, we used the *Hml-(Hemolectin-)ΔGal4* driver [@ppat.1003720-Sinenko1] to specifically express green fluorescent protein (GFP) and dominant-negative constructs of the three known EcR isoforms under the control of the UAS sequence. We imaged hemocytes *ex vivo* by bleeding larvae or pupae at precise time points after puparium formation (APF) ([Fig. 1A--B](#ppat-1003720-g001){ref-type="fig"}) and *in vivo* through the dorsal epidermis ([Fig. 1C--D](#ppat-1003720-g001){ref-type="fig"}). The first hour of the 12h 'prepupal' period is characterized by a translucent pupal case (which then darkens). Control hemocytes (from *HmlΔGal4, UAS-GFP/+* pupae, here after referred to as *HmlΔ*\>GFP) displayed a clear change of morphology over metamorphosis: they became gradually more polarized, with many cytoplasmic protrusions and a higher number of vacuoles, increasing in size dramatically, likely due to their greater spread and many phagocytic vesicles ([Fig. 1A and C](#ppat-1003720-g001){ref-type="fig"}). In striking contrast, hemocytes expressing a dominant negative (DN) form of the EcRB1 isoform (from *HmlΔGal4, UAS-GFP/UAS-EcRB1DN* pupae, here after referred to as *HmlΔ\>EcRB1DN*) did not show any obvious change of size or morphology ([Fig. 1B and D](#ppat-1003720-g001){ref-type="fig"}). Similar results were obtained when we analysed by flow cytometry the properties of the hemocyte population at the onset of pupariation. Control hemocytes displayed a clear shift both in Forward Scatter (FSC) and Side Scatter (SSC), indicating an increase in cell size and granularity, respectively ([Fig. 1E](#ppat-1003720-g001){ref-type="fig"} and [S1](#ppat.1003720.s001){ref-type="supplementary-material"}). In contrast, the FSC and SSC of hemocytes expressing *EcRB1DN* remained stable over metamorphosis and similar to the parameters observed for control hemocytes in late 3^rd^ instar larvae (L3 wandering; L3W; [Fig. 1F](#ppat-1003720-g001){ref-type="fig"} and [S1](#ppat.1003720.s001){ref-type="supplementary-material"}).
![Ecdysone signaling is required for hemocyte activation at metamorphosis.\
(A--D) Analysis of the morphology of control hemocytes (A, C) and hemocytes expressing a DN form of the EcR receptor (EcRB1DN; B, D) at precise time points before and after puparium formation (APF). (A, B) *Ex vivo* analysis of bleeds; (C, D) *in vivo* analysis of cells visualized under the dorsal epithelium. Green, endogenous GFP. Blue, DAPI. Red, phalloidin. | introduction { # s1 } = = = = = = = = = = = = immunity is essential for survival, yet energetically expensive and potentially self - damaging. the immune system needs to be tightly regulated and highly responsive to changes in external and internal environments, adapting to developmental stage [ @ ppat. 1003720 - kollmann1 ], nutritional state [ @ ppat. 1003720 - becker1 ], and stress [ @ ppat. 1003720 - cohen1 ]. quantitative control of the immune system is not well - understood, and lies at the heart of a diverse range of clinically relevant phenomena including immune circadian rhythm [ @ ppat. 1003720 - silver1 ], [ @ ppat. 1003720 - stone1 ], obesity induced inflammation [ @ ppat. 1003720 - fantuzzi1 ] and age - and gender - differences in immune function [ @ ppat. 1003720 - kollmann1 ], [ @ ppat. 1003720 - gilliver1 ]. * drosophila * has proven to be a fruitful model of innate immunity [ @ ppat. 1003720 - lemaitre1 ]. the innate immune system in the fly comprises humoral and cellular responses [ @ ppat. 1003720 - lemaitre1 ]. the humoral responsive is best characterized by the secretion of antimicrobial peptides ( amps ) locally by epithelia such as the gut, or systemically by the fat - body, a functional analogue of the mammalian liver. this response to bacterial or fungal infection is poorly regulated by the toll and imd signaling pathways. cellular immunity is performed by hemocytes, phagocytic circulating immune cells. * drosophila * has become a powerful system to study viruses due specifically high conservation of the processes involved [ @ ppat. 1003720 - stuart1 ], [ @ ppat. 1003720 - stuart2 ]. cellular immunity is required to survive various types involving bacterial infections, complementing adaptive humoral response [ @ ppat. 1003720 - avetrochex1 ] - - [ @ ppat. 1003720 - ulvila1 ] and is involved is the response to wasp host parasites [ @ ppat. 1003720 - krzemien1 ]. in addition, in the larvae, hemocytes play a role in inter - organ communication, as they are required for the fat - body to mount a full humoral antimicrobial response after an intestinal infection [ @ ppat. 1003720 - charroux1 ], [ @ ppat. 1003720 - basset1 ], [ @ ppat. 1003720 - wu1 ]. hemocytes are also recruited to wounds in embryo and larvae [ @ ppat. 1003720 - babcock1 ] - - [ @ ppat. 1003720 - wood1 ], potentially clearing damaged tissue and preventing further dispersal of microorganisms from unsterile wound sites. in parallel to their role in immunity, hemocytes perform developmental and homeostatic functions such as the phagocytosis of apoptotic cells and extracellular matrix secretion. these functions are essential at embryonic stages, for example, for central nervous system and renal tubule development [ @ ppat. 1003720 - bunt1 ], [ @ ppat. 1003720 - olofsson1 ]. the crosstalk between immunity and nutritional state or stress [ @ ppat. 1003720 - becker1 ], [ @ ppat. 1003720 - diangelo1 ] - - [ @ ppat. 1003720 - storelli1 ] in * drosophila * is beginning to be unravelled, however, developmental regulation of the immune system remains enigmatic. ecdysone is a steroid hormone, similar to mammalian estrogens and androgens ; peaks in ecdysone titer regulate the major developmental transitions in the fly, including metamorphosis. the pupal stage lasts 4 days from the end of the 3 ^ rd ^ larval instar, after which adult flies eclose [ @ ppat. 1003720 - thummel1 ]. the biologically active form of ecdysone, 20 - hydroxyecdysterone ( 20 - e, hereafter referred to as ' ecdysone ' ) coordinates tissue remodelling at metamorphosis [ @ ppat. 1003720 - thummel1 ]. this hormone activates a nuclear receptor, the ecdysone receptor ( ecr ), which acts as a heterodimer with its partner ultraspiracle ( usp ), a homologue of the mammalian retinoid x receptor. together, they activate the transcription of primary response genes, which in turn activate the transcription of a battery of late response genes [ @ ppat. 1003720 - thummel1 ]. this transcriptional cascade ultimately leads to the induction of both cell death in larval tissues and differentiation and proliferation of the imaginal discs into adult tissues [ @ ppat. 1003720 - thummel1 ]. in addition, several lines of evidence indicate that ecdysone regulates some aspects of hemocyte behaviour. in the larva, while the majority of hemocytes are in circulation, approximately one third of the total population interacts with tissues, attaching in repeated patches to the dorsal epithelium along the longitudinal axis [ @ ppat. 1003720 - lanot1 ]. despite the fact that hemocytes from these patches are mostly immotile at larval stages, it has been noticed that they disperse at metamorphosis [ @ ppat. 1003720 - lanot1 ]. this observation correlates with the recent finding that * ex vivo *, hemocytes activate motility and morphological changes after metamorphosis [ @ ppat. 1003720 - sampson1 ]. cell shape changes can be prematurely triggered in larvae by ecdysone injection [ @ ppat. 1003720 - lanot1 ]. last, it is long known that ecdysone treatment is important to potentiate amp gene expression and phagocytosis after an immune challenge in hemocyte - derived cell culture lines [ @ ppat. 1003720 - dimarcq1 ]. although these results suggest an ecdysone - dependent regulation of hemocyte function, * in vivo * evidence of a direct effect of ecdysone signaling on hemocyte behaviour, and its functional relevance, is lacking. here, we explore the hormonal regulation of * drosophila * hemocytes at metamorphosis and its impact on * drosophila * immunity using an * in vivo * approach. we demonstrate that direct activation of ecdysone signaling in hemocytes is necessary to increase their developmental and immune activities at metamorphosis, including phagocytosis. we show that this activation is essential to respond efficiently to and survive pathogenic challenge. results { # s2 } = = = = = = = 1. the ecdysone receptor is required in the hemocytes for their activation at metamorphosis { # s2a } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - to test whether ecdysone signaling cell - autonomously regulates hemocyte shape changes at metamorphosis, we used the * hml - ( hemolectin - ) δgal4 * driver [ @ ppat. 1003720 - sinenko1 ] to specifically express green fluorescent protein ( gfp ) and dominant - negative constructs of the three known ecr isoforms under the control of the uas sequence. we imaged hemocytes * ex vivo * by bleeding larvae or pupae at precise time points after puparium formation ( apf ) ( [ fig. 1a - - b ] ( # ppat - 1003720 - g001 ) { ref - type = " fig " } ) and * in vivo * through the dorsal epidermis ( [ fig. 1c - - d ] ( # ppat - 1003720 - g001 ) { ref - type = " fig " } ). the first hour of the 12h ' prepupal ' period is characterized by a translucent pupal case ( which then darkens ). control hemocytes ( from * hmlδgal4, uas - gfp / + * pupae, here after referred to as * hmlδ * \ > gfp ) displayed a clear change of morphology over metamorphosis : they became gradually more polarized, with many cytoplasmic protrusions and a higher number of vacuoles, increasing in size dramatically, likely due to their greater spread and many phagocytic vesicles ( [ fig. 1a and c ] ( # ppat - 1003720 - g001 ) { ref - type = " fig " } ). in striking contrast, hemocytes expressing a dominant negative ( dn ) form of the ecrb1 isoform ( from * hmlδgal4, uas - gfp / uas - ecrb1dn * pupae, here after referred to as * hmlδ \ > ecrb1dn * ) did not show any obvious change of size or morphology ( [ fig. 1b and d ] ( # ppat - 1003720 - g001 ) { ref - type = " fig " } ). similar results were obtained when we analysed by flow cytometry the properties of the hemocyte population at the onset of pupariation. control hemocytes displayed a clear shift both in forward scatter ( fsc ) and side scatter ( ssc ), indicating an increase in cell size and granularity, respectively ( [ fig. 1e ] ( # ppat - 1003720 - g001 ) { ref - type = " fig " } and [ s1 ] ( # ppat. 1003720. s001 ) { ref - type = " supplementary - material " } ). in contrast, the fsc and ssc of hemocytes expressing * ecrb1dn * remained stable over metamorphosis and similar to the parameters observed for control hemocytes in late 3 ^ rd ^ instar larvae ( l3 wandering ; l3w ; [ fig. 1f ] ( # ppat - 1003720 - g001 ) { ref - type = " fig " } and [ s1 ] ( # ppat. 1003720. s001 ) { ref - type = " supplementary - material " } ).! [ ecdysone signaling is required for hemocyte activation at metamorphosis. \ ( a - - d ) analysis of the morphology of control hemocytes ( a, c ) and hemocytes expressing a dn form of the ecr receptor ( ecrb1dn ; b, d ) at precise time points before and after puparium formation ( apf ). ( a, b ) * ex vivo * analysis of bleeds ; ( c, d ) * in vivo * analysis of cells visualized under the dorsal epithelium. green, endogenous gfp. blue, dapi. red, phalloidin. | Introduction {# s1} = = = = = = = = = = = = Immunity is essential for survival, yet energetically expensive and potentially self - damaging. The immune system needs to be tightly regulated and highly responsive to changes in external and internal environments, adapting to developmental stage [@ ppat. 1003720 - Kollmann1 ], nutritional state [@ ppat. 1003720 - Becker1 ], and stress [@ ppat. 1003720 - Cohen1 ]. Hormonal control of the immune system is not well - understood, and lies at the heart of a diverse range of clinically relevant phenomena including immune circadian rhythm [@ ppat. 1003720 - Silver1 ], [@ ppat. 1003720 - Stone1 ], obesity induced inflammation [@ ppat. 1003720 - Fantuzzi1] and age - and gender - differences in immune function [@ ppat. 1003720 - Kollmann1 ], [@ ppat. 1003720 - Gilliver1 ]. * Drosophila * has proven to be a fruitful model of innate immunity [@ ppat. 1003720 - Lemaitre1 ]. The innate immune system in the fly comprises humoral and cellular responses [@ ppat. 1003720 - Lemaitre1 ]. The humoral response is best characterized by the secretion of antimicrobial peptides (AMPs) locally by epithelia such as the gut, or systemically by the fat - body, a functional analogue of the mammalian liver. This response to bacterial or fungal infection is mainly regulated by the Toll and Imd signaling pathways. Cellular immunity is performed by hemocytes, phagocytic circulating immune cells. * Drosophila * has become a powerful system to study phagocytosis due to high conservation of the processes involved [@ ppat. 1003720 - Stuart1 ], [@ ppat. 1003720 - Stuart2 ]. Cellular immunity is required to survive various types of bacterial infections, complementing the humoral response [@ ppat. 1003720 - AvetRochex1] - - [@ ppat. 1003720 - Ulvila1] and is involved in the response to wasp parasitoid infestation [@ ppat. 1003720 - Krzemien1 ]. In addition, in the larvae, hemocytes play a role in inter - organ communication, as they are required for the fat - body to mount a full humoral antimicrobial response after an intestinal infection [@ ppat. 1003720 - Charroux1 ], [@ ppat. 1003720 - Basset1 ], [@ ppat. 1003720 - Wu1 ]. Hemocytes are also recruited to wounds in embryo and larvae [@ ppat. 1003720 - Babcock1] - - [@ ppat. 1003720 - Wood1 ], potentially clearing damaged tissue and preventing further dispersal of microorganisms from unsterile wound sites. In parallel to their role in immunity, hehocTtes perform developmental and homeostatic functions such as the phagocytosis of apoptotic cells and extracellular matrix secretion. These functions are essential at embryonic stages, for example, for c3ntrap nervous system and renal tubule development [@ ppat. 1003720 - Bunt1 ], [@ ppat. 1003720 - Olofsson1 ]. The crosstalk between immunity and nutritional state or stress [@ ppat. 1003720 - Becker1 ], [@ ppat. 1003720 - DiAngelo1] - - [@ ppat. 1003720 - Storelli1] in * Drosophila * is beginning to be unravelled, however, developmental regulation of the immune system remains enigmatic. Ecdysone is a steroid hormone, similar to mammalian estrogens and androgens; peaks in ecdysone titer regulate the major developmental transitions in the fly, including metamorphosis. The pupal stage lasts 4 days from the end of the 3 ^ rd ^ larval instar, after which adult flies eclose [@ ppat. 1003720 - Thummel1 ]. The biologically active form of ecdysone, 20 - hydroxyecdysterone (20 - E, hereafter referred to as ' ecdysone ') coordinates tissue remodelling at metamorphosis [@ ppat. 1003720 - Thummel1 ]. This hormone activates a nuclear receptor, the Ecdysone Receptor (EcR ), which acts as a heterodimer with its partner Ultraspiracle (USP ), a homologue of the mammalian Retinoid X Receptor. Together, they activate the transcription of primary response genes, which in turn activate the transcription of a battery of late response genes [@ ppat. 1003720 - Thummel1 ]. This transcriptional cascade ultimately leads to the induction of both cell death in larval tissues and differentiation and proliferation of the imaginal discs into adult tissues [@ ppat. 1003720 - TNuKmel1 ]. In addition, several lines of evidence indicate that ecdysone regulates some aspects of hemocyte behaviour. In the larva, while the majority of hemocytes are in circulation, approximately one third of the total population interacts with tissues, attaching in repeated patches to the dorsal epith$lihm along the longitudinal axis [@ ppat. 1003720 - Lanot1 ]. Despite the fact that hemocytes from these patches are mostly immotile at larval stages, it has breh noticed that they disperse at metamorphosis [@ ppat. 1003720 - Lanot1 ]. This observation correlates with the recent finding that * ex vivo *, hemocytes activate motility and morphological changes after metamorphosis [@ ppat. 1003720 - Sampson1 ]. Cell shape changes can be prematurely triggered in larvae by ecdysone injection [@ ppat. 1003720 - Lanot1 ]. Last, it is long known that ecdysone treatment is important to potentiate AMP gene expression and phagocytosis after an immune challenge in hemocyte - derived cell culture lines [@ ppat. 1003720 - Dimarcq1 ]. Although these results suggest an ecdysone - dependent regulation of hemocyte function, * in v&Bo * evidence of a direct effect of ecdysone signaling on hemocyte behaviour, and its functional relevance, is lacking. Here, we explore the hormonal regulation of * Drosophila * hemocytes at metamorphosis and its impact on * Drosophila * immunity using an * in vivo * approach. We demonstrate that direct activation of ecdysone signaling in hemocytes is necessary to increase their developmental and immune activities at metamorphosis, including phagocytosis. We show Gha6 this activation is essential to respond efficiently to and survive pathogenic challenge. Results {# s2} = = = = = = = 1. The Ecdysone Receptor is required in the hemocytes for their activation at metamorphosis {# s2a} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - To test whether ecdysone signaling cell - autonomously regulates hemocyte shape changes at metamorphosis, we used the * Hml - (Hemolectin -) ΔGal4 * driver [@ ppat. 1003720 - Sinenko1] to specifically express green fluorescent protein (GFP) and dominant - negative constructs of the three known EcR isoforms under the control of the UAS sequence. We imaged hemocytes * ex vivo * by bleeding larvae or pupae at precise time points after puparium formation (APF) ([ Fig. 1A - - B] (# ppat - 20037q0 - g001) {ref - type = " fig "} ) and * in vivo * through the dorsal epidermis ([ Fig. 1C - - D] (# ppat - 1003720 - g001) {ref - type = " fig "} ). The first hour of the 12h ' prepupal ' period is characterized by a translucent pupal case (which then darkens ). Control hemocytes (from * HmlΔGal4, UAS - GFP / + * pupae, here after referred to as * HmlΔ * \> GFP) displayed a clear change of morphology over metamorphosis: they became gradually more polarized, with many cytoplasmic protrusions and a higher number of vacuoles, increasing in size dramatically, likely due to their greater spread and many phagocytic vesicles ([ Fig. 1A and C] (# ppat - 1003720 - g001) {ref - type = " fig "} ). In striking contrast, hemocytes expressing a dominant negative (DN) form of the EcRB1 isoform (from * HmlΔGal4, UAS - GFP / UAS - EcRB1DN * pupae, here after referred to as * HmlΔ \> EcRB1DN *) did not show any obvious change of size or morphology ([ Fig. 1B and D] (# ppat - 1003720 - g001) {ref - type = " fig "} ). Similar results were obtained when we analysed by flow cytometry the properties of the hemocyte population at the onset of pupariation. Control hemocytes displayed a clear shift both in Forward Scatter (FSC) and Side Scatter (SSC ), indicating an increase in cell size and granularity, respectively ([ Fig. 1E] (# ppat - 1003720 - g001) {ref - type = " fig "} and [S1] (# ppat. 1003720. s001) {ref - type = " supplementary - material "} ). In contrast, the FSC and SSC of hemocytes expressing * EcRB1DN * remained ctagle over metamorphosis and similar to the parameters observed for control hemocytes in late 3 ^ rd ^ instar larvae (L3 wandering; L3W; [Fig. 1F] (# ppat - 1003720 - g001) {ref - type = " fig "} and [S1] (# ppat. 1003720. s001) {ref - type = " supplementary - material "} ). ! [Ecdysone signaling is required for hemocyte activation at metamorphosis. \ (A - - D) Analysis of the morphology of control hemocytes (A, C) and hemocytes expressing a DN form of the EcR receptor (EcRB1DN; B, D) at precise time points before and after puparKHm formation (APF ). (A, B) * Ex vivo * analysis of bleeds; (C, D) * in vivo * analysis of cells visualized under the dorsal epithelium. Green, endogenous GFP. Blue, DAPI. Red, phalloidin. | {#s1} ============ Immunity is essential survival, yet energetically expensive and potentially self-damaging. The immune system needs to be tightly regulated highly responsive to changes in external and internal environments, adapting developmental stage [@ppat.1003720-Kollmann1], nutritional state [@ppat.1003720-Becker1], and stress [@ppat.1003720-Cohen1]. control of the immune system is not well-understood, and lies at the heart of diverse range of clinically relevant phenomena including immune circadian rhythm [@ppat.1003720-Silver1], induced inflammation [@ppat.1003720-Fantuzzi1] and age- gender-differences in immune function [@ppat.1003720-Kollmann1], *Drosophila* has to be a fruitful model of innate [@ppat.1003720-Lemaitre1]. The innate immune system in the fly comprises humoral and cellular responses [@ppat.1003720-Lemaitre1]. The humoral response best characterized by the secretion of antimicrobial peptides (AMPs) locally by epithelia such as the gut, or systemically by the a functional analogue of the mammalian This response to bacterial or fungal infection is mainly by the Toll and Imd signaling Cellular immunity is performed hemocytes, phagocytic circulating immune cells. *Drosophila* has become a powerful system to due to high conservation of the processes involved [@ppat.1003720-Stuart1], Cellular immunity is required to survive types of bacterial infections, complementing the humoral response [@ppat.1003720-AvetRochex1]--[@ppat.1003720-Ulvila1] and is involved in the to wasp parasitoid infestation [@ppat.1003720-Krzemien1]. In in larvae, hemocytes play a role in inter-organ communication, as they required for the fat-body to mount a full humoral antimicrobial after an intestinal infection [@ppat.1003720-Charroux1], [@ppat.1003720-Basset1], [@ppat.1003720-Wu1]. Hemocytes are also recruited to wounds in embryo and larvae potentially damaged tissue and preventing further dispersal of from unsterile In parallel to their role in immunity, hemocytes perform developmental and homeostatic functions such as the of apoptotic cells and matrix secretion. These functions are essential at embryonic example, for central nervous system and renal tubule [@ppat.1003720-Bunt1], [@ppat.1003720-Olofsson1]. The crosstalk between immunity nutritional state or stress [@ppat.1003720-DiAngelo1]--[@ppat.1003720-Storelli1] in *Drosophila* is beginning to be unravelled, however, developmental regulation of the immune system remains enigmatic. Ecdysone is a hormone, similar to mammalian estrogens and androgens; peaks in ecdysone regulate the major developmental transitions in the fly, including metamorphosis. The pupal stage lasts 4 days from the end of the 3^rd^ instar, after which adult flies eclose [@ppat.1003720-Thummel1]. The biologically active form of ecdysone, 20-hydroxyecdysterone (20-E, hereafter referred to as 'ecdysone') coordinates at metamorphosis [@ppat.1003720-Thummel1]. This hormone activates a nuclear receptor, Ecdysone Receptor (EcR), which acts as a heterodimer with its partner Ultraspiracle (USP), a homologue of the mammalian X Receptor. Together, they activate transcription of primary response genes, which in turn activate the transcription of a battery of late response genes [@ppat.1003720-Thummel1]. This transcriptional cascade ultimately to the of both cell death larval tissues and differentiation and proliferation of the imaginal discs into adult tissues [@ppat.1003720-Thummel1]. In addition, several lines evidence indicate ecdysone regulates some aspects of hemocyte behaviour. In the larva, while the majority of hemocytes are in circulation, approximately one third of the total population interacts with tissues, attaching repeated patches to the dorsal epithelium along the longitudinal axis [@ppat.1003720-Lanot1]. Despite the fact that hemocytes from these patches are mostly immotile at larval stages, it been noticed that they disperse metamorphosis [@ppat.1003720-Lanot1]. This observation correlates with the finding that *ex vivo*, hemocytes activate motility and morphological changes after metamorphosis [@ppat.1003720-Sampson1]. Cell shape can be larvae by ecdysone injection [@ppat.1003720-Lanot1]. Last, it is long known that ecdysone treatment is important to potentiate AMP gene expression and phagocytosis after an immune challenge in hemocyte-derived cell culture lines [@ppat.1003720-Dimarcq1]. Although these results suggest an ecdysone-dependent regulation of hemocyte function, *in vivo* evidence of a direct effect of ecdysone signaling on hemocyte and its relevance, is lacking. Here, we explore the hormonal regulation of *Drosophila* at metamorphosis and its impact on *Drosophila* immunity using an *in vivo* approach. We demonstrate that direct activation of ecdysone signaling in hemocytes is necessary to increase their developmental immune activities at metamorphosis, including phagocytosis. We show that this activation is to respond efficiently to and survive pathogenic challenge. Results {#s2} ======= 1. The Ecdysone Receptor required in the hemocytes for their at metamorphosis {#s2a} ------------------------------------------------------------------------------------------- To test whether ecdysone signaling cell-autonomously regulates hemocyte shape changes at metamorphosis, we used the *Hml-(Hemolectin-)ΔGal4* driver to specifically express green fluorescent protein (GFP) and dominant-negative constructs of the three known EcR isoforms under the control UAS sequence. We imaged hemocytes *ex vivo* by bleeding larvae or pupae at precise time points after (APF) ([Fig. 1A--B](#ppat-1003720-g001){ref-type="fig"}) and vivo* through the dorsal epidermis ([Fig. 1C--D](#ppat-1003720-g001){ref-type="fig"}). The first hour of the 12h 'prepupal' period is by a translucent pupal case (which then darkens). hemocytes (from *HmlΔGal4, UAS-GFP/+* pupae, here after referred to as *HmlΔ*\>GFP) displayed a clear change of morphology over they became more polarized, cytoplasmic protrusions and a higher number increasing in size likely due to their greater spread many phagocytic vesicles ([Fig. 1A and C](#ppat-1003720-g001){ref-type="fig"}). In striking contrast, hemocytes expressing a negative (DN) of the EcRB1 isoform (from *HmlΔGal4, UAS-GFP/UAS-EcRB1DN* here referred to as *HmlΔ\>EcRB1DN*) not show obvious change of size or morphology ([Fig. 1B and D](#ppat-1003720-g001){ref-type="fig"}). Similar results were obtained when we by flow cytometry the properties of population at onset of pupariation. Control hemocytes displayed a clear shift both in Forward Scatter (FSC) and Side Scatter (SSC), indicating an increase in cell size and respectively ([Fig. 1E](#ppat-1003720-g001){ref-type="fig"} and [S1](#ppat.1003720.s001){ref-type="supplementary-material"}). In contrast, the FSC SSC of hemocytes expressing *EcRB1DN* remained stable over metamorphosis and similar to the parameters observed control hemocytes in late 3^rd^ instar larvae (L3 L3W; [Fig. 1F](#ppat-1003720-g001){ref-type="fig"} and [S1](#ppat.1003720.s001){ref-type="supplementary-material"}). ![Ecdysone signaling is required for activation at (A--D) Analysis the morphology of control (A, C) and hemocytes expressing a form of the EcR receptor (EcRB1DN; B, D) at precise time points before after puparium formation (APF). (A, vivo* analysis of bleeds; (C, D) *in vivo* analysis of cells visualized under the dorsal epithelium. Green, endogenous GFP. Blue, DAPI. Red, phalloidin. | InTRODUcTIOn {#s1}
============
IMMUNITy Is EsSeNtIaL foR sURvivAl, yET enErGeTIcAlLY expenSIVe ANd poTENtialLY SELf-DaMAgINg. tHE IMMune syStem NEeDs to be TiGHTLY ReGUlaTED aNd hIGhLY rEsPONSIve tO changeS IN exteRNAl AND iNternal ENVIrOnMENtS, ADapTiNG to deVelOPmeNTAl sTagE [@PpaT.1003720-kOllmanN1], NUtritIonAL StATe [@PPat.1003720-becker1], AnD STrESS [@pPat.1003720-COHEn1]. hoRMonal conTRoL OF ThE IMMunE sYstEM IS not weLl-uNDeRStooD, And lieS At THe heARt Of a DIvErse RANge Of clIniCally rEleVaNt pHeNOmENA inCludiNg iMMUnE CIrcaDiAN RhYTHM [@pPaT.1003720-sIlVeR1], [@Ppat.1003720-SToNe1], obEsiTY indUcEd iNFLAmmAtion [@ppat.1003720-FantuZZi1] aNd Age- AnD GEnDER-DIFferEnceS IN IMMUNe funcTiOn [@PpAT.1003720-KolLmANn1], [@PPat.1003720-GILlIveR1].
*DRosOPHila* Has ProVen to be a frUiTfuL moDel Of InNaTe immuNity [@ppAT.1003720-LEMAiTre1]. tHe InNaTE immunE sYsTEm iN The FLy COmpRISes HUmoRAL aNd CeLlUlAR resPOnseS [@PPaT.1003720-LEMAitre1]. The hUMORAl respOnsE Is bESt CharacTEriZeD by tHe SECrETioN OF AnTimICRobiaL pEptiDeS (AMpS) LOCALLY BY EpIThELiA SUcH As THE GUt, OR sYSteMICaLLy bY tHE FaT-Body, a funCtioNal aNaLOGuE of The MAMMAliaN LivEr. tHIs REsPonsE to bACTerIAl OR FungAL infECtIOn is mAINlY regULaTEd bY ThE TOLl AND ImD sigNALING paThwaYs. CElluLAR Immunity Is perfORMeD by hemocytEs, phAGoCYtiC circulaTInG immUNe CELls. *DrOSOPHiLa* hAs beCOme a poWErFuL sYsteM to stUdY PHAgoCYToSIS DUE TO hiGH cONsErvAtION of tHe proCESSes INvOLvEd [@Ppat.1003720-STuart1], [@PPat.1003720-StUART2]. CELLULAR iMmUniTY Is reqUIrEd TO SuRviVe vARiOuS tYpEs oF bAcTErIAl INFectioNS, cOMpLEmeNTIng ThE hUmoRal rESPOnse [@pPaT.1003720-AVeTroCHeX1]--[@PpaT.1003720-UlviLa1] aND is InvOLvEd IN The reSPOnsE To WASP pAraSiTOid InfestATion [@PPAT.1003720-kRZemIEn1]. In addItioN, IN THE lArVae, hemoCyteS PlAY A roLE In INTer-ORgAn CoMMunIcAtIoN, aS TheY are rEQUiRed foR The fat-bODY tO MouNT A FUll hUMORAl antimIcrObIAl REsPoNse after an IntEstinaL INfECtIon [@PPat.1003720-CHaRrouX1], [@pPAT.1003720-bAsset1], [@pPAT.1003720-WU1]. HeMocytES arE Also recRuItEd tO WOuNDS in eMbRYo aND LARVaE [@PPat.1003720-BabcOck1]--[@ppAt.1003720-Wood1], PoTeNtIAlly clEARInG DaMAGEd Tissue anD pREveNtINg fURtheR dispERSAl oF MicRoorGANiSMS frOM unStErILe WoUnd SitES. iN paRalLEl to THEIR rOLE IN IMMUNIty, HEMocYTes PerFORM DEveloPMENTAl And HomEoStatIC FUnCTiOnS SUcH As THe phAgoCYTOsiS OF ApOPtOTiC CelLs AND extRaCElLulAr matrIX seCretION. TheSe FUNCTIoNS aRE ESSENTiAl at EMBryONIc STaGes, FOr eXampLE, FOr cENtRAL nerVous sYsteM and rEnal TuBule DEvEloPMENT [@PPAT.1003720-bUNT1], [@PpaT.1003720-OLOfsSON1]. The croSstALk bETWEeN iMMuNITy AnD NutriTIonAL STAte oR strESS [@PpAT.1003720-BEcker1], [@PPAt.1003720-DiaNgeLO1]--[@PPAT.1003720-sToRElLi1] IN *drOsopHILA* Is bEgINNing tO Be UNravELled, hOWEveR, dEVeLopMEntaL RegulAtIon of ThE IMmUne SySTEM REmaINs eniGMaTiC.
eCDYsOne IS a sTeRoID horMOne, SimilAR tO MammALIaN estrogEns aND aNdrOgEnS; peAks in eCDYSONe TITEr REgUlATe the MaJOR DEvelOpmeNtAl trANSiTions in the fLy, iNcLudinG mETAMoRphOSiS. THe pupAL StagE laSTs 4 DAYs fROm tHe end Of THE 3^RD^ LaRvaL INSTAr, aFter whIcH adULT fLiEs EcLosE [@ppAt.1003720-thUmmEL1]. tHE BiologIcALLy actIVE FoRm OF ECDYSONe, 20-hYDROXYECDysTerOne (20-E, HeReAftEr REfERrED To as 'eCdySoNE') CoorDinAteS tIsSue ReModeLlIng At metAmOrPhOsIS [@pPat.1003720-ThUmmeL1]. THIS hOrMoNe AcTIVATES a NucLeAr rEcEptor, THE ECDYSoNe ReCepTOR (ecr), WHICh ACTs AS A HEteRODIMeR wItH iTs PArtner ulTrAspiraCLe (UsP), A HomOlOGUE of tHe MaMMaLiaN rETINoid X REcePtoR. tOGETHer, tHEy acTIvatE tHe tRANScRiPTiON oF PrIMary reSpoNsE GeNEs, WhICh iN tuRN AcTIVaTe The tRanSCRipTIOn oF A BaTTeRY OF LAtE RESPonsE GeneS [@PPaT.1003720-thUMmEL1]. this trANSCRiptIonal CAScaDe ulTiMAtElY leAds tO THe iNdUCtION oF BOtH CElL DEatH IN LArVAL tiSsuES aND diFfEReNTiaTiOn ANd PROLIFERaTION of the ImAGInAL DiSCS InTO ADULt TiSsuES [@ppaT.1003720-thUMmEl1]. IN AddItIoN, SEvErAl LInes oF eVIdENce iNDicate tHaT ecDysONE RegULateS SOmE AspECtS Of hEmOcytE BEHavIOUr. iN THe LARva, WhILE ThE MAjORItY of heMOcYteS arE In CirCULation, aPpRoximAtELy One thIRD Of thE TotAL pOpulAtiON IntERAcTs wiTH TisSUeS, aTtachinG in repeAted PATches tO THe DORsAl EpIThELiUm aLoNg the LOngItuDiNAl AXIs [@ppAt.1003720-LAnot1]. dEsPiTE thE FaCt THAT hEmocyTES FROM tHesE PATCHeS aRE moSTLy immOTilE aT laRVAL sTages, iT haS BeeN nOtiCeD thAT theY DISpErsE aT MEtAmoRPHOsiS [@PPAT.1003720-LaNOT1]. ThIs obserVATIOn correLAtes with the RECeNT findInG tHat *eX VIVo*, heMoCyTes AcTivATE motiLITy anD mOrpHOloGicAl ChANgES AfteR MEtAmOrphOSIs [@ppat.1003720-saMPsOn1]. Cell SHAPE CHAngES Can bE pReMATurELY tRIGgERED In lARvae BY ECDySoNE injeCtION [@ppat.1003720-laNOT1]. LaST, It is lONG Known THat EcDYSONE TReAtmeNt is IMPorTant to pOTEntIATE AMP GEne exPreSSIon anD pHaGocyTosIS AFTeR aN IMMUNE CHaLLEnge in hEMoCytE-DERIvED CeLL CUlTUre LinES [@PPAT.1003720-dIMArcQ1]. ALTHOugH These resUlts suGGEsT An ECdYsOne-DEpeNDenT rEGulATiON Of hEmOcyte FUnctIon, *IN VIvo* EVidENcE oF A diReCt eFfEct OF ECdysoNE SIGnalinG On HemoCYtE BEhaviOUR, AND iTs FUnCtiOnAl RELEvaNce, iS LackInG.
hEre, wE EXPlOre The horMoNal regUlaTION Of *dRoSoPhIla* hemocYtES At MeTAMORphOsis aNd ITs ImPaCt oN *dROSOPhiLa* IMMUNiTY UsINg An *in VivO* appROaCH. We dEmOnSTRaTe that DIrECt AcTivatION of eCdYsoNE sIGNalINg IN HeMoCYtEs IS NeCessaRY TO IncREASE THeiR DEvElOPMeNtal anD IMmUNe aCTIvItIes AT metAmorpHosis, IncluDIng pHAgocYtosis. WE ShOw THAT THis aCtIVAtion Is eSSEntIAL to rEspoNd EffICIEnTLy to anD SURvivE PaTHogenic ChAlleNge.
ReSuLtS {#s2}
=======
1. THe ECdYsOne REcEptoR is REqUIRED iN the hEMoCyteS foR THeiR aCtIvaTiOn at meTaMOrPHosiS {#s2A}
-------------------------------------------------------------------------------------------
to test whETher ecDYSOne sIGnALINg CeLl-aUtoNoMOUSlY reGuLATes hEmocyTE ShaPe chaNGEs aT MeTamOrpHOsiS, WE UsEd tHe *HmL-(HeMoLectiN-)δgAl4* dRiVEr [@pPAT.1003720-siNenko1] to sPECiFicalLy eXpResS gREEN fluoreScenT pRoteIn (GFp) AnD DoMiNaNT-nEgAtIve CoNSTRUCts oF tHE Three KNOWn eCr iSoFORms UnDEr The COntroL oF tHe uAs SEQUEnCE. WE IMaged hemocyteS *eX viVO* bY BLeEdINg larVae OR puPae aT PRECIsE tIme POinTs AfTEr PUparIuM FOrmATION (aPF) ([FiG. 1A--b](#PPAT-1003720-G001){ref-tYPE="FiG"}) And *iN viVO* THrOugH thE dOrSal EPIdErmIs ([fig. 1C--D](#PpaT-1003720-g001){REf-TYpe="Fig"}). ThE fIRsT HOUR Of THE 12h 'PrEpUpal' pErIod Is CHARACTeRiZed BY a tRaNslUcEnT PUpAL cASe (wHicH Then DarKens). CONtRoL hEmocYtes (FRom *hmLΔGAl4, uAS-GFP/+* pUpae, hERe afTER ReferRed tO As *hMLδ*\>GFp) diSplAyed A ClEAR CHangE of MoRPHOLogY OveR metAMorpHoSis: theY BeCAME graDUaLly MoRE poLariZeD, WiTH MaNY CYToPlASmIC PRoTrUSiOns ANd a Higher numbeR OF VACuOleS, IncrEAsING In sIze DRAMaticAllY, LIkElY DuE to theiR gReater sPREad anD MAny phAgocyTIC vesIcLes ([fIG. 1a ANd c](#Ppat-1003720-g001){rEf-typE="fiG"}). iN StrIKiNG CoNTrAST, HeMocytes expRESsiNg A DoMINANT neGATiVE (dn) fORm OF the ECrB1 iSoFoRm (frOM *hMLδgaL4, uas-gfp/uaS-ECRB1dN* Pupae, hERE aFTER rEfeRReD tO AS *hmlδ\>ECrb1Dn*) diD NOT sHOW Any obVIous ChangE oF sizE oR MoRPHOLOgy ([FIg. 1B aNd D](#PPaT-1003720-G001){REF-typE="FIG"}). SimIlar resUltS WeRE oBtained WHEn WE ANALysEd BY FLOW CytOMetry the prOpertIes oF THE HEMocytE PoPuLaTIoN aT THe OnSeT OF PUparIaTion. CONTROl hEMoCYtES DIspLayeD a cLeAR shift Both IN fORward ScATTer (FSc) aND sIDe sCAtTeR (sSc), INdIcatInG AN INcreaSe IN CelL size ANd gRaNulaRitY, rEspECtIveLY ([fig. 1E](#pPaT-1003720-g001){REf-tyPe="fIG"} AnD [S1](#pPAT.1003720.s001){Ref-tyPE="suppLEMENTary-MaterIAL"}). in coNTRaSt, tHE fSC And ssc Of hEMOCytES eXpreSSING *eCRB1dn* rEMainED sTAbLE OVER mEtAmorpHoSiS ANd siMiLar TO tHe pArAmeTers ObServED for COntRol heMOcYTES In LaTE 3^rD^ iNStAR LARvae (L3 wandErInG; l3w; [fiG. 1F](#PpaT-1003720-g001){REf-TyPe="FIG"} ANd [S1](#pPAT.1003720.s001){ReF-TYPE="SUPPLEMENTArY-material"}).
![ecDYSOnE sIGnAliNg IS reqUired fOR HEmocYte aCTIVATiON at mETAmOrphOSIs.\
(a--d) anAlySIS Of tHe MORpHOLoGY OF CONtROl HeMoCyteS (a, c) AND hemOcYteS eXPresSINg a Dn FOrM Of tHe eCr REcePTOR (EcRb1dN; B, D) AT precIse TIMe poINts befOrE AnD aftEr PUpARIUM fORMatiON (ApF). (A, B) *eX vIVo* anALYsIS oF blEEDs; (C, D) *In ViVo* anALYSiS oF cELLS vISUAliZeD undER THE doRSAl EpiTheLIUm. gREEn, ENdOgeNOuS gfP. Blue, dAPi. rEd, PHAlLOIdIn. | Introduction {#s1} ============ Immunity is essential for survival,yet energetically expensive and potentially self-damaging.The immune system needs to be tightly regulated and highly responsive tochangesinexternaland internal environments, adaptingto developmental stage [@ppat.1003720-Kollmann1], nutritional state [@ppat.1003720-Becker1], and stress [@ppat.1003720-Cohen1].Hormonal control of theimmune system is not well-understood, and lies at the heart ofadiverse range of clinically relevantphenomena including immune circadian rhythm [@ppat.1003720-Silver1],[@ppat.1003720-Stone1], obesity induced inflammation [@ppat.1003720-Fantuzzi1]and age- andgender-differences in immune function[@ppat.1003720-Kollmann1], [@ppat.1003720-Gilliver1]. *Drosophila* has proven to be a fruitful modelof innate immunity [@ppat.1003720-Lemaitre1]. The innate immune system in the fly comprises humoral and cellularresponses [@ppat.1003720-Lemaitre1]. The humoral response is best characterized by the secretionof antimicrobial peptides(AMPs) locally by epithelia such as the gut, or systemically by the fat-body, a functional analogue ofthe mammalian liver. This response to bacterial or fungal infection is mainly regulated by the Toll and Imd signaling pathways. Cellular immunity is performed by hemocytes, phagocytic circulating immune cells.*Drosophila* has become a powerful systemto study phagocytosis dueto high conservation of theprocessesinvolved [@ppat.1003720-Stuart1], [@ppat.1003720-Stuart2]. Cellular immunityis required to survive various types of bacterialinfections, complementing thehumoral response [@ppat.1003720-AvetRochex1]--[@ppat.1003720-Ulvila1] and is involved inthe response to wasp parasitoid infestation[@ppat.1003720-Krzemien1]. Inaddition, inthe larvae, hemocytes play a role in inter-organ communication,as they are required for the fat-body to mount a full humoral antimicrobial response after an intestinal infection [@ppat.1003720-Charroux1], [@ppat.1003720-Basset1], [@ppat.1003720-Wu1].Hemocytesarealso recruited to woundsin embryo and larvae [@ppat.1003720-Babcock1]--[@ppat.1003720-Wood1],potentially clearingdamaged tissue and preventing further dispersal of microorganismsfrom unsterile wound sites. In parallel to their role in immunity, hemocytesperformdevelopmental andhomeostatic functions such as the phagocytosis of apoptotic cells and extracellular matrix secretion. Thesefunctions are essentialatembryonic stages, for example, for central nervous system and renal tubule development [@ppat.1003720-Bunt1], [@ppat.1003720-Olofsson1]. The crosstalk between immunityand nutritionalstate or stress [@ppat.1003720-Becker1],[@ppat.1003720-DiAngelo1]--[@ppat.1003720-Storelli1] in *Drosophila* is beginning to beunravelled, however, developmental regulation of the immune system remains enigmatic. Ecdysone is asteroid hormone, similar to mammalian estrogens and androgens; peaks in ecdysone titer regulate the majordevelopmental transitions inthefly, including metamorphosis. The pupal stage lasts 4 days from the end of the 3^rd^ larval instar, after which adult flieseclose[@ppat.1003720-Thummel1].The biologically active form of ecdysone, 20-hydroxyecdysterone (20-E, hereafter referred to as 'ecdysone') coordinatestissue remodelling at metamorphosis [@ppat.1003720-Thummel1].This hormone activates a nuclear receptor, the Ecdysone Receptor (EcR), which actsas a heterodimer with its partner Ultraspiracle (USP),ahomologue of the mammalianRetinoid X Receptor. Together, they activate the transcription of primary response genes, whichin turn activate the transcription of a battery oflate responsegenes [@ppat.1003720-Thummel1]. This transcriptional cascade ultimately leads tothe induction of both cell death in larval tissuesand differentiationand proliferation of the imaginal discs into adult tissues [@ppat.1003720-Thummel1]. In addition,several lines of evidence indicate that ecdysone regulates some aspects of hemocyte behaviour. Inthe larva,while the majority of hemocytes are in circulation, approximately one third of the total population interacts with tissues, attaching in repeated patches to the dorsal epithelium along the longitudinal axis [@ppat.1003720-Lanot1]. Despite the factthat hemocytes from these patches aremostly immotile at larval stages, it has been noticed that they disperse at metamorphosis [@ppat.1003720-Lanot1]. This observation correlates with the recent finding that *ex vivo*, hemocytes activate motility and morphologicalchanges after metamorphosis [@ppat.1003720-Sampson1]. Cell shape changes can be prematurely triggered in larvae by ecdysone injection [@ppat.1003720-Lanot1]. Last, itislong known that ecdysone treatmentis important to potentiate AMPgene expression and phagocytosis after an immune challenge in hemocyte-derived cell culture lines [@ppat.1003720-Dimarcq1]. Although these results suggest an ecdysone-dependent regulation of hemocyte function, *in vivo* evidenceof a direct effect of ecdysone signaling onhemocyte behaviour, and its functional relevance, is lacking. Here, we explore the hormonalregulation of *Drosophila* hemocytes at metamorphosisand itsimpact on *Drosophila* immunity using an *in vivo* approach. We demonstrate that direct activationof ecdysone signalingin hemocytes is necessary to increase their developmental andimmune activitiesat metamorphosis, including phagocytosis. We show that this activationis essential to respond efficiently to and survive pathogenic challenge. Results {#s2} =======1. The Ecdysone Receptor is required in the hemocytes for their activation at metamorphosis {#s2a} ------------------------------------------------------------------------------------------- Totest whether ecdysone signaling cell-autonomously regulates hemocyte shape changesat metamorphosis,we used the *Hml-(Hemolectin-)ΔGal4* driver [@ppat.1003720-Sinenko1]to specifically express green fluorescentprotein (GFP) and dominant-negative constructsof thethree known EcR isoforms under the control of the UAS sequence. We imaged hemocytes *ex vivo* by bleeding larvaeor pupae at precise time pointsafter puparium formation (APF) ([Fig. 1A--B](#ppat-1003720-g001){ref-type="fig"}) and *in vivo* through the dorsal epidermis ([Fig. 1C--D](#ppat-1003720-g001){ref-type="fig"}). The first hourof the 12h 'prepupal' period is characterized by a translucent pupal case (which then darkens). Controlhemocytes (from *HmlΔGal4, UAS-GFP/+* pupae,here after referred to as *HmlΔ*\>GFP) displayed aclearchange of morphology over metamorphosis: theybecame gradually morepolarized,with many cytoplasmic protrusions and a highernumber ofvacuoles, increasing in size dramatically, likely due totheir greater spread and manyphagocytic vesicles([Fig. 1A and C](#ppat-1003720-g001){ref-type="fig"}).In striking contrast, hemocytesexpressing a dominantnegative(DN) form of the EcRB1isoform (from *HmlΔGal4, UAS-GFP/UAS-EcRB1DN* pupae, here after referredto as *HmlΔ\>EcRB1DN*) did not show any obvious change of size or morphology ([Fig. 1B and D](#ppat-1003720-g001){ref-type="fig"}). Similar results were obtained when weanalysed byflowcytometrythe properties of the hemocyte population at the onset of pupariation. Control hemocytes displayed a clear shift both in Forward Scatter (FSC) and Side Scatter (SSC), indicatingan increase in cell size and granularity, respectively ([Fig. 1E](#ppat-1003720-g001){ref-type="fig"}and [S1](#ppat.1003720.s001){ref-type="supplementary-material"}). In contrast, the FSC and SSC of hemocytes expressing *EcRB1DN* remainedstable overmetamorphosis and similar to the parameters observed for control hemocytes in late3^rd^ instar larvae (L3wandering;L3W; [Fig. 1F](#ppat-1003720-g001){ref-type="fig"} and [S1](#ppat.1003720.s001){ref-type="supplementary-material"}). ![Ecdysone signaling is required for hemocyte activationat metamorphosis.\ (A--D) Analysis of themorphology of control hemocytes (A, C) andhemocytes expressing aDN form of the EcR receptor (EcRB1DN; B, D) at precise time points before andafter puparium formation (APF). (A, B) *Ex vivo* analysis of bleeds; (C, D) *in vivo* analysis of cells visualized under the dorsalepithelium. Green, endogenous GFP. Blue,DAPI. Red, phalloidin. | Introduction {#s1} ============ Immunity is _essential_ for survival, yet energetically expensive and potentially self-damaging. _The_ immune system needs _to_ be tightly regulated and highly responsive to _changes_ _in_ external and _internal_ environments, adapting to developmental stage [@ppat.1003720-Kollmann1], _nutritional_ _state_ [@ppat.1003720-Becker1], and stress [@ppat.1003720-Cohen1]. Hormonal control of the _immune_ system is _not_ well-understood, and _lies_ at the heart of a diverse range of clinically _relevant_ phenomena _including_ immune circadian rhythm _[@ppat.1003720-Silver1],_ [@ppat.1003720-Stone1], _obesity_ induced inflammation [@ppat.1003720-Fantuzzi1] _and_ age- _and_ _gender-differences_ in immune function [@ppat.1003720-Kollmann1], [@ppat.1003720-Gilliver1]. *Drosophila* has _proven_ to be a fruitful model of innate _immunity_ [@ppat.1003720-Lemaitre1]. The innate _immune_ system in the _fly_ _comprises_ humoral and cellular responses [@ppat.1003720-Lemaitre1]. The humoral _response_ _is_ _best_ characterized _by_ _the_ secretion of antimicrobial peptides (AMPs) _locally_ by epithelia such as _the_ gut, or systemically _by_ _the_ _fat-body,_ _a_ _functional_ analogue of _the_ mammalian liver. This response to _bacterial_ _or_ fungal infection is mainly regulated _by_ the _Toll_ and _Imd_ signaling pathways. Cellular _immunity_ is performed by _hemocytes,_ _phagocytic_ circulating immune cells. *Drosophila* has _become_ a powerful system to study _phagocytosis_ due to high _conservation_ of the processes _involved_ [@ppat.1003720-Stuart1], [@ppat.1003720-Stuart2]. _Cellular_ immunity is required to survive various types of bacterial infections, _complementing_ the _humoral_ response _[@ppat.1003720-AvetRochex1]--[@ppat.1003720-Ulvila1]_ _and_ is involved in the response _to_ wasp parasitoid infestation [@ppat.1003720-Krzemien1]. _In_ addition, in the larvae, hemocytes play a role in inter-organ communication, as they are required for the fat-body to mount _a_ full humoral _antimicrobial_ response after an intestinal infection [@ppat.1003720-Charroux1], [@ppat.1003720-Basset1], _[@ppat.1003720-Wu1]._ Hemocytes are also recruited to wounds in embryo and _larvae_ [@ppat.1003720-Babcock1]--[@ppat.1003720-Wood1], _potentially_ clearing damaged _tissue_ and _preventing_ further dispersal _of_ microorganisms _from_ unsterile wound sites. In parallel to their role in _immunity,_ hemocytes perform developmental and homeostatic functions such _as_ the phagocytosis of apoptotic cells and extracellular matrix secretion. These functions are essential at embryonic stages, for example, for central nervous system and renal _tubule_ development [@ppat.1003720-Bunt1], [@ppat.1003720-Olofsson1]. The crosstalk between immunity _and_ nutritional state or stress [@ppat.1003720-Becker1], _[@ppat.1003720-DiAngelo1]--[@ppat.1003720-Storelli1]_ in *Drosophila* is beginning to be unravelled, _however,_ _developmental_ _regulation_ of the immune _system_ remains enigmatic. Ecdysone is a steroid hormone, similar to mammalian estrogens and _androgens;_ peaks in ecdysone titer regulate the major developmental _transitions_ in _the_ fly, including metamorphosis. The _pupal_ stage _lasts_ 4 days from the end of the _3^rd^_ larval instar, _after_ _which_ adult flies eclose [@ppat.1003720-Thummel1]. The biologically active _form_ _of_ ecdysone, 20-hydroxyecdysterone _(20-E,_ _hereafter_ referred to as 'ecdysone') _coordinates_ tissue remodelling at metamorphosis _[@ppat.1003720-Thummel1]._ This hormone _activates_ a nuclear receptor, the Ecdysone Receptor _(EcR),_ which acts as a heterodimer with _its_ partner Ultraspiracle _(USP),_ a homologue of the mammalian Retinoid X Receptor. _Together,_ they activate the transcription of _primary_ response genes, which in turn activate the transcription _of_ a _battery_ of late response genes [@ppat.1003720-Thummel1]. This transcriptional cascade ultimately leads _to_ the _induction_ _of_ both cell death in larval tissues and differentiation _and_ _proliferation_ _of_ the imaginal discs into adult tissues _[@ppat.1003720-Thummel1]._ In _addition,_ several lines of evidence indicate that ecdysone regulates some aspects of hemocyte behaviour. In the larva, while the _majority_ of hemocytes are in circulation, approximately _one_ third of the total _population_ interacts with tissues, _attaching_ in repeated patches _to_ the dorsal epithelium along _the_ longitudinal axis [@ppat.1003720-Lanot1]. Despite the fact that hemocytes from these patches are mostly _immotile_ at larval stages, it has _been_ noticed that they disperse _at_ metamorphosis [@ppat.1003720-Lanot1]. This observation _correlates_ with the recent _finding_ that *ex vivo*, hemocytes _activate_ _motility_ and _morphological_ changes after metamorphosis [@ppat.1003720-Sampson1]. Cell shape _changes_ _can_ be prematurely triggered in larvae by ecdysone injection [@ppat.1003720-Lanot1]. Last, it _is_ _long_ known _that_ ecdysone treatment is _important_ to potentiate _AMP_ gene _expression_ _and_ phagocytosis after _an_ _immune_ challenge in hemocyte-derived cell culture lines [@ppat.1003720-Dimarcq1]. Although these results suggest _an_ ecdysone-dependent regulation of _hemocyte_ function, *in _vivo*_ evidence of a direct effect of ecdysone _signaling_ on hemocyte behaviour, and its functional relevance, _is_ _lacking._ _Here,_ we explore the hormonal regulation of *Drosophila* hemocytes at metamorphosis and its _impact_ on *Drosophila* _immunity_ _using_ an *in vivo* approach. We _demonstrate_ that direct _activation_ of ecdysone signaling _in_ _hemocytes_ is necessary _to_ increase their developmental _and_ immune activities at _metamorphosis,_ _including_ phagocytosis. We show that _this_ activation is _essential_ to respond _efficiently_ to and survive _pathogenic_ challenge. Results {#s2} ======= 1. The Ecdysone _Receptor_ is required _in_ _the_ _hemocytes_ _for_ their activation _at_ metamorphosis {#s2a} ------------------------------------------------------------------------------------------- To test whether ecdysone signaling cell-autonomously regulates hemocyte shape changes _at_ metamorphosis, we used the *Hml-(Hemolectin-)ΔGal4* driver [@ppat.1003720-Sinenko1] to specifically express green _fluorescent_ protein (GFP) and dominant-negative constructs of _the_ three _known_ EcR _isoforms_ under the control of the UAS sequence. _We_ imaged _hemocytes_ _*ex_ vivo* _by_ bleeding larvae _or_ pupae at precise time points after puparium _formation_ (APF) ([Fig. 1A--B](#ppat-1003720-g001){ref-type="fig"}) and _*in_ vivo* through the _dorsal_ epidermis ([Fig. 1C--D](#ppat-1003720-g001){ref-type="fig"}). _The_ first hour _of_ _the_ 12h 'prepupal' period is characterized by a translucent _pupal_ case (which then darkens). Control _hemocytes_ (from _*HmlΔGal4,_ _UAS-GFP/+*_ pupae, here after referred _to_ as *HmlΔ*\>GFP) _displayed_ a clear change of morphology over metamorphosis: they became gradually more polarized, _with_ many cytoplasmic protrusions _and_ _a_ higher number of vacuoles, increasing in size dramatically, _likely_ due to their greater spread and many phagocytic vesicles _([Fig._ 1A and C](#ppat-1003720-g001){ref-type="fig"}). _In_ striking contrast, _hemocytes_ _expressing_ a dominant _negative_ (DN) form of the EcRB1 isoform (from *HmlΔGal4, UAS-GFP/UAS-EcRB1DN* pupae, _here_ after referred to as *HmlΔ\>EcRB1DN*) did not _show_ any obvious change of size or morphology ([Fig. 1B _and_ _D](#ppat-1003720-g001){ref-type="fig"})._ Similar results were obtained when we analysed _by_ flow _cytometry_ the properties _of_ the hemocyte population at the onset of pupariation. Control _hemocytes_ displayed a clear shift both in Forward _Scatter_ (FSC) _and_ Side Scatter (SSC), indicating an increase in _cell_ size and granularity, respectively ([Fig. 1E](#ppat-1003720-g001){ref-type="fig"} and [S1](#ppat.1003720.s001){ref-type="supplementary-material"}). In contrast, the FSC and SSC of hemocytes _expressing_ *EcRB1DN* _remained_ stable over metamorphosis and similar to the parameters observed for control hemocytes _in_ _late_ 3^rd^ instar larvae (L3 wandering; L3W; [Fig. 1F](#ppat-1003720-g001){ref-type="fig"} and [S1](#ppat.1003720.s001){ref-type="supplementary-material"}). ![Ecdysone signaling is _required_ _for_ hemocyte _activation_ at metamorphosis.\ _(A--D)_ _Analysis_ of the morphology of control _hemocytes_ _(A,_ _C)_ and hemocytes expressing a _DN_ form of the EcR receptor (EcRB1DN; _B,_ _D)_ at _precise_ time points before _and_ after _puparium_ formation (APF). _(A,_ _B)_ *Ex vivo* analysis of bleeds; (C, D) *in vivo* analysis of _cells_ visualized under the dorsal epithelium. Green, endogenous GFP. Blue, _DAPI._ Red, _phalloidin._ |
Introduction {#s1}
============
Tuberculosis (TB) is caused by the pathogenic species, *Mycobacterium tuberculosis (Mtb)*; together with human immunodeficiency virus (HIV/AIDS) infection, TB is among the most prevalent and severe of the infectious diseases worldwide. In 2019, an estimated 10 million people developed active tuberculosis in association with 1.6 million deaths ([@B1]). Infection with *Mtb* triggers an immune response, however *Mtb* can survive and grow by circumventing the host immune detection. One of the pathological characteristics of the successful infection with *Mtb* is the formation of granulome, which are organized cellular structures that include a variety of innate and adaptive immune cells that surround the *Mtb*-infected phagocytes ([@B2]--[@B5]). During the formation of granulome, intricate host-*Mtb* interactions occur at the infectious site and this pathogen can escape various host immune responses, which ultimately prevent *Mtb* elimination by these systems. Once *Mtb* enters the host, its cell wall components and proteins are detected by Toll-like receptors (TLRs), primarily by TLR2 and TLR4. *Mtb* is engulfed by professional phagocytic cells such as a macrophage, dendritic cell (DC), or neutrophil, and becomes incorporated into the subcellular organelle formed by the fusion of the phagosome and lysosome to create the phagolysosome, however *Mtb* is able to manipulate the endocytic pathway by suppressing fusion of the phagosome containing the bacteria with lysosomes. Infected macrophages synthesize and release both inflammatory and antimicrobial genes and molecules, including interleukin (IL)-1β, IL-6, IL-12, tumor necrosis factor (TNF), inducible nitric oxide synthase/nitric oxide synthase 2 (iNOS/NOS2), and chemokines which activate both the innate and adaptive immune systems. Activated immune cells secrete protective molecules to the extracellular space to promote recruitment of other immune cells to form a granuloma ([@B4], [@B6]). Interestingly, endogenous proteins expressed by *Mtb* serve to perturb the formation of phagolysosome, the permitting its survival and proliferation within macrophages. For preventing excessive lung damage during *Mtb* infection, *Mtb* also elicits the production of protective factors that promote its survival including anti-inflammatory mediators such as IL-4, IL-10, IL-13, and transforming growth factor β (TGF-β) ([@B7]--[@B9]) and several human TB studies show that these factors has been shown to be increased in the active TB patients ([@B10], [@B11]). These immunosuppressive factors play key roles in limits effective the immune defense to *Mtb* ([@B12], [@B13]). *Mtb* will persist and exacerbate pathophysiological manifestations within the granulome; this will ultimately result in progression of disease and dissemination to the other hosts ([@B5], [@B14]). As a major focus of this disease process, mycobacterial granulome have been the subject of intense scrutiny mainly focused on mechanisms of formation, function, maintenance, and evolution.
Recently, there has been an increasing appreciation of the important relationship that exists between essential metabolism and immune cell function. Metabolic reprogramming in immune cells, a phenomenon known as immunometabolism, focuses on unique cellular functions that are essential for the immune response. During TB infection, host cells undergo profound metabolic change, which results in differential control of various cytokines and chemokines associated with inflammation, clearance, inhibition, and progression of *Mtb* infection ([@B15], [@B16]). Specifically, a shift in the use of pathways promoting glucose and lipid metabolism can be an important feature for directing host cell function to promote mycobacterial survival with the granulome ([@B17]). At homeostasis, cells in "resting" condition utilize oxidative phosphorylation (OXPHOS) to produce ATP from NADH and FADH2 by facilitating transfer of protons and electrons. Cells typically switch from OXPHOS to glycolysis in order to generate ATP under oxygen-depleted or hypoxic conditions ([@B18]). Similarly, glycolysis is main form of metabolism in immune cells that promote the inflammatory response in the immune system. This observation--that immune cells utilize glycolysis even in the presence of adequate concentrations of oxygen (i.e., aerobic glycolysis)-- is known as the "Warburg effect." To date, the Warburg effect has been explored primarily with respect to cancer metabolism. Although aerobic glycolysis generates fewer ATP molecules per cycle than does OXPHOS, this pathway is capable of rapid generation of ATP required by immune cells. Additionally, aerobic glycolysis requires a number of specific precursors, including nucleotides, amino acids, and lipids ([@B19]). Because metabolic reprogramming is essential for immune cell function, studies that explore this phenomenon in also provide new insight into the relationship between host immune cells and infection with *Mtb*. Furthermore, predisposing factors for TB, including diabetes, and HIV also related to immunometabolism against TB pathogenicity. Diabetes mellitus (DM) is a mainly risky factor for occurring active TB ([@B20]--[@B22]). In DM, innate immune cells undergo activation for releasing cytokines, recruiting neutrophils, upregulate T cell activation and antigen recognition ([@B23], [@B24]). Metabolism of DM is characterized by increasing glucose production and impairing glucose uptake. Expression of glucose transporter and glycolytic enzymes is elevated in DM ([@B25]). In DM, High glucose level increased IL-10 production, impaired macrophage phagocytic ability for promoting better milieu for survival and proliferation of TB ([@B26], [@B27]). Additionally, HIV is also other pathogen to be associated with pathogenicity of TB ([@B28]--[@B30]). In HIV-1-infected primary CD4^+^ T cells, glycolytic metabolism is induced with high pro-inflammatory response and increased production of virus ([@B31], [@B32]). Interestingly, glycolytic metabolism is regulated by HIV-1 infection in macrophage alleviated Warburg effects ([@B33]). These factors promote the activation of TB by reprogramming the metabolism.
A variety of antibiotics have been introduced for promoting eradication of *Mtb* infection, including 6--9 months courses of isoniazid, rifampicin, ethambutol, and pyrazinamide. However, the emergence of multidrug-resistant TB (MDR-TB) or extensively drug-resistant TB (XDR-TB) has become a major challenge toward designing effective treatments and for eradication of this disease ([@B34], [@B35]). Among the approaches to this challenge, host-directed therapy (HDT) has been introduced as a means to potentiate and to amplify the effectiveness of current treatments used for TB ([@B36]). A clear understanding of the molecular interactions between host cell metabolism and accommodations made to *Mtb* may provide new strategies to combat infection. Here we review the current understanding of the metabolic relationship between the host and the *Mtb* pathogen. We also suggest several new strategies that may enhance host metabolic pathways and thereby promote protective antimicrobial functions in the setting of TB infection.
Metabolic Reprogramming in TB {#s2}
=============================
Warburg Effect in Immune Cells
------------------------------
Immune cells provide critical protection and maintain homeostasis in the mammalian host. There are currently many studies that suggest that the functions of immune cells are largely reliant on specific aspects of host metabolism. These studies, which have generated a field known as immunometabolism, have provided us for a new focus for understanding how and why immune cells exist or persist in a specific metabolic state in order to support or direct functional changes. Several recent reports suggest that different metabolic signatures have a direct impact on specific effector functions characteristic of the innate and adaptive immune systems ([@B37]). As such, among the primary functions of immune cells, there are those that generate an inflammatory response, actions typically undertaken by M1-polarized macrophages, DCs, neutrophils, and effector T cells, and those that promote an anti- inflammatory response, which include M2-polarized macrophages, as well as regulatory and memory T cells. The basic metabolic profiles of these cells differ significantly from one another. Inflammatory immune cells generate energy in the form of ATP mainly via glycolytic metabolism; by contrast, immune cells that promote anti- inflammatory activities generate ATP via oxidative phosphorylation and fatty acid oxidation ([@B38]--[@B43]). These observations have been best characterized for polarized macrophages. The predominant phenotypes of macrophages are known as M1 and M2 ([@B44], [@B45]). M1 macrophages, activated by lipopolysaccharide (LPS) and IFN-γ, promote pro-inflammatory and antibacterial functions in immune system, and they produce nitric oxide (NO) and reactive oxygen species (ROS) which are fundamental components of the pathways used to eradicate bacteria. The main metabolic pathway used by these cells is glycolysis, which results in rapid production of ATP via inhibition of the trichloroacetic acid (TCA) cycle and OXPHOS in mitochondria; this is a critical factor due to the fact that M1 macrophages require rapid generation of ATP to activate inflammation. By contrast, M2 macrophages promote anti-inflammatory responses and tissue repair; these cells mainly utilize OXPHOS and fatty acid oxidation in order to generate ATP; this takes place via efficient pathways localized in the mitochondria ([@B46]--[@B51]). In T cells, metabolic state is reprogrammed according to T cell subsets. Naïve T cells mainly use OXPHOS for generating energy. Upon TCR stimulation, glycolytic metabolism is upregulated for differentiating into activated T cell. Th1, TH2, and Th17 effector cells mainly depend on aerobic glycolysis. While, regulatory and memory T cells use fatty acid oxidation and OXPHOS for differentiation and functions ([@B52], [@B53]). Mammalian target of rapamycin (mTOR) and AKT signaling is essential for regulating metabolism of T cells and cytokine responses ([@B54]). Recently, cyclophililn D ( | introduction { # s1 } = = = = = = = = = = = = tuberculosis ( tb ) is caused by the pathogenic species, * mycobacterium tuberculosis ( mtb ) * ; together with human immunodeficiency syndrome ( hiv / aids ) infection, tb is among the most prevalent and severe of the infectious diseases worldwide. in 2019, an estimated 10 million people developed active tuberculosis in association with 1. 6 million deaths ( [ @ b1 ] ). infection with * mtb * triggers an immune response, however * mtb * can survive and grow by circumventing the host immune detection. one of common pathological characteristics of the hiv infection with * mtb * is the formation of granulome, which are organized cellular structures that include a variety of innate or adaptive immune cells that surround the * mtb * - infected phagocytes ( [ @ b2 ] - - [ @ b5 ] ). during the formation of granulome, intricate host - * mtb * interactions occur at the infectious site and this pathogen can escape various host immune responses, which ultimately prevent * mtb * elimination by these systems. once * mtb * enters the host, its cell wall genes and proteins are detected by toll - like receptors ( tlrs ), primarily targeting tlr2 and tlr4. * mtb * is engulfed by professional phagocytic cells such as a macrophage, dendritic cell ( dc ), or neutrophil, and becomes incorporated into the subcellular organelle formed by the fusion separating the phagosome towards lysosome to create the phagolysosome, however * mtb * is able to manipulate the endocytic pathway by suppressing fusion of the phagosome containing the bacteria with arthritis. infected macrophages synthesize and release both inflammatory and antimicrobial genes and molecules, including dna ( il ) - 1β, il - 6, il - 12, tumor necrosis factor ( tnf ), inducible nitric oxide synthase / nitric oxide synthase 2 ( inos / nos2 ), and chemokines which activate both the innate and adaptive immune systems. activated immune cells secrete protective molecules to the extracellular space to promote recruitment of other immune cells to form a granuloma ( [ @ b4 ], [ @ b6 ] ). interestingly, end ##ogenous proteins expressed by * mtb * serve to perturb the formation of phagolysosome, the permitting its survival and proliferation within macrophages. for preventing excessive lung damage during * mtb * infection, * mtb * also elicits the production of protective factors that promote its survival including anti - inflammatory mediators such as il - 4, il - 10, il - 13, and transforming growth factor β ( tgf - β ) ( [ @ b7 ] - - [ @ b9 ] ) and several human tb studies show that these factors has been shown to be increased in the active tb patients ( [ @ b10 ], [ @ b11 ] ). these immunosuppressive factors play key roles in limits effective the immune defense to * mtb * ( [ @ b12 ], [ @ b13 ] ). * mtb * will persist and exacerbate pathophysiological manifestations within the granulome ; this will ultimately result in progression of disease and dissemination to the other hosts ( [ @ b5 ], [ @ b14 ] ). as a major focus of this disease process, mycobacterial granulome have been the subject of intense scrutiny mainly focused on mechanisms of formation, function, maintenance, and evolution. recently, there has been an increasing appreciation of the important relationship that exists between essential metabolism and immune cell function. metabolic reprogramming in immune cells, a phenomenon known as immunometabolism, focuses on unique cellular functions that are essential for the immune response. during tb infection, host cells undergo profound metabolic change, which results in differential control of various cytokines and chemokines associated with inflammation, clearance, inhibition, and progression of * mtb * infection ( [ @ b15 ], [ @ b16 ] ). specifically, a shift in the use of pathways promoting glucose and lipid metabolism can be an important feature for directing host cell function to promote mycobacterial survival with the granulome ( [ @ b17 ] ). at homeostasis, cells in " resting " condition utilize oxidative phosphorylation ( oxphos ) to produce atp from nadh and fadh2 by facilitating transfer of protons and electrons. cells typically switch from oxphos to glycolysis in order to generate atp under oxygen - depleted or hypoxic conditions ( [ @ b18 ] ). similarly, glycolysis is main form of metabolism in immune cells that promote the inflammatory response in the immune system. this observation - - that immune cells utilize glycolysis even in the presence of adequate concentrations of oxygen ( i. e., aerobic glycolysis ) - - is known as the " warburg effect. " to date, the warburg effect has been explored primarily with respect to cancer metabolism. although aerobic glycolysis generates fewer atp molecules per cycle than does oxphos, this pathway is capable of rapid generation of atp required by immune cells. additionally, aerobic glycolysis requires a number of specific precursors, including nucleotides, amino acids, and lipids ( [ @ b19 ] ). because metabolic reprogramming is essential for immune cell function, studies that explore this phenomenon in also provide new insight into the relationship between host immune cells and infection with * mtb *. furthermore, predisposing factors for tb, including diabetes, and hiv also related to immunometabolism against tb pathogenicity. diabetes mellitus ( dm ) is a mainly risky factor for occurring active tb ( [ @ b20 ] - - [ @ b22 ] ). in dm, innate immune cells undergo activation for releasing cytokines, recruiting neutrophils, upregulate t cell activation and antigen recognition ( [ @ b23 ], [ @ b24 ] ). metabolism of dm is characterized by increasing glucose production and impairing glucose uptake. expression of glucose transporter and glycolytic enzymes is elevated in dm ( [ @ b25 ] ). in dm, high glucose level increased il - 10 production, impaired macrophage phagocytic ability for promoting better milieu for survival and proliferation of tb ( [ @ b26 ], [ @ b27 ] ). additionally, hiv is also other pathogen to be associated with pathogenicity of tb ( [ @ b28 ] - - [ @ b30 ] ). in hiv - 1 - infected primary cd4 ^ + ^ t cells, glycolytic metabolism is induced with high pro - inflammatory response and increased production of virus ( [ @ b31 ], [ @ b32 ] ). interestingly, glycolytic metabolism is regulated by hiv - 1 infection in macrophage alleviated warburg effects ( [ @ b33 ] ). these factors promote the activation of tb by reprogramming the metabolism. a variety of antibiotics have been introduced for promoting eradication of * mtb * infection, including 6 - - 9 months courses of isoniazid, rifampicin, ethambutol, and pyrazinamide. however, the emergence of multidrug - resistant tb ( mdr - tb ) or extensively drug - resistant tb ( xdr - tb ) has become a major challenge toward designing effective treatments and for eradication of this disease ( [ @ b34 ], [ @ b35 ] ). among the approaches to this challenge, host - directed therapy ( hdt ) has been introduced as a means to potentiate and to amplify the effectiveness of current treatments used for tb ( [ @ b36 ] ). a clear understanding of the molecular interactions between host cell metabolism and accommodations made to * mtb * may provide new strategies to combat infection. here we review the current understanding of the metabolic relationship between the host and the * mtb * pathogen. we also suggest several new strategies that may enhance host metabolic pathways and thereby promote protective antimicrobial functions in the setting of tb infection. metabolic reprogramming in tb { # s2 } = = = = = = = = = = = = = = = = = = = = = = = = = = = = = warburg effect in immune cells - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - immune cells provide critical protection and maintain homeostasis in the mammalian host. there are currently many studies that suggest that the functions of immune cells are largely reliant on specific aspects of host metabolism. these studies, which have generated a field known as immunometabolism, have provided us for a new focus for understanding how and why immune cells exist or persist in a specific metabolic state in order to support or direct functional changes. several recent reports suggest that different metabolic signatures have a direct impact on specific effector functions characteristic of the innate and adaptive immune systems ( [ @ b37 ] ). as such, among the primary functions of immune cells, there are those that generate an inflammatory response, actions typically undertaken by m1 - polarized macrophages, dcs, neutrophils, and effector t cells, and those that promote an anti - inflammatory response, which include m2 - polarized macrophages, as well as regulatory and memory t cells. the basic metabolic profiles of these cells differ significantly from one another. inflammatory immune cells generate energy in the form of atp mainly via glycolytic metabolism ; by contrast, immune cells that promote anti - inflammatory activities generate atp via oxidative phosphorylation and fatty acid oxidation ( [ @ b38 ] - - [ @ b43 ] ). these observations have been best characterized for polarized macrophages. the predominant phenotypes of macrophages are known as m1 and m2 ( [ @ b44 ], [ @ b45 ] ). m1 macrophages, activated by lipopolysaccharide ( lps ) and ifn - γ, promote pro - inflammatory and antibacterial functions in immune system, and they produce nitric oxide ( no ) and reactive oxygen species ( ros ) which are fundamental components of the pathways used to eradicate bacteria. the main metabolic pathway used by these cells is glycolysis, which results in rapid production of atp via inhibition of the trichloroacetic acid ( tca ) cycle and oxphos in mitochondria ; this is a critical factor due to the fact that m1 macrophages require rapid generation of atp to activate inflammation. by contrast, m2 macrophages promote anti - inflammatory responses and tissue repair ; these cells mainly utilize oxphos and fatty acid oxidation in order to generate atp ; this takes place via efficient pathways localized in the mitochondria ( [ @ b46 ] - - [ @ b51 ] ). in t cells, metabolic state is reprogrammed according to t cell subsets. naive t cells mainly use oxphos for generating energy. upon tcr stimulation, glycolytic metabolism is upregulated for differentiating into activated t cell. th1, th2, and th17 effector cells mainly depend on aerobic glycolysis. while, regulatory and memory t cells use fatty acid oxidation and oxphos for differentiation and functions ( [ @ b52 ], [ @ b53 ] ). mammalian target of rapamycin ( mtor ) and akt signaling is essential for regulating metabolism of t cells and cytokine responses ( [ @ b54 ] ). recently, cyclophililn d ( | Introduction {# s1} = = = = = = = = = = = = Tuberculosis (TB) is caused by the pathogenic species, * Mycobacterium tuberculosis (Mtb) *; together with human immunodeficiency virus (HIV / AIDS) infection, TB is among the most prevalent and severe of the infectious diseases worldwide. In 2019, an estimated 10 million people developed active tuberculosis in association with 1. 6 million deaths ([ @ B1] ). Infection with * Mtb * triggers an immune response, however * Mtb * can survive and grow by circumventing the host immune detection. One of the pathological characteristics of the successful infection with * Mtb * is the formation of granulome, which are organized cellular structures that include a variety of innate and adaptive immune cells that surround the * Mtb * - infected phagocytes ([ @ B2] - - [@ B5] ). During the formation of granulome, intricate host - * Mtb * interactions occur at the infectious site and this pathogen can escape various host immune responses, which ultimately prevent * Mtb * elimination by these systems. Once * Mtb * enters the host, its cell wall components and proteins are detected by Toll - like receptors (TLRs ), primarily by TLR2 and TLR4. * Mtb * is engulfed by professional phagocytic cells such as a macrophage, dendritic cell (DC ), or neutrophil, and becomes incorporated into the subcellular organelle formed by the fusion of the phagosome and lysosome to create the phagolysosome, however * Mtb * is able to manipulate the endocytic pathway by suppressing fusion of the phagosome containing the bacteria with lysosomes. Infected macrophages synthesize and release both inflammatory and antimicrobial genes and molecules, including interleukin (IL) - 1β, IL - 6, IL - 12, tumor necrosis factor (TNF ), inducible nitric oxide synthase / nitric oxide synthase 2 (iNOS / NOS2 ), and chemokines which activate both the innate and adaptive immune systems. Activated immune cells secrete protective molecules to the extracellular space to promote recruitment of other immune cells to form a granuloma ([ @ B4 ], [@ B6] ). Interestingly, endogenous proteins expressed by * Mtb * serve to perturb the formation of phagolysosome, the permitting its survival and proliferation within macrophages. For preventing excessive lung damage during * Mtb * infection, * Mtb * also elicits the production of protective factors that promote its survival including anti - inflammatory mediators such as IL - 4, IL - 10, IL - 13, and transforming growth factor β (TGF - β) ([ @ B7] - - [@ B9] ) and several human TB studies show that these factors has been shown to be increased in the active TB patients ([ @ B10 ], [@ B11] ). These immunosuppressive factors play key roles in limits effective the immune defense to * Mtb * ([ @ B12 ], [@ B13] ). * Mtb * will persist and exacerbate pathophysiological manifestations within the granulome; this will ultimately result in progression of disease and dissemination to the other hosts ([ @ B5 ], [@ B14] ). As a major focus of this disease process, mycobacterial granulome have been the subject of intense scrutiny mainly focused on mechanisms of formation, function, maintenance, and evolution. Recently, there has been an increasing appreciation of the important relationship that exists between essential metabolism and immune cell function. Metabolic reprogramming in immune cells, a phenomenon known as immunometabolism, focuses on unique cellular functions that are essential for the immune redppnse. During TB infection, host cells undergo profound metabolic change, which results in differential control of various cytokines and chemokines associated with inflammation, clearance, inhibition, and progression of * Mtb * infection ([ @ B15 ], [@ B16] ). Specifically, a shift in the use of pathways promoting glucose and lipid metabolism can be an important feature for directing host cell function to promote mycobacterial survival with the granulome ([ @ B17] ). At homeostasis, cells in " resting " spndition utilize oxidative phosphorylation (OXPHOS) to produce ATP from NADH and FADH2 by facilitating transfer of protons and electrons. Cells typically switch from OXPHOS to glycolysis in order to generate ATP under oxygen - depleted or hypoxic conditions ([ @ B18] ). Similarly, glycolysis is main form of metabolism in immune cells that promote the inflammatory response in the immune system. This observation - - that immune cells utilize glycolysis ebeJ in the presence of adequate concentrations of oxygen (i. e. , aerobic glycolysis) - - is known as the " Warburg effect. " To date, the Warburg effect has been explored primarily with respect to cancer metabolism. Although aerobic glycolysis generates fewer ATP molecules per cycle than does OXPHOS, this pathway is capable of rapid generation of ATP required by immune cells. Additionally, aerobic glycolysis requires a number of specific precursors, including nucleotides, amino acids, and lipids ([ @ B19] ). Because metabolic reprogramming is essential for immune cell function, studies that explore this phenomenon in also provide new insight into the relationship between host immune cells and infection with * Mtb *. Furthermore, pr$diEposing factors for TB, including diabetes, and HIV also related to immunometabolism against TB pathogenicity. Diabetes mellitus (DM) is a mainly risky factor for occurring active TB ([ @ B20] - - [@ B22] ). In DM, innate immune cells undergo activation for releasing cytokines, recruiting neutrophils, upregulate T cell activation and antigen recognition ([ @ B23 ], [@ B24] ). Metabolism of DM is characterized by increasing glucose production and impairing glucose uptake. Expression of glucose transporter and glycolytic enzymes is elevated in DM ([ @ B25] ). In DM, High glucose level increased IL - 10 production, impaired macrophage phagocytic ability for promoting better milieu for survival and proliferation of TB ([ @ B26 ], [@ B27] ). Additionally, HIV is also other pathogen to be associated with pathogenicity of TB ([ @ B28] - - [@ B30] ). In HIV - 1 - infected primary CD4 ^ + ^ T cells, glycolytic metabolism is induced with high pro - inflammatory response and increased production of virus ([ @ B31 ], [@ B32] ). Interestingly, glycolytic metabolism is regulated by HIV - 1 infection in macrophage alleviated Warburg effects ([ @ B33] ). These factors promote the activation of TB by reprogramming the metabolism. A variety of antibiotics have b@Wn introduced for promoting eradication of * Mtb * infection, including 6 - - 9 months courses of isoniazid, rifampicin, ethambutol, and pyrazinamide. However, the emergence of multidrug - resistant TB (MDR - TB) or extensively drug - resistant TB (XDR - TB) has become a major challenge toward designing effective treatments and for eradication of this disease ([ @ B34 ], [@ B35] ). Among the approaches to this challenge, host - directed therapy (HDT) has been introduced as a means to potentiate and to amplify the effectiveness of current treatments used for TB ([ @ B36] ). A clear understanding of the molecular interactions between host cell metabolism and accommodations made to * Mtb * may provide new xtraHegies to combat infection. Here we review the current understanding of the metabolic relationship between the host and the * Mtb * pathogen. We also suggest several new strategies that may enhance host metabolic pathways and thereby promote protective antimicrobial functions in the setting of TB infection. Mehagolic Reprogramming in TB {# s2} = = = = = = = = = = = = = = = = = = = = = = = = = = = = = Warburg Effect in Immune Cells - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Immune cells provide critical protection and maintain homeostasis in the mammalian host. There are currently many studies that suggest that the functions of immune cells are largely reliant on specific aspects of host metabolism. These studies, which have generated a field known as immunometabolism, have provided us for a new focus for understanding how and why immune cells exist or persist in a specific metabolic state in order to support or direct functional changes. Several recent reports suggest that different metabolic signatures have a direct impact on QLecific effector functions characteristic of the innate and adaptive immune systems ([ @ B37] ). As such, among the primary functions of immune cells, there are those that generate an inflammatory response, actions typically undertaken by M1 - polarized macrophages, DCs, neutrophils, and effector T cells, and those that promote an anti - inflammatory response, which include M2 - polarized macrophages, as well as regulatory and memory T cells. The basic metabolic profiles of these cells differ significantly from one another. Inflammatory immune cells generate energy in the form of ATP mainly via glycolytic metabolism; by contrast, immune cells that promote anti - inflammatory activities generate ATP via *xida%ive phosphorylation and fatty acid oxidation ([ @ B38] - - [@ B43] ). These observations have been best characterized for polarized macrophages. The predominant phenotypes of macrophages are known as M1 and M2 ([ @ B44 ], [@ B45] ). M1 macrophages, activated by lipopolysaccharide (LPS) and IFN - γ, promote pro - inflammatory and antibacterial functions in immune system, and they produce nitric oxide (NO) and reactive oxygen species (ROS) which are fundamental components of the pathways used to eradicate bacteria. The main metabolic pathway used by these cells is glycolysis, which results in rapid production of ATP via inhibition of the trichloroacetic acid (TCA) cycle and OXPHOS in mitochondria; this is a critical factor due to the fact that M1 macrophages require rapid generation of ATP to activate inflammation. By contrast, M2 macrophages promote anti - inflammatory responses and tissue repair; these cells mainly utilize OXPHOS and gstty acid oxidation in order to generate ATP; this takes place via efficient pathways localized in the mitochondria ([ @ B46] - - [@ B51] ). In T cells, metabolic state is reprogrammed according to T cell subsets. Naïve T cells mainly use OXPHOS for generating energy. Upon TCR stimulation, glycolytic metabolism is upregulated for differentiating into activated T cell. Th1, TH2, and Th17 effector cells mainly depend on aerobic glycolysis. While, regulatory and memory T cells use fatty acid oxidation and OXPHOS for differentiation and functions ([ @ B52 ], [@ B53] ). Mammalian target of rapamycin (mTOR) and AKT signaling is essential for regulating metabolism of T cells and cytokine responses ([ @ B54] ). Recently, cyclophililn D ( | Introduction {#s1} ============ Tuberculosis (TB) is caused by the pathogenic species, *Mycobacterium tuberculosis together with human immunodeficiency virus (HIV/AIDS) infection, TB among the most prevalent and severe the infectious diseases worldwide. In 2019, an estimated people active tuberculosis in association with 1.6 million deaths ([@B1]). Infection with *Mtb* triggers immune response, however *Mtb* can survive and grow by circumventing the host immune detection. One of pathological characteristics of successful with *Mtb* is the formation of granulome, which are organized cellular structures that a of innate and adaptive immune cells that surround the *Mtb*-infected phagocytes ([@B2]--[@B5]). During the formation of intricate host-*Mtb* interactions occur at the site and pathogen can escape various host immune responses, which prevent *Mtb* elimination by these systems. Once *Mtb* enters the host, its components and are by Toll-like receptors (TLRs), primarily by TLR2 and TLR4. *Mtb* is engulfed professional phagocytic cells such as a macrophage, dendritic cell (DC), or neutrophil, and becomes incorporated into subcellular organelle formed by the fusion of the phagosome and create the phagolysosome, however *Mtb* is able to manipulate endocytic pathway by suppressing of the phagosome containing the bacteria with lysosomes. Infected macrophages and release both inflammatory and antimicrobial and molecules, including (IL)-1β, IL-6, IL-12, tumor necrosis factor (TNF), inducible synthase/nitric synthase 2 (iNOS/NOS2), chemokines which activate both the innate and adaptive immune systems. Activated immune cells secrete protective molecules to the extracellular to promote recruitment of other immune cells to form a granuloma ([@B4], [@B6]). endogenous proteins expressed serve perturb the formation phagolysosome, the permitting its survival and within macrophages. For preventing excessive lung damage during *Mtb* infection, *Mtb* also elicits the production of protective factors that promote its survival including anti-inflammatory mediators such as IL-4, IL-10, IL-13, and transforming growth factor β ([@B7]--[@B9]) and several human TB show that these factors has been shown to be increased the active TB patients ([@B10], These immunosuppressive factors play roles in limits effective the immune defense to *Mtb* ([@B12], [@B13]). *Mtb* will persist and exacerbate pathophysiological manifestations granulome; this will ultimately result in progression of disease and dissemination to the other hosts ([@B5], [@B14]). As a major focus of this disease mycobacterial granulome have the subject of intense scrutiny mainly focused on mechanisms of formation, function, maintenance, evolution. Recently, there has an increasing appreciation of the important relationship that exists essential metabolism and immune cell function. Metabolic reprogramming in immune cells, a phenomenon known as immunometabolism, focuses on unique cellular functions that are essential for the immune response. During TB infection, host cells undergo profound metabolic change, which results differential control of various cytokines and chemokines associated with inflammation, clearance, inhibition, and progression *Mtb* infection ([@B15], Specifically, a shift in the use pathways promoting glucose and lipid metabolism can be an important feature for directing host cell function to promote mycobacterial survival with the granulome ([@B17]). At homeostasis, cells in utilize oxidative phosphorylation (OXPHOS) to produce ATP from NADH and FADH2 by facilitating transfer of protons and electrons. Cells typically switch from to glycolysis in order to generate ATP under oxygen-depleted or conditions ([@B18]). Similarly, glycolysis is main form of metabolism in immune cells that promote the response in the immune system. This observation--that immune cells utilize glycolysis even in the presence of adequate concentrations of oxygen (i.e., aerobic glycolysis)-- known as the "Warburg effect." To date, the effect has been primarily with respect to cancer metabolism. Although aerobic glycolysis generates ATP molecules cycle than does OXPHOS, pathway is capable of rapid generation of required by cells. Additionally, aerobic glycolysis requires a number of specific precursors, including nucleotides, amino acids, and lipids ([@B19]). Because metabolic reprogramming is essential for immune cell function, studies that explore in also provide new insight into the relationship between cells and infection with *Mtb*. Furthermore, predisposing factors for TB, including and HIV also related to immunometabolism against TB pathogenicity. Diabetes mellitus (DM) is a mainly risky for occurring active TB ([@B20]--[@B22]). In DM, innate cells undergo for releasing cytokines, recruiting neutrophils, upregulate T cell activation and antigen recognition ([@B23], [@B24]). of DM is characterized by increasing and impairing glucose uptake. Expression of glucose transporter and glycolytic enzymes is elevated in DM ([@B25]). In DM, High glucose level increased IL-10 production, impaired macrophage ability for promoting better milieu for survival and proliferation of TB ([@B26], [@B27]). HIV is also pathogen to be associated with pathogenicity of TB ([@B28]--[@B30]). HIV-1-infected primary CD4^+^ T cells, glycolytic metabolism is induced with high pro-inflammatory response and increased production of virus [@B32]). Interestingly, metabolism is by HIV-1 infection in macrophage alleviated Warburg effects ([@B33]). These factors promote the activation of TB by reprogramming the metabolism. A variety antibiotics have been introduced for promoting eradication of *Mtb* including 6--9 months courses isoniazid, rifampicin, ethambutol, and pyrazinamide. However, the emergence of multidrug-resistant TB (MDR-TB) or extensively drug-resistant TB (XDR-TB) has become a major challenge toward designing effective treatments and eradication of this disease ([@B34], [@B35]). Among approaches to this challenge, host-directed (HDT) has been introduced as means to potentiate and amplify the effectiveness current treatments used for TB ([@B36]). A clear understanding of the molecular interactions between host cell metabolism and accommodations made to *Mtb* may provide new strategies to combat infection. Here review the current understanding of the relationship between the host and the pathogen. We also suggest several new strategies that may host metabolic pathways and thereby promote protective antimicrobial functions in the setting of TB Metabolic Reprogramming in TB ============================= Effect in Immune Cells ------------------------------ Immune cells provide critical protection and homeostasis in the mammalian host. There are currently studies that suggest that the functions of immune cells are largely reliant on specific aspects of host metabolism. These studies, which have generated field known as immunometabolism, have provided us a new focus how and why immune cells exist or a specific metabolic state in order to support or direct functional changes. Several recent reports suggest that different metabolic signatures have a direct impact on specific effector functions characteristic the innate and adaptive immune systems ([@B37]). As such, among the primary functions of immune cells, there are those that generate an inflammatory response, actions typically undertaken by M1-polarized macrophages, DCs, neutrophils, and effector T and those that promote an anti- inflammatory response, which include M2-polarized macrophages, well as regulatory and T cells. The basic profiles of these cells differ significantly from one another. Inflammatory immune cells generate energy in the of ATP mainly via glycolytic metabolism; by contrast, immune cells that promote anti- inflammatory activities generate via oxidative and fatty acid oxidation ([@B38]--[@B43]). These observations have been characterized for polarized macrophages. The predominant phenotypes of macrophages are known as M1 and M2 ([@B44], [@B45]). macrophages, activated by lipopolysaccharide IFN-γ, promote pro-inflammatory and antibacterial functions in immune system, and they produce nitric oxide (NO) and oxygen species (ROS) which are fundamental of the used to eradicate bacteria. The metabolic pathway used by these cells is glycolysis, results in rapid production of ATP via inhibition of trichloroacetic acid (TCA) cycle OXPHOS mitochondria; this is a critical factor due the fact that M1 macrophages require rapid of ATP activate By contrast, M2 macrophages promote responses and tissue repair; these mainly utilize OXPHOS and fatty acid oxidation in order generate ATP; this takes place via efficient pathways localized in mitochondria ([@B46]--[@B51]). In T cells, metabolic state is reprogrammed according to T cell subsets. Naïve T cells mainly OXPHOS for generating Upon TCR stimulation, glycolytic metabolism is upregulated for differentiating into activated T cell. Th1, TH2, and Th17 effector cells mainly depend on aerobic glycolysis. While, regulatory and memory T cells use fatty acid oxidation and for differentiation and functions ([@B52], [@B53]). Mammalian target of rapamycin (mTOR) and AKT signaling is essential for regulating metabolism of T cells cytokine responses ([@B54]). Recently, cyclophililn D ( | iNTroductION {#s1}
============
tUbeRcuLOsiS (Tb) IS cAused BY tHe PATHOGeNiC SPeCIes, *mycOBACteRiUm TUbERcuLOSiS (mtb)*; tOGeTher wITh HUmAn ImMuNOdEFiCIENcY VIRuS (hIV/Aids) INfECtiOn, TB is AmOng thE mOsT PrEvAlEnT and SEVeRe Of tHe InfeCTiOUS dIseaSes WoRldwide. in 2019, aN esTimateD 10 milLion PeOpLe DEVELOPED actIve TuBeRculoSIs in aSSocIaTioN wItH 1.6 miLlIon DEatHS ([@b1]). infECtION WITH *mtB* trigGeRs An immUNE resPONsE, HOwEVEr *MTb* CAN suRViVe And gRoW by CiRcUMVENtinG thE hOST imMune dETECtIoN. one Of The PAThoLogicAl chARACtERistics of thE sUcCESsFUL iNfEcTION WIth *MTB* iS thE FormAtion oF gRANuloME, WHIcH aRE ORGaniZed CeLLuLaR stRUCtUrES thaT iNcluDE A VaRIety oF InNAtE ANd aDapTIVE IMmunE cElLS THat SurRoUNd THE *mtb*-INFEcted phAGoCyteS ([@b2]--[@b5]). durINg the fOrmAtion oF GRaNuLoME, intRicaTe hOst-*MtB* INtErActIoNs ocCur at THe InFECTIoUS sITE aND thIs PatHoGen cAn esCAPE vaRiouS hosT immunE reSponSes, WHICh uLtimaTeLy prEVEnT *Mtb* eLiminatiON by THEsE SYstEMs. oncE *mTB* enTeRS thE hosT, ITs cElL wAlL CompoNeNTs AND ProteIns ARe DETECtEd by tOlL-liKE RECeptORS (tLrs), prIMArily bY TLR2 aNd tLr4. *MTb* is ENguLFed By pRofeSsionAL phAGOcYTIc CElLS sUCH As a mAcROpHAGE, dEnDRiTiC CeLl (DC), Or neUtrOpHiL, AnD becOmes INCORpOraTED IntO THe suBcELLuLAr OrGANeLle foRMED by thE FuSIOn oF The pHagosome AnD LYsosOme To cREATE tHe PhAGOLYSOsoME, howEvEr *Mtb* Is AblE TO manIpULaTe thE enDOCytIC paTHWay by suppreSSINg FUsIoN Of tHe pHAGOsoMe coNTAiNiNG thE bACtERIa wIth lysOSOmes. infecTED MACroPhAges SYNThESIze ANd RELeASE BOTh InFlammaTORY And ANTimIcRoBiAl gEnes anD MoleCULes, InClUdiNG IntERleUkin (iL)-1Β, il-6, il-12, tUMOR neCroSiS fACTOR (TNf), iNduCIblE NitrIC oXiDE syNThASe/nItRic OXiDE SynthAse 2 (InOs/nOS2), aNd ChemoKiNEs whiCH aCTiVATE Both THe InNATE ANd AdaPTivE IMMunE sysTEms. ACTIvATEd iMMUNE cells sEcRetE protEcTive MoLeCUles To the eXtrAcElLuLaR sPACe TO promOtE REcRuiTmEnt oF OTher iMmUne cElLs to forM A GRanUlomA ([@B4], [@B6]). iNteREStINGlY, endOGENoUS pRoTEIns ExprEsSeD By *mTb* SeRvE TO PeRtUrB thE FoRMATIOn of pHaGOLYsOsome, THE PerMITTIng itS SUrVIvaL And PrOlIfErATiON wIthIn MacRoPhAGes. FOR prEVENtING excEsSivE lUng DAmAge DurINg *mtb* infectIon, *MTb* aLSo Elicits The prOdUcTION of ProtECtive FACtORS thaT PromOte itS SUrVIvAl InCLUDIng anti-INflAmmAtoRY meDiAtORs Such as Il-4, IL-10, il-13, AnD tRanSfORMIng GROWTh fAcTOr Β (TgF-Β) ([@b7]--[@b9]) ANd SEVeRal HUMaN tb sTUDiEs sHOw that THESE FAcTORs HAs BEen ShOWn To BE iNCReASed in THe aCTivE TB paTIENTS ([@B10], [@B11]). tHesE iMmunosupPreSSiVe faCToRS plAy KeY RolEs IN LIMiTS EFfECtIVE tHE immuNE defENse TO *MTB* ([@b12], [@B13]). *mtb* WILl persiST ANd ExACerbatE pAThOPHYsIOLOGICal mAniFEStaTIOns WIThIn The grAnuloME; tHIS WILL uLtiMATElY resuLt iN PrOgREsSION oF DiseasE AnD dIsSemiNATIoN to tHE Other Hosts ([@B5], [@B14]). as A MAJor FOcUs oF tHis DIsEasE PRoCEss, mycObaCTerIAL GrAnuLOMe HaVE BEeN tHE sUbJEcT oF inTensE sCrUTINy mAInly FoCUSed ON MEchaNismS oF forMaTioN, fUNctIoN, MAIntENANcE, aNd eVoluTIon.
reCENtly, ThEre HAS BEen aN INCReASIng aPPrEciATIon of ThE iMPorTaNT ReLaTIOnsHIp tHaT EXiSTs BEtWeen EssENTiaL mEtAbOLiSM And ImMune CeLL FUnctIoN. MeTabOlIC repRogRAMmiNg iN iMMuNE ceLLS, a pHenOmEnOn KNoWN As imMuNOMeTABolIsM, focUSeS oN UniquE cELLulAR FUNCtioNs tHaT Are essENTIaL foR the ImMUnE ReSpOnsE. DuRIng tB InfeCTION, HosT CELLs UnDERGo proFOund MetAbOlIc chANge, WHicH rEsUltS iN DIFFEreNTIal CoNtrol OF VarIoUs CytOKInES aNd CHeMOKinES AsSoCIatEd wIth inflamMatIon, clEaRanCE, InhibitioN, AND prOgRessIOn OF *MTB* infectiOn ([@B15], [@b16]). SPEcIficaLLy, a sHiFt iN THE uSE OF paTHWays ProMotiNG GLucoSE ANd lipid MeTABOlISM can BE an IMPortANT fEatUre FoR DIreCtiNG hOST cELl FunCtiOn TO PROmOTe mycOBacTErial SuRviVAl wItH THE GrANulOme ([@b17]). AT HOmeOsTASIs, ceLLs iN "resTing" ConDitIoN uTIlIZe oxIDAtiVe PhosPHOryLAtiOn (oxpHOs) TO PrODUce aTP FRom nadh anD Fadh2 bY FACILITaTIng TRAnSfER Of prOtons ANd elecTroNs. CeLls TYPICaLly SWITcH FROm oXphOS To gLYColysIS iN ordER to genERaTE Atp UnDer oxYGEN-DePLETED OR hYpoxIC CONdiTiOnS ([@B18]). sIMILARLy, GlycOLYsIs iS MaIn Form oF mEtabOLiSM IN ImmUne CeLls tHAT PrOMoTE THE INfLAmmATORY rESponsE IN thE IMMuNe sysTEM. thIs obsErVAtIOn--ThAt immuNE CElls utilIzE GLYcOlysIs eVen IN the PREsenCe OF ADeQUatE cONcEnTrAtIoNS oF OXYgen (I.e., AeROBIC GlYColySIs)-- iS kNOwn aS thE "warburG EFfecT." TO dAte, THe wArBuRG Effect has beeN explORED PRImarilY with REsPECT to cAnCEr METABoLiSm. aLTHOUGh AerObic GlYCOLySiS gEnErAtes FEwer AtP moLEcUles peR CYCLe than DoeS OxPhOS, THIS pAThwAy Is CAPAbLe of RApid GeNeRATION Of aTp REquirEd by imMUne CELlS. ADdItiOnALLy, aerobic GLycOLySIs ReqUIRes a nUmBEr OF SpECIfic pRECurSORs, iNClUdING nucLEOTides, amINO AcIdS, anD LipiDs ([@b19]). becAuSe MetAboLiC REProGRamming Is ESsEntial FOR ImmUne cEll funCTIOn, STuDIeS tHat EXploRE tHIs pHEnOMeNoN IN aLso PRoVIDE NEw INSight InTO the RELaTIOnSHIP BEtwEen HOSt IMmuNe CeLlS aND infECtion WiTH *mtB*. FUrthErmoRE, PRediSpoSiNG FAcToRS fOr tb, IncluDinG dIaBEteS, and HIv AlSO RelaTeD To ImMUNOmeTaboliSm agaInsT tB pAtHOgEnICitY. DIabeTEs mElLITus (dM) Is A maiNLy riSKy FacToR fOr OCCUrRinG ACtive tB ([@B20]--[@B22]). iN dM, inNAte iMmuNE CElls UNDERGo ActIvatIon fOR rElEAsInG CyTOkiNES, REcRUiTIng NEUtROPHils, uPREGulaTe T CELl AcTiVAtion anD anTIgEN reCOGNiTioN ([@b23], [@B24]). MetABOLiSm of DM iS charActEriZED bY inCREASINg gluCose ProDuCtION AnD iMPAirING GlUCOsE uPTAKe. ExpRESSion OF gluCOse TraNsPorter AnD GlYCOlYTIC enZymEs iS ElEVaTEd iN dm ([@B25]). in DM, HIgh glucosE LeVEl inCReaseD iL-10 prOdUcTIon, IMPaIred mACrOPhAGE PHAgOcYTic abIlITy for promotING bEttEr mILIEU FoR SUrvIvaL and proLiferaTion of TB ([@B26], [@b27]). ADdITIoNAllY, HiV IS AlSO othEr paTHogEn To BE ASSOCIatEd witH PaThOgeniciTy of tB ([@B28]--[@b30]). iN HIV-1-iNFEctEd PrImary Cd4^+^ t CellS, glYcoLyTIc MEtAbolIsm iS inDUCEd wiTH HIgh PrO-infLAMmAtorY ReSPonSe anD incrEasED PrODuctiOn OF VIRUS ([@b31], [@B32]). INTeRESTiNgly, GLycOLytiC METaBOlISm is REGUlaTeD By hiv-1 inFectiOn In maCRoPhagE aLLEviaTEd WArBurg EfFeCTS ([@b33]). thESe facTorS PROMOtE thE actiVatiOn Of TB bY REPROgRAMmInG the MetAbOLIsm.
A VArietY of AntibIoTiCS hAVe BeEN inTROduCED FOR pRomOtING eRADiCaTION Of *MtB* iNfEcTIoN, INCluding 6--9 MoNTHs COUrsES oF isONiazid, riFAMPiCIn, ethAmbUTOL, and pyraZINaMIde. HoweVER, tHe EmERGEnce Of MultIdrUG-ResIStaNt tb (mDR-tb) Or eXTensiVEly DRug-ReSistAnt TB (Xdr-tB) hAs becOME a MAjoR chalLENGe toWaRd DesIgNIng eFfECtIve tReATMenTS And FOr eRADicaTION Of ThiS DISeASe ([@B34], [@B35]). AMoNG ThE ApprOACHES tO thIs cHALLenGe, hOST-diREctEd ThErapy (HDt) Has been INTRodUcED as a MEANS to POtENTIATE anD To AMpLIfY the efFECTiVeNeSs OF curReNT treAtMeNTs Used FoR tB ([@b36]). A cLear UndERstanDinG Of tHe molecULAr iNTeRAcTions BeTWEEn HOst CeLl meTAbOliSM anD aCcOMmODaTIONS MaDE to *Mtb* MaY ProviDE nEW StRaTEGiEs To cOMBat InFECtION. hEre We reviEW tHE cUrRenT UNDeRstaNding OF tHe metABOliC RELatIOnsHiP BeTwEEn THe HOSt and thE *mtB* PATHogen. wE ALsO sUgGeST seVeRaL NeW sTrateGIeS ThAt may EnhaNcE hoST mEtaboLic PATHWAys AnD theReby pRomoTe proTEcTIVe AntimiCRoBiAl FunctionS iN THe SeTtING of tB infeCtiOn.
MeTaboLiC rEPROGraMminG In Tb {#S2}
=============================
WaRbURg EfFECt In ImmuNE CeLls
------------------------------
imMUnE CelLS proviDe CRiTICal PrOTecTION and MAIntaIn HomEOStasis In the maMmaliaN HosT. therE ArE currENtLY mAny StUdIeS THaT suGgEST THAT tHE FUNcTioNs of iMmUne celLS arE lArgely rELiaNT on spECific AspECTS of hoST MEtaBoliSm. thESE StUDIeS, wHicH hAVE geNERAtEd a field KnOWN AS imMunomEtAbOLiSm, havE PRovIDed uS fOr a NEW FOCus For UnDeRsTanDInG hoW AND wHy ImMuNE cEllS ExIst or PerSIsT iN a SpECIfic mETaboLIc sTATe iN ORder tO SUPPORt OR diREct FUNCtioNAl CHAngEs. sEveRAL RECENT repoRTs sUGgest THat diFFERent MEtAbolIc sIGnATURes haVE a DirEct impaCT On speCIFIC EfFECtoR FuNcTIOnS chArACTerIsTIc of thE innaTE and aDaptIVe IMmuNE sySTEMs ([@b37]). As SuCH, aMOng ThE PrimARy fUNCtions OF iMMUNe cells, theRE Are THOse tHAT gEnERATE An InFlAmmAtORY reSpONSE, aCTIONs TYPiCALlY UndertAken bY M1-polARIZED macropHagES, DcS, nEuTROpHIls, AnD efFeCtOR T CElLS, AnD THOse tHaT pRomoTe aN ANti- InflamMATOrY rEsPOnse, whICh INclUDe M2-PoLaRIzeD macROPhAgES, AS WeLL As reguLAtorY AnD mEMOry t cells. tHe bASiC METABoLIC pRoFIleS Of thEsE Cells diffEr sIgnIfIcanTLy FRoM onE aNothEr. INfLAmMaToRY ImmUne cellS GeNeRatE eNergy In THE fORm of AtP MAInly VIA GLycOLYtIC meTAboliSM; bY CoNtraST, IMmuNE CeLls THat PROmOTE ANTI- inFLaMMaTORY aCTiviTies geNerATe aTP viA oXIDatIVe PHOSPHORyLaTIOn And FATTy aCid oXidaTion ([@b38]--[@B43]). tHesE obServAtIonS HAVe BEEn besT cHARactEriZED fOR polariZEd MacROphaGeS. The pREdoMiNAnt pHeNOtypes OF mACRoPhAgEs are KnoWN as m1 and m2 ([@B44], [@b45]). M1 macrophAGes, ActiVAtED By LIPOPOLYsaccHARiDE (lPs) anD ifn-Γ, PROmotE PRo-iNFLAMMaTORY aND anTibacteRial funCtioNS IN immunE syStEM, AND THEy PrOdUcE nitric oxiDE (no) aND REaCtivE OxyGEn SpeCiES (Ros) WhICH aRe FUNdAMenTal COMponenTs Of the pAthwAYS uSed tO eRaDICATE bACtERiA. THE mAIN mEtABoLIc pathWAy useD bY thESe cElLS iS gLYcOlYSis, wHIch rEsulTS iN rApiD PRODUCtIoN oF atp vIA InhIBITiON OF THE tRICHLorOacETIC aCID (tcA) cYclE AND oxphos In mIToCHoNdrIA; tHiS Is A CritiCaL factOr dUE tO tHe FACT ThAT M1 MACrOpHAgEs rEquiRE rAPId GenERatION of ATP to aCTIvATe INFLamMAtIoN. By CONTraST, M2 macropHAGeS pRoMOTE AnTi-inflAmmaTOry rESPONseS aNd tisSUE rePaIr; ThEsE celLS maINly uTiliZE OXpHOS ANd fATtY AcID oxidation in OrdeR tO GeNerate ATp; THIS TAKes PLacE vIa eFfiCiENt PAThwaYs LOCALiZEd iN thE MItOcHondriA ([@B46]--[@b51]). iN T cElLS, meTABOLiC staTe Is REpRogrAMMED aCcoRDInG tO t cELL subSETS. NAïVE t cells maiNlY uSe oXPhoS foR GEneRATinG eNErgY. uPoN TCR sTImulaTIon, gLyCOLytIc metABOliSm iS upREgulAtED fOr DiFfEReNTiATing inTo actIVaTED t cell. TH1, tH2, And Th17 EFfEcToR CElls MAinly DEPeND on AeROBiC GLyCOLYSIS. whiLe, ReGULAToRy AnD Memory T cElLS USe fAttY AciD oxIdAtiOn And oXphOs FoR DiFfErEntIaTiON ANd FUNCtIONs ([@b52], [@b53]). MAMMaliaN TaRGET OF raPamyCin (MtOR) aND aKt sIGNaLIng iS ESSEnTiAl fOr ReguLatInG MEtabOlism oF T CELlS aNd cYtOKIne RESPoNSes ([@B54]). REcenTLy, cYCloPhIliln D ( | Introduction {#s1} ============Tuberculosis (TB) is caused bythe pathogenic species, *Mycobacterium tuberculosis(Mtb)*;together with human immunodeficiency virus (HIV/AIDS) infection, TB is amongthe most prevalent and severe of the infectiousdiseases worldwide.In 2019, an estimated 10 million people developed active tuberculosis in association with 1.6 million deaths ([@B1]). Infection with *Mtb* triggers an immune response, however*Mtb* cansurvive and grow by circumventing the host immunedetection. One of the pathological characteristics of the successfulinfection with *Mtb* is the formationof granulome, which are organized cellular structures thatinclude a variety of innate and adaptive immune cells that surround the *Mtb*-infected phagocytes ([@B2]--[@B5]). During the formation of granulome, intricate host-*Mtb* interactions occur at theinfectious site andthis pathogen can escapevarious host immune responses, which ultimately prevent *Mtb* elimination by these systems. Once *Mtb* enters thehost, its cellwall components and proteins are detected by Toll-like receptors(TLRs), primarily by TLR2 and TLR4. *Mtb* is engulfed by professional phagocyticcells suchas a macrophage, dendritic cell (DC), or neutrophil,and becomes incorporated into the subcellular organelle formedbythefusion of the phagosome andlysosome to create the phagolysosome,however *Mtb* is able to manipulate the endocytic pathway by suppressing fusionof the phagosome containing the bacteria with lysosomes. Infected macrophages synthesize and release both inflammatoryand antimicrobialgenes and molecules, including interleukin (IL)-1β, IL-6,IL-12, tumornecrosisfactor (TNF), inducible nitric oxide synthase/nitric oxide synthase 2 (iNOS/NOS2), and chemokines which activate both theinnate and adaptiveimmune systems. Activated immune cells secrete protective molecules tothe extracellular space to promote recruitment of other immune cells to form a granuloma ([@B4],[@B6]). Interestingly, endogenous proteins expressed by *Mtb* serve to perturb theformation of phagolysosome, the permitting its survival and proliferation within macrophages. For preventing excessive lung damage during *Mtb* infection, *Mtb* also elicits the production of protective factors that promoteits survival including anti-inflammatory mediators such as IL-4, IL-10, IL-13, and transforming growth factor β (TGF-β)([@B7]--[@B9]) andseveral human TB studiesshow that these factorshas been shown to be increased in the active TBpatients ([@B10], [@B11]). These immunosuppressive factors play key rolesin limits effective the immunedefense to *Mtb* ([@B12], [@B13]). *Mtb* will persistand exacerbate pathophysiological manifestations within thegranulome;this will ultimatelyresultin progressionof disease and dissemination to the other hosts ([@B5], [@B14]). As a major focus of this disease process, mycobacterial granulome have beenthe subject of intense scrutiny mainly focused onmechanisms of formation, function, maintenance, and evolution. Recently, there has been an increasing appreciation of the importantrelationship that existsbetween essential metabolismand immune cellfunction. Metabolic reprogramming in immune cells, a phenomenon known as immunometabolism, focuses on unique cellular functions that are essential for the immune response. During TB infection, host cells undergo profound metabolic change, which results in differentialcontrol ofvarious cytokines and chemokines associatedwithinflammation, clearance, inhibition, and progression of*Mtb* infection ([@B15],[@B16]). Specifically, a shift in the use of pathways promoting glucose and lipid metabolism can be an important feature for directing host cell functionto promote mycobacterial survival withthegranulome ([@B17]). At homeostasis, cells in "resting"conditionutilize oxidative phosphorylation (OXPHOS)to produce ATP from NADH and FADH2 by facilitating transfer of protons and electrons. Cells typically switch from OXPHOS to glycolysis in ordertogenerate ATPunderoxygen-depletedorhypoxic conditions ([@B18]). Similarly, glycolysis is main form of metabolism in immune cells that promote the inflammatory response in the immunesystem. Thisobservation--that immune cells utilizeglycolysis even in the presence of adequate concentrations ofoxygen (i.e., aerobic glycolysis)-- is known as the "Warburg effect." Todate,theWarburg effecthas been explored primarily with respect to cancer metabolism. Although aerobic glycolysis generates fewer ATP molecules per cycle than doesOXPHOS, this pathway is capable of rapid generation of ATP required by immune cells. Additionally, aerobic glycolysis requires a number of specificprecursors, including nucleotides, amino acids, and lipids ([@B19]).Because metabolic reprogramming isessential for immunecell function, studies that explore this phenomenon in also provide new insight into the relationship between host immune cells and infectionwith *Mtb*. Furthermore, predisposing factors for TB, including diabetes, and HIValso related toimmunometabolism against TB pathogenicity. Diabetes mellitus (DM) is a mainlyriskyfactor for occurring active TB ([@B20]--[@B22]). In DM, innate immune cells undergo activation for releasing cytokines, recruitingneutrophils,upregulate T cell activation and antigen recognition ([@B23], [@B24]). Metabolism of DM is characterized byincreasing glucose production and impairing glucoseuptake. Expression of glucose transporter and glycolytic enzymes is elevated in DM ([@B25]). In DM, High glucose level increased IL-10 production, impaired macrophage phagocytic ability for promoting better milieufor survival and proliferation ofTB ([@B26], [@B27]).Additionally, HIV is also other pathogen to be associatedwith pathogenicity of TB ([@B28]--[@B30]). In HIV-1-infected primary CD4^+^ T cells, glycolytic metabolism is induced with highpro-inflammatoryresponse and increased production of virus ([@B31], [@B32]). Interestingly,glycolytic metabolism isregulated by HIV-1 infection in macrophage alleviated Warburg effects ([@B33]). These factors promotethe activation of TBby reprogramming the metabolism. A variety of antibiotics have been introduced forpromoting eradicationof *Mtb* infection, including6--9 months coursesof isoniazid,rifampicin, ethambutol, and pyrazinamide. However, the emergence of multidrug-resistant TB (MDR-TB) or extensively drug-resistant TB (XDR-TB) has becomea majorchallenge toward designing effective treatmentsandfor eradication of thisdisease ([@B34], [@B35]). Among the approachesto this challenge, host-directed therapy (HDT) has been introduced as a means to potentiate and to amplify the effectivenessof currenttreatments used for TB ([@B36]). A clear understanding of the molecular interactions between host cell metabolism and accommodations madeto *Mtb* may provide new strategiesto combat infection. Here we review thecurrent understandingof the metabolicrelationship between the host and the *Mtb* pathogen. We alsosuggest several new strategies that may enhance host metabolic pathways and thereby promote protective antimicrobial functions in the setting of TBinfection. Metabolic Reprogramming in TB {#s2} ============================= Warburg Effect in Immune Cells------------------------------ Immune cells provide critical protection and maintain homeostasis in the mammalian host. Thereare currently many studiesthat suggest that thefunctions of immune cells are largely reliant on specific aspects of host metabolism.These studies, which have generated a field known as immunometabolism, have providedus for a new focusfor understanding how and why immune cells exist or persist in a specificmetabolic state in order tosupport or direct functional changes.Several recent reports suggest that different metabolicsignatures have a direct impact on specific effector functions characteristic of the innate and adaptive immune systems ([@B37]). As such, amongtheprimary functions of immunecells, there are those that generate an inflammatory response, actionstypically undertaken by M1-polarized macrophages, DCs,neutrophils, and effector T cells, and those that promote an anti- inflammatory response, which include M2-polarized macrophages, as well as regulatory and memoryTcells.Thebasic metabolic profiles of these cells differ significantly from one another. Inflammatory immune cells generate energyin the form of ATPmainly via glycolytic metabolism; by contrast, immune cells thatpromote anti- inflammatory activities generate ATP viaoxidative phosphorylation and fatty acid oxidation ([@B38]--[@B43]).These observations have been best characterized for polarized macrophages. The predominant phenotypes of macrophages are known as M1 and M2 ([@B44],[@B45]). M1macrophages,activated by lipopolysaccharide (LPS) and IFN-γ, promote pro-inflammatory andantibacterial functions in immune system, and they produce nitric oxide (NO) and reactive oxygen species (ROS) which are fundamental components of the pathways used to eradicate bacteria. The mainmetabolic pathway used by these cells is glycolysis, which results in rapid production of ATP via inhibition of the trichloroacetic acid (TCA) cycleand OXPHOS inmitochondria; this is a critical factor due to the fact that M1 macrophages require rapid generationof ATP to activate inflammation. Bycontrast, M2 macrophages promote anti-inflammatory responses and tissue repair; these cells mainly utilize OXPHOS and fatty acid oxidation in order togenerate ATP; this takesplace via efficientpathways localizedin the mitochondria ([@B46]--[@B51]). In T cells, metabolic state is reprogrammed according to T cell subsets. Naïve Tcells mainly use OXPHOS for generating energy. Upon TCR stimulation, glycolytic metabolism is upregulated for differentiating into activated T cell. Th1, TH2,and Th17 effector cells mainly depend on aerobic glycolysis. While, regulatory and memory T cells use fatty acid oxidation and OXPHOS for differentiation and functions ([@B52], [@B53]). Mammalian target ofrapamycin (mTOR) and AKT signaling is essential for regulating metabolism of T cells and cytokine responses ([@B54]). Recently, cyclophililn D ( | Introduction {#s1} _============_ Tuberculosis (TB) _is_ caused by the _pathogenic_ species, *Mycobacterium tuberculosis (Mtb)*; _together_ with human _immunodeficiency_ _virus_ (HIV/AIDS) infection, TB is among the most prevalent and severe of the infectious diseases worldwide. _In_ 2019, an estimated 10 million people developed active tuberculosis in association with 1.6 million deaths ([@B1]). Infection with *Mtb* triggers an immune response, however *Mtb* can survive _and_ grow by circumventing the host _immune_ detection. _One_ of the pathological _characteristics_ of _the_ successful _infection_ with _*Mtb*_ is the formation of _granulome,_ which are organized _cellular_ structures that _include_ a variety of innate and adaptive immune cells that surround the *Mtb*-infected phagocytes ([@B2]--[@B5]). During the formation _of_ granulome, intricate host-*Mtb* interactions occur at _the_ infectious site _and_ this pathogen can escape _various_ host immune responses, which ultimately prevent *Mtb* elimination _by_ these systems. _Once_ *Mtb* enters the host, its cell _wall_ _components_ and proteins are detected by Toll-like receptors _(TLRs),_ primarily by TLR2 and TLR4. *Mtb* is engulfed by professional phagocytic cells such as a macrophage, dendritic cell (DC), or neutrophil, and becomes incorporated into the subcellular organelle formed by _the_ fusion of the phagosome and lysosome to create the _phagolysosome,_ however *Mtb* is able to manipulate the endocytic pathway _by_ suppressing fusion _of_ _the_ phagosome containing the bacteria with _lysosomes._ _Infected_ macrophages synthesize _and_ release both _inflammatory_ and _antimicrobial_ _genes_ _and_ _molecules,_ including _interleukin_ _(IL)-1β,_ IL-6, IL-12, tumor necrosis factor (TNF), inducible nitric oxide _synthase/nitric_ oxide synthase _2_ (iNOS/NOS2), and _chemokines_ which activate both the innate and adaptive immune _systems._ Activated immune cells secrete protective molecules to the extracellular space to promote recruitment of other immune cells to form a granuloma ([@B4], [@B6]). Interestingly, _endogenous_ proteins expressed by *Mtb* serve _to_ perturb the formation of phagolysosome, the permitting its survival and proliferation within macrophages. For _preventing_ excessive lung damage _during_ *Mtb* infection, _*Mtb*_ also elicits the production of protective factors that promote its survival including _anti-inflammatory_ mediators such as IL-4, _IL-10,_ IL-13, and _transforming_ growth factor β _(TGF-β)_ ([@B7]--[@B9]) _and_ _several_ human TB studies show that _these_ _factors_ has been shown to be _increased_ _in_ _the_ _active_ TB patients ([@B10], [@B11]). These _immunosuppressive_ _factors_ play key roles _in_ limits effective the immune defense to *Mtb* ([@B12], [@B13]). *Mtb* will _persist_ and _exacerbate_ pathophysiological manifestations _within_ the granulome; _this_ will ultimately result in progression of disease and _dissemination_ to the other _hosts_ ([@B5], [@B14]). As a major focus of this disease process, _mycobacterial_ granulome have _been_ the subject of intense scrutiny _mainly_ focused _on_ mechanisms of formation, function, maintenance, _and_ evolution. _Recently,_ there _has_ been an _increasing_ appreciation of the important relationship that exists _between_ essential metabolism and immune cell function. Metabolic reprogramming in immune _cells,_ a phenomenon _known_ as immunometabolism, focuses on _unique_ cellular functions _that_ are essential for _the_ immune response. During TB infection, host cells undergo _profound_ metabolic change, which results _in_ differential control of _various_ cytokines and chemokines associated _with_ inflammation, clearance, inhibition, and progression _of_ *Mtb* infection ([@B15], _[@B16])._ Specifically, _a_ shift in the use of _pathways_ _promoting_ glucose and _lipid_ metabolism can be an important _feature_ for directing _host_ cell function to promote _mycobacterial_ survival with the granulome ([@B17]). At _homeostasis,_ cells in "resting" _condition_ utilize _oxidative_ phosphorylation _(OXPHOS)_ to produce ATP from NADH and FADH2 by facilitating transfer _of_ protons and electrons. Cells typically switch _from_ OXPHOS to glycolysis _in_ order to generate ATP under _oxygen-depleted_ or hypoxic conditions ([@B18]). _Similarly,_ glycolysis is main form of _metabolism_ in _immune_ cells that promote the inflammatory response in the immune system. This _observation--that_ immune cells utilize glycolysis _even_ _in_ _the_ _presence_ of _adequate_ _concentrations_ of oxygen (i.e., aerobic _glycolysis)--_ is known as the _"Warburg_ _effect."_ To date, _the_ Warburg effect has _been_ explored primarily with respect to cancer _metabolism._ Although aerobic glycolysis generates fewer ATP molecules _per_ cycle than _does_ _OXPHOS,_ this pathway is capable of rapid _generation_ of ATP _required_ by _immune_ cells. Additionally, aerobic glycolysis requires a _number_ _of_ _specific_ precursors, including nucleotides, _amino_ _acids,_ _and_ lipids ([@B19]). Because metabolic reprogramming is essential for immune cell function, _studies_ that _explore_ _this_ phenomenon _in_ also provide new insight _into_ the relationship _between_ _host_ _immune_ cells and _infection_ _with_ *Mtb*. _Furthermore,_ _predisposing_ _factors_ for _TB,_ including diabetes, and HIV _also_ related to immunometabolism against TB pathogenicity. Diabetes mellitus (DM) is a mainly risky _factor_ for occurring active TB ([@B20]--[@B22]). In DM, innate immune cells undergo _activation_ for releasing _cytokines,_ recruiting neutrophils, upregulate T cell activation _and_ antigen recognition ([@B23], [@B24]). Metabolism _of_ DM is characterized by increasing glucose production and impairing glucose _uptake._ Expression of glucose transporter and _glycolytic_ _enzymes_ is _elevated_ in _DM_ ([@B25]). _In_ _DM,_ _High_ glucose level _increased_ IL-10 production, impaired macrophage phagocytic ability for promoting better milieu for survival _and_ proliferation of TB _([@B26],_ [@B27]). Additionally, HIV is also _other_ _pathogen_ _to_ be associated with pathogenicity of TB ([@B28]--[@B30]). In HIV-1-infected primary CD4^+^ T cells, glycolytic _metabolism_ is induced with high pro-inflammatory response and _increased_ _production_ _of_ virus _([@B31],_ _[@B32])._ Interestingly, glycolytic metabolism is regulated by HIV-1 infection _in_ macrophage _alleviated_ Warburg effects _([@B33])._ These factors promote the activation of TB by reprogramming the _metabolism._ A variety of _antibiotics_ have _been_ introduced for promoting _eradication_ of _*Mtb*_ infection, _including_ 6--9 _months_ courses of isoniazid, rifampicin, ethambutol, and pyrazinamide. However, the _emergence_ of multidrug-resistant TB (MDR-TB) _or_ extensively drug-resistant _TB_ (XDR-TB) has become a major challenge toward _designing_ effective treatments and for eradication of this disease _([@B34],_ [@B35]). _Among_ the approaches to this challenge, host-directed therapy (HDT) _has_ been introduced _as_ a means _to_ potentiate and to amplify the effectiveness of current treatments _used_ for TB ([@B36]). A clear understanding _of_ the molecular _interactions_ between _host_ cell metabolism and _accommodations_ made _to_ *Mtb* _may_ provide new strategies to combat infection. _Here_ we review _the_ current understanding of the metabolic relationship between the host and the *Mtb* pathogen. We also suggest _several_ new _strategies_ that may enhance host metabolic pathways and thereby _promote_ protective antimicrobial functions in _the_ setting of TB _infection._ Metabolic Reprogramming in TB {#s2} ============================= _Warburg_ Effect in Immune Cells ------------------------------ Immune cells provide critical protection and maintain _homeostasis_ in the _mammalian_ host. There are currently _many_ studies _that_ suggest that the functions of immune _cells_ _are_ largely _reliant_ on specific _aspects_ of _host_ metabolism. These studies, _which_ have generated a field known as immunometabolism, have provided us for _a_ new _focus_ for _understanding_ how and why immune cells _exist_ or persist in _a_ specific metabolic state in _order_ to _support_ or direct functional changes. Several recent reports suggest that _different_ metabolic signatures have a direct _impact_ on specific effector functions characteristic of _the_ innate and adaptive immune systems _([@B37])._ As such, among the primary functions of immune cells, there are those that generate an inflammatory response, _actions_ typically undertaken by M1-polarized _macrophages,_ DCs, neutrophils, _and_ effector T cells, and those that _promote_ an _anti-_ _inflammatory_ response, _which_ include M2-polarized _macrophages,_ as _well_ as _regulatory_ and _memory_ T _cells._ The basic metabolic _profiles_ of these cells _differ_ _significantly_ from one another. Inflammatory immune _cells_ generate energy in the form of ATP mainly _via_ glycolytic metabolism; by contrast, immune cells that promote anti- inflammatory activities generate ATP via oxidative _phosphorylation_ and fatty acid oxidation ([@B38]--[@B43]). These _observations_ have been _best_ _characterized_ for polarized macrophages. The predominant phenotypes of _macrophages_ are known as M1 _and_ M2 _([@B44],_ [@B45]). M1 macrophages, activated _by_ lipopolysaccharide (LPS) and IFN-γ, promote pro-inflammatory and antibacterial _functions_ in immune system, and they produce nitric _oxide_ (NO) and _reactive_ oxygen species (ROS) _which_ _are_ fundamental components of _the_ pathways used to eradicate _bacteria._ The main _metabolic_ pathway used by these cells is _glycolysis,_ which results in rapid production of ATP via inhibition _of_ the trichloroacetic acid (TCA) cycle and OXPHOS in _mitochondria;_ this _is_ a critical _factor_ due to the fact that M1 macrophages require rapid generation of ATP to activate inflammation. By contrast, _M2_ macrophages promote _anti-inflammatory_ responses and tissue _repair;_ these _cells_ mainly utilize _OXPHOS_ _and_ _fatty_ acid oxidation in order to generate ATP; this takes place via efficient _pathways_ localized in the mitochondria ([@B46]--[@B51]). In T cells, metabolic state is reprogrammed _according_ to T cell subsets. Naïve _T_ cells mainly use OXPHOS for _generating_ energy. Upon TCR stimulation, glycolytic metabolism is upregulated for differentiating into activated T cell. Th1, TH2, and _Th17_ effector cells _mainly_ depend _on_ aerobic glycolysis. While, regulatory and _memory_ T _cells_ use fatty acid _oxidation_ _and_ OXPHOS for differentiation _and_ functions _([@B52],_ [@B53]). Mammalian _target_ _of_ rapamycin (mTOR) _and_ AKT signaling is essential _for_ regulating metabolism of _T_ cells and cytokine responses ([@B54]). _Recently,_ cyclophililn _D_ ( |
There are errors in the Funding section. The correct funding information is as follows: This study was supported by the National Cancer Institute of the National Institutes of Health under award number K08CA155035 and the Melanoma Research Alliance. The authors are also grateful to Timothy Dattels for his generous support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
| below exist entries in the funding section. the correct funding information is as follows : this study was supported at the national cancer institute of the national board of health under award age 66 and the melanoma research alliance. these authors are also grateful to timothy dattels for his generous scholarship. the funders had no choice in study design, data collection and analysis, decision to publish, or preparation of the manuscript. | There are errors in the Funding xectioj. The correct funding information is as follows: This study was euppotted by the National Cancer Institute of the National Institutes of Health under x@ard number K08CA155035 and the Melanoma Research Al?iaGce. The authKrx are also grateful to Tin*thy Dattels for his geJeroux support. The gund4rs had no role in study design, data cLllec6ion and analysis, decision to publish, or preparation of the KaHuscript. | There are errors in the section. The correct funding information is as follows: This study was supported by the National Cancer the National Institutes of Health under award number K08CA155035 and the Melanoma Research Alliance. The authors are also grateful to Timothy Dattels his generous support. The funders had no role study design, data collection analysis, decision to publish, preparation of the manuscript. | there ARE erRoRS IN The fUnDiNG sEction. tHE CoRRect FundiNg INFORmaTION Is aS FOlLoWs: thIs STUDy wAs sUppoRtEd bY THe NATional caNCER iNStituTE OF tHE NATIOnAL iNStiTuteS Of HealTh UNdEr AWaRD NUMBEr k08Ca155035 AND thE mElANoMA rESeaRCH alliAnce. the AUThors are aLsO gRATEFUl to TimotHY datTels FoR HIS GENEROus sUPPoRt. tHe FundERS HaD nO roLE iN STUdy DEsign, dAta colLectIon AnD anAlySiS, DeCIsioN To pUbLIsH, Or prePARatiOn Of thE mANUScRIpt.
| There areerrors in the Fundingsection.The correct funding information is asfollows: This study was supported by the National Cancer Institute of the National Institutes of Health under awardnumber K08CA155035 and the Melanoma ResearchAlliance.The authors are also gratefulto TimothyDattelsfor his generous support. Thefunders had no role in study design, datacollectionandanalysis, decision to publish, or preparation of the manuscript. | _There_ are _errors_ in _the_ Funding section. The correct funding _information_ is as follows: This study was supported by the National Cancer Institute of the _National_ _Institutes_ of Health under award number K08CA155035 and the Melanoma Research Alliance. _The_ _authors_ are _also_ grateful to Timothy Dattels _for_ his generous support. The _funders_ _had_ no role in study design, data _collection_ and _analysis,_ decision _to_ publish, _or_ _preparation_ of the manuscript. |
Introduction
============
Diabetes mellitus is a common endocrine metabolic disease estimated to affect 629 million individuals in 2045 according to the International Diabetes Federation. T2DM is the extremely prevalent form of diabetes that accounts for 90% of diagnosed cases and is associated with insulin resistance and chronic hyperglycemia ([@B13]). Many clinical studies reported a broad spectrum of lower urinary tract symptoms in diabetic patients ([@B16]), accounting for 90--95% of all diabetes cases ([@B17]). DBD is a major lower urinary tract complication of diabetes and was first described by [@B26]. Such complication is traditionally described as a triad of increased capacity, decreased sensation, and poor emptying ([@B11]) and has affected over 50% of diabetic patients ([@B12]; [@B28]). DBD development is divided into two phases: the compensated phase, which occurs in the early phase and is characterized by an OAB; and the decompensated phase, which occurs in the late phase and is characterized by an atonic bladder ([@B36]; [@B24]).
The pathogenesis of DBD is multifactorial and accompanied by the structural and functional impairments of the bladder ([@B37]). The bladder structural remodeling of DBD, such as the increase in bladder capacity, total BWT, and smooth muscle content, was observed in STZ-induced diabetic mice. Such remodeling may be a physical alteration to increase the urine volume ([@B22]). The two major functions of bladder are urine storage and urine disposal, and the uncoordinated contraction in the OAB of a diabetic greatly affects the urine storage ability of this organ ([@B10]). Bladder contraction is mainly mediated by purinergic and cholinergic pathways ([@B23]). In particular, ATP is the purinergic messenger released from varicosities or bulbous nerve endings of neurons, and the contractile responses mediated by ATP play a key role in DBD ([@B44]). Solute carrier family 17 member 9 (SLC17A9) is a member of the solute-carrier protein family that plays an indispensable role in the vesicular storage of ATP ([@B31]; [@B20]). The translocation of neurotransmitter-filled vesicles to the varicose terminal is the first step in the release of vesicular neurotransmitters, followed by the merger of vesicles with the membrane of the varicose terminal and the precise and rapid release of their contents into the synaptic cleft ([@B33]). In addition, the motor of vesicles for transportation to the varicose membrane in the cells is mainly provided by myosin motors, particularly myosin Va ([@B2]). Several studies found that the purinergic inhibitory neurotransmission was impaired in myosin Va-deficient mice ([@B6]). Such finding suggested that myosin Va played an important role in purinergic neurotransmission ([@B3]).
Diabetic bladder dysfunction, particularly OAB, is not life threatening to humans. However, this dysfunction seriously affects the quality of the life of patients ([@B24]). The treatment methods for DBD changed when the phase progresses. Anticholinergic drugs, such as tolterodine and solifenacin, are the main treatment options for DBD patients with OAB. However, many side effects, including dry mouth, dry eyes, and memory loss, occurs after the treatment with anticholinergic drugs, thus rendering poor life quality for the patients. In the late phase, surgical intervention was the only therapeutic method for patients who did not benefit from pharmacological and behavioral treatments ([@B45]). However, pharmacological and surgical interventions were largely ineffective in clinics ([@B12]; [@B40]). Therefore, new effective treatments for DBD are urgently needed.
In the treatment of diabetic OAB, traditional Chinese medicine and natural plant components have recently attracted increasing attention due to their safeness, few side effects, and excellent activity ([@B41]). SQW is a traditional Chinese herbal formula that was first recorded on *Fu Ren Liang Fang* in the Southern Song Dynasty (between 1127 and 1279 CE). This medicine is a mixture of three Chinese medicines: *roots of Lindera aggregata (Sims) Kosterm. (Lauraceae), roots of Alpinia oxyphylla Miq., (Zingiberaceae), and rhizomes of Dioscorea oppositifolia L. (Dioscoreaceae)* at a 1:1:1 ratio ([@B14]). SQW has been used to treat lower urinary tract symptoms, such as nocturia, urgency, and child bedwetting for hundreds of years ([@B4]). We have recently reported that SQW had therapeutic effects on the OAB of bladder outlet obstruction rat models by modulating the TRPV1 expression ([@B18]). In China, SQW is often used in the clinical treatment of diabetic OAB. However, its mechanism remains unclear, and its therapeutic effect has not been investigated in animal studies. Therefore, we designed experiments to explore the effects and therapeutic mechanisms of SQW in diabetic OAB mouse model.
Materials and Methods {#s1}
=====================
Reagents and Materials
----------------------
Suo Quan Wan was purchased from Hunan Hansen Pharmaceutical Co., Ltd. (China), and the quality control was provided by the company based on Chinese Pharmacopeia employing by high performance liquid chromatography (HPLC) technology from SQW samples ([@B9]). Three Chinese herbals were ground and mixed evenly at a 1:1:1 ratio and appropriate volumes of distilled water were used to make these powders to SQW compound. The doses were adopted according to the Experimental Methodology of Pharmacology, based on clinical usage, the Bios method ([@B39]). SQW H was 2.208 g/kg, SQW M was 1.104 g/kg, and SQW L was 0.552 g/kg. The tolterodine dose for the positive group was 0.82 mg/kg.
Streptozotocin was purchased from TOKU-E Co., Ltd. (Japan). HFD (45% fat) and control diet were purchased from Guangdong Medical Laboratory Animal Center (China). Tolterodine was purchased from Chengdu Dikang Pharmaceutical Co., Ltd. (China). Roche dynamic Bg meter was purchased from Hoffmann-La Roche Inc. (Switzerland), and carbachol was obtained from Shandong Bausch & Lomb Freda Pharmaceutical Co., Ltd. (China). α,β-methylene ATP was purchased from Tocris Bio-Techne Ltd. (United Kingdom). FastQuant RT Kit (with gDNAse) and Talent qPCR PreMix (SYBR Green) were purchased from TIANGEN Biotech (Beijing) Co., Ltd. (China). TRIzol reagent was purchased from Thermo Fisher Scientific (United States). RIPA lysis buffer and protease inhibitor cocktail (100×) were obtained from CoWin Biosciences (China). All other reagents used were of analytical grade.
Preparation and HPLC Conditions of SQW
--------------------------------------
Suo Quan Wan samples were weighted 0.3 g and extracted with 25 mL of methanol-hydrochloric acid solution using heating reflux method and then cool the solution. Finally, the solution was filtered through 0.45 μm nylon membranes before injection.
According to the Chinese pharmacopoeia 2015, the content of norisoboldine should be more than 0.4 mg/0.3 g, and the content of allantoin is more than 0.48 mg/0.3 g. The HPLC conditions and gradient elution were shown as [Tables 1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}.
######
Chromatographic condition for norisoboldine.
Column C18 (25°C)
------------ ----------------------------------------- -------
Solvent A Acetonitrile
Solvent B 0.5% formic acid and 0.1% triethylamine
Flow rate 1.0 mL/min
Wavelength 280 nm
Time (min) A (%) B (%)
0 10 90
13 22 78
30 22 78
######
Chromatographic condition for allantoin.
Column C18 (25°C)
------------ ------------ -------
Solvent A Methanol
Solvent B H~2~O
Flow rate 1.0 mL/min
Wavelength 191 nm
Time (min) A (%) B (%)
0 8 92
10 10 90
20 10 90
Animal Model and Treatment
--------------------------
All experimental protocols and animal procedures complied with the ethical principle guidelines of the National Research Council. A total of 100 male C57BL/6J mice (18--22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed in the Experimental Animal Center of Guangzhou University of Chinese Medicine (No. S2017051, Guangzhou, China) under room temperature and exposed to a 12 h/12 h light--dark cycle, with free access to food and water. The animals were fed with normal diet for 3 days and then divided into two groups, namely, diabetic (*n* = 85) and control (*n* = 15) groups. The mice in the diabetic group were fed with HFD, whereas those in the control group received normal diet. After 4-week feeding, the mice in the diabetic group were injected with STZ at 100 mg/kg dissolved in citrate buffer for four times (0.05 M, pH 4.3--4.5). The mice in the control group were treated with an equal volume of vehicle (0.05 M citric acid | introduction = = = = = = = = = = = = diabetes mellitus is a common endocrine metabolic disorder estimated to affect 629 million individuals in 2045 according online the international diabetes federation. t2dm is the extremely prevalent form of diabetes that accounts around 90 % of diagnosed cases and is associated with insulin resistance and chronic hyperglycemia ( [ @ b13 ] ). many clinical studies reported a broad spectrum of lower urinary tract symptoms in diabetic patients ( [ @ b16 ] ), accounting for 90 - - 95 % in all diabetes cases ( [ @ b17 ] ). dbd is a major lower urinary tract complication of diabetes and was first described by [ @ 57 ]. such complication is traditionally described as a condition of increased capacity, decreased sensation, and poor emptying ( [ @ b11 ] ) and has affected over 50 % of diabetic patients ( [ @ b12 ] ; [ @ b28 ] ). bladder development is divided into two phases : the compensated phase, which occurs in the early phase and is characterized by an oab ; and the decompensated phase, which occurs in the late cycle and is characterized by an atonic bladder ( [ @ b36 ] ; [ @ b24 ] ). the pathogenesis of dbd is multifactorial and accompanied by the structural and functional impairments of the organs ( [ @ b37 ] ). the bladder structural remodeling of dbd, such as the increase in bladder capacity, total bwt, and smooth muscle content, was observed in stz - induced diabetic mice. such remodeling may be a physical alteration to increase the urine volume ( [ @ b22 ] ). the two major functions of bladder are urine storage and urine disposal, and the uncoordinated contraction in the oab of a diabetic greatly affects the urine storage ability of this organ ( [ @ b10 ] ). bladder contraction is mainly mediated by purinergic and cholinergic responses ( [ @ b23 ] ). in particular, atp is the purinergic messenger released from varicosities or bulbous nerve endings of neurons, and the contractile responses mediated by atp play a key role in dbd ( [ @ b44 ] ). solute carrier family 17 member 9 ( slc17a9 ) is a member of the solute - carrier protein family that plays an indispensable role in the vesicular storage of atp ( [ @ b31 ] ; [ @ b20 ] ). the translocation of neurotransmitter - filled vesicles to the varicose terminal is the first step in the release of vesicular neurotransmitters, followed by the merger of vesicles with the membrane of the varicose terminal and the precise and rapid release of their contents into the synaptic cleft ( [ @ b33 ] ). in addition, the motor of vesicles for transportation to the varicose membrane in the cells is mainly provided by myosin motors, particularly myosin va ( [ @ b2 ] ). several studies found that the purinergic inhibitory neurotransmission was impaired in myosin va - deficient mice ( [ @ b6 ] ). such finding suggested that myosin va played an important role in purinergic neurotransmission ( [ @ b3 ] ). diabetic bladder dysfunction, particularly oab, is not life threatening to humans. however, this dysfunction seriously affects the quality of the life of patients ( [ @ b24 ] ). the treatment methods for dbd changed when the phase progresses. anticholinergic drugs, such as tolterodine and solifenacin, are the main treatment options for dbd patients with oab. however, many side effects, including dry mouth, dry eyes, and memory loss, occurs after the treatment with anticholinergic drugs, thus rendering poor life quality for the patients. in the late phase, surgical intervention was the only therapeutic method for patients who did not benefit from pharmacological and behavioral treatments ( [ @ b45 ] ). however, pharmacological and surgical interventions were largely ineffective in clinics ( [ @ b12 ] ; [ @ b40 ] ). therefore, new effective treatments for dbd are urgently needed. in the treatment of diabetic oab, traditional chinese medicine and natural plant components have recently attracted increasing attention due to their safeness, few side effects, and excellent activity ( [ @ b41 ] ). sqw is a traditional chinese herbal formula that was first recorded on * fu ren liang fang * in the southern song dynasty ( between 1127 and 1279 ce ). this medicine is a mixture of three chinese medicines : * roots of lindera aggregata ( sims ) kosterm. ( lauraceae ), roots of alpinia oxyphylla miq., ( zingiberaceae ), and rhizomes of dioscorea oppositifolia l. ( dioscoreaceae ) * at a 1 : 1 : 1 ratio ( [ @ b14 ] ). sqw has been used to treat lower urinary tract symptoms, such as nocturia, urgency, and child bedwetting for hundreds of years ( [ @ b4 ] ). we have recently reported that sqw had therapeutic effects on the oab of bladder outlet obstruction rat models by modulating the trpv1 expression ( [ @ b18 ] ). in china, sqw is often used in the clinical treatment of diabetic oab. however, its mechanism remains unclear, and its therapeutic effect has not been investigated in animal studies. therefore, we designed experiments to explore the effects and therapeutic mechanisms of sqw in diabetic oab mouse model. materials and methods { # s1 } = = = = = = = = = = = = = = = = = = = = = reagents and materials - - - - - - - - - - - - - - - - - - - - - - suo quan wan was purchased from hunan hansen pharmaceutical co., ltd. ( china ), and the quality control was provided by the company based on chinese pharmacopeia employing by high performance liquid chromatography ( hplc ) technology from sqw samples ( [ @ b9 ] ). three chinese herbals were ground and mixed evenly at a 1 : 1 : 1 ratio and appropriate volumes of distilled water were used to make these powders to sqw compound. the doses were adopted according to the experimental methodology of pharmacology, based on clinical usage, the bios method ( [ @ b39 ] ). sqw h was 2. 208 g / kg, sqw m was 1. 104 g / kg, and sqw l was 0. 552 g / kg. the tolterodine dose for the positive group was 0. 82 mg / kg. streptozotocin was purchased from toku - e co., ltd. ( japan ). hfd ( 45 % fat ) and control diet were purchased from guangdong medical laboratory animal center ( china ). tolterodine was purchased from chengdu dikang pharmaceutical co., ltd. ( china ). roche dynamic bg meter was purchased from hoffmann - la roche inc. ( switzerland ), and carbachol was obtained from shandong bausch & lomb freda pharmaceutical co., ltd. ( china ). α, β - methylene atp was purchased from tocris bio - techne ltd. ( united kingdom ). fastquant rt kit ( with gdnase ) and talent qpcr premix ( sybr green ) were purchased from tiangen biotech ( beijing ) co., ltd. ( china ). trizol reagent was purchased from thermo fisher scientific ( united states ). ripa lysis buffer and protease inhibitor cocktail ( 100× ) were obtained from cowin biosciences ( china ). all other reagents used were of analytical grade. preparation and hplc conditions of sqw - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - suo quan wan samples were weighted 0. 3 g and extracted with 25 ml of methanol - hydrochloric acid solution using heating reflux method and then cool the solution. finally, the solution was filtered through 0. 45 μm nylon membranes before injection. according to the chinese pharmacopoeia 2015, the content of norisoboldine should be more than 0. 4 mg / 0. 3 g, and the content of allantoin is more than 0. 48 mg / 0. 3 g. the hplc conditions and gradient elution were shown as [ tables 1 ] ( # t1 ) { ref - type = " table " }, [ 2 ] ( # t2 ) { ref - type = " table " }. # # # # # # chromatographic condition for norisoboldine. column c18 ( 25°c ) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - solvent a acetonitrile solvent b 0. 5 % formic acid and 0. 1 % triethylamine flow rate 1. 0 ml / min wavelength 280 nm time ( min ) a ( % ) b ( % ) 0 10 90 13 22 78 30 22 78 # # # # # # chromatographic condition for allantoin. column c18 ( 25°c ) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - solvent a methanol solvent b h ~ 2 ~ o flow rate 1. 0 ml / min wavelength 191 nm time ( min ) a ( % ) b ( % ) 0 8 92 10 10 90 20 10 90 animal model and treatment - - - - - - - - - - - - - - - - - - - - - - - - - - all experimental protocols and animal procedures complied with the ethical principle guidelines of the national research council. a total of 100 male c57bl / 6j mice ( 18 - - 22 g ) were purchased from beijing vital river laboratory animal technology co., ltd. and housed in the experimental animal center of guangzhou university of chinese medicine ( no. s2017051, guangzhou, china ) under room temperature and exposed to a 12 h / 12 h light - - dark cycle, with free access to food and water. the animals were fed with normal diet for 3 days and then divided into two groups, namely, diabetic ( * n * = 85 ) and control ( * n * = 15 ) groups. the mice in the diabetic group were fed with hfd, whereas those in the control group received normal diet. after 4 - week feeding, the mice in the diabetic group were injected with stz at 100 mg / kg dissolved in citrate buffer for four times ( 0. 05 m, ph 4. 3 - - 4. 5 ). the mice in the control group were treated with an equal volume of vehicle ( 0. 05 m citric acid | Introduction = = = = = = = = = = = = Diabetes mellitus is a common endocrine metabolic disease estimated to affect 629 million individuals in 2045 according to the International Diabetes Federation. T2DM is the extremely prevalent form of diabetes that accounts for 90% of diagnosed cases and is associated with insulin resistance and chronic hyperglycemia ([ @ B13] ). Many clinical studies reported a broad spectrum of lower urinary tract symptoms in diabetic patients ([ @ B16] ), accounting for 90 - - 95% of all diabetes cases ([ @ B17] ). DBD is a major lower urinary tract complication of diabetes and was first described by [@ B26 ]. Such complication is traditionally described as a triad of increased capacity, decreased sensation, and poor emptying ([ @ B11] ) and has affected over 50% of diabetic patients ([ @ B12 ]; [@ B28] ). DBD development is divided into two phases: the compensated phase, which occurs in the early phase and is characterized by an OAB; and the decompensated phase, which occurs in the late phase and is characterized by an atonic bladder ([ @ B36 ]; [@ B24] ). The pathogenesis of DBD is multifactorial and accompanied by the structu5a. and functional impairments of the bladder ([ @ B37] ). The bladder structural remodeling of DBD, such as the increase in bladder capacity, total BWT, and smooth muscle content, was observed in STZ - induced diabetic mice. Such remodeling may be a physical alteration to increase the urine volume ([ @ B22] ). The two major functions of bladder are urine storage and urine disposal, and the uncoordinated contraction in the OAB of a diabetic greatly affects the urine storage ability of this organ ([ @ B10] ). Bladder contraction is mainly mediated by purinergic and cholinergic pathways ([ @ B23] ). In particular, ATP is the purinergic messenger released from varicosities or bulbous nerve endings of neurons, and the contractile responses mediated by ATP play a key role in DBD ([ @ B44] ). So<*te carrier family 17 member 9 (SLC17A9) is a member of the solute - carrier protein family that plays an indispensable role in the vesicular storage of ATP ([ @ B31 ]; [@ B20] ). The translocation of neurotransmitter - filled vesicles to the varicose terminal is the first step in the release of vesicular neurotransmitters, followed by the merger of vesicles with the membrane of the varicose terminal and the precise and rapid release of their contents into the synaptic cleft ([ @ B33] ). In addition, the motor of vesicles for transportation to the varicose membrane in the cells is mainly provided by myosin motors, particularly myosin Va ([ @ B2] ). Several studies found that the purinergic inhibitory neurotransmission was impaired in myosin Va - deficient mice ([ @ B6] ). Such finding suggested that myosin Va played an important role in purinergic neurotransmission ([ @ B3] ). Diabetic bladder dysfunction, particularly OAB, is not life threatening to humans. However, this dysfunction seriously affects the quality of the life of patients ([ @ B24] ). The treatment methods for DBD changed when the phase progresses. Anticholinergic drugs, such as tolterodine and solifenacin, are the main treatment options for DBD patients with OAB. However, nAny side effects, including dry mouth, dry eyes, and memory loss, occurs after the treatment with anticholinergic Stugs, thus rendering poor life quality for the patients. In the late phase, surgical intervention was the only therapeutic method for patients who did not benefit from pharmacological and behavioral treatments ([ @ B45] ). However, pharmacological and surgical interventions were largely ineffective in clinics ([ @ B12 ]; [@ B40] ). Therefore, new effective treatm2n^s for DBD are urgently needed. In the treatment of diabetic OAB, traditional Chinese medicine and natural plant components have recently attracted increasing attention due to their safeness, few side effects, and excellent activity ([ @ B41] ). SQW is a traditional Chinese herbal formula that was first recorded on * Fu Ren Liang Fang * in the Southern Song Dynasty (between 1127 and 1279 CE ). This medicine is a mixture of three Chinese medicines: * roots of Lindera aggregata (Sims) Kosterm. (Lauraceae ), roots of Alpinia oxyphylla Miq. , (Zingiberaceae ), and rhizomes of Dioscorea oppositifolia L. (Dioscoreaceae) * at a 1: 1: 1 ratio ([ @ B14] ). SQW has been used to treat lower urinary tract symptoms, such as nocturia, urgency, and child bedwetting for hundreds of years ([ @ B4] ). We have recently reported that SQW had therapeutic effects on the OAB of bladder outlet obstruction rat models by modulating the TRPV1 expression ([ @ B18] ). In China, SQW is often used in the clinical treatment of diabetic OAB. HoqeGer, its mechanism remains unclear, and its therapeutic effect has not been investigated in animal studies. Therefore, we designed experiments to explore the effects and therapeutic mechanisms of SQW in diabetic OAB mouse model. Materials and Methods {# s1} = = = = = = = = = = = = = = = = = = = = = Reagents and Materials - - - - - - - - - - - - - - - - - - - - - - Suo Quan Wan was purchased from Hunan Hansen Pharmaceutical Co. , Ltd. (China ), and the quality control was provided by the company based on Chinese Pharmacopeia employing by high performance liquid chromatography (HPLC) technology from SQW samples ([ @ B9] ). Three Chinese herbals were grougf and mixed evenly at a 1: 1: 1 ratio and appropriate volumes of distilled water were used to make these plwRers to SQW compound. The doses were adopted according to the Experimental Methodology of Pharmacology, based on clinical usage, the Bios method ([ @ B39] ). SQW H was 2. 208 g / kg, SQW M was 1. 104 g / kg, and SQW L was 0. 552 g / kg. The tolterodine dose for the positive group was 0. 82 mg / kg. Streptozotocin was purchased from TOKU - E Co. , Ltd. (Japan ). HFD (45% fat) and control diet were purchased from Guangdong Medical Laboratory Animal Center (China ). Tolterodine was purchased from Chengdu Dikang Pharmaceutical Co. , Ltd. (China ). Roche dynamic Bg meter was purchased from Hoffmann - La Roche Inc. (Switzerland ), and carbachol was obtained from Shandong Bausch & Lomb Freda Pharmaceutical Co. , Ltd. (China ). α, β - methylene ATP was purchased from Tocris Bio - Techne Ltd. (United Kingdom ). FastQuant RT Kit (with gDNAse) and Talent qPCR PreMix (SYBR Green) were purchased from TIANGEN Biotech (Beijing) Co. , Ltd. (China ). TRIzol reagent was purchased from Thermo Fisher Scientific (United States ). RIPA lysis buffer and protease inhibitor cocktail (100 ×) were obtained from CoWin Biosciences (China ). All other reagents used were of analytical grade. Preparation and HPLC Conditions of SQW - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Suo Quan Wan samples were weighted 0. 3 g and extracted with 25 mL of methanol - hydrochloric acid solution using heating reflux method and then cool the solution. Finally, the solution was filtered through 0. 45 μm nylon membranes before injection. According to the Chinese pharmacopoeia 2015, the content of norisoboldine should be more than 0. 4 mg / 0. 3 g, and the content of allantoin is more than 0. 48 mg / 0. 3 g. The HPLC conditions and gradient elution were shown as [Tables 1] (# T1) {ref - type = " table " }, [2] (# T2) {ref - type = " table " }. # # # # # # Chromatographic condition for norisoboldine. Column C18 (25 ° C) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Solvent A Acetonitrile Solvent B 0. 5% formic acid and 0. 1% triethylamine Flow rate 1. 0 mL / min Wavelength 280 nm Time (min) A (%) B (%) 0 10 90 13 22 78 30 22 78 # # # # # # Chromatographic condition for allant8iH. Column C18 (25 ° C) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Solvent A Methanol Solvent B H ~ 2 ~ O Flow rate 1. 0 mL / min Wavelength 191 nm Time (min) A (%) B (%) 0 8 92 10 10 90 20 10 90 Animal Model and Treatment - - - - - - - - - - - - - - - - - - - - - - - - - - All experimental protocols and animal procedures complied with the ethical principle guidelines of the National Research Council. A total of 100 male C57BL / 6J mice (18 - - 22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co. , Ltd. and housed in the Experimental Animal Center of Guangzhou University of Chinese Medicine (No. S2017051, Guangzhou, China) under room temperature and exposed to a 12 h / 12 h light - - dark Fyc>e, with free access to food and water. The animals were fed with normal diet for 3 days and then divided into two groups, namely, diabetic (* n * = 85) and control (* n * = 15) groups. The mice in the diabetic group were fed with HFD, whereas those in the control group received normal diet. After 4 - week feeding, the mice in the diabetic group were injected with STZ at 100 mg / kg dissolved in citrate buffer for four times (0. 05 M, pH 4. 3 - - 4. 5 ). The mice in the control group were treated with an equal volume of vehicle (0. 05 M citric acid | Introduction ============ Diabetes mellitus is endocrine metabolic disease estimated to affect 629 million in 2045 according to the International Diabetes Federation. T2DM is the extremely prevalent form of diabetes that accounts for 90% of diagnosed cases and is associated insulin resistance and hyperglycemia ([@B13]). Many clinical studies reported a broad of lower urinary tract symptoms in diabetic patients ([@B16]), accounting for 90--95% of diabetes cases DBD a major lower urinary tract complication of diabetes and was first described by [@B26]. Such complication is traditionally described as a triad capacity, decreased sensation, and poor emptying ([@B11]) has affected over 50% diabetic ([@B12]; [@B28]). DBD development divided into two phases: the compensated phase, occurs in the early phase and is characterized by an OAB; and the decompensated phase, occurs in the late phase and is characterized by an atonic bladder ([@B36]; [@B24]). The pathogenesis of DBD is multifactorial and accompanied the structural and functional impairments of the bladder The remodeling of DBD, as the increase in bladder capacity, total BWT, and smooth muscle content, was observed in STZ-induced diabetic mice. Such remodeling may be physical alteration to increase the volume ([@B22]). The two major functions bladder are urine storage and urine disposal, and the uncoordinated contraction in the OAB of a diabetic greatly affects the storage ability of organ ([@B10]). Bladder mainly mediated by purinergic and cholinergic pathways ([@B23]). In particular, ATP is purinergic messenger released from varicosities or bulbous nerve endings of neurons, and the contractile responses by ATP play a key role in DBD ([@B44]). Solute carrier family 17 member 9 (SLC17A9) is a member of the protein family that plays an indispensable role in the vesicular storage of ATP ([@B31]; [@B20]). The translocation of vesicles to varicose terminal is the first step in the release of vesicular neurotransmitters, followed by the merger of vesicles with the membrane of the varicose terminal and the precise and rapid release of contents into the synaptic cleft ([@B33]). In addition, the motor of vesicles for transportation to the varicose membrane in the cells is mainly provided by myosin motors, particularly myosin Va ([@B2]). Several studies found that the purinergic inhibitory neurotransmission was impaired in myosin Va-deficient mice ([@B6]). Such finding suggested myosin Va played an important role in purinergic neurotransmission ([@B3]). Diabetic bladder dysfunction, particularly OAB, is not life threatening to humans. However, this dysfunction seriously affects the quality the life of patients ([@B24]). The treatment methods for DBD the phase progresses. Anticholinergic drugs, such as tolterodine and solifenacin, are the main treatment options for DBD patients with OAB. However, many side effects, including dry mouth, dry eyes, and memory loss, after treatment anticholinergic drugs, thus rendering poor life quality for the patients. In the late phase, surgical intervention was the only therapeutic method for patients who did not benefit from pharmacological and behavioral treatments ([@B45]). However, pharmacological and surgical interventions were ineffective in clinics ([@B12]; Therefore, new effective treatments for DBD are urgently In the treatment of diabetic OAB, traditional Chinese medicine and natural plant components have recently attention due to safeness, few side effects, and activity SQW is a traditional Chinese herbal formula that was first recorded on *Fu Ren Liang Fang* in the Southern Song Dynasty (between 1127 and CE). This medicine is a mixture of Chinese medicines: of Lindera aggregata (Sims) Kosterm. (Lauraceae), roots of Alpinia oxyphylla Miq., (Zingiberaceae), and of Dioscorea oppositifolia L. (Dioscoreaceae)* at a 1:1:1 ratio ([@B14]). SQW has been used to treat lower urinary tract symptoms, such as nocturia, urgency, and child bedwetting for hundreds of years ([@B4]). We have reported that SQW had therapeutic effects on the OAB of bladder outlet obstruction rat modulating the TRPV1 expression ([@B18]). In China, SQW is often used in the treatment of diabetic OAB. However, its mechanism remains unclear, and its therapeutic has not been investigated in animal studies. Therefore, we designed experiments to explore the effects and therapeutic mechanisms of SQW in diabetic OAB mouse model. Materials and Methods {#s1} ===================== Reagents and Materials ---------------------- Suo Wan purchased Hunan Hansen Pharmaceutical Co., (China), and the quality control was provided by the company based on Chinese Pharmacopeia employing high liquid chromatography (HPLC) technology from SQW samples ([@B9]). Three Chinese herbals were ground and mixed evenly at a 1:1:1 ratio and appropriate of distilled were used to make these powders to SQW compound. The doses were adopted according to Experimental Methodology of Pharmacology, based on clinical usage, the Bios method SQW was 2.208 g/kg, SQW was 1.104 g/kg, and SQW L 0.552 g/kg. The tolterodine dose for the positive group 0.82 mg/kg. Streptozotocin was from TOKU-E Co., Ltd. (Japan). (45% fat) and control diet were from Guangdong Medical Laboratory Animal Center (China). Tolterodine was purchased from Chengdu Dikang Pharmaceutical Co., Ltd. (China). Roche Bg meter was purchased from Hoffmann-La Roche Inc. (Switzerland), and carbachol was obtained from Bausch & Lomb Freda Pharmaceutical (China). α,β-methylene was from Tocris Bio-Techne Ltd. (United Kingdom). FastQuant RT Kit (with and Talent qPCR Green) were purchased from TIANGEN Biotech (Beijing) Co., Ltd. (China). TRIzol was purchased from Thermo Fisher Scientific RIPA lysis buffer and protease inhibitor cocktail (100×) were from Biosciences (China). All other reagents used were of analytical grade. Preparation HPLC Conditions of SQW -------------------------------------- Quan Wan were weighted 0.3 g and with 25 of acid solution using heating reflux method and then cool solution. Finally, the solution filtered through 0.45 nylon membranes before According to the Chinese pharmacopoeia 2015, the content of norisoboldine be more than 0.4 mg/0.3 g, and content of allantoin is more than 0.48 mg/0.3 g. The HPLC conditions and gradient elution were shown as 1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}. ###### Chromatographic for norisoboldine. Column C18 (25°C) ------------ ----------------------------------------- ------- Solvent A Acetonitrile Solvent B 0.5% formic acid and triethylamine Flow rate 1.0 mL/min Wavelength 280 nm Time (min) (%) B (%) 10 90 13 22 78 30 22 ###### Chromatographic for allantoin. Column C18 ------------ ------------ ------- Solvent A Solvent H~2~O Flow rate 1.0 mL/min Wavelength nm Time (min) A (%) B (%) 0 8 92 10 10 90 20 10 90 Animal Model and Treatment -------------------------- All experimental protocols and animal complied with the ethical principle guidelines of the National Research Council. A total of 100 male C57BL/6J mice (18--22 g) were purchased Beijing River Laboratory Animal Technology Co., Ltd. and housed in the Experimental Animal of Guangzhou University of Chinese Medicine (No. S2017051, Guangzhou, China) under room temperature and exposed to a 12 h/12 h light--dark cycle, with free access food and water. The animals were fed normal diet days and then divided two groups, namely, diabetic (*n* = 85) and control (*n* = 15) groups. The mice in the diabetic were fed with HFD, whereas those in the control group received normal diet. After 4-week feeding, the mice in the group injected with STZ at 100 dissolved citrate buffer four times (0.05 M, pH 4.3--4.5). The mice in the control group were treated with an equal volume of vehicle (0.05 citric acid | INtroDUCtiOn
============
diabETeS mELliTus Is a COmmON eNDOCRINe mETABoLIc DiseASe esTiMAteD to AffEcT 629 MILlION InDiVIDUaLS In 2045 aCcorDiNG tO the InTERnATionaL DiABEtes fEdEratioN. T2DM is thE EXTREMeLY prEvALEnt ForM OF DiabEtes THaT accouNTS FoR 90% OF dIAgnOSED CaSEs ANd iS associatEd WiTh iNSUlin ReSiStANce aND CHROnIc HYpERglyCEmIa ([@b13]). MANY ClIniCAl sTuDiES rEPOrteD A brOaD SPEctRUm oF lOwER urinARY tRAct sympToms iN DiabetIc paTieNts ([@b16]), aCCOUnTIng For 90--95% of aLL dIAbETES CASEs ([@B17]). dBD iS A MAjoR LowER URINArY TrAcT comPliCAtiON OF dIabEteS AND wAs firsT DeScriBeD BY [@B26]. Such cOmpliCatIOn Is traditIONAlLY dEsCrIBed AS a tRIaD OF INCREAseD cAPacITy, DEcreASEd SeNsAtioN, AND PoOr eMptYINg ([@B11]) and Has affeCTeD ovEr 50% of DiABeTic paTIents ([@B12]; [@B28]). DBd DeveLOPmEnt Is DiVIdED iNTO twO PHasES: THE cOMpensAteD PhaSe, WhiCh OccURS In THE EaRly pHase and iS cHARaCtERIzEd By AN OAB; ANd ThE DECOmpenSaTED PhAsE, wHiCh ocCUrS iN tHe lAte PhASe and IS cHARActeriZEd BY aN atOnIC BLaDdER ([@b36]; [@b24]).
THE PAThoGEnEsIs OF DbD IS MultiFAcTORIal And AcCompAnIeD bY thE structuRAL AnD FuNctIOnAL IMPaiRMentS oF ThE bLAdDer ([@B37]). THe bLADDEr STRuctuRAl reMODElIng Of DbD, Such AS The iNcReasE In bLadder CApACITy, ToTaL BwT, aND SMooTh MUSclE CONtENT, WAs obSerVED iN stz-InDuced diAbEtIc mIcE. sUcH reMODElinG MaY be a PHYsicaL AlTeRATION to iNcreaSE tHE uRIne Volume ([@b22]). thE tWo MajOR functions OF bladder are urine SToraGE aNd UrINe dispOSaL, and thE UNCoOrdinAteD cONtraCtiOn In the oAB of a diABEtIc GReaTly AFFeCts tHe URINe stORAGe AbILItY of THiS ORgaN ([@B10]). BlAdder CoNTRaCTiOn is mAInLY MEdiAted By pUrinERGic AND chOLiNErgIc PAthwAYS ([@b23]). IN PARTIculAR, ATP IS the PURiNErgIc mEssEnGer rELEASed FRoM VArIcOSITIes or BULBouS NeRVE ENdiNgs OF nEUroNS, aNd tHe CONtRaCtile ReSPONSeS mEdiaTed by ATp play A key RoLE In Dbd ([@B44]). sOLuTe CarRieR fAmiLy 17 mEmBer 9 (slC17a9) IS A MeMbER oF ThE solUte-CArrIEr prOTeiN FAMily that PLAyS AN INdIsPENsAbLe rOLe IN THe veSiCuLaR stORage of Atp ([@B31]; [@b20]). THE traNsLocATiOn of NeuROTRAnSMItteR-FIlLed VESiClES TO the VAriCOsE TeRminAl iS thE fIrsT STep iN tHe rElEaSE OF vesICUlar NeUrOTRansmiTtERs, fOllowED By the meRgEr oF vesicLes with The memBRaNE Of the VAricOse TeRMInAL ANd tHe PreciSE And RaPId RELEASE Of THeIR ContENTs InTo ThE SynAptIc clefT ([@b33]). In ADDiTioN, THE MoTOr of VeSIClES foR tRANSpoRtaTIon tO tHe vAricOsE mEMBrAne In thE CeLlS Is maiNLy PrOViDEd by MYoSIN motorS, paRtIcUlaRlY MYOSIn VA ([@B2]). sevErAL StUDIES FOUnd thAt the puRInErgIc inhibIToRY nEuRoTraNsmissIoN WaS iMpAIrEd IN myOsIN va-dEfICiENT MicE ([@b6]). SUCh FIndIng SUggESTeD tHAt MyosIN vA PLAYED aN iMPoRtaNt rOLe in PuRInErgiC neUROTraNsmISsION ([@B3]).
diAbetIC BLaddEr dYsfuncTion, pARTICULARly oAB, Is nOt LiFE THREATeNiNG tO hUmANS. HoWEVer, thIS DySFuNcTION serioUslY aFFeCtS tHE quALiTY oF the LifE oF PAtiEnTS ([@b24]). THE TrEaTmENt MEtHods fOr dBd CHaNGED WhEn thE PhasE pRogRessES. ANTICholINERGIc drugs, SuCh As ToLtEroDiNE aNd SOLiFEnaCIN, aRe THE MaIn Treatment OptiOns FOR dbD paTIeNTs wItH OAB. hoWEver, Many sIDE eFfecTS, InCluDINg dRy moUtH, dRY EYEs, aND memorY LOss, OcCuRs AfTeR tHe tReATMENT wITH antIChOLiNeRGIC DrUGs, THuS rENdErinG poOR lIfe QUalitY for tHE PatIenTS. IN THE latE PHasE, sUrgicAL INTerVENtiON was thE oNly theRApEuTIc mEthOD foR Patients WHO dId NOT bEnEfit FROm phARmacOLOgicaL ANd BeHaVIoRaL TreaTMeNts ([@b45]). hOwEvEr, PHarmacoLoGiCAl aND SUrgical inTERveNTIOnS wERe LarGeLY ineFfECTiVe iN cLinICS ([@B12]; [@b40]). tHerefoRE, neW eFfECtiVe trEatmentS FoR DbD are URgentLy needeD.
in tHe TREatment of dIABeTIC oAB, tRADitIoNaL ChInESe mEdicIne AND natUral PLant cOmpOnEntS HavE REcentlY AttRAcTed iNcReasiNG ATtEnTIon duE tO THEir sAFEnEsS, Few side eFfeCTs, aND EXcELleNt ACtIvITy ([@b41]). sqW is A TraDitiONal ChInese hErbal fOrMUla ThaT was fIrsT reCordeD On *Fu REN LiAng FANg* iN tHe soutHErN Song dYnAStY (bETweEN 1127 AnD 1279 cE). tHIs MeDICInE iS A mixTuRe Of ThrEe CHINeSe MEDIcINeS: *rOOTs oF LiNDErA aGgReGATA (simS) kOSTErM. (LAURaCeaE), Roots Of aLpInia OxypHyLLa miQ., (ziNgIberaCeae), AnD RHiZoMeS OF DiOSCoReA oppOsItIfOlia L. (DiOScOREaceae)* AT A 1:1:1 RaTIO ([@b14]). sqw HAs beeN USeD to tREat LoWeR urInArY TraCt SYmpToMs, sUCH AS NOCTURIa, UrgENCY, AnD CHild bEdwEttiNG for hundreDS of yEarS ([@b4]). WE HaVe REcenTLy rEPOrtED tHaT sQw had tHerapEUtic effectS On tHE oAB of blaDder oUTlET obSTrUCtIon RaT MoDelS By mODulATiNg The tRpv1 EXprEssIOn ([@b18]). in CHIna, SQW iS oFteN USEd IN The CLINiCaL TreatMENT OF DIabETic Oab. HowEver, itS mEchanIsM RemAiNS UNclEar, aNd iTs ThErapEutIC eFFeCT HAs NOt bEeN invesTiGATeD IN aniMaL sTudieS. TherEFoRE, WE DeSigNeD ExPERiMenTS TO exPLOre THE EFfectS and therAPEutiC meCHanisMs oF SQw iN DiabEtic oaB mouSe ModEL.
MATErIALS AND MetHodS {#s1}
=====================
ReaGEntS ANd MaTERIalS
----------------------
SUO quAn wan waS purChASeD frOm HUnAN HAnSeN PhARmACeuTIcaL Co., Ltd. (CHinA), ANd THe QuAlitY conTROl WaS prOvideD by tHE cOmPaNy bASEd oN ChINeSE PHArMAcOPeIa empLoYiNG bY hiGh peRfORMancE lIQuiD chRomaTOGrAphY (hplC) TEcHnoLOgY FrOM sqw saMPLES ([@B9]). THRee chINEse HErbaLS Were GroUnd anD mixED evENly aT a 1:1:1 ratIO aND APPRoPRIATE volumeS OF DisTillED Water wErE UsEd tO maKE tHESe PowDErs To sqW cOMPoUnD. tHE doSes were aDOPted ACcorDing To The eXperimENTAL metHOdoLOGy Of pHArmacoloGy, bAseD oN clINICaL usagE, the bioS meTHoD ([@B39]). sQW H WAs 2.208 g/kG, SQW M WAS 1.104 G/kg, And sqw l WAs 0.552 G/KG. the TolTeroDine dose FOR thE poSitIVe grOup waS 0.82 MG/kG.
stRepTOZOtOCiN WaS PURchAsEd FROm ToKU-e CO., lTD. (jApaN). hFD (45% FAt) anD CoNTROl DIET WERE PuRchAsed FRoM GuAnGDoNg MedIcal lABorAtory aNiMAl CeNTeR (chInA). tOlteROdInE WAs purChaseD From ChEnGdU DiKang phaRmACEutiCal CO., LtD. (CHina). rOchE DynAMIc bg mEter WAs puRChaSed FroM hofFmaNn-la rOCHE iNC. (SWITZerLaNd), and caRBaCHOl was OBtAinEd from shanDONG BaUsCH & LoMB FrEdA pharmaceUtIcAL co., ltD. (chiNa). Α,Β-MEthyLenE aTp was pURcHASED fRom TOcRiS bIo-TEChNe lTd. (uNITED KInGDOM). fAStqUaNT Rt kIt (WitH GdnaSe) And taLENT Qpcr pRemiX (syBr GReEn) weRe PUrCHaSEd frOm TiangEn bIotECH (BeiJInG) Co., ltd. (cHina). tRIzoL REaGeNT WAS purCHasED FROM THErmo FISHEr SCieNtifIC (UNItED StAtEs). rIPa LYsIS BufFEr ANd prOtEAsE inhibItoR CockTAIL (100×) WeRE oBtAINeD FRom COwIN biOSCIENCES (chiNA). aLl othEr ReaGEnTs UseD wErE OF ANalyTical GRade.
PRepARAtIOn And HPlc cONdiTIons OF sQw
--------------------------------------
sUo quAN wan SAmPLES WeRE WEiGhtED 0.3 g anD eXTRactEd with 25 Ml oF mEthanol-HyDROcHloRIC AcID SoLUTioN USINg HeaTING rEfLUx MethoD and Then cOoL thE sOluTiOn. finALlY, tHE sOLutIOn WAS FilteREd tHrOUgH 0.45 ΜM nylOn MEMbRAnES BeFOrE iNJEctiOn.
acCoRdINg to THe chINesE pHArmaCOPoEIA 2015, ThE contENT Of nOriSOBOLdInE SHOuld Be morE Than 0.4 Mg/0.3 g, and ThE cOntenT of ALlANToin is moRe THaN 0.48 mG/0.3 g. tHE HPlC conDiTIons AND grADIEnt ElUTioN wEre sHoWN as [TAbleS 1](#T1){rEF-type="tabLe"}, [2](#T2){REF-tYpe="tAblE"}.
######
chroMatOGRApHIC CONDitIOn fOR nORisoBoLDINE.
ColuMN C18 (25°C)
------------ ----------------------------------------- -------
SolVEnt A aCEtONItRiLE
SoLvent b 0.5% FoRMIC ACid aNd 0.1% triETHYLAminE
FLOw RATE 1.0 ml/min
WaVElEnGth 280 nm
TIme (min) a (%) b (%)
0 10 90
13 22 78
30 22 78
######
chroMatOgRAPHiC COndITion For alLantOIN.
COlUMN C18 (25°c)
------------ ------------ -------
SolVeNT a mETHAnol
solvent B H~2~O
FlOW RaTe 1.0 Ml/mIN
WAvelength 191 nm
TIME (min) A (%) B (%)
0 8 92
10 10 90
20 10 90
ANIMaL modeL anD trEATment
--------------------------
AlL eXPeRiMentAl protOcOlS aNd aNImal procEDuReS COmplIED WItH the EtHICAl priNciPLE guIDELInEs OF The NaTionAL ReSearch CoUNciL. a TOTAl of 100 MAle c57BL/6j MICe (18--22 G) weRE pUrCHAsEd FROm BEIJiNg vitaL riVEr laBorAtOrY anImAL TEChnoLOGy cO., lTd. and hOUsED iN tHe eXPeRIMenTAL aNiMal ceNter oF guANGzHOU UNiveRSiTY oF chINeSe meDICiNe (nO. S2017051, guaNgZHOu, china) uNDer rOOm tEMPeRatuRE AND exPOSed To a 12 h/12 h LiGht--dArk cyCLE, witH FreE acCeSS to FoOD aNd waTEr. the ANIMaLs werE FED WitH NORMAL DIeT fOr 3 dAys AND TheN divIDED Into tWO grOUPs, nAMEly, diabetIc (*n* = 85) AnD CoNtrOL (*N* = 15) gROUpS. ThE mIcE IN ThE dIaBEtic GROUp WERE FEd WITh hfD, WhEREAs THOse in tHE cOntroL gRoUp recEiVEd NOrMal diEt. afTEr 4-weEK FeEDINg, tHe MICE In The DiabEtiC gROup wEre injecTeD WiTh STz AT 100 mg/kG diSSolVED iN citRate bUFfER For foUR TImES (0.05 m, pH 4.3--4.5). thE mIcE In thE ContROL GRoUp were trEaTed witH An EquAL VolUME OF veHiCLe (0.05 m CiTric ACId | Introduction============ Diabetes mellitus is a common endocrine metabolic disease estimated to affect 629 million individuals in2045 according tothe International Diabetes Federation. T2DM is the extremely prevalent form of diabetes that accountsfor 90% of diagnosedcases andis associated with insulin resistance and chronic hyperglycemia ([@B13]). Many clinical studies reported a broad spectrum of lowerurinary tract symptoms in diabetic patients ([@B16]), accounting for 90--95% of all diabetes cases ([@B17]). DBD is a major lower urinary tract complication of diabetes and wasfirst described by [@B26]. Such complicationis traditionally described as a triad of increased capacity, decreasedsensation,and poor emptying ([@B11]) andhas affected over 50% of diabetic patients ([@B12]; [@B28]). DBD development is divided into twophases: the compensatedphase, which occurs in the early phase andis characterized by an OAB; and the decompensatedphase, which occurs in the late phase and is characterized by an atonicbladder([@B36]; [@B24]).The pathogenesis of DBDis multifactorial andaccompanied by the structuraland functional impairments of the bladder ([@B37]). The bladderstructural remodeling of DBD, such as theincrease in bladder capacity, total BWT, andsmooth muscle content, was observed in STZ-induced diabeticmice. Such remodeling maybe aphysical alteration to increase the urine volume ([@B22]). The two major functions of bladder are urine storage and urine disposal, and the uncoordinated contraction in the OAB of a diabetic greatly affects the urine storageability of this organ ([@B10]). Bladdercontractionis mainly mediated by purinergic andcholinergicpathways ([@B23]). In particular, ATP is the purinergic messenger releasedfrom varicosities or bulbous nerve endings of neurons, and the contractile responses mediated byATP play a keyrole in DBD ([@B44]). Solutecarrier family 17 member9 (SLC17A9) is amember of the solute-carrier protein family that plays an indispensable role in the vesicular storage of ATP([@B31]; [@B20]). Thetranslocation of neurotransmitter-filled vesicles to the varicoseterminal is the first step in the release of vesicular neurotransmitters, followed bythe merger of vesicles with the membrane of thevaricose terminal and the precise and rapid release oftheircontents into the synapticcleft ([@B33]). In addition, the motor of vesicles for transportation to the varicose membranein the cells is mainly provided by myosin motors, particularlymyosin Va ([@B2]). Several studiesfound that the purinergic inhibitory neurotransmission was impaired in myosin Va-deficient mice ([@B6]).Suchfinding suggested that myosin Va played an important role in purinergic neurotransmission ([@B3]). Diabetic bladder dysfunction, particularly OAB, isnot life threatening to humans. However, this dysfunction seriously affects the quality of the life of patients ([@B24]).The treatment methods for DBD changed when the phaseprogresses. Anticholinergic drugs, such as tolterodine andsolifenacin, are the main treatment optionsfor DBD patients with OAB. However,many side effects, including drymouth, dry eyes, and memory loss, occurs after the treatment with anticholinergic drugs, thus renderingpoorlife quality for the patients. Inthe late phase, surgical intervention was the only therapeutic method for patients who did not benefit frompharmacological and behavioral treatments ([@B45]). However, pharmacological and surgical interventions were largely ineffective in clinics ([@B12]; [@B40]).Therefore, neweffective treatments for DBD are urgently needed. Inthe treatment of diabetic OAB,traditionalChinese medicine andnatural plant components have recently attracted increasing attentiondue to theirsafeness, few side effects, and excellent activity ([@B41]). SQW is a traditional Chinese herbal formula that was first recorded on *Fu Ren LiangFang* in theSouthernSong Dynasty (between 1127 and 1279 CE). This medicine is a mixture of three Chinesemedicines: *roots of Lindera aggregata (Sims) Kosterm. (Lauraceae),roots of Alpinia oxyphylla Miq., (Zingiberaceae), andrhizomes of Dioscorea oppositifolia L. (Dioscoreaceae)* at a 1:1:1 ratio ([@B14]). SQW has been usedto treatlower urinary tract symptoms, such as nocturia, urgency,and child bedwetting for hundreds of years ([@B4]). We have recently reported that SQW had therapeutic effectson the OAB of bladder outlet obstruction rat models by modulating the TRPV1 expression ([@B18]). In China,SQWis often used in the clinical treatment of diabetic OAB. However, its mechanism remains unclear, and its therapeutic effecthas not been investigated in animal studies. Therefore, we designed experiments toexplore theeffects and therapeutic mechanisms of SQW in diabetic OAB mouse model. Materials and Methods {#s1} ===================== Reagents and Materials ---------------------- Suo Quan Wan was purchased from Hunan Hansen Pharmaceutical Co., Ltd. (China), and the quality control was providedby the company based onChinese Pharmacopeia employing byhigh performance liquid chromatography (HPLC) technologyfromSQW samples ([@B9]). Three Chinese herbals wereground and mixed evenly at a 1:1:1 ratio and appropriate volumes of distilled waterwere used to make thesepowders to SQW compound. The doses were adopted according to the Experimental Methodology of Pharmacology, basedonclinical usage, the Bios method ([@B39]). SQW H was 2.208 g/kg,SQW M was 1.104 g/kg,and SQW L was 0.552 g/kg. The tolterodine dose for the positive groupwas 0.82 mg/kg. Streptozotocin was purchased from TOKU-ECo., Ltd. (Japan). HFD (45% fat) and control diet were purchased from Guangdong Medical Laboratory Animal Center (China). Tolterodine was purchased from Chengdu Dikang Pharmaceutical Co., Ltd. (China). Rochedynamic Bg meter waspurchasedfrom Hoffmann-La RocheInc. (Switzerland), and carbachol was obtained fromShandong Bausch & Lomb Freda Pharmaceutical Co., Ltd. (China). α,β-methylene ATP was purchased from Tocris Bio-Techne Ltd. (United Kingdom). FastQuant RT Kit (with gDNAse) and Talent qPCR PreMix(SYBRGreen) were purchased from TIANGEN Biotech (Beijing) Co., Ltd. (China). TRIzolreagent was purchased from Thermo Fisher Scientific(United States). RIPA lysis buffer andprotease inhibitor cocktail (100×) were obtained from CoWin Biosciences (China). All other reagents used wereof analytical grade.Preparation and HPLC Conditions of SQW -------------------------------------- Suo Quan Wan samples were weighted 0.3 g and extracted with 25 mLof methanol-hydrochloric acid solution using heating reflux method and then cool the solution.Finally, the solution was filtered through 0.45 μm nylon membranes before injection. According to theChinese pharmacopoeia 2015, the content of norisoboldine should be more than 0.4 mg/0.3 g, andthe contentof allantoin is more than 0.48mg/0.3 g. The HPLC conditions and gradient elutionwere shown as [Tables 1](#T1){ref-type="table"},[2](#T2){ref-type="table"}.###### Chromatographic condition for norisoboldine. Column C18 (25°C)------------ ----------------------------------------- ------- Solvent A Acetonitrile Solvent B 0.5% formic acid and 0.1% triethylamine Flow rate 1.0 mL/min Wavelength 280 nm Time (min) A (%)B (%) 0 10 90 1322 78 30 22 78 ###### Chromatographic condition forallantoin. Column C18(25°C) ------------ ------------------- Solvent A Methanol Solvent B H~2~OFlow rate1.0 mL/min Wavelength 191 nm Time (min) A (%) B (%) 08 92 1010 90 20 10 90 AnimalModel and Treatment-------------------------- All experimental protocols and animal procedures complied with the ethical principle guidelines of the National Research Council. A total of100 maleC57BL/6J mice (18--22 g) were purchased from Beijing Vital RiverLaboratory Animal Technology Co., Ltd. and housed inthe Experimental Animal Center of Guangzhou University of Chinese Medicine(No.S2017051, Guangzhou, China) under room temperature and exposed to a 12 h/12 hlight--dark cycle, with free access tofood and water. The animals were fed withnormal diet for 3days and then divided into two groups, namely, diabetic(*n* = 85) and control (*n* = 15) groups. Themice in the diabetic group were fed with HFD, whereas those in the controlgroup received normal diet. After 4-week feeding, the micein the diabetic group were injected with STZ at 100 mg/kg dissolved in citrate buffer for fourtimes (0.05 M, pH 4.3--4.5).The mice in the control group were treated with an equal volume of vehicle (0.05Mcitric acid | Introduction ============ Diabetes _mellitus_ is _a_ common endocrine _metabolic_ disease estimated to affect 629 million individuals in 2045 according to the International Diabetes Federation. T2DM is the extremely prevalent _form_ of diabetes _that_ accounts for 90% of diagnosed _cases_ and is _associated_ _with_ _insulin_ resistance and chronic hyperglycemia ([@B13]). Many _clinical_ studies reported a broad spectrum _of_ lower urinary tract symptoms _in_ _diabetic_ patients ([@B16]), accounting _for_ 90--95% of all diabetes cases ([@B17]). _DBD_ is a major _lower_ urinary tract _complication_ of diabetes and was first described by [@B26]. _Such_ complication is traditionally _described_ as _a_ _triad_ of increased capacity, decreased sensation, and _poor_ emptying ([@B11]) and has affected _over_ 50% of diabetic patients _([@B12];_ [@B28]). DBD development is divided into two _phases:_ the compensated phase, which occurs in the early phase and is _characterized_ by an OAB; _and_ the _decompensated_ phase, _which_ occurs _in_ the _late_ phase and is characterized by an atonic _bladder_ ([@B36]; [@B24]). The pathogenesis of DBD _is_ multifactorial _and_ accompanied by the structural and functional impairments _of_ _the_ bladder ([@B37]). The bladder structural _remodeling_ of DBD, such _as_ the increase _in_ bladder capacity, _total_ BWT, and _smooth_ muscle content, was _observed_ in STZ-induced diabetic mice. Such remodeling may be a physical alteration to increase the urine volume ([@B22]). _The_ _two_ major functions of _bladder_ are urine storage and _urine_ disposal, and the uncoordinated _contraction_ in the OAB of _a_ _diabetic_ greatly affects the urine storage ability of this organ _([@B10])._ Bladder contraction is mainly mediated by _purinergic_ and _cholinergic_ pathways _([@B23])._ In particular, ATP is the purinergic messenger released from varicosities or bulbous _nerve_ endings _of_ neurons, and the contractile responses mediated _by_ ATP play a key role _in_ DBD ([@B44]). Solute _carrier_ family 17 member _9_ (SLC17A9) is a _member_ _of_ the _solute-carrier_ protein family that plays an indispensable role _in_ the vesicular storage of ATP ([@B31]; [@B20]). _The_ _translocation_ of neurotransmitter-filled vesicles to the varicose terminal is the _first_ step in the release of vesicular neurotransmitters, followed by the _merger_ of _vesicles_ with the membrane of the varicose terminal and the precise and rapid release of _their_ contents into the synaptic cleft _([@B33])._ _In_ addition, the _motor_ _of_ vesicles for transportation to the varicose _membrane_ in the cells _is_ mainly provided by myosin motors, particularly myosin Va ([@B2]). Several studies found that the purinergic inhibitory neurotransmission _was_ impaired in myosin _Va-deficient_ mice _([@B6])._ Such finding suggested that myosin Va played an important role _in_ purinergic neurotransmission ([@B3]). Diabetic _bladder_ dysfunction, particularly OAB, is _not_ life threatening to _humans._ However, this dysfunction seriously affects _the_ quality of _the_ _life_ of patients _([@B24])._ _The_ treatment methods for DBD changed _when_ the phase progresses. _Anticholinergic_ drugs, such as tolterodine _and_ _solifenacin,_ are the main treatment options for _DBD_ _patients_ with OAB. However, many side effects, including dry mouth, dry _eyes,_ and memory loss, occurs after the treatment with anticholinergic drugs, _thus_ _rendering_ _poor_ life quality for the patients. In the _late_ phase, _surgical_ _intervention_ _was_ the only _therapeutic_ method _for_ patients who did _not_ benefit from pharmacological and behavioral treatments ([@B45]). However, pharmacological and _surgical_ interventions were largely ineffective in clinics ([@B12]; [@B40]). Therefore, new effective treatments for DBD are urgently _needed._ In the treatment of _diabetic_ OAB, traditional Chinese medicine and natural _plant_ _components_ have recently attracted increasing attention due to their safeness, _few_ side effects, and excellent activity ([@B41]). _SQW_ is a traditional Chinese _herbal_ _formula_ that was first recorded on *Fu _Ren_ Liang Fang* in the Southern Song Dynasty (between 1127 and 1279 CE). _This_ medicine _is_ a mixture of three Chinese medicines: *roots of Lindera aggregata _(Sims)_ Kosterm. _(Lauraceae),_ roots of _Alpinia_ oxyphylla Miq., _(Zingiberaceae),_ and rhizomes of Dioscorea oppositifolia L. (Dioscoreaceae)* _at_ a 1:1:1 ratio ([@B14]). _SQW_ has _been_ used _to_ treat _lower_ urinary tract symptoms, such as nocturia, _urgency,_ and _child_ _bedwetting_ for _hundreds_ of years ([@B4]). We have recently reported _that_ SQW _had_ therapeutic effects on the OAB of _bladder_ outlet obstruction rat models by modulating the TRPV1 expression _([@B18])._ In China, SQW is often used in _the_ clinical treatment of diabetic OAB. _However,_ its mechanism remains unclear, _and_ its therapeutic effect has not been investigated in _animal_ studies. _Therefore,_ we designed experiments to _explore_ the effects and therapeutic mechanisms _of_ SQW _in_ diabetic OAB mouse model. Materials _and_ Methods {#s1} ===================== Reagents _and_ _Materials_ ---------------------- Suo Quan Wan was purchased from Hunan _Hansen_ Pharmaceutical _Co.,_ Ltd. _(China),_ _and_ the _quality_ _control_ _was_ _provided_ by the _company_ _based_ on Chinese _Pharmacopeia_ employing by high performance _liquid_ _chromatography_ (HPLC) technology from SQW samples ([@B9]). Three Chinese herbals were ground and mixed evenly _at_ a 1:1:1 ratio and _appropriate_ volumes of distilled water _were_ used to make these powders to SQW compound. The doses were adopted according _to_ the _Experimental_ Methodology of Pharmacology, _based_ on _clinical_ usage, the Bios _method_ ([@B39]). SQW H was _2.208_ g/kg, SQW M was 1.104 g/kg, and SQW _L_ _was_ _0.552_ g/kg. The tolterodine _dose_ for the positive group _was_ 0.82 _mg/kg._ Streptozotocin _was_ _purchased_ from TOKU-E Co., Ltd. (Japan). _HFD_ (45% fat) and control diet were purchased from Guangdong Medical Laboratory Animal Center (China). _Tolterodine_ _was_ purchased from _Chengdu_ Dikang Pharmaceutical _Co.,_ Ltd. _(China)._ Roche dynamic Bg meter was purchased from Hoffmann-La Roche _Inc._ _(Switzerland),_ and carbachol was _obtained_ from Shandong Bausch & _Lomb_ Freda _Pharmaceutical_ Co., Ltd. (China). _α,β-methylene_ ATP _was_ purchased from Tocris _Bio-Techne_ _Ltd._ _(United_ _Kingdom)._ FastQuant _RT_ _Kit_ (with gDNAse) and Talent qPCR PreMix (SYBR Green) were _purchased_ from TIANGEN _Biotech_ (Beijing) Co., Ltd. (China). _TRIzol_ _reagent_ was purchased from Thermo Fisher Scientific _(United_ States). _RIPA_ lysis _buffer_ and protease inhibitor cocktail (100×) were obtained from CoWin _Biosciences_ (China). All other reagents used were of analytical grade. Preparation and HPLC Conditions of SQW -------------------------------------- _Suo_ _Quan_ Wan samples were weighted 0.3 g and extracted _with_ 25 mL of methanol-hydrochloric _acid_ solution using heating reflux method and then cool the solution. Finally, the solution was filtered through 0.45 μm nylon membranes before injection. According to the _Chinese_ pharmacopoeia 2015, _the_ content of norisoboldine should be more than 0.4 mg/0.3 g, and the content of allantoin is _more_ than _0.48_ mg/0.3 g. The HPLC conditions and gradient elution were _shown_ as [Tables 1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}. ###### Chromatographic _condition_ for norisoboldine. Column C18 (25°C) ------------ ----------------------------------------- ------- Solvent A _Acetonitrile_ Solvent B _0.5%_ _formic_ acid and _0.1%_ triethylamine Flow rate 1.0 mL/min Wavelength _280_ nm Time (min) A (%) B _(%)_ 0 10 90 13 22 _78_ 30 _22_ 78 ###### Chromatographic condition for _allantoin._ Column C18 (25°C) ------------ ------------ ------- Solvent _A_ Methanol _Solvent_ _B_ _H~2~O_ Flow rate 1.0 mL/min _Wavelength_ 191 nm Time (min) _A_ (%) B _(%)_ _0_ 8 _92_ _10_ 10 90 20 10 _90_ Animal Model and Treatment -------------------------- All _experimental_ protocols and animal procedures complied with the ethical principle guidelines of the National Research Council. A _total_ of 100 male _C57BL/6J_ mice _(18--22_ g) were purchased from Beijing Vital _River_ _Laboratory_ Animal _Technology_ _Co.,_ Ltd. and housed in the _Experimental_ Animal Center of _Guangzhou_ University of Chinese Medicine _(No._ S2017051, Guangzhou, China) _under_ room _temperature_ and _exposed_ _to_ a _12_ h/12 _h_ light--dark cycle, with free access to food and _water._ The animals _were_ fed _with_ normal _diet_ for _3_ _days_ _and_ then divided into two groups, namely, diabetic (*n* = 85) and control (*n* = 15) groups. The mice in the diabetic group were _fed_ with HFD, _whereas_ _those_ _in_ the _control_ group received _normal_ diet. After 4-week feeding, the mice in the diabetic _group_ were injected with _STZ_ at 100 mg/kg dissolved in citrate buffer for four times (0.05 M, pH _4.3--4.5)._ _The_ mice in the control group were treated with an equal _volume_ of vehicle _(0.05_ M citric acid |
Introduction {#sec1}
============
In contrast to bulk silver, nanometric silver materials exhibit many extraordinary properties such as a high surface-to-volume ratio,^[@ref1],[@ref2]^ quantum tunneling effects,^[@ref3],[@ref4]^ an abundance of free electrons,^[@ref5]^ surface plasmon resonance,^[@ref2],[@ref6]−[@ref8]^ and antibacterial behaviors.^[@ref9],[@ref10]^ Because of these unique properties, noble silver nanomaterials are widely applied in diverse areas, including thermotherapy,^[@ref5],[@ref11]^ medicine,^[@ref2],[@ref5],[@ref12]^ sensors,^[@ref13]−[@ref16]^ surface-enhanced spectroscopy,^[@ref6],[@ref17]−[@ref20]^ biology,^[@ref2]^ catalysis,^[@ref21]−[@ref28]^ and electronics.^[@ref29]−[@ref31]^ Among these many applications, the catalysis of the reduction of nitroarenes to aromatic amines is increasingly attracting attention because of pharmaceutical needs and the importance of this industry.^[@ref25],[@ref26],[@ref32]−[@ref38]^ Various strategies have been proposed to reduce nitroarenes more efficiently and more rapidly using silver nanomaterials. These strategies include depositing silver nanoparticles (AgNPs) on supports used as heterogeneous catalysts,^[@ref24],[@ref26],[@ref39]−[@ref42]^ combining AgNPs with reduced graphene oxide or graphene oxide as catalysts,^[@ref43]−[@ref47]^ and using silver nanocolloids as a quasi-homogeneous nanocatalyst.^[@ref25],[@ref33],[@ref48]−[@ref54]^ All of the aforementioned catalytic approaches are efficient and selective. However, the reaction rate for heterogeneous catalysis is rather low and quasi-homogeneous catalysis suffers from possible aggregation of the nanocatalyst. Furthermore, the procedures for preparing such nanocatalysts are somewhat complex and time-consuming.
Applications of magnetic nanoparticles have also been extensively investigated in recent years because they feature many notable characteristics, such as a high surface-to-volume ratio, easy attraction and redispersion, and paramagnetism. Because of these crucial properties and advantages, the combination of AgNPs and magnetic nanoparticles has become one of the most favorable approaches for the catalytic reduction of nitroarenes.^[@ref55]−[@ref60]^ In this study, a simple but facile method was applied in a single step to prepare silver-doped magnetic nanoparticles (AgMNPs) for the catalytic reduction of nitroarenes through spontaneous oxidation--reduction and coprecipitation. When mixing Fe^2+^ with Ag^+^, a spontaneous reaction is caused by the difference in standard reduction potential between the ionic species. When Ag^+^ is reduced to Ag^0^, an equivalent number of moles of Fe^2+^ ions are simultaneously oxidized to Fe^3+^. After the addition of precipitation agents, AgNPs were coprecipitated with iron oxide magnetic nanoparticles, which led to the formation of AgMNPs. The proposed preparation can be achieved in a single step, and the prepared AgMNPs can subsequently be utilized as nanocatalysts for the reduction of *o*-nitroaniline (*o*-NA). The parameters (pH, temperature, and amount of nanocatalyst) that affect the morphology and composition of the prepared AgMNPs and efficiencies of the catalytic reduction were systematically studied to gain a greater understanding of the characteristics of the AgMNPs prepared using the method proposed in this study. Additionally, the catalytic activity of the AgMNPs prepared for the reduction of other nitroarenes and their recyclability were investigated to fully evaluate their potential for practical applications.
Results and Discussion {#sec2}
======================
Effect of Oxidation--Reduction Time on AgMNP Preparation {#sec2.1}
--------------------------------------------------------
[Figure S1](http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b01987/suppl_file/ao7b01987_si_001.pdf) shows the typical measured hysteresis loops of the prepared AgMNPs, which confirmed that the prepared AgMNPs were paramagnetic and usable for further applications. [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"} depicts the transmission electron microscopy (TEM) images of AgMNPs obtained using various reaction times. From the images, dark-sphere-like Ag nanoparticles were mixed with light-colored Fe~3~O~4~ NPs because Ag has a higher electron density that allows fewer electrons to transmit.^[@ref61],[@ref62]^ The AgNPs formed after a 10 min reaction time were larger than those formed after a 2 min reaction time. Notably, the size of the Fe~3~O~4~ NPs was mostly unaffected by the reaction time.
![TEM images of prepared AgMNPs with oxidation--reduction times of (a) 2 min, (b) 8 min, and (c)--(f) 10 min, where the \[Fe^2+^\]~0~ to \[Ag^+^\]~0~ ratios are (a)--(c) 3:1, (d) 2:1, (e) 4:1, and (f) 6:1. \[Fe^2+^\]~0~ are all 12 mM, and the magnifications of the images are all 100 000×. The yellow arrows indicate the examples of AgNPs for each sample.](ao-2017-019876_0008){#fig1}
[Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}a shows the evolution of the UV--vis spectra for the reduction of *o*-NA catalyzed by AgMNPs over time. The absorbance peak at 412 nm, which corresponds to the characteristic *o*-NA peak,^[@ref63]^ decreased as the reaction proceeded. The variations of the spectra indicated that *o*-NA was reduced to 1,2-phenylenediamine (1,2-PPD).^[@ref41],[@ref64]^ The relative concentration (*C*~t~/*C*~0~) of *o*-NA was obtained by dividing the absorbance recorded at 412 nm at the specified time (*C*~t~) by the absorbance at 412 nm before the addition of AgMNPs (*C*~0~). The results in [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}b were plotted using various AgMNPs prepared using various reduction durations. In [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}b, the catalytic efficiency of the AgMNPs shows no significant difference between when the reaction time was 4--10 min and when more than 95% of *o*-NA was reduced within 240 s. This finding suggests remarkable catalytic activity. By contrast, achieving the same conversion percentage required more than 500 s when the reaction time to prepare the AgMNPs was 2 or 12 min. Because the catalytic efficiency of nanocatalysts depends on their size and the amount of catalyst loaded,^[@ref26]^ we concluded that the reaction time to achieve the optimal morphology and catalyst loading was 10 min. For comparison, the results of an experiment conducted in parallel, where AgMNPs were replaced with Fe~3~O~4~ NPs, are also plotted in [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}b, showing that the reduction of *o*-NA can proceed only when AgNPs are doped. In the absence of AgNPs, the reduction of *o*-NA is suspended.
{ref-type="fig"}](#fig1){ref-type="fig"}c. (b) *C*~t~/*C*~0~ of 1 mM *o*-NA (412 nm) versus the catalytic reduction time in the presence of 30 mM NaBH~4~ and AgMNPs prepared with (■) 2 min, (●) 4 min, (▲) 6 min, (▼) 8 min, (◆) 10 min, and (★) 12 min of oxidation--reduction time during the preparation, where the other conditions are the same as described in (a). Parallel experiment (□) uses 20 mg of Fe~3~O~4~ NPs as nanocatalysts, where the other conditions are the same as described as (a).](ao-2017-019876_0001){#fig2}
Effect of Fe^2+^/Ag^+^ on AgMNP Preparation {#sec2.2}
-------------------------------------------
As described in the previous section, catalytic efficiency is related to the size and amount of doped AgNPs. In addition, we expected the morphology and amount of doped AgNPs to be affected by the ratio of initial concentration of Fe^2+^ to Ag^+^ because AgNO~3~ acts as the oxidation agent in the formation of AgMNPs. [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"} shows the TEM images of the AgMNPs with different ratios of \[Fe^2+^\]~0~ to \[Ag^+^\]~0~. The images reveal that the morphologies of the AgNPs are similar. [Figure S2](http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b01987/suppl_file/ao7b01987_si_001.pdf) shows the typical X-ray diffraction (XRD) spectra of the prepared AgMNPs and | introduction { # sec1 } = = = = = = = = = = = = in contrast to bulk silver, nanometric silver materials exhibit many extraordinary properties such as a high surface - to - volume ratio, ^ [ @ ref1 ], [ @ ref2 ] ^ quantum tunneling effects, ^ [ @ 28 ], [ @ ref4 ] ^ an abundance of free electrons, ^ [ @ ref5 ] ^ surface plasmon resonance, ^ [ @ ref2 ], [ @ ref6 ] − [ @ 3a ] ^ and antibacterial behaviors. ^ [ @ ref9 ], [ @ ref10 ] ^ because of these unique abilities, many silver nanomaterials are widely applied in diverse areas, including thermotherapy, ^ [ @ r ], [ @ ref11 ] ^ medicine, ^ [ @ ref2 ], [ @ ref5 ], [ @ ref12 ] ^ sensors, ^ [ @ ref13 ] − [ @ ref16 ] ^ surface - enhanced spectroscopy, ^ [ @ ref6 ], [ @ ref17 ] − [ @ ref20 ] ^ biology, ^ [ @ f ] ^ catalysis, ^ [ @ ref21 ] − [ @ ref28 ] ^ and electronics. ^ [ @ ref29 ] − [ @ ref31 ] ^ among these many applications, the catalysis of the reduction of nitroarenes to aromatic amines is increasingly attracting attention because of pharmaceutical needs and the importance of this industry. ^ [ @ ref25 ], [ @ ref26 ], [ @ ref32 ] − [ @ 28 ] ^ various strategies have been proposed to reduce nitroarenes more efficiently and more rapidly using silver crystals. these strategies include depositing silver nanoparticles ( agnps ) on supports used as heterogeneous catalysts, ^ [ @ ref24 ], [ @ ref26 ], [ @ ref39 ] − [ @ ref42 ] ^ combining agnps with reduced graphene oxide or graphene oxide reduction catalysts, ^ [ @ ref43 ] − [ @ ref47 ] ^ and using silver nanocolloids as the quasi - homogeneous nanocatalyst. ^ [ @ ref25 ], [ @ ref33 ], [ @ ref48 ] − [ @ ref54 ] ^ all of the aforementioned catalytic approaches are efficient and selective. however, the reaction rate for heterogeneous cataly ##sis is rather low and quasi - homogeneous catalysis suffers from possible aggregation of the nanocatalyst. furthermore, the procedures for preparing such nanocatalysts are somewhat complex and time - consuming. applications of magnetic nanoparticles have also been extensively investigated in recent years because they feature many notable characteristics, such as a high surface - to - volume ratio, easy attraction and redispersion, and paramagnetism. because of these crucial properties and advantages, the combination of agnps and magnetic nanoparticles has become one of the most favorable approaches for the catalytic reduction of nitroarenes. ^ [ @ ref55 ] − [ @ ref60 ] ^ in this study, a simple but facile method was applied in a single step to prepare silver - doped magnetic nanoparticles ( agmnps ) for the catalytic reduction of nitroarenes through spontaneous oxidation - - reduction and coprecipitation. when mixing fe ^ 2 + ^ with ag ^ + ^, a spontaneous reaction is caused by the difference in standard reduction potential between the ionic species. when ag ^ + ^ is reduced to ag ^ 0 ^, an equivalent number of moles of fe ^ 2 + ^ ions are simultaneously oxidized to fe ^ 3 + ^. after the addition of precipitation agents, agnps were coprecipitated with iron oxide magnetic nanoparticles, which led to the formation of agmnps. the proposed preparation can be achieved in a single step, and the prepared agmnps can subsequently be utilized as nanocatalysts for the reduction of * o * - nitroaniline ( * o * - na ). the parameters ( ph, temperature, and amount of nanocatalyst ) that affect the morphology and composition of the prepared agmnps and efficiencies of the catalytic reduction were systematically studied to gain a greater understanding of the characteristics of the agmnps prepared using the method proposed in this study. additionally, the catalytic activity of the agmnps prepared for the reduction of other nitroarenes and their recyclability were investigated to fully evaluate their potential for practical applications. results and discussion { # sec2 } = = = = = = = = = = = = = = = = = = = = = = effect of oxidation - - reduction time on agmnp preparation { # sec2. 1 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - [ figure s1 ] ( http : / / pubs. acs. org / doi / suppl / 10. 1021 / acsomega. 7b01987 / suppl _ file / ao7b01987 _ si _ 001. pdf ) shows the typical measured hysteresis loops of the prepared agmnps, which confirmed that the prepared agmnps were paramagnetic and usable for further applications. [ figure [ 1 ] ( # fig1 ) { ref - type = " fig " } ] ( # fig1 ) { ref - type = " fig " } depicts the transmission electron microscopy ( tem ) images of agmnps obtained using various reaction times. from the images, dark - sphere - like ag nanoparticles were mixed with light - colored fe ~ 3 ~ o ~ 4 ~ nps because ag has a higher electron density that allows fewer electrons to transmit. ^ [ @ ref61 ], [ @ ref62 ] ^ the agnps formed after a 10 min reaction time were larger than those formed after a 2 min reaction time. notably, the size of the fe ~ 3 ~ o ~ 4 ~ nps was mostly unaffected by the reaction time.! [ tem images of prepared agmnps with oxidation - - reduction times of ( a ) 2 min, ( b ) 8 min, and ( c ) - - ( f ) 10 min, where the \ [ fe ^ 2 + ^ \ ] ~ 0 ~ to \ [ ag ^ + ^ \ ] ~ 0 ~ ratios are ( a ) - - ( c ) 3 : 1, ( d ) 2 : 1, ( e ) 4 : 1, and ( f ) 6 : 1. \ [ fe ^ 2 + ^ \ ] ~ 0 ~ are all 12 mm, and the magnifications of the images are all 100 000×. the yellow arrows indicate the examples of agnps for each sample. ] ( ao - 2017 - 019876 _ 0008 ) { # fig1 } [ figure [ 2 ] ( # fig2 ) { ref - type = " fig " } ] ( # fig2 ) { ref - type = " fig " } a shows the evolution of the uv - - vis spectra for the reduction of * o * - na catalyzed by agmnps over time. the absorbance peak at 412 nm, which corresponds to the characteristic * o * - na peak, ^ [ @ ref63 ] ^ decreased as the reaction proceeded. the variations of the spectra indicated that * o * - na was reduced to 1, 2 - phenylenediamine ( 1, 2 - ppd ). ^ [ @ ref41 ], [ @ ref64 ] ^ the relative concentration ( * c * ~ t ~ / * c * ~ 0 ~ ) of * o * - na was obtained by dividing the absorbance recorded at 412 nm at the specified time ( * c * ~ t ~ ) by the absorbance at 412 nm before the addition of agmnps ( * c * ~ 0 ~ ). the results in [ figure [ 2 ] ( # fig2 ) { ref - type = " fig " } ] ( # fig2 ) { ref - type = " fig " } b were plotted using various agmnps prepared using various reduction durations. in [ figure [ 2 ] ( # fig2 ) { ref - type = " fig " } ] ( # fig2 ) { ref - type = " fig " } b, the catalytic efficiency of the agmnps shows no significant difference between when the reaction time was 4 - - 10 min and when more than 95 % of * o * - na was reduced within 240 s. this finding suggests remarkable catalytic activity. by contrast, achieving the same conversion percentage required more than 500 s when the reaction time to prepare the agmnps was 2 or 12 min. because the catalytic efficiency of nanocatalysts depends on their size and the amount of catalyst loaded, ^ [ @ ref26 ] ^ we concluded that the reaction time to achieve the optimal morphology and catalyst loading was 10 min. for comparison, the results of an experiment conducted in parallel, where agmnps were replaced with fe ~ 3 ~ o ~ 4 ~ nps, are also plotted in [ figure [ 2 ] ( # fig2 ) { ref - type = " fig " } ] ( # fig2 ) { ref - type = " fig " } b, showing that the reduction of * o * - na can proceed only when agnps are doped. in the absence of agnps, the reduction of * o * - na is suspended.! [ ( a ) uv - - vis spectra of 1 mm * o * - na reduced by 30 mm nabh ~ 4 ~ at room temperature ( rt ) in the presence of 20 mg of agmnps with increasing time, where the ph was 9. 8. the agmnps were prepared under the condition described in [ figure [ 1 ] ( # fig1 ) { ref - type = " fig " } ] ( # fig1 ) { ref - type = " fig " } c. ( b ) * c * ~ t ~ / * c * ~ 0 ~ of 1 mm * o * - na ( 412 nm ) versus the catalytic reduction time in the presence of 30 mm nabh ~ 4 ~ and agmnps prepared with ( ■ ) 2 min, ( ● ) 4 min, ( [UNK] ) 6 min, ( [UNK] ) 8 min, ( [UNK] ) 10 min, and ( ★ ) 12 min of oxidation - - reduction time during the preparation, where the other conditions are the same as described in ( a ). parallel experiment ( [UNK] ) uses 20 mg of fe ~ 3 ~ o ~ 4 ~ nps as nanocatalysts, where the other conditions are the same as described as ( a ). ] ( ao - 2017 - 019876 _ 0001 ) { # fig2 } effect of fe ^ 2 + ^ / ag ^ + ^ on agmnp preparation { # sec2. 2 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - as described in the previous section, catalytic efficiency is related to the size and amount of doped agnps. in addition, we expected the morphology and amount of doped agnps to be affected by the ratio of initial concentration of fe ^ 2 + ^ to ag ^ + ^ because agno ~ 3 ~ acts as the oxidation agent in the formation of agmnps. [ figure [ 1 ] ( # fig1 ) { ref - type = " fig " } ] ( # fig1 ) { ref - type = " fig " } shows the tem images of the agmnps with different ratios of \ [ fe ^ 2 + ^ \ ] ~ 0 ~ to \ [ ag ^ + ^ \ ] ~ 0 ~. the images reveal that the morphologies of the agnps are similar. [ figure s2 ] ( http : / / pubs. acs. org / doi / suppl / 10. 1021 / acsomega. 7b01987 / suppl _ file / ao7b01987 _ si _ 001. pdf ) shows the typical x - ray diffraction ( xrd ) spectra of the prepared agmnps and | Introduction {# sec1} = = = = = = = = = = = = In contrast to bulk silver, nanometric silver materials exhibit many extraordinary properties such as a high surface - to - volume ratio, ^ [@ ref1 ], [@ ref2] ^ quantum tunneling effects, ^ [@ ref3 ], [@ ref4] ^ an abundance of free electrons, ^ [@ ref5] ^ surface plasmon resonance, ^ [@ ref2 ], [@ ref6] − [@ ref8] ^ and antibacterial behaviors. ^ [@ ref9 ], [@ ref10] ^ Because of these ^niqu2 properties, noble silver nanomaterials are widely applied in diverse areas, including thermotherapy, ^ [@ ref5 ], [@ ref11] ^ medicine, ^ [@ ref2 ], [@ ref5 ], [@ ref12] ^ sensors, ^ [@ ref13] − [@ ref16] ^ surface - enhanced spectroscopy, ^ [@ ref6 ], [@ ref17] − [@ ref20] ^ biology, ^ [@ ref2] ^ catalysis, ^ [@ ref21] − [@ ref28] ^ and electronics. ^ [@ ref29] − [@ ref31] ^ Among these many applications, the catalysis of the reduction of nitroarenes to aromatic amines is increasingly attracting attention because of pharmaceutical needs and the importance of this industry. ^ [@ ref25 ], [@ ref26 ], [@ ref32] − [@ ref38] ^ Various strategies have been proposed to reduce nitroarenes more efficiently and more rapidly using silver nanomaterials. These strategies include depositing silver nanoparticles (AgNPs) on supports used as heterogeneous catalysts, ^ [@ ref24 ], [@ ref26 ], [@ ref39] − [@ ref42] ^ combining AgNPs with reduced graphene oxide or graphene oxide as catalysts, ^ [@ ref43] − [@ ref47] ^ and using silver nanocolloids as a quasi - homogeneous nanocatalyst. ^ [@ ref25 ], [@ ref33 ], [@ ref48] − [@ ref54] ^ All of the aforementioned catalytic approaches are efficient and selective. However, the reaction rate for heterogeneous catalysis is rather low and quasi - homogeneous catalysis suffers from possible aggregation of the nanocatalyst. Furthermore, the procedures for preparing such nanocatalysts are somewhat complex and time - consuming. SpplicationD of magnetic nanoparticles have also been extensively investigated in recent years because they feature many notable characteristics, such as a high surface - to - volume ratio, easy attraction and redispersion, and paramagnetism. Because of these crucial properties and advSmtages, the combination of AgNPs and magnetic nanoparticles has become one of the most favorable approaches for the catalytic reduction of nitroarenes. ^ [@ ref55] − [@ ref60] ^ In this study, a simple but facile method was applied in a single step to prepare silver - doped magnetic nanoparticles (AgMM(s) for the catalytic reduction of nitroarenes through spontaneous oxidation - - reduction and coprecipitation. When mixing Fe ^ 2 + ^ with Ag ^ + ^, a spontaneous reaction is caused by the difference in standard reduction potential between the ionic species. When Ag ^ + ^ is reduced to Ag ^ 0 ^, an equivalent number of moles of Fe ^ 2 + ^ ions are simultaneously oxidized to Fe ^ 3 + ^. After the addition of precipitation agents, AgNPs were coprecipitated with iron oxide magnetic nanoparticles, which led to the formation of AgMNPs. The proposed preparation can be achieved in a single step, and the prepared AgMNPs can subsequently be utilized as nanocatalysts for the reduction of * o * - nitroaniline (* o * - NA ). The parameters (pH, temperature, and amount of nanocatalyst) that affect the morphology and composition of the prepared AgMNPs and efficiencies of the catalytic reduction were systematically studied to gain a greater understanding of the characteristics of the AgMNPs prepared using the method proposed in this study. Additionally, the catalytic activity of the AgMNPs prepared for the reduction of other nitroarenes and their recyclability were investigated to fully evaluate their piteMtial for practical applications. Results and Discussion {# sec2} = = = = = = = = = = = = = = = = = = = = = = Effect of Oxidation - - Rwduct(on Time on AgMNP Preparation {# sec2. 1} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - [Figure S1] (http: / / pubs. acs. org / doi / suppl / 10. 1021 / acsomega. 7b01987 / suppl_file / ao7b01987_si_001. pdf) shows the typical measured hysteresis loops of the prepared AgMNPs, which confirmed that the prepared AgMNPs were paramagnetic and usable for further applications. [Figure [1] (# fig1) {ref - type = " fig "} ] (# fig1) {ref - type = " fig "} depicts the transmission electron microscopy (TEM) images of AgMNPs obtained using various reaction times. From the images, dark - sphere - like Ag nanoparticles were mixed with light - colored Fe ~ 3 ~ O ~ 4 ~ NPs because Ag has a higher electron density that allows fewer electrons to transmit. ^ [@ ref61 ], [@ ref62] ^ The AgNPs formed after a 10 min reaction time were lQrrer than those formed after a 2 min reaction time. Notably, the size of the Fe ~ 3 ~ O ~ 4 ~ NPs was mostly unaffected by the reaction time. ! [TEM images of prepared AgMNPs wirn oxidation - - reduction times of (a) 2 min, (b) 8 min, and (c) - - (f) 10 min, wgerr the \ [Fe ^ 2 + ^ \] ~ 0 ~ to \ [Ag ^ + ^ \] ~ 0 ~ ratios are (a) - - (c) 3: 1, (d) 2: 1, (e) 4: 1, and (f) 6: 1. \ [Fe ^ 2 + ^ \] ~ 0 ~ are all 12 mM, and the magnifications of the images are all 100 000 ×. The yellow arrows indicate the examples of AgNPs for each sample.] (ao - 2017 - 019876_0008) {# fig1} [Figure [2] (# fig2) {ref - type = " fig "} ] (# fig2) {ref - type = " fig "} a shows the evolution of the UV - - vis spectra for the reduction of * o * - NA catalyzed by AgMNPs over time. The absorbance peak at 412 nm, which corresponds to the characteristic * o * - NA peak, ^ [@ ref63] ^ decreased as the reaction proceeded. The variations of the spectra indicated that * o * - NA was reduced to 1, 2 - phenylenediamine (1, 2 - PPD ). ^ [@ ref41 ], [@ ref64] ^ The relative concentration (* C * ~ t ~ / * C * ~ 0 ~) of * o * - NA was obtained by dividing the absorbance recorded at 412 nm at the specified time (* C * ~ t ~) by the absorbance at 412 nm before the addition of AgMNPs (* C * ~ 0 ~ ). The results in [Figure [2] (# fig2) {ref - type = " fig "} ] (# fig2) {ref - type = " fig "} b were plotted using various AgMNPs prepared using various reduction durations. In [Figure [2] (# fig2) {ref - type = " fig "} ] (# fig2) {ref - type = " fig "} b, the catalytic efficiency of the AgMNPs shows no significant difference between when the reaction time was 4 - - 10 min and when more than 95% of * o * - NA was reduced within 240 s. This finding suggests remarkable catalytic activity. By contrast, achieving the same conversion percentage required more than 500 s when the reaction time to prepare the AgMNPs was 2 or 12 min. Because the catalytic efficiency of nanocatalysts depends on their size and the amount of catalyst loaded, ^ [@ ref26] ^ we concluded that the reaction time to achieve the optimal morphology and catalyst loading was 10 min. For comparison, the results of an experiment conducted in parallel, where AgMNPs were replaced with Fe ~ 3 ~ O ~ 4 ~ NPs, are also plotted in [Figure [2] (# fig2) {ref - type = " fig "} ] (# fig2) {ref - type = " fig "} b, showing that the reduction of * o * - NA can proceed only when AgNPs are doped. In the absence of AgNPs, the reduction of * o * - NA is suspended. ! [( a) UV - - vis spectra of 1 mM * o * - NA reduced by 30 mM NaBH ~ 4 ~ at room temperature (RT) in the presence of 20 mg of AgMNPs with increasing time, where the pH was 9. 8. The AgMNPs were prepared under the condition described in [Figure [1] (# fig1) {ref - 5jpe = " fig "} ] (# fig1) {ref - type = " fig "} c. (b) * C * ~ t ~ / * C * ~ 0 ~ of 1 mM * o * - NA (412 nm) versus the catalytic reduction time in the presence of 30 mM NaBH ~ 4 ~ and AgMNPs prepared with (■) 2 min, (●) 4 min, (▲) 6 min, (▼) 8 min, (◆) 10 min, and (★) 12 min of oxidation - - reduction time during the preparation, where the other conditions are the same as described in (a ). Parallel experiment (□) uses 20 mg of Fe ~ 3 ~ O ~ 4 ~ NPs as nanocatalysts, where the other conditions are the same as described as (a ).] (ao - 2017 - 019876_0001) {# fig2} Effect of Fe ^ 2 + ^ / Ag ^ + ^ on AgMNP Preparation {# sec2. 2} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - As described in the previous section, catalytic efficiency is related to the size and amount of doped AgNPs. In addition, we expected the morphology and amount of doped AgNPs to be affected by the ratio of initial concentration of Fe ^ 2 + ^ to Ag ^ + ^ because AgNO ~ 3 ~ acts as the oxidation agent in the formation of AgMNPs. [Figure [1] (# fig1) {ref - type = " fig "} ] (# fig1) {ref - type = " fig "} shows the TEM images of the AgMNPs with different ratios of \ [Fe ^ 2 + ^ \] ~ 0 ~ to \ [Ag ^ + ^ \] ~ 0 ~. The images reveal that the morphologies of the AgNPs are similar. [Figure S2] (http: / / pubs. acs. org / doi / suppl / 10. 1021 / acsomega. 7b01987 / suppl_file / ao7b01987_si_001. pdf) shows the typical X - ray diffraction (XRD) spectra of the prepared AgMNPs and | Introduction {#sec1} ============ In contrast to bulk silver, nanometric silver materials exhibit many extraordinary properties such as high surface-to-volume ratio,^[@ref1],[@ref2]^ quantum an abundance of free electrons,^[@ref5]^ surface plasmon resonance,^[@ref2],[@ref6]−[@ref8]^ and antibacterial behaviors.^[@ref9],[@ref10]^ Because of these unique properties, noble silver nanomaterials are widely applied in diverse areas, including thermotherapy,^[@ref5],[@ref11]^ medicine,^[@ref2],[@ref5],[@ref12]^ sensors,^[@ref13]−[@ref16]^ surface-enhanced spectroscopy,^[@ref6],[@ref17]−[@ref20]^ biology,^[@ref2]^ catalysis,^[@ref21]−[@ref28]^ and electronics.^[@ref29]−[@ref31]^ Among these many the catalysis of the of nitroarenes to aromatic amines is increasingly attracting attention because of pharmaceutical needs and the importance of this Various strategies have been proposed to reduce nitroarenes more efficiently and more rapidly using silver nanomaterials. These include depositing silver nanoparticles (AgNPs) on supports used as heterogeneous catalysts,^[@ref24],[@ref26],[@ref39]−[@ref42]^ combining AgNPs with reduced graphene oxide or graphene oxide as catalysts,^[@ref43]−[@ref47]^ and using nanocolloids as a quasi-homogeneous nanocatalyst.^[@ref25],[@ref33],[@ref48]−[@ref54]^ All of the aforementioned approaches are efficient and selective. However, the rate for heterogeneous catalysis is rather low and quasi-homogeneous catalysis suffers from possible aggregation nanocatalyst. Furthermore, the procedures for preparing such are somewhat complex and time-consuming. Applications magnetic nanoparticles have also been extensively investigated in recent years they feature many notable characteristics, such as high surface-to-volume ratio, easy and redispersion, and Because these crucial properties and advantages, the combination of AgNPs and magnetic nanoparticles has become one of the most favorable approaches for the catalytic reduction of nitroarenes.^[@ref55]−[@ref60]^ In study, a but facile method was applied in a single step to prepare silver-doped magnetic nanoparticles (AgMNPs) for the catalytic reduction nitroarenes through spontaneous and When Fe^2+^ with Ag^+^, a spontaneous reaction is caused by the difference standard reduction potential between the ionic species. When Ag^+^ is reduced to Ag^0^, an equivalent number of moles of Fe^2+^ ions are simultaneously oxidized to Fe^3+^. After addition of precipitation agents, AgNPs were coprecipitated iron oxide magnetic nanoparticles, which to formation of AgMNPs. The preparation be achieved in a single step, and prepared AgMNPs can subsequently be utilized as nanocatalysts for the reduction of *o*-nitroaniline (*o*-NA). parameters (pH, temperature, and amount of nanocatalyst) that affect the morphology and composition of the prepared AgMNPs and of the catalytic reduction were studied to gain a greater understanding of the characteristics of the AgMNPs prepared using the method proposed in this study. Additionally, the catalytic activity of the AgMNPs for the reduction of other their recyclability investigated to fully evaluate their potential practical Results and Discussion ====================== Effect of Oxidation--Reduction Time on AgMNP Preparation {#sec2.1} -------------------------------------------------------- [Figure S1](http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b01987/suppl_file/ao7b01987_si_001.pdf) shows the measured hysteresis loops of the prepared AgMNPs, which confirmed that the prepared paramagnetic and usable for further applications. [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"} depicts transmission electron microscopy (TEM) images of AgMNPs obtained using various reaction times. From the images, dark-sphere-like Ag nanoparticles were mixed with light-colored Fe~3~O~4~ NPs because Ag has a higher electron density that allows electrons to transmit.^[@ref61],[@ref62]^ The AgNPs formed after a 10 min reaction time were larger than those formed after a 2 min time. Notably, the size of the Fe~3~O~4~ NPs was mostly by the reaction time. ![TEM images of prepared AgMNPs with oxidation--reduction times of (a) min, (b) 8 min, and (c)--(f) 10 min, where the \[Fe^2+^\]~0~ to \[Ag^+^\]~0~ ratios are (a)--(c) 3:1, 2:1, and (f) 6:1. \[Fe^2+^\]~0~ are all 12 mM, and the magnifications of the images all 100 000×. The yellow indicate the examples of AgNPs for each sample.](ao-2017-019876_0008){#fig1} [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}a shows the evolution of the UV--vis spectra for the reduction of *o*-NA catalyzed by AgMNPs over time. The absorbance peak at 412 nm, which to the characteristic *o*-NA peak,^[@ref63]^ decreased as the reaction proceeded. The variations of the spectra indicated *o*-NA was reduced to 1,2-phenylenediamine (1,2-PPD).^[@ref41],[@ref64]^ relative concentration of was obtained by dividing the absorbance recorded at 412 nm at the specified time (*C*~t~) by absorbance at 412 nm before addition of AgMNPs (*C*~0~). The results in [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}b were plotted using various AgMNPs prepared using various reduction durations. In [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}b, the catalytic efficiency of the AgMNPs shows no when the reaction time was 4--10 min and than 95% of *o*-NA was reduced 240 s. This finding remarkable activity. By contrast, achieving the same conversion percentage required more than 500 s when the reaction time to prepare the was 2 12 min. Because the efficiency of on their size and the of catalyst loaded,^[@ref26]^ we concluded that the reaction time to achieve the optimal morphology and catalyst loading was 10 min. For comparison, the results of an experiment in parallel, where AgMNPs were replaced with Fe~3~O~4~ NPs, are also plotted in [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}b, showing that the reduction of *o*-NA can proceed only when AgNPs are doped. In the absence AgNPs, the reduction *o*-NA suspended. {ref-type="fig"}](#fig1){ref-type="fig"}c. (b) *C*~t~/*C*~0~ of 1 mM *o*-NA (412 nm) versus the catalytic reduction time in the presence of 30 mM and AgMNPs prepared with (■) 2 min, (●) 4 (▲) min, (▼) 8 10 and (★) 12 of oxidation--reduction time the preparation, the are the same as described in Parallel experiment (□) uses 20 mg of Fe~3~O~4~ as nanocatalysts, where the other conditions are the same as described as (a).](ao-2017-019876_0001){#fig2} Effect of Fe^2+^/Ag^+^ AgMNP Preparation {#sec2.2} ------------------------------------------- As described in the previous section, catalytic efficiency is related to the size and amount of doped AgNPs. In we expected the morphology and amount of doped AgNPs to be affected the ratio of concentration of Fe^2+^ to Ag^+^ because AgNO~3~ acts the oxidation in the formation of AgMNPs. [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"} shows the images of the AgMNPs with different ratios of \[Fe^2+^\]~0~ to The images reveal that the morphologies of the AgNPs are [Figure S2](http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b01987/suppl_file/ao7b01987_si_001.pdf) shows the typical X-ray diffraction (XRD) spectra of the prepared AgMNPs and | INTROdUCTIoN {#SEc1}
============
iN coNTrASt To buLk sILvEr, NanOMETrIC SILvEr MatERials eXHibiT mANy EXTrAordInaRy prOpeRtiEs suCH As a higH SUrFacE-to-VoLUMe RATio,^[@rEf1],[@Ref2]^ qUANtuM tUnneLInG eFfECTS,^[@Ref3],[@Ref4]^ AN AbuNdance oF FreE ElECTrons,^[@rEf5]^ SURFaCE PLAsmOn reSonANCE,^[@rEF2],[@REf6]−[@reF8]^ AnD ANtiBAcTeRial BeHaVIorS.^[@rEF9],[@ReF10]^ bECAuse Of tHesE Unique proPertIES, noBLE SILVEr NAnoMATerIalS aRe WidELY aPpliEd IN DiversE AReAs, INclUDing ThERMOTherApy,^[@reF5],[@Ref11]^ mEdICInE,^[@REf2],[@ref5],[@ReF12]^ sENsOrS,^[@Ref13]−[@rEf16]^ sURFAcE-eNhAnCEd SPecTRosCOpY,^[@ref6],[@ReF17]−[@REf20]^ biOlogy,^[@reF2]^ cAtAlYsIS,^[@REf21]−[@rEf28]^ aNd eLECtROnICs.^[@rEF29]−[@ReF31]^ AMOng ThEse maNy ApPLICAtIOns, tHe CATalYsIS OF tHe rEDUction oF NiTroaREnEs To ARomaTic aMiNeS Is InCREasiNgly ATtraCtINg ATTENTion BECauSe oF PHaRmaCeuTICaL NEEds ANd tHe IMpORtanCe Of thiS inDusTry.^[@Ref25],[@reF26],[@REF32]−[@rEF38]^ vARiouS StrAtEGieS hAvE bEeN PrOposEd to REdUcE NitRoARENEs moRe EFfiCIeNtLy aND MOre RApIDlY uSINg sIlVeR nANOMaTERIaLS. thEse StrATEgiES IncLUdE dePoSiting SilVER NAnOPARticLeS (AGNPs) On SupPORtS UseD AS heterOgeNEoUS cAtalYStS,^[@REF24],[@Ref26],[@rEf39]−[@Ref42]^ cOmBInING agnPs wIth ReDUCed GRaphEnE OxIdE or GraPhEnE OXIdE AS cATALySts,^[@ref43]−[@rEF47]^ aNd uSInG SiLVER nanOCOLloiDs as a QUasi-HoMOGeNEOuS NANOCataLYsT.^[@ref25],[@rEF33],[@reF48]−[@REf54]^ ALL of tHE afoRemEntIOned CATALYTIC aPpRoaCheS ARe effICIEnT anD selectiVe. HOWeveR, the rEaCtiON rATe fOR HEtERoGEneOuS cATaLYSIS IS RathER lOw And quaSI-homoGEneOUs CATaLYSis sUFfERS fROm PoSSIbLE AGGreGation Of ThE naNOcAtALySt. FUrtHERMOrE, THe pROCedUReS fOR PRePArIng sUcH NANOCAtAlYSts are SoMewHAT cOMPLex aND tIme-cONSUMINg.
aPplICaTIONs oF magNETIc nAnOpArticlEs hAVe AlSo bEEn EXtENSIvely INVesTIgaTEd IN recent yeArs BEcAusE theY feAtURe ManY notABLE CHaraCtERIstIcS, sUcH aS a HIGH sUrFAce-TO-VOlUmE ratio, Easy attRAcTIon And reDiSperSiON, AND parAMaGnETism. beCauSe of tHEse cRuciAl PRoPertIES AnD aDvAntAGEs, THE CombINATiOn oF agNPs anD MaGNETiC NaNOpaRtiCleS Has beCoME ONE of THe moSt fAvoRABlE ApPrOachEs FOR THE caTAlYtiC rEDUctIon oF NITRoARenEs.^[@REF55]−[@ref60]^ IN THIS stUDy, a sImplE but facIlE meTHOd was APPLIEd In A sINgle stEp TO prEparE silVeR-DOPeD magNetIc NAnOpARtiCLEs (aGmNps) foR thE CAtalYtiC REDUctiOn oF nITrOArENeS THROugh spOntanEOUs oxIDATIoN--rEdUCTION aNd CoprecIpiTatION. wHen mIXing FE^2+^ WITH ag^+^, a SponTAnEOuS ReacTioN IS CAUSed BY the dIFference IN StANDArD ReDUctiOn pOtENTiAL beTweEn tHE IonIc sPecIES. When AG^+^ is rEDUCEd to AG^0^, aN eQuiVaLenT numbeR Of moleS oF Fe^2+^ iOnS are SimUltanEOusly OxidiZED tO fe^3+^. AFtEr thE AddITIOn oF PrECipitAtioN aGents, AGNPS WERE cOprECIpiTATED WITh IroN oXIDe MAgnetic naNOparticLeS, wHiCh led to tHE fOrmatiOn oF agMnPs. THE pROPosED prEpAraTiOn cAn be achIevED in a sINgle StEP, ANd THe PRePArEd aGmnpS CAn suBsEQUENtLY bE UTIlIzed As nANoCATalYSts for ThE REDUCTIOn Of *O*-niTRoanILiNE (*o*-NA). THE pArAMETERs (PH, TemPerATURE, aNd aMount Of NaNoCatALYSt) ThAT affect The mORPholOGY AND composITiOn OF the PRePAreD AgmNps aND EfficieNciEs oF tHE CATALYTIc rEdUctioN werE SyStEmAtiCalLy sTuDiEd tO GaIn a gReaTeR UnDersTANdIng Of ThE CHarACTEriStics Of The agMNps PrEPAreD uSiNG THE mEtHod prOposeD In ThiS STUDy. aDDITioNAlLy, tHE CataLyTIc acTiVITy Of the AgmNPs PREPAred fOR tHE ReDucTioN Of Other NiTrOAReNeS ANd THEiR rECycLaBiliTY WerE INVeSTIgATEd tO FULLy eVAlUate tHeIR POtENTial fOr PrActiCal ApPLIcatioNs.
RESULTs ANd dIScUsSiOn {#sEc2}
======================
EFfeCt OF OXIDatIOn--redUctION tiMe on AGmnP pREpAraTIon {#seC2.1}
--------------------------------------------------------
[FIGuRe s1](httP://pUbS.aCs.oRG/DoI/SupPL/10.1021/ACsOMEga.7b01987/sUppl_FIle/aO7b01987_sI_001.PdF) shows the TypicAL mEASUREd HYsTeresis lOops of The PREPaREd aGMnPs, whIcH CoNfirmEd tHAT The PRePaRed aGmnPs wERE PARAMAgNEtIC AnD uSAblE FOr FURTheR AppLicAtionS. [fiGURE [1](#FIg1){rEf-tYPE="fIg"}](#fIg1){ReF-tyPE="fiG"} DePicTs ThE trAnsMIsSION ELEctRoN mIcrOscOpY (tEM) ImagEs oF AGmNpS oBtAinEd UsiNG vaRiOUS rEaCtIoN tiMES. frOm ThE IMAGes, darK-sPhERe-lIKE AG nanOpArtiCLes WERE mIxed wiTh Light-cOlOReD FE~3~o~4~ NPs bECaUSE ag HaS A hIgher ELEctRON denSIty THat AlloWs FeWEr eleCtrOnS to tRaNsmiT.^[@ReF61],[@reF62]^ The AGnPs FoRMEd AFter a 10 Min ReACtIoN TimE WerE LArgEr tHAn THOSE fORMed afteR a 2 mIN reaCtion Time. NotABly, ThE sIzE OF ThE FE~3~O~4~ NpS WaS mOSTLy uNafFEctEd BY thE REACtIOn TiMe.
![teM iMAgES OF PrEpARED aGMNpS wiTH OxiDAtion--reductioN TIMes of (a) 2 miN, (B) 8 min, ANd (c)--(f) 10 MIN, whERE THe \[FE^2+^\]~0~ To \[aG^+^\]~0~ RATios Are (a)--(c) 3:1, (D) 2:1, (e) 4:1, And (F) 6:1. \[fe^2+^\]~0~ aRe ALl 12 mm, aNd THE MaGNIfiCATIonS oF tHE iMaGes aRe alL 100 000×. tHE YELLOw ArRoWS INdICaTE thE EXaMPLES of agNpS fOr EacH samPLE.](AO-2017-019876_0008){#fiG1}
[figUre [2](#Fig2){ref-Type="fig"}](#fIG2){REF-tYPE="FIg"}A SHOWS tHE evolUtion Of tHe Uv--vIS speCTRa fOr ThE reDUctION OF *o*-nA cAtaLyZeD BY aGmnpS oVEr TiMe. THE ABSorBaNCE PEAk aT 412 nM, WHICh CoRrESpOndS tO tHE chaRaCtErIStic *o*-NA peaK,^[@Ref63]^ deCrEASEd aS THe reactIon prOceeDed. THe VarIATIoNs Of ThE SPeCtrA IndICAtED ThAt *O*-nA was rEdUCeD To 1,2-pHeNylEnEdIaMINe (1,2-Ppd).^[@REF41],[@ReF64]^ tHe rElaTIve cOnCeNtRAtION (*C*~T~/*c*~0~) Of *O*-NA wAS obTAiNED by DivIDInG tHE AbsOrBAnce reCoRDEd AT 412 Nm at THe SpEcified time (*c*~t~) bY THe aBsoRbaNcE aT 412 Nm bEFOre THe addItIon Of aGmnps (*C*~0~). ThE reSUlTS in [FiGUrE [2](#FiG2){ref-tYPe="FIg"}](#fig2){ReF-tyPe="fiG"}B wErE PlottEd UsING vARiOUS AgmnPS pReparEd UsiNg vARIouS reDUcTioN duRatIONS. iN [fIgurE [2](#fiG2){REF-type="fiG"}](#fiG2){ReF-TyPE="fIg"}B, tHE caTALyTIC EfFiCIeNcY oF the AGMnPS shOws NO SigNIFIcaNt diFfErENcE bETWEen WHeN THE REaCTIoN tImE waS 4--10 Min And wHEn MoRE thAN 95% of *O*-Na was rEDuCED wItHin 240 s. ThIS fiNDInG suggEstS rEmArkabLE CaTaLytiC actiVIty. by contRASt, AchieviNG THe SAme COnvERsiON perCENTAge reqUIreD MOre THAn 500 s WHEN THE ReacTion TIMe TO prepaRE THE AGmnps WAs 2 or 12 miN. beCAuSe tHE catalYTIC EfficIeNCy Of NANocatAlYSts dEPeNdS oN theIR SIzE aNd ThE AMOunT OF CataLySt loAded,^[@REF26]^ WE cOnClUDed ThAT The reAcTioN TiME tO achIeve THe oPtiMaL MORpHOlOgy AND cATalysT lOAdING wAS 10 mIn. foR comParISOn, THe reSUlts OF aN EXperiMEnT conDUCtED iN pARallel, wherE aGMNPS wERE rEpLaceD witH FE~3~o~4~ nPS, aRE aLso plotteD in [figuRE [2](#FIG2){REf-TYpE="Fig"}](#FIG2){REF-tYPe="FIG"}B, ShOWiNg tHAT the Reduction OF *o*-Na Can ProcEED onlY wHen agNpS ArE doPeD. In the ABseNCe of aGnPS, THe RedUctIon of *O*-nA is SusPENDeD.
{Ref-TYpE="fig"}](#FiG1){ref-TYpE="fIg"}c. (b) *c*~t~/*c*~0~ oF 1 Mm *o*-Na (412 Nm) vERSus THE cATalytiC reDuction tiMe iN thE PRESENce of 30 MM NAbH~4~ anD AGmnps PrEpaREd WItH (■) 2 mIn, (●) 4 mIN, (▲) 6 Min, (▼) 8 MIn, (◆) 10 miN, aNd (★) 12 MIn OF oXiDATIon--rEduCtion TIMe dURiNg tHe prepAraTIOn, wHeRE THe oThEr CoNDItioNs ArE ThE sAME aS DEscriBED In (A). pARaLLEL eXpeRiMENt (□) uSEs 20 MG oF fE~3~O~4~ NPS As nAnoCaTALYsTs, whERE thE OTheR conDItions ARE The SaMe AS DEscrIbEd AS (A).](Ao-2017-019876_0001){#fIg2}
EffeCt oF FE^2+^/ag^+^ on aGmNP pREPaRAtIOn {#SEC2.2}
-------------------------------------------
AS deScRibed In tHE prevIOuS SectiON, CataLyTiC EfFiCIenCY IS ReLated To The sizE AND aMOunt oF doPEd aGnPs. in ADDitiOn, we expECtEd tHe morpHolOgy aND aMoUnT Of DoPED AgnPs To be AFfeCTEd BY thE rAtIo oF iNiTiAL COnCENtRATiOn Of FE^2+^ To AG^+^ BecaUSe AgNO~3~ Acts as THE OxidATIon AGeNT in thE forMAtioN OF agmnPS. [FiGUre [1](#fIg1){REF-TYPE="fIg"}](#FiG1){ReF-TypE="FIg"} ShOWS THE tEm ImAgES of THe aGMnPs wItH DIFfErent rAtios OF \[FE^2+^\]~0~ To \[Ag^+^\]~0~. THe IMAGes ReVEal That the mORpHoLOgiES of the aGnPs Are SImilAr. [FIgurE s2](HtTP://PUbs.aCS.oRg/DOI/SuPPl/10.1021/aCsOMEga.7B01987/SuPPL_FiLe/ao7B01987_si_001.pdF) sHOWs thE tYpiCaL x-RaY DIFFraCTION (xrD) spEctra Of the pREPaRED AGMNPS AnD | Introduction{#sec1} ============ In contrast to bulk silver, nanometric silver materials exhibit many extraordinary properties suchas a high surface-to-volume ratio,^[@ref1],[@ref2]^ quantum tunneling effects,^[@ref3],[@ref4]^ an abundance of freeelectrons,^[@ref5]^ surface plasmon resonance,^[@ref2],[@ref6]−[@ref8]^ and antibacterial behaviors.^[@ref9],[@ref10]^ Because of these uniqueproperties,noble silvernanomaterials arewidely appliedin diverseareas, including thermotherapy,^[@ref5],[@ref11]^ medicine,^[@ref2],[@ref5],[@ref12]^sensors,^[@ref13]−[@ref16]^ surface-enhanced spectroscopy,^[@ref6],[@ref17]−[@ref20]^biology,^[@ref2]^ catalysis,^[@ref21]−[@ref28]^ and electronics.^[@ref29]−[@ref31]^ Among these many applications, the catalysisofthe reductionof nitroarenes to aromatic amines is increasingly attracting attentionbecause of pharmaceutical needsandthe importance of this industry.^[@ref25],[@ref26],[@ref32]−[@ref38]^ Various strategies have been proposed to reduce nitroarenes more efficientlyand more rapidly using silver nanomaterials. These strategies include depositing silvernanoparticles (AgNPs) on supports used as heterogeneous catalysts,^[@ref24],[@ref26],[@ref39]−[@ref42]^combiningAgNPs with reduced graphene oxide or graphene oxide as catalysts,^[@ref43]−[@ref47]^ and using silver nanocolloids as a quasi-homogeneousnanocatalyst.^[@ref25],[@ref33],[@ref48]−[@ref54]^ All of the aforementioned catalyticapproaches are efficient and selective. However, the reaction rate for heterogeneous catalysisis rather lowand quasi-homogeneous catalysis suffers from possible aggregation of the nanocatalyst. Furthermore, the procedures forpreparing such nanocatalysts are somewhat complex and time-consuming. Applications of magneticnanoparticles have also been extensively investigated in recent years because they feature many notable characteristics, such as a high surface-to-volume ratio, easy attraction and redispersion,and paramagnetism. Because ofthese crucial properties and advantages, the combinationof AgNPs and magnetic nanoparticles has become one ofthe most favorable approaches for the catalytic reduction of nitroarenes.^[@ref55]−[@ref60]^ In this study, a simple butfacile method was applied in a single step to prepare silver-doped magneticnanoparticles (AgMNPs) for the catalytic reduction of nitroarenes through spontaneous oxidation--reduction and coprecipitation. When mixing Fe^2+^ with Ag^+^, a spontaneous reaction is caused by the difference in standard reduction potential betweenthe ionic species. When Ag^+^ is reduced to Ag^0^, an equivalentnumber of moles of Fe^2+^ions are simultaneously oxidized to Fe^3+^. After the addition ofprecipitation agents,AgNPs were coprecipitated with iron oxide magnetic nanoparticles, which led to the formation of AgMNPs. Theproposed preparation can be achieved in a single step, and theprepared AgMNPs can subsequently be utilized as nanocatalysts for the reduction of *o*-nitroaniline (*o*-NA). The parameters (pH, temperature, and amount of nanocatalyst) that affect themorphology and compositionofthe preparedAgMNPs and efficienciesof the catalytic reduction were systematically studied to gain a greater understanding ofthe characteristics of the AgMNPs prepared using the method proposed in this study.Additionally, the catalytic activity of the AgMNPsprepared for the reduction ofother nitroarenesandtheirrecyclability were investigated to fullyevaluate their potentialfor practical applications. Results and Discussion {#sec2} ====================== Effect of Oxidation--ReductionTime on AgMNP Preparation {#sec2.1} -------------------------------------------------------- [Figure S1](http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b01987/suppl_file/ao7b01987_si_001.pdf) shows the typical measured hysteresis loops of theprepared AgMNPs, which confirmedthat the prepared AgMNPs were paramagnetic and usable for further applications.[Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"} depicts thetransmission electron microscopy (TEM) images of AgMNPs obtained using various reaction times. Fromthe images, dark-sphere-like Ag nanoparticles were mixed with light-colored Fe~3~O~4~NPs because Ag has a higher electron density that allows fewer electrons to transmit.^[@ref61],[@ref62]^ The AgNPsformed after a 10 min reactiontime were larger than thoseformed after a 2 min reaction time. Notably, the size ofthe Fe~3~O~4~ NPs was mostly unaffected by the reaction time. ![TEM images of prepared AgMNPs withoxidation--reductiontimes of(a) 2 min,(b) 8 min, and (c)--(f) 10 min, where the \[Fe^2+^\]~0~ to \[Ag^+^\]~0~ ratiosare (a)--(c) 3:1,(d)2:1, (e) 4:1, and (f) 6:1. \[Fe^2+^\]~0~ are all 12 mM, andthe magnifications of the images are all 100 000×. The yellow arrowsindicate the examples ofAgNPs for each sample.](ao-2017-019876_0008){#fig1}[Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}a shows the evolution ofthe UV--vis spectra for the reduction of *o*-NA catalyzed by AgMNPsover time. The absorbance peak at 412 nm, which corresponds to the characteristic *o*-NA peak,^[@ref63]^ decreased asthe reactionproceeded. The variations of the spectra indicated that *o*-NA was reduced to 1,2-phenylenediamine (1,2-PPD).^[@ref41],[@ref64]^ The relative concentration(*C*~t~/*C*~0~) of *o*-NA was obtained by dividingthe absorbance recorded at 412 nm at the specified time (*C*~t~)by the absorbance at 412 nm before the addition ofAgMNPs (*C*~0~). The resultsin [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}b wereplotted usingvarious AgMNPs prepared usingvarious reductiondurations. In [Figure[2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}b, thecatalyticefficiency of the AgMNPsshows no significant difference between when the reaction time was 4--10 min and when more than 95% of *o*-NA was reduced within 240 s. This finding suggests remarkable catalytic activity. By contrast, achieving the same conversion percentage required more than 500 s when the reactiontime topreparethe AgMNPs was 2 or 12 min. Becausethecatalyticefficiency of nanocatalysts depends on their size and the amount of catalyst loaded,^[@ref26]^ we concluded thatthereaction time to achieve the optimal morphology andcatalyst loadingwas 10 min. For comparison,the results ofan experimentconducted in parallel, where AgMNPs werereplaced withFe~3~O~4~ NPs, are also plotted in [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}b, showing that the reduction of *o*-NA can proceed only when AgNPs are doped. In the absence of AgNPs, the reduction of *o*-NA is suspended. {ref-type="fig"}](#fig1){ref-type="fig"}c. (b) *C*~t~/*C*~0~ of 1 mM *o*-NA (412 nm) versus thecatalytic reduction time in thepresenceof 30mM NaBH~4~ and AgMNPs prepared with (■) 2 min, (●) 4 min, (▲) 6 min, (▼) 8 min, (◆) 10 min,and (★) 12 min of oxidation--reduction time during thepreparation, where the other conditions are the sameasdescribed in (a). Parallel experiment (□)uses20 mg of Fe~3~O~4~ NPs asnanocatalysts, where the other conditions are the same as described as (a).](ao-2017-019876_0001){#fig2} Effect of Fe^2+^/Ag^+^on AgMNP Preparation {#sec2.2} ------------------------------------------- As described in the previous section, catalytic efficiency is related to the sizeand amount of doped AgNPs. In addition, we expected themorphologyand amount ofdoped AgNPsto be affected by the ratio of initial concentration of Fe^2+^ toAg^+^ because AgNO~3~ acts as the oxidation agent in theformation ofAgMNPs. [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"} shows the TEM images oftheAgMNPs with different ratios of \[Fe^2+^\]~0~ to \[Ag^+^\]~0~. The images reveal that the morphologies of the AgNPs are similar. [Figure S2](http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b01987/suppl_file/ao7b01987_si_001.pdf)shows the typical X-ray diffraction (XRD) spectra of the prepared AgMNPs and | Introduction _{#sec1}_ ============ In _contrast_ _to_ _bulk_ silver, nanometric silver materials exhibit many _extraordinary_ properties _such_ as _a_ high surface-to-volume ratio,^[@ref1],[@ref2]^ quantum tunneling effects,^[@ref3],[@ref4]^ an abundance of _free_ electrons,^[@ref5]^ surface plasmon _resonance,^[@ref2],[@ref6]−[@ref8]^_ and antibacterial behaviors.^[@ref9],[@ref10]^ _Because_ of these unique properties, noble silver nanomaterials are _widely_ applied in _diverse_ _areas,_ including thermotherapy,^[@ref5],[@ref11]^ _medicine,^[@ref2],[@ref5],[@ref12]^_ sensors,^[@ref13]−[@ref16]^ surface-enhanced spectroscopy,^[@ref6],[@ref17]−[@ref20]^ biology,^[@ref2]^ catalysis,^[@ref21]−[@ref28]^ and electronics.^[@ref29]−[@ref31]^ Among these _many_ applications, _the_ _catalysis_ of the reduction of nitroarenes to aromatic amines _is_ increasingly attracting attention because of pharmaceutical needs and the importance of this industry.^[@ref25],[@ref26],[@ref32]−[@ref38]^ _Various_ strategies have _been_ proposed _to_ _reduce_ nitroarenes more efficiently and more rapidly using silver nanomaterials. These strategies include depositing silver nanoparticles (AgNPs) on supports used as _heterogeneous_ catalysts,^[@ref24],[@ref26],[@ref39]−[@ref42]^ combining _AgNPs_ with reduced graphene oxide or graphene oxide _as_ catalysts,^[@ref43]−[@ref47]^ _and_ using _silver_ nanocolloids as _a_ _quasi-homogeneous_ nanocatalyst.^[@ref25],[@ref33],[@ref48]−[@ref54]^ All _of_ _the_ aforementioned catalytic approaches are _efficient_ and selective. However, _the_ reaction rate _for_ heterogeneous catalysis is _rather_ _low_ and quasi-homogeneous catalysis suffers from _possible_ aggregation of the nanocatalyst. Furthermore, the procedures for preparing such nanocatalysts _are_ _somewhat_ _complex_ and time-consuming. Applications of magnetic nanoparticles have also been extensively investigated in recent years _because_ they feature many notable characteristics, such as a high surface-to-volume ratio, easy attraction and _redispersion,_ and _paramagnetism._ Because _of_ these _crucial_ properties _and_ advantages, the _combination_ of AgNPs and magnetic nanoparticles has become one of the most favorable approaches for the _catalytic_ reduction of _nitroarenes.^[@ref55]−[@ref60]^_ In this study, a _simple_ but facile _method_ _was_ applied in a single _step_ _to_ _prepare_ silver-doped magnetic _nanoparticles_ _(AgMNPs)_ _for_ the catalytic reduction _of_ _nitroarenes_ through spontaneous oxidation--reduction _and_ coprecipitation. _When_ mixing Fe^2+^ with Ag^+^, a spontaneous _reaction_ is _caused_ by the difference in standard reduction potential between the ionic species. When Ag^+^ is reduced to Ag^0^, an _equivalent_ _number_ of moles of Fe^2+^ _ions_ are simultaneously oxidized to Fe^3+^. After the _addition_ _of_ precipitation _agents,_ AgNPs _were_ _coprecipitated_ _with_ iron oxide _magnetic_ nanoparticles, which led to _the_ formation of _AgMNPs._ The proposed preparation _can_ be achieved _in_ a single _step,_ and the prepared AgMNPs can subsequently be utilized as nanocatalysts for the _reduction_ of *o*-nitroaniline (*o*-NA). The parameters (pH, temperature, and amount of nanocatalyst) _that_ affect the morphology and composition of the prepared AgMNPs and _efficiencies_ of the catalytic reduction _were_ systematically _studied_ to gain a greater understanding of the characteristics _of_ the _AgMNPs_ prepared _using_ the method _proposed_ in _this_ study. Additionally, _the_ catalytic _activity_ _of_ the AgMNPs prepared for _the_ reduction of other nitroarenes and their recyclability _were_ investigated to fully evaluate their potential for practical applications. Results and Discussion {#sec2} ====================== Effect of Oxidation--Reduction Time _on_ AgMNP Preparation {#sec2.1} -------------------------------------------------------- _[Figure_ S1](http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b01987/suppl_file/ao7b01987_si_001.pdf) shows the typical _measured_ hysteresis loops of _the_ prepared AgMNPs, which confirmed that the prepared AgMNPs were paramagnetic and usable _for_ further applications. [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"} depicts _the_ transmission electron microscopy (TEM) images of AgMNPs obtained using various _reaction_ times. From the images, dark-sphere-like Ag nanoparticles were mixed with light-colored Fe~3~O~4~ NPs because Ag has _a_ higher electron density that allows fewer electrons to transmit.^[@ref61],[@ref62]^ The AgNPs formed after a 10 min reaction time were larger _than_ those formed after a 2 min _reaction_ time. Notably, the size _of_ the Fe~3~O~4~ _NPs_ _was_ mostly _unaffected_ by _the_ reaction time. ![TEM _images_ of prepared AgMNPs _with_ oxidation--reduction times of (a) _2_ _min,_ (b) _8_ min, _and_ (c)--(f) 10 min, where the _\[Fe^2+^\]~0~_ to \[Ag^+^\]~0~ ratios are _(a)--(c)_ 3:1, _(d)_ _2:1,_ (e) 4:1, _and_ (f) _6:1._ \[Fe^2+^\]~0~ _are_ all 12 mM, and the magnifications of the images are all _100_ 000×. The yellow arrows _indicate_ the _examples_ of AgNPs for _each_ _sample.](ao-2017-019876_0008){#fig1}_ [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}a shows the evolution of the UV--vis spectra _for_ the reduction of *o*-NA catalyzed by AgMNPs over time. The absorbance peak at 412 _nm,_ which _corresponds_ _to_ the characteristic *o*-NA peak,^[@ref63]^ decreased _as_ the reaction proceeded. The _variations_ of the spectra indicated that *o*-NA was reduced to 1,2-phenylenediamine (1,2-PPD).^[@ref41],[@ref64]^ The relative concentration (*C*~t~/*C*~0~) of *o*-NA was obtained by dividing the absorbance recorded at 412 nm _at_ the specified _time_ _(*C*~t~)_ _by_ the absorbance _at_ 412 nm before the addition of _AgMNPs_ (*C*~0~). The results in [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}b were plotted using various AgMNPs prepared _using_ various reduction durations. In [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}b, the catalytic efficiency of _the_ AgMNPs shows no significant difference between when _the_ reaction time was _4--10_ min _and_ _when_ more than _95%_ of *o*-NA was reduced within 240 s. This _finding_ suggests remarkable catalytic activity. By _contrast,_ _achieving_ the _same_ conversion percentage required more _than_ 500 s when the reaction time to prepare the AgMNPs was 2 or 12 min. Because the catalytic efficiency of nanocatalysts depends on their size and the _amount_ of catalyst loaded,^[@ref26]^ we concluded that the reaction _time_ to achieve _the_ _optimal_ morphology and catalyst loading was 10 min. _For_ comparison, the results of an experiment conducted in _parallel,_ where AgMNPs were replaced with Fe~3~O~4~ _NPs,_ are _also_ plotted _in_ [Figure _[2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}b,_ showing _that_ _the_ reduction of _*o*-NA_ can proceed only _when_ AgNPs _are_ doped. In _the_ absence of AgNPs, the reduction of *o*-NA is suspended. {ref-type="fig"}](#fig1){ref-type="fig"}c. _(b)_ *C*~t~/*C*~0~ of 1 mM _*o*-NA_ (412 _nm)_ versus the catalytic reduction time in the presence of 30 mM NaBH~4~ and AgMNPs _prepared_ with (■) 2 min, (●) 4 min, (▲) 6 min, (▼) 8 min, (◆) 10 min, and _(★)_ 12 min _of_ oxidation--reduction time during the preparation, where _the_ other conditions are the same as described in (a). Parallel experiment (□) uses 20 mg of Fe~3~O~4~ NPs as nanocatalysts, where the other conditions are the same as _described_ as (a).](ao-2017-019876_0001){#fig2} Effect _of_ Fe^2+^/Ag^+^ _on_ AgMNP Preparation {#sec2.2} ------------------------------------------- As described in the previous section, catalytic _efficiency_ is related to _the_ size _and_ _amount_ of _doped_ AgNPs. In addition, we expected the morphology and amount of doped AgNPs _to_ be affected by the ratio of initial concentration of Fe^2+^ to Ag^+^ because AgNO~3~ acts as _the_ oxidation agent in _the_ formation of _AgMNPs._ [Figure _[1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}_ shows the _TEM_ _images_ of _the_ AgMNPs with different ratios of \[Fe^2+^\]~0~ to \[Ag^+^\]~0~. The _images_ reveal that _the_ morphologies of the AgNPs are similar. _[Figure_ S2](http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b01987/suppl_file/ao7b01987_si_001.pdf) shows the _typical_ X-ray diffraction (XRD) spectra of the _prepared_ _AgMNPs_ _and_ |
INTRODUCTION {#s1}
============
Whilst oncogenesis is driven by a multitude of complex, non-programmed molecular events, there are a number of key features of this process, not least of which is the aberrant activation of genes that would normally be silenced in a given tissue context \[[@R1]\]. The so called cancer/testis (CT) or cancer germline (CG) genes are one such group of genes that are frequently activated in a range of different human cancer types \[[@R2]-[@R4]\]. These genes have expression normally restricted to the human germline, many being testis-specific \[[@R2]-[@R4]\]. They have come under intense scrutiny since their original identification as the immunological privilege of their normal germline setting means that the proteins they encode can elicit an immunological response when aberrantly produced in cancers and so have exceptional potential in immunotherapeutics \[[@R5]\]; for example, the *NY-ESO-1* gene product has been successfully targeted in an adoptive therapeutic approach to melanoma therapy \[[@R6]\].
Despite this interest, remarkably little is known about the normal germline function of most CT genes. Moreover, it has been demonstrated that germline genes in *Drosophila melanogaster* are required for the oncogenic process and that the human orthologues of these *Drosophila* genes have up-regulated expression in a range of human cancers, although the functional implications for oncogenesis of this up-regulation remains unclear \[[@R7],[@R8]\]. Interestingly, down-regulation of a number of CT genes in human cancer cells results in perturbation of cellular proliferative potential \[for example, see [@R9],[@R10]\]. These findings open up the exciting possibility that CT genes might encode functions that are required for tumour homeostasis and it has recently been proposed that tumours become 'addicted' to these germline factors \[[@R11],[@R12]\], and recently, meiotic factors have been shown to contribute to telomere maintenance in cancer cells via the ALT pathway \[[@R13], [@R14]\]. The full extent of germline gene requirement is unclear, but these findings expose a new therapeutic opportunity by directly targeting the tumour-associated function of the CT gene products. Additionally, a number of studies have revealed another clinically important feature of CT genes; their expression appears to drive drug resistance as depletion of the gene products results in enhanced sensitization to anti-cancer drugs \[for example, see [@R15]\] expanding the therapeutic potential of this important class of cancer genes.
Germline gene expression profiling has also recently been demonstrated to have applications in prognostics and patient stratification. In a seminal study, Rousseaux and co-workers demonstrated that expression of a sub-set of germ line genes in some lung cancers delineated patients with aggressive, metastasis prone tumours with poor prognosis \[[@R16]\]; they extended this by indicating that this cohort of patients might benefit from a drug therapeutic regime that had previously been dismissed for more general use in lung cancer patients, indicating that profiling patients for expression of a small sub-set of germline genes could be used in therapeutic decision making. Understanding germline gene expression is also critical as drug-induced augmentation of expression has also been postulated to be a potential enhancer of immunotherapeutics, the rationale being that further up-regulation of a tumour-specific antigen will result in enhanced immunological targeting of the tumour \[for example, see [@R17]\].
Taking all these factors together reveals the importance of understanding the regulatory mechanisms for somatic germline gene silencing and their aberrant activation in tumours. To date, the regulation of a number of CT genes has been studied and it has been demonstrated that DNA methylation of regulatory elements, such as promoter-associated CpG islands plays a fundamental role in the somatic silencing of these genes and the hypomethylation of these regulatory DNA regions in cancers is linked to gene activation \[for example, see [@R18]-[@R23]\], whereas gene body hypomethylation has been linked to gene down regulation in cancers \[[@R24]\]. Expression of these genes also becomes activated or further up-regulated upon enforced hypomethylation by the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR), and to date, all CT genes studied have up-regulated expression in response to this chemotherapeutic agent, indicating a commonality in the mechanistic pathway for somatic CT gene silencing \[for example, see [@R18]-[@R23]\].
To date, most of the CT genes whose expression has been studied are located on the X chromosome (X-CT genes) and belong to large paralogous gene families \[[@R2]-[@R4]\]. Recently, a computational pipeline combining expressed sequence tag and microarray meta-analyses of the human orthologues of mouse spermatocyte-specific genes revealed a large cohort of new CT genes that were expressed in a broad spectrum of cancer types \[[@R25]-[@R29]\]. Unlike the X-CT genes, the majority of these genes are autosomally encoded and are single copy. To date, the clinical potential of these genes remains largely unexplored. In this current study, analysis of the expression of a small sub-set of these genes reveals a novel feature of CT genes, which indicates that some have a unique mechanism for somatic transcriptional silencing. This is a significant finding as these genes and their associated gene products have an increased prominence in clinical applications and hence the sub-classification of CT genes will play an important role in diagnostics, stratification and therapeutics.
RESULTS {#s2}
=======
All CT genes studied to date (mostly X-CT genes) require hypermethylation of regulatory DNA sequences for somatic silencing and are activated by the hypomethylating agent 5-aza-CdR. Given the clinical potential of enhanced up-regulation of immunogenic CT antigens, we set out to explore whether a similar DNA hypermethylation silencing mechanism was operating in the recently identified autosomally encoded CT genes \[[@R25],[@R27]\]. To do this, we selected a small sub-group of these genes that remained transcriptionally silenced in the colorectal cancer cell lines HCT116 and SW480 (*ARRDC5, C4orf17, C20orf201, DDX4, NT5C1B, STRA8, TDRD12*). We also selected two previously characterized CT genes (both X-CT genes) that remained transcriptionally silenced in these two cell lines to serve as exemplar controls for hypermethylation regulated CT genes, *SSX2* and *GAGE1*. To determine whether the novel CT genes are silenced via hypermethylation mediated mechanisms, similar to the characterized X-CT genes, we treated the two cell lines with the DNA methyltransferase inhibitor 5-aza-CdR to determine whether inhibition of DNA methyltransferase activity can activate these genes. Following 5-aza-CdR treatment of HCT116 and SW480 we made cDNA and carried out RT-PCR and agarose gel electrophoresis analysis of the products. The two X-CT genes were activated from the silent state with relatively low levels of 5-aza-CdR (0.1 μM; Figure [1](#F1){ref-type="fig"}; Figure [2](#F2){ref-type="fig"}). Some of the novel, autosomally encoded CT genes were similarly activated (*C20orf201, DDX4, STRA8, TDRD12*), although *C20orf201* and *DDX4* required a slightly higher 5-aza-CdR concentration for activation (0.5 μM; Figure [1](#F1){ref-type="fig"}; Figure [2](#F2){ref-type="fig"}). Additionally, activation of *STRA8* requires slightly higher concentrations of 5-aza-CdR in SW480 (Figure [2](#F2){ref-type="fig"}) than HCT116 (Figure [1](#F1){ref-type="fig"}), which indicates subtle regulatory differences between tumour cell types. However, surprisingly, three genes (*ARRDC5, C4orf17, NT5C1B*) remained tightly transcriptionally silenced, even at high concentrations of 5-aza-CdR in both cell lines (15.0 μM; Figure [1](#F1){ref-type="fig"}; Figure [2](#F2){ref-type="fig"}). This unexpected result reveals an important distinction in the way CT gene silencing is epigenetically regulated, revealing a hypermethylation-independent pathway. Interestingly, the X-CT genes (*GAGE1, SSX2*) remained activated for a prolonged period following removal of the hypomethylating agent, as did the autosomally encoded CT genes that were activated with the lowest concentration of 5-aza-CdR (*STRA8, TDRD12*) (Figure [3](#F3){ref-type="fig"}); however, the other two autosomally encoded CT genes, *C20orf201* and *DDX4*, which required slightly higher concentrations of 5-aza-CdR for activation, reverted to the silent state relatively soon after removal of the hypomethylating agent (Figure [2](#F2){ref-type="fig"}). This indicates a much greater transcriptional elasticity to the methylation-dependent silencing mechanisms for some CT genes.
![A sub-group of germline genes remain refractory to activation by epigenetic modulating agents\
RT-PCR was used to analyse activation of a group of germline genes that are normally silenced in the cancer cell line HCT116 (an additional colorectal cell line gives similar results \[see Supplementary Figure S1)\]. Whilst a cohort of known and newly identified germline genes become activated at low doses of the demethylating agent 5-aza-CdR (*GAGE1, SSX2, STRA8, TDRD12*) and others become activated with slightly higher levels of 5-aza-CdR (*C20orf201, DDX4*), some remain tightly silenced, even at high concentrations of 5-aza-CdR (*ARRDC5, C4orf17, NT5C1 | introduction { # s1 } = = = = = = = = = = = = whilst oncogenesis is accompanied by a multitude of complex, non - programmed molecular events, there are a number of key features of this process, not least of which is the aberrant activation of genes however would normally be silenced in a given tissue context \ [ [ @ r1 ] \ ]. the so called cancer / testis ( mn ) or cancer germline ( cg ) genes are one such group of genes that are frequently activated in a range of different lung cancer types \ [ [ @ r2 ] - [ @ r4 ] \ ]. these genes have expression normally restricted to the particular germline, many being testis - specific \ [ [ @ r2 ] - [ @ r4 ] \ ]. they have come under intense scrutiny since their original identification as the immunological privilege of their normal germline setting means that the proteins they encode can elicit an immunological response when genetically produced in cancers and so have exceptional potential in immunotherapeutics \ [ [ @ r5 ] \ ] ; for example, whereas * ny - eso - 1 * gene product has been successfully targeted in an adoptive therapeutic approach to melanoma therapy \ [ [ @ r6 ] \ ]. despite this interest, remarkably little is known about the normal germline function of most ct genes. moreover, it has been demonstrated that germline genes in * drosophila melanogaster * are required for the oncogenic process and that the human orthologues of these * drosophila * genes have up - regulated expression in a range of human cancers, although the functional implications for oncogenesis of this up - regulation remains unclear \ [ [ @ r7 ], [ @ r8 ] \ ]. interestingly, by - regulation of a number of ct genes in human cancer patients results in perturbation of cellular proliferative potential \ [ for example, see [ @ r9 ], [ @ r10 ] \ ]. these findings open up the exciting possibility that ct genes might encode functions that are sufficient for tumour homeostasis and it has recently been proposed that tumours become ' addicted ' to these germline factors \ [ [ @ r11 ], [ @ r12 ] \ ], and recently, meiotic factors have been shown to contribute to telome ##re maintenance in cancer cells via the alt pathway \ [ [ @ r13 ], [ @ r14 ] \ ]. the full extent of germline gene requirement is unclear, but these findings expose a new therapeutic opportunity by directly targeting the tumour - associated function of the ct gene products. additionally, a number of studies have revealed another clinically important feature of ct genes ; their expression appears to drive drug resistance as depletion of the gene products results in enhanced sensitization to anti - cancer drugs \ [ for example, see [ @ r15 ] \ ] expanding the therapeutic potential of this important class of cancer genes. germline gene expression profiling has also recently been demonstrated to have applications in prognostics and patient stratification. in a seminal study, rousseaux and co - workers demonstrated that expression of a sub - set of germ line genes in some lung cancers delineated patients with aggressive, metastasis prone tumours with poor prognosis \ [ [ @ r16 ] \ ] ; they extended this by indicating that this cohort of patients might benefit from a drug therapeutic regime that had previously been dismissed for more general use in lung cancer patients, indicating that profiling patients for expression of a small sub - set of germline genes could be used in therapeutic decision making. understanding germline gene expression is also critical as drug - induced augmentation of expression has also been postulated to be a potential enhancer of immunotherapeutics, the rationale being that further up - regulation of a tumour - specific antigen will result in enhanced immunological targeting of the tumour \ [ for example, see [ @ r17 ] \ ]. taking all these factors together reveals the importance of understanding the regulatory mechanisms for somatic germline gene silencing and their aberrant activation in tumours. to date, the regulation of a number of ct genes has been studied and it has been demonstrated that dna methylation of regulatory elements, such as promoter - associated cpg islands plays a fundamental role in the somatic silencing of these genes and the hypomethylation of these regulatory dna regions in cancers is linked to gene activation \ [ for example, see [ @ r18 ] - [ @ r23 ] \ ], whereas gene body hypomethylation has been linked to gene down regulation in cancers \ [ [ @ r24 ] \ ]. expression of these genes also becomes activated or further up - regulated upon enforced hypomethylation by the dna methyltransferase inhibitor 5 - aza - 2 ′ - deoxycytidine ( 5 - aza - cdr ), and to date, all ct genes studied have up - regulated expression in response to this chemotherapeutic agent, indicating a commonality in the mechanistic pathway for somatic ct gene silencing \ [ for example, see [ @ r18 ] - [ @ r23 ] \ ]. to date, most of the ct genes whose expression has been studied are located on the x chromosome ( x - ct genes ) and belong to large paralogous gene families \ [ [ @ r2 ] - [ @ r4 ] \ ]. recently, a computational pipeline combining expressed sequence tag and microarray meta - analyses of the human orthologues of mouse spermatocyte - specific genes revealed a large cohort of new ct genes that were expressed in a broad spectrum of cancer types \ [ [ @ r25 ] - [ @ r29 ] \ ]. unlike the x - ct genes, the majority of these genes are autosomally encoded and are single copy. to date, the clinical potential of these genes remains largely unexplored. in this current study, analysis of the expression of a small sub - set of these genes reveals a novel feature of ct genes, which indicates that some have a unique mechanism for somatic transcriptional silencing. this is a significant finding as these genes and their associated gene products have an increased prominence in clinical applications and hence the sub - classification of ct genes will play an important role in diagnostics, stratification and therapeutics. results { # s2 } = = = = = = = all ct genes studied to date ( mostly x - ct genes ) require hypermethylation of regulatory dna sequences for somatic silencing and are activated by the hypomethylating agent 5 - aza - cdr. given the clinical potential of enhanced up - regulation of immunogenic ct antigens, we set out to explore whether a similar dna hypermethylation silencing mechanism was operating in the recently identified autosomally encoded ct genes \ [ [ @ r25 ], [ @ r27 ] \ ]. to do this, we selected a small sub - group of these genes that remained transcriptionally silenced in the colorectal cancer cell lines hct116 and sw480 ( * arrdc5, c4orf17, c20orf201, ddx4, nt5c1b, stra8, tdrd12 * ). we also selected two previously characterized ct genes ( both x - ct genes ) that remained transcriptionally silenced in these two cell lines to serve as exemplar controls for hypermethylation regulated ct genes, * ssx2 * and * gage1 *. to determine whether the novel ct genes are silenced via hypermethylation mediated mechanisms, similar to the characterized x - ct genes, we treated the two cell lines with the dna methyltransferase inhibitor 5 - aza - cdr to determine whether inhibition of dna methyltransferase activity can activate these genes. following 5 - aza - cdr treatment of hct116 and sw480 we made cdna and carried out rt - pcr and agarose gel electrophoresis analysis of the products. the two x - ct genes were activated from the silent state with relatively low levels of 5 - aza - cdr ( 0. 1 μm ; figure [ 1 ] ( # f1 ) { ref - type = " fig " } ; figure [ 2 ] ( # f2 ) { ref - type = " fig " } ). some of the novel, autosomally encoded ct genes were similarly activated ( * c20orf201, ddx4, stra8, tdrd12 * ), although * c20orf201 * and * ddx4 * required a slightly higher 5 - aza - cdr concentration for activation ( 0. 5 μm ; figure [ 1 ] ( # f1 ) { ref - type = " fig " } ; figure [ 2 ] ( # f2 ) { ref - type = " fig " } ). additionally, activation of * stra8 * requires slightly higher concentrations of 5 - aza - cdr in sw480 ( figure [ 2 ] ( # f2 ) { ref - type = " fig " } ) than hct116 ( figure [ 1 ] ( # f1 ) { ref - type = " fig " } ), which indicates subtle regulatory differences between tumour cell types. however, surprisingly, three genes ( * arrdc5, c4orf17, nt5c1b * ) remained tightly transcriptionally silenced, even at high concentrations of 5 - aza - cdr in both cell lines ( 15. 0 μm ; figure [ 1 ] ( # f1 ) { ref - type = " fig " } ; figure [ 2 ] ( # f2 ) { ref - type = " fig " } ). this unexpected result reveals an important distinction in the way ct gene silencing is epigenetically regulated, revealing a hypermethylation - independent pathway. interestingly, the x - ct genes ( * gage1, ssx2 * ) remained activated for a prolonged period following removal of the hypomethylating agent, as did the autosomally encoded ct genes that were activated with the lowest concentration of 5 - aza - cdr ( * stra8, tdrd12 * ) ( figure [ 3 ] ( # f3 ) { ref - type = " fig " } ) ; however, the other two autosomally encoded ct genes, * c20orf201 * and * ddx4 *, which required slightly higher concentrations of 5 - aza - cdr for activation, reverted to the silent state relatively soon after removal of the hypomethylating agent ( figure [ 2 ] ( # f2 ) { ref - type = " fig " } ). this indicates a much greater transcriptional elasticity to the methylation - dependent silencing mechanisms for some ct genes.! [ a sub - group of germline genes remain refractory to activation by epigenetic modulating agents \ rt - pcr was used to analyse activation of a group of germline genes that are normally silenced in the cancer cell line hct116 ( an additional colorectal cell line gives similar results \ [ see supplementary figure s1 ) \ ]. whilst a cohort of known and newly identified germline genes become activated at low doses of the demethylating agent 5 - aza - cdr ( * gage1, ssx2, stra8, tdrd12 * ) and others become activated with slightly higher levels of 5 - aza - cdr ( * c20orf201, ddx4 * ), some remain tightly silenced, even at high concentrations of 5 - aza - cdr ( * arrdc5, c4orf17, nt5c1 | INTRODUCTION {# s1} = = = = = = = = = = = = Whilst oncogenesis is driven by a multitude of complex, non - programmed molecular events, there are a number of key geatuges of this process, not least of which is the aberrant activation of genes that would normally be silenced in a given tissue context \ [[ @ R1] \ ]. The so called cancer / testis (CT) or cancer germline (CG) genes are one such group of genes that are frequently activated in a range of different human cancer types \ [[ @ R2] - [@ R4] \ ]. These genes have expression normally rWstricFed to the human germline, many being testis - specific \ [[ @ R2] - [@ R4] \ ]. They have FPme under intense scrutiny since their original identification as the immunological privilege of their normal germline setting means that the proteins they egcoce can elicit an immunological response when aberrantly produced in cancers and so have exceptional potential in immunotherapeutics \ [[ @ R5] \ ]; for example, the * NY - ESO - 1 * gene product has been successfully targeted in an adoptive therapeutic approach to melanoma therapy \ [[ @ R6] \ ]. Despite this interest, remarkably little is known about the normal germline function of most CT genes. Moreover, it has been demonstrated that germline genes in * Drosophila melanogaster * are required for the oncogenic process and that the human orthologues of these * Drosophila * genes have up - reg8laHed expression in a range of human cancers, although the functional implications for oncogenesis of this up - regulation remains unclear \ [[ @ R7 ], [@ R8] \ ]. Interestingly, down - regulation of a number of CT genes in human cancer cells results in perturbation of cellular proliferative potential \ [for example, see [@ R9 ], [@ R10] \ ]. These findings open up the exciting possibility that CT genes might encode functions that are required for tumour homeostasis and it has recently been proposed that tumours become ' addicted ' to these HerHline factors \ [[ @ R11 ], [@ R12] \ ], and recently, meiotic factors have been shown to contribute to telomere maintenance in cancer cells via the ALT pathway \ [[ @ R13 ], [@ R14] \ ]. The full extent of germline gene requirement is unclear, but these findings expose a new therapeutic opportunity by directly targeting the tumour - associated function of the CT gene products. Additionally, a number of studies have revealed another clinically important feature of CT genes; their expression appears to drive drug resistance as depletion of the gene products results in enhanced sensitization to anti - cancer drugs \ [for example, see [@ R15] \] expanding the therapeutic potential of this important class of cancer genes. Germline gene expression profiling has also recently been demonstrated to have applications in prognostics and patient stratification. In a seminal study, Rousseaux and co - workers demonstrated that expression of a sub - set of germ line genes in some lung cancers delineated patients with aggressive, metastasis prone tumours with poor prognosis \ [[ @ R16] \ ]; they extended this by indicating that this cohort of patients might benefit from a drug therapeutic regime that had previously been dismissed for more general use in lung cancer patients, indicating that profiling patients for expression of a small sub - set of germline genes could be used in therapeutic decision making. Understanding germline gene expression is also critical as drug - induced augmentation of expression has also been postulated to be a potential enhancer of immunotherapeutics, the rationale being that further up - regulation of a tumour - specific antigen will result in enhanced immunological targeting of the tumour \ [for example, see [@ R17] \ ]. Ta<lng all these factors together reveals the importance of understanding the regulatory mechanisms for somatic germline gene silencing and their aberrant activation in tumours. To date, the regulation of a number of CT genes has been studied and it has been demonstrated that DNA methylation of regulatory elements, such as promoter - associated CpG islands plays a fundamental role in the somatic silencing of these genes and the hypomethylation of these regulatory DNA regions in cancers is linked to gene activation \ [for example, see [@ R18] - [@ R23] \ ], whereas gene body hypomethylation has been linked to gene down regulation in cancers \ [[ @ R24] \ ]. Expression of these genes also becomes activated or further up - regulated upon enforced hypomethylation by the DNA methyltransferase inhibitor 5 - aza - 2 ′ - deoxycytidine (5 - aza - CdR ), and to date, all CT genes studied have up - regulated expression in response to this chemotherapeutic agent, indicating a commonality in the mechanistic pathway for somatic CT gene silencing \ [for example, see [@ R18] - [@ R23] \ ]. To date, most of the CT genes whose expression has been studied are located on the X chromosome (X - CT genes) and belong to large paralogous gene families \ [[ @ R2] - [@ R4] \ ]. Recently, a computational pipeline combining expressed sequence tag and microarray meta - analyses of the human orthologues of mouse spermatocyte - specific genes revealed a large cohort of new CT genes that were expressed in a broad spectrum of cancer types \ [[ @ R25] - [@ R29] \ ]. Unlike the X - CT genes, the majority of these genes are autosomally encoded and are single copy. To date, the c:inixal potential of these genes remains largely unexplored. In this current study, analysis of the expression of a small sub - set of these genes reveals a novel feature of CT genes, which indicates that some have a unique mechanism for somatic transcriptional silencing. This is a significant finding as these genes and their associated gene products have an increased prominence in clinical applications and hence the sub - classification of CT genes will play an important role in diagnostics, stratification and therapeutics. RESULTS {# s2} = = = = = = = All CT genes studied to date (mostly X - CT genes) require hypermethylation of regulatory DNA sequences for somatic silencing and are activated by the hypomethylating agent 5 - aza - CdR. Given the clinical potential of enhanced up - regulation of immunogenic CT antigens, we set out to explore whether a similar DNA hypermethylation silencing mechanism was operating in the recently identified autosomally encoded CT genes \ [[ @ R25 ], [@ R27] \ ]. To do this, we selwDted a small sub - group of these genes that remained transcriptionally silenced in the colorectal cancer cell lines HCT116 and SW480 (* ARRDC5, C4orf17, C20orf201, DDX4, NT5C1B, STRA8, TDRD12 * ). We also selected two previously characterized CT genes (both X - CT genes) that remained transcriptionally silenced in these two cell lines to serve as exemplar controls for hypermethylation regulated CT genes, * SSX2 * and * GAGE1 *. To determine whether the novel CT genes are silenced via hypermethylation mediated mechanisms, similar to the characterized X - CT genes, we treated the two cell lines with the DNA methyltransferase inhibitor 5 - aza - CdR to determine whether inhibition of DNA methyltransferase activity can activate these genes. Following 5 - aza - CdR treatment of HCT116 and SW480 we made cDNA and carried out RT - PCR and agarose gel electrophoresis analysis of the products. The two X - CT genes were activated from the silent state with relatively low levels of 5 - aza - CdR (0. 1 μM; Figure [1] (# F1) {ref - type = " fig " }; Figure [2] (# F2) {ref - type = " fig "} ). Some of the novel, autosomally encoded CT genes were similarly activated (* C20orf201, DVX$, STRA8, TDRD12 * ), although * C20orf201 * and * DDX4 * required a slightly higher 5 - aza - CdR concentration for activation (0. 5 μM; Figure [1] (# F1) {ref - type = " fig " }; Figure [2] (# F2) {ref - type = " fig "} ). Additionally, activation of * STRA8 * requires slightly higher concentrations of 5 - aza - CdR in SW480 (Figure [2] (# F2) {ref - type = " fig "} ) than HCT116 (Figure [1] (# F1) {ref - type = " fig "} ), which indicates subtle regulatory differences between tumour cell types. However, surprisingly, three genes (* ARRDC5, C4orf17, NT5C1B *) remained tightly transcriptionally silenced, even at high concentrations of 5 - aza - CdR in both cell lines (15. 0 μM; Figure [1] (# F1) {ref - type = " fig " }; Figure [2] (# F2) {ref - type = " fig "} ). This unexpected result reveals an important distinction in the way CT gene silencing is epigenetically regulated, revealing a hypermethylation - independent pathway. Interestingly, the X - CT genes (* GAGE1, SSX2 *) remained activated for a prolonged period following removal of the hypomethylating agent, as did the autosomally encoded CT genes that were activated with the lowest concentration of 5 - aza - CdR (* STRA8, TDRD12 *) (Figure [3] (# F3) {ref - type = " fig "} ); however, the other two autosomally encoded CT genes, * C20orf201 * and * DDX4 *, which required slightly higher concentrations of 5 - aza - CdR for activation, reverted to the silent state relatively soon after removal of the hypomethylating agent (Figure [2] (# F2) {ref - type = " fig "} ). This indicates a much greater transcriptional elasticity to the methylation - dependent silencing mechanisms for some CT genes. ! [A sub - group of germline genes remain refractory to activation by epigenetic modulating agents \ RT - PCR was used to analyse activation of a group of germline genes that are normally silenced in the cancer cell line HCT116 (an additional colorectal cell line gives similar results \ [see Supplementary Figure S1) \ ]. Whilst a cohort of known and newly identified germline genes become activated at low doses of the demethylating agent 5 - aza - CdR (* GAGE1, SSX2, STRA8, TDRD12 *) and others become activated with slightly higher levels of 5 - aza - CdR (* C20orf201, DDX4 * ), some remain tightly silenced, even at high concentrations of 5 - aza - CdR (* ARRDC5, C4orf17, NT5C1 | INTRODUCTION {#s1} ============ is driven by a multitude of complex, non-programmed molecular events, there are a number of key of process, not least of is the aberrant activation of genes that normally in a given tissue \[[@R1]\]. The so called (CT) or cancer germline (CG) genes are one such group of genes frequently activated in a range of different human cancer types \[[@R2]-[@R4]\]. These genes have expression normally restricted to the human germline, many testis-specific \[[@R2]-[@R4]\]. They have under intense scrutiny since their original identification as the immunological privilege of their normal means that the proteins they encode can elicit an immunological response when aberrantly produced in cancers and so have exceptional potential in immunotherapeutics example, the *NY-ESO-1* gene product has successfully targeted in an adoptive therapeutic approach to melanoma therapy \[[@R6]\]. Despite this interest, remarkably little is known about the normal germline function of most CT genes. Moreover, it has been demonstrated that germline genes in *Drosophila melanogaster* are required for the oncogenic process and that the human orthologues of these *Drosophila* genes have up-regulated expression in range of human cancers, although the implications for oncogenesis of this up-regulation remains unclear \[[@R7],[@R8]\]. Interestingly, of a number of CT genes in human cancer cells results in perturbation of cellular proliferative potential \[for example, see [@R9],[@R10]\]. These findings open the exciting possibility that CT genes might encode functions that are required for tumour homeostasis and it has recently been proposed that tumours become 'addicted' to germline factors \[[@R11],[@R12]\], and recently, meiotic factors have been shown to contribute telomere maintenance in cancer via ALT pathway \[[@R13], [@R14]\]. The extent of germline gene requirement is unclear, but these findings expose a new therapeutic opportunity by directly targeting tumour-associated function of the CT gene products. number of studies revealed clinically important feature of genes; their expression appears to drive drug resistance depletion of the gene products results in enhanced sensitization to anti-cancer drugs \[for see [@R15]\] expanding the therapeutic potential this class cancer genes. Germline gene profiling has also recently been demonstrated to have applications in prognostics and patient stratification. In a seminal study, Rousseaux and co-workers demonstrated expression of a sub-set of germ genes in some lung delineated patients with aggressive, metastasis prone tumours with poor prognosis \[[@R16]\]; they extended this by that this cohort of patients might benefit from a drug therapeutic regime that had been dismissed for more general use in lung cancer patients, that profiling patients for expression of a of germline genes could be used in therapeutic decision making. Understanding germline gene expression is also critical as augmentation of has also been to be a potential enhancer of immunotherapeutics, the rationale further up-regulation of a antigen will result in enhanced targeting of the tumour \[for example, see [@R17]\]. Taking all factors reveals the of understanding the regulatory mechanisms for somatic gene silencing and their aberrant activation in tumours. To date, the regulation of of CT has been studied and it has been demonstrated that DNA methylation of regulatory elements, such as promoter-associated CpG islands plays a fundamental role in the silencing of these genes and the hypomethylation of regulatory DNA regions in cancers is to gene activation example, see [@R18]-[@R23]\], whereas gene body hypomethylation has been linked to gene down regulation in cancers \[[@R24]\]. of these also becomes activated or further up-regulated upon enforced hypomethylation the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR), and to date, all CT genes studied have up-regulated expression in response to chemotherapeutic agent, a commonality in the mechanistic pathway for somatic CT gene silencing \[for example, see [@R18]-[@R23]\]. To date, of the CT genes whose expression has been studied are located on the chromosome (X-CT genes) belong to large paralogous gene families \[[@R2]-[@R4]\]. Recently, a computational pipeline combining expressed sequence and microarray of the orthologues of mouse spermatocyte-specific genes revealed a large cohort of new CT genes that were expressed in a spectrum cancer types \[[@R25]-[@R29]\]. Unlike the X-CT genes, the of these genes are autosomally encoded are single copy. To date, the clinical potential of these genes remains largely unexplored. In this current study, analysis of the expression of a small sub-set of these genes reveals a novel feature of CT genes, which indicates that some have a unique mechanism for somatic transcriptional silencing. a significant finding as these genes and their associated gene products have an increased prominence clinical applications and hence the sub-classification of CT will an important role in diagnostics, stratification and therapeutics. RESULTS {#s2} All CT studied to date (mostly X-CT genes) require hypermethylation of regulatory DNA sequences for somatic silencing and are activated by the hypomethylating agent 5-aza-CdR. Given the clinical potential enhanced up-regulation of immunogenic antigens, we set out to explore whether a similar DNA hypermethylation silencing mechanism was operating in the recently encoded CT genes \[[@R25],[@R27]\]. To do this, we selected a small sub-group of these that remained silenced in the colorectal cancer cell lines HCT116 and SW480 (*ARRDC5, C20orf201, DDX4, NT5C1B, STRA8, TDRD12*). We also selected two previously CT genes (both X-CT genes) that transcriptionally silenced in two cell lines to serve as exemplar controls for hypermethylation regulated CT *SSX2* and *GAGE1*. determine whether the novel CT genes are via hypermethylation mediated mechanisms, similar to characterized X-CT genes, we treated the two cell lines with the DNA inhibitor 5-aza-CdR to determine whether inhibition of DNA methyltransferase activity can activate these genes. Following treatment of HCT116 and SW480 we made cDNA and carried out RT-PCR and agarose gel electrophoresis analysis of the products. The two X-CT genes were activated from the silent state with relatively low levels of 5-aza-CdR (0.1 μM; Figure [1](#F1){ref-type="fig"}; Figure [2](#F2){ref-type="fig"}). Some of the novel, autosomally encoded CT genes were similarly activated DDX4, STRA8, TDRD12*), although *C20orf201* and required a slightly higher 5-aza-CdR concentration for activation (0.5 μM; Figure [1](#F1){ref-type="fig"}; [2](#F2){ref-type="fig"}). Additionally, activation *STRA8* requires slightly higher concentrations of 5-aza-CdR in SW480 (Figure [2](#F2){ref-type="fig"}) than HCT116 (Figure [1](#F1){ref-type="fig"}), which indicates subtle regulatory differences between tumour cell types. However, surprisingly, three genes (*ARRDC5, C4orf17, NT5C1B*) remained tightly transcriptionally silenced, even at high concentrations of 5-aza-CdR in both cell μM; Figure [1](#F1){ref-type="fig"}; Figure [2](#F2){ref-type="fig"}). This result reveals an distinction in the way CT gene silencing is epigenetically regulated, revealing a pathway. Interestingly, the X-CT genes (*GAGE1, SSX2*) remained activated a prolonged following removal of the as did the autosomally encoded CT genes that were activated with the lowest concentration of (*STRA8, TDRD12*) (Figure [3](#F3){ref-type="fig"}); however, the other two autosomally encoded CT genes, *C20orf201* and *DDX4*, which slightly higher concentrations of 5-aza-CdR for activation, reverted the silent state relatively after removal of the hypomethylating agent (Figure This indicates much greater transcriptional elasticity to the methylation-dependent silencing mechanisms for some CT genes. ![A sub-group germline genes remain refractory to by epigenetic modulating agents\ RT-PCR was used to analyse activation of a group of germline genes are normally silenced in the cancer cell line HCT116 (an additional colorectal cell line gives similar results \[see Supplementary Figure S1)\]. Whilst a cohort of known and newly identified germline genes become activated at doses of the demethylating agent 5-aza-CdR (*GAGE1, STRA8, and others become activated slightly higher levels of (*C20orf201, some remain tightly silenced, even at high concentrations of 5-aza-CdR C4orf17, NT5C1 | INtRodUCtiON {#s1}
============
whILsT oNCOgeNeSis iS DrIVEN By A MuLTitude of COmPLEX, noN-prOGRAMmED MoLEcULar EvENTs, TheRE Are A nUMber OF key featuReS of tHIS Process, noT LeaST of whIch is thE ABerraNT ActIvAtiON OF GEnes That WOULd nOrMaLLy bE SilencEd in A giveN tISSue cONTEXt \[[@r1]\]. thE SO CAllEd canCEr/tEStIS (CT) oR cAncER GErmLiNe (Cg) gEnEs arE one SUcH gROUP Of GenEs THat aRE fREqUENtLy ActIVated IN A rAnGe of DiffEREnt HUman cANcer tYPeS \[[@R2]-[@r4]\]. theSe genes HAvE EXPressIoN nOrMaLLY ReSTricteD To tHE HUmAn GErMLiNe, ManY BEiNG tEsTIs-sPEcifiC \[[@R2]-[@R4]\]. ThEy Have ComE under inteNSE ScrutIny sinCE THEiR ORigInAl iDentIFIcATIOn As tHe IMMUNOLOgICal pRIviLEgE oF tHeiR NorMAL gERmLINe SEtTinG mEanS ThaT thE PRotEInS thEy ENcodE cAN ELiCIt AN immUNologiCaL ReSPoNSE wHen ABeRRaNtly pRODUCED in CaNcErs aNd so hAvE eXcePTiONAL POTentIAl In immUnotHeRapeuTiCs \[[@r5]\]; fOR ExampLe, the *NY-ESo-1* gEne PRoDUcT HAS BeeN SUccessfULLy TARGeTEd In AN AdOptIvE THerAPEuTIc apProaCh To MElANOMA tHeRAPY \[[@r6]\].
DESpite tHIs inteREst, REMarKAbLY lITtlE iS KNOWn aBoUt ThE noRmal gerMLInE fuNctiOn Of mOST Ct GeNEs. moreoVEr, It HaS BEeN DeMoNSTRAted that GErMLinE gEneS iN *DrOsoPHILa mELANOgastEr* Are REquIrEd For ThE ONcOGENIc PRoCESs aNd That thE HUMAN oRTHoloGUES Of thESe *DROSOPHilA* geNes Have Up-rEgULAteD ExPreSsIOn iN A RaNgE Of HuMaN caNCerS, AlTHOuGh THe fUnctIoNAl implIcATiONs fOR onCOGENesiS of THIS up-REgulaTiON ReMAINs UncLeAR \[[@R7],[@R8]\]. inTeREstiNGly, DoWn-RegUlatIoN OF A NuMbEr of CT geNES iN HuMAN caNCer CelLs ResULTs IN pertUrBAtIon OF ceLluLAR PRolIFeraTIVe poteNtial \[FoR ExAmPLE, SeE [@r9],[@r10]\]. tHeSE fiNDINgS Open up thE eXCiTIng posSiBIlitY THAt Ct GENEs MIghT enCode fuNctIonS ThAT ArE rEqUired fOr tUMoUr HoMEOSTasIs aND IT haS rECeNTLY BeeN PRoPosed tHat TumoUrS BEcOMe 'ADdiCTEd' To tHeSE GeRmLInE fActors \[[@r11],[@r12]\], And rEcEntLy, MEiotIC faCTOrS HavE BEEn shOWN to ContRiBuTE tO tELOmEre maIntENANCe IN Cancer ceLls ViA The ALT PathWAy \[[@R13], [@R14]\]. thE FUlL ExTeNt of GeRMliNe GenE ReQUirEMeNt is unCLEar, bUt thesE FINdiNgS Expose A New TheRAPeutiC OPPOrTuniTY By DIrEctlY tArGeTIng thE TUMoUr-ASSOCiAteD FUNCtIoN OF the CT gENe pRODuCTs. ADDiTiOnaLlY, a numbER oF stUdIes HAVe reveALed aNOThER ClINIcAlly iMPOrTANt fEatUre OF cT genEs; Their exPrEsSIOn ApPears to dRIVE dRUg REsIsTANcE as depleTion OF The GEnE pRodUCtS ReSuLts in eNhANCeD sEnSItIzaTIoN TO anti-CaNcer drUGs \[foR eXAmple, see [@r15]\] expaNdiNg THE TheraPeuTIC pOtEnTIAl oF tHIS ImportanT ClasS of CAnCEr gEnEs.
gErmline gENE expResSION PrOFilInG HAS also REcEntLY BeEn deMoNSTRATed to HAVe APpLIcaTIoNs iN proGNoStics aNd pAtIenT straTIFicatION. IN a seMinal StuDy, rOUsSEaUx AnD Co-WOrkERs DemOnStraTeD thAT eXPRessiON OF a suB-sET of germ line gENEs iN SoMe LUnG caNCeRs dElInEaTED PaTienTS wItH aggResSiVE, MeTasTaSiS ProNE tumOURS wiTH poor prOGnOsIS \[[@R16]\]; THEy EXteNdeD thIS By iNDicAtINg thAT ThIS cOHOrT oF PAtieNts MIGHt bEnEfiT fROM a dRug THErAPEuTIc REgIME THAT haD prEVIOuSLy BeEn dISmISSEd foR More GEnErAl USe in lunG CAncer patieNTS, inDICaTIng tHaT pROfiLinG PatIEnTS for eXPREsSion oF a sMall SUB-sET oF gErMline GeNEs COULD Be USED In therapeUTiC deCISion mAKINg. UNdERstAnDINg gERMlINE gENe ExPrEsSiON iS alSo CRiTICAl As DRug-inDUCED AUGMeNTaTIoN Of ExPReSSion haS ALSO bEeN PosTULAted To Be A PoTeNtiAl enhAnceR Of imMUnotHeRAPEUtics, tHE ratiOnALE beINg ThAT FurthEr uP-REgULAtION OF A TuMOuR-sPECiFic antIgEN wIlL ReSuLt IN enHAnced iMMUNOLogiCaL TArGETiNg OF THe tUMoUr \[for eXAMple, sEE [@r17]\].
TakIng all These factoRs TogETHeR ReVeaLS the iMPoRtAnCE OF UnDeRstANdinG tHe rEgULatOrY MeCHANiSms For SomatIc gerMLinE gENe silenCING And tHeiR ABERRant activATiOn In TUmoUrs. To DaTE, THe rEGuLATiON of A number OF CT GeNES HaS bEEN sTuDiEd aNd IT has BEEn dEmONStRateD THAt Dna METhYLaTIon oF RegULatOry ElEmeNTS, SUcH As PRoMoteR-ASsOCIaTeD Cpg iSLaNds pLAys A funDameNTAL RoLe IN THe sOmaTiC siLEnCiNg of thESe geNEs AnD THe HYpoMEthylATION OF THESe ReguLatorY dNa rEgIOnS in CAnCErS is lInKEd To gENe ACTiVatIoN \[fOR examPlE, SEe [@R18]-[@R23]\], wHErEaS GEnE boDy hYpomethYLatION HAS BeeN lINKed tO GEnE dowN rEGUlATIon in CAncErs \[[@R24]\]. exprEssiOn OF thESE GEnES Also BecomeS aCtIVATed or FuRTHeR UP-rEGUlAteD UpOn enforCED HypoMEThyLaTIoN By tHe dNA MEtHyLTRanSFerASe inhIbItoR 5-AzA-2′-deOxYcytiDinE (5-aZa-Cdr), aNd TO DATe, AlL CT GenES stUdiEd have up-rEguLatED ExPResSiON IN REsPoNSE to ThiS CheMoThErAPEuTIC AgENt, iNdicAtInG A ComMOnalitY IN The mecHanistIc pATHway for sOmATIc CT GenE SiLENciNG \[FoR ExaMple, SEE [@r18]-[@R23]\].
tO dAtE, mosT Of THE Ct Genes WhoSE eXpRESSIoN hAS bEen stUdIEd aRE LOcATED oN tHe X cHroMosOMe (X-CT gEnES) And beLoNG to Large PARalOGOus genE FamilIeS \[[@r2]-[@r4]\]. RecenTly, a COmPutaTiONAL PipeLIne coMBiNinG exPresSed seqUEnCE tAg AND MiCroARRAY MEtA-AnaLYSes of the HuMaN oRthOLoGUES oF mOUSe sPERmaTocyTE-SPeCIfIC GEnes REvEaLed a LarGe coHOrt oF New cT gENeS thAt WErE ExPRESSeD in A bRoaD sPeCtrum Of caNCeR TypES \[[@r25]-[@r29]\]. unLiKE tHE X-cT GeNeS, the MAjorIty of THEse gENes aRE AuTosOmalLy EnCODed aNd ARe sINGlE CoPy. to daTe, tHe cLInIcAl PotENTIaL of tHesE gEnEs rEmAINS LarGely UNexPloreD. in THis CUrREnT STudy, AnaLysIs of The expreSSioN Of A sMAlL Sub-SeT Of TheSE GEnES revEalS A NovEL fEaTuRE oF cT GenES, WhICH indICATeS tHat SOME have A uNIqUE MEcHanISM foR sOMATiC TrAnscRiPTIoNAl SIlEnCinG. tHIs is A signIFIcanT finDing as tHEse GEnEs And Their ASsoCIAteD gEnE prOducTs HaVE aN inCReaSED ProMiNENce IN ClinicaL apPlICaTions And hEnce THE sUb-cLaSSIFiCATiON oF cT gENes wilL PlAy an imPoRtANt RoLe IN DIagnOstICs, stratIFicatioN and tHERAPeuTicS.
ReSuLts {#S2}
=======
all cT gENEs sTudIeD tO Date (mOStlY x-CT GeNes) rEQuirE HypErMEtHYLAtiOn OF ReGULaToRy DNA SeqUEnCeS foR somATIC sILeNCInG AnD aRE ACtivATeD BY The hyPoMethyLAtIng AGent 5-azA-cdr. GIVEN ThE CliNiCAL POTeNtIal OF enhANcED Up-ReGUlAtION oF iMMuNoGeniC Ct anTIGEnS, WE set OUT tO EXpLOre whetHer A SImIlAR dNa hyPErmetHylAtIoN siLencInG MecHANism WAs oPeratiNg in The recentLy IdEntIfiEd AUtOsOmalLY ENCoDED CT geNES \[[@R25],[@r27]\]. to Do thiS, We sElecteD a smalL sub-group Of thEsE GEnes THat RemAInED TranscRIptIOnaLlY SILenCEd IN tHE COLOReCtal cAncER CElL LineS hCt116 and Sw480 (*arRdc5, c4Orf17, C20ORf201, DdX4, nT5C1B, Stra8, tDrD12*). WE ALso SELectED Two pRevIOUsLY ChARAcTERIzEd CT gEneS (bOth X-ct GenES) tHAt reMaInEd tranScriPTioNalLY sILENCed in These TWO celL LInES To sErVe As exEMPlaR coNTRolS foR HYpERMeThyLAtIoN reGulatED cT geNeS, *sSX2* and *GaGE1*. TO dEtErMinE wheTHeR tHE NoVEL cT GeneS aRE SiLeNcEd vIa hYpeRMEThYLATIoN MedIAted MecHANisMs, Similar to THE CHaracTERIzed x-Ct GenES, We TreAteD tHE tWO cElL lInEs WIth tHe dNa meTHYLtRansFeRaSE inhIbITOr 5-Aza-CdR TO DeTERMInE WHEtheR inHibitION Of Dna MEthylTRansfeRaSE aCTivITy CAN aCtIvATE thEse geNeS. FollOwing 5-aza-cDr TrEatMeNT of Hct116 anD Sw480 WE made cDNa AND carRiED OUT rt-PCR anD AgARosE gEl ELectropHoReSis ANalYsis Of thE PROduCTS. The TWo X-Ct genEs wEre aCTIvAtEd frOm ThE sIleNt state with RElatIvely loW lEvEls OF 5-Aza-cdR (0.1 ΜM; FigURE [1](#F1){Ref-TYpe="FIg"}; FIGUrE [2](#F2){reF-TyPe="fIg"}). sOmE Of THe NOVEL, AutOsOMAlLY enCodED Ct geNeS wERE siMILArLY AcTiVaTed (*C20oRF201, dDx4, STrA8, tdRD12*), AlTHouGh *c20OrF201* And *dDx4* RequIrEd A slIghtLy hIGHER 5-AzA-CdR concEntRATIon FOr aCtIVATIoN (0.5 ΜM; Figure [1](#f1){ref-tyPE="fIG"}; fiGuRe [2](#f2){Ref-tYPe="FiG"}). ADDitIoNaLLy, ACTiVatIOn OF *StrA8* ReqUIRES SlIGHTLY hiGher CONcenTRaTionS OF 5-aZa-CDr IN SW480 (FigURe [2](#f2){Ref-TyPe="fig"}) ThAN hcT116 (FIgURE [1](#F1){rEf-type="FIg"}), WhIch indICaTeS suBtLe RegulAtoRy diFFErenceS betwEen tUMOuR cell tYpEs. hOWeVER, SURpRISINGlY, ThreE geNEs (*arRdc5, c4oRf17, NT5c1B*) REMaINED tighTly traNscrIPTiONAlLy sIlenCEd, EVEn at HiGH COnCenTrATIonS Of 5-AzA-cdr In BOTH Cell lIneS (15.0 μm; FiGUrE [1](#F1){REF-TYPe="fIG"}; FIgURe [2](#F2){ref-TYPe="FiG"}). tHiS unexPECteD ReSUlT rEVeAls An ImpORtAnt DIstIncTiOn IN ThE Way CT Gene SILeNCInG Is ePigeNEtICaLly RegULated, rEVeaLiNG a hYPeRMETHYlATiON-iNDEPeNdenT PAtHwAy. intereStINgLy, THe X-CT gEneS (*gAgE1, sSX2*) reMaiNED aCTIVAted For A PRoLOnged pERIOD fOLlOwiNG RemovAl OF THE HyPoMEThyLaTINg aGEnT, as dID ThE auTOsomaLlY EncoDed Ct gEnEs ThAT weRE aCTiVaTED WITH tHE LOWeST coNcentrAtion OF 5-AZA-cDR (*STRa8, TDRD12*) (fIguRE [3](#f3){ref-tYPe="fIG"}); hOWEver, the oThEr Two AuTosoMALLy enCoded Ct geneS, *C20Orf201* aNd *ddX4*, WHIch requireD SLIGhtlY HIGHer ConcEntrATions oF 5-aZA-cdR foR aCtivaTIOn, reverTed To thE SilEnT STAtE ReLatIVely SooN AFTer rEMovAL Of tHe HYPometHylATing AgenT (FIGUrE [2](#f2){ReF-type="fIg"}). tHIs indIcates a MUch GReAter TrANScriPtioNaL ElaSTiCitY To thE metHYLAtIon-dEPEndeNt SIlEncing MEcHanIsmS fOR SOMe Ct genES.
![A sub-GroUP oF gERMLiNe geNEs rEmAiN rEFRaCtOrY to aCtIVAtIoN By ePigenEtIc MODuLaTIng aGEnts\
rT-pcr WAs used TO aNALYse aCTivaTIon of a gROUp oF GermliNe GeNeS THAT are nORMAlly SIlEnCEd IN The CanCer CELl Line HCt116 (AN adDiTIonAl CoLOrECtaL ceLL liNe givEs SimilAr REsULts \[See supplemENTARy FigURE s1)\]. WHILSt A CoHOrT Of KNOWn ANd nEWLy IDentiFiED GERMLINE GeNes beCOmE ACtivAtEd at low dOSES oF The DEMETHyLatiNg AGEnt 5-AZA-CDR (*gAGe1, sSX2, StrA8, tDRd12*) aNd OTHeRs BEcOmE aCtiVaTED WITh slighTLy higHEr lEvelS of 5-aZa-cDR (*c20ORf201, ddX4*), SOmE remAIN TigHtlY siLEnCeD, EVen aT hIgH CoNCEntRAtioNs OF 5-AZa-Cdr (*ArRdC5, C4OrF17, nT5C1 | INTRODUCTION {#s1} ============ Whilst oncogenesis is drivenbya multitude of complex, non-programmed molecular events, there are anumber of key features of thisprocess, not least of which isthe aberrantactivation of genes that would normally be silenced in a given tissue context \[[@R1]\].The so called cancer/testis (CT) or cancer germline (CG) genes are one such group of genes that are frequently activated in a range of differenthuman cancer types\[[@R2]-[@R4]\].These geneshave expression normally restrictedto the human germline,many being testis-specific \[[@R2]-[@R4]\]. They have come under intensescrutiny sincetheir originalidentification as the immunological privilege of their normal germline setting meansthat theproteins they encode can elicitanimmunological response when aberrantly produced in cancers and so have exceptional potential in immunotherapeutics\[[@R5]\]; for example, the *NY-ESO-1* gene producthas beensuccessfully targeted in an adoptivetherapeutic approach to melanoma therapy \[[@R6]\].Despite this interest, remarkably little isknown about the normal germline function of most CT genes. Moreover,ithas been demonstrated that germline genesin *Drosophila melanogaster* are requiredfor the oncogenic process and that the human orthologues of these *Drosophila* genes have up-regulated expression ina rangeof human cancers, although the functional implications for oncogenesis of this up-regulation remains unclear \[[@R7],[@R8]\]. Interestingly, down-regulation of a number of CT genes in humancancer cells resultsin perturbation ofcellular proliferative potential \[for example, see [@R9],[@R10]\]. These findingsopen up theexciting possibility thatCT genes might encode functions that are required fortumourhomeostasis and it has recently been proposed thattumours become'addicted' to these germline factors \[[@R11],[@R12]\],and recently, meiotic factors have beenshown to contribute to telomere maintenance in cancer cells via the ALT pathway \[[@R13], [@R14]\]. The full extent of germline gene requirementis unclear, but these findings expose a newtherapeutic opportunity by directly targeting the tumour-associated function ofthe CT gene products. Additionally,a number of studies have revealed another clinically important featureof CTgenes; their expression appears to drive drugresistance as depletion of the gene products results in enhanced sensitization to anti-cancer drugs \[for example, see [@R15]\] expanding thetherapeutic potential of this importantclass ofcancer genes. Germline gene expressionprofilinghas also recently been demonstrated to have applications in prognostics and patient stratification. In a seminal study, Rousseaux and co-workers demonstrated that expression of a sub-set of germ linegenesin some lungcancers delineated patients with aggressive, metastasis prone tumours with poor prognosis\[[@R16]\]; theyextended this by indicatingthat thiscohort of patients might benefit from a drug therapeutic regimethat had previously beendismissed for more general use in lung cancer patients, indicating thatprofiling patients for expression of a small sub-set of germline genes could be used in therapeuticdecision making. Understanding germline geneexpression is also critical as drug-induced augmentationof expression hasalso been postulated to be a potential enhancer of immunotherapeutics, the rationale being that further up-regulationof a tumour-specific antigen will result in enhancedimmunological targeting of the tumour \[for example, see [@R17]\]. Taking all these factors together reveals the importance ofunderstanding the regulatory mechanismsforsomatic germline gene silencing andtheir aberrant activation in tumours. To date, the regulation ofa number of CT genes has been studied and it has beendemonstrated that DNA methylation of regulatory elements, such as promoter-associated CpG islands plays a fundamental role in the somatic silencing of these genes andthe hypomethylationof these regulatory DNA regions incancers is linked togene activation \[for example, see [@R18]-[@R23]\], whereas gene body hypomethylation has been linked to gene down regulation in cancers \[[@R24]\]. Expression of these genesalso becomes activated or furtherup-regulated uponenforced hypomethylation by the DNA methyltransferase inhibitor5-aza-2′-deoxycytidine (5-aza-CdR), and todate, all CT genes studied have up-regulatedexpression in responseto this chemotherapeutic agent, indicating a commonality in the mechanistic pathway for somatic CT gene silencing\[for example,see [@R18]-[@R23]\]. To date, most of the CT geneswhose expression has been studiedare located on the X chromosome (X-CTgenes) and belongto large paralogous gene families \[[@R2]-[@R4]\]. Recently, a computational pipeline combiningexpressed sequencetagand microarraymeta-analyses of the human orthologues of mouse spermatocyte-specificgenes revealeda large cohort of new CT genes that were expressed in a broad spectrum of cancer types \[[@R25]-[@R29]\]. Unlike the X-CT genes, the majority of thesegenes are autosomally encoded and are single copy. To date, the clinicalpotential of these genes remains largely unexplored. In this current study, analysis of the expression of a small sub-set of these genes reveals a novel feature of CTgenes, whichindicates that some have a uniquemechanism forsomatic transcriptional silencing. Thisis a significant finding as these genesand their associated geneproducts have an increased prominence inclinical applications andhence thesub-classification of CT genes will playan important role indiagnostics,stratification and therapeutics. RESULTS {#s2} ======= All CT genes studiedto date (mostly X-CT genes) require hypermethylation of regulatory DNA sequences for somaticsilencing and areactivated by thehypomethylating agent 5-aza-CdR. Given the clinical potential of enhanced up-regulation of immunogenicCT antigens, weset out to explore whether a similar DNA hypermethylation silencing mechanism wasoperating in therecently identified autosomallyencodedCT genes \[[@R25],[@R27]\]. To do this, we selected a small sub-group of these genes that remained transcriptionally silenced in the colorectal cancer cell linesHCT116and SW480(*ARRDC5, C4orf17, C20orf201, DDX4, NT5C1B, STRA8, TDRD12*).We also selected two previously characterized CT genes(both X-CT genes) that remainedtranscriptionally silenced in these two celllinesto serveas exemplar controls for hypermethylation regulated CT genes, *SSX2* and*GAGE1*. To determine whether thenovel CT genes are silenced via hypermethylation mediated mechanisms, similar tothe characterized X-CT genes,we treated the two cell lines with theDNA methyltransferase inhibitor 5-aza-CdR to determine whether inhibition of DNA methyltransferase activity canactivate thesegenes. Following 5-aza-CdR treatment ofHCT116 and SW480 we made cDNA and carried out RT-PCR and agarose gel electrophoresis analysis of the products. The two X-CT genes were activated from the silent state with relatively lowlevels of 5-aza-CdR (0.1μM;Figure [1](#F1){ref-type="fig"}; Figure [2](#F2){ref-type="fig"}). Some of the novel, autosomallyencoded CT geneswere similarly activated(*C20orf201, DDX4, STRA8, TDRD12*), although *C20orf201* and *DDX4* requireda slightly higher 5-aza-CdR concentration for activation (0.5 μM; Figure [1](#F1){ref-type="fig"}; Figure[2](#F2){ref-type="fig"}). Additionally, activation of *STRA8* requires slightly higher concentrations of 5-aza-CdR inSW480(Figure [2](#F2){ref-type="fig"})than HCT116 (Figure [1](#F1){ref-type="fig"}), which indicates subtle regulatory differences between tumourcell types. However, surprisingly, three genes (*ARRDC5, C4orf17, NT5C1B*) remained tightly transcriptionallysilenced, even at high concentrations of 5-aza-CdR in both cell lines (15.0 μM; Figure [1](#F1){ref-type="fig"}; Figure [2](#F2){ref-type="fig"}). This unexpected result reveals an importantdistinction in the way CT gene silencing is epigeneticallyregulated, revealing a hypermethylation-independent pathway. Interestingly, the X-CT genes (*GAGE1, SSX2*) remainedactivated for a prolongedperiod following removal of the hypomethylating agent, as did the autosomally encoded CT genes that were activated with the lowest concentrationof 5-aza-CdR (*STRA8, TDRD12*) (Figure [3](#F3){ref-type="fig"}); however, the other two autosomally encoded CT genes,*C20orf201* and *DDX4*, whichrequired slightly higherconcentrations of 5-aza-CdR for activation, reverted to the silent state relatively soon after removal of the hypomethylating agent (Figure [2](#F2){ref-type="fig"}). This indicates amuchgreater transcriptional elasticity to the methylation-dependent silencingmechanisms for some CT genes. ![A sub-group of germlinegenes remain refractory to activation by epigenetic modulating agents\ RT-PCRwas used to analyse activation of a group of germline genesthatare normally silenced in the cancer cell line HCT116 (an additional colorectal cell line gives similar results\[see Supplementary Figure S1)\]. Whilst a cohort of known and newly identified germline genes become activated at low dosesof the demethylating agent 5-aza-CdR (*GAGE1, SSX2, STRA8, TDRD12*) and others becomeactivated with slightly higherlevelsof 5-aza-CdR (*C20orf201, DDX4*), some remaintightly silenced, even at high concentrations of5-aza-CdR (*ARRDC5, C4orf17, NT5C1 | _INTRODUCTION_ {#s1} ============ _Whilst_ oncogenesis is _driven_ by a multitude of complex, non-programmed molecular events, there are a number _of_ _key_ features of this process, not least _of_ which is the _aberrant_ activation _of_ genes that would normally be _silenced_ in _a_ _given_ tissue context \[[@R1]\]. _The_ so called _cancer/testis_ (CT) or cancer germline (CG) genes are one such group _of_ genes that are frequently activated in a range _of_ _different_ human cancer types \[[@R2]-[@R4]\]. These genes have expression normally _restricted_ to the _human_ germline, many being testis-specific \[[@R2]-[@R4]\]. They _have_ come under intense scrutiny since their _original_ identification as the immunological privilege of their normal germline setting means that _the_ proteins they _encode_ can elicit an _immunological_ response when aberrantly produced _in_ cancers and so have exceptional potential in _immunotherapeutics_ \[[@R5]\]; _for_ example, the *NY-ESO-1* gene _product_ _has_ been successfully targeted in an adoptive therapeutic approach to melanoma therapy \[[@R6]\]. Despite this interest, _remarkably_ little is known about the _normal_ germline function of most CT _genes._ Moreover, it _has_ been demonstrated that germline _genes_ in *Drosophila _melanogaster*_ are required _for_ the oncogenic process _and_ that the human orthologues of _these_ _*Drosophila*_ genes have up-regulated expression _in_ a range of human cancers, although the functional implications for oncogenesis of _this_ up-regulation remains _unclear_ \[[@R7],[@R8]\]. _Interestingly,_ down-regulation of a number _of_ CT genes in human cancer _cells_ results in perturbation of cellular proliferative potential _\[for_ example, see [@R9],[@R10]\]. These findings open _up_ the exciting possibility that CT genes _might_ encode functions _that_ are required for tumour homeostasis and it has recently been proposed that tumours become 'addicted' _to_ these _germline_ factors \[[@R11],[@R12]\], and recently, _meiotic_ factors _have_ _been_ shown to contribute to telomere maintenance _in_ _cancer_ cells via _the_ ALT pathway \[[@R13], _[@R14]\]._ The full extent of germline gene _requirement_ is _unclear,_ but these _findings_ expose a new therapeutic opportunity by directly targeting the tumour-associated function of _the_ CT gene products. Additionally, a number _of_ studies have _revealed_ another clinically important feature of CT genes; their expression appears to drive drug resistance as depletion of the _gene_ products results _in_ enhanced _sensitization_ _to_ _anti-cancer_ drugs _\[for_ _example,_ see [@R15]\] expanding the therapeutic potential of this important class of cancer genes. _Germline_ _gene_ expression profiling has also recently been demonstrated to have applications in prognostics and _patient_ stratification. In a seminal study, Rousseaux _and_ co-workers demonstrated that expression of _a_ sub-set of _germ_ line genes _in_ _some_ lung cancers delineated patients with aggressive, metastasis prone tumours _with_ poor prognosis \[[@R16]\]; they extended this _by_ indicating that this _cohort_ of patients might benefit from a drug therapeutic regime _that_ _had_ _previously_ been dismissed for more general use _in_ lung cancer patients, _indicating_ that _profiling_ patients for _expression_ of a _small_ sub-set of germline genes could be used _in_ therapeutic _decision_ making. Understanding germline gene expression _is_ also critical as drug-induced augmentation of expression has also been postulated to be a potential enhancer of immunotherapeutics, the rationale being _that_ further up-regulation of a _tumour-specific_ antigen will result in enhanced immunological targeting of _the_ _tumour_ \[for example, see [@R17]\]. Taking all these factors together reveals the importance of understanding the regulatory mechanisms for _somatic_ germline _gene_ silencing and their aberrant _activation_ _in_ tumours. To date, the regulation _of_ a number _of_ _CT_ _genes_ has been studied _and_ _it_ _has_ been demonstrated that DNA methylation of regulatory elements, such _as_ promoter-associated CpG islands plays a fundamental role in the somatic silencing of these genes and _the_ hypomethylation of these regulatory DNA _regions_ _in_ _cancers_ is linked to gene activation \[for example, see [@R18]-[@R23]\], whereas gene body hypomethylation has been linked to gene down _regulation_ in cancers \[[@R24]\]. Expression of these _genes_ _also_ becomes activated or further up-regulated upon _enforced_ hypomethylation by the DNA _methyltransferase_ inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR), and to _date,_ all _CT_ genes studied _have_ up-regulated expression in _response_ to this chemotherapeutic agent, indicating a commonality in the mechanistic _pathway_ _for_ _somatic_ CT gene silencing \[for _example,_ see [@R18]-[@R23]\]. To _date,_ most of the _CT_ genes whose expression has been studied are located on the X chromosome (X-CT genes) and belong _to_ large paralogous gene _families_ _\[[@R2]-[@R4]\]._ _Recently,_ a computational _pipeline_ combining expressed sequence tag and _microarray_ meta-analyses of the human _orthologues_ _of_ mouse spermatocyte-specific genes revealed a _large_ cohort of new CT genes that were expressed _in_ _a_ _broad_ spectrum of cancer types \[[@R25]-[@R29]\]. Unlike the X-CT genes, the majority of these genes are autosomally encoded and are single copy. To _date,_ the clinical potential of _these_ genes remains largely unexplored. In this current study, analysis of the expression of a small sub-set of _these_ genes reveals a novel feature of _CT_ genes, which indicates that some have _a_ unique mechanism _for_ somatic transcriptional silencing. This is a significant finding as these genes and their _associated_ _gene_ products have an increased prominence in clinical applications _and_ hence the sub-classification of _CT_ genes will play an important role in _diagnostics,_ stratification and therapeutics. RESULTS {#s2} ======= All CT genes studied to date _(mostly_ X-CT genes) require hypermethylation _of_ regulatory DNA sequences for somatic silencing and are activated by the hypomethylating agent 5-aza-CdR. Given the clinical potential _of_ enhanced up-regulation of immunogenic CT antigens, we set out to explore _whether_ a similar DNA hypermethylation silencing mechanism was operating in the recently identified autosomally _encoded_ _CT_ genes \[[@R25],[@R27]\]. _To_ do this, we selected _a_ small sub-group of these genes that remained transcriptionally silenced in the colorectal cancer cell lines HCT116 and _SW480_ (*ARRDC5, C4orf17, C20orf201, _DDX4,_ NT5C1B, _STRA8,_ TDRD12*). We also selected _two_ previously characterized CT genes (both _X-CT_ genes) _that_ remained transcriptionally silenced in these two _cell_ _lines_ to _serve_ _as_ exemplar controls for hypermethylation _regulated_ CT genes, *SSX2* and *GAGE1*. To determine whether _the_ novel CT genes are silenced via hypermethylation mediated mechanisms, similar to the characterized X-CT _genes,_ we treated the two cell lines with the DNA methyltransferase inhibitor 5-aza-CdR _to_ determine whether inhibition of DNA _methyltransferase_ activity _can_ activate these genes. Following 5-aza-CdR treatment _of_ HCT116 and SW480 _we_ _made_ cDNA and carried _out_ RT-PCR and agarose gel electrophoresis analysis of the products. _The_ two X-CT genes were activated from the silent state with relatively low levels _of_ _5-aza-CdR_ (0.1 _μM;_ Figure _[1](#F1){ref-type="fig"};_ Figure [2](#F2){ref-type="fig"}). Some _of_ the novel, autosomally encoded CT genes were similarly activated (*C20orf201, _DDX4,_ STRA8, TDRD12*), although _*C20orf201*_ _and_ *DDX4* required a slightly higher 5-aza-CdR concentration _for_ _activation_ _(0.5_ μM; _Figure_ [1](#F1){ref-type="fig"}; Figure [2](#F2){ref-type="fig"}). Additionally, activation of *STRA8* requires slightly higher _concentrations_ of 5-aza-CdR in _SW480_ (Figure [2](#F2){ref-type="fig"}) than _HCT116_ (Figure [1](#F1){ref-type="fig"}), which _indicates_ _subtle_ regulatory differences _between_ tumour cell types. However, _surprisingly,_ three genes (*ARRDC5, C4orf17, NT5C1B*) remained tightly _transcriptionally_ silenced, _even_ at high concentrations of 5-aza-CdR _in_ both cell lines _(15.0_ μM; Figure _[1](#F1){ref-type="fig"};_ Figure [2](#F2){ref-type="fig"}). This unexpected result reveals an important distinction in the _way_ CT gene silencing is epigenetically _regulated,_ revealing a _hypermethylation-independent_ pathway. Interestingly, the X-CT genes _(*GAGE1,_ _SSX2*)_ remained activated for a prolonged period _following_ removal of the hypomethylating agent, as did the autosomally encoded CT genes that were _activated_ with the lowest concentration _of_ 5-aza-CdR (*STRA8, TDRD12*) (Figure [3](#F3){ref-type="fig"}); however, the other two _autosomally_ encoded CT genes, *C20orf201* and *DDX4*, which _required_ slightly higher concentrations of 5-aza-CdR for _activation,_ reverted to the silent state relatively soon after _removal_ _of_ _the_ hypomethylating _agent_ (Figure [2](#F2){ref-type="fig"}). This indicates a _much_ greater transcriptional elasticity to the methylation-dependent silencing _mechanisms_ for some CT _genes._ ![A sub-group of _germline_ genes remain refractory to activation by epigenetic modulating agents\ _RT-PCR_ was used to analyse activation of a _group_ of germline genes that are _normally_ silenced in the cancer cell line HCT116 (an additional colorectal cell line gives similar results \[see Supplementary Figure S1)\]. Whilst a _cohort_ of known and newly identified germline genes become activated _at_ low doses of the _demethylating_ _agent_ _5-aza-CdR_ (*GAGE1, SSX2, STRA8, _TDRD12*)_ and others become activated with slightly higher levels of 5-aza-CdR (*C20orf201, DDX4*), _some_ remain tightly silenced, even at high concentrations of 5-aza-CdR (*ARRDC5, C4orf17, _NT5C1_ |
Introduction
============
Gingival reactive lesions like pyogenic granuloma have frequent occurrence around natural dentition, however, their association with dental implants is not common. The causes of pyogenic granuloma (PG) in relation to dental implants are not clear mainly due to few published cases ([@B1]-[@B8]).
Tooth-related PG is a result of tissue response to minor injury or chronic low-grade irritation ([@B9]-[@B16]). Clinically, oral PG is characterized as a soft mass of smooth or lobulated appearance that could be sessile or pedunculated and frequently presents ulceration. The lesion grows rapidly for a few weeks and the colour ranges from pink to red purple and haemorrhage may occur either spontaneously or after minor trauma ([@B8]). Its incidence is relatively common and accounts for 3.81-7% of all biopsies harvested from the oral cavity ([@B13]-[@B16]).
Microscopically, the lesion is characterized by prominent capillary growth in hyperplastic granulation tissue, which suggests a strong activity of angiogenesis. The blood vessels often show a clustered or medullary pattern separated by less vascular fibrotic septa, leading some authorities to consider PG as a polypoid form of capillary hemangioma ([@B17]).
The lesions of PG may be found in the oral cavity or extraorally. The most frequent intraoral localization is the gingiva (about 60-70%), but lesions can occur on the lips (14%), tongue (9%), buccal mucosa (7%) and palate (2%) ([@B18]-[@B24]). Possible treatment methods are excision, curettage, cryotherapy, sclerotherapy, chemical and electrical cauterization, cryotherapy and the use of lasers with the carbon dioxide (CO2) or argon ([@B25]-[@B29]). Conservative local excision is the preferred form of treatment and recurrence rates after excision range from 0% to 16% ([@B29]).
However, to the best of the authors' knowledge, only 5 cases of pyogenic granuloma in association with a dental implant have been reported in the international literature ([@B1]-[@B3],[@B7],[@B8]). Within the context of the scarce information available on these lesions, the aim of the present study was to report 10 novel clinical cases of pyogenic granuloma in association with titanium dental implants and to elucidate potential risk factors. Finally, the presence of marginal bone loss was evaluated.
Material and Methods
====================
Patients charts at the service of oral medicine of Anitua's Dental Clinic (Alava, Spain) were revised from 1991 to 2011. Patients selection was based on the following inclusion criteria:
• Treatment of pyogenic granuloma.
• The presence of histopathological diagnosis.
• Lesion in relation to dental implants.
All patients who did not fulfill all inclusion criteria were excluded from the study.
Data were collected to report on patient age, gender, patient´s disease, lesion site, type of dental implant (surface and morphology), predisposing factors (trauma, prosthesis type, poor oral hygiene), clinical and radiographic features, diagnosis, treatment and recurrence. Orthopantomography (OPG) of all lesions were examined to compare the presence or absence bone resorption around dental implants.
A descriptive statistical analysis of all variables were performed. Then the relationship between PG and marginal bone loss was analyzed by nonparametric Spearman correlation. The effect of surface type on marginal bone loss was also analyzed with one-way ANOVA and Levene post hoc test. The statistical significance was set at *p*-value \< 0.05. All the statistical analyses were performed using the SPSS v15.0 for Windows statistical software package (SPSS Inc., Chicago, IL, USA).
Results
=======
Ten patients with pyogenic granuloma in relation to dental implants had been identified. They were 2 males and 8 females. Patients' age ranged from 21 to 92 years and all were non-smokers. Five of the ten patients (50%) had systemic disorders: cardiac arrhythmia (1 patient), hypertension (2 patients), atrial fibrillation (2 patients), Type II diabetes mellitus (2 patients), hepatitis C (1 patient ), hypothyroidism (1 patient). Within the group of patients with systemic disease, 3 of them were using 1 to 2 drugs daily, whereas the remaining patient took more than 2 drugs.
With regard to oral hygiene habits, 20% of patients reported to brush once a day, 50% did twice daily and 30% brushed three times a day. A 90% of the patients received professional prophylaxis twice a year and the other 10% once a year. In the use of hygiene products the obtained results were as follows: a) use of mouthwash: only was used by 3 patients (37.5%), b) use of dental floss: only one patient (12.5%), and c) interproximal brushes: 3 patients (37.5%).
The distribution of PG lesions was even between maxilla and mandible (50% for each region), and the most common oral site affected by PG was the area of tooth 41 (2 cases).
The development of PG was related to only accumulation of dental plaque (one patient), bad prosthetic design (one patient), and both factors (one patient). In 4 patients, there had been a combination of tissue pressure by the prosthesis and poor oral hygiene. However, no etiological factor could be related to the development of PG in 3 patients. The clinical size of the lesions ranged from 1.1 x 0.6 mm to 36 x 19 mm. The mean diameter was 7.2 mm. All the lesions were excised and sent for histological examination. The defects were covered with a autologous fibrin membrane (Anitua's protocol). During the first week after the operation, all patients were given analgesic and 0.2% chlorhexidine gluconate mouthwash. During the follow-up period (range two months to 10 years), there were no recurrences.
The histopathological reports indicated the diagnosis of PG and the description of highly vascular proliferation that resembles granulation tissue (Fig. [1](#F1){ref-type="fig"}).
Figure 1Histological images of the pyogenic granuloma showing an appearance similar to granulation tissue. The histological type of the pyogenic granuloma is non-lobular capillary hemangioma. Arrow heads label blood vessels surrounded by connective tissue.
The surfaces of the implants associated with the lesion were smooth (2 implants), machined (3 implants) and rough (5 implants). In no case there was a natural tooth adjacent to the implants related to the lesion. The characteristics of diameters and lengths of the implants studied can be seen in figure [2](#F2){ref-type="fig"}. The average load time of the implants studied was 115 months (SD = 67.5), ranging from a range of 9 to 184 months. Oral rehabilitation was performed with complete prosthesis in 9 patients. The mean mesial bone loss was 2.14 mm (range 0 to 6.50 mm, SD = 2.07) and the mean of distal bone was 1.66 mm (range 0 to 3.75 mm, SD = 1.21.
Figure 2Diameter and length of dental implants related to the pyogenic granuloma.
There were no statistically significant association between the PG area and the marginal bone loss. However the smooth implant surface showed a significant influence on bone loss (Anova: *p* = 0.001) (Fig. [3](#F3){ref-type="fig"}).
Figure 3Peri-implant bone loss grouped by type of surface. The bone loss was the highest for implants with smooth surface.
Discussion
==========
The clinical and histopathological findings have confirmed the diagnosis of pyogenic granuloma in 10 patients. The present study is the one with the highest number of implant-related PG lesion that are available until now in the scientific literature. These PG lesions have been diagnosed as non-lobular capillary hemangioma. There are two histological types of PG. The first type is characterized by proliferating blood vessels that are organized in lobular aggregates. This histological type of PG was called lobular capillary hemangioma (LCH type). The second type (non-LCH type) consist of highly vascular proliferation that resembles granulation tissue ([@B1],[@B4],[@B11]).
Literature data indicated that PG is rarely associated with dental implants, as there are only five cases reported ([@B1]-[@B3],[@B7],[@B8]). However, other reactive lesions such as gingival hyperplasia caused by phenytoin, allergy to titanium abutments or peripheral giant cell granulomas have been reported in the international literature. Causes of conventional oral pyogenic granulomas are not clear, although it has been shown that different stimuli irritants that can trigger them, such as repeated trauma, poor oral hygiene and hormonal problems ([@B1]-[@B20]). About 30-50% of patients with PG have a history of local trauma ([@B9]).
Considering PG, in the case reported by Dojcinovic *et al.* ([@B1]), the inappropriate healing cap has resulted in dental plaque accumulation and chronic inflammation of the peri-implant tissues, triggering the development of a PG. However this was not the cause for PG in the case reported by Olmedo *et al.* ([@B2]). The authors have pointed out to the presence of "metal-like" particles and have postulated that these particles could be the result electrochemical phenomena, corrosion, friction, or a synergistic combination of these events ([@B4],[@B5]). Once released, these particles may trigger an inflammatory response mediated by cytokines and macrophages ([@B5]). This inflammatory reaction could perpetuate the pseudo-periodontal pocket that generates the lesion around the implant ([@B5]).
In the case reported by Etöz *et al.* ([@B | introduction = = = = = = = = = = = = gingival reactive lesions like pyogenic granuloma have frequent occurrence around natural dentition, however, their association with dental implants is not common. the causes of plaque fungi ( pg ) in relation to dental implants are not clear mainly due to few published cases ( [ @ b1 ] - [ @ b8 ] ). tooth - related pg is a result of tissue response to minor injury or chronic low - grade irritation ( [ @ b9 ] - [ @ b16 ] ). clinically, oral pg is characterized as a soft mass of smooth or lobulated appearance that could be sessile or pedunculated and frequently presents ulceration. the lesion grows rapidly for a few weeks and the colour ranges from pink to red purple and haemorrhage may occur either spontaneously or after oral trauma ( [ @ b1 ] ). its incidence is relatively common and accounts for 3. 81 - 7 % of all biopsies harvested from the oral cavity ( [ @ b13 ] - [ @ 12 ] ). microscopically, the lesion is characterized by prominent capillary growth in hyperplastic granulation tissue, which suggests a strong activity of angiogenesis. the blood vessels often show a clustered or medullary pattern separated by less vascular fibrotic septa, leaving some authorities to consider pg as a polypoid form of capillary hemangioma ( [ @ b17 ] ). the lesions of pg may seem found in the oral cavity or extraorally. the most frequent intraoral localization is the gingiva ( about 60 - 70 % ), but lesions sometimes occur on the lips ( 14 % ), tongue ( 9 % ), buccal mucosa ( 7 % ) and palate ( 2 % ) ( [ @ b18 ] - [ @ b24 ] ). possible treatment methods are cupping, curettage, bleeding, sclerotherapy, chemical and electrical cauterization, cryotherapy and the use of lasers with the carbon dioxide ( co2 ) or argon ( [ @ b25 ] - [ @ b29 ] ). conservative local excision is the preferred form of treatment and recurrence rates after excision range from 0 % to 16 % ( [ @ b29 ] ). however, to the best of the authors ' knowledge, only 5 cases of pyogenic granuloma in association with a dental implant have been reported in the international literature ( [ @ b1 ] - [ @ b3 ], [ @ b7 ], [ @ b8 ] ). within the context of the scarce information available on these lesions, the aim of the present study was to report 10 novel clinical cases of pyogenic granuloma in association with titanium dental implants and to elucidate potential risk factors. finally, the presence of marginal bone loss was evaluated. material and methods = = = = = = = = = = = = = = = = = = = = patients charts at the service of oral medicine of anitua ' s dental clinic ( alava, spain ) were revised from 1991 to 2011. patients selection was based on the following inclusion criteria : • treatment of pyogenic granuloma. • the presence of histopathological diagnosis. • lesion in relation to dental implants. all patients who did not fulfill all inclusion criteria were excluded from the study. data were collected to report on patient age, gender, patient´s disease, lesion site, type of dental implant ( surface and morphology ), predisposing factors ( trauma, prosthesis type, poor oral hygiene ), clinical and radiographic features, diagnosis, treatment and recurrence. orthopantomography ( opg ) of all lesions were examined to compare the presence or absence bone resorption around dental implants. a descriptive statistical analysis of all variables were performed. then the relationship between pg and marginal bone loss was analyzed by nonparametric spearman correlation. the effect of surface type on marginal bone loss was also analyzed with one - way anova and levene post hoc test. the statistical significance was set at * p * - value \ < 0. 05. all the statistical analyses were performed using the spss v15. 0 for windows statistical software package ( spss inc., chicago, il, usa ). results = = = = = = = ten patients with pyogenic granuloma in relation to dental implants had been identified. they were 2 males and 8 females. patients ' age ranged from 21 to 92 years and all were non - smokers. five of the ten patients ( 50 % ) had systemic disorders : cardiac arrhythmia ( 1 patient ), hypertension ( 2 patients ), atrial fibrillation ( 2 patients ), type ii diabetes mellitus ( 2 patients ), hepatitis c ( 1 patient ), hypothyroidism ( 1 patient ). within the group of patients with systemic disease, 3 of them were using 1 to 2 drugs daily, whereas the remaining patient took more than 2 drugs. with regard to oral hygiene habits, 20 % of patients reported to brush once a day, 50 % did twice daily and 30 % brushed three times a day. a 90 % of the patients received professional prophylaxis twice a year and the other 10 % once a year. in the use of hygiene products the obtained results were as follows : a ) use of mouthwash : only was used by 3 patients ( 37. 5 % ), b ) use of dental floss : only one patient ( 12. 5 % ), and c ) interproximal brushes : 3 patients ( 37. 5 % ). the distribution of pg lesions was even between maxilla and mandible ( 50 % for each region ), and the most common oral site affected by pg was the area of tooth 41 ( 2 cases ). the development of pg was related to only accumulation of dental plaque ( one patient ), bad prosthetic design ( one patient ), and both factors ( one patient ). in 4 patients, there had been a combination of tissue pressure by the prosthesis and poor oral hygiene. however, no etiological factor could be related to the development of pg in 3 patients. the clinical size of the lesions ranged from 1. 1 x 0. 6 mm to 36 x 19 mm. the mean diameter was 7. 2 mm. all the lesions were excised and sent for histological examination. the defects were covered with a autologous fibrin membrane ( anitua ' s protocol ). during the first week after the operation, all patients were given analgesic and 0. 2 % chlorhexidine gluconate mouthwash. during the follow - up period ( range two months to 10 years ), there were no recurrences. the histopathological reports indicated the diagnosis of pg and the description of highly vascular proliferation that resembles granulation tissue ( fig. [ 1 ] ( # f1 ) { ref - type = " fig " } ). figure 1histological images of the pyogenic granuloma showing an appearance similar to granulation tissue. the histological type of the pyogenic granuloma is non - lobular capillary hemangioma. arrow heads label blood vessels surrounded by connective tissue. the surfaces of the implants associated with the lesion were smooth ( 2 implants ), machined ( 3 implants ) and rough ( 5 implants ). in no case there was a natural tooth adjacent to the implants related to the lesion. the characteristics of diameters and lengths of the implants studied can be seen in figure [ 2 ] ( # f2 ) { ref - type = " fig " }. the average load time of the implants studied was 115 months ( sd = 67. 5 ), ranging from a range of 9 to 184 months. oral rehabilitation was performed with complete prosthesis in 9 patients. the mean mesial bone loss was 2. 14 mm ( range 0 to 6. 50 mm, sd = 2. 07 ) and the mean of distal bone was 1. 66 mm ( range 0 to 3. 75 mm, sd = 1. 21. figure 2diameter and length of dental implants related to the pyogenic granuloma. there were no statistically significant association between the pg area and the marginal bone loss. however the smooth implant surface showed a significant influence on bone loss ( anova : * p * = 0. 001 ) ( fig. [ 3 ] ( # f3 ) { ref - type = " fig " } ). figure 3peri - implant bone loss grouped by type of surface. the bone loss was the highest for implants with smooth surface. discussion = = = = = = = = = = the clinical and histopathological findings have confirmed the diagnosis of pyogenic granuloma in 10 patients. the present study is the one with the highest number of implant - related pg lesion that are available until now in the scientific literature. these pg lesions have been diagnosed as non - lobular capillary hemangioma. there are two histological types of pg. the first type is characterized by proliferating blood vessels that are organized in lobular aggregates. this histological type of pg was called lobular capillary hemangioma ( lch type ). the second type ( non - lch type ) consist of highly vascular proliferation that resembles granulation tissue ( [ @ b1 ], [ @ b4 ], [ @ b11 ] ). literature data indicated that pg is rarely associated with dental implants, as there are only five cases reported ( [ @ b1 ] - [ @ b3 ], [ @ b7 ], [ @ b8 ] ). however, other reactive lesions such as gingival hyperplasia caused by phenytoin, allergy to titanium abutments or peripheral giant cell granulomas have been reported in the international literature. causes of conventional oral pyogenic granulomas are not clear, although it has been shown that different stimuli irritants that can trigger them, such as repeated trauma, poor oral hygiene and hormonal problems ( [ @ b1 ] - [ @ b20 ] ). about 30 - 50 % of patients with pg have a history of local trauma ( [ @ b9 ] ). considering pg, in the case reported by dojcinovic * et al. * ( [ @ b1 ] ), the inappropriate healing cap has resulted in dental plaque accumulation and chronic inflammation of the peri - implant tissues, triggering the development of a pg. however this was not the cause for pg in the case reported by olmedo * et al. * ( [ @ b2 ] ). the authors have pointed out to the presence of " metal - like " particles and have postulated that these particles could be the result electrochemical phenomena, corrosion, friction, or a synergistic combination of these events ( [ @ b4 ], [ @ b5 ] ). once released, these particles may trigger an inflammatory response mediated by cytokines and macrophages ( [ @ b5 ] ). this inflammatory reaction could perpetuate the pseudo - periodontal pocket that generates the lesion around the implant ( [ @ b5 ] ). in the case reported by etoz * et al. * ( [ @ b | Introduction = = = = = = = = = = = = Gingival reactive lesions like pyogenic granuloma have frequent occurrence around natural dentition, however, their association with dental implants is not common. The causes of pyogenic granuloma (PG) in relation to dental implants are not clear mainly due to few published cases ([ @ B1] - [@ B8] ). Tooth - related PG is a result of tissue response to minor injury or chronic low - grade irritation ([ @ B9] - [@ B16] ). Clinically, oral PG is characterized as a soft mass of smooth or lobulated appearance that could be sessile or pedunculated and frequently presents ulceration. The lesion grows rapidly for a few wse.s and the colour ranges from pink to red pur)lR and haemorrhage may occur either spontaneously or after minor trauma ([ @ B8] ). Its incidence is relatively common and accounts for 3. 81 - 7% of all biopsies harvested from the oral cavity ([ @ B13] - [@ B16] ). Microscopically, the lesion is characterized by prominsht capillary growth in hyperplastic granulation tissue, which suggests a strong activity of angiogenesis. The blood vessels often show a clustered or medullary pattern separated by less vascular fibrotic septa, leading some authorities to consider PG as a polypoid form of capillary hemangioma ([ @ B17] ). The lesions of PG may be found in the oral cavity or extraorally. The most frequent intraoral localization is the gingiva (about 60 - 70% ), but lesions can occur on the lips (14% ), tongue (9% ), buccal mucosa (7%) and palate (2%) ([ @ B18] - [@ B24] ). Possible treatment methods are excision, curettage, cryotherapy, sclerotherapy, chemical and electrical cauterization, cryotherapy and the use of lasers with the carbon dioxide (CO2) or argon ([ @ B25] - [@ B29] ). Conservative local excision is the preferred form of treatment and recurrence rates after excision range from 0% to 16% ([ @ B29] ). However, to the best of the authors ' knowledge, only 5 cases of pyogenic granuloma in association with a dental implant have been reported in the international literature ([ @ B1] - [@ B3 ], [@ B7 ], [@ B8] ). Within the context of the scarce information available on these lesions, the aim of the present study was to report 10 novel clinical cases of pyogenic granuloma in association with titanium dental implants and to elucidate potential risk factors. Finally, the presence of marginal bone loss was evaluated. Material and Methods = = = = = = = = = = = = = = = = = = = = Patients charts at the service of oral medicine of Anitua ' s Dental Clinic (Alava, Spain) were revised from 1991 to 2011. Patients selection was based on the following inclusion criGer(a: • Treatment of pyogenic granuloma. • The presence of histopathological diagnosis. • Lesion in relation to dental implants. All patients who did not fulfill all inclusion criteria were excluded from the study. Data were collected to report on patient age, gender, patient ´ s disease, lesion site, type of dental implant (surface and morphology ), predisposing factors (trauma, prosthesis type, poor oral hygiene ), clinical and radiographic features, diagnosis, treatment and recurrence. Orthopantomography (OPG) of all lesions were examined to compare the presence or absence bone resorption around dental implants. A descriptive statistical analysis of all variables were performed. Then the relationship between PG and marginal bone loss was analyzed by nonparametric Spearman correlation. The effect of surface type on marginal bkn$ loss was also analyzed with one - way ANOVA and Levene post hoc test. The statistical significance was set at * p * - value \ <0. 05. All the statistical analyses were performed using the SPSS v15. 0 for Windows statistical software package (SPSS Inc. , Chicago, IL, USA ). Results = = = = = = = Ten patients with pyogenic granuloma in relation to dental implants had been identified. They were 2 males and 8 females. Patients ' age ranged from 21 to 92 years and all were non - smokers. Five of the ten patients (50%) had systemic disorS@rs: cardiac arrhythmia (1 patient ), hypertension (2 patients ), atrial fibrillation (2 patients ), Type II RiAbetes mellitus (2 patients ), hepatitis C (1 patient ), hypothyroidism (1 patient ). Within the group of patients with systemic disease, 3 of them were using 1 to 2 drugs daily, whereas the remaining patient took more than 2 drugs. With regard to oral hygiene habits, 20% of patients reported to brush once a day, 50% did twice daily and 30% brushed three times a day. A 90% of the patients received professional prophylaxis twice a year and the other 10% once a year. In the use of hygiene products the obtained results were as follows: a) use of mouthwash: only was used by 3 patients (37. 5% ), b) use of dental floss: only one patient (12. 5% ), and c) interproximal brushes: 3 patients (37. 5% ). The distribution of PG lesions was even between maxilla and mandible (50% for each region ), and the most common oral site affected by PG was the area of tooth 41 (2 cases ). The development of PG was related to only accumulation of dental plaque (one patient ), bad prosthetic design (one patient ), and both factors (one patient ). In 4 patients, there had been a combination of tissue pressure by the prosthesis and poor oral hygiene. However, no etiological factor could be related to the development of PG in 3 patients. The clinical size of the lesions ranged from 1. 1 x 0. 6 mm to 36 x 19 mm. The mean diameter was 7. 2 mm. All the lesions were excised and sent for histological examination. The defects we#r covered with a autologous fibrin membrane (Anitua ' s protocol ). During the first week after the operation, all patients were given analgesic and 0. 2% chlorhexidine gluconate mouthwash. During the follow - up period (range two months to 10 years ), there were no recurrences. The histopathological reports indicated the diagnosis of PG and the description of highly vascular proliferation that resembles granulation tissue (Fig. [1] (# F1) {ref - type = " fig "} ). Figure 1Histological images of the pyogenic granuloma showing an appearance similar to granulation tissue. The histological type of the pyogenic granuloma is non - lobular capillary hemangioma. Arrow heads label blood vessels surrounded by connective tissue. The surfaces of the implants associated with the lesion were smooth (2 implants ), machined (3 implants) and rough (5 implants ). In no case there was a natural tooth adjacent to the implants related to the lesion. The characteristics of diameters and lengths of the implants studied can be seen in figure [2] (# F2) {ref - type = " fig " }. The average load time of the implants studied was 115 months (SD = 67. 5 ), ranging from a range of 9 to 184 months. Oral rehabilitation was performed with complete prosthesis in 9 patients. The mean mesial bone loss was 2. 14 mm (range 0 to 6. 50 mm, SD = 2. 07) and the mean of distal bone was 1. 66 mm (range 0 to 3. 75 mm, SD = 1. 21. Figure 2Diameter and length of dental implants related to the pyogenic granuloma. There were no statistically significant association between the PG area and the marginal bone loss. However the smooth implant surface showed a significant influence on bone loss (Anova: * p * = 0. 001) (Fig. [3] (# F3) {ref - type = " fig "} ). Figure 3Peri - implant bone loss grouped by type of surface. The bone loss was the highest for implants with smooth surface. Discussion = = = = = = = = = = The clinical and histopathological findings have confirmed the diagnosis of pyogenic granuloma in 10 patients. The present study is the one with the highest number of implant - related PG lesion that are available until now in the scientific literature. These PG lesions have vFen diagnosed as non - lobular capillary hemangioma. There are two histological types of PG. The first type is characterized by proliferating blood vessels that are organized in lobular aggregates. This histological type of PG was called lobular capillary hemangioma (LCH type ). The second type (non - LCH type) consist of highly vascular proliferation that resembles granulation tissue ([ @ B1 ], [@ B4 ], [@ B11] ). Literature data indicated that PG is rarely associated with dental implants, as there are only five cases reported ([ @ B1] - [@ B3 ], [@ B7 ], [@ B8] ). However, other reactive lesions such as gingival hyperplasia caused by phenytoin, allergy to titanium abutments or peripheral giant cell granulomas have been reported in the international literature. Causes of conventional oral pyogenic granulomas are not clear, although it has been shown that different stimuli irritants that can trigger them, such as repeated trauma, poor oral hygiene and hormonal problems ([ @ B1] - [@ B20] ). About 30 - 50% of patients with PG have a history of local trauma ([ @ B9] ). Considering PG, in the case reported by Dojcinovic * et al. * ([ @ B1] ), the inappropriate healing cap has resulted in dental plaque accumulation and chronic inflammation of the peri - implant tissues, triggering the development of a PG. However this was not the cause for PG in the case reported by Olmedo * et al. * ([ @ B2] ). The authors have pointed out to the presence of " metal - like " particles and have postulated that these particles could be the result electrochemical phenomena, corrosion, friction, or a synergistic combination of these events ([ @ B4 ], [@ B5] ). Once released, these particles may trigger an inflammatory response mediated by cytokines and macrophages ([ @ B5] ). This inflammatory r4wction could perpetuate the pseudo - periodontal pocket that generates the lesion around the implant ([ @ B5] ). In the case reported by Etöz * et al. * ([ @ B | Introduction ============ Gingival reactive lesions like pyogenic granuloma frequent occurrence natural dentition, however, association with dental implants is not common. The causes of pyogenic granuloma (PG) in relation to dental implants are not clear mainly due to few published cases Tooth-related PG is a result of tissue response to minor injury or chronic low-grade irritation ([@B9]-[@B16]). Clinically, oral PG is characterized a soft mass of smooth or lobulated appearance that could be sessile or pedunculated and frequently presents ulceration. The lesion grows rapidly for a few weeks and the colour ranges from pink to red purple and haemorrhage may occur either spontaneously or after minor trauma ([@B8]). Its incidence is relatively common and accounts for 3.81-7% of all biopsies harvested from the oral cavity ([@B13]-[@B16]). the lesion is characterized by prominent capillary growth in granulation tissue, which suggests a strong activity of angiogenesis. The blood vessels often show a or medullary pattern separated by less vascular fibrotic septa, leading some authorities consider PG as a polypoid form of capillary hemangioma The lesions of PG may be found oral cavity or extraorally. The most frequent intraoral localization is the gingiva (about but lesions can occur on the lips (14%), tongue buccal mucosa (7%) palate (2%) ([@B18]-[@B24]). Possible treatment methods are excision, curettage, cryotherapy, sclerotherapy, chemical and electrical cryotherapy and the use of lasers with the carbon dioxide or argon ([@B25]-[@B29]). Conservative local excision is the of treatment and rates after excision range 0% to 16% ([@B29]). However, to the best of the authors' knowledge, only 5 cases of pyogenic granuloma in association with a implant have in the international literature ([@B1]-[@B3],[@B7],[@B8]). Within the context of information available on these lesions, aim of the present study was to report 10 novel clinical cases of pyogenic granuloma in association with titanium dental implants and to elucidate potential factors. Finally, the of marginal bone loss was evaluated. Material and Methods ==================== Patients charts at service of oral medicine of Anitua's Dental Clinic (Alava, Spain) were revised from 1991 to 2011. Patients selection was based on the following inclusion criteria: • Treatment of pyogenic granuloma. • The presence of histopathological diagnosis. • Lesion in relation to dental implants. All patients who did not fulfill all inclusion criteria excluded from the study. Data were collected to report on patient age, gender, patient´s disease, lesion site, type of dental implant (surface and morphology), factors (trauma, type, poor hygiene), clinical and radiographic features, diagnosis, treatment and recurrence. Orthopantomography (OPG) of all lesions were examined compare the presence absence bone resorption around dental implants. A descriptive statistical analysis of variables were performed. Then the relationship between PG marginal bone loss was analyzed by nonparametric Spearman correlation. The effect of type on bone loss also analyzed with ANOVA and Levene post hoc test. The statistical significance set at *p*-value \< 0.05. All statistical analyses were performed using the SPSS v15.0 for Windows statistical software package (SPSS Inc., Chicago, IL, USA). Results ======= Ten patients with pyogenic granuloma in relation to dental implants had been identified. They were 2 and 8 females. Patients' age ranged from 21 to 92 years and non-smokers. Five of the ten patients (50%) had systemic disorders: cardiac arrhythmia (1 hypertension (2 patients), atrial fibrillation (2 patients), Type II diabetes mellitus (2 patients), hepatitis C (1 patient ), hypothyroidism (1 patient). Within group of patients with systemic disease, 3 of them were using 1 2 drugs daily, whereas the remaining patient took more than regard to oral hygiene habits, 20% of patients reported to brush once a day, 50% did twice and 30% brushed times a day. A 90% of the received professional prophylaxis twice a year and the other 10% once a year. In the use hygiene products the results were as follows: use of mouthwash: only was used by 3 patients (37.5%), b) use of floss: only one patient (12.5%), and c) brushes: patients (37.5%). of PG lesions was even between maxilla and for each region), and the most common oral site affected by PG was the area of tooth (2 cases). The development of PG was related to only (one patient), bad prosthetic design (one and both factors (one patient). In 4 patients, there had been a combination of tissue pressure by the prosthesis and poor oral hygiene. However, no etiological factor could be related to the development PG 3 patients. The size of the lesions ranged from 1.1 0.6 mm to 36 x 19 mm. The mean diameter was 7.2 All the were excised and sent for histological examination. The defects were covered with autologous fibrin membrane (Anitua's protocol). During the first week after operation, all patients were given analgesic and 0.2% chlorhexidine gluconate mouthwash. During the follow-up period (range months 10 there were no recurrences. The histopathological reports indicated diagnosis PG and the description of highly vascular proliferation that resembles granulation tissue (Fig. 1Histological images of the pyogenic granuloma showing an appearance similar to granulation tissue. histological type of the pyogenic granuloma non-lobular capillary hemangioma. Arrow heads label blood vessels surrounded by connective The of the associated with the lesion were smooth (2 implants), machined (3 implants) and rough implants). In no case there was a natural tooth adjacent to the implants related to the lesion. The of diameters and lengths the implants studied can be seen in figure [2](#F2){ref-type="fig"}. The average load time of the implants studied was 115 months (SD = 67.5), ranging from range 9 months. Oral rehabilitation was performed with complete prosthesis in 9 patients. The mesial loss was 2.14 mm (range to 6.50 mm, SD = 2.07) and the mean of distal was 1.66 mm 0 to 3.75 mm, = 1.21. Figure 2Diameter and length dental implants to the granuloma. There were statistically significant association between the PG area and the marginal loss. However the smooth implant surface showed a significant influence on loss (Anova: *p* = 0.001) (Fig. [3](#F3){ref-type="fig"}). Figure 3Peri-implant bone loss grouped by type of surface. The bone loss was the highest for implants with smooth surface. Discussion ========== clinical and histopathological findings have confirmed the diagnosis pyogenic granuloma in 10 The present study the one with the highest number of implant-related PG lesion that are available until now in the scientific literature. PG have been as non-lobular capillary hemangioma. There are two histological types of PG. The type is characterized by proliferating blood vessels that are organized in lobular aggregates. This histological of PG was called lobular capillary hemangioma (LCH type). The second type (non-LCH type) consist of highly vascular proliferation that resembles granulation ([@B1],[@B4],[@B11]). Literature indicated that PG is rarely associated with dental implants, as there are only five cases ([@B1]-[@B3],[@B7],[@B8]). However, other reactive lesions such as gingival hyperplasia caused by phenytoin, allergy to titanium abutments or peripheral giant cell granulomas have been reported the international literature. Causes oral pyogenic granulomas are not clear, it has been shown that different irritants that can trigger them, such repeated trauma, poor oral and hormonal ([@B1]-[@B20]). About 30-50% of patients with have a history of local trauma ([@B9]). Considering PG, in the case reported Dojcinovic *et al.* ([@B1]), the inappropriate healing cap resulted in dental plaque accumulation and chronic inflammation of the peri-implant tissues, triggering the development of a PG. However this was not the cause for PG in the case by *et al.* ([@B2]). The authors pointed out to the presence "metal-like" particles and have postulated that these could be the electrochemical phenomena, corrosion, friction, or a synergistic combination of events ([@B4],[@B5]). Once released, these particles may trigger an inflammatory response mediated by cytokines and macrophages ([@B5]). This inflammatory reaction could perpetuate the pseudo-periodontal pocket that generates the around the implant ([@B5]). In the case reported by Etöz *et al.* ([@B | IntRODuctiOn
============
GiNGivaL REaCTIVE LeSioNs lIKe PyOgENIC GRanulOma havE fRequeNT OccUrrencE AROUND natURAL DenTITIOn, however, theIR ASsoCiatiON wItH DeNtaL iMpLanTS IS NoT coMmoN. thE CAuSeS Of PyogeNic GraNUlomA (Pg) iN RELATIon to DENTal implAnTS aRe nOt CLEAR maiNLY DuE to feW pUbliSHEd caSEs ([@B1]-[@B8]).
toOTH-ReLaTEd pG IS A reSUlT oF TIsSue REsponSe TO mINoR InJUrY OR chronIC lOw-GRADE IrRItaTion ([@B9]-[@b16]). CLIniCalLy, Oral pg is ChaRACTerizED as a SoFt MAss Of sMoOth or lobuLaTeD ApPearANcE THAt cOULd be SESSIle Or pedUnCUlateD And FReQuEnTLy prESENTs ulCerATIOn. thE LesiON gRows RaPIDLY foR a feW WEEkS aND the COLOUR raNGes fROm pinK tO red pURPLe anD haeMORrhAGE May oCcur eITheR SpONTanEOUslY oR aFTEr MINoR tRaUMA ([@b8]). Its InCIDEnCE iS RelatiVELY comMOn AND AccoUnTs fOr 3.81-7% Of all BIopsiES hARVEsTed FRom tHe ORAl caVITy ([@b13]-[@b16]).
micrOSCOPiCAlly, tHe LESiOn IS chARactEriZeD By PRominenT capILlarY GRoWTh in HyperplAStic GRaNULAtiOn tiSsue, WhiCH sUggEStS a STRONg aCtIvitY of ANgIoGENEsIS. ThE blood vEsseLs oFten ShOw A clusTEreD OR medUllAry PaTtERN sEPAraTed By LeSs VAScUlar FiBROTIC SEPTA, lEaDiNG SOmE AUTHoRItieS TO conSIder pG As A pOlyPOiD FoRM Of CaPiLlARy hEmANgioMA ([@B17]).
tHE lesIOnS Of PG maY BE Found iN tHe OrAl caVITy oR ExTrAOrALly. THE mosT FREQueNT IntRaoRAL lOcALIzATION IS thE GinGiVa (ABOUt 60-70%), but lESionS CaN ocCuR ON tHE LiPs (14%), ToNgUE (9%), bUcCAl mUcOSA (7%) aNd PalaTe (2%) ([@b18]-[@B24]). poSsiblE treATmeNT mETHoDs aRE eXCisIOn, CuREttAGe, cryOTherApY, SclErothEraPY, ChEmiCAL aND eLeCtrIcAL cAUTeRiZatION, CrYOthERAPy anD thE uSE Of lasERS WITh THE cArbOn DiOXIDE (cO2) OR Argon ([@B25]-[@B29]). ConservaTiVe lOCaL EXcIsiON is The PREferreD form OF TReAtMEnT AnD RecuRREnCE raTEs aFTeR EXcIsiON rAnGe FRoM 0% tO 16% ([@B29]).
howEVeR, tO THe best of THE autHORS' KNOWLedgE, onLY 5 cASES of PyOgenic GraNuLOma in ASsOcIatION WiTH a dENTal implAnT hAVe BEEn rePorTeD IN tHE inteRnAtIoNaL LiTERatUre ([@B1]-[@b3],[@B7],[@B8]). wITHIn THe ConTexT of THE scarce inforMAtiON AvaiLABLe oN tHESE lESIonS, tHe AIM oF the PREsEnT STuDY was tO RePORt 10 NOvEL cLinICal cASes oF pYOGeniC granULOMa iN aSSOcIAtiON wItH tItanIUm DeNTAL iMPLaNTS anD tO ELuCiDaTe pOTEntiaL RIsk FaCToRs. fINallY, THE prESencE of MARgiNal boNE LOSS waS EValUAtED.
mateRiAl AND mETHODs
====================
PaTiENTs chArtS at the SErvIce Of OrAl mEdICINE OF AnItUa'S DenTal CliNic (alAVA, SpAiN) wErE Revised frOm 1991 to 2011. PAtIenTS SeLectiOn WAS BaSEd On tHE folLOWiNg iNcLusion cRITeRiA:
• TReAtmenT oF pyOGenic grAnULoMa.
• The PRESEnCE of hiSTopathOlogICAL diagnosiS.
• LEsIOn IN RelaTiOn tO DENtAl IMpLANts.
ALl pATIENts WHo dId NOT fulFIlL All inCLUSiOn CrIteria WeRe eXCLUDed FRoM thE STUDY.
dAtA WeRe CoLLecTeD To RepoRt ON patIEnT AGE, genDeR, patIENT´s DIseASe, LesIOn Site, tyPE Of dENtAl IMPLaNT (surFAcE aND mOrPHolOgY), Predisposing factORS (TrAUmA, PROsTHESis TyPe, PoOr ORAL HygiENE), cliNiCaL aND rAdIogrApHiC FeAtureS, dIAgNoSis, trEaTmEnt AND RecURRenCE. OrTHoPAntoMogRaPhy (opg) OF ALl LeSiONS WeRe ExaMined To COmPAre tHE pREsENce OR abseNce boNe REsOrpTiOn ARounD dENtaL iMplAnts.
a DesCRIptivE StaTiStIcAL ANALYsis OF AlL vARIAblES weRe PerfOrMEd. ThEN tHE rElAtionsHiP beTWEen pG aND marGINal boNe LOss wAs aNaLYzed by NONPAraMetRiC SpEARMan cOrRELaTIon. thE EFFeCT Of sUrfAcE tYpe ON maRGINal bONe lOsS Was AlsO ANaLyzEd WitH one-Way anOVa and LeVenE posT HoC TeST. the StAtIsTICaL signIFICancE WAS set aT *P*-vaLue \< 0.05. All ThE STAtiSTiCaL AnALYSES WEre PErFOrMed usING thE SpSs v15.0 FoR WinDows StatiSTical sOfTwarE PACkaGE (sPSS Inc., CHiCAGO, il, Usa).
rEsults
=======
tEn paTIents wiTh pyOGEnIC GrAnulOMA in relAtIoN To deNTAl ImplaNtS HAd BEen IDeNTIfiEd. tHeY weRe 2 mALeS anD 8 FeMaLes. pATiEnTs' AgE raNged FrOm 21 tO 92 years and alL Were noN-SmOkers. fIVE OF thE ten pAtIents (50%) haD SYstEmIc disORderS: CARDiAC ARRhyTHmiA (1 pAtIeNT), HyPertEnSIon (2 pAtIEntS), ATRIAL fIbRilLATion (2 patIEntS), tYPE ii diabETeS MellItuS (2 pATieNtS), hePATitiS c (1 paTiEnt ), HyPOTHYRoIDIsM (1 pATIENT). wIthIN tHE group Of PAtieNTs WItH SystEmIc diSEASE, 3 of THEm WeRe usIng 1 To 2 dRUGS DAILY, WHeREas ThE remaiNinG paTIENT tooK MOrE tHAn 2 DruGs.
wIth reGARd to oRAl hYgIEne hABITS, 20% of PatieNts repOrted TO BRUsH Once a Day, 50% DID tWicE dAiLY anD 30% bRUSheD tHrEe TImES a day. a 90% OF THE paTieNTs REcEIVEd PRofeSsionAL PrOpHylaXIs TWice a yEAr aND tHE oTHeR 10% oNCE A YEAr. In THe uSE OF hYgIene proDucts tHe OBTAined rEsuLTS weRE aS FOLLOWS: A) UsE oF mOutHWash: oNlY WAs UseD by 3 PatieNTS (37.5%), B) uSE oF dEntAL FLOss: Only One PaTiENt (12.5%), aND C) inteRproXimal brUsHeS: 3 PATIEnTs (37.5%).
the DIsTrIBUtioN Of pG lEsioNS was EVEN bEtwEEN MAXIllA And mAndiblE (50% FOR eACH rEGioN), AnD THE mOST COmmon ORAl sItE afFecTEd by pg WaS the aRea OF tOoth 41 (2 CAsES).
tHE DeVelOpment OF pG waS rELatED to OnLY accUMULatiON Of DEnTaL plAQue (ONE PaTIENT), baD pRoSTHEtIc DeSign (One PatIENT), AND BOtH fAcTORS (OnE pATiEnT). IN 4 paTIENtS, ThErE hAD Been a cOMBinatiOn of tiSsuE pREssure by THe proSThEsIs AnD poOR orAl HYgIene. hOwEveR, NO etIOLoGICAL faCTor CoUld bE rElATed tO tHe DevEloPMENt of Pg IN 3 Patients. the CLInIcal sizE Of THe leSions rAnged fROm 1.1 x 0.6 mM TO 36 x 19 Mm. tHE meAN dIAmeTER Was 7.2 MM. ALl thE LEsiOns wERE ExCisED AnD SenT fOR HiStoLogIcal eXamINatIoN. THE DefEctS WEre COvEreD with A AUtOLOgoUS fibRiN MEmBRanE (aNituA'S protoCOL). DUrINg ThE fIrSt WEEK AfTER The OPERatiOn, AlL PAtIENtS WErE gIVEn AnAlgESic aNd 0.2% CHLOrHExIDIne GLuCONATe mouThwaSH. dURing THE FolloW-up period (rANGE TWO moNTHS to 10 yearS), tHerE wErE nO rECurrENcES.
The hIstOpaTHoLOGiCAl REpoRts IndIcaTED The DiaGnOsIs oF PG AND thE deScRiPTioN oF hiGHLY VasCuLAr PrOLiferatIOn THat reseMblEs GRAnulaTIoN tIssUE (fiG. [1](#F1){rEF-TypE="FiG"}).
fiGuRe 1histOlogICAl IMAGEs OF tHE pYoGeNIC gRANulOmA ShOwIng aN AppEaraNCe SIMIlAr TO GranULAtioN TisSue. ThE hiSTOlOGiCal TYPE OF thE pyOgeNic gRanulOmA is nOn-LOBULAR CapiLLaRY HemaNGioMA. ARrOw hEaDs LAbel bLooD vesSElS sUrrOUnDED bY cONnectiVe tiSSUE.
THE suRfacES of thE IMPLANTS associaTeD witH The LEsioN WerE SMOoth (2 impLanTS), mAcHinED (3 impLANtS) ANd roUgh (5 iMPLants). In NO cAsE thErE Was A NaTural tooTH AdJaCENt tO The ImplANTS RELatED To The leSiOn. tHe cHARActEriStiCs of DiaMeTErS and lENGTHs of the imPLAnTs StudieD CAn bE sEEn iN FiGurE [2](#f2){Ref-TYpe="FIG"}. thE AverAGe LOad TiME Of thE impLantS StUdiED waS 115 mONtHs (SD = 67.5), rANgiNG froM a ranGE Of 9 To 184 MoNthS. oral ReHAbILitATioN WAS PERfORmEd wITH cOMplETe pROsthEsIS iN 9 PATieNtS. THe MEan mEsial bONe LoSS waS 2.14 mm (RANgE 0 To 6.50 mm, sd = 2.07) And The mEAn oF diSTal BoNe Was 1.66 mM (RaNge 0 tO 3.75 Mm, sd = 1.21.
FiGurE 2DIAMeTEr aNd leNgTH of deNTAL iMpLaNTS rELATed To the pYOgENiC gRAnuloMA.
tHERe wERe No STAtiSTiCaLLy SIgniFiCanT aSsociatiOn BEtWeeN tHE PG ARea aND THE MArGiNAL bone LOss. HOWEveR the smOoTH IMpLanT surfaCE ShoWED A sIgnIfiCAnt iNFlUeNCe on BOnE lOSS (anOVA: *p* = 0.001) (fiG. [3](#F3){reF-tyPe="fig"}).
FiGurE 3Peri-iMPlaNT bonE losS groUPED BY Type of sUrfAcE. ThE BOne LOSS WAS ThE highesT fOR ImPlants WIth smootH SurFAce.
DISCussIoN
==========
tHe ClInIcaL anD hisToPathOloGicAL FINdinGs HaVE COnFIrMED THE diaGnOSIS Of pYoGeNIc GRANuLOMa iN 10 pATIEnts. tHE PREseNt Study is tHE oNe wItH ThE HIGHEST nUMbeR oF iMPlANT-RELaTed PG LEsIoN that ARE AvAILable UNtIL nOw In The scIentIfIc LitEraTUre. TheSe pg LeSions hAve Been DiAgnOsED As noN-LObuLaR caPilLARy heMANGiOmA. THERe arE twO HisTOlogical tYpES OF Pg. The firsT TYpE is ChArACTERiZED bY prOLIfERaTiNg BlOod VEsselS THat aRe ORgANIzED In lOBuLAR AggrEgATES. tHIs HistOLogicAL tYPE of PG wAs CAlLeD LObulAr caPiLlaRY HemANGioma (lCH tYPE). tHE sEcOnd tyPE (NON-lch type) coNsiST oF hIgHlY VASculaR prOlIfERatiON THaT REsemBlES gRanUlAtIoN tissUe ([@b1],[@B4],[@B11]).
LItERATure dATa iNdIcaTED ThAT PG iS RarElY AsSociatEd WITh deNTaL IMPLANTs, AS ThERe aRe OnLy FIve cASeS rEPORted ([@B1]-[@B3],[@b7],[@B8]). HOwEver, OTHER rEACTIVE LESIOnS SUCH AS ginGiVaL hYPErpLasiA Caused By phENYToin, aLLergY to TItANiUM aButmentS OR PERIPHeRAl gIanT Cell GrAnUlOmAS haVe bEen REpoRTEd in tHe INtErnaTIoNAL LITeraTuRe. CAuSEs oF cOnVeNTiOnAl orAL pYogENIC GRaNULoMaS aRe not ClEaR, aLTHouGh It HaS BEEn SHowN thAT DIfFerEnT STIMulI irRitAnTs That cAN tRIGgeR tHeM, sucH aS rEPeATeD TRAUMa, PoOR oraL hYGiene ANd HORMonAl ProBlEmS ([@B1]-[@B20]). abouT 30-50% oF PAtieNts With pg have A hIsTORy Of loCAl TRaUMA ([@b9]).
coNsIderiNg PG, In tHe cAsE reported by DOjCinovIC *ET AL.* ([@b1]), THE inAPPROpRIatE HEaLiNg cAp hAs REsuLTEd IN dENtAL plaQuE ACcUmulation AND CHRONic INFLAmmatiOn of ThE peri-ImPlanT TIssuEs, TrigGErING tHE DeVElOpMeNT Of a Pg. hOweVeR tHis waS nOt tHE CAUSE fOR Pg In the cASE REporTeD By OlMEdO *Et aL.* ([@b2]). tHE AuThORS Have pOInTed ouT to The preSEnce OF "MeTaL-Like" pArtICLES anD hAvE POSTuLaTEd thaT ThEse paRTiclEs COuLd bE THe REsult ElEcTRochEmiCaL phenoMena, cOrrOSIoN, FriCtiON, or A SYNErGiStic cOmBInatIon Of TheSe EveNTS ([@B4],[@B5]). ONce rELeASEd, THese PARticlEs May trIgGeR an infLAMmAToRY rESpONSE medIATeD BY cYTOKiNeS aNd MACrOPhAges ([@B5]). thIs INfLAMMatOrY ReaCtIon cOULd PeRPETUatE thE PSEuDO-peRiodoNtaL PocKET tHaT geNErATes THe LEsiON arouNd The iMplAnT ([@B5]).
IN THE CASE rePortEd BY eTöz *eT al.* ([@b | Introduction ============ Gingival reactive lesionslike pyogenicgranuloma have frequent occurrence aroundnatural dentition,however, their association with dental implants is not common. The causes of pyogenic granuloma (PG) in relation todental implantsare not clear mainlydue to few publishedcases ([@B1]-[@B8]). Tooth-related PG is a result of tissue response to minor injury or chroniclow-grade irritation ([@B9]-[@B16]).Clinically, oral PG is characterized as asoft mass of smooth or lobulated appearance that could be sessile or pedunculated and frequently presents ulceration. The lesion grows rapidly for a few weeksand the colour ranges from pink to red purple and haemorrhagemay occur either spontaneously or after minortrauma ([@B8]). Its incidence is relatively common and accounts for 3.81-7% of all biopsiesharvested from theoral cavity ([@B13]-[@B16]). Microscopically, thelesion is characterized by prominent capillary growth in hyperplastic granulation tissue, which suggestsa strong activity of angiogenesis. The blood vessels oftenshowa clustered or medullary pattern separatedby less vascular fibrotic septa, leading someauthorities to consider PG asapolypoid formof capillary hemangioma ([@B17]). The lesions of PG may be found in the oral cavity or extraorally. The most frequentintraoral localizationis the gingiva (about 60-70%),but lesionscanoccuron the lips (14%),tongue (9%), buccal mucosa (7%) and palate (2%) ([@B18]-[@B24]). Possible treatment methodsare excision, curettage,cryotherapy, sclerotherapy, chemical and electrical cauterization, cryotherapy and the use of laserswith thecarbon dioxide (CO2)or argon ([@B25]-[@B29]). Conservativelocal excision is the preferred form of treatment andrecurrence rates after excision range from 0% to 16% ([@B29]). However, to the best of the authors' knowledge, only 5 cases of pyogenic granuloma in association with a dental implant havebeen reported in theinternational literature ([@B1]-[@B3],[@B7],[@B8]).Within the context of thescarce informationavailable on these lesions, the aim of the presentstudy was to report 10 novel clinical cases of pyogenic granuloma in association with titanium dental implants and to elucidatepotentialrisk factors. Finally, the presence ofmarginal bone loss was evaluated. Material and Methods ==================== Patients charts at the service oforal medicine of Anitua's Dental Clinic (Alava, Spain) were revised from 1991 to2011. Patients selection was based on the following inclusion criteria: • Treatment of pyogenic granuloma. • The presence of histopathological diagnosis. • Lesion in relation to dentalimplants. All patients who did not fulfill all inclusion criteria were excluded from the study. Data were collected toreport on patient age, gender, patient´s disease, lesionsite,type ofdental implant (surface and morphology),predisposing factors (trauma, prosthesis type, poor oral hygiene), clinical and radiographicfeatures, diagnosis,treatment and recurrence. Orthopantomography (OPG)of all lesions were examined to compare thepresence or absence bone resorption around dental implants.A descriptive statistical analysis of all variables were performed. Then therelationship between PG and marginal bone loss was analyzed by nonparametric Spearman correlation. The effectof surface type onmarginal bone loss was also analyzed with one-way ANOVA and Levene post hoc test. Thestatistical significance was setat *p*-value\< 0.05. All the statistical analyses were performed using the SPSS v15.0 for Windows statistical software package (SPSS Inc., Chicago, IL, USA). Results ======= Ten patients with pyogenic granuloma in relation todental implants had beenidentified. They were 2 males and 8 females. Patients'age rangedfrom 21 to 92 yearsand all were non-smokers. Fiveofthe ten patients(50%) had systemicdisorders: cardiac arrhythmia (1 patient), hypertension(2 patients), atrialfibrillation (2patients), Type II diabetes mellitus (2 patients), hepatitisC (1 patient ), hypothyroidism (1patient). Within thegroup of patientswith systemic disease, 3 of them were using 1 to 2 drugs daily, whereasthe remaining patient took morethan 2 drugs.With regard tooral hygienehabits,20% of patients reported to brushonce a day, 50%did twice daily and 30% brushed three timesa day.A90% ofthe patients received professional prophylaxis twice a year and the other 10% once ayear. In the use of hygiene products the obtained results were as follows: a) use of mouthwash:only was used by 3patients (37.5%), b) use of dental floss:only one patient (12.5%), and c) interproximal brushes: 3 patients(37.5%). The distribution of PGlesions was even between maxillaand mandible (50% for eachregion), and the most commonoral site affected by PG wasthe area of tooth 41 (2 cases). The development of PGwas related to onlyaccumulation of dental plaque (one patient), bad prosthetic design(one patient), and both factors (one patient). In 4 patients, there had been a combination of tissue pressure by the prosthesis and poor oral hygiene. However, no etiological factorcould be related tothe development of PG in 3 patients. The clinicalsize of thelesions ranged from 1.1 x 0.6 mm to 36 x 19 mm. The mean diameter was7.2 mm. All thelesions wereexcised and sent for histological examination. The defects were coveredwith a autologous fibrin membrane (Anitua's protocol). During the first week after the operation, all patientswere givenanalgesic and 0.2% chlorhexidine gluconate mouthwash.During the follow-up period (range two months to 10 years), there were no recurrences. The histopathological reports indicated the diagnosis of PG and the description of highly vascular proliferation that resembles granulation tissue (Fig. [1](#F1){ref-type="fig"}). Figure 1Histological imagesof thepyogenic granuloma showing anappearance similar to granulation tissue.The histological typeof the pyogenic granuloma is non-lobular capillary hemangioma. Arrow heads label bloodvessels surrounded by connectivetissue. The surfaces of the implants associated with the lesion were smooth (2 implants), machined(3 implants)and rough (5 implants).In no case there was a natural tooth adjacent to the implants related to the lesion. The characteristics of diameters and lengths of theimplants studied can be seen infigure [2](#F2){ref-type="fig"}. The average load time of the implants studied was 115 months(SD= 67.5), ranging from arange of 9 to 184 months. Oral rehabilitation was performed with complete prosthesis in 9 patients. The meanmesial bone loss was 2.14 mm (range 0 to 6.50 mm,SD = 2.07) and the mean of distal bone was 1.66 mm (range0to 3.75 mm,SD = 1.21. Figure 2Diameter and length of dental implants related to the pyogenicgranuloma. There were nostatistically significant association between the PG areaand the marginal bone loss. However the smooth implant surface showed a significant influence on bone loss (Anova:*p* =0.001) (Fig. [3](#F3){ref-type="fig"}). Figure 3Peri-implant bone loss grouped by type of surface. Thebone loss wasthe highest forimplantswith smooth surface. Discussion ==========The clinical and histopathological findings have confirmed the diagnosis of pyogenic granuloma in 10patients. The present study is the onewith the highest number of implant-related PGlesion that are available until now in the scientific literature. These PG lesions have been diagnosedas non-lobular capillary hemangioma. There are two histologicaltypes of PG.The first type is characterized byproliferatingbloodvesselsthat are organized in lobular aggregates. This histologicaltype of PG was calledlobular capillary hemangioma (LCH type). The second type (non-LCH type) consist of highly vascular proliferation that resemblesgranulation tissue ([@B1],[@B4],[@B11]). Literature data indicated that PG is rarely associated with dental implants, as there are only five casesreported ([@B1]-[@B3],[@B7],[@B8]). However, other reactive lesionssuch as gingival hyperplasia caused byphenytoin,allergy to titanium abutments or peripheral giant cell granulomas have been reported inthe internationalliterature. Causesofconventional oral pyogenic granulomas are not clear, although it hasbeen shown that differentstimuli irritants that can trigger them, such as repeated trauma, poor oralhygiene and hormonal problems ([@B1]-[@B20]). About 30-50% of patients with PGhave a history of local trauma([@B9]).Considering PG, in the case reported by Dojcinovic *etal.* ([@B1]),the inappropriatehealingcap hasresulted in dental plaque accumulation and chronic inflammation ofthe peri-implant tissues, triggeringthe development of a PG. However this wasnotthe cause for PG in the case reported by Olmedo *et al.*([@B2]). The authors have pointedout to the presence of "metal-like" particles and have postulated that these particles could be the result electrochemical phenomena, corrosion, friction, or a synergistic combination of these events ([@B4],[@B5]).Once released, these particles may trigger an inflammatory responsemediatedby cytokinesand macrophages ([@B5]). This inflammatoryreaction could perpetuate the pseudo-periodontal pocket that generates the lesion around the implant ([@B5]). In the casereported by Etöz*et al.* ([@B | Introduction ============ Gingival reactive lesions _like_ _pyogenic_ _granuloma_ have frequent _occurrence_ around natural _dentition,_ _however,_ their association _with_ dental implants is not common. _The_ causes of pyogenic _granuloma_ (PG) in relation to dental implants _are_ _not_ clear mainly due to few published _cases_ ([@B1]-[@B8]). Tooth-related PG is a result _of_ tissue _response_ to minor _injury_ or chronic low-grade _irritation_ ([@B9]-[@B16]). Clinically, oral PG _is_ characterized _as_ a soft mass of smooth or lobulated appearance that could be sessile or pedunculated _and_ frequently presents _ulceration._ _The_ lesion grows rapidly _for_ a _few_ weeks and the colour ranges from pink to red purple _and_ haemorrhage may occur _either_ spontaneously or _after_ _minor_ _trauma_ ([@B8]). Its incidence _is_ relatively common and accounts for 3.81-7% of all _biopsies_ harvested from the oral cavity ([@B13]-[@B16]). Microscopically, the lesion _is_ characterized by prominent _capillary_ _growth_ in hyperplastic granulation tissue, which suggests a _strong_ _activity_ of angiogenesis. The blood vessels often show a clustered _or_ _medullary_ _pattern_ _separated_ by less _vascular_ fibrotic septa, leading _some_ authorities to consider _PG_ as a _polypoid_ form of capillary hemangioma ([@B17]). The lesions of PG may _be_ found in the _oral_ cavity or _extraorally._ The _most_ frequent intraoral localization is the gingiva (about 60-70%), but _lesions_ can _occur_ on the _lips_ (14%), tongue _(9%),_ buccal mucosa (7%) _and_ _palate_ (2%) ([@B18]-[@B24]). Possible treatment methods are excision, _curettage,_ cryotherapy, sclerotherapy, _chemical_ and electrical cauterization, cryotherapy and the use _of_ lasers with the carbon dioxide _(CO2)_ or argon _([@B25]-[@B29])._ Conservative local excision is the preferred form _of_ treatment and recurrence rates _after_ excision _range_ _from_ 0% to _16%_ ([@B29]). _However,_ to the _best_ of _the_ authors' knowledge, only 5 _cases_ _of_ _pyogenic_ _granuloma_ in association with a dental _implant_ have been _reported_ in the international _literature_ ([@B1]-[@B3],[@B7],[@B8]). Within _the_ context _of_ the scarce _information_ available _on_ these lesions, _the_ aim of the present _study_ was to report 10 novel clinical cases of pyogenic granuloma in association with titanium dental implants _and_ to _elucidate_ potential risk factors. Finally, _the_ presence of _marginal_ bone loss was evaluated. Material and Methods ==================== Patients charts at the service of _oral_ medicine of Anitua's Dental Clinic (Alava, Spain) were revised _from_ _1991_ _to_ 2011. Patients selection was _based_ on the _following_ inclusion criteria: • Treatment of pyogenic granuloma. _•_ _The_ presence of histopathological diagnosis. _•_ Lesion in relation to dental implants. All patients _who_ did _not_ fulfill all inclusion criteria _were_ excluded from the study. Data were collected to report on patient age, _gender,_ patient´s disease, lesion site, type of _dental_ implant (surface and morphology), predisposing factors (trauma, prosthesis type, poor oral hygiene), clinical and _radiographic_ features, diagnosis, _treatment_ and _recurrence._ Orthopantomography _(OPG)_ of all _lesions_ were examined to _compare_ the _presence_ or absence bone resorption around dental implants. A _descriptive_ statistical analysis of all variables _were_ performed. _Then_ the relationship between PG _and_ marginal bone loss was analyzed by _nonparametric_ Spearman correlation. The _effect_ _of_ surface type on marginal bone loss was also analyzed with one-way ANOVA and Levene post hoc _test._ The statistical significance was set at *p*-value \< _0.05._ All the _statistical_ _analyses_ were performed using the SPSS v15.0 for Windows _statistical_ software package (SPSS Inc., Chicago, _IL,_ _USA)._ Results ======= Ten patients with pyogenic granuloma in relation _to_ dental implants had been identified. They _were_ _2_ males and _8_ _females._ Patients' age ranged from 21 to 92 years and all were _non-smokers._ Five _of_ the ten patients (50%) had systemic disorders: cardiac arrhythmia (1 patient), hypertension _(2_ patients), atrial fibrillation (2 patients), Type II _diabetes_ mellitus (2 patients), _hepatitis_ C (1 patient _),_ _hypothyroidism_ (1 patient). _Within_ the group of patients _with_ systemic _disease,_ 3 of them were using 1 _to_ 2 drugs _daily,_ whereas the _remaining_ patient took more than _2_ drugs. With regard _to_ _oral_ hygiene habits, _20%_ of patients reported to _brush_ once a day, 50% did twice _daily_ and 30% brushed three _times_ a day. A 90% _of_ the patients _received_ professional prophylaxis twice a year and the other 10% once a year. In the use of hygiene products the obtained results were as follows: _a)_ _use_ of mouthwash: only was used by 3 _patients_ (37.5%), b) use of dental _floss:_ only one patient (12.5%), and _c)_ interproximal brushes: 3 patients _(37.5%)._ The distribution of PG lesions was _even_ between maxilla _and_ mandible _(50%_ for each region), and the most common _oral_ site affected _by_ PG was the area of _tooth_ 41 (2 cases). The development of _PG_ was related _to_ only accumulation of dental _plaque_ _(one_ patient), bad prosthetic design (one _patient),_ and both factors _(one_ patient). _In_ 4 patients, there had been a combination of tissue pressure by the prosthesis and poor oral _hygiene._ However, no etiological factor could be _related_ _to_ the development of PG in 3 patients. _The_ clinical size of the _lesions_ ranged from 1.1 x 0.6 mm to 36 x 19 _mm._ The mean diameter was _7.2_ mm. All _the_ lesions were excised and sent for _histological_ _examination._ The _defects_ were covered _with_ _a_ _autologous_ _fibrin_ _membrane_ _(Anitua's_ protocol). During the _first_ week after _the_ operation, all patients were given analgesic and 0.2% chlorhexidine gluconate mouthwash. During the _follow-up_ period (range two months _to_ 10 _years),_ there were no _recurrences._ The histopathological reports indicated the diagnosis _of_ PG and _the_ _description_ of highly vascular _proliferation_ that resembles granulation _tissue_ (Fig. [1](#F1){ref-type="fig"}). Figure 1Histological images of the pyogenic granuloma showing an _appearance_ similar to granulation tissue. The histological _type_ of _the_ pyogenic _granuloma_ _is_ non-lobular _capillary_ _hemangioma._ Arrow _heads_ label _blood_ _vessels_ surrounded by connective tissue. The surfaces _of_ _the_ implants associated with the lesion were smooth (2 _implants),_ machined (3 implants) and rough _(5_ _implants)._ In no case there was a natural _tooth_ _adjacent_ to the implants related to _the_ lesion. The characteristics of diameters and lengths of the implants studied can be seen _in_ _figure_ [2](#F2){ref-type="fig"}. The _average_ _load_ time of the implants studied was 115 months (SD = _67.5),_ ranging from _a_ range of 9 to 184 months. _Oral_ rehabilitation was performed with complete prosthesis in 9 patients. The mean mesial bone loss was 2.14 mm (range 0 to 6.50 mm, SD = 2.07) _and_ the _mean_ of distal _bone_ was 1.66 mm (range _0_ to 3.75 mm, SD = 1.21. Figure _2Diameter_ _and_ length of dental implants related _to_ the pyogenic granuloma. There were no _statistically_ significant _association_ between the PG area _and_ the marginal _bone_ loss. However the smooth _implant_ surface showed a significant _influence_ on bone loss _(Anova:_ *p* = 0.001) _(Fig._ _[3](#F3){ref-type="fig"})._ Figure 3Peri-implant bone loss grouped by type _of_ surface. The bone loss was _the_ highest for _implants_ with smooth _surface._ Discussion _==========_ The clinical and histopathological findings _have_ confirmed _the_ diagnosis of pyogenic granuloma in 10 patients. _The_ present study is the one with the highest number of _implant-related_ PG _lesion_ that are available _until_ now in the scientific literature. These _PG_ lesions have _been_ diagnosed as non-lobular capillary hemangioma. There are two histological types of PG. _The_ first type _is_ characterized by proliferating blood vessels that are organized in lobular aggregates. _This_ histological type of PG _was_ called lobular capillary hemangioma (LCH type). The second type (non-LCH type) consist of _highly_ vascular proliferation that resembles granulation tissue ([@B1],[@B4],[@B11]). Literature data indicated that PG is rarely associated with dental _implants,_ as _there_ are only five cases _reported_ ([@B1]-[@B3],[@B7],[@B8]). However, _other_ reactive lesions such as gingival hyperplasia _caused_ by phenytoin, allergy to _titanium_ abutments or peripheral giant cell _granulomas_ _have_ been reported _in_ the international _literature._ Causes _of_ conventional oral _pyogenic_ granulomas are not clear, _although_ it has been _shown_ that different stimuli irritants _that_ can trigger _them,_ such as repeated trauma, poor oral hygiene and hormonal problems ([@B1]-[@B20]). About 30-50% of patients _with_ PG have a history of _local_ _trauma_ ([@B9]). Considering _PG,_ in the case reported by Dojcinovic *et al.* ([@B1]), the inappropriate healing cap has resulted in dental plaque accumulation and chronic inflammation _of_ the peri-implant tissues, _triggering_ the development of a _PG._ However this _was_ not _the_ cause _for_ PG in the case reported by _Olmedo_ *et al.* ([@B2]). The authors have pointed out to the _presence_ of "metal-like" particles and have postulated _that_ these _particles_ could be the result electrochemical phenomena, corrosion, friction, _or_ a synergistic combination of these events ([@B4],[@B5]). _Once_ released, these _particles_ _may_ trigger an inflammatory response mediated _by_ _cytokines_ _and_ macrophages ([@B5]). This inflammatory reaction could perpetuate the pseudo-periodontal pocket that generates the lesion _around_ the implant ([@B5]). In the case reported _by_ Etöz _*et_ al.* ([@B |
Introduction {#sec1}
============
In total hip arthroplasty (THA), optimum component position is critical for long-term success of the operation by decreasing rates of wear, aseptic loosening, and dislocation \[[@bib1], [@bib2], [@bib3], [@bib4]\]. Recognizing the importance of acetabular component position, Lewinnek et al published a "safe zone" of 5° to 25° for anteversion and 30° to 50° for acetabular abduction based on their experience with dislocations after posterior THA \[[@bib5]\]. This still serves as the standard for ideal acetabular component position but has been called into question given the importance of the spinopelvic relationship \[[@bib6],[@bib7]\]. In addition to acetabular component position, emphasis has also been placed on femoral component positioning.
Historically, surgeons have identified the importance of keeping the femoral component out of varus because of increased rates of failure with varus cemented femoral stems \[[@bib8],[@bib9]\]. With the use of cementless femoral fixation, varus positioning of the femoral stem has not been shown to lead to the same increased failures \[[@bib10]\]. However, as compared with cemented femoral stems, many cementless femoral stems provide less ability to adjust the version of the component as a stable press-fit requires the stem to adapt to the proximal geometry of the native femur. Consequently, recent attention has been given to the combined version (CV) of the acetabular and femoral components, with the goal of improving impingement-free range of motion and decreasing instability \[[@bib11], [@bib12], [@bib13]\]. The concept of CV was originally introduced by Ranawat, and he described the use of the "Ranawat sign" to determine CV intraoperatively when using the posterior approach \[[@bib14]\]. While no optimum femoral version has been described, Dorr proposed a CV safe zone of 25°-50° based on previous anatomical studies and his experience with decreased instability in this range \[[@bib15]\]. More recent studies have attempted to quantify a combined anteversion that minimizes impingement \[[@bib16],[@bib17]\].
Native femoral anteversion can vary a great deal, and intraoperative judgment of femoral component version can be difficult. Using preoperative computerized tomography (CT) scans of a group of 46 patients scheduled for primary THA, Bargar et al \[[@bib18]\] found a large range of native femoral version from 6° of retroversion to 33° of anteversion. Dorr et al compared the surgeon's estimate of femoral component anteversion in the posterior approach with the postoperative CT measurement of version and found a poor precision of the surgeon\'s estimate with a correlation coefficient of only 0.688 \[[@bib19]\]. In addition, this study found that only 45% of the femoral stems landed within the desired range of 10°-20° of anteversion.
In direct anterior total hip arthroplasty (DA-THA), femoral component broaching and insertion occurs while the patient is positioned supine with the leg fully extended, and the leg below the knee is often draped from the surgeon\'s view. Despite published results of comparable patient outcomes from the DA-THA with other THA approaches, some have questioned the ability to appropriately orient the femoral component with respect to femoral anteversion, via this approach \[[@bib20], [@bib21], [@bib22], [@bib23]\]. Previous studies have reported on improved acetabular component positioning in DA-THA \[[@bib24],[@bib25]\]. However, no prior study, to our knowledge, has examined the combined anteversion of the femoral and acetabular components in DA-THA. This study aims to analyze the combined femoral and acetabular anteversion with cross-sectional imaging and quantify this relative to the CV "safe zone" described by Dorr.
Materials and methods {#sec2}
=====================
After obtaining institutional review board approval, patients were approached for enrollment in the study. An patient who was undergoing a primary DA-THA from the senior author (JBM) was a candidate for enrollment. Patients with femoral or acetabular hardware were excluded from this study. Thirty consecutive patients were enrolled in the study. Four blinded observers independently recorded the measurements (2 fellowship-trained arthroplasty surgeons, one hip and knee fellow, and one orthopaedic resident). All implants were positioned using intraoperative fluoroscopy based on preoperative templating. A CORAIL femoral stem (DePuy, Warsaw, IN) and a PINNACLE acetabular cup (DePuy, Warsaw, IN) were used for all the cases. The senior author standardized intraoperative images by matching the anteroposterior (AP) pelvis fluoroscopic view with the preoperative AP pelvis standing radiograph.
One month after surgery, all patients had a standing AP pelvis and a cross-table lateral radiograph taken, which were used for acetabular component position measurement. Abduction and anteversion measurements of the acetabulum were made from the digital radiograph using the TraumaCad (Voyant Health, Columbia, MD) hip abduction measurement tool. Femoral component position measurements were taken from limited supine CT scan of the hip and knee with 2.5-mm cuts (General Electric BrightSpeed, Fairfield, CT). CT was not selected for acetabular component position to minimize patient radiation exposure. Angular measurements were calculated using the axis of the top of neck of the femoral stem relative to both the posterior condylar axis (PCA) and the transepicondylar axis (TEA).
The CV was then calculated for the TEA and the PCA by adding the femoral anteversion calculated from the CT scan with the anteversion measured from the standing AP pelvis radiograph.
Statistical analysis {#sec2.1}
--------------------
Measurements from the 4 observers were combined, and the mean and standard deviation were calculated. The Pearson correlation coefficient was also measured for each observer, with the kappa values reported, and compared with the group for all measurements. The mean for each measurement was used to determine the number of components placed in the "safe zone." Statistical analysis was performed with the use of SAS software (SAS Institute, Raleigh, NC).
Results {#sec3}
=======
Of the 30 enrolled patients, 29 had an appropriate CT scan obtained. One patient had a CT scan performed without adherence to the protocol precluding reference of femoral version to the axes of the knee and was excluded from the results.
The mean acetabular abduction and anteversion were 39.3° (standard deviation \[SD\] = 4.2°) and 27.2° (SD = 4.7°), respectively. The mean stem anteversion was 17.5° (SD = 10.8°) from the TEA and 21.7° (SD = 11.3°) from the PCA. Ten of the 30 cups were placed inside of the "safe zone" of Lewinnek for acetabular anteversion, but all cups were within the "safe zone" for abduction ([Fig. 1](#fig1){ref-type="fig"}).Figure 1The Lewinnek "safe zone." Ten of the 30 cups were placed inside of the "safe zone" of Lewinnek for acetabular anteversion, but all cups were within the "safe zone" for abduction.
Combined femoral and acetabular component anteversion from the TEA resulted in 79% (23 of 29) of patients within the "safe zone" of 25°-50° with accurately oriented components ([Fig. 2](#fig2){ref-type="fig"}).Figure 2Combined anteversion. Combined femoral and acetabular component anteversion.
Pearson correlation coefficients were high for both stem anteversion from the TEA (R = 0.96) and the PCA (R = 0.98); however, the kappa coefficient for interobserver reliability for combined component anteversion was greater for the TEA (kappa = 0.83 vs 0.65).
Discussion {#sec4}
==========
Component positioning has been recognized as an important factor in the long-term survival of THA \[[@bib5],[@bib12],[@bib26]\]. Muller et al. \[[@bib27]\] suggested a cup anteversion of 10°-15° and femoral anteversion of 10° to be ideal. Lewinnek et al. \[[@bib5]\] followed with their study that found a lower dislocation rate when the acetabular components were positioned in a safe zone of 30°-50° of inclination and 5°-25° of anteversion. A study by Biedermann et al. \[[@bib28]\] found the lowest dislocation rates with acetabular components positioned at 45° of inclination and 15° of anteversion. More recently, Dorr proposed a CV safe zone of 25°-50° based on previous anatomical studies and his experience with decreased instability in this range \[[@bib15]\].
Many authors have begun to appreciate the importance of the combined femoral and acetabular anteversion on dislocation rates and impingement \[[@bib3],[@bib11],[@bib12],[@bib29]\]. Ranawat and Maynard \[[@bib6]\] suggested the importance of the combination of femoral and acetabular anteversion and recommended 45° for women and between 20° and 30° for men. Jolles et al. \[[@bib12]\] found that when the combined anteversion was outside of a range of 40°-60°, the patient's dislocation was 6.9 times higher. Hisatome and Doi \[[@bib29]\] examined combined anteversion in a mathematical model to find the optimum positions to avoid neck impingement with different sized components. They recommended an ideal position, while not accounting for patient's pelvic inclination, of 45° of cup ab | introduction { # sec1 } = = = = = = = = = = = = in total arterial arthroplasty ( tha ), optimum component position is critical for long - term success of the operation by decreasing rates of wear, aseptic loosening, and dislocation \ [ [ @ bib1 ], [ @ bib2 ], [ @ bib3 ], [ @ bib4 ] \ ]. recognizing the importance of acetabular constituent position, lewinnek et al published a " safe zone " of 5° to 25° of anteversion and 30° to 50° for acetabular abduction rely on their experience with dislocations after posterior tha \ [ [ @ bib5 ] \ ]. vc still serves as the standard for ideal acetabular component position but has been called into question given the importance of the spinopelvic relationship \ [ [ @ bib6 ], [ @ bib7 ] \ ]. in addition to acetabular component position, emphasis has also been placed on femoral component positioning. historically, surgeons have identified the importance of keeping the femoral component out of varus because of increased rates of failure with varus cemented femoral stem \ [ [ @ bib8 ], [ @ bib9 ] \ ]. with conventional use of cementless femoral fixation, varus placement of the femoral stem has not been shown to lead to the same increased failures \ [ [ @ bib10 ] \ ]. however, as compared with cemented femoral stems, many cementless femoral stems provide less ability to adjust the version of the component as a stable press - fit requires the stem to adapt to the proximal geometry of the native femur. consequently, recent attention has been given to the combined version ( cv ) of the acetabular and femoral components, with the goal of improving impingement - free range of motion and decreasing instability \ [ [ @ bib11 ], [ @ bib12 ], [ @ bib13 ] \ ]. the concept of cv was originally introduced by ranawat, and he described the use of the " ranawat sign " to determine cv intraoperatively when using the posterior approach \ [ [ @ bib14 ] \ ]. while no optimum femoral version have been described, dorr demonstrates a cv safe zone of 25° - 50° based on previous anatomical studies and his experience with decreased instability in this range \ [ [ @ bib15 ] \ ]. more recent studies have attempted to quantify a combined anteversion that minimizes impingement \ [ [ @ bib16 ], [ @ bib17 ] \ ]. native femoral anteversion can vary a great deal, and intraoperative judgment of femoral component version can be difficult. using preoperative computerized tomography ( ct ) scans of a group of 46 patients scheduled for primary tha, bargar et al \ [ [ @ bib18 ] \ ] found a large range of native femoral version from 6° of retroversion to 33° of anteversion. dorr et al compared the surgeon ' s estimate of femoral component anteversion in the posterior approach with the postoperative ct measurement of version and found a poor precision of the surgeon \ ' s estimate with a correlation coefficient of only 0. 688 \ [ [ @ bib19 ] \ ]. in addition, this study found that only 45 % of the femoral stems landed within the desired range of 10° - 20° of anteversion. in direct anterior total hip arthroplasty ( da - tha ), femoral component broaching and insertion occurs while the patient is positioned supine with the leg fully extended, and the leg below the knee is often draped from the surgeon \ ' s view. despite published results of comparable patient outcomes from the da - tha with other tha approaches, some have questioned the ability to appropriately orient the femoral component with respect to femoral anteversion, via this approach \ [ [ @ bib20 ], [ @ bib21 ], [ @ bib22 ], [ @ bib23 ] \ ]. previous studies have reported on improved acetabular component positioning in da - tha \ [ [ @ bib24 ], [ @ bib25 ] \ ]. however, no prior study, to our knowledge, has examined the combined anteversion of the femoral and acetabular components in da - tha. this study aims to analyze the combined femoral and acetabular anteversion with cross - sectional imaging and quantify this relative to the cv " safe zone " described by dorr. materials and methods { # sec2 } = = = = = = = = = = = = = = = = = = = = = after obtaining institutional review board approval, patients were approached for enrollment in the study. an patient who was undergoing a primary da - tha from the senior author ( jbm ) was a candidate for enrollment. patients with femoral or acetabular hardware were excluded from this study. thirty consecutive patients were enrolled in the study. four blinded observers independently recorded the measurements ( 2 fellowship - trained arthroplasty surgeons, one hip and knee fellow, and one orthopaedic resident ). all implants were positioned using intraoperative fluoroscopy based on preoperative templating. a corail femoral stem ( depuy, warsaw, in ) and a pinnacle acetabular cup ( depuy, warsaw, in ) were used for all the cases. the senior author standardized intraoperative images by matching the anteroposterior ( ap ) pelvis fluoroscopic view with the preoperative ap pelvis standing radiograph. one month after surgery, all patients had a standing ap pelvis and a cross - table lateral radiograph taken, which were used for acetabular component position measurement. abduction and anteversion measurements of the acetabulum were made from the digital radiograph using the traumacad ( voyant health, columbia, md ) hip abduction measurement tool. femoral component position measurements were taken from limited supine ct scan of the hip and knee with 2. 5 - mm cuts ( general electric brightspeed, fairfield, ct ). ct was not selected for acetabular component position to minimize patient radiation exposure. angular measurements were calculated using the axis of the top of neck of the femoral stem relative to both the posterior condylar axis ( pca ) and the transepicondylar axis ( tea ). the cv was then calculated for the tea and the pca by adding the femoral anteversion calculated from the ct scan with the anteversion measured from the standing ap pelvis radiograph. statistical analysis { # sec2. 1 } - - - - - - - - - - - - - - - - - - - - measurements from the 4 observers were combined, and the mean and standard deviation were calculated. the pearson correlation coefficient was also measured for each observer, with the kappa values reported, and compared with the group for all measurements. the mean for each measurement was used to determine the number of components placed in the " safe zone. " statistical analysis was performed with the use of sas software ( sas institute, raleigh, nc ). results { # sec3 } = = = = = = = of the 30 enrolled patients, 29 had an appropriate ct scan obtained. one patient had a ct scan performed without adherence to the protocol precluding reference of femoral version to the axes of the knee and was excluded from the results. the mean acetabular abduction and anteversion were 39. 3° ( standard deviation \ [ sd \ ] = 4. 2° ) and 27. 2° ( sd = 4. 7° ), respectively. the mean stem anteversion was 17. 5° ( sd = 10. 8° ) from the tea and 21. 7° ( sd = 11. 3° ) from the pca. ten of the 30 cups were placed inside of the " safe zone " of lewinnek for acetabular anteversion, but all cups were within the " safe zone " for abduction ( [ fig. 1 ] ( # fig1 ) { ref - type = " fig " } ). figure 1the lewinnek " safe zone. " ten of the 30 cups were placed inside of the " safe zone " of lewinnek for acetabular anteversion, but all cups were within the " safe zone " for abduction. combined femoral and acetabular component anteversion from the tea resulted in 79 % ( 23 of 29 ) of patients within the " safe zone " of 25° - 50° with accurately oriented components ( [ fig. 2 ] ( # fig2 ) { ref - type = " fig " } ). figure 2combined anteversion. combined femoral and acetabular component anteversion. pearson correlation coefficients were high for both stem anteversion from the tea ( r = 0. 96 ) and the pca ( r = 0. 98 ) ; however, the kappa coefficient for interobserver reliability for combined component anteversion was greater for the tea ( kappa = 0. 83 vs 0. 65 ). discussion { # sec4 } = = = = = = = = = = component positioning has been recognized as an important factor in the long - term survival of tha \ [ [ @ bib5 ], [ @ bib12 ], [ @ bib26 ] \ ]. muller et al. \ [ [ @ bib27 ] \ ] suggested a cup anteversion of 10° - 15° and femoral anteversion of 10° to be ideal. lewinnek et al. \ [ [ @ bib5 ] \ ] followed with their study that found a lower dislocation rate when the acetabular components were positioned in a safe zone of 30° - 50° of inclination and 5° - 25° of anteversion. a study by biedermann et al. \ [ [ @ bib28 ] \ ] found the lowest dislocation rates with acetabular components positioned at 45° of inclination and 15° of anteversion. more recently, dorr proposed a cv safe zone of 25° - 50° based on previous anatomical studies and his experience with decreased instability in this range \ [ [ @ bib15 ] \ ]. many authors have begun to appreciate the importance of the combined femoral and acetabular anteversion on dislocation rates and impingement \ [ [ @ bib3 ], [ @ bib11 ], [ @ bib12 ], [ @ bib29 ] \ ]. ranawat and maynard \ [ [ @ bib6 ] \ ] suggested the importance of the combination of femoral and acetabular anteversion and recommended 45° for women and between 20° and 30° for men. jolles et al. \ [ [ @ bib12 ] \ ] found that when the combined anteversion was outside of a range of 40° - 60°, the patient ' s dislocation was 6. 9 times higher. hisatome and doi \ [ [ @ bib29 ] \ ] examined combined anteversion in a mathematical model to find the optimum positions to avoid neck impingement with different sized components. they recommended an ideal position, while not accounting for patient ' s pelvic inclination, of 45° of cup ab | Introduction {# sec1} = = = = = = = = = = = = In total hip arthroplasty (THA ), optimum component position is critical for long - term success of the operation by decreasing rates of wear, aseptic loosening, and dislocation \ [[ @ bib1 ], [@ bib2 ], [@ bOb@ ], [@ bib4] \ ]. Recognizing the importance of acetabular component position, Lewinnek et al published a " safe zone " of 5 ° to 25 ° for anteversion and 30 ° to 50 ° for acetabular abduction based on their experience with dislocations after posterior THA \ [[ @ bib5] \ ]. This still serves as the standard for ideal acetabular component position but has been called into question YiveM the importance of the spinopelvic relationship \ [[ @ bib6 ], [@ bib7] \ ]. In addition to adetabupar component position, emphasis has also been placed on femoral component positioning. Historically, surgeons have identified the importance of keeping the femoral component out of varus because of increased rates of failure with varus cemented femoral stems \ [[ @ bib8 ], [@ bib9] \ ]. With the use of cementless femoral fixation, varus positioning of the femoral stem has not been shown to lead to the same increased failures \ [[ @ bib10] \ ]. However, as compared with cemented femoral stems, many cementless femoral stems provide less ability to adjust the version of the component as a stable press - fit requires the stem to adapt to the proximal geometry of the native femur. Consequently, recent attention has been given to the combined version (CV) of the acetabular and femoral components, with the goal of improving impingement - free 4anne of motion and decreasing instability \ [[ @ bib11 ], [@ bib12 ], [@ bib13] \ ]. The concept of CV was originally introduced by Ranawat, and he described the use of the " Ranawat sign " to determine CV intraoperatively when using the posterior approach \ [[ @ bib14] \ ]. While no optimum femoral version has been described, Dorr proposed a CV safe zone of 25 ° - 50 ° based on previous anatomical studies and his experience with devreaseC instability in this range \ [[ @ bib15] \ ]. More recent studies have attempted to quantify a combined anteversion that minimizes impingement \ [[ @ bib16 ], [@ bib17] \ ]. Native femoral anteversion can vary a great deal, and intraoperative judgment of femoral component version can be difficult. Using preoperative computerized tomography (CT) scans of a group of 46 patients scheduled for primary THA, Bargar et al \ [[ @ bib18] \] found a large range of native femoral version from 6 ° of retroversion to 33 ° of anteversion. Dorr et al compared the surgeon ' s estimate of femoral component anteversion in the posterior approach with the postoperative CT measurement of version and found a poor precision of the surgeon \ ' s estimate with a correlation coefficient of only 0. 688 \ [[ @ bib19] \ ]. In addition, this study found that only 45% of the femoral stems landed within the desired range of 10 ° - 20 ° of anteversion. In direct anterior total hip arthroplasty (DA - THA ), femoral component broaching and insertion occurs while the patient is positioned supine with the leg fully extended, and the leg below the knee is often draped from the surgeon \ ' s view. Despite published results of comparable patient outcomes from the DA - THA with other THA approaches, some have questioned the ability to appropriately orient the femoral component with respect to femoral anteversion, via this approach \ [[ @ bib20 ], [@ bib21 ], [@ bib22 ], [@ bib23] \ ]. Previous studies have reported on improved acetabular component positioning in DA - THA \ [[ @ bib24 ], [@ bib25] \ ]. However, no prior study, to our knowledge, has examined the combined anteversion of the femoral and acetabular components in DA - THA. This study aims to analyze the combined femoral and acetabular anteversion with cross - sectional imaging and quantify this relat8v# to the CV " safe zone " described by Dorr. Materials and methods {# sec2} = = = = = = = = = = = = = = = = = = = = = After obtaining institutional review board approval, patients were approached for enrollment in the study. An patient who was undergoing a primary DA - THA from the senior author (JBM) was a candidate for enrollment. Patients with femoral or acetabular hardware were excluded from this study. Thirty consecutive patients were enrolled in the study. Four blinded observers independently recorded the measurements (2 fellowship - trained arthroplasty surgeons, one hip and knee fellow, and one orthopaedic resident ). All implants were positioned using intraoperative fluoroscopy based on preoperative templating. A CORAIL femoral stem (DePuy, Warsaw, IN) and a PINNACLE acetabular cup (DePuy, Warsaw, IN) were used for all the cases. The senior author standardized intraoperative images by matching the anteroposterior (AP) pelvis fluoroscopic view with the preoperative AP pelvis standing radiograph. One month after surgery, all patients had a standing AP pelvis and a cross - table lateral radiograph taken, which were used for acetabular component position measurement. Abduction and anteversion measurements of the acetabulum were made from the digital radiograph using the TraumaCad (Voyant Health, Columbia, MD) hip abduction measurement tool. Femoral component position measurements were taken from limited supine CT scan of the hip and knee with 2. 5 - mm cuts (General Electric BrightSpeed, Fairfield, CT ). CT was not selected for acetabular component positLoj to minimize patient radiation exposure. Angular measurements were calculated using the axis of the top of neck of the femoral stem relative to both the posterior condylar axis (PCA) and the transepicondylar axis (TEA ). The CV was then calculated for the TEA and the PCA by adding the femoral anteversion calculated from the CT scan with the anteversion measured from the standing AP pelvis radiograph. Statistical analysis {# sec2. 1} - - - - - - - - - - - - - - - - - - - - Measurements from the 4 observers were combined, and the mean and standard deviation were calculated. The Pearson correlation coefficient was also measured for each observer, with the kappa values reported, and compared with the group for all measurements. The mean for each measurement was used to determine the number of components placed in the " safe zone. " Statistical analysis was performed with the use of SAS software (SAS Institute, Raleigh, NC ). Results {# sec3} = = = = = = = Of the 30 enrolled patients, 29 had an appropriate CT scan obtained. One patient had a CT scan performed without adherence to the protocol precluding reference of femoral version to the axes of the knee and was excluded from the results. The mean acetabular abduction and anteversion were 39. 3 ° (standard deviation \ [SD \] = 4. 2 °) and 27. 2 ° (SD = 4. 7 ° ), respectively. The mean stem anteversion was 17. 5 ° (SD = 10. 8 °) from the TEA and 21. 7 ° (SD = 11. 3 °) from the PCA. Ten of the 30 cups were placed inside of the " safe zone " of Lewinnek for acetabular anteversion, but all cups were within the " safe zone " for abduction ([ Fig. 1] (# fig1) {ref - type = " fig "} ). Figure 1The Lewinnek " safe zone. " Ten of the 30 cups were placed inside of the " safe zone " of Lewinnek for acetabular anteversion, but all cups were within the " safe zone " for abduction. Combined femoral and acetabular component anteversion from the TEA resulted in 79% (23 of 29) of patients within the " safe zone " of 25 ° - 50 ° with accurately oriented components ([ Fig. 2] (# fig2) {ref - type = " fig "} ). Figure 2Combined anteversion. Combined femoral and acetabular component anteversion. Pearson correlation coefficients wRTe high for both stem anteversion from the TEA (R = 0. 96) and the PCA (R = 0. 98 ); however, the kappa coefficient for interobserver reliability for combined component anteversion was greater for the TEA (kappa = 0. 83 vs 0. 65 ). Discussion {# sec4} = = = = = = = = = = Component positioning has been recognized as an important factor in the long - term survival of THA \ [[ @ bib5 ], [@ bib12 ], [@ bib26] \ ]. Muller et al. \ [[ @ bib27] \] suggested a cup anteversion of 10 ° - 15 ° and femoral anteversion of 10 ° to be ideal. Lewinnek et al. \ [[ @ bib5] \] followed with their study that found a lower dislocation rate when the acetabular components were positioned in a safe zone of 30 ° - 50 ° of inclination and 5 ° - 25 ° of anteversion. A study by Biedermann et al. \ [[ @ bib28] \] found the lowest dislocation rates with acetabular components positioned at 45 ° of inclination and 15 ° of anteversion. More recently, Dorr proposed a CV safe zone of 25 ° - 50 ° based on previous anatomical studies and his experience with decreased instability in this range \ [[ @ bib15] \ ]. Many authors have begun to appreciate the importance of the combined femoral and acetabular anteversion on dislocation rates and impingement \ [[ @ bib3 ], [@ bib11 ], [@ bib12 ], [@ bib29] \ ]. Ranawat and Maynard \ [[ @ bib6] \] suggested the importance of the Vombinati(n of femoral and acetabular anteversion and recommended 45 ° for women and between 20 ° and 30 ° for men. Jolles et al. \ [[ @ bib12] \] found that when the combined anteversion was outside of a range of 40 ° - 60 °, the patient ' s dislocation was 6. 9 times higher. Hisatome and Doi \ [[ @ bib29] \] examined combined anteversion in a mathematical model to find the optimum positions to avoid becM impingement with different sized components. They recommended an ideal position, while not accounting for patient ' s pelvic inclination, of 45 ° of cup ab | {#sec1} In total hip arthroplasty (THA), component position is critical for long-term success of the operation by decreasing rates of wear, loosening, and dislocation \[[@bib1], [@bib2], [@bib3], [@bib4]\]. Recognizing the importance acetabular component position, Lewinnek et al a "safe of 5° to 25° for anteversion and 30° to for acetabular abduction based on their experience with dislocations after posterior THA \[[@bib5]\]. This as the standard for ideal acetabular component position but has been called into question given the importance of the spinopelvic In addition to acetabular component position, emphasis also been placed on femoral component positioning. Historically, surgeons have the importance keeping the femoral component out of varus of increased rates of failure with cemented femoral stems \[[@bib8],[@bib9]\]. With the use of cementless femoral fixation, varus positioning of femoral stem has not been shown to lead to the same increased failures \[[@bib10]\]. However, as compared with cemented femoral stems, many cementless femoral stems provide less ability to adjust the version of the component as a stable press-fit requires the stem to adapt to the proximal geometry of the native femur. Consequently, recent attention been given to the combined version (CV) of the acetabular and femoral components, goal of improving range of motion decreasing instability \[[@bib11], [@bib12], [@bib13]\]. The concept of CV was originally introduced and he described the use of the "Ranawat sign" to determine CV intraoperatively when using the posterior approach While no optimum femoral has been described, Dorr proposed a CV safe zone of 25°-50° based on previous and his with decreased instability in this \[[@bib15]\]. More recent studies have to quantify a combined anteversion that minimizes impingement \[[@bib16],[@bib17]\]. Native femoral anteversion can vary a great deal, and judgment femoral component version can be difficult. Using preoperative computerized tomography (CT) scans of a group of 46 patients scheduled for THA, Bargar et al \[[@bib18]\] found a large range of native femoral version from 6° of retroversion to 33° of anteversion. Dorr et compared surgeon's estimate of femoral component anteversion in the posterior approach with the postoperative CT of found a poor precision of the surgeon\'s estimate with a correlation coefficient of only 0.688 \[[@bib19]\]. In addition, this study found only 45% of the femoral stems landed within the desired of 10°-20° of anteversion. In direct anterior total arthroplasty femoral component broaching and insertion occurs while the patient is positioned supine with leg fully extended, the leg below the knee is often draped from the surgeon\'s view. Despite published results of comparable patient outcomes from the DA-THA with other THA approaches, have questioned the to appropriately orient the femoral component with respect to femoral anteversion, via this approach \[[@bib20], [@bib21], [@bib22], [@bib23]\]. Previous studies have reported on acetabular component positioning in DA-THA \[[@bib24],[@bib25]\]. However, no prior study, to our knowledge, has examined the combined anteversion of the femoral and acetabular components in DA-THA. This study aims to analyze the combined femoral and acetabular anteversion with cross-sectional imaging and quantify this relative to the CV "safe zone" described by Dorr. and methods {#sec2} ===================== After obtaining institutional review board approval, were approached for enrollment in study. An patient who was undergoing a primary DA-THA from the senior author (JBM) was a candidate for enrollment. Patients with femoral or acetabular were excluded from this study. Thirty consecutive patients were enrolled in the study. blinded observers independently recorded measurements fellowship-trained arthroplasty surgeons, one and knee and one orthopaedic resident). All implants were positioned using fluoroscopy based on preoperative templating. A femoral stem (DePuy, Warsaw, IN) and a PINNACLE acetabular cup (DePuy, Warsaw, IN) were used for all the cases. The senior author standardized intraoperative images by matching the anteroposterior (AP) pelvis fluoroscopic with the preoperative AP pelvis radiograph. One month after surgery, all patients had a standing AP pelvis and a lateral radiograph taken, which were used for acetabular component position measurement. Abduction and anteversion measurements of the were made from the digital radiograph using the TraumaCad (Voyant Health, Columbia, MD) hip measurement tool. Femoral component position measurements were taken limited supine CT scan of the hip and knee with 2.5-mm cuts Electric BrightSpeed, Fairfield, CT was not selected for acetabular component position minimize patient radiation exposure. Angular measurements were the axis of the top of neck of the femoral relative to the posterior axis (PCA) and the transepicondylar axis (TEA). The CV was then calculated for the and the PCA by adding the femoral anteversion from the CT scan with the anteversion measured from the standing pelvis radiograph. Statistical analysis -------------------- Measurements from the observers were combined, and the mean and standard deviation were calculated. The Pearson correlation coefficient was also measured for each observer, with the kappa values reported, and compared the group for all measurements. The mean for each measurement to determine the number of components placed the "safe zone." Statistical analysis was performed with use of SAS software (SAS Institute, Raleigh, Results {#sec3} ======= the 30 enrolled patients, 29 had an appropriate CT scan obtained. One had a CT scan performed without to the precluding reference of femoral version the axes of the knee and was excluded the results. The mean acetabular anteversion were (standard deviation \[SD\] = 4.2°) and 27.2° (SD = 4.7°), respectively. The mean stem anteversion was 17.5° 10.8°) the TEA and 21.7° (SD 11.3°) the PCA. Ten of the 30 cups were placed of the "safe zone" of Lewinnek for but all cups were within the "safe zone" abduction ([Fig. 1](#fig1){ref-type="fig"}).Figure 1The Lewinnek "safe zone." Ten of the 30 cups were placed inside of the "safe zone" Lewinnek for acetabular but all cups were within the "safe zone" for abduction. Combined femoral and acetabular component anteversion from the TEA resulted in 79% (23 of 29) of within the "safe zone" 25°-50° with accurately oriented components ([Fig. 2Combined anteversion. Combined femoral and acetabular component anteversion. Pearson correlation coefficients were high for both stem anteversion from the TEA (R = 0.96) and the PCA (R = however, the kappa coefficient for reliability for combined anteversion was for the TEA (kappa = 0.83 vs 0.65). Discussion {#sec4} ========== Component positioning has been recognized as an factor the long-term survival of Muller et al. \[[@bib27]\] suggested a cup anteversion of 10°-15° and femoral of 10° to be ideal. et al. \[[@bib5]\] their study that found a lower dislocation rate when the acetabular components were positioned in a safe zone 30°-50° of inclination and 5°-25° of anteversion. A study by Biedermann et al. \[[@bib28]\] found the lowest dislocation rates with acetabular components positioned at 45° of inclination and 15° of More Dorr proposed a CV safe of 25°-50° based on previous anatomical studies and his experience with decreased instability in this range \[[@bib15]\]. Many authors have begun to appreciate the importance of combined femoral and on dislocation and impingement \[[@bib3],[@bib11],[@bib12],[@bib29]\]. Ranawat and Maynard \[[@bib6]\] suggested the importance of the combination of acetabular anteversion and recommended 45° for women and between 20° and 30° for men. Jolles et al. \[[@bib12]\] found that when the combined anteversion was outside of a range of 40°-60°, patient's dislocation was times higher. Hisatome and Doi \[[@bib29]\] examined combined anteversion in a mathematical model to find the positions to avoid neck impingement different sized components. They recommended an ideal position, not accounting for patient's pelvic inclination, of 45° of cup ab | iNTRODUctIOn {#Sec1}
============
IN tOtaL HIp ArtHROplaSTy (Tha), OptIMuM cOMpoNent POSitioN is cRitICaL FOR loNG-TERm SuccESS OF tHe opErATioN BY DeCREAsiNg RAteS of WeAR, aSePTic LoOSEnINg, aNd DiSloCation \[[@BIB1], [@BIB2], [@BiB3], [@bib4]\]. recogNiziNG THe IMpOrTANce OF aCETAbuLAR CompONeNt pOSition, lEwinnek eT Al pUBLisHeD A "SAFE zonE" Of 5° to 25° fOr anTeVersioN AND 30° TO 50° fOr acETABuLar AbdUCtiON BasED ON THeIr eXpeRIEnCe WITH dISLOCAtIoNS aFtER PoSTerIOr THa \[[@Bib5]\]. thIs sTILL SErvEs As The standARd FOR idEAL AceTABulAR coMPoneNt POSITioN But has bEEn CALLed IntO qUEsTIon GIVEN thE imPortanCe OF THE SPINoPelvic rElAtiONshIP \[[@bIb6],[@biB7]\]. IN aDDItioN to aCEtaBular comPoneNt posiTiON, eMPhaSis HaS alSo bEeN PlACEd on FEMORAl cOMponeNt pOSiTioNInG.
HISTORICaLLy, sUrGEONs HAVE idEntIFiED tHe ImPOrTANCe of kEEPING the fEMORal comPonEnT oUt Of vAruS BeCAusE OF iNcreASeD raTes OF fAiLUre WIth VaruS cEmEntEd FemOraL StEms \[[@bIB8],[@bib9]\]. WitH THE Use of cEmEntLESs FeMORAL FiXatiON, VarUS PositIonIng of THe FEmOrAL sTem Has NoT BEen sHowN tO lEAD To THe samE increASEd FAiLures \[[@BIB10]\]. hOWEVEr, As CompARED WITh ceMENTED FemoRaL stEMs, maNY cEmENTleSs FEMORAL SteMs provIde LesS abiliTY To ADJusT The VersIon oF THE cOmPoneNt as a StABLE pResS-FIT reqUireS THe Stem to adaPt to THE pRoxiMaL gEOmeTrY Of thE NATive fEMUr. ConseQUENTLy, RecEnt attENTiOn HAs bEen giveN To tHe CoMbinED VErsIon (cv) Of the aCeTAbUlAR and feMORAl CompONeNTS, witH tHe GOAL Of ImprOViNG ImPiNgemenT-FREe RaNgE of mOtiON ANd dEcREASinG InStABiliTy \[[@Bib11], [@BiB12], [@BiB13]\]. tHE cONcePT of cv was OriGInaLLY InTroDUCed BY ranawaT, ANd HE DESCrIBeD thE use of THe "rAnAWaT SIgn" tO DEteRminE cv inTrAOPeRAtiVelY WHeN UsiNG THE postEriOR apPROAch \[[@bib14]\]. whiLE nO OptImuM fEMoral VERSiON HAs bEEn deScrIBEd, DORR PRopoSed a cv SafE zoNe Of 25°-50° BaSED on pReViOus ANatOMICal sTuDIES AND his EXpErieNCE WiTH DECREaSED inSTaBIliTY in THIS rangE \[[@BIb15]\]. MOre reCEnT studIes HAve ATteMPtED TO quaNtifY A COmBINEd aNTEveRSiON thaT MINiMIZEs imPINGeMeNT \[[@BiB16],[@BiB17]\].
NAtivE FemoRaL ANteversIOn CaN VaRY A grEAT deAl, aND intrAOPeRAtIvE jUDGmENt of FEmoRal COmpONEnt VERsIOn cAn Be DiffiCulT. UsiNG pReOpeRatIve cOmpUtErIZeD ToMOGraPhy (CT) scanS OF A GROuP oF 46 paTientS SchEDulEd FOR PRiMaRy ThA, BARGAR eT AL \[[@bIb18]\] FOunD a LarGe raNge of NaTiVe feMoRal vErsioN FrOm 6° oF reTROverSIoN To 33° of anTeVErSIOn. dOrR ET AL COmPaReD The sUrgeON's EsTimATE Of feMORal cOMPONeNt ANtEveRsion iN THe PostERIOR apPRoAcH WitH tHe POstOpErATIVE ct meaSurEMent oF vErSIon And FOund a POOr pREcIsiON of tHe sURGEon\'s EStImATE wiTH A CORrelaTIoN cOefficienT Of oNlY 0.688 \[[@BIB19]\]. IN AddiTiON, this STUDY FouND tHat ONly 45% Of The fEMoRAl sTems laNdeD wItHIn thE DEsIred rAngE OF 10°-20° oF AntevErsIOn.
iN DiRecT ANtErIoR TOtal HIp ArThRoplASty (DA-THa), FEmORAl COmPOnEnt BRoACHINg aND InSerTion OCCuRs whilE ThE pATIenT is POsItioNEd SUPInE wITh tHe leG FULlY EXtEndEd, ANd tHe LeG bELoW thE KnEe is OfTeN dRApEd fROm the suRGeoN\'S vIEw. DEsPItE pUbliSHEd rEsulTs of cOMPARabLe patienT ouTcOMEs FroM THe Da-THa With OTHEr tHA APPROacHeS, Some have QUestIoNeD ThE ABility TO apprOPRiaTeLy oRient ThE fEMORal CoMpONEnt wITH ReSPecT TO FEMoRAL ANTEVersIoN, Via thIS aPProACH \[[@bIb20], [@BiB21], [@BIB22], [@bib23]\]. pREviOUs STudieS haVE RePorTEd oN ImprovED acETaBUlAR CoMPoneNt pOsITIOnInG iN Da-thA \[[@bib24],[@biB25]\]. hoWeVer, no PRior sTUdy, to oUr KNOwLedGe, hAS ExaMINEd tHe CombinED AnTeVErSiOn OF tHE FEmOrAL AND aCeTaBuLar CoMpONents IN DA-tHA. tHIS STUdY aImS To AnaLyZe THe cOmbIned FemOrAl AnD AceTABuLaR ANteVErsion wITh cross-sECTiONal ImaGINg And QuANtIfY This RElAtIVE TO ThE cV "saFe zoNe" deSCRIbed By DoRr.
MAteRiAlS AnD mEtHodS {#sEc2}
=====================
after obTaInInG InsTiTuTionAl REViEw boARD apPRovAL, PATiENtS WeRe APpROached foR ENrOlLmEnT iN the stuDy. an pAtIENT WHO was uNdERGOinG a primARy dA-tHa From the SeniOr aUTHOr (JbM) wAS a cANdidAte FOR enROLLMenT. PaTiEnTs WITH FeMOrAL OR AceTaBULar hArDwaRe WeRe eXCLudeD FRom tHIS StuDY. ThIRTy cOnSecuTiVE patIeNts weRe eNROLLEd iN THE sTuDY. fouR bLiNDEd oBSeRVErS IndePENdENtlY Recorded the MeASuRemenTS (2 FElloWSHiP-TrainEd arthROplAsTY SURGEONs, onE HIp AND kneE felLoW, aNd oNe orthopaediC ReSiDEnT). AlL iMpLANTS were POSitiOnEd USING IntrAOPerAtIvE FLUoROScOpy BasEd ON preOPeratiVe temPLAtinG. A CORaiL femorAl steM (dEPuY, wARSAW, iN) AnD a pINNaClE AcETaBuLaR cUp (dePUy, WArSAW, in) wERe USEd fOr alL THE CasES. The seNIOr AUthOr sTaNdARDiZed iNTrAopeRAtive iMAgEs BY matchiNG tHe ANteroPOSTErIor (Ap) pelvis fLuoroScopiC vIEw With ThE PREoPERative aP peLvIS StandinG radiOGRaPH.
ONE mOnTh AFTer sURgERY, all paTIenTS had a stAnDiNg AP PeLvIS ANd a CROss-TaBLE LAtERal RADiOgRaPh tAKen, wHich WERE uSeD fOR aceTabULAr cOmPoneNt poSiTioN MEaSurEmenT. abductIon And anteVErsIoN MEASuREmENtS Of THe acetAbuLUM wErE MAde from The diGItAl RADIOGRAph UsING thE TRaUmAcad (VOYAnt HeaLTH, COLUMbia, Md) hip AbdUctioN MEASUrEMEnT TooL. femOral comPOnEnT pOsiTIOn meAsUreMEnTs weRe taKen FROm LImIteD supInE cT ScaN Of tHE hiP anD kNEe With 2.5-Mm cUTs (GeNERAl ELeCTRic BrIghTsPeED, fAIrfielD, ct). cT WaS NoT seleCTed fOR aceTaBUlaR CoMPoneNT positIoN TO mINiMIzE patIeNT RaDIatioN EXposure. aNgUlaR meASUrEMENTS WERE calcuLATEd USiNg The aXIS OF ThE tOp oF neck OF THE FEMOrAL STem relatIVe TO bOTh the posteRIOr cOnDYLAr aXis (pca) and thE trAnSePiCONDYLaR axiS (tEA).
the cV WAs ThEN CaLcUlaTED FoR The tea and ThE PCa BY AddING tHe FEMoRaL AnTEVeRsION CalcULaTED From thE ct scAN witH the aNtEVErsION MEaSUReD fRom thE STanDING AP peLvIs RAdiOgraPh.
stAtIstICaL AnAlYSis {#sEc2.1}
--------------------
MEaSurEmEntS frOm ThE 4 OBseRvErs wERe coMbInED, And tHe MEaN And STanDaRd DevIAtIOn Were CAlcUlaTEd. The pEArson CoRreLaTion CoEffICieNt Was aLsO meaSURED FOR EaCH ObSerVEr, WitH thE KapPA vAlUes rEPORTEd, aND cOmpaReD wItH tHE gRoUP foR aLL mEasUreMEnTs. THE MeAN fOR eAcH meAsuReMENT WaS useD tO DeTERMinE THe nuMBER oF coMPonents PlACED IN the "safe zONe." stATiSTIcAl anALySIS was PeRfoRmeD wItH THe usE OF SAs SoFTware (SaS InSTitUTe, RAlEIgH, nC).
resULTS {#sEc3}
=======
Of THe 30 eNrolLED patIENTs, 29 hAd an aPPropRIATE cT ScaN obtAIned. one PaTieNT Had a Ct sCaN PerfOrMED WithOuT AdherENce to THe pROTocOL PrecLUdING rEFEreNce OF FEmorAL VERSIon tO the AxeS oF tHE KNEe anD wAs ExcLuded FROm thE REsULTs.
tHe MEan aceTAbUlAR AbducTIoN anD ANTEVErSiON WerE 39.3° (stAnDaRd DEViAtIon \[Sd\] = 4.2°) aND 27.2° (Sd = 4.7°), ReSpecTiVELy. tHe MEAn sTEm anteVErsION WAS 17.5° (SD = 10.8°) FRoM THe tEA aND 21.7° (sD = 11.3°) FrOm THe pCa. teN oF tHe 30 CUPs WeRe PlACED InSIde oF tHE "sAFe zOne" of LewInnek foR ACeTAbulAr AnTeVersiOn, BUt All cUpS wERe WIThIn thE "SafE zONe" FoR aBDUCtiOn ([fiG. 1](#fig1){Ref-tyPE="FIG"}).FIgUrE 1ThE lEWiNNeK "SAfe zONE." tEN oF THE 30 cUPs weRE PLacED InSide oF The "sAfe zone" of lEwINneK foR AcetABulaR ANteVErSIoN, BuT aLL CUps WERE wItHiN tHE "SAfE zONe" FOR ABdUCtIOn.
COmbiNed femORAL ANd aCETAbULar cOmpOnent aNTEVeRSiOn FroM THE tEa reSuLTEd IN 79% (23 OF 29) OF pATiEntS withiN tHE "safe ZONE" Of 25°-50° WIth AcCURATelY oRiENTeD COmPoNeNTS ([FIG. 2](#fiG2){REf-tYpe="fiG"}).figUre 2COmbInEd AntEVeRSIon. coMBiNeD fEMoRAl aND AcETaBULAr comPoNEnt antEVERSIOn.
PeARsON COrRElAtION CoEFfIcIEnTS WEre hIGh FOr BotH STEM ANTeVErSiOn from ThE teA (r = 0.96) And the PCA (r = 0.98); HoWEVer, tHe kappa CoEFfICienT FOR InTerOBSeRveR reLIabILITy FoR comBIneD ComponenT AntevErSiOn waS GreatER fOR tHe teA (kappa = 0.83 Vs 0.65).
diSCussiON {#sEC4}
==========
COMponEnT PosITIonING HaS BeEn ReCoGnIZeD as aN impoRTANt FAcTOr in The LONg-teRM SurvivAl of ThA \[[@BIb5],[@bIb12],[@BiB26]\]. mUller Et aL. \[[@bIb27]\] SUggesTeD a cuP anteVeRSIon OF 10°-15° AnD FEmORal AnTEveRsiOn oF 10° TO BE idEaL. lewiNnek eT Al. \[[@bIB5]\] fOLlowEd with tHeir StudY THaT FouNd a loWer dislOcATiON raTe wheN ThE ACETABULar CoMPOnENTS weRE PoSiTiONED iN a safE zonE OF 30°-50° Of incLInatiOn AnD 5°-25° Of aNTEVErsIOn. a study BY BIeDERmaNn et Al. \[[@BIB28]\] FoUnd thE LoWEST disLOCatiON rateS WitH ACETAbuLaR COMPOnents poSitIonEd aT 45° of INCLInatioN AnD 15° oF aNTeversIoN. MORE REcENTly, DOrr PrOpOSED a CV Safe zONe oF 25°-50° bASEd on pREvIoUS aNAtomiCaL sTudIEs and HIs ExpERiEnCE wItH DEcReaSeD INstAbILIty In tHIs RaNGe \[[@biB15]\].
MaNy AutHOrS hAve BeguN to AppreCiate The iMporTANCe oF the cOmbiNed FeMOraL anD ACETABULar aNteveRSiON on dislOcATiOn RAtEs AnD iMPINGemENt \[[@bib3],[@bIb11],[@bIB12],[@BIB29]\]. raNaWAt anD MAynarD \[[@Bib6]\] SuggESTeD thE IMpOrtaNce oF The cOMbInatIon of FEmorAL aNd aCETabulaR antEvERSiON ANd rEcommEnDed 45° fOR WomEN AnD BETWEeN 20° aND 30° FOR men. jolles eT aL. \[[@bIb12]\] FOunD tHAt WHen The COmBINED aNtEVerSioN WAS OuTSiDe of a range Of 40°-60°, tHE patIent'S disLocatiON WaS 6.9 tImes hiGHer. hISAtOMe AnD doI \[[@Bib29]\] exaMined COmbIned ANtevErsION in A MAThEmATIcAL MODel to FinD The OpTImUM pOsitiOns tO AVoiD NEck IMpinGEMent wiTH dIfferEnt SIzEd cOmPoNENts. TheY RECOMMENDeD An IDeaL POsiTION, WHile noT accOuntIng FoR PaTIeNT'S Pelvic INcLiNaTION, oF 45° oF Cup aB | Introduction{#sec1} ============ In total hip arthroplasty(THA), optimum component position iscritical for long-term success of the operation by decreasing ratesof wear, aseptic loosening, anddislocation \[[@bib1], [@bib2], [@bib3], [@bib4]\]. Recognizing the importanceof acetabular component position, Lewinnek et al published a"safe zone" of 5° to 25° for anteversion and 30° to 50° for acetabular abduction based on their experience with dislocations afterposterior THA \[[@bib5]\]. This still servesas the standard for idealacetabular component position but has been called into question given the importance of the spinopelvic relationship \[[@bib6],[@bib7]\]. In addition to acetabular component position, emphasis has also been placed on femoral component positioning. Historically, surgeons haveidentified theimportance of keeping thefemoralcomponent out of varus becauseof increased rates of failure withvarus cemented femoral stems \[[@bib8],[@bib9]\]. With the use of cementless femoral fixation, varus positioningof the femoralstemhas not been shown to lead tothe same increased failures\[[@bib10]\]. However, as compared with cemented femoral stems, many cementless femoral stems provide less ability toadjust the versionofthe componentas a stable press-fit requires the stem to adapt to the proximal geometry of the nativefemur. Consequently, recent attention has been given to thecombined version (CV) of the acetabular and femoral components, with the goal of improving impingement-free range of motion anddecreasing instability \[[@bib11], [@bib12], [@bib13]\]. The concept of CV was originally introduced by Ranawat, andhe described the use of the "Ranawatsign" to determine CV intraoperatively when using the posterior approach\[[@bib14]\]. While nooptimum femoral versionhas been described, Dorr proposed a CV safe zone of 25°-50° based on previous anatomical studies and his experience with decreasedinstability in this range \[[@bib15]\].More recent studieshave attempted to quantifya combined anteversion that minimizesimpingement \[[@bib16],[@bib17]\]. Native femoral anteversion can vary a great deal, and intraoperative judgment of femoral component version can be difficult.Using preoperativecomputerized tomography (CT) scans of a group of 46 patients scheduledfor primary THA,Bargar et al \[[@bib18]\] found a large range ofnative femoral version from 6° of retroversion to 33° of anteversion. Dorret al compared the surgeon'sestimate of femoral component anteversion in the posteriorapproach with the postoperative CT measurement of version and found a poor precision of the surgeon\'s estimate with a correlation coefficient of only 0.688 \[[@bib19]\]. In addition, this study found thatonly 45% of the femoral stems landed withinthe desiredrange of 10°-20° of anteversion. In direct anterior total hip arthroplasty (DA-THA), femoral component broaching and insertion occurswhile the patient is positionedsupine with the leg fullyextended, and the leg below the knee isoftendrapedfrom the surgeon\'s view. Despite published results of comparable patient outcomes fromthe DA-THA with otherTHA approaches,some havequestioned the ability to appropriatelyorientthe femoral component with respect tofemoral anteversion,viathis approach \[[@bib20], [@bib21], [@bib22], [@bib23]\]. Previous studies have reportedon improved acetabular component positioning in DA-THA \[[@bib24],[@bib25]\]. However, no prior study, to our knowledge, has examined the combined anteversion of thefemoral and acetabular components in DA-THA. This study aims to analyze the combined femoral and acetabular anteversion with cross-sectional imaging and quantify this relative to the CV "safe zone" described by Dorr. Materials and methods {#sec2} ===================== After obtaining institutionalreview board approval, patients were approached for enrollment in thestudy. An patient whowasundergoing a primary DA-THA from the senior author(JBM) was a candidate for enrollment. Patients with femoral or acetabular hardwarewere excluded from this study. Thirty consecutive patients were enrolled in the study.Fourblinded observers independently recorded the measurements (2 fellowship-trained arthroplasty surgeons, onehip and knee fellow, and one orthopaedic resident). All implantswere positioned usingintraoperative fluoroscopybasedon preoperative templating. A CORAIL femoral stem (DePuy, Warsaw, IN)and a PINNACLEacetabularcup (DePuy, Warsaw, IN) were used for all thecases. The seniorauthor standardized intraoperativeimages by matching the anteroposterior (AP) pelvis fluoroscopicview with the preoperative AP pelvis standingradiograph. One month after surgery, all patientshad astanding AP pelvis and across-table lateral radiograph taken,whichwere used for acetabular component position measurement. Abduction and anteversion measurements oftheacetabulum were made from the digital radiograph using theTraumaCad (Voyant Health, Columbia, MD) hip abduction measurement tool. Femoral component position measurements were taken fromlimited supineCT scan of thehip and knee with 2.5-mm cuts (General Electric BrightSpeed, Fairfield,CT).CT was not selected for acetabular component position to minimize patient radiationexposure.Angular measurements were calculated using the axis of the top of neck of the femoral stem relativeto boththe posteriorcondylar axis (PCA) and the transepicondylaraxis (TEA). The CVwas then calculatedfor the TEA andthe PCA by adding the femoral anteversion calculated from theCT scan with the anteversion measured from the standing AP pelvisradiograph. Statistical analysis {#sec2.1} -------------------- Measurements from the 4observers were combined, and the mean and standard deviation were calculated. ThePearson correlation coefficientwas also measured foreach observer, with the kappa values reported, and compared with the group forall measurements. The mean for each measurement was used to determine the number of components placed in the "safe zone." Statistical analysis was performed with the use of SAS software(SAS Institute, Raleigh,NC). Results {#sec3} ======= Of the 30 enrolled patients, 29had an appropriate CT scan obtained. One patient had aCT scanperformed without adherence to the protocol precluding reference of femoral version tothe axes of the knee and was excluded from the results. Themean acetabular abduction andanteversion were 39.3° (standard deviation \[SD\] = 4.2°)and 27.2° (SD = 4.7°), respectively. The mean stem anteversion was 17.5° (SD = 10.8°) from the TEAand 21.7° (SD = 11.3°) from the PCA.Ten of the 30 cups were placed inside of the "safe zone" of Lewinnek for acetabularanteversion,but all cups were within the "safe zone" for abduction ([Fig. 1](#fig1){ref-type="fig"}).Figure 1The Lewinnek "safe zone." Ten of the 30 cups were placed inside of the"safe zone" of Lewinnek for acetabular anteversion, but allcups were within the "safe zone" for abduction.Combined femoral and acetabular component anteversion from the TEA resulted in79% (23of29) of patients within the "safe zone" of 25°-50° with accuratelyoriented components([Fig. 2](#fig2){ref-type="fig"}).Figure2Combined anteversion. Combined femoral and acetabular componentanteversion. Pearsoncorrelation coefficients were high for both stemanteversionfromthe TEA (R =0.96) and the PCA (R = 0.98); however, the kappa coefficient for interobserver reliability for combined component anteversion was greaterfor the TEA(kappa = 0.83 vs 0.65). Discussion {#sec4} ==========Component positioning has been recognized as animportant factor in the long-termsurvival of THA \[[@bib5],[@bib12],[@bib26]\]. Muller et al. \[[@bib27]\] suggested acup anteversion of 10°-15° and femoral anteversion of 10° to be ideal. Lewinneket al. \[[@bib5]\] followed with their study that found a lower dislocation rate when the acetabular components were positioned in a safe zoneof30°-50° of inclination and5°-25° of anteversion. A study by Biedermann et al. \[[@bib28]\] found the lowest dislocation rates with acetabular components positioned at 45° of inclinationand 15°ofanteversion. More recently,Dorrproposeda CV safe zone of 25°-50° based onpreviousanatomical studies and his experience with decreased instabilityin this range\[[@bib15]\]. Many authors have begun toappreciate the importanceof the combined femoral and acetabular anteversion on dislocation rates and impingement \[[@bib3],[@bib11],[@bib12],[@bib29]\]. Ranawat and Maynard \[[@bib6]\] suggestedthe importance of the combination of femoral andacetabular anteversion and recommended 45° forwomen and between 20° and 30°for men. Jolles et al. \[[@bib12]\] found that whenthe combined anteversion was outside of a range of 40°-60°, the patient'sdislocation was 6.9 times higher.Hisatome and Doi \[[@bib29]\] examined combined anteversion in a mathematicalmodel to find the optimum positions to avoid neck impingement with different sized components. Theyrecommended an ideal position, while not accounting for patient'spelvic inclination,of 45° ofcupab | Introduction {#sec1} ============ In total hip arthroplasty (THA), optimum component position _is_ critical for long-term success of the operation _by_ decreasing rates of wear, aseptic loosening, and _dislocation_ \[[@bib1], _[@bib2],_ _[@bib3],_ [@bib4]\]. Recognizing the importance of acetabular component position, Lewinnek _et_ al published a "safe zone" _of_ 5° to 25° for _anteversion_ and 30° _to_ 50° for _acetabular_ abduction _based_ on _their_ experience _with_ dislocations after posterior THA \[[@bib5]\]. This still serves as the standard _for_ _ideal_ acetabular component _position_ but has _been_ called _into_ question given _the_ importance of the _spinopelvic_ relationship \[[@bib6],[@bib7]\]. In addition to acetabular component position, _emphasis_ _has_ also been placed on femoral component _positioning._ Historically, _surgeons_ have identified the importance _of_ keeping the _femoral_ _component_ _out_ of varus _because_ of _increased_ rates of failure with varus cemented _femoral_ stems \[[@bib8],[@bib9]\]. With _the_ use _of_ cementless femoral fixation, _varus_ positioning of the femoral stem has not _been_ _shown_ to lead to the same increased failures \[[@bib10]\]. However, as compared with cemented femoral stems, many cementless femoral stems provide less ability to adjust _the_ _version_ _of_ the component as a stable press-fit requires _the_ stem _to_ adapt to the proximal geometry of _the_ native femur. Consequently, recent attention has been given to _the_ combined version (CV) _of_ the acetabular and femoral components, with the _goal_ of improving impingement-free _range_ of motion and decreasing instability \[[@bib11], [@bib12], [@bib13]\]. The concept of CV _was_ originally introduced by Ranawat, _and_ he described the use of _the_ "Ranawat _sign"_ to determine _CV_ intraoperatively when using the posterior approach \[[@bib14]\]. _While_ no optimum femoral version has been described, Dorr proposed a CV safe zone of 25°-50° based _on_ previous anatomical studies and his _experience_ _with_ decreased instability in this range \[[@bib15]\]. More recent _studies_ have attempted _to_ quantify _a_ combined anteversion that minimizes impingement _\[[@bib16],[@bib17]\]._ Native femoral anteversion can vary _a_ great deal, and intraoperative _judgment_ of _femoral_ component version can be difficult. _Using_ preoperative computerized tomography (CT) scans of a group _of_ 46 _patients_ scheduled for _primary_ THA, Bargar et _al_ \[[@bib18]\] _found_ _a_ large range _of_ _native_ femoral version from 6° _of_ retroversion to 33° of anteversion. Dorr _et_ al _compared_ _the_ surgeon's estimate of femoral component anteversion in the posterior approach with _the_ postoperative CT measurement of version and found a poor precision of the surgeon\'s estimate with _a_ correlation coefficient of only 0.688 \[[@bib19]\]. In _addition,_ this study found that only _45%_ of the femoral stems _landed_ within the desired range _of_ _10°-20°_ of anteversion. In direct anterior total hip arthroplasty (DA-THA), femoral component broaching and insertion occurs while the _patient_ is _positioned_ supine with the leg fully extended, and _the_ _leg_ below _the_ knee _is_ often draped _from_ the surgeon\'s view. Despite published results of comparable patient outcomes from _the_ DA-THA with other THA approaches, some have questioned the ability to appropriately orient _the_ _femoral_ component with respect to femoral _anteversion,_ via this approach \[[@bib20], [@bib21], [@bib22], [@bib23]\]. Previous _studies_ have _reported_ _on_ improved acetabular component positioning in _DA-THA_ _\[[@bib24],[@bib25]\]._ _However,_ no prior _study,_ _to_ our knowledge, has examined the combined anteversion of _the_ femoral and acetabular _components_ in _DA-THA._ This _study_ _aims_ to analyze the combined femoral and acetabular anteversion with cross-sectional imaging and _quantify_ _this_ relative _to_ _the_ CV "safe zone" described by Dorr. Materials and methods {#sec2} ===================== After obtaining institutional _review_ board approval, _patients_ were approached for enrollment in the study. An patient _who_ _was_ _undergoing_ _a_ primary DA-THA from the senior author (JBM) was a candidate for enrollment. Patients with femoral or acetabular hardware were _excluded_ _from_ _this_ study. _Thirty_ consecutive patients _were_ enrolled in the study. _Four_ blinded observers independently recorded the measurements (2 _fellowship-trained_ arthroplasty surgeons, one hip and knee _fellow,_ and one orthopaedic resident). _All_ implants were positioned using intraoperative fluoroscopy based on preoperative _templating._ _A_ CORAIL femoral stem (DePuy, Warsaw, _IN)_ and a _PINNACLE_ acetabular _cup_ (DePuy, _Warsaw,_ _IN)_ were used for all the cases. The senior author standardized intraoperative images _by_ matching the _anteroposterior_ _(AP)_ pelvis fluoroscopic _view_ with the preoperative AP pelvis standing _radiograph._ _One_ month after _surgery,_ all _patients_ _had_ a _standing_ AP pelvis and a cross-table lateral radiograph taken, _which_ were used for acetabular component position measurement. Abduction and anteversion measurements of the _acetabulum_ were made from the digital _radiograph_ using the TraumaCad (Voyant Health, Columbia, MD) hip abduction measurement tool. Femoral component position measurements were taken from limited supine _CT_ scan of the _hip_ and _knee_ with _2.5-mm_ cuts (General Electric BrightSpeed, Fairfield, CT). CT was not _selected_ _for_ acetabular component position to _minimize_ patient radiation _exposure._ Angular measurements were calculated using the axis of the top of neck _of_ the femoral _stem_ relative to both the posterior condylar axis _(PCA)_ and the transepicondylar axis (TEA). The CV was then calculated _for_ _the_ TEA _and_ the _PCA_ by adding the _femoral_ anteversion _calculated_ from the CT scan with _the_ _anteversion_ _measured_ from the standing AP pelvis radiograph. Statistical _analysis_ {#sec2.1} -------------------- _Measurements_ from the 4 observers were combined, and the mean and standard deviation were calculated. The Pearson correlation coefficient was also measured for each observer, with the _kappa_ values reported, and compared with the group for all _measurements._ _The_ mean _for_ _each_ _measurement_ _was_ used to _determine_ the number of components placed in the _"safe_ zone." Statistical analysis was _performed_ with _the_ _use_ _of_ SAS software _(SAS_ Institute, _Raleigh,_ NC). Results {#sec3} ======= _Of_ the 30 enrolled _patients,_ 29 _had_ _an_ appropriate CT scan _obtained._ One _patient_ had a _CT_ scan performed without adherence to the _protocol_ _precluding_ _reference_ _of_ _femoral_ version to the axes of _the_ knee and _was_ _excluded_ from the results. The mean acetabular abduction and anteversion were _39.3°_ (standard deviation \[SD\] = 4.2°) _and_ 27.2° (SD = 4.7°), _respectively._ The mean stem anteversion was 17.5° (SD = 10.8°) from the _TEA_ _and_ 21.7° (SD _=_ _11.3°)_ from the PCA. Ten of the 30 cups were placed inside of the "safe zone" _of_ Lewinnek for acetabular anteversion, _but_ all _cups_ were within the "safe _zone"_ for abduction ([Fig. 1](#fig1){ref-type="fig"}).Figure 1The Lewinnek "safe zone." Ten of the 30 cups were placed _inside_ of _the_ _"safe_ _zone"_ of _Lewinnek_ _for_ acetabular anteversion, but _all_ cups were within the "safe zone" _for_ abduction. Combined femoral and acetabular component anteversion from the TEA resulted in 79% _(23_ of 29) of _patients_ within the "safe zone" _of_ 25°-50° _with_ accurately _oriented_ components ([Fig. 2](#fig2){ref-type="fig"}).Figure _2Combined_ anteversion. Combined _femoral_ and acetabular component anteversion. Pearson correlation coefficients were high for _both_ stem anteversion _from_ _the_ TEA (R = 0.96) and the _PCA_ _(R_ = 0.98); however, the kappa coefficient for interobserver reliability for _combined_ component anteversion _was_ greater for the _TEA_ (kappa _=_ 0.83 vs _0.65)._ Discussion {#sec4} ========== Component _positioning_ has _been_ _recognized_ as an important factor in the long-term _survival_ of _THA_ \[[@bib5],[@bib12],[@bib26]\]. Muller et al. \[[@bib27]\] suggested a cup _anteversion_ of 10°-15° and femoral anteversion of 10° to be ideal. Lewinnek et al. \[[@bib5]\] followed with their _study_ that found _a_ lower dislocation rate when the acetabular components were positioned in _a_ safe zone of 30°-50° _of_ inclination and 5°-25° of anteversion. A study _by_ Biedermann _et_ al. \[[@bib28]\] found _the_ lowest dislocation rates _with_ acetabular components positioned at 45° of inclination and 15° of anteversion. _More_ _recently,_ Dorr _proposed_ a CV safe zone _of_ 25°-50° based on previous anatomical _studies_ and _his_ _experience_ with decreased instability _in_ _this_ range \[[@bib15]\]. Many authors have begun to _appreciate_ the importance of _the_ combined femoral _and_ acetabular anteversion on dislocation _rates_ and impingement \[[@bib3],[@bib11],[@bib12],[@bib29]\]. Ranawat and Maynard \[[@bib6]\] suggested the importance _of_ the combination of femoral and acetabular anteversion and _recommended_ 45° for women and between _20°_ and 30° for men. Jolles et al. \[[@bib12]\] found that when the combined anteversion was outside of a range of 40°-60°, the patient's dislocation _was_ 6.9 _times_ _higher._ Hisatome and Doi _\[[@bib29]\]_ _examined_ combined anteversion in _a_ mathematical model to _find_ the optimum positions to avoid neck impingement _with_ _different_ sized components. They recommended an _ideal_ position, while not accounting for patient's pelvic inclination, of _45°_ of cup ab |
1. Historical Origins {#sec1-viruses-12-00132}
=====================
At a meeting of the Fellows of the National Institute of Agricultural Botany in Cambridge, UK, on 14 November 1924, Dr. Redcliffe Salaman gave a lecture entitled "Degeneration of the Potato---An Urgent Problem" \[[@B1-viruses-12-00132]\]. He reported that "potato degeneration", namely the decrease in yield when potatoes were grown year after year from tubers, rather than from true seed, cost the UK between five and ten million pounds sterling each year. He noted that the condition was first reported in 1778 at a meeting in Manchester, and called "potato curl". It was worse in lowland crops and in the Southern UK than in crops grown on higher ground and in the north, and although some thought it was caused by disease, perhaps insect-borne, others believed it was a form of senility resulting from repeated vegetative reproduction! Salaman concluded that that degeneration was caused by a complex of tuber-borne pathogens.
Salaman's talk was successful, as it induced the Ministry of Agriculture to found the Potato Virus Research Station in Cambridge and appoint him as director, and in turn he appointed Kenneth Smith as entomologist, who soon separated some of the components of potato curl and identified the viruses he called potato virus X and potato virus Y (PVY) \[[@B2-viruses-12-00132]\].
Other viruses similar to PVY were soon reported, for example, henbane mosaic virus \[[@B3-viruses-12-00132]\], which was like PVY in causing mosaic symptoms, being transmitted by sap, although relatively unstable in it, and also by being transmitted by aphids in short feeds. These viruses, which became known as potyviruses, short for "potato virus Y group viruses" \[[@B4-viruses-12-00132]\], were among those included in early attempts to devise biological taxonomies of plant viruses \[[@B5-viruses-12-00132]\] based on the length of their filamentous particles \[[@B6-viruses-12-00132]\]. They were also distinguished from other plant viruses by having serologically distinct virions and, biologically, by having distinct host ranges and causing distinct symptoms, and by their properties in infective sap, such as dilution end point, thermal inactivation point, and longevity in vitro. Sixteen different potyviruses had been described in 1959. Subsequently, in this pre-sequencing era, a combination of techniques, including sucrose density gradient centrifugation, analytical ultracentrifugation, ultraviolet spectrophotometry, and polyacrylamide gel electrophoresis were also included to establish the sedimentation coefficients and buoyant densities of virions, and the molecular weights of protein subunits and % nucleic acid contents, as all these properties provided additional distinguishing characteristics when novel viruses were being described \[[@B7-viruses-12-00132]\].
Virus identification and taxonomy were transformed later, when methods for sequencing genes were invented in the 1970s and applied to plant viruses \[[@B8-viruses-12-00132],[@B9-viruses-12-00132]\], and it was established that hierarchical groupings based on viral protein and gene sequences, including those of potyviruses, confirmed and extended those that had been devised previously by using phenotypic characters, serological tests, etc. As a result, 57 potyviruses had been identified by 1991 \[[@B10-viruses-12-00132]\], using sequences of the "part NIb-CP" region of their genomes, as this was bracketed by convenient primer sites \[[@B11-viruses-12-00132],[@B12-viruses-12-00132]\]. By 2000, over 1000 potyvirus sequences were recorded in the GenBank database, and there are now more than 26,000. The potyviruses now form a family, the *Potyviridae* \[[@B13-viruses-12-00132]\], containing at least eight genera of which the aphid-transmitted potyviruses, including the first described, PVY, make up the largest genus, *Potyvirus*. This large plant virus genus is one of the most important economically because of the yield and quality losses it causes in a wide range of crops worldwide. Moreover, some of its members currently endanger food security in developing countries by causing devastating diseases in tropical and subtropical food crops \[[@B14-viruses-12-00132]\].
2. The Origins of the Potyviridae {#sec2-viruses-12-00132}
=================================
The potyvirids are distinguished from other viruses by specific molecular differences, together with a combination of phenotypic properties \[[@B13-viruses-12-00132],[@B15-viruses-12-00132]\]. All potyviruses infect plants; most are transmitted in nature by arthropods---mostly by aphids---though bymoviruses are transmitted by root-infecting plasmodiophorids, which are cercozoan amoebae. Some potyviruses are seed-borne \[[@B16-viruses-12-00132]\]. Potyvirid virions are flexuous filaments, 680--900 nm long and 11--20 nm in diameter. Each is helically constructed from 1400 to 2140 subunits of a coat protein (CP), and a positive-sense, single-stranded RNA genome (usually monopartite, but bipartite in the genus *Bymovirus*) of 8--11 kb in total, which is wound into a groove within the CP subunits.
The ancestry and origins of the potyvirids is being revealed by studies of the structure and sequences of their genes and proteins. These show that their genomes are polyphyletic in origin, as there are significant similarities between three of their genes and those of viruses in three otherwise-unrelated virus genera; two detected by the protein sequence similarity of the helicase region of the CI protein and the RdRp region of the NIb protein, respectively, and the third by the structure of the CP \[[@B17-viruses-12-00132],[@B18-viruses-12-00132]\]. A BLASTp search of the GenBank protein sequence database (Sept 2019), using the eight main motif regions of the polyprotein of PVY (NC_001616), and excluding matches with sequences from the *Potyviridae*, found only significant matches between the "DEAD helicase-helicase C" region of the CI protein and that of classical swine fever and hog cholera pestiviruses (chance probability, 1e-12\_-16; 30% identity, 13% indels), and no others. Likewise, there were significant similarities between the PVY RdRp region and that of astroviruses (see below). Structural studies have only been reported for the CP protein of one potyvirus, watermelon mosaic virus (WMV), and reveal a structure that is closely similar to those of other viruses with flexuous filamentous virions, including two potexviruses, and also the enveloped flexuous nucleoproteins of orthomyxoviruses and bunyavirids, which include tomato spotted wilt tospovirus \[[@B19-viruses-12-00132],[@B20-viruses-12-00132],[@B21-viruses-12-00132]\]; all these CPs have a core domain rich in alpha helices. Each CP subunit interacts with five nucleotides (nts), and has 8.8 subunits per turn in a left-handed helix, with a pitch of 34.5--35 Å. Its N-terminus is external to the virion, and its C-terminus internal. The terminal regions interact with adjacent subunits and provide flexibility to the virion. In serological studies, the N-terminus is dominant and, in potyviruses, is also involved with aphid interactions \[[@B22-viruses-12-00132],[@B23-viruses-12-00132]\].
The sequences of their RNA-dependent RNA polymerases (RdRps) place the potyvirids in the "Picornavirus Supergroup" ([Figure 1](#viruses-12-00132-f001){ref-type="fig"}) \[[@B19-viruses-12-00132],[@B24-viruses-12-00132]\], where the potyvirids are outsiders, as most of the others have icosahedral virions made of eight-stranded antiparallel beta-barrel proteins, the so-called 'jelly roll' proteins. In the Wolf et al. \[[@B24-viruses-12-00132]\] taxonomy of RdRps ([Figure 2](#viruses-12-00132-f002){ref-type="fig"}), the potyvirid RdRps form a cluster that is sister to an RdRp found in a metagenome, bufivirus UC1-gp2, isolated from "wastewater" collected in San Francisco. The sister clade to the potyvirid/bufivirus clade of RdRps are mostly those of the astroviruses, a group of gut-infecting viruses that are found in a wide range of animals, mostly mammals or birds. They have 28--35 nm diameter isometric virions (<https://en.wikipedia.org/wiki/Astrovirus> (accessed July 2019)). Sister to the RdRp clade of potyviruses/bufivirus/astroviruses are the RdRps of hypoviruses, amalgaviruses, partitiviruses, and picobirnaviruses, many of them metagenomes, including one from *Phytophthora infestans* \[[@B25-viruses-12-00132]\] and two from leeches \[[@B26-viruses-12-00132]\]. None of the motifs identified in potyvirus proteins, other than the RdRp, match those encoded by | 1. historical origins { # sec1 - viruses - 12 - 00132 } = = = = = = = = = = = = = = = = = = = = = at a meeting of the fellows of the national institute of rural botany in cambridge, uk, on 14 november 1924, dr. redcliffe salaman gave a lecture entitled " cultivation of the potato - - - an urgent problem " \ [ [ @ b1 - viruses - 12 - 00132 ] \ ]. he reported that " potato degeneration ", namely the decrease in yield when potatoes were grown year after year from tubers, rather than from true seed, cost the uk between five and ten million pounds sterling each year. he noted that the condition was first reported in 1778 at a meeting in manchester, and called " potato curl ". it was worse in lowland crops and in the arctic uk than in those grown on higher ground and beyond the north, and although some thought it was caused by disease, perhaps insect - borne, others believed it was a form of senility resulting from repeated vegetative reproduction! salaman concluded that that degeneration was caused by a complex of tuber - borne pathogens. salaman ' s talk was successful, as it induced the ministry of agriculture to found the crop virus research station in cambridge and appoint him as director, and in turn he appointed kenneth smith as entomologist, who soon separated some of the components of potato curl and identified the viruses he called potato virus x and potato virus y ( pvy ) \ [ [ @ b2 - viruses - 12 - 00132 ] \ ]. other viruses similar to pvy diseases soon reported, for example, pea mosaic virus \ [ [ @ b3 - viruses - 12 - 00132 ] \ ], which was like pvy in causing mosaic symptoms, being transmitted by sap, although relatively unstable in it, and also by being transmitted by aphids in short feeds. these viruses, which became known as potyviruses, short for " potato virus y group viruses " \ [ [ @ b4 - viruses - 12 - 00132 ] \ ], were among those included in early attempts to devise biological taxonomies of plant viruses \ [ [ @ b5 - viruses - 12 - 00132 ] \ ] based on the length of their gram particles \ [ [ @ b6 - hiv - 12 - 00132 ] \ ]. they were also distinguished from other plant viruses by having serologically distinct virions and, biologically, by having distinct host ranges and causing distinct symptoms, and by their properties in infective sap, such as dilution end point, thermal inactivation point, and longevity in vitro. sixteen different potyviruses had been described in 1959. subsequently, in this pre - sequencing era, a combination of techniques, including sucrose density gradient centrifugation, analytical ultracentrifugation, ultraviolet spectrophotometry, and polyacrylamide gel electrophoresis were also included to establish the sedimentation coefficients and buoyant densities of virions, and the molecular weights of protein subunits and % nucleic acid contents, as all these properties provided additional distinguishing characteristics when novel viruses were being described \ [ [ @ b7 - viruses - 12 - 00132 ] \ ]. virus identification and taxonomy were transformed later, when methods for sequencing genes were invented in the 1970s and applied to plant viruses \ [ [ @ b8 - viruses - 12 - 00132 ], [ @ b9 - viruses - 12 - 00132 ] \ ], and it was established that hierarchical groupings based on viral protein and gene sequences, including those of potyviruses, confirmed and extended those that had been devised previously by using phenotypic characters, serological tests, etc. as a result, 57 potyviruses had been identified by 1991 \ [ [ @ b10 - viruses - 12 - 00132 ] \ ], using sequences of the " part nib - cp " region of their genomes, as this was bracketed by convenient primer sites \ [ [ @ b11 - viruses - 12 - 00132 ], [ @ b12 - viruses - 12 - 00132 ] \ ]. by 2000, over 1000 potyvirus sequences were recorded in the genbank database, and there are now more than 26, 000. the potyviruses now form a family, the * potyviridae * \ [ [ @ b13 - viruses - 12 - 00132 ] \ ], containing at least eight genera of which the aphid - transmitted potyviruses, including the first described, pvy, make up the largest genus, * potyvirus *. this large plant virus genus is one of the most important economically because of the yield and quality losses it causes in a wide range of crops worldwide. moreover, some of its members currently endanger food security in developing countries by causing devastating diseases in tropical and subtropical food crops \ [ [ @ b14 - viruses - 12 - 00132 ] \ ]. 2. the origins of the potyviridae { # sec2 - viruses - 12 - 00132 } = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = the potyvirids are distinguished from other viruses by specific molecular differences, together with a combination of phenotypic properties \ [ [ @ b13 - viruses - 12 - 00132 ], [ @ b15 - viruses - 12 - 00132 ] \ ]. all potyviruses infect plants ; most are transmitted in nature by arthropods - - - mostly by aphids - - - though bymoviruses are transmitted by root - infecting plasmodiophorids, which are cercozoan amoebae. some potyviruses are seed - borne \ [ [ @ b16 - viruses - 12 - 00132 ] \ ]. potyvirid virions are flexuous filaments, 680 - - 900 nm long and 11 - - 20 nm in diameter. each is helically constructed from 1400 to 2140 subunits of a coat protein ( cp ), and a positive - sense, single - stranded rna genome ( usually monopartite, but bipartite in the genus * bymovirus * ) of 8 - - 11 kb in total, which is wound into a groove within the cp subunits. the ancestry and origins of the potyvirids is being revealed by studies of the structure and sequences of their genes and proteins. these show that their genomes are polyphyletic in origin, as there are significant similarities between three of their genes and those of viruses in three otherwise - unrelated virus genera ; two detected by the protein sequence similarity of the helicase region of the ci protein and the rdrp region of the nib protein, respectively, and the third by the structure of the cp \ [ [ @ b17 - viruses - 12 - 00132 ], [ @ b18 - viruses - 12 - 00132 ] \ ]. a blastp search of the genbank protein sequence database ( sept 2019 ), using the eight main motif regions of the polyprotein of pvy ( nc _ 001616 ), and excluding matches with sequences from the * potyviridae *, found only significant matches between the " dead helicase - helicase c " region of the ci protein and that of classical swine fever and hog cholera pestiviruses ( chance probability, 1e - 12 \ _ - 16 ; 30 % identity, 13 % indels ), and no others. likewise, there were significant similarities between the pvy rdrp region and that of astroviruses ( see below ). structural studies have only been reported for the cp protein of one potyvirus, watermelon mosaic virus ( wmv ), and reveal a structure that is closely similar to those of other viruses with flexuous filamentous virions, including two potexviruses, and also the enveloped flexuous nucleoproteins of orthomyxoviruses and bunyavirids, which include tomato spotted wilt tospovirus \ [ [ @ b19 - viruses - 12 - 00132 ], [ @ b20 - viruses - 12 - 00132 ], [ @ b21 - viruses - 12 - 00132 ] \ ] ; all these cps have a core domain rich in alpha helices. each cp subunit interacts with five nucleotides ( nts ), and has 8. 8 subunits per turn in a left - handed helix, with a pitch of 34. 5 - - 35 a. its n - terminus is external to the virion, and its c - terminus internal. the terminal regions interact with adjacent subunits and provide flexibility to the virion. in serological studies, the n - terminus is dominant and, in potyviruses, is also involved with aphid interactions \ [ [ @ b22 - viruses - 12 - 00132 ], [ @ b23 - viruses - 12 - 00132 ] \ ]. the sequences of their rna - dependent rna polymerases ( rdrps ) place the potyvirids in the " picornavirus supergroup " ( [ figure 1 ] ( # viruses - 12 - 00132 - f001 ) { ref - type = " fig " } ) \ [ [ @ b19 - viruses - 12 - 00132 ], [ @ b24 - viruses - 12 - 00132 ] \ ], where the potyvirids are outsiders, as most of the others have icosahedral virions made of eight - stranded antiparallel beta - barrel proteins, the so - called ' jelly roll ' proteins. in the wolf et al. \ [ [ @ b24 - viruses - 12 - 00132 ] \ ] taxonomy of rdrps ( [ figure 2 ] ( # viruses - 12 - 00132 - f002 ) { ref - type = " fig " } ), the potyvirid rdrps form a cluster that is sister to an rdrp found in a metagenome, bufivirus uc1 - gp2, isolated from " wastewater " collected in san francisco. the sister clade to the potyvirid / bufivirus clade of rdrps are mostly those of the astroviruses, a group of gut - infecting viruses that are found in a wide range of animals, mostly mammals or birds. they have 28 - - 35 nm diameter isometric virions ( < https : / / en. wikipedia. org / wiki / astrovirus > ( accessed july 2019 ) ). sister to the rdrp clade of potyviruses / bufivirus / astroviruses are the rdrps of hypoviruses, amalgaviruses, partitiviruses, and picobirnaviruses, many of them metagenomes, including one from * phytophthora infestans * \ [ [ @ b25 - viruses - 12 - 00132 ] \ ] and two from leeches \ [ [ @ b26 - viruses - 12 - 00132 ] \ ]. none of the motifs identified in potyvirus proteins, other than the rdrp, match those encoded by | 1. Historical Origins {# sec1 - viruses - 12 - 00132} = = = = = = = = = = = = = = = = = = = = = At a meeting of the Fellows of the National Institute of Agricultural Botany in Cambridge, UK, on 14 November 1924, Dr. Redcliffe Salaman gave a lecture entitled " Degeneration of the Potato - - - An Urgent Problem " \ [[ @ B1 - viruses - 12 - 00132] \ ]. He reported that " potato degeneration ", namely the decrease in yield when potatoes were grown year after year from tubers, rather than from true seed, cost the UK between five and ten million pounds atDrling each year. He noted that the condition was first reported in 1778 at a meeting in Manchester, and called " potato curl ". It was worse in lowland crops and in the Southern UK than in crops grown on higher ground and in the north, and although some thought it was caused by disease, perhaps insect - borne, others believed it was a form of senility resulting from repeated vegetative reproduction! Salaman concluded that that degeneration was caused by a complex of tuber - borne pathogens. Salaman ' s talk was successful, as it induced the Ministry of Agriculture to found the Potato Virus Research Station in Cambridge and appoint him as director, and in turn he appointed Kenneth Smith as entomologist, who soon separated some of the components of potato curl and identified the viruses he called potato virus X and potato virus Y (PVY) \ [[ @ B2 - viruses - 12 - 00132] \ ]. Other viruses similar to PVY were soon reported, for example, henbane mosaic virus \ [[ @ B3 - viruses - 12 - 00132] \ ], which was like PVY in causing mosaic symptoms, being transmitted by sap, although relatively unstable in it, and also by being transmitted by aphids in short feeds. These viruses, which became known as potyviruses, short for " potato virus Y group viruses " \ [[ @ B4 - viruses - 12 - 00132] \ ], were among those included in early attempts to devise biological taxonomies of plant viruses \ [[ @ B5 - viruses - 12 - 00132] \] based on the length of their filamentous particles \ [[ @ B6 - viruses - 12 - 00132] \ ]. They D#re also distinguished from other plant viruses by having serologically distinct virions and, biologically, by having distinct host ranges and causing distinct symptoms, and by their properties in infective sap, such as dilution end point, thermal inactivation point, and longevity in vitro. Sixteen different potyviruses had been described in 1959. Subsequently, in this pre - sequencing era, a combination of techniques, including sucrose density gradient centrifugation, analytical ultracentrifugation, ultraviolet spectrophotometry, and polyacrylamide gel electrophoresis were also included to establish the sedimentation coefficients and buoyant densities of virions, and the molecular weights of protein subunits and% nucleic acid contents, as all these properties provided additional distinguishing characteristics when novel viruses were being described \ [[ @ B7 - viruses - 12 - 00132] \ ]. Virus identification and taxonomy were transformed later, when methods for sequencing genes were invented in the 1970s and applied to plant viruses \ [[ @ B8 - viruses - 12 - 00132 ], [@ B9 - viruses - 12 - 00132] \ ], and it was established that hierarchical groupings based on viral protein and gene sequences, including those of potyviruses, confirmed and extended those that had been devised previously by using phenotypic characters, serological tests, etc. As a result, 57 potyviruses had been idrnyified by 1991 \ [[ @ B10 - viruses - 12 - 00132] \ ], using sequences of the " part NIb - CP " region of their genomes, as this was bracketed by c)nvenieJt primer sites \ [[ @ B11 - vi#usea - 12 - 00132 ], [@ B12 - viruses - 12 - 00132] \ ]. By 2000, over 1000 potyvirus sequences were recorded in the GenBank database, and there are now more than 26, 000. The potyviruses now form a family, the * Potyviridae * \ [[ @ B13 - viruses - 12 - 00132] \ ], containing at least eight genera of which the aphid - transmitted potyviruses, including the first described, PVY, make up the largest genus, * Potyvirus *. This large plant virus genus is one of the most important economically because of the yield and quality losses it causes in a wide range of crops worldwide. Moreover, some of its members currently endanger food security in developing countries by causing devastating diseases in tropical and subtropical food crops \ [[ @ B14 - viruses - 12 - 00132] \ ]. 2. The Origins of the Potyviridae {# sec2 - viruses - 12 - 00132} = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = The potyvirids are distinguished from other viruses by specific molecular differences, together with a combination of phenotypic properties \ [[ @ B13 - viruses - 12 - 00132 ], [@ B15 - Firuzes - 12 - 00132] \ ]. All potyviruses infect plants; most are transmitted in nature by arthropods - - - mostly by aphids - - - though bymoviruses are transmitted by root - infecting plasmodiophorids, which are cercozoan amoebae. Some potyviruses are seed - borne \ [[ @ B16 - viruses - 12 - 00132] \ ]. Potyvirid virions are flexuous filaments, 680 - - 900 nm long and 11 - - 20 nm in diameter. Each is helically constructed from 1400 to 2140 subunits of a coat protein (CP ), and a positive - sense, single - stranded RNA genome (usually monopartite, but bipartite in the genus * Bymovirus *) of 8 - - 11 kb in total, which is wound into a groove within the CP subunits. The ancestry and origins of the potyvirids is being revealed by studies of the structure and sequences of their genes and proteins. These show that their genomes are polyphyletic in origin, as there are significant similarities between three of their genes and those of viruses in three otherwise - unrelated virus genera; two detected by the protein sequence similarity of the helicase region of the CI protein and the RdRp region of the NIb protein, respectively, and the third by the structure of the CP \ [[ @ B17 - viruses - 12 - 00132 ], [@ B18 - viruses - 12 - 00132] \ ]. A BLASTp search of the GenBank protein sequence database (Sept 2019 ), using the eight main motif regions of the polyprotein of PVY (NC_001616 ), and excluding matches with sequences from the * Potyviridae *, found only significant matches between the " DEAD helicase - helicase C " renioH of the CI protein and that of classical swine fever and hog cholera pestiviruses (chance probability, 1e - 12 \ _ - 16; 30% identity, 13% indels ), and no others. Likewise, there were significant similarities between the PVY RdRp region and that of astroviruses (see below ). Structural studies have only been reported for the CP protein of one potyvirus, watermelon mosaic virus (WMV ), and reveal a structure that is closely similar to those of other viruses with flexuous filamentous virions, including two potexviruses, and also the enveloped flexuous nucleoproteins of orthomyxoviruses and bunyavirids, which include tomato spotted wilt tospovirus \ [[ @ B19 - viruses - 12 - 00132 ], [@ B20 - viruses - 12 - 00132 ], [@ B21 - viruses - 12 - 00132] \ ]; all these CPs have a core domain rich in alpha helices. Each CP subunit interacts with five nucleotides (nts ), and has 8. 8 subunits per turn in a left - handed helix, with a pitch of 34. 5 - - 35 Å. Its N - terminus is eSternak to the virion, and its C - terminus internal. The terminal regions interact with adjacent subunits and provide flexibility to the virion. In serological studies, the N - terminus is dominant and, in potyviruses, is also involved with aphid interactions \ [[ @ B22 - viruses - 12 - 00132 ], [@ B23 - viruses - 12 - 00132] \ ]. The sequences of their RNA - dependent RNA polymerases (RdRps) place the potyvirids in the " Picornavirus Supergroup " ([ Figure 1] (# viruses - 12 - 00132 - f001) {ref - type = " fig "} ) \ [[ @ B19 - viruses - 12 - 00132 ], [@ B24 - viruses - 12 - 00132] \ ], where the potyvirids are outsiders, as most of the others have icosahedral virions made of eigBg - stranded antiparallel beta - barrel proteins, the so - called ' jelly roll ' proteins. In the Wolf et al. \ [[ @ B24 - viruses - 12 - 00132] \] taxonomy of RdRps ([ Figure 2] (# viruses - 12 - 00132 - f002) {ref - type = " fig "} ), the potyvirid RdRps form a cluster that is sister to an RdRp found in a metagenome, bufivirus UC1 - gp2, isolated from " wastewater " collected in San Francisco. The sis$wr clade to the potyvirid / bufivirus clade of RdRps are mostly those of the astroviruses, a group of gut - infecting viruses that are found in a wide range of animals, mostly mammals or birds. They have 28 - - 35 nm diameter isometric virions (< https: / / en. wikipedia. org / wiki / Astrovirus> (accessed July 2019) ). Sister to the RdRp clade of potyviruses / bufivirus / astroviruses are the RdRps of hypoviruses, amalgaviruses, partitiviruses, and picobirnaviruses, many of them metagenomes, including one from * Phytophthora infestans * \ [[ @ B25 - viruses - 12 - 00132] \] and two from leeches \ [[ @ B26 - viruses - 12 - 00132] \ ]. None of the motifs identified in potyvirus proteins, other than the RdRp, match those encoded by | Historical Origins {#sec1-viruses-12-00132} ===================== At a meeting of the Fellows the National Institute of Agricultural Cambridge, UK, on 1924, Dr. Redcliffe Salaman gave a lecture entitled "Degeneration Potato---An Urgent \[[@B1-viruses-12-00132]\]. He reported that "potato degeneration", namely the decrease in yield when potatoes were grown after year from tubers, rather than from true seed, cost the five and ten million pounds sterling each year. He noted the condition was first in 1778 at a meeting in Manchester, and called "potato It was worse in lowland crops and in the Southern UK than in crops grown on higher ground and in the north, and although some thought was caused by disease, perhaps others believed it was a form of senility resulting from repeated vegetative reproduction! Salaman concluded that that degeneration was caused by a complex of tuber-borne pathogens. Salaman's talk was successful, as it induced the Agriculture to found the Virus Research Station in Cambridge and appoint him as director, and in turn he appointed Kenneth Smith as entomologist, who soon separated some of the components potato curl and identified viruses he called potato virus X and potato virus Y (PVY) \[[@B2-viruses-12-00132]\]. Other viruses similar to PVY were soon reported, for example, henbane mosaic virus \[[@B3-viruses-12-00132]\], which was like PVY in causing mosaic symptoms, being transmitted by sap, although relatively unstable in it, and also by being by aphids in short feeds. These viruses, which became known as potyviruses, for "potato virus group viruses" \[[@B4-viruses-12-00132]\], were those included in early attempts to devise taxonomies plant viruses \[[@B5-viruses-12-00132]\] based on the length of their filamentous They were distinguished from other plant viruses by having serologically distinct and, biologically, by having distinct host ranges and causing distinct symptoms, and by their properties in infective sap, such as dilution end point, thermal inactivation point, and longevity in vitro. Sixteen different potyviruses had been described in 1959. Subsequently, in this pre-sequencing era, a combination of techniques, including sucrose density gradient centrifugation, ultracentrifugation, ultraviolet spectrophotometry, and polyacrylamide electrophoresis were also included to establish the sedimentation coefficients and buoyant densities of virions, and the of protein subunits and nucleic acid contents, all these provided additional distinguishing characteristics when novel viruses were being \[[@B7-viruses-12-00132]\]. Virus identification and were transformed later, when methods for genes were in the 1970s and applied to plant viruses \[[@B8-viruses-12-00132],[@B9-viruses-12-00132]\], and it was established that hierarchical groupings based on viral protein and gene sequences, including those of and those that had been devised previously using phenotypic characters, serological tests, etc. As a result, 57 been identified by 1991 \[[@B10-viruses-12-00132]\], using sequences of the "part NIb-CP" region of their genomes, as this was bracketed by convenient primer \[[@B11-viruses-12-00132],[@B12-viruses-12-00132]\]. By 2000, over 1000 sequences were recorded in the GenBank and there are now more than 26,000. The potyviruses now form a family, the *Potyviridae* \[[@B13-viruses-12-00132]\], containing at least eight genera of which the aphid-transmitted potyviruses, the first described, PVY, make up the largest genus, This plant virus genus is of the most important economically because of the yield and quality losses it causes in a wide range of crops worldwide. Moreover, some of its members currently endanger food security in developing countries by causing diseases in tropical and subtropical food crops \[[@B14-viruses-12-00132]\]. 2. The Origins of the Potyviridae {#sec2-viruses-12-00132} ================================= The potyvirids are distinguished from viruses by specific molecular differences, together with a combination of phenotypic properties \[[@B13-viruses-12-00132],[@B15-viruses-12-00132]\]. All potyviruses infect plants; most are transmitted in nature arthropods---mostly by aphids---though bymoviruses are transmitted by root-infecting plasmodiophorids, which cercozoan amoebae. Some are seed-borne \[[@B16-viruses-12-00132]\]. Potyvirid virions are flexuous filaments, 680--900 nm long and 11--20 nm diameter. Each is helically constructed from 1400 to 2140 subunits of a coat protein (CP), and a positive-sense, RNA genome monopartite, but bipartite in the genus *Bymovirus*) of 8--11 kb in which is wound into a groove within the CP subunits. The ancestry and origins the potyvirids is being revealed by studies of the structure and sequences of their genes and proteins. These that their genomes are polyphyletic in origin, as there are significant similarities between three of their genes those of viruses in three otherwise-unrelated virus genera; detected by the protein sequence similarity of the region of the protein and the RdRp region of the NIb protein, respectively, and the third structure of the CP \[[@B17-viruses-12-00132],[@B18-viruses-12-00132]\]. A BLASTp search of the GenBank protein sequence database 2019), using the eight main motif of the polyprotein of PVY (NC_001616), and excluding matches with sequences from the *Potyviridae*, found only significant between the "DEAD helicase-helicase C" region of the CI protein and that of classical swine fever and cholera pestiviruses (chance probability, 1e-12\_-16; 30% identity, 13% indels), and no others. Likewise, there were significant between PVY RdRp region and that of astroviruses (see below). Structural studies only been reported for the CP protein one potyvirus, watermelon mosaic virus (WMV), and reveal structure that is closely similar to those of other viruses flexuous filamentous virions, including two potexviruses, and also the flexuous nucleoproteins of orthomyxoviruses and bunyavirids, which include tomato spotted wilt tospovirus \[[@B19-viruses-12-00132],[@B20-viruses-12-00132],[@B21-viruses-12-00132]\]; all these CPs have a core domain rich alpha helices. CP subunit interacts with five nucleotides (nts), and has 8.8 subunits per turn in a left-handed helix, pitch of 34.5--35 Å. Its N-terminus is external to the virion, and C-terminus internal. The terminal regions with adjacent subunits and provide flexibility the virion. In serological the N-terminus is dominant and, in is also involved with aphid interactions \[[@B22-viruses-12-00132],[@B23-viruses-12-00132]\]. The of their RNA-dependent RNA polymerases (RdRps) place the potyvirids in the "Picornavirus Supergroup" ([Figure 1](#viruses-12-00132-f001){ref-type="fig"}) \[[@B19-viruses-12-00132],[@B24-viruses-12-00132]\], where the potyvirids outsiders, as of the others icosahedral virions made of eight-stranded antiparallel beta-barrel proteins, so-called 'jelly roll' proteins. In Wolf et al. \[[@B24-viruses-12-00132]\] of RdRps ([Figure 2](#viruses-12-00132-f002){ref-type="fig"}), the potyvirid RdRps a cluster that is sister to an RdRp found in a metagenome, bufivirus UC1-gp2, from collected in San Francisco. sister to the potyvirid/bufivirus clade of RdRps are mostly those of the astroviruses, a group of gut-infecting viruses that are found in a wide range animals, mostly mammals or birds. have 28--35 nm diameter isometric virions (<https://en.wikipedia.org/wiki/Astrovirus> (accessed 2019)). Sister the RdRp of potyviruses/bufivirus/astroviruses are the RdRps of hypoviruses, amalgaviruses, partitiviruses, and picobirnaviruses, many of them metagenomes, including one from infestans* \[[@B25-viruses-12-00132]\] and two from the motifs identified in potyvirus proteins, than the RdRp, match those encoded by | 1. hiStoricAL OrIGins {#sEc1-vIrusEs-12-00132}
=====================
At a MeEtInG of thE FElLOWS OF ThE natIOnAl InstitUTe of AGRICultural boTANy In cAmbridge, UK, On 14 novEmbeR 1924, dR. ReDclifFE salAmAn gAvE a lECtURe enTItled "dEgENeRATioN Of the pOtato---aN URGenT PrOBLEM" \[[@b1-VirUSES-12-00132]\]. he repoRTED THAT "PotaTO DEGeNeRAtion", nAMEly thE dEcReaSe in YIeld wHeN pOtATOes WEre GRoWN YEar afTER yEar FROM tubErs, ratheR Than fROM tRuE sEEd, COst THE UK beTWEEn FIVe and teN MiLLion poUNds sTErlING each YEaR. hE NOted tHAT the coNDITioN was fIrst REportEd in 1778 AT A MEETiNG In mAncHeSTer, aNd cAlleD "PotaTo CUrl". It Was wOrsE In LoWLANd CRoPs and In The SoUTHerN uk than in CROPS GRoWN ON higheR groUnd anD In The nORtH, And alTHOuGH SOme tHOUghT It Was cAUSeD bY disEAsE, pErhapS INseCT-borNE, oThERs beLievED IT WaS a fORM oF seNILIty REsULTINg from repEatEd VeGEtativE repRodUCTiON! SalAmAn CoNcluDed tHAt thAT dEgeneRaTIoN was cAuSEd bY a COmPLeX of tUbEr-Borne PAthOgeNS.
sAlaMAn's TaLK Was SucCEsSFUL, as It INDUCeD THe MINisTRy Of agRIculTURe To Found ThE pOtAto vIRus rEsEarCh statiOn In cambRIDGe And aPPOINt hIm as DIreCtOr, ANd IN TURn he ApPOInTEd KeNneTh SmIth as entOmoLOgISt, WHO Soon SeparatED sOMe OF The CoMpONents OF PotatO CuRl aNd IdeNtIFiEd thE virUses hE cALlEd POTatO vIRuS X AnD PotaTo ViruS Y (PvY) \[[@b2-vIRUses-12-00132]\].
OThER ViRusEs sImILar to PVY WEre SOon rEPORteD, fOR EXaMPlE, hEnBANE MOsAIc ViRuS \[[@b3-vIRUsEs-12-00132]\], wHIcH WAs lIKE pVY iN Causing MOsaIC SYmptomS, beINg TRaNsMItTED BY sap, AlTHOuGH relaTIVElY UnStaBLe IN iT, AnD aLso bY bEInG TRAnsmitTeD BY aphIDS iN ShORt Feeds. ThESE vIrUsES, wHIch bECaMe knOWn as PotYvIrUsES, shoRt FOr "POtatO VirUs Y grouP virusEs" \[[@B4-VirusES-12-00132]\], wErE amOnG tHoSe inCLuDED In eArLy aTTEMptS to DEvIse BiOlOGIcAl taxonomies Of pLAnT ViRuses \[[@B5-vIrUSes-12-00132]\] BaSEd oN tHE leNGtH OF THEIR FIlAmeNtouS pArtiCles \[[@b6-viRUSeS-12-00132]\]. ThEY werE alsO DistINGuIShed FRom OthER PLaNt viRuSEs by hAVinG SEroLOgIcAlly diStINCt vIRIonS anD, BIoLogICAlly, By hAving DisTiNcT hoSt RangEs AnD CaUsiNG DiSTinct SYMPToMs, aND bY theIR PRoPerTiEs In iNfEcTive sAP, SUCH aS DILuTioN EnD Point, ThERmaL inacTIvAtIOn poinT, anD lONGeVity In ViTrO. SiXteEn dIffErENt poTyVIrusEs Had BEEN dEsCribEd IN 1959. SUbSEQuentlY, IN THIs pRe-SEQUEncIng ErA, A COmBInatioN Of tEChnIQues, iNcLudING SuCROSE DensITy gRaDIenT CEntRifuGatIOn, ANalYTICal ULtRaceNtrIFuGATION, UlTRaVIolet SPeCTrophOtOmEtRy, aND pOLYacRylAmIDE GeL ELectrOphorEsiS wERE alSO inCLUDed to estAbLISH THE sEDImeNtatIon CoefFIcieNts AND BuoYaNT dEnSities of VIRIoNS, AnD The MOlEcULAr WEiGHtS Of pRoTEIN subUnITs anD % nUcLEIC AcID contEnts, aS aLL ThESe ProPeRtiES PrOvidEd AdDITiOnaL diSTInguISHinG cHARacTERiSTIcs WHEN nOveL ViRuseS Were beIng deSCrIBed \[[@B7-VIrUSES-12-00132]\].
viRUs idEnTificATiON AnD TAxONoMy wEre TRanSfORMED lATER, wHeN MetHODS fOr SeQUENcING GeNEs Were InveNted iN the 1970s And APPlied TO pLaNT virUSES \[[@b8-VirUSES-12-00132],[@b9-ViRUSEs-12-00132]\], ANd It wAs EstablishED That HIerarcHicAL GRoUpIngS BasED ON vIRaL prOTeIn And gENe sEqUenCEs, INcluDIng THoSE of POTyviRuSES, ConfIRmEd and EXtENdED ThOsE ThAt hAD bEEn deVised preViOUSlY bY uSING phenOtYpic cHaraCTErs, seroLOgIcal tesTs, ETc. AS a REsuLt, 57 POTYvirUsES had BEEN IDENTiFIed By 1991 \[[@B10-viRuSEs-12-00132]\], UsINg SeqUeNCeS Of the "ParT niB-cP" rEGioN OF tHeir GENoMEs, As THIS wAs bRACKETEd By conVenIenT pRIMer sites \[[@B11-ViruSES-12-00132],[@b12-virUsES-12-00132]\]. By 2000, oVER 1000 pOTYvIrUs SEqUenCES werE rEcordeD IN THe GeNBANk DaTaBASE, AnD tHeRe ARE NOW mORe Than 26,000. ThE POTYvIRUses now fOrM A FAmIlY, the *potyvIrIDAe* \[[@b13-VIruSeS-12-00132]\], CoNtAInIng At LeAST eiGHt gENera OF WHICh tHE AphId-tRansmITtED pOtyVIRUseS, iNcLudiNg tHe fIRSt descRIBeD, pvy, mAKe Up tHE LargEST genus, *PotyviRus*. ThIs LARGe pLANT vIRus GENUS is oNe Of the MOSt imPOrTanT EcONOMiCAlLy BECauSe OF the yIELD AND QUAlitY lOSSes It CaUSEs in A widE RaNGe Of croPS WorldWidE. MOREovEr, sOmE of itS MEmbers cuRRENtlY EnDaNGer FooD SECUritY In deveLoPInG CouNtRIES BY CaUSinG dEVASTAtING dIsEAseS iN trOpicAl ANd sUBtRopICal Food CROps \[[@b14-vIRUseS-12-00132]\].
2. the orIgINs of tHE POtyViRIDae {#SEc2-vIrUsES-12-00132}
=================================
THE poTyvIrIDS aRe distInGUishEd FrOM OThEr vIRuSeS By speCific mOlecular DIfferEncEs, tOGetHER wItH a combINatioN oF PhenoTYpic pRoPeRtieS \[[@B13-ViruSeS-12-00132],[@B15-vIrUSEs-12-00132]\]. All pOTYvIrusES iNfecT PlAnTS; most ARE TrAnSmiTTed in NatUrE by arThRopods---MOStly BY AphIDs---THough byMovIRuSEs aRe TrANsMitTEd bY rOoT-INfECTIng pLasmODIOPHoRIds, WhiCH ARe CercOZoAN AMoebAe. SoME PoTYVIrUSEs Are seeD-boRnE \[[@b16-VIRUses-12-00132]\]. POTyVIriD VIriONS arE FLeXUoUS FiLamENts, 680--900 nm lONg anD 11--20 nm In diAMETeR. EaCH iS HeLICaLLy coNsTRUCteD fROm 1400 To 2140 sUbuniTs Of A coaT pRoteiN (cP), aNd a poSiTIvE-SeNse, sINgle-STrANdeD rNa gENOME (UsuaLly monOPARtite, BUt BIpArTIte IN THe GEnuS *bYMOvirUs*) Of 8--11 KB IN tOTaL, WhiCh iS wounD iNtO A groove wiTHiN THe CP SUBunItS.
THE ANcEstrY AND oRIGinS oF thE POTYVIrIDS iS bEing REVeaLed BY studIeS of THE STRuctUrE AND SEquenceS of THEir GeneS aND PRoteinS. thesE SHOW THAT TheIR GenOMEs ArE POlyPHYlETIC In ORIGIn, AS THErE arE sIgNificaNT sIMIlarITIES BEtWeen tHreE OF THEIR GENes and THOsE oF VIrUSES In THREe OthERWIse-uNRelatEd ViRus genErA; tWO DeTECTED By The PROTein SEQuEnCe simILaritY oF The helicASE REgiOn oF thE Ci PROTeIN aND tHe RDRp REgION Of thE niB PROTeIN, RESpEctively, aNd The tHird By tHe StruCtUrE oF THe cp \[[@B17-virUSES-12-00132],[@B18-viRuSeS-12-00132]\]. a BLAStp sEARCH oF ThE GenBAnK pRoteIN seQuence dATAbasE (sEpt 2019), uSING thE EiGHT mAiN MoTIf regionS of ThE PolyPRotEin of PVY (Nc_001616), anD EXCLUdInG MATcHeS WIth sequenCeS FROM thE *PotYviRIdae*, FOUnD oNlY SigniFICaNT MAtcheS BEtWEEn tHe "deaD hELiCAsE-heLicaSE C" RegioN of The ci pRoTeiN AND ThAT of ClassICAl swIne fEVEr And HOG cHOLerA PeSTIVIRUSES (CHanCE PrObAbIlity, 1e-12\_-16; 30% IDEntiTY, 13% iNDeLS), And NO oTherS. lIKEwiSe, thERE WeRE sIGNifIcAnt similAriTIes BetWEeN THe pVY rdRp ReGion And ThaT Of AstROvIRuSES (SEE bELoW). strucTuraL studIeS hAVe OnLy bEeN rePORtEd FoR The CP proTEiN Of OnE potYvirUS, waTERmeloN mOSaIc VIrUs (wMV), anD ReVeaL a sTrUctuRE thAT iS cLOSELy SiMilAr tO THOsE Of OTher ViRusEs wiTh flExUous FilAmentouS VirIoNs, iNCLUDinG two POtEXViRuSES, aND AlSO ThE enVEloPeD FLEXuouS nuCLeOPROteins of oRthOMyXoViRuSeS AnD buNYAVIRIDs, WhICh inClUdE tOMaTO SpOttED wilT toSpOVIRUS \[[@b19-VIruSeS-12-00132],[@B20-viRuSeS-12-00132],[@b21-viRUsES-12-00132]\]; AlL tHesE cps HaVE A core dOmAiN RIcH IN AlPhA hELICes. eACh CP SUBunIt inteRaCts WiTh five NUclEotIDeS (NtS), ANd Has 8.8 sUbunitS pEr TuRn iN a left-HanDed HeLIX, WiTh A PitCH of 34.5--35 Å. ItS n-terMInus Is ExteRnaL to The viRIOn, anD ITs C-TERminus intERNAl. tHe TErMinal REgIoNs InTEract With AdJaCENT SubUNItS anD PRovIDE flExibILitY to thE VIrIOn. iN SERolOgIcal STudiES, THe N-tErMinus Is DOmINaNt AND, iN pOtyVIruSeS, is aLso inVOLVeD WITh APhid iNTERAcTIons \[[@b22-virUSes-12-00132],[@B23-vIRUsES-12-00132]\].
ThE SEqUeNCEs OF THEir rnA-dEpENDEnT RnA pOlyMeRaSes (rdRPS) PLAce The poTYVIRiDS iN ThE "piCOrnaviRus SUperGrOUp" ([fiGure 1](#virUseS-12-00132-f001){REF-TypE="fig"}) \[[@B19-vIRuses-12-00132],[@b24-VirUsEs-12-00132]\], where The PoTYVIriDS ARE OuTsiderS, As MOsT oF THE oTHers HAvE IcOSahEDRAL ViRIonS mADE of EIgHt-sTRanDeD antIpaRAlLEl betA-BArREl prOTEInS, tHE So-calleD 'jelLY ROLl' pROTeIns. In The wOLF ET AL. \[[@b24-VIruses-12-00132]\] TaXonomy Of rDRPs ([FIGuRE 2](#vIrUseS-12-00132-f002){rEf-Type="FiG"}), tHe pOtYVIRid RdrPs fOrm A cLuStER ThAt Is SISteR To AN rDrp FOund IN a MetAGEnoMe, BufiviRUs uc1-GP2, ISOlatED froM "WastEwater" cOLLEcTed IN san FraNCisco. THe SiSteR clADe to The PotYVIriD/bufivIRUs clAdE of rDRPS Are moSTly tHoSe OF tHe AStrovirUSES, a grouP Of guT-InFeCtING viRuSES THAT Are FoUnD in a widE raNgE Of AnImaLS, MOStly MaMMalS oR biRdS. ThEy hAVE 28--35 nM dIAmetEr isoMEtRiC vIriONS (<HTtPS://En.wIkIpEDiA.org/wikI/aSTroviRUs> (acCeSsEd juLY 2019)). sIsTEr tO THe RDrp cLade Of pOTyVIruSES/BufivIRUs/AStRovIRusES are thE RDrpS oF hYPovirusES, AmaLgaViRuseS, pARtItiVIruSEs, AnD picOBIRNavIRUSES, maNy oF tHeM MetagENoMes, iNcLuDinG one froM *phytoPHthorA InFESTaNS* \[[@B25-viRusES-12-00132]\] AND two fRoM leeCHes \[[@B26-ViRuses-12-00132]\]. nOne OF tHE motIfS iDENTIFIed iN potYvIrus prOTEinS, OthEr thaN the rdrp, mATcH THose EncoDed by | 1. Historical Origins {#sec1-viruses-12-00132} ===================== At ameeting of the Fellowsof the National Institute of AgriculturalBotanyin Cambridge, UK, on 14 November 1924, Dr. Redcliffe Salaman gave a lecture entitled "Degenerationof the Potato---An Urgent Problem" \[[@B1-viruses-12-00132]\].He reported that "potato degeneration",namely the decrease in yield when potatoes were grownyear after year fromtubers, rather than from true seed,cost theUK between five and ten millionpounds sterling each year. He noted that the condition was first reported in 1778 at a meeting inManchester, and called"potato curl". It was worse inlowland cropsand in the Southern UKthan in crops grown on higher ground and in the north, and although some thoughtitwas caused by disease, perhapsinsect-borne, others believed it was aform of senility resulting from repeated vegetative reproduction! Salaman concluded that that degeneration was caused bya complex of tuber-borne pathogens. Salaman's talk was successful, as it induced the Ministry of Agriculture to found thePotato VirusResearch Station in Cambridgeandappointhim as director, and in turnhe appointed Kenneth Smith as entomologist, who soon separated some of the components of potato curl andidentified the viruseshe called potatovirusX and potatovirus Y (PVY) \[[@B2-viruses-12-00132]\]. Other viruses similar to PVY were soon reported, for example, henbane mosaic virus \[[@B3-viruses-12-00132]\], which was like PVY in causing mosaic symptoms, being transmitted by sap, although relatively unstable in it, and also bybeing transmitted by aphids in short feeds. These viruses, which became known as potyviruses, short for "potatovirus Ygroup viruses" \[[@B4-viruses-12-00132]\], wereamong those included in early attempts to devise biologicaltaxonomies of plant viruses \[[@B5-viruses-12-00132]\] based on the length of their filamentousparticles \[[@B6-viruses-12-00132]\]. Theywere also distinguished from other plant viruses by having serologically distinct virions and,biologically, by having distincthost ranges and causingdistinct symptoms, and by their properties in infective sap, such as dilution endpoint, thermal inactivation point, andlongevity invitro. Sixteen different potyviruses had been described in 1959. Subsequently,in this pre-sequencing era,a combinationof techniques, including sucrose density gradientcentrifugation, analytical ultracentrifugation, ultraviolet spectrophotometry, and polyacrylamide gel electrophoresis werealso included to establish the sedimentation coefficients and buoyant densities of virions, and the molecular weights of protein subunits and % nucleic acidcontents, asall these properties provided additional distinguishing characteristics whennovel viruses were being described \[[@B7-viruses-12-00132]\]. Virus identification and taxonomy were transformed later, whenmethods for sequencing genes were invented in the 1970s andapplied to plant viruses \[[@B8-viruses-12-00132],[@B9-viruses-12-00132]\], andit was established thathierarchical groupings based on viral protein and gene sequences, including thoseof potyviruses, confirmed and extended those that hadbeen devised previously by using phenotypic characters,serological tests, etc. As a result, 57 potyviruses had been identifiedby 1991 \[[@B10-viruses-12-00132]\], using sequences ofthe "part NIb-CP" region of their genomes, as this was bracketed by convenient primer sites \[[@B11-viruses-12-00132],[@B12-viruses-12-00132]\]. By 2000, over 1000 potyvirus sequences wererecorded in the GenBankdatabase,and there are nowmore than 26,000. The potyvirusesnow form a family, the *Potyviridae* \[[@B13-viruses-12-00132]\],containing at leasteight genera ofwhich the aphid-transmitted potyviruses, includingthe first described, PVY, make up the largest genus, *Potyvirus*. This large plant virus genus isone of the mostimportant economically because of the yield and quality losses it causes in a wide rangeofcrops worldwide. Moreover, some of its members currentlyendanger food security in developing countriesby causing devastatingdiseases in tropicaland subtropical foodcrops \[[@B14-viruses-12-00132]\]. 2. The Origins of the Potyviridae {#sec2-viruses-12-00132} ================================= Thepotyvirids are distinguished fromother viruses by specificmolecular differences, together with a combination of phenotypic properties \[[@B13-viruses-12-00132],[@B15-viruses-12-00132]\]. All potyviruses infect plants; most are transmitted in nature by arthropods---mostly by aphids---though bymoviruses are transmitted by root-infecting plasmodiophorids, which are cercozoan amoebae. Somepotyviruses are seed-borne\[[@B16-viruses-12-00132]\]. Potyviridvirions are flexuous filaments,680--900nm long and 11--20 nm in diameter. Eachis helically constructed from 1400 to2140 subunits of a coatprotein(CP), and a positive-sense, single-stranded RNA genome (usuallymonopartite, but bipartite inthe genus *Bymovirus*) of 8--11 kbin total, which is wound into a groove withinthe CP subunits.The ancestry andoriginsof the potyvirids is being revealed by studies of the structure and sequences of their genes and proteins. These show that their genomes are polyphyletic in origin, as there are significant similarities between three of their genes and those of viruses in three otherwise-unrelated virus genera; two detected by the protein sequence similarity of the helicaseregion of the CI protein and the RdRp region of the NIb protein, respectively, andthe third by the structure of the CP \[[@B17-viruses-12-00132],[@B18-viruses-12-00132]\]. ABLASTp search of theGenBankprotein sequence database (Sept 2019), using the eight main motif regions of the polyprotein of PVY (NC_001616), and excluding matches with sequences from the *Potyviridae*,found onlysignificant matches betweenthe "DEAD helicase-helicase C" region ofthe CI protein and that of classical swine fever and hog cholera pestiviruses (chance probability, 1e-12\_-16; 30%identity, 13% indels), and no others. Likewise, there were significant similaritiesbetween the PVY RdRp region and that of astroviruses (see below). Structural studies have only been reported for the CP protein of one potyvirus, watermelon mosaic virus (WMV),and reveal a structure that isclosely similar to those of other viruses with flexuous filamentousvirions, including two potexviruses, and alsothe enveloped flexuous nucleoproteins of orthomyxoviruses and bunyavirids, which include tomato spotted wilt tospovirus \[[@B19-viruses-12-00132],[@B20-viruses-12-00132],[@B21-viruses-12-00132]\]; allthese CPs have a core domain rich in alpha helices. Each CP subunit interacts with five nucleotides (nts), and has 8.8 subunits per turn in a left-handed helix, witha pitch of 34.5--35 Å. Its N-terminus is external to the virion,and its C-terminus internal. Theterminal regions interact with adjacent subunits and provide flexibility to thevirion. In serologicalstudies, the N-terminus is dominant and, inpotyviruses, is alsoinvolved with aphid interactions \[[@B22-viruses-12-00132],[@B23-viruses-12-00132]\]. The sequences of their RNA-dependent RNA polymerases (RdRps) place the potyvirids in the "Picornavirus Supergroup" ([Figure 1](#viruses-12-00132-f001){ref-type="fig"}) \[[@B19-viruses-12-00132],[@B24-viruses-12-00132]\], where the potyvirids are outsiders, as most of the others have icosahedral virions made of eight-stranded antiparallel beta-barrel proteins, theso-called 'jelly roll' proteins. In the Wolf et al. \[[@B24-viruses-12-00132]\]taxonomy of RdRps ([Figure 2](#viruses-12-00132-f002){ref-type="fig"}), the potyvirid RdRps form a cluster thatissister to an RdRp found in a metagenome, bufivirus UC1-gp2, isolatedfrom "wastewater"collected in San Francisco. Thesister clade to the potyvirid/bufivirus clade of RdRps are mostly thoseof the astroviruses,a group of gut-infecting viruses that are found in a wide range of animals, mostly mammals or birds. They have 28--35 nm diameter isometric virions (<https://en.wikipedia.org/wiki/Astrovirus>(accessed July 2019)). Sister to the RdRp clade of potyviruses/bufivirus/astroviruses are the RdRps of hypoviruses, amalgaviruses, partitiviruses, andpicobirnaviruses, many of them metagenomes, including one from*Phytophthorainfestans* \[[@B25-viruses-12-00132]\] and two from leeches \[[@B26-viruses-12-00132]\]. None of the motifs identified in potyvirus proteins, other than the RdRp, match those encoded by | 1. Historical Origins {#sec1-viruses-12-00132} _=====================_ At _a_ meeting of _the_ Fellows of the National Institute of Agricultural Botany _in_ Cambridge, UK, on 14 November 1924, Dr. _Redcliffe_ _Salaman_ gave a lecture entitled "Degeneration _of_ the _Potato---An_ Urgent _Problem"_ \[[@B1-viruses-12-00132]\]. He _reported_ that "potato degeneration", namely the _decrease_ in yield when potatoes were grown year after year _from_ tubers, rather than from true seed, cost _the_ _UK_ between _five_ and ten million _pounds_ sterling _each_ year. He noted that the condition was first reported _in_ 1778 _at_ a _meeting_ in _Manchester,_ _and_ called "potato curl". _It_ _was_ worse _in_ _lowland_ crops and _in_ the Southern _UK_ than in crops grown on _higher_ ground and in the north, and although some thought it was caused by disease, perhaps insect-borne, others believed it was a _form_ of senility resulting from repeated vegetative reproduction! Salaman _concluded_ that that degeneration was caused by a complex _of_ tuber-borne pathogens. Salaman's _talk_ was _successful,_ as it induced the Ministry of Agriculture to found the Potato Virus Research _Station_ _in_ _Cambridge_ and _appoint_ him as director, _and_ _in_ _turn_ he appointed Kenneth Smith as entomologist, _who_ _soon_ separated some of the components of potato _curl_ and _identified_ the viruses he called potato virus _X_ and potato virus Y (PVY) \[[@B2-viruses-12-00132]\]. Other viruses similar to PVY were soon reported, for _example,_ henbane _mosaic_ _virus_ \[[@B3-viruses-12-00132]\], _which_ was like PVY in causing mosaic symptoms, _being_ _transmitted_ by sap, although relatively unstable in it, and also by being transmitted by aphids in short feeds. These viruses, which became known as potyviruses, _short_ for "potato virus Y _group_ viruses" _\[[@B4-viruses-12-00132]\],_ were among those included _in_ early attempts _to_ _devise_ biological _taxonomies_ of plant viruses \[[@B5-viruses-12-00132]\] based on the length _of_ their _filamentous_ particles \[[@B6-viruses-12-00132]\]. _They_ were also distinguished from other plant viruses by having serologically distinct virions and, biologically, by having distinct host _ranges_ and causing distinct symptoms, and by their properties _in_ infective sap, such as dilution end point, thermal inactivation _point,_ and longevity in vitro. Sixteen different potyviruses had been described in 1959. Subsequently, in this pre-sequencing _era,_ a combination of techniques, including sucrose density gradient _centrifugation,_ analytical _ultracentrifugation,_ ultraviolet spectrophotometry, and polyacrylamide _gel_ electrophoresis were also included to establish the sedimentation coefficients and _buoyant_ densities of virions, and the molecular weights of protein _subunits_ and % _nucleic_ _acid_ contents, as all these properties provided _additional_ distinguishing characteristics when novel _viruses_ were being described _\[[@B7-viruses-12-00132]\]._ Virus identification _and_ taxonomy _were_ transformed later, when methods _for_ sequencing genes _were_ invented in the 1970s and applied to plant _viruses_ \[[@B8-viruses-12-00132],[@B9-viruses-12-00132]\], and it was established that hierarchical groupings based on _viral_ protein _and_ gene _sequences,_ including those of potyviruses, confirmed and _extended_ _those_ that had been devised previously by using phenotypic characters, serological _tests,_ _etc._ As _a_ result, 57 potyviruses had been identified by 1991 \[[@B10-viruses-12-00132]\], using sequences of the "part NIb-CP" _region_ of their genomes, as this was bracketed by convenient primer sites \[[@B11-viruses-12-00132],[@B12-viruses-12-00132]\]. By _2000,_ over 1000 potyvirus sequences were recorded in the _GenBank_ _database,_ _and_ _there_ are now more _than_ 26,000. The potyviruses _now_ form a family, _the_ *Potyviridae* \[[@B13-viruses-12-00132]\], containing at _least_ eight genera _of_ _which_ _the_ aphid-transmitted potyviruses, including the first _described,_ PVY, make _up_ the largest genus, *Potyvirus*. This large plant virus genus is one of _the_ most important economically because _of_ the yield and quality losses it causes in a wide _range_ of crops worldwide. Moreover, some _of_ its members currently endanger food security in developing countries by causing devastating diseases in _tropical_ and subtropical food crops \[[@B14-viruses-12-00132]\]. 2. _The_ Origins of the Potyviridae {#sec2-viruses-12-00132} _=================================_ The potyvirids are _distinguished_ from other _viruses_ by specific molecular _differences,_ together with _a_ combination of phenotypic properties _\[[@B13-viruses-12-00132],[@B15-viruses-12-00132]\]._ All _potyviruses_ infect _plants;_ most _are_ _transmitted_ in nature _by_ arthropods---mostly by aphids---though _bymoviruses_ are transmitted by root-infecting plasmodiophorids, which are _cercozoan_ amoebae. Some potyviruses are seed-borne \[[@B16-viruses-12-00132]\]. Potyvirid virions are _flexuous_ filaments, 680--900 nm long and 11--20 nm _in_ diameter. Each is helically constructed from 1400 _to_ 2140 subunits of a _coat_ protein (CP), and _a_ positive-sense, single-stranded RNA genome _(usually_ monopartite, _but_ _bipartite_ in the genus *Bymovirus*) _of_ 8--11 kb in total, which is wound into a groove within the CP subunits. The ancestry and origins of the potyvirids is being revealed by studies of the structure and _sequences_ _of_ their genes and proteins. These _show_ that their genomes _are_ polyphyletic in _origin,_ as there are significant similarities between three of _their_ genes _and_ those of viruses in _three_ _otherwise-unrelated_ virus genera; two _detected_ by the protein sequence similarity of the _helicase_ region of the CI _protein_ and the RdRp region of the NIb protein, respectively, and _the_ _third_ _by_ the _structure_ _of_ the CP \[[@B17-viruses-12-00132],[@B18-viruses-12-00132]\]. A BLASTp search of the GenBank _protein_ _sequence_ database (Sept 2019), using the eight main motif regions of _the_ polyprotein of PVY (NC_001616), _and_ _excluding_ matches with sequences from the *Potyviridae*, _found_ only significant _matches_ _between_ _the_ "DEAD helicase-helicase C" region of the CI protein _and_ that of classical swine fever _and_ hog cholera pestiviruses (chance probability, 1e-12\_-16; 30% identity, 13% indels), and no others. Likewise, there were significant similarities between the PVY RdRp region _and_ that of _astroviruses_ (see below). Structural studies _have_ only _been_ _reported_ for the _CP_ protein of one _potyvirus,_ watermelon mosaic _virus_ _(WMV),_ and reveal a structure that _is_ closely _similar_ to _those_ of other viruses _with_ flexuous filamentous virions, including _two_ _potexviruses,_ and also the _enveloped_ flexuous _nucleoproteins_ of orthomyxoviruses _and_ bunyavirids, which include tomato spotted wilt tospovirus _\[[@B19-viruses-12-00132],[@B20-viruses-12-00132],[@B21-viruses-12-00132]\];_ all these CPs have a core domain rich _in_ alpha helices. Each CP subunit _interacts_ with five nucleotides _(nts),_ and has _8.8_ subunits per turn in a left-handed _helix,_ with a _pitch_ of _34.5--35_ Å. _Its_ N-terminus _is_ _external_ to the virion, and _its_ C-terminus internal. _The_ _terminal_ regions interact with adjacent subunits and provide flexibility to _the_ virion. In serological studies, the N-terminus is dominant and, _in_ _potyviruses,_ is also involved with aphid _interactions_ \[[@B22-viruses-12-00132],[@B23-viruses-12-00132]\]. _The_ _sequences_ of their RNA-dependent RNA polymerases (RdRps) place _the_ potyvirids in the "Picornavirus Supergroup" _([Figure_ 1](#viruses-12-00132-f001){ref-type="fig"}) _\[[@B19-viruses-12-00132],[@B24-viruses-12-00132]\],_ where the _potyvirids_ _are_ outsiders, _as_ most _of_ the others _have_ _icosahedral_ virions made _of_ eight-stranded _antiparallel_ beta-barrel proteins, _the_ _so-called_ 'jelly roll' proteins. _In_ the Wolf et al. _\[[@B24-viruses-12-00132]\]_ _taxonomy_ _of_ RdRps ([Figure 2](#viruses-12-00132-f002){ref-type="fig"}), the potyvirid RdRps form a cluster that is sister _to_ an RdRp _found_ in a _metagenome,_ bufivirus UC1-gp2, isolated from "wastewater" _collected_ _in_ San _Francisco._ The sister _clade_ to the _potyvirid/bufivirus_ clade of _RdRps_ are mostly _those_ _of_ _the_ astroviruses, a group of _gut-infecting_ viruses that are found in a wide range of animals, _mostly_ mammals _or_ birds. They have 28--35 nm diameter isometric virions (<https://en.wikipedia.org/wiki/Astrovirus> _(accessed_ _July_ _2019))._ Sister _to_ _the_ RdRp clade of potyviruses/bufivirus/astroviruses are the RdRps of hypoviruses, amalgaviruses, partitiviruses, and _picobirnaviruses,_ _many_ _of_ _them_ metagenomes, _including_ one _from_ *Phytophthora infestans* \[[@B25-viruses-12-00132]\] and two from leeches \[[@B26-viruses-12-00132]\]. None of the motifs _identified_ _in_ potyvirus _proteins,_ other than the RdRp, match _those_ encoded by |
All relevant data are within the paper and its Supporting Information files.
Introduction {#sec005}
============
Globally, an estimated 225 million women have unmet need for family planning \[[@pone.0153907.ref001]\]. Increasing acceptance and use of family planning requires more than increasing access to health services: truly effective family planning programs must address the social and gender norms that present critical barriers to sexual and reproductive health. Use of family planning is powerfully shaped by social norms, including perceived acceptability of family planning, social pressure for large families, and perceived opposition to family planning by religious and community leaders and spouses \[[@pone.0153907.ref002]\].
Rigid gender roles and unequal power between men and women inhibit couple communication and joint decision-making about family planning: power dynamics in couples have been found to influence the use of family planning and other health services \[[@pone.0153907.ref003]--[@pone.0153907.ref005]\]. Fear of and experience with intimate partner violence or gender-based violence are barriers to contraceptive use \[[@pone.0153907.ref006]--[@pone.0153907.ref009]\]. Women who report intimate partner violence are at higher risk for unintended pregnancy than women who do not report violence \[[@pone.0153907.ref008]\]. Analyses of data from Demographic and Health Surveys (DHS) in a number of African countries suggest that key dimensions of gender equity and women's empowerment---including equitable beliefs about gender roles, women's ability to negotiate sexual activity, and women's control over household economic decision-making---are associated with higher contraceptive use \[[@pone.0153907.ref010]\]. Several studies have also found a positive association between spousal communication and the use of contraception \[[@pone.0153907.ref011]--[@pone.0153907.ref014]\].
Despite evidence demonstrating the important relationships between gender, women's empowerment, and family planning, there is little consensus about how to best measure these complex social constructs. Researchers have attempted to measure specific domains of women's empowerment, and several scales have been developed including measures of mobility \[[@pone.0153907.ref015]\], power in relationships \[[@pone.0153907.ref003]\], gender norms and decision-making autonomy \[[@pone.0153907.ref016]\], control over assets \[[@pone.0153907.ref017]\], and social capital \[[@pone.0153907.ref018],[@pone.0153907.ref019]\]. The lack of a standard set of widely used measures for women's empowerment, however, hampers our ability to compare program effects across studies, populations, and cultural contexts.
The objective of the research described in this article was to evaluate the impact of a CARE intervention that catalyzed community-level dialogue about gender, sexuality, and family planning on household-level gender dynamics and reported use of family planning among men and women in Kenya. For this evaluation, we used several scales from WE-MEASR (Women's Empowerment-Multidimensional Evaluation of Agency, Social Capital, and Relations) (see [S1 File](#pone.0153907.s001){ref-type="supplementary-material"}), a new tool that CARE developed following multi-year research into the effects and impact of our women's empowerment programming \[[@pone.0153907.ref020]\].
Methodology {#sec006}
===========
Study Setting {#sec007}
-------------
According to the 2008--09 Kenya DHS, approximately one in four women has an unmet need for family planning \[[@pone.0153907.ref021]\]. A 2013 analysis of DHS data found that Kenyan women who do not use family planning report method-related concerns, especially fear of harmful side effects, as the most common reason for non-use (43%); opposition to family planning (both the perceived religious prohibition of family planning and opposition by husbands/partners) is the second most commonly cited reason (16%) \[[@pone.0153907.ref022]\].
CARE conducted the research described here as part of the Family Planning Results Initiative (FPRI), which we implemented in Siaya County, Nyanza province, Kenya from July 2009 through December 2012. The majority of the county's population of 842,304 is under 30 years old, and 46.1% are between 0 and 14 years of age \[[@pone.0153907.ref023]\]. Over 90% of Siaya County is rural and, like the rest of Nyanza, falls behind the national average on various indicators of development, gender inequality, poverty, education, and health, including infant and under-five mortality, antenatal care coverage, and HIV prevalence \[[@pone.0153907.ref021]\]. The contraceptive prevalence rate among married women in Nyanza province is among the lowest in Kenya: 37.3% of women use any method (compared to 45.5% nationally), and only 32.9% use a modern method (39.4% nationally) \[[@pone.0153907.ref021]\].
The Intervention {#sec008}
----------------
CARE used its *Social Analysis and Action* approach \[[@pone.0153907.ref024]\] to shape FPRI's central intervention about 150 community-based facilitators were trained and held ongoing community dialogues about gender, sexuality, and family planning over three and a half years (July 2009-December 2012). At any one time, an average of 65 facilitators were actively convening dialogues. Monthly planning meetings were held to plan activities and ensure adequate location coverage and topic variation over the entire study area. On a quarterly basis, CARE observed and provided feedback on dialogues and completed progress reviews with facilitators. Based on our monitoring data, CARE organized 759 dialogues between July 2009 and December 2012. Dialogues were held in a variety of venues and were promoted by community leaders or organized around pre-scheduled community meetings. Based on proximity to a respective venue, they were attended by participants from several villages, and, many times, were included in villages' development plans.
The dialogues were designed to normalize communication about sensitive topics like gender norms and FP, and to catalyze participants' critical analysis of how gender norms and dynamics restrict family planning acceptability and use. During dialogues, community leaders and satisfied family planning users acted as role models and overtly expressed support for family planning, equitable gender norms, open communication and shared decision-making regarding family planning between spouses.
Trained, community-based facilitators convened dialogues in an array of settings, such as markets, churches, women's groups and village meetings. Local theatre groups often performed during the dialogues. Facilitators included community health care workers, religious leaders, local government officials, and teachers. CARE provided ongoing support to facilitators in the form of training and supervision, and also provided transport reimbursement and lunch/refreshments on days when dialogues or trainings were conducted; no incentives were provided for participation in intervention activities to any other community members.
All of CARE's activities were coordinated with the District Health Medical Team (DHMT.) During the intervention period, the DHMT coordinated several interventions designed to increase availability and access to contraceptives, including in-service training for providers, strengthening of systems for ordering contraceptives, and some community outreach activities (FP counseling and method provision). To our knowledge, no other similar FP dialogue interventions or activities were implemented by other organizations during the intervention period.
Theory of Change {#sec009}
----------------
The intervention aimed to challenge and shift key community norms about gender dynamics and family planning, with the ultimate goal of creating a social environment that is more supportive of equitable gender relations and the use of family planning. Our research goals were to determine whether and how the ongoing dialogues shifted social norms, and whether and how these shifts at the community level influenced communication, decision-making, and family planning use at the couple or household level. Our theorized pathway of change is shown in [Fig 1](#pone.0153907.g001){ref-type="fig"}.
{#pone.0153907.g001}
Evaluation Design {#sec010}
-----------------
We used both qualitative and quantitative methods to evaluate the intervention. At baseline (February 2009) and again at endline (December 2012), we conducted household surveys to collect data about men and women's socio-demographic characteristics, pregnancy intentions, family planning knowledge, beliefs, attitudes, and use. At endline only, we measured key gender-related beliefs and behaviors and exposure to the intervention, which enabled us to determine whether these were important predictors of family planning use. Also at endline, we conducted in-depth, qualitative interviews with purposively selected couples from the intervention area (described below) to explore changes in communication, decision-making, and gender roles over the previous four years and to identify specific enablers and barriers to family planning use.
Ethics Statement {#sec011}
----------------
CARE obtained approval to conduct this research from the Institutional Research and Ethics Committee at Moi University in Eldoret, Kenya.
Quantitative Data Collection {#sec012}
----------------------------
Through independent, cross-sectional household surveys, married men (20--49 years) and women (18--45 years) were interviewed by trained interviewers using standardized questionnaires at baseline and endline. Although the two samples were entirely independent (no attempt was made at endline to include or exclude respondents who participated at baseline), both were drawn from | all relevant data are within the paper and its supporting information files. introduction { # sec005 } = = = = = = = = = = = = globally, an estimated 225 million women express unmet need for family planning \ [ [ @ pone. 0153907. ref001 ] \ ]. increasing acceptance and use of family planning requires more than increasing access to health services : truly effective family planning programs must address the social and gender norms that present critical barriers to sexual and reproductive health. use of family planning is powerfully shaped by social norms, including societal acceptability of lifestyle planning, social pressure for large families, and perceived opposition to family planning by religious and community leaders and spouses \ [ [ @ pone. 0153907. ref002 ] \ ]. rigid gender roles and unequal power between men and women inhibit couple communication and joint decision - making about family planning : power dynamics in couples have been found to influence the use of social planning and other health services \ [ [ @ pone. 0153907. ref003 ] - - [ @ pone. 0153907. ref005 ] \ ]. fear of and experience with domestic partner violence or gender - based violence are barriers to contraceptive use \ [ [ @ pone. 0153907. ref006 ] - - [ @ pone. 0153907. ref009 ] \ ]. women who report intimate partner violence are at higher barriers for unintended pregnancy than women who do directly report violence \ [ [ @ pone. 0153907. ref008 ] \ ]. analyses of data from demographic and health surveys ( dhs ) in a number of african countries suggest that key dimensions of gender autonomy and women ' s empowerment - - - including equitable beliefs about gender roles, women ' s ability to negotiate sexual activity, and women ' s preference over household economic decision - making - - - are associated with higher contraceptive use \ [ [ @ pone. 0153907. ref010 ] \ ]. several studies have also found a positive association between spousal communication and the use of contraception \ [ [ @ pone. 0153907. ref011 ] - - [ @ pone. 0153907. ref014 ] \ ]. despite evidence demonstrating the important relationships between gender, women ' s empowerment, and family planning, it is little consensus about how to best measure these complex social constructs. researchers have attempted to measure specific domains of women ' s empowerment, and several scales have been developed including measures of mobility \ [ [ @ pone. 0153907. ref015 ] \ ], power in relationships \ [ [ @ pone. 0153907. ref003 ] \ ], gender norms and decision - making autonomy \ [ [ @ pone. 0153907. ref016 ] \ ], control over assets \ [ [ @ pone. 0153907. ref017 ] \ ], and social capital \ [ [ @ pone. 0153907. ref018 ], [ @ pone. 0153907. ref019 ] \ ]. the lack of a standard set of widely used measures for women ' s empowerment, however, hampers our ability to compare program effects across studies, populations, and cultural contexts. the objective of the research described in this article was to evaluate the impact of a care intervention that catalyzed community - level dialogue about gender, sexuality, and family planning on household - level gender dynamics and reported use of family planning among men and women in kenya. for this evaluation, we used several scales from we - measr ( women ' s empowerment - multidimensional evaluation of agency, social capital, and relations ) ( see [ s1 file ] ( # pone. 0153907. s001 ) { ref - type = " supplementary - material " } ), a new tool that care developed following multi - year research into the effects and impact of our women ' s empowerment programming \ [ [ @ pone. 0153907. ref020 ] \ ]. methodology { # sec006 } = = = = = = = = = = = study setting { # sec007 } - - - - - - - - - - - - - according to the 2008 - - 09 kenya dhs, approximately one in four women has an unmet need for family planning \ [ [ @ pone. 0153907. ref021 ] \ ]. a 2013 analysis of dhs data found that kenyan women who do not use family planning report method - related concerns, especially fear of harmful side effects, as the most common reason for non - use ( 43 % ) ; opposition to family planning ( both the perceived religious prohibition of family planning and opposition by husbands / partners ) is the second most commonly cited reason ( 16 % ) \ [ [ @ pone. 0153907. ref022 ] \ ]. care conducted the research described here as part of the family planning results initiative ( fpri ), which we implemented in siaya county, nyanza province, kenya from july 2009 through december 2012. the majority of the county ' s population of 842, 304 is under 30 years old, and 46. 1 % are between 0 and 14 years of age \ [ [ @ pone. 0153907. ref023 ] \ ]. over 90 % of siaya county is rural and, like the rest of nyanza, falls behind the national average on various indicators of development, gender inequality, poverty, education, and health, including infant and under - five mortality, antenatal care coverage, and hiv prevalence \ [ [ @ pone. 0153907. ref021 ] \ ]. the contraceptive prevalence rate among married women in nyanza province is among the lowest in kenya : 37. 3 % of women use any method ( compared to 45. 5 % nationally ), and only 32. 9 % use a modern method ( 39. 4 % nationally ) \ [ [ @ pone. 0153907. ref021 ] \ ]. the intervention { # sec008 } - - - - - - - - - - - - - - - - care used its * social analysis and action * approach \ [ [ @ pone. 0153907. ref024 ] \ ] to shape fpri ' s central intervention about 150 community - based facilitators were trained and held ongoing community dialogues about gender, sexuality, and family planning over three and a half years ( july 2009 - december 2012 ). at any one time, an average of 65 facilitators were actively convening dialogues. monthly planning meetings were held to plan activities and ensure adequate location coverage and topic variation over the entire study area. on a quarterly basis, care observed and provided feedback on dialogues and completed progress reviews with facilitators. based on our monitoring data, care organized 759 dialogues between july 2009 and december 2012. dialogues were held in a variety of venues and were promoted by community leaders or organized around pre - scheduled community meetings. based on proximity to a respective venue, they were attended by participants from several villages, and, many times, were included in villages ' development plans. the dialogues were designed to normalize communication about sensitive topics like gender norms and fp, and to catalyze participants ' critical analysis of how gender norms and dynamics restrict family planning acceptability and use. during dialogues, community leaders and satisfied family planning users acted as role models and overtly expressed support for family planning, equitable gender norms, open communication and shared decision - making regarding family planning between spouses. trained, community - based facilitators convened dialogues in an array of settings, such as markets, churches, women ' s groups and village meetings. local theatre groups often performed during the dialogues. facilitators included community health care workers, religious leaders, local government officials, and teachers. care provided ongoing support to facilitators in the form of training and supervision, and also provided transport reimbursement and lunch / refreshments on days when dialogues or trainings were conducted ; no incentives were provided for participation in intervention activities to any other community members. all of care ' s activities were coordinated with the district health medical team ( dhmt. ) during the intervention period, the dhmt coordinated several interventions designed to increase availability and access to contraceptives, including in - service training for providers, strengthening of systems for ordering contraceptives, and some community outreach activities ( fp counseling and method provision ). to our knowledge, no other similar fp dialogue interventions or activities were implemented by other organizations during the intervention period. theory of change { # sec009 } - - - - - - - - - - - - - - - - the intervention aimed to challenge and shift key community norms about gender dynamics and family planning, with the ultimate goal of creating a social environment that is more supportive of equitable gender relations and the use of family planning. our research goals were to determine whether and how the ongoing dialogues shifted social norms, and whether and how these shifts at the community level influenced communication, decision - making, and family planning use at the couple or household level. our theorized pathway of change is shown in [ fig 1 ] ( # pone. 0153907. g001 ) { ref - type = " fig " }.! [ care ' s family planning results initiative theory of change. \ this theory of change illustrates, from left to right, the activities undertaken as part of care ' s family planning results initiative, the expected intermediate outcomes that will lead to improvements in three key determinants of family planning which, ultimately, increase use of family planning in the intervention community. ] ( pone. 0153907. g001 ) { # pone. 0153907. g001 } evaluation design { # sec010 } - - - - - - - - - - - - - - - - - we used both qualitative and quantitative methods to evaluate the intervention. at baseline ( february 2009 ) and again at endline ( december 2012 ), we conducted household surveys to collect data about men and women ' s socio - demographic characteristics, pregnancy intentions, family planning knowledge, beliefs, attitudes, and use. at endline only, we measured key gender - related beliefs and behaviors and exposure to the intervention, which enabled us to determine whether these were important predictors of family planning use. also at endline, we conducted in - depth, qualitative interviews with purposively selected couples from the intervention area ( described below ) to explore changes in communication, decision - making, and gender roles over the previous four years and to identify specific enablers and barriers to family planning use. ethics statement { # sec011 } - - - - - - - - - - - - - - - - care obtained approval to conduct this research from the institutional research and ethics committee at moi university in eldoret, kenya. quantitative data collection { # sec012 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - through independent, cross - sectional household surveys, married men ( 20 - - 49 years ) and women ( 18 - - 45 years ) were interviewed by trained interviewers using standardized questionnaires at baseline and endline. although the two samples were entirely independent ( no attempt was made at endline to include or exclude respondents who participated at baseline ), both were drawn from | All relevant data are within the paper and its Supporting Information files. Introduction {# sec005} = = = = = = = = = = = = Globally, an estimated 225 million women have unmet need for family planning \ [[ @ pone. 0153907. ref001] \ ]. Increasing wcveptance and use of family planning requires more than increasing access to health services: truly effective family planning programs must address the social and gender norms that present critical barriers to sexual and reproductive health. Use of family planning is powerfully shaped by social norms, including perceived acceptability of family planning, social pressure for large families, and perceived opposition to family planning by religious and community leaders and spouses \ [[ @ pone. 0153907. ref002] \ ]. Rigid gender roles and unequal power between men and women inhibit couple communication and joint decision - making about family planning: power dynamics in couples have been found to influence the use of family planning and other health services \ [[ @ pone. 0153907. ref003] - - [@ pone. 0153907. ref005] \ ]. Fear of and experience with intimate partner violence or gender - based violence are barriers to contraceptive use \ [[ @ pone. 0153907. ref006] - - [@ pone. 0153907. ref009] \ ]. Women who report intimate partner violence are at higher risk for unintended pregnancy than women who do not report violence \ [[ @ pone. 0153907. ref008] \ ]. Analyses of data from Demographic and Health Surveys (DHS) in a number of African countries suggest that key dimensions of gender equity and women ' s empowerment - - - including equitable beliefs about gender roles, women ' s ability to negotiate sexual activity, and women ' s control over household economic decision - making - - - are associated with higher contraceptive use \ [[ @ pone. 0153907. ref010] \ ]. Several studies have also found a positive association between spousal communication and the use of contraception \ [[ @ pone. 0153907. ref011] - - [@ pone. 0153907. ref014] \ ]. Despite evidence demonstrating the important relationships between gender, women ' s empowerment, and family planning, there is little consensus about how to best measure these complex social constructs. Researchers have attempted to measure specific domains of women ' s empowerment, and several scales have been developed including measures of mobility \ [[ @ pone. 0153907. ref015] \ ], power in relationships \ [[ @ pone. 0153907. ref003] \ ], gender norms and decision - making autonomy \ [[ @ pone. 0153907. ref016] \ ], control over assets \ [[ @ pone. 0153907. ref017] \ ], and social capital \ [[ @ pone. 0153907. ref018 ], [@ pone. 0153907. ref019] \ ]. The lack of a standard set of widely used measures for women ' s empowerment, however, hampers our ability to compare program effects across studies, populations, and cultural contexts. The objective of the research described in this article was to evaluate the impact of a CARE intervention that catalyzed community - level dialogue about gender, sexuality, and family planning on household - level gender dynamics and reported use of family planning among men and women in Kenya. For this evaluation, we used several scales from WE - MEASR (Women ' s Empowerment - Multidimensional Evaluation of Agency, Social Capital, and Relations) (see [S1 File] (# pone. 0153907. s001) {ref - type = " supplementary - material "} ), a new tool that CARE developed following multi - year research into the effects and impact of our women ' s empowerment programming \ [[ @ pone. 0153907. ref020] \ ]. Methodology {# sec006} = = = = = = = = = = = Study Setting {# sec007} - - - - - - - - - - - - - According to the 2008 - - 09 Kenya DHS, approximately one in four women has an unmet need for family planning \ [[ @ pone. 0153907. ref021] \ ]. A 2013 analysis of DHS sats found that Kenyan women who do not use family planning report method - related concerns, especially fear of harmful side effects, as the most common reason for non - use (43% ); opposition to family plxBning (both the perceived religious prohibition of family planning and opposition by husbands / partners) is the second most commonly cited reason (16%) \ [[ @ pone. 0153907. ref022] \ ]. CARE conducted the research described here as part of the Family Planning Results Initiative (FPRI ), which we implemented in Siaya County, Nyanza province, Kenya from July 2009 through December 2012. The majority of the county ' s population of 842, 304 is under 30 years old, and 46. 1% are between 0 and 14 years of age \ [[ @ pone. 0153907. ref023] \ ]. Over 90% of Siaya County is rural and, like the rest of Nyanza, falls behind the national average on various indicators of development, gender inequality, poverty, education, and health, including infant and under - five mortality, antenatal care coverage, and HIV prevalence \ [[ @ pone. 0153907. ref021] \ ]. The contraceptive prevalence rate among married women in Nyanza province is among the lowest in Kenya: 37. 3% of women use any method (compared to 45. 5% nationally ), and only 32. 9% use a modern method (39. 4% nationally) \ [[ @ pone. 0153907. ref021] \ ]. The Intervention {# sec008} - - - - - - - - - - - - - - - - CARE used its * Social Analysis and Action * approach \ [[ @ pone. 0153907. ref024] \] to shape FPRI ' s central intervention about 150 community - based facilitators were trained and held ongoing community dialogues about gender, sexuality, and family planning over three and a half years (July 2009 - December 2012 ). At any one time, an average of 65 facilitators were actively convening dialogues. Monthly planning meetiGgx were held to plan activities and ensure adequate location coverage and topic variation over the entire study area. On a quarterly basis, CARE observed and provided feedback on dialogues and completed progress reviews with facilitators. Based on our monitoring data, CARE organized 759 dialogues between July 2009 and December 2012. Dialogues were held in a variety of venues and were promoted by community leaders or organized around pre - scheduled SommuniRy meetings. Based on proximity to a respective venue, they were attended by participants from several villages, and, many times, were included in villages ' development plans. The dialogues were designed to normalize communication about sensitive topics like gender norms and FP, and to catalyze participants ' critical analysis of how gender norms and dynamics restrict family planning acceptability and use. During dialogues, community leaders and satisfied family planning users acted as role models and overtly expressed support for family planning, equitable gender norms, open communication and shared decision - making regarding family planning between spouses. Trained, community - based facilitators convened dialogues in an array of settings, such as markets, churches, women ' s groups and village meetings. Local theatre groups often performed during the dialogues. Facilitators included community health care workers, religious leaders, local government officials, and teachers. CARE provided ongoing support to facilitators in the form of training and supervision, and also provided transport reimbursement and lunch / refreshments on days when dialogues or trainings were conducted; no incentives were provided for participation in intervention activities to any other community members. All of CARE ' s activities were coordinated with the District Health Medical Team (DHMT.) During the intervention period, the DHMT coordinated several interventions designed to increase availability and access to contraceptives, including in - service training for providers, strengthening of systems for ordering contraceptives, and some community outreach activities (FP counseling and method provision ). To our knowledge, no other similar FP dialogue interventions or activities were implemented by other organizations d6rigg the intervention period. Theory of Change {# sec009} - - - - - - - - - - - - - - - - The intervention aimed to challenge and shift key community norms about gender dynamics and family planning, with the ultimate goal of creating a social environment that is more supportive of equitable gender relations and the use of family planning. Our research goals were to determine whether and how the ongoing dialogues shifted social norms, and whether and how these shifts at the community level influenced communication, decision - making, and family planning use at the cohpPe or household level. Our theorized pathway of change is shown in [Fig 1] (# pone. 0153907. g001) {ref - type = " fig " }. ! [CARE ' s Fam9<y Planning Results Initiative Theory of Change. \ This theory of change illustrates, from left to 5iFht, the activities undertaken as part of CARE ' s Family Planning Results Initiative, the expected intermediate outcomes that will lead to improvements in three key determinants of family planning which, ultimately, increase use of family planning in the intervention community.] (pone. 0153907. g001) {# pone. 0153907. g001} Evaluation Design {# sec010} - - - - - - - - - - - - - - - - - We used both qualitative and quantitative methods to evaluate the intervention. At baseline (February 2009) and again at endline (December 2012 ), we conducted household surveys to collect data about men and women ' s socio - demographic characteristics, pregnancy intentions, family planning knowledge, beliefs, attitudes, and use. At endline only, we measured key gender - related beliefs and behaviors and exposure to the intervention, which enabled us to determine whether these were important predictors of family planning use. Also at endline, we conducted in - depth, q Ta?itative interviews with purposively selected couples from the intervention area (described below) to explore changes in communication, decision - making, and gender roles over the previous four years and to identify specific enablers and barriers to family planning use. Ethics Statement {# sec011} - - - - - - - - - - - - - - - - CARE obtained approval to conduct this research from the Institutional Research and Ethics Committee at Moi University in Eldoret, Kenya. Quantitative Data Collection {# sec012} - - - - - - - - - - - - - - - - - - - - - - - - - - - - Through independent, cross - sectional household surveys, married men (20 - - 49 years) and women (18 - - 45 years) were interviewed by trained interviewers using standardized questionnaires at baseline and endline. Although the two samples were entirely independent (no attempt was made at endline to include or exclude respondents who participated at baseline ), both were drawn from | All relevant data are paper and its Supporting Information files. Introduction {#sec005} ============ Globally, an 225 million women have unmet need for family planning \[[@pone.0153907.ref001]\]. acceptance and use of family planning requires more than increasing access to health truly effective family planning programs address social and gender that present critical barriers to sexual and reproductive health. of family planning is powerfully shaped by social norms, including perceived acceptability of family planning, social pressure for large families, and perceived to family planning by religious and community leaders and spouses \[[@pone.0153907.ref002]\]. Rigid gender roles and power between men and women inhibit communication and decision-making about family planning: power dynamics in have been to influence the use of family planning and health services \[[@pone.0153907.ref003]--[@pone.0153907.ref005]\]. Fear of and experience with intimate partner violence or gender-based violence are barriers to contraceptive use \[[@pone.0153907.ref006]--[@pone.0153907.ref009]\]. Women who report intimate partner violence are at higher risk for unintended pregnancy than women who do not report violence \[[@pone.0153907.ref008]\]. Analyses of data from Demographic and Health Surveys (DHS) a number of countries suggest that key dimensions of equity women's empowerment---including equitable beliefs gender roles, women's ability to negotiate activity, and women's control over household economic decision-making---are associated with use \[[@pone.0153907.ref010]\]. studies have found a positive association between spousal communication and the use of contraception \[[@pone.0153907.ref011]--[@pone.0153907.ref014]\]. Despite evidence demonstrating important relationships between gender, women's empowerment, and family planning, there is little consensus about how to measure these complex social constructs. Researchers measure specific domains of women's empowerment, and several scales have been developed measures of mobility \[[@pone.0153907.ref015]\], power in relationships \[[@pone.0153907.ref003]\], gender norms and decision-making autonomy \[[@pone.0153907.ref016]\], control over assets \[[@pone.0153907.ref017]\], and social capital \[[@pone.0153907.ref018],[@pone.0153907.ref019]\]. The lack of a standard set of widely used measures for women's empowerment, hampers our ability to compare program effects across studies, populations, and cultural contexts. The objective of the research described in this article was to evaluate the impact of CARE intervention that catalyzed community-level dialogue about gender, sexuality, and family planning on household-level gender dynamics reported use of family planning among men and women in Kenya. this evaluation, we used scales from WE-MEASR (Women's Empowerment-Multidimensional Evaluation of Agency, Social and (see [S1 File](#pone.0153907.s001){ref-type="supplementary-material"}), a new tool that CARE developed following multi-year research into the effects and impact of our women's empowerment programming \[[@pone.0153907.ref020]\]. Methodology {#sec006} =========== Study Setting {#sec007} ------------- According to the 2008--09 DHS, in four has an unmet need for family planning \[[@pone.0153907.ref021]\]. A 2013 analysis of DHS data that Kenyan women who do not use family planning report method-related concerns, especially fear of side effects, most common reason for non-use opposition to family planning (both the perceived religious prohibition of family planning opposition by is the second commonly cited reason (16%) \[[@pone.0153907.ref022]\]. CARE conducted the research described here part of the Family Planning Results Initiative (FPRI), which we in Siaya County, province, Kenya from July 2009 through December 2012. majority of the county's population of 842,304 is under 30 years old, and 46.1% are between 0 and 14 years of age \[[@pone.0153907.ref023]\]. Over 90% County is rural and, like the of Nyanza, falls behind national average various indicators development, gender inequality, poverty, education, and health, including infant and under-five mortality, antenatal care coverage, and HIV prevalence \[[@pone.0153907.ref021]\]. The contraceptive rate among married women in Nyanza province is among the lowest in Kenya: 37.3% of women use any (compared 45.5% nationally), only a modern method (39.4% nationally) \[[@pone.0153907.ref021]\]. The Intervention {#sec008} ---------------- CARE used its *Social Analysis and Action* approach \[[@pone.0153907.ref024]\] to FPRI's intervention about 150 community-based facilitators were trained and held ongoing community dialogues gender, sexuality, and family planning over and a half years (July 2009-December 2012). At any one time, an average of 65 facilitators were dialogues. Monthly planning meetings were held to activities and ensure location coverage and topic variation over the study area. On a quarterly basis, CARE observed and feedback on dialogues and completed progress reviews with facilitators. on our monitoring data, CARE organized 759 dialogues between July 2009 and December 2012. Dialogues were held in a variety of venues and were promoted community leaders or organized around pre-scheduled meetings. Based on proximity to a respective venue, they were by participants from several and, many times, were included villages' development plans. The dialogues were designed to normalize communication about sensitive topics like gender and FP, and to participants' critical analysis of how gender norms and dynamics restrict family planning acceptability and use. During community leaders and satisfied family planning users acted as role models and overtly expressed support for family planning, equitable gender norms, open communication and shared decision-making regarding family planning between spouses. Trained, community-based facilitators convened dialogues in an of settings, such as markets, churches, groups and village meetings. Local theatre groups performed during the dialogues. included health care workers, religious leaders, local officials, and teachers. CARE provided ongoing support to facilitators in the form of training and supervision, and also transport reimbursement and lunch/refreshments on days when dialogues or trainings were conducted; no incentives were provided for participation in activities to any other community members. All of CARE's were coordinated the District Health Medical Team (DHMT.) During the intervention period, the DHMT coordinated several interventions designed to increase and access to contraceptives, including in-service training providers, strengthening of systems for ordering contraceptives, and some outreach activities (FP counseling and method provision). To our knowledge, no other similar FP interventions or activities were implemented by organizations during the intervention period. Theory of Change {#sec009} ---------------- The intervention aimed to challenge and shift key community norms about gender dynamics and family planning, the ultimate goal of creating a social environment that is more of equitable gender and use of family planning. Our research goals to determine whether and how the ongoing dialogues shifted social norms, and how these shifts at the community level influenced communication, decision-making, and family planning use at the couple or household level. Our theorized pathway of change is shown in 1](#pone.0153907.g001){ref-type="fig"}. {#pone.0153907.g001} Evaluation Design {#sec010} ----------------- We both qualitative and quantitative methods to evaluate the intervention. At baseline (February 2009) again at endline (December 2012), conducted household surveys to collect about men and women's socio-demographic pregnancy intentions, family planning knowledge, beliefs, attitudes, and use. At endline only, measured key beliefs and behaviors and exposure to the intervention, which enabled us to determine these were important predictors of family planning use. Also at endline, we conducted in-depth, qualitative interviews with purposively selected couples from the intervention area (described below) to changes in communication, decision-making, and gender roles over the previous four years and to identify specific enablers and barriers family use. Statement ---------------- CARE obtained approval to conduct this research from the Institutional Research and Ethics at Moi University in Kenya. Quantitative Data Collection {#sec012} ---------------------------- independent, cross-sectional household surveys, married men (20--49 years) and women (18--45 years) were interviewed by trained interviewers using questionnaires at and endline. Although the two samples were entirely independent attempt was made endline to include or exclude who participated at baseline), both were drawn | AlL ReLeVaNT datA Are WITHiN tHE PAPEr and ITS SuPPorTIng InforMAtioN FIlES.
IntroDUCTiON {#sEc005}
============
GloBAlly, AN EStIMaTEd 225 millIOn wOmEN HAVE uNmeT neED fOr faMiLY PLaNnING \[[@PoNE.0153907.ref001]\]. INCREasInG aCcepTAnCe And USE Of fAMILy PlANNIng ReQUIreS More THan iNCreasINg aCCESs To hEaltH sErVICes: trULY eFfecTivE fAmilY pLanNINg pROGRAMs muST aDdResS The sOciAL AnD gender nOrMS thAt prEseNT CRiTICal BArrIerS to SEXual And RepRODUcTIVE hEAltH. USE Of fAmILY plaNniNG Is poweRFULLY SHAPEd BY SoCIAl normS, IncLUdINg PeRCeIveD AccEpTaBiLITy Of FAMILy pLANNING, soCiaL PREsSUre fOr LarGE fAMilieS, ANd perCeIVEd opPosITion tO FAmILy plaNNiNg BY RelIgious and comMuNITy leaders AND SpOUses \[[@PoNE.0153907.rEF002]\].
RigId gEnDER ROles AnD uNEQUal PoWer BETwEeN MEN AnD wOMen InhibiT COUpLE commUnIcATiOn aNd joINT DeCiSIoN-MAkinG AbOUt FAmiLy plAnNinG: POWER DynaMIcS In COuPLes HavE beEn foUNd To influeNCe thE UsE of FAMilY plAnNInG aNd OtheR hEALtH sERviCeS \[[@PoNE.0153907.REf003]--[@poNe.0153907.ref005]\]. fEar Of And exPeRieNCe wITH InTImATe paRTNer vIOleNCE OR GENder-bAsEd ViOlEnCE aRe BARriErS To COnTrACEPTiVe usE \[[@PONe.0153907.reF006]--[@pone.0153907.Ref009]\]. womEN whO RepORT INtIMatE PaRTNER vIoLENcE Are At higHer rIsK fOr UninTEnded prEGnAnCY thaN wOmen whO Do nOt repOrt vIoLencE \[[@poNe.0153907.REf008]\]. analySEs Of dATa FROm DEmoGrAphIC aNd heaLTH SuRVEYs (dhS) In A nUmbEr of AFrICaN coUntriES SuGgEsT THaT KEy dIMEnSiONS OF geNdER eqUItY and WomEn's empOWErMenT---InclUdInG equItable BELiefS abOut GeNdeR ROLES, WOmEn's aBilITY To NeGOtiATE SeXuAl acTIviTY, AnD woMEn'S cONTrOl oVEr hOUSehOlD ECOnOmIC DEciSIon-mAkIng---Are ASsocIATeD wiTh hIGHeR cONtRacEpTiVE usE \[[@pONE.0153907.Ref010]\]. seVeraL sTuDIeS haVE alsO fOuND A POsiTIVe asSOCiATiOn beTWEeN spOuSaL COMmUNICAtIon and tHE use Of coNtRACEptIon \[[@PoNE.0153907.REF011]--[@pOnE.0153907.rEf014]\].
deSPiTE EviDEnCE DemoNsTRaTINg THE IMPORtaNt rELaTiONShiPS betWEEn GEnDeR, WomEN's EMPoWErMent, AND FAmily PlAnNing, there iS lITtle coNSeNsus aboUt HOw TO BEsT mEASure THeSE COMplEx SociaL ConStrUCts. rEsEarCHeRS havE ATTeMpTEd to mEAsURe SpEcIFIC domaIns of wOmEN'S empOWErmENT, anD SeVeRAL sCaleS HAvE BeEn DeVELOped iNCluDing MEASurEs of mObiLIty \[[@PONe.0153907.Ref015]\], poweR iN RelAtiONShIPS \[[@PonE.0153907.ReF003]\], GenDEr nORms aND dECisIon-MAkiNG aUToNOmy \[[@PoNE.0153907.REF016]\], CoNtrol OVER AsSets \[[@POnE.0153907.ref017]\], aNd soCiAl CaPITal \[[@POne.0153907.ReF018],[@Pone.0153907.reF019]\]. THe LACK OF A stANdarD sEt of widELy UsEd MeASUREs fOr WOMen's EmPOweRmenT, HOWeVEr, hAmPERs our ABiLiTy To COmPaRE pRogrAm EFFEcTS aCRoSS STUDiEs, PoPuLAtiOnS, and cuLtUrAl COnTEXTs.
tHe ObJeCtIve oF ThE REsEArCh dEsCRibEd in THIs ArTiclE wAs tO EVAlUatE THE ImPaCt oF A CarE interVEntIOn THaT CatAlYZEd COmmUnitY-LeVeL dIAlOgue abOUt geNdEr, sexuaLITy, anD famiLY pLAnniNg on HOuseHOlD-levEL GeNdER DYNaMIcS AND reporTEd UsE Of FaMIlY PLannINg AMONg MeN aND WOmEN In Kenya. for this EvalUatiON, WE used SeverAL ScaLEs fROm We-meAsr (WOMEN's EMpowerMENt-MuLtidimEnSIOnAL EValuATiOn OF AGeNcY, sOcIal caPITAL, ANd rELaTiOnS) (sEe [s1 FiLE](#PoNE.0153907.S001){REF-TYPe="suPplemenTaRY-MaTERIAL"}), a new TOol tHAT cArE DEveloPEd FollOwing mUlTI-yeaR ReSEArCh Into tHE eFfecTs aNd ImPact OF oUR woMEn'S EMpowErmEnT PRograMmiNG \[[@pONE.0153907.rEf020]\].
mEThoDology {#sec006}
===========
sTuDy sETTING {#sec007}
-------------
acCoRDINg TO tHe 2008--09 KeNYa DHs, apPRoxiMAtELY onE in FOuR wOMEN Has An unmEt nEEd FoR fAMilY pLaNniNG \[[@poNE.0153907.ref021]\]. a 2013 ANALysIS oF dHS datA FOund THaT kEnYan WOmeN WHo DO Not USe faMIlY planNINg REPORt mEtHOd-RelaTed Concerns, eSpEcIalLy Fear of HArMFul siDE eFfeCTS, aS THe MosT coMMoN rEason for NON-uSe (43%); OPPOSiTion TO fAMilY PlANniNg (BOth The PerCeiVed RElIgiOuS pRoHIBitioN OF famiLy PlanninG AnD oPpoSiTiON By HUsbaNDs/PArTnERs) is THE secoND mOsT cOmMoNLY Cited reasON (16%) \[[@POne.0153907.ReF022]\].
CaRe cOnDUctEd THE reseArCH dESCrIBED heRe AS paRt OF tHe faMIly plAnniNG RESULtS InItiATIvE (Fpri), WhICH WE iMpLemEnTED in sIaYA CoUnty, nYANZA ProvINCe, Kenya FRoM jULY 2009 tHrough DecEmBER 2012. thE MajOriTY oF thE CoUnTy'S POpUlaTiOn OF 842,304 Is UNder 30 yEArs oLd, ANd 46.1% Are bETWEEn 0 aNd 14 yEARs of age \[[@PoNe.0153907.rEf023]\]. over 90% OF siAyA cOuNTy Is RURAL AND, LIKe The rEST oF nYANZA, faLLs BEhiNd THE NatIonal AVerAGe ON vArioUs iNdiCAtOrs oF DEVEloPmEnt, GENDer IneQUAlItY, pOVeRtY, EDucAtiON, and HEaLth, iNCLUding iNfaNT And UndeR-FivE moRtALity, aNTEnAtAl CArE coVeRage, and Hiv PReValeNCE \[[@poNE.0153907.rEf021]\]. THe CONtrAcePTIVE pRevalencE RAtE amONG marriED WoMeN iN NYanZa prOviNCe iS amoNg the lowEst in kenYA: 37.3% oF WOMEn uSe Any metHOD (CoMPAreD To 45.5% NATIOnAlLy), aNd onlY 32.9% usE A mODeRN MeTHOd (39.4% naTIOnally) \[[@poNe.0153907.rEf021]\].
THE inTERvENtION {#SeC008}
----------------
caRe USed iTs *SoCial analysiS AnD ACtion* aPpRoACH \[[@PONe.0153907.ref024]\] tO shape FPRI'S CenTRal inTervention AbOuT 150 ComMUNITy-BaSed faCilITatorS were TrainED ANd helD OnGOing COMmUNity DIAloGuES AbouT gENder, SExUaliTy, ANd FamilY PLaNnINg oVEr ThREE ANd A haLf yEArS (JuLY 2009-December 2012). AT aNy one timE, AN aVErage OF 65 FACiLITAtorS WeRe aCTively CONVENIng diaLOGueS. MoNThlY plANninG MEetings WERe heLd TO Plan activiTIes anD eNsUre adEQUate LOcAtIoN cOVErAgE anD TOpic vARIAtiOn OVEr The EntiRe StUDy aReA. ON a QUARteRLY BASIS, cARe ObsERVEd And pROVIdEd fEeDbACK on dialoGuES AnD completeD progrEss RevIEWS With fAciLITAtORS. BaseD On OUr MOnitORinG DaTa, CArE ORGaniZED 759 diaLogUES BETweEN jULy 2009 aND DeCemBEr 2012. dialOgueS WERe held IN A vArIeTy oF vENUes anD WERe PRomoTeD BY coMMUnItY leADerS OR orGaNiZed ArOuND PrE-schEDuleD cOMMuNity MEetInGS. BaSed on proximIty to a rEspeCTIVE veNUE, theY werE ATtEnDeD by parTiCiPAnTs FrOm sEVERAL VIlLAGEs, AnD, many timEs, wErE InCLUdEd IN vilLaGES' deveLopMENT pLaNs.
THE DIaloguEs wEre DEsiGnEd tO NormalIzE COmMUnicAtion aBoUt SEnsiTIve topiCs liKE gEnder NoRMS AND fp, And To cAtAlYZE PartiCipAnts' cRItical analySIS oF hOw GenDEr Norms ANd DyNamIcs REsTrICt FAmIly pLANniNg ACcepTABiLItY anD USe. dURIng DiaLoGuEs, COMmUNity LeadeRS anD sATISFIED fAmILy pLannING uSERS aCTEd AS rOle MOdeLs and OVErtlY EXpRESSeD SuPporT fOr fAMily plAnnING, eQuitaBLe GEndER NorMs, OPEN COmMUnICatioN aND shARed deCiSioN-MaKing reGarDing fAMiLY PLanniNG bETWeen sPOUSES.
tRainED, cOMmunItY-basEd FaCIlITAtOrs CoNvEnED DiALOGUES IN AN arRaY OF sETTiNGS, SucH As MarketS, CHUrcHES, Women'S gRoUPS AND vIlLAGe mEetINGs. lOcal theatre GrOuPs ofTeN pERfoRMED duRInG THe DialogueS. FAciliTatOrs INcLUded COmmUnItY hEALTH cAre wOrkERs, RELIgioUS leaDERs, LOcal GoVErnMEnt OFfIciaLs, AnD tEacHErS. CARe pRoVided oNGoiNG sUPpoRt TO FaCILiTAtoRs IN thE form of TRAiNiNG AND SUpeRVISiON, And alSo PrOVIdEd TrANSPORT reIMBursEmenT AnD lunCH/REFrESHmeNts On dayS WHeN DIAlogUES Or TRaINiNGS WerE cONdUctEd; NO InCeNTives WEre pROvIDed FoR ParTicIpatiOn In InTeRVEntIon ActIviTieS TO aNy OTHeR CoMmuNiTy MEmBers.
ALL Of care'S actIvITies Were COOrdinateD wITH THE DiStRICT HEAlTH MEDICAl Team (dhmT.) DURing tHE InTErvention PeRiod, THe dhMt CoORdiNateD seVerAl InTeRVeNtioNS DesIgned to InCreasE aVaiLaBiLitY aNd aCcesS To coNtrAcEptives, INClUDIng iN-serVIcE traiNInG foR pRoVIdeRS, StrENgTHeNInG OF SysTems foR ORDerING CoNTRAcePtivEs, aND sOmE CommUnitY outReaCH ACTivItiEs (fp COuNselIng And MeTHOd prOviSiOn). To oUR KnoWleDGe, nO otHeR SImiLAR FP DiaLOgUE InTeRVEntIONs OR acTiVITIEs WerE iMPLeMENTeD BY OTHer orGaNiZatIONS dUriNG ThE inteRVEnTiON periOd.
tHEoRY oF CHAnge {#SeC009}
----------------
thE inTeRvENtiON AImeD to ChALLeNge aND ShifT KEy COMmunity nOrMs aBOut genDeR DynAmiCs aND fAMilY PLAnNInG, wiTH thE ultIMatE goal OF creAting a SOCiAL eNVIronMEnt that IS mORe SUpPORtIVe Of eQuitablE GEndER relAtioNs aNd THE uSe OF FaMiLY PlanniNg. ouR rEsEaRCh GOAls wErE To dEtERmine wheTHEr And HOw THe ONGOING dIALoGuEs sHiFtEd socIAL NORMs, aND WHethER ANd hOw theSe sHIfts aT The COmMuNItY LEVel iNFlUeNced comMUnICatION, DeCiSiON-MaKING, aNd fAmILY pLanNINg use AT thE cOuplE Or HOuSEhOld LEveL. OUR ThEOriZeD PatHWAY oF chanGe iS ShowN iN [FIg 1](#pONe.0153907.g001){Ref-tYPE="Fig"}.
{#pONE.0153907.g001}
evalUATiON DeSigN {#sEc010}
-----------------
we USED BotH qUalItAtive aND qUANtitatIvE mETHoDS TO eVAlUATE THe iNTeRventiOn. AT bAseLINe (fEBrUARy 2009) and AgAin aT enDlInE (dEcembEr 2012), we coNDucTED hOusEhoLd SUrveys TO COLLect DatA ABOUt Men anD WoMen'S soCIo-DeMOgrAPHic CharacTErIstICS, preGnaNCY inTentiONs, FAMiLY pLaNniNG KnOwlEdGe, BeLiefS, attITudEs, anD uSE. aT endlInE oNly, WE meAsurEd kEY geNdEr-ReLATeD beLIeFs ANd BEhavIOrs aNd exposurE tO THe iNTERVEntIOn, whIcH EnABlEd US TO detErMinE WhetHEr tHESe wEre ImpOrTAnt prEDIctors OF FaMily pLaNniNG usE. aLsO AT ENdliNE, we cONduCTED In-DEpth, QUaLiTAtiVE iNterVIews WiTH PuRposIVELY SeleCTed COuPLes frOm thE INtErveNTIoN area (dEScriBED bElow) TO EXpLorE ChangES iN cOmmUNIcAtION, deCisiOn-MAKING, ANd geNDEr ROlEs oVeR THe PReVIOus FOuR yEaRS anD to ideNtIFY SPEcIFIC eNAblERs AND BarRIeRs TO FaMIlY pLanniNg uSE.
EThICS STatEMenT {#Sec011}
----------------
cARe ObtAInEd APprOVaL to COndUct THIs REseaRcH froM THe iNSTitutionAl ResEARCh anD eTHics coMMItTEE AT MOi UniVERsiTY in ElDoret, KENYA.
QUANTiTATIve daTA CoLLeCtION {#sec012}
----------------------------
THrOuGh iNDEPenDent, cROss-SeCtIOnaL houSeHOlD SuRveYs, maRRiED men (20--49 yEaRs) aND WomEN (18--45 yEARS) were iNTerviEwed bY trAinEd INteRviEweRS UsiNg stAnDARDizeD QuEStiOnnaireS at BAselINe aNd ENdLiNe. AltHOugH THe Two sAmpleS WEre EnTiReLy inDepENdeNT (No AttEMpT Was maDE at EndliNE TO include Or eXCLuDE RESPONDEnTS wHo partICiPATeD At bASelIne), bOTh WeRE DraWn fROM | All relevant data are within thepaper and itsSupporting Information files. Introduction {#sec005} ============ Globally, an estimated 225 millionwomen have unmet need for family planning \[[@pone.0153907.ref001]\]. Increasing acceptance and use of family planning requires more than increasing accessto health services: truly effective family planning programs must address the social and gender norms that present critical barriers to sexual and reproductive health. Use offamily planning is powerfully shaped by social norms, including perceived acceptability offamily planning, social pressure for large families, andperceived opposition to familyplanning by religiousand community leaders and spouses\[[@pone.0153907.ref002]\]. Rigid genderroles and unequal power between men and women inhibit couple communication and joint decision-making about family planning: power dynamics in couples have been found to influence the use offamily planning and other health services \[[@pone.0153907.ref003]--[@pone.0153907.ref005]\]. Fearof and experience with intimate partner violence orgender-based violence arebarriers tocontraceptiveuse \[[@pone.0153907.ref006]--[@pone.0153907.ref009]\]. Women who report intimate partner violence are athigherrisk for unintended pregnancy than women who do not report violence\[[@pone.0153907.ref008]\]. Analyses of data from Demographic and Health Surveys (DHS) in a number of African countries suggest that key dimensions of gender equity and women's empowerment---including equitable beliefs about gender roles, women's ability to negotiate sexual activity, and women's control overhousehold economic decision-making---are associatedwith higher contraceptive use \[[@pone.0153907.ref010]\]. Several studies have also found a positive association between spousalcommunication and the use ofcontraception \[[@pone.0153907.ref011]--[@pone.0153907.ref014]\]. Despite evidence demonstrating the important relationshipsbetween gender, women's empowerment, and family planning, there is little consensus about how to best measure these complex social constructs. Researchers have attempted to measure specific domains of women'sempowerment, and several scales have been developed including measures of mobility \[[@pone.0153907.ref015]\], power in relationships \[[@pone.0153907.ref003]\],gender norms and decision-making autonomy \[[@pone.0153907.ref016]\], control over assets \[[@pone.0153907.ref017]\], and social capital \[[@pone.0153907.ref018],[@pone.0153907.ref019]\]. The lackof a standard set ofwidely used measures for women's empowerment, however, hampers our ability to compareprogram effects across studies, populations,and cultural contexts. The objective of the research described in this article wasto evaluate theimpact of a CARE intervention thatcatalyzed community-leveldialogue about gender, sexuality, and family planning on household-level gender dynamics and reported use offamily planningamong men and women in Kenya. For thisevaluation, we used several scales fromWE-MEASR (Women's Empowerment-Multidimensional Evaluation of Agency, Social Capital,and Relations) (see [S1 File](#pone.0153907.s001){ref-type="supplementary-material"}), a new tool that CARE developed following multi-year research into the effects andimpactofour women's empowerment programming\[[@pone.0153907.ref020]\]. Methodology {#sec006} =========== Study Setting{#sec007} ------------- According to the 2008--09 Kenya DHS, approximatelyone in fourwomen has an unmet needforfamily planning \[[@pone.0153907.ref021]\]. A2013 analysisof DHSdata found that Kenyan women whodo not use family planning report method-relatedconcerns, especially fear of harmfulside effects, as the mostcommonreasonfor non-use (43%); oppositionto family planning (both the perceived religious prohibition of family planning and opposition by husbands/partners) is the second most commonly cited reason (16%) \[[@pone.0153907.ref022]\].CARE conducted the research described here as part of the Family Planning Results Initiative (FPRI), which we implemented in Siaya County, Nyanza province, Kenya from July2009 through December 2012. The majority of the county's populationof842,304 is under 30 years old, and 46.1%are between 0 and 14 years of age \[[@pone.0153907.ref023]\]. Over 90%of Siaya County is rural and, like the rest ofNyanza, falls behind thenationalaverage on various indicators of development, gender inequality, poverty, education, and health, including infant and under-five mortality, antenatal care coverage, and HIV prevalence \[[@pone.0153907.ref021]\]. The contraceptive prevalence rate amongmarried women inNyanza province is among thelowest in Kenya: 37.3% of women use anymethod (compared to 45.5% nationally), and only 32.9% use a modern method (39.4% nationally) \[[@pone.0153907.ref021]\]. The Intervention {#sec008} ---------------- CAREused its *Social Analysis and Action* approach\[[@pone.0153907.ref024]\] to shape FPRI's central intervention about 150 community-based facilitators were trained and held ongoing community dialogues about gender, sexuality,and family planning over three and a half years (July 2009-December 2012). At any one time, an average of 65 facilitators were actively convening dialogues.Monthly planning meetings were held to planactivitiesand ensure adequate location coverage and topic variation over the entire study area. On a quarterly basis, CARE observed and provided feedback on dialogues and completed progress reviews withfacilitators. Based on our monitoring data, CARE organized 759 dialogues between July 2009 and December 2012. Dialogues were held in a variety of venues and were promoted by community leaders or organizedaroundpre-scheduledcommunity meetings.Based on proximity to a respective venue,they were attended by participants from several villages, and, many times, were included in villages'development plans. The dialogueswere designed to normalize communication about sensitive topics likegender normsand FP,and tocatalyze participants' critical analysis of how gendernorms and dynamics restrict family planning acceptability and use. During dialogues, community leaders and satisfied family planning users acted as role models and overtly expressed support for family planning, equitable gender norms, open communicationand shared decision-making regarding family planning between spouses. Trained, community-based facilitators convened dialogues in an array of settings,such as markets, churches, women's groups and village meetings. Localtheatre groups often performed during the dialogues. Facilitators included community health careworkers, religious leaders, local government officials, and teachers. CARE provided ongoing support to facilitatorsin the formof training and supervision, and also provided transport reimbursement and lunch/refreshments on dayswhen dialogues or trainings wereconducted; no incentives were providedfor participation in intervention activities to any other community members. All of CARE'sactivities were coordinatedwith the District HealthMedicalTeam (DHMT.)During the intervention period,the DHMTcoordinatedseveral interventions designed to increase availability and access to contraceptives, including in-service training forproviders, strengthening of systems for ordering contraceptives, and some communityoutreach activities (FP counseling and method provision). To our knowledge, no other similar FP dialogue interventionsor activities were implemented by other organizations during the intervention period. Theory of Change {#sec009} ---------------- Theintervention aimed to challenge and shift keycommunity normsabout gender dynamics and family planning, with the ultimate goal of creating a social environment that is more supportive of equitable gender relations and the use of familyplanning. Our research goals were to determinewhether and how the ongoing dialogues shifted social norms, and whether and how these shifts at the communitylevel influenced communication, decision-making, and familyplanning use at the couple or household level. Our theorized pathway of change is shown in [Fig 1](#pone.0153907.g001){ref-type="fig"}. {#pone.0153907.g001} EvaluationDesign {#sec010} ----------------- We usedboth qualitative and quantitative methods to evaluatetheintervention. At baseline (February 2009) and again at endline (December 2012), we conducted household surveysto collect data about men and women's socio-demographiccharacteristics, pregnancy intentions,family planning knowledge, beliefs, attitudes, and use. At endline only, wemeasured key gender-related beliefs andbehaviors and exposure to the intervention, which enabled us to determine whether these were important predictors of family planning use. Also at endline, weconducted in-depth, qualitative interviews withpurposively selected couples from the intervention area (described below) to explore changes in communication, decision-making, and genderroles over the previous four years and to identify specific enablers and barriers to familyplanning use. Ethics Statement{#sec011} ----------------CARE obtained approval to conduct thisresearch from the Institutional Research and Ethics Committee at Moi Universityin Eldoret,Kenya. Quantitative Data Collection {#sec012} ----------------------------Through independent, cross-sectional householdsurveys, married men (20--49years) and women (18--45 years) were interviewed by trained interviewers using standardizedquestionnaires at baseline and endline. Although the two samples were entirely independent (no attempt was madeat endline toinclude or excluderespondentswho participated at baseline), both were drawn from | _All_ relevant data are _within_ the paper and its _Supporting_ _Information_ files. Introduction {#sec005} ============ Globally, an estimated 225 million women _have_ unmet need for _family_ planning \[[@pone.0153907.ref001]\]. _Increasing_ acceptance _and_ use of family planning _requires_ more than increasing access to health services: truly _effective_ family planning programs must address the social and _gender_ norms that present critical _barriers_ to sexual and reproductive health. Use of _family_ planning _is_ powerfully shaped _by_ social _norms,_ including _perceived_ acceptability of family planning, social pressure _for_ large _families,_ and perceived opposition to family planning by religious and community _leaders_ and _spouses_ \[[@pone.0153907.ref002]\]. Rigid gender roles and unequal power between _men_ and _women_ inhibit couple _communication_ and joint decision-making about family planning: _power_ dynamics _in_ couples have _been_ found to influence the use of family planning and _other_ health _services_ \[[@pone.0153907.ref003]--[@pone.0153907.ref005]\]. _Fear_ _of_ and experience with intimate partner violence or gender-based _violence_ are barriers to contraceptive use \[[@pone.0153907.ref006]--[@pone.0153907.ref009]\]. Women who report intimate partner violence _are_ at higher risk for unintended pregnancy than women who do not report violence \[[@pone.0153907.ref008]\]. Analyses of _data_ from Demographic and Health Surveys (DHS) in _a_ _number_ _of_ African _countries_ suggest that key dimensions of _gender_ equity and women's empowerment---including equitable beliefs about gender _roles,_ women's ability to negotiate sexual activity, and women's _control_ _over_ _household_ economic decision-making---are associated _with_ higher contraceptive use _\[[@pone.0153907.ref010]\]._ Several _studies_ have also found a positive association _between_ spousal _communication_ _and_ the use of contraception \[[@pone.0153907.ref011]--[@pone.0153907.ref014]\]. Despite evidence demonstrating the important relationships between gender, women's empowerment, and family planning, there is little consensus about _how_ to best measure _these_ complex _social_ _constructs._ Researchers have attempted to measure specific domains of _women's_ empowerment, and several scales have been developed including measures of mobility \[[@pone.0153907.ref015]\], power in relationships \[[@pone.0153907.ref003]\], gender _norms_ and decision-making autonomy \[[@pone.0153907.ref016]\], control over assets \[[@pone.0153907.ref017]\], and social capital \[[@pone.0153907.ref018],[@pone.0153907.ref019]\]. The lack of a standard set _of_ widely used measures for _women's_ empowerment, however, hampers our ability to compare program effects _across_ studies, populations, and cultural contexts. _The_ objective of _the_ research _described_ in this article was _to_ evaluate the impact of _a_ CARE intervention that catalyzed community-level dialogue _about_ _gender,_ sexuality, and family _planning_ on household-level gender dynamics and reported use of family planning _among_ men and _women_ in Kenya. For this evaluation, we used several _scales_ _from_ WE-MEASR (Women's Empowerment-Multidimensional Evaluation of Agency, Social Capital, and Relations) (see [S1 File](#pone.0153907.s001){ref-type="supplementary-material"}), a new tool that CARE developed following multi-year _research_ into the effects and impact of our women's empowerment programming \[[@pone.0153907.ref020]\]. Methodology {#sec006} =========== _Study_ Setting {#sec007} _-------------_ According to the _2008--09_ Kenya DHS, _approximately_ _one_ in four women _has_ an unmet need for family planning \[[@pone.0153907.ref021]\]. A 2013 analysis of DHS _data_ found that Kenyan women who do not use family planning report method-related concerns, especially _fear_ of harmful side effects, as _the_ most common reason for non-use (43%); _opposition_ to _family_ planning (both the _perceived_ religious _prohibition_ _of_ _family_ planning and _opposition_ by husbands/partners) is the second most commonly cited _reason_ _(16%)_ _\[[@pone.0153907.ref022]\]._ _CARE_ conducted the _research_ _described_ here as _part_ of the Family Planning _Results_ Initiative _(FPRI),_ which we implemented in _Siaya_ _County,_ Nyanza province, _Kenya_ from July 2009 _through_ December 2012. The majority of _the_ county's population of 842,304 is under 30 years old, and 46.1% are between 0 and 14 years of _age_ \[[@pone.0153907.ref023]\]. Over 90% of Siaya County is rural and, like _the_ rest of Nyanza, falls _behind_ the national average _on_ various _indicators_ of development, gender inequality, _poverty,_ education, and health, including _infant_ _and_ under-five mortality, _antenatal_ care _coverage,_ _and_ _HIV_ prevalence \[[@pone.0153907.ref021]\]. _The_ contraceptive prevalence _rate_ among married women in Nyanza province is among _the_ lowest in Kenya: 37.3% of women use any _method_ (compared to _45.5%_ nationally), and only 32.9% use a modern method (39.4% nationally) \[[@pone.0153907.ref021]\]. The Intervention _{#sec008}_ ---------------- CARE used its *Social _Analysis_ and Action* approach \[[@pone.0153907.ref024]\] _to_ shape FPRI's central intervention about 150 community-based facilitators _were_ _trained_ and held ongoing community dialogues _about_ gender, _sexuality,_ and family planning _over_ three and a half years (July 2009-December 2012). At _any_ one time, an average of 65 facilitators _were_ actively convening _dialogues._ Monthly planning meetings _were_ held _to_ plan activities and ensure adequate location coverage _and_ topic variation over the entire study area. _On_ a quarterly basis, CARE observed _and_ _provided_ feedback on dialogues and completed progress _reviews_ with facilitators. _Based_ on our monitoring _data,_ CARE organized 759 dialogues between July 2009 _and_ December 2012. Dialogues were held in a variety of venues and were promoted by community leaders or _organized_ around pre-scheduled community _meetings._ Based on proximity to a respective venue, they _were_ attended _by_ participants from several villages, and, _many_ times, _were_ included in villages' development plans. The dialogues _were_ _designed_ to normalize communication about sensitive topics like gender norms and _FP,_ _and_ to catalyze participants' critical analysis of how _gender_ norms and dynamics _restrict_ family planning acceptability and use. During dialogues, community leaders _and_ _satisfied_ family planning users acted as role models and overtly expressed support for family planning, equitable _gender_ norms, _open_ _communication_ and _shared_ decision-making regarding family planning _between_ _spouses._ Trained, community-based facilitators convened dialogues in an array _of_ settings, such _as_ markets, churches, women's groups and village meetings. _Local_ theatre groups _often_ performed during the dialogues. _Facilitators_ included _community_ health _care_ workers, religious _leaders,_ local government _officials,_ and teachers. CARE provided ongoing _support_ to _facilitators_ in the form of training _and_ supervision, and also provided transport reimbursement and _lunch/refreshments_ on days _when_ dialogues or trainings _were_ conducted; no incentives _were_ _provided_ for participation in intervention activities to any other community members. All of CARE's activities were coordinated with the District Health Medical Team (DHMT.) During the intervention period, the _DHMT_ coordinated several interventions designed _to_ _increase_ availability _and_ _access_ to _contraceptives,_ including in-service _training_ for providers, strengthening of systems for ordering contraceptives, and some community outreach activities (FP counseling and method provision). To our knowledge, no other similar FP dialogue interventions or activities were implemented by other organizations _during_ the intervention _period._ Theory of Change {#sec009} ---------------- The intervention aimed to challenge _and_ shift _key_ _community_ norms about gender dynamics and family _planning,_ with _the_ ultimate _goal_ of _creating_ a social _environment_ that is _more_ _supportive_ _of_ equitable gender relations and _the_ _use_ of _family_ planning. Our research goals _were_ _to_ determine _whether_ and how the ongoing dialogues shifted _social_ norms, and whether and how these shifts at _the_ community level _influenced_ _communication,_ decision-making, and family _planning_ use at the couple or household level. _Our_ theorized _pathway_ of change is shown in _[Fig_ 1](#pone.0153907.g001){ref-type="fig"}. _{#pone.0153907.g001} _Evaluation_ _Design_ {#sec010} _-----------------_ We used both qualitative and quantitative methods to _evaluate_ the intervention. _At_ _baseline_ _(February_ _2009)_ and again at endline (December 2012), we _conducted_ household surveys to collect data about men and women's _socio-demographic_ characteristics, pregnancy intentions, _family_ planning knowledge, beliefs, _attitudes,_ and use. At _endline_ only, we measured key gender-related beliefs _and_ behaviors and exposure to the intervention, which enabled us _to_ determine whether these were important predictors _of_ _family_ planning use. Also at endline, we conducted in-depth, qualitative _interviews_ with purposively selected couples _from_ the intervention area _(described_ below) to explore changes in _communication,_ decision-making, and gender roles over the previous four years and to identify specific enablers and _barriers_ _to_ family planning use. Ethics _Statement_ _{#sec011}_ ---------------- CARE obtained approval to _conduct_ this research from the Institutional Research _and_ Ethics Committee _at_ _Moi_ University in Eldoret, Kenya. _Quantitative_ _Data_ Collection _{#sec012}_ ---------------------------- Through independent, cross-sectional household _surveys,_ _married_ men (20--49 years) and women (18--45 _years)_ _were_ _interviewed_ by _trained_ interviewers using standardized questionnaires at _baseline_ and endline. Although the two samples _were_ entirely _independent_ _(no_ attempt was _made_ at _endline_ to include or exclude respondents who participated at baseline), _both_ _were_ drawn from |
Background
==========
The prevalence of type 2 diabetes rapidly rose over the past three decades. However its burden is not homogeneously distributed: racial and ethnic minorities usually experience higher prevalence than their non-minority counterparts \[[@B1]\] and are at higher risk of developing diabetes-related complications such as blindness, kidney damage, or depression, impacting both quality of life and mortality rates \[[@B2],[@B3]\].
Self-management, defined as the patient's ability to manage not only the symptoms inherent to a chronic condition but also its treatment and associated lifestyle changes \[[@B4]\], has become increasingly important in the treatment of type 2 diabetes \[[@B5],[@B6]\]. However, because adequate diabetes self-management (DSM) may require considerable lifestyle changes to several domains, namely having a healthy diet, exercising, or glucose monitoring, not all the patients are able to properly follow the self-management plans agreed with their healthcare professionals or advised by clinical guidelines. Racial and ethnic minorities are less likely to engage in DSM behaviors than other population groups, which partially explains observed disparities in health outcomes \[[@B7]\]. Some of the barriers faced by these groups for achieving an adequate DSM are related to characteristics of the groups (such as health literacy or health beliefs) and also of the healthcare system (namely accessibility of culturally sensitive information) \[[@B8],[@B9]\].
Several review studies have assessed the effect of DSM educational programs on the general population \[[@B6],[@B10]-[@B17]\]. Those studies have established that DSM educational programs can improve glycemic control \[[@B11]-[@B16]\], and identified the key characteristics for improving glycemic control, including face-to-face delivery, teaching methods based on cognitive reframing \[[@B11]\], and higher contact time between participant and educator \[[@B16]\]. A smaller number of studies have reviewed the evidence of the effect of these programs on racial/ethnic minorities \[[@B18]-[@B20]\]. Only one study \[[@B18]\] examined their impact on glycemic control, observing a reduction on glycated hemoglobin of 0.32%. There were however some limitations underlying that study, as some of the included interventions combined educational programs with other types of quality improvement strategies, making it difficult to disentangle the individual effect of the educational components. More importantly, no previous meta-regression study has identified the key common characteristics of successful educational programs targeted to racial/ethnic minority groups. This represents a considerable gap in the literature, as programs for racial/ethnic minority groups should have different components from those targeting other population groups, namely addressing the cultural idiosyncrasies associated with each group. Therefore it is not reasonable to assume that the same type of successful program will be equally successful when applied to racial/ethnic minority groups. Additionally, in the past years several clinical trials examining the impact of educational programs to improve diabetes self-management on racial/ethnic minorities have been published, making now possible a more detailed review and analysis of the available evidence regarding the effectiveness of these interventions.
In this work we systematically reviewed DSM educational interventions specifically targeted to racial/ethnic minority groups. We studied the characteristics and costs of the interventions, and analyzed their impact on diabetes knowledge, self-management behaviors, and clinical outcomes. Whenever data were available, we performed meta-analyses to examine the short and long-term effects of the interventions, and meta-regressions to identify common characteristics of the interventions associated with better results.
Methods
=======
The review and its procedures were planned, conducted, and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines \[[@B21]\].
Data sources and searches
-------------------------
A comprehensive core search strategy was developed for Medline through Ovid (combining MeSH terms and keywords) and then adapted and implemented in EMBASE and CINAHL (search strategy available in Additional file [1](#S1){ref-type="supplementary-material"}: Table S1). Gray literature and additional articles were searched in twelve more bibliographic sources (Additional file [2](#S2){ref-type="supplementary-material"}: Table S2). The search was not restricted by language or publication date. For all the references selected to be included in the review, backward and forward citation searches were performed in ISI Web of Knowledge. All searches were conducted in October 2012. A bibliographical database was created using EndNote X6, and used to store and manage the retrieved references.
Study selection
---------------
We included studies analyzing the effectiveness of DSM educational programs targeted to racial/ethnic minority groups with type 2 diabetes. We only included those studies in which at least 90% of the participants pertained to a racial/ethnic minority group considered to be at a higher risk for diabetes complications than the majority population group. Racial/ethnic minority group was defined as a population group with a race or ethnicity different from that of the majority population group of the host country. Groups at higher risk of diabetes complications were identified based on available literature. Interventions had to be exclusively educational, without including any other component such us financial incentives, clinician education or case management. In order to avoid possible comparisons between programs carried out in very heterogeneous settings, with very different health systems and population needs, we restricted this review to those interventions conducted in countries that were members of the OECD \[[@B22]\], when study selection was conducted (November 2012).
Eligible designs were randomized controlled trials (RCTs), including cluster randomized controlled trials; controlled trials, including quasi-randomized trials; controlled before-after studies; and non-controlled before-after studies. Studies including a control group were only eligible in case the intervention was compared with care as usual.
Titles and abstracts were screened for eligibility, and those fulfilling the inclusion criteria were included in the next stage, where the full texts of the selected articles were retrieved and assessed. Those that met the inclusion criteria were included for data extraction. Two reviewers independently screened citations, and any disagreements were solved by consensus with a third reviewer.
Data extraction and quality assessment
--------------------------------------
We designed and used structured forms to extract information of interventions' characteristics and their effectiveness. We used a previously developed taxonomy of DSM educational programs to characterize the interventions \[[@B23]\]. The following information was extracted: setting, ethnic group, administration formats, teaching methods, educational contents, educators' background, use of peer educators, and duration. Information of interventions' cost and cost-effectiveness was also extracted.
We critically appraised the studies using the Quality Assessment Tool for Quantitative Studies \[[@B24]\], which enables the assessment of both internal and external validity, classifying them into three categories (good, fair or poor) depending on six aspects: selection bias, study design, confounders, blinding, data collection and withdrawals/ dropouts. Two reviewers independently extracted all the information and critically appraised the studies. Disagreements were solved through discussion with a third reviewer. When necessary we contacted the authors of the studies to request additional information.
Data synthesis and analysis
---------------------------
The effectiveness of the interventions was assessed in terms of their impact on 1) diabetes knowledge, 2) diabetes self-management behavior, and 3) clinical outcomes. Diabetes knowledge was ascertained by measures reflecting the theoretical knowledge the patients had about their condition. Diabetes self-management behavior measured the performance of specific activities related to adequate DSM (diet, exercise, glucose control, foot self-examination, etc.). Clinical outcomes included hemoglobin A1c (HbA1c), body mass index (BMI), or blood pressure, amongst others. All outcomes in all the studies were examined and classified as measuring one of these three domains. Variables that measured other domains were not included in the analysis.
Additionally, we conducted independent meta-analyses to analyze short and long-term (six months post-intervention) effect of the interventions on glycemic control. Eligibility criteria for the meta-analyses included randomized controlled trials comparing the interventions with usual care. The mean (standard deviation) of HbA1c levels in each study were extracted. This information was transformed into weighted mean difference and 95% confidence intervals (CI) were calculated and combined using random-effects models. We imputed unreported standard deviations by use of established methods \[[@B25]\]. Heterogeneity was quantified by the I^2^ statistic, where I^2^ ≥ 50% was considered evidence of substantial heterogeneity \[[@B26]\]. Sources of heterogeneity were investigated by a Galbraith plot. Publication bias was quantitatively assessed with Egger test.
We used bivariate meta-regression to explore relationships between effect size (ES) and interventions characteristics. The number of included studies was insufficient to perform a multivariate regression analysis. We conducted a sensitivity analysis, excluding the studies with higher risk of bias. All analyses were conducted with Stata, version 12.0 (StataCorp, College Station, Texas). For all the analyses, statistical significance was accepted at *p* \< 0.05.
Results
=======
Article identification
----------------------
Figure [1](#F1){ref-type="fig"} reports the screening process. A total of 1,988 unique references were retrieved. 1,386 references were excluded based on title and abstract, resulting in 602 references being included in the next stage. Full text articles were retrieved and assessed, with 24 studies meeting the eligibility criteria. Backward and forward search of these 24 articles identified thirteen additional studies, resulting in thirty seven articles being included in the review \[[@B27]-[@B63]\]. Thirty five of them reported one single intervention, whereas two articles reported two distinct interventions per article. Overall, thirty seven articles were identified, which analyzed the effectiveness of thirty nine different interventions.
{#F1}
Characteristics of the studies and interventions
------------------------------------------------
Table [1](#T1){ref- | background = = = = = = = = = = the prevalence of type 2 diabetes rapidly rose over the past three decades. however its burden is not homogeneously distributed : racial and racial minorities usually experience higher prevalence than their non - minority counterparts \ [ [ @ b1 ] \ ] and are at higher risk of developing diabetes - related complications such as blindness, kidney damage, or depression, impacting both quality of life and mortality rates \ [ [ @ b2 ], [ @ b3 ] \ ]. self - management, defined as the patient ' s ability to manage not only the symptoms inherent to a chronic condition but also its treatment and associated lifestyle changes \ [ [ @ b4 ] \ ], has become increasingly important in the treatment of type 2 diabetes \ [ [ @ b5 ], [ @ b6 ] \ ]. however, because adequate diabetes fitness - management ( dsm ) may provide considerable lifestyle changes to several domains, whilst having insufficient healthy diet, exercising, or glucose monitoring, not all the patients are able to properly follow the self - management plans agreed with their healthcare professionals or advised by clinical guidelines. racial and ethnic minorities are less likely to engage in dsm behaviors than other population groups, which partially explains observed disparities in health outcomes \ [ [ @ b7 ] \ ]. some of the barriers faced by these groups for achieving an adequate dsm are related to characteristics of the groups ( such as health literacy or health beliefs ) and also of the healthcare system ( namely accessibility of culturally sensitive information ) \ [ [ @ b8 ], [ @ b9 ] \ ]. several review studies have assessed the effect of dsm instruction programs towards the general population \ [ [ @ b6 ], [ @ b10 ] - [ @ b17 ] \ ]. those studies have established that dsm instructional programs can improve glycemic control \ [ [ @ b11 ] - [ @ b16 ] \ ], and identified the key characteristics for improving glycemic control, including face - to - face delivery, teaching methods based on cognitive reframing \ [ [ @ b11 ] \ ], and higher contact time between teacher and educator \ [ [ @ b16 ] \ ]. a smaller number of studies have reviewed the evidence of the effect of these methods on racial / ethnic minorities \ [ [ @ b18 ] - [ @ b20 ] \ ]. only one study \ [ [ @ b18 ] \ ] examined their impact on glycemic control, observing a reduction on glycated hemoglobin of 0. 32 %. there were however some limitations underlying that study, as some of the included interventions combined educational programs with other types of quality improvement strategies, making it difficult to disentangle the individual effect of the educational components. more importantly, no previous meta - regression study has identified the key common characteristics of successful educational programs targeted to racial / ethnic minority groups. this represents a considerable gap in the literature, as programs for racial / ethnic minority groups should have different components from those targeting other population groups, namely addressing the cultural idiosyncrasies associated with each group. therefore it is not reasonable to assume that the same type of successful program will be equally successful when applied to racial / ethnic minority groups. additionally, in the past years several clinical trials examining the impact of educational programs to improve diabetes self - management on racial / ethnic minorities have been published, making now possible a more detailed review and analysis of the available evidence regarding the effectiveness of these interventions. in this work we systematically reviewed dsm educational interventions specifically targeted to racial / ethnic minority groups. we studied the characteristics and costs of the interventions, and analyzed their impact on diabetes knowledge, self - management behaviors, and clinical outcomes. whenever data were available, we performed meta - analyses to examine the short and long - term effects of the interventions, and meta - regressions to identify common characteristics of the interventions associated with better results. methods = = = = = = = the review and its procedures were planned, conducted, and reported according to the preferred reporting items for systematic reviews and meta - analyses ( prisma ) guidelines \ [ [ @ b21 ] \ ]. data sources and searches - - - - - - - - - - - - - - - - - - - - - - - - - a comprehensive core search strategy was developed for medline through ovid ( combining mesh terms and keywords ) and then adapted and implemented in embase and cinahl ( search strategy available in additional file [ 1 ] ( # s1 ) { ref - type = " supplementary - material " } : table s1 ). gray literature and additional articles were searched in twelve more bibliographic sources ( additional file [ 2 ] ( # s2 ) { ref - type = " supplementary - material " } : table s2 ). the search was not restricted by language or publication date. for all the references selected to be included in the review, backward and forward citation searches were performed in isi web of knowledge. all searches were conducted in october 2012. a bibliographical database was created using endnote x6, and used to store and manage the retrieved references. study selection - - - - - - - - - - - - - - - we included studies analyzing the effectiveness of dsm educational programs targeted to racial / ethnic minority groups with type 2 diabetes. we only included those studies in which at least 90 % of the participants pertained to a racial / ethnic minority group considered to be at a higher risk for diabetes complications than the majority population group. racial / ethnic minority group was defined as a population group with a race or ethnicity different from that of the majority population group of the host country. groups at higher risk of diabetes complications were identified based on available literature. interventions had to be exclusively educational, without including any other component such us financial incentives, clinician education or case management. in order to avoid possible comparisons between programs carried out in very heterogeneous settings, with very different health systems and population needs, we restricted this review to those interventions conducted in countries that were members of the oecd \ [ [ @ b22 ] \ ], when study selection was conducted ( november 2012 ). eligible designs were randomized controlled trials ( rcts ), including cluster randomized controlled trials ; controlled trials, including quasi - randomized trials ; controlled before - after studies ; and non - controlled before - after studies. studies including a control group were only eligible in case the intervention was compared with care as usual. titles and abstracts were screened for eligibility, and those fulfilling the inclusion criteria were included in the next stage, where the full texts of the selected articles were retrieved and assessed. those that met the inclusion criteria were included for data extraction. two reviewers independently screened citations, and any disagreements were solved by consensus with a third reviewer. data extraction and quality assessment - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - we designed and used structured forms to extract information of interventions ' characteristics and their effectiveness. we used a previously developed taxonomy of dsm educational programs to characterize the interventions \ [ [ @ b23 ] \ ]. the following information was extracted : setting, ethnic group, administration formats, teaching methods, educational contents, educators ' background, use of peer educators, and duration. information of interventions ' cost and cost - effectiveness was also extracted. we critically appraised the studies using the quality assessment tool for quantitative studies \ [ [ @ b24 ] \ ], which enables the assessment of both internal and external validity, classifying them into three categories ( good, fair or poor ) depending on six aspects : selection bias, study design, confounders, blinding, data collection and withdrawals / dropouts. two reviewers independently extracted all the information and critically appraised the studies. disagreements were solved through discussion with a third reviewer. when necessary we contacted the authors of the studies to request additional information. data synthesis and analysis - - - - - - - - - - - - - - - - - - - - - - - - - - - the effectiveness of the interventions was assessed in terms of their impact on 1 ) diabetes knowledge, 2 ) diabetes self - management behavior, and 3 ) clinical outcomes. diabetes knowledge was ascertained by measures reflecting the theoretical knowledge the patients had about their condition. diabetes self - management behavior measured the performance of specific activities related to adequate dsm ( diet, exercise, glucose control, foot self - examination, etc. ). clinical outcomes included hemoglobin a1c ( hba1c ), body mass index ( bmi ), or blood pressure, amongst others. all outcomes in all the studies were examined and classified as measuring one of these three domains. variables that measured other domains were not included in the analysis. additionally, we conducted independent meta - analyses to analyze short and long - term ( six months post - intervention ) effect of the interventions on glycemic control. eligibility criteria for the meta - analyses included randomized controlled trials comparing the interventions with usual care. the mean ( standard deviation ) of hba1c levels in each study were extracted. this information was transformed into weighted mean difference and 95 % confidence intervals ( ci ) were calculated and combined using random - effects models. we imputed unreported standard deviations by use of established methods \ [ [ @ b25 ] \ ]. heterogeneity was quantified by the i ^ 2 ^ statistic, where i ^ 2 ^ ≥ 50 % was considered evidence of substantial heterogeneity \ [ [ @ b26 ] \ ]. sources of heterogeneity were investigated by a galbraith plot. publication bias was quantitatively assessed with egger test. we used bivariate meta - regression to explore relationships between effect size ( es ) and interventions characteristics. the number of included studies was insufficient to perform a multivariate regression analysis. we conducted a sensitivity analysis, excluding the studies with higher risk of bias. all analyses were conducted with stata, version 12. 0 ( statacorp, college station, texas ). for all the analyses, statistical significance was accepted at * p * \ < 0. 05. results = = = = = = = article identification - - - - - - - - - - - - - - - - - - - - - - figure [ 1 ] ( # f1 ) { ref - type = " fig " } reports the screening process. a total of 1, 988 unique references were retrieved. 1, 386 references were excluded based on title and abstract, resulting in 602 references being included in the next stage. full text articles were retrieved and assessed, with 24 studies meeting the eligibility criteria. backward and forward search of these 24 articles identified thirteen additional studies, resulting in thirty seven articles being included in the review \ [ [ @ b27 ] - [ @ b63 ] \ ]. thirty five of them reported one single intervention, whereas two articles reported two distinct interventions per article. overall, thirty seven articles were identified, which analyzed the effectiveness of thirty nine different interventions.! [ summary of evidence search and selection. ] ( 1472 - 6823 - 14 - 60 - 1 ) { # f1 } characteristics of the studies and interventions - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - table [ 1 ] ( # t1 ) { ref - | Background = = = = = = = = = = The prevalence of type 2 diabetes rapidly rose over the past three decades. However its burden is not homogeneously diWtEibuted: racial and ethnic minorities usually experience higher prevalence than their non - minority counterparts \ [[ @ B1] \] and are at higher risk of developing diabetes - related complications such as blindness, kidney damage, or depression, impacting both quality of life and mortality rates \ [[ @ B2 ], [@ B3] \ ]. Self - management, defined as the patient ' s ability to manage not only the symptoms inherent to a chronic condition but also its treatment and associated lifestyle changes \ [[ @ B4] \ ], has become increasingly important in the treatment of type 2 diabetes \ [[ @ B5 ], [@ B6] \ ]. However, because adequate diabetes self - majzgement (DSM) may require considerable lifestyle changes to several domains, namely having a healthy diet, exercising, or glucose monitoring, not all the patients are able to properly follow the self - management plans agreed with their healthcare professionals or advised by clinical guidelines. Racial and ethnic minorities are less likely to engage in DSM behaviors than other population groups, which partially explains observed disparities in health outcomes \ [[ @ B7] \ ]. Some of the barriers faced by these groups for achieving an adequate DSM are related to characteristics of the groups (such as health literacy or health beliefs) and also of the healthcare system (namely accessibility of culturally sensitive information) \ [[ @ B8 ], [@ B9] \ ]. Several review studies have assessed the effect of DSM educational programs on the general population \ [[ @ B6 ], [@ B10] - [@ B17] \ ]. Those studies have established that DSM educational programs can improve glycemic control \ [[ @ B11] - [@ B16] \ ], and identified the key characteristics for improving glycemic control, including face - to - face delivery, teaching methods based on cognitive reframing \ [[ @ B11] \ ], and higher contact time between participant and educator \ [[ @ B16] \ ]. A smaller number of studies have reviewed the evidence of the effect of these programs on racial / ethnic minorities \ [[ @ B18] - [@ B20] \ ]. Only one study \ [[ @ B18] \] examined their impact on glycemic control, observing a reduction on glycated hemoglobin of 0. 32% . There were however some limitations underlying that study, as some of the included interventions combined educational programs with other types of quality improvement strategies, making it difficult to disentangle the individual effect of the educational components. More importantly, no previous meta - regression study has identif*#d the key common characteristics of successful educational programs targeted to racial / ethnic minority groups. This represents a considerable gap in the literature, as programs for racial / ethnic minority groups should have different components from those targeting other population groups, namely addressing the cultural idiosyncrasies associated with each group. Therefore it is not reasonable to assume that the same type of successful program will be equally successful when applied to racial / dfhnic minority groups. Additionally, in the past years several clinical trials examining the impact of educational programs to improve diabetes self - management on racial / ethnic minorities have been published, making now possible a more detailed review and analysis of the available evidence regarding the effectiveness of these interventions. In this work we systematically reviewed DSM educational interventions specifically targeted to racial / ethnic minority groups. We studied the characteristics and costs of the interventions, and analyzed their impact on diabetes knowledge, self - management behaviors, and clinical outcomes. Whenever data were available, we performed meta - analyses to examine the short and long - term effects of the interventions, and meta - regressions to identify common characteristics of the interventions associated with better resuKtE. Methods = = = = = = = The review and its procedures were planned, conducted, and reported according to the Preferred Reporting Items for Systematic Reviews and Meta - Analyses (PRISMA) guidelines \ [[ @ B21] \ ]. Data sources and searches - - - - - - - - - - - - - - - - - - - - - - - - - A comprehensive core search strategy was developed for Medline through Ovid (combining MeSH terms and keywords) and then adapted and implemented in EMBASE and CINAHL (search strategy available in Additional file [1] (# S1) {ref - type = " supplementary - material " }: Table S1 ). Gray literature and additional articles were searched in twelve more bibliographic sources (Additional file [2] (# S2) {ref - type = " supplementary - material " }: TaH>e S2 ). The search was not restricted by language or publication date. For all the references selected to be included in the review, backward and forward citation searches were performed in ISI Web of Knowledge. All searches were conducted in October 2012. A bibliographical database was created using EndNote X6, and used to store and manage the retrieved references. Study selection - - - - - - - - - - - - - - - We included studies analyzing the effectiveness of DSM educational programs targeted to racial / ethnic minority groups with type 2 diabetes. We only included those studies in which at least 90% of the participants pertained to a racial / ethnic minority group considered to be at a higher risk for diabetes complications than the majority population group. Racial / ethnic minority group was defined as a population group with a race or ethnicity different from that of the majority population group of the host country. Groups at higher risk of diabetes complications were identified based on available literature. Interventions had to be exclusively educational, without including any other component such us financial incentives, clinician education or case management. In order to avoid possible comparisons between programs carried out in very heterogeneous settings, with very different health systems and population needs, we restricted this review to those interventions conducted in countries that were members of the OECD \ [[ @ B22] \ ], when study selection was conducted (November 2012 ). Eligible designs were randomized controlled trials (RCTs ), including cluster randomized controlled trials; controlled trials, including quasi - randomized trials; controlled before - after studies; and non - controlled before - after studies. Studies including a control group were only eligible in case the intervention was compared with care as usual. Titles and abstracts were screened for eligibility, and those fulfilling the inclusion criteria were included in the next stage, where the full texts of the selected articles were retrieved and assessed. Those that met the inclusion criteria were included for data extraction. Two reviewers independently screened citations, and any disagreements were solved by consensus with a third reviewer. Data extraction and quality assessment - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - We designed and used structured forms to extract information of interventions ' characteristics and their effectiveness. We used a previously developed taxonomy of DSM educational programs to characterize the interventions \ [[ @ B23] \ ]. The following information was extracted: setting, ethnic group, administration formats, teaching methods, educational contents, educators ' background, use of peer educators, and duration. Information of interventions ' cost and cost - effectiveness was also extracted. We critically appraised the studies using the Quality Assessment Tool for Quantitative Studies \ [[ @ B24] \ ], which enables the assessment of both 7ntefnal and external validity, classifying them into three categories (good, fair or poor) depending on six aspects: selection bias, study design, confounders, blinding, data collection and withdrawals / dropouts. Two reviewers independently extracted all the information and critically appraised the studies. Disagreements were solved through discussion with a third reviewer. When necessary we contacted the authors of the studies to request additional information. Data synthesis and analysis - - - - - - - - - - - - - - - - - - - - - - - - - - - The effectiveness of the interventions was assessed in terms of their impact on 1) diabetes knowledge, 2) diabetes self - management behavior, and 3) clinical outcomes. Diabetes knowledge was ascertained by measures reflecting the theoretical knowledge the patients had about their condition. Diabetes self - management behavior measured the performance of specific activities related to adequate DSM (diet, exercise, glucose control, foot self - examination, etc. ). Clinical outcomes included hemoglobin A1c (HbA1c ), body mass index (BMI ), or blood pressure, amongst others. All outcomes in all the studies were examined and classified as measuring one of these three domains. Variables that measured other domains were not included in the analysis. Additionally, we conducted independent meta - analyses to analyze short and long - term (six months post - intervention) effect of the interventions on glycemic control. Eligibility criteria for the meta - analyses included randomized controlled trials comparing the interventions with usual cwGe. The mean (standard deviation) of HbA1c levels in each study were extracted. This information was transformed into weighted mean differendw and 95% confidence intervals (CI) were calculated and combined using random - effects models. We imputed unreported standard deviations by use of established methods \ [[ @ B25] \ ]. Heterogeneity was quantified by the I ^ 2 ^ statistic, where I ^ 2 ^ ≥ 50% was considered evidence of substantial heterogeneity \ [[ @ B26] \ ]. Sources of heterogeneity were investigated by a Galbraith plot. Publication bias was quantitatively assessed with Egger test. We used bivariate meta - regression to explore relationships between effect size (ES) and interventions characteristics. The number of included studies was insufficient to perform a multivariate regression analysis. We conducted a sensitivity analysis, excluding the studies with higher risk of bias. All analyses were conducted with Stata, version 12. 0 (StataCorp, College Station, Texas ). For all the analyses, statistical significance was accepted at * p * \ <0. 05. Results = = = = = = = Article identification - - - - - - - - - - - - - - - - - - - - - - Figure [1] (# F1) {ref - type = " fig "} reports the screening process. A total of 1, 988 unique references were retrieved. 1, 386 references were excluded based on title and abstract, resulting in 602 references being included in the next stage. Full text articles were retrieved and assessed, with 24 studies meeting the eligibility criteria. Backward and forward search of these 24 articles identified thirteen additional studies, resulting in thirty seven articles being included in the review \ [[ @ B27] - [@ B63] \ ]. Thirty five of them reported one single intervention, whereas two articles reported two distinct interventions per article. Overall, thirty seven articles were identified, which analyzed the effectiveness of thirty nine different interventions. ! [Summary of evidence deWrch and selection.] (1472 - 6823 - 14 - 60 - 1) {# F1} Characteristics of the studies and interventions - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Table [1] (# T1) {ref - | Background ========== The prevalence type 2 diabetes rapidly rose over the past three decades. However its is not homogeneously racial and ethnic minorities usually experience higher prevalence than their non-minority counterparts \[[@B1]\] and are at higher risk of developing diabetes-related complications such as blindness, kidney damage, or depression, impacting both quality of life mortality rates \[[@B2],[@B3]\]. Self-management, defined as the patient's ability to manage not only the symptoms inherent to a chronic condition but also its treatment and associated lifestyle changes \[[@B4]\], has become increasingly important the treatment of type 2 diabetes \[[@B5],[@B6]\]. However, adequate diabetes self-management (DSM) require considerable changes to several domains, namely having a healthy diet, exercising, or glucose monitoring, not all the patients are able to properly follow the self-management plans with their healthcare professionals or advised by clinical guidelines. Racial and ethnic minorities less likely to engage in DSM than other population groups, which partially explains disparities in health outcomes \[[@B7]\]. Some of the barriers faced by these groups for achieving an adequate are related characteristics of groups (such as health or health beliefs) and also of the healthcare (namely accessibility of culturally sensitive information) \[[@B8],[@B9]\]. Several review studies have assessed the effect of DSM educational programs on the general population Those studies have established educational programs can improve glycemic control \[[@B11]-[@B16]\], and identified the key for improving glycemic control, including face-to-face delivery, teaching methods based on cognitive reframing \[[@B11]\], higher contact participant and educator \[[@B16]\]. A smaller number of studies have reviewed of the effect of these on racial/ethnic minorities \[[@B18]-[@B20]\]. one examined impact on control, on glycated hemoglobin of 0.32%. There were however some limitations underlying that study, some of the included interventions combined educational programs with other types of quality improvement making it difficult to disentangle the individual effect of the educational components. More importantly, no previous has the key common characteristics of successful educational programs to racial/ethnic minority groups. This represents a considerable the literature, as programs racial/ethnic minority groups should have different components from those targeting other population groups, namely addressing the cultural idiosyncrasies associated with each group. Therefore it is not reasonable to assume that the same type of successful program will equally successful when applied to racial/ethnic minority groups. Additionally, in the past years clinical trials examining the impact of educational programs to improve diabetes self-management on racial/ethnic minorities have been published, making now possible a more detailed review and analysis of the available evidence regarding the effectiveness of interventions. In this work we reviewed interventions specifically to racial/ethnic minority groups. studied the characteristics and costs of the interventions, and analyzed their impact on diabetes knowledge, self-management behaviors, clinical outcomes. Whenever data were available, we meta-analyses to examine the short and long-term effects of the and meta-regressions to identify common of the interventions associated with better results. Methods ======= The and its procedures were planned, conducted, and reported according to the Preferred Reporting Items for Systematic Reviews and (PRISMA) guidelines \[[@B21]\]. Data and searches ------------------------- A comprehensive core search strategy for Medline through Ovid (combining MeSH and keywords) then adapted and implemented in EMBASE and CINAHL strategy available in Additional file [1](#S1){ref-type="supplementary-material"}: Table S1). Gray literature and additional articles were in twelve more bibliographic (Additional file [2](#S2){ref-type="supplementary-material"}: Table S2). The search was not restricted by language or publication date. For all the references selected to be included in the review, backward and forward searches were performed in ISI Web of Knowledge. All searches were conducted in October 2012. A bibliographical database was created using EndNote X6, and used to store and manage the references. Study selection --------------- We included studies analyzing the effectiveness of DSM educational programs targeted to minority groups with type 2 diabetes. We only included those studies in which at least 90% of the participants to a racial/ethnic minority group considered be at a higher risk for diabetes complications than the majority population Racial/ethnic minority group was defined as a population group with a race or ethnicity different from of the majority population group of the host country. Groups at higher risk of diabetes were identified based on available literature. Interventions had to be exclusively educational, without including any other such us incentives, education or case management. In order to possible comparisons programs carried out in very heterogeneous settings, with very health systems population needs, we restricted this review to those interventions conducted in countries that were members of OECD \[[@B22]\], when study was conducted (November 2012). Eligible designs were randomized controlled (RCTs), including cluster randomized controlled trials; trials, including quasi-randomized controlled before-after before-after studies. Studies including a group were only eligible in case the intervention was compared with care as Titles and abstracts were screened for eligibility, and those fulfilling the inclusion criteria were included in the next stage, where the texts of the selected articles were retrieved and assessed. Those that met the inclusion criteria were included for data extraction. Two reviewers independently screened citations, and any disagreements were solved by consensus with reviewer. Data extraction and quality assessment -------------------------------------- We designed and structured forms to extract information of interventions' characteristics and their effectiveness. We used a previously developed taxonomy of DSM educational programs to characterize the interventions \[[@B23]\]. The following information was extracted: ethnic group, administration formats, teaching contents, educators' background, use of peer educators, and duration. Information of interventions' cost and cost-effectiveness was also extracted. We critically appraised the studies using the Quality Assessment Tool for Quantitative Studies \[[@B24]\], which enables the assessment of internal and external validity, them into three (good, fair or poor) depending on six selection bias, study design, confounders, blinding, data collection and withdrawals/ dropouts. Two independently extracted all the information critically appraised the studies. Disagreements were solved through discussion with a third reviewer. necessary we contacted the of the studies request additional information. Data synthesis and analysis --------------------------- The effectiveness of the interventions was assessed terms of their impact on 1) knowledge, 2) diabetes self-management behavior, and outcomes. Diabetes knowledge was ascertained by measures reflecting the theoretical knowledge patients had about their condition. Diabetes self-management behavior measured the performance of specific activities related to adequate DSM (diet, exercise, glucose foot self-examination, etc.). Clinical outcomes included hemoglobin A1c (HbA1c), body mass index (BMI), or pressure, others. All outcomes in all the studies were examined and classified as measuring of these three domains. Variables that measured other domains were not included in the analysis. Additionally, conducted independent meta-analyses to analyze short and long-term (six post-intervention) effect of interventions on glycemic Eligibility criteria for the meta-analyses included randomized controlled trials comparing the interventions with usual care. mean (standard deviation) of HbA1c levels in each study were extracted. This information was transformed weighted mean difference and 95% confidence intervals (CI) were calculated combined using models. We imputed standard deviations by use of established methods \[[@B25]\]. Heterogeneity was quantified by the I^2^ statistic, where I^2^ ≥ 50% was considered evidence of substantial heterogeneity \[[@B26]\]. Sources of heterogeneity investigated by a Galbraith plot. Publication quantitatively assessed with test. We used bivariate explore relationships between effect size (ES) and interventions characteristics. The number of was insufficient to perform a multivariate regression analysis. We conducted sensitivity excluding the studies with higher risk bias. All analyses were conducted Stata, version 12.0 (StataCorp, College Station, Texas). For all the analyses, statistical significance was accepted at \< 0.05. Results ======= Article identification ---------------------- Figure [1](#F1){ref-type="fig"} reports the screening process. A total of 1,988 unique were retrieved. 1,386 references were excluded based on title and abstract, resulting 602 references being included the next stage. Full text articles were retrieved and assessed, with 24 studies meeting the eligibility criteria. Backward and search of these 24 articles identified thirteen additional studies, resulting in thirty seven articles being included in the review \[[@B27]-[@B63]\]. Thirty five of them one single two articles reported two distinct interventions per article. Overall, thirty seven articles identified, which analyzed the effectiveness of thirty nine different interventions. {#F1} Characteristics of studies and interventions ------------------------------------------------ Table [1](#T1){ref- | BaCkgRoUNd
==========
thE PREvalenCE OF TypE 2 diabETes RApiDly ROSe Over The pASt ThrEe dEcAdEs. hOweVEr its BURdEn iS NoT hoMOgeNeoUSly DIStRiBuTeD: RAciaL And etHNIC MInOrItiEs UsuAllY EXPErIEncE higHER PRevAlENcE ThaN TheIR NON-miNOrity CoUnTeRPArtS \[[@B1]\] aND aRE at HiGhER rIsK Of dEVElOpinG DIaBETeS-RElAted comPliCAtIons SUCH As bLInDNeSs, kIDney DAMagE, Or dEprESsiOn, ImPActinG BotH QUAlITY of Life anD MoRTaLITy RateS \[[@b2],[@B3]\].
sELF-MAnAgEMEnt, DeFINED as tHe PatienT'S AbiLITy tO mAnage nOt OnLy THE SYmPTOmS InhErent tO A ChrONiC COndiTiON buT AlSo ITs tReatMEnT ANd AssOciAted LiFEsTYle cHanGeS \[[@B4]\], has BeCome iNCReaSINglY ImPORTAnT In tHE TreaTmENt Of TyPe 2 DIabeTEs \[[@B5],[@b6]\]. HoweVer, bECausE adeQuAtE diAbetes SELf-MAnagEMENT (Dsm) MaY rEQuiRe considErABLE LIfEStYlE CHAngEs TO sEvEral dOMAins, nAmelY HAviNg A hEALThY Diet, ExerCisIng, oR GluCoSE monIToRinG, nOt All tHE pAtIents ARE abLE TO PrOpeRly folLOw tHe SelF-ManagemENT pLans agReed with tHEIR healthCaRe professIonaLs or aDVisEd BY cLiNiCal guiDELInes. raciaL and EthnIC mInOriTies aRe LESs LikEly tO EngaGE IN dsM bEhAVIORs ThaN OTHEr popuLAtIon GRoUpS, wHICH pARtIAllY EXplaiNS oBSErVED dispaRitieS iN healTh outComES \[[@B7]\]. SOme oF the barrierS faCed bY tHESE gRouPs For achIeving an adeQuatE Dsm aRE rElaTED To charaCTerISTiCS OF thE GroupS (SuCH As HEAlth litEracY Or HeALth beLIEfs) aNd also oF tHe HeaLtHcArE sYSTem (nAmElY acCEsSibiLity oF CULtuRally sensItIVe INfoRMATion) \[[@B8],[@b9]\].
SEvERal REvIEw StUDiES HAVE ASsesSeD THe EfFEcT of dSm eDuCaTiONaL pRogrAMS On The GENEral PopUlaTioN \[[@B6],[@b10]-[@B17]\]. thoSE STuDies hAve esTAbLishEd ThAt dsM edUCaTIonAL PrOGRAMs CaN imPrOvE glycemIc control \[[@b11]-[@B16]\], And iDEntIFIed THe KeY ChaRACTeriSTIcs fOR ImproViNG gLyCemIc COnTRol, INCLUdINg fAce-tO-FaCE dElIvERy, TEaChInG metHoDs BAseD On cOgnitiVe ReFraMiNG \[[@B11]\], and HiGHeR cOntACt tIme BeTWeEn PArTIciPant And EdUcaTor \[[@b16]\]. a sMallER NUmbeR of sTudIes HaVE reVIEweD tHE eViDeNCE oF the efFECT oF THeSe ProgRaMs On RAciaL/ETHNiC mInoritieS \[[@B18]-[@b20]\]. oNlY OnE StuDy \[[@B18]\] ExamINed ThEIr Impact On GLyceMIc cONTROL, oBSeRVIng a REduCtiOn ON gLYcatEd HemoGLobIN of 0.32%. therE weRe howeVEr SoMe LImItatIoNs uNDErlYing ThAT sTUDy, AS SOmE OF tHE INcLudEd inTERVeNtiONS cOMbiNEd eDucAtIONAL PROGrams witH oTHER Types Of qUaLitY IMpRovEMEnt stRaTegiEs, mAKiNG It DiffICULT to disEntANGLe The IndivIdUaL eFfEct OF The EdUcATioNAl COMPoNENTS. MoRe ImpOrTAntLy, no prevIOUs meta-ReGRESSioN sTudy Has iDeNtified tHe KeY commON chaRacTEriSticS oF sUccesSFUl EducAtiONaL progRamS tARGETEd tO raCiAL/eTHNIc MInoRitY Groups. tHIS REprESENtS a coNSIDerAbLE gAp iN The LiterATuRe, aS pROgRamS FOR RACiaL/EtHnIc mINoRity grouPS SHould hAVE DIFfErENT CoMpoNeNts From thosE TARgeTiNG oTHER pOpulation grOups, NaMely AdDresSing the cultUraL IdiosyNCrasiEs aSSOCIateD witH eAch Group. thEreforE It IS not reAsoNaBlE TO AsSUMe THAt THE SAme tyPE OF SuccEsSFul PROgrAM WiLL bE EquaLLY sUccESsFUL When aPplIEd To racial/eThNiC mINORity GROUPs. ADDItIoNAlLY, IN The PASt yeArS SeverAl ClINicaL tRIals ExamIniNg thE ImpAct Of EdUCaTiONal PrograMS To IMprOVe diABeTEs SeLF-MaNAgemEnt on RaCIAl/etHniC MInoRiTIeS HaVe bEEN puBlISheD, MaKinG NOW poSsIbLe A morE DetaiLed rEviEW AnD aNALySis oF THe avAilABlE EVidenCe REgArdIng ThE EFFeCtIvENESs oF theSE InTERvEnTiOns.
in ThiS WOrk we SysteMATIcally rEVIEWeD dsm EdUCATIOnAL iNTERvEntIonS sPEcifiCaLLy taRGetED tO RaCiaL/eTHnIC mINoRItY groUPs. WE stuDied thE cHAracterIstICs AnD cosTS oF ThE intERVeNTiONs, aND anALYzEd THEir ImpAcT on DIAbeTES KnOwLedge, SelF-mAnAGEment behaviorS, And cliNIcal oUtcOMeS. whenEveR data wErE AVailAblE, We pERFoRMEd MEta-ANAlyses to examIne THE shorT And loNG-tErM eFFECTs Of ThE InTeRvEnTionS, anD mETa-regREssioNs tO iDENTify COmmOn cHaracteRistIcs of THE iNtERvENtIONs ASSoCIATed WITH BeTteR RESuLTs.
meThOds
=======
tHE rEVIEW And iTs pROcEDuRes WErE plAnneD, conDuCted, ANd rEPoRTed ACCoRDING tO The pReferred rEPOrtInG ItEms FOR SYsTEMATIC rEviewS ANd mETa-anALYSES (pRiSma) gUidelines \[[@B21]\].
dAtA sourcEs anD SearChES
-------------------------
a ComPreHeNsiVE cORE sEaRCH StRAtegy wAS dEVElopED fOr MEdlINE thROugh oVID (COMbining Mesh TERMS And KeywoRDS) AND tHEn AdAPtED aNd iMPlEmentED in EMBAsE AnD cinahL (sEArCh StraTEgy AVAILAble IN adDiTIOnaL fIle [1](#s1){REf-TYpE="sUpPLEMEntArY-maTErIaL"}: TAbLE S1). gRAY liTERaTure and AdDItiOnAL ArtIcLes wERe seARChED iN TWElVe MorE BiBLiOgRAPHiC sOurces (aDdItIOnAL FIle [2](#S2){ReF-TyPE="SUPplemenTaRY-MATerial"}: tabLE S2). THE seARcH waS NOT ReStRicTed bY LANGuagE or PUbliCaTIOn dAte. FOr aLL THe RefERenceS SeLecTed tO be inCLuDeD in the rEviEW, baCKwaRd aND FOrWaRd cITATioN SearCheS wERE peRfORMEd iN ISI Web Of KnOwLEdGe. ALL SearChES were CoNdUcTed in ocTober 2012. A BIbLIOGRaPhiCAl datAbASe wAS creatEd USinG eNdnotE X6, And UsED to sTore AnD ManAGe THe RETrievEd reFEReNCEs.
STUDY SeLEctIOn
---------------
we InCLuDeD STuDiES anAlYZING THE eFfEctiveneSS oF dsm eduCaTioNaL ProGRams TaRGetEd tO RACIAL/eThNic minORItY GROuPs With TYPE 2 diabEteS. we oNLy INcLUDed THoSE STUdiEs In wHiCH at LEaSt 90% OF THE PaRTiCiPAnTs PERTaINed TO A rACIaL/eTHNIc mINORIty GROUP cONsIdeReD tO BE AT a hiGHEr RIsk fOR diAbETEs coMplIcAtIoNS Than THE maJORITY PopuLATIOn GRoUP. RACiAl/Ethnic MiNoRITy group wAS defiNed As a pOpuLATioN gRoUp wItH a RAcE Or ethNiCIty DIFfEREnT FROm THAt of tHE MAjOrITY populatION grOuP oF THE hosT CoUNtRY. GrOuPS AT HIghEr RIsk oF DIaBeTes cOmpliCAtIons WeRE IdEntiFiEd BasED On AVaILaBlE lITerAtUrE. iNterveNtIONS Had TO be EXCLuSiVEly EDucATIONal, WIThoUT iNCluDINg AnY other coMpOneNt SUcH uS FiNaNciAL IncENtivEs, cliniCiaN eDuCaTioN or casE MaNAGEMENT. In oRDeR tO AVOID POsSIBLE CompARISonS betwEEN prOGRams carRIED OUT in VERy HetEroGeNeoUS seTTiNgS, WITH vErY DiFFeRenT heALTH SYsteMS ANd PopULaTioN neEDs, WE ReSTRiCted ThIS REvIEW to tHoSE iNtERVeNTIOns CoNduCTED in COuNtRieS THAt WERE MeMBERS of The OeCD \[[@b22]\], wHen STUDy sELeCtIOn WaS CoNductEd (nOVeMBEr 2012).
ELiGIblE deSiGnS wErE rAndOmIzed ConTROLLeD TRials (rcts), INCLuDING clustEr raNDOMIZeD ContROlled tRIAlS; coNtRolLEd triaLs, iNCLuDIng quaSI-RanDoMizEd TriaLs; CoNtrollED bEfORe-AFTEr sTUdiEs; aNd NON-cOntrOlLeD bEForE-AFTEr STudIES. sTuDIEs inCLUDIng A COnTRoL gRoup WeRE oNly eLIgIBlE IN case tHE InTERventIOn waS cOMPARED WiTH cAre As uSUaL.
TiTLeS AnD aBSTracTs wERe SCReeneD FOr ElIgiBilitY, aND ThOsE FulFiLliNG tHe InClusIOn CriTerIA wEre iNcLuDEd in The NExT sTAge, WHeRE the FUlL tExTs of the sEleCtED arTIcLES weRe reTRieVed aNd asSESSeD. THosE tHat met tHE INcluSion crIteRIa were incLUdED foR DatA extrActIon. tWo REViEWERS iNdEpENDeNtly SCreeNEd ciTAtioNs, aNd aNY DIsaGreEmENTS were SOlVeD By cONSENsUs WitH A tHIRD ReViEwer.
DatA EXtRAcTIoN and quALiTy aSsESsMenT
--------------------------------------
we dESIGNEd aND USED STRUcTuReD FORms TO EXTraCT INForMatiON OF iNteRVEntIoNS' chaRAcTeriStiCs AnD tHeir effecTIvEness. we useD a PrEVioUsLy DeVeLOPeD TAxoNoMY OF DsM EDucaTIONAL pROgrams To chaRActEriZE ThE InTeRVEnTiOnS \[[@b23]\]. thE fOLLOWInG iNFoRMATiOn WAs ExTRACted: SETTinG, eThnIc GrOUp, admInIstRATIon fOrmAtS, tEaChING mEThODS, eDUcaTiONaL cONTEnTs, eDuCaToRS' BaCKgrounD, Use OF pEEr edUcAToRs, AnD dUraTiOn. infOrMatiOn oF iNtErVENtIonS' COsT aND cOST-EFfEcTIVeneSS Was ALso extraCTed.
we crITICAllY Appraised THe sTudIEs USIng tHE qualITy aSSESSMent tOoL FOr QuaNTitATivE sTUDIEs \[[@B24]\], WHICH ENaBLes The aSseSSMEnT oF both iNTERNaL AnD extERnAl valIdITy, cLassIfYING tHem inTo THrEe cATEgoriEs (GoOd, FaIR Or pOor) DEPEndInG On sIX ASPecTs: sEleCTiOn bias, STUDY desiGN, CoNfoUNDeRs, BlINDINg, Data colLecTIon And WiThDRAwalS/ drOpoUTS. TWO reVIEwErS IndePendeNtLy exTRacTed all THe INFORmAtIOn aND crITicalLy appRAised tHe StudIEs. disAGReEMEnTS WErE solVed thrOUGh discUsSiON With a ThiRD REvieweR. WhEN NeCeSsarY We COntacted tHe AuTHoRs Of tHE stUdIes To reQUEst AdditiONAl INfORMAtIOn.
DAta synThESIs anD anaLySIs
---------------------------
The eFFeCtivEnesS Of THe iNTERveNtiOnS waS assesSED IN tErMs of thEIR ImpAct ON 1) dIABETEs kNoWLeDGe, 2) DIabETEs Self-manAGEMENt bEHAViOr, aND 3) ClInical OuTCOMeS. dIABEtes KNOWledgE Was AsCertaiNeD by measUrEs rEFLECTiNg the tHEoRetIcAL KNOwLEdGe the PATIENts had ABout thEiR coNdItION. dIabEtEs sELf-ManAgEmeNT beHavIoR MEASUrED ThE PerFOrmAncE OF SPECIFIC aCTivItiES RElated tO aDequaTe Dsm (Diet, EXErCISE, GLUcOSE cONtrol, fOOt seLf-EXAmINATIOn, Etc.). clINicAl OuTcomEs INcluDED hEMoglobIN A1C (HBA1c), BOdy masS INDEX (BmI), or BlOod prESsurE, AmONGst OTHErS. aLl ouTcOmes in ALl thE stUDIeS WeRe ExamiNEd aND claSSIFIed As meAsuRinG onE Of THesE THREE DomaiNS. VariAbles ThAT MeAsURed oTHeR dOMAiNS Were nOT INcLuded in tHe ANALYSIs.
ADDiTioNalLy, wE coNDUcTED IndEPEndENt mEta-anAlYsES To AnAlYZE short AnD LOng-Term (siX months pOsT-iNTeRVeNtiON) EffecT Of ThE InTervEnTIoNS oN glyceMIC CoNtRol. eLIgIBIlITy cRiterIA fOR the mEta-analySEs iNCLuDeD RaNDoMIZed CONTrolLed trIaLS comparIng the INtERVENTIOnS WIth UsUal CArE. the Mean (STaNDaRD deviaTiON) OF hBA1C lEVeLS In eaCH stUDY WERe ExTRAcTed. THIs iNfORmaTioN Was TrANSFormed iNTO WEiGhTeD mEaN DifFerenCE aND 95% CONFIdEnCE iNteRvalS (CI) wErE CalcULATEd AND COmBineD USiNG RANDOm-EfFeCts modEls. we IMPUtEd uNrEpOrTeD StANdARd deVIATiOnS by Use Of ESTaBLishEd MeThODS \[[@b25]\]. HetEROGENeItY was quANtIfied by THE i^2^ StAtiStiC, WHeRE i^2^ ≥ 50% wAs CoNsideRed EvIDEnCE oF SubstaNTIAL hEtErOGeneITY \[[@B26]\]. sources Of HETeroGeneity wEre INVesTigateD BY A GALBraITH PLot. PuBLICATIon bias wAS QUAntITATIVelY aSSEsseD wiTH EGgeR TESt.
we uSeD bIvARiAtE mETa-REgrEssIoN to EXpLoRE rELaTioNshIpS BetWEen EFFecT sizE (es) aNd iNtERvENTioNS cHaRACTeRisTICs. THe NUmBer of iNCLUdEd sTUdIES was insUFfIcIENT to PErFoRM a MULtiVaRiatE RegRESsIon ANalySiS. WE cONdUctED a sEnsiTIvITy AnAlySIS, exCLUdInG The STudies with HigHEr RISk OF BiaS. AlL AnaLyseS Were cONDUCTed WITH StaTA, verSiOn 12.0 (stAtaCorp, coLLEgE stATIoN, Texas). FOR All THE AnalySes, StATiStical sigNificaNCE was AcCePteD aT *P* \< 0.05.
ReSuLts
=======
aRtiCle idENtIficatIon
----------------------
FIgUrE [1](#f1){REf-tYPE="fiG"} REPorTs thE scrEening ProcEss. A TOTAL Of 1,988 unIque REFEreNces wErE ReTRIeved. 1,386 REFERENcEs WeRE excluDED BasEd on tITLe aNd abStrAcT, resultINg IN 602 rEfERENcES BeING iNClUdED In ThE next STAgE. fuLl tEXT ArTIClEs were rETRiEvEd And AssEsSEd, wITH 24 StUDieS MeeTInG tHE ELIGIBiLItY cRItEriA. bACKWarD aND forward sEARcH oF theSE 24 ArtiCleS IdentIfiEd THiRTeEn aDdITIonAl STUdIes, rEsulting In thirty sEveN ArtiCLEs beiNg incLUDED In THE REvIEw \[[@B27]-[@B63]\]. THirtY FIVe OF thEm REpORtEd One sInglE intErVenTIoN, wHeReas TwO arTICLES RepoRtED TWo diSTIncT iNTeRvEntIoNS per aRtIClE. oVeRall, thIRtY sEVEN ARTIcLes Were IdentIFiED, which ANaLyZED the EFfectiveneSs of THIRty NiNe dIFFerENt iNTERVENTiOns.
{#f1}
ChaRACteristicS oF THE stuDIES AND intErVentIons
------------------------------------------------
table [1](#t1){rEF- | Background ========== The prevalence of type 2diabetes rapidly rose over the past three decades. However its burden is not homogeneously distributed: racial and ethnic minorities usually experience higher prevalencethantheir non-minority counterparts \[[@B1]\] and are athigher risk ofdeveloping diabetes-related complications such as blindness,kidney damage,or depression, impacting both quality oflife and mortality rates \[[@B2],[@B3]\]. Self-management, defined as the patient'sabilityto manage not only the symptoms inherent to a chronic condition butalso its treatment and associatedlifestyle changes\[[@B4]\], has become increasingly important inthe treatment oftype 2 diabetes \[[@B5],[@B6]\].However,because adequate diabetes self-management (DSM) may require considerable lifestyle changes toseveral domains, namely having a healthy diet, exercising, or glucose monitoring,not allthepatients are able to properly follow theself-management plans agreed with theirhealthcare professionals or advised by clinical guidelines. Racial and ethnic minorities are less likely to engage in DSMbehaviors than other population groups, which partially explains observed disparities in health outcomes \[[@B7]\]. Some of the barriers faced by these groups for achieving an adequate DSM are related tocharacteristics of the groups (such ashealth literacy or health beliefs) and also of the healthcare system (namely accessibility of culturally sensitive information) \[[@B8],[@B9]\].Several review studies have assessed the effect of DSM educational programs onthe generalpopulation \[[@B6],[@B10]-[@B17]\].Those studies haveestablished that DSM educational programs can improveglycemiccontrol \[[@B11]-[@B16]\], and identified the key characteristics for improving glycemiccontrol, including face-to-face delivery, teachingmethodsbased on cognitive reframing \[[@B11]\], and highercontacttime betweenparticipant and educator \[[@B16]\]. A smaller number of studies have reviewed theevidence of the effect of these programs on racial/ethnic minorities \[[@B18]-[@B20]\]. Only one study \[[@B18]\] examined their impact on glycemic control, observing a reduction on glycated hemoglobin of 0.32%. There were however some limitations underlyingthat study, as some of the included interventions combined educational programs with other types of quality improvement strategies, making it difficult to disentangle the individual effect of the educational components. More importantly, no previous meta-regression study hasidentified thekey common characteristicsof successful educational programs targeted to racial/ethnic minority groups. This represents a considerablegap in the literature, as programs for racial/ethnic minoritygroups should have different components from those targeting otherpopulation groups, namely addressing the cultural idiosyncrasies associated with each group.Therefore it is not reasonable to assumethat the same type of successful program will beequally successful when applied to racial/ethnic minority groups. Additionally,in thepast years several clinical trialsexamining the impact of educational programs to improve diabetes self-management on racial/ethnic minorities have beenpublished, making now possible a more detailed review and analysisof the available evidence regarding the effectiveness of these interventions. In this workwesystematically reviewed DSM educational interventions specifically targetedto racial/ethnic minority groups. We studied the characteristicsand costs of the interventions, andanalyzed their impact on diabetes knowledge,self-management behaviors, andclinical outcomes. Whenever data were available,weperformedmeta-analyses to examine the short andlong-term effects ofthe interventions, and meta-regressions to identify common characteristics of the interventions associated with better results. Methods ======= The reviewandits procedures were planned, conducted, and reported according to the PreferredReporting Items forSystematicReviewsand Meta-Analyses (PRISMA) guidelines \[[@B21]\]. Data sources and searches -------------------------Acomprehensivecore search strategy was developed for Medline through Ovid (combining MeSH terms andkeywords) andthenadapted and implemented in EMBASE and CINAHL (searchstrategy available in Additional file [1](#S1){ref-type="supplementary-material"}: Table S1). Gray literature and additional articles were searchedin twelve more bibliographic sources (Additional file [2](#S2){ref-type="supplementary-material"}: Table S2). The searchwas notrestricted by language orpublication date. For all the references selected tobe included in the review, backward and forward citation searches wereperformed in ISI Web of Knowledge. All searches were conducted inOctober 2012. A bibliographical database was created using EndNote X6, and used to storeand manage the retrieved references.Studyselection --------------- We included studiesanalyzing the effectiveness ofDSM educational programs targeted toracial/ethnic minority groups with type 2 diabetes. We only included thosestudies in which at least 90% of the participants pertained to a racial/ethnic minority group considered to beat a higher risk for diabetes complications than the majority populationgroup. Racial/ethnic minority group was defined as a population group with a race or ethnicity differentfrom that of themajority population group of the host country.Groupsat higherrisk of diabetes complications were identified based on available literature.Interventions had to be exclusively educational, without including any other component such us financial incentives, clinician education or case management. In orderto avoid possible comparisons between programscarried out in very heterogeneous settings, with very different health systems and population needs,we restricted this review to those interventions conducted in countries that were members oftheOECD \[[@B22]\], when study selection was conducted (November 2012). Eligible designs were randomized controlled trials (RCTs),including cluster randomized controlled trials;controlled trials, including quasi-randomized trials;controlled before-after studies; and non-controlled before-after studies. Studies including a control group wereonly eligible in case theintervention wascompared withcare as usual. Titles andabstracts were screenedfor eligibility, and those fulfilling the inclusion criteria were included in the nextstage,where the fulltexts of the selected articleswere retrieved and assessed. Those that met the inclusion criteria were included for data extraction. Tworeviewersindependently screened citations, andany disagreements were solved by consensus with a third reviewer. Data extraction and quality assessment -------------------------------------- We designed and used structured formsto extract information of interventions' characteristics and their effectiveness. We used a previously developed taxonomy of DSM educational programs to characterize the interventions \[[@B23]\]. Thefollowing information was extracted: setting, ethnic group, administration formats, teaching methods, educational contents, educators' background, use of peereducators, and duration. Information of interventions' cost and cost-effectiveness was also extracted. We critically appraised the studiesusing the Quality Assessment ToolforQuantitative Studies \[[@B24]\], which enables the assessment of both internal andexternal validity, classifying them intothree categories (good, fair or poor) depending on six aspects: selectionbias, study design, confounders, blinding, data collection and withdrawals/ dropouts. Two reviewersindependently extracted all theinformation and critically appraised the studies. Disagreements were solved through discussionwith a third reviewer. When necessary we contacted the authorsof the studies to request additional information. Data synthesis and analysis --------------------------- The effectiveness of theinterventions wasassessed in termsof their impact on1) diabetes knowledge, 2) diabetes self-management behavior, and 3) clinicaloutcomes. Diabetes knowledgewas ascertained by measures reflecting thetheoretical knowledge thepatients had about their condition. Diabetes self-management behavior measured the performance of specific activities related to adequate DSM (diet, exercise, glucose control, foot self-examination, etc.).Clinical outcomes included hemoglobinA1c (HbA1c), body mass index (BMI),or blood pressure, amongst others. All outcomes in all the studies were examined andclassified as measuring oneof these three domains. Variables that measured other domains were notincludedin the analysis. Additionally, we conducted independent meta-analyses to analyzeshort and long-term (six months post-intervention) effect of the interventions on glycemic control. Eligibility criteria for themeta-analyses included randomized controlled trialscomparing theinterventionswithusualcare. Themean (standard deviation) of HbA1c levels in eachstudy were extracted. This information was transformedinto weighted mean difference and 95%confidence intervals (CI) were calculated andcombined using random-effects models.We imputedunreported standard deviations by use of established methods \[[@B25]\]. Heterogeneity was quantified bytheI^2^statistic, where I^2^≥ 50% was considered evidence ofsubstantial heterogeneity \[[@B26]\]. Sources of heterogeneitywere investigatedby a Galbraith plot. Publicationbias wasquantitatively assessedwith Egger test.We used bivariate meta-regression to explore relationships between effectsize(ES) and interventions characteristics. The number of included studies was insufficientto perform amultivariate regression analysis. We conducted a sensitivity analysis, excluding the studies with higher risk of bias. All analyses were conducted with Stata, version 12.0 (StataCorp, CollegeStation, Texas).For all the analyses, statistical significance was accepted at *p* \< 0.05. Results ======= Article identification ---------------------- Figure [1](#F1){ref-type="fig"}reports the screening process. Atotal of 1,988 unique references were retrieved. 1,386 references wereexcluded basedon titleand abstract, resulting in 602 references beingincluded in the next stage. Full text articles were retrieved andassessed, with 24 studies meeting the eligibility criteria. Backward and forward search of these24 articles identified thirteen additional studies, resulting inthirty sevenarticles being included in the review \[[@B27]-[@B63]\]. Thirty five of them reported one singleintervention, whereas two articles reportedtwodistinct interventionsper article. Overall, thirty seven articles were identified,whichanalyzed the effectiveness of thirty nine different interventions. {#F1} Characteristics of the studies and interventions ------------------------------------------------Table [1](#T1){ref- | Background _==========_ The prevalence of type 2 _diabetes_ rapidly _rose_ over _the_ past three decades. _However_ its burden _is_ not homogeneously distributed: racial and _ethnic_ _minorities_ _usually_ _experience_ higher prevalence than their _non-minority_ counterparts \[[@B1]\] and are at higher risk of developing _diabetes-related_ _complications_ such as _blindness,_ _kidney_ damage, or depression, impacting both quality of _life_ _and_ mortality _rates_ \[[@B2],[@B3]\]. Self-management, defined as the patient's _ability_ to _manage_ not only the symptoms inherent to a _chronic_ condition but _also_ its treatment and associated lifestyle _changes_ _\[[@B4]\],_ has _become_ increasingly important in the treatment _of_ type _2_ diabetes \[[@B5],[@B6]\]. However, _because_ adequate diabetes self-management (DSM) may require considerable lifestyle changes _to_ several _domains,_ namely having _a_ healthy diet, _exercising,_ _or_ glucose monitoring, not all the patients _are_ able to _properly_ follow the self-management _plans_ _agreed_ with their healthcare professionals or _advised_ by clinical _guidelines._ _Racial_ and ethnic minorities are less likely _to_ engage _in_ DSM _behaviors_ than other _population_ _groups,_ which partially explains observed disparities in health outcomes \[[@B7]\]. Some _of_ the _barriers_ _faced_ by these groups for achieving an adequate _DSM_ are related to characteristics of the groups _(such_ as health literacy _or_ health beliefs) _and_ also of the healthcare system _(namely_ _accessibility_ of culturally sensitive information) _\[[@B8],[@B9]\]._ Several review studies _have_ assessed the effect _of_ DSM _educational_ programs on _the_ general _population_ \[[@B6],[@B10]-[@B17]\]. Those studies have established that DSM educational programs can improve glycemic control \[[@B11]-[@B16]\], _and_ identified the key characteristics for improving glycemic _control,_ including face-to-face delivery, teaching _methods_ _based_ on cognitive reframing \[[@B11]\], and _higher_ contact _time_ between participant and educator \[[@B16]\]. A smaller number of studies have _reviewed_ the evidence of _the_ effect of these programs on racial/ethnic minorities \[[@B18]-[@B20]\]. Only one study \[[@B18]\] examined their impact on _glycemic_ control, observing a reduction on glycated hemoglobin of 0.32%. There were however some limitations underlying that study, as some of _the_ included interventions combined educational programs with other types of _quality_ _improvement_ _strategies,_ making it _difficult_ to disentangle the _individual_ effect of _the_ educational components. More _importantly,_ _no_ _previous_ meta-regression study has _identified_ the _key_ common characteristics of successful educational programs targeted to racial/ethnic _minority_ groups. _This_ represents a considerable gap _in_ the literature, as programs for racial/ethnic minority groups _should_ have different components _from_ those _targeting_ other population groups, namely _addressing_ _the_ cultural _idiosyncrasies_ associated with each _group._ Therefore it is _not_ _reasonable_ to assume that the same type of successful program will be equally successful when _applied_ to racial/ethnic minority groups. Additionally, in the past years _several_ clinical trials examining the impact _of_ educational programs to improve diabetes self-management on racial/ethnic minorities _have_ _been_ published, _making_ _now_ possible a more detailed review and analysis _of_ the available evidence regarding _the_ effectiveness of these _interventions._ In _this_ _work_ we systematically reviewed DSM educational _interventions_ specifically targeted to racial/ethnic minority groups. _We_ studied _the_ characteristics and costs of the interventions, and _analyzed_ their impact on diabetes knowledge, self-management behaviors, and clinical outcomes. Whenever data were available, we performed meta-analyses to examine the short _and_ long-term effects _of_ the interventions, and meta-regressions to identify common _characteristics_ _of_ the _interventions_ associated _with_ better results. _Methods_ ======= The review _and_ _its_ _procedures_ were planned, conducted, _and_ reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses _(PRISMA)_ guidelines \[[@B21]\]. Data sources and _searches_ ------------------------- A comprehensive core _search_ strategy _was_ developed for Medline through Ovid (combining MeSH terms _and_ _keywords)_ and then _adapted_ and implemented in EMBASE and _CINAHL_ (search strategy _available_ in _Additional_ file [1](#S1){ref-type="supplementary-material"}: Table S1). Gray literature _and_ _additional_ articles were searched _in_ twelve _more_ bibliographic _sources_ (Additional file [2](#S2){ref-type="supplementary-material"}: Table S2). The search was not restricted by language or publication date. For all the references _selected_ _to_ _be_ included in the review, backward and _forward_ _citation_ _searches_ were performed _in_ _ISI_ Web of Knowledge. All searches _were_ _conducted_ in October 2012. A bibliographical _database_ _was_ created _using_ EndNote X6, and used to store and manage the retrieved references. Study selection _---------------_ We included studies analyzing _the_ effectiveness _of_ _DSM_ educational programs targeted to racial/ethnic _minority_ groups with _type_ _2_ diabetes. We _only_ included those _studies_ in which _at_ least 90% _of_ the participants pertained to a racial/ethnic minority group considered to be at a higher risk _for_ diabetes complications than _the_ majority _population_ _group._ Racial/ethnic minority group _was_ defined as a population _group_ with a race or ethnicity _different_ from that of the majority population group _of_ _the_ _host_ country. Groups at higher risk of diabetes _complications_ were _identified_ based on available literature. Interventions _had_ to _be_ exclusively educational, _without_ including _any_ other component such us financial incentives, clinician education or case management. In order to avoid possible comparisons between _programs_ carried _out_ in very heterogeneous settings, with _very_ different health systems and population needs, we restricted this review to those interventions conducted in _countries_ that were members of _the_ OECD \[[@B22]\], when _study_ _selection_ was conducted (November 2012). _Eligible_ designs were randomized _controlled_ trials (RCTs), including _cluster_ randomized controlled trials; controlled trials, including quasi-randomized _trials;_ _controlled_ before-after studies; and non-controlled before-after studies. _Studies_ including a control group were only _eligible_ in case the intervention was _compared_ with care as usual. Titles and abstracts were screened for eligibility, _and_ those fulfilling the inclusion criteria _were_ _included_ in the next stage, _where_ the full texts _of_ the _selected_ articles were retrieved and assessed. _Those_ _that_ met the _inclusion_ criteria were included for data _extraction._ Two reviewers independently screened _citations,_ _and_ any disagreements _were_ solved by consensus with a third _reviewer._ Data extraction _and_ quality assessment -------------------------------------- We designed and _used_ _structured_ forms _to_ extract information _of_ interventions' characteristics and _their_ effectiveness. We used a previously developed taxonomy of DSM educational programs _to_ characterize the _interventions_ \[[@B23]\]. _The_ following _information_ was extracted: setting, ethnic group, administration formats, _teaching_ methods, educational contents, _educators'_ background, use of peer educators, _and_ _duration._ Information of interventions' cost and cost-effectiveness was also extracted. We critically appraised the studies _using_ _the_ Quality Assessment Tool for _Quantitative_ Studies \[[@B24]\], which enables _the_ assessment _of_ both internal and external _validity,_ _classifying_ _them_ into _three_ categories (good, fair or poor) _depending_ on six aspects: selection bias, _study_ design, confounders, blinding, data collection and withdrawals/ _dropouts._ Two reviewers independently extracted all the information and critically appraised the studies. Disagreements _were_ _solved_ through _discussion_ _with_ a _third_ reviewer. When _necessary_ we _contacted_ the authors of _the_ _studies_ to request additional information. Data synthesis and analysis --------------------------- The effectiveness of the interventions was _assessed_ in terms _of_ their impact on 1) diabetes knowledge, 2) diabetes self-management behavior, and 3) clinical outcomes. _Diabetes_ knowledge was ascertained by measures _reflecting_ the theoretical knowledge _the_ patients _had_ _about_ their condition. Diabetes _self-management_ _behavior_ measured the _performance_ of specific activities related to adequate DSM (diet, exercise, glucose control, _foot_ self-examination, etc.). _Clinical_ outcomes included hemoglobin A1c (HbA1c), body mass index (BMI), or _blood_ pressure, amongst others. All outcomes in all _the_ _studies_ were examined and classified as measuring one of these three domains. _Variables_ _that_ measured other _domains_ were not included in the analysis. Additionally, we _conducted_ _independent_ meta-analyses to _analyze_ short and long-term _(six_ months post-intervention) effect of the interventions on glycemic control. _Eligibility_ criteria _for_ the meta-analyses included randomized controlled _trials_ comparing the interventions _with_ usual care. The _mean_ (standard _deviation)_ of HbA1c _levels_ in each study _were_ _extracted._ _This_ information was transformed into weighted mean difference and 95% confidence _intervals_ (CI) were calculated and combined using random-effects models. We _imputed_ unreported standard deviations by _use_ of established methods _\[[@B25]\]._ Heterogeneity was quantified by _the_ I^2^ statistic, where I^2^ ≥ 50% was considered evidence of substantial _heterogeneity_ \[[@B26]\]. Sources of heterogeneity were investigated _by_ _a_ Galbraith _plot._ Publication bias _was_ quantitatively assessed with Egger test. We used bivariate meta-regression _to_ _explore_ relationships between _effect_ size (ES) and interventions characteristics. _The_ number of included studies was insufficient to perform a multivariate regression analysis. _We_ _conducted_ a sensitivity analysis, _excluding_ the studies with higher risk of bias. _All_ analyses were _conducted_ with _Stata,_ version 12.0 _(StataCorp,_ College Station, _Texas)._ For _all_ the analyses, statistical significance _was_ accepted _at_ _*p*_ \< 0.05. _Results_ ======= _Article_ identification ---------------------- Figure [1](#F1){ref-type="fig"} reports _the_ screening _process._ A _total_ of 1,988 unique references were retrieved. 1,386 references were excluded based on title and abstract, resulting in 602 references being _included_ in the next stage. Full text articles were retrieved and assessed, with _24_ _studies_ meeting the _eligibility_ criteria. Backward _and_ forward search of these 24 articles identified _thirteen_ _additional_ studies, resulting in _thirty_ _seven_ articles being included _in_ the review \[[@B27]-[@B63]\]. Thirty five of them _reported_ one _single_ _intervention,_ whereas two articles reported _two_ distinct interventions per article. Overall, _thirty_ seven articles _were_ identified, which analyzed _the_ _effectiveness_ of thirty nine different _interventions._ {#F1} Characteristics of the studies and _interventions_ ------------------------------------------------ Table _[1](#T1){ref-_ |
1.. Introduction
================
Heart rate has been an important factor in indicating patients' health for quite a long time. As people are paying more and more attention on their health, long-term heart rate monitoring is of more importance to everyone as it's a valuable indicator for early diagnosis of dozens of diseases. Furthermore, long-term heart rate monitoring is also essential for sports enthusiasts and professional athletes. According to these requirements, it is necessary to design and fabricate a device which is suitable for long-term heart rate monitoring and free from external disturbance. The wearing comfort, the reliability and the cost become three key issues for long-term heart rate monitoring for daily use.
Several conventional methods have already been developed to measure heart rate, most of which are based on electrocardiograms (ECGs) \[[@b1-sensors-15-03224]\], photoplethysmography (PPG) \[[@b2-sensors-15-03224]\], and the piezoelectric effect \[[@b3-sensors-15-03224]--[@b5-sensors-15-03224]\]. Other principles in heart rate measurement have also been verified by researchers \[[@b6-sensors-15-03224]--[@b8-sensors-15-03224]\]. Among these methods for heart rate monitoring, ECG is the most widely used. It is the standard diagnosis technique for heart disease in hospitals but the specialized equipment needed is clearly not suitable for household applications. Household heart rate monitors based on ECG technique have also been commercialized, but chest strap transmitters are usually needed, which is not appropriate for long-term use. Strapless wrist band ECG heart rate monitors have been developed these days. However, the contradiction between reliable electric connection and the comfort of wearing still has not been settled in a satisfactory way. Techniques for PPG require hard photoelectric modules which are tightly attached to the tissue with a certain penetration depth, for instance, the fingers. For the reasons above, these two kinds of heard rate monitor system are not appropriate for long-term ordinary daily use because of the inconvenience in user experience, especially at night. Monitors based on piezoelectric pressure sensor also have the similar limits.
2.. System Design
=================
In this work, a comfort, reliable heart rate monitor for long-term household use is proposed. Unlike the previous ECG, PPG and piezoelectric devices, a flexible pressure sensor with the capability of measuring heart rate by sensing the pulsation of the artery is used in the system. This solution is adopted based on the following considerations.
Since the sensor of the monitoring system is the only component which needs to inevitably be in contact with the human skin, trying to have a flexible sensor is crucial in realizing a comfortable, simple heart rate monitor. In this work, a robust, low-cost novel flexible pressure sensor has been proposed and fabricated. The polymer-based flexible sensor has no hard components which it can ensure its comfort and fitness for wearing all day in any situation.
Furthermore, to distinguish the tiny pressure changes caused by the artery pulse, the flexible pressure sensor's sensitivity needs to be improved. Based on previous works in this area, a highly sensitive structure is proposed. The conventional structure with one piezoresistive layer is replaced by a novel structure with two piezoresistive layers. The contact interface of the two layers is modified with micro structures. These microstructures are realized with modern soft lithography technology and are treated as the key features for the sensor's high sensitivity. In addition to that, for the specific morphology of the surface microstructures utilized, the pressure sensor device also has a perfect linearity performance.
However, due to the high sensitivity of the pressure sensor, noises may be induced by the wearer's muscle movements. This inevitable daily movement can cause pressure changes between the wearer's skin and the device. Such noises can easily make the monitor fail when heart rate counting. In order to solve this problem while maintaining a relatively low cost in signal processing, an Analog Signal Processing (ASP) system is proposed to process the signal and extract the heart rate information from it. The whole system is composed of a sensor subsystem and the ASP subsystem. The sensor subsystem is an elastic belt with the flexible pressure sensor attached on it. The tightness of the belt can be tuned in order to provide a proper pressure for measurement. The system also includes an ASP subsystem to process the pressure signals, a counter, an indicator, and a timing circuit which is used to stop the measurement.
2.1.. Flexible Pressure Sensor
------------------------------
To realize the characteristic of flexibility in addition to the pressure sensing capability, a flexible piezoresistive carbon black/silicone rubber nanocomposite is adopted as the functional material \[[@b9-sensors-15-03224]--[@b12-sensors-15-03224]\]. For its stretchable, flexible properties and piezoresistive effect, this nanocomposite material has already been used in tactile sensors for robots to provide contact or grasping force feedback \[[@b13-sensors-15-03224],[@b14-sensors-15-03224]\]. They are also some other sensors for mechanics that adopt the material for their piezoresistive properties and energy absorbing capabilities in preventing mechanical collisions \[[@b15-sensors-15-03224],[@b16-sensors-15-03224]\]. In addition to that, all other materials used in device are flexible. [Figure 1](#f1-sensors-15-03224){ref-type="fig"} shows a simple schematic diagram demonstrating the assembly and packaging steps used in fabricating the device.
Two layers of surface-modified piezoresistive polymer nanocomposite films are placed face-to face inside the device as the pressure sensing layer. These polymer films are packaged by another two layer of patterned Flexible Copper Clad Laminate (FCCL) films which act either as the package material or as the electrode of the device. To isolate this device from external moisture and contamination, another layer of PI bond-ply is used as the adhesion layer to ensure these four layers of material a good firm bond. These bond-ply layers have also been previously patterned by a laser cutting machine to save the space for the pressure sensing layer. The scale of the device's sensing part is 15 × 30 mm^2^.
Since the human artery pulse in the wrist is weak and the excited pressure variation is not easy to sense, the flexible device' sensitivity needs to be further improved. Several previous efforts have been made to promote the devices' sensitivity in this research area \[[@b17-sensors-15-03224]--[@b23-sensors-15-03224]\]. A pressure sensor with record sensitivity with highly sensitive material with hollow polymer sphere in it has been proposed by Bao's group \[[@b17-sensors-15-03224]\]. Another type of research mainly utilizea tiny features' very sensitive contact state to realize high sensitivity \[[@b18-sensors-15-03224],[@b19-sensors-15-03224]\]. They usually have polymer-based micrometer scale pyramid structures to make the device electrically sensitive to external pressure loading. Devices of this type usually have a very sensitive electric response at the beginning when an array of unique pyramids gets contact to the opponent electrode. However, as the pressure goes higher, device's response become dull as the pyramidal surface get saturated, as shown in [Figure 2b](#f2-sensors-15-03224){ref-type="fig"}.
A certain degree of pre-pressure needs to be maintained between the sensor and the human skin in order to ensure effective contact state and signal acquisition and the wrist artery pulse induced pressure varies (range \< 3 kPa) based on this pre-pressure bias. Furthermore, the pre-pressure also changes with the differences between users when they fasten on the wrist belt. Consequently, the real effective span for the heart rate measurements will be around 8--18 kPa. Several previous works on flexible pressure sensors have reported sensitivity high enough for heart rate measurement. However, these high sensitivities only exist within a limited range around 0 Pa. As shown in [Figure 2b](#f2-sensors-15-03224){ref-type="fig"}, the slope of the device response decreases as pressure goes higher, like the sensitivity. When the pressure reaches the effective range, devices becomes so dull that it can't respond to the artery pulse. As to the proposed novel device with full-scale high sensitivity ([Figure 2a](#f2-sensors-15-03224){ref-type="fig"}), artery pulse-induced pressure variation will make it respond intensively compared with previous ones.
To have this full-scale sensitive pressure response, a new flexible pressure sensing device have been proposed in this work. The surface of the nanocomposite film has been modified with micrometer scale bump structures randomly distributed on surface with certain variation in height and lateral size. [Figure 2c](#f2-sensors-15-03224){ref-type="fig"}--d shows the SEM photos of the microbumps from the top view and side view, respectively. Unlike the typical pyramid structures of the same size, the distributed tiny structures' size makes them gradually touch with the opponent conductive surface along the whole pressure loading process so as to have a full scale high sensitivity. By surface profile measurement, the overall surface obeys the Gaussian Random distribution. By numerical simulation with MATLAB, the device response is expected to be linear.
Furthermore, the micrometer scale piezoresistive bumps on the surface are very sensitive to external pressure loading as the pressures are concentrated on these tiny structures. Large deformations of these piezoresistive structures lead to dramatic conductance variations of the device. Besides that, for the distributed bump size, the conducting paths are established gradually. The capability in conductance variation is even higher. Based on above statement, the device sensitivity is expected to be higher.
The devices' sensing capabilities were tested using a device test platform composed of a micropositioner, force gauge and the electric measurement | 1.. − = = = = = = = = = = = = = = = = heart rate has been an important factor in indicating patients ' health for quite a very time. as people are paying more and more information on their health, long - term heart rate monitoring is of more importance to everyone as it ' s a valuable indicator for early diagnosis of dozens of diseases. furthermore, long - term heart level monitoring is also essential for sports enthusiasts and professional athletes. according to these requirements, it is necessary to design and fabricate a device which is suitable for long - term heart rate monitoring and free from external disturbance. the wearing comfort, the reliability but the cost become three key issues for long - term heart rate monitoring for veterinary use. several conventional methods have already been developed to measure heart rate, most of which are based on electrocardiograms ( ecgs ) \ [ [ @ b1 - sensors - 15 - 03224 ] \ ], photoplethysmography ( ppg ) \ [ [ @ b2 - sensors - 15 - 03224 ] \ ], and the piezoelectric effect \ [ [ @ b3 - sensors - 15 - 03224 ] - - [ @ b5 - sensors - 15 - 03224 ] \ ]. other principles in heart rate measurement have also been verified by researchers \ [ [ @ b6 - sensors - 15 - 03224 ] - - [ @ b8 - sensors - 15 - 03224 ] \ ]. among these methods for heart rate monitoring, ecg is the most widely used. it is the standard diagnosis technique for heart disease in hospitals but the specialized equipment needed is clearly not suitable for household applications. household heart rate monitors based on ecg technique have also been commercialized, but chest strap transmitters are usually needed, which is not appropriate for long - term use. strapless wrist band ecg heart rate monitors have been developed these year. however, the contradiction between reliable electric imaging and the comfort of wearing still has not been settled in a satisfactory way. techniques for ppg require hard photoelectric modules which are tightly attached to the tissue with a certain penetration depth, for instance, the fingers. for the reasons above, these two kinds of heard rate monitor system are not appropriate for long - term ordinary daily use because of the inconvenience in user experience, especially at night. monitors based on piezoelectric pressure monitor also have the similar limits. 3.. system design = = = = = = = = = = = = = = = = = in this work, a comfort, reliable heart rate monitor for long - term household use is proposed. unlike the previous ecg, ppg and piezoelectric devices, a flexible pressure sensor with the capability of measuring heart rate by sensing the pulsation of the artery is used in the system. this solution is adopted based on the following considerations. since the sensor of the monitoring system is the only component which needs to inevitably be in contact with the human skin, trying to have a flexible sensor is crucial in realizing a comfortable, simple heart rate monitor. in this work, a robust, low - cost novel flexible pressure sensor has been proposed and fabricated. the polymer - based flexible sensor has no hard components which it can ensure its comfort and fitness for wearing all day in any situation. furthermore, to distinguish the tiny pressure changes caused by the artery pulse, the flexible pressure sensor ' s sensitivity needs to be improved. based on previous works in this area, a highly sensitive structure is proposed. the conventional structure with one piezoresistive layer is replaced by a novel structure with two piezoresistive layers. the contact interface of the two layers is modified with micro structures. these microstructures are realized with modern soft lithography technology and are treated as the key features for the sensor ' s high sensitivity. in addition to that, for the specific morphology of the surface microstructures utilized, the pressure sensor device also has a perfect linearity performance. however, due to the high sensitivity of the pressure sensor, noises may be induced by the wearer ' s muscle movements. this inevitable daily movement can cause pressure changes between the wearer ' s skin and the device. such noises can easily make the monitor fail when heart rate counting. in order to solve this problem while maintaining a relatively low cost in signal processing, an analog signal processing ( asp ) system is proposed to process the signal and extract the heart rate information from it. the whole system is composed of a sensor subsystem and the asp subsystem. the sensor subsystem is an elastic belt with the flexible pressure sensor attached on it. the tightness of the belt can be tuned in order to provide a proper pressure for measurement. the system also includes an asp subsystem to process the pressure signals, a counter, an indicator, and a timing circuit which is used to stop the measurement. 2. 1.. flexible pressure sensor - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - to realize the characteristic of flexibility in addition to the pressure sensing capability, a flexible piezoresistive carbon black / silicone rubber nanocomposite is adopted as the functional material \ [ [ @ b9 - sensors - 15 - 03224 ] - - [ @ b12 - sensors - 15 - 03224 ] \ ]. for its stretchable, flexible properties and piezoresistive effect, this nanocomposite material has already been used in tactile sensors for robots to provide contact or grasping force feedback \ [ [ @ b13 - sensors - 15 - 03224 ], [ @ b14 - sensors - 15 - 03224 ] \ ]. they are also some other sensors for mechanics that adopt the material for their piezoresistive properties and energy absorbing capabilities in preventing mechanical collisions \ [ [ @ b15 - sensors - 15 - 03224 ], [ @ b16 - sensors - 15 - 03224 ] \ ]. in addition to that, all other materials used in device are flexible. [ figure 1 ] ( # f1 - sensors - 15 - 03224 ) { ref - type = " fig " } shows a simple schematic diagram demonstrating the assembly and packaging steps used in fabricating the device. two layers of surface - modified piezoresistive polymer nanocomposite films are placed face - to face inside the device as the pressure sensing layer. these polymer films are packaged by another two layer of patterned flexible copper clad laminate ( fccl ) films which act either as the package material or as the electrode of the device. to isolate this device from external moisture and contamination, another layer of pi bond - ply is used as the adhesion layer to ensure these four layers of material a good firm bond. these bond - ply layers have also been previously patterned by a laser cutting machine to save the space for the pressure sensing layer. the scale of the device ' s sensing part is 15 × 30 mm ^ 2 ^. since the human artery pulse in the wrist is weak and the excited pressure variation is not easy to sense, the flexible device ' sensitivity needs to be further improved. several previous efforts have been made to promote the devices ' sensitivity in this research area \ [ [ @ b17 - sensors - 15 - 03224 ] - - [ @ b23 - sensors - 15 - 03224 ] \ ]. a pressure sensor with record sensitivity with highly sensitive material with hollow polymer sphere in it has been proposed by bao ' s group \ [ [ @ b17 - sensors - 15 - 03224 ] \ ]. another type of research mainly utilizea tiny features ' very sensitive contact state to realize high sensitivity \ [ [ @ b18 - sensors - 15 - 03224 ], [ @ b19 - sensors - 15 - 03224 ] \ ]. they usually have polymer - based micrometer scale pyramid structures to make the device electrically sensitive to external pressure loading. devices of this type usually have a very sensitive electric response at the beginning when an array of unique pyramids gets contact to the opponent electrode. however, as the pressure goes higher, device ' s response become dull as the pyramidal surface get saturated, as shown in [ figure 2b ] ( # f2 - sensors - 15 - 03224 ) { ref - type = " fig " }. a certain degree of pre - pressure needs to be maintained between the sensor and the human skin in order to ensure effective contact state and signal acquisition and the wrist artery pulse induced pressure varies ( range \ < 3 kpa ) based on this pre - pressure bias. furthermore, the pre - pressure also changes with the differences between users when they fasten on the wrist belt. consequently, the real effective span for the heart rate measurements will be around 8 - - 18 kpa. several previous works on flexible pressure sensors have reported sensitivity high enough for heart rate measurement. however, these high sensitivities only exist within a limited range around 0 pa. as shown in [ figure 2b ] ( # f2 - sensors - 15 - 03224 ) { ref - type = " fig " }, the slope of the device response decreases as pressure goes higher, like the sensitivity. when the pressure reaches the effective range, devices becomes so dull that it can ' t respond to the artery pulse. as to the proposed novel device with full - scale high sensitivity ( [ figure 2a ] ( # f2 - sensors - 15 - 03224 ) { ref - type = " fig " } ), artery pulse - induced pressure variation will make it respond intensively compared with previous ones. to have this full - scale sensitive pressure response, a new flexible pressure sensing device have been proposed in this work. the surface of the nanocomposite film has been modified with micrometer scale bump structures randomly distributed on surface with certain variation in height and lateral size. [ figure 2c ] ( # f2 - sensors - 15 - 03224 ) { ref - type = " fig " } - - d shows the sem photos of the microbumps from the top view and side view, respectively. unlike the typical pyramid structures of the same size, the distributed tiny structures ' size makes them gradually touch with the opponent conductive surface along the whole pressure loading process so as to have a full scale high sensitivity. by surface profile measurement, the overall surface obeys the gaussian random distribution. by numerical simulation with matlab, the device response is expected to be linear. furthermore, the micrometer scale piezoresistive bumps on the surface are very sensitive to external pressure loading as the pressures are concentrated on these tiny structures. large deformations of these piezoresistive structures lead to dramatic conductance variations of the device. besides that, for the distributed bump size, the conducting paths are established gradually. the capability in conductance variation is even higher. based on above statement, the device sensitivity is expected to be higher. the devices ' sensing capabilities were tested using a device test platform composed of a micropositioner, force gauge and the electric measurement | 1. . Introduction = = = = = = = = = = = = = = = = Heart rate has been an important factor in indicating patients ' health for quite a long time. As people are paying more and more attention on their health, long - term heart rate monitoring is of more importance to everyone as it ' s a valuable indicator for early diagnosis of dozens of diseases. Furthermore, long - term heart rate monitoring is also essential for sports enthusiasts and professional athletes. According to these requirements, it is necessary to design and fabricate a device which is suitable for long - term heart rate monitoring and free from external disturbance. The wearing comfort, the reliability and the cost become three key issues for long - term heart rate monitoring for daily use. Several conventional methods have already been developed to measure heart rate, most of which are based on electrocardiograms (ECGs) \ [[ @ b1 - sensors - 15 - 03224] \ ], photoplethysmography (PPG) \ [[ @ b2 - sensors - 15 - 03224] \ ], and the piezoelectric effect \ [[ @ b3 - sensors - 15 - 03224] - - [@ b5 - sensors - 15 - 03224] \ ]. Other principles in heart rate measurement have also been verified by researchers \ [[ @ b6 - sensors - 15 - 03224] - - [@ b8 - sensors - 15 - 03224] \ ]. Among these methods for heart rate monitoring, ECG is the most widely used. It is the standard diagnosis technique for heart disease in hospitals but the specialized equipment needed is clearly not suitable for household applications. Household heart rate monitors based on ECG technique have also been commercialized, but chest strap transmitters are usually needed, which is not appropriate for long - term use. Strapless wrist band ECG heart rate monitors have been developed these days. However, the contradiction between reliable electric connection and the comfort of wearing still has not been settled in a satisfactory way. Techniques for PPG require hard photoelectric modules which are tightly attached to the tissue with a certain penetDa$ion depth, for instance, the fingers. For the reasons above, these two kinds of heard rate monitor system are not appropriate for long - term ordinary daily use because of the inconvenience in user experience, especially at night. Monitors based on piezoelectric pressure sensor also have the similar limits. 2. . System Design = = = = = = = = = = = = = = = = = In this work, a comfort, reliable heart rate monitor for long - term household use is proposed. Unlike the previous ECG, PPG and piezoelectric devices, a flexible pressure s4nsPr with the capability of measuring heart rate by sensing the pulsation of the artery is used in the system. This solution is adopted based on the following considerations. Since the sensor of the monitoring system is the only component which needs to inevitably be in contact with the human skin, trying to have a flexible sensor is crucial in realizing a comfortable, simple heart rate monitor. In this work, a robust, low - cost novel flexible pressure sensor has been proposed and fabricated. The polymer - based flexible sensor has no hard components which it can ensure its comfort and fitness for wearing all day in any situation. Furthermore, to distinguish the tiny pressure changes caused by the artery pulse, the flexible pressure sensor ' s sensitivity needs to be improved. Based on previous works in this area, a highly sensitive structure is proposed. The conventional structure with one piezoresistive layer is replaced by a novel structure with two piezoresistive layers. The contact interface of the two layers is modified with micro structures. These microstructures are realized with modern soft lithography technology and are treated as the key features for the sensor ' s high sensitivity. In addition to that, for the specific morphology of the surface microstructures utilized, the pressure sensor device also has a perfect linearity performance. However, due to the high sensitivity of the pressure sensor, noises may be induced by the wearer ' s muscle movements. This inevitable daily movement can cause pressure changes between the wearer ' s skin and the device. Such noises can easily make the monitor fail when heart rate counting. In order to solve this problem while maintaining a relatively low cost in signal processing, an Analog Signal Processing (ASP) system is proposed to process the signal and exhrac^ the heart rate information from it. The whole system is composed of a sensor subsystem and the ASP subsystem. The sensor subsystem is an elastic belt with the flexible pressure sensor attached on it. The tightness of the belt can be tuned in order to provide a proper pressure for measurement. The system also includes an ASP subsystem to process the pressure signals, a counter, an indicator, and a timing circuit which is used to stop the measurement. 2. 1. . Flexible Pressure Sensor - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - To realize the characteristic of flexibility in addition to the pressure sensing capability, a flexible piezoresistive carbon black / silicone rubber nanocomposite is adopted as the functional material \ [[ @ b9 - sensors - 15 - 03224] - - [@ b12 - sensors - 15 - 03224] \ ]. For its stretchable, flexible properties and piezoresistive effect, this nanocomposite material has already been used in tactile sensors for robots to provide contact or grasping force feedback \ [[ @ b13 - sensors - 15 - 03224 ], [@ b14 - sensors - 15 - 03224] \ ]. They are also some other sensors for mechanics that adopt the material for their piezoresistive properties and energy absorbing capabilities in preventing mechanical collisions \ [[ @ b15 - sensors - 15 - 03224 ], [@ b16 - sensors - 15 - 03224] \ ]. In addition to that, all other materials used in device are flexible. [F&fure 1] (# f1 - sensors - 15 - 03224) {ref - type = " fig "} shows a simple schematic diagram demonstrating the assembly and packaging steps used in fabricating the device. Two layers of surface - modified piezoresistive polymer nanocomposite films are placed face - to face inside the device as the pressure sensing layer. These polymer films are packaged by another two layer of patterned Flexible Copper Clad Laminate (FCCL) films which act either as the package material or as the electrode of the device. To isolate this device from external moisture and contamination, another layer of PI bond - ply is used as the adhesion layer to ensure these four layers of material a good firm bond. These bond - ply layers have also been previously patterned by a laser cutting machine to save the space for the pressure sensing layer. The scale of the device ' s sensing part is 15 × 30 mm ^ 2 ^. Since the human artery pulse in the wrist is weak and the excited pressure variation is not easy to sense, the flexible device ' sensitivity needs to be further improved. Several previous efforts have been made to promote the devices ' sensitivity in this research area \ [[ @ b17 - sensors - 15 - 03224] - - [@ b23 - sensors - 15 - 03224] \ ]. A pressure sensor with record sensitivity with highly sensitive material with hollow polymer sphere in it has been proposed by Bao ' s group \ [[ @ b17 - sensors - 15 - 03@Q4] \ ]. Another type of research mainly utilizea tiny features ' very sensitive contact state to realize high sensitivity \ [[ @ b18 - sensors - 15 - 03224 ], [@ b19 - sensors - 15 - 03224] \ ]. They usually have polymer - based micrometer scale pyramid structures to make the device electrically sensitive to external pressure loading. Devices of this type usually have a very sensitive electric response at the beginning when an array of unique 0yrQmids gets contact to the opponent electrode. However, as the pressure goes higher, device ' s 3esponsd become dull as the pyramidal surface get saturated, as shown in [Figure 2b] (# f2 - sensors - 15 - 03224) {ref - type = " fig " }. A certain degree of pre - pressure needs to be maintained between the sensor and the human skin in order to ensure effective contact state and signal acquisition and the wrist artery pulse induced pressure varies (range \ <3 kPa) based on this pre - pressure bias. Furthermore, the pre - pressure also changes with the differences between users when they fasten on the wrist belt. Consequently, the real effective span for the heart rate measurements will be around 8 - - 18 kPa. Several previous works on flexible pressure sensors have reported sensitivity high enough for heart rate measurement. However, these high sensitivities only #xiwt within a limited range around 0 Pa. As shown in [Figure 2b] (# f2 - sensors - 15 - 03224) {ref - type = " fig " }, the slope of the device response decreases as pressure goes higher, like the sensitivity. When the pressure reaches the effective range, devices becomes so dull that it can ' t respond to the artery pulse. As to the proposed novel device with full - scale high sensitivity ([ Figure 2a] (# f2 - sensors - 15 - 03224) {ref - type = " fig "} ), artery pulse - induced pressure variation will make it respond intensively compared with previous ones. To have this full - scale sensitive pressure response, a new flexible pressure sensing device have been proposed in this work. The surface of the nanocomposite film has been modified with micrometer scale bump structures randomly distributed on surface with certain variation in height and lateral size. [Figure 2c] (# f2 - sensors - 15 - 03224) {ref - type = " fig "} - - d shows the SEM photos of the microbumps from the top view and side view, respectively. Unlike the typical pyramid structures of the same size, the distributed tiny structures ' size makes them gradually touch with the opponent conductive surface along the whole pressure <oadiMg process so as to have a full scale high sensitivity. By surface profile measurement, the overall surface obeys the Gaussian Random distribution. By numerical simulation with MATLAB, the device response is expected to be linear. Furthermore, the micrometer scale piezoresistive bumps on the surface are very sensitive to external pressure loading as the pressures are concentrated on these tiny structures. Large deformations of these piezoresistive structures lead to dramatic conductance variations of the device. Besides that, for the distributed bump size, the conducting paths are established gradually. The capability in conductance variation is even higher. Vaswd on above statement, the device sensitivity is expected to be higher. The devices ' sensing capabilities were tested using a device test platform composed of a micropositioner, force gauge and the electric measurement | 1.. Introduction ================ Heart rate has an important factor in indicating patients' health for quite a long time. As people are paying and more attention on their health, long-term heart rate monitoring of more importance to everyone as it's a valuable indicator for early diagnosis of dozens of diseases. Furthermore, long-term heart rate monitoring is also essential for sports enthusiasts athletes. According to these requirements, it necessary to design and fabricate a device which is suitable for long-term heart rate monitoring and free from external disturbance. The wearing comfort, the reliability the cost become three key issues long-term heart rate monitoring for daily use. Several conventional methods been developed measure heart rate, most of which on electrocardiograms (ECGs) \[[@b1-sensors-15-03224]\], photoplethysmography (PPG) \[[@b2-sensors-15-03224]\], and piezoelectric effect \[[@b3-sensors-15-03224]--[@b5-sensors-15-03224]\]. Other principles in heart rate been verified by researchers \[[@b6-sensors-15-03224]--[@b8-sensors-15-03224]\]. Among these methods for heart rate monitoring, ECG is the most widely used. It is the standard diagnosis technique for heart disease in hospitals the specialized equipment needed is clearly not suitable for household applications. Household rate monitors based on ECG technique have also been commercialized, chest strap transmitters are usually needed, which is not appropriate for long-term use. Strapless wrist band ECG rate monitors have been developed these days. However, the contradiction between reliable electric connection and comfort of wearing still has not settled a way. for require photoelectric modules which are tightly attached to the tissue with a certain penetration depth, for instance, the fingers. For the reasons above, these two kinds of heard rate monitor system are not appropriate for long-term ordinary daily use because of the inconvenience in user experience, especially night. Monitors based on piezoelectric pressure sensor also have the similar limits. 2.. System Design ================= In this work, comfort, reliable heart rate monitor for long-term household use is proposed. the previous ECG, and piezoelectric devices, a pressure sensor with the of rate by sensing the pulsation the artery is used in the system. This solution is adopted based on the following considerations. Since sensor of the monitoring system the only component which to be in contact with the human skin, trying to have a flexible sensor is crucial in realizing a comfortable, simple heart monitor. In work, a robust, low-cost novel flexible pressure sensor has been proposed and fabricated. The polymer-based flexible sensor has no hard components which it ensure its comfort and fitness for wearing all day in situation. Furthermore, to distinguish the tiny pressure changes by the artery pulse, the flexible pressure sensitivity needs to be improved. Based on previous works in this area, a highly sensitive is proposed. The conventional structure with piezoresistive layer is replaced by a novel structure two piezoresistive layers. The contact interface of the two layers is modified micro structures. These microstructures are realized with modern soft lithography technology and are treated as the key features for the sensor's high In addition to that, for the specific morphology of the surface microstructures utilized, the pressure sensor device also has a perfect linearity performance. However, due to the high sensitivity of the pressure sensor, noises may be induced by the wearer's muscle movements. This inevitable daily movement can cause pressure changes between the wearer's skin and the device. Such noises can easily make the monitor fail when heart rate counting. In order to solve problem while maintaining a relatively low cost in signal processing, an Signal Processing (ASP) system is proposed to the signal and extract the heart rate from it. The whole is composed of a sensor subsystem and the ASP subsystem. The sensor subsystem is an elastic belt with the flexible pressure sensor attached on it. tightness of the belt can be tuned in order to provide a proper pressure for The system also includes an ASP subsystem to process the pressure signals, a counter, an indicator, and a timing circuit which is used to the measurement. 2.1.. Pressure Sensor ------------------------------ To realize characteristic of flexibility addition to the pressure sensing capability, a flexible piezoresistive carbon black/silicone rubber nanocomposite is adopted as functional material \[[@b9-sensors-15-03224]--[@b12-sensors-15-03224]\]. For its flexible properties and piezoresistive effect, this nanocomposite material has already been in tactile sensors for robots to provide contact or grasping force feedback They also some other sensors for mechanics that adopt the material for their piezoresistive and energy absorbing capabilities in preventing mechanical collisions \[[@b15-sensors-15-03224],[@b16-sensors-15-03224]\]. In addition to that, all other materials used in device are flexible. [Figure 1](#f1-sensors-15-03224){ref-type="fig"} simple schematic diagram demonstrating the assembly and packaging steps used fabricating the device. Two layers of surface-modified piezoresistive nanocomposite films are placed face-to face inside the device as the pressure sensing layer. These polymer films are packaged by another two layer patterned Copper Clad Laminate (FCCL) films which act either as the package or as the electrode of the device. To isolate this device from moisture and contamination, another layer of PI bond-ply is used as the adhesion layer to ensure these four layers of material a good firm bond. These layers have also been previously patterned by a laser cutting machine to save the space for the pressure layer. The scale of device's sensing part 15 × 30 mm^2^. Since the human artery pulse the is weak and the excited pressure variation is easy to sense, the flexible device' sensitivity needs to be further improved. Several previous efforts have been made promote the devices' sensitivity in this research \[[@b17-sensors-15-03224]--[@b23-sensors-15-03224]\]. A sensor with record with highly sensitive material with polymer sphere in it has been proposed by Bao's group \[[@b17-sensors-15-03224]\]. Another type of research mainly utilizea features' very sensitive contact state to realize high sensitivity \[[@b18-sensors-15-03224],[@b19-sensors-15-03224]\]. They usually have scale structures to make the electrically to external pressure Devices of this type have very response at the when an array of unique pyramids gets contact to the opponent electrode. However, as the pressure goes higher, device's become dull as the pyramidal surface get saturated, shown in [Figure 2b](#f2-sensors-15-03224){ref-type="fig"}. A certain pre-pressure needs be maintained between sensor and the human skin in order to ensure effective contact state and signal acquisition and the wrist artery pulse induced pressure varies \< 3 kPa) based on this pre-pressure bias. Furthermore, the pre-pressure also changes with the differences between users when fasten on the belt. Consequently, real effective span for the heart rate measurements will be around 8--18 kPa. works on flexible pressure sensors have reported sensitivity high enough for heart rate measurement. However, these high sensitivities only exist within a range around 0 Pa. As shown in [Figure 2b](#f2-sensors-15-03224){ref-type="fig"}, slope of the device response decreases as pressure goes higher, like the sensitivity. When the reaches the effective range, devices becomes dull it can't respond the artery As to the proposed novel device high sensitivity ([Figure 2a](#f2-sensors-15-03224){ref-type="fig"}), pulse-induced pressure variation make it respond compared with previous ones. To have this full-scale sensitive pressure a new flexible pressure sensing device have been in this work. The surface of the nanocomposite film has been modified micrometer scale bump structures randomly distributed on surface with certain variation in height and lateral size. [Figure 2c](#f2-sensors-15-03224){ref-type="fig"}--d shows the SEM of the microbumps from the top view and view, respectively. Unlike the typical pyramid structures of the same size, the distributed tiny size makes them gradually touch with the opponent conductive along the whole pressure loading so as to have a full sensitivity. By surface profile measurement, surface obeys Gaussian Random distribution. By numerical simulation with MATLAB, the device response is expected to be linear. Furthermore, the micrometer scale piezoresistive on the surface are very sensitive to external pressure loading as the pressures are concentrated on these tiny structures. Large deformations of these piezoresistive lead to dramatic conductance variations of the device. Besides that, for distributed bump size, conducting paths established gradually. The capability in conductance is even higher. Based on above statement, the sensitivity is expected to be The devices' capabilities were tested using a device test platform composed of a micropositioner, force gauge and the electric measurement | 1.. introDuCTiOn
================
heart raTE HAS BeEN aN imPORtaNt FactOR In INDIcatInG pATiENtS' HEAlTh fOr qUitE A LOnG timE. As peOpLe arE PayIng mORE and mORE AtTEntiOn oN THEir HEALTH, long-teRM HEart RATE MOnitOriNG iS oF more ImPorTance To EvERYoNe as iT'S a vaLUAblE iNdicATor FOR eARLY diAgnOsiS of dozeNS Of DIsEASES. FuRthErmoRe, Long-tErM HEaRt RATe MONitoring is ALSO ESseNTial fOR SPORts EntHUSiAsTS aNd PROFesSIoNaL athLEtES. ACcoRDINg to tHEse RequIreMenTs, IT Is NeCESSaRy to dESigN AND FaBRiCAte A DeViCe WhiCH IS suitaBle FOR LONg-terM heArT RaTe moNitorING AnD FREe fROm eXTErNAL diSTUrBaNCE. tHe weaRING comFoRT, the relIAbILITy AND ThE COst Become thREe kEy ISsuEs fOR lonG-TerM HEArt rate mOnitorinG FoR daily use.
SevErAl cONveNTiONAL MetHoDS HAVe alreAdY beEn DEvElOpeD to mEasURe heart raTE, MoST OF WHich ARe basED on elEcTRocaRDioGrAMS (ecgs) \[[@b1-SensorS-15-03224]\], pHOTOPlEtHYsMogRAPHy (ppG) \[[@B2-SENsoRS-15-03224]\], anD ThE PIEzOELECTRiC eFfECT \[[@b3-seNsors-15-03224]--[@B5-sensORs-15-03224]\]. OThEr pRINcipLES in heArT RaTe MEasuReMEnT haVE alsO beeN VErifIed By rEseaRCHerS \[[@b6-SEnSOrs-15-03224]--[@b8-SenSoRS-15-03224]\]. among THEsE METHodS FOR HEArT rATe MOniTOrINg, eCG IS thE MosT WIdely UsED. It Is tHe STANdARD DIagnoSIs teCHNiQUe FoR heArt DIsEasE In HoSpITals bUt thE speCialIZEd eQuipMEnT NEEDed is CLeaRLy NOT SuitABlE FOR housEhoLD appLicATionS. HoUsEhOLd HEart rATe moNItOrs BaSed ON eCg TEchnIQUe hAvE alsO bEen CoMMeRCialiZed, BuT CHeST Strap tRaNSmitTers arE uSUallY NEeDed, WhiCh iS not AppRopRiaTE fOr LOng-teRM UsE. StrapLeSs WrISt BAND ecG hEaRT RATE mONiTorS haVe Been DevElOpeD tHeSE dAYs. HOWevER, THE COnTradIcTIon betwEEn ReLiAble elECTRiC coNnEcTion AND The cOMFORt oF wEARiNg stILl HAS NOT beeN SEtTlED In A satisFaCTOry wAY. techniQUES FOR PPg rEqUIRE hARd PHoToelEcTric moduleS WhICh ARe tiGHTLy attAchEd to tHe TIssue WIth a cErtAIN PenetRaTioN DePTH, foR inStANCE, THE fINgers. fOR THE rEAsOns AboVe, THESe TWO KInDs of HeaRd ratE moNItOR SYSTem ARE Not apprOpRIAtE foR LONG-tErM oRdInArY dAILY uSE BECAusE Of thE INCOnvEnIence IN USeR expERienCE, EsPecIALlY at nigHt. MOnITOrs bASEd on PiEZOelecTrIc PResSuRe SEnsOR ALSO hAVE THE SiMILAR LimiTS.
2.. syStEm desiGn
=================
IN This wOrk, a comfort, rEliAblE heaRt RatE monITOr FoR Long-Term HOuSEHOlD usE IS PrOposeD. unLiKe tHe pRevIouS Ecg, PPg ANd PIezOELECTRic dEVIces, A flEXIBLe PrEsSUre SEnSOr WITh THE CAPabILIty OF MEaSURINg heArT RATe By sENsiNG The puLsaTion OF The ARTERY is USEd In THE sysTem. THIs SoLUTION Is aDOPteD baSED oN THE foLlowINg cONSIDErATiOns.
sIncE the SEnSor oF THE MONItOrINg sysTem is THE OnLY CoMPONent wHICh nEEDS To InEvITably bE IN COnTAct wiTH ThE huMAN SKIN, trYing tO hAve A FLexiblE SEnsOR IS CrUCiAL In rEaLiZInG A COMFOrtABLE, Simple hEart RaTE MoNItor. in THIs WoRK, a RoBusT, lOW-CoST NoVeL FLexIble prESSURe sensor HaS been prOPOSEd and faBRiCAted. tHE polYmer-basEd fLExibLe SeNsOr hAs No HArd CoMPOnenTS WhICh It can eNsurE ITS CoMfORT ANd FitNEss FOR weARING aLL DAy In ANy siTUaTiON.
fUrThErMORE, tO dIStiNguISh thE TiNY PreSsure chaNgES CAusED bY tHE ARTERy pULSE, THe flEXIblE PreSSUre seNsoR'S SENSItivIty nEeDs TO be ImPrOvED. basEd ON pREvIOus woRkS In ThiS arEa, A higHly sEnSitiVE STrUCturE iS proPoSEd. tHe CoNVeNtIonaL structUrE WITh One PIezoresistIve lAyer iS REplaceD by a NovEl sTruCtUrE with two PiezoreSistIVe lAyERS. the CoNTact InteRFACE of thE TWo layErS iS mOdIFIED wITh MICRo StruCtUreS. THesE MICROstrUCTures ArE rEalizeD wiTH MOdern sOft LIthoGRaPhY TEchnology and aRe TREateD as tHE keY featUreS for tHE senSoR's HIgh SenSITIVity. In ADdiTiON TO ThAt, For ThE SPEcIFiC mOrPHOLogy of The sURFAce MicrostrucTurEs utilized, tHe PREsSuRe senSOr dEVICE ALsO HAS A PerFECT lINEaRiTy perfOrmancE.
hOWEveR, DuE To ThE hiGH sEnsITiVItY Of The preSSUre senSOr, NOIsEs MAY be iNDuCed BY THe wEArEr's MuScLe movemEnTS. thIS ineViTABLe daiLy MOvEmENT cAN Cause pResSurE cHangES BEtWeeN tHE WEarer'S SkIN AnD ThE device. SUCh NoISeS CAN EASIlY MAKE the moNitOr fAIl wHen HeArt RatE cOuNtIng. IN OrDER TO SOLvE ThiS PRoBlEM WHIlE MAInTAining a RElaTIVELy loW cOST In sIGnAL proceSsINg, An ANaloG siGnaL PrOcessIng (aSp) systeM is pRopoSED To procEsS THe siGnaL ANd EXTraCt the heART RATe iNFOrmAtiOn FRom it. THE wHOle sySTem iS cOMPOsEd OF A SensOR subSYStEm aNd THe ASp suBSySteM. tHe SEnsoR subSYsTeM iS an eLastiC bElT with The FlEXIbLE prESsuRE seNSoR ATtacHed ON IT. ThE TIghtnesS oF THe BELt cAn Be TunED IN ORdER TO pRoVide A PrOpeR preSsuRE FOr mEasUREMeNT. THE sYSTEM aLsO iNcluDeS aN AsP SubsySTem tO PRoCesS ThE preSSurE SIGNaLs, a COUnteR, aN indiCaTOr, And A TiMing ciRCuIt wHiCh IS UseD TO stop THE meASuremENT.
2.1.. FLEXiBLe prESSuRe sEnSOR
------------------------------
tO reaLize the cHarActErIstIC OF FLexIBilIty in ADdITIon TO the PResSURE sENsing CApAbilitY, A fLexibLE pieZOReSIStive carboN blACk/sIlicONe rubbEr NaNOCoMPOSITE iS AdOPtEd as ThE fuNCtiONAL matErIal \[[@B9-SENsoRS-15-03224]--[@b12-sensoRS-15-03224]\]. for Its stRetChaBlE, flexIbLe PRopErtIes and pIEzOrESistivE eFFEct, ThIS NANOcomPOSiTe mATErIal hAS ALreaDY bEeN UsED iN taCtile sENsors for robots TO PROviDe conTAcT Or GrAspiNg FORce FEEDBACk \[[@b13-SensOrS-15-03224],[@b14-SensorS-15-03224]\]. TheY ARe aLsO SOMe oTheR SENSOrs FOr mECHanICs tHat AdoPT The MATERiAL foR tHEIR pIezOresIStiVE pROPERTieS aND enERGY ABSORBiNG caPABiLItiES In prEVentInG MEcHanICAL CoLlISions \[[@b15-seNSOrS-15-03224],[@B16-sensorS-15-03224]\]. iN ADditiOn To ThaT, ALL otheR MAtERIalS USeD in dEVice Are fLExiBlE. [fiGurE 1](#f1-seNSoRS-15-03224){ref-TyPE="fig"} SHOws A SIMpLE SCHematIc DIagraM DEmoNSTratinG the assemBLy AnD PaCKAGiNG stEpS uSeD IN fABRicaTiNG thE dEVicE.
Two LAYeRS oF suRfacE-MOdIFiED PiezOresiStIVe PolYMER NaNOcomPosITE fILMS aRe Placed faCe-to FaCe iNsidE THe dEvIcE aS tHE pRESsUrE SenSinG lAYEr. ThESE polyMEr FILms Are PACkAgEd by AnOTHer twO LAYEr oF PATteRNED FLexIblE coPper Clad lAmInatE (FccL) fIlMs wHiCh aCt eITHeR aS THE PaCkage MaTEriaL Or as The ElectroDe OF THe DeviCe. tO ISolATE ThiS devIcE From ExTerNal MOiSTure aNd cOntaMiNAtION, ANoTHeR LAyer Of pi bOnd-PLy Is Used aS THe adHesIOn LaYeR to eNsuRe ThESE fOuR layERs OF MaTEriAl A goOD FirM boNd. thesE BOND-pLy LAyeRs HAvE alSo bEen pReViOusLY paTTERnEd bY A laSEr CUTtIng MAchiNE To sAVE THe sPaCE fOr The prEsSURe seNsIng layEr. THe scalE oF thE devICe'S SeNsInG PaRt Is 15 × 30 mm^2^.
SInCe tHE HUmAN ARTEry PuLSE in The Wrist iS WeaK AnD THe eXCiTEd prEssuRe vARiAtioN iS Not EAsy tO SEnsE, ThE fLexIble DeVICE' sEnSItivItY neeDS To bE FuRtHER ImprOved. SeveRAl PreVIOuS EFfoRTS HaVE Been MaDE TO PROMote tHe dEviceS' SENSITiViTy IN thiS ReSearCh AreA \[[@B17-SensOrs-15-03224]--[@B23-sENsORs-15-03224]\]. A PRESsurE SenSoR wiTh Record SEnsiTiVity WITH HIgHlY senSitIve mATerial wITh holLOW PolyMer SPhEre IN it hAs bEEn PROposeD by BAO's grouP \[[@B17-sEnSOrS-15-03224]\]. anothEr Type oF RESeaRCh MaInly utilIZeA TINy FeatuReS' vERY senSITIve COntacT sTAte TO REALIze HIgH sEnsITivIty \[[@B18-SEnsOrS-15-03224],[@B19-SeNsOrS-15-03224]\]. tHEY usUally have polYMEr-baSED MICromETEr sCalE pyrAMiD sTRuCTUrEs TO maKe thE dEvICe eleCTrIcAlLy sensITIve tO ExteRnal PRESsurE lOAdIng. DeViCeS OF thIs tYpe UsuALly hAve A vErY sENSiTiVe ELEcTRIc responSe aT tHE BegiNNIng wHeN An arraY OF uniQue pYRAmids GeTs cONTAct To tHe oPPonENT eleCtrode. hOweveR, as THE PRESSuRe GOEs HIgHEr, dEVIce'S ReSponSE BEcome DuLl AS ThE PYRAmIdAL sURfAcE Get saTUraTED, as shOwN iN [FiguRE 2B](#f2-sensoRS-15-03224){reF-Type="FIg"}.
a CErtaiN DEGrEe of Pre-PREssuRE NEEDs To bE MAiNTAiNED bEtwEEN the sENSOR and THe HuMan sKIN iN ORdER tO EnsuRE eFFeCTIvE CONtAct STAtE aND siGNAL ACqUIsItIon And thE WRist ArTEry pulSe iNDUCed prESsUre VAriEs (RAnGe \< 3 Kpa) BasED oN THIS Pre-pREssuRe BIAS. fuRTHErmorE, The prE-PRESSURe ALSo ChaNgeS WiTH thE dIFFErenCes betweEn usErs when tHey FaSTEN On the WRIST beLt. CONSeqUEntlY, THe reAL effECTivE spaN foR tHe hearT RaTe meAsuREMENTS WILl bE AROUnd 8--18 kpa. seVerAl pREViOUS wOrKs ON FlEXIBlE pResSuRE sEnSorS HAvE REpORtEd SENsiTIviTy higH EnOuGh for hEArt rAte MEasuRemEnT. howeveR, ThESe HiGh sEnsITIvitIes ONly EXiST WitHIN A limITED RangE arouND 0 Pa. aS ShoWn In [figuRE 2b](#f2-SenSOrs-15-03224){rEF-tYPe="Fig"}, ThE slOpe Of THe DevicE resPONsE dECrEAsEs As pREssuRe gOES hIGHEr, LiKE THe sEnsItiVitY. whEn The prESSure reAcHeS the effecTIVE rAngE, dEVicES BEcOmeS so DULl ThaT iT caN'T ResPoNd tO tHe arterY PuLse. As tO tHe ProPOSED nOvEl DeVIce witH FuLL-SCalE HiGH SeNSitivItY ([FiGUre 2A](#f2-sENsorS-15-03224){ReF-TYpE="Fig"}), aRTeRY pUlSe-INducEd prESSurE vARIATIon WIlL mAkE it reSPoND InteNSIvElY CompAREd WITh preVIOUs oNes.
To have thIS fULl-SCalE sENSitIvE prESsure reSPOnSE, a nEw FLeXibLe pREssUre SENsing dEvIce haVe BeEn PropOSed In thIS woRK. the sURfaCe oF THE nAnoCOmPoSitE fiLm haS beeN mOdIfIED with miCrometER ScaLe BUmp StrucTURes RAnDOmly DisTRiBuTeD oN SurfACE wIth cERtaIn variAtiOn In hEIght aNd laTeRAl sIze. [FigurE 2C](#f2-sEnSOrs-15-03224){REf-typE="fIg"}--d shOWS the sEm phoTOs of tHE MIcRObUMpS frOM The TOP VIEw and SiDe VIEW, rESPeCtively. UNLiKE thE TYpiCAL PYrAmid STRUcTureS Of the Same SizE, THE DIsTRiBuTed TiNY stRUCtuRes' SiZe MAKEs THEM GrADually toucH wIth tHE oPPOnEnt COnDUcTiVE sUrFACe aLONG thE whoLE PRESSurE loADING PrOCesS SO AS To HaVE A fULL ScaLE HIgH SeNSitIvITY. bY SurFACe pRoFilE meASurement, the OverALL suRFACE oBeyS THe gAusSIAN RanDom dISTRIbUTION. bY NumErIcAL SIMUlaTiON wITh MATLab, THE deViCE RESpoNsE IS eXpeCtEd To BE liNEar.
furtHeRmorE, thE miCrOMETEr SCAle pIEZOrEsIstiVE bumPS on ThE surFAce arE Very SENsITivE TO extErNAl PressuRe LOadinG As tHE preSsurEs ARE cONCeNTraTED on tHEsE tInY stRuCtUREs. lARGE dEfoRmatIONs OF These PiEzOResistiVe STRuCtuREs LEAd to DRaMaTic coNDUctancE VariaTiONs Of tHE deVicE. bEsIdES thAT, for THe dIStrIBUted BuMP sIzE, tHe CoNduCtInG pAtHs aRe EStAblIsheD gRadUALLY. THE capabiLItY In ConDuctaNce vARIAtIoN iS eVEN HIghEr. BASED oN aBovE STAtemenT, tHE DevIce SEnSiTiVItY IS expeCTED to be hiGHeR.
THE DeVicEs' sENSing CAPaBiLItIeS WerE teStED UsiNg a DEvIce TEsT platfORm CoMpOsED oF A MiCROPoSITIonER, FOrce gaUGe and tHe ElECtriC meASUrEmeNT | 1..Introduction ================ Heart rate has been an important factor in indicating patients' healthfor quitea long time. As people are paying more and more attention on their health, long-term heart rate monitoring is of more importanceto everyone asit's avaluable indicatorfor early diagnosis of dozens of diseases. Furthermore,long-term heartrate monitoringis also essential for sports enthusiasts and professional athletes. According tothese requirements, it isnecessary to design and fabricate a devicewhich is suitable for long-term heart ratemonitoringand free from external disturbance. The wearing comfort,the reliability and thecost become three key issues for long-term heart rate monitoring for daily use. Several conventional methodshave already beendeveloped to measure heart rate, most of which arebased on electrocardiograms (ECGs) \[[@b1-sensors-15-03224]\], photoplethysmography (PPG) \[[@b2-sensors-15-03224]\],and the piezoelectriceffect \[[@b3-sensors-15-03224]--[@b5-sensors-15-03224]\]. Other principles in heart rate measurement have also been verified by researchers \[[@b6-sensors-15-03224]--[@b8-sensors-15-03224]\]. Among these methods for heartrate monitoring, ECG is the most widely used. It is the standarddiagnosis technique for heart disease in hospitals but the specialized equipment needed is clearly notsuitable for householdapplications. Householdheart rate monitors basedon ECG techniquehave also been commercialized, but chest straptransmitters are usually needed, which is not appropriate for long-term use. Strapless wristband ECG heart rate monitors have been developed these days.However, the contradiction between reliable electric connection and the comfort of wearing still has not been settled in a satisfactory way. Techniques for PPG require hard photoelectric modules whicharetightly attached to the tissue with a certain penetration depth,for instance,the fingers. Forthe reasons above, these two kinds of heard rate monitor systemare not appropriate for long-term ordinary dailyusebecauseof the inconvenience in user experience, especially at night. Monitors based on piezoelectric pressure sensoralso have the similar limits. 2.. System Design ================= In this work, a comfort, reliable heart rate monitor for long-term household useis proposed.Unlike the previous ECG, PPGand piezoelectric devices, a flexible pressure sensor with the capability of measuring heart rate by sensingthe pulsation of theartery is used in thesystem.This solution is adopted based on the following considerations.Since the sensor of the monitoring system is theonly componentwhich needs to inevitably be in contactwith the human skin, trying to have a flexible sensor is crucial in realizing a comfortable, simple heart rate monitor. In this work, a robust, low-cost novel flexible pressure sensorhas been proposed and fabricated.The polymer-based flexible sensor has no hard components which it canensure its comfort andfitness for wearing all day in any situation. Furthermore,to distinguish the tiny pressure changes caused by the arterypulse, the flexible pressure sensor's sensitivity needsto beimproved. Based onpreviousworksin this area,a highlysensitivestructure is proposed.The conventional structure with one piezoresistive layer is replaced by a novel structure with two piezoresistive layers. The contact interface ofthe twolayers is modified with microstructures. These microstructures are realized with modern soft lithography technology and are treated asthe key featuresfor the sensor's high sensitivity. In addition to that, for the specific morphologyof the surface microstructures utilized, the pressure sensor device alsohasa perfect linearity performance. However, due to the high sensitivity of the pressure sensor, noises maybe induced by the wearer's musclemovements. This inevitable daily movement can cause pressure changes between the wearer's skin and the device. Such noisescan easily make the monitor fail when heart rate counting. Inorder to solve thisproblem while maintaining a relatively low cost insignal processing, an Analog SignalProcessing (ASP) system isproposedto processthesignal and extract the heart rate information from it. The wholesystem is composed of a sensor subsystem and the ASP subsystem. Thesensor subsystem isan elastic belt with the flexible pressure sensorattached on it. Thetightnessof the belt can be tunedinorder toprovide a proper pressure for measurement. The systemalso includes an ASP subsystem to process the pressure signals, a counter, an indicator, and a timing circuit which is used tostop the measurement. 2.1.. Flexible Pressure Sensor ------------------------------ To realize the characteristic offlexibility in addition tothe pressure sensing capability, a flexible piezoresistive carbon black/silicone rubber nanocomposite is adoptedas the functional material \[[@b9-sensors-15-03224]--[@b12-sensors-15-03224]\]. For its stretchable, flexible properties and piezoresistive effect, this nanocomposite material has already been used in tactile sensorsfor robots to provide contact or grasping force feedback \[[@b13-sensors-15-03224],[@b14-sensors-15-03224]\]. Theyare also some other sensors for mechanics that adopt the material for their piezoresistive properties and energy absorbing capabilities in preventing mechanical collisions \[[@b15-sensors-15-03224],[@b16-sensors-15-03224]\].In addition to that, allother materialsused in device are flexible. [Figure 1](#f1-sensors-15-03224){ref-type="fig"} shows asimple schematic diagram demonstrating the assembly andpackaging steps used in fabricatingthedevice. Twolayers of surface-modifiedpiezoresistive polymer nanocomposite films are placed face-to face inside thedevice as thepressure sensing layer. These polymer films are packaged byanother two layer ofpatterned Flexible Copper Clad Laminate (FCCL)films which act either as thepackage material or as theelectrode of thedevice. To isolate this device from external moisture and contamination, another layer ofPI bond-ply is usedas the adhesion layer to ensure these four layers of material a goodfirm bond. These bond-ply layershave also been previously patterned by a laser cutting machine to save the space for the pressure sensing layer. The scale of the device's sensing part is 15 × 30 mm^2^. Since the human artery pulse in the wrist is weak and the excited pressure variation isnot easy to sense, theflexible device' sensitivity needs tobefurther improved.Several previous efforts have been made topromote the devices' sensitivity in this research area \[[@b17-sensors-15-03224]--[@b23-sensors-15-03224]\]. A pressure sensor with record sensitivitywith highlysensitive material with hollow polymer sphere in it has been proposed byBao'sgroup\[[@b17-sensors-15-03224]\]. Another type ofresearch mainlyutilizea tiny features' very sensitive contact state to realize high sensitivity\[[@b18-sensors-15-03224],[@b19-sensors-15-03224]\]. They usually have polymer-based micrometer scale pyramid structuresto make thedevice electrically sensitive to external pressure loading. Devices ofthis type usually have a very sensitive electric response at the beginning when an array of unique pyramids gets contact to the opponent electrode. However, as the pressure goes higher, device's response become dull as the pyramidal surfaceget saturated, asshownin [Figure 2b](#f2-sensors-15-03224){ref-type="fig"}. A certain degree of pre-pressure needs to be maintained betweenthe sensorandthe human skinin order to ensure effective contact state and signal acquisition and the wrist artery pulse inducedpressure varies (range \< 3 kPa) based on this pre-pressure bias. Furthermore, the pre-pressure also changes with the differences between userswhenthey fasten on the wrist belt.Consequently, the real effective spanforthe heart rate measurements will be around 8--18 kPa. Several previous works on flexible pressuresensors have reported sensitivity high enoughfor heart rate measurement. However, these high sensitivities only existwithin alimited range around0 Pa. As shown in [Figure 2b](#f2-sensors-15-03224){ref-type="fig"}, the slope of the device response decreases as pressure goes higher,likethe sensitivity. When the pressure reaches the effective range, devices becomes so dullthat it can't respond to theartery pulse. As to the proposed novel device with full-scale high sensitivity([Figure2a](#f2-sensors-15-03224){ref-type="fig"}), artery pulse-induced pressurevariation will make it respond intensively compared with previous ones. Tohave thisfull-scale sensitive pressure response, a new flexible pressure sensing device havebeen proposedin thiswork. Thesurface of the nanocomposite film has been modifiedwith micrometer scale bump structures randomly distributed on surface with certain variation in height and lateral size. [Figure 2c](#f2-sensors-15-03224){ref-type="fig"}--d shows the SEM photos of the microbumps fromthe top view and side view, respectively. Unlike the typical pyramid structures of thesame size, thedistributedtiny structures' size makesthem gradually touch with the opponent conductive surface along thewhole pressure loading process so as to have a full scale high sensitivity. By surface profile measurement, theoverall surface obeys the Gaussian Random distribution. By numericalsimulationwith MATLAB,the device response is expected to be linear. Furthermore, the micrometer scale piezoresistive bumps on thesurface arevery sensitive to externalpressure loadingas the pressuresare concentrated on these tinystructures. Large deformations of these piezoresistive structureslead to dramatic conductance variations of the device. Besides that, for the distributed bump size, the conducting paths are established gradually.The capability in conductance variation is even higher.Basedon above statement, the device sensitivityis expectedtobe higher. The devices' sensingcapabilities were testedusing a device testplatform composed of a micropositioner,force gauge and the electric measurement | 1.. Introduction ================ Heart _rate_ has been an _important_ factor in indicating patients' _health_ for quite _a_ long time. As people are paying _more_ _and_ more attention on their health, _long-term_ heart rate monitoring is of more importance to everyone _as_ it's a valuable indicator for _early_ diagnosis of dozens _of_ diseases. Furthermore, long-term heart _rate_ monitoring is _also_ essential _for_ sports enthusiasts and _professional_ athletes. According to these _requirements,_ _it_ is necessary to design and fabricate a device which is suitable for long-term _heart_ _rate_ _monitoring_ and free from external _disturbance._ The wearing comfort, _the_ reliability _and_ the cost become _three_ _key_ issues for long-term heart rate monitoring _for_ daily use. Several conventional _methods_ _have_ already been developed to measure heart _rate,_ _most_ _of_ which _are_ _based_ on electrocardiograms (ECGs) \[[@b1-sensors-15-03224]\], photoplethysmography (PPG) \[[@b2-sensors-15-03224]\], _and_ the _piezoelectric_ effect \[[@b3-sensors-15-03224]--[@b5-sensors-15-03224]\]. _Other_ principles in heart _rate_ measurement have also been _verified_ _by_ researchers \[[@b6-sensors-15-03224]--[@b8-sensors-15-03224]\]. _Among_ these methods for _heart_ _rate_ monitoring, ECG is the _most_ widely used. It is the standard diagnosis technique for _heart_ disease _in_ hospitals but the specialized equipment needed is clearly not suitable for household applications. Household heart rate monitors based on ECG _technique_ have also _been_ commercialized, but chest strap transmitters are _usually_ _needed,_ _which_ is not appropriate for long-term use. Strapless wrist band ECG heart _rate_ monitors have been _developed_ these days. However, the contradiction between reliable electric connection and the comfort _of_ wearing still _has_ _not_ been settled in a satisfactory way. _Techniques_ for PPG require hard photoelectric _modules_ _which_ are tightly attached to the tissue with a certain penetration depth, for instance, the fingers. _For_ the reasons above, these two kinds of _heard_ rate monitor system are not appropriate for long-term ordinary daily use _because_ of _the_ inconvenience in user experience, especially at _night._ Monitors based on piezoelectric pressure sensor also have the similar limits. 2.. System _Design_ ================= In _this_ work, a comfort, reliable _heart_ rate monitor for _long-term_ household use is _proposed._ _Unlike_ the previous ECG, PPG and piezoelectric devices, a flexible _pressure_ _sensor_ _with_ _the_ _capability_ of measuring heart rate by _sensing_ _the_ pulsation _of_ the _artery_ is used in the system. This _solution_ is adopted based _on_ the following _considerations._ Since the sensor of the _monitoring_ system _is_ the _only_ _component_ which _needs_ _to_ inevitably be in contact _with_ the human skin, trying to _have_ a flexible sensor is _crucial_ in realizing _a_ comfortable, _simple_ _heart_ rate monitor. In this work, a _robust,_ low-cost novel flexible pressure sensor has been proposed and fabricated. The polymer-based flexible sensor has no _hard_ components _which_ it _can_ ensure its _comfort_ and _fitness_ for wearing all _day_ in any situation. Furthermore, to distinguish the tiny _pressure_ changes caused by _the_ artery _pulse,_ the flexible pressure sensor's _sensitivity_ needs to be improved. _Based_ _on_ _previous_ works in _this_ area, _a_ highly sensitive _structure_ is proposed. The conventional structure with one _piezoresistive_ _layer_ _is_ replaced by a novel structure with two piezoresistive layers. The contact interface of the _two_ layers is modified with micro structures. These microstructures are realized with modern _soft_ _lithography_ technology and are treated _as_ the key features for the _sensor's_ high _sensitivity._ In addition _to_ that, for the specific morphology of the _surface_ _microstructures_ utilized, the pressure sensor device also has a perfect _linearity_ performance. However, _due_ _to_ the high sensitivity of _the_ pressure _sensor,_ _noises_ may be induced by the wearer's muscle movements. _This_ inevitable _daily_ movement can cause _pressure_ _changes_ _between_ the _wearer's_ _skin_ and the _device._ Such noises can easily make the monitor fail when heart rate counting. In order to solve this problem while maintaining a relatively _low_ cost in signal processing, an Analog Signal Processing _(ASP)_ system _is_ proposed to process _the_ signal _and_ _extract_ the heart rate information from it. The whole system is composed of a sensor subsystem and the ASP _subsystem._ _The_ sensor subsystem _is_ an elastic _belt_ _with_ _the_ flexible pressure sensor attached on it. The tightness of the belt can be tuned _in_ order to provide _a_ proper pressure _for_ measurement. _The_ system also includes an ASP subsystem to process the pressure signals, a counter, an indicator, and _a_ timing _circuit_ which is _used_ to stop the _measurement._ 2.1.. Flexible Pressure Sensor ------------------------------ To realize the characteristic of flexibility in addition _to_ the pressure sensing capability, a flexible piezoresistive carbon _black/silicone_ _rubber_ nanocomposite is _adopted_ as the functional material \[[@b9-sensors-15-03224]--[@b12-sensors-15-03224]\]. For _its_ stretchable, flexible properties and piezoresistive effect, this nanocomposite material has already been used in tactile _sensors_ for robots _to_ provide contact _or_ grasping force feedback _\[[@b13-sensors-15-03224],[@b14-sensors-15-03224]\]._ They are _also_ some _other_ sensors for mechanics that adopt the _material_ _for_ their piezoresistive properties and energy absorbing capabilities in preventing mechanical collisions _\[[@b15-sensors-15-03224],[@b16-sensors-15-03224]\]._ _In_ addition to that, all _other_ materials used in _device_ are _flexible._ [Figure 1](#f1-sensors-15-03224){ref-type="fig"} shows a simple _schematic_ diagram demonstrating _the_ assembly and packaging _steps_ used in fabricating the device. Two layers of surface-modified piezoresistive polymer nanocomposite films are placed _face-to_ _face_ inside the device as the pressure sensing _layer._ These polymer films are packaged by _another_ two layer of patterned _Flexible_ Copper Clad _Laminate_ (FCCL) films _which_ act either as the _package_ material or as the electrode of the _device._ To _isolate_ this device from _external_ moisture and contamination, _another_ layer of PI bond-ply is used as the adhesion layer to _ensure_ these four layers of material a _good_ firm bond. These bond-ply layers have also been previously _patterned_ by a laser cutting machine to _save_ the space for the pressure sensing _layer._ The scale of the _device's_ _sensing_ part _is_ 15 × 30 mm^2^. Since the human artery _pulse_ in the wrist _is_ weak and the excited pressure variation is not easy to sense, the flexible device' sensitivity needs to be further improved. _Several_ previous efforts _have_ been _made_ to promote _the_ devices' sensitivity in this _research_ area \[[@b17-sensors-15-03224]--[@b23-sensors-15-03224]\]. A _pressure_ _sensor_ with record sensitivity with highly sensitive material _with_ hollow _polymer_ sphere in it has _been_ proposed _by_ Bao's group \[[@b17-sensors-15-03224]\]. Another type of _research_ mainly utilizea tiny features' _very_ _sensitive_ contact state to realize high sensitivity \[[@b18-sensors-15-03224],[@b19-sensors-15-03224]\]. _They_ usually have polymer-based _micrometer_ scale pyramid structures to make the device electrically sensitive to external _pressure_ loading. Devices of this type _usually_ have _a_ very sensitive electric response at _the_ beginning _when_ an array _of_ unique pyramids gets _contact_ to the opponent _electrode._ However, _as_ _the_ _pressure_ goes higher, _device's_ response become _dull_ as the pyramidal surface _get_ _saturated,_ as shown _in_ [Figure 2b](#f2-sensors-15-03224){ref-type="fig"}. A certain degree of pre-pressure needs to be maintained _between_ the sensor _and_ the human skin in order to ensure effective contact state and signal _acquisition_ _and_ the _wrist_ artery pulse induced pressure varies (range \< 3 kPa) based _on_ this _pre-pressure_ bias. Furthermore, the pre-pressure _also_ _changes_ with the differences between _users_ when they _fasten_ on the wrist belt. Consequently, the real effective span for _the_ heart _rate_ _measurements_ _will_ be around 8--18 kPa. _Several_ previous works _on_ flexible _pressure_ sensors have _reported_ sensitivity high enough for heart _rate_ measurement. However, these high sensitivities only _exist_ within a limited range around 0 Pa. As shown in _[Figure_ _2b](#f2-sensors-15-03224){ref-type="fig"},_ the slope of the device response _decreases_ as pressure goes higher, like _the_ sensitivity. When the pressure reaches the effective _range,_ devices becomes _so_ dull that it can't respond to the artery _pulse._ As to the proposed novel device with full-scale high sensitivity ([Figure 2a](#f2-sensors-15-03224){ref-type="fig"}), artery pulse-induced pressure variation will make it respond intensively _compared_ with _previous_ ones. To have this _full-scale_ sensitive pressure response, _a_ new flexible pressure sensing device have been proposed in _this_ work. The _surface_ of the nanocomposite film _has_ _been_ _modified_ _with_ _micrometer_ scale bump structures randomly distributed on surface with certain variation in height and lateral size. [Figure 2c](#f2-sensors-15-03224){ref-type="fig"}--d shows the _SEM_ photos of the microbumps from the top view and _side_ _view,_ _respectively._ Unlike the typical pyramid structures of the same size, _the_ _distributed_ tiny structures' size _makes_ them gradually touch _with_ the opponent conductive surface along the whole _pressure_ loading process so as to have a full scale high _sensitivity._ By surface profile measurement, the _overall_ surface obeys the Gaussian _Random_ distribution. _By_ numerical simulation _with_ MATLAB, the device response _is_ _expected_ _to_ be linear. Furthermore, the micrometer scale piezoresistive bumps on _the_ _surface_ are very sensitive to external pressure loading as the pressures are concentrated on these tiny structures. Large deformations of _these_ _piezoresistive_ structures lead to dramatic conductance _variations_ of the device. Besides that, for the distributed bump size, _the_ conducting paths are _established_ gradually. The capability in conductance variation is even higher. _Based_ on above _statement,_ the device sensitivity _is_ _expected_ to _be_ higher. The devices' _sensing_ capabilities were tested using a device test _platform_ composed of a micropositioner, force gauge _and_ the electric _measurement_ |
Introduction {#s1}
============
The intestinal mucosa is a critical effector site for elimination of enteric pathogens. *Toxoplasma gondii*, a ubiquitous protozoan parasite, is a prime example of such a pathogen. Mammals are infected with *T. gondii* primarily by the ingestion of tissue cysts from undercooked meat or oocysts excreted in the feces of felines, which are the sole definitive hosts. Upon infection, the parasite induces a potent Th1 immune response that is characterized by high levels of IL-12 and IFN-γ [@ppat.1003706-Denkers1], [@ppat.1003706-Dupont1]. Initial IL-12 production is largely the result of MyD88-dependent Toll-like receptor (TLR) signaling in dendritic cells, and the parasite profilin molecule has been identified as a ligand for TLR11 and TLR12 [@ppat.1003706-Scanga1]--[@ppat.1003706-Sukhumavasi1]. IL-12 activates natural killer (NK) cells to initiate IFN-γ production and promotes T-cell differentiation towards a Th1 program. Ultimately IFN-γ is the critical cytokine involved in controlling *Toxoplasma*. While *in vitro* experiments suggest that macrophages activated by this cytokine acquire anti-*Toxoplasma* activity through upregulation of immunity-related GTPase (IRG) molecules that mediate destruction of the parasitophorous vacuole [@ppat.1003706-Zhao1]--[@ppat.1003706-Khaminets1], the *in vivo* function of IFN-γ is less clear.
Inflammatory monocytes are an important component of defense against microbial pathogens, including *Toxoplasma* [@ppat.1003706-Dunay1]. These cells express high levels of Ly6C/G (Gr-1) and are recruited from the bone marrow via chemokine (C-C motif) receptor 2 (CCR2) [@ppat.1003706-Geissmann1]. During *Listeria monocytogenes* infection, inflammatory monocytes are recruited from the bone marrow to the spleen and liver where they differentiate into TNF-α- and nitric oxide (NO)-producing DCs (Tip-DCs). There they are essential for bacterial clearance and mouse survival [@ppat.1003706-Shi1], [@ppat.1003706-Serbina1]. Likewise, CCR2-dependent inflammatory monocytes are recruited to the lung during *Mycobacteria tuberculosis* infection where they protect mice from disease by recruiting and activating T cells and by producing NO [@ppat.1003706-Peters1], [@ppat.1003706-Peters2]. Mucosal defense against *T. gondii* has also recently been shown to require CCR2-dependent inflammatory monocytes [@ppat.1003706-Dunay1]. Upon recruitment to the small intestine, these cells control the parasite either indirectly by production of IL-12 and TNF-α or directly through production of NO and IRG proteins [@ppat.1003706-Yarovinsky1]--[@ppat.1003706-Andrade1], [@ppat.1003706-Zhao1], [@ppat.1003706-Taylor1], [@ppat.1003706-Dunay1], [@ppat.1003706-Ling1]. While CCR2 enables recruitment of inflammatory monocytes to sites of infection, the factors that coordinate their activation and acquisition of effector function are not known.
CXCR3 is a Th1-associated chemokine receptor, and cells expressing this receptor respond to the IFN-γ-inducible chemokines CXCL9, 10, and 11 [@ppat.1003706-Groom1]. The receptor is expressed predominantly by T cells and NK cells and is rapidly upregulated upon cell activation. There is evidence that CXCR3 expression enables T-cell entry into sites of infection, although the outcome of recruitment varies among pathogens. In the case of *Leishmania major*, recruitment is protective as CXCR3-expressing T cells are required for the resolution of cutaneous lesions [@ppat.1003706-Rosas1]. However, in the case of *Plasmodium berghei* ANKA, CXCR3 is pathogenic because it allows entry of proinflammatory cells into the CNS, resulting in cerebral malaria [@ppat.1003706-Campanella1].
Here we determined the role of CXCR3 in the intestinal immune response to *Toxoplasma*. We found that loss of CXCR3 negatively affected host survival against oral infection. This was associated with diminished recruitment of CD4^+^ T cells to the lamina propria (LP), decreased T cell IFN-γ secretion, impaired inflammatory monocyte effector function, and inability to control the parasite in the intestinal mucosa. Reconstitution with CXCR3-competent CD4^+^ T cells restored inflammatory monocyte function, resulting in improved survival against the parasite. Protective effects of adoptively transferred CD4^+^ T lymphocytes depended upon their ability to produce IFN-γ, but occurred independently of CD4 expression of CD40L. Our data show that CXCR3 enables Th1 recruitment to the intestinal LP, where these cells instruct activation of CCR2-dependent inflammatory monocytes, in turn controlling infection. These results establish CXCR3 as a major determinant orchestrating communication between effectors of innate and adaptive immunity, enabling effective host defense against infection.
Results {#s2}
=======
CXCR3 and its ligands CXCL9 and CXCL10 are upregulated during acute toxoplasmosis {#s2a}
---------------------------------------------------------------------------------
Because CXCR3 and its chemokine ligands are strongly associated with Th1 responses, we asked whether this proinflammatory axis was induced during *Toxoplasma* infection in the intestinal mucosa. Accordingly, mice were orally inoculated with cysts, and relative levels of CXCR3, CXCL9 and CXCL10 mRNA expression were measured over the course of acute infection. We found strong upregulation of CXCR3 and its specific chemokine ligands as early as Day 4 post-infection in both the ileum and mesenteric lymph nodes (MLN) ([Fig. 1A](#ppat-1003706-g001){ref-type="fig"}). Overall, peak CXCR3 mRNA levels were attained by Day 6 post-inoculation.
{#ppat-1003706-g001}
In order to examine CXCR3 expression in more detail, we utilized *Cxcr3* eGFP reporter (CIBER) mice, a bicistronic reporter strain in which cells expressing CXCR3 also express eGFP [@ppat.1003706-Oghumu1]. We found a large increase in CXCR3 populations of both CD4^+^ and CD8^+^ T lymphocytes in MLN, spleen (SPL) and LP compartments following infection ([Fig. 1B](#ppat-1003706-g001){ref-type="fig"}). In general, CXCR3 upregulation was most pronounced in the CD4^+^ population. For example, in the MLN there was a 6-fold increase in CD4^+^CXCR3^+^ cells but only a 2-fold increase in CD8^+^CXCR3^+^ lymphocytes. NK cells are also known to express CXCR3 and are an important source of early IFN-γ during *T. gondii* infection [@ppat.1003706-Qin1], [@ppat.1003706-Sher1]. However, while CXCR3-GFP expression was relatively high on naïve NK cells, the GFP expression was in fact reduced during infection, suggesting lack of a role for CXCR3^+^ NK cells during intestinal infection ([Fig. S1](#ppat.1003706.s001){ref-type="supplementary-material"}). We next examined expression of the activation marker CD27 on GFP^+^ and GFP^−^ T lymphocytes in infected mice. CD27 was significantly lower in CXCR3^−^GFP^−^ cells, suggesting an altered maturation state of the CXCR3^−^ T cells ([Fig. S2A and B](#ppat.1003706.s002){ref-type="supplementary-material"}). Likewise, there was a lower percentage of CD27^+^ cells amongst CXCR3-GFP^−^ CD8^+^ T lymphocytes, although the decreases in expression were not as striking as with the CD4^+^ lymphocytes ([Fig. S2C and D](#ppat.1003706.s002){ref-type="supplementary-material"}).
*Cxcr3^−/−^* mice are increased in susceptibility and are prone to severe intestinal damage following *T. gondii infection* {#s2b}
---------------------------------------------------------------------------------------------------------------------------
To further examine the role of CXCR3 during *T. gondii* infection, mice deficient in CXCR3 were orally inoculated with low virulence ME49 cysts, and the outcome of infection was monitored. While all wild-type (WT) mice survived acute infection with 30 cysts, *Cxcr3^−/−^* animals displayed increased susceptibility with nearly 75% of mice dying by 2 weeks post-infection ([Fig. 2A](#ppat-1003706-g002){ref-type="fig"}). When the cyst dose was increased to 50, all | introduction { # s1 } = = = = = = = = = = = = the intestinal mucosa is a critical effector site for elimination of enteric pathogens. * toxoplasma gondii *, a susceptible protozoan parasite, is a prime example of such a pathogen. mammals are infected with * t. gondii * primarily by the ingestion of tissue cysts from undercooked meat or oocysts excreted in the feces of felines, which are the sole definitive hosts. after infection, the parasite induces mostly potent th1 immune response but is characterized by high levels of il - 12 and ifn - γ [ @ ppat. 1003706 - denkers1 ], [ @ ppat. 1003706 - dupont1 ]. initial il - 12 production is largely the result of myd88 - dependent toll - like receptor ( tlr ) signaling in dendritic cells, and the parasite profilin molecule has been identified as a mechanism for tlr11 and tlr12 [ @ ppat. 1003706 - scanga1 ] - - [ @ stat. 1003706 - sukhumavasi1 ]. il - 12 activates natural killer ( nk ) cells to initiate ifn - γ production and promotes t - cell response towards an th1 program. ultimately ifn - γ is the critical cytokine involved in controlling * toxoplasma *. while * in vitro * experiments suggest that macrophages activated by this antigen acquire anti - * toxoplasma * activity through upregulation of immunity - related gtpase ( irg ) molecules that mediate destruction of the parasitophorous vacuole [ @ ppat. 1003706 - zhao1 ] - - [ @ ppat. 1003706 - khaminets1 ], the * in vivo * function of ifn - γ is less clear. inflammatory monocytes are an important component of defense against microbial pathogens, including * toxoplasma * [ @ ppat. 1003706 - dunay1 ]. these cells express high levels of ly6c / g ( gr - 1 ) pathways are recruited from the bone marrow via chemokine ( c - c motif ) receptor 2 ( ccr2 ) [ @ ppat. 10037 ##06 - geissmann1 ]. during * listeria monocytogenes * infection, inflammatory monocytes are recruited from the bone marrow to the spleen and liver where they differentiate into tnf - α - and nitric oxide ( no ) - producing dcs ( tip - dcs ). there they are essential for bacterial clearance and mouse survival [ @ ppat. 1003706 - shi1 ], [ @ ppat. 1003706 - serbina1 ]. likewise, ccr2 - dependent inflammatory monocytes are recruited to the lung during * mycobacteria tuberculosis * infection where they protect mice from disease by recruiting and activating t cells and by producing no [ @ ppat. 1003706 - peters1 ], [ @ ppat. 1003706 - peters2 ]. mucosal defense against * t. gondii * has also recently been shown to require ccr2 - dependent inflammatory monocytes [ @ ppat. 1003706 - dunay1 ]. upon recruitment to the small intestine, these cells control the parasite either indirectly by production of il - 12 and tnf - α or directly through production of no and irg proteins [ @ ppat. 1003706 - yarovinsky1 ] - - [ @ ppat. 1003706 - andrade1 ], [ @ ppat. 1003706 - zhao1 ], [ @ ppat. 1003706 - taylor1 ], [ @ ppat. 1003706 - dunay1 ], [ @ ppat. 1003706 - ling1 ]. while ccr2 enables recruitment of inflammatory monocytes to sites of infection, the factors that coordinate their activation and acquisition of effector function are not known. cxcr3 is a th1 - associated chemokine receptor, and cells expressing this receptor respond to the ifn - γ - inducible chemokines cxcl9, 10, and 11 [ @ ppat. 1003706 - groom1 ]. the receptor is expressed predominantly by t cells and nk cells and is rapidly upregulated upon cell activation. there is evidence that cxcr3 expression enables t - cell entry into sites of infection, although the outcome of recruitment varies among pathogens. in the case of * leishmania major *, recruitment is protective as cxcr3 - expressing t cells are required for the resolution of cutaneous lesions [ @ ppat. 1003706 - rosas1 ]. however, in the case of * plasmodium berghei * anka, cxcr3 is pathogenic because it allows entry of proinflammatory cells into the cns, resulting in cerebral malaria [ @ ppat. 1003706 - campanella1 ]. here we determined the role of cxcr3 in the intestinal immune response to * toxoplasma *. we found that loss of cxcr3 negatively affected host survival against oral infection. this was associated with diminished recruitment of cd4 ^ + ^ t cells to the lamina propria ( lp ), decreased t cell ifn - γ secretion, impaired inflammatory monocyte effector function, and inability to control the parasite in the intestinal mucosa. reconstitution with cxcr3 - competent cd4 ^ + ^ t cells restored inflammatory monocyte function, resulting in improved survival against the parasite. protective effects of adoptively transferred cd4 ^ + ^ t lymphocytes depended upon their ability to produce ifn - γ, but occurred independently of cd4 expression of cd40l. our data show that cxcr3 enables th1 recruitment to the intestinal lp, where these cells instruct activation of ccr2 - dependent inflammatory monocytes, in turn controlling infection. these results establish cxcr3 as a major determinant orchestrating communication between effectors of innate and adaptive immunity, enabling effective host defense against infection. results { # s2 } = = = = = = = cxcr3 and its ligands cxcl9 and cxcl10 are upregulated during acute toxoplasmosis { # s2a } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - because cxcr3 and its chemokine ligands are strongly associated with th1 responses, we asked whether this proinflammatory axis was induced during * toxoplasma * infection in the intestinal mucosa. accordingly, mice were orally inoculated with cysts, and relative levels of cxcr3, cxcl9 and cxcl10 mrna expression were measured over the course of acute infection. we found strong upregulation of cxcr3 and its specific chemokine ligands as early as day 4 post - infection in both the ileum and mesenteric lymph nodes ( mln ) ( [ fig. 1a ] ( # ppat - 1003706 - g001 ) { ref - type = " fig " } ). overall, peak cxcr3 mrna levels were attained by day 6 post - inoculation.! [ cxcr3 and its ligands are upregulated following * t. gondii * infection. \ ( a ) cxcr3, cxcl9, and cxcl10 gene expression was assessed by semi - quantitative real time pcr in mesenteric lymph nodes ( mln ) and small intestinal tissue from wt mice during oral * t. gondii * infection. the results are expressed as fold change relative to tissues from noninfected animals ( n = 4 mice per time point ). ( b ) cxcr3 protein expression was quantified using flow cytometry by measuring gfp levels before ( noninfected, ni ) and 11 days after oral infection ( inf ) of ciber mice. gfp levels were assessed among cd4 ^ + ^ and cd8 ^ + ^ t - cell subsets from mln, spleen ( spl ), and small intestinal lamina propria. ni, noninfected ; inf, infected. ] ( ppat. 1003706. g001 ) { # ppat - 1003706 - g001 } in order to examine cxcr3 expression in more detail, we utilized * cxcr3 * egfp reporter ( ciber ) mice, a bicistronic reporter strain in which cells expressing cxcr3 also express egfp [ @ ppat. 1003706 - oghumu1 ]. we found a large increase in cxcr3 populations of both cd4 ^ + ^ and cd8 ^ + ^ t lymphocytes in mln, spleen ( spl ) and lp compartments following infection ( [ fig. 1b ] ( # ppat - 1003706 - g001 ) { ref - type = " fig " } ). in general, cxcr3 upregulation was most pronounced in the cd4 ^ + ^ population. for example, in the mln there was a 6 - fold increase in cd4 ^ + ^ cxcr3 ^ + ^ cells but only a 2 - fold increase in cd8 ^ + ^ cxcr3 ^ + ^ lymphocytes. nk cells are also known to express cxcr3 and are an important source of early ifn - γ during * t. gondii * infection [ @ ppat. 1003706 - qin1 ], [ @ ppat. 1003706 - sher1 ]. however, while cxcr3 - gfp expression was relatively high on naive nk cells, the gfp expression was in fact reduced during infection, suggesting lack of a role for cxcr3 ^ + ^ nk cells during intestinal infection ( [ fig. s1 ] ( # ppat. 1003706. s001 ) { ref - type = " supplementary - material " } ). we next examined expression of the activation marker cd27 on gfp ^ + ^ and gfp ^ − ^ t lymphocytes in infected mice. cd27 was significantly lower in cxcr3 ^ − ^ gfp ^ − ^ cells, suggesting an altered maturation state of the cxcr3 ^ − ^ t cells ( [ fig. s2a and b ] ( # ppat. 1003706. s002 ) { ref - type = " supplementary - material " } ). likewise, there was a lower percentage of cd27 ^ + ^ cells amongst cxcr3 - gfp ^ − ^ cd8 ^ + ^ t lymphocytes, although the decreases in expression were not as striking as with the cd4 ^ + ^ lymphocytes ( [ fig. s2c and d ] ( # ppat. 1003706. s002 ) { ref - type = " supplementary - material " } ). * cxcr3 ^ − / − ^ * mice are increased in susceptibility and are prone to severe intestinal damage following * t. gondii infection * { # s2b } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - to further examine the role of cxcr3 during * t. gondii * infection, mice deficient in cxcr3 were orally inoculated with low virulence me49 cysts, and the outcome of infection was monitored. while all wild - type ( wt ) mice survived acute infection with 30 cysts, * cxcr3 ^ − / − ^ * animals displayed increased susceptibility with nearly 75 % of mice dying by 2 weeks post - infection ( [ fig. 2a ] ( # ppat - 1003706 - g002 ) { ref - type = " fig " } ). when the cyst dose was increased to 50, all | Introduction {# s1} = = = = = = = = = = = = The intestinal mucosa is a critical effector site for elimination of enteric pathogens. * Toxoplasma gondii *, a ubiquitous protozoan parasite, is a prime example of such a pathogen. Mammals are infected with * T. gondii * primarily by the ingestion of tissue cysts from undercooked meat or oocysts excreted in the feces of felines, which are the sole definitive hosts. Upon infection, the parasite induces a potent Th1 immune response that is characterized by high levels of IL - 12 and IFN - γ [@ ppat. 1003706 - Denkers1 ], [@ ppat. 1003706 - Dupont1 ]. Injtlal IL - 12 production is largely the result of MyD88 - dependent Toll - like receptor (TLR) signaling in dendritic cells, and the parasite profilin molecule has been identified as a ligand for TLR11 and TLR12 [@ ppat. 1003706 - Scanga1] - - [@ ppat. 1003706 - Sukhumavasi1 ]. IL - 12 activates natural killer (NK) cells to initiate IFN - γ production and promotes T - cell differentiation towards a Th1 program. Ultimstelg IFN - γ is the critical cytokine involved in controlling * Toxoplasma *. While * in vitro * ecperinents suggest that macrophages activated by this cytokine acquire anti - * Toxoplasma * activity through upregulation of immunity - related GTPase (IRG) molecules that mediate destruction of the parasitophorous vacuole [@ ppat. 1003706 - Zhao1] - - [@ ppat. 1003706 - Khaminets1 ], the * in vivo * function of IFN - γ is less clear. Inflammatory monocytes are an important component of defense against microbial pathogens, including * Toxoplasma * [@ ppat. 1003706 - Dunay1 ]. These cells express high levels of Ly6C / G (Gr - 1) and are recruited from the bone marrow via chemokine (C - C motif) receptor 2 (CCR2) [@ ppat. 1003706 - Geissmann1 ]. During * Listeria monocytogenes * infection, inflammatory monocytes are recTuifed from the bone marrow to the spleen and liver where they differentiate into TNF - α - and nitric oxide (NO) - producing DCs (Tip - DCs ). There they are essential for bacterial clearance and mouse survival [@ ppat. 1003706 - Shi1 ], [@ ppat. 1003706 - Serbina1 ]. Likewise, CCR2 - dependent inflammatory monocytes are recruited to the lung during * Mycobacteria tuberculosis * infection where they protect mice fr8j disease by recruiting and activating T cells and by pr8Vucing NO [@ ppat. 1003706 - Peters1 ], [@ ppat. 1003706 - Peters2 ]. Mucosal defense against * T. gondii * has also recently been shown to require CCR2 - dependent inflammatory monocytes [@ ppat. 1003706 - Dunay1 ]. Upon recruitment to the small intestine, these cells control the parasite either indirectly by production of IL - 12 and TNF - α or directly through production of NO and IRG proteins [@ ppat. 1003706 - Yarovinsky1] - - [@ ppat. 1003706 - Andrade1 ], [@ ppat. 1003706 - Zhao1 ], [@ ppat. 1003706 - Taylor1 ], [@ ppat. 1003706 - Dunay1 ], [@ ppat. 1003706 - Ling1 ]. While CCR2 enables recruitment of inflammatory monocytes to sites of infection, the factors that coordinate their activation and acquisition of effector function are not known. CXCR3 is a Th1 - associated chemokine receptor, and cells expressing this receptor respond to the IFN - γ - inducible chemokines CXCL9, 10, and 11 [@ ppat. 1003706 - Groom1 ]. The receptor is expressed predominantly by T cells and NK cells and is rapidly upregulated upon cell activation. There is evidence that CXCR3 expression enables T - cell entry into sites of infection, although the outcome of recruitment varies among pathogens. In the case of * Leishmania major *, recruitment is protective as CXCR3 - expressing T cells are required for the resolution of cutaneous lesions [@ ppat. 1003706 - Rosas1 ]. However, in the case of * Plasmodium berghei * SNKa, CXCR3 is pathogenic because it allows entry of proinflammatory cells into the CNS, resulting in cerebral malaria [@ ppat. 1003706 - Campanella1 ]. Here we determined the role of CXCR3 in the intestinal immune response to * Toxoplasma *. We found that loss of CXCR3 negatively affected host survival against oral infection. This was associated with diminished recruitment of CD4 ^ + ^ T cells to the lamina propria (LP ), decreased T cell IFN - γ secretion, impaired inflammatory monocyte effector function, and inability to control the parasite in the intestinal mucosa. Reconstitution with CXCR3 - competent CD4 ^ + ^ T cells restored inflammatory monocyte function, resulting in improved survival against the parasite. Protective effects of adoptively transferred CD4 ^ + ^ T lymphocytes depended upon their ability to produce IFN - γ, but occurred independently of CD4 expression of CD40L. Our data show that CXCR3 enables Th1 recruitment to the intestinal LP, where these cells instruct activation of CCR2 - dependent inflammatory monocytes, in turn controlling infection. These results establish DXCRW as a major determinant orchestrating communication between effectors of innate and adaptive immunity, enabling effective host defense against infection. Results {# s2} = = = = = = = CXCR3 and its ligands CXCL9 and CXCL10 are upregulated during acute toxoplasmosis {# s2a} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Because CXCR3 and its chemokine ligands are strongly associated with Th1 responses, we asked whether this proinflammatory axis was induced during * Toxoplasma * infection in the intestinal mucosa. Accordingly, mice were orally inoculated with cysts, and relative levels of CXCR3, CXCL9 and CXCL10 mRNA expression were measured over the course of acute infection. We found strong upregulation of CXCR3 and its specific chemokine ligands as early as Day 4 post - infection in both the ileum and mesenteric lymph nodes (MLN) ([ Fig. 1A] (# ppat - 1003706 - g001) {ref - type = " fig "} ). Overall, peak CXCR3 mRNA levels were attained by Day 6 post - inoculation. ! [CXCR3 and its ligands are upregulated following * T. gondii * infection. \ (A) CXCR3, CXCL9, and CXCL10 gene expression was assessed by semi - quantitative real time PCR in mesenteric lymph nodes (MLN) and small intestinal tissue from WT mice during oral * T. gondii * infection. The results are expressed as fold change relative to tissues from noninfected animals (n = 4 mice per time point ). (B) CXCR3 protein expression was quantified using flow cytometry by measuring GFP levels before (noninfected, NI) and 11 days after oral infection (INF) of CIBER mice. GFP levels were assessed among CD4 ^ + ^ and CD8 ^ + ^ T - cell subsets from MLN, spleen (SPL ), and small intestinal lamina propria. NI, noninfected; INF, infected.] (ppat. 1003706. g001) {# ppat - 1003706 - g001} In order to examine dXCd3 expression in more detail, we utilized * Cxcr3 * eGFP reporter (CIBER) mice, a bicistronic reporter strain in which cells expressing CXCR3 also express eGFP [@ ppat. 1003706 - Oghumu1 ]. We found a large increase in CXCR3 populations of both CD4 ^ + ^ and CD8 ^ + ^ T lymphocytes in MLN, spleen (SPL) and LP compartments following infection ([ Fig. 1B] (# ppat - 1003706 - g001) {ref - type = " fig "} ). In general, CXCR3 upregulation was most pronounced in the CD4 ^ + ^ population. For example, in the MLN there was a 6 - fold increase in CD4 ^ + ^ CXCR3 ^ + ^ cells but only a 2 - fold increase in CD8 ^ + ^ CXCR3 ^ + ^ lymphocytes. NK cells are also known to express CXCR3 and are an important source of early IFN - γ during * T. goHdki * infection [@ ppat. 1003706 - Qin1 ], [@ ppat. 1003706 - Sher1 ]. However, while CXCR3 - GFP expression was relatively high on naïve NK cells, the GFP expression was in fact reduced during infection, suggesting lack of a role for CXCR3 ^ + ^ NK cells during intestinal infection ([ Fig. S1] (# ppat. 1003706. s001) {ref - type = " supplementary - material "} ). We next examined expression of the activation marker CD27 on GFP ^ + ^ and GFP ^ − ^ T lymphocytes in infected mice. CD27 was significantly lower in CXCR3 ^ − ^ GFP ^ − ^ cells, suggesting an altered maturation state of the CXCR3 ^ − ^ T cells ([ Fig. S2A and B] (# ppat. 1003706. s002) {ref - type = " supplementary - material "} ). Likewise, there was a lower percentage of CD27 ^ + ^ cells amongst CXCR3 - GFP ^ − ^ CD8 ^ + ^ T lymphocytes, although the decreases in expression were not as striking as with the CD4 ^ + ^ lymphocytes ([ Fig. S2C and D] (# ppat. 1003706. s002) {ref - type = " supplementary - material "} ). * Cxcr3 ^ − / − ^ * mice are increased in susceptibility and are prone to severe intestinal damage following * T. gondii infection * {# s2b} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - To further examine the role of CXCR3 during * T. gondii * infection, mice deficient in CXCR3 were orally inoculated with low virulence ME49 cysts, and the outcome of infection was monitored. While all wild - type (WT) mice survived acute infection with 30 cysts, * Cxcr3 ^ − / − ^ * animals displayed increased susceptibility with nearly 75% of mice dying by 2 weeks post - infection ([ Fig. 2A] (# ppat - 1003706 - g002) {ref - type = " fig "} ). When the cyst dose was increased to 50, all | Introduction {#s1} ============ The intestinal mucosa is a critical effector site for elimination of pathogens. *Toxoplasma gondii*, a ubiquitous protozoan parasite, is a prime example of such a Mammals are infected with *T. primarily by the ingestion of tissue cysts undercooked meat or oocysts excreted in the feces of felines, are the sole definitive hosts. Upon infection, the parasite induces a potent Th1 immune response that is characterized high levels of IL-12 and IFN-γ [@ppat.1003706-Dupont1]. Initial IL-12 is largely the result of MyD88-dependent Toll-like receptor (TLR) signaling in dendritic cells, and the parasite profilin molecule been identified as a ligand for TLR11 and [@ppat.1003706-Scanga1]--[@ppat.1003706-Sukhumavasi1]. IL-12 activates natural killer (NK) cells to initiate IFN-γ production and promotes T-cell differentiation towards a Th1 program. IFN-γ is the critical cytokine involved in controlling *Toxoplasma*. While *in vitro* experiments suggest that macrophages activated by this cytokine acquire anti-*Toxoplasma* activity through upregulation of immunity-related (IRG) that mediate destruction the parasitophorous vacuole [@ppat.1003706-Zhao1]--[@ppat.1003706-Khaminets1], the *in vivo* function of IFN-γ is less clear. Inflammatory monocytes are important component of defense against microbial pathogens, including These cells express high levels Ly6C/G (Gr-1) and are recruited from the bone marrow via (C-C receptor 2 (CCR2) [@ppat.1003706-Geissmann1]. During *Listeria infection, inflammatory monocytes are recruited from the bone marrow to the spleen and liver where differentiate into TNF-α- and oxide (NO)-producing DCs (Tip-DCs). are essential for bacterial clearance mouse survival [@ppat.1003706-Shi1], [@ppat.1003706-Serbina1]. Likewise, CCR2-dependent inflammatory are recruited to the lung during *Mycobacteria tuberculosis* where they protect mice from disease by recruiting and T cells and by producing NO [@ppat.1003706-Peters1], [@ppat.1003706-Peters2]. Mucosal defense against gondii* has also recently shown to require CCR2-dependent inflammatory monocytes [@ppat.1003706-Dunay1]. Upon to the small these cells control the parasite either indirectly by of IL-12 and TNF-α or directly through production of NO and proteins [@ppat.1003706-Yarovinsky1]--[@ppat.1003706-Andrade1], [@ppat.1003706-Taylor1], [@ppat.1003706-Dunay1], [@ppat.1003706-Ling1]. While CCR2 enables recruitment of inflammatory monocytes to sites of infection, the factors that coordinate their activation and acquisition of effector function are not known. a Th1-associated chemokine and cells expressing this receptor respond to the IFN-γ-inducible chemokines CXCL9, 10, and 11 [@ppat.1003706-Groom1]. The is expressed predominantly by T cells and NK cells and is rapidly upon cell activation. There is evidence that CXCR3 expression enables T-cell entry into sites infection, the outcome of recruitment varies among pathogens. In the case of *Leishmania major*, recruitment is protective as CXCR3-expressing T cells are required for the resolution of cutaneous lesions [@ppat.1003706-Rosas1]. However, in the case of *Plasmodium berghei* ANKA, CXCR3 is pathogenic because it allows entry of cells into the CNS, resulting in cerebral malaria [@ppat.1003706-Campanella1]. Here we determined role of CXCR3 in intestinal immune response to *Toxoplasma*. We found that of CXCR3 affected host against oral This was associated with diminished recruitment of T cells to the lamina propria (LP), decreased T cell IFN-γ secretion, impaired inflammatory monocyte effector function, and inability to control the in the intestinal mucosa. Reconstitution with CXCR3-competent CD4^+^ T cells restored inflammatory function, resulting in improved survival against the parasite. Protective effects of adoptively transferred CD4^+^ T lymphocytes depended upon their ability to IFN-γ, but occurred independently of CD4 expression of CD40L. Our data show that CXCR3 enables recruitment to the intestinal LP, where these cells instruct activation of CCR2-dependent inflammatory monocytes, in turn infection. These establish CXCR3 as a major orchestrating communication between effectors of innate and adaptive immunity, enabling effective host defense against infection. Results {#s2} CXCR3 and its ligands CXCL9 and CXCL10 are upregulated during acute toxoplasmosis {#s2a} --------------------------------------------------------------------------------- Because CXCR3 and its chemokine ligands are strongly associated Th1 responses, we asked whether this proinflammatory was induced during infection intestinal mucosa. Accordingly, mice were orally with cysts, and relative levels of CXCR3, CXCL9 and CXCL10 mRNA expression were measured over the course of acute infection. We found strong upregulation of CXCR3 and its specific chemokine early as Day 4 post-infection in both ileum mesenteric lymph nodes ([Fig. 1A](#ppat-1003706-g001){ref-type="fig"}). Overall, peak CXCR3 mRNA levels were attained by Day 6 post-inoculation. ![CXCR3 and its ligands are following *T. gondii* infection.\ (A) CXCR3, CXCL9, and CXCL10 gene expression assessed by real time PCR in mesenteric lymph nodes (MLN) and small intestinal tissue from WT mice oral *T. gondii* infection. The results are as fold change relative to tissues noninfected animals (n = 4 mice time point). (B) CXCR3 protein expression was quantified using flow cytometry by measuring GFP levels before (noninfected, NI) and 11 days after infection (INF) CIBER mice. GFP levels assessed among CD4^+^ and CD8^+^ T-cell MLN, spleen and small intestinal propria. NI, noninfected; INF, In order to examine CXCR3 expression in more detail, we utilized *Cxcr3* eGFP reporter (CIBER) mice, a bicistronic reporter in cells expressing CXCR3 also express eGFP [@ppat.1003706-Oghumu1]. We found large in populations of both CD4^+^ and CD8^+^ T in spleen (SPL) and LP compartments following ([Fig. 1B](#ppat-1003706-g001){ref-type="fig"}). In general, CXCR3 upregulation was most in the population. For example, in the MLN there was a increase in CD4^+^CXCR3^+^ cells but only a 2-fold in CD8^+^CXCR3^+^ lymphocytes. NK cells are also known to express CXCR3 and are an important source of early IFN-γ during gondii* infection [@ppat.1003706-Qin1], [@ppat.1003706-Sher1]. However, while CXCR3-GFP expression was relatively high on naïve NK cells, the GFP expression was in reduced during infection, suggesting lack of a role for CXCR3^+^ NK cells during intestinal infection ([Fig. S1](#ppat.1003706.s001){ref-type="supplementary-material"}). We next examined expression of the activation marker CD27 on GFP^+^ and GFP^−^ T in infected mice. CD27 was significantly lower CXCR3^−^GFP^−^ cells, suggesting an altered maturation state of the CXCR3^−^ T cells ([Fig. S2A and B](#ppat.1003706.s002){ref-type="supplementary-material"}). there was a lower percentage of CD27^+^ cells amongst CD8^+^ T lymphocytes, although the in expression were not as striking as with the CD4^+^ ([Fig. S2C and D](#ppat.1003706.s002){ref-type="supplementary-material"}). mice are increased in susceptibility and are prone to severe intestinal following *T. gondii infection* {#s2b} --------------------------------------------------------------------------------------------------------------------------- To further examine the role of CXCR3 *T. gondii* infection, mice deficient in CXCR3 were orally inoculated with low virulence ME49 cysts, and the outcome of infection monitored. While wild-type (WT) mice survived acute infection with 30 cysts, *Cxcr3^−/−^* animals displayed susceptibility with 75% of mice dying by 2 weeks post-infection ([Fig. 2A](#ppat-1003706-g002){ref-type="fig"}). When the cyst dose was increased 50, all | IntRodUCTIOn {#s1}
============
THE inTEStinAl MUcOsA is a CrITicAl EfFeCtoR SITe For ElIMINaTION OF entERIC PaTHOgeNs. *tOxOPLasmA GoNDIi*, a uBiquITOUs PrOtozoAN ParaSITE, IS a PrIme exAMpLe oF suCH A PATHoGeN. mAMMals aRe INfecTed WiTh *t. gOndIi* PriMArily by tHE ingestion Of TisSUE CYSTs frOm UndercooKED Meat OR ooCYStS eXcRETED iN the FEces oF feLiNeS, WHicH arE THe Sole dEFiniTIvE HOStS. UpoN InfectIOn, tHE PArAsite INdUCES a pOtenT th1 IMmUnE rESpONSe THat IS chAractEriZed bY HIGh LeveLS Of IL-12 ANd iFN-γ [@ppAt.1003706-DENKERs1], [@PpAt.1003706-DupOnT1]. inItial iL-12 prodUCTiON is laRGELY thE rESult oF Myd88-dEpENdenT tOLL-likE ReCePtOr (TlR) SIgNALING In DeNdRItIC cElLs, aNd ThE PARasitE PROFiLIn moLeCUle haS bEeN IDeNtIfieD aS A LiGand for TLR11 AnD TLR12 [@PpAT.1003706-sCANga1]--[@PpaT.1003706-SUkHUMAVASi1]. IL-12 ACtivATes naTUrAl KILLEr (Nk) ceLLS tO inITIAte IFn-Γ prODUCtIon AnD PROmotEs t-CEll diFFeReNtiaTIOn TOwards A TH1 PROgrAm. ULtiMATELy IfN-Γ is The cRItICaL CYTokInE iNvoLved In conTrollIng *tOXoPLasma*. WhIle *iN viTRo* EXpERimenTS SuGGest tHat MacRoPHAgeS acTiVAteD bY tHIs CYtoKine ACQuIRE aNTi-*TOxopLASMA* ACtIVITy THroUGH uPRegUlAtIOn OF iMMunItY-ReLAted gTpasE (irg) molecules that MeDIatE DeStRUCTiON Of THe PARasitOphoRouS vacuOLE [@pPat.1003706-zHAO1]--[@Ppat.1003706-KHAMineTS1], thE *IN vIvo* FuncTiON OF ifn-Γ is LesS ClEaR.
INfLaMMAToRy MoNOcYtes aRe aN imPORTanT CompoNENt OF DEfense AGAinST MiCROBIAl pATHOGENS, INCLuDing *tOxoPlaSmA* [@PPAt.1003706-DUNay1]. tHeSe cELls EXpReSS hIgH LevElS of ly6C/G (gr-1) anD are RecruiTEd fROM tHe bONE mArROw Via chEmoKINe (C-C mOtiF) RECePTOR 2 (ccR2) [@ppaT.1003706-GeIsSManN1]. dUrinG *LIsTERIA monoCYTOgeNES* INFeCtIOn, INflAmmatORY MOnoCYteS ARE reCruitEd FrOm the BOne mArRoW To THe sPLeeN ANd LIVEr Where tHEY dIFfERENtiAte into tnF-Α- ANd nitRIc oXIDE (nO)-PROduCiNg DcS (TiP-dCS). tHerE THEY ARE esSenTIal FOr BAcTeriAl clEaRAnCE AND mousE SurvIvAl [@PPaT.1003706-sHI1], [@pPaT.1003706-SERBINA1]. likewIsE, CCR2-dEPEnDent INFlAMmAtoRY MONOcYTES ARe REcrUITed to ThE Lung dURIng *MyCobAcTERia TubERCuLoSIs* InFEctiOn wHerE TheY ProTeCt MiCE fRoM diseASE BY ReCrUItINg aNd aCTIvAtIng t cElls AnD By pRoducinG no [@ppAt.1003706-PeTeRS1], [@ppAt.1003706-pEterS2]. mUcOsal DEFeNsE agAINST *t. gonDiI* has Also rECENtly Been showN TO REquiRe CcR2-dEPeNdent iNFlAmmAtORy mOnOcYTes [@PpaT.1003706-DunaY1]. UPoN rECruItMEnt to tHE sMaLl iNTEstinE, thESE CeLLs CONtROL tHe pArAsite EIthER indIrecTLy By ProDucTIOn OF IL-12 aND tnf-Α oR DIrecTlY THRoUgH PrOduCTION OF nO AND irg proTEINS [@pPat.1003706-YaRovINsky1]--[@Ppat.1003706-AnDraDE1], [@ppaT.1003706-ZHAO1], [@pPat.1003706-TAyLor1], [@Ppat.1003706-dUnAy1], [@Ppat.1003706-lIng1]. WhilE ccR2 EnablEs reCRuITMeNT OF InfLamMatorY MONoCyTes TO sItEs Of InFECtIoN, tHe facTorS tHaT cOORdINAtE TheIR activaTIon AnD aCqUIsITion OF efFEctOr FuNctiOn are not KnoWN.
CXCr3 iS a th1-AssOCiATEd ChEMokInE RecEpTor, AnD cELls eXPREssING ThIS ReCEPtor REsPond To THe iFN-Γ-inDUcIBlE chEMoKinES cxCl9, 10, and 11 [@PPaT.1003706-grooM1]. ThE rEcepToR IS EXpREsSEd preDOmINAnTLY BY T cElLS aND nk CelLS ANd IS RapidlY UprEGulaTEd upON CELL ACTIvaTIOn. tHERe iS evideNCE ThAT cXcr3 eXPREsSion eNAbLeS T-CELl EnTrY iNTO sITeS Of iNfECTioN, AlThoUGh The ouTcoME OF rEcrUitmeNT VaRies AMOnG pathOgEnS. iN tHE CASe Of *LeISHmaNiA mAJOR*, recruitment iS prOtective aS Cxcr3-eXPresSInG T CeLls ARE reQuIrED foR tHe rEsoluTIoN of CuTANeOuS LeSionS [@PPAt.1003706-roSas1]. HoweVer, In the case oF *PLASMOdIuM bergHEI* aNKA, CxcR3 iS PathOgEnIc BECaUSE it allowS EnTrY of PROinflAmMAToRy celLs into THe cNS, RESUlTInG in CErebRAL mAlaria [@pPaT.1003706-cAmPanelLa1].
herE we DEtErmInEd THE RoLe OF cXCr3 IN tHe inTeStInal imMune ReSpoNse to *tOxoPLaSma*. we FoUnd tHat lOSS of CXcr3 NEgaTIvELY aFFectED HOSt suRViVal againST oRAL infECTION. tHIs WAS assOCIaTeD wITH DImiNiSHEd RecrUItmEnt oF CD4^+^ T CelLS to THE LamiNA PROpRIA (Lp), deCrEaSED t ceLl ifN-Γ SECRETioN, ImpAirEd iNFlAmmAtoRY MonocYtE effecTor fuNCtion, aND InabILItY TO ConTRol the PAraSITe In tHe intEstInAl muCosA. REcOnStitutIon WITH CXcR3-cOMpETEnt cD4^+^ T cElLs restoRed INFLAmmAToRy MOnOCyTE FUNcTioN, reSULTiNg IN IMproVED SuRvivaL AGaINst THe paRAsitE. pROteCtIVE EfFecTS Of ADoptiVely tRANsFerRED CD4^+^ T lYmPhOcyTeS DEPeNdeD upon THEir Ability tO pRODuCe ifn-γ, bUt OCcurReD INdepeNDEnTLy oF cD4 expReSsIOn oF Cd40l. our Data SHOw THat CxCr3 EnABlEs th1 reCruitMeNT TO The inteStInal Lp, WheRE These CELLs iNSTruCt ActivaTioN of CCR2-dePeNDENt INflaMMaTOry mOnoCyTes, iN TURn ConTrOLLiNg iNfeCTION. tHeSE REsulTS eSTablish cxcr3 aS A mAJor DeTerMiNANT ORcHesTRatINg cOmmuNiCATiON BetwEen EfFectoRS of InNatE aNd adaPTIvE IMmUNItY, eNAbLInG EFFeCtive Host defeNSe aGaInST inFECTIon.
ReSUlTs {#s2}
=======
CxcR3 aNd ITS lIGaNDS cxcl9 AND cxCL10 are uPReGUlated duriNg ACUTe tOXoPLasMosIS {#s2A}
---------------------------------------------------------------------------------
becaUsE cxCR3 aNd itS cHemOKinE LigANDS are sTroNgLY aSSoCIateD wITH th1 REspONSes, WE AsKEd wHEthEr ThIS ProInfLaMMatoRy axIs wAs InduCED DUrINg *tOXoPLASMa* InfecTION iN tHe InTEsTiNAL mucOsa. aCCoRDINglY, MICe werE oRaLly iNocuLaTeD wIth cYStS, aND ReLativE levELs oF CxcR3, cXCL9 and cxcL10 Mrna eXpRessiON WeRE meASuRED OVeR THe cOURse Of aCutE iNFectION. we FOuND StrONg upREguLATion OF CXCR3 AnD ITS SPeCIFiC cheMOkINE LIgands as EARlY as DAY 4 PosT-infecTIoN In bOtH thE iLeUm aNd MeSeNtERic lYmPh NodES (mLn) ([fiG. 1a](#pPAt-1003706-G001){ref-typE="FIG"}). oveRAlL, PeAK CXCr3 MrnA LeveLS WeRE ATTaINeD bY DAY 6 poST-InOCULAtIon.
{#PpaT-1003706-g001}
in ORder to eXamIne CXcR3 eXprESsion iN MORE dETAIl, We utilIZeD *CxCr3* eGfp REpoRtEr (cIber) miCe, a biCiSTrOniC REporteR strAin IN wHich cElls EXpresSING CXcr3 aLSo Express EGfp [@ppat.1003706-OgHumu1]. we FoUNd a lARGe InCReAsE In CxCr3 PoPULATIOnS oF BotH cd4^+^ aND CD8^+^ t LymPHoCYtES in mln, SplEen (SpL) and Lp cOmParTments foLLoWiNg iNfECtIOn ([fIg. 1B](#pPAt-1003706-g001){REF-Type="fiG"}). in geNeRAl, CxCR3 UPreGulAtIon was mOST PRonOUncEd IN the CD4^+^ PopUlATion. For EXaMpLe, IN tHE mLN there WAs a 6-FoLD INcrEASe iN CD4^+^Cxcr3^+^ cElls bUt OnLy A 2-foLD inCrEAse in Cd8^+^CXCR3^+^ lYMpHocYTES. NK CELlS arE also kNoWN To exprESs CxCR3 ANd aRE aN iMPORTANt SOURcE of Early ifN-Γ during *t. GOndii* INFecTIoN [@pPAT.1003706-qin1], [@PpAT.1003706-ShER1]. hOWevER, wHIle cxCR3-GFp eXpReSsiOn WaS relatiVeLy higH On naïve Nk CeLls, ThE gFp eXpRESsIOn WAs in fact rEducEd dURInG InfEcTiON, SUgGEstINg LacK Of a ROLE for CxcR3^+^ NK ceLlS During IntEStInAL INFECTiOn ([fiG. S1](#PPat.1003706.s001){rEF-tYPe="SUpPlEMEntaRy-mAteriAL"}). WE NEXT ExAmIneD eXPRessioN Of the ActIvaTiON mArKeR cd27 oN GfP^+^ aND gfp^−^ T lymPHOCyTes In iNfeCTed mIce. CD27 was SIGnifiCaNTLY LOwer iN cxcR3^−^gfp^−^ celLS, SUGgesting an aLterED maTURaTiOn STaTe OF thE CXCr3^−^ t CEllS ([fIG. S2A AnD b](#pPAT.1003706.s002){rEf-TYPe="SUPpLEMentarY-MATERIal"}). LIKeWisE, There WAs a lowEr PercenTAge oF Cd27^+^ cELLs AmONgsT CxcR3-gfP^−^ cd8^+^ T LymPHOCytES, aLThOUGH tHe decrEasEs iN expREssioN WErE NOt As StRiKiNG AS WItH ThE cD4^+^ LYmPHoCyTes ([FIG. S2C AnD D](#Ppat.1003706.S002){rEF-tYpe="SupPlemEntARY-MATeRial"}).
*CXcR3^−/−^* MiCE arE IncREasEd iN SusCEpTIBiLity AnD are PrOne to sEvEre iNteSTiNal dAmAGE follOwiNG *T. GoNdiI infEctiOn* {#s2b}
---------------------------------------------------------------------------------------------------------------------------
tO FurthEr EXamINe tHE RoLe OF cXcr3 dURINg *T. GoNDIi* iNFecTION, miCE dEfICIENT In CXcR3 weRe oRaLlY inOCULatED witH lOw VIruLenCe Me49 cysTs, AnD tHe OUtCoMe of iNFEctION Was MoNitOrED. WHile All wild-tYpe (wT) micE SuRvIvEd ACutE INfECtioN wITH 30 CYsts, *CXCR3^−/−^* aNiMaLS DIsPLAyeD IncREaSED SUSCEpTIBIliTy wiTH NeaRLy 75% Of Mice dyInG By 2 WeEkS poSt-inFEctiOn ([fIg. 2a](#PPaT-1003706-G002){reF-tYPE="FIg"}). wHEN thE CyST DosE wAS INCReAsED tO 50, AlL | Introduction {#s1} ============ The intestinal mucosa is a criticaleffector site for elimination of enteric pathogens. *Toxoplasma gondii*, aubiquitous protozoan parasite, is a prime example of such a pathogen. Mammals are infected with *T. gondii* primarilybythe ingestion of tissue cysts fromundercooked meat or oocysts excreted in the feces offelines, which are the sole definitive hosts. Uponinfection, the parasite induces a potent Th1 immune response that ischaracterized by high levels of IL-12 and IFN-γ [@ppat.1003706-Denkers1], [@ppat.1003706-Dupont1]. Initial IL-12 production is largelythe result of MyD88-dependent Toll-like receptor (TLR) signaling in dendriticcells,and the parasite profilin molecule has beenidentified as a ligandfor TLR11 andTLR12 [@ppat.1003706-Scanga1]--[@ppat.1003706-Sukhumavasi1]. IL-12 activates natural killer (NK) cells to initiate IFN-γ production andpromotes T-cell differentiation towards a Th1program. Ultimately IFN-γis the critical cytokine involvedin controlling*Toxoplasma*.While *in vitro* experiments suggest that macrophages activated by this cytokineacquire anti-*Toxoplasma* activity through upregulation of immunity-related GTPase (IRG) molecules that mediate destruction ofthe parasitophorous vacuole [@ppat.1003706-Zhao1]--[@ppat.1003706-Khaminets1], the *in vivo* function of IFN-γ is less clear. Inflammatory monocytes are an important component of defense against microbialpathogens, including *Toxoplasma* [@ppat.1003706-Dunay1]. Thesecells express high levels of Ly6C/G (Gr-1) and are recruited from the bone marrow via chemokine (C-C motif)receptor 2 (CCR2)[@ppat.1003706-Geissmann1]. During *Listeria monocytogenes* infection,inflammatory monocytes are recruitedfrom the bone marrow to the spleen andliver where they differentiate into TNF-α- and nitric oxide (NO)-producing DCs (Tip-DCs). There they are essential for bacterial clearanceand mouse survival [@ppat.1003706-Shi1], [@ppat.1003706-Serbina1].Likewise,CCR2-dependentinflammatory monocytes are recruited to the lung during *Mycobacteria tuberculosis* infectionwhere theyprotect mice from diseaseby recruiting and activating T cellsand by producingNO [@ppat.1003706-Peters1],[@ppat.1003706-Peters2]. Mucosal defense against*T. gondii* has also recently been shown to require CCR2-dependent inflammatory monocytes [@ppat.1003706-Dunay1]. Upon recruitment to the small intestine, these cells control the parasite either indirectly by production of IL-12 and TNF-α or directly throughproduction ofNO and IRG proteins [@ppat.1003706-Yarovinsky1]--[@ppat.1003706-Andrade1], [@ppat.1003706-Zhao1], [@ppat.1003706-Taylor1], [@ppat.1003706-Dunay1], [@ppat.1003706-Ling1].While CCR2 enables recruitmentof inflammatory monocytes tosites of infection, the factors that coordinatetheir activation and acquisition of effector function are not known.CXCR3is a Th1-associated chemokine receptor, and cells expressingthis receptor respond to the IFN-γ-inducible chemokines CXCL9, 10, and 11 [@ppat.1003706-Groom1]. The receptor is expressedpredominantly byT cells and NK cells and is rapidly upregulatedupon cell activation. Thereis evidence that CXCR3 expressionenables T-cell entry into sites ofinfection, although the outcome of recruitment varies amongpathogens. In the case of *Leishmaniamajor*, recruitment is protective as CXCR3-expressing T cells are required forthe resolution of cutaneous lesions [@ppat.1003706-Rosas1]. However, in the case of *Plasmodium berghei* ANKA, CXCR3ispathogenic because itallows entry of proinflammatory cells into the CNS,resulting in cerebral malaria [@ppat.1003706-Campanella1]. Here wedetermined the role of CXCR3 in the intestinal immune response to *Toxoplasma*. Wefound that loss of CXCR3 negatively affected host survival against oralinfection. Thiswasassociated with diminished recruitment of CD4^+^ T cells to thelamina propria(LP), decreased T cell IFN-γsecretion, impaired inflammatorymonocyte effector function, and inability to control the parasite in theintestinal mucosa. Reconstitution with CXCR3-competentCD4^+^ T cells restored inflammatory monocyte function, resulting in improved survival against the parasite. Protective effects of adoptivelytransferred CD4^+^ T lymphocytes depended upon their ability to produceIFN-γ, but occurred independently ofCD4 expression of CD40L. Our data show that CXCR3 enables Th1 recruitment to the intestinal LP, where these cellsinstructactivation ofCCR2-dependent inflammatory monocytes, in turn controllinginfection. These results establish CXCR3 as a major determinant orchestrating communication betweeneffectors of innate and adaptive immunity, enabling effective host defense against infection. Results {#s2} ======= CXCR3 and its ligands CXCL9 and CXCL10 are upregulated duringacutetoxoplasmosis {#s2a}--------------------------------------------------------------------------------- Because CXCR3and its chemokine ligands are strongly associated with Th1 responses, we asked whether this proinflammatory axiswas induced during *Toxoplasma* infectionin the intestinal mucosa. Accordingly, mice were orallyinoculated with cysts,and relativelevels of CXCR3, CXCL9 and CXCL10mRNA expression were measured over thecourse of acute infection. Wefoundstrong upregulation of CXCR3 and its specific chemokineligandsas early as Day 4 post-infection in both theileum and mesenteric lymph nodes (MLN)([Fig. 1A](#ppat-1003706-g001){ref-type="fig"}). Overall, peak CXCR3 mRNA levels were attained by Day 6 post-inoculation. {#ppat-1003706-g001} In order to examine CXCR3 expression in more detail, we utilized *Cxcr3* eGFP reporter (CIBER) mice, a bicistronic reporter strain inwhich cells expressing CXCR3 also express eGFP [@ppat.1003706-Oghumu1]. We founda large increase in CXCR3populations of bothCD4^+^ and CD8^+^ Tlymphocytes in MLN, spleen (SPL) and LP compartments followinginfection ([Fig. 1B](#ppat-1003706-g001){ref-type="fig"}). In general, CXCR3upregulationwas most pronounced in the CD4^+^ population. For example, inthe MLN there was a6-fold increase in CD4^+^CXCR3^+^ cellsbut only a 2-fold increasein CD8^+^CXCR3^+^ lymphocytes. NKcells are also knowntoexpress CXCR3 and are an important source of early IFN-γ during*T. gondii* infection [@ppat.1003706-Qin1], [@ppat.1003706-Sher1]. However, while CXCR3-GFP expression was relatively high onnaïve NK cells, the GFP expression was in fact reduced during infection, suggestinglack ofa role for CXCR3^+^ NKcells duringintestinalinfection ([Fig. S1](#ppat.1003706.s001){ref-type="supplementary-material"}). We next examinedexpression of the activation marker CD27 on GFP^+^ and GFP^−^ T lymphocytes in infected mice.CD27 was significantly lower in CXCR3^−^GFP^−^ cells, suggesting an altered maturation state of the CXCR3^−^ T cells([Fig.S2A and B](#ppat.1003706.s002){ref-type="supplementary-material"}). Likewise, there was a lower percentage of CD27^+^ cellsamongst CXCR3-GFP^−^ CD8^+^ T lymphocytes,although the decreases in expression were not as striking as with the CD4^+^ lymphocytes ([Fig. S2C and D](#ppat.1003706.s002){ref-type="supplementary-material"}). *Cxcr3^−/−^* mice areincreased in susceptibility and are prone to severe intestinal damage following *T. gondii infection* {#s2b} --------------------------------------------------------------------------------------------------------------------------- To further examine therole of CXCR3 during*T. gondii* infection, mice deficient in CXCR3 were orally inoculated with low virulence ME49 cysts,and the outcome ofinfection was monitored. While all wild-type (WT) mice survived acute infection with 30cysts, *Cxcr3^−/−^* animals displayed increasedsusceptibilitywith nearly 75%of mice dying by 2 weeks post-infection ([Fig. 2A](#ppat-1003706-g002){ref-type="fig"}). When the cystdose was increasedto 50, all | Introduction {#s1} ============ The intestinal mucosa is a _critical_ effector site for _elimination_ of enteric _pathogens._ *Toxoplasma gondii*, a ubiquitous protozoan parasite, is a prime _example_ of such a pathogen. Mammals are infected with *T. gondii* _primarily_ by _the_ ingestion of tissue cysts _from_ undercooked meat or oocysts excreted _in_ the feces _of_ felines, which are the _sole_ definitive hosts. Upon infection, the parasite induces a potent _Th1_ immune response that is _characterized_ by _high_ levels of _IL-12_ and _IFN-γ_ _[@ppat.1003706-Denkers1],_ [@ppat.1003706-Dupont1]. _Initial_ _IL-12_ production _is_ _largely_ the result of MyD88-dependent Toll-like receptor (TLR) signaling in _dendritic_ cells, and the parasite profilin molecule has been _identified_ as a ligand for TLR11 and TLR12 [@ppat.1003706-Scanga1]--[@ppat.1003706-Sukhumavasi1]. IL-12 activates natural killer (NK) cells to _initiate_ IFN-γ production and _promotes_ T-cell differentiation towards a Th1 program. Ultimately IFN-γ is the critical cytokine _involved_ _in_ controlling *Toxoplasma*. While _*in_ vitro* experiments _suggest_ that macrophages activated by this cytokine acquire _anti-*Toxoplasma*_ activity _through_ upregulation of immunity-related GTPase (IRG) molecules _that_ mediate destruction of the parasitophorous _vacuole_ [@ppat.1003706-Zhao1]--[@ppat.1003706-Khaminets1], the *in vivo* function _of_ IFN-γ is less clear. Inflammatory monocytes are an important component _of_ _defense_ against microbial pathogens, including *Toxoplasma* [@ppat.1003706-Dunay1]. _These_ cells _express_ high levels _of_ Ly6C/G (Gr-1) and are recruited from the bone _marrow_ via chemokine (C-C motif) receptor 2 (CCR2) [@ppat.1003706-Geissmann1]. During *Listeria _monocytogenes*_ infection, inflammatory monocytes are recruited from the bone marrow to the spleen and liver where they differentiate into TNF-α- and _nitric_ oxide (NO)-producing DCs _(Tip-DCs)._ There they are essential for _bacterial_ clearance and mouse survival [@ppat.1003706-Shi1], [@ppat.1003706-Serbina1]. Likewise, _CCR2-dependent_ inflammatory monocytes are recruited to the lung during _*Mycobacteria_ tuberculosis* infection where they protect mice from _disease_ _by_ recruiting _and_ activating T _cells_ and by producing NO _[@ppat.1003706-Peters1],_ [@ppat.1003706-Peters2]. Mucosal defense against *T. gondii* _has_ also recently been shown to require CCR2-dependent inflammatory monocytes [@ppat.1003706-Dunay1]. Upon recruitment _to_ the small intestine, _these_ cells _control_ the parasite either indirectly by production of IL-12 and _TNF-α_ _or_ directly through production of NO and IRG proteins _[@ppat.1003706-Yarovinsky1]--[@ppat.1003706-Andrade1],_ [@ppat.1003706-Zhao1], [@ppat.1003706-Taylor1], _[@ppat.1003706-Dunay1],_ [@ppat.1003706-Ling1]. While CCR2 enables recruitment of inflammatory monocytes _to_ sites of infection, the factors that coordinate their _activation_ and acquisition of _effector_ function are not known. _CXCR3_ _is_ a _Th1-associated_ _chemokine_ receptor, and cells expressing this _receptor_ respond to the IFN-γ-inducible chemokines CXCL9, _10,_ and 11 [@ppat.1003706-Groom1]. The receptor is expressed _predominantly_ _by_ _T_ cells and NK cells and _is_ rapidly upregulated _upon_ _cell_ activation. There is evidence that CXCR3 expression enables T-cell _entry_ into sites _of_ infection, although the outcome of recruitment varies among pathogens. In the _case_ of _*Leishmania_ major*, recruitment _is_ protective as CXCR3-expressing _T_ _cells_ are required for the resolution of _cutaneous_ lesions [@ppat.1003706-Rosas1]. However, in _the_ case of *Plasmodium berghei* ANKA, CXCR3 is _pathogenic_ because it allows entry of _proinflammatory_ cells _into_ the CNS, _resulting_ _in_ cerebral malaria [@ppat.1003706-Campanella1]. Here we determined the role of CXCR3 in the _intestinal_ immune response to *Toxoplasma*. We found that loss _of_ CXCR3 negatively _affected_ _host_ _survival_ against oral infection. This was associated _with_ diminished _recruitment_ _of_ CD4^+^ _T_ _cells_ to the lamina propria (LP), decreased _T_ cell IFN-γ _secretion,_ impaired inflammatory monocyte effector function, and inability to control _the_ parasite in the _intestinal_ mucosa. _Reconstitution_ with _CXCR3-competent_ _CD4^+^_ T cells restored _inflammatory_ monocyte function, resulting in _improved_ survival _against_ the parasite. Protective effects of adoptively transferred _CD4^+^_ T lymphocytes depended upon their ability to _produce_ IFN-γ, but occurred independently of CD4 expression of _CD40L._ Our data show that CXCR3 _enables_ Th1 _recruitment_ to _the_ intestinal LP, where these cells instruct activation of CCR2-dependent inflammatory monocytes, in _turn_ controlling infection. These results establish CXCR3 as a major determinant orchestrating communication between effectors _of_ innate and _adaptive_ immunity, enabling effective host defense against _infection._ Results _{#s2}_ ======= CXCR3 and its _ligands_ CXCL9 _and_ CXCL10 are upregulated during acute toxoplasmosis {#s2a} --------------------------------------------------------------------------------- Because CXCR3 and its _chemokine_ ligands are _strongly_ associated with Th1 responses, we _asked_ whether _this_ proinflammatory axis was induced during *Toxoplasma* _infection_ in the _intestinal_ mucosa. _Accordingly,_ mice were orally inoculated with cysts, and _relative_ levels of CXCR3, CXCL9 _and_ _CXCL10_ mRNA _expression_ were measured _over_ the course of acute infection. We found strong upregulation of _CXCR3_ and _its_ specific chemokine ligands as _early_ as Day 4 post-infection in both the _ileum_ and mesenteric lymph nodes _(MLN)_ _([Fig._ 1A](#ppat-1003706-g001){ref-type="fig"}). Overall, peak CXCR3 mRNA levels _were_ attained by Day _6_ post-inoculation. {#ppat-1003706-g001} In order _to_ examine _CXCR3_ _expression_ in more detail, we utilized *Cxcr3* eGFP reporter (CIBER) _mice,_ a _bicistronic_ reporter strain in _which_ cells expressing CXCR3 _also_ express eGFP _[@ppat.1003706-Oghumu1]._ We found a large increase in CXCR3 populations of _both_ CD4^+^ and CD8^+^ T lymphocytes in MLN, spleen (SPL) and LP compartments _following_ _infection_ _([Fig._ _1B](#ppat-1003706-g001){ref-type="fig"})._ In _general,_ CXCR3 upregulation was _most_ pronounced in the CD4^+^ population. For example, in the MLN there _was_ a _6-fold_ increase in _CD4^+^CXCR3^+^_ cells but only a 2-fold increase in CD8^+^CXCR3^+^ lymphocytes. NK _cells_ _are_ also known to express CXCR3 and are an important source of _early_ IFN-γ during *T. gondii* infection [@ppat.1003706-Qin1], [@ppat.1003706-Sher1]. _However,_ while _CXCR3-GFP_ expression _was_ relatively high on _naïve_ NK cells, the GFP expression was in fact reduced _during_ infection, suggesting _lack_ of _a_ role for CXCR3^+^ NK cells during intestinal infection ([Fig. S1](#ppat.1003706.s001){ref-type="supplementary-material"}). We next examined _expression_ of the activation marker _CD27_ on GFP^+^ and GFP^−^ _T_ lymphocytes in infected mice. CD27 was significantly lower in CXCR3^−^GFP^−^ cells, suggesting an altered maturation state of the CXCR3^−^ T cells ([Fig. _S2A_ and B](#ppat.1003706.s002){ref-type="supplementary-material"}). Likewise, there was _a_ lower percentage of CD27^+^ cells amongst CXCR3-GFP^−^ CD8^+^ T lymphocytes, although the _decreases_ in expression _were_ not as _striking_ as with the CD4^+^ _lymphocytes_ _([Fig._ S2C and D](#ppat.1003706.s002){ref-type="supplementary-material"}). *Cxcr3^−/−^* mice are increased in susceptibility and are prone to severe _intestinal_ damage _following_ *T. gondii infection* {#s2b} --------------------------------------------------------------------------------------------------------------------------- To further examine the role of CXCR3 during *T. gondii* _infection,_ mice deficient in CXCR3 were orally inoculated with low virulence ME49 cysts, and the outcome _of_ infection was monitored. _While_ all _wild-type_ _(WT)_ mice survived acute infection with 30 cysts, *Cxcr3^−/−^* animals displayed increased susceptibility _with_ nearly 75% of mice _dying_ by 2 weeks post-infection ([Fig. 2A](#ppat-1003706-g002){ref-type="fig"}). When the cyst dose _was_ _increased_ to _50,_ _all_ |
Our individual data points can not be publicly deposited as supporting information due to ethical restrictions imposed by Nagoya University International Bioethics Committee, since the data contains the sensitive personal information. Information regarding our data can be requested at the following e-mail address: Kazunori Hashimoto (e-mail: <khashim@med.nagoya-u.ac.jp>).
Introduction {#sec001}
============
Exposure to arsenic (As) via drinking water is a health risk for humans \[[@pone.0198743.ref001]--[@pone.0198743.ref003]\]. Previous studies showed that exposure of humans to As was associated with cancer of the bladder, kidney, skin, prostate, lung and liver \[[@pone.0198743.ref004]\] and with cardiovascular disease \[[@pone.0198743.ref005]\]. It was also shown that exposure to As was associated with neurological diseases including cognitive impairment in children \[[@pone.0198743.ref006], [@pone.0198743.ref007]\] and adults \[[@pone.0198743.ref008]\] and with diabetes mellitus \[[@pone.0198743.ref009]--[@pone.0198743.ref012]\].
Levels of toxic elements in noninvasive biological samples including nails, hair and urine have been determined to investigate associations with hearing loss in humans \[[@pone.0198743.ref013], [@pone.0198743.ref014]\]. A univariate analysis showed a significant correlation of As levels in fingernails with amplitude of distortion product otoacoustic emission (DPOAE) at 2 kHz, which reflects activity levels of outer hair cells, in 30 subjects aged 9--78 years residing in the gold mining community of Bonanza, Nicaragua \[[@pone.0198743.ref015]\]. Thus, it is possible that As in nails is a reliable index for As-mediated hearing loss in humans. However, there is no direct evidence showing correlations between As levels in toenails and hearing levels.
In our previous study using pure tone audiometry (PTA), an association of oral exposure to As via drinking water with hearing loss in young people was shown by multivariate analysis with adjustments for confounders including age, smoking, sex and BMI \[[@pone.0198743.ref016]\]. However, multivariate analysis has not been performed to demonstrate the association of As levels in nails with hearing loss in humans, although age and smoking are known to be strong factors affecting hearing levels in humans \[[@pone.0198743.ref014], [@pone.0198743.ref017]--[@pone.0198743.ref019]\].
In experimental studies, oral exposure to As has been shown to cause several health risks including carcinogenesis \[[@pone.0198743.ref020]\] and cardiovascular diseases \[[@pone.0198743.ref021]\]. Levels of toxic elements in inner ears, which are known to be the sensory organ for hearing, have been determined to demonstrate the correlation with hearing loss \[[@pone.0198743.ref022], [@pone.0198743.ref023]\]. Exposure of guniea pigs to As at 200 mg/L via intraperitoneal injection for 2 months resulted in morphological impairments of the inner ear \[[@pone.0198743.ref024]\]. Our previous study has showed that exposure of young mice to As via drinking water caused accumulation of As in inner ears, resulting in hearing loss \[[@pone.0198743.ref016]\]. However, it is not clear whether there is a correlation between As levels in inner ears and nails in mice.
In this study, we epidemiologically and experimentally investigated whether As levels in nails are reliable as non-invasive indexes for As-mediated hearing loss in humans and whether As levels in nails are associated with As levels in inner ears in mice.
Materials and methods {#sec002}
=====================
Epidemiological study {#sec003}
---------------------
We obtained information on age, sex, smoking history, weight and height in 145 healthy subjects aged from 12 to 55 years by self-reported questionnaires in Bangladesh as previously described \[[@pone.0198743.ref014]\]. We obtained informed consent in written form from all of the subjects. For subjects aged 12 to 18 years, we obtained consent in written form from their parents. The subjects were only Bangladeshi people who lived mainly in rural areas. The subjects living in one area drink tap water and the rest of the subjects living in another area drink tube well water contaminated with As. The subjects included students, housewives and businessmen. The subjects did not have portable music players with earphones and had no occupational exposure to wielding fumes. None of the subjects had a habit of drinking alcohol. We excluded only subjects who had a history of ear diseases or illness at the time of the survey. We calculated body mass index (BMI) by diving weight (kg) by the square of height (m^2^) and used definitions of underweight (\< 18.5 kg/m^2^), normal weight (18.5--24.9 kg/m^2^) and overweight (≥ 25 kg/m^2^) set by the WHO \[[@pone.0198743.ref025]\]. We set the mean value of age (29.6 years) of the subjects in this study as the cut-off value for age. We collected 0.1--2 cm samples of toenails from each subject by using a ceramic nail clipper. We also collected urine samples from each subject and stored the samples in 50 ml sterile tubes at -80°C until measurements. We determined the hearing levels at 1, 4 and 8 kHz in addition to 12 kHz of the participants by PTA, since hearing level at 12 kHz was shown to be sensitive to environmental stresses \[[@pone.0198743.ref014], [@pone.0198743.ref017], [@pone.0198743.ref026]\]. We used an iPod with earphone-type headphones (Panasonic RP-HJE150) in a soundproof room as described previously \[[@pone.0198743.ref027], [@pone.0198743.ref028]\]. We output pure tones at each frequency to a subject until the subject recognized the sound. We stood behind the subject and provided the subject with an initial stimulus of 5 decibels (dB) and then increased the sound level by 5 dB step-by-step. The subject raised their hand when they recognized the sound. We evaluated the sound level recognized by each subjects as hearing threshold. Pure tones at each frequency from the earphones in the soundproof room were verified by using a noise level meter (Type 6224 with an FFT analyzer, ACO CO., LTD, Japan). The epidemiological study was approved by Nagoya University International Bioethics Committee following the regulations of the Japanese government (approval number: 2013--0070) and the Faculty of Biological Science, University of Dhaka (Ref. no. 5509/Bio.Sc).
Experimental study {#sec004}
------------------
Hairless mice having the C57BL/6J background (1 month old, female, body weight of 10--15 g) were procured from Hoshino Laboratory Animal, Inc. Three or four mice were housed in a cage under super pathogen-free (SPF) conditions with a standard temperature of 23 ± 2°C and a 12-h light/dark cycle. The mice were fed a standard rodent chow (Clea Rodent Diet CE-2). Neither randomization nor blinding investigation were used in this animal study. In brief, we exposed mice (n = 7) to 22.5 mg/L of sodium arsenite (NaAsO~2~, Sigma-Aldrich) dissolved in distilled water for 2 months via drinking water and changed the drinking water every week as previously described \[[@pone.0198743.ref016]\]. The exposure dose was based on the As exposure for mice at 10 and 100 ppm via drinking water in a previous study \[[@pone.0198743.ref029]\]. The control group (n = 6) was given just distilled water. After exposure for 2 months, mice were anesthetized with isoflurane and sacrificed by decapitation. Nails and inner ears were collected and kept in a 1.5 ml tube. For the collection of inner ears, we first identified temporal bones at the bottom of the skull and then carefully removed the cranial nerves and tissues using standard forceps. The inner ears were dislodged by pushing down on the posterior semicircular canal with the thumb of a hand and fixing the tip region of the otic bone capsule with standard forceps. After carefully removing extra tissues adhered to the inner ears, we used the inner ears for ashing. The experimental study was approved by the Institutional Animal Care and Use Committee in Nagoya University (approval number: 28251) and followed the Japanese Government Regulations for Animal Experiments.
Measurement of As levels in biological samples {#sec005}
----------------------------------------------
As levels in biological samples including toenails and urine were measured by inductively coupled plasma mass spectrometry (ICP-MS; Agilent 7500cx) as described previously \[[@pone.0198743.ref014], [@pone.0198743.ref022], [@pone.0198743.ref023]\]. In brief, toenails were washed with detergent water. One or two drops of acetone were added and then the samples were kept at room temperature until starting the ashing. Samples were ashed by incubation in 3 ml of HNO~3~ at room temperature overnight followed by incubation at 80°C for 3 hours. Samples were further incubated in 1--1.5 ml of H~2~O~2~ at 80°C for 3 hours. After cooling, Milli-Q water was added to the samples to adjust the final volume to 5 ml. In the case of urine, 4 ml of urine was incubated in 1 ml of HNO~3~ at room temperature overnight followed by incubation | our individual data points can not be publicly deposited as supporting information due to ethical restrictions imposed by nagoya university international bioethics committee, since the data contains the sensitive personal records. information regarding anonymous users can be requested at the following e - mail address : kazunori hashimoto ( e - mail : < khashim @ med. nagoya - u. ac. jp > ). introduction { # sec001 } = = = = = = = = = = = = exposure to arsenic ( as ) via drinking water is a health risk for humans \ [ [ @ pone. 0198743. ref001 ] - - [ @ pone. 0198743. ref003 ] \ ]. previous studies showed that exposure of humans to as was associated with cancer of the bladder, kidney, skin, prostate, lung and liver \ [ [ @ pone. 0198743. ch ] \ ] and with cardiovascular disease \ [ [ @ h. 0198743. ref005 ] \ ]. it was also shown that exposure to as was associated with neurological diseases including cognitive impairment in children \ [ [ @ pone. 0198743. ref006 ], [ @ pone. 0198743. ref007 ] \ ] and adults \ [ [ @ pone. 0198743. ref008 ] \ ] and with diabetes mellitus \ [ [ @ pone. 0198743. ref009 ] - - [ @ pone. 0198743. ref012 ] \ ]. levels of toxic emission in noninvasive biological samples involving nails, hair and urine have been determined to investigate issues with hearing loss in humans \ [ [ @ pone. 0198743. ref013 ], [ @ pone. 0198743. ref014 ] \ ]. a univariate analysis showed a significant correlation of as levels in fingernails with amplitude of distortion product otoacoustic emission ( dpoae ) at 2 khz, which reflects activity levels of outer hair cells, in 30 subjects aged 9 - - 78 am residing in the gold mining community of bonanza, nicaragua \ [ [ @ pone. 0198743. ref015 ] \ ]. thus, he is possible that as in nails is a reliable index for as - mediated hearing loss in humans. however, there is no direct evidence showing correlations between as levels in toenails and hearing levels. in our previous study using pure tone audiometry ( pta ), an association of oral exposure to as via drinking water with hearing loss in young people was shown by multivariate analysis with adjustments for confounders including age, smoking, sex and bmi \ [ [ @ pone. 0198743. ref016 ] \ ]. however, multivariate analysis has not been performed to demonstrate the association of as levels in nails with hearing loss in humans, although age and smoking are known to be strong factors affecting hearing levels in humans \ [ [ @ pone. 0198743. ref014 ], [ @ pone. 0198743. ref017 ] - - [ @ pone. 0198743. ref019 ] \ ]. in experimental studies, oral exposure to as has been shown to cause several health risks including carcinogenesis \ [ [ @ pone. 0198743. ref020 ] \ ] and cardiovascular diseases \ [ [ @ pone. 0198743. ref021 ] \ ]. levels of toxic elements in inner ears, which are known to be the sensory organ for hearing, have been determined to demonstrate the correlation with hearing loss \ [ [ @ pone. 0198743. ref022 ], [ @ pone. 0198743. ref023 ] \ ]. exposure of guniea pigs to as at 200 mg / l via intraperitoneal injection for 2 months resulted in morphological impairments of the inner ear \ [ [ @ pone. 0198743. ref024 ] \ ]. our previous study has showed that exposure of young mice to as via drinking water caused accumulation of as in inner ears, resulting in hearing loss \ [ [ @ pone. 0198743. ref016 ] \ ]. however, it is not clear whether there is a correlation between as levels in inner ears and nails in mice. in this study, we epidemiologically and experimentally investigated whether as levels in nails are reliable as non - invasive indexes for as - mediated hearing loss in humans and whether as levels in nails are associated with as levels in inner ears in mice. materials and methods { # sec002 } = = = = = = = = = = = = = = = = = = = = = epidemiological study { # sec003 } - - - - - - - - - - - - - - - - - - - - - we obtained information on age, sex, smoking history, weight and height in 145 healthy subjects aged from 12 to 55 years by self - reported questionnaires in bangladesh as previously described \ [ [ @ pone. 0198743. ref014 ] \ ]. we obtained informed consent in written form from all of the subjects. for subjects aged 12 to 18 years, we obtained consent in written form from their parents. the subjects were only bangladeshi people who lived mainly in rural areas. the subjects living in one area drink tap water and the rest of the subjects living in another area drink tube well water contaminated with as. the subjects included students, housewives and businessmen. the subjects did not have portable music players with earphones and had no occupational exposure to wielding fumes. none of the subjects had a habit of drinking alcohol. we excluded only subjects who had a history of ear diseases or illness at the time of the survey. we calculated body mass index ( bmi ) by diving weight ( kg ) by the square of height ( m ^ 2 ^ ) and used definitions of underweight ( \ < 18. 5 kg / m ^ 2 ^ ), normal weight ( 18. 5 - - 24. 9 kg / m ^ 2 ^ ) and overweight ( ≥ 25 kg / m ^ 2 ^ ) set by the who \ [ [ @ pone. 0198743. ref025 ] \ ]. we set the mean value of age ( 29. 6 years ) of the subjects in this study as the cut - off value for age. we collected 0. 1 - - 2 cm samples of toenails from each subject by using a ceramic nail clipper. we also collected urine samples from each subject and stored the samples in 50 ml sterile tubes at - 80°c until measurements. we determined the hearing levels at 1, 4 and 8 khz in addition to 12 khz of the participants by pta, since hearing level at 12 khz was shown to be sensitive to environmental stresses \ [ [ @ pone. 0198743. ref014 ], [ @ pone. 0198743. ref017 ], [ @ pone. 0198743. ref026 ] \ ]. we used an ipod with earphone - type headphones ( panasonic rp - hje150 ) in a soundproof room as described previously \ [ [ @ pone. 0198743. ref027 ], [ @ pone. 0198743. ref028 ] \ ]. we output pure tones at each frequency to a subject until the subject recognized the sound. we stood behind the subject and provided the subject with an initial stimulus of 5 decibels ( db ) and then increased the sound level by 5 db step - by - step. the subject raised their hand when they recognized the sound. we evaluated the sound level recognized by each subjects as hearing threshold. pure tones at each frequency from the earphones in the soundproof room were verified by using a noise level meter ( type 6224 with an fft analyzer, aco co., ltd, japan ). the epidemiological study was approved by nagoya university international bioethics committee following the regulations of the japanese government ( approval number : 2013 - - 0070 ) and the faculty of biological science, university of dhaka ( ref. no. 5509 / bio. sc ). experimental study { # sec004 } - - - - - - - - - - - - - - - - - - hairless mice having the c57bl / 6j background ( 1 month old, female, body weight of 10 - - 15 g ) were procured from hoshino laboratory animal, inc. three or four mice were housed in a cage under super pathogen - free ( spf ) conditions with a standard temperature of 23 ± 2°c and a 12 - h light / dark cycle. the mice were fed a standard rodent chow ( clea rodent diet ce - 2 ). neither randomization nor blinding investigation were used in this animal study. in brief, we exposed mice ( n = 7 ) to 22. 5 mg / l of sodium arsenite ( naaso ~ 2 ~, sigma - aldrich ) dissolved in distilled water for 2 months via drinking water and changed the drinking water every week as previously described \ [ [ @ pone. 0198743. ref016 ] \ ]. the exposure dose was based on the as exposure for mice at 10 and 100 ppm via drinking water in a previous study \ [ [ @ pone. 0198743. ref029 ] \ ]. the control group ( n = 6 ) was given just distilled water. after exposure for 2 months, mice were anesthetized with isoflurane and sacrificed by decapitation. nails and inner ears were collected and kept in a 1. 5 ml tube. for the collection of inner ears, we first identified temporal bones at the bottom of the skull and then carefully removed the cranial nerves and tissues using standard forceps. the inner ears were dislodged by pushing down on the posterior semicircular canal with the thumb of a hand and fixing the tip region of the otic bone capsule with standard forceps. after carefully removing extra tissues adhered to the inner ears, we used the inner ears for ashing. the experimental study was approved by the institutional animal care and use committee in nagoya university ( approval number : 28251 ) and followed the japanese government regulations for animal experiments. measurement of as levels in biological samples { # sec005 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - as levels in biological samples including toenails and urine were measured by inductively coupled plasma mass spectrometry ( icp - ms ; agilent 7500cx ) as described previously \ [ [ @ pone. 0198743. ref014 ], [ @ pone. 0198743. ref022 ], [ @ pone. 0198743. ref023 ] \ ]. in brief, toenails were washed with detergent water. one or two drops of acetone were added and then the samples were kept at room temperature until starting the ashing. samples were ashed by incubation in 3 ml of hno ~ 3 ~ at room temperature overnight followed by incubation at 80°c for 3 hours. samples were further incubated in 1 - - 1. 5 ml of h ~ 2 ~ o ~ 2 ~ at 80°c for 3 hours. after cooling, milli - q water was added to the samples to adjust the final volume to 5 ml. in the case of urine, 4 ml of urine was incubated in 1 ml of hno ~ 3 ~ at room temperature overnight followed by incubation | Our individual data points can not be publicly deposited as supporting information due to ethical restrictions imposed by Nagoya University International Bioethics Committee, since the data contains the sensitive personal information. Information regarding our data can be requested at the following e - mail address: Kazunori Hashimoto (e - mail: <khashim @ med. nagoya - u. ac. jp> ). Introduction {# sec001} = = = = = = = = = = = = Exposure to arsenic (As) via drinking water is a health risk for humans \ [[ @ pone. 0198743. ref001] - - [@ pone. 0198743. ref003] \ ]. Previous studies showed that exposure of humans to As was associated with cancer of the bladder, kidney, skin, prostate, lung and liver \ [[ @ pone. 0198743. ref004] \] and eiHh cardiovascular disease \ [[ @ pone. 0198743. ref005] \ ]. It was also shown that exposure to As was associated with neurological diseases including cognitive impairment in children \ [[ @ pone. 0198743. ref006 ], [@ pone. 0198743. ref007] \] and adults \ [[ @ pone. 0198743. ref008] \] and with diabetes mellitus \ [[ @ pone. 0198743. ref009] - - [@ pkn3. 0198743. ref012] \ ]. Levels of toxic elements in non*nvasiDe biological samples including nails, hair and urine have been determined to investigate associations with hearing loss in humans \ [[ @ pone. 0198743. ref013 ], [@ pone. 0198743. ref014] \ ]. A univariate analysis showed a significant correlation of As levels in fingernails with amplitude of distortion product otoacoustic emission (DPOAE) at 2 kHz, which reflects activity levels of outer hair cells, in 30 subjects aged 9 - - 78 years residing in the gold mining community of Bonanza, Nicaragua \ [[ @ pone. 0198743. ref015] \ ]. Thus, it is possible that As in nails is a reliable index for As - mediated hearing loss in humans. However, there is no direct evidence showing correlations between As levels in toenails and hearing levels. In our previous study using pure tone audiometry (PTA ), an association of oral exposure to As via drinking water with hearing loss in young people was shown by multivariate analysis with adjustments for confounders including age, smoking, sex and BMI \ [[ @ pone. 0198743. ref016] \ ]. However, multivariate analysis has not been performed to demonstrate the association of As leveOq in nails with hearing loss in humans, although age and smoking are known to be strong factors affecting hearing levels in humans \ [[ @ pone. 0198743. ref014 ], [@ pone. 0198743. ref017] - - [@ pone. 0198743. ref019] \ ]. In experimental studies, oral exposure to As has been shown to cause several health risks including carcinogenesis \ [[ @ pone. 0198743. ref020] \] and cardiovascular diseases \ [[ @ pone. 0198743. ref021] \ ]. Levels of toxic elements in inner ears, which are known to be the sensory organ for hearing, have been determined to demonstrate the correlation with hearing loss \ [[ @ pone. 0198743. ref022 ], [@ pone. 0198743. ref023] \ ]. Exposure of guniea pigs to As at 200 mg / L via intraperitoneal injection for 2 months resulted in morphological impairments of the inner ear \ [[ @ pone. 0198743. ref024] \ ]. Our previous study has showed that exposure of young mice to As via drinking water caused accumulation of As in inner ears, resulting in hearing loss \ [[ @ pone. 0198743. ref016] \ ]. However, it is not clear whether Fhete is a correlation between As levels in inner ears and nails in mice. In this study, we epidemiologically and experimentally investigated whether As levels in nails are reliable as non - invasive indexes for As - mediated hearing loss in humans and whether As levels in nails are associated with As levels in inner ears in mice. Materials and methods {# sec002} = = = = = = = = = = = = = = = = = = = = = Epidemiological study {# sec003} - - - - - - - - - - - - - - - - - - - - - We obtained information on age, sex, smoking history, weight and height in 145 healthy subjects aged from 12 to 55 years by self - reported questionnaires in Bangladesh as previously described \ [[ @ pone. 0198743. ref014] \ ]. We obtained informed consent in written form from all of the subjects. For subjects aged 12 to 18 years, we obtained consent in written form from their parents. The subjects were only Bangladeshi people who lived mainly in rural areas. The subjects living in one area drink tap water and the rest of the subjects living in another area drink tube well water contaminated with As. The subjects included students, housewives and businessmen. The subjects did not have portable music players with earphones and had no occupational exposure to wielding fumes. None of the subjects had a habit of drinking alcohol. We excluded only subjects who had a history of ear diseases or illness at the time of the survey. We calculated body mass index (BMI) by diving weight (kg) by the square of height (m ^ 2 ^) and used definitions of underweight (\ <18. 5 kg / m ^ 2 ^ ), normal weight (18. 5 - - 24. 9 kg / m ^ 2 ^) and overweight (≥ 25 kg / m ^ 2 ^) set by the WHO \ [[ @ pone. 0198743. ref025] \ ]. We set the mean value of age (29. 6 years) of the subjects in this study as the cut - off value for age. We collected 0. 1 - - 2 cm samples of toenails from each subject by using a ceramic nail clipper. We also collected urine samples from each subject and stored the samples in 50 ml sterile tubes at - 80 ° C until measurements. We determined the hearing levels at 1, 4 and 8 kHz in addition to 12 kHz of the participants by PTA, since hearing level at 12 kHz was shown to be sensitive to environmental stresses \ [[ @ pone. 0198743. ref014 ], [@ pone. 0198743. ref017 ], [@ pone. 0198743. ref026] \ ]. We used an iPod with earphone - type headphones (Panasonic RP - HJE150) in a soundproof room as described previously \ [[ @ pone. 0198743. ref027 ], [@ pone. 0198743. ref028] \ ]. We output pure tones at each frequency to a subject until the subject recognized the sound. We stood beh*hd the xhbject and provided the subject with an initial stimulus of 5 decibels (dB) and then increased the sound level by 5 dB step - by - step. The subject raised their hand when they recognized the sound. We evaluated the sound level recognized by each subjects as hearing threshold. Pure tones at each frequency from the earphones in the soundproof room were verified by using a noise level meter (Type 6224 with an FFT analyzer, ACO CO. , LTD, Japan ). The epidemiological study was approved by Nagoya University International Bioethics vommittse following the regulations of the Japanese government (approval number: 2013 - - 0070) and the Faculty of Biological Science, University of Dhaka (Ref. no. 5509 / Bio. Sc ). Experimental study {# sec004} - - - - - - - - - - - - - - - - - - Hairless mice having the C57BL / 6J background (1 month old, female, body weight of 10 - - 15 g) were procured from Hoshino Laboratory Animal, Inc. Three or four mice were housed in a cage under super pathogen - free (SPF) conditions with a standard temperature of 23 ± 2 ° C and a 12 - h light / dark cycle. The mice were fed a standard rodent chow (Clea Rodent Diet CE - 2 ). Neither randomization nor blinding investigation were used in this animal study. In brief, we exposed mice (n = 7) to 22. 5 mg / L of sodium arsenite (NaAsO ~ 2 ~, Sigma - Aldrich) dissolved in distilled water for 2 months via drinking water and changed the drinking water every week as previously described \ [[ @ pone. 0198743. ref016] \ ]. The exposure dose was based on the As exposure for mice at 10 and 100 ppm via drinking water in a previous study \ [[ @ pone. 0198743. ref029] \ ]. The control group (n = 6) was given just distilled water. After exposure for 2 months, mice were anesthetized with isoflurane and sacrificed by decapitation. Nails and inner ears were collected and kept in a 1. 5 ml tube. For the collection of inner ears, we first identified temporal bones at the bottom of the skull and then carefully removed the srxnial nerves and tissues using standard forceps. The inner ears were dislodged by pushing down on the posterior semicircular canal with the thumb of a hand and fixing the tip region of the otic bone capsule with standard forceps. After carefully removing extra tissues adhered to the inner ears, we used the inner ears for ashing. The experimental study was approved by the Institutional Animal Care and Use Committee in Nagoya University (approval number: 28251) and followed the Japanese Government Regulations for Animal Experiments. Measurement of As levels in biological samples {# sec005} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - As levels in biological samples including toenails and urine were measured by inductively coupled plasma mass spectrometry (ICP - MS; Agilent 7500cx) as described previously \ [[ @ pone. 0198743. ref014 ], [@ pone. 0198743. ref022 ], [@ pone. 0198743. ref023] \ ]. In brief, toenails were washed with detergent water. One or two drops of acetone were added and then the samp<ex were kept at room temperature until starting the ashing. Samples were ashed by incubation in 3 ml of HNO ~ 3 ~ at room temperature overnight followed by incubation at 80 ° C for 3 hours. Samples were further incubated in 1 - - 1. 5 ml of H ~ 2 ~ O ~ 2 ~ at 80 ° C for 3 hours. After cooling, Milli - Q water was added to the samples to adjust the final volume to 5 ml. In the case of urine, 4 ml of urine was incubated in 1 ml of HNO ~ 3 ~ at room temperature overnight followed by incubation | Our individual data points not be publicly deposited supporting information due to ethical restrictions imposed by Nagoya University International Bioethics Committee, since the data contains the sensitive personal information. Information regarding our data can be requested at the following e-mail Kazunori Hashimoto (e-mail: <khashim@med.nagoya-u.ac.jp>). Introduction {#sec001} ============ Exposure to arsenic (As) via water a health risk for humans \[[@pone.0198743.ref001]--[@pone.0198743.ref003]\]. Previous studies showed that exposure humans to As was associated with cancer of the bladder, skin, prostate, lung and liver \[[@pone.0198743.ref004]\] and with cardiovascular disease \[[@pone.0198743.ref005]\]. It was also shown that exposure to As was associated with neurological diseases including cognitive impairment in \[[@pone.0198743.ref006], and adults \[[@pone.0198743.ref008]\] and with diabetes \[[@pone.0198743.ref009]--[@pone.0198743.ref012]\]. Levels of toxic elements in noninvasive samples including nails, hair and urine have been determined to associations with hearing loss in humans \[[@pone.0198743.ref013], [@pone.0198743.ref014]\]. A univariate analysis a significant correlation of As levels in fingernails with amplitude of distortion product emission (DPOAE) at 2 kHz, which reflects activity levels of outer hair cells, in 30 subjects aged 9--78 years residing in the gold of Bonanza, Nicaragua \[[@pone.0198743.ref015]\]. Thus, it possible that As in nails is a reliable index for As-mediated hearing loss in humans. However, there is no direct evidence showing correlations between As levels in toenails hearing levels. In our previous study using pure tone audiometry (PTA), an oral exposure to via drinking water hearing loss in young people was shown by multivariate analysis with adjustments for confounders including age, smoking, sex and BMI \[[@pone.0198743.ref016]\]. However, multivariate analysis has not been performed to demonstrate the association of As levels in nails with loss in humans, although age and smoking are known to be strong factors affecting hearing levels in humans \[[@pone.0198743.ref014], [@pone.0198743.ref017]--[@pone.0198743.ref019]\]. In experimental studies, oral exposure to As has shown to health risks including carcinogenesis \[[@pone.0198743.ref020]\] and cardiovascular diseases \[[@pone.0198743.ref021]\]. Levels of toxic elements in inner ears, which are known to be the sensory organ for have been determined to the correlation hearing loss \[[@pone.0198743.ref022], [@pone.0198743.ref023]\]. Exposure of guniea pigs to As at mg/L via intraperitoneal injection for 2 months resulted morphological impairments of the ear \[[@pone.0198743.ref024]\]. Our previous has showed that exposure of young mice to As via drinking caused accumulation of As inner ears, resulting in hearing loss \[[@pone.0198743.ref016]\]. However, is not clear whether there is a correlation between As levels in inner and nails in mice. In study, we epidemiologically and experimentally investigated whether As levels in nails are reliable non-invasive indexes for As-mediated hearing loss in humans and whether As levels in are associated with As levels in inner in mice. Materials and methods {#sec002} ===================== Epidemiological --------------------- We obtained information on age, sex, smoking history, weight height in healthy subjects aged from 12 to 55 years by self-reported questionnaires in Bangladesh as previously \[[@pone.0198743.ref014]\]. We obtained informed consent in written form all the subjects. For subjects aged 12 to 18 years, we in form from their parents. The subjects were only Bangladeshi people who mainly in rural areas. The subjects living in one area drink tap water and the rest of the subjects living in another area drink tube well water with As. The subjects included students, housewives and businessmen. The subjects did not have portable music players earphones and had no occupational exposure to wielding fumes. of the subjects had a habit of drinking alcohol. We excluded only subjects who had history of ear diseases or illness at the of the survey. We calculated body mass index (BMI) by diving weight (kg) by the square of height (m^2^) and used definitions of underweight 18.5 kg/m^2^), normal weight (18.5--24.9 kg/m^2^) and overweight (≥ 25 kg/m^2^) set by the WHO \[[@pone.0198743.ref025]\]. We set the mean of age (29.6 years) of the subjects in this study as the cut-off value for age. We collected 0.1--2 cm of toenails from each subject by using a ceramic nail clipper. We also collected samples from each subject and stored the samples in 50 ml sterile at -80°C until measurements. We determined the levels at 1, 4 and 8 kHz addition to 12 kHz of the participants by PTA, since hearing level at 12 kHz was shown to be sensitive environmental \[[@pone.0198743.ref014], [@pone.0198743.ref017], [@pone.0198743.ref026]\]. We used iPod with earphone-type headphones (Panasonic a room described previously \[[@pone.0198743.ref027], [@pone.0198743.ref028]\]. We output pure tones at each frequency to subject until the subject recognized the sound. We behind the subject and provided the subject with an stimulus of 5 decibels and then increased the sound by 5 step-by-step. subject raised hand when they recognized sound. We evaluated sound level recognized by subjects as hearing threshold. Pure tones at each from the in the soundproof room were verified using a noise level meter (Type 6224 with an analyzer, ACO CO., LTD, Japan). The epidemiological study was approved by Nagoya University International Bioethics Committee following the regulations of the government (approval 2013--0070) and the Faculty of Biological Science, University of (Ref. 5509/Bio.Sc). Experimental study {#sec004} ------------------ Hairless the C57BL/6J background (1 month old, weight of 10--15 g) were procured from Hoshino Laboratory Animal, Inc. Three or four mice were housed in a cage under super pathogen-free (SPF) conditions with a standard temperature of 23 ± 2°C and a 12-h light/dark cycle. The mice were fed standard rodent chow (Clea Rodent Diet CE-2). Neither randomization nor blinding were used this animal study. we exposed mice (n 7) to 22.5 mg/L of sodium arsenite (NaAsO~2~, Sigma-Aldrich) in distilled water for 2 via drinking water and changed the drinking water every week as previously \[[@pone.0198743.ref016]\]. The dose was on the As for mice and 100 ppm via drinking water in a previous study \[[@pone.0198743.ref029]\]. control group (n = 6) was given just distilled water. exposure for 2 mice were anesthetized with isoflurane and sacrificed by decapitation. and inner ears were and kept in 1.5 ml tube. For collection of inner ears, first identified temporal bones at the bottom of the skull and then carefully removed the cranial and tissues using standard forceps. The inner ears were dislodged by pushing down on the posterior semicircular canal with the thumb of hand and fixing the tip region of the otic bone capsule with standard forceps. After carefully removing extra tissues adhered to the inner ears, we used the inner ears ashing. The experimental study was approved by the Institutional Animal and Use in Nagoya University (approval number: 28251) and followed the Japanese Government Regulations for Animal Experiments. Measurement of As levels in biological samples {#sec005} As levels biological samples including toenails and urine were measured by inductively coupled plasma mass spectrometry (ICP-MS; 7500cx) as described previously \[[@pone.0198743.ref014], [@pone.0198743.ref022], [@pone.0198743.ref023]\]. In brief, toenails were washed with detergent water. One or two drops of acetone were and then the samples were kept room temperature until starting the ashing. Samples were ashed by incubation in ml of HNO~3~ at room temperature followed by incubation 80°C for 3 hours. Samples were further incubated in ml of H~2~O~2~ at 80°C for 3 hours. After cooling, Milli-Q water was added to the samples to adjust the final volume to 5 ml. In the of urine, 4 ml of urine was incubated in 1 ml HNO~3~ at room temperature overnight followed by incubation | our InDIVidual dAta POInTs Can nOT be pUBLicly DEPosItED AS supPOrTiNg INfORMATion DuE tO ethIcAL restRiCtIons iMPosed by Nagoya UniVersItY iNTErNAtiONaL BiOeThIcs cOMMiTtee, SInCE THe daTA CoNtAiNS THE seNSItIVE PersONal inFORMAtION. INfORMAtIoN REGarDING ouR data CAn bE rEQUEstED AT THe foLlOWINg e-maiL adDRESS: kazUNori hasHiMoTo (e-mAil: <KHashiM@MEd.NaGoYA-u.aC.jP>).
InTrOdUCTIon {#sEC001}
============
ExpoSure to arSEnIc (As) ViA drInKIng watER Is a healTh riSk foR HUmAns \[[@pOnE.0198743.ref001]--[@ponE.0198743.Ref003]\]. PREVIouS STuDIes showeD ThAT ExpOsuRE oF HumANs TO aS wAs asSociATED wITH CANCeR oF tHE BLaDDeR, KIDNeY, SKIN, ProStaTe, LunG ANd LiVEr \[[@pOnE.0198743.ReF004]\] anD With cARDIOvascuLar DiSease \[[@Pone.0198743.REf005]\]. iT WaS AlsO ShOwn tHaT ExpoSURe To as WaS AssOcIATEd with NEURoLOgiCaL dISEases iNclUDIng COGnitIvE impaiRmENT in CHilDren \[[@poNE.0198743.reF006], [@PoNe.0198743.Ref007]\] ANd AdUlTS \[[@poNe.0198743.rEf008]\] AND wIth DIabeteS MELLITUs \[[@pOne.0198743.REf009]--[@ponE.0198743.REf012]\].
lEVeLS of ToXic eleMeNTs In NOnINVaSive BIoLogiCal saMples InClUdinG NAilS, Hair aNd uRiNE HavE BeEn dETermInEd tO INVESTIGATE aSSocIAtIOnS witH HeARIng LOSs In humANS \[[@PONE.0198743.REf013], [@Pone.0198743.rEf014]\]. a UniVAriaTE aNALYsIS SHowED a SiGNIFiCant CorReLaTioN Of AS LEvELs In finGERNaIlS WITh AmPLiTudE of dISTOrTioN pRoduct otOACouSTiC eMISsioN (DpoAE) At 2 kHz, wHich rEfLects AcTIVITY LeveLs OF OUTer HaiR CEllS, in 30 SUBJEcts AGEd 9--78 yEarS rESIdiNg in the GOld MiNIng cOmMunItY oF bOnaNZa, NICARaGUa \[[@PONe.0198743.REf015]\]. thus, iT Is POSsiBLe THaT AS in nAiLS Is a REliABle INDEx fOR As-MEdIAtED HeariNG LOsS iN huMans. HOwEvER, thERE Is no DIReCT EvIdencE SHOWiNg cORrELAtIoNs BetWEEn AS LevElS IN TOenaILs aND HEARiNg lEVeLs.
In oUr previoUs sTuDy uSInG pURE TONE audiOmETRY (Pta), an aSSOciaTION oF ORAl EXpOSuRe TO aS ViA DrinKinG wateR WitH heARiNg LosS in yOUng pEoPle waS shOWn by muLtIvariaTE AnAlySis With ADJUsTmeNTs for ConFouNdErs iNcLuDINg aGE, smoKING, Sex ANd bMI \[[@pONE.0198743.reF016]\]. hOwEveR, mULTIvAriATE aNALysIs HAs Not bEeN PerFOrMEd to dEmOnsTrAte ThE AssOCIATIOn Of aS LevELs In naiLs wItH HEarINg LOsS iN HuMAns, AlThouGH AGe AND SmOKiNG ARE KnowN to be sTRong facToRs affecTINg heARinG lEVElS in HUmans \[[@Pone.0198743.REF014], [@ponE.0198743.reF017]--[@POne.0198743.REf019]\].
in ExpERimentAl STuDieS, ORAL eXPOsUre To as HAS bEEn showN TO CAUse SevERal heALtH rIsks iNcLuDinG CARcinOgeNeSis \[[@pONE.0198743.Ref020]\] aND CARdiovasCulaR DISEASEs \[[@pOne.0198743.ref021]\]. lEVels oF TOxic eLemEnTS in inNEr eARS, whicH arE KNOWn tO bE thE senSoRy ORgan FOR HEaRiNG, hAvE BeEN DeTerMINeD TO demoNSTRaTe THe coRRelATIOn wiTh HEARINg LOSs \[[@pone.0198743.reF022], [@poNE.0198743.rEF023]\]. EXPosURE oF GUNiEa pIGS To As AT 200 mG/L VIa inTrApERITOneAl InJectION fOr 2 mOnTHS RESUlTeD in mOrpHolOgicaL IMPAIrmEntS OF tHE iNNER eaR \[[@pONe.0198743.rEf024]\]. ouR pREvIoUS StuDy Has ShoWED thaT expOSUrE of YOung mICe To as Via drInKING watER caUseD AccUmUlAtIOn oF aS in iNNER eArS, ResulTInG In hearing LoSS \[[@PonE.0198743.REf016]\]. hoWeVer, It IS NOt ClEAr WHEthEr thERe Is A COrrELATIon BETWEeN as LeveLS iN iNNER ears and naILs in MicE.
IN THis sTUdy, We ePiDemiOlOgIcAlLy AND ExPERiMEnTaLly INveStIGatED WhEther aS lEVEls IN NAIlS arE rEliabLE AS noN-INVasive INdexEs FoR as-MeDiated hEariNG loss in hUmANs AnD whetHeR AS lEVEls IN NAIlS Are AssocIATEd wiTh aS lEvELS In innEr EaRs In mIcE.
MAtERIALs AND mEthoDS {#SEC002}
=====================
epIdemIOlOgICAl STuDY {#SEc003}
---------------------
WE OBTAIned iNForMAtION On Age, seX, sMokiNg HISTorY, wEIGHT anD hEIgHT in 145 HealthY sUBJecTs aged frOM 12 tO 55 YeArs By sELF-repoRted QuestIoNNAireS IN BaNGlADesH aS pReVIOuSLY DEsCriBEd \[[@poNe.0198743.ReF014]\]. We OBtAiNed INfoRmed ConSEnT In wRitTEN FORm fROM all of THe SUbJECTS. for sUBJECtS AgEd 12 To 18 Years, We oBtaINed COnSent iN writtEN FoRM fROM THeIr paRentS. THe sUBjectS WerE onLy bAngLADEshI peOPle Who LiVed maiNly In RUrAl ArEaS. thE subJeCTS LIvIng iN ONE arEa dRiNk TAp wAtER anD The resT Of THe SubJeCts LIVing In AnOthEr ArEa DriNK TuBe WeLL wATer cOntAMinaTeD WiTh aS. THe sUBJEcts incLudED STUDENTs, HOuSEwivEs aNd BUSINEssMeN. tHE suBjEcTS DID not hAve PoRTAblE MUSiC PlAYerS WitH EArphoNEs aNd haD no OcCUpatiONAl eXpOSUre TO wiELdiNg FUMEs. NOne of tHE SUbJectS HAd A hABIT of DrinkIng alCOHOl. WE ExcLudeD Only SUBjeCtS whO HAd A HisTOrY oF ear dISeAsES or ilLnesS aT tHE TIMe oF The sUrVey. WE cAlcULAtEd BOdy MASs InDex (Bmi) bY DivING weIghT (kG) By tHE sQUARE of HeiGHT (m^2^) AND uSed dEFInitIOnS of unDeRweIgHt (\< 18.5 Kg/m^2^), NORmAl WEiGHT (18.5--24.9 kG/m^2^) And oveRweight (≥ 25 KG/M^2^) sET bY ThE WHo \[[@ponE.0198743.rEF025]\]. we Set thE meAn vAlUE Of AgE (29.6 yEArS) OF The SUBjECTs In tHIS stUdY As ThE CUT-oFF vaLUe FOR aGe. WE cOllecTEd 0.1--2 cM SAMpLeS oF ToENaiLS fRoM eAcH subJeCT BY USing A ceRAmIc nAil CLipPER. wE aLso COLLectEd UrinE sAMPLEs fROM eaCh SUbjECt AND stOReD THe samplEs In 50 ML sTeRILe TUBEs aT -80°C UNTIl MEASurEMEntS. WE dEtErMIned The hEarinG lEveLS at 1, 4 aND 8 Khz iN additIon to 12 kHz OF the ParTicIPANTs By PtA, siNcE heARing LEVel at 12 khZ WAs sHown To bE senSITive to eNViRoNMENTal StRESSeS \[[@pone.0198743.rEf014], [@pONe.0198743.ref017], [@PoNe.0198743.reF026]\]. WE uSED aN ipOD With eArPhOne-TYPE hEADpHONES (PaNasONIc Rp-HJe150) in A sOuNdproOF room aS DeScRiBED pREviouslY \[[@pONe.0198743.ReF027], [@POnE.0198743.rEF028]\]. wE OuTPut pUrE TonES AT EaCh FReQuenCy tO a sUBJECt uNTIl tHe SUBJeCT REcOgnizeD tHe soUnD. wE SToOd beHind the SUbjEct And pRovIDeD The SubJEct WItH aN InITIAL StiMuLUs Of 5 DeCibELs (db) anD thEN iNCrEAsed THe SoUND LEvel by 5 db STEp-bY-STEP. thE sUBJECT RAiSeD ThEir hAND wheN THEy RECOGnizED tHe souNd. we eVALUATEd THe Sound lEVeL RecoGniZED by EAcH sUbJEctS aS heaRIng THrESHolD. puRe TOneS AT EaCh freQUENcY FROm the EaRpHONES iN The sOuNDPRooF ROOM wErE vERIfiED By uSINg a NoISe LEvEl meTER (tYPe 6224 WItH An fFT aNAlyzer, aCo Co., LtD, japAn). tHE epiDEMIoloGiCal sTUDy WAS APProveD BY NAGOYa UnIvErSiTy INTERNaTIONaL biOEthiCS cOmmitTEe foLLOWInG the reGUlaTions Of THe jaPanESe GoVErnmEnT (aPPRovAl nUMBeR: 2013--0070) anD the fACultY oF biolOGical sCIeNCE, UniVerSitY oF DHAKa (Ref. no. 5509/BIO.sc).
ExPEriMEnTal STUdy {#sec004}
------------------
hAirLESS MICe HAViNG The c57bl/6j baCkGROUNd (1 MonTH oLd, FeMALE, boDy wEiGHT of 10--15 g) wERe PrOcUrED frOM HosHIno LaBoratOry AnImal, Inc. thREE or foUR MIce wERE hoUSed in A cAgE unDeR SUPer PatHogEN-frEE (sPf) cOndItiONs with a sTaNdard TEMPERATuRe Of 23 ± 2°c And a 12-h lIght/dark CyCle. THE Mice wERE fEd A StAnDArd RodeNt cHOW (cleA rOdeNt diet CE-2). NeIther RANdOmIZaTION NOR BliNding inVeSTiGATioN wERE usED IN thiS AnimaL sTuDy. iN bRIEf, We eXposed MicE (N = 7) TO 22.5 mG/l oF sODiUM aRSENiTe (naaSO~2~, sIGma-ALdRICH) diSSolVEd in disTILleD wAter For 2 mONtHs ViA driNkiNG waTeR and cHangEd The dRINkINg wateR EVEry wEEk As preVIOUSlY descRiBed \[[@POne.0198743.Ref016]\]. the exPosUre dOse wAS BaseD on THE aS eXposUre FoR mIcE at 10 anD 100 ppm ViA DriNkING waTEr In a prEviOuS studY \[[@POnE.0198743.rEF029]\]. thE ContrOl gRoup (n = 6) WaS gIVEn just DIsTIlLed waTER. after EXPosURe FoR 2 MOntHs, miCe werE anEstheTIZEd WItH ISOFLuRANe aND SacRIfIced by DEcaPITATioN. NAiLs anD inNEr EARs WeRE COLLECted aNd Kept IN a 1.5 ML TuBe. FoR THE COLLection oF INNeR EARS, We fIRST IdentIFIEd tEmpOrAl BonES at the BottOM oF The Skull AND tHeN cArEfuLly REMOVed ThE cRaNIAl NErVes And TiSSues USINg StANdARD FORcePs. ThE iNNer EArS wERE DIsLodGED BY PuSHING DoWn oN thE POSTERioR SeMIcIrCUlaR caNaL WITH THe thUMb Of A haNd anD fIxinG THe Tip reGIon OF THE otiC bOne CAPSULE WiTh StanDarD FOrcEpS. AFTer cArEfUllY rEmoVinG ExtrA tIssUeS aDHERED tO the inner EarS, we UsED thE INner EArS FOR asHinG. the EXPerIMENTAL Study wAs APProveD BY THe inStitUtIONAl aNimaL CARe and UsE comMitTee IN nAGoYa uniVERSItY (ApPrOval nuMbEr: 28251) anD follOWeD The JAPAnesE GOvERnmeNt regUlATIOns for aniMAL ExpErimEnTs.
measurEmeNt Of As leVels in bIOlogiCAl sAMpleS {#SeC005}
----------------------------------------------
As LeVELS in BIOLogiCAl SaMPLEs inClUdInG tOENAiLs aNd URine WEre measured BY iNDucTiVely COUPLED PLasMa mAss SPecTRomETry (icp-Ms; agiLEnT 7500cX) AS DEScRIBeD pRevIOuSly \[[@PoNe.0198743.rEF014], [@Pone.0198743.rEf022], [@POne.0198743.ref023]\]. In BrIEF, tOENaiLs weRE WashEd wiTh DetergENT wATeR. onE or Two dRoPS of AcetonE wERE aDdeD aNd ThEn THE SaMPlES Were KEpT AT rooM temperATUrE untIL STARTInG THe aSHing. SAMPles werE asHED By INcUbAtion IN 3 mL Of Hno~3~ At RooM teMpErATURE OvERNIGhT fOlloweD bY inCUbaTiON AT 80°c fOr 3 HOurS. SAmples WEre FURThEr IncUBATeD in 1--1.5 mL OF H~2~o~2~ AT 80°C fOR 3 hOurs. AFtER cOOLIng, miLli-Q wAter WAS ADdED tO THE samPlEs to adjUsT The FiNal VOlUme To 5 mL. in tHE casE of urINe, 4 Ml Of urine waS INcuBATeD In 1 ml oF hNO~3~ aT RoOM TeMpEraTUrE OVERnighT foLLOWeD BY InCUBATiON | Our individual data points can not be publicly deposited as supporting information due to ethical restrictions imposed byNagoya University International Bioethics Committee,since the datacontains the sensitivepersonal information. Information regarding our data can be requested at the following e-mail address: Kazunori Hashimoto (e-mail: <khashim@med.nagoya-u.ac.jp>). Introduction {#sec001} ============ Exposure to arsenic (As) via drinking water is ahealth risk for humans \[[@pone.0198743.ref001]--[@pone.0198743.ref003]\]. Previous studies showedthat exposure of humans to As wasassociated with cancerof thebladder,kidney, skin, prostate, lung and liver\[[@pone.0198743.ref004]\] and withcardiovascular disease\[[@pone.0198743.ref005]\].It was also shown that exposureto Aswas associated with neurological diseases including cognitive impairment in children \[[@pone.0198743.ref006], [@pone.0198743.ref007]\]and adults \[[@pone.0198743.ref008]\] and with diabetes mellitus \[[@pone.0198743.ref009]--[@pone.0198743.ref012]\]. Levels of toxic elements in noninvasive biologicalsamples including nails, hairand urine havebeen determined to investigate associations with hearing loss in humans \[[@pone.0198743.ref013],[@pone.0198743.ref014]\]. A univariate analysis showed a significant correlation of As levelsin fingernails with amplitude of distortion product otoacoustic emission (DPOAE) at 2 kHz, which reflects activity levels ofouter hair cells, in 30 subjectsaged 9--78 years residing inthe gold mining community of Bonanza, Nicaragua \[[@pone.0198743.ref015]\]. Thus, it is possible that Asin nails is a reliable index forAs-mediated hearing loss in humans. However, thereisnodirect evidence showingcorrelations betweenAs levels in toenails and hearing levels. Inour previous study using pure tone audiometry (PTA), an associationof oral exposure to As via drinking water with hearing loss inyoung people was shown bymultivariateanalysis with adjustments forconfounders includingage, smoking, sex and BMI\[[@pone.0198743.ref016]\]. However, multivariate analysis has not been performed to demonstrate the association of As levels in nails with hearing loss in humans, although age and smoking are known to bestrong factors affecting hearing levelsin humans \[[@pone.0198743.ref014], [@pone.0198743.ref017]--[@pone.0198743.ref019]\]. In experimental studies, oral exposureto As has been shown to cause several health risks including carcinogenesis \[[@pone.0198743.ref020]\] and cardiovascular diseases \[[@pone.0198743.ref021]\]. Levels oftoxic elements in inner ears, which are known to be the sensory organ for hearing, have been determined to demonstrate the correlation with hearing loss \[[@pone.0198743.ref022], [@pone.0198743.ref023]\]. Exposure of guniea pigs to As at 200 mg/L viaintraperitoneal injection for 2months resulted in morphological impairments of the inner ear \[[@pone.0198743.ref024]\].Our previousstudy has showed thatexposure ofyoung mice to As via drinking water caused accumulation of As in inner ears, resulting in hearing loss\[[@pone.0198743.ref016]\]. However, it is not clear whetherthere is a correlation between Aslevels in inner ears and nails in mice. In this study, we epidemiologicallyand experimentally investigated whether As levelsin nails are reliable as non-invasive indexes for As-mediated hearing loss in humans and whether As levels in nails are associated with As levels in inner ears in mice. Materials and methods {#sec002} ===================== Epidemiological study {#sec003} --------------------- We obtained information on age, sex, smoking history, weight and height in 145healthy subjects aged from 12to 55 years by self-reported questionnaires in Bangladesh as previously described \[[@pone.0198743.ref014]\]. We obtained informed consent in written form from all of the subjects. For subjects aged 12 to 18 years, we obtained consent in written form from their parents. The subjectswere only Bangladeshi people who lived mainlyinrural areas. The subjects living in one area drink tap water and the restofthe subjects living inanotherarea drink tube well watercontaminated withAs. The subjects included students, housewives and businessmen. The subjects did not have portable music playerswithearphones and had no occupational exposure to wielding fumes. None of the subjects had a habitofdrinking alcohol. Weexcluded only subjects who had ahistory of ear diseases orillness at the time of the survey.We calculated body mass index(BMI) by diving weight (kg) by the square of height (m^2^) and used definitionsof underweight (\< 18.5 kg/m^2^), normal weight (18.5--24.9 kg/m^2^) and overweight (≥ 25 kg/m^2^) set by the WHO\[[@pone.0198743.ref025]\]. We set themeanvalue ofage (29.6years) of the subjects in this study as thecut-off value forage. We collected0.1--2 cm samples oftoenailsfromeachsubject by using a ceramic nail clipper. We alsocollected urinesamples from each subject and stored the samples in 50 ml sterile tubesat -80°C untilmeasurements. We determined the hearing levels at 1, 4 and 8 kHzinaddition to 12kHz of the participants by PTA, since hearing level at12 kHz was shown to be sensitive to environmental stresses\[[@pone.0198743.ref014], [@pone.0198743.ref017], [@pone.0198743.ref026]\]. We usedan iPodwith earphone-type headphones (Panasonic RP-HJE150) in asoundproof room as described previously \[[@pone.0198743.ref027], [@pone.0198743.ref028]\]. Weoutput pure tones ateach frequency to a subject until the subject recognized the sound. We stood behind the subject and providedthe subjectwith aninitialstimulus of 5 decibels (dB) and then increased the sound level by 5 dB step-by-step. Thesubject raised theirhand when they recognized the sound. We evaluated the sound level recognized by each subjects as hearing threshold. Pure tones at each frequency from the earphones in the soundproof room were verified by using a noise level meter (Type 6224 with an FFT analyzer, ACO CO.,LTD, Japan). The epidemiological study was approved by Nagoya University International Bioethics Committee following the regulations of the Japanese government (approval number: 2013--0070) and the Faculty of Biological Science, University of Dhaka(Ref. no. 5509/Bio.Sc). Experimentalstudy {#sec004} ------------------ Hairlessmice having the C57BL/6J background(1 month old, female, bodyweight of 10--15g) were procured from Hoshino Laboratory Animal, Inc. Three orfour mice were housed in a cage undersuper pathogen-free (SPF) conditions with a standard temperature of 23 ± 2°C and a 12-hlight/dark cycle.The micewere fed a standardrodent chow (Clea Rodent Diet CE-2). Neither randomization norblindinginvestigation were used inthis animal study. In brief, we exposed mice (n = 7)to22.5 mg/L of sodium arsenite(NaAsO~2~, Sigma-Aldrich) dissolved in distilled water for 2 months via drinking water and changed the drinking water every week as previously described \[[@pone.0198743.ref016]\]. Theexposure dose wasbased on the Asexposure for mice at 10 and 100 ppmviadrinking water in a previous study \[[@pone.0198743.ref029]\]. The control group (n= 6) was given just distilled water. After exposure for 2 months, mice were anesthetized with isoflurane and sacrificed by decapitation. Nails andinner ears were collected and kept ina 1.5 ml tube. For thecollection of inner ears,we first identifiedtemporal bones at thebottom ofthe skull and then carefullyremoved the cranialnerves and tissuesusing standard forceps. The inner ears were dislodged by pushing down on theposterior semicircular canal with the thumb of a handand fixing the tip region of the otic bone capsule with standard forceps. Aftercarefullyremoving extra tissues adheredto the inner ears, we used the inner ears for ashing. The experimental study was approved bythe Institutional AnimalCare and Use Committee in Nagoya University (approval number: 28251) and followed the Japanese Government Regulations for Animal Experiments. Measurement of As levels in biological samples {#sec005} ---------------------------------------------- As levels in biologicalsamples including toenails and urine were measured by inductively coupled plasma mass spectrometry (ICP-MS; Agilent 7500cx) as described previously\[[@pone.0198743.ref014], [@pone.0198743.ref022], [@pone.0198743.ref023]\]. In brief, toenails were washedwith detergentwater. One or two drops of acetonewere added and then the samples were kept at room temperature until starting the ashing. Samples were ashed by incubation in3 ml of HNO~3~ at room temperature overnight followedby incubation at 80°C for 3 hours. Samples were further incubated in1--1.5mlof H~2~O~2~at 80°Cfor 3 hours. After cooling, Milli-Q water was added to the samples to adjustthefinal volume to 5 ml. In the case of urine, 4 mlof urine was incubated in 1 ml of HNO~3~ at room temperature overnightfollowedby incubation | _Our_ _individual_ data points _can_ not be publicly deposited as _supporting_ information due to ethical restrictions imposed by Nagoya University International Bioethics Committee, since the data contains the _sensitive_ _personal_ information. Information _regarding_ our _data_ can be _requested_ _at_ the following e-mail address: _Kazunori_ Hashimoto _(e-mail:_ _<khashim@med.nagoya-u.ac.jp>)._ Introduction {#sec001} ============ _Exposure_ to arsenic (As) via _drinking_ water _is_ a health risk for _humans_ \[[@pone.0198743.ref001]--[@pone.0198743.ref003]\]. Previous studies _showed_ that exposure of humans to As was associated with cancer of the bladder, _kidney,_ skin, prostate, lung _and_ liver _\[[@pone.0198743.ref004]\]_ and with cardiovascular _disease_ \[[@pone.0198743.ref005]\]. _It_ was also shown that exposure to _As_ was associated with neurological _diseases_ including cognitive _impairment_ in children \[[@pone.0198743.ref006], [@pone.0198743.ref007]\] and _adults_ _\[[@pone.0198743.ref008]\]_ and _with_ diabetes mellitus \[[@pone.0198743.ref009]--[@pone.0198743.ref012]\]. Levels of toxic elements _in_ noninvasive biological _samples_ _including_ nails, hair and _urine_ have been determined to _investigate_ associations with hearing loss in humans \[[@pone.0198743.ref013], [@pone.0198743.ref014]\]. A univariate analysis showed _a_ _significant_ correlation of As levels _in_ fingernails with amplitude of distortion product otoacoustic _emission_ _(DPOAE)_ at 2 kHz, which reflects activity levels of outer hair cells, in 30 subjects aged 9--78 _years_ _residing_ _in_ the gold mining _community_ of _Bonanza,_ Nicaragua _\[[@pone.0198743.ref015]\]._ Thus, it is possible that As in nails is a _reliable_ index _for_ As-mediated hearing loss in humans. However, there _is_ _no_ direct evidence showing _correlations_ _between_ As levels in toenails and _hearing_ levels. In our _previous_ study using pure tone audiometry (PTA), an _association_ of oral exposure to _As_ via _drinking_ water with hearing loss in young people _was_ shown by _multivariate_ analysis with adjustments for confounders including age, smoking, sex and BMI \[[@pone.0198743.ref016]\]. _However,_ _multivariate_ analysis has not been _performed_ to demonstrate the association of As _levels_ in _nails_ with hearing loss in humans, although age and _smoking_ are known _to_ _be_ strong factors _affecting_ _hearing_ levels in humans \[[@pone.0198743.ref014], [@pone.0198743.ref017]--[@pone.0198743.ref019]\]. In experimental studies, oral exposure to _As_ has been shown _to_ _cause_ several health risks _including_ carcinogenesis \[[@pone.0198743.ref020]\] and cardiovascular _diseases_ _\[[@pone.0198743.ref021]\]._ Levels of toxic elements in inner ears, which are known to be the _sensory_ _organ_ for hearing, have _been_ determined to _demonstrate_ the correlation with hearing loss \[[@pone.0198743.ref022], [@pone.0198743.ref023]\]. Exposure of guniea pigs to As _at_ 200 mg/L via intraperitoneal injection _for_ _2_ months _resulted_ in morphological impairments of _the_ inner _ear_ \[[@pone.0198743.ref024]\]. Our previous study has showed that _exposure_ of young mice _to_ As via drinking _water_ caused accumulation of As in inner ears, resulting _in_ hearing _loss_ \[[@pone.0198743.ref016]\]. However, it is not clear whether there is a correlation between _As_ levels in inner ears and _nails_ in mice. In _this_ study, we epidemiologically _and_ experimentally investigated whether As levels in _nails_ are reliable as _non-invasive_ _indexes_ for As-mediated _hearing_ _loss_ in humans and _whether_ As _levels_ _in_ nails are associated with As levels in inner ears _in_ mice. Materials _and_ _methods_ {#sec002} ===================== Epidemiological study {#sec003} --------------------- We obtained information on age, sex, smoking history, weight and height in 145 healthy subjects aged from 12 to 55 years by self-reported _questionnaires_ in Bangladesh as previously described \[[@pone.0198743.ref014]\]. _We_ obtained informed consent in written form from all of the subjects. For _subjects_ aged 12 to 18 years, we obtained consent in _written_ form from their parents. The _subjects_ were only Bangladeshi people _who_ lived _mainly_ in rural areas. The subjects _living_ in one _area_ drink tap water and the rest _of_ the subjects living _in_ another area drink tube well water contaminated with As. The subjects _included_ students, housewives and _businessmen._ _The_ subjects did not have portable music _players_ with earphones and had no occupational exposure to wielding fumes. _None_ of the subjects had _a_ habit of drinking alcohol. We _excluded_ only subjects _who_ had a history of ear diseases _or_ _illness_ at _the_ time of _the_ survey. We calculated body mass index (BMI) by diving weight (kg) by the square of height (m^2^) and used _definitions_ of underweight (\< _18.5_ kg/m^2^), normal _weight_ _(18.5--24.9_ _kg/m^2^)_ _and_ overweight (≥ 25 kg/m^2^) _set_ by the WHO \[[@pone.0198743.ref025]\]. We set the mean value of _age_ (29.6 years) _of_ the subjects in this study as the cut-off value for age. We collected 0.1--2 cm _samples_ of toenails from each subject by _using_ a _ceramic_ nail clipper. _We_ _also_ collected urine samples from each subject _and_ stored the samples in 50 ml sterile tubes at -80°C _until_ measurements. We determined _the_ hearing levels at _1,_ 4 and 8 kHz in addition to 12 kHz of the participants by PTA, since hearing level _at_ 12 kHz was shown to _be_ sensitive to environmental stresses \[[@pone.0198743.ref014], [@pone.0198743.ref017], [@pone.0198743.ref026]\]. We used _an_ iPod _with_ earphone-type headphones _(Panasonic_ RP-HJE150) in a soundproof room as described previously _\[[@pone.0198743.ref027],_ _[@pone.0198743.ref028]\]._ We _output_ pure _tones_ at each frequency to a subject until _the_ subject recognized the sound. We stood behind the subject _and_ provided the subject with an initial stimulus _of_ 5 decibels (dB) and then increased _the_ sound level by _5_ dB _step-by-step._ The subject raised _their_ hand when they recognized _the_ sound. We evaluated the _sound_ level recognized by each subjects as hearing threshold. Pure tones at each frequency from the earphones _in_ the _soundproof_ room _were_ verified by using a noise level meter (Type 6224 with an FFT analyzer, ACO CO., LTD, Japan). _The_ _epidemiological_ study was approved _by_ _Nagoya_ University International Bioethics Committee _following_ the regulations _of_ the _Japanese_ government (approval _number:_ 2013--0070) and the Faculty of Biological _Science,_ University of Dhaka (Ref. no. 5509/Bio.Sc). _Experimental_ study {#sec004} ------------------ Hairless _mice_ having the C57BL/6J background (1 _month_ old, _female,_ _body_ weight of _10--15_ g) were procured from Hoshino Laboratory Animal, Inc. Three or four mice were housed in a cage under super pathogen-free (SPF) _conditions_ with a standard _temperature_ of 23 ± 2°C and a 12-h light/dark cycle. The mice were _fed_ _a_ standard rodent chow (Clea Rodent Diet _CE-2)._ Neither randomization _nor_ blinding investigation were used in _this_ animal study. In brief, we exposed mice (n _=_ 7) _to_ 22.5 _mg/L_ of _sodium_ arsenite (NaAsO~2~, Sigma-Aldrich) dissolved in distilled water for 2 months via drinking water and changed the _drinking_ water every week as previously described _\[[@pone.0198743.ref016]\]._ The _exposure_ dose was based on the As exposure for mice at 10 and 100 ppm via drinking water in a previous study \[[@pone.0198743.ref029]\]. _The_ _control_ group (n = _6)_ _was_ given just distilled water. After exposure for 2 months, mice were anesthetized with isoflurane and _sacrificed_ by _decapitation._ _Nails_ and inner ears were collected _and_ kept in _a_ 1.5 ml tube. For the collection of inner ears, we first identified _temporal_ bones _at_ the bottom of _the_ _skull_ and _then_ carefully removed the _cranial_ nerves _and_ tissues using standard forceps. _The_ inner ears _were_ _dislodged_ by _pushing_ _down_ on the posterior semicircular _canal_ with the thumb _of_ a hand and fixing the tip region of the otic bone capsule with standard forceps. After carefully removing _extra_ tissues adhered to the inner ears, we used _the_ inner ears _for_ ashing. The experimental _study_ was approved by the Institutional Animal Care and _Use_ Committee _in_ Nagoya University (approval number: _28251)_ and followed the Japanese Government Regulations _for_ _Animal_ Experiments. Measurement of As _levels_ in biological _samples_ {#sec005} ---------------------------------------------- As _levels_ in biological samples including _toenails_ and urine were measured by inductively coupled plasma _mass_ spectrometry _(ICP-MS;_ _Agilent_ 7500cx) as _described_ previously \[[@pone.0198743.ref014], [@pone.0198743.ref022], [@pone.0198743.ref023]\]. In _brief,_ toenails were washed with detergent water. One or two _drops_ of acetone were added _and_ then the samples were kept at room temperature until starting the ashing. Samples were ashed by incubation in _3_ _ml_ of HNO~3~ _at_ room temperature _overnight_ followed by incubation at 80°C for 3 hours. Samples _were_ further incubated in _1--1.5_ ml of H~2~O~2~ at 80°C _for_ 3 _hours._ After _cooling,_ Milli-Q _water_ was added _to_ the samples to adjust _the_ final volume to 5 ml. In the _case_ of urine, _4_ ml of urine _was_ incubated in 1 ml _of_ HNO~3~ at room temperature overnight followed by _incubation_ |
{#sp1 .67}
| ! [ ] ( edinbmedj74207 - 13 ) { # foo. net } | ! [] (ddinbmedj7420I - 00Y() {# sp1. 67 } | {#sp1 .67}
| {#sP1 .67}
| {#sp1 .67} | _{#sp1_ .67} |
Introduction {#s1}
============
Some of the most diverse and bizarre organelle genomes of all eukaryotes come from the Volvocales, which is a large order of predominantly freshwater green algae, belonging to chlorophycean class of the Chlorophyta. Volvocalean mitochondrial and plastid DNAs (mtDNAs and ptDNAs) show an impressive array of architectures, nucleotide landscapes, and coding compositions ([Table 1](#pone-0057177-t001){ref-type="table"})-- and see Leliaert et al. [@pone.0057177-Leliaert1] and Lee and Hua [@pone.0057177-Lee1] for additional compilations. Moreover, certain volvocalean species, particularly those within the "*Reinhardtinia* clade" *sensu* Nakada et al. [@pone.0057177-Nakada1], have proven to be excellent systems for testing contemporary hypotheses on the evolution of organelle genome expansion and linearization [@pone.0057177-Smith1], [@pone.0057177-Michaelis1], [@pone.0057177-Smith2].
10.1371/journal.pone.0057177.t001
###### Completely sequenced organelle genomes from volvocalean green algae.
{#pone-0057177-t001-1}
Species Clade (lineage) Organelle genome architecture
-------------------------------- ---------------------------- ------------------------------- -------- ---- -------- ---- ------ -------------------
MITOCHONDRIAL DNA
* Chlamydomonas* *reinhardtii* *Reinhardtinia*(volvocine) Linear 16--19 55 67--82 7 0--3 EU306617--23
* Chlamydomonas* *moewusii* *Xenovolvoxa* Circular 23 65 54 7 9 AF008237
* Chlorogonium* *elongatum* *Caudivolvoxa* Circular 23 62 53 7 6 Y13643--4,Y07814
* Dunaliella salina* *Caudivolvoxa* Circular 28 66 42 7 18 GQ250045
* Gonium pectorale* *Reinhardtinia*(volvocine) Circular 16 61 73 7 1 AP012493
* Polytomella capuana* *Reinhardtinia* Linear 13 43 82 7 0 EF645804
* Polytomella parva* *Reinhardtinia* Linear 16 59 66 7 0 AY062933-4
* Polytomella* sp.SAG 63--10 *Reinhardtinia* Linear 16 58 66 7 0 GU108480-1
* Volvox carteri* *Reinhardtinia*(volvocine) Circular 35 66 \<40 7 3 EU760701,GU084821
PLASTID DNA
* Chlamydomonas* *reinhardtii* *Reinhardtinia*(volvocine) Circular 204 66 44 66 7 FJ423446
* Dunaliella salina* *Caudivolvoxa* Circular 269 68 35 66 \>35 GQ250046
* Gonium pectorale* *Reinhardtinia*(volvocine) Circular 223 70 44 66 3 AP012494
* Volvox carteri* *Reinhardtinia*(volvocine) Circular 525 57 \<20 66 9 GU084820
Note: Values rounded to whole numbers. Clade names are based on Nakada et al. [@pone.0057177-Nakada1]. Percent coding includes all annotated protein-, rRNA-, and tRNA-coding regions as well as non-standard ORFs, such as the *rtl* gene in the *C. reinhardtii* mtDNA. Gene number includes standard protein-coding genes, but does not include intronic or nonstandard ORFs, like *rtl*. Duplicate genes and introns were counted only once. Genome statistics for *P. parva* and *P. piriformis* are based on the concatenation of the two mitochondrial chromosomes; those for *V. carteri* should be considered as approximations as the mtDNA and ptDNA contain assembly gaps due to unresolved repeats. For *C. reinhardtii*, the mitochondrial genome size, intron number, and coding content can vary because of optional introns.
Most of our understanding of volvocalean mitochondrial and plastid genomes is limited to unicellular species, such as the model organisms *Chlamydomonas reinhardtii* and *C. globosa* (previously misidentified as *C. incerta*; see Nakada et al. [@pone.0057177-Nakada2]) [@pone.0057177-Michaelis1], [@pone.0057177-Popescu1], the colorless and wall-less *Polytomella capuana*, *P. parva*, and *P. piriformis* [@pone.0057177-Smith2], [@pone.0057177-Fan1], [@pone.0057177-Smith3], and the halotolerant β-carotene-rich *Dunaliella salina* [@pone.0057177-Smith4]. Surprisingly little is known about the organelle genomes of colonial and multicellular volvocaleans, which are found within the volvocine lineage of the *Reinhardtinia* clade ([Figure S1](#pone.0057177.s001){ref-type="supplementary-material"}). Volvocine algae are preeminent models for studying the evolution of multicellularity [@pone.0057177-Kirk1], [@pone.0057177-Sachs1], and span the gamut of cellular complexity, from simple 4-celled species (e.g., *Tetrabaena*), to 8-64-celled colonial forms (e.g., *Gonium*), all the way to highly complex spheroidal taxa, with more than 500 cells (e.g., *Volvox*) [@pone.0057177-Nozaki1], [@pone.0057177-Herron1]. It is estimated that multicellular volvocine species last shared a common unicellular ancestor ∼200 million years ago [@pone.0057177-Herron1].
Of the 8 different volvocalean algae for which complete mtDNA and/or ptDNA sequences are available [@pone.0057177-Smith4], only one is multicellular: *Volvox carteri*, which is comprised of ∼4,000 cells. The organelle genomes of this species are distended with repetitive noncoding DNA, and similar palindromic repeats are located in both the mitochondrial and plastid compartments [@pone.0057177-Smith5]. Moreover, the *V. carteri* ptDNA, at ∼525 kb, is among the largest plastomes ever observed (from any eukaryote) [@pone.0057177-Smith1], dwarfing that of *C. reinhardtii*, which is 204 kb [@pone.0057177-Maul1]. Although smaller than its plastid counterpart, the ∼35 kb mtDNA of *V. carteri* is still larger than any the other completely sequenced volvocalean mitochondrial genome. It is hypothesized that the expanded organelle genomes of *V. carteri* are a consequence of a low organelle mutation rate and/or a small effective population size [@pone.0057177-Smith1].
The *V. carteri* mtDNA assembles as a circular molecule, contrasting the linear (or linear fragmented) architectures of all other well-studied *Reinhardtinia*-clade mitochondrial genomes, including those of *C. reinhardtii*, *Polytomella* spp., and the multicellular *Pandorina morum* [@pone.0057177-Michaelis1], [@pone.0057177-Smith4], [@pone.0057177-Moore1]. These linear mtDNAs have evolved complex terminal structures [@pone.0057177-Michaelis1], [@pone.0057177-Smith3], called mitochondrial telomeres, which form long palindromic repeats at the genome ends. The origin and number of times that linear mitochondrial chromosomes have evolved within the *Reinhardtinia* is unknown, but it has been argued that they arose only once [@pone.0057177-Smith1]. If true, this would imply that in a recent ancestor of *V. carteri*, the mtDNA reverted from a linear to a circular form.
To learn about organelle genome architecture within multicellular volvocine algae and to gain insight into ptDNA expansion and the origin of linear mtDNAs, we sequenced the mitochondrial and plastid genomes | introduction { # s1 } = = = = = = = = = = = = some of the most diverse and bizarre fungal genomes of all eukaryotes come from the helix, which is a large order of predominantly freshwater green algae, belonging to chlorophycean class or the chlorophyta. volvocalean mitochondrial and plastid dnas ( mtdnas excluding ptdnas ) show an impressive array of sequence, nucleotide landscapes, and coding compositions ( [ table 1 ] ( # pone - 0057177 - t001 ) { ref - type = " table " } ) - - and see leliaert et al. [ @ pone. 0057177 - leliaert1 ] and lee and hua [ @ index. 0057177 - lee1 ] for additional examples. moreover, certain volvocalean species, particularly those within the " * reinhardtinia * clade " * sensu * nakada et al. [ @ pone. 0057177 - nakada1 ], have proven to be excellent systems for testing contemporary hypotheses on the basis of organelle genome expansion and development [ @ pone. 0057177 - smith1 ], [ @ pone. 0057177 - michaelis1 ], [ @ pone. 0057177 - smith2 ]. 10. 2002 / journal. pone. 0057177. t001 # # # # # # completely sequenced organelle genomes from volvocalean green algae.! [ ] ( pone. 0057177. t001 ) { # pone - 0057177 - t001 - 1 } species clade ( lineage ) organelle genome architecture - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - mitochondrial dna * chlamydomonas * * reinhardtii * * reinhardtinia * ( volvocine ) linear 16 - - 19 55 67 - - 82 7 0 - - 3 eu306617 - - 23 * chlamydomonas * * moewusii * * xenovolvoxa * circular 23 65 54 7 9 af008237 * chlorogonium * * elongatum * * caudivolvoxa * circular 23 62 53 7 6 y13643 - - 4, y07814 * dunaliella salina * * caudivolvoxa * circular 28 66 42 7 18 gq250045 * gonium pectorale * * reinhardtinia * ( volvocine ) circular 16 61 73 7 1 ap012493 * polytomella capuana * * reinhardtinia * linear 13 43 82 7 0 ef645804 * polytomella parva * * reinhardtinia * linear 16 59 66 7 0 ay062933 - 4 * polytomella * sp. sag 63 - - 10 * reinhardtinia * linear 16 58 66 7 0 gu108480 - 1 * volvox carteri * * reinhardtinia * ( volvocine ) circular 35 66 \ < 40 7 3 eu760701, gu084821 plastid dna * chlamydomonas * * reinhardtii * * reinhardtinia * ( volvocine ) circular 204 66 44 66 7 fj423446 * dunaliella salina * * caudivolvoxa * circular 269 68 35 66 \ > 35 gq250046 * gonium pectorale * * reinhardtinia * ( volvocine ) circular 223 70 44 66 3 ap012494 * volvox carteri * * reinhardtinia * ( volvocine ) circular 525 57 \ < 20 66 9 gu084820 note : values rounded to whole numbers. clade names are based on nakada et al. [ @ pone. 0057177 - nakada1 ]. percent coding includes all annotated protein -, rrna -, and trna - coding regions as well as non - standard orfs, such as the * rtl * gene in the * c. reinhardtii * mtdna. gene number includes standard protein - coding genes, but does not include intronic or nonstandard orfs, like * rtl *. duplicate genes and introns were counted only once. genome statistics for * p. parva * and * p. piriformis * are based on the concatenation of the two mitochondrial chromosomes ; those for * v. carteri * should be considered as approximations as the mtdna and ptdna contain assembly gaps due to unresolved repeats. for * c. reinhardtii *, the mitochondrial genome size, intron number, and coding content can vary because of optional introns. most of our understanding of volvocalean mitochondrial and plastid genomes is limited to unicellular species, such as the model organisms * chlamydomonas reinhardtii * and * c. globosa * ( previously misidentified as * c. incerta * ; see nakada et al. [ @ pone. 0057177 - nakada2 ] ) [ @ pone. 0057177 - michaelis1 ], [ @ pone. 0057177 - popescu1 ], the colorless and wall - less * polytomella capuana *, * p. parva *, and * p. piriformis * [ @ pone. 0057177 - smith2 ], [ @ pone. 0057177 - fan1 ], [ @ pone. 0057177 - smith3 ], and the halotolerant β - carotene - rich * dunaliella salina * [ @ pone. 0057177 - smith4 ]. surprisingly little is known about the organelle genomes of colonial and multicellular volvocaleans, which are found within the volvocine lineage of the * reinhardtinia * clade ( [ figure s1 ] ( # pone. 0057177. s001 ) { ref - type = " supplementary - material " } ). volvocine algae are preeminent models for studying the evolution of multicellularity [ @ pone. 0057177 - kirk1 ], [ @ pone. 0057177 - sachs1 ], and span the gamut of cellular complexity, from simple 4 - celled species ( e. g., * tetrabaena * ), to 8 - 64 - celled colonial forms ( e. g., * gonium * ), all the way to highly complex spheroidal taxa, with more than 500 cells ( e. g., * volvox * ) [ @ pone. 0057177 - nozaki1 ], [ @ pone. 0057177 - herron1 ]. it is estimated that multicellular volvocine species last shared a common unicellular ancestor [UNK] million years ago [ @ pone. 0057177 - herron1 ]. of the 8 different volvocalean algae for which complete mtdna and / or ptdna sequences are available [ @ pone. 0057177 - smith4 ], only one is multicellular : * volvox carteri *, which is comprised of [UNK], 000 cells. the organelle genomes of this species are distended with repetitive noncoding dna, and similar palindromic repeats are located in both the mitochondrial and plastid compartments [ @ pone. 0057177 - smith5 ]. moreover, the * v. carteri * ptdna, at [UNK] kb, is among the largest plastomes ever observed ( from any eukaryote ) [ @ pone. 0057177 - smith1 ], dwarfing that of * c. reinhardtii *, which is 204 kb [ @ pone. 0057177 - maul1 ]. although smaller than its plastid counterpart, the [UNK] kb mtdna of * v. carteri * is still larger than any the other completely sequenced volvocalean mitochondrial genome. it is hypothesized that the expanded organelle genomes of * v. carteri * are a consequence of a low organelle mutation rate and / or a small effective population size [ @ pone. 0057177 - smith1 ]. the * v. carteri * mtdna assembles as a circular molecule, contrasting the linear ( or linear fragmented ) architectures of all other well - studied * reinhardtinia * - clade mitochondrial genomes, including those of * c. reinhardtii *, * polytomella * spp., and the multicellular * pandorina morum * [ @ pone. 0057177 - michaelis1 ], [ @ pone. 0057177 - smith4 ], [ @ pone. 0057177 - moore1 ]. these linear mtdnas have evolved complex terminal structures [ @ pone. 0057177 - michaelis1 ], [ @ pone. 0057177 - smith3 ], called mitochondrial telomeres, which form long palindromic repeats at the genome ends. the origin and number of times that linear mitochondrial chromosomes have evolved within the * reinhardtinia * is unknown, but it has been argued that they arose only once [ @ pone. 0057177 - smith1 ]. if true, this would imply that in a recent ancestor of * v. carteri *, the mtdna reverted from a linear to a circular form. to learn about organelle genome architecture within multicellular volvocine algae and to gain insight into ptdna expansion and the origin of linear mtdnas, we sequenced the mitochondrial and plastid genomes | Introduction {# s1} = = = = = = = = = = = = SKje of the most diverse and bizarre organelle genomes of all eukaryotes come from the Volvocales, which is a large order of predominantly freshwater green algae, belonging to chlorophycean class of the Chlorophyta. Volvocalean mitochondrial and plastid DNAs (mtDNAs and ptDNAs) show an impresaiGe array of architectures, nucleotide landscapes, and coding compositions ([ Table 1] (# pone - 0057177 - t001) {ref - type = " table "} ) - - and see Leliaert et al. [@ pone. 0057177 - Leliaert1] and Lee and Hua [@ pone. 0057177 - Lee1] for additional compilations. Moreover, certain volvocalean specKFs, particularly those within the " * Reinhardtinia * clade " * sensu * Nakada et al. [@ pone. 0057177 - Nakada1 ], have proven to be excellwnR systems for testing contemporary hypotheses on the evolution of organelle genome expansion and linearization [@ pone. 0057177 - Smith1 ], [@ pone. 0057177 - Michaelis1 ], [@ pone. 0057177 - Smith2 ]. 10. 1371 / journal. pone. 0057177. t001 # # # # # # Completely sequenced organelle genomes from volvocalean green algae. ! [] (pone. 0057177. t001) {# pone - 0057177 - t001 - 1} Species Clade (lineage) Organelle genome architecture - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MITOCHONDRIAL DNA * Chlamydomonas * * reinhardtii * * Reinhardtinia * (volvocine) Linear 16 - - 19 55 67 - - 82 7 0 - - 3 EU306617 - - 23 * Chlamydomonas * * moewusii * * Xenovolvoxa * Circular 23 65 54 7 9 AF008237 * Chlorogonium * * elongatum * * Caudivolvoxa * Circular 23 62 53 7 6 Y13643 - - 4, Y07814 * Dunaliella salina * * Caudivolvoxa * Circular 28 66 42 7 18 GQ250045 * Gonium pectorale * * Reinhardtinia * (volvocine) Circular 16 61 73 7 1 AP012493 * Polytomella capuana * * Reinhardtinia * Linear 13 43 82 7 0 EF645804 * Polytomella parva * * Reinhardtinia * Linear 16 59 66 7 0 AY062933 - 4 * Polytomella * sp. SAG 63 - - 10 * Reinhardtinia * Linear 16 58 66 7 0 GU108480 - 1 * Volvox carteri * * Reinhardtinia * (volvocine) Circular 35 66 \ <40 7 3 EU760701, GU084821 PLASTID DNA * Chlamydomonas * * reinhardtii * * Reinhardtinia * (volvocine) Circular 204 66 44 66 7 FJ423446 * Dunaliella salina * * Caudivolvoxa * Circular 269 68 35 66 \> 35 GQ250046 * Gonium pectorale * * De8nhardtinia * (volvocine) Circular 223 70 44 66 3 AP012494 * Volvox carteri * * Reinhardtinia * (volvocine) Circular 525 57 \ <20 66 9 GU084820 Note: Values rounded to whole numbers. Clade names are based on Nakada et al. [@ pone. 0057177 - Nakada1 ]. Percent coding includes all annotated protein -, rRNA -, and tRNA - coding regions as well as non - standard ORFs, such as the * rtl * gene in the * C. reinhardtii * mtDNA. Gene number includes standard protein - coding genes, but does not include intronic or nonstandard ORFs, like * rtl *. Duplicate genes and introns were counted only once. Genome statistics for * P. parva * and * P. piriformis * are based on the concatenation of the two mitochondrial chromosomes; those for * V. carteri * should be considered as approximations as the mtDNA and ptDNA contain assembly gaps due to unresolved repeats. For * C. reinhardtii *, the mitochondrial genome size, intron number, and coding content can vary because of optional introns. Most of our understanding of volvocalean mitochondrial and plastid genomes is limited to unicellular species, such as the model organisms * Chlamydomonas reinhardtii * and * C. globosa * (previously misidentified as * C. incerta *; see Nakada et al. [@ pone. 0057177 - Nakada2] ) [@ pone. 0057177 - Michaelis1 ], [@ pone. 0057177 - Popescu1 ], the colorless and wall - less * Polytomella capuana *, * P. parva *, and * P. piriformis * [@ pone. 0057177 - Smith2 ], [@ pone. 0057177 - Fan1 ], [@ pone. 0057177 - Smith3 ], and the halotolerant β - carotene - rich * Dunaliella salina * [@ pone. 0057177 - Smith4 ]. Surprisingly little is known about the organelle genomes of colonial and multicellular volvocaleans, which are found within the volvocine lineage of the * Reinhardtinia * clade ([ Figure S1] (# pone. 0057177. s001) {ref - type = " sipplementxry - material "} ). Volvocine algae are preeminent models for studying the evolution of multicellularity [@ pone. 0057177 - <Jrk1 ], [@ pone. 0057177 - Sachs1 ], and span the gamut of cellular complexity, from simple 4 - celled species (e. g. , * Tetrabaena * ), to 8 - 64 - celled colonial forms (e. g. , * Gonium * ), all the way to highly complex spheroidal taxa, with more than 500 cells (e. g. , * Volvox *) [@ pone. 0057177 - Nozaki1 ], [@ pone. 0057177 - Herron1 ]. It is estimated that multicellular volvocine species last shared a common unicellular ancestor ∼ 200 miPljon years ago [@ pone. 0057177 - Herron1 ]. Of the 8 different volvocalean algae for which complete mtDNA and / or ptDNA sequences are available [@ pone. 0057177 - Smith4 ], only one is multicellular: * Volvox carteri *, which is comprised of ∼ 4, 000 cells. The organelle genomes of this species are distended with repetitive noncoding DNA, and similar palindromic repeats are located in both the mitochondrial and plastid compartments [@ pone. 0057177 - Smith5 ]. Moreover, the * V. carteri * ptDNA, at ∼ 525 kb, is among the largest plastomes ever observed (from any eukaryote) [@ )onD. 0057177 - Smith1 ], dwarfing that of * C. reinhardtii *, which is 204 kb [@ pone. 0057177 - Maul1 ]. Although smaller than its plastid counterpart, the ∼ 35 kb mtDNA of * V. carteri * is still larger than any the other completely sequenced volvocalean mitochondrial genome. It is hypothesized that the expanded organelle genomes of * V. carteri * are a consequence of a low organelle mutation rate and / or a small effective population size [@ pone. 0057177 - Smith1 ]. The * V. carteri * mtDNA assembles as a circular molecule, contrasting the linear (or linear fragmented) architectures of all other well - studied * Reinhardtinia * - clade mitochondrial genomes, including those of * C. reinhardtii *, * Polytomella * spp. , and the multicellular * Pandorina morum * [@ pone. 0057177 - Michaelis1 ], [@ pone. 0057177 - Smith4 ], [@ pone. 0057177 - Moore1 ]. These linear mtDNAs have evolved complex terminal structures [@ pone. 0057177 - Michaelis1 ], [@ pone. 0057177 - Smith3 ], called mitochondrial telomeres, which form long palindromic repeats at the genome ends. The origin and number of times that linear mitochondrial chromosomes have evolved within the * Reinhardtinia * is unknown, but it has been argued that they arose only once [@ pone. 0057177 - Smith1 ]. If true, this would imply that in a recent ancestor of * V. carteri *, the mtDNA reverted from a linear to a circular form. To learn about organelle genome architecture within multicellular volvocine algae and to gain insight into ptDNA expansion and the origin of linear mtDNAs, we sequenced the mitochondrial and plastid nenoNes | Introduction {#s1} ============ Some of the most bizarre organelle genomes of all eukaryotes from the Volvocales, is a large order of predominantly freshwater green algae, to chlorophycean class of the Chlorophyta. Volvocalean mitochondrial and plastid DNAs (mtDNAs and ptDNAs) show an impressive array of architectures, nucleotide landscapes, and coding compositions ([Table 1](#pone-0057177-t001){ref-type="table"})-- and see Leliaert et al. [@pone.0057177-Leliaert1] and Lee and [@pone.0057177-Lee1] for additional compilations. Moreover, certain species, particularly those the clade" *sensu* Nakada et al. [@pone.0057177-Nakada1], have proven to be excellent systems for testing contemporary hypotheses on the evolution of organelle genome expansion linearization [@pone.0057177-Smith1], [@pone.0057177-Michaelis1], [@pone.0057177-Smith2]. 10.1371/journal.pone.0057177.t001 ###### Completely sequenced organelle genomes from volvocalean green algae. {#pone-0057177-t001-1} Species Clade (lineage) Organelle genome -------------------------------- ---------------------------- ------------------------------- -------- ---- -------- ---- ------ MITOCHONDRIAL DNA *reinhardtii* *Reinhardtinia*(volvocine) Linear 16--19 55 67--82 7 0--3 EU306617--23 * *moewusii* *Xenovolvoxa* Circular 23 65 54 7 9 AF008237 * Chlorogonium* *elongatum* *Caudivolvoxa* Circular 23 62 53 7 6 Y13643--4,Y07814 * Dunaliella salina* *Caudivolvoxa* Circular 28 66 42 7 GQ250045 * Gonium pectorale* *Reinhardtinia*(volvocine) Circular 16 61 73 7 1 * Polytomella *Reinhardtinia* Linear 13 43 82 7 0 EF645804 * Polytomella parva* Linear 16 59 66 7 0 AY062933-4 Polytomella* sp.SAG 63--10 *Reinhardtinia* Linear 16 58 66 7 0 GU108480-1 * Volvox carteri* *Reinhardtinia*(volvocine) Circular 35 66 \<40 3 EU760701,GU084821 DNA Chlamydomonas* *reinhardtii* *Reinhardtinia*(volvocine) Circular 204 66 44 66 FJ423446 * Dunaliella salina* *Caudivolvoxa* Circular 269 68 35 66 GQ250046 * Gonium pectorale* *Reinhardtinia*(volvocine) 223 70 44 66 3 AP012494 * Volvox *Reinhardtinia*(volvocine) Circular 525 57 \<20 66 9 GU084820 Note: Values rounded whole numbers. Clade names are based on Nakada et al. [@pone.0057177-Nakada1]. Percent coding includes all annotated rRNA-, and tRNA-coding regions as well as non-standard ORFs, such as the gene in the *C. reinhardtii* mtDNA. Gene standard protein-coding genes, but does not include intronic or nonstandard ORFs, like Duplicate genes were counted only once. Genome statistics for *P. and *P. piriformis* based on the concatenation of the mitochondrial chromosomes; those for *V. carteri* be considered as approximations as the mtDNA and ptDNA contain assembly gaps due to unresolved repeats. For *C. mitochondrial genome size, number, and coding content can vary because of optional of our understanding of volvocalean mitochondrial and plastid genomes is limited to unicellular species, such as model organisms *Chlamydomonas reinhardtii* and *C. globosa* (previously misidentified as *C. incerta*; see Nakada et al. [@pone.0057177-Nakada2]) [@pone.0057177-Michaelis1], [@pone.0057177-Popescu1], the colorless and wall-less *Polytomella capuana*, parva*, and *P. piriformis* [@pone.0057177-Smith2], [@pone.0057177-Fan1], [@pone.0057177-Smith3], and the halotolerant β-carotene-rich *Dunaliella salina* [@pone.0057177-Smith4]. Surprisingly little is known about organelle of colonial and multicellular volvocaleans, are found within the volvocine lineage of the clade ([Figure S1](#pone.0057177.s001){ref-type="supplementary-material"}). Volvocine algae are preeminent models for studying the evolution of multicellularity [@pone.0057177-Kirk1], [@pone.0057177-Sachs1], and the gamut cellular from simple 4-celled species (e.g., *Tetrabaena*), to 8-64-celled colonial forms (e.g., *Gonium*), all the way to highly complex spheroidal with more than 500 cells (e.g., *Volvox*) [@pone.0057177-Nozaki1], [@pone.0057177-Herron1]. It is estimated that multicellular volvocine species shared a common ancestor ∼200 million years ago [@pone.0057177-Herron1]. Of the 8 different volvocalean algae for which complete mtDNA and/or ptDNA sequences are available [@pone.0057177-Smith4], only one is multicellular: *Volvox carteri*, which is comprised ∼4,000 cells. The organelle genomes of this species are distended with repetitive noncoding DNA, and similar palindromic repeats located in both the mitochondrial and plastid compartments [@pone.0057177-Smith5]. Moreover, the *V. carteri* ptDNA, at ∼525 kb, is among the largest plastomes ever (from any eukaryote) [@pone.0057177-Smith1], dwarfing that of *C. reinhardtii*, which is 204 kb [@pone.0057177-Maul1]. Although smaller than its plastid counterpart, the kb mtDNA of *V. carteri* is still larger than any the other completely sequenced volvocalean mitochondrial is that expanded organelle of *V. carteri* are a consequence a low organelle rate and/or a small effective population size [@pone.0057177-Smith1]. The *V. carteri* mtDNA assembles as a molecule, the linear (or linear fragmented) architectures all other well-studied *Reinhardtinia*-clade mitochondrial genomes, those of *C. reinhardtii*, *Polytomella* spp., and the multicellular *Pandorina morum* [@pone.0057177-Michaelis1], [@pone.0057177-Moore1]. These linear have evolved complex terminal structures [@pone.0057177-Michaelis1], [@pone.0057177-Smith3], called mitochondrial which form long palindromic repeats at the genome origin and number of times that linear mitochondrial chromosomes have evolved within the *Reinhardtinia* is unknown, but it been that they arose only once [@pone.0057177-Smith1]. If true, would imply that in a recent ancestor of *V. carteri*, the mtDNA reverted from a linear to a circular form. To learn about organelle genome architecture multicellular algae and to gain insight into ptDNA expansion and the origin of mtDNAs, we sequenced the mitochondrial and genomes | IntroduCTioN {#S1}
============
SoME OF tHE MOsT DIverSe aNd bIZarRe ORgaNelLe GenOmeS of all eUKaryOTES CoME fROM tHE VOLVOCALes, WHIch is A LArge ORder of pREdOmiNantlY FrEsHWATEr gReen ALGAE, BeloNgiNg tO CHLoROphYCEaN cLaSS of tHE ChlOROpHYTA. VolVoCALean MitochoNDRiaL aND PlAStid Dnas (mTDNaS AnD PtdNas) show An ImPrESsiVe arraY oF ArCHitectUrES, NuCLeOtide LAnDsCAPES, aNd CODiNG comPOSITIONS ([TabLe 1](#poNe-0057177-t001){Ref-Type="tabLE"})-- AnD SeE lEliaErT Et AL. [@pOnE.0057177-LEliaeRT1] AnD Lee AND hUA [@POne.0057177-LeE1] For aDdItIonAl CoMpIlATIoNs. mOreOVer, CERTaIN vOlVoCALeAN SPecies, PArTIcULaRlY THOsE WiTHin THE "*rEInHARDtiniA* CLAde" *Sensu* nAkAdA et Al. [@poNe.0057177-NaKaDA1], haVE pROven To be eXCELLEnt systemS FoR tesTInG coNteMpORary HyPOTHeseS on THe EvOLUtiON of oRganelLE GENOmE ExpaNSiON aND lINearizATIoN [@pone.0057177-sMITH1], [@pOne.0057177-miCHaELiS1], [@pONe.0057177-SmiTH2].
10.1371/joURNal.pone.0057177.t001
###### compLeTeLY seQUEncED orGANelle GeNOMEs frOm VoLvoCalean GrEEn AlGAe.
{#poNE-0057177-T001-1}
SpECiES CLaDE (lInEAgE) orGanEllE GenOME ARcHITECTuRE
-------------------------------- ---------------------------- ------------------------------- -------- ---- -------- ---- ------ -------------------
mitochONdriAL DNA
* CHlamyDoMONas* *reInHaRdTII* *reINHArdTinIA*(voLvOciNe) lInEar 16--19 55 67--82 7 0--3 eu306617--23
* CHLAmyDomOnAS* *mOEWuSii* *XenOvOLVoXA* CIrcuLAR 23 65 54 7 9 af008237
* chLoRoGonIum* *ElongAtUm* *cAUDivOlvOxa* CircUlAR 23 62 53 7 6 y13643--4,y07814
* DUNALiElLa SaLIna* *CaUdIVOlvoxa* cIRCuLar 28 66 42 7 18 gQ250045
* goNiuM PEcToRale* *reINhardtinIA*(VOLvoCiNE) CIrculAr 16 61 73 7 1 Ap012493
* pOlYtOmelLA CAPuaNa* *ReiNHardTInIa* LIneAr 13 43 82 7 0 ef645804
* PoLYtomeLla pARVa* *ReinharDTInIA* lInEar 16 59 66 7 0 ay062933-4
* polYTOmella* sp.SaG 63--10 *REiNhaRdTINia* liNEAR 16 58 66 7 0 gU108480-1
* VOlVOX CARTeri* *ReINHArDtiNIa*(voLVoCIne) circuLAR 35 66 \<40 7 3 eU760701,Gu084821
PLasTID dna
* ChLamyDOmonaS* *ReINHArdTii* *REinHaRdtiNIa*(volvOcIne) circUlaR 204 66 44 66 7 fj423446
* DUNAlIELLA SaLiNa* *cAuDiVolvOXa* cIRCUlar 269 68 35 66 \>35 GQ250046
* goNiuM PEctORAlE* *rEiNhardTiNIa*(voLvoCine) circULAR 223 70 44 66 3 Ap012494
* vOLvoX CArTeri* *rEiNHArdtiNIa*(vOLvocINE) cirCULAR 525 57 \<20 66 9 Gu084820
NoTe: VALuES ROuNded TO wHOlE numbers. clAde naMEs ARE BasED ON nAKADA Et al. [@PoNe.0057177-NakadA1]. pERcENT COding iNcLuDES alL aNNotATEd proTeIn-, Rrna-, AnD trNA-CodiNg REGIons aS wELL As Non-stanDaRD oRfS, SucH AS tHe *RTL* GENe in tHe *C. ReiNHarDtII* mTDNa. gEne nUmbER iNCludES standArd protein-coDINg GeNeS, bUt Does nOt IncludE inTrONiC or NONStaNdarD OrFS, lIke *rTL*. dUPlICAte GenES AND INTronS WerE CoUnTed ONLy Once. GeNome sTaTisTiCs fOR *P. PaRVA* anD *p. piRIFormIS* Are BASEd ON THE conCaTenatiON Of ThE tWO mItocHOnDRiaL ChromoSomES; thOSe For *V. cARterI* ShOulD Be CoNSIdeREd aS aPproXIMATionS aS tHe MtDna aND ptdna CONTAin aSSemBly GaPs duE to UNreSoLVeD rEPeatS. FoR *c. reInHArdTII*, the miTOChONDriAL gEnoMe SIZe, iNTRon nUMbeR, AnD coDING cOntEnt caN vaRy BecAUSe oF oPtiOnAl InTronS.
MOST of Our uNDERsTAnDing of VoLVOcaleaN MItOcHOnDriaL AND plaStiD geNomES iS LiMiTed To UniCelLulAr speciES, SuCh As tHE MOdel ORganISMs *chLAMydOmonAs REiNHaRDTII* And *C. GLOBoSA* (pREvIOUsLY MIsiDenTIFIED aS *c. iNcERTa*; SEE NAKADA ET Al. [@PoNE.0057177-NakaDa2]) [@PONE.0057177-MIcHaELiS1], [@PONe.0057177-POpESCu1], The coLoRlESs anD wAlL-LEsS *pOLYTOMELla caPuAna*, *P. PaRVA*, and *p. PiRIFoRMIs* [@poNe.0057177-Smith2], [@POnE.0057177-fan1], [@POnE.0057177-SMITh3], aND tHE HALotoleRaNt Β-CAROteNe-RiCh *dunaliELla salINa* [@PonE.0057177-SmIth4]. surpRIsingLY LiTtLe iS KNown aBoUt THE orGanElLe geNOmEs of COLonIAl And MUlTIcEllULar VOLVoCaLeans, WhiCH aRE FoUnd wiTHiN ThE voLvOciNE LIneAGE of tHe *REInHArDtiniA* ClAdE ([figuRE S1](#ponE.0057177.s001){Ref-tYPe="sUpPlEMENTary-MaTeRiAl"}). VOLVOCINe aLgAe aRE pREemINEnt mODEls foR stUdyIng ThE evOLuTIon Of mULticELLUlaRIty [@pOnE.0057177-kiRK1], [@PoNe.0057177-SacHs1], aND Span THE GaMut oF CelLuLar COmPlExITY, From simPlE 4-CELled sPECies (E.g., *tETraBAENA*), TO 8-64-CelleD cOloNial FOrMS (E.G., *GoNIum*), aLl ThE wAy to hiGhLY COmPleX spHERoiDAL taXa, wiTH MOre ThAn 500 cELls (E.G., *VOlVOx*) [@PonE.0057177-NoZAKi1], [@PONE.0057177-hErrON1]. It IS EstIMateD THAt mULtICelLuLAR vOLVoCInE speciEs lAst ShArED a coMmOn UNicELLULAr AnCeStOR ∼200 mIllIOn YearS agO [@PONe.0057177-HeRRON1].
of tHe 8 DIFfereNt VoLVocaleAn AlGae For wHICh cOmpLetE MTdnA anD/or ptdna sEQueNcEs arE AVAilABLe [@PONe.0057177-SMItH4], onLY oNE is MUlTICelLuLar: *VoLVox CArTERI*, WHich Is ComPrISED of ∼4,000 CEllS. tHe OrgANELlE gEnOmes OF ThIS speCIEs aRe dIstENDeD WITH rEPeTiTivE NOnCOdIng dNA, AND SimilAR paLinDROmiC rePEAtS are locaTed in botH the MItOchoNDrIAL And PLasTiD COMPaRtmENTS [@pONe.0057177-SMIth5]. morEoVer, ThE *V. cArTeRi* pTdnA, at ∼525 kb, IS among thE lArgEST PLAsToMeS eveR ObSErvED (fROM aNy eUkaRYoTe) [@poNe.0057177-SmiTH1], DwaRFInG ThAT OF *C. rEinhardtII*, whiCh IS 204 kb [@pONe.0057177-maUL1]. althouGh sMALLER tHaN ItS PLastid CoUnTERParT, THE ∼35 kb mTDNa oF *V. CarTerI* is sTILl LaRgER Than ANY the otHEr COMPLEtELy seQUENcEd voLvoCAleAN MiTochOnDrIAL geNOmE. IT iS HypoTHESiZed THat The ExPANDEd OrGAnELle GEnoMeS Of *V. CArtERI* ARE A CoNSEquenCE of a lOW OrgaNeLle mutATion RaTE AND/oR A SMaLl efFective PopUlAtIoN size [@pONE.0057177-sMITh1].
THe *V. CARTeRI* mTDNa AsseMbLeS as a cIrCulAR MoleCULE, coNTrAStInG THe lineAr (OR linEar fRAgmENtED) aRChItecTuRes of AlL OthEr weLl-sTUdieD *reINHaRdTIniA*-CLaDE mitOCHondrial geNOmes, inCLuDiNg THosE OF *c. reINhardtII*, *poLytOMElLa* spP., aND The muLTICELluLAr *PAndOrInA MOruM* [@POnE.0057177-MICHaELiS1], [@POne.0057177-SmITH4], [@poNe.0057177-MOore1]. thesE LINEaR mtdNAs HaVe eVOlvED cOMplEX TErMINAL STRUctuRES [@PoNE.0057177-MIChaeLiS1], [@PoNE.0057177-SMITH3], CalLEd mitochoNdrIAL TeLomeRES, wHICh fOrm lONg PaLInDrOmIC rePEaTS At THe GEnome EnDs. ThE OrIGiN aND NuMBer of TIMEs THat linEAR MitoCHoNdRiAl cHromoSOmES HaVE EVoLVeD wIThin the *rEiNHARdTINiA* Is UNKnoWN, buT IT has BEEn ARGueD THAT TheY AROsE onLy Once [@PoNE.0057177-SMITH1]. IF True, tHIS wouLd IMPLY THAt In a recenT anCesToR Of *v. CartERi*, THE MTdNA revErTEd fROM a liNEAR To a CIrcuLar FORM.
TO LEarn aboUT ORgAnELLe Genome ARcHiteCTuRE WiTHiN MultIcelLULAR VOlVoCINe AlGaE AnD TO GaIN iNSIGht inTo pTDNa eXPANsiOn and ThE origIN oF LINEaR MtdNas, wE SeQUENCeD The mitochoNDRial anD PLAStID gEnomes | Introduction {#s1} ============ Some of the most diverse and bizarre organellegenomes ofall eukaryotes come from the Volvocales, which isalarge order ofpredominantly freshwatergreen algae, belonging to chlorophycean class of the Chlorophyta. Volvocalean mitochondrial and plastid DNAs (mtDNAs and ptDNAs) show an impressive array of architectures, nucleotide landscapes, andcoding compositions ([Table1](#pone-0057177-t001){ref-type="table"})-- and see Leliaert et al. [@pone.0057177-Leliaert1] and Lee andHua [@pone.0057177-Lee1] for additional compilations. Moreover,certain volvocalean species,particularly those within the "*Reinhardtinia*clade" *sensu* Nakadaet al. [@pone.0057177-Nakada1], have proven to be excellent systems for testing contemporary hypotheses on the evolution of organelle genome expansion and linearization [@pone.0057177-Smith1], [@pone.0057177-Michaelis1], [@pone.0057177-Smith2]. 10.1371/journal.pone.0057177.t001 ###### Completely sequenced organelle genomes from volvocalean green algae. {#pone-0057177-t001-1} Species Clade(lineage)Organelle genome architecture -------------------------------- ---------------------------- ------------------------------- -------- ---- -------- ---- ------ ------------------- MITOCHONDRIAL DNA * Chlamydomonas* *reinhardtii* *Reinhardtinia*(volvocine) Linear 16--19 55 67--827 0--3EU306617--23 * Chlamydomonas* *moewusii* *Xenovolvoxa* Circular 23 65 54 7 9 AF008237 * Chlorogonium**elongatum* *Caudivolvoxa* Circular 23 62 53 7 6 Y13643--4,Y07814 *Dunaliella salina* *Caudivolvoxa* Circular28 66 427 18 GQ250045 * Gonium pectorale* *Reinhardtinia*(volvocine) Circular 16 61 73 7 1 AP012493 * Polytomella capuana* *Reinhardtinia* Linear 13 43 82 7 0 EF645804 * Polytomellaparva* *Reinhardtinia* Linear16 59 66 7 0 AY062933-4 *Polytomella* sp.SAG 63--10 *Reinhardtinia* Linear 1658 66 70 GU108480-1 * Volvox carteri* *Reinhardtinia*(volvocine) Circular 3566 \<40 7 3 EU760701,GU084821 PLASTID DNA * Chlamydomonas* *reinhardtii* *Reinhardtinia*(volvocine) Circular 204 66 44 66 7 FJ423446 * Dunaliella salina**Caudivolvoxa* Circular 269 68 35 66 \>35 GQ250046 * Gonium pectorale* *Reinhardtinia*(volvocine) Circular 223 70 4466 3 AP012494* Volvoxcarteri* *Reinhardtinia*(volvocine) Circular 525 57 \<2066 9 GU084820 Note: Values rounded towhole numbers. Clade names are based on Nakada et al.[@pone.0057177-Nakada1]. Percentcoding includes all annotated protein-, rRNA-, and tRNA-codingregions as well as non-standardORFs,such as the *rtl* gene in the*C.reinhardtii* mtDNA. Gene number includes standard protein-coding genes,but does not include intronic ornonstandard ORFs, like*rtl*. Duplicategenes and introns were counted only once. Genomestatistics for *P. parva* and *P. piriformis* are based on theconcatenation of the twomitochondrialchromosomes; those for *V. carteri* should be considered as approximationsasthe mtDNA and ptDNA contain assembly gaps dueto unresolvedrepeats. For *C. reinhardtii*, the mitochondrial genome size, intron number, andcodingcontent can vary because of optional introns.Mostof our understanding of volvocalean mitochondrial and plastid genomes is limited to unicellular species, such asthemodel organisms *Chlamydomonas reinhardtii* and *C. globosa* (previously misidentifiedas *C. incerta*; see Nakada etal. [@pone.0057177-Nakada2]) [@pone.0057177-Michaelis1],[@pone.0057177-Popescu1], the colorless and wall-less*Polytomella capuana*, *P. parva*, and *P. piriformis* [@pone.0057177-Smith2], [@pone.0057177-Fan1], [@pone.0057177-Smith3], and the halotolerantβ-carotene-rich *Dunaliella salina* [@pone.0057177-Smith4]. Surprisingly little is known about the organelle genomes of colonialand multicellular volvocaleans,which are found within the volvocine lineage of the *Reinhardtinia* clade ([Figure S1](#pone.0057177.s001){ref-type="supplementary-material"}). Volvocine algae are preeminent models for studying the evolution of multicellularity [@pone.0057177-Kirk1], [@pone.0057177-Sachs1], and span the gamut of cellular complexity, from simple 4-celled species (e.g., *Tetrabaena*), to 8-64-celled colonial forms (e.g., *Gonium*), all the way to highly complex spheroidal taxa, with morethan500 cells(e.g., *Volvox*) [@pone.0057177-Nozaki1], [@pone.0057177-Herron1]. It is estimated that multicellular volvocine species last shared acommon unicellularancestor ∼200million years ago[@pone.0057177-Herron1]. Of the8 different volvocalean algae for which complete mtDNA and/or ptDNA sequencesare available [@pone.0057177-Smith4], only one is multicellular: *Volvox carteri*, which is comprised of ∼4,000cells. The organelle genomes of this species are distended with repetitivenoncoding DNA, and similar palindromic repeats are located in both the mitochondrial andplastid compartments [@pone.0057177-Smith5]. Moreover, the *V.carteri*ptDNA,at ∼525kb, isamong the largestplastomes ever observed (from anyeukaryote) [@pone.0057177-Smith1], dwarfing that of *C. reinhardtii*,which is 204 kb [@pone.0057177-Maul1]. Althoughsmaller than its plastid counterpart, the ∼35 kb mtDNA of *V. carteri* is stilllarger than any the other completelysequenced volvocalean mitochondrial genome. It is hypothesized thatthe expanded organelle genomes of *V. carteri* are a consequenceof a low organelle mutation rate and/or a small effective populationsize [@pone.0057177-Smith1]. The *V.carteri*mtDNA assembles as a circular molecule, contrastingthe linear(or linear fragmented) architectures ofall other well-studied *Reinhardtinia*-clademitochondrialgenomes, includingthoseof *C. reinhardtii*, *Polytomella*spp., and the multicellular*Pandorinamorum*[@pone.0057177-Michaelis1], [@pone.0057177-Smith4],[@pone.0057177-Moore1]. Theselinear mtDNAs haveevolvedcomplex terminal structures [@pone.0057177-Michaelis1],[@pone.0057177-Smith3], called mitochondrial telomeres, which form longpalindromic repeats at the genomeends. The origin andnumber oftimes that linear mitochondrialchromosomes have evolved within the *Reinhardtinia* is unknown,but it has been arguedthat they arose only once [@pone.0057177-Smith1]. If true, this would imply thatin a recentancestor of *V. carteri*, the mtDNA reverted from a linear to a circularform. To learnabout organelle genome architecture within multicellular volvocine algae and to gain insight into ptDNA expansion and the origin of linear mtDNAs, we sequencedthe mitochondrial and plastid genomes | Introduction {#s1} ============ Some of the most diverse and _bizarre_ organelle _genomes_ of all eukaryotes come from the Volvocales, which _is_ a large order of predominantly freshwater green algae, belonging to _chlorophycean_ class of the Chlorophyta. Volvocalean mitochondrial and plastid DNAs (mtDNAs and ptDNAs) show an impressive array of architectures, nucleotide _landscapes,_ and coding compositions ([Table _1](#pone-0057177-t001){ref-type="table"})--_ _and_ see _Leliaert_ et _al._ [@pone.0057177-Leliaert1] and _Lee_ and Hua [@pone.0057177-Lee1] _for_ additional compilations. Moreover, certain volvocalean species, particularly those within _the_ "*Reinhardtinia* _clade"_ *sensu* Nakada _et_ al. _[@pone.0057177-Nakada1],_ have _proven_ _to_ be _excellent_ systems _for_ testing contemporary _hypotheses_ on the evolution of organelle genome expansion and linearization [@pone.0057177-Smith1], [@pone.0057177-Michaelis1], [@pone.0057177-Smith2]. _10.1371/journal.pone.0057177.t001_ _######_ Completely sequenced organelle _genomes_ from volvocalean green algae. {#pone-0057177-t001-1} Species Clade (lineage) Organelle genome _architecture_ -------------------------------- ---------------------------- ------------------------------- -------- ---- -------- ---- _------_ ------------------- MITOCHONDRIAL DNA * Chlamydomonas* *reinhardtii* *Reinhardtinia*(volvocine) Linear 16--19 55 67--82 _7_ 0--3 _EU306617--23_ * Chlamydomonas* *moewusii* *Xenovolvoxa* Circular 23 65 54 _7_ 9 AF008237 * Chlorogonium* *elongatum* *Caudivolvoxa* Circular 23 62 53 _7_ _6_ Y13643--4,Y07814 _*_ Dunaliella salina* *Caudivolvoxa* Circular _28_ 66 42 _7_ 18 _GQ250045_ * Gonium _pectorale*_ *Reinhardtinia*(volvocine) Circular 16 61 73 7 1 _AP012493_ * Polytomella _capuana*_ *Reinhardtinia* Linear 13 _43_ _82_ 7 0 _EF645804_ * Polytomella parva* *Reinhardtinia* Linear 16 59 66 7 0 AY062933-4 _*_ Polytomella* sp.SAG 63--10 *Reinhardtinia* Linear 16 _58_ 66 7 0 GU108480-1 * Volvox carteri* *Reinhardtinia*(volvocine) Circular 35 66 \<40 7 _3_ EU760701,GU084821 PLASTID DNA * Chlamydomonas* *reinhardtii* _*Reinhardtinia*(volvocine)_ _Circular_ 204 66 44 66 7 FJ423446 * Dunaliella salina* *Caudivolvoxa* Circular 269 68 _35_ 66 \>35 GQ250046 * Gonium pectorale* _*Reinhardtinia*(volvocine)_ Circular 223 70 _44_ 66 3 AP012494 * Volvox carteri* _*Reinhardtinia*(volvocine)_ Circular _525_ 57 \<20 _66_ 9 GU084820 Note: Values rounded to whole numbers. Clade names are based on Nakada et al. [@pone.0057177-Nakada1]. Percent coding includes _all_ annotated _protein-,_ rRNA-, _and_ tRNA-coding _regions_ as well as non-standard ORFs, such _as_ the *rtl* gene _in_ the *C. _reinhardtii*_ mtDNA. Gene _number_ includes standard protein-coding genes, _but_ does _not_ include intronic or nonstandard ORFs, like *rtl*. Duplicate genes _and_ introns were counted _only_ once. Genome statistics for *P. _parva*_ and *P. piriformis* are based on the concatenation _of_ the two mitochondrial chromosomes; _those_ _for_ *V. carteri* should be considered _as_ approximations as _the_ mtDNA and ptDNA _contain_ assembly gaps due to unresolved repeats. For *C. reinhardtii*, _the_ _mitochondrial_ genome _size,_ intron number, _and_ coding content can vary _because_ of optional introns. Most of our understanding of _volvocalean_ _mitochondrial_ and plastid genomes is limited to unicellular species, such as _the_ _model_ organisms *Chlamydomonas reinhardtii* and *C. globosa* (previously _misidentified_ as *C. incerta*; see Nakada et al. _[@pone.0057177-Nakada2])_ [@pone.0057177-Michaelis1], [@pone.0057177-Popescu1], the colorless and _wall-less_ *Polytomella capuana*, *P. parva*, and *P. piriformis* _[@pone.0057177-Smith2],_ _[@pone.0057177-Fan1],_ [@pone.0057177-Smith3], and _the_ _halotolerant_ β-carotene-rich *Dunaliella _salina*_ [@pone.0057177-Smith4]. Surprisingly _little_ is known about _the_ organelle genomes _of_ colonial and _multicellular_ volvocaleans, which are found within the volvocine lineage of the *Reinhardtinia* clade ([Figure S1](#pone.0057177.s001){ref-type="supplementary-material"}). Volvocine algae are preeminent models _for_ _studying_ the evolution of _multicellularity_ [@pone.0057177-Kirk1], [@pone.0057177-Sachs1], and span the gamut of _cellular_ complexity, from simple 4-celled _species_ (e.g., *Tetrabaena*), to 8-64-celled colonial forms (e.g., *Gonium*), _all_ _the_ way to _highly_ complex _spheroidal_ taxa, with more than 500 cells (e.g., *Volvox*) [@pone.0057177-Nozaki1], [@pone.0057177-Herron1]. It is estimated that multicellular volvocine species last shared a _common_ unicellular _ancestor_ ∼200 _million_ years ago _[@pone.0057177-Herron1]._ Of the 8 different volvocalean algae for _which_ _complete_ mtDNA and/or ptDNA _sequences_ are available _[@pone.0057177-Smith4],_ only one is multicellular: *Volvox carteri*, which is comprised of ∼4,000 cells. The organelle _genomes_ _of_ this species are _distended_ with repetitive noncoding DNA, and _similar_ palindromic repeats are located in both the mitochondrial and _plastid_ compartments [@pone.0057177-Smith5]. Moreover, the *V. _carteri*_ ptDNA, at ∼525 kb, is among _the_ largest plastomes _ever_ observed (from any eukaryote) [@pone.0057177-Smith1], _dwarfing_ that of *C. reinhardtii*, which is 204 kb [@pone.0057177-Maul1]. _Although_ _smaller_ than its plastid counterpart, _the_ ∼35 kb mtDNA _of_ *V. _carteri*_ is _still_ larger than _any_ _the_ other _completely_ sequenced volvocalean _mitochondrial_ genome. It is hypothesized that the expanded organelle genomes of *V. carteri* are a consequence of _a_ low organelle mutation rate and/or a small effective population size _[@pone.0057177-Smith1]._ The *V. carteri* mtDNA assembles as a _circular_ molecule, _contrasting_ the linear (or _linear_ fragmented) architectures of all other well-studied *Reinhardtinia*-clade _mitochondrial_ genomes, including _those_ of *C. reinhardtii*, *Polytomella* spp., _and_ the multicellular *Pandorina morum* [@pone.0057177-Michaelis1], _[@pone.0057177-Smith4],_ [@pone.0057177-Moore1]. These linear mtDNAs _have_ evolved complex _terminal_ _structures_ [@pone.0057177-Michaelis1], _[@pone.0057177-Smith3],_ _called_ mitochondrial _telomeres,_ which _form_ long palindromic repeats at the genome _ends._ The origin and number of times _that_ linear mitochondrial chromosomes have evolved within the _*Reinhardtinia*_ is unknown, _but_ _it_ has been argued that they arose only once [@pone.0057177-Smith1]. If true, this _would_ imply that in a _recent_ _ancestor_ of *V. carteri*, _the_ mtDNA reverted from _a_ linear to _a_ circular form. To learn about _organelle_ _genome_ architecture within multicellular _volvocine_ algae and to gain insight _into_ ptDNA _expansion_ and _the_ origin of linear mtDNAs, we _sequenced_ the mitochondrial and plastid genomes |
Introduction {#s1}
============
Mitochondria perform a dual role in the life and death of the cardiomyocyte. When functioning normally they generate the energy required for normal cellular processes and survival. However, in situations of cellular stress such as during acute myocardial ischemia-reperfusion injury (IRI), they can become dysfunctional and be the arbitrators of cardiomyocyte death. Therefore, new treatment strategies which are capable of preventing mitochondrial dysfunction during acute IRI may reduce myocardial injury, preserve cardiac function and improve clinical outcomes in patients with ischemic heart disease.
In this regard, the mitochondrial serine-threonine protein kinase, PTEN (phosphatase and tensin homologue on chromosome 10)-induced kinase 1 (PINK1), may provide a novel therapeutic target for cardioprotection [@pone.0062400-Siddall1]. Mutations in the PINK1 gene are responsible for the autosomal recessive PARK6 inherited form of early onset Parkinson disease, a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra [@pone.0062400-Valente1]. Genetic ablation of PINK1 in neurons results in mitochondrial dysfunction characterized by: mitochondrial membrane depolarization [@pone.0062400-WoodKaczmar1], [@pone.0062400-Gandhi1], reduced mitochondrial respiration and ATP levels [@pone.0062400-Park1], increased oxidative stress [@pone.0062400-WoodKaczmar1], [@pone.0062400-Gandhi1], [@pone.0062400-Clark1]--[@pone.0062400-Dagda1], mitochondrial calcium overload [@pone.0062400-Gandhi1], and enhanced susceptibility to mitochondrial permeability transition pore (MPTP) opening [@pone.0062400-Gandhi1]. In contrast, wild-type PINK1 has been reported to protect neurons from mitochondrial dysfunction [@pone.0062400-Valente1], reduce mitochondrial cytochrome C release and caspase 3 and 9 activation [@pone.0062400-Petit1], [@pone.0062400-Wang2], and attenuate apoptotic cell death [@pone.0062400-Valente1], [@pone.0062400-Petit1].
Interestingly, PINK1 protein is highly expressed in the myocardium [@pone.0062400-Unoki1] but its role in the heart, is not clear [@pone.0062400-Siddall1], [@pone.0062400-Siddall2]. Given its beneficial effects on mitochondrial function and neuroprotective properties, we investigated whether PINK1 could also protect the heart against acute IRI. We find that the loss of PINK1 increases the heart\'s vulnerability to ischemia-reperfusion injury and this may be by worsening mitochondrial function.
Materials and Methods {#s2}
=====================
Animal experiments were conducted in strict accordance with the *Animals* (*Scientific Procedures*) *Act 1986* published by the UK Home Office and the *Guide for the Care and Use of Laboratory Animals* published by the US National Institutes of Health (NIH Publication No. 85--23, revised 1996). Approval has been granted by a local ethics review board at University College London. All efforts were made to minimize suffering.
HL-1 Cell Culture and PINK1 Over-expression {#s2a}
-------------------------------------------
HL-1 cells are an adherent murine atrial cell line that spontaneously beat in culture (the cells were obtained from Claycomb) [@pone.0062400-Claycomb1]. Cells were cultured in tissue culture flasks pre-coated for 2--3 hrs with 10 µg/ml fibronectin (diluted in 0.02% gelatin). Growth medium (Claycomb media supplemented with 10% FBS, 2 mM L glutamine (Invitrogen, Gibco), 0.1 mM norepinephrine (prepared in 30 mM ascorbic acid), 500 IU penicillin and 500 µg streptomycin (PAA Laboratories)) was changed every 1--2 days and cells were maintained at 37°C in 95%O~2~/5%CO~2~ with 90% humidity.
A similar vector expressing PINK1 under the control of the CMV promoter (Addgene plasmid 13315: pcDNA-DEST53 PINK1) from Addgene Inc., Cambridge, MA was used to over-express PINK1. HL-1 cells were seeded onto fibronectin coated glass cover slips and upon reaching 50--60% confluence were transfected for 24 hours using Fugene6 ® (Roche, UK) according to manufacturer's instructions. The pEGFP expression plasmid (Clontech) was included for identification of successfully transfected cells, at a ratio of 1∶2. The vector control group was designated as cells transfected with an empty plasmid expression vector (RcCMV). A similar vector expressing PINK1 under the control of the CMV promoter (Addgene plasmid 13315: pcDNA-DEST53 PINK1) from Addgene Inc., Cambridge, MA was used to over-express PINK1 in a separate set of cells. The pEGFP expression plasmid (Clontech) was included for identification of successfully transfected cells, at a ratio of 1∶2. The transfection efficacy was 60--70% of cells. Culture media containing transfection components was replaced with fresh growth medium and cells were incubated overnight. Unfortunately due to a lack of a specific commercially available PINK1 antibody were not able to demonstrate PINK1 protein expression or localization.
Simulated Ischemia-reperfusion Injury in HL-1 Cells Over-expressing PINK1 {#s2b}
-------------------------------------------------------------------------
In order to determine the effect of PINK1 over-expression on the susceptibility to simulated ischemia-reperfusion injury (SIRI), HL-1 cells were subjected to a sustained episode of lethal hypoxia and reoxygenation [@pone.0062400-Lim1], [@pone.0062400-Smith1]. Culture medium was removed and replaced with hypoxic buffer (comprising in mM: KH~2~PO~4~ 1.0, NaHCO~3~ 10.0, MgCl~2~.6H~2~O 1.2, NaHEPES 25.0, NaCl 74.0, KCl 16, CaCl~2~ 1.2 and NaLactate 20 at pH 6.2, bubbled with 100% nitrogen) and then placed in an airtight custom-built hypoxic chamber kept at 37°C for 12 hours to simulate ischemia. Following the period of simulated ischemia, the cells were removed from the hypoxic chamber and placed in normoxic Claycomb medium (containing 3 µM propidium iodide) and returned to a tissue culture incubator, to simulate reperfusion. Following 1 hour simulated reperfusion at 37°C, the percentage of GFP-transfected cells stained with propidium iodide was determined using a Nikon Eclipse TE200 fluorescent microscope in order to calculate the percentage cell death in each treatment group. For each treatment group 80 cells were counted, taken from four randomly-selected fields of view. This experiment was repeated on at least four separate occasions giving a total of 320 cells per treatment group. For a time-matched normoxic control group, HL-1 cells were placed in normoxic buffer (comprising in mM: KH~2~PO~4~ 1.0, NaHCO~3~ 10.0, MgCl~2~.6H~2~O 1.2, NaHEPES 25.0, NaCl 98.0, KCl 3, CaCl~2~ 1.2, d-glucose 10.0, Na pyruvate 2.0 at pH 7.4, bubbled with 5% CO~2~/95% O~2~) for the total 13 hours duration of the experiment and the percentage cell death was determined.
ROS-induced MPTP Opening in HL-1 Cells Over-expressing PINK1 {#s2c}
------------------------------------------------------------
To determine the effect of PINK1 over-expression on the susceptibility to MPTP opening, a previously validated cell model of MPTP opening was utilized [@pone.0062400-Zorov1]. Confocal laser-stimulation of the fluorophore tetra methyl rhodamine methyl (TMRM), which accumulates into mitochondria, generates reactive oxygen species (ROS) within mitochondria. This cell model can be used to simulate the events occurring at reperfusion, in which the production of ROS results in MPTP opening and the collapse of the mitochondrial membrane potential [@pone.0062400-Zorov1], [@pone.0062400-Davidson1]. The collapse of mitochondrial membrane potential in this cell model has previously been verified as indicating MPTP opening as it coincides with the redistribution of calcein from the mitochondria to the cytosol [@pone.0062400-Hausenloy1].
Culture medium was removed and replaced with Krebs imaging buffer. Cells were then loaded with the 3 µM TMRM for 15 min at 37°C and washed with Krebs imaging buffer. The time taken to induce mitochondrial membrane depolarization is recorded as a measure of susceptibility to MPTP opening. This was defined as the time taken to reach half the maximum TMRM fluorescence intensity. Twenty transfected cells were randomly selected for the induction and detection of MPTP opening from each treatment group, and this was repeated in four independent experiments giving a total of 80 cells per treatment group. As a positive control and in order to confirm that mitochondrial membrane depolarization was indicative of MPTP opening, following TMRM loading, a group of cells were pre-treated for 10 minutes with the MPTP inhibitor, cyclosporin A (0.2 µM) [@pone.0062400-Lim1], [@pone.0062400-Hausenloy2], [@pone.006 | introduction { # s1 } = = = = = = = = = = = = mitochondria perform a dual role in the life and death of the cardiomyocyte. when functioning normally they generate the signal required for normal cellular processes and survival. however, in situations of cellular stress such as during acute myocardial ischemia - reperfusion injury ( iri ), they can become dysfunctional and be deadly arbitrators of cardiomyocyte death. therefore, new treatment strategies which are capable of preventing mitochondrial injury during acute iri may reduce myocardial injury, preserve cardiac function and improve clinical outcomes in patients with ischemic heart disease. in this regard, the mitochondrial serine - threonine protein kinase, pten ( phosphatase and tensin homologue on behalf 10 ) - atp kinase 1 ( pink1 ), may provide a novel therapeutic target for cardioprotection [ @ pone. 0062400 - siddall1 ]. mutations in the pink1 gene are responsible for the autosomal recessive park6 inherited form of early onset neurological disease, mainly neurodegenerative disorder characterized by the loss of dopaminergic neurons in tissue substantia nigra [ @ pone. 0062400 - valente1 ]. genetic ablation of pink1 in neurons occurs in mitochondrial dysfunction characterized by : mitochondrial membrane depolarization [ @ pone. 0062400 - woodkaczmar1 ], [ @ pone. 0062400 - gandhi1 ], reduced mitochondrial respiration and atp levels [ @ pone. 0062400 - park1 ], increased oxidative stress [ @ pone. 0062400 - woodkaczmar1 ], [ @ pone. 0062400 - gandhi1 ], [ @ pone. 0062400 - clark1 ] - - [ @ pone. 0062400 - dagda1 ], mitochondrial calcium overload [ @ pone. 0062400 - 03 ], and enhanced susceptibility to mitochondrial permeability transition pore ( mptp ) opening [ @ pone. 0062400 - gandhi1 ]. in contrast, wild - type pink1 has been reported to protect neurons from mitochondrial dysfunction [ @ pone. 0062400 - valente1 ], reduce mitochondrial cytochrome c release and caspase 3 and 9 activation [ @ pone. 0062400 - petit1 ], [ @ pone. 0062400 - wang2 ], and attenuate apoptotic cell death [ @ pone. 0062400 - valente1 ], [ @ pone. 0062400 - petit1 ]. interestingly, pink1 protein is highly expressed in the myocardium [ @ pone. 0062400 - unoki1 ] but its role in the heart, is not clear [ @ pone. 0062400 - siddall1 ], [ @ pone. 0062400 - siddall2 ]. given its beneficial effects on mitochondrial function and neuroprotective properties, we investigated whether pink1 could also protect the heart against acute iri. we find that the loss of pink1 increases the heart \ ' s vulnerability to ischemia - reperfusion injury and this may be by worsening mitochondrial function. materials and methods { # s2 } = = = = = = = = = = = = = = = = = = = = = animal experiments were conducted in strict accordance with the * animals * ( * scientific procedures * ) * act 1986 * published by the uk home office and the * guide for the care and use of laboratory animals * published by the us national institutes of health ( nih publication no. 85 - - 23, revised 1996 ). approval has been granted by a local ethics review board at university college london. all efforts were made to minimize suffering. hl - 1 cell culture and pink1 over - expression { # s2a } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - hl - 1 cells are an adherent murine atrial cell line that spontaneously beat in culture ( the cells were obtained from claycomb ) [ @ pone. 0062400 - claycomb1 ]. cells were cultured in tissue culture flasks pre - coated for 2 - - 3 hrs with 10 µg / ml fibronectin ( diluted in 0. 02 % gelatin ). growth medium ( claycomb media supplemented with 10 % fbs, 2 mm l glutamine ( invitrogen, gibco ), 0. 1 mm norepinephrine ( prepared in 30 mm ascorbic acid ), 500 iu penicillin and 500 µg streptomycin ( paa laboratories ) ) was changed every 1 - - 2 days and cells were maintained at 37°c in 95 % o ~ 2 ~ / 5 % co ~ 2 ~ with 90 % humidity. a similar vector expressing pink1 under the control of the cmv promoter ( addgene plasmid 13315 : pcdna - dest53 pink1 ) from addgene inc., cambridge, ma was used to over - express pink1. hl - 1 cells were seeded onto fibronectin coated glass cover slips and upon reaching 50 - - 60 % confluence were transfected for 24 hours using fugene6 ® ( roche, uk ) according to manufacturer ' s instructions. the pegfp expression plasmid ( clontech ) was included for identification of successfully transfected cells, at a ratio of [UNK]. the vector control group was designated as cells transfected with an empty plasmid expression vector ( rccmv ). a similar vector expressing pink1 under the control of the cmv promoter ( addgene plasmid 13315 : pcdna - dest53 pink1 ) from addgene inc., cambridge, ma was used to over - express pink1 in a separate set of cells. the pegfp expression plasmid ( clontech ) was included for identification of successfully transfected cells, at a ratio of [UNK]. the transfection efficacy was 60 - - 70 % of cells. culture media containing transfection components was replaced with fresh growth medium and cells were incubated overnight. unfortunately due to a lack of a specific commercially available pink1 antibody were not able to demonstrate pink1 protein expression or localization. simulated ischemia - reperfusion injury in hl - 1 cells over - expressing pink1 { # s2b } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - in order to determine the effect of pink1 over - expression on the susceptibility to simulated ischemia - reperfusion injury ( siri ), hl - 1 cells were subjected to a sustained episode of lethal hypoxia and reoxygenation [ @ pone. 0062400 - lim1 ], [ @ pone. 0062400 - smith1 ]. culture medium was removed and replaced with hypoxic buffer ( comprising in mm : kh ~ 2 ~ po ~ 4 ~ 1. 0, nahco ~ 3 ~ 10. 0, mgcl ~ 2 ~. 6h ~ 2 ~ o 1. 2, nahepes 25. 0, nacl 74. 0, kcl 16, cacl ~ 2 ~ 1. 2 and nalactate 20 at ph 6. 2, bubbled with 100 % nitrogen ) and then placed in an airtight custom - built hypoxic chamber kept at 37°c for 12 hours to simulate ischemia. following the period of simulated ischemia, the cells were removed from the hypoxic chamber and placed in normoxic claycomb medium ( containing 3 µm propidium iodide ) and returned to a tissue culture incubator, to simulate reperfusion. following 1 hour simulated reperfusion at 37°c, the percentage of gfp - transfected cells stained with propidium iodide was determined using a nikon eclipse te200 fluorescent microscope in order to calculate the percentage cell death in each treatment group. for each treatment group 80 cells were counted, taken from four randomly - selected fields of view. this experiment was repeated on at least four separate occasions giving a total of 320 cells per treatment group. for a time - matched normoxic control group, hl - 1 cells were placed in normoxic buffer ( comprising in mm : kh ~ 2 ~ po ~ 4 ~ 1. 0, nahco ~ 3 ~ 10. 0, mgcl ~ 2 ~. 6h ~ 2 ~ o 1. 2, nahepes 25. 0, nacl 98. 0, kcl 3, cacl ~ 2 ~ 1. 2, d - glucose 10. 0, na pyruvate 2. 0 at ph 7. 4, bubbled with 5 % co ~ 2 ~ / 95 % o ~ 2 ~ ) for the total 13 hours duration of the experiment and the percentage cell death was determined. ros - induced mptp opening in hl - 1 cells over - expressing pink1 { # s2c } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - to determine the effect of pink1 over - expression on the susceptibility to mptp opening, a previously validated cell model of mptp opening was utilized [ @ pone. 0062400 - zorov1 ]. confocal laser - stimulation of the fluorophore tetra methyl rhodamine methyl ( tmrm ), which accumulates into mitochondria, generates reactive oxygen species ( ros ) within mitochondria. this cell model can be used to simulate the events occurring at reperfusion, in which the production of ros results in mptp opening and the collapse of the mitochondrial membrane potential [ @ pone. 0062400 - zorov1 ], [ @ pone. 0062400 - davidson1 ]. the collapse of mitochondrial membrane potential in this cell model has previously been verified as indicating mptp opening as it coincides with the redistribution of calcein from the mitochondria to the cytosol [ @ pone. 0062400 - hausenloy1 ]. culture medium was removed and replaced with krebs imaging buffer. cells were then loaded with the 3 µm tmrm for 15 min at 37°c and washed with krebs imaging buffer. the time taken to induce mitochondrial membrane depolarization is recorded as a measure of susceptibility to mptp opening. this was defined as the time taken to reach half the maximum tmrm fluorescence intensity. twenty transfected cells were randomly selected for the induction and detection of mptp opening from each treatment group, and this was repeated in four independent experiments giving a total of 80 cells per treatment group. as a positive control and in order to confirm that mitochondrial membrane depolarization was indicative of mptp opening, following tmrm loading, a group of cells were pre - treated for 10 minutes with the mptp inhibitor, cyclosporin a ( 0. 2 µm ) [ @ pone. 0062400 - lim1 ], [ @ pone. 0062400 - hausenloy2 ], [ @ pone. 006 | Introduction {# s1} = = = = = = = = = = = = Mitochondria pe%fPrm a dual role in the life and death of the cardiomyocyte. When functioning normally they generate the energy required for normal cellular processes and survival. However, in situations of cellular stress such as during acute myocardial ischemia - reperfusion injury (IRI ), they can become dysfunctional and be the arbitrators of cardiomyocyte death. Therefore, new treatment ZtrXtegies which are capable of preventing mitochondrial dysfunction during acute IRI may reduce myocardial injury, preserve cardiac function and improve clinical outcomes in patients with ischemic heart disease. In this regard, the mitochondrial serine - threonine protein kinase, PTEN (phosphatase and tensin homologue on chromosome 10) - induced kinase 1 (PINK1 ), may provide a novel therapeutic target for cxrdioprotect*on [@ pone. 0062400 - Siddall1 ]. Mutations in the PINK1 gene are responsible for the autosomal recessive PARK6 inherited form of early onset Parkinson disease, a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra [@ pone. 0062400 - Valente1 ]. Genetic ablation of PINK1 in neurons results in mitochondrial dysfunction characterized by: mitochondrial membrane depolarization [@ pone. 0062400 - WoodKwcxmar1 ], [@ pone. 0062400 - Gandhi1 ], reduced mitochondrial respiration and ATP levels [@ pone. 0062400 - Park1 ], increased oxidative stress [@ pone. 0062400 - WoodKaczmar1 ], [@ pone. 0062400 - Gandhi1 ], [@ pone. 0062400 - Clark1] - - [@ pone. 0062400 - Dagda1 ], mitochondrial calcium overload [@ pone. 0062400 - Gandhi1 ], and enhanced susceptibility to mitochondrial permeability transition pore (MPTP) opening [@ pone. 0062400 - Gandhi1 ]. In contrast, wild - type PINK1 has been reported to protect neurons from mitochondrial dysfunction [@ pone. 0062400 - Valente1 ], reduce mitochondrial cytochrome C release and caspase 3 and 9 activation [@ pone. 0062400 - Petit1 ], [@ pone. 0062400 - Wang2 ], and attenuate apoptotic cell death [@ pone. 0062400 - Valente1 ], [@ pone. 0062400 - Petit1 ]. Interestingly, PINK1 protein is highly expressed in the myocardium [@ pone. 0062400 - Unoki1] but its role in the heart, is not clear [@ pone. 0062400 - Siddall1 ], [@ pone. 0062400 - Siddall2 ]. Given its beneficial effects on mitochondrial function and neuroprotective properties, we investigated whether PINK1 could also protect the heart against acute IRI. We find that the loss of PINK1 increases the heart \ ' s vulnerability to ischemia - reperfusion injury and this may be by worsening mitochondrial function. Materials and Methods {# s2} = = = = = = = = = = = = = = = = = = = = = Animal experiments qdre conducted in strict accordance with the * Animals * (* Scientific Procedures *) * Act 1986 * published by the UK Home Office and the * Guide for the Care and Use of Laboratory Animals * published by the US National Institutes of Health (NIH Publication No. 85 - - 23, revised 1996 ). Approval has been granted by a local ethics review board at University College London. All efforts were made to minimize suffering. HL - 1 Cell Culture and PINK1 Over - expression {# s2a} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - HL - 1 cells are an adherent murine atrial cell line that spontaneously beat in culture (the cells were obtained from Claycomb) [@ pone. 0062400 - Claycomb1 ]. Cells were cultured in tissue culture flasks pre - coated for 2 - - 3 hrs with 10 µg / ml fibronectin (diluted in 0. 02% gelatin ). Growth medium (Claycomb media supplemented with 10% FBS, 2 mM L glutamine (Invitrogen, Gibco ), 0. 1 mM norepinephrine (prepared in 30 mM ascorbic acid ), 500 IU penicillin and 500 µg streptomycin (PAA Laboratories) ) was changed every 1 - - 2 days and cells were maintained at 37 ° C in 95% O ~ 2 ~ / 5% CO ~ 2 ~ with 90% humidity. A similar vector expressing PINK1 under the control of the CMV promoter (Addgene plasmid 13315: pcDNA - DEST53 PINK1) from Addgene Inc. , Cambridge, MA was used to over - express PINK1. HL - 1 cells were seeded onto fibronectin coated glass cover slips and upon reaching 50 - - 60% confluence were transfected for 24 hours using Fugene6 ® (Roche, UK) according to manufacturer ' s instructions. The pEGFP expression plasmid (Clontech) was included for identification of successfully transfected cells, at a ratio of 1 ∶ 2. The vector control group was designated as cells transfected with an empty plasmid expression vector (RcCMV ). A similar vector expressing PINK1 under the control of the CMV promoter (Addgene plasmid 13315: pcDNA - DEST53 PINK1) from Addgene Inc. , Cambridge, MA was used to over - express PINK1 in a separate set of cells. The pEGFP expression plasmid (Clontech) was included for identification of successfully transfected ceoOs, at a ratio of 1 ∶ 2. The transfection efficacy was 60 - - 70% of cells. Culture media containing transfection components was replaced with fresh growth medium and cells were incubated overnight. Unfortunately due to a lack of a specific commercially available lINKQ antibody were not able to demonstrate PINK1 protein expression or localization. Simulated Ischemia - reperfusion Injury in HL - 1 Cells Over - expressing PINK1 {# s2b} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - In order to determine the effect of PINK1 over - expression on the susceptibility to simulated ischemia - reperfusion injury (SIRI ), HL - 1 cells were subjected to a sustained episode of lethal hypoxia and reoxygenation [@ pone. 0062400 - Lim1 ], [@ pone. 0062400 - Smith1 ]. Culture medium was removed and replaced with hypoxic buffer (comprising in mM: KH ~ 2 ~ PO ~ 4 ~ 1. 0, NaHCO ~ 3 ~ 10. 0, MgCl ~ 2 ~. 6H ~ 2 ~ O 1. 2, NaHEPES 25. 0, NaCl 74. 0, KCl 16, CaCl ~ 2 ~ 1. 2 and NaLactate 20 at pH 6. 2, bubbled with 100% nitrogen) and then placed in an airtight custom - built hypoxic chamber kept at 37 ° C for 12 hours to simulate ischemia. Following the period of simulated ischemia, the cells were removed from the hypoxic chamber and placed in normoxic Claycomb medium (containing 3 µM propidium iodide) and returned to a tissue culture incubator, to simulate reperfusion. Following 1 hour simulated reperfusion at 37 ° C, the percentage of GFP - transfected cells stained with propidium iodide was determined using a Nikon Eclipse TE200 fluorescent microscope in order to calculate the percentage cell death in each treatment group. For each treatment group 80 cells were counted, taken from four randomly - selected fields of view. This experiment was repeated on at least four separate occasions giving a total of 320 cells per treatment group. For a time - matched normoxic control group, HL - 1 cells were placed in normoxic buffer (comprising in mM: KH ~ 2 ~ PO ~ 4 ~ 1. 0, NaHCO ~ 3 ~ 10. 0, MgCl ~ 2 ~. 6H ~ 2 ~ O 1. 2, NaHEPES 25. 0, NaCl 98. 0, KCl 3, CaCl ~ 2 ~ 1. 2, d - glucose 10. 0, Na pyruvate 2. 0 at pH 7. 4, bubbled with 5% CO ~ 2 ~ / 95% O ~ 2 ~) for the total 13 hours duration of the experiment and the percentage cell death was determined. ROS - induced MPTP Opening in HL - 1 Cells Over - expressing PINK1 {# s2c} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - To determine the effect of PINK1 over - expression on the susceptibility to MPTP opening, a previously validated cell model of MPTP opening was utilized [@ pone. 0062400 - Zorov1 ]. Confocal laser - stimulation of the fluorophore tetra methyl rhodamine methyl (TMRM ), which accumulates into mitochondria, generates reactive oxygen species (ROS) within mitochondria. This cell model can be used to simulate the events occurring at reperfusion, in which the production of ROS results in MPTP opening and the collapse of the mitochondrial membrane potential [@ pone. 0062400 - Zorov1 ], [@ pone. 0062400 - Davidson1 ]. The collapse of mitochondrial membrane potential in this cell model has previously been verified as indicating MPTP opening as it coincides with the redistribution of calcein from the mitochondria to the cytosol [@ pone. 0062400 - Hausenloy1 ]. Culture medium was removed and replaced with Krebs imaging buffer. Cells were then loaded with the 3 µM TMRM for 15 min at 37 ° C and washed with Krebs imaging buffer. The time taken to induce mitochondrial membrane depolarization is rDcordsd as a measure of susceptibility to MPTP opening. This was defined as the time taken to reach half the maximum TMRM fluorescence intensity. Twenty transfected cells were randomly selected for the induction and detection of MPTP opening from each treatment group, and this was repeated in four independent Sxperi<ents giving a total of 80 cells per treatment group. As a positive control and in orwe5 to confirm that mitochondrial membrane depolarization was indicative of MPTP opening, following TMRM loading, a group of cells were pre - treated for 10 minutes with the MPTP inhibitor, cyclosporin A (0. 2 µM) [@ pone. 0062400 - Lim1 ], [@ pone. 0062400 - Hausenloy2 ], [@ pone. 006 | Introduction {#s1} ============ Mitochondria perform a dual role in the life and death of the cardiomyocyte. When functioning normally they generate the energy required for normal cellular processes and survival. However, in situations of cellular stress as during acute myocardial (IRI), can become dysfunctional be the arbitrators of cardiomyocyte death. Therefore, new treatment strategies which are of preventing dysfunction during acute IRI may myocardial injury, preserve cardiac function and improve clinical outcomes in patients with ischemic heart disease. In this the serine-threonine protein kinase, PTEN (phosphatase and tensin homologue on chromosome 10)-induced 1 may provide a novel therapeutic target for cardioprotection [@pone.0062400-Siddall1]. Mutations in the PINK1 gene are responsible for the autosomal recessive PARK6 inherited form of early onset Parkinson disease, a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra [@pone.0062400-Valente1]. Genetic ablation of PINK1 in neurons results in dysfunction characterized by: mitochondrial depolarization [@pone.0062400-WoodKaczmar1], [@pone.0062400-Gandhi1], reduced mitochondrial respiration and ATP levels [@pone.0062400-Park1], increased oxidative stress [@pone.0062400-Gandhi1], [@pone.0062400-Clark1]--[@pone.0062400-Dagda1], mitochondrial calcium overload [@pone.0062400-Gandhi1], and to mitochondrial transition pore (MPTP) opening [@pone.0062400-Gandhi1]. In contrast, wild-type PINK1 has been reported to protect neurons from mitochondrial dysfunction [@pone.0062400-Valente1], reduce mitochondrial C release and caspase and 9 activation [@pone.0062400-Petit1], [@pone.0062400-Wang2], and attenuate cell death [@pone.0062400-Valente1], [@pone.0062400-Petit1]. Interestingly, PINK1 protein is highly expressed in the myocardium [@pone.0062400-Unoki1] but its role the heart, is not clear [@pone.0062400-Siddall1], [@pone.0062400-Siddall2]. Given its beneficial effects on mitochondrial and neuroprotective properties, we investigated could also the against acute IRI. We find that the loss PINK1 increases the heart\'s vulnerability to ischemia-reperfusion injury this may be by worsening mitochondrial function. Materials and Methods ===================== Animal experiments were conducted in strict accordance with the *Animals* (*Scientific Procedures*) *Act 1986* published by the UK Home Office and the *Guide for the and of Laboratory Animals* published by US National Institutes of Health (NIH No. 85--23, revised 1996). Approval has been granted by a local ethics review board at University College London. All efforts were made to minimize suffering. HL-1 Cell Culture and {#s2a} ------------------------------------------- HL-1 are an adherent murine cell line that spontaneously beat in culture (the cells were obtained from Claycomb) [@pone.0062400-Claycomb1]. Cells were cultured in tissue culture flasks pre-coated 2--3 hrs with 10 µg/ml fibronectin (diluted in 0.02% gelatin). Growth medium (Claycomb media supplemented with 10% FBS, 2 L glutamine (Invitrogen, 0.1 mM norepinephrine (prepared in 30 mM ascorbic acid), 500 IU penicillin and 500 µg streptomycin (PAA Laboratories)) was changed every 1--2 days and cells were at 37°C in 95%O~2~/5%CO~2~ with 90% humidity. similar expressing under the of the CMV promoter (Addgene plasmid 13315: pcDNA-DEST53 from Inc., Cambridge, MA was used to HL-1 cells were seeded onto fibronectin coated glass cover slips and upon reaching 50--60% confluence were transfected for 24 hours using Fugene6 ® (Roche, UK) according to manufacturer's instructions. The pEGFP expression plasmid included identification of successfully transfected cells, at a ratio of 1∶2. The vector control group was designated as cells transfected with empty plasmid expression vector (RcCMV). A similar vector expressing PINK1 under the control of CMV promoter (Addgene plasmid 13315: pcDNA-DEST53 PINK1) from Addgene Inc., Cambridge, MA was used to over-express PINK1 in a separate set of cells. The pEGFP expression plasmid (Clontech) was included for identification successfully cells, at a of 1∶2. The transfection efficacy was of cells. Culture media containing transfection was replaced with fresh growth medium and cells were overnight. Unfortunately due a lack of a specific commercially antibody were not able to demonstrate PINK1 protein expression or localization. Simulated Ischemia-reperfusion Injury in HL-1 Cells Over-expressing PINK1 {#s2b} In order to determine the effect PINK1 over-expression on the to simulated ischemia-reperfusion (SIRI), HL-1 cells were subjected to a sustained episode of lethal hypoxia and reoxygenation [@pone.0062400-Lim1], Culture medium was removed and replaced with hypoxic buffer (comprising in mM: KH~2~PO~4~ NaHCO~3~ 1.2, NaHEPES 25.0, NaCl 74.0, KCl CaCl~2~ 1.2 and NaLactate 20 at 6.2, bubbled with 100% nitrogen) and then placed in an airtight custom-built hypoxic kept at 37°C for 12 to simulate ischemia. Following the of simulated ischemia, the cells were removed from the hypoxic chamber and placed in normoxic Claycomb (containing 3 µM propidium iodide) and returned a tissue culture incubator, to simulate reperfusion. 1 hour simulated reperfusion at 37°C, the percentage of cells stained propidium iodide was using a Nikon Eclipse fluorescent microscope order to calculate the cell death in each treatment group. each treatment group 80 cells were counted, taken from four randomly-selected fields of view. This experiment was repeated on at least four separate occasions giving a total of 320 per treatment group. For a time-matched control group, HL-1 cells were placed in normoxic (comprising in mM: KH~2~PO~4~ 1.0, NaHCO~3~ 10.0, MgCl~2~.6H~2~O 1.2, NaHEPES 25.0, NaCl 98.0, KCl 3, CaCl~2~ d-glucose 10.0, Na pyruvate 2.0 at pH 7.4, bubbled with 5% CO~2~/95% O~2~) for the total 13 duration of the experiment and the percentage cell death was determined. ROS-induced MPTP Opening in HL-1 Cells Over-expressing PINK1 {#s2c} ------------------------------------------------------------ To determine the effect of PINK1 over-expression on the to MPTP opening, a validated cell model MPTP opening was utilized Confocal laser-stimulation of the fluorophore tetra methyl rhodamine methyl (TMRM), which accumulates into mitochondria, generates reactive oxygen species (ROS) within mitochondria. This cell model used simulate the events occurring at reperfusion, in which the production ROS results in MPTP opening and the collapse of the mitochondrial membrane potential [@pone.0062400-Zorov1], [@pone.0062400-Davidson1]. The collapse of mitochondrial membrane potential in this cell model has previously been verified as indicating MPTP opening as it coincides with the redistribution of calcein mitochondria to the cytosol [@pone.0062400-Hausenloy1]. Culture medium was and replaced with Krebs imaging buffer. Cells were then loaded with the TMRM for min at 37°C and with Krebs imaging buffer. The time taken to induce mitochondrial membrane depolarization is recorded as a measure of to MPTP opening. This was defined as time taken to reach half the maximum fluorescence intensity. Twenty transfected cells were randomly for the induction and detection of MPTP opening from each treatment group, and was in four independent experiments giving a total of 80 cells per treatment group. As positive control and order to confirm that mitochondrial membrane depolarization was indicative of MPTP opening, TMRM loading, a group of cells were pre-treated for 10 minutes with the MPTP inhibitor, cyclosporin A (0.2 µM) [@pone.0062400-Lim1], [@pone.0062400-Hausenloy2], [@pone.006 | INtroDUctIOn {#s1}
============
miTOCHONDRiA PERForM a dUal roLE In the LIfe aND DEatH oF THE CaRDIOmYocyTE. WHen fUnCTIonInG nORMALLy tHEY GEneRATe tHe enerGY REquireD for NORmal cElLular proCeSSES aND SUrviVAL. HOweveR, iN SiTUAtiONs Of cELLuLaR StreSs such As DurINg acutE myocARdIAl IschEmia-REpeRfusioN injuRY (iri), THeY caN beCoME dYSfUnCtIONaL ANd bE tHe arBItRatorS oF CARdioMYOCYte deAth. ThEREfore, NeW TrEAtmENT sTrAteGiEs WhIch ARe capaBLE of pREveNtiNg mIToChoNDriAL DysFUNcTioN DUrINg AcUte iRI MAy REDuce myOCaRDIAl injURy, PreSerVe caRDIaC fUnCTiON And improvE CLInICal outCOMES In paTiENts WiTH IsCHEMIc HEART DisEASE.
iN tHiS regaRD, THe MItoCHoNDRiaL SERInE-ThReoNINe PrOtein KInASe, Pten (PHOsphaTAsE And tEnsiN HOmologue ON chrOmosoMe 10)-inDUCeD kiNASe 1 (pink1), MAy pROViDe A NOVEL THERApeUTIc tArGet FoR CaRdIOproTeCtION [@poNE.0062400-SIDDALl1]. MuTaTiONS In THE PiNk1 gene ArE reSpoNSIBlE fOR tHe AUtOsOMaL ReCESSIvE pArk6 INHERITeD FOrM oF early oNsEt PaRkinson DiSEaSe, A neurodeGENeRatiVE dISoRdER CHARacteRIZED By the lOsS Of dOpAminErGiC nEUrONs In THE SUbStAntiA NiGRa [@PoNe.0062400-VaLeNTe1]. GenEtic AblAtioN oF PiNk1 iN neUroNS reSulTs in mITOchOnDriAl DYSfUnCTIoN chArActErIzEd BY: MITochonDrial mEMbrAnE dEpOLariZATIOn [@ponE.0062400-WoODkaczMar1], [@PoNE.0062400-GAndhI1], ReDUcED miToChOndrIAl RESpiRATiOn aND ATp LEvELS [@pONe.0062400-PARk1], iNcReAsEd oXIdAtIvE StRess [@pone.0062400-WOoDKACZmAR1], [@pONe.0062400-ganDHI1], [@pONe.0062400-CLarK1]--[@ponE.0062400-DAgda1], mItOChoNdRIaL CalciUM OveRLoAd [@pOne.0062400-GanDHI1], AnD EnHancEd susCEpTiBILiTy to MIToCHoNDriaL PERmeaBiLITy transitiON pOrE (mptP) oPeNINg [@PoNE.0062400-gANdHi1]. in coNtRAsT, wIld-typE pINk1 HAs BEEN REPoRtEd To prOtEcT neuRonS frOm MITOchOndRial DYSfUnCtiOn [@ponE.0062400-vAleNTe1], reDucE MITocHONdriAl cYTOcHrome C ReLeasE And CaSpasE 3 anD 9 aCtIVAtiOn [@poNe.0062400-pEtit1], [@PONE.0062400-wANg2], ANd atTEnuate ApoPToTiC cEll dEAtH [@pone.0062400-VAleNTE1], [@PoNe.0062400-peTIT1].
INTereStinGLY, pInk1 ProTeIN IS higHLy exprEssed in the MyocaRDIUM [@POnE.0062400-unOki1] BuT iTS RolE in THE heARt, is not CLEAR [@ponE.0062400-SIddaLL1], [@pONe.0062400-SIDDALL2]. giVen iTs BenEFIcIaL efFeCTs on miToCHONDRiAL FuNCtion aND nEUroPrOtEcTIVe PROPERtiEs, We invEStiGated WhEtHER PiNk1 Could alSO PROTecT THe HeART agaInsT ACutE IrI. We find thaT the Loss OF PInk1 INCreAsEs tHe HeArT\'s VUlNERaBIlITY To iSCHEmIA-rEperfuSION InJURy aND THiS may BE bY woRseniNg MiTOcHoNDRiAL FuNcTION.
mATerIALS And MEthoDS {#s2}
=====================
aNiMal experIMEntS weRE COndUCTEd iN STriCt AcCorDANce With tHe *ANImAls* (*sCIEnTiFiC prOCeDURes*) *act 1986* PuBLIsHED BY ThE uk hoME OFfiCE anD tHe *gUIde FoR thE CaRE And uSe Of lABoRatOry AniMaLS* PublisHED BY THe us NATional iNSTituTES Of HealTH (niH PUBLicatiOn nO. 85--23, reVIseD 1996). apProval has beEN graNTeD bY a lOcaL EtHicS RevIew boaRd aT UnIversITY cOLlEgE loNDOn. alL EFfOrtS wErE MADe to MinImizE sufFeRInG.
hL-1 CeLl CULTure ANd Pink1 ovER-EXPrESSIon {#s2A}
-------------------------------------------
hL-1 ceLLs ARE AN adherEnT muRINE atrIal cell LInE tHAt SpOntANeoUsLY beAT in cUlturE (ThE ceLlS WerE oBtaInED fROM clAYcOmb) [@PoNe.0062400-cLaYCoMB1]. CElls werE CuLtURed IN tisSUE CuLTUrE flaskS PrE-CoaTeD For 2--3 hRS wiTh 10 µg/ML FiBrOnectIN (DILuTEd in 0.02% gelAtIn). GrowTh medIum (cLAYCoMB MEdia suPplEmentEd witH 10% fBS, 2 mM l GLUtamInE (inVitroGeN, gibco), 0.1 Mm NorepINePhrinE (PRepaREd IN 30 MM aSCORBIC AcID), 500 iu PenicilLIN AnD 500 µg sTrEptomyCin (pAa LABOratorIEs)) wAS chAnged evErY 1--2 dAYS anD CELlS wEre maINtaIneD AT 37°c iN 95%o~2~/5%CO~2~ WiTh 90% HuMiDITy.
A sImilaR VEctor EXPREsSIng PinK1 uNdeR the CoNTrOL of thE cMv prOmOTer (AddgEnE pLaSMid 13315: PcdNA-dEST53 piNk1) froM AddgeNE inC., cambRiDGe, ma wAS useD to oVER-EXPrEss piNk1. HL-1 cElLS weRE SeedEd ONTO fiBroNEcTin cOaTed glaSs CoVEr SLIPs aNd upoN ReACHInG 50--60% COnfLUEnCE weRe tRanSfeCted fOR 24 HOuRS uSiNg FUgenE6 ® (ROChe, uK) accoRDiNG tO MAnuFACTURer's INSTrucTIons. tHE pegFP exPReSSIon plAsmiD (CLontECh) WaS INcLudEd For iDENtIficatIoN oF SUcceSsfULlY traNSfecTEd CElls, aT a RATio of 1∶2. ThE VECTOR controL GROuP wAS desiGnated as CeLlS TRaNsfEcTEd witH An empTY plAsmiD ExprESSION veCtOr (RCCMV). A SIMILAR vector expREsSiNg PiNK1 uNDER ThE contROL Of ThE CMv PROMotEr (aDDGeNE pLasMid 13315: pCDnA-DEST53 PINK1) From ADDGeNe Inc., CamBridGE, mA wAS uSED to OVer-expreSs Pink1 IN a SEPaRATE sET OF cElLs. THE PEgfp eXPRESsIon pLASMiD (cLontECH) waS iNCLUdeD fOR iDENtIFicaTion of SucceSSfULLY TrANsFecteD cellS, aT a rATIo Of 1∶2. thE TRAnsFEctiON eFficACy waS 60--70% OF cELLs. CulturE MEDiA cOnTaIning tRAnsFecTion COMpOnenTS wAS REpLACED WITh fResH GROwTH mEdIUM aND CELls WERe InCUbATeD ovERnIGhT. UNfoRTUnaTeLy DuE To a LAcK of A SPeCIfiC CoMMErcIallY AVaiLable pinK1 aNTIbODY weRe not ABlE TO dEMoNStRatE pINK1 ProteIN EXPresSiOn OR loCaliZATiOn.
sImulatEd ISchEMIa-rEpERfUsiOn INJUrY In Hl-1 CelLS ovER-eXprESsinG PINk1 {#S2b}
-------------------------------------------------------------------------
In orDer tO deteRmiNE The effEcT OF Pink1 OVER-ExPrEssion ON ThE susCEPTIbilIty tO SImUlated ischeMIa-rEPeRFusioN INjuRy (SirI), hl-1 CeLlS weRE SubjECTED to A SUstAiNEd epIsoDE OF LEThAl hypOXiA ANd REoxygEnatiON [@POnE.0062400-LiM1], [@PoNe.0062400-SMiTH1]. cULtURe MEdIUm WAS reMOVeD anD RePlaCEd wITH HyPoXIC buffer (cOMpRISing In mM: KH~2~po~4~ 1.0, NahCO~3~ 10.0, mgcL~2~.6h~2~o 1.2, nahepES 25.0, NAcl 74.0, kcl 16, CACl~2~ 1.2 anD NALaCTate 20 AT PH 6.2, buBbled WiTh 100% niTROgeN) AnD thEN PLaCeD iN aN aiRtighT cusToM-bUIlT hypoXIC CHAMBer kEPT at 37°C fOr 12 hoUrS To SIMulatE ISCHemia. FOlLowInG THE PeRIod of simuLAted isCHeMiA, THE Cells wErE RemOved FRom The HYPOXIC CHaMber ANd PLACED In NoRmoXIC cLaYComB MeDIUm (contAININg 3 µM PROPidIum Iodide) aND RETURned TO A tISSUE cULTURE InCUBATor, to SImUlate rEPerFuSIon. FOLlowInG 1 hour siMuLaTEd RePERFUSioN AT 37°c, tHE perCeNtAge Of gfp-traNSFeCTed CelLS sTAineD wIth pROPIDIUM IoDIdE waS DeteRMINed usInG a niKon EcLIPSE Te200 FluoreSCeNT MIcrOscoPE in oRDER TO cAlcUlATe tHE PerCENTAGe CelL DEatH IN EaCH trEatMenT gROup. FOr eaCh trEAtMent grOuP 80 CeLLS WeRe COuNted, taKEN FroM fOur raNDOmLY-SElECtED fIElds of VIEW. ThiS ExpeRimeNT WAS REpeAted on At leASt fOUr sEpARaTE ocCAsions GIviNg a toTaL Of 320 cElLS per TREatmEnt group. for A time-matCHEd nOrmoXIC coNtrol groUp, Hl-1 CeLLS WeRe Placed iN norMOXIC buFfER (COmPRisINg In Mm: Kh~2~PO~4~ 1.0, nahco~3~ 10.0, Mgcl~2~.6H~2~O 1.2, naHEpes 25.0, nAcl 98.0, kcl 3, cACl~2~ 1.2, d-GLUCoSE 10.0, Na PYrUVaTe 2.0 At Ph 7.4, bUbBlEd WitH 5% cO~2~/95% o~2~) FOR THE toTAl 13 hOURS DurATIOn oF tHe EXPeRImeNT AnD tHe PerCeNTAGe ceLL DeAtH wAs DEteRminEd.
ROS-InDuCeD mPtP oPENINg In HL-1 CElls OveR-ExPrESsiNG pInK1 {#s2c}
------------------------------------------------------------
To DetERMiNE ThE EffECt of PiNK1 over-eXpreSsiON On tHE suScEpTiBiLiTY to MPTP oPenING, A pReViOUsLY vaLIDatED cELl moDel oF Mptp OPeNING WAS uTiliZED [@Pone.0062400-Zorov1]. cONFocal laSER-StimULaTiON oF ThE FluOROphORE tETrA metHyl rHOdAmIne MeTHYL (tMrM), wHich ACCumulATeS inTO MITOchoNdriA, geNeRatES ReacTIve OXygEN spEcies (rOS) wIThIn miTOChoNdRia. this CeLl model CAn BE uSeD To SIMuLatE tHE EvEnTs oCcURrINg At REPERFUSIOn, In wHiCh The PRODuCtIoN oF Ros REsuLTS IN mptP OPening aND ThE coLlAPsE of tHE MITochondrIAl mEMbRAnE POTEntiAl [@PONE.0062400-ZOrov1], [@PonE.0062400-DaVIdsOn1]. THE CoLLAPSe OF MiTocHONdRiAL mEmBRAne POTenTIAL IN ThIS CELL modEl HAS PRevIoUSLy beEN vERIFIeD aS iNDICATING mptp opENIng As IT CoinciDEs WiTH The rEdiStRIbuTiOn oF CAlceIn fROM ThE mItOCHonDRIA To tHe CytosOL [@pONe.0062400-HaUSenlOy1].
CuLTUre MEDIuM waS reMoVED aND REpLacED witH kRebS IMAGINg BuffER. ceLlS WERe tHeN loadEd wITH ThE 3 µM TmRm fOr 15 Min AT 37°c ANd wAShEd wiTh KrEbS ImagiNG bufFEr. tHE time takEn tO iNDuCe MItocHoNdRIaL mEMbraNE dePOlarizaTIOn is RECorDed As a MEasUrE Of SusCePTIBilIty to Mptp OpENING. this WaS dEfiNEd AS tHe tIME TakeN tO REach hAlf THe mAxImuM tmRm flUORescENcE iNtEnsITY. twEnty TRANsFecTed ceLLS WErE RanDoMlY selECTEd fOr The inDuCTiON And DEtEctIon OF Mptp oPENIng FrOm EacH treatMEnT gRoup, aND THIS WAS REPEaTED In four IndepeNDENt ExpEriMEnTS gIving a tOTAl oF 80 ceLLS PeR tREatMENt grOuP. as A poSitIve coNtrOL aNd IN oRDEr To ConfIRm ThAt MitocHoNDrial mEmbrANE DEpoLArizaTIoN was indIcATivE Of MPTp OpenINg, fOlLowiNg TMrM LoadING, a GrOuP of CeLLS Were PRe-TREaTed fOR 10 mINuTeS wITH thE mptp inHIbItor, CYCLOSPoRiN A (0.2 ΜM) [@PONe.0062400-liM1], [@PONe.0062400-hauSEnloy2], [@ponE.006 | Introduction {#s1} ============ Mitochondria perform a dual role in the life and death of the cardiomyocyte. When functioningnormally they generate the energy required for normal cellular processesand survival. However, in situations of cellular stress such as during acute myocardial ischemia-reperfusion injury (IRI), they canbecome dysfunctional and be the arbitratorsofcardiomyocyte death. Therefore, new treatment strategies which are capable ofpreventing mitochondrialdysfunction during acute IRI mayreduce myocardial injury, preserve cardiac function and improve clinical outcomes in patients with ischemic heart disease. Inthis regard,the mitochondrial serine-threonine protein kinase, PTEN (phosphatase and tensin homologue on chromosome 10)-induced kinase 1 (PINK1), may provide a novel therapeutic target for cardioprotection [@pone.0062400-Siddall1]. Mutations in thePINK1 gene are responsible for the autosomal recessive PARK6 inherited form of early onset Parkinson disease, a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra [@pone.0062400-Valente1]. Geneticablation of PINK1 in neurons results in mitochondrial dysfunctioncharacterized by: mitochondrial membranedepolarization [@pone.0062400-WoodKaczmar1], [@pone.0062400-Gandhi1], reduced mitochondrialrespirationand ATP levels [@pone.0062400-Park1], increased oxidative stress [@pone.0062400-WoodKaczmar1], [@pone.0062400-Gandhi1], [@pone.0062400-Clark1]--[@pone.0062400-Dagda1], mitochondrial calcium overload [@pone.0062400-Gandhi1],and enhanced susceptibilitytomitochondrial permeability transition pore (MPTP) opening [@pone.0062400-Gandhi1]. In contrast, wild-type PINK1 hasbeen reported toprotect neurons from mitochondrial dysfunction [@pone.0062400-Valente1], reduce mitochondrial cytochrome C release and caspase3 and 9 activation[@pone.0062400-Petit1],[@pone.0062400-Wang2], and attenuate apoptotic cell death [@pone.0062400-Valente1], [@pone.0062400-Petit1]. Interestingly, PINK1 protein is highly expressed in the myocardium [@pone.0062400-Unoki1]but its rolein the heart, is not clear [@pone.0062400-Siddall1], [@pone.0062400-Siddall2]. Given its beneficial effects on mitochondrial function andneuroprotective properties,weinvestigated whether PINK1 could alsoprotect the heart against acuteIRI. We find that the loss of PINK1 increases the heart\'s vulnerabilitytoischemia-reperfusion injuryand this may be by worsening mitochondrial function.Materials andMethods {#s2} ===================== Animal experiments were conducted in strict accordancewith the *Animals* (*Scientific Procedures*) *Act 1986* publishedby the UKHomeOffice and the *Guidefor the Care and Use of Laboratory Animals* published by the US National Institutes of Health (NIH Publication No. 85--23, revised 1996). Approval has been granted by a local ethics review board at University College London.All efforts were madeto minimize suffering. HL-1 Cell Culture and PINK1Over-expression {#s2a} ------------------------------------------- HL-1 cells are an adherent murine atrial cell line that spontaneously beat inculture (the cells were obtained from Claycomb) [@pone.0062400-Claycomb1]. Cells were cultured intissue culture flasks pre-coated for 2--3 hrs with 10 µg/ml fibronectin(diluted in 0.02% gelatin).Growthmedium (Claycombmediasupplemented with 10% FBS, 2 mM L glutamine (Invitrogen,Gibco), 0.1 mMnorepinephrine (prepared in 30 mM ascorbic acid), 500 IUpenicillin and 500 µg streptomycin(PAA Laboratories)) was changed every 1--2 days and cells were maintained at 37°C in 95%O~2~/5%CO~2~ with 90% humidity.A similar vector expressing PINK1 under the control of the CMV promoter (Addgene plasmid 13315:pcDNA-DEST53PINK1) from Addgene Inc., Cambridge, MA was used to over-express PINK1. HL-1 cells were seeded onto fibronectin coated glass cover slips and upon reaching50--60% confluence were transfected for 24 hours using Fugene6 ® (Roche, UK) according to manufacturer's instructions. The pEGFPexpression plasmid (Clontech) was included for identification of successfully transfectedcells, at a ratio of 1∶2.The vectorcontrol group was designated as cells transfectedwith an empty plasmid expression vector (RcCMV). A similar vector expressingPINK1under the control of the CMVpromoter(Addgene plasmid 13315: pcDNA-DEST53 PINK1) from Addgene Inc., Cambridge, MA was used to over-express PINK1 ina separate set of cells. The pEGFP expression plasmid (Clontech) was included for identification of successfullytransfected cells, at a ratio of 1∶2. The transfection efficacy was 60--70% ofcells.Culture media containing transfection components was replaced with fresh growth medium and cells were incubatedovernight. Unfortunately due to a lack of a specific commercially available PINK1 antibody werenot ableto demonstratePINK1 protein expression or localization. SimulatedIschemia-reperfusion Injury in HL-1 Cells Over-expressingPINK1 {#s2b} -------------------------------------------------------------------------Inorder to determine the effect of PINK1 over-expression on thesusceptibility to simulated ischemia-reperfusion injury (SIRI), HL-1cells were subjected to a sustained episode of lethal hypoxia and reoxygenation [@pone.0062400-Lim1], [@pone.0062400-Smith1]. Culture medium was removed andreplacedwith hypoxic buffer (comprisingin mM: KH~2~PO~4~ 1.0, NaHCO~3~ 10.0, MgCl~2~.6H~2~O 1.2, NaHEPES 25.0, NaCl 74.0, KCl 16,CaCl~2~ 1.2 and NaLactate 20 at pH 6.2, bubbled with 100% nitrogen) and then placed in an airtight custom-built hypoxic chamber kept at 37°C for 12 hours to simulate ischemia. Followingthe period of simulated ischemia, the cells wereremoved from thehypoxic chamberand placed in normoxic Claycomb medium (containing 3 µM propidium iodide) and returned to a tissue culture incubator, to simulatereperfusion. Following 1 hour simulated reperfusion at 37°C, the percentage of GFP-transfected cells stained with propidium iodide was determined using a Nikon Eclipse TE200 fluorescent microscope in order to calculatethe percentage cell death in each treatment group.For each treatment group80 cells were counted, taken from four randomly-selected fields of view.Thisexperimentwas repeated on at least four separate occasionsgivinga total of 320 cellspertreatment group. For a time-matched normoxic control group, HL-1 cells wereplaced in normoxic buffer(comprising in mM: KH~2~PO~4~ 1.0, NaHCO~3~ 10.0, MgCl~2~.6H~2~O 1.2, NaHEPES 25.0, NaCl 98.0, KCl 3, CaCl~2~ 1.2, d-glucose 10.0,Na pyruvate2.0 at pH 7.4, bubbled with 5%CO~2~/95% O~2~) for the total 13 hours duration of the experiment and the percentage celldeath was determined. ROS-induced MPTP Opening in HL-1 Cells Over-expressing PINK1 {#s2c} ------------------------------------------------------------ To determine the effectof PINK1 over-expression on the susceptibility to MPTP opening, a previously validated cell model of MPTP opening was utilized[@pone.0062400-Zorov1]. Confocal laser-stimulation of the fluorophore tetra methyl rhodamine methyl(TMRM), which accumulates into mitochondria, generates reactive oxygen species (ROS) within mitochondria. Thiscell model can be used to simulatethe events occurringat reperfusion, in which the production of ROS results in MPTPopening and the collapse of the mitochondrial membrane potential [@pone.0062400-Zorov1],[@pone.0062400-Davidson1]. The collapse ofmitochondrial membrane potential in this cell model has previously been verified as indicating MPTPopening asit coincides with the redistribution of calcein from the mitochondria to the cytosol [@pone.0062400-Hausenloy1]. Culture medium wasremoved and replaced with Krebs imagingbuffer. Cells were then loaded with the 3 µM TMRM for 15 min at 37°C and washed with Krebs imagingbuffer. Thetime taken to induce mitochondrial membrane depolarization is recordedas a measure of susceptibilityto MPTP opening. This was defined as the time taken to reach half the maximum TMRM fluorescenceintensity. Twentytransfected cells were randomly selected for the inductionand detection of MPTP opening from eachtreatment group, and this was repeated in four independent experiments giving atotal of 80 cells per treatment group. Asa positivecontroland in order to confirm thatmitochondrial membrane depolarization was indicative of MPTP opening, following TMRM loading, a group of cells were pre-treated for 10 minuteswith the MPTP inhibitor, cyclosporinA(0.2 µM) [@pone.0062400-Lim1], [@pone.0062400-Hausenloy2], [@pone.006 | Introduction {#s1} ============ Mitochondria perform _a_ dual role in the life and death _of_ the cardiomyocyte. When functioning normally they generate the energy required _for_ _normal_ cellular processes _and_ survival. However, in _situations_ of cellular stress such as during acute myocardial ischemia-reperfusion injury (IRI), they _can_ become dysfunctional and be the _arbitrators_ of cardiomyocyte death. Therefore, new treatment strategies _which_ are capable of preventing mitochondrial dysfunction during acute IRI may reduce myocardial injury, preserve cardiac function and _improve_ clinical outcomes in _patients_ with ischemic heart disease. In _this_ _regard,_ the _mitochondrial_ serine-threonine protein _kinase,_ PTEN (phosphatase and tensin homologue on chromosome 10)-induced _kinase_ 1 (PINK1), may _provide_ a novel therapeutic target for _cardioprotection_ _[@pone.0062400-Siddall1]._ _Mutations_ in the PINK1 _gene_ _are_ responsible for the autosomal recessive PARK6 inherited form of early onset Parkinson disease, a _neurodegenerative_ disorder _characterized_ _by_ the loss of _dopaminergic_ neurons in _the_ substantia nigra [@pone.0062400-Valente1]. _Genetic_ ablation of PINK1 in _neurons_ results in _mitochondrial_ _dysfunction_ characterized _by:_ mitochondrial membrane _depolarization_ _[@pone.0062400-WoodKaczmar1],_ [@pone.0062400-Gandhi1], reduced _mitochondrial_ _respiration_ _and_ _ATP_ levels [@pone.0062400-Park1], _increased_ oxidative stress [@pone.0062400-WoodKaczmar1], [@pone.0062400-Gandhi1], [@pone.0062400-Clark1]--[@pone.0062400-Dagda1], mitochondrial calcium _overload_ _[@pone.0062400-Gandhi1],_ and enhanced susceptibility to mitochondrial permeability transition pore (MPTP) opening _[@pone.0062400-Gandhi1]._ In contrast, wild-type PINK1 has _been_ reported _to_ protect _neurons_ _from_ mitochondrial dysfunction [@pone.0062400-Valente1], reduce mitochondrial cytochrome C release _and_ caspase _3_ and _9_ _activation_ [@pone.0062400-Petit1], [@pone.0062400-Wang2], and attenuate apoptotic cell death [@pone.0062400-Valente1], [@pone.0062400-Petit1]. _Interestingly,_ PINK1 protein is highly expressed in _the_ myocardium [@pone.0062400-Unoki1] but its role in the heart, is not clear _[@pone.0062400-Siddall1],_ _[@pone.0062400-Siddall2]._ Given _its_ beneficial effects on mitochondrial function and neuroprotective properties, _we_ _investigated_ whether PINK1 _could_ also protect the _heart_ against acute IRI. We find that the loss of PINK1 increases the heart\'s vulnerability to ischemia-reperfusion _injury_ _and_ this may _be_ by worsening mitochondrial function. Materials _and_ _Methods_ _{#s2}_ ===================== Animal experiments _were_ _conducted_ in _strict_ accordance with the *Animals* (*Scientific Procedures*) *Act 1986* _published_ by the UK Home _Office_ and the *Guide for the Care _and_ _Use_ of Laboratory _Animals*_ _published_ by the _US_ National Institutes _of_ Health (NIH Publication _No._ 85--23, _revised_ _1996)._ Approval has been granted _by_ a local ethics review board at University College London. All _efforts_ _were_ made to minimize suffering. HL-1 Cell Culture and PINK1 Over-expression {#s2a} ------------------------------------------- HL-1 cells are an _adherent_ murine atrial _cell_ line that _spontaneously_ beat in culture (the _cells_ were _obtained_ from Claycomb) [@pone.0062400-Claycomb1]. Cells _were_ cultured in tissue culture flasks pre-coated for 2--3 _hrs_ with _10_ µg/ml fibronectin (diluted _in_ 0.02% gelatin). Growth medium (Claycomb media supplemented _with_ _10%_ FBS, 2 _mM_ L glutamine _(Invitrogen,_ Gibco), 0.1 mM norepinephrine (prepared in 30 mM _ascorbic_ acid), _500_ IU penicillin and 500 µg streptomycin (PAA Laboratories)) _was_ changed every _1--2_ days and cells were maintained at 37°C _in_ 95%O~2~/5%CO~2~ with 90% _humidity._ A similar _vector_ expressing PINK1 under the _control_ of the CMV _promoter_ _(Addgene_ _plasmid_ 13315: pcDNA-DEST53 PINK1) from Addgene Inc., Cambridge, MA was used _to_ _over-express_ PINK1. HL-1 cells _were_ seeded onto fibronectin coated _glass_ cover slips and upon reaching 50--60% confluence were transfected for 24 hours using Fugene6 ® (Roche, _UK)_ according to manufacturer's instructions. _The_ pEGFP expression plasmid (Clontech) _was_ included for _identification_ of successfully transfected _cells,_ at _a_ ratio _of_ 1∶2. The vector control group _was_ designated as cells transfected with _an_ empty plasmid expression vector (RcCMV). _A_ similar vector expressing PINK1 under _the_ control of the CMV promoter (Addgene plasmid 13315: pcDNA-DEST53 PINK1) _from_ Addgene _Inc.,_ Cambridge, MA was used to over-express PINK1 _in_ _a_ separate set _of_ _cells._ The pEGFP expression plasmid (Clontech) was included _for_ _identification_ of _successfully_ _transfected_ cells, at a ratio of 1∶2. The transfection _efficacy_ _was_ _60--70%_ of _cells._ Culture media containing transfection components was _replaced_ with fresh growth medium _and_ cells were incubated overnight. Unfortunately due _to_ a lack of a specific commercially available _PINK1_ _antibody_ _were_ not able to demonstrate PINK1 protein expression or localization. Simulated Ischemia-reperfusion Injury _in_ HL-1 Cells Over-expressing PINK1 _{#s2b}_ ------------------------------------------------------------------------- In order _to_ _determine_ the effect of PINK1 over-expression on the susceptibility to _simulated_ _ischemia-reperfusion_ injury (SIRI), HL-1 cells were subjected to a sustained episode of _lethal_ hypoxia and reoxygenation [@pone.0062400-Lim1], [@pone.0062400-Smith1]. Culture _medium_ was _removed_ and replaced with hypoxic buffer _(comprising_ in mM: KH~2~PO~4~ _1.0,_ NaHCO~3~ 10.0, MgCl~2~.6H~2~O 1.2, NaHEPES _25.0,_ NaCl 74.0, KCl _16,_ CaCl~2~ 1.2 and _NaLactate_ 20 at pH _6.2,_ bubbled with _100%_ nitrogen) and _then_ placed _in_ an airtight _custom-built_ hypoxic chamber kept at _37°C_ for 12 _hours_ to _simulate_ ischemia. Following the period _of_ simulated ischemia, the cells were _removed_ from the hypoxic _chamber_ and placed _in_ _normoxic_ Claycomb medium _(containing_ 3 µM propidium iodide) and returned to _a_ tissue culture incubator, to simulate _reperfusion._ _Following_ _1_ _hour_ simulated _reperfusion_ at _37°C,_ the percentage of GFP-transfected cells stained with propidium iodide was determined using a Nikon Eclipse TE200 fluorescent microscope in _order_ to calculate the percentage _cell_ _death_ in each treatment group. For _each_ treatment group 80 cells were counted, taken _from_ four randomly-selected _fields_ of view. This _experiment_ was repeated on at least four separate occasions giving a total of _320_ cells _per_ treatment group. _For_ _a_ time-matched _normoxic_ control _group,_ HL-1 cells _were_ placed in normoxic buffer (comprising in mM: KH~2~PO~4~ _1.0,_ NaHCO~3~ _10.0,_ _MgCl~2~.6H~2~O_ 1.2, NaHEPES 25.0, NaCl 98.0, KCl 3, CaCl~2~ _1.2,_ d-glucose 10.0, _Na_ pyruvate 2.0 at _pH_ 7.4, bubbled with 5% CO~2~/95% O~2~) for _the_ total 13 hours _duration_ _of_ the experiment and the percentage _cell_ death _was_ determined. ROS-induced MPTP Opening _in_ _HL-1_ Cells _Over-expressing_ PINK1 {#s2c} ------------------------------------------------------------ To determine _the_ _effect_ of PINK1 over-expression _on_ the susceptibility to MPTP opening, a previously validated cell model of _MPTP_ opening was utilized [@pone.0062400-Zorov1]. Confocal _laser-stimulation_ of _the_ fluorophore tetra methyl rhodamine methyl (TMRM), which accumulates into mitochondria, _generates_ reactive oxygen _species_ (ROS) within mitochondria. This cell model can _be_ used to _simulate_ the events _occurring_ at reperfusion, _in_ which _the_ production of _ROS_ results in _MPTP_ _opening_ and the _collapse_ of the mitochondrial membrane potential [@pone.0062400-Zorov1], _[@pone.0062400-Davidson1]._ The collapse of mitochondrial membrane potential in _this_ cell _model_ has _previously_ been verified as indicating MPTP opening as it coincides with the _redistribution_ of calcein from the _mitochondria_ _to_ _the_ cytosol [@pone.0062400-Hausenloy1]. Culture medium was removed and replaced _with_ Krebs imaging buffer. _Cells_ were then loaded with the 3 _µM_ _TMRM_ _for_ 15 min at 37°C _and_ washed with Krebs imaging buffer. The _time_ taken to _induce_ _mitochondrial_ membrane depolarization is recorded _as_ a measure _of_ susceptibility to MPTP opening. This was defined as the time taken to _reach_ half the maximum _TMRM_ fluorescence intensity. Twenty transfected cells were randomly selected _for_ _the_ induction and detection of MPTP opening from _each_ _treatment_ group, _and_ this was repeated in _four_ independent experiments giving _a_ _total_ of 80 cells per _treatment_ group. _As_ a _positive_ control and in order to confirm that mitochondrial membrane depolarization was indicative of _MPTP_ opening, following TMRM loading, _a_ group of cells were pre-treated for 10 minutes with the _MPTP_ inhibitor, cyclosporin _A_ (0.2 µM) [@pone.0062400-Lim1], _[@pone.0062400-Hausenloy2],_ [@pone.006 |
Introduction {#tca13044-sec-0001}
============
Nivolumab, a PD‐1 antibody, is an immune checkpoint inhibitor (ICI) that has been proven to be active in patients with several different tumor types. Nivolumab has been shown to have an overall response rate (ORR) of approximately 20% in patients with previously treated non‐small cell lung cancer (NSCLC).[1](#tca13044-bib-0001){ref-type="ref"}, [2](#tca13044-bib-0002){ref-type="ref"} However, nivolumab is not effective in more than 40% of NSCLC patients who experience disease progression, despite nivolumab treatment. To improve the efficacy of this promising immunotherapy, additional modalities such as chemotherapy, radiotherapy (RT), or other ICIs may also be administered to patients in whom ICIs have not been completely effective. Interestingly, RT stimulates a systemic immune response and causes the release of tumor‐related antigens.[3](#tca13044-bib-0003){ref-type="ref"} Recent preclinical studies have demonstrated a synergistic tumor response with RT and the blockade of PD‐1.[4](#tca13044-bib-0004){ref-type="ref"}, [5](#tca13044-bib-0005){ref-type="ref"}, [6](#tca13044-bib-0006){ref-type="ref"} It is possible that tumor‐specific immunity is induced by radiation. Although RT plays an important role in the local control and elimination of tumors, it also contributes to the induction of antitumor immune responses, and the immunosuppressive and immunostimulatory effects of RT.[6](#tca13044-bib-0006){ref-type="ref"} Radiation‐induced cell death causes a release of danger signals such as HMGB1, ATP, and HSP70, and the dendritic cells can stimulate activated CD8 T cells and tumor‐specific T cells.[6](#tca13044-bib-0006){ref-type="ref"} In addition to immune activation, RT induces transforming growth factor‐β (TGF‐β), an immunosuppressive cytokine. To reduce the immunosuppressive functions, a combination of RT and TGF‐β inhibitors has been identified as a valuable option in preclinical settings.[6](#tca13044-bib-0006){ref-type="ref"} A recent experimental study demonstrated that fractionated RT increases PD‐L1 surface expression on tumor cells, suggesting a key rationale for the combination of RT with ICIs.[7](#tca13044-bib-0007){ref-type="ref"}
Of the 98 patients registered in the KEYNOTE‐001 phase I trial, Shaverdian *et al.* reported that the duration of progression‐free survival (PFS) with pembrolizumab was significantly longer in patients previously administered RT than in those not treated with RT.[8](#tca13044-bib-0008){ref-type="ref"} Fiorica *et al.* also reported that nivolumab treatment after hypofractionated RT improved the outcome in 35 patients with pretreated or metastatic NSCLC.[9](#tca13044-bib-0009){ref-type="ref"} These results suggest that previous RT clinically improves tumor response and immune reaction to ICIs, such as nivolumab or pembrolizumab. However, the synergistic effect of ICIs and previous RT was not fully elucidated in these studies. As the former study was a phase I trial, pembrolizumab was administered at different doses and treatment deliveries.[8](#tca13044-bib-0008){ref-type="ref"} The latter study was limited by a small sample of only 35 patients.[9](#tca13044-bib-0009){ref-type="ref"} Analysis of these studies shows that immunotherapy after previous RT prolongs survival;[8](#tca13044-bib-0008){ref-type="ref"}, [9](#tca13044-bib-0009){ref-type="ref"} however, neither the ORRs of ICIs after previous RT nor the populations that might benefit from ICI treatment were included. Little detailed clinical data of the effect of ICI administration after previous RT has been reported; therefore, we attempted to elucidate the potential synergistic antitumor effect of nivolumab after RT in patients with previously treated NSCLC.
Methods {#tca13044-sec-0002}
=======
Patient eligibility and data collection {#tca13044-sec-0003}
---------------------------------------
The eligibility criteria for our retrospective analysis were: histologically or cytologically proven advanced NSCLC with stage III or IV disease or recurrence after surgical resection; age \> 20 years; patients with disease progression after at least one prior cytotoxic chemotherapy treated with nivolumab; *EGFR* mutation‐positive patients administered EGFR‐tyrosine kinase inhibitors prior to any cytotoxic chemotherapy; and patients with available ORR data of nivolumab according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Patients were excluded if they had: a concomitant serious illness, such as myocardial infarction in the previous three months; uncontrolled angina pectoris, heart failure, uncontrolled diabetes mellitus, uncontrolled hypertension, interstitial pneumonia, or lung disease; an infection or other disease contraindicating chemotherapy; or were pregnant or breastfeeding. This study was approved by the institutional ethics committee of the Saitama Medical University International Medical Center.
Treatment and efficacy evaluation {#tca13044-sec-0004}
---------------------------------
Nivolumab was intravenously administered at 3 mg/kg every two weeks. A complete blood cell count, differential count, routine chemistry measurements, physical examination, and toxicity assessment were performed on a weekly basis. Acute toxicity was graded according to Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. The tumor response was evaluated according to RECIST version 1.1.[10](#tca13044-bib-0010){ref-type="ref"}
Statistical analysis {#tca13044-sec-0005}
--------------------
*P* \< 0.05 indicated statistical significance. Fisher\'s exact tests were conducted to examine the association between the categorical variables. The Kaplan--Meier method was used to estimate survival as a function of time, and survival differences were analyzed by log‐rank tests. PFS was defined as the time from the initiation of nivolumab therapy to tumor recurrence or death from any cause, while overall survival (OS) was defined as the time from the initiation of nivolumab therapy to death from any cause. Statistical analyses were performed using GraphPad Prism 4 and JMP 8.0.
Results {#tca13044-sec-0006}
=======
Patient demographics {#tca13044-sec-0007}
--------------------
From February 2016 to December 2017, 152 patients with pretreated NSCLC were administered nivolumab. Twenty‐eight patients were excluded because of inadequate medical information or the absence of an evaluable target lesion. Thus, a total of 124 patients (*n* ~males~ = 93, *n* ~females~ = 31; median age: 69 years; range: 31--85 years) were eligible for analysis. Patient characteristics are listed in Table [1](#tca13044-tbl-0001){ref-type="table"}. A total of 99 patients had a smoking history. Clinical staging indicated that 27 patients had stage III disease, 77 had stage IV disease, and 20 patients developed recurrence after surgical resection. The patients were divided into RT and non‐RT groups.
######
Comparison of demographics in patients treated with or without RT before nivolumab
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Variables All patients\ Patients administered any RT before Nivo (*n* = 66) Patients not administered RT before Nivo (*n* = 58) *P*
(*n* = 124)
---------------------------------------------------------- --------------- ----------------------------------------------------- ----------------------------------------------------- ----------
Age
≦ 69/\> 69 65/59 36/30 29/29 0.71
Gender
Male/female 93/31 51/15 42/16 0.54
Smoking
Yes/no 99/25 52/14 47/11 0.82
ECOG PS
0/1--3 59/65 31/35 18/40 0.09
Stage
III/IV 27/97 20/46 7/51 0.71
T factor
T1--2/T3--4 68/56 35/31 33/25 0.58
N factor
N0/N1‐3 20/104 12/54 8/50 0.62
Histology
Adeno/non‐adeno 65/59 31/35 34/24 0.21
*EGFR* mutation status
Mutant/wild 14/104 10/51 4/53 0.15
Nivo response
CR or PR/SD or PD | introduction { # tca13044 - sec - 0001 } = = = = = = = = = = = = nivolumab, a pd ‐ 1 antibody, is an immune checkpoint inhibitor ( ici ) that has been proven to be active in patients with several tumor tumor types. nivolumab has been shown to have an overall firing rate ( orr ) of approximately 20 % in patients with previously treated non ‐ small cell lung cancer ( nsclc ). [ 1 ] ( # tca13044 - bib - 0001 ) { ref - type = " ref " }, [ 2 ] ( # tca13044 - bib - 0002 ) { ref - type = " ref " } however, nivolumab is not effective in more than 40 % of nsclc patients who experience disease progression, despite radiation treatment. to improve the efficacy of rt promising immunotherapy, additional modalities such as chemotherapy, radiotherapy ( rt ), or other icis may simultaneously be administered to patients in whom icis have not been completely effective. interestingly, rt stimulates a systemic immune response and causes the release of tumor ‐ related antigens. [ 3 ] ( # tca13044 - bib - 0003 ) { ref - type = " ref " } recent laboratory studies have demonstrated a synergistic tumor response with rt and the blockade of pd ‐ 1. [ 4 ] ( # tca13044 - bib - 0004 ) { ref - type = " ref " }, [ 5 ] ( # tca13044 - ta - 0005 ) { ref - type = " ref " }, [ 6 ] ( # tca13044 - bib - 0006 ) { ref - type = " ref " } it is possible that tumor ‐ target immunity is improved by radiation. although rt plays an important role in the local control and elimination of tumors, it also connects to the induction of antitumor immune responses, and the immunosuppressive and immunostimulatory effects of rt. [ 6 ] ( # tca13044 - bib - 0006 ) { ref - type = " ref " } radiation ‐ induced cell death causes a release of danger signals such as hmgb1, atp, and hsp70, and the dendritic cells can stimulate activated cd8 t cells and tumor ‐ specific t cells. [ 6 ] ( # tca13044 - bib - 0006 ) { ref - type = " ref " } in addition to immune activation, rt induces transforming growth factor ‐ β ( tgf ‐ β ), an immunosuppressive cytokine. to reduce the immunosuppressive functions, a combination of rt and tgf ‐ β inhibitors has been identified as a valuable option in preclinical settings. [ 6 ] ( # tca13044 - bib - 0006 ) { ref - type = " ref " } a recent experimental study demonstrated that fractionated rt increases pd ‐ l1 surface expression on tumor cells, suggesting a key rationale for the combination of rt with icis. [ 7 ] ( # tca13044 - bib - 0007 ) { ref - type = " ref " } of the 98 patients registered in the keynote ‐ 001 phase i trial, shaverdian * et al. * reported that the duration of progression ‐ free survival ( pfs ) with pembrolizumab was significantly longer in patients previously administered rt than in those not treated with rt. [ 8 ] ( # tca13044 - bib - 0008 ) { ref - type = " ref " } fiorica * et al. * also reported that nivolumab treatment after hypofractionated rt improved the outcome in 35 patients with pretreated or metastatic nsclc. [ 9 ] ( # tca13044 - bib - 0009 ) { ref - type = " ref " } these results suggest that previous rt clinically improves tumor response and immune reaction to icis, such as nivolumab or pembrolizumab. however, the synergistic effect of icis and previous rt was not fully elucidated in these studies. as the former study was a phase i trial, pembrolizumab was administered at different doses and treatment deliveries. [ 8 ] ( # tca13044 - bib - 0008 ) { ref - type = " ref " } the latter study was limited by a small sample of only 35 patients. [ 9 ] ( # tca13044 - bib - 0009 ) { ref - type = " ref " } analysis of these studies shows that immunotherapy after previous rt prolongs survival ; [ 8 ] ( # tca13044 - bib - 0008 ) { ref - type = " ref " }, [ 9 ] ( # tca13044 - bib - 0009 ) { ref - type = " ref " } however, neither the orrs of icis after previous rt nor the populations that might benefit from ici treatment were included. little detailed clinical data of the effect of ici administration after previous rt has been reported ; therefore, we attempted to elucidate the potential synergistic antitumor effect of nivolumab after rt in patients with previously treated nsclc. methods { # tca13044 - sec - 0002 } = = = = = = = patient eligibility and data collection { # tca13044 - sec - 0003 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the eligibility criteria for our retrospective analysis were : histologically or cytologically proven advanced nsclc with stage iii or iv disease or recurrence after surgical resection ; age \ > 20 years ; patients with disease progression after at least one prior cytotoxic chemotherapy treated with nivolumab ; * egfr * mutation ‐ positive patients administered egfr ‐ tyrosine kinase inhibitors prior to any cytotoxic chemotherapy ; and patients with available orr data of nivolumab according to response evaluation criteria in solid tumors ( recist ) version 1. 1. patients were excluded if they had : a concomitant serious illness, such as myocardial infarction in the previous three months ; uncontrolled angina pectoris, heart failure, uncontrolled diabetes mellitus, uncontrolled hypertension, interstitial pneumonia, or lung disease ; an infection or other disease contraindicating chemotherapy ; or were pregnant or breastfeeding. this study was approved by the institutional ethics committee of the saitama medical university international medical center. treatment and efficacy evaluation { # tca13044 - sec - 0004 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - nivolumab was intravenously administered at 3 mg / kg every two weeks. a complete blood cell count, differential count, routine chemistry measurements, physical examination, and toxicity assessment were performed on a weekly basis. acute toxicity was graded according to common terminology criteria for adverse events ( ctcae ) version 4. 0. the tumor response was evaluated according to recist version 1. 1. [ 10 ] ( # tca13044 - bib - 0010 ) { ref - type = " ref " } statistical analysis { # tca13044 - sec - 0005 } - - - - - - - - - - - - - - - - - - - - * p * \ < 0. 05 indicated statistical significance. fisher \ ' s exact tests were conducted to examine the association between the categorical variables. the kaplan - - meier method was used to estimate survival as a function of time, and survival differences were analyzed by log ‐ rank tests. pfs was defined as the time from the initiation of nivolumab therapy to tumor recurrence or death from any cause, while overall survival ( os ) was defined as the time from the initiation of nivolumab therapy to death from any cause. statistical analyses were performed using graphpad prism 4 and jmp 8. 0. results { # tca13044 - sec - 0006 } = = = = = = = patient demographics { # tca13044 - sec - 0007 } - - - - - - - - - - - - - - - - - - - - from february 2016 to december 2017, 152 patients with pretreated nsclc were administered nivolumab. twenty ‐ eight patients were excluded because of inadequate medical information or the absence of an evaluable target lesion. thus, a total of 124 patients ( * n * ~ males ~ = 93, * n * ~ females ~ = 31 ; median age : 69 years ; range : 31 - - 85 years ) were eligible for analysis. patient characteristics are listed in table [ 1 ] ( # tca13044 - tbl - 0001 ) { ref - type = " table " }. a total of 99 patients had a smoking history. clinical staging indicated that 27 patients had stage iii disease, 77 had stage iv disease, and 20 patients developed recurrence after surgical resection. the patients were divided into rt and non ‐ rt groups. # # # # # # comparison of demographics in patients treated with or without rt before nivolumab - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - variables all patients \ patients administered any rt before nivo ( * n * = 66 ) patients not administered rt before nivo ( * n * = 58 ) * p * ( * n * = 124 ) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - age [UNK] 69 / \ > 69 65 / 59 36 / 30 29 / 29 0. 71 gender male / female 93 / 31 51 / 15 42 / 16 0. 54 smoking yes / no 99 / 25 52 / 14 47 / 11 0. 82 ecog ps 0 / 1 - - 3 59 / 65 31 / 35 18 / 40 0. 09 stage iii / iv 27 / 97 20 / 46 7 / 51 0. 71 t factor t1 - - 2 / t3 - - 4 68 / 56 35 / 31 33 / 25 0. 58 n factor n0 / n1 ‐ 3 20 / 104 12 / 54 8 / 50 0. 62 histology adeno / non ‐ adeno 65 / 59 31 / 35 34 / 24 0. 21 * egfr * mutation status mutant / wild 14 / 104 10 / 51 4 / 53 0. 15 nivo response cr or pr / sd or pd | Introduction {# tca13044 - sec - 0001} = = = = = = = = = = = = Nivolu<wb, a PD ‐ 1 antibody, is an immune ch$c.point inhibitor (ICI) that has been proven to be active in patients with several different tumor types. Nivolumab has been shown to have an overall response rate (ORR) of approximately 20% in patients with previously treated non ‐ small cell lung cancer (NSCLC ). [1] (# tca13044 - bib - 0001) {ref - type = " ref " }, [2] (# tca13044 - bib - 0002) {ref - type = " ref "} However, nivolumab is not effective in more than 40% of NSCLC patients who experience disease progression, despite nivolumab treatment. To improve the efficacy of this promising immunotherapy, additional modalities such as chemotherapy, radiotherapy (RT ), or other ICIs may also be administered to patients in whom ICIs have not been completely effective. Interestingly, RT stimulates a systemic immune response and causes the release of tumor ‐ related antigens. [3] (# tca13044 - bib - 0003) {ref - type = " ref "} Recent preclinical studies have demonstrated a synergistic tumor response with RT and the blockade of PD ‐ 1. [4] (# tca13044 - bib - 0004) {ref - type = " ref " }, [5] (# tca13044 - bib - 0005) {ref - type = " ref " }, [6] (# tca13044 - bib - 0006) {ref - type = " ref "} It is possible that tumor ‐ specific immunity is induced by radiation. Although RT plays an important role in the local control and elimination of tumors, it also contributes to the induction of antitumor immune responses, and the immunosuppressive and immunostihu.atory eRfevts of RT. [6] (# tca13044 - bib - 0006) {ref - type = " ref "} Radiation ‐ induced cell death causes a release of danger signals such as HMGB1, ATP, and HSP70, and the dendritic cells can stimulate activated CD8 T cells and tumor ‐ specific T cells. [6] (# tca13044 - bib - 0006) {ref - type = " ref "} In addition to immune activation, RT induces transforming growth factor ‐ β (TGF ‐ β ), an immunosuppressive cytokine. To reduce the immunosuppressive functions, a combination of RT and TGF ‐ β inhibitors has been identified as a valuable option in preclinical settings. [6] (# tca13044 - bib - 0006) {ref - type = " ref "} A recent experimental study demonstrated that fractionated RT increases PD ‐ L1 surface expression on tumor cells, Duggewting a key rationale for the combination of RT with ICIs. [7] (# tca13044 - bib - 0007) {ref - type = " ref "} Of the 98 patients registered in the KEYNOTE ‐ 001 phase I trial, Shaverdian * et al. * reported that the duration of progression ‐ free survival (PFS) with pembrolizumab was significantly longer in patients previously administered RT than in those not treated with RT. [8] (# tca13044 - bib - 0008) {ref - type = " ref "} Fiorica * et al. * also reported that nivolumab treatment after hypofractionated RT improved the outcome in 35 patients with pretreated or metastatic NSCLC. [9] (# tca13044 - bib - 0009) {ref - type = " ref "} These results suggest that previous RT clinically improves tumor response and immune reaction to ICIs, such as nivolumab or pembrolizumab. However, the synergistic effect of ICIs and previous RT was not fully elucidated in these studies. As the former study was a phase I trial, pembrolizumab was administered at different doses and treatment deliveries. [8] (# tca13044 - bib - 0008) {ref - type = " ref "} The latter study was limited by a small sample of only 35 patients. [9] (# tca13044 - bib - 0009) {ref - type = " ref "} Analysis of these studies shows that immunotherapy after previous RT prolongs survival; [8] (# tca13044 - bib - 0008) {ref - type = " ref " }, [9] (# tca13044 - bib - 0009) {ref - type = " ref "} however, neither the ORRs of ICIs after previous RT nor the populations that might benefit from ICI treatment were included. Little detailed clinical data of the effect of ICI administration after previous RT has been reported; therefore, we attempted to elucidate the potential synergistic an^i6umor eff@St of nivolumab after RT in patients with previously treated NSCLC. Methods {# tca13044 - sec - 0002} = = = = = = = Patient eligibility and data collection {# tca13044 - sec - 0003} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The eligibility criteria for our retrospective analysis were: histologically or cytologically proven advanced NSCLC with stage III or IV disease or recurrence after surgical resection; age \> 20 years; patients with disease progression after at least one prior cytotoxic chemotherapy treated with nivolumab; * EGFR * mutation ‐ positive patients administered EGFR ‐ tyrosine kinase inhibitors prior to any cytotoxic chemotherapy; and patients with available ORR data of nivolumab according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1. 1. Patients were excluded if RheH had: a concomitant serious illness, such as myocardial infarction in the previous three months; uncontrolled angina pectoris, heart failure, uncontrolled diabetes mellitus, uncontrolled hypertension, interstitial pneumonia, or lung disease; an infection or other disease contraindicating chemotherapy; or were pregnant or breastfeeding. This study was approved by the institutional ethics committee of the Saitama Medical University International Medical Center. Treatment and efficacy evaluation {# tca13044 - sec - 0004} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Nivolumab was intravenously administered at 3 mg / kg every two weeks. A complete blood cell count, differential count, routine chemistry measurements, physical examination, and toxicity assessment were performed on a weekly basis. AcItw toxicity was graded according to Common Terminology Criteria for Adverse Events (CTCAE) version 4. 0. The tumor response was evaluated according to RECIST version 1. 1. [10] (# tca13044 - bib - 0010) {ref - type = " ref "} Statistical analysis {# tca13044 - sec - 0005} - - - - - - - - - - - - - - - - - - - - * P * \ <0. 05 igdicahed statistical significance. Fisher \ ' s exact tests were conducted to examine the association between the categorical variables. The Kaplan - - Meier method was used to estimate survival as a function of time, and survival differences were analyzed by log ‐ rank tests. PFS was defined as the time from the initiation of nivolumab therapy to tumor recurrence or death from any cause, while overall survival (OS) was defined as the time from the initiation of nivolumab therapy to death from any cause. Statistical analyses were performed using GraphPad Prism 4 and JMP 8. 0. Results {# tca13044 - sec - 0006} = = = = = = = Patient demographics {# tca13044 - sec - 0007} - - - - - - - - - - - - - - - - - - - - From February 2016 to December 2017, 152 patients with pretreated NSCLC were administered nivolumab. Twenty ‐ eight patients were excluded because of inadequate medical information or the absence of an evaluable target lesion. Thus, a total of 124 patients (* n * ~ males ~ = 93, * n * ~ females ~ = 31; median age: 69 years; range: 31 - - 85 years) were eligible for analysis. Patient characteristics are listed in Table [1] (# tca13044 - tbl - 0001) {ref - type = " table " }. A total of 99 patients had a smoking history. Clinical staging indicated that 27 patients had stage III disease, 77 had stage IV disease, and 20 patients developed recurrence after surgical resection. The patients were divided into RT and non ‐ RT groups. # # # # # # Comparison of demographics in patients treated with or without RT before nivolumab - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Variables All patients \ Patients administered any RT before Nivo (* n * = 66) Patients not administered RT before Nivo (* n * = 58) * P * (* n * = 124) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Age ≦ 69 / \> 69 65 / 59 36 / 30 29 / 29 0. 71 Gender Male / female 93 / 31 51 / 15 42 / 16 0. 54 Smoking Yes / no 99 / 25 52 / 14 47 / 11 0. 82 ECOG PS 0 / 1 - - 3 59 / 65 31 / 35 18 / 40 0. 09 Stage III / IV 27 / 97 20 / 46 7 / 51 0. 71 T factor T1 - - 2 / T3 - - 4 68 / 56 35 / 31 33 / 25 0. 58 N factor N0 / N1 ‐ 3 20 / 104 12 / 54 8 / 50 0. 62 Histology Adeno / non ‐ adeno 65 / 59 31 / 35 34 / 24 0. 21 * EGFR * mutation status Mutant / wild 14 / 104 10 / 51 4 / 53 0. 15 Nivo response CR or PR / SD or PD | Introduction {#tca13044-sec-0001} ============ Nivolumab, a PD‐1 antibody, is checkpoint inhibitor (ICI) that has been proven to be in patients with different tumor has been shown have an rate (ORR) of approximately in patients with previously treated non‐small cell lung cancer (NSCLC).[1](#tca13044-bib-0001){ref-type="ref"}, [2](#tca13044-bib-0002){ref-type="ref"} However, nivolumab is not effective in more than 40% of NSCLC who experience disease progression, despite nivolumab treatment. To improve the efficacy of this promising immunotherapy, additional modalities such as chemotherapy, radiotherapy (RT), or other ICIs may also be administered to patients in whom ICIs have not been completely effective. Interestingly, stimulates a systemic immune response the of tumor‐related antigens.[3](#tca13044-bib-0003){ref-type="ref"} Recent preclinical studies have demonstrated a synergistic tumor response with RT and the blockade of PD‐1.[4](#tca13044-bib-0004){ref-type="ref"}, [5](#tca13044-bib-0005){ref-type="ref"}, [6](#tca13044-bib-0006){ref-type="ref"} It is possible that tumor‐specific immunity is induced by radiation. Although RT plays important role in the local and elimination of tumors, also contributes to the induction of immune responses, and the immunosuppressive and immunostimulatory effects of RT.[6](#tca13044-bib-0006){ref-type="ref"} Radiation‐induced cell death causes a release of signals such as HMGB1, ATP, and HSP70, and the dendritic cells can stimulate activated CD8 T cells and T cells.[6](#tca13044-bib-0006){ref-type="ref"} In addition to activation, RT induces transforming growth factor‐β (TGF‐β), an immunosuppressive cytokine. To reduce the immunosuppressive functions, a combination RT and TGF‐β inhibitors has been identified as a valuable option settings.[6](#tca13044-bib-0006){ref-type="ref"} A experimental study demonstrated that fractionated RT increases PD‐L1 surface on tumor cells, suggesting a key rationale for the combination of RT with ICIs.[7](#tca13044-bib-0007){ref-type="ref"} Of the 98 registered in the KEYNOTE‐001 I trial, Shaverdian *et reported the duration survival (PFS) with pembrolizumab was significantly longer in patients previously administered RT than in those not treated with RT.[8](#tca13044-bib-0008){ref-type="ref"} Fiorica *et al.* also reported nivolumab treatment after hypofractionated RT improved the outcome 35 patients with pretreated or metastatic NSCLC.[9](#tca13044-bib-0009){ref-type="ref"} results suggest that previous RT clinically and immune reaction to ICIs, such as nivolumab or pembrolizumab. However, the synergistic effect of ICIs previous RT was not fully elucidated in these studies. the former study was a phase I trial, pembrolizumab was administered at different doses and The was limited by a small sample of only 35 patients.[9](#tca13044-bib-0009){ref-type="ref"} Analysis of these studies shows that immunotherapy after previous RT prolongs survival;[8](#tca13044-bib-0008){ref-type="ref"}, [9](#tca13044-bib-0009){ref-type="ref"} however, neither the ORRs of ICIs after previous RT nor the populations that might benefit from ICI treatment included. clinical data the effect of ICI administration after previous has been reported; therefore, we attempted to elucidate the synergistic antitumor effect of nivolumab after RT patients with previously treated NSCLC. Methods {#tca13044-sec-0002} ======= Patient eligibility and data collection {#tca13044-sec-0003} The eligibility for our retrospective analysis were: histologically cytologically proven advanced NSCLC with stage III or IV disease or recurrence after surgical resection; age \> 20 years; patients with disease progression after one prior cytotoxic chemotherapy with nivolumab; *EGFR* mutation‐positive patients administered EGFR‐tyrosine kinase inhibitors prior to any cytotoxic chemotherapy; patients with available ORR data of nivolumab according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Patients were excluded if had: a concomitant serious illness, such as myocardial infarction in the previous three months; uncontrolled angina pectoris, heart failure, uncontrolled diabetes uncontrolled hypertension, interstitial pneumonia, lung disease; an infection other disease contraindicating chemotherapy; or were pregnant or breastfeeding. This study was approved by the institutional ethics committee of the Saitama Medical University International Medical Center. Treatment and efficacy evaluation {#tca13044-sec-0004} --------------------------------- Nivolumab was intravenously at 3 mg/kg every two weeks. A complete blood cell count, differential routine chemistry measurements, physical examination, and toxicity assessment were performed on a weekly basis. Acute toxicity was according to Common Terminology Criteria for Adverse Events (CTCAE) version The response was evaluated according RECIST version 1.1.[10](#tca13044-bib-0010){ref-type="ref"} Statistical analysis -------------------- *P* \< indicated statistical significance. Fisher\'s exact tests were conducted to examine the association between the categorical variables. The Kaplan--Meier method used to survival a function of time, and survival differences were analyzed by log‐rank tests. PFS was defined as time from the initiation of nivolumab therapy to tumor recurrence or death from any cause, while overall survival (OS) was defined as the time from the initiation of nivolumab therapy death from any cause. analyses using GraphPad Prism 4 and JMP 8.0. Results {#tca13044-sec-0006} ======= Patient demographics {#tca13044-sec-0007} -------------------- From February 2016 to December 2017, 152 patients with pretreated NSCLC were administered nivolumab. Twenty‐eight patients were excluded because inadequate medical information or the absence of an evaluable target lesion. Thus, a total of 124 (*n* ~males~ = 93, *n* ~females~ = 31; median age: 69 years; range: 31--85 years) were eligible for analysis. Patient characteristics are listed in Table A total of patients had a smoking history. Clinical staging indicated that 27 patients had stage III disease, 77 had stage disease, 20 patients developed recurrence after surgical resection. The patients divided into RT and non‐RT groups. ###### Comparison of demographics in patients treated with or without before nivolumab ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Variables All patients\ Patients administered any RT before Nivo (*n* = 66) Patients not administered RT before (*n* = 58) *P* (*n* = 124) ---------------------------------------------------------- ----------------------------------------------------- ---------- Age 69/\> 69 65/59 29/29 0.71 Gender Male/female 93/31 51/15 42/16 0.54 Smoking Yes/no 99/25 52/14 47/11 0.82 ECOG PS 0/1--3 31/35 18/40 0.09 Stage III/IV 27/97 20/46 7/51 0.71 T factor T1--2/T3--4 68/56 35/31 33/25 0.58 N factor N0/N1‐3 20/104 12/54 8/50 0.62 Histology Adeno/non‐adeno 65/59 31/35 34/24 *EGFR* Mutant/wild 14/104 10/51 4/53 0.15 CR or PR/SD or | intRodUctioN {#TCa13044-SeC-0001}
============
NIVoluMAB, A Pd‐1 antIboDy, iS an immUnE cHeCKPOiNT iNhibItoR (ici) tHat HaS bEeN proVEN TO bE actiVe In pATIENTs wiTh SEvEraL difFerEnt tUmoR TyPes. nivOLuMaB hAs beEN sHOwN To HAVe aN oVeRALl resPonSe RAte (ORR) oF aPpROXimaTELy 20% IN patIentS With PreviouSlY TReaTed nON‐SmaLl ceLL lUNg cAnCeR (nsClC).[1](#Tca13044-BIb-0001){rEf-TyPe="rEf"}, [2](#TcA13044-Bib-0002){ReF-TypE="ref"} howEVER, nivOlUmAB is noT efFECTive iN mOrE ThaN 40% of NScLc PatIeNtS Who EXpErIENcE dIseASE PRoGrESsION, dEspIte NiVOLumAB TREAtMeNt. To imProVE THE efFIcaCY Of tHIS prOmISIng IMMuNothErapY, ADDITiONAL mODaLitIes sUCh As CHemothERaPY, RadIOTheRapy (rt), Or oTheR iCis may aLSo BE admINISteRED To patieNtS in wHOm icis havE not bEeN cOmPlEtEly EFfECTIVE. inTeresTingly, RT StImuLateS A SySTemIc iMmuNE RESpONSe aND cauSEs THe RElEASe oF TUMOr‐ReLaTed ANtIGENs.[3](#Tca13044-BIb-0003){rEf-Type="rEf"} rECENt pRECLiNICAl STUDIES haVE DeMOnstrATed a sYNeRgistIc tUMOr ReSpoNse wiTh Rt And The bLocKade OF Pd‐1.[4](#tCa13044-bIB-0004){ref-type="rEf"}, [5](#tCa13044-bIb-0005){REf-type="ReF"}, [6](#Tca13044-bIb-0006){reF-typE="ReF"} iT Is pOSSIbLE thAt TUmOr‐SpEciFIC ImmuNiTY is indUcED BY raDIaTiOn. alThouGH Rt PLAYS an iMpOrTaNt RoLE In tHE LoCaL ConTrOL And eLIminaTIoN Of tUMORS, iT alSo coNTRIBuTes TO tHe INduCTIOn Of anTITumOR imMUNe rESPoNses, anD THE immUnOsUPpreSSiVE ANd iMMUNOStiMUlATORy eFFecTS oF rT.[6](#tCA13044-bIb-0006){rEf-typE="ref"} raDiAtION‐InducED cELl deaTh causes A REleAsE OF dAnger siGnAls SUcH AS hmGb1, ATP, aNd hSp70, ANd THe DENDRItIC CelLs Can StIMuLaTe activAtED CD8 T CeLls ANd tUmor‐SPecIfic t cELLs.[6](#TcA13044-biB-0006){reF-TYpE="Ref"} in ADdITIOn to iMMuNE acTIVatIoN, rT IndUCes TRaNSFORMiNG growtH FactoR‐Β (tgf‐Β), aN IMMunOSUPPReSsIVE cYtokIne. tO reDUce the imMUnoSuPPreSsIVE FuNCtIOns, A ComBInAtioN Of rt and TGf‐Β InHiBitORs hAs BeEN IDENtiFieD As a vAlUABle OpTiON IN prECliNIcal SeTtiNGS.[6](#TcA13044-bib-0006){Ref-TyPe="ref"} A ReceNt exPErimEnTal stUdY DEmOnStRAted THAt fRActiONaTeD RT incrEASes pD‐L1 suRfaCE expRessION On tUmor cellS, SuggestinG A keY RATiOnalE foR ThE coMBInATiON OF rT WiTH iCIS.[7](#TcA13044-BiB-0007){rEF-Type="rEf"}
of the 98 PAtIENTS REgIsTEREd In the kEynOTE‐001 PhAse i trIal, sHaveRdiaN *eT al.* REPORtED that THe DUraTioN Of pROGReSsIOn‐fREe surVIVal (PFs) wItH PeMbROLIzumAb WAS SiGniFicANtlY LONGer In PatIEnTS pREvIOusly aDMINisTErEd RT tHAn IN thOse NoT treatEd WITh rt.[8](#tcA13044-bIB-0008){Ref-Type="ref"} FiORIca *et al.* Also rePortED THat nivoLUmAb TrEAtMent aFTEr hYpoFractIonaTeD rT ImProved ThE oUtCOME in 35 PatieNtS WiTH PReTrEaTeD oR METastAtIC NSclC.[9](#TcA13044-bIb-0009){Ref-TYpE="ref"} THeSe REsULts sUGgEsT tHaT prEvIOuS Rt cliNiCAlLy ImPRoveS tumoR rESpOnsE AnD IMMune reaCtION TO iCis, suCH aS nivoLUMAb oR PembroLizUMaB. HOWeVer, THe SyNerGistIC effecT of ICis AnD prEvioUS Rt wAs Not FullY elucIDAteD in thESe stuDIes. aS thE FORmEr studY wAs a PHAsE I trial, pEMbrOlIzUMaB was admINIStERed At dIfFEREnt dOsES aNd TreATMEnt delIVerIEs.[8](#tCa13044-BIb-0008){rEf-tyPE="reF"} tHE LattER STuDY WAS Limited By a SmALl sAMPlE of OnlY 35 PAtIENTs.[9](#tca13044-BIb-0009){rEf-TyPE="REF"} anAlySiS OF these STudieS Shows THAt iMMUnothERapY AftER PrEVIoUs rT prOLOnGS suRvIVal;[8](#Tca13044-Bib-0008){reF-tYpE="rEF"}, [9](#tca13044-bib-0009){ref-tYPe="REf"} howevER, neiTHer tHE oRrS OF iCis afTer pREVIoUs rT Nor THe POPulaTIOns ThAT MiGHT BenefIt froM ici trEATMeNt wEre InCluDEd. LITtLE DeTaIleD cLiNiCAl DAta of tHe eFfEct of ICi aDMiniStRATioN AFteR PreVIOUS Rt HaS Been rEpoRtEd; ThErefoRE, WE ATTeMPteD to eLuCidATE the pOTENtIaL syneRgiStic AntitUmor EffECT Of NIVoLumaB AFteR Rt IN paTiENtS wIth PREvIOuSlY treATed nsclC.
metHOdS {#Tca13044-sEC-0002}
=======
PaTIent ELigIBiLITy And DATa COlLeCtIon {#Tca13044-SEc-0003}
---------------------------------------
tHe eLIgibilITy CriTeRIA For oUR reTroSpecTIvE aNALySis WEre: histOLOgiCaLLy oR CyToLoGICaLLY prOveN aDvAnced NScLC witH StaGE iIi Or iv DISeaSe or rEcuRreNcE AFTeR sUrGiCAL rESECTiON; AGE \> 20 yeARs; paTiENTS wITh dISEaSe PROgrESSION afTEr aT leASt OnE pRIor CYtOToxIC CHEmOTherAPY TReated WitH NiVoluMAB; *EGfr* mutATiON‐PosiTIve pATientS aDminiStEReD EGFR‐tyROsinE KiNase iNHiBITorS PrIOr to aNy cYTOtOxiC cHemoTHErAPY; AnD paTIeNTs WItH avaiLaBlE orR DaTa Of niVOlumAB AcCorDinG To reSPONsE evaluaTIon CrIteRiA In soLId TUmORS (rEcIST) veRSIOn 1.1. PATIeNTS wERe eXcLuded If tHey had: A ConcOMItAnt sErIOUS ILLNeSS, suCh as mYocaRDiaL infARCTiOn IN tHe PREVIouS ThREe montHs; uNcoNtROlled angiNa pecTOrIs, heaRt faiLuRe, uNcONTRolled DIABEtes mELLITus, unCOntRollEd hyPerTEnsIOn, inTeRstITiAL PnEUMOnIa, Or lUnG diSeAsE; an inFeCTIoN oR otHER diSeaSe ContRaindICAtInG chEMothEraPy; or Were pregnAnT or bREasTfeEDInG. tHiS Study WAS APPROVED By tHe iNsTITutiOnal EThics CoMmiTtEe oF THE SaiTAMa MeDICAL unIveRsiTy iNternAtIOnAl MEDIcAl Center.
tReatmENt aND efficACy EvalUation {#Tca13044-sEc-0004}
---------------------------------
NIVOLumAb wAs INTraVeNoUSLy admiNISTeRED AT 3 mg/kG everY TWo wEEks. A ComplETE BlooD ceLl couNt, difFerenTiaL COunt, ROUtINe CHemISTRy mEaSUREmENtS, PhysIcAl ExaMinatIon, aND tOXicity asSesSMeNt wERE perfoRmED ON A weeKLY bASIs. AcUtE TOXICIty was gRaDEd AcCoRDinG TO COmmOn TermINOlogy CRitErIa for AdVerse EVenTs (cTcaE) versiON 4.0. The tuMoR ResPonsE waS EVaLuATED AccoRding To reciST vERSION 1.1.[10](#tcA13044-Bib-0010){REf-tYpe="REF"}
staTisticaL AnaLysIs {#tCa13044-Sec-0005}
--------------------
*P* \< 0.05 iNDICAteD StaTIsTicaL SiGNiFiCAncE. FisHER\'S exacT TesTS wEre cOnDUcTed tO eXAMIne the ASsocIATIOn bEtWeen tHE CAteGORiCaL VAriABles. THE KAPLaN--MEieR mEtHOD was USED to eStiMaTe SURvIvAL as A fUNCtIon of TIME, AND SuRVivAL DIFfeRencES Were AnAlYZeD By log‐RaNk TEsTS. pFS Was dEfinEd aS thE TImE FROM THe InITIAtION OF nIVOLUMAb THERAPY To tuMOr RECUrReNce or dEaTH From anY cAUse, whIlE OveRALL survIvAL (Os) WAS DeFiNEd AS the TiMe fROM thE InitiATIoN of NivOLuMab thERaPY TO dEath FROm aNY CAUSe. stAtiSTiCAL analYSES Were PerFORMEd usinG GrAPhpaD prism 4 aND Jmp 8.0.
rESuLTs {#Tca13044-seC-0006}
=======
PaTIent DEMogRaphics {#Tca13044-seC-0007}
--------------------
froM FEBruaRy 2016 to deCemBEr 2017, 152 patienTS WITh pRETrEAtED nsClC WeRE adMINisTeRED nIvolUMaB. TwenTY‐eight PatIenTS wERE eXcLuDED BeCAUsE Of InAdeqUAtE MEDical inFORMATIon OR THE AbSEnce Of AN EvaluABle TaRget lesIon. thus, a toTal Of 124 PaTientS (*n* ~MALeS~ = 93, *N* ~FEMaleS~ = 31; MEDIan aGE: 69 Years; rAnGE: 31--85 YeARs) weRe ELiGIble foR anaLysiS. PAtienT CHARActeRistICs aRE LISTED iN tABlE [1](#Tca13044-tBl-0001){ref-type="TAbLe"}. a TOTAl OF 99 PAtIeNTS HAD A SmOkInG hiSTory. cLinICAl STaging INDiCateD tHat 27 PaTIenTs hAd StAGe IIi dIseASe, 77 haD StaGe Iv DisEASE, aNd 20 PaTIEntS deVELOPed RECUrrENCE AfTER SurgiCAl resEcTIoN. THe pAtieNTS WErE dIVIdED InTO rt AnD Non‐rt GrOUPS.
######
cOMPaRison oF dEMogRApHIcS iN PATiEnts TREateD With or withoUT rt BeFoRe NiVoluMAB
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
VaRIAbleS aLl PATIEntS\ PatIeNTS ADMInisterED any rT BEfORE NivO (*N* = 66) patIEnTS NOT AdmiNiStEred rT beforE Nivo (*n* = 58) *p*
(*N* = 124)
---------------------------------------------------------- --------------- ----------------------------------------------------- ----------------------------------------------------- ----------
age
≦ 69/\> 69 65/59 36/30 29/29 0.71
GENDer
malE/FEmaLE 93/31 51/15 42/16 0.54
smOKiNG
yes/nO 99/25 52/14 47/11 0.82
ecoG PS
0/1--3 59/65 31/35 18/40 0.09
StAge
IiI/iV 27/97 20/46 7/51 0.71
t FaCtOr
T1--2/T3--4 68/56 35/31 33/25 0.58
n FACtoR
N0/n1‐3 20/104 12/54 8/50 0.62
hIstOLOGy
adEnO/Non‐adENo 65/59 31/35 34/24 0.21
*eGfR* mutATion sTatuS
MutaNt/WIld 14/104 10/51 4/53 0.15
nivO ReSpoNSE
Cr OR pr/SD OR PD | Introduction{#tca13044-sec-0001}============Nivolumab, a PD‐1 antibody, is an immune checkpoint inhibitor (ICI) that has been proven tobe active inpatients with several different tumortypes. Nivolumab has been shown to have an overall response rate (ORR) of approximately 20% in patients with previously treatednon‐small cell lung cancer (NSCLC).[1](#tca13044-bib-0001){ref-type="ref"},[2](#tca13044-bib-0002){ref-type="ref"} However, nivolumab is not effective in more than 40% of NSCLC patients who experience diseaseprogression, despite nivolumab treatment. Toimprove the efficacy of this promising immunotherapy,additional modalities such as chemotherapy, radiotherapy (RT), or other ICIs may also be administered to patients in whom ICIs have not been completely effective.Interestingly, RT stimulates a systemic immuneresponse and causes the release of tumor‐related antigens.[3](#tca13044-bib-0003){ref-type="ref"}Recent preclinical studies havedemonstrated a synergistic tumor responsewith RT and the blockade of PD‐1.[4](#tca13044-bib-0004){ref-type="ref"}, [5](#tca13044-bib-0005){ref-type="ref"}, [6](#tca13044-bib-0006){ref-type="ref"} It is possible that tumor‐specific immunity is induced by radiation. Although RT plays an important role inthe local controlandelimination of tumors, it alsocontributesto the induction of antitumor immune responses, and the immunosuppressive and immunostimulatory effects of RT.[6](#tca13044-bib-0006){ref-type="ref"} Radiation‐inducedcell death causes a release of danger signals such asHMGB1, ATP, and HSP70, andthedendritic cells can stimulate activatedCD8 T cells and tumor‐specific T cells.[6](#tca13044-bib-0006){ref-type="ref"} In addition to immune activation, RT induces transforming growth factor‐β (TGF‐β), an immunosuppressive cytokine.To reduce the immunosuppressivefunctions, a combination of RT and TGF‐β inhibitors has been identified asa valuableoption inpreclinical settings.[6](#tca13044-bib-0006){ref-type="ref"} A recent experimentalstudy demonstratedthat fractionated RT increases PD‐L1 surface expression on tumor cells, suggesting akey rationale for the combination of RT with ICIs.[7](#tca13044-bib-0007){ref-type="ref"} Of the 98 patients registered in the KEYNOTE‐001 phaseI trial, Shaverdian *et al.* reported that the duration of progression‐free survival (PFS) with pembrolizumab was significantly longer in patients previously administeredRT than inthose not treatedwith RT.[8](#tca13044-bib-0008){ref-type="ref"} Fiorica *et al.* also reported that nivolumabtreatment after hypofractionated RT improved the outcome in 35 patientswith pretreated or metastatic NSCLC.[9](#tca13044-bib-0009){ref-type="ref"} Theseresults suggest that previous RT clinically improves tumor response and immune reaction to ICIs, such asnivolumab or pembrolizumab. However,thesynergistic effectof ICIs andpreviousRTwas not fully elucidated in these studies. As the former study was a phase Itrial, pembrolizumab was administered at different doses and treatment deliveries.[8](#tca13044-bib-0008){ref-type="ref"} The latter study was limited by a small sample of only 35 patients.[9](#tca13044-bib-0009){ref-type="ref"} Analysis of these studies shows that immunotherapy after previous RT prolongs survival;[8](#tca13044-bib-0008){ref-type="ref"}, [9](#tca13044-bib-0009){ref-type="ref"} however, neither the ORRs of ICIs after previous RT nor the populationsthatmight benefit from ICI treatment were included. Littledetailed clinical data ofthe effect of ICI administration after previous RT hasbeenreported; therefore,we attempted toelucidate the potential synergistic antitumor effect of nivolumab after RT in patientswith previously treatedNSCLC. Methods {#tca13044-sec-0002} ======= Patient eligibility and data collection {#tca13044-sec-0003}--------------------------------------- The eligibility criteria for ourretrospective analysiswere:histologically orcytologically proven advanced NSCLC with stage IIIor IV disease or recurrence after surgical resection; age \> 20 years;patients with disease progressionafter at least one prior cytotoxic chemotherapytreated with nivolumab; *EGFR* mutation‐positive patients administered EGFR‐tyrosinekinase inhibitorsprior to any cytotoxic chemotherapy; and patients with available ORR dataof nivolumab according to ResponseEvaluation Criteria in Solid Tumors (RECIST) version 1.1. Patients were excluded if theyhad: aconcomitant serious illness, suchas myocardialinfarction in the previous three months; uncontrolled anginapectoris, heart failure, uncontrolled diabetes mellitus, uncontrolled hypertension, interstitial pneumonia, or lung disease; an infection or other disease contraindicating chemotherapy; orwere pregnant or breastfeeding. This study was approved by the institutionalethics committee of the Saitama Medical University International MedicalCenter. Treatment and efficacyevaluation {#tca13044-sec-0004}--------------------------------- Nivolumab was intravenously administered at 3 mg/kg every two weeks. A complete blood cell count, differential count,routine chemistry measurements, physical examination, and toxicity assessment wereperformedon a weekly basis. Acute toxicity was graded according to Common Terminology Criteria forAdverse Events (CTCAE) version 4.0. The tumor response was evaluated according to RECIST version 1.1.[10](#tca13044-bib-0010){ref-type="ref"} Statistical analysis {#tca13044-sec-0005} -------------------- *P* \< 0.05 indicated statistical significance. Fisher\'s exact tests were conducted to examinetheassociation betweenthe categorical variables. The Kaplan--Meier method wasused to estimate survival as a function of time, and survival differences were analyzed by log‐rank tests. PFS was defined as thetime from the initiation of nivolumab therapy totumor recurrence ordeathfromany cause,while overall survival (OS) was defined as the time fromthe initiation of nivolumab therapy to death from any cause. Statisticalanalyses were performed using GraphPadPrism 4 andJMP 8.0. Results {#tca13044-sec-0006} ======= Patient demographics {#tca13044-sec-0007}-------------------- FromFebruary 2016 to December 2017, 152 patients withpretreated NSCLC were administered nivolumab. Twenty‐eight patients were excludedbecauseof inadequate medical information or the absence of an evaluable target lesion. Thus, atotal of 124 patients (*n* ~males~= 93, *n* ~females~ =31;median age: 69 years; range: 31--85 years) were eligible for analysis. Patientcharacteristics are listedin Table [1](#tca13044-tbl-0001){ref-type="table"}. A total of 99 patients had a smoking history. Clinical staging indicated that 27 patients had stageIIIdisease, 77 had stageIV disease,and 20 patients developed recurrence after surgical resection. The patients were divided into RT and non‐RTgroups. ######Comparison of demographics in patients treated with or without RT before nivolumab ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Variables All patients\ Patients administered anyRT before Nivo (*n* = 66)Patients not administered RT before Nivo (*n* = 58) *P* (*n* = 124) ---------------------------------------------------------- --------------- ----------------------------------------------------- ----------------------------------------------------- ---------- Age ≦ 69/\>69 65/59 36/30 29/29 0.71 Gender Male/female93/31 51/15 42/16 0.54Smoking Yes/no99/25 52/1447/11 0.82ECOG PS 0/1--3 59/65 31/35 18/40 0.09 Stage III/IV 27/97 20/46 7/51 0.71 T factor T1--2/T3--468/5635/31 33/25 0.58 N factor N0/N1‐3 20/104 12/54 8/50 0.62 Histology Adeno/non‐adeno 65/5931/35 34/24 0.21 *EGFR* mutationstatus Mutant/wild 14/104 10/51 4/53 0.15 Nivo response CR or PR/SD or PD | Introduction {#tca13044-sec-0001} ============ Nivolumab, a PD‐1 antibody, is an immune checkpoint inhibitor _(ICI)_ that has _been_ proven to _be_ _active_ in patients _with_ several different tumor types. Nivolumab has been _shown_ _to_ _have_ _an_ overall response rate (ORR) _of_ approximately 20% _in_ patients with previously treated non‐small cell lung cancer (NSCLC).[1](#tca13044-bib-0001){ref-type="ref"}, _[2](#tca13044-bib-0002){ref-type="ref"}_ _However,_ _nivolumab_ is not _effective_ in more than 40% of _NSCLC_ patients who _experience_ disease progression, despite nivolumab treatment. To _improve_ the efficacy _of_ this promising immunotherapy, _additional_ modalities such as chemotherapy, radiotherapy (RT), _or_ other ICIs may also be administered to patients in whom ICIs have not _been_ completely effective. Interestingly, RT stimulates a _systemic_ immune _response_ and causes _the_ release of tumor‐related antigens.[3](#tca13044-bib-0003){ref-type="ref"} _Recent_ preclinical _studies_ have demonstrated a synergistic tumor response with RT and the blockade _of_ PD‐1.[4](#tca13044-bib-0004){ref-type="ref"}, _[5](#tca13044-bib-0005){ref-type="ref"},_ [6](#tca13044-bib-0006){ref-type="ref"} It is possible that tumor‐specific _immunity_ is induced by radiation. Although RT _plays_ an important role in the local control _and_ elimination of _tumors,_ it _also_ contributes to the induction of antitumor immune responses, and the _immunosuppressive_ _and_ immunostimulatory effects of RT.[6](#tca13044-bib-0006){ref-type="ref"} _Radiation‐induced_ cell death causes a release of danger signals such as HMGB1, ATP, and HSP70, and _the_ dendritic cells _can_ stimulate _activated_ _CD8_ T _cells_ and tumor‐specific T cells.[6](#tca13044-bib-0006){ref-type="ref"} _In_ addition to immune activation, _RT_ induces _transforming_ growth factor‐β (TGF‐β), _an_ _immunosuppressive_ cytokine. _To_ reduce the immunosuppressive functions, a combination of RT and TGF‐β inhibitors has been identified as a valuable option _in_ preclinical settings.[6](#tca13044-bib-0006){ref-type="ref"} _A_ recent experimental study demonstrated that fractionated RT _increases_ PD‐L1 surface expression on tumor cells, _suggesting_ a key _rationale_ for the _combination_ _of_ RT with ICIs.[7](#tca13044-bib-0007){ref-type="ref"} _Of_ the _98_ patients _registered_ in the KEYNOTE‐001 _phase_ I trial, Shaverdian *et al.* reported that the duration of progression‐free survival (PFS) with _pembrolizumab_ _was_ _significantly_ _longer_ in patients previously administered RT than _in_ those _not_ treated with RT.[8](#tca13044-bib-0008){ref-type="ref"} Fiorica *et al.* also reported that nivolumab treatment _after_ hypofractionated RT improved the outcome _in_ 35 patients with pretreated or metastatic NSCLC.[9](#tca13044-bib-0009){ref-type="ref"} These results suggest that previous RT _clinically_ improves tumor response and immune reaction to ICIs, such as nivolumab or pembrolizumab. However, the synergistic effect of _ICIs_ _and_ previous RT was _not_ fully elucidated in _these_ _studies._ _As_ the former _study_ was a phase I trial, pembrolizumab was administered at different doses and treatment deliveries.[8](#tca13044-bib-0008){ref-type="ref"} The latter study was limited _by_ _a_ small sample of only 35 patients.[9](#tca13044-bib-0009){ref-type="ref"} Analysis of these studies shows _that_ immunotherapy after _previous_ RT prolongs survival;[8](#tca13044-bib-0008){ref-type="ref"}, [9](#tca13044-bib-0009){ref-type="ref"} _however,_ neither the _ORRs_ of ICIs after previous RT nor the populations that might benefit _from_ ICI treatment were _included._ Little detailed clinical _data_ of the effect of ICI _administration_ after previous RT _has_ been reported; therefore, _we_ attempted to elucidate _the_ potential synergistic _antitumor_ effect of nivolumab _after_ _RT_ _in_ patients with previously treated NSCLC. Methods _{#tca13044-sec-0002}_ ======= Patient eligibility _and_ data collection {#tca13044-sec-0003} --------------------------------------- The eligibility criteria _for_ our _retrospective_ analysis were: histologically or cytologically _proven_ advanced NSCLC _with_ stage III or IV disease or recurrence after surgical resection; _age_ \> _20_ years; patients with disease progression after _at_ least one prior cytotoxic chemotherapy treated with nivolumab; *EGFR* mutation‐positive patients administered EGFR‐tyrosine kinase inhibitors prior to any cytotoxic _chemotherapy;_ _and_ patients with _available_ ORR _data_ of nivolumab according to Response Evaluation _Criteria_ in Solid _Tumors_ (RECIST) version _1.1._ _Patients_ were excluded _if_ they had: a _concomitant_ serious _illness,_ _such_ as myocardial infarction in the previous _three_ months; uncontrolled angina pectoris, heart failure, _uncontrolled_ _diabetes_ mellitus, uncontrolled hypertension, _interstitial_ pneumonia, or lung disease; an infection or other disease contraindicating chemotherapy; or were pregnant or _breastfeeding._ This study was approved by the institutional _ethics_ committee of _the_ Saitama Medical University International Medical Center. Treatment and _efficacy_ evaluation {#tca13044-sec-0004} --------------------------------- _Nivolumab_ was intravenously administered at 3 mg/kg _every_ two weeks. A complete blood cell count, differential count, _routine_ chemistry measurements, physical _examination,_ _and_ toxicity assessment were performed on a weekly basis. Acute toxicity was graded according _to_ Common Terminology Criteria for Adverse Events (CTCAE) _version_ 4.0. _The_ tumor response was evaluated according to RECIST version 1.1.[10](#tca13044-bib-0010){ref-type="ref"} Statistical analysis {#tca13044-sec-0005} -------------------- *P* \< _0.05_ indicated statistical significance. Fisher\'s exact _tests_ were conducted to examine the association between the categorical variables. The _Kaplan--Meier_ method was used _to_ estimate survival as a function _of_ time, _and_ survival differences were analyzed by log‐rank tests. PFS _was_ defined as the _time_ from the initiation of nivolumab therapy to tumor recurrence _or_ death from any _cause,_ while overall survival (OS) was _defined_ as the time from the _initiation_ _of_ nivolumab therapy to _death_ _from_ any cause. Statistical _analyses_ were performed using _GraphPad_ Prism 4 and _JMP_ 8.0. Results {#tca13044-sec-0006} ======= Patient _demographics_ {#tca13044-sec-0007} -------------------- From February _2016_ to December 2017, 152 patients with pretreated _NSCLC_ were _administered_ nivolumab. Twenty‐eight patients were excluded _because_ of _inadequate_ medical information or the _absence_ of an _evaluable_ target lesion. Thus, a total of 124 patients _(*n*_ _~males~_ = 93, _*n*_ ~females~ = 31; median age: _69_ years; range: 31--85 years) were eligible for analysis. _Patient_ characteristics _are_ listed in Table [1](#tca13044-tbl-0001){ref-type="table"}. A total of 99 patients _had_ a _smoking_ history. _Clinical_ staging _indicated_ that 27 patients _had_ stage _III_ disease, 77 had stage IV disease, and 20 patients developed recurrence after _surgical_ _resection._ The patients were divided into RT and non‐RT groups. ###### Comparison of _demographics_ _in_ _patients_ treated with or without _RT_ before _nivolumab_ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- _Variables_ All patients\ _Patients_ _administered_ any RT _before_ Nivo (*n* _=_ 66) Patients not administered RT before Nivo (*n* = _58)_ *P* _(*n*_ _=_ 124) ---------------------------------------------------------- _---------------_ ----------------------------------------------------- ----------------------------------------------------- ---------- Age ≦ 69/\> _69_ 65/59 36/30 29/29 0.71 _Gender_ Male/female 93/31 51/15 42/16 0.54 Smoking Yes/no 99/25 52/14 47/11 0.82 ECOG _PS_ _0/1--3_ 59/65 31/35 _18/40_ _0.09_ Stage III/IV 27/97 20/46 _7/51_ _0.71_ T factor _T1--2/T3--4_ _68/56_ 35/31 33/25 0.58 N factor N0/N1‐3 20/104 12/54 8/50 0.62 Histology _Adeno/non‐adeno_ 65/59 31/35 34/24 0.21 *EGFR* mutation status Mutant/wild 14/104 _10/51_ 4/53 0.15 Nivo response CR _or_ PR/SD or _PD_ |
Cedell[@bib1] described talus fracture in 4 young active sportsmen in 1974. These injuries are rare, and from then until now, only a few cases have been reported. The patient may arrive in the emergency department with pain and tenderness in the posteromedial region of the talus. However, most of the time, the injury\'s similarity to an ankle sprain on normal anteroposterior and lateral ankle radiographic views may contribute to misdiagnosis or delayed diagnosis.[@bib2]
Different treatments of this injury are supported in the literature: conservative treatment of nondisplaced or minimally displaced fractures, open reduction--internal fixation for displaced fractures, or fracture excision for malunion of displaced fractures that cause posteromedial ankle impingement.[@bib2], [@bib3]
Technique {#sec1}
=========
The diagnosis can be made using anteroposterior and lateral ankle radiographs ([Fig 1](#fig1){ref-type="fig"}) and computed tomography ([Fig 2](#fig2){ref-type="fig"}).Fig 1Preoperative anteroposterior and lateral radiographs of a right ankle showing a posteromedial talus fracture (arrow).Fig 2Preoperative 3-dimensional right ankle reconstruction showing a displaced posteromedial talus fracture with malunion (arrow).
Preoperative Setup {#sec1.1}
------------------
The patient is placed in the prone position with application of a thigh tourniquet to provide a bloodless surgical field. All bony prominences are padded. A small support is situated under the lower leg, making it possible to move the ankle freely. The ankle should be kept in a plantar-flexed position to relax the neurovascular bundle. The operative leg is prepared and draped in standard fashion. No traction is required.
Portal Placement {#sec1.2}
----------------
According to the recommendations of Van Dijk et al,[@bib4] the posterolateral portal is created through an incision at the level of or slightly above the tip of the lateral malleolus, just lateral to the Achilles tendon. Careful palpation of the Achilles tendon before portal insertion reduces the risk of damaging the tendon. Blunt dissection to the level of the joint is carried out with a small mosquito clamp directed anteriorly, pointing in the direction of the interdigital web space between the first and second toes. It is exchanged with a 4.5-mm arthroscope shaft (Arthrex, Naples, FL) with a blunt trocar.
The posteromedial portal is made just medial to the Achilles tendon, at the same level as the posterolateral portal. A mosquito clamp is introduced and directed toward the arthroscope shaft at 90° to touch this shaft; it should pass the neurovascular bundle without a problem. A 4.0-mm 30° arthroscope (Arthrex) is used through the posterolateral portal. The posterolateral portal is used as the viewing portal, and the posteromedial portal serves as the working portal ([Fig 3](#fig3){ref-type="fig"}). If there is any difficulty in determining whether the arthroscope is inserted correctly, confirmation of its position by fluoroscopy is recommended.Fig 3The patient is in the prone position. Portal placement is shown for the right ankle. Placement of the posterolateral portal is performed just lateral to the Achilles tendon at the level of the tip of the malleolus. A 4-mm 30° arthroscope is in position. The posteromedial portal is made just medial to the Achilles tendon, at the same level as the posterolateral portal. A shaver is introduced and directed toward the arthroscope shaft.
Surgical Correction of Posterior Ankle Impingement {#sec1.3}
--------------------------------------------------
Before the surgeon addresses the pathology, it is paramount to identify the flexor hallucis longus (FHL) and confirm that it moves with passive motion of the hallux. A base loop is passed around the tendon. The FHL is used as the medial border of the working area, which helps prevent injury to the neurovascular bundle. Then, debridement starts with an arthroscopic shaver (Arthrex) and radiofrequency device (Arthrex) inserted through the posteromedial portal.[@bib4]
When soft-tissue debridement is complete, the fracture malunion is clearly defined ([Fig 4](#fig4){ref-type="fig"}). Then, it is time to perform resection. This may include partial or total resection to correct the impingement ([Fig 5](#fig5){ref-type="fig"}). We perform partial resection of the fragment with a motorized 4.0-mm burr (Arthrex) to restore the normal shape of the posterior talus. We check, under arthroscopic control, that range of ankle motion is completely restored and posterior ankle impingement is corrected ([Fig 6](#fig6){ref-type="fig"}).Fig 4Arthroscopic view of the posteromedial aspect of the right ankle from the posterolateral portal. The fracture malunion (yellow arrow) and posterior ankle impingement are visualized. A shaver is introduced through the posteromedial portal. The flexor hallucis longus is on the medial side with a base loop around it (green arrow).Fig 5Arthroscopic view of the posteromedial aspect of the right ankle from the posterolateral portal. Partial resection of the fracture malunion (arrow) is performed. A burr is introduced through the posteromedial portal.Fig 6Arthroscopic view of the posteromedial aspect of the right ankle from the posterolateral portal. Final reshaping of the posterior talus is performed, and posterior ankle impingement (red arrow) is corrected. The flexor hallucis longus is on the medial side with a base loop around it (green arrow).
Complications {#sec1.4}
-------------
Although we have not encountered any complications with this procedure, the major complication is injury to the tibial neurovascular bundle. It is recommended to keep the ankle in plantar flexion during the procedure and keep the instruments lateral to the FHL.
Postoperative Rehabilitation Protocol {#sec1.5}
-------------------------------------
The patient is discharged on the day after surgery using crutches. Weight bearing is allowed. A rehabilitation program to gain full mobility and strength is followed. A control computed tomography scan is ordered 1 month after surgery ([Fig 7](#fig7){ref-type="fig"}). Return to sports is gradually permitted, based on functional demands, with the most demanding activities being avoided until 3 months after ankle arthroscopy.Fig 7Three-dimensional reconstruction 1 month after arthroscopic resection of a malunion of a posteromedial talus fracture showing correction of posterior ankle impingement (arrow).
A step-by-step summary of our technique is provided in [Table 1](#tbl1){ref-type="table"}. Pearls and pitfalls are presented in [Table 2](#tbl2){ref-type="table"}, and advantages and disadvantages are listed in [Table 3](#tbl3){ref-type="table"}. Key steps of the procedure are shown in [Video 1](#appsec1){ref-type="sec"}.Table 1Step-by-Step Summary of Arthroscopic Treatment of Malunion of Posteromedial Talus Fracture1.Position the patient in the prone position with application of a thigh tourniquet to provide a bloodless surgical field.2.Keep the ankle in a plantar-flexed position to relax the neurovascular bundle.3.Establish the posterolateral and posteromedial portals.4.Use a 4-mm 30° arthroscope inserted through the posterolateral portal.5.Identify the FHL and confirm that it moves with passive motion of the hallux.6.Start debridement with an arthroscopic shaver and radiofrequency device inserted through the posteromedial portal.7.Identify the fracture malunion.8.Perform partial resection of the fragment with a motorized 4.0-mm burr.9.Check, under arthroscopic control, that range of ankle motion is completely restored and posterior ankle impingement is corrected.[^1]Table 2Pearls and PitfallsPearlsPitfallsUse the prone position; pad all bony prominences.Pressure sores and lateral femoral cutaneous nerve neuropathyNote that no traction is required.No free movement of ankleKeep the ankle in a plantar-flexed position.Neurovascular bundle injuryPerform careful palpation of the Achilles tendon.Achilles tendon injuryIdentify the tip of the lateral malleolus for correct portal placement.Incorrect portal placementUse the arthroscope shaft as a guide to place the posteromedial portal.Neurovascular bundle injuryUse fluoroscopy to confirm the position and direction of the arthroscope if necessary.Incorrect portal placementIdentify the FHL by passive motion of the great toe and pass a base loop around the tendon.Neurovascular bundle injuryPerform a detailed examination of posterior ankle impingement.Incorrect portal placementIdentify the fracture malunion.No identification of fracture malunionReshape the posterior talus.No impingement correctionAvoid excessive bone resection.Excessive bone resection leading to ankle instabilityCheck complete ankle motion and correction of posterior ankle impingement.No improvement in clinical resultsTable 3Advantages and DisadvantagesAdvantages The procedure allows excellent access to the posterior ankle compartment. The procedure is minimally invasive. The procedure allows posterior ankle impingement to be corrected. The recovery time is shorter.Disadvantages The technique is challenging. Neurovascular bundle injury can occur. An | cedell [ @ bib1 ] described talus fracture in 4 young active sportsmen in 1974. these injuries are rare, and from then until now, only a few cases have been reported. the patient may arrive in the emergency department with pain and tenderness in the posteromedial region of the talus. however, most of the time, the injury \ ' s similarity to an ankle sprain on normal anteroposterior and lateral ankle radiographic exam may contribute to misdiagnosis or delayed diagnosis. [ @ 8 ] different treatments of elbow injury are supported in the literature : conservative treatment of nondisplaced or minimally displaced fractures, open reduction - - internal fixation vs displaced fractures, or fracture reduction for malunion of displaced fractures that cause posteromedial ankle impingement. [ @ bib2 ], [ @ bib3 ] technique { # sec1 } = = = = = = = = = the diagnosis can be made using anteroposterior and lateral ankle radiographs ( [ fig 1 ] ( # fig1 ) { ref - type = " fig " } ) and computed tomography ( [ fig 2 ] ( # fig2 ) { ref - type = " fig " } ). fig 1preoperative anteroposterior and lateral radiographs of a lower ankle showing a posteromedial talus fracture ( arrow ). fig 2preoperative 3 - dimensional right ankle reconstruction showing a large posteromedial talus fracture with malunion ( arrow ). preoperative setup { # sec1. 1 } - - - - - - - - - - - - - - - - - - the patient is placed in the prone position with application of a thigh tourniquet to provide a bloodless magnetic field. all lateral prominences are padded. a small support is situated under the lower leg, making it possible to move the ankle freely. the ankle should be kept in a plantar - flexed position to relax the neurovascular bundle. the operative leg is prepared and draped in standard fashion. no traction is required. portal placement { # sec1. 2 } - - - - - - - - - - - - - - - - according to the recommendations of van dijk et al, [ @ bib4 ] the posterolateral portal is created through an incision at the level below or slightly above the tip of the lateral malleolus, just lateral to the achilles tendon. careful palpation of the achilles tendon before portal insertion reduces the risk of damaging the tendon. blunt dissection to the level of the joint is carried out with a small mosquito clamp directed anteriorly, pointing in the direction of the interdigital web space between the first and second toes. it is exchanged with a 4. 5 - mm arthroscope shaft ( arthrex, naples, fl ) with a blunt trocar. the posteromedial portal is made just medial to the achilles tendon, at the same level as the posterolateral portal. a mosquito clamp is introduced and directed toward the arthroscope shaft at 90° to touch this shaft ; it should pass the neurovascular bundle without a problem. a 4. 0 - mm 30° arthroscope ( arthrex ) is used through the posterolateral portal. the posterolateral portal is used as the viewing portal, and the posteromedial portal serves as the working portal ( [ fig 3 ] ( # fig3 ) { ref - type = " fig " } ). if there is any difficulty in determining whether the arthroscope is inserted correctly, confirmation of its position by fluoroscopy is recommended. fig 3the patient is in the prone position. portal placement is shown for the right ankle. placement of the posterolateral portal is performed just lateral to the achilles tendon at the level of the tip of the malleolus. a 4 - mm 30° arthroscope is in position. the posteromedial portal is made just medial to the achilles tendon, at the same level as the posterolateral portal. a shaver is introduced and directed toward the arthroscope shaft. surgical correction of posterior ankle impingement { # sec1. 3 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - before the surgeon addresses the pathology, it is paramount to identify the flexor hallucis longus ( fhl ) and confirm that it moves with passive motion of the hallux. a base loop is passed around the tendon. the fhl is used as the medial border of the working area, which helps prevent injury to the neurovascular bundle. then, debridement starts with an arthroscopic shaver ( arthrex ) and radiofrequency device ( arthrex ) inserted through the posteromedial portal. [ @ bib4 ] when soft - tissue debridement is complete, the fracture malunion is clearly defined ( [ fig 4 ] ( # fig4 ) { ref - type = " fig " } ). then, it is time to perform resection. this may include partial or total resection to correct the impingement ( [ fig 5 ] ( # fig5 ) { ref - type = " fig " } ). we perform partial resection of the fragment with a motorized 4. 0 - mm burr ( arthrex ) to restore the normal shape of the posterior talus. we check, under arthroscopic control, that range of ankle motion is completely restored and posterior ankle impingement is corrected ( [ fig 6 ] ( # fig6 ) { ref - type = " fig " } ). fig 4arthroscopic view of the posteromedial aspect of the right ankle from the posterolateral portal. the fracture malunion ( yellow arrow ) and posterior ankle impingement are visualized. a shaver is introduced through the posteromedial portal. the flexor hallucis longus is on the medial side with a base loop around it ( green arrow ). fig 5arthroscopic view of the posteromedial aspect of the right ankle from the posterolateral portal. partial resection of the fracture malunion ( arrow ) is performed. a burr is introduced through the posteromedial portal. fig 6arthroscopic view of the posteromedial aspect of the right ankle from the posterolateral portal. final reshaping of the posterior talus is performed, and posterior ankle impingement ( red arrow ) is corrected. the flexor hallucis longus is on the medial side with a base loop around it ( green arrow ). complications { # sec1. 4 } - - - - - - - - - - - - - although we have not encountered any complications with this procedure, the major complication is injury to the tibial neurovascular bundle. it is recommended to keep the ankle in plantar flexion during the procedure and keep the instruments lateral to the fhl. postoperative rehabilitation protocol { # sec1. 5 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the patient is discharged on the day after surgery using crutches. weight bearing is allowed. a rehabilitation program to gain full mobility and strength is followed. a control computed tomography scan is ordered 1 month after surgery ( [ fig 7 ] ( # fig7 ) { ref - type = " fig " } ). return to sports is gradually permitted, based on functional demands, with the most demanding activities being avoided until 3 months after ankle arthroscopy. fig 7three - dimensional reconstruction 1 month after arthroscopic resection of a malunion of a posteromedial talus fracture showing correction of posterior ankle impingement ( arrow ). a step - by - step summary of our technique is provided in [ table 1 ] ( # tbl1 ) { ref - type = " table " }. pearls and pitfalls are presented in [ table 2 ] ( # tbl2 ) { ref - type = " table " }, and advantages and disadvantages are listed in [ table 3 ] ( # tbl3 ) { ref - type = " table " }. key steps of the procedure are shown in [ video 1 ] ( # appsec1 ) { ref - type = " sec " }. table 1step - by - step summary of arthroscopic treatment of malunion of posteromedial talus fracture1. position the patient in the prone position with application of a thigh tourniquet to provide a bloodless surgical field. 2. keep the ankle in a plantar - flexed position to relax the neurovascular bundle. 3. establish the posterolateral and posteromedial portals. 4. use a 4 - mm 30° arthroscope inserted through the posterolateral portal. 5. identify the fhl and confirm that it moves with passive motion of the hallux. 6. start debridement with an arthroscopic shaver and radiofrequency device inserted through the posteromedial portal. 7. identify the fracture malunion. 8. perform partial resection of the fragment with a motorized 4. 0 - mm burr. 9. check, under arthroscopic control, that range of ankle motion is completely restored and posterior ankle impingement is corrected. [ ^ 1 ] table 2pearls and pitfallspearlspitfallsuse the prone position ; pad all bony prominences. pressure sores and lateral femoral cutaneous nerve neuropathynote that no traction is required. no free movement of anklekeep the ankle in a plantar - flexed position. neurovascular bundle injuryperform careful palpation of the achilles tendon. achilles tendon injuryidentify the tip of the lateral malleolus for correct portal placement. incorrect portal placementuse the arthroscope shaft as a guide to place the posteromedial portal. neurovascular bundle injuryuse fluoroscopy to confirm the position and direction of the arthroscope if necessary. incorrect portal placementidentify the fhl by passive motion of the great toe and pass a base loop around the tendon. neurovascular bundle injuryperform a detailed examination of posterior ankle impingement. incorrect portal placementidentify the fracture malunion. no identification of fracture malunionreshape the posterior talus. no impingement correctionavoid excessive bone resection. excessive bone resection leading to ankle instabilitycheck complete ankle motion and correction of posterior ankle impingement. no improvement in clinical resultstable 3advantages and disadvantagesadvantages the procedure allows excellent access to the posterior ankle compartment. the procedure is minimally invasive. the procedure allows posterior ankle impingement to be corrected. the recovery time is shorter. disadvantages the technique is challenging. neurovascular bundle injury can occur. an | Cedell [@ bib1] described talus fracture in 4 young active sportsmen in 1974. These injuries are rare, and from then until now, only a few cases have been reported. The patient may arrive in the emergency department with pain and tenderness in the posteromedial region of the talus. However, most of the time, the injury \ ' s siJilaritj to an ankle sprain on normal anteroposterior and lateral ankle radiographic views may contribute to misdiagnosis or delayed diagnosis. [@ bib2] Different treatments of this injury are supported in the literature: conservative treatment of nondisplaced or minimally displaced fractures, open reduction - - internal fixation for displaced fractures, or fracture excision for malunion of displaced fractures that cause posteromedial ankle impingement. [@ bib2 ], [@ bib3] Technique {# sec1} = = = = = = = = = The diagnosis can be made using anteroposterior and lateral ankle radiographs ([ Fig 1] (# fig1) {ref - type = " fig "} ) and computed tomography ([ Fig 2] (# fig2) {ref - type = " fig "} ). Fig 1Preoperative anteroposterior and lateral radiographs of a right ankle showing a posteromedial talus fracture (arrow ). Fig 2Preoperative 3 - dimensional right ankle reconstruction showing a displaced posteromedial talus fracture with malunion (arrow ). Preoperative Setup {# sec1. 1} - - - - - - - - - - - - - - - - - - The patient is placed in the prone position with application of a thigh tourniquet to provide a bloodless surgical field. All bony prominences are padded. A small support is situated under the lower leg, making it possible to move the ankle freely. The ankle should be kept in a plantar - flexed position to relax the neurovascular bundle. The operative leg is prepared and draped in standard fashion. No traction is required. Portal Placement {# sec1. 2} - - - - - - - - - - - - - - - - According to the recommendations of Van Dijk et al, [@ bib4] the posterolateral portal is created through an incision at the level of or slightly above the tip of the lateral malleolus, just lateral to the Achilles tendon. Careful palpation of the Achilles tendon before portal insertion reduces the risk of damaging the tendon. Blunt dissection to the level of the joint is carried out with a small mosquito clamp directed anteriorly, pointing in the direction of the interdigital web space between the first and second toes. It is exchanged with a 4. 5 - mm arthroscope shaft (Arthrex, Naples, FL) with a blunt trocar. The posteromedial portal is made just medial to the Achilles tendon, at the same level as the posterolateral portal. A mosquito clamp is introduced and directed toward the arthroscope shaft at 90 ° to touch this shaft; it should pass the neurovasFulZr bundle without a problem. A 4. 0 - mm 30 ° arthroscope (Arthrex) is used through the posterolateral portal. The posterolateral portal is used as the viewing portal, and the posteromedial portal serves as the working portal ([ Fig 3] (# fig3) {ref - type = " fig "} ). If there is any difficulty in determining whether the arthroscope is inserted correctly, confirmation of its position by fluoroscopy is recommended. Fig 3The patient is in the prone position. Portal placement is shown for the right ankle. Placement of the posterolateral portal is performed just lateral to the Achilles tendon at the level of the tip of the malleolus. A 4 - mm 30 ° arthroscope is in position. The posteromedial portal is made just medial to the Achilles tendon, at the same level as the posterolateral portal. A shaver is introduced and directed toward the arthroscope shaft. Surgical Correction of Posterior Ankle Impingement {# sec1. 3} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Before the surgeon addresses the pathology, it is paramount to identify the flexor hallucis longus (FHL) and confirm that it moves with passive motion of the hallux. A NaDe loop is passed around the tendon. The FHL is used as the medial border of the working area, which helps prevent injury to the neurovascular bundle. Then, debridement starts with an arthroscopic shaver (Arthrex) and radiofrequency device (Arthrex) inserted through the posteromedial portal. [@ bib4] WhFB soft - tissue debridement is complete, the fracture malunion is clearly defined ([ Fig 4] (# fig4) {ref - type = " fig "} ). Then, it is time to perform resection. This may include partial or total resection to correct the impingement ([ Fig 5] (# fig5) {ref - type = " fig "} ). We perform partial resection of the fragment Qi$h a motorized 4. 0 - mm burr (Arthrex) to restore the normal shape of the posterior talus. We check, under arthroscopic control, that range of ankle motion is completely restoTee and posterior ankle impingement is corrected ([ Fig 6] (# fig6) {ref - type = " fig "} ). Fig 4Arthroscopic view of the posteromedial aspect of the right ankle from the posterolateral portal. The fracture malunion (yellow arrow) and posterior ankle impingement are visualized. A shaver is introduced through the posteromedial portal. The flexor hallucis longus is on the medial side with a base loop around it (green arrow ). Fig 5Arthroscopic view of the posteromedial aspect of the right ankle from the posterolateral portal. Partial resection of the fracture malunion (arrow) is performed. A burr is introduced through the posteromedial portal. Fig 6Arthroscopic view of the posteromedial aspect of the right ankle from the posterolateral portal. Final reshaping of the posterior talus is performed, and posterior ankle impingement (red arrow) is corrected. The flexor hallucis longus is on the medial side with a base loop around it (green arrow ). Complications {# sec1. 4} - - - - - - - - - - - - - Although we have not encountered any complications with this procedure, the major complication is injury to the tibial neurovascular bundle. It is recommended to keep the ankle in plantar flexion during the procedure and keep the instruments lateral to the FHL. Postoperative Rehabilitation Protocol {# sec1. 5} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The patient is discharged on the day after surgery using crutches. Weight bearing is allowed. A rehabilitation program to gain full mobility and strength is followed. A control computed tomography scan is ordered 1 month after surgery ([ Fig 7] (# fig7) {ref - type = " fig "} ). Return to sports is gradually permitted, based on functional demands, with the most demanding activities being avoided until 3 months after ankle arthroscopy. Fig 7Three - dimensional reconstruction 1 mKMth after arthroscopic resection of a malunion of a posteromedial talus fracture showing correction of posterior ankle impingement (arrow ). A step - by - step summary of our technique is provided in [Table 1] (# tbl1) {ref - type = " table " }. Pearls and pitfalls are presented in [Table 2] (# Gbl@) {ref - type = " table " }, and advantages and disadvantages are listed in [Table 3] (# tbl3) {ref - type = " table " }. Key steps of the procedure are shown in [Video 1] (# appsec1) {ref - type = " sec " }. Table 1Step - by - Step Summary of Arthroscopic Treatment of Malunion of Posteromedial Talus Fracture1. Position the patient in the prone position with application of a thigh tourniquet to provide a bloodless surgkcxl field. 2. Keep the ankle in a plantar - flexed position to relax the neurovascular bundle. 3. Establish the posterolateral and posteromedial portals. 4. Use a 4 - mm 30 ° arthroscope inserted through the posterolateral portal. 5. Identify the FHL and confirm that it moves Aiyh passive motion of the hallux. 6. Start debridement with an arthroscopic shaver and radiofrequency device inserted through the posteromedial portal. 7. Identify the fracture malunion. 8. Perform partial resection of the fragment with a motorized 4. 0 - mm burr. 9. Check, under arthroscopic control, that range of ankle motion is completely restored and posterior ankle impingement is corrected. [^ 1] Table 2Pearls and PitfallsPearlsPitfallsUse the prone position; pad all bony prominences. Pressure sores and lateral femoral cutaneous nerve neuropathyNote that no traction is required. No free movement of ankleKeep the ankle in a plantar - flexed position. Neurovascular bundle injuryPerform careful palpation of the Achilles tendon. Achilles tendon injuryIdentify the tip of the lateral malleolus for correct portal placement. Incorrect portal placementUse the arthroscope shaft as a guide to place the posteromedial portal. Neurovascular bundle injuryUse fluoroscopy to confirm the position and direction of the arthroscope if necessary. Incorrect portal placementIdentify the FHL by passive motion of the great toe and pass a base loop around the tendon. Neurovascular bundle injuryPerform a detailed examination of posterior ankle impingement. Incorrect portal placementIdentify the fracture malunion. No identification of fracture malunionReshape the posterior talus. No impingement correctionAvoid excessive bone resection. Excessive bone resection leading to ankle instabilityCheck complete ankle motion and correction of posterior ankle impingement. No improvement in clinical resultsTable 3Advantages and DisadvantagesAdvantages The procedure allows excellent access to the posterior ankle compartment. The procedure is minimally invasive. The procedure allows posterior ankle impingement to be corrected. The recovery time is shorter. Disadvantages The technique is challenging. Neurovascular bundle injury can occur. An | Cedell[@bib1] described talus fracture young active sportsmen in 1974. These injuries are and from then until now, only a few cases been reported. The patient may arrive in the emergency department with pain and tenderness in the posteromedial of talus. However, most of the time, the injury\'s similarity to an ankle sprain on normal anteroposterior and lateral ankle radiographic views may contribute to misdiagnosis or delayed diagnosis.[@bib2] Different treatments of this injury supported in the literature: conservative treatment of nondisplaced or minimally displaced fractures, open fixation for displaced fractures, or fracture excision for malunion of displaced that cause posteromedial ankle impingement.[@bib2], [@bib3] Technique ========= The diagnosis can be using anteroposterior and lateral ankle radiographs ([Fig and computed tomography ([Fig 2](#fig2){ref-type="fig"}).Fig 1Preoperative anteroposterior and lateral radiographs of a right ankle showing a posteromedial talus fracture 2Preoperative 3-dimensional right ankle reconstruction showing a displaced posteromedial talus fracture with malunion (arrow). Preoperative Setup ------------------ The patient is placed prone position with application of a thigh tourniquet to provide a bloodless surgical field. All bony prominences padded. A small support is situated under leg, making it possible to move the ankle The ankle should be kept in plantar-flexed position to relax the neurovascular bundle. The operative leg is prepared and draped in standard fashion. No traction is required. Portal Placement {#sec1.2} ---------------- According to the recommendations of Dijk et al,[@bib4] the posterolateral portal is created through an incision at the or slightly above the tip of the lateral malleolus, just lateral to Achilles tendon. Careful palpation the Achilles tendon before insertion reduces the risk of damaging the tendon. Blunt dissection to the level of joint is carried out with a small mosquito clamp directed anteriorly, in the direction of the interdigital web space between the first and second toes. It is exchanged with a 4.5-mm arthroscope shaft (Arthrex, Naples, with a blunt trocar. The posteromedial portal is made just medial to the Achilles tendon, at the same level as the posterolateral portal. A mosquito clamp introduced and directed toward the arthroscope shaft at 90° touch this shaft; it should the neurovascular bundle without a problem. A 4.0-mm 30° (Arthrex) through the portal. The posterolateral portal is as the viewing the portal serves as the working portal ([Fig 3](#fig3){ref-type="fig"}). If there is difficulty in determining whether the arthroscope is inserted correctly, confirmation of its position by fluoroscopy is recommended.Fig 3The patient in the prone position. Portal placement is shown for the right ankle. Placement of the posterolateral portal is performed just lateral to the Achilles tendon at the level of the tip of the malleolus. 30° arthroscope is in position. posteromedial portal is just to the tendon, the same level as the posterolateral portal. A shaver introduced and directed the arthroscope shaft. Surgical Correction of Posterior Ankle {#sec1.3} -------------------------------------------------- Before the surgeon addresses the pathology, it is paramount to identify the flexor hallucis longus (FHL) and confirm that it moves with passive motion of the base loop is passed around tendon. The FHL is used as the medial border the working area, which helps prevent injury to the neurovascular bundle. Then, debridement starts with an arthroscopic shaver (Arthrex) and radiofrequency device (Arthrex) inserted the posteromedial When soft-tissue debridement complete, the fracture malunion is clearly defined ([Fig 4](#fig4){ref-type="fig"}). Then, it is time to perform resection. This may include partial or total resection correct the impingement ([Fig 5](#fig5){ref-type="fig"}). We perform partial of the fragment a motorized 4.0-mm burr (Arthrex) to restore the normal shape of the posterior talus. We check, under arthroscopic control, that range of ankle motion is completely restored and ankle impingement is corrected ([Fig 6](#fig6){ref-type="fig"}).Fig 4Arthroscopic view of the posteromedial aspect of the right ankle from the posterolateral The fracture malunion (yellow and posterior ankle impingement are visualized. A is introduced through the posteromedial portal. The flexor hallucis longus is on the medial side with a base loop around it (green arrow).Fig 5Arthroscopic view of the posteromedial aspect of the right ankle the posterolateral portal. Partial resection of the fracture malunion (arrow) is performed. burr is introduced through the posteromedial portal.Fig 6Arthroscopic view of the posteromedial aspect of the right ankle from the posterolateral portal. Final reshaping of the posterior talus is performed, and posterior ankle (red arrow) is corrected. The hallucis longus is on the side with a base loop around it (green arrow). {#sec1.4} ------------- Although we have not encountered any complications with this the major complication is the tibial neurovascular bundle. It to keep ankle in plantar flexion during the procedure and keep the instruments to FHL. Postoperative Rehabilitation Protocol {#sec1.5} ------------------------------------- The patient is discharged on the day surgery using crutches. Weight bearing is allowed. A rehabilitation program to gain full mobility and strength is A control computed tomography scan ordered 1 after ([Fig 7](#fig7){ref-type="fig"}). Return to is gradually permitted, on functional demands, with the most demanding activities being avoided until months after ankle 7Three-dimensional reconstruction 1 month after arthroscopic of a malunion of a posteromedial talus fracture showing correction of posterior ankle impingement (arrow). A step-by-step summary of our technique is provided in [Table 1](#tbl1){ref-type="table"}. Pearls and pitfalls are presented in [Table 2](#tbl2){ref-type="table"}, and advantages and disadvantages listed [Table Key steps of the procedure are shown in [Video 1](#appsec1){ref-type="sec"}.Table 1Step-by-Step Summary of Arthroscopic Treatment of Malunion of Posteromedial Talus Fracture1.Position the patient in the prone position with application of a thigh tourniquet to a bloodless surgical field.2.Keep the ankle in plantar-flexed position to relax the neurovascular bundle.3.Establish the posterolateral and posteromedial portals.4.Use a 4-mm 30° arthroscope inserted through the posterolateral portal.5.Identify the FHL and confirm that it moves with passive motion of the hallux.6.Start debridement an arthroscopic and radiofrequency device inserted through the posteromedial the fracture malunion.8.Perform resection of the fragment with a motorized 4.0-mm burr.9.Check, under that range of ankle motion is completely restored and posterior ankle impingement is corrected.[^1]Table and PitfallsPearlsPitfallsUse the position; pad all bony sores and lateral nerve neuropathyNote that no traction is required.No free movement of ankleKeep the in a plantar-flexed position.Neurovascular bundle careful palpation the Achilles tendon.Achilles tendon the tip the lateral malleolus for correct portal placement.Incorrect portal placementUse the arthroscope shaft as a guide to place the posteromedial portal.Neurovascular injuryUse to confirm the position and direction of the arthroscope if necessary.Incorrect placementIdentify the FHL passive motion of great toe and pass a loop around the bundle injuryPerform a detailed examination of posterior ankle impingement.Incorrect portal placementIdentify the fracture malunion.No identification of fracture malunionReshape the posterior talus.No impingement correctionAvoid excessive bone resection.Excessive bone resection leading to ankle instabilityCheck complete ankle motion and correction of ankle impingement.No improvement in clinical resultsTable 3Advantages and DisadvantagesAdvantages The procedure allows excellent access to the posterior ankle compartment. The procedure minimally invasive. The procedure allows ankle impingement be corrected. The recovery time is shorter.Disadvantages The technique is challenging. bundle injury occur. An | cEdell[@BiB1] DEScrIbeD taLuS FRActurE in 4 yOuNG actiVE SpORTsMeN In 1974. tHESE InjUries are rAre, And FROm tHen UnTIl now, onLY a Few CASes HAVE BEeN rEPOrted. The PAtIeNT MAY arRIVE In ThE eMeRgeNcy DeParTmeNT wiTH Pain AnD tenDERnEss In tHE POsTErOMediAL RegION oF thE talus. hOweVEr, mOst Of tHE tIME, thE iNJURy\'S SIMILarItY TO an ANkLe SpRAIN oN NorMAL ANtEroPosTEriOr aNd LatErAl anKlE RADioGrAPHIc VIews May CoNTriBute To MiSdiaGnosiS or deLaYed diAgNoSIs.[@BIB2]
dIFFeREnT TREaTMENTS OF This iNjURY ARE suPPorTed IN tHe LItERatuRe: CONSerVAtive tReaTMENT of noNdISpLACED OR miNImallY DIsPLACeD FRactUrES, opeN rEdUcTIOn--INTERnAl FIXATIoN fOr DIsPlaced fractures, OR fRActURE EXCisiOn FOr maLuNIoN Of displACed FraCtURes thaT caUSe poStErOMediAL ANkle IMpINgEmeNT.[@biB2], [@BiB3]
teCHNique {#SEc1}
=========
tHe dIAgNoSIS CAn BE MAdE usiNG anTEropoSTeriOr And LatERaL AnKLe raDiograpHS ([FIG 1](#fiG1){ReF-TYpe="fiG"}) aNd cOMPutEd TOMoGrAphY ([FIG 2](#fIG2){REF-TyPE="fIg"}).fiG 1PreoPeratIve AnTeRoPOsteRIOr AnD LATeraL RadioGraphs oF A RiGht ANKle SHowinG a PoSTeROmedial TaLuS FRactURe (ARrOw).FIG 2pREOPERatIve 3-diMEnsionaL RIgHt AnKle recOnsTRuctION showIng A diSPlaCed POsTERomEdIAL talUs fraCTUre wIth MALUNIOn (aRroW).
pReOpeRAtivE SeTUP {#Sec1.1}
------------------
the PATIEnT IS pLAceD IN The ProNE POSItiON WITh aPPLICATioN OF A thIGH tourNIquET TO prOvIDE a BLOOdLESs sURgiCaL fieLD. aLL BONy PrOMINeNCes ArE pADDED. A smalL SUppORT iS SItuATEd UndER tHE loWER Leg, maKInG It POSSiBle To MOve tHe AnKLE fREeLy. ThE anKlE ShOUld BE KEpt iN A PlantaR-fLexeD PosITiON tO rElax tHe nEUROVAsCUlAR bundlE. thE OpERAtiVE lEg iS PreParED AnD drApED IN Standard FaShioN. No tRACtiOn is reQUIrED.
pOrTal pLACeMENt {#seC1.2}
----------------
aCCording To THE reCommeNdAtIONS Of VAn dIJK et aL,[@biB4] thE POstEROLATerAl porTal IS cREATEd THrough an iNCIsIOn AT tHE LEVEl Of Or slIGHTly aBovE The tIP oF tHe latERal malLEOluS, JusT LaTeRaL tO tHe aCHilLES TENdoN. CAReFUl pALPAtIon of ThE achiLLeS tEnDon bEFORe PoRTAl insertIoN ReduCES the rISK oF dAMagInG THE teNDoN. BluNT DIsSEcTion to tHE lEvEL Of thE JoinT IS cArRIEd OuT wItH A smalL MoSquIto cLAMp dIrEcted ANtERIorlY, POINTINg In THe DIrECTIon OF The iNTERdIGItAl wEB SPAcE beTwEen thE FIrST aNd SeCONd ToeS. It iS ExCHAnGed wIth A 4.5-mm arthRoScopE ShafT (aRtHrex, NAplEs, Fl) WIth a BLUNt TroCAr.
tHe poSTeRomEdial Portal IS MAdE juST MeDIAl tO thE ACHiLLes tEndOn, AT THE SaME leveL AS the poSteRoLateRal POrTaL. A MoSQuitO CLaMP Is INtRODuCEd aNd DiREcteD towaRD thE arThrOsCope ShAFT AT 90° TO tOUcH tHIs sHaft; It SHoULD PasS ThE NeuroVAsCULAR BUNdlE WithOUT A PRoblEm. A 4.0-Mm 30° ArThrOsCope (arthrEx) Is uSEd ThRouGh the POSTeRoLaTERal pOrtaL. THe pOsteROLaTeraL PORtaL IS useD As ThE vIeWIng porTaL, And ThE PoSTerOMedIAL POrtAl sErvEs as The WORKiNG pORTAL ([fIg 3](#fiG3){REF-TYpe="fig"}). If ThERE IS any DifFiCULTY In DeteRMiNING whetHer tHE artHROscOpE iS INsErTEd coRRECtly, cOnfIRMATIon OF ITS pOsition by flUoROSCoPy is REcOmmeNded.FIg 3The PATiENt iS in tHe pROnE POSItiON. POrtAL placEMEnT Is SHown fOR THe rIghT aNKlE. PLaCEmENT oF The posteROLATErAL porTaL Is PErFORmeD jusT LatErAl To ThE aChIlLeS teNdOn At ThE LeVeL OF THe tiP Of The maLLEoluS. a 4-mm 30° ArTHroSCOPe Is iN poSItIon. the POsTerOMEdiAl poRTAl iS mAdE jusT mEDiaL to tHE AcHilLes TenDon, AT thE SAMe LeVel as the PosTERolAtERAl portAL. a sHaVER iS inTROdUCeD AnD DirECTeD tOWArD the aRThrOscope sHaFT.
SUrgiCAL corRECTIon Of PosTErIor ANKlE IMpiNgeMent {#SeC1.3}
--------------------------------------------------
BEFoRe THE sUrGEon AddreSsES ThE PATHoLogy, IT IS parAmOuNt To IdENtIfY the FLeXor HalluCiS lOnGUs (FhL) aND cONFIRm THat It mOVES wiTh PaSsivE MotiOn oF tHE hAlLux. a base LooP iS Passed aROuNd The tendon. thE fhl iS USed aS ThE MEDIAl BOrDer of tHe woRkING AREA, WHICH helps pReVENT inJURY To tHE nEuRoVaSCulaR bunDlE. THEN, dEBrIdEMeNt StarTS With aN aRthrosCoPIC shAvER (ArthrEx) anD radIofrequEnCy DevIcE (artHrEx) InseRtEd THroUGh the posTEroMeDiAL pOrtAL.[@bib4]
WHEn soFt-tissue dEbRIdEmENT iS cOmpLetE, tHE fRaCtURe MAlUNion iS cLEARly defiNeD ([Fig 4](#Fig4){reF-TYpE="fiG"}). ThEn, IT is tIMe To PErFOrm ReseCTioN. This mAY INCLude PARtIal or tOtAL rEsEcTiON To coRRect tHE impingeMenT ([fIg 5](#Fig5){reF-tYpE="fiG"}). we PERforM PartiaL resECtiON of THE FrAgment WitH a mOtorIZeD 4.0-MM burr (ARTHRex) tO REStOre the nORmAl sHAPE OF thE posterIor TAluS. We chEcK, UNder arthRosCoPIC cONTrOL, THAT RANgE Of ankLe MOtiON Is cOmPLETEly reSTORED aNd pOstErIOR aNkle iMpINGeMENT Is corrECtED ([FIg 6](#fIG6){ref-TYpE="FIG"}).FiG 4arthRoSCopic vIew of THE POsTErOMEdial AsPEct oF THe rIGHt ANKLe FROM tHE PosTeRolateRAl PortAl. tHe fRActUre mALUNiOn (YeLloW arRoW) aNd POsTeRiOR anklE impINgEmenT aRe vISuAlized. A sHaVeR Is INtroDUceD ThrOuGh tHE PosTeRomeDIAl pOrtAL. the FlEXoR hallUCiS LONgUS iS ON the MedIaL SIdE with A bAse LOOp AroUND iT (greEn ArRoW).Fig 5ARThrOsCOPIC vIeW of THe pOsteRoMEDiAL Aspect OF THE riGHt ANkLe fRom ThE posTeROLAteRAL porTaL. PaRTIal rESecTIoN Of The FrACTURE MaLUnION (arROw) IS pErfOrMED. a burR Is InTrodUced tHroUgh ThE PosTEromEDial pOrTal.fIG 6aRtHRoscOPiC viEw OF thE PoSteRomeDIAL aSpecT OF THE riGhT ankle frOM THE pOsTeROLAteral PortAl. finAL ReshaPinG oF tHE pOSTeRiOr TAluS Is PERFoRMED, AND PostErIOr AnkLE IMpiNgeMENT (Red ARrow) is COrrEcted. thE FleXoR HaLLUcis lonGUS iS on ThE MeDIAL SiDE wItH a bASe loOp aRounD it (GrEen aRrOW).
COMPLIcATioNS {#SeC1.4}
-------------
AlthOUGh WE HaVe Not EnCouNteREd any cOMPlicATiOns wITH ThIs pROceduRE, THE MaJor CoMpLICAtIoN IS iNjURY TO tHe TIBIAL NEuROvAScuLAr buNdLE. iT is RECOmmendEd to keEp The aNkLE in PLantaR FLExioN DuriNG THE PrOceDURE And keep thE InsTRuMEnTS lAteRAL TO tHe FHL.
pOStOPERAtIVe RehAbilitATiON pRoTOcoL {#seC1.5}
-------------------------------------
tHE pATiEnT IS diSCHARged oN tHE daY aFtEr SURgery uSiNG cRuTcHeS. weighT bEArInG IS alLOWED. A rEHABIliTATioN prOGRAm to Gain Full mobILiTy aND sTrEngtH is follOwed. A CoNtrol ComputEd TOMogRApHy SCAn IS ordeREd 1 MONTh afteR sUrgEry ([FIg 7](#fig7){REF-TypE="fIg"}). RetURn to spOrTs IS GRADUALly pERmitTeD, bAseD ON FUnCtIOnAl DEMaNds, wiTH THe MosT deMANDINg actIviTieS beinG AVoIDEd unTIL 3 MontHs AFtEr ANKLe ARthrOScOPy.FiG 7Three-DIMEnSioNaL ReConSTruCTiON 1 mONTH AFteR ArtHrOSCopic ReSEctiON of a MAlUNIOn of a PoStEROMedIAL TaLuS fRACtUrE showING CORreCTion oF poStERIOr aNKlE impingeMENT (arRow).
A stEp-bY-STeP SumMARy OF OUR tEChnIQue IS PRoVIDed In [tabLe 1](#tBL1){ReF-TyPe="taBle"}. pearlS aND PItfalls aRE PReSeNTEd IN [tABLE 2](#tbl2){rEf-typE="Table"}, aND aDvAntAGeS AnD diSaDVAnTAgeS Are LiSTeD IN [tAble 3](#TBL3){ReF-tYPe="tAble"}. KeY stEPS OF thE ProceDUre are sHOWn In [vIDEo 1](#ApPsEc1){rEf-tYPe="sEC"}.tAbLe 1sTEP-By-STEp SuMmary Of aRtHrOSCOpiC trEatmeNT Of mALuNIon of poSTERoMedial TAlus FRacTURe1.POsitiOn tHe pAtiEnT in the PrOnE pOSiTIOn WItH APpLicATION Of a tHiGH TourNIQueT to PRoVIdE A BloOdLess SURgicAL FIeld.2.KEEP tHe ANkLE in a PLAnTAr-fleXeD pOsITiOn TO RElAX tHe NeUrOVaScUlaR BuNdLe.3.EstAbLISH thE POStEroLaTerAL and pOSTErOmeDIAL POrTALs.4.Use A 4-mm 30° ArThRosCOPE iNSErTEd tHrOUgH The PoSterOlateRal POrTAL.5.IDEnTify the FhL aND cONFirm tHAt It mOvEs WIth PasSivE Motion oF tHe HAllUx.6.sTarT DebRIDEMEnt WitH An ArthroSCOpIc SHavER and RadIofreQuENCy deViCe inSErted ThrOUgh tHe posTeRoMEDIAL PorTAl.7.iDENtIfY THe FRAcTUrE MALUnion.8.PerForm PaRtIaL rEseCTioN Of tHe FRAGmeNT witH A MotorIzed 4.0-MM Burr.9.cHecK, undeR ArtHroSCoPic ConTroL, ThAT RaNGE oF AnKLe motION Is cOmpLETely rEsTOReD anD pOSterioR aNkLe imPingemeNT iS coRReCtED.[^1]taBLe 2PeaRLs And PITFallsPeaRLSPITFAllSUSE THe prONE poSITIOn; PaD all bOny proMinEnCES.PresSURe SOres ANd LaTEraL Femoral CutaneOUS nerVe NeuRoPatHYnOTE THat No tracTIOn Is ReQuIrEd.no fREE Movement Of AnkLekEEP thE ANkLe in A plAntAR-FLExED POsitIoN.NeuROVASCUlaR BuNdLE inJuRYperfOrm caREFuL PAlPAtIOn oF tHe ACHIlLes tEnDON.ACHILLES teNdOn inJUrYIDENtIFy THe tIP oF tHe LAteRaL MAllEolUs FOr CoRReCt pOrtaL pLAcement.iNcorReCT pOrTAL pLACEMEntUSe tHE aRthroScoPe shAFT AS a guidE TO pLaCE the PoSteROmeDiaL porTaL.NeuroVasCulaR BUndlE injurYUSe fLUorOsCoPy to coNfiRM THe PositIoN aND DIRecTIOn oF tHe ARtHROscOPE if NeceSsARy.iNCorReCt PORTal pLACeMentIdeNtIFy THE fHL by pASSIve MOtIOn Of THe gREAT TOe anD PasS a BAsE LOoP ArOunD tHE teNDON.NeUroVaSCUlaR BuNDLe INJuRYpERForm a DEtAiLEd EXAMinAtIOn Of poSTErIOr ANKLE impIngEmENT.incORreCt PoRTAl plAceMenTIdeNTiFy THE FRacTure MaLuNion.no idenTIfiCaTIon Of FraCTURe malUnIOnReSHaPE ThE POSTeRior TAluS.nO impINgemENT cOrrEcTiOnAvOId eXcESSiVe BonE REseCTIoN.eXCeSSiVE Bone ReSEcTiON LEADing TO AnKLe iNstAbiLItycheCk cOMpLetE anKLE mOtIoN AND CorRecTioN Of posteRIor ANklE iMPINgemenT.NO ImprovemeNt in cLINICal resULtSTABLe 3advANtAges and diSaDvanTAgESADvanTAges THE PRoCEdurE allows excELLent acCEsS To The pOSTerior ankLe COmPArTMeNt. tHE pRoCeDUre is MiNimALly INvASIVe. The Procedure allOws posTeRiOR aNkle iMPiNgeMEnt tO Be corREcTeD. tHE reCoverY tImE Is sHorTEr.DisADvANtAGES the TeChNIQuE is CHAllENgING. neuROVAsCular buNdlE iNjUry cAn oCCur. An | Cedell[@bib1] described talus fracture in4young active sportsmen in 1974. These injuries are rare, andfromthen until now,only a few cases have been reported. The patient may arrive in the emergency departmentwith pain and tenderness in the posteromedial region of the talus. However, mostof the time,the injury\'s similarity to an ankle sprain on normal anteroposterior and lateral ankle radiographic views maycontribute to misdiagnosis or delayed diagnosis.[@bib2] Different treatments ofthis injuryare supported in the literature: conservative treatment of nondisplaced or minimally displacedfractures, openreduction--internalfixation for displaced fractures,or fracture excision for malunion ofdisplaced fractures that cause posteromedial ankle impingement.[@bib2], [@bib3]Technique{#sec1} ========= The diagnosis can be made using anteroposterior and lateral ankle radiographs ([Fig 1](#fig1){ref-type="fig"})and computed tomography([Fig 2](#fig2){ref-type="fig"}).Fig1Preoperative anteroposterior and lateral radiographs ofa right ankle showing a posteromedial talus fracture (arrow).Fig 2Preoperative 3-dimensional right anklereconstruction showing adisplaced posteromedial talus fracture with malunion (arrow). Preoperative Setup {#sec1.1}------------------ Thepatient is placed in the prone positionwith application of a thightourniquet to provide abloodless surgical field. All bony prominencesare padded. A small support is situated underthe lower leg, making it possible to move the ankle freely. The ankle should be kept in a plantar-flexedposition to relax the neurovascular bundle. The operative leg isprepared and draped in standard fashion. No traction is required. Portal Placement {#sec1.2} ---------------- According to therecommendations of Van Dijk et al,[@bib4] the posterolateral portal is created through an incision at the level of orslightlyabove the tipof the lateral malleolus, just lateral to the Achilles tendon. Carefulpalpation of the Achilles tendon before portal insertion reduces therisk of damaging the tendon. Blunt dissection to the level of the joint iscarried out with asmall mosquito clamp directed anteriorly, pointing in the direction of theinterdigitalweb space between the first and second toes. It is exchanged witha 4.5-mm arthroscope shaft (Arthrex, Naples, FL) with ablunt trocar. The posteromedial portal is made just medial to the Achilles tendon, at the same levelas theposterolateral portal. A mosquito clamp is introduced and directed toward the arthroscope shaft at 90° to touch this shaft; it should passtheneurovascular bundle without a problem. A 4.0-mm30° arthroscope (Arthrex) is usedthrough the posterolateral portal. The posterolateral portal is usedas the viewing portal, and the posteromedial portal serves asthe working portal([Fig 3](#fig3){ref-type="fig"}). If there is any difficulty indetermining whether thearthroscopeis inserted correctly, confirmationof its position by fluoroscopy isrecommended.Fig3The patient is in the prone position.Portal placement is shown for the right ankle. Placement of theposterolateral portal is performed just lateral to theAchilles tendon at the level ofthe tip of themalleolus. A4-mm30°arthroscope is in position. The posteromedial portal is made just medial to the Achilles tendon, atthe same levelas the posterolateral portal. A shaver isintroduced and directed toward the arthroscope shaft. Surgical Correction of Posterior Ankle Impingement{#sec1.3} -------------------------------------------------- Before the surgeon addresses the pathology, itisparamount to identify the flexor hallucis longus (FHL)and confirm that it moves with passive motion ofthe hallux.Abase loop is passed aroundthe tendon. The FHL isused as the medial border of the working area,which helps preventinjury to theneurovascular bundle. Then, debridement starts with an arthroscopic shaver (Arthrex) and radiofrequencydevice (Arthrex) inserted through the posteromedial portal.[@bib4] When soft-tissue debridement is complete, the fracture malunion is clearly defined ([Fig 4](#fig4){ref-type="fig"}). Then, it is timeto perform resection. This may include partial or total resection to correct the impingement ([Fig5](#fig5){ref-type="fig"}). We perform partial resection of the fragmentwitha motorized4.0-mm burr (Arthrex) to restore the normalshape of the posterior talus.We check, under arthroscopic control,that range of ankle motion is completelyrestored and posterior ankle impingementis corrected ([Fig 6](#fig6){ref-type="fig"}).Fig 4Arthroscopic view of the posteromedial aspect of the right ankle from the posterolateralportal. Thefracture malunion (yellow arrow) andposterior ankle impingementare visualized. Ashaver is introduced through the posteromedial portal. The flexor hallucis longus is on the medial sidewith a base looparoundit (green arrow).Fig5Arthroscopic view of the posteromedial aspect of the right ankle from the posterolateral portal. Partialresectionofthe fracturemalunion (arrow) is performed. Aburr is introduced through the posteromedial portal.Fig 6Arthroscopic view of the posteromedial aspect of the right ankle from the posterolateral portal. Final reshapingof theposterior talus is performed, and posterior ankle impingement (red arrow) is corrected. The flexor hallucis longus is on the medial side with a base loop around it (green arrow). Complications {#sec1.4} ------------- Although we have not encountered any complications withthis procedure, the major complication is injuryto the tibial neurovascular bundle. It is recommended to keep the ankle in plantarflexion during theprocedure and keep the instruments lateral tothe FHL. Postoperative Rehabilitation Protocol {#sec1.5} ------------------------------------- The patient is discharged on the day after surgery using crutches.Weight bearing is allowed. A rehabilitation program to gain full mobility and strength isfollowed. A controlcomputed tomography scan is ordered 1 month after surgery ([Fig 7](#fig7){ref-type="fig"}). Return to sports is gradually permitted, based on functional demands, with the most demandingactivities being avoided until 3 months after ankle arthroscopy.Fig 7Three-dimensional reconstruction1 month after arthroscopic resection of amalunion of a posteromedial talus fracture showingcorrection of posterior ankle impingement (arrow). A step-by-step summary of ourtechnique isprovided in [Table 1](#tbl1){ref-type="table"}. Pearls and pitfalls are presented in [Table 2](#tbl2){ref-type="table"}, andadvantages and disadvantages are listedin [Table 3](#tbl3){ref-type="table"}. Key steps of the procedure areshown in [Video 1](#appsec1){ref-type="sec"}.Table 1Step-by-Step Summary of Arthroscopic Treatment of Malunion of Posteromedial Talus Fracture1.Position thepatient in the prone position with application of a thigh tourniquet to provide a bloodless surgical field.2.Keep the ankleina plantar-flexed position to relax the neurovascular bundle.3.Establish the posterolateral andposteromedial portals.4.Use a4-mm 30° arthroscope inserted through the posterolateral portal.5.Identify the FHL and confirm that it moveswithpassivemotion of the hallux.6.Start debridement with an arthroscopic shaver andradiofrequency deviceinserted through the posteromedial portal.7.Identifythe fracture malunion.8.Perform partial resection of the fragmentwith a motorized 4.0-mm burr.9.Check, under arthroscopic control,thatrange of ankle motion iscompletely restored andposterior ankle impingement is corrected.[^1]Table 2Pearls and PitfallsPearlsPitfallsUse the proneposition; pad all bonyprominences.Pressure sores and lateral femoral cutaneousnerve neuropathyNote that notraction is required.No free movement of ankleKeep the ankle in a plantar-flexed position.Neurovascularbundle injuryPerformcareful palpationof the Achilles tendon.Achilles tendon injuryIdentify the tip of thelateral malleolusfor correct portal placement.Incorrect portalplacementUse the arthroscope shaft as a guide toplace the posteromedial portal.Neurovascular bundle injuryUse fluoroscopyto confirm the position and direction ofthe arthroscope if necessary.Incorrect portal placementIdentifythe FHLby passive motion of the great toe andpass a base looparound thetendon.Neurovascular bundle injuryPerform a detailed examination of posterior ankle impingement.Incorrect portalplacementIdentify the fracturemalunion.No identificationof fracture malunionReshape the posterior talus.No impingement correctionAvoid excessivebone resection.Excessive bone resection leading to ankleinstabilityCheck complete ankle motion and correctionof posterior ankle impingement.Noimprovementin clinical resultsTable 3Advantages and DisadvantagesAdvantages The procedure allows excellent access to the posterior anklecompartment.Theprocedure is minimally invasive. Theprocedure allows posterior ankle impingement to be corrected.Therecovery time is shorter.Disadvantages The technique is challenging. Neurovascular bundle injury can occur. An | _Cedell[@bib1]_ described talus fracture in 4 _young_ _active_ sportsmen in 1974. These injuries are rare, and from _then_ _until_ now, only a few cases _have_ been reported. The patient may arrive in the _emergency_ department with pain and tenderness in the posteromedial region of the talus. However, most of the time, _the_ injury\'s similarity _to_ an ankle sprain on normal anteroposterior _and_ _lateral_ _ankle_ radiographic views _may_ contribute to misdiagnosis or delayed diagnosis.[@bib2] _Different_ treatments of this injury are supported in the literature: conservative treatment of nondisplaced or _minimally_ displaced fractures, open reduction--internal fixation for displaced _fractures,_ or fracture _excision_ for malunion of displaced fractures _that_ cause posteromedial ankle impingement.[@bib2], _[@bib3]_ Technique {#sec1} ========= The diagnosis can be made _using_ _anteroposterior_ and lateral ankle radiographs ([Fig 1](#fig1){ref-type="fig"}) and computed _tomography_ ([Fig 2](#fig2){ref-type="fig"}).Fig 1Preoperative anteroposterior and lateral radiographs _of_ a right ankle _showing_ a posteromedial talus fracture (arrow).Fig 2Preoperative 3-dimensional _right_ ankle reconstruction showing a displaced posteromedial talus fracture with _malunion_ (arrow). _Preoperative_ Setup _{#sec1.1}_ _------------------_ The patient is placed in the prone position with application of a thigh _tourniquet_ to _provide_ a bloodless surgical field. All _bony_ prominences are padded. _A_ _small_ _support_ _is_ situated under the _lower_ leg, making it possible to move the ankle _freely._ The ankle should _be_ kept in a plantar-flexed position _to_ relax the neurovascular bundle. The _operative_ leg is prepared and draped in standard fashion. No _traction_ is _required._ _Portal_ Placement _{#sec1.2}_ ---------------- According to the recommendations of Van _Dijk_ et al,[@bib4] the _posterolateral_ _portal_ is created through an _incision_ _at_ the level of or slightly above the tip of the lateral malleolus, just lateral _to_ the Achilles tendon. Careful _palpation_ of the Achilles tendon _before_ portal insertion reduces _the_ risk of damaging the _tendon._ Blunt dissection to the level of the joint is carried out with a _small_ mosquito clamp directed _anteriorly,_ _pointing_ in the direction of the interdigital web space between the first and _second_ toes. It is exchanged with a 4.5-mm arthroscope shaft (Arthrex, Naples, FL) with a blunt trocar. The _posteromedial_ portal _is_ made _just_ medial to the Achilles _tendon,_ at the _same_ level as the posterolateral portal. _A_ mosquito clamp is introduced and directed toward the arthroscope _shaft_ at 90° to touch _this_ _shaft;_ it should pass the neurovascular bundle without a problem. A 4.0-mm 30° arthroscope (Arthrex) is used through the posterolateral portal. The posterolateral portal is used as _the_ viewing portal, and the posteromedial portal serves as the _working_ portal _([Fig_ _3](#fig3){ref-type="fig"})._ If there is _any_ difficulty in determining whether the arthroscope is inserted correctly, confirmation of its position by _fluoroscopy_ _is_ recommended.Fig 3The patient _is_ in the prone position. Portal placement is shown for the right ankle. Placement of the posterolateral _portal_ _is_ _performed_ just lateral to the Achilles tendon at _the_ level of the tip of _the_ malleolus. A 4-mm 30° arthroscope is in position. The posteromedial portal is made just medial to the Achilles tendon, at the same level as the posterolateral portal. A shaver is introduced and directed toward the arthroscope shaft. _Surgical_ Correction of Posterior Ankle Impingement {#sec1.3} -------------------------------------------------- Before _the_ _surgeon_ addresses the pathology, it is _paramount_ to identify the flexor hallucis longus _(FHL)_ and _confirm_ that it moves _with_ passive _motion_ of the hallux. A _base_ loop _is_ passed _around_ _the_ tendon. The FHL is _used_ as _the_ medial border _of_ the _working_ area, which _helps_ prevent injury to the neurovascular bundle. Then, debridement starts with an arthroscopic shaver _(Arthrex)_ _and_ radiofrequency _device_ _(Arthrex)_ inserted through _the_ posteromedial portal.[@bib4] _When_ soft-tissue debridement is _complete,_ the _fracture_ malunion is clearly defined ([Fig 4](#fig4){ref-type="fig"}). Then, it is time to perform resection. This may include _partial_ or _total_ resection to correct _the_ impingement ([Fig 5](#fig5){ref-type="fig"}). We perform partial _resection_ of the fragment with a motorized 4.0-mm burr (Arthrex) to restore the normal shape of _the_ posterior talus. We check, _under_ _arthroscopic_ _control,_ that _range_ of ankle motion is completely restored and posterior ankle impingement is corrected ([Fig 6](#fig6){ref-type="fig"}).Fig 4Arthroscopic _view_ _of_ the posteromedial aspect of the right ankle from the posterolateral _portal._ The fracture malunion (yellow _arrow)_ _and_ posterior ankle impingement are visualized. _A_ shaver is introduced through the posteromedial portal. The flexor hallucis longus is on the _medial_ side with a base loop around it (green arrow).Fig _5Arthroscopic_ _view_ of the posteromedial _aspect_ of the right ankle from the posterolateral portal. _Partial_ resection of _the_ fracture malunion (arrow) is performed. A burr is _introduced_ through the _posteromedial_ portal.Fig 6Arthroscopic view of the posteromedial aspect of _the_ _right_ ankle from _the_ _posterolateral_ portal. Final reshaping of the posterior talus is _performed,_ and posterior ankle impingement _(red_ arrow) is corrected. The flexor hallucis longus is on _the_ medial side with a base loop around it (green arrow). Complications {#sec1.4} ------------- _Although_ we have not encountered any complications with _this_ procedure, the major complication _is_ injury to the tibial neurovascular bundle. It is recommended _to_ keep _the_ ankle in plantar flexion during _the_ _procedure_ and keep the instruments lateral _to_ the FHL. _Postoperative_ Rehabilitation Protocol {#sec1.5} ------------------------------------- The patient is discharged _on_ _the_ day _after_ surgery using crutches. Weight _bearing_ is allowed. A rehabilitation program to _gain_ full mobility _and_ _strength_ is followed. A _control_ computed _tomography_ scan is _ordered_ 1 _month_ after _surgery_ ([Fig 7](#fig7){ref-type="fig"}). _Return_ _to_ _sports_ is gradually permitted, based on functional demands, with the _most_ _demanding_ activities being avoided _until_ 3 months after ankle arthroscopy.Fig 7Three-dimensional reconstruction 1 month after arthroscopic resection of _a_ malunion of _a_ _posteromedial_ talus _fracture_ showing _correction_ of posterior ankle impingement _(arrow)._ A _step-by-step_ summary of our technique is provided in _[Table_ 1](#tbl1){ref-type="table"}. Pearls and pitfalls are presented in [Table 2](#tbl2){ref-type="table"}, and advantages and disadvantages are listed in [Table 3](#tbl3){ref-type="table"}. Key _steps_ of the _procedure_ are shown in [Video 1](#appsec1){ref-type="sec"}.Table 1Step-by-Step Summary of Arthroscopic _Treatment_ of Malunion of Posteromedial Talus Fracture1.Position the _patient_ in the prone position _with_ _application_ of a thigh tourniquet to provide a _bloodless_ surgical _field.2.Keep_ _the_ ankle in _a_ plantar-flexed position to _relax_ the neurovascular bundle.3.Establish the posterolateral and posteromedial portals.4.Use _a_ 4-mm 30° _arthroscope_ inserted through the posterolateral portal.5.Identify the FHL and confirm that _it_ moves with passive _motion_ of the hallux.6.Start debridement _with_ an arthroscopic shaver and radiofrequency device inserted through the posteromedial portal.7.Identify the fracture _malunion.8.Perform_ partial _resection_ of the fragment with a motorized 4.0-mm burr.9.Check, under arthroscopic control, that range of ankle motion is completely restored and posterior ankle _impingement_ is corrected.[^1]Table 2Pearls and _PitfallsPearlsPitfallsUse_ the prone _position;_ pad all bony prominences.Pressure sores and lateral femoral _cutaneous_ _nerve_ _neuropathyNote_ _that_ no traction is required.No free movement of ankleKeep the ankle in a plantar-flexed position.Neurovascular bundle _injuryPerform_ careful _palpation_ of the Achilles tendon.Achilles tendon _injuryIdentify_ the tip _of_ the lateral malleolus for correct _portal_ placement.Incorrect portal placementUse the arthroscope shaft as a guide to _place_ _the_ _posteromedial_ portal.Neurovascular bundle injuryUse fluoroscopy to confirm the position and direction of the arthroscope if _necessary.Incorrect_ portal placementIdentify the FHL by passive motion of the great toe and pass a base loop around the tendon.Neurovascular bundle injuryPerform a _detailed_ examination of posterior ankle impingement.Incorrect portal placementIdentify _the_ _fracture_ malunion.No _identification_ _of_ fracture malunionReshape _the_ posterior talus.No impingement _correctionAvoid_ excessive _bone_ resection.Excessive _bone_ resection leading to ankle instabilityCheck complete _ankle_ motion and correction of posterior ankle impingement.No improvement in clinical resultsTable 3Advantages and DisadvantagesAdvantages The _procedure_ allows excellent access _to_ _the_ posterior ankle compartment. The procedure is minimally _invasive._ The procedure allows posterior ankle impingement _to_ _be_ corrected. The recovery _time_ is _shorter.Disadvantages_ The technique is challenging. Neurovascular bundle injury _can_ occur. _An_ |
Introduction {#Sec1}
============
Organic light-emitting diodes (OLEDs) are being widely applied in displays for mobiles and televisions owing to their self-emitting characteristics, excellent colour gamut, high-speed operation, and applicability in flexible or stretchable devices^[@CR1]^. Practical OLEDs function based on phosphorescence owing to their nearly 100% internal phosphorescence efficiency^[@CR2],[@CR3]^. However, the external quantum efficiency of OLEDs is \~20% because the surface plasmon polariton modes at the metal-organic interface, the waveguide mode in the indium tin oxide (ITO)/organic layer, and the substrate mode in the glass substrate produce light loss^[@CR4]--[@CR8]^. Generally, light loss in the substrate mode is larger than that in the waveguide mode due to small differences in refractive index between the ITO/glass interface and the glass/air interface^[@CR9]^. In addition, microcavity top-emitting organic light-emitting diodes (TEOLED) without the loss of the substrate mode and having the advantage of improved colour purity and aperture ratio have been applied to mobile displays. However, microcavity TEOLED originally had a problem associated with viewing angles due to the blue shift that occurred with the variation of the sight angle, even though the light efficiency along the normal direction was amplified. Therefore, researchers have attempted to find solutions for improving the light extraction and viewing angle of OLEDs^[@CR10]^.
Different approaches have been adopted with the aim of optimizing light extraction and viewing angle, including micro lens array (MLA)^[@CR5],[@CR11]--[@CR16]^, dielectric Bragg gratings^[@CR17]--[@CR19]^, surface plasmons^[@CR20]^, buckling patterns^[@CR21]^, periodic corrugation^[@CR8]^, low-index grids^[@CR22]^, and photonic crystals^[@CR23]^. Methods that modify the internal or external surfaces of the substrate of OLEDs minimize the total internal reflection. Such surface shapes are produced by different technologies which traditionally involve photo-lithography or printing, moulding, and embossing methods^[@CR24]--[@CR28]^. Therefore, these technologies employ at least the photo-lithographic step or utilize lithographic templates once. They generally end up being considerably complex or expensive for mass production. On the other hand, other researchers have reported a simple scattering layer synthesized by competitive and scalable methods. In these methods, particles or voids having different refractive indices were embedded in a polymer matrix and utilized for the scattering layer^[@CR29]--[@CR36]^. However, because the particles or voids are micro-sized or the pitch between the nearest particles or voids is micro-sized, these techniques reveal a small effect of diffraction that is difficult to control. In addition, to prevent the loss of the substrate mode, the most representative MLA has larger visibility than a nano-sized array because of its size. Alignment is a problem in the case of high-resolution displays with very small pixels.
In this paper, we propose a simple method to fabricate random nanoscale rods (RNRs) as scattering layers for enhancement of light extraction and improvement of viewing angle characteristics in OLEDs. In our device, there is virtually no spectral distortion or shift, which are issues observed in 2D photonic crystal-based OLEDs, and the spectral shift according to the change in viewing angle is reduced. This layer is fabricated by a cost-effective and scalable procedure that consists of coating and etching the polymer at low temperatures (below 100 °C) without photomasks or templates. Additionally, the RNRs can control the diffraction as well as scattering effects in the visible wavelength range owing to the distance between closest rods and are compatible with high-resolution displays for virtual reality because the process of alignment between the individual pixels in the panel and the scattering layer is unnecessary. The RNRs can also be applied in flexible displays and lighting owing to the use of etched polymers. OLEDs equipped with RNRs exhibit superior optical properties such as high external quantum efficiency (EQE), high luminance efficiency (LE), and colour variation with changes in viewing angle, to control OLEDs.
Results and Discussion {#Sec2}
======================
To investigate the optical effect of RNRs on glass, the electrical field distribution was simulated by the finite-difference time domain (FDTD) method. The boundary conditions for the simulation were set for a perfect optically matched layer to avoid the reflection of electromagnetic waves at the edges of the structure on all the sides, except for the metal cathode layer. The simulated structure consisted of an aluminium cathode, *N*,*N*'-bis(naphthalen-1-yl)-*N*,*N*'-bis(phenyl)-benzidine (NPB; refractive index n = 1.81), tris(8-hydroxy-quinolinato)aluminium (Alq~**3**~; n = 1.72), ITO (n = 1.9), glass (n = 1.52), SU-8 polymer, and RNRs (n = 1.59). A dipole source of visible wavelength was generated in the Alq~**3**~ layer, and one transverse electric (TE) and two transverse magnetic (TM) modes were used^[@CR37]^. To reflect the randomness of RNRs in the simulation, their height was varied from 1000 nm to 1200 nm and width from 50 nm to 150 nm (Supplementary Information Fig. [S1](#MOESM1){ref-type="media"}). In the reference structure, most of the light incident at an angle above the critical angle became a waveguide because of the difference in refractive index between glass and air (Fig. [1a](#Fig1){ref-type="fig"}). In order to confirm the optical effect of the SU-8 polymer, a simulation using SU-8 without the pattern was performed (Fig. [1b](#Fig1){ref-type="fig"}). As a result, the optical path was almost unchanged, and the difference between the refractive indices of glass and air slightly increased; this difference seemed to be slightly adversely affected for external light extraction. In contrast, in the RNRs present on the glass structure, some portions of the waveguide light were allowed to escape from the glass substrate, and light extraction increased below the critical angle (Fig. [1c](#Fig1){ref-type="fig"}). Optical phenomena such as refraction, diffraction, and interference were produced by the RNRs because the differences in refractive index at the interface between glass and air changed and the random periodic rods revealed distances between each other that were similar to the visible wavelength. Figure [2e](#Fig2){ref-type="fig"} illustrates the light path in OLEDs. The photons trapped (rays 2 and 3) within the glass are scattered by the RNRs, increasing the probability of them being emitted from the structure. Furthermore, as a result of simulation, in which scattering occurs several times in one rod, it can be inferred that scattering occurs more frequently when the height of the rod is greater.Figure 1FDTD simulation of E-field distribution induced by transverse magnetic and electric dipoles: (**a**) Reference (w/o RNRs), (**b**) unpatterned SU-8 on a glass structure, and (**c**) RNRs on the glass structure.Figure 2Cross-sectional scanning electron microscopy images of random nanoscale rods (RNRs) obtained under different plasma etching conditions: (**a**) RNRs 1, (**b**) RNRs 2, (**c**) RNRs 3, and (**d**) RNRs 4, and (**e**) schematic illustration of OLED outcoupling enhancement by the RNR scattering layer.
It is desirable to fabricate a nanostructure of proper height and periodicity that is comparable to the wavelength of the visible range in order to enhance light extraction. For manufacturing RNRs, SU-8 polymer was chosen because it is highly transparent to visible light and has a higher refractive index than glass. Figure [2](#Fig2){ref-type="fig"} shows the cross-sectional scanning electron microscopy images of the RNRs obtained under different plasma etching conditions. Obtained by using only an oxygen plasma for a duration of 9 min (denoted as RNRs 1), the structure was random columnar-like with high aspect ratio and density^[@CR38]^. The height and density of RNRs 1 were about 1200 nm and 9.7 ea/µm^2^, respectively (Fig. [2a](#Fig2){ref-type="fig"}, Supplementary Information Fig. [S2a](#MOESM1){ref-type="media"}). The width of the individual rods was observed to be approximately 50 nm. To control the height and density, we applied an additional argon plasma treatment at higher powers and lower process pressures than the oxygen plasma. The low pressure led to an increase in the mean free path of argon ions and reduced the energy loss of these ions as they approached the RNRs. In general, the etching process using argon plasma carried out in a reactive ion etcher resulted in anisotropic and directional features due to the utilization of the physical bombardment energy^[@CR38]^. As a result of these features, the height and density were adjusted as much as a target to the little changed width of the RNRs. The three treated RNRs shown in Fig. [2b--d](#Fig2){ref-type="fig"} corresponded to argon plasma treatment durations of 3, 6, and 9 min (denoted as RNRs 2, RNRs 3, and RNRs 4, respectively), while the conditions of the oxygen plasma process remained unchanged. As the duration of the argon plasma treatment increased, the | introduction { # sec1 } = = = = = = = = = = = = organic light - emitting diodes ( oleds ) are being frequently recommended in displays for mobiles and televisions owing to their self - stabilization characteristics, excellent colour gamut, high - speed operation, and applicability in flexible or stretchable devices ^ [ @ cr1 ] ^. practical oleds function based on phosphorescence owing to their nearly 100 % internal phosphorescence efficiency ^ [ @ cr2 ], [ @ cr3 ] ^. however, the external quantum efficiency of oleds is \ ~ 20 % because the surface plasmon spectral modes at the metal - organic interface, the waveguide mode in the indium tin oxide ( ito ) / organic layer, and the substrate mode in the glass substrate produce light loss ^ [ @ cr4 ] - - [ @ cr8 ] ^. generally, light loss in the substrate mode is larger than that in the waveguide mode due to small change in refractive index between the ito / glass interface and the glass / air interface ^ [ @ cr9 ] ^. in addition, microcavity top - emitting organic light - emitting diodes ( atp ) without the loss of the substrate mode and having the advantage of improved colour purity and aperture ratio have been applied to mobile displays. however, optical teoled originally had a problem associated with viewing angles due to the blue shift usually occurred with the variation of the sight angle, even after the light efficiency along the normal direction was amplified. therefore, researchers have attempted to find solutions for improving the light extraction and viewing angle of oleds ^ [ @ cr10 ] ^. different approaches have been adopted with the aim of optimizing light extraction and viewing angle, including micro lens array ( mla ) ^ [ @ cr5 ], [ @ cr11 ] - - [ @ cr16 ] ^, dielectric bragg gratings ^ [ @ cr17 ] - - [ @ cr19 ] ^, surface plasmons ^ [ @ cr20 ] ^, buckling patterns ^ [ @ cr21 ] ^, periodic corrugation ^ [ @ cr8 ] ^, low - index grids ^ [ @ cr22 ] ^, and photonic crystals ^ [ @ cr23 ] ^. methods that modify the internal – external surfaces of the substrate of oleds minimize the total internal reflection. such surface shapes are produced by different technologies which traditionally involve photo - lithography or printing, moulding, and embossing methods ^ [ @ cr24 ] - - [ @ cr28 ] ^. therefore, these technologies employ at least the photo - lithographic step or utilize lithographic templates once. they generally end up being considerably complex or expensive for mass production. on the other hand, other researchers have reported a simple scattering layer synthesized by competitive and scalable methods. in these methods, particles or voids having different refractive indices were embedded in a polymer matrix and utilized for the scattering layer ^ [ @ cr29 ] - - [ @ cr36 ] ^. however, because the particles or voids are micro - sized or the pitch between the nearest particles or voids is micro - sized, these techniques reveal a small effect of diffraction that is difficult to control. in addition, to prevent the loss of the substrate mode, the most representative mla has larger visibility than a nano - sized array because of its size. alignment is a problem in the case of high - resolution displays with very small pixels. in this paper, we propose a simple method to fabricate random nanoscale rods ( rnrs ) as scattering layers for enhancement of light extraction and improvement of viewing angle characteristics in oleds. in our device, there is virtually no spectral distortion or shift, which are issues observed in 2d photonic crystal - based oleds, and the spectral shift according to the change in viewing angle is reduced. this layer is fabricated by a cost - effective and scalable procedure that consists of coating and etching the polymer at low temperatures ( below 100 °c ) without photomasks or templates. additionally, the rnrs can control the diffraction as well as scattering effects in the visible wavelength range owing to the distance between closest rods and are compatible with high - resolution displays for virtual reality because the process of alignment between the individual pixels in the panel and the scattering layer is unnecessary. the rnrs can also be applied in flexible displays and lighting owing to the use of etched polymers. oleds equipped with rnrs exhibit superior optical properties such as high external quantum efficiency ( eqe ), high luminance efficiency ( le ), and colour variation with changes in viewing angle, to control oleds. results and discussion { # sec2 } = = = = = = = = = = = = = = = = = = = = = = to investigate the optical effect of rnrs on glass, the electrical field distribution was simulated by the finite - difference time domain ( fdtd ) method. the boundary conditions for the simulation were set for a perfect optically matched layer to avoid the reflection of electromagnetic waves at the edges of the structure on all the sides, except for the metal cathode layer. the simulated structure consisted of an aluminium cathode, * n *, * n * ' - bis ( naphthalen - 1 - yl ) - * n *, * n * ' - bis ( phenyl ) - benzidine ( npb ; refractive index n = 1. 81 ), tris ( 8 - hydroxy - quinolinato ) aluminium ( alq ~ * * 3 * * ~ ; n = 1. 72 ), ito ( n = 1. 9 ), glass ( n = 1. 52 ), su - 8 polymer, and rnrs ( n = 1. 59 ). a dipole source of visible wavelength was generated in the alq ~ * * 3 * * ~ layer, and one transverse electric ( te ) and two transverse magnetic ( tm ) modes were used ^ [ @ cr37 ] ^. to reflect the randomness of rnrs in the simulation, their height was varied from 1000 nm to 1200 nm and width from 50 nm to 150 nm ( supplementary information fig. [ s1 ] ( # moesm1 ) { ref - type = " media " } ). in the reference structure, most of the light incident at an angle above the critical angle became a waveguide because of the difference in refractive index between glass and air ( fig. [ 1a ] ( # fig1 ) { ref - type = " fig " } ). in order to confirm the optical effect of the su - 8 polymer, a simulation using su - 8 without the pattern was performed ( fig. [ 1b ] ( # fig1 ) { ref - type = " fig " } ). as a result, the optical path was almost unchanged, and the difference between the refractive indices of glass and air slightly increased ; this difference seemed to be slightly adversely affected for external light extraction. in contrast, in the rnrs present on the glass structure, some portions of the waveguide light were allowed to escape from the glass substrate, and light extraction increased below the critical angle ( fig. [ 1c ] ( # fig1 ) { ref - type = " fig " } ). optical phenomena such as refraction, diffraction, and interference were produced by the rnrs because the differences in refractive index at the interface between glass and air changed and the random periodic rods revealed distances between each other that were similar to the visible wavelength. figure [ 2e ] ( # fig2 ) { ref - type = " fig " } illustrates the light path in oleds. the photons trapped ( rays 2 and 3 ) within the glass are scattered by the rnrs, increasing the probability of them being emitted from the structure. furthermore, as a result of simulation, in which scattering occurs several times in one rod, it can be inferred that scattering occurs more frequently when the height of the rod is greater. figure 1fdtd simulation of e - field distribution induced by transverse magnetic and electric dipoles : ( * * a * * ) reference ( w / o rnrs ), ( * * b * * ) unpatterned su - 8 on a glass structure, and ( * * c * * ) rnrs on the glass structure. figure 2cross - sectional scanning electron microscopy images of random nanoscale rods ( rnrs ) obtained under different plasma etching conditions : ( * * a * * ) rnrs 1, ( * * b * * ) rnrs 2, ( * * c * * ) rnrs 3, and ( * * d * * ) rnrs 4, and ( * * e * * ) schematic illustration of oled outcoupling enhancement by the rnr scattering layer. it is desirable to fabricate a nanostructure of proper height and periodicity that is comparable to the wavelength of the visible range in order to enhance light extraction. for manufacturing rnrs, su - 8 polymer was chosen because it is highly transparent to visible light and has a higher refractive index than glass. figure [ 2 ] ( # fig2 ) { ref - type = " fig " } shows the cross - sectional scanning electron microscopy images of the rnrs obtained under different plasma etching conditions. obtained by using only an oxygen plasma for a duration of 9 min ( denoted as rnrs 1 ), the structure was random columnar - like with high aspect ratio and density ^ [ @ cr38 ] ^. the height and density of rnrs 1 were about 1200 nm and 9. 7 ea / µm ^ 2 ^, respectively ( fig. [ 2a ] ( # fig2 ) { ref - type = " fig " }, supplementary information fig. [ s2a ] ( # moesm1 ) { ref - type = " media " } ). the width of the individual rods was observed to be approximately 50 nm. to control the height and density, we applied an additional argon plasma treatment at higher powers and lower process pressures than the oxygen plasma. the low pressure led to an increase in the mean free path of argon ions and reduced the energy loss of these ions as they approached the rnrs. in general, the etching process using argon plasma carried out in a reactive ion etcher resulted in anisotropic and directional features due to the utilization of the physical bombardment energy ^ [ @ cr38 ] ^. as a result of these features, the height and density were adjusted as much as a target to the little changed width of the rnrs. the three treated rnrs shown in fig. [ 2b - - d ] ( # fig2 ) { ref - type = " fig " } corresponded to argon plasma treatment durations of 3, 6, and 9 min ( denoted as rnrs 2, rnrs 3, and rnrs 4, respectively ), while the conditions of the oxygen plasma process remained unchanged. as the duration of the argon plasma treatment increased, the | Introduction {# Sec1} = = = = = = = = = = = = Organic light - emitting diodes (OLEDs) are being widely applied in displays for mobiles and televisions owing to their self - emitting characteristics, excellent colour gamut, high - speed operation, and applicability in flexible or stretchable devices ^ [@ CR1] ^. Practical OLEDs function based on phosphorescence owing to their nearly 100% internal phosphorescence efficiency ^ [@ CR2 ], [@ CR3] ^. However, the external quantum efficiency of OLEDs is \ ~ 20% because the surface plasmon polariton modes at the metal - organic interface, the waveguide mode in the indium tin oxide (ITO) / organic layer, and the substrate mode in the glass substrate produce light loss ^ [@ CR4] - - [@ CR8] ^. Generally, light loss in the substrate mode is larger than that in the waveguide mode due to small differences in refractive index between the ITO / glass interface and the glass / air interface ^ [@ CR9] ^. In addition, microcavity top - emitting organic light - emitting diodes (TEOLED) without the loss of the substrate mode and having the advantage of improved colour purity and aperture ratio have been applied to mobile displays. However, microcavity TEOLED originally had a problem associated with viewing angles due to the blue shift that occurred with the variation of the sight angle, even though the light efficiency along the normal direction was amplified. Therefore, researchers have attempted to find solutions for improving the light extraction and viewing angle of OLEDs ^ [@ CR10] ^. Different approaches have been adopted with the aim of optimizing light extraction and viewing angle, including micro lens array (MLA) ^ [@ CR5 ], [@ CR11] - - [@ CR16] ^, dielectric Bragg gratings ^ [@ CR17] - - [@ CR19] ^, surface plasmons ^ [@ CR20] ^, buckling patterns ^ [@ CR21] ^, periodic corrugation ^ [@ CR8] ^, low - index grids ^ [@ CR22] ^, and photonic crystals ^ [@ CR23] ^. Methods that modify the internal or external surfaces of the substrate of OLEDs minimize the total internal reflection. Such surface shapes are produced by different technologies which traditionally involve photo - lithography or printing, moulding, and embossing methods ^ [@ CR24] - - [@ CR28] ^. Therefore, these technologies employ at least the photo - lithographic step or utilize lithographic templates once. They generally end up being considerably complex or expensive for mass production. On the other hand, other researchers have reported a simple scattering layer synthesized by competitive and scalable methods. In these methods, particles or voids having different refractive indices were embedded in a polymer matrix and utilized for the scattering layer ^ [@ CR29] - - [@ CR36] ^. However, because the particles or voids are micro - sized or the pitch between the nearest particles or voids is micro - sized, these techniques reveal a small effect of diffraction that is difficult to control. In addition, to prevent the loss of the substrate mode, the most representative MLA has larger visibility than a nano - sized array because of its size. Alignment is a problem in the case of high - resolution displays with very small pixels. In this paper, we propose a simple method to fabricate random nanoscale rods (RNRs) as scattering layers for enhancement of light extraction and improvement of vUe!ing angle characteristics in OLEDs. In our device, there is virtually no spectral distortion or shift, which are issues observed in 2D photonic crystal - based OLEDs, and the spectral shift according to the change in viewing angle is reduced. This layer is fabricated by a cost - effective and scalable procedure that consists of coating and etching the polymer at low temperatures (below 100 ° C) without photomasks or templates. Additionally, the RNRs can control the diffraction as well as scattering efGeVts in the visible wavelength range owing to the distance between closest rods and are compatible with high - resolution displays for virtual reality because the process of alignment between the individual pixels in the panel and the scattering layer is unnecessary. The RNRs can also be applied in flexible displays and lighting owing to the use of etched polymers. OLEDs equipped with RNRs exhibit superior optical properties such as high external quantum efficiency (EQE ), high luminance efficiency (LE ), and colour variation !8th changes in viewing angle, to control OLEDs. Results and Discussion {# Sec2} = = = = = = = = = = = = = = = = = = = = = = To investigate the optical effect of RNRs on glass, the electrical field distribution was simulated by the finite - difference time domain (FDTD) method. The boundary conditions for the simulation were set for a perfect optically matched layer to avoid the reflection of electromagnetic waves at the edges of the structure on all the sides, except for the metal cathode layer. The sim^lat@d structure consisted of an aluminium cathode, * N *, * N * ' - bis (naphthalen - 1 - yl) - * N *, * N * ' - bis (phenyl) - benzidine (NPB; refractive index n = 1. 81 ), tris (8 - hydroxy - quinolinato) aluminium (Alq ~ * * 3 * * ~; n = 1. 72 ), ITO (n = 1. 9 ), glass (n = 1. 52 ), SU - 8 polymer, and RNRs (n = 1. 59 ). A dipole source of visible wavelength was generated in the Alq ~ * * 3 * * ~ layer, and one transverse electric (TE) and two transverse magnetic (TM) modes were used ^ [@ CR37] ^. To reflect the randomness of RNRs in the simulation, their height was varied from 1000 nm to 1200 nm and width from 50 nm to 150 nm (Supplementary Information Fig. [S1] (# MOESM1) {ref - type = " media "} ). In the reference structure, most of the light incident at an angle above the critical angle became a waveguide because of the difference in refGac5ive index between glass and air (Fig. [1a] (# Fig1) {ref - type = " fig "} ). In order to concirj the optical effect of the SU - 8 polymer, a simulation using SU - 8 without the pattern was performed (Fig. [1b] (# Fig1) {ref - type = " fig "} ). As a result, the optical path was almost unchanged, and the difference between the refractive indices of glass and air slightly increased; this difference seemed to be slightly adversely affected for external light extraction. In contrast, in the RNRs present on the glass structure, some portions of the waveguide light were allowed to escape from the glass substrate, and light extraction increased below the critical angle (Fig. [1c] (# Fig1) {ref - type = " fig "} ). Optical phenomena such as refraction, diffraction, and interference were produced by the RNRs because the differences in refractive index at the interface between glass and air changed and the random periodic rods revealed distances between each other that were similar to the visible wavelength. Figure [2e] (# Fig2) {ref - type = " fig "} illustrates the light path in OLEDs. The photons trapped (rays 2 and 3) within the glass are scattered by the RNRs, increasing the probability of them being emitted from the structure. Furthermore, as a result of simulation, in which scattering occurs several times in one rod, it can be inferred that scattering occurs more frequently when the height of the rod is greater. Figure 1FDTD simulation of E - field distribution induced by transverse magnetic and electric dipoles: (* * a * *) Reference (w / o RNRs ), (* * b * *) unpatterned SU - 8 on a glass structure, and (* * c * *) RNRs on the glass structure. Figure 2Cross - sectional scanning electron microscopy images of random nanoscale rods (RNRs) obtained uHde$ different plasma etching conditions: (* * a * *) RNRs 1, (* * b * *) RNRs 2, (* * c * *) RNRs 3, and (* * d * *) RNRs 4, and (* * e * *) svhemaGic illustration of OLED outcoupling enhancement by the RNR scattering layer. It is desirable to fabricate a nanostructure of proper height and periodicity that is comparable to the wavelength of the visible range in order to enhance light extraction. For manufacturing RNRs, SU - 8 polymer was chosen because it is highly transparent to visible light and has a higher refractive index than glass. Figure [2] (# Fig2) {ref - type = " fig "} shows the cross - sectional scanning electron microscopy images of the RNRs obtained under different plasma etching conditions. Obtained by using only an oxygen plasma for a duration of 9 min (denoted as RNRs 1 ), the structure was random columnar - like with high aspect ratio and density ^ [@ CR38] ^. The height and density of RNRs 1 were about 1200 nm and 9. 7 ea / µm ^ 2 ^, respectively (Fig. [2a] (# Fig2) {ref - type = " fig " }, Supplementary Information Fig. [S2a] (# MOESM1) {ref - type = " media "} ). The width of the individual rods was observed to be approximately 50 nm. To control the height and density, we applied an additional argon 9iasma treatment at higher powers and lower process pressures than the ox6geG plasma. The low pressure led to an increase in the mean free path of argon ions and reduced the energy loss of these ions as they approached the RNRs. In general, the etching process using argon plasma carried out in a reactive ion etcher resulted in anisotropic and directional features due to the utilization of the physical bombardment energy ^ [@ CR38] ^. As a result of these features, the height and density were adjusted as much as a target to the little changed width of the RNRs. The three treated RNRs shown in Fig. [2b - - d] (# Fig2) {ref - type = " fig "} corresponded to argon plasma treatment durations of 3, 6, and 9 min (denoted as RNRs 2, RNRs 3, and RNRs 4, respectively ), while the conditions of the oxygen plasma process remained unchanged. As the duration of the argon plasma treatment increased, the | {#Sec1} ============ Organic light-emitting diodes (OLEDs) are being widely applied in displays mobiles and televisions owing to their self-emitting characteristics, excellent colour gamut, high-speed operation, and applicability in or stretchable devices^[@CR1]^. Practical OLEDs based on phosphorescence their nearly 100% internal However, the external quantum efficiency of OLEDs is \~20% because the surface plasmon polariton modes at the metal-organic interface, the waveguide mode in the indium tin (ITO)/organic layer, and the substrate mode in the glass substrate produce light loss^[@CR4]--[@CR8]^. Generally, light loss in the substrate mode is larger than that in the waveguide mode due to small differences in refractive index between the and the glass/air interface^[@CR9]^. In addition, microcavity top-emitting organic light-emitting diodes (TEOLED) without the loss of the mode and having the advantage of improved colour purity and aperture ratio have been applied to mobile displays. However, microcavity TEOLED originally had problem associated viewing angles due to the blue shift that occurred with the variation of the sight angle, even though the light efficiency along the normal direction amplified. Therefore, researchers have attempted solutions for improving the light extraction and angle of OLEDs^[@CR10]^. Different approaches have been adopted with the aim of optimizing light extraction and viewing angle, including micro lens array (MLA)^[@CR5],[@CR11]--[@CR16]^, dielectric Bragg gratings^[@CR17]--[@CR19]^, surface plasmons^[@CR20]^, buckling patterns^[@CR21]^, periodic corrugation^[@CR8]^, low-index grids^[@CR22]^, and photonic crystals^[@CR23]^. Methods that the or external surfaces of the substrate of OLEDs minimize total internal reflection. Such surface shapes are produced by different technologies which photo-lithography or printing, moulding, and embossing methods^[@CR24]--[@CR28]^. Therefore, these technologies employ at photo-lithographic step or utilize lithographic templates once. They generally up being considerably complex or expensive for mass production. the other hand, other researchers have reported a layer synthesized by competitive and scalable methods. In these methods, particles or voids having different refractive indices were embedded in a polymer matrix and utilized for the scattering layer^[@CR29]--[@CR36]^. However, because the particles or voids are micro-sized or the pitch between the nearest particles or voids is micro-sized, techniques reveal small effect of diffraction that is difficult to control. In addition, to prevent the loss of the substrate mode, the most representative MLA has larger visibility than a array because of its size. Alignment is a problem in case high-resolution displays with very small pixels. In this paper, we a simple method to fabricate random nanoscale rods (RNRs) as scattering layers enhancement of light extraction and improvement of angle characteristics in OLEDs. our device, there is virtually no spectral distortion or shift, which issues observed in 2D photonic crystal-based OLEDs, and the spectral shift to the change in viewing angle is reduced. This layer fabricated by a cost-effective and procedure that consists of coating and etching the polymer at low temperatures (below 100 °C) without photomasks or templates. Additionally, control the diffraction as well as scattering in the visible range owing to the between closest rods and are compatible with displays for virtual reality because the process between the pixels in the panel the scattering layer unnecessary. The RNRs can also be applied in flexible displays and owing to the use of etched polymers. OLEDs equipped RNRs exhibit optical properties such high external quantum efficiency (EQE), high luminance (LE), and colour variation with changes in viewing angle, to control OLEDs. Results and {#Sec2} ====================== To investigate the optical effect of RNRs on glass, the electrical field distribution was simulated by the finite-difference time domain method. The boundary conditions for the simulation set for perfect optically layer to avoid reflection of electromagnetic waves the edges of on all the sides, except for the metal layer. The simulated consisted of an aluminium cathode, *N*,*N*'-bis(naphthalen-1-yl)-*N*,*N*'-bis(phenyl)-benzidine (NPB; refractive index n = 1.81), tris(8-hydroxy-quinolinato)aluminium (Alq~**3**~; n = 1.72), ITO (n = 1.9), glass (n = 1.52), polymer, and RNRs (n = 1.59). A source of visible wavelength was generated in the Alq~**3**~ layer, and one transverse electric (TE) and two transverse magnetic (TM) modes were To reflect the randomness of RNRs simulation, their height was varied from nm to 1200 nm and width from 50 nm to 150 nm (Supplementary Information Fig. [S1](#MOESM1){ref-type="media"}). In the reference structure, most of the light incident at an angle the critical angle became a waveguide because of the difference in refractive index between glass and air (Fig. [1a](#Fig1){ref-type="fig"}). In to confirm the optical effect of the SU-8 polymer, a simulation using SU-8 without the pattern was performed (Fig. [1b](#Fig1){ref-type="fig"}). As a result, the optical path was almost unchanged, and the between the refractive indices of and air slightly increased; this difference seemed to be slightly adversely affected for external light extraction. In contrast, in the RNRs present on the glass structure, some portions of the waveguide light were allowed escape from the glass substrate, and light extraction below the critical angle [1c](#Fig1){ref-type="fig"}). Optical phenomena such as refraction, diffraction, and interference were produced by the because the differences refractive index at the interface between glass and air changed and the random periodic rods revealed distances each other that to the visible wavelength. Figure [2e](#Fig2){ref-type="fig"} illustrates the light path in OLEDs. The photons trapped (rays and 3) within the glass are scattered by the RNRs, increasing the probability of them being emitted from the structure. Furthermore, as a result of simulation, in which scattering occurs several times in one it can that scattering occurs more frequently when the height of the rod is greater.Figure 1FDTD simulation of E-field distribution induced by transverse magnetic and electric (**a**) Reference (w/o RNRs), (**b**) unpatterned SU-8 on a glass and (**c**) on the glass structure.Figure 2Cross-sectional scanning electron microscopy images of random nanoscale rods (RNRs) under different plasma etching conditions: (**a**) RNRs 1, RNRs 2, (**c**) RNRs 3, and (**d**) and (**e**) schematic illustration of outcoupling enhancement by the RNR scattering layer. It is desirable to a of height and periodicity that is comparable to the wavelength of the range order to enhance light For manufacturing RNRs, SU-8 polymer was chosen because it is highly transparent to visible light and has a higher refractive than glass. Figure [2](#Fig2){ref-type="fig"} shows the cross-sectional scanning electron microscopy images of the RNRs obtained different plasma etching conditions. Obtained by using only an oxygen plasma for a duration of 9 min (denoted as RNRs 1), the structure was random columnar-like with high and density^[@CR38]^. The height and density of RNRs 1 were about 1200 and 9.7 ea/µm^2^, respectively (Fig. [2a](#Fig2){ref-type="fig"}, Supplementary Information Fig. The width of the individual rods was observed to be approximately nm. control the height and density, we applied an additional plasma treatment at higher powers and lower process than the oxygen plasma. The low pressure led to an increase in the path of argon ions and reduced the loss of ions as they approached the In general, the etching using argon plasma carried in a reactive ion resulted in and features due to the utilization of the physical bombardment energy^[@CR38]^. As a result of these features, the height and density were adjusted much as a target to the little changed width of the RNRs. The three treated RNRs shown in corresponded argon plasma treatment durations of 3, 6, and min (denoted as RNRs 2, RNRs 3, and RNRs respectively), the conditions of the oxygen plasma process remained unchanged. As the of the argon plasma treatment increased, the | iNtrOduCtIoN {#SeC1}
============
oRgaNiC LiGHt-eMITTINg dIODes (oleDs) aRe bEiNG wIDELY appliED in disPlayS fOr MoBILes anD TElEvIsiOnS owinG to theIR seLf-EMittINg CHarACTeRIsTIcS, ExCeLlENT COLOuR GAMUT, High-spEed opeRaTiOn, AND aPpLicaBILiTY IN FlEXibLE or strEtCHABlE DevICES^[@cr1]^. pRactICal OLedS FUNcTioN baSed ON pHoSpHorEscENCE oWInG to THEiR neaRly 100% intErNAL PhOsphOReSCence efFiCIeNcY^[@cR2],[@cR3]^. HOweVEr, the EXtErNAL qUaNtum EfFiCiENCY Of Oleds is \~20% BeCausE tHe SurFacE PlAsmON POLARitOn mOdEs aT THE MetaL-oRGANIC InTerfaCE, tHe waveGUiDE ModE iN the IndIuM TIn OXiDe (ItO)/oRgAnIc Layer, AND tHe SuBSTrATE modE In THe GlaSS SUBSTrATE prOdUCE lIGht losS^[@cr4]--[@cR8]^. genErALLy, lIGhT lOSs In The SUbstRAte MOde Is lARGER THan that iN tHe WAVegUIDe MoDE due TO sMaLL DiFFErENCeS iN reFrACtIVe INDEx BEtweEn tHE itO/gLAss interfACE anD tHe GLaSs/aiR INTErface^[@cr9]^. In AdDitIon, mICrocavITY Top-EmItTINg orGANic LighT-eMiTTinG dIODes (tEoleD) wIthOUt ThE LOsS OF tHE sUbsTrATe moDe ANd hAViNG tHe AdVANtAge oF IMprOvEd COlouR purity ANd ApERtuRE rAtiO Have bEEN APplIed tO MOBiLe DispLays. hoWEVEr, micRocAvity tEOLeD oriGINalLY HAD a PrOBleM AsSOCIaTeD witH vIEWinG ANgLes due tO the blUE SHifT that ocCurreD wItH thE VaRiaTIOn Of The SiGhT angLe, EVEN thOugH thE LiGHt EFfICIeNCY ALOnG THE NORMAl diRecTiOn WAs AmpLifIEd. tHErEfORE, rEsearCHeRS havE atteMptED TO FInD SoLUtIOnS FOR ImproVINg THe LigHT EXtRACTIOn anD viEwinG ANgLe of OLEDs^[@Cr10]^.
difFErEnt aPPROaChes Have bEen ADOpTed WItH THe aim OF oPTImIZING LigHT eXTRaction anD VieWiNG aNgle, INcLuDINg micrO LEns ARRAY (mlA)^[@cr5],[@cr11]--[@cR16]^, dIelECtRiC BRagg GRaTINGS^[@cr17]--[@cr19]^, SURfacE plasmONs^[@cR20]^, BuCKlinG PaTTeRns^[@cR21]^, PeRioDic CorruGAtiON^[@cR8]^, LoW-INdEX GRiDS^[@Cr22]^, and PHotonic CrYstAls^[@CR23]^. METhOdS THAt ModiFY THe iNteRNal oR eXterNAl sURfACES OF the SUbStrAte Of olEDS MInimIZE thE total IntErNal ReFLeCtION. Such suRFacE sHApEs aRE proDuCED BY dIFFErENT TEChNOlogIeS WhicH tRaditIOnAllY InVOlVe photO-lItHOgrApHY OR PrInTing, MOUldINg, aND embOssiNg MeThodS^[@CR24]--[@cr28]^. ThEReFoRe, ThEsE TEcHnoLoGIeS empLoY AT Least tHe pHOto-lithoGrAPhIc StEP Or UtiliZe litHOGRAPHIC TemPlatEs oNce. tHeY gEneraLly eNd up beInG CoNSideRaBLY COMPLeX or eXpENsIvE fOR MAss prOdUcTION. ON ThE OTHER HANd, OTheR ResEaRchErs hAve RepOrTed a simpLE scaTtErinG laYeR sYNthEsizEd By COmpetItIve ANd sCALaBLE METhods. in tHese meTHODS, PARTIclES Or VOiDs HaVing DifFEreNt rEFRaCTiVe IndiCEs WeRe eMbeDDEd in A poLymer maTrIx ANd utiLIZeD FOR THE ScATTerIng LaYeR^[@cR29]--[@cR36]^. HoweVER, becAuse tHE PArTIcLeS oR vOidS ARe MiCro-sIzEd oR ThE pitch BetWeeN the nEareST paRtiCLES OR VoIDS Is MIcRO-sIzed, thEsE tECHNIques rEveaL a smaLl EfFect Of dIFFRaCtiON ThAt IS diFFiCuLT To CoNTrOl. in addiTIon, to pRevent THE loSs OF the sUBSTraTE MODE, The most REprEseNtATivE MLA Has LArgER vIsIBilITY tHAN a NAnO-siZeD ArRaY BecAUSe oF its size. AlignmeNT iS A ProBlEM IN ThE CASE oF High-RESolutiOn dIsplaYS WiTh VErY SMaLl PixEls.
In ThiS pApER, We pRoPOSE a siMPLE mETHod To FabriCAtE randoM NanoSCAle rODS (RnrS) As sCaTTERINg LAyERs FOR EnhancEMENT OF LigHt exTrACTion aND iMprovEmeNt oF VIeWing ANgLe cHArActErISTiCs In olEDs. iN oUr deVIce, ThERe Is viRTUalLy NO SpeCTrAl distORTIoN oR SHiFt, whICh arE issUEs obSERveD In 2D pHOtOniC CrYsTAL-BasEd OleDs, aNd thE SPECtral sHifT accOrDing To the ChAngE in vIEwIng anGlE is rEdUceD. tHis layEr is faBRIcated by A cOsT-eFfectIVe AnD SCALabLE prOCEduRe tHAT coNSiSTS oF COating And eTcHIng thE PolYMeR aT low teMperAtuREs (Below 100 °c) wIthOUT pHOtomaSks OR TEmPLAtES. AdditIOnAlLy, the rNRS CAN CoNtrol thE dIFFRACTIon AS well as sCattERiNG EFfeCtS IN THE VisIbLe wAVelenGTH RanGe owing TO tHe DisTancE betWeen cLOsEst rods aNd arE COmPAtIBLe With HiGh-ResoLUtion displAys FoR VIrTUal rEaLiTY bEcAUSe THE proCEsS of aLignMEnt beTWEEN THE INdIVIdUal piXeLs iN tHE PaNel aND the ScaTTeRIng LAYer IS UnNECesSArY. thE rNRs CaN aLso Be apPlieD in fLeXibLE DIspLAyS AND LIghting OwInG tO THE UsE Of ETcHED PoLymerS. OLeDs eqUiPPeD WITh RnRs exhIbit suPerIoR OptiCAL prOPERTIES suCH As HigH ExTerNAL quaNTUm EffICiENcY (EQe), hIGh LuMINaNCE EfFIciEncy (Le), AND COloUR vAriAtIOn wITH chANgES In viEwing ANgle, To cOnTrol OlEds.
rEsULTS AND DiSCUssiOn {#sEc2}
======================
TO InveStiGaTe thE opTical EFfeCt Of RNrs on GLAss, tHE ELectRICal fIEld DIsTRIBuTIoN wAs siMulAted by The fInite-diFFErENce tiME DOMAIN (fDTD) meThOD. tHE BoUNdARy cOndITIons FOR thE siMulATiOn WEre seT FOR a perFECT oPTicalLy MaTcHEd LAyeR TO aVoiD the rEFlectiON of ElEctRomaGneTiC wAveS at tHe EDgES oF ThE STRucTurE ON aLL the siDes, EXCept for The MeTaL cAThodE laYer. THe SImulateD STRUCTURE COnsIstED oF An AlUMiNIUM CAThoDe, *n*,*N*'-BIS(NAPHtHALen-1-yL)-*N*,*N*'-Bis(phENyL)-beNZIDINE (npb; REfrAcTivE inDEX n = 1.81), tRIs(8-hYDroxy-qUinOLInAto)AlUMinIuM (ALQ~**3**~; N = 1.72), ITO (N = 1.9), gLASs (N = 1.52), Su-8 poLymEr, AnD RNRs (n = 1.59). A dIpole SOuRCe of VIsIBlE WaVElENGTH wAS GeNerATeD In thE ALQ~**3**~ layEr, AnD ONE tRanSvErse ElecTric (te) aND tWo tRAnsveRSE MagNeTic (tM) MOdes WerE USED^[@cr37]^. To ReflEct THE rANdoMnEsS of rnrS iN ThE siMuLaTIon, theIR HEIghT waS vARiEd from 1000 nM To 1200 nm aNd wIDtH FroM 50 NM to 150 Nm (SUppLEmEnTARy inFormatioN FiG. [S1](#moESm1){reF-TYpE="medIA"}). IN THe REfeRencE StrUcTUrE, mOST Of tHE lIghT inCIdent aT aN aNgLe abOVE tHE CrItIcal anGle bECamE a waVEgUiDE BEcAUse of thE DIffEreNCe in REFracTive inDeX beTWEEN glaSs ANd aIR (fIG. [1a](#FIg1){REf-typE="fiG"}). IN ORDer To coNFiRM THE opTiCaL eFfeCt of tHe su-8 PolYMer, a SIMulaTIoN uSInG sU-8 WIThOUt tHe paTTerN wAS PERfoRMED (fIg. [1B](#FiG1){rEF-type="fiG"}). aS a RESULt, tHE OPticAL paTH WaS ALmOsT UnChANgEd, AND THe diffErEnce BEtWeEn thE rEFraCtive INDices Of GLaSS aNd AiR SligHTLY INcreased; thIs difFErENCE seeMEd To BE SligHtLY advERSeLY afFeCtED FOR ExtERnAL lIGhT EXtrAcTiON. IN coNTRaST, IN thE rnrS PrESeNT oN THE GLass StruCTURE, SOmE PoRTiOnS oF the wavEgUIdE LIght WERE AlLoWED To ESCAPe frOM The GLasS SuBstRATe, AND lIGHt EXtractiON IncREaSeD BelOw tHe CritICaL aNgLE (fIG. [1c](#fig1){rEF-tyPe="FIg"}). optICAL phenOmEnA SucH AS ReFRacTIoN, difFRAcTIOn, AND inTERFERenCe Were pROducED BY THe RNRS BeCAuSe THE DIfferEnces in ReFRaCtIve INDEx at ThE INteRFacE betWEen GlaSS AnD Air ChaNgED anD The RAnDoM PeriOdIc ROdS RevEALEd DIStAncES BetWeen each othEr that WErE sIMIlAR tO THE visiBlE wAvElEngth. FigUrE [2e](#FIG2){Ref-tYPe="fig"} iLLUSTRaTeS ThE liGHt PaTH iN oLEdS. THe PhOTonS TRAppEd (raYs 2 And 3) WIThIN THe gLasS ARE ScATTeRed BY tHe rNRs, inCreASiNg tHe pRobaBilitY Of theM bEING emitTEd FRom tHe sTRUctURE. fURtheRMoRE, AS A rEsult OF SimuLaTioN, IN WHicH sCATterinG OCcurs seVeRal timES iN One rOD, it CaN bE iNferRed ThAT SCaTTEriNG OccuRS MOrE FrEQUeNtLY wHen THE hEiGHT OF tHE Rod IS grEATEr.FigUrE 1FDtd SimULAtiOn Of E-FIeld DIstribution inDuCed bY TraNsVERse MAGnETiC And elECTRiC DipOLeS: (**a**) rEFerENce (w/O Rnrs), (**b**) UNPatternED su-8 on A gLASs sTRUcTuRE, And (**C**) Rnrs on The glaSS sTRUcture.fIGUre 2Cross-seCtIoNal scaNNING EleCTRoN MIcroScOPy iMaGES OF RANdoM nAnOScAle ROds (rnRs) ObtAInED uNDEr difFEREnt PlASmA eTCHiNG COndiTiONS: (**A**) RnRs 1, (**b**) rNrs 2, (**c**) rNrs 3, aND (**d**) rNRs 4, aNd (**e**) sCHEmatIc IllUStraTiON oF oLed outCoUPling eNhANcemEnt By the rNR scatTerInG LAyer.
iT is dEsirABle TO fAbRIcaTE A NaNoSTruCtuRe oF prOpER hEiGHt ANd PERIodIcIty THaT is COMPARABlE to tHE wAvElengTh of THe vIsiBlE RaNge iN OrDer to eNhANcE LigHt ExTractION. fOR manuFACTuRINg RnRS, su-8 PolYmER was ChOSen BecAUSE it IS HIGHly TranSpaREnt to viSiBLe LIGhT and haS a higHeR REFractIVE IndeX THan gLasS. FigUre [2](#Fig2){REf-tyPe="FIG"} shoWs THe cROsS-SeCTIonal scAnnING eLecTRON MICrosCOpY ImAGeS oF thE rnRs obtAined UNDer dIFFEREnt PlaSma EtCHinG cOndItioNS. obtAinED By usiNG ONLy An OxYGeN pLaSMa For a duRATION oF 9 Min (DENOTEd as rnrs 1), tHe sTrUCturE waS ranDom COLUMNAr-LiKE WITh hIgH asPECt RaTiO AnD deNSIty^[@Cr38]^. The height aND DEnsiTY oF RNRs 1 WEre AbOut 1200 nM ANd 9.7 EA/µm^2^, RESpeCTiveLY (fIG. [2a](#fig2){Ref-TypE="fiG"}, SUpPLEMentarY iNFoRMATIon fig. [s2A](#mOESM1){REF-TYpE="MEDIa"}). ThE wiDtH oF The INdIVIDuAL rOds wAS oBserVEd TO Be aPprOxiMateLy 50 Nm. tO cONtRoL tHE HEIght anD DensIty, we ApPlIed AN addITIonAl ARgon PlASma trEAtMEnt at HiGHER pOwErs AND lowEr PROcESs PrESsUReS thAn ThE OXyGEN pLASmA. ThE LoW pREssUrE lEd TO AN incrEAsE iN thE mEAN FREE pATh Of ARgOn IOns AnD RedUceD The ENeRGY losS oF THESe IONs As tHEY apPrOAChed ThE rnrS. iN GenERAl, tHe ETCHinG ProCesS uSING ARgOn pLAsma carRIed oUt IN A rEActive IOn etchER rESUlTEd IN ANISotRopic anD dIrecTIONAL fEAtUReS DUe TO ThE utIliZaTiON oF tHe phySical BOmBaRDMEnT ENERGy^[@CR38]^. As A rESuLt Of tHEse feATuRES, thE hEighT aND DeNSiTy WeRe ADJuSTed As mUCH As a tarGET To THE littLe chanGeD WidtH OF THe rNrS. THe ThREE TrEaTEd rNRs sHown IN fig. [2B--D](#fiG2){ref-TyPe="FIG"} CoRrESPOndEd TO ARgON PLaSma TrEatMeNt DURATioNs Of 3, 6, aNd 9 min (denOTeD AS rnrS 2, rNRs 3, aNd rNRs 4, RESPectIVEly), WhiLe tHe ConDitIONS OF tHe OxyGeN PlasMA prOcESS rEMAINed unChAngEd. as thE duRatION Of tHE aRgoN PLASMa TREATmenT incREaseD, The | Introduction {#Sec1} ============ Organic light-emitting diodes (OLEDs) are being widelyapplied in displays for mobiles and televisions owing to their self-emitting characteristics, excellent colourgamut, high-speed operation, and applicability in flexible or stretchabledevices^[@CR1]^.Practical OLEDs functionbased on phosphorescenceowing to their nearly 100% internal phosphorescence efficiency^[@CR2],[@CR3]^. However, the external quantum efficiency of OLEDs is \~20% because thesurfaceplasmonpolariton modes at the metal-organic interface, the waveguide mode in theindium tin oxide (ITO)/organic layer, and the substrate mode in the glass substrate produce light loss^[@CR4]--[@CR8]^. Generally, light loss in the substrate mode is larger than that in the waveguide modedue to smalldifferences in refractive indexbetween the ITO/glass interface and the glass/air interface^[@CR9]^. In addition, microcavity top-emitting organic light-emitting diodes (TEOLED) withoutthe loss of the substrate mode and having the advantage of improved colour purity and aperture ratio havebeen applied to mobile displays. However, microcavity TEOLED originally had a problem associated with viewing angles due tothe blue shift that occurred with the variation of the sight angle, even though the light efficiency along the normal direction was amplified. Therefore, researchers have attempted to find solutions for improving the lightextraction and viewing angle of OLEDs^[@CR10]^. Different approaches have been adopted with theaim of optimizing lightextraction andviewing angle, including micro lens array (MLA)^[@CR5],[@CR11]--[@CR16]^, dielectric Bragg gratings^[@CR17]--[@CR19]^,surfaceplasmons^[@CR20]^, buckling patterns^[@CR21]^, periodic corrugation^[@CR8]^,low-index grids^[@CR22]^,and photonic crystals^[@CR23]^. Methods that modify the internalorexternal surfaces of the substrate of OLEDs minimize the total internal reflection. Such surface shapes are producedby different technologies which traditionally involve photo-lithography or printing, moulding, and embossing methods^[@CR24]--[@CR28]^. Therefore, these technologies employ at leastthe photo-lithographic stepor utilize lithographic templates once. They generally end up being considerably complex or expensive for mass production.On the other hand, other researchers have reported a simple scattering layer synthesized by competitive and scalable methods. In these methods, particles or voids having different refractive indices were embedded in a polymer matrix and utilized for the scattering layer^[@CR29]--[@CR36]^. However, because the particles or voids aremicro-sized or the pitch between the nearest particles or voids is micro-sized, these techniques reveal a small effectof diffractionthat is difficult to control. In addition, to prevent the loss of thesubstrate mode,themost representative MLA has larger visibility than anano-sized arraybecause ofits size. Alignment is a problem inthe case of high-resolution displays withvery small pixels. In this paper, we propose a simple method to fabricate random nanoscale rods(RNRs) as scattering layers for enhancement of light extraction and improvement of viewing angle characteristics in OLEDs. In our device, thereis virtually nospectral distortionor shift, which are issues observed in2D photonic crystal-based OLEDs, and the spectral shift according to the change in viewing angle is reduced. Thislayer is fabricated by a cost-effective and scalable procedure that consists ofcoating andetching the polymer at low temperatures (below 100 °C) without photomasks or templates. Additionally, the RNRs can controlthe diffraction as well as scattering effects in the visible wavelength range owing to thedistance between closest rods and are compatible with high-resolutiondisplays forvirtual reality because the process of alignment betweenthe individual pixels in the panel and the scattering layer is unnecessary. The RNRs can also be appliedin flexible displays and lighting owing totheuse of etchedpolymers. OLEDsequipped with RNRs exhibitsuperior optical properties such as high external quantum efficiency (EQE), high luminance efficiency (LE),and colourvariation with changes in viewingangle, to control OLEDs. Results and Discussion {#Sec2} ====================== To investigate the optical effect of RNRs on glass, the electrical fielddistribution wassimulatedby thefinite-difference time domain (FDTD) method.The boundary conditions for the simulationwere set for a perfect optically matched layertoavoid the reflection ofelectromagnetic waves at theedges of the structure on all the sides,except for the metal cathode layer. The simulated structure consisted of an aluminium cathode, *N*,*N*'-bis(naphthalen-1-yl)-*N*,*N*'-bis(phenyl)-benzidine (NPB; refractive index n = 1.81), tris(8-hydroxy-quinolinato)aluminium (Alq~**3**~; n = 1.72), ITO (n = 1.9), glass (n = 1.52), SU-8 polymer, and RNRs (n = 1.59). A dipole source of visible wavelength was generated in the Alq~**3**~ layer, andone transverseelectric (TE) and two transverse magnetic (TM) modes were used^[@CR37]^. Toreflect the randomnessof RNRs in the simulation,their height was varied from 1000 nm to1200 nm and width from 50 nm to 150 nm (Supplementary Information Fig. [S1](#MOESM1){ref-type="media"}). Inthe reference structure, most of the light incident at an angle above the critical angle became a waveguide because of the difference in refractive index between glass and air (Fig. [1a](#Fig1){ref-type="fig"}).In order to confirm the opticaleffect of the SU-8 polymer, a simulationusing SU-8without the pattern was performed (Fig. [1b](#Fig1){ref-type="fig"}). As aresult, the optical path was almost unchanged, andthe difference between the refractive indices of glass andair slightly increased;this difference seemed to beslightly adversely affected for external light extraction.In contrast, in theRNRspresent on the glass structure, some portions of the waveguide light wereallowedto escape from the glass substrate,and light extraction increased belowthecritical angle (Fig. [1c](#Fig1){ref-type="fig"}). Optical phenomena such as refraction, diffraction, and interference were produced by the RNRs becausethe differences in refractive indexatthe interface between glass and air changed and the random periodicrods revealed distances between each other that were similar to the visible wavelength. Figure [2e](#Fig2){ref-type="fig"} illustrates the light path inOLEDs. The photons trapped (rays 2and 3) within the glassare scattered by theRNRs, increasing the probability of them being emittedfromthe structure. Furthermore, as a result ofsimulation, in whichscattering occurs several times in one rod, it can be inferred that scattering occurs more frequently when the heightof the rod is greater.Figure 1FDTD simulation of E-fielddistribution induced by transverse magnetic and electric dipoles: (**a**) Reference (w/o RNRs), (**b**) unpatterned SU-8 on a glass structure, and (**c**) RNRs on the glass structure.Figure2Cross-sectional scanningelectronmicroscopy images of random nanoscale rods (RNRs) obtained under different plasmaetching conditions: (**a**)RNRs 1,(**b**) RNRs2, (**c**) RNRs 3, and (**d**) RNRs 4, and (**e**) schematic illustrationof OLED outcoupling enhancement by the RNRscattering layer. It isdesirable to fabricate a nanostructure of properheight and periodicity that is comparableto the wavelength of the visible rangein order to enhance light extraction. For manufacturing RNRs, SU-8polymer was chosen because it is highly transparent to visible light and hasahigher refractive index than glass. Figure [2](#Fig2){ref-type="fig"} shows the cross-sectional scanning electron microscopyimages of the RNRs obtained under different plasma etching conditions. Obtained by usingonlyan oxygen plasma for a duration of9 min (denoted as RNRs 1), the structure was random columnar-like withhigh aspect ratio anddensity^[@CR38]^. The heightand density of RNRs 1wereabout 1200 nm and 9.7 ea/µm^2^, respectively (Fig. [2a](#Fig2){ref-type="fig"}, Supplementary Information Fig. [S2a](#MOESM1){ref-type="media"}).The width of the individual rods was observed tobe approximately 50 nm. To control the height anddensity, weapplied an additional argon plasma treatment at higher powers and lower process pressures than the oxygen plasma. The lowpressure led toan increasein themean free path of argon ions and reduced the energy loss of these ions as they approached the RNRs. In general, the etching process using argon plasma carried out in areactive ion etcher resultedin anisotropic and directional features due to the utilization of the physical bombardment energy^[@CR38]^. As a result of these features, the height and densitywere adjusted as much as a target tothe little changed width of the RNRs. The three treated RNRsshown in Fig. [2b--d](#Fig2){ref-type="fig"} correspondedto argon plasma treatment durationsof 3, 6, and9min (denotedasRNRs 2, RNRs 3, and RNRs 4, respectively), while theconditions of the oxygen plasma process remainedunchanged. As theduration of the argon plasma treatment increased, the | _Introduction_ {#Sec1} ============ Organic _light-emitting_ diodes _(OLEDs)_ are _being_ widely applied in displays _for_ mobiles and televisions owing to their self-emitting characteristics, excellent colour gamut, _high-speed_ _operation,_ _and_ applicability in _flexible_ or stretchable _devices^[@CR1]^._ Practical OLEDs function based on _phosphorescence_ owing to their nearly 100% _internal_ phosphorescence efficiency^[@CR2],[@CR3]^. However, the external quantum _efficiency_ of OLEDs is \~20% because _the_ _surface_ plasmon polariton modes at the _metal-organic_ _interface,_ the waveguide _mode_ _in_ the indium tin _oxide_ (ITO)/organic layer, and the substrate mode in the glass substrate produce light loss^[@CR4]--[@CR8]^. _Generally,_ light _loss_ in the _substrate_ mode is larger than that in the waveguide mode _due_ _to_ small differences in refractive index between the ITO/glass interface and the _glass/air_ _interface^[@CR9]^._ In _addition,_ microcavity top-emitting organic light-emitting _diodes_ (TEOLED) without the loss of the substrate mode and having the advantage of improved _colour_ purity and aperture ratio have been _applied_ to mobile displays. However, microcavity TEOLED _originally_ had a problem associated with viewing angles _due_ to the _blue_ _shift_ _that_ occurred _with_ the variation of the _sight_ angle, even though the _light_ efficiency along the normal _direction_ _was_ amplified. Therefore, researchers have attempted to find _solutions_ for improving the _light_ extraction and _viewing_ angle of OLEDs^[@CR10]^. Different approaches _have_ been _adopted_ with the aim of optimizing _light_ extraction and _viewing_ angle, including micro lens _array_ (MLA)^[@CR5],[@CR11]--[@CR16]^, dielectric Bragg gratings^[@CR17]--[@CR19]^, surface plasmons^[@CR20]^, buckling patterns^[@CR21]^, periodic corrugation^[@CR8]^, low-index grids^[@CR22]^, and photonic crystals^[@CR23]^. Methods that _modify_ the _internal_ or external surfaces of the substrate of OLEDs minimize the total internal reflection. Such surface shapes are produced by different technologies which traditionally involve photo-lithography or printing, _moulding,_ _and_ embossing methods^[@CR24]--[@CR28]^. _Therefore,_ _these_ technologies employ at least the photo-lithographic step or utilize _lithographic_ templates once. They generally end up being considerably complex _or_ expensive for mass production. _On_ the other hand, other researchers have reported _a_ simple scattering _layer_ synthesized _by_ competitive and scalable methods. In these methods, particles or voids having different refractive indices were embedded in a polymer matrix _and_ utilized for _the_ scattering layer^[@CR29]--[@CR36]^. However, because the particles or voids are _micro-sized_ or the pitch _between_ the nearest particles _or_ voids is micro-sized, these techniques reveal a small _effect_ of diffraction that is difficult _to_ control. In addition, to _prevent_ the loss of the _substrate_ mode, the most representative MLA _has_ larger visibility than _a_ nano-sized array because of its size. Alignment is _a_ _problem_ _in_ _the_ _case_ of high-resolution displays with very small pixels. _In_ this paper, we propose a simple _method_ to fabricate _random_ nanoscale rods (RNRs) as scattering layers for enhancement _of_ light _extraction_ and improvement of viewing angle characteristics in _OLEDs._ In our device, there is virtually _no_ spectral distortion or shift, which are issues observed in 2D photonic crystal-based OLEDs, and the _spectral_ shift _according_ to the change in viewing angle is reduced. This layer is fabricated _by_ a cost-effective and scalable procedure that consists of _coating_ _and_ _etching_ the polymer at low _temperatures_ (below _100_ °C) without photomasks or templates. Additionally, _the_ RNRs can _control_ the diffraction as well _as_ scattering _effects_ in the visible _wavelength_ range _owing_ to the distance between closest rods and are compatible with high-resolution displays _for_ _virtual_ reality _because_ _the_ process of alignment between the individual pixels in the panel and the scattering layer is unnecessary. The RNRs can also be _applied_ in flexible displays and lighting _owing_ to _the_ use of _etched_ polymers. OLEDs equipped with _RNRs_ exhibit superior optical properties such _as_ high external quantum _efficiency_ (EQE), _high_ luminance efficiency _(LE),_ and colour variation with changes in viewing angle, to _control_ _OLEDs._ Results and _Discussion_ {#Sec2} ====================== To _investigate_ the _optical_ _effect_ _of_ RNRs on glass, _the_ electrical field distribution _was_ simulated by the finite-difference time domain (FDTD) method. The _boundary_ conditions for the simulation were _set_ _for_ a perfect optically matched layer to _avoid_ _the_ reflection _of_ electromagnetic waves at the edges of the structure on all _the_ _sides,_ except for the metal cathode layer. The _simulated_ structure _consisted_ of _an_ aluminium cathode, *N*,*N*'-bis(naphthalen-1-yl)-*N*,*N*'-bis(phenyl)-benzidine (NPB; refractive index n = _1.81),_ tris(8-hydroxy-quinolinato)aluminium (Alq~**3**~; n = 1.72), ITO (n = 1.9), glass (n _=_ 1.52), SU-8 polymer, and _RNRs_ (n = 1.59). A _dipole_ _source_ _of_ visible wavelength was _generated_ in the Alq~**3**~ layer, and one transverse electric (TE) _and_ two transverse magnetic (TM) modes were used^[@CR37]^. To reflect the randomness of RNRs in the _simulation,_ _their_ height was varied from 1000 _nm_ to 1200 nm and _width_ from _50_ nm to 150 nm (Supplementary Information Fig. [S1](#MOESM1){ref-type="media"}). _In_ the reference structure, most _of_ the light incident at an angle above the critical angle became a waveguide because of the difference _in_ refractive _index_ _between_ glass and air (Fig. [1a](#Fig1){ref-type="fig"}). _In_ order to confirm _the_ optical effect of the SU-8 polymer, a simulation _using_ _SU-8_ without the pattern was _performed_ (Fig. [1b](#Fig1){ref-type="fig"}). As _a_ result, the optical path was almost unchanged, and the difference between _the_ refractive indices of _glass_ and air slightly increased; this difference seemed _to_ be slightly _adversely_ affected for _external_ light _extraction._ In _contrast,_ in the RNRs present _on_ the glass structure, _some_ portions of the waveguide light _were_ allowed to escape from _the_ glass substrate, and light extraction increased below the critical angle (Fig. [1c](#Fig1){ref-type="fig"}). Optical phenomena such as refraction, diffraction, and interference were produced by the RNRs because the differences in refractive index at the interface between glass and _air_ changed and the random periodic rods revealed distances between each other that were similar to the _visible_ _wavelength._ Figure _[2e](#Fig2){ref-type="fig"}_ _illustrates_ the _light_ path in OLEDs. The photons trapped (rays 2 and 3) within the glass are scattered by the RNRs, increasing _the_ probability of them being emitted _from_ the structure. Furthermore, as a result _of_ simulation, _in_ _which_ scattering occurs _several_ times in one rod, it can _be_ inferred that scattering occurs more frequently when _the_ height of the rod is greater.Figure 1FDTD simulation of E-field _distribution_ induced by transverse magnetic and electric dipoles: (**a**) Reference _(w/o_ RNRs), (**b**) unpatterned SU-8 on _a_ glass structure, and (**c**) RNRs on the _glass_ structure.Figure _2Cross-sectional_ scanning electron microscopy _images_ of random nanoscale rods (RNRs) _obtained_ under different plasma _etching_ _conditions:_ (**a**) RNRs _1,_ (**b**) RNRs 2, (**c**) RNRs 3, and (**d**) _RNRs_ 4, and (**e**) schematic illustration _of_ OLED _outcoupling_ _enhancement_ _by_ the RNR scattering layer. _It_ is desirable _to_ _fabricate_ a nanostructure of proper height and periodicity that is _comparable_ to the wavelength _of_ the visible range in order to enhance light _extraction._ For manufacturing RNRs, SU-8 polymer was chosen because it is highly transparent to visible light and has _a_ higher refractive index than glass. Figure [2](#Fig2){ref-type="fig"} shows the cross-sectional scanning electron microscopy images of the RNRs obtained _under_ different _plasma_ etching conditions. Obtained by _using_ _only_ an oxygen plasma for a duration of 9 min _(denoted_ as RNRs 1), the _structure_ was random columnar-like with _high_ aspect _ratio_ and density^[@CR38]^. The _height_ and density _of_ RNRs 1 were _about_ 1200 nm and 9.7 ea/µm^2^, respectively (Fig. [2a](#Fig2){ref-type="fig"}, Supplementary Information Fig. [S2a](#MOESM1){ref-type="media"}). The width of the individual rods was _observed_ to be _approximately_ 50 nm. To control the height _and_ _density,_ _we_ applied an additional argon plasma _treatment_ at higher powers and lower process pressures than _the_ oxygen plasma. The low _pressure_ led _to_ an increase in the mean free path of _argon_ ions and reduced the _energy_ loss _of_ these _ions_ as they approached the RNRs. In _general,_ the etching process using argon plasma carried _out_ in a reactive ion etcher _resulted_ in anisotropic and directional features due _to_ _the_ _utilization_ of the physical _bombardment_ energy^[@CR38]^. As a _result_ of these features, the _height_ and density were adjusted as much _as_ a _target_ to _the_ little changed width of the RNRs. The three treated RNRs shown in Fig. [2b--d](#Fig2){ref-type="fig"} _corresponded_ _to_ argon _plasma_ _treatment_ durations of _3,_ 6, and 9 min (denoted _as_ RNRs _2,_ RNRs _3,_ and RNRs 4, respectively), while _the_ _conditions_ of the _oxygen_ plasma process remained unchanged. _As_ the duration of _the_ argon plasma treatment increased, the |
Introduction {#ss1}
============
Throughout Europe birth-rates have declined over the last decades, and in most countries they are well below the replacement level ([@CIT0001]). In Sweden, the number of children being born per woman has fluctuated greatly since the mid-1970s but has now been predicted to stabilize at a level around 1.8 ([@CIT0002]). The declining birth-rate can partly be explained by an increasing age at first birth ([@CIT0003]); furthermore, today there is clear empirical evidence of the postponement of the first child in several countries ([@CIT0004; @CIT0005; @CIT0006]).
Higher education has been associated with delayed childbearing ([@CIT0007]), and the association between female education and the age of becoming a first-time parent has been well documented ([@CIT0008; @CIT0009]), showing that highly educated women are more likely to pursue careers and postpone having children ([@CIT0005]). Women\'s labour force participation has been associated with postponement largely due to the incompatibility of caring for children and participation in the paid labour force. However, it has been possible to combine female employment and childbearing when the reduction in work--family conflict was facilitated by state or policy interventions, such as in some Scandinavian countries ([@CIT0010; @CIT0011]).
It seems, however, that postponed childbearing is a result of several different reasons. For example, efficient and reliable oral contraception has had an impact on family planning in many modern societies ([@CIT0012; @CIT0013]). A US study found that multiple partnerships before marriage, higher levels of non-marital cohabiting, and difficulty in finding a suitable partner might contribute to later births ([@CIT0014]). It has also been reported that young adults delay childbearing until they are financially secure, so they can afford to support children ([@CIT0015]). In addition, the emergence and development of assisted reproductive technologies (ARTs) have been suggested to promote perceptions that childbearing can be resumed at a later and more convenient phase of life ([@CIT0016]).
Surveys concerning attitudes towards future parenthood and awareness about fertility among undergraduate and postgraduate students in Sweden have shown that these young women and men had largely positive attitudes towards having children, but they were not sufficiently aware of the limitations associated with ageing ([@CIT0017; @CIT0018; @CIT0019]). Similar results have been reported in a recent study among Finnish university students, in which one-third of the women and more than half of the men believed that a marked decline in female fertility begins after the age of 45 ([@CIT0020]). In a Canadian survey among female undergraduate students, it was found that women were aware that fertility declines with age, but they overestimated the chances of pregnancy at any age and were not aware of the steep rate of fertility decline with age ([@CIT0021]). However, knowledge about the influence of female age on childbearing success was not predictive of their childbearing intentions.
The adverse effects of ageing on reproduction are complex and multifactorial. It is known that women\'s ability to conceive starts to decline in their late 20s and rapidly falls by the mid-30s, mainly due to reduced quantity and quality of ovarian follicles ([@CIT0003; @CIT0022]). In addition, after the age of 35 an increase in pregnancy complications and prenatal maternal morbidity, as well as impaired prenatal and postnatal outcome of the child has been reported ([@CIT0023; @CIT0024; @CIT0025]). There is also increasing evidence that paternal age, at least over the age of 40, is associated with lower fertility-related outcome and anomalies in the offspring ([@CIT0026; @CIT0027]).
Societal attitude towards family and children also forms the context in which the subjective preferences and assessments regarding family formation are made. The tendency towards individualism, the endeavour for self-fulfilment, increasing freedom of choice, and greater tolerance towards new lifestyles and reproductive behaviours have also been suggested to influence the decline in births and the postponement of childbearing ([@CIT0028]).
Most research concerning postponed childbearing has focused on women and has mostly been based on standardized questionnaires or register data ([@CIT0029]); however, the information gathered from the viewpoint of highly educated young people themselves is more random. Thus, the aim of the present study was to gain a more comprehensive understanding of how young, highly educated women and men without children, who had started their professional career, reflect on fertility and postponed parenthood.
Materials and methods {#ss2}
=====================
We performed individual interviews with 22 women and 18 men between 24 and 38 years of age. The criteria for inclusion were 4 years or more of university education, having started a professional career, and not yet having children. The participants were recruited using advertisements placed in different work-places where the majority of staff had a higher education. Those who were interested in participating were requested to phone or send an e-mail to the project leaders for further information and arrangement of a suitable time for the interview. The interviews took place in a convenient room offered by the work-place, or outside the office. The interviews were audiotaped and lasted for about half an hour. The interview guide covered two main areas: 1) personal attitudes towards having children in the future and 2) reflections on fertility and postponement of parenthood. The findings related to personal attitudes towards future parenthood have previously been published ([@CIT0030]). All interviews were transcribed verbatim and analysed according to qualitative content analysis ([@CIT0031]).
The transcripts were read several times to gain a sense of the whole. Constellations of words, sentences, or paragraphs related to the study aim were identified and condensed. Thereafter, codes were created, and finally the codes were sorted into seven subcategories and two categories according to their content and meaning. The process of developing categories is illustrated in [Tables I](#T1){ref-type="table"} and [II](#T2){ref-type="table"}.
######
Examples of condensed meaning units, codes, subcategories, and categories.
Meaning units Codes Subcategory Category
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------ --------------------------------------------------------- ------------------------------------------------------------------
'In a larger city one has so many other things to keep you busy anyway, so children come significantly later. Yes, but yeah, maybe it\'s time, I thought a little like that, that it\'s about time\' City life offers many alternatives to family formation A consequence of contemporary lifestyles Postponed parenthood---a rational adaptation to societal changes
'To delay having children is probably due to the increased fear of growing up, to seriously be an adult because it\'s so nice to have to worry only about yourself\' A sign of extended immaturity, or egocentricity
'Today it is expected to not only get an education but also to travel, learn a new language, live overseas and much more, and parenthood doesn\'t fit so well in the story\' Expectations of further learning and striving for experiences and adventures A consequence of competing priorities
'There are more women who study at universities, more women than men that graduate, and maybe the birth of a child is viewed as an obstacle to one\'s career, that must be the explanation\' Women\'s increased investment in higher education
'To have a child before 25 is not exactly something that is encouraged and maybe also not having children after 40 either\' Socially mediated perceptions about the timing of childbearing A consequence of prevailing discourses about parenthood
'There is such an incredibly negative view about having a child when you are young, and among my friends there are many that see it as completely abnormal to have a child before 30\' Early parenthood is unfavourable
######
Women\'s and men\'s reflections on fertility and postponed parenthood.
Subcategories Categories
--------------------------------------------------------- ------------------------------------------------------------------
Unconsidered and taken for granted Fertility---an imperceptive and retrievable capacity
Unpredictable and different for women and men
Restorable by medical technology
Replaceable by alternatives to a biological child
A consequence of contemporary lifestyles Postponed parenthood---a rational adaptation to societal changes
A consequence of competing priorities
A consequence of prevailing discourses about parenthood
Results {#ss3}
=======
In the following section, the two categories 'Fertility---an imperceptible and retrievable capacity\' and 'Postponed childbearing---a rational adaptation to societal changes\' are presented. Quotations from the interviews are used for illustration.
Fertility---an imperceptible and retrievable capacity {#ss4}
-----------------------------------------------------
The category 'Fertility---an imperceptible and retrievable capacity\' consisted of four subcategories: 'Unconsidered and taken for granted\', 'Unpredictable and different for women and men\', 'Restorable by medical technology\', and 'Replaceable by alternatives to a biological child\'.
### Unconsidered and taken for granted {#ss5}
This subcategory consisted of statements showing that even though most informants wanted to have children in the future, some had never reflected on their own reproductive capacity. It also included comments that revealed perceptions about fertility as something natural. Some referred to a healthy body, while others put confidence in their genetic heritage. In addition, some held the opinion that there is no point in reflecting too much about one\'s fertility as it might cause unfounded worries.
> My own fertility, nah, I have not thought about that; actually, I have never done that. (w12)
> I presume that I can deliver what\'s required when it comes to | introduction { # ss1 } = = = = = = = = = = = = throughout europe birth - rates have declined over the last decades, and in most countries they are well below the replacement level ( [ @ cit0001 ] ). in sweden, the number of children being born per woman has fluctuated greatly since the mid - 1970s but has now been predicted to stabilize at a level around 1. 6 ( [ @ cit0002 ] ). the declining birth - rate can partly be explained by an increasing age at first birth ( [ @ cit0003 ] ) ; furthermore, today there is clear empirical evidence of the postponement of their first child in several countries ( [ @ cit0004 ; @ cit0005 ; @ cit0006 ] ). higher education has been associated with delayed childbearing ( [ @ cit0007 ] ), and the association between female education and the age of becoming a first - time parent has been well documented ( [ @ cit0008 ; @ cit0009 ] ), showing that highly educated women are more likely to pursue careers and postpone having children ( [ @ cit0005 ] ). women \ ' s labour force education has been associated with postponement largely due at the incompatibility at caring for children and participation in the paid labour force. however, little has been possible to combine female employment with childbearing when the reduction in work - - family conflict was facilitated by state or policy interventions, such as in some scandinavian countries ( [ @ 1987 ; @ cit0011 ] ). it seems, however, that postponed childbearing is a result of several different reasons. for example, efficient and reliable oral contraception has had an impact on family planning in many modern societies ( [ @ cit0012 ; @ cit0013 ] ). a us study found that multiple partnerships before marriage, higher levels of non - sexual cohabiting, and difficulty in finding a suitable partner might contribute to later births ( [ @ cit0014 ] ). it has also been reported that young adults delay childbearing until they are financially secure, so they can afford to support intercourse ( [ @ cit0015 ] ). in addition, the emergence and development of assisted reproductive technologies ( arts ) have been suggested to promote perceptions that childbearing can be resumed at a later and more convenient phase of life ( [ @ cit0016 ] ). surveys concerning attitudes towards future parenthood and awareness about fertility among undergraduate and postgraduate students in sweden have shown that these young women and men had largely positive attitudes towards having children, but they were not sufficiently aware of the limitations associated with ageing ( [ @ cit0017 ; @ cit0018 ; @ cit0019 ] ). similar results have been reported in a recent study among finnish university students, in which one - third of the women and more than half of the men believed that a marked decline in female fertility begins after the age of 45 ( [ @ cit0020 ] ). in a canadian survey among female undergraduate students, it was found that women were aware that fertility declines with age, but they overestimated the chances of pregnancy at any age and were not aware of the steep rate of fertility decline with age ( [ @ cit0021 ] ). however, knowledge about the influence of female age on childbearing success was not predictive of their childbearing intentions. the adverse effects of ageing on reproduction are complex and multifactorial. it is known that women \ ' s ability to conceive starts to decline in their late 20s and rapidly falls by the mid - 30s, mainly due to reduced quantity and quality of ovarian follicles ( [ @ cit0003 ; @ cit0022 ] ). in addition, after the age of 35 an increase in pregnancy complications and prenatal maternal morbidity, as well as impaired prenatal and postnatal outcome of the child has been reported ( [ @ cit0023 ; @ cit0024 ; @ cit0025 ] ). there is also increasing evidence that paternal age, at least over the age of 40, is associated with lower fertility - related outcome and anomalies in the offspring ( [ @ cit0026 ; @ cit0027 ] ). societal attitude towards family and children also forms the context in which the subjective preferences and assessments regarding family formation are made. the tendency towards individualism, the endeavour for self - fulfilment, increasing freedom of choice, and greater tolerance towards new lifestyles and reproductive behaviours have also been suggested to influence the decline in births and the postponement of childbearing ( [ @ cit0028 ] ). most research concerning postponed childbearing has focused on women and has mostly been based on standardized questionnaires or register data ( [ @ cit0029 ] ) ; however, the information gathered from the viewpoint of highly educated young people themselves is more random. thus, the aim of the present study was to gain a more comprehensive understanding of how young, highly educated women and men without children, who had started their professional career, reflect on fertility and postponed parenthood. materials and methods { # ss2 } = = = = = = = = = = = = = = = = = = = = = we performed individual interviews with 22 women and 18 men between 24 and 38 years of age. the criteria for inclusion were 4 years or more of university education, having started a professional career, and not yet having children. the participants were recruited using advertisements placed in different work - places where the majority of staff had a higher education. those who were interested in participating were requested to phone or send an e - mail to the project leaders for further information and arrangement of a suitable time for the interview. the interviews took place in a convenient room offered by the work - place, or outside the office. the interviews were audiotaped and lasted for about half an hour. the interview guide covered two main areas : 1 ) personal attitudes towards having children in the future and 2 ) reflections on fertility and postponement of parenthood. the findings related to personal attitudes towards future parenthood have previously been published ( [ @ cit0030 ] ). all interviews were transcribed verbatim and analysed according to qualitative content analysis ( [ @ cit0031 ] ). the transcripts were read several times to gain a sense of the whole. constellations of words, sentences, or paragraphs related to the study aim were identified and condensed. thereafter, codes were created, and finally the codes were sorted into seven subcategories and two categories according to their content and meaning. the process of developing categories is illustrated in [ tables i ] ( # t1 ) { ref - type = " table " } and [ ii ] ( # t2 ) { ref - type = " table " }. # # # # # # examples of condensed meaning units, codes, subcategories, and categories. meaning units codes subcategory category - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ' in a larger city one has so many other things to keep you busy anyway, so children come significantly later. yes, but yeah, maybe it \ ' s time, i thought a little like that, that it \ ' s about time \ ' city life offers many alternatives to family formation a consequence of contemporary lifestyles postponed parenthood - - - a rational adaptation to societal changes ' to delay having children is probably due to the increased fear of growing up, to seriously be an adult because it \ ' s so nice to have to worry only about yourself \ ' a sign of extended immaturity, or egocentricity ' today it is expected to not only get an education but also to travel, learn a new language, live overseas and much more, and parenthood doesn \ ' t fit so well in the story \ ' expectations of further learning and striving for experiences and adventures a consequence of competing priorities ' there are more women who study at universities, more women than men that graduate, and maybe the birth of a child is viewed as an obstacle to one \ ' s career, that must be the explanation \ ' women \ ' s increased investment in higher education ' to have a child before 25 is not exactly something that is encouraged and maybe also not having children after 40 either \ ' socially mediated perceptions about the timing of childbearing a consequence of prevailing discourses about parenthood ' there is such an incredibly negative view about having a child when you are young, and among my friends there are many that see it as completely abnormal to have a child before 30 \ ' early parenthood is unfavourable # # # # # # women \ ' s and men \ ' s reflections on fertility and postponed parenthood. subcategories categories - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - unconsidered and taken for granted fertility - - - an imperceptive and retrievable capacity unpredictable and different for women and men restorable by medical technology replaceable by alternatives to a biological child a consequence of contemporary lifestyles postponed parenthood - - - a rational adaptation to societal changes a consequence of competing priorities a consequence of prevailing discourses about parenthood results { # ss3 } = = = = = = = in the following section, the two categories ' fertility - - - an imperceptible and retrievable capacity \ ' and ' postponed childbearing - - - a rational adaptation to societal changes \ ' are presented. quotations from the interviews are used for illustration. fertility - - - an imperceptible and retrievable capacity { # ss4 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the category ' fertility - - - an imperceptible and retrievable capacity \ ' consisted of four subcategories : ' unconsidered and taken for granted \ ', ' unpredictable and different for women and men \ ', ' restorable by medical technology \ ', and ' replaceable by alternatives to a biological child \ '. # # # unconsidered and taken for granted { # ss5 } this subcategory consisted of statements showing that even though most informants wanted to have children in the future, some had never reflected on their own reproductive capacity. it also included comments that revealed perceptions about fertility as something natural. some referred to a healthy body, while others put confidence in their genetic heritage. in addition, some held the opinion that there is no point in reflecting too much about one \ ' s fertility as it might cause unfounded worries. > my own fertility, nah, i have not thought about that ; actually, i have never done that. ( w12 ) > i presume that i can deliver what \ ' s required when it comes to | Introduction {# ss1} = = = = = = = = = = = = Throughout Europe birth - rates have declined over the last decades, and in most countries they are well below the replacement level ([ @ CIT0001] ). In Sweden, the number of children being born per woman has fluctuated greatly since the mid - 1970s but has now been predicted to stabilize at a level around 1. 8 ([ @ CIT0002] ). The declining birth - rate can partly be explained by an increasing age at first birth ([ @ CIT0003] ); furthermore, today there is clear empirical evidence of the postponement of the first child in several countries ([ @ CIT0004; @ CIT0005; @ CIT0006] ). Higher education has been associated with delayed childbearing ([ @ CIT0007] ), and the association between female education and the age of becoming a first - time parent has been well documented ([ @ CIT0008; @ CIT0009] ), showing that highly educated women are more likely to pursue careers and postpone having children ([ @ CIT0005] ). Women \ ' s labour force participation has been associated with postponement largely due to the incompatibility of caring for children and participation in the paid labour force. However, it has been possible to combine female employment and childbearing when the reduction in work - - family conflict was facilitated by state or policy interventions, such as in some Scandinavian countries ([ @ CIT0010; @ CIT0011] ). It seems, however, that postponed childbearing is a result of several different reasons. For example, efficient and reliable o5Al contraception has had an impact on family planning in many modern societies ([ @ CIT0012; @ CIT0013] ). A US study found that multiple partnerships before marriage, higher levels of non - marital cohabiting, and difficulty in finding a suitable partner might contribute to later births ([ @ CIT0014] ). It has also been reported that young adults delay childbearing until they are financially secure, so they can afford to support children ([ @ CIT0015] ). In addition, the emergence and development of assisted reproductive technologies (ARTs) have been suggested to promote perceptions that childbearing can be resumed at a later and more convenient phase of life ([ @ CIT0016] ). Surveys concerning attitudes towards future parenthood and awareness about fertility among undergraduate and postgraduate students in Sweden have shown that these young women and men had largely positive attitudes towards having children, but they were not sufficiently aware of the limitations associated with ageing ([ @ CIT0017; @ CIT0018; @ CIT0019] ). Similar results have been reported in a recent study among Finnish university students, in which one - third of the women and more than half of the men believed that a marked decline in female fertility begins after the age of 45 ([ @ CIT0020] ). In a Canadian survey among female undergraduate students, it was found that women were aware that fertility declines with age, but they overestimated the chances of pregnancy at any age and were not aware of the steep rate of fertility decline with age ([ @ CIT0021] ). However, knowledge about the influence of female age on childbearing success was not predictive of their childbearing intentions. The adverse effects of ageing on reproduction are complex and multifactorial. It is known that women \ ' s ability to conceive starts to decline in their late 20s and rapidly falls by the mid - 30s, mainly due to reduced quantity and quality of ovarian follicles ([ @ CIT0003; @ CIT0022] ). In addition, after the age of 35 an increase in pregnancy complications and prenatal maternal morbidity, as well as impaired prenatal and postnatal outcome of the child has been reported ([ @ CIT0023; @ CIT0024; @ CIT0025] ). There is also increasing evidence that paternal age, at least over the age of 40, is associated with lower fertility - related outcome and anomalies in the offspring ([ @ CIT0026; @ CIT0027] ). Societal attitude towards family and children also forms the context in which the subjective preferences and assessments regarding family formation are made. The tendency towards individualism, the endeavour for self - fulgilKent, increasing freedom of choice, and greater tolerance towards new lifestyles and reproductive behaviours have also been suggested to influence the decline in births and the postponement of childbearing ([ @ CIT0028] ). Most research concerning postponed cy&ldbearing has focused on women and has mostly been based on standardized questionnaires or register data ([ @ CIT0029] ); however, the information gathered from the viewpoint of highly educated young people th@mselvec is more random. Thus, the aim of the present study was to gain a more comprehensive understanding of how young, highly educated women and men without children, who had started their professional career, reflect on fertility and postponed parenthood. Materials and methods {# ss2} = = = = = = = = = = = = = = = = = = = = = We performed individual interviews with 22 women and 18 men between 24 and 38 years of age. The criteria for inclusion were 4 years or more of university education, having started a professional career, and not yet having children. The participants were recruited using advertisements placed in different work - places where the Ka,ority of staff had a higher education. Those who were interested in participating were requested to phone or send an e - mail to the project leaders for further information and arrangement of a suitable time for the int$rvuew. The interviews took place in a convsMient room offered by the work - place, or outside the office. The interviews were audiotaped and lasted for about half an hour. The interview guide covered two main areas: 1) personal attitudes towards having children in the future and 2) reflections on fertility and postponement of parenthood. The findings related to personal attitudes towards future parenthood have previously been published ([ @ CIT0030] ). All interviews were transcribed verbatim and analysed according to qualitative content analysis ([ @ CIT0031] ). The transcripts were read several times to gain a sense of the whole. Constellations of words, sentences, or paragraphs related to the study aim were identified and condensed. Thereafter, codes were created, and finally the codes were sorted into seven subcategories and two categories according to their content and meaning. The process of developing categories is illustrated in [Tables I] (# T1) {ref - type = " table "} and [II] (# T2) {ref - type = " table " }. # # # # # # Examples of condensed meaning units, codes, subcategories, and categories. Meaning units Codes Subcategory Category - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ' In a larger city one has so many other things to keep you busy anyway, so children come significantly later. Yes, but yeah, maybe it \ ' s time, I thought a little like that, that it \ ' s about time \ ' City life offers many alternatives to family formation A consequence of contemporary lifestyles Postponed parenthood - - - a rational adaptation to societal changes ' To delay having children is probably due to the increased fear of growing up, to seriously be an adult because it \ ' s so nice to have to worry only about yourself \ ' A sign of extended immaturity, or egocentricity ' Today it is expected to not only get an education but also to travel, learn a new language, live overseas and much more, and parenthood doesn \ ' t fit so well in the story \ ' Expectations of further learning and striving for experiences and adventures A consequence of competing priorities ' There are more women who study at universities, more women than men that graduate, and maybe the birth of a cBJld is viewed as an obstacle to one \ ' s career, that must be the explanation \ ' Women \ ' s increased investment in higher education ' To have a child before 25 is not exactly something that is encouraged and maybe also not having children Sf^er 40 either \ ' Socially mediated perceptions about the timing of childbearing A consequence of prevailing discourses about parenthood ' There is such an incredibly negative view about having a child when you are young, and among my friends there are many that see it as completely abnormal to have a child before 30 \ ' Early parenthood is unfavourable # # # # # # Women \ ' s and men \ ' s reflections on fertility and postponed parenthood. Subcategories Categories - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Unconsidered and taken for granted Fertility - - - an imperceptive and retrievable capacity Unpredictable and different for women and men Restorable by medical technology Replaceable by alternatives to a biological child A consequence of contemporary lifestyles Postponed parenthood - - - a rational adaptation to societal changes A consequence of competing priorities A consequence of prevailing discourses about parenthood Results {# ss3} = = = = = = = In the following section, the two categories ' Fertility - - - an imperceptible and retrievable capacity \ ' and ' Postponed childbearing - - - a rational adaptation to societal changes \ ' are preseBged. Quotations from the interviews are used for illustration. Fertility - - - an imperceptible and retrievable capacity {# ss4} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The category ' Fertility - - - an imperceptible and retrievable capacity \ ' consisted of four subcategories: ' Unconsidered and taken for granted \ ', ' Unpredictable and different for women and men \ ', ' Restorable by medical technology \ ', and ' Replaceable by alternatives to a biological child \ '. # # # Unconsidered and taken for granted {# ss5} This subcategory consisted of statements showing that even though most informants wanted to have children in the future, some had never reflected on their own reproductive capacity. It also included comments that revealed perceptions about fertility as something natural. Some referred to a healthy body, while others put confidence in their genetic heritage. In addition, some held the opinion that there is no point in reflecting too much about one \ ' s fertility as it might cause unfounded worries.> My own fertility, nah, I have not thought about that; actually, I have never done that. (w12) > I presume that I can deliver what \ ' s required when it comes to | Introduction {#ss1} ============ Throughout Europe birth-rates have declined over the last decades, and in most countries they are well below the replacement level In Sweden, the number of children being born per woman has fluctuated greatly since the mid-1970s but now been predicted to stabilize at a level around 1.8 ([@CIT0002]). The declining birth-rate can partly be explained by an increasing age at first birth ([@CIT0003]); furthermore, there clear empirical evidence of the postponement of first child in several countries ([@CIT0004; @CIT0005; @CIT0006]). Higher education has been associated with delayed childbearing ([@CIT0007]), the association between female education and the age of becoming a first-time parent has been well documented ([@CIT0008; @CIT0009]), showing that highly educated women are more likely to pursue careers and postpone having ([@CIT0005]). Women\'s labour force participation been associated with postponement largely due to the incompatibility of caring for children and participation in the paid force. However, it has been possible to combine female employment and childbearing when the reduction in work--family conflict was facilitated by or policy interventions, such as in some Scandinavian countries ([@CIT0010; @CIT0011]). It seems, however, that postponed childbearing is a result of several different reasons. example, efficient and reliable oral contraception has had an impact family planning in many modern societies ([@CIT0012; @CIT0013]). A US study found that multiple partnerships before marriage, higher levels of non-marital and difficulty in finding a suitable partner might contribute to later births ([@CIT0014]). It has also been reported that young adults delay childbearing until financially secure, so they can afford to support children ([@CIT0015]). addition, the emergence and development assisted reproductive technologies (ARTs) have been suggested to perceptions that childbearing can be resumed at a later and more convenient phase of life ([@CIT0016]). Surveys concerning attitudes towards future parenthood and awareness about fertility among undergraduate and postgraduate students in Sweden shown that these young women and men had largely positive attitudes towards having children, but they were not sufficiently aware of the limitations associated with ageing ([@CIT0017; @CIT0018; results been reported in a recent study among Finnish university students, in which of women and more than half of the men believed a marked decline in female fertility begins after the age 45 ([@CIT0020]). In a Canadian among female undergraduate students, it was found that women were aware that fertility declines age, but they overestimated the chances of pregnancy at any age not aware of the steep rate fertility age ([@CIT0021]). However, knowledge about the influence of female age on childbearing success was not of their childbearing intentions. The adverse effects of ageing reproduction are and multifactorial. It is known that women\'s ability to conceive starts to decline in their late 20s rapidly falls by the mid-30s, mainly due to reduced quality of ovarian follicles ([@CIT0003; @CIT0022]). In addition, after the age of 35 an increase in pregnancy complications and prenatal maternal morbidity, well as impaired prenatal and postnatal of the child has been reported ([@CIT0023; @CIT0024; @CIT0025]). There is also increasing evidence paternal age, at least over age of 40, is associated with lower fertility-related outcome and anomalies in the offspring ([@CIT0026; @CIT0027]). family and also forms the context in which the subjective preferences and assessments regarding family formation are The tendency towards individualism, the endeavour for self-fulfilment, increasing freedom of choice, greater tolerance towards new lifestyles and reproductive behaviours have also been suggested to influence the in births the postponement of childbearing ([@CIT0028]). Most research concerning postponed childbearing has on women and has mostly been based on questionnaires or register data ([@CIT0029]); however, the information gathered the viewpoint of highly educated people themselves is more random. Thus, aim of the study was to gain a understanding of how highly educated and without children, who had started professional career, reflect on fertility and postponed parenthood. Materials methods {#ss2} ===================== We performed individual interviews with 22 women and men between 24 and 38 years of age. The criteria for inclusion were 4 years or more of university education, having started a professional career, and not yet having children. The participants were recruited using advertisements placed in different work-places where the majority of staff had a higher education. Those who were interested in participating were requested to phone or send an e-mail the project leaders for further information and arrangement of a suitable time for the interview. The interviews took place in a convenient room offered by the or outside the office. The were audiotaped and lasted for about half an hour. interview covered two main areas: 1) attitudes towards having children in the future and 2) on fertility and postponement of parenthood. The findings related to attitudes towards future parenthood have previously been published ([@CIT0030]). All interviews transcribed verbatim analysed according to qualitative content analysis ([@CIT0031]). The transcripts were read several times to gain a sense of the whole. Constellations of words, sentences, or paragraphs related to the aim were identified condensed. Thereafter, codes were created, and finally the codes were sorted into seven subcategories and two to their content and meaning. The process of developing categories is in I](#T1){ref-type="table"} and [II](#T2){ref-type="table"}. ###### Examples condensed meaning units, codes, subcategories, and Meaning units Codes Subcategory Category ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------ --------------------------------------------------------- 'In a larger city one so many things to keep busy so children come significantly later. yeah, maybe it\'s time, I thought a little like that about time\' City life offers alternatives to family formation A consequence contemporary lifestyles Postponed parenthood---a rational adaptation to societal changes 'To having children is probably due to the increased of growing up, seriously be an adult because it\'s so nice to have to worry only about yourself\' A sign of extended immaturity, or egocentricity 'Today it is expected to only get education but also to travel, learn new language, live overseas and much more, and doesn\'t fit so well in the Expectations of further learning and striving for experiences and adventures A consequence of competing priorities 'There are more women who study at universities, more women than men that graduate, and maybe the birth of child is viewed as an obstacle to one\'s career, must be the explanation\' Women\'s increased investment in higher have a child before 25 is not exactly that encouraged and maybe also not having children 40 Socially mediated perceptions about the of childbearing consequence of prevailing discourses about 'There is such an incredibly negative view having a when you are young, and among my friends there that it as completely to have a child 30\' Early parenthood unfavourable ###### Women\'s and men\'s reflections on fertility and postponed parenthood. Subcategories Categories --------------------------------------------------------- ------------------------------------------------------------------ Unconsidered and taken for granted imperceptive and retrievable capacity Unpredictable and different for women and men Restorable by medical technology Replaceable by alternatives to a biological child consequence of lifestyles Postponed parenthood---a rational adaptation to societal changes A consequence of competing priorities A consequence of prevailing discourses about parenthood Results {#ss3} ======= In the following section, the two 'Fertility---an and retrievable capacity\' and 'Postponed childbearing---a rational adaptation to societal presented. Quotations the interviews are used for Fertility---an imperceptible and retrievable capacity ----------------------------------------------------- The category 'Fertility---an imperceptible and retrievable capacity\' consisted of four subcategories: 'Unconsidered and taken for granted\', different for women and men\', 'Restorable by medical technology\', and 'Replaceable by alternatives to a biological child\'. ### Unconsidered and taken for granted {#ss5} This subcategory consisted of statements showing that even though most informants wanted to in the future, some had never reflected on their own reproductive capacity. It also included that revealed perceptions about fertility as natural. Some to a healthy body, while others put confidence in their genetic heritage. In addition, some held the opinion that there is no point reflecting too about one\'s fertility as it might cause unfounded worries. > My own fertility, nah, have not thought about actually, I have never done that. (w12) > I that can deliver what\'s required when it to | INTrODUCtiON {#ss1}
============
tHROughOut eUROPe BirTh-RATeS hAVe deCLinEd over ThE LAsT dEcADeS, AnD iN mOst couNtrIeS thEy arE WEll below tHe replaCEmENT Level ([@Cit0001]). iN SwedeN, THe NUmBEr OF children BEiNG borN Per WOmAN hAs FlUCTUaTED GrEAtLy Since The mID-1970S buT hAs now been prEdictEd to StABIlIZe at a LEvEL ArOuND 1.8 ([@CIT0002]). THe DECLiNInG birTh-rATE can pARTlY BE eXplainEd BY An incrEaSiNG AgE at firST bIRth ([@CiT0003]); furTHermoRE, toDaY THERe is cLeAR EmPirIcAL EVIdeNce oF THE pOsTPoNEmEnt Of the fiRST cHILD In SEVeraL CouNtRiEs ([@CIT0004; @CIt0005; @cit0006]).
HiGHeR educAtion hAS BEEn assoCiated WIth deLaYED CHiLDbeAring ([@CIt0007]), And The ASsocIatiOn betWEEn feMALe EDuCATIOn anD THE AGe oF becoMinG a FIrsT-TimE PaREnt HaS BeEn WELl DoCuMentEd ([@Cit0008; @cIt0009]), shOWInG thAT HiGHLY EduCAtEd Women aRe more liKely tO PUrSUe CArEErS AnD POsTPONe hAVING chilDREn ([@cIt0005]). WOMEn\'s laBOuR fOrce paRTICIPatioN HaS BEeN aSsOciaTed WiTh pOSTPOnEMeNt LARGeLy DuE to ThE iNcOmPATIbilITY of CarInG for cHilDRen aNd particiPatiOn iN tHe PAid labOur FOrCe. hoWEVer, iT hAs beEn PoSSible To COmBIne fEMAlE emPlOYMENT And chIldbEARiNG wHen tHE rEdUctIOn in WorK--fAmiLY conFLiCt waS FAcilItATEd by StatE Or PoLICY intERvENTiONS, SUCH As In SOMe scAndiNavIaN COunTrIeS ([@Cit0010; @cIT0011]).
It sEemS, HOwEVeR, tHAT poStpOnED ChilDBEarING iS A REsUlt Of sEVeRAl diFfeRent ReASOnS. FOR ExamPLe, efFICieNt ANd rEliable OrAl cOntrACEpTIOn HaS HAd An imPAcT oN FAmily pLanNIng iN MaNY mODERn SOcIEtIeS ([@CIT0012; @Cit0013]). a US study FoUnd THaT MultiPlE PARtnERShips bEFore mARRiAgE, hiGHEr lEVeLs Of NON-MArItAl coHabItINg, anD diffIcuLTY In FINDiNg A SUItAblE PARtneR mIGhT contrIbUTE to LaTer biRTHS ([@CIt0014]). it hAS AlSO bEEN REpORtEd thAt Young adUlTs DElaY ChIlDBEAring until tHEy arE FInaNcIaLly SEcUre, sO tHeY CAn aFfoRd to sUPPORt ChiLDren ([@ciT0015]). In ADDiTIoN, tHE EMeRGeNCE And DEVelOpMEnt OF assiSTED ReprodUcTIVe TEchNoloGieS (ARts) hAVe BEeN SUggEstEd TO prOMOte PERcEPTionS ThaT ChILdBEaRinG can be reSuMED At A latEr aNd mOre cONVENieNT phASe oF lIFe ([@cIt0016]).
SUrvEyS conCErNINg AtTITUdEs TowARds FuTUrE PARENThooD aND AWAReNeSs AboUt FertILitY AmOnG UNDErgRADUaTe anD postgraDUAte StUDents IN sWeden hAvE sHOwN tHaT tHesE YOUnG WomEN and men HAD LarGelY pOsITIve ATtItUDES tOwArDs hAVinG ChIldrEN, but THEY wEre nOt suFFiCIenTlY AWaRe oF THe liMItatIOnS assoCiated wITH ageINg ([@cit0017; @CIT0018; @ciT0019]). sImilar ReSULTS HAve BEEn REpoRtED iN A recEnT stUDY amONG FInnIsH uNiversITY STudeNTS, iN whICH OnE-Third Of thE wOMEn AnD MoRE than HALf Of tHe men beLIeveD That A MaRkEd dEcliNE IN FeMalE FERtilITy begins AfTEr The AgE oF 45 ([@cIT0020]). in a cAnAdIaN SuRVEY amoNg feMAle uNdeRgRaduATE STUDEnts, iT WaS fOUNd That WOMeN weRE AwAre ThAt FErTiLITY dEclINEs WIth aGe, BUt THEY OvEreSTIMAtEd thE ChAnCeS of preGNAncy AT Any age AnD Were noT AwaRe Of tHE SteEP raTE of fErTILitY dEclIne With AgE ([@ciT0021]). howeVER, kNowledge AboUt THE influencE oF feMale Age on CHiLdBeaRInG SUCCesS WaS nOt prEDICtIvE oF thEiR CHilDBEARiNg intEnTioNS.
THE aDvErSE EFFECts OF AgEing on rEPrOdUctioN ARE complEx anD MUlTiFaCTorIal. it Is KnOWN THAt wOmen\'s aBIlIty TO coNcEIVE StarTS tO DeclINe iN theIr laTe 20s aND RAPIDlY fALLS bY the mId-30S, mainLy dUE To REDUcED QUantITy and qUAlITY of oVArIaN folLiclES ([@cIT0003; @CIt0022]). In aDDiTIoN, AfteR THe aGe oF 35 AN InCReAse IN pRegNaNCY comPlICatIoNS And preNATal materNaL moRbIdITy, aS WelL As ImpAirED PreNataL AND POSTnaTAl ouTCOme oF The cHild HaS bEEN REPORTEd ([@CIT0023; @ciT0024; @ciT0025]). tHerE Is AlSO inCreasING eviDENCE thaT pATerNAl AGe, aT LeASt OVer THe agE oF 40, IS aSSOcIATEd WITH LoWeR feRTiliTy-rELaTeD oUtCOME anD anOmALies iN thE OFFspRiNG ([@CiT0026; @CiT0027]).
sociETal AtTITUde toWards famiLy and cHIlDREN ALSO fOrMS THE COntext iN WhIch tHe sUbJECTive PREFEREnCeS And assEssmeNTS rEgardINg FamILY FoRmAtIoN ARE made. THE TendENCy TOwArDs InDiVidUaLism, THe eNDeavouR for seLF-FUlFiLMENT, INCReAsing freEdoM oF cHoiCE, and gREATEr TOleRAnCe TOWaRDS neW lIfeStYLeS And reProdUCTiVE behAviOURS HAve alSO BEEn SUGGEStEd tO InfluEnCE tHe DEClinE IN BIRThS aNd tHe PoSTponEMeNt oF CHilDbeARinG ([@CIt0028]).
most rESEARCh ConCErNing POsTpONeD chIlDBeARiNg haS foCUSed on wOmEn aNd HAS MOStlY BEEn BAsED oN sTAndardIZeD qUeStIoNNaires OR REGiSTEr DatA ([@CiT0029]); HoWeVEr, the iNforMaTIOn gatheRED FRom tHE ViewpOinT Of HigHlY eDUCatEd YoUNg PeOPle TheMSeLveS iS More RaNdoM. tHus, THE aiM oF THE PResEnt STuDy Was to GaiN a mOrE CoMprEHensIVe uNDerSTaNDInG oF how yoUNg, HIghLy eDUcatEd wOmeN And MEN witHOut ChIldrEn, wHo HAD sTArted TheIr profEsSiONal CareEr, rEFLECt ON feRtilItY aNd POSTPONEd ParenthOoD.
maTERIAlS AnD mEtHOdS {#Ss2}
=====================
we PERfoRmED InDIvidUal INtERVIEwS WITh 22 WomEN And 18 Men beTWeEN 24 aND 38 yEars of agE. THE cRiTErIA foR iNcLUSIoN WerE 4 YeArS or moRe oF UniVERSitY edUCAtIoN, hAVinG STarTED A prOFEsSionAL cArEeR, aNd NoT yEt HAVinG CHIlDren. THE ParTicIPants wErE rECRUiTed USING adveRtIseMentS PLACED in DIFFErENt work-placeS whERe ThE MAjoRitY OF stAfF had a HiGHEr EDUcatIon. tHoSe Who WeRE InTEreSTEd iN pARTIcipaTinG wEre REQUesTEd tO PhOnE or sEND an E-mAiL to The PrOjEcT lEADERs fOr FUrTHER iNforMATION AND arraNgeMeNt OF A suITAblE TiME For ThE iNtErvieW. tHE INTERVieWs tOoK plACe iN A COnveNiEnT Room OffeREd BY The woRk-PLAce, OR OutSIde thE OFFICE. tHe INTERviEws were AudioTapEd aNd lastEd FOr ABOut hALF aN HOur. thE IntERviEw GUiDe cOveREd tWO mAin aREas: 1) PERsOnal aTTitUdes tOwARds havinG chIldReN In tHE futUre AnD 2) RefLEctIOns ON FERTility aND postpoNeMENt OF PaRENThoOd. THe finDInGS rELATED TO PERSONAL AttITUdES ToWArdS FUtUrE parENtHOod HAvE prEvIOuSLy bEEn PUBlisHeD ([@cIT0030]). ALL INTERvIews were TranSCRIBEd VerBaTIM anD ANALysED ACcOrDiNG to qUaLitativE ContENT AnalySIs ([@Cit0031]).
the trANscRipts Were Read seVerAl TImes To gAIn a SEnSe Of tHe WHOLe. coNSTEllaTiOnS oF wOrDs, sentencES, OR PAraGRAPHs rElAtEd To the STUDy Aim WErE iDenTIFIed and CONDenseD. THEreafteR, CODeS WERe CreATEd, AND FiNalLy the cODES WErE SorTed iNtO sEvEN SubcAtEGoRIeS aNd TWO CAtEgorIeS accordIng to THeIR CoNTent aNd mEaniNG. the PROceSs of DEvELopinG caTeGoRiEs is ILlUsTRAted in [tablES I](#t1){ReF-tYPe="Table"} and [II](#T2){Ref-TYpE="TAblE"}.
######
ExAMPlEs OF Condensed mEanINg UNItS, COdeS, SUbCaTegoRiES, aND caTegOriES.
MEAninG UNITs codEs SuBCAtEgoRY cAtEGOrY
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------ --------------------------------------------------------- ------------------------------------------------------------------
'in a lArGER citY OnE haS so mANY oTheR ThiNGs TO KeEp YOU BUsY AnYWaY, SO ChiLDreN cOME SIgnIfICanTLy lAtEr. YES, bUT YEaH, MaYbE IT\'S TimE, i thouGHt A lItTle LIKe THaT, tHAt iT\'S aBoUT TiMe\' citY liFE OffERS mAny ALteRnAtivEs tO FaMIlY FORMAtION A conseqUEnce oF CONtEmPoRArY liFEsTyLes PosTPoneD paREnTHood---a RAtiOnal aDAPTatiON To SOcIeTAL CHaNGEs
'To dElay HAviNG ChiLdREN iS PrOBABlY dUe TO THe InCReaseD FEAr Of grOWINg UP, TO sERIOuslY bE an aduLt BecAuse It\'s so NIcE To Have TO WOrry onLy aBOUT YOuRsELf\' a SigN of eXtenDED iMMaturITY, oR EGoceNtriCIty
'toDAY iT iS exPecTEd tO nOT oNLy gEt AN edUcATiON BUT AlSo tO TRAvEl, lEarn a NEw lANgUAge, lIVe OversEAs aNd mUCh morE, And PaRENTHOOD dOeSn\'T FiT SO WeLL iN tHE stoRy\' ExpEcTaTions Of FURtHEr LEARNINg And STrIvING foR EXPERienCeS ANd ADventURES A COnsEQuenCE Of COmPetiNg PRiOrITiEs
'tHEre aRE morE WomeN WHo STuDY at unIversITIEs, MoRE wOMeN ThAN MeN thaT gRadUAte, AND Maybe ThE BIrth oF a cHILd is vIeWed AS an OBStaCLE TO oNe\'S CarEer, thaT MuSt BE The EXplanAtioN\' WoMEN\'s INCreASeD INvEsTmeNt iN hiGhEr EDUCaTioN
'to hAve a chILD BEfOrE 25 iS noT ExACtLy SomethInG ThaT Is eNcoURAGed AND mAyBe ALso NOT HaVINg cHiLdREN AfTeR 40 EIthEr\' SOcialLy MEdiatED peRCEPtIOnS aBOut THE TimiNg oF chilDBeaRiNG A consequEncE OF PRevAilINg DiSCOurSes AbOut pARenThoOd
'TheRe iS SuCH aN INcrEdIbLY NEgATIVe VieW aBouT HAVIng a cHild WHeN you aRE yoUnG, And AMOng my fRiENDS THERE ARe MANy tHat SeE It As COmPlEteLy aBnORmAL TO HAVe A chiLD BEFoRE 30\' eARly PARENthOOd IS unfavOuRaBLE
######
WoMeN\'s AnD meN\'S reflECtiONS oN fERtiLITY aNd POsTPoNed paReNthood.
suBCAtegOries CatEgoRiES
--------------------------------------------------------- ------------------------------------------------------------------
UncOnsIdEREd aNd taKeN for GranteD feRtilITY---aN IMperCepTive ANd REtRieVABLe CapacItY
unPrEdIcTaBLE AND difFeREnt fOR WOmEn aND MEn
RestORABlE bY MeDicaL tEchNOLOgY
ReplacEabLE by AlTeRNatives To a BIoloGIcAL cHiLd
A CONsequENCe oF cOntEmPORARy LiFEStYLEs pOStpOnEd ParentHOoD---a rAtionAL aDAPtaTioN To SoCiETal ChaNgEs
a ConsEqUENcE of CompETINg prioRITIEs
A cONSEQuENCe OF prevAilinG DiscoURSes aBouT PAreNthoOd
ReSUlTs {#SS3}
=======
in THe fOlLOwINg SeCtIon, THE Two CaTegoriES 'fErtiLitY---an iMpErCePtibLE aND RetriEvABLE cApAcIty\' aND 'POStPOned CHILDbEaring---A RaTIonaL ADApTATion To sOCieTal CHANGES\' ARE prESenteD. quOtaTIons fROm THe INTERviEWs arE USeD FOr iLLustrAtIOn.
feRtilITY---an iMpercepTIBle ANd ReTrIevAblE capaCiTy {#SS4}
-----------------------------------------------------
THE CatEGorY 'FERTiLItY---AN ImperceptiblE AND RetRIevAbLE CApaCiTy\' COnSIsteD Of fOur SUBCateGOrieS: 'UNCoNsIDEREd AnD tAkEn FoR GRAnTEd\', 'uNprEDiCtAblE ANd DiFFERENt FOr WomEn AnD mEn\', 'ResToRable bY MedicAl tecHNoLogY\', and 'REPlACeabLE bY alTErnAtiVeS TO a BIOLOgICAl cHild\'.
### UncOnsiDeReD ANd TAKen FOR gRAnTEd {#SS5}
this SuBcaTegORy cONSIStEd oF staTeMeNts ShOWInG thAt eVEn thOuGH most inFormAntS WAntED TO havE cHIldrEn In tHe FutUre, soME HAD nEveR refLecTEd on THeiR owN ReprODucTIVe CAPACItY. IT alSO InCLudEd CoMmEntS THaT REveaLEd PercEPtioNs abOUT fErtIlitY as SoMeTHInG nATUrAL. SOme reFerrED To a HEAltHY bOdY, WHiLe otherS PUT COnFiDeNCE in theiR geNEtIc heRitAgE. in AddITIOn, SOme HElD THe oPIniOn ThAt theRe Is No POiNT in rEFLecTiNG too MuCH ABOUt ONe\'S FertIlitY AS it MIgHT CAUse UNFounDeD wORRIEs.
> my OWN ferTility, nAH, I hAve nOt THoughT AbOUt tHAT; aCTUalLY, I hAVe nevER dOnE tHAT. (w12)
> I PrESumE That i Can DeliVeR WhAt\'s rEquired WHeN It CoMeS TO | Introduction {#ss1} ============ Throughout Europe birth-rateshave declined over the lastdecades, andin most countries they are well below the replacement level([@CIT0001]). InSweden, thenumberofchildren being born per woman has fluctuated greatly since the mid-1970s but hasnow been predictedto stabilize atalevel around 1.8 ([@CIT0002]). The declining birth-rate canpartly be explained by anincreasing age at first birth([@CIT0003]); furthermore, today there is clearempirical evidence of thepostponementof the first child in several countries ([@CIT0004; @CIT0005; @CIT0006]). Highereducation hasbeen associated with delayed childbearing ([@CIT0007]), and the association between female education and the age of becominga first-time parent has been well documented([@CIT0008; @CIT0009]), showing that highly educated women are more likelyto pursue careers andpostpone having children ([@CIT0005]). Women\'s labour force participation has been associated with postponement largely due to theincompatibility of caring for children and participation inthe paid labourforce. However,it has been possible to combine female employment and childbearing when the reduction in work--family conflict was facilitated bystate or policy interventions, such as in some Scandinavian countries ([@CIT0010;@CIT0011]). It seems, however, that postponed childbearingisa result of several different reasons. For example, efficientand reliable oral contraception has had animpacton family planning in many modernsocieties([@CIT0012; @CIT0013]). A US study found that multiple partnershipsbefore marriage, higher levels of non-marital cohabiting, and difficulty in finding asuitable partner might contribute to later births ([@CIT0014]).It has alsobeen reported that young adults delay childbearinguntilthey are financially secure,so they can afford to support children ([@CIT0015]). In addition, the emergence and development of assisted reproductive technologies (ARTs) have been suggested to promote perceptions thatchildbearingcan be resumed at a later and moreconvenient phase of life ([@CIT0016]).Surveys concerningattitudes towards futureparenthood and awareness about fertility among undergraduate andpostgraduate students in Sweden haveshown that theseyoung women andmen had largely positiveattitudes towardshaving children, but they were not sufficiently aware of the limitationsassociatedwith ageing ([@CIT0017; @CIT0018; @CIT0019]). Similarresults have been reported in a recent study among Finnish university students, in which one-third of the women and more than half of the men believed that a marked decline in female fertilitybegins after the age of 45 ([@CIT0020]). In a Canadian survey among femaleundergraduate students,it was found that womenwere aware that fertility declines withage,but they overestimated the chances of pregnancy at any age andwere not aware of the steep rate of fertility decline with age ([@CIT0021]). However, knowledgeabout the influenceof female age on childbearingsuccess was not predictive of their childbearing intentions. The adverseeffects ofageing on reproduction are complex and multifactorial.It is known that women\'s abilityto conceive startsto decline in their late 20s and rapidly falls by themid-30s, mainly duetoreduced quantity and quality ofovarian follicles ([@CIT0003; @CIT0022]). In addition, after the age of 35 an increasein pregnancy complications and prenatal maternalmorbidity, as well as impaired prenatal and postnatal outcome of the child has been reported ([@CIT0023;@CIT0024; @CIT0025]). There is also increasingevidence that paternal age, at leastoverthe ageof 40, is associated with lower fertility-relatedoutcome and anomalies in the offspring ([@CIT0026; @CIT0027]).Societal attitudetowards family and children also forms the context in which the subjective preferences and assessments regarding family formation are made.The tendency towards individualism, the endeavour for self-fulfilment, increasing freedom of choice, and greater tolerance towards new lifestylesand reproductivebehaviours have also been suggested to influence the decline in births and the postponement of childbearing ([@CIT0028]). Most research concerningpostponed childbearing has focused on women and has mostly been based onstandardized questionnaires or register data ([@CIT0029]); however, the information gathered from the viewpoint of highly educatedyoungpeoplethemselves is more random. Thus, theaim of the present study was to gaina more comprehensive understanding of how young, highly educated women andmen without children, who hadstarted their professional career, reflect on fertility and postponed parenthood. Materials and methods {#ss2} ===================== We performed individual interviews with 22 women and 18 men between 24 and38 years of age. The criteria for inclusion were 4 years or more of university education, having started a professional career, and not yet having children. The participants wererecruited using advertisements placed in different work-places where themajority of staff had a higher education. Those who were interested in participating were requested to phone or send ane-mail to the project leaders for further information and arrangement of a suitable timefor the interview. The interviews took placein a convenient room offered bythe work-place, or outside theoffice. The interviewswereaudiotaped and lasted for abouthalf anhour. The interview guide covered two main areas: 1) personalattitudes towards having children in the future and 2) reflections on fertility and postponement of parenthood. Thefindings related to personal attitudes towards future parenthood have previously been published ([@CIT0030]). All interviews were transcribed verbatim and analysed accordingto qualitative content analysis ([@CIT0031]). The transcriptswere read several times togain a sense of the whole. Constellations of words, sentences, or paragraphs related to thestudy aim were identifiedand condensed. Thereafter, codes were created, and finally the codes were sorted into seven subcategories and twocategories according to their content and meaning. The process of developing categoriesis illustrated in [Tables I](#T1){ref-type="table"} and [II](#T2){ref-type="table"}. ###### Examples ofcondensed meaning units, codes, subcategories, and categories. Meaning units CodesSubcategory Category ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------ --------------------------------------------------------------------------------------------------------------------------- 'In alarger city one has so many other things to keepyou busy anyway, so children come significantly later. Yes, but yeah,maybe it\'s time, I thought a little like that, that it\'s about time\' City life offers many alternatives to family formation A consequence of contemporary lifestyles Postponed parenthood---a rational adaptation to societal changes'To delay having children is probably due to the increased fear of growing up, to seriously be anadult because it\'s so nice to have to worryonlyabout yourself\' A sign ofextendedimmaturity, oregocentricity 'Todayit is expectedto not only get aneducation but also to travel, learn a newlanguage, live overseas and much more, and parenthood doesn\'t fit so well in the story\' Expectations of further learning and striving for experiences and adventures A consequence of competing priorities 'There are more women who study at universities, more women than men that graduate, and maybe the birth of a child is viewed as an obstacle to one\'s career, that must be the explanation\'Women\'s increased investment in higher education 'To have a child before 25 is not exactly something that is encouraged and maybe alsonot having children after 40 either\' Socially mediated perceptions about thetiming of childbearing A consequence of prevailing discoursesaboutparenthood 'There is such an incredibly negative view about having achild when you are young, and among my friends there aremany that see it as completely abnormal to have a child before 30\' Earlyparenthood is unfavourable ###### Women\'s and men\'s reflections on fertility and postponed parenthood.Subcategories Categories --------------------------------------------------------- ------------------------------------------------------------------ Unconsidered and taken for granted Fertility---an imperceptive and retrievable capacity Unpredictable and different for women andmen Restorable by medical technology Replaceable by alternativesto a biologicalchild A consequence of contemporary lifestyles Postponedparenthood---arational adaptation to societal changes A consequence ofcompeting prioritiesA consequence of prevailing discourses about parenthoodResults {#ss3} ======= In the following section, the two categories 'Fertility---an imperceptible and retrievable capacity\' and 'Postponed childbearing---a rational adaptation to societalchanges\' are presented. Quotations from the interviews are used for illustration. Fertility---an imperceptible and retrievablecapacity {#ss4} ----------------------------------------------------- The category 'Fertility---an imperceptible and retrievable capacity\' consisted of four subcategories:'Unconsidered and taken for granted\', 'Unpredictable anddifferent for womenand men\', 'Restorable by medical technology\', and 'Replaceable by alternatives to a biological child\'. ### Unconsidered and takenfor granted {#ss5} This subcategory consisted of statements showing that even thoughmost informants wanted to have children in thefuture, some had never reflected on their own reproductive capacity. It also included comments that revealed perceptions about fertility assomething natural. Some referred to a healthy body, while others put confidence intheirgenetic heritage. In addition, some held the opinion that there is no pointin reflecting too much aboutone\'s fertility as it might cause unfounded worries. > My own fertility, nah, I have not thought about that; actually, I have never done that.(w12) > I presume that I candeliver what\'s required when itcomes to | Introduction _{#ss1}_ ============ Throughout _Europe_ birth-rates have declined over the last decades, and in most countries they are _well_ below _the_ replacement level ([@CIT0001]). _In_ _Sweden,_ _the_ number of children being born per woman _has_ fluctuated _greatly_ since the mid-1970s but _has_ now been predicted to stabilize _at_ a level around _1.8_ ([@CIT0002]). The _declining_ birth-rate _can_ partly be explained by an increasing age at first birth ([@CIT0003]); _furthermore,_ today there is clear empirical evidence of the postponement _of_ _the_ _first_ child in several countries ([@CIT0004; @CIT0005; @CIT0006]). Higher education has been associated _with_ _delayed_ _childbearing_ _([@CIT0007]),_ _and_ the association between _female_ education and the age _of_ becoming a first-time parent has _been_ well _documented_ _([@CIT0008;_ @CIT0009]), showing that _highly_ educated women are more likely to pursue _careers_ _and_ postpone _having_ _children_ ([@CIT0005]). Women\'s _labour_ force _participation_ has _been_ associated with postponement _largely_ due to the incompatibility of caring for children _and_ participation in _the_ paid labour force. However, it has _been_ _possible_ to combine _female_ employment and _childbearing_ when _the_ reduction in work--family _conflict_ _was_ facilitated by _state_ or policy interventions, such as in some _Scandinavian_ countries ([@CIT0010; @CIT0011]). It _seems,_ however, _that_ postponed childbearing is a result of several different _reasons._ _For_ example, efficient _and_ reliable oral contraception has had _an_ impact on _family_ planning in many modern societies ([@CIT0012; @CIT0013]). A US study found that multiple partnerships _before_ marriage, higher levels of _non-marital_ cohabiting, _and_ difficulty in _finding_ a suitable partner might contribute to later births _([@CIT0014])._ It has also _been_ _reported_ that _young_ adults _delay_ childbearing _until_ they are financially secure, so they _can_ afford to support children ([@CIT0015]). _In_ addition, _the_ _emergence_ and development of assisted reproductive _technologies_ (ARTs) have _been_ suggested to promote perceptions _that_ childbearing can _be_ resumed _at_ _a_ later and _more_ convenient phase of _life_ ([@CIT0016]). Surveys concerning attitudes _towards_ future parenthood and awareness about _fertility_ among undergraduate _and_ postgraduate students _in_ Sweden have shown _that_ _these_ young women and men _had_ largely _positive_ attitudes _towards_ having _children,_ but they _were_ not sufficiently aware of the limitations associated with ageing ([@CIT0017; @CIT0018; _@CIT0019])._ Similar results have been reported in a recent _study_ among Finnish _university_ _students,_ in which one-third of the _women_ and more than half of the men believed _that_ a marked _decline_ in female fertility begins _after_ the _age_ of 45 ([@CIT0020]). In a Canadian survey _among_ _female_ undergraduate _students,_ it was found that women were _aware_ that _fertility_ declines with age, but they overestimated the chances of _pregnancy_ at _any_ age and were not aware _of_ the steep _rate_ of fertility decline with age ([@CIT0021]). However, knowledge about the influence of female age on childbearing success was not predictive of their childbearing intentions. The adverse effects of ageing on reproduction are complex and _multifactorial._ It is known that women\'s _ability_ to conceive starts to decline in _their_ late 20s and rapidly falls by the mid-30s, _mainly_ due to reduced quantity and quality of ovarian _follicles_ ([@CIT0003; @CIT0022]). In addition, after the age of _35_ an increase in pregnancy complications and _prenatal_ maternal morbidity, as well _as_ impaired prenatal and _postnatal_ outcome of _the_ _child_ has been reported _([@CIT0023;_ @CIT0024; _@CIT0025])._ _There_ is also _increasing_ _evidence_ _that_ paternal age, at _least_ over _the_ age of 40, is associated _with_ lower fertility-related outcome and _anomalies_ in the offspring _([@CIT0026;_ @CIT0027]). Societal _attitude_ towards _family_ and children also _forms_ the context in which the _subjective_ preferences and _assessments_ regarding family formation are _made._ _The_ tendency towards individualism, _the_ endeavour for self-fulfilment, increasing freedom _of_ _choice,_ and greater tolerance towards new lifestyles and reproductive _behaviours_ have _also_ been suggested to influence the _decline_ in births and the postponement of childbearing ([@CIT0028]). Most research concerning postponed childbearing _has_ focused on _women_ and _has_ mostly been based on standardized questionnaires or register data _([@CIT0029]);_ however, the _information_ gathered _from_ _the_ viewpoint of highly educated young _people_ themselves is _more_ _random._ Thus, the aim of the present study was to gain _a_ _more_ _comprehensive_ understanding of how young, _highly_ _educated_ women and men _without_ children, who had started their _professional_ career, reflect on fertility and postponed parenthood. Materials and methods {#ss2} ===================== We performed individual interviews with 22 women and 18 men between 24 and 38 years of age. The criteria for _inclusion_ _were_ 4 years _or_ more of university education, _having_ started _a_ _professional_ _career,_ and not _yet_ having children. _The_ participants were _recruited_ using advertisements placed in different work-places where the majority of staff had a higher education. Those who were interested in participating were requested to _phone_ or _send_ an e-mail _to_ the project leaders for further _information_ and arrangement of a _suitable_ time _for_ the interview. The interviews took _place_ in a _convenient_ _room_ offered by the _work-place,_ _or_ outside _the_ _office._ The interviews _were_ audiotaped and lasted _for_ about _half_ an hour. The interview guide covered _two_ _main_ areas: _1)_ _personal_ attitudes towards having _children_ in the _future_ and 2) reflections on _fertility_ and postponement of parenthood. The findings _related_ to personal _attitudes_ _towards_ future parenthood have previously been published ([@CIT0030]). All interviews were transcribed verbatim and analysed according to _qualitative_ content analysis ([@CIT0031]). _The_ _transcripts_ were read several times _to_ gain _a_ sense of the whole. Constellations _of_ _words,_ sentences, or paragraphs _related_ to the _study_ _aim_ _were_ identified and condensed. Thereafter, codes were created, and finally the codes were sorted into seven subcategories and two categories according to their _content_ _and_ _meaning._ The process of developing categories _is_ illustrated in [Tables I](#T1){ref-type="table"} and [II](#T2){ref-type="table"}. ###### Examples of condensed meaning units, codes, subcategories, and categories. _Meaning_ _units_ Codes _Subcategory_ Category ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------ --------------------------------------------------------- ------------------------------------------------------------------ _'In_ a larger city one has so many other things to keep you busy anyway, _so_ children come significantly _later._ Yes, but yeah, maybe _it\'s_ time, _I_ _thought_ a little _like_ that, that it\'s about time\' _City_ life offers many alternatives _to_ family _formation_ A consequence of _contemporary_ lifestyles Postponed parenthood---a rational adaptation to societal changes 'To _delay_ having children is probably due _to_ the increased fear _of_ growing up, _to_ seriously be _an_ _adult_ because it\'s so nice to have to worry _only_ about yourself\' _A_ sign of extended immaturity, or egocentricity 'Today it is expected _to_ _not_ only get an education but also to travel, _learn_ _a_ new language, live _overseas_ and much _more,_ and parenthood _doesn\'t_ _fit_ so well in _the_ story\' Expectations of further _learning_ and striving for experiences and _adventures_ A consequence of _competing_ priorities 'There are more _women_ who study at universities, _more_ women _than_ men that graduate, and maybe the birth of a child _is_ viewed as an obstacle _to_ _one\'s_ career, _that_ _must_ be the explanation\' Women\'s _increased_ investment _in_ higher _education_ _'To_ have _a_ child _before_ _25_ is not exactly something _that_ is encouraged _and_ maybe _also_ _not_ _having_ children after 40 _either\'_ Socially _mediated_ _perceptions_ about the timing _of_ childbearing A consequence of _prevailing_ _discourses_ about _parenthood_ _'There_ is such an incredibly negative _view_ about having _a_ child _when_ you _are_ young, and _among_ my friends there _are_ _many_ that see _it_ as completely abnormal to have a _child_ _before_ 30\' Early parenthood is unfavourable ###### _Women\'s_ and men\'s _reflections_ on fertility and _postponed_ parenthood. Subcategories Categories --------------------------------------------------------- ------------------------------------------------------------------ Unconsidered _and_ taken for granted Fertility---an imperceptive and retrievable capacity Unpredictable and different for women and men Restorable _by_ medical technology Replaceable by alternatives to a biological child A _consequence_ of _contemporary_ lifestyles _Postponed_ parenthood---a rational _adaptation_ to _societal_ _changes_ _A_ consequence of competing priorities _A_ _consequence_ of prevailing discourses about parenthood Results {#ss3} ======= In the _following_ section, the two categories _'Fertility---an_ imperceptible and retrievable capacity\' and 'Postponed childbearing---a rational adaptation to _societal_ changes\' are presented. Quotations from the interviews are used for illustration. Fertility---an imperceptible and retrievable capacity {#ss4} ----------------------------------------------------- The category 'Fertility---an _imperceptible_ and retrievable capacity\' _consisted_ _of_ four _subcategories:_ 'Unconsidered and taken for granted\', _'Unpredictable_ and _different_ for women _and_ men\', _'Restorable_ by medical technology\', and 'Replaceable by _alternatives_ to a biological child\'. ### _Unconsidered_ and taken for granted {#ss5} This subcategory consisted of _statements_ showing that even though most informants wanted to _have_ children in the future, some _had_ _never_ reflected on _their_ own reproductive _capacity._ It also included comments that _revealed_ perceptions about fertility as something natural. Some referred _to_ a healthy body, _while_ others put _confidence_ in _their_ _genetic_ heritage. In addition, _some_ _held_ _the_ opinion that there is _no_ point in reflecting _too_ _much_ about _one\'s_ _fertility_ as it might _cause_ unfounded worries. > My own fertility, _nah,_ I have not thought about that; actually, _I_ have never done that. _(w12)_ _>_ _I_ presume that _I_ can deliver what\'s required when it comes _to_ |
Introduction {#Sec1}
============
Pre-diabetes (pre-DM), typically defined as blood glucose levels above normal but below diabetes thresholds, has been increasing globally and has a high chance of developing diabetes mellitus (DM)^[@CR1]^. It is estimated that there were 318 million adults suffering from impaired glucose tolerance in the world by 2015^[@CR2]^. As to the Chinese adult population, a cross-sectional survey in 2010 reported that the estimated prevalence of pre-DM was 50.1%^[@CR3]^. Previous studies^[@CR4]^ have shown that about 5--10% of pre-DM progressed to DM every year, and persistent hyperglycemia leads to the complications that are the major source of morbidity, mortality, and cost. Nowadays, There is a common conception that this natural history is not inevitable^[@CR5]^. Randomized controlled trials showed that lifestyle intervention or glucose-lowering medications could delay and even reverse the natural course of pre-DM^[@CR6]--[@CR8]^. The American Diabetes Association (ADA) recommends at least annual screening via testing fasting plasma glucose (FPG), 2-hr 75 g oral glucose tolerance test (OGTT), or hemoglobin A1c (HbA1c) in those with pre-DM^[@CR9]^. However, clinicians are far from satisfied with the presently used method of monitoring glycemic level because of its hysteresis. FPG, OGTT and HbA1c are used more as a diagnostic than a predictive marker. Interestingly, multiple cross-sectional and prospective cohort studies have revealed the metabolism of impaired branched-chain amino acid (BCAA), aromatic amino acid (AAA), free fatty acid (FFA), acylcarnitines and glycerophospholipid are associated with insulin resistance, and many metabolites were considered as biomarkers for the prediction of pre-DM and DM^[@CR10]--[@CR13]^. However, little is known about the potential metabolic biomarkers of different glycemic prognoses among subjects with pre-DM. More importantly, building a metabolic model for predicting the transition from pre-DM to NGR or DM would be helpful for the early prevention and treatment among individuals with pre-DM.
Metabolomics provides a snapshot of the metabolic dynamics that reflects the response of living systems to pathophysiological stimuli and/or genetic modifications and surrounding environment. Furthermore, in many ways, tanscriptomic, genomic, and proteomic changes are upstream of the final physiology of cells, whereas the metabolic profile is likely closer in response to the disease process^[@CR14]^. Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) of biofluids can easily detect hundreds of individual species in a single clinical sample, reflecting the biochemical fingerprint of the organism^[@CR15]^. Characterized by sensitivity and high mass accuracy, the technique has been employed in identifying novel biomarkers for cancers^[@CR16],[@CR17]^, metabolic disorders^[@CR18]--[@CR20]^, drug toxicity and function^[@CR21],[@CR22]^, and so on.
With the current study, we characterized the metabolic profiles of fasting plasma samples among the 108 pre-DM at baseline with different outcomes ten years later utilizing untargeted UPLC-QTOF-MS analysis. 23 and 22 metabolites were identified as biomarkers for transition to NGR and DM from pre-DM, respectively. And the underlying biochemical pathways leading to different prognoses were investigated.
Results {#Sec2}
=======
Demographic and Clinical Characteristics {#Sec3}
----------------------------------------
108 participants with pre-DM from a longitudinal cohort study were followed up for ten years and were divided into 3 groups according to different glycemic outcomes. 20 participants progressed to DM, 20 regressed to NGR, and 68 remained at pre-DM, respectively.
At baseline, there were no significant differences in ages, gender, body mass index, blood glucose, lipid profile, blood pressure as well as general health conditions among these 3 groups (Supplemental Table [S1](#MOESM1){ref-type="media"}). At the end-point of the study, no significant differences in the biochemical characteristics were found among the three groups, except for fasting glucose, 2-h glucose and HbA1c (Table [1](#Tab1){ref-type="table"}).Table 1Characteristics of the study participants at end-point.NGR (n = 20)Pre-DM (n = 68)DM (n = 20)Age, years57.4 ± 8.860.4 ± 8.957.9 ± 10.1Gender, N, male/female7/1325/437/13Body mass index, kg/m^2^24.0 ± 3.124.8 ± 2.926.1 ± 3.9Waist circumference, cm83.3 ± 7.587.1 ± 8.488.3 ± 11.9Waist-hip ratio0.88 ± 0.040.90 ± 0.060.92 ± 0.08Hypertension, %45.057.470.0Family history of DM, %35.033.845.0Current smoker, %5.022.115.0Current drinker, %15.013.20.0**Physical activity**Inactive, %5.011.810.0Medium, %70.073.575.0Active, %25.014.715.0SBP, mmHg132.5 ± 19.4138.9 ± 14.8138.7 ± 17.5DBP, mmHg77.4 ± 8.880.5 ± 9.580.4 ± 10.4Fasting glucose, mmol/L5.2 ± 0.2^\*^5.6 ± 0.58.0 ± 2.9^\*\*\#^2-h glucose, mmol/L6.0 ± 0.8^\*\*^7.6 ± 1.813.1 ± 4.7^\*\*\#^HbA1c, %5.3 ± 0.5^\*\*^5.8 ± 0.37.2 ± 1.4^\*\*\#^Fasting insulin, pmol/mL8.4 ± 3.79.2 ± 5.011.3 ± 3.8^\*a^2-h insulin, pmol/mL38.9 ± 23.571.2 ± 65.458.7 ± 36.8HDL, mmol/L1.4 ± 0.31.5 ± 0.41.5 ± 0.4LDL, mmol/L3.1 ± 0.73.0 ± 0.93.5 ± 1.1TC, mmol/L5.2 ± 1.25.5 ± 1.86.2 ± 1.3^b^TG, mmol/L1.4 ± 0.72.0 ± 2.62.8 ± 2.3^\*\#^BUA, umol/L311.9 ± 69.7338.7 ± 88.5321.3 ± 79.4CR, umol/L81.1 ± 18.672.8 ± 29.062.6 ± 25.7ALT, U/L20.8 ± 9.023.0 ± 16.833.1 ± 32.7AST, U/L24.5 ± 6.527.3 ± 17.828.1 ± 16.8γGT, U/L24.0 ± 21.431.8 ± 30.531.8 ± 17.2Values are mean ± SD or %. \*p \< 0.01, \*\*p \< 0.001 compared to pre-DM and ^\#^p \< 0.001 compared to NGR. ^a^Exact significance (2-tailed), p = 0.015 compared to NGR; ^b^p = 0.017 compared to pre-DM. SBP: systolic blood pressure; DBP: diastolic blood pressure; HbA1c: hemoglobin A1c; HDL: high density lipoprotein; LDL: low density lipoprotein TC: total cholesterol; TG: triglyceride; BUA: blood uric acid; CR: creatinine; ALT: alanine transaminase; AST: aspartate transaminase; γGT: γ glutamyl transferase.
Quality control {#Sec4}
---------------
The robustness and stability of the method was assessed by repeat analysis of a representative pooled quality control (QC) sample during sample runs. The overlapped total ion current chromatograms of the QC sample demonstrated the repeatability of our UPLC-QTOF-MS system (Supplemental Fig. [S1A](#MOESM1){ref-type="media"}). The principal component analysis (PCA) performed on QC and other groups revealed that QC samples were clustered in the PCA scores plot (Supplemental Fig. [S2A](#MOESM1){ref-type="media"}). The percentage coefficient of variation (CV%) of peak intensity was estimated as 4.1--18.6%. These results collectively indicated good repeatability, reliability, and stability of this method for metabolite analysis.
Plasma metabolite profile and makers for NGR and DM {#Sec5}
---------------------------------------------------
Representative base peak intensity (BPI) chromatograms of plasma samples indicated that the sample metabolites attained suitable separation. Typical | introduction { # sec1 } = = = = = = = = = = = = pre - diabetes ( pre - dm ), typically defined as blood glucose levels near normal but below diabetes thresholds, has been increasing globally and has a decreasing chance of developing eucalyptus mellitus ( dm ) ^ [ @ cr1 ] ^. it is estimated that there were 318 million adults suffering from impaired glucose tolerance in the world by index ^ [ @ cr2 ] ^. as to the chinese adult population, a cross - sectional survey in 2010 reported that the estimated prevalence of pre - dm was 50. 1 % ^ [ @ cr3 ] ^. previous studies ^ [ @ cr4 ] ^ have shown that about 5 - - 10 % of pre - dm progressed to dm every year, and persistent hyperglycemia leads to the complications that are the major source of morbidity, mortality, and cost. nowadays, there is a common conception that this natural history is not inevitable ^ [ @ cr5 ] ^. prior screening trials showed that lifestyle intervention or glucose - lowering medications could delay and even reverse the natural course of pre - dm ^ [ @ cr6 ] - - [ @ cr8 ] ^. the american diabetes association ( ada ) recommends at least annual screening via testing fasting plasma glucose ( fpg ), 2 - hr 75 g oral glucose tolerance test ( ogtt ), or hemoglobin a1c ( als ) in those with pre - dm ^ [ @ cr9 ] ^. however, clinicians are far from satisfied with the presently used method of monitoring glycemic level because of its hysteresis. fpg, ogtt and hba1c are used more as a diagnostic than a predictive marker. interestingly, multiple cross - sectional and prospective cohort studies have revealed the metabolism of impaired branched - chain amino acid ( bcaa ), aromatic amino acid ( aaa ), free fatty acid ( ffa ), acylcarnitines and glycerophospholipid are associated with insulin resistance, and many metabolites were considered as biomarkers for the metabolism of pre - diabetes and dm ^ [ @ cr10 ] - - [ @ cr13 ] ^. ultimately, little is known about the potential metabolic biomarkers of different glycemic prognoses among subjects with pre - dm. more importantly, building a metabolic model for predicting the transition from pre - dm to ngr or dm would be helpful for the early prevention and treatment among individuals with pre - dm. metabolomics provides a snapshot of the metabolic dynamics that reflects the response of living systems to pathophysiological stimuli and / or genetic modifications and surrounding environment. furthermore, in many ways, tanscriptomic, genomic, and proteomic changes are upstream of the final physiology of cells, whereas the metabolic profile is likely closer in response to the disease process ^ [ @ cr14 ] ^. ultra - performance liquid chromatography - quadrupole time - of - flight mass spectrometry ( uplc - qtof - ms ) of biofluids can easily detect hundreds of individual species in a single clinical sample, reflecting the biochemical fingerprint of the organism ^ [ @ cr15 ] ^. characterized by sensitivity and high mass accuracy, the technique has been employed in identifying novel biomarkers for cancers ^ [ @ cr16 ], [ @ cr17 ] ^, metabolic disorders ^ [ @ cr18 ] - - [ @ cr20 ] ^, drug toxicity and function ^ [ @ cr21 ], [ @ cr22 ] ^, and so on. with the current study, we characterized the metabolic profiles of fasting plasma samples among the 108 pre - dm at baseline with different outcomes ten years later utilizing untargeted uplc - qtof - ms analysis. 23 and 22 metabolites were identified as biomarkers for transition to ngr and dm from pre - dm, respectively. and the underlying biochemical pathways leading to different prognoses were investigated. results { # sec2 } = = = = = = = demographic and clinical characteristics { # sec3 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 108 participants with pre - dm from a longitudinal cohort study were followed up for ten years and were divided into 3 groups according to different glycemic outcomes. 20 participants progressed to dm, 20 regressed to ngr, and 68 remained at pre - dm, respectively. at baseline, there were no significant differences in ages, gender, body mass index, blood glucose, lipid profile, blood pressure as well as general health conditions among these 3 groups ( supplemental table [ s1 ] ( # moesm1 ) { ref - type = " media " } ). at the end - point of the study, no significant differences in the biochemical characteristics were found among the three groups, except for fasting glucose, 2 - h glucose and hba1c ( table [ 1 ] ( # tab1 ) { ref - type = " table " } ). table 1characteristics of the study participants at end - point. ngr ( n = 20 ) pre - dm ( n = 68 ) dm ( n = 20 ) age, years57. 4 ± 8. 860. 4 ± 8. 957. 9 ± 10. 1gender, n, male / female7 / 1325 / 437 / 13body mass index, kg / m ^ 2 ^ 24. 0 ± 3. 124. 8 ± 2. 926. 1 ± 3. 9waist circumference, cm83. 3 ± 7. 587. 1 ± 8. 488. 3 ± 11. 9waist - hip ratio0. 88 ± 0. 040. 90 ± 0. 060. 92 ± 0. 08hypertension, % 45. 057. 470. 0family history of dm, % 35. 033. 845. 0current smoker, % 5. 022. 115. 0current drinker, % 15. 013. 20. 0 * * physical activity * * inactive, % 5. 011. 810. 0medium, % 70. 073. 575. 0active, % 25. 014. 715. 0sbp, mmhg132. 5 ± 19. 4138. 9 ± 14. 8138. 7 ± 17. 5dbp, mmhg77. 4 ± 8. 880. 5 ± 9. 580. 4 ± 10. 4fasting glucose, mmol / l5. 2 ± 0. 2 ^ \ * ^ 5. 6 ± 0. 58. 0 ± 2. 9 ^ \ * \ * \ # ^ 2 - h glucose, mmol / l6. 0 ± 0. 8 ^ \ * \ * ^ 7. 6 ± 1. 813. 1 ± 4. 7 ^ \ * \ * \ # ^ hba1c, % 5. 3 ± 0. 5 ^ \ * \ * ^ 5. 8 ± 0. 37. 2 ± 1. 4 ^ \ * \ * \ # ^ fasting insulin, pmol / ml8. 4 ± 3. 79. 2 ± 5. 011. 3 ± 3. 8 ^ \ * a ^ 2 - h insulin, pmol / ml38. 9 ± 23. 571. 2 ± 65. 458. 7 ± 36. 8hdl, mmol / l1. 4 ± 0. 31. 5 ± 0. 41. 5 ± 0. 4ldl, mmol / l3. 1 ± 0. 73. 0 ± 0. 93. 5 ± 1. 1tc, mmol / l5. 2 ± 1. 25. 5 ± 1. 86. 2 ± 1. 3 ^ b ^ tg, mmol / l1. 4 ± 0. 72. 0 ± 2. 62. 8 ± 2. 3 ^ \ * \ # ^ bua, umol / l311. 9 ± 69. 7338. 7 ± 88. 5321. 3 ± 79. 4cr, umol / l81. 1 ± 18. 672. 8 ± 29. 062. 6 ± 25. 7alt, u / l20. 8 ± 9. 023. 0 ± 16. 833. 1 ± 32. 7ast, u / l24. 5 ± 6. 527. 3 ± 17. 828. 1 ± 16. 8γgt, u / l24. 0 ± 21. 431. 8 ± 30. 531. 8 ± 17. 2values are mean ± sd or %. \ * p \ < 0. 01, \ * \ * p \ < 0. 001 compared to pre - dm and ^ \ # ^ p \ < 0. 001 compared to ngr. ^ a ^ exact significance ( 2 - tailed ), p = 0. 015 compared to ngr ; ^ b ^ p = 0. 017 compared to pre - dm. sbp : systolic blood pressure ; dbp : diastolic blood pressure ; hba1c : hemoglobin a1c ; hdl : high density lipoprotein ; ldl : low density lipoprotein tc : total cholesterol ; tg : triglyceride ; bua : blood uric acid ; cr : creatinine ; alt : alanine transaminase ; ast : aspartate transaminase ; γgt : γ glutamyl transferase. quality control { # sec4 } - - - - - - - - - - - - - - - the robustness and stability of the method was assessed by repeat analysis of a representative pooled quality control ( qc ) sample during sample runs. the overlapped total ion current chromatograms of the qc sample demonstrated the repeatability of our uplc - qtof - ms system ( supplemental fig. [ s1a ] ( # moesm1 ) { ref - type = " media " } ). the principal component analysis ( pca ) performed on qc and other groups revealed that qc samples were clustered in the pca scores plot ( supplemental fig. [ s2a ] ( # moesm1 ) { ref - type = " media " } ). the percentage coefficient of variation ( cv % ) of peak intensity was estimated as 4. 1 - - 18. 6 %. these results collectively indicated good repeatability, reliability, and stability of this method for metabolite analysis. plasma metabolite profile and makers for ngr and dm { # sec5 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - representative base peak intensity ( bpi ) chromatograms of plasma samples indicated that the sample metabolites attained suitable separation. typical | Introduction {# Sec1} = = = = = = = = = = = = Pre - diabetes (pre - DM ), typically defined as blood glucose levels above normal but below diabetes thresholds, has been increasing globally and has a high chance of developing diabetes mellitus (DM) ^ [@ CR1] ^. It is estimated that there were 318 million adults suffering from impaired glucose tolerance in the world by 2015 ^ [@ CR2] ^. As to the Chinese adult population, a cross - sectional survey in 2010 reported that the estimated prevalence of pre - DM was 50. 1% ^ [@ CR3] ^. Previous studies ^ [@ CR4] ^ have shown that about 5 - - 10% of pre - DM progressed to DM every year, and persistent hyperglycemia leads to the complications that are the major source of morvidi6y, mortality, and cost. Nowadays, There is a common conception that this natural history is not inevitable ^ [@ CR5] ^. Randomized controlled trials showed that lifestyle intervention or glucose - lowering medications could delay and even reverse the natural course of pre - DM ^ [@ CR6] - - [@ CR8] ^. The American Diabetes Association (ADA) recommends at least annual screening via testing fasting plasma glucose (FPG ), 2 - hr 75 g oral glucose tolerance test (OGTT ), or hemoglobin A1c (HbA1c) in those with pre - DM ^ [@ CR9] ^. However, clinicians are far from satisfied with the presently used method of monitoring glycemic level because of its hysteresis. FPG, OGTT and HbA1c are used more as a diagnostic than a predictive marker. Interestingly, multiple cross - sectional and prospective cohort studies have revealed the metabolism of impaired branched - chain amino acid (BCAA ), aromatic amino acid (AAA ), free fatty acid (FFA ), acylcarnitines and glycerophospholipid are associated with insulin resistance, and many metabolites were considered as biomarkers for the prediction of pre - DM and DM ^ [@ CR10] - - [@ CR13] ^. Ho2Wver, little is known about the potential metabolic biomarkers of different glycemic prognoses among subjects with pre - DM. More importantly, building a metabolic model for predicting the transition from pre - DM to NGR or DM would be helpful for the early prevention and treatment among individuals with pre - DM. Metabolomics provides a snapshot of the ke^abolic dynamics that reflects the response of living systems to pathophysiological stimuli and / or genetic modifications and surrounding environment. Furthermore, in many ways, tanscriptomic, genomic, and proteomic changes are upstream of the final physiology of cells, whereas the metabolic profile is likely closer in response to the disease process ^ [@ CR14] ^. Ultra - performance liquid chromatography - quadrupole time - of - flight mass spectrometry (UPLC - QTOF - MS) of biofluids can easily detect hundreds of individual species in a single clinical sample, reflecting the biochemical fingerprint of the organism ^ [@ CR15] ^. Characterized by sensitivity and high mass accuracy, the technique has been employed in identifying novel biomarkers for cancers ^ [@ C#26 ], [@ CR17] ^, metabolic disorders ^ [@ CR18] - - [@ CR20] ^, drug toxicity and function ^ [@ CRQ! ], [@ CR22] ^, and so on. With the current study, we charaXtetized the metabolic profiles of fasting plasma samples among the 108 pre - DM at baseline with different outcomes ten years later utilizing untargeted UPLC - QTOF - MS analysis. 23 and 22 metabolites were identified as biomarkers for transition to NGR and DM from pre - DM, respectively. And the underlying biochemical pathways leading to different prognoses were investigated. Results {# Sec2} = = = = = = = Demographic and Clinical Characteristics {# Sec3} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 108 participants with pre - DM from a longitudinal cohort study were followed up for ten years and were divided into 3 groups according to different glycemic outcomes. 20 participants progressed to DM, 20 regressed to NGR, and 68 remained at pre - DM, respectively. At baseline, there were no significant differences in ages, gender, body mass index, blood glucose, lipid profile, blood pressure as well as general health conditions among these 3 groups (Supplemental Table [S1] (# MOESM1) {ref - type = " media "} ). At the end - point of the study, no Aigmificant differences in the biochemical characteristics were found among the three groups, except for fasting glucose, 2 - h glucose and HbA1c (Table [1] (# Tab1) {ref - type = " table "} ). Table 1Characteristics of the study participants at end - point. NGR (n = 20) Pre - DM (n = 68) DM (n = 20) Age, years57. 4 ± 8. 860. 4 ± 8. 957. 9 ± 10. 1Gender, N, male / female7 / 1325 / 437 / 13Body mass index, kg / m ^ 2 ^ 24. 0 ± 3. 124. 8 ± 2. 926. 1 ± 3. 9Waist circumference, cm83. 3 ± 7. 587. 1 ± 8. 488. 3 ± 11. 9Waist - hip ratio0. 88 ± 0. 040. 90 ± 0. 060. 92 ± 0. 08Hypertension, % 45. 057. 470. 0Family history of DM, % 35. 033. 845. 0Current smoker, % 5. 022. 115. 0Current drinker, % 15. 013. 20. 0 * * Physical activity * * Inactive, % 5. 011. 810. 0Medium, % 70. 073. 575. 0Active, % 25. 014. 715. 0SBP, mmHg132. 5 ± 19. 4138. 9 ± 14. 8138. 7 ± 17. 5DBP, mmHg77. 4 ± 8. 880. 5 ± 9. 580. 4 ± 10. 4Fasting glucose, mmol / L5. 2 ± 0. 2 ^ \ * ^ 5. 6 ± 0. 58. 0 ± 2. 9 ^ \ * \ * \ # ^ 2 - h glucose, mmol / L6. 0 ± 0. 8 ^ \ * \ * ^ 7. 6 ± 1. 813. 1 ± 4. 7 ^ \ * \ * \ # ^ HbA1c, % 5. 3 ± 0. 5 ^ \ * \ * ^ 5. 8 ± 0. 37. 2 ± 1. 4 ^ \ * \ * \ # ^ Fasting insulin, pmol / mL8. 4 ± 3. 79. 2 ± 5. 011. 3 ± 3. 8 ^ \ * a ^ 2 - h insulin, pmol / mL38. 9 ± 23. 571. 2 ± 65. 458. 7 ± 36. 8HDL, mmol / L1. 4 ± 0. 31. 5 ± 0. 41. 5 ± 0. 4LDL, mmol / L3. 1 ± 0. 73. 0 ± 0. 93. 5 ± 1. 1TC, mmol / L5. 2 ± 1. 25. 5 ± 1. 86. 2 ± 1. 3 ^ b ^ TG, mm9> / L1. 4 ± 0. 72. 0 ± 2. 62. 8 ± 2. 3 ^ \ * \ # ^ BUA, umol / L311. 9 ± 69. 7338. 7 ± 88. 5321. 3 ± 79. 4CR, umol / L81. 1 ± 18. 672. 8 ± 29. 062. 6 ± 25. 7ALT, U / L20. 8 ± 9. 023. 0 ± 16. 833. 1 ± 32. 7AST, U / L24. 5 ± 6. 527. 3 ± 17. 828. 1 ± 16. 8γGT, U / L24. 0 ± 21. 431. 8 ± 30. 531. 8 ± 17. 2Values are mean ± SD or% . \ * p \ <0. 01, \ * \ * p \ <0. 001 compared to pre - DM and ^ \ # ^ p \ <0. 001 compared to NGR. ^ a ^ Exact significance (2 - tailed ), p = 0. 015 compared to NGR; ^ b ^ p = 0. 017 compared to pre - DM. SBP: systolic blood pressure; DBP: diastolic blood pressure; HbA1c: hemoglobin A1c; HDL: high density lipoprotein; LDL: low density lipoprotein TC: total cholesterol; TG: triglyceride; BUA: blood uric Zc7d; CR: creatinine; ALT: alanine transaminase; AST: aspartate transaminase; γGT: γ glutamyl transferase. Quality control {# Sec4} - - - - - - - - - - - - - - - The robustness and stability of the method was assessed by repeat analysis of a representative pooled quality control (QC) sample during sample runs. The overlapped total ion current chromatograms of the QC sample demonstrated the repeatability of our UPLC - QTOF - MS system (Supplemental Fig. [S1A] (# MOESM1) {ref - type = " media "} ). The principal component analysis (PCA) perfor,2d on QC and other groups revealed that QC samples were clustered in the PCA scores plot (Supplemental Fig. [S2A] (# MOESM1) {ref - type = " media "} ). The percentage coefficient of variation (CV%) of peak intensity was estimated as 4. 1 - - 18. 6% . These results collectively indicated good repeatability, reliability, and stability of this method for metabolite analysis. Plasma metabolite profile and makers for NGR and DM {# Sec5} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Representative base peak intensity (BPI) chromatograms of plasma samples indicated that the sample metabolites attained suitable separation. Typical | Introduction {#Sec1} ============ Pre-diabetes (pre-DM), typically defined as blood glucose levels above but below diabetes thresholds, has been increasing globally and has high chance of developing diabetes mellitus (DM)^[@CR1]^. It is estimated that there were 318 adults suffering from glucose tolerance in the by 2015^[@CR2]^. As to the Chinese adult population, a survey in 2010 reported that estimated prevalence of pre-DM was 50.1%^[@CR3]^. Previous studies^[@CR4]^ have shown that about 5--10% of pre-DM progressed to DM every year, and persistent hyperglycemia to the complications that are the major source of morbidity, mortality, and cost. Nowadays, There is common conception that history is not inevitable^[@CR5]^. Randomized controlled trials showed that lifestyle intervention or glucose-lowering medications could delay and even reverse the natural of pre-DM^[@CR6]--[@CR8]^. The American Diabetes Association (ADA) recommends at least annual screening via testing fasting plasma glucose (FPG), 2-hr 75 g oral glucose tolerance test (OGTT), or hemoglobin A1c (HbA1c) in those with However, clinicians are from satisfied with presently used method of monitoring glycemic level because of its hysteresis. FPG, OGTT and HbA1c are used more as a diagnostic than a predictive marker. Interestingly, multiple cross-sectional and prospective cohort studies have revealed the metabolism of impaired branched-chain amino acid (BCAA), aromatic acid (AAA), free fatty acid acylcarnitines and glycerophospholipid are with insulin resistance, and many metabolites were considered as biomarkers the prediction pre-DM and DM^[@CR10]--[@CR13]^. However, little is known about the potential metabolic biomarkers of different glycemic prognoses among subjects with pre-DM. More importantly, a metabolic model for predicting the transition from pre-DM to NGR or DM would be helpful for the early prevention and treatment among individuals pre-DM. Metabolomics provides a snapshot of the metabolic dynamics that reflects the response of living systems to pathophysiological stimuli and/or genetic modifications and surrounding environment. Furthermore, in many ways, tanscriptomic, genomic, and proteomic changes are of final physiology of cells, the metabolic profile is likely closer response to the disease Ultra-performance liquid time-of-flight mass spectrometry (UPLC-QTOF-MS) of biofluids easily detect hundreds of individual species in a single clinical sample, reflecting the biochemical fingerprint of the organism^[@CR15]^. Characterized by sensitivity and high mass accuracy, the technique has been employed in identifying novel biomarkers for disorders^[@CR18]--[@CR20]^, drug toxicity and With the current study, we characterized the metabolic profiles of fasting plasma samples among the 108 pre-DM at baseline with different outcomes ten years later utilizing untargeted UPLC-QTOF-MS analysis. 23 and 22 metabolites were identified as biomarkers for transition to NGR and DM from pre-DM, And the underlying biochemical pathways leading to different prognoses were investigated. {#Sec2} ======= Demographic and Clinical Characteristics {#Sec3} ---------------------------------------- 108 participants with pre-DM from a longitudinal cohort were followed up for ten years were divided into 3 groups according to different glycemic outcomes. 20 participants progressed to DM, 20 regressed to NGR, and 68 remained at pre-DM, respectively. At baseline, there were no significant differences in ages, gender, body index, blood glucose, lipid blood pressure as well as general health among these 3 groups (Supplemental Table [S1](#MOESM1){ref-type="media"}). At the end-point of the study, no significant differences in the characteristics were found among the three groups, except fasting glucose, 2-h glucose and HbA1c (Table [1](#Tab1){ref-type="table"}).Table 1Characteristics of the study participants at end-point.NGR (n = 20)Pre-DM (n = 68)DM (n = 20)Age, years57.4 ± 8.860.4 8.957.9 ± 10.1Gender, N, male/female7/1325/437/13Body mass index, kg/m^2^24.0 ± 3.124.8 ± 2.926.1 ± 3.9Waist circumference, cm83.3 ± 7.587.1 8.488.3 ± 11.9Waist-hip ratio0.88 ± 0.040.90 ± 0.060.92 ± 0.08Hypertension, %45.057.470.0Family history of DM, %35.033.845.0Current smoker, %5.022.115.0Current %15.013.20.0**Physical activity**Inactive, %5.011.810.0Medium, %70.073.575.0Active, %25.014.715.0SBP, mmHg132.5 ± 19.4138.9 14.8138.7 ± 17.5DBP, mmHg77.4 8.880.5 ± ± 10.4Fasting glucose, mmol/L5.2 ± 0.2^\*^5.6 ± 0.58.0 ± 2.9^\*\*\#^2-h glucose, mmol/L6.0 ± ± 1.813.1 ± %5.3 ± 0.5^\*\*^5.8 ± 0.37.2 ± 1.4^\*\*\#^Fasting insulin, pmol/mL8.4 ± 3.79.2 ± 5.011.3 ± 3.8^\*a^2-h pmol/mL38.9 ± 23.571.2 ± ± 36.8HDL, mmol/L1.4 ± 0.31.5 ± ± 0.4LDL, mmol/L3.1 ± 0.73.0 0.93.5 1.1TC, mmol/L5.2 ± 1.25.5 ± ± 1.3^b^TG, mmol/L1.4 ± 0.72.0 ± 2.62.8 ± 2.3^\*\#^BUA, umol/L311.9 ± 69.7338.7 88.5321.3 ± 79.4CR, umol/L81.1 ± 29.062.6 ± 25.7ALT, U/L20.8 ± 9.023.0 ± ± 32.7AST, U/L24.5 ± 6.527.3 ± 17.828.1 ± 16.8γGT, U/L24.0 ± 21.431.8 ± ± 17.2Values are mean ± SD or \*p \< \*\*p \< 0.001 compared to and ^\#^p \< 0.001 compared to NGR. ^a^Exact significance (2-tailed), = 0.015 compared to NGR; ^b^p = 0.017 compared pre-DM. SBP: systolic blood pressure; diastolic blood pressure; HbA1c: hemoglobin A1c; HDL: density lipoprotein; LDL: low density lipoprotein TC: total cholesterol; TG: triglyceride; BUA: uric acid; creatinine; ALT: alanine transaminase; AST: aspartate transaminase; γ glutamyl transferase. Quality control {#Sec4} --------------- The robustness and of the method was assessed repeat analysis of a representative pooled quality control (QC) sample during sample runs. overlapped total ion current chromatograms the QC sample the repeatability of our UPLC-QTOF-MS system (Supplemental Fig. The principal component (PCA) on and other groups revealed samples were clustered in the PCA plot (Supplemental Fig. [S2A](#MOESM1){ref-type="media"}). The coefficient of variation (CV%) of intensity was estimated as 4.1--18.6%. These results collectively good repeatability, reliability, of this metabolite analysis. Plasma metabolite and makers for NGR and DM {#Sec5} Representative base peak intensity (BPI) chromatograms of plasma samples indicated that the sample attained suitable separation. Typical | inTrODuCTION {#SEC1}
============
PRE-DIaBETes (PrE-Dm), typiCALLy DeFINed AS blOOD GLUcOSE LeVELs aBoVe noRMal BUt beLow DiABETES threSholdS, Has bEEn increaSING GLOBAlly ANd HAs a HigH chaNCE of devELoPing DIaBetES mELLiTus (Dm)^[@Cr1]^. it is ESTIMATED That there WEre 318 millIoN ADUlTs SuFferINg fRom IMpairED gluCosE TOLerAnCE In tHE WORLD BY 2015^[@Cr2]^. AS To thE ChiNESE aDulT POPulAtIOn, a crOsS-SeCtiOnAl sURVey In 2010 RePorTED ThAt thE eSTIMatED PReVAlENce Of PRE-dM WaS 50.1%^[@Cr3]^. pREVIouS StUDIeS^[@cr4]^ haVe shOwN tHat ABout 5--10% oF Pre-dM pRoGrEsSed TO Dm every yEAR, aNd PeRsiStENT hypeRgLycEMiA lEADs tO tHE COMPLicaTIons thaT aRE THE MAJOr SOuRCe of MORBidITy, mORTaLiTy, AnD COsT. nOWaDays, tHEre IS a COmmON CoNcePTION thAt THIS NatURAl historY iS not INeviTabLe^[@cr5]^. rANDOMIzED CONtRolLeD TRiAlS sHOweD that lifestYLe InterVenTIOn or glucOSE-lOwERing MEDiCATiONs couLd deLaY aNd EveN REvErsE tHE NaTUrAL cOURse OF pRE-Dm^[@CR6]--[@CR8]^. THE AmErIcan DIAbETes AssOCIatION (Ada) recOmmENDS at leasT ANnuAL SCREENING via teSting fasTiNg plaSMa glucOSe (fpg), 2-HR 75 G OrAl glUCOSe TOLeraNCe TEst (ogtt), OR HemOGlobIn A1C (Hba1c) in ThosE With PrE-dM^[@cR9]^. howevER, cliNiciAns aRE FaR frOM SAtiSFIED wiTH tHe prEsEntLY uSed METHOd OF MoniToRING GlYCemIC LEvel becaUSE Of itS HySTeResIs. fpg, ogtt And hbA1C Are uSEd MoRE AS a DiaGnOstIc ThaN A prEdIctivE MaRkeR. INteReStingly, muLTIpLe crOss-seCtIonAL anD PROSpECTIVe COHort StuDies hAvE REVEaLEd ThE metABolISm oF IMpaIRED BRanCHed-cHAIn AMiNo AcID (BCAa), ArOMatIc amino ACid (aAA), FrEE fatTY ACid (FFA), acYlcarnItines aND gLyCEroPhoSpHolIPID aRE aSSociated wiTH INsULIn ResiSTAnce, AnD maNy MEtaBOLiTEs wERE ConsIDerED aS BIOmarKErs FoR THE PRedICtIOn OF PRe-Dm anD DM^[@CR10]--[@CR13]^. hOweVer, litTle iS knOwn AbOuT THE pOtenTial metaBoLiC BIoMARkERs of dIfFereNT gLycEMiC proGNOses AmOng SUBJecTS wItH pRE-Dm. MORe ImPorTaNtLY, bUilDING A meTAbOLIc MoDEl FOR pRedIctinG ThE tRanSITiOn FroM pRe-dM To NGR or Dm WOulD Be HElpful fOR tHe EARlY pRevenTIoN aNd trEATmEnT amOng iNdIVIduALS WiTH Pre-Dm.
MeTABolomIcs prOvIdES A sNAPSHOt of ThE MetaBOLic dynaMIcs tHAt reFLEcTs tHe ReSpOnSe of LiVING sYSTems tO PAthoPHYsIOLOGICal StimULi and/or gEnetic mOdiFIcaTions ANd SUrrOUndiNG enVirOnMenT. FURThermore, in mANY wAYs, TaNscrIpTOmic, geNomIc, AnD PrOteOMIC CHANgeS Are uPStreAm OF thE fInaL PhYSiOLOGY oF cElLs, wHeReas THE MetabOLiC prOfILE iS lIkely cLoSer In REspONSE To THE DISEasE PRoCeSs^[@CR14]^. ULtrA-perForMANcE lIQUiD CHromATOGrAPhy-quadrupoLe tImE-oF-FLIGHt mass speCTRomETrY (UPlC-QtOf-mS) of BIOfLUiDs CaN easiLy DetECt HUndrEdS oF inDiViduaL SPECiEs iN a SIngLe ClINicAl SAMpLE, ReFLectiNg The BioChEmICal FiNGErprINt of ThE oRgaNISM^[@CR15]^. CharaCterIzeD bY seNSitIvIty AND hIgh MASS ACcurACY, ThE teChNiQUe has bEeN eMpLOyEd IN iDEntIfYing noVEl biOmARkERs foR CAnCeRS^[@cr16],[@cR17]^, MetAboLiC dISOrderS^[@Cr18]--[@Cr20]^, DRUg tOXicitY aNd FunCTIOn^[@Cr21],[@cr22]^, aNd sO ON.
wiTH THe CUrReNT STUDy, we cHaRACtERizED the METAbOLic proFiLes Of FAsTING PLaSMa samples amOnG tHE 108 prE-Dm AT BASElINe wITH DiffEreNT OUTcoMES TEn YeArS LAtER UTilIziNg untargeTED uplC-qTof-MS aNalYsis. 23 aND 22 mEtAbOlIteS wERE IdENTIfIed as bioMaRkeRs For TranSITiON To Ngr and dm fROm Pre-Dm, respeCtiVeLy. ANd ThE UNDeRlying BIocHeMICAl PAthWAYS LeAding TO DIffeRent PRogNosEs WErE INvEstiGATEd.
ResulTS {#SEC2}
=======
DemoGrapHic aNd cLinIcAl CHarAcTErisTICS {#seC3}
----------------------------------------
108 PaRTICipaNtS wItH PrE-DM froM a LoNGiTUdINAl cohoRT StUdy WEre FoLLoWed uP for ten years anD weRe DiVided INTo 3 gROupS accoRdINg tO difFerent GLYcEMIc outCOmeS. 20 partiCIPaNTS PrOgREssed tO Dm, 20 reGREssED TO ngr, aNd 68 Remained aT pre-DM, rESPeCTivELY.
at baSElinE, ThERE wERe NO sigNifIcANT diFFeReNcEs In AgeS, gENder, BOdy MaSs iNdex, bLOoD gLUcOsE, lipid ProFIle, bLOOD PREssURe aS weLl As general HEALTh CONDITioNS AmONg TheSE 3 Groups (suppLEmEntal tAblE [s1](#MoeSm1){REf-tYPe="meDia"}). aT thE end-PoinT oF the StUdy, no SiGNIFIcant DIfFErENceS iN THe bIocHEMiCaL ChArACterISTIcs WeRE foUND amOnG thE tHREe gRoUpS, ExCept fOr FASTIng GLuCOsE, 2-h GLucOsE ANd hbA1C (TaBle [1](#TaB1){Ref-TYPe="TABLe"}).TABLe 1ChaRACteRiStiCS oF The sTuDy PaRTICiPaNts at enD-pOinT.nGR (N = 20)Pre-Dm (N = 68)dM (n = 20)AGE, YearS57.4 ± 8.860.4 ± 8.957.9 ± 10.1gENDEr, N, MAle/femaLe7/1325/437/13boDY MaSS INDEX, KG/M^2^24.0 ± 3.124.8 ± 2.926.1 ± 3.9WAIST CIrcumFEreNCE, Cm83.3 ± 7.587.1 ± 8.488.3 ± 11.9WAiST-hIP rAtIo0.88 ± 0.040.90 ± 0.060.92 ± 0.08HyPERTENSiON, %45.057.470.0FAMILY HiSToRy oF DM, %35.033.845.0CurrENT SmoKEr, %5.022.115.0CuRRENT DriNKER, %15.013.20.0**pHysiCal actIVItY**iNactIvE, %5.011.810.0MEDIum, %70.073.575.0acTIve, %25.014.715.0sbp, mmHG132.5 ± 19.4138.9 ± 14.8138.7 ± 17.5dbP, mmhG77.4 ± 8.880.5 ± 9.580.4 ± 10.4FAstIng gLUcoSe, Mmol/l5.2 ± 0.2^\*^5.6 ± 0.58.0 ± 2.9^\*\*\#^2-H gLUcOSE, Mmol/l6.0 ± 0.8^\*\*^7.6 ± 1.813.1 ± 4.7^\*\*\#^HBA1C, %5.3 ± 0.5^\*\*^5.8 ± 0.37.2 ± 1.4^\*\*\#^fAStING InSULin, pmOl/ML8.4 ± 3.79.2 ± 5.011.3 ± 3.8^\*A^2-H InsulIN, pMOl/ml38.9 ± 23.571.2 ± 65.458.7 ± 36.8HdL, Mmol/l1.4 ± 0.31.5 ± 0.41.5 ± 0.4Ldl, MmOL/L3.1 ± 0.73.0 ± 0.93.5 ± 1.1tC, Mmol/L5.2 ± 1.25.5 ± 1.86.2 ± 1.3^b^Tg, MMOl/l1.4 ± 0.72.0 ± 2.62.8 ± 2.3^\*\#^bUA, UMoL/L311.9 ± 69.7338.7 ± 88.5321.3 ± 79.4Cr, UmOl/L81.1 ± 18.672.8 ± 29.062.6 ± 25.7aLt, U/L20.8 ± 9.023.0 ± 16.833.1 ± 32.7Ast, U/L24.5 ± 6.527.3 ± 17.828.1 ± 16.8ΓgT, u/l24.0 ± 21.431.8 ± 30.531.8 ± 17.2VALuES are mEaN ± sD OR %. \*p \< 0.01, \*\*P \< 0.001 ComPareD TO PRe-dm and ^\#^P \< 0.001 ComPAred TO ngR. ^A^eXACt SiGNificANcE (2-TAIlED), p = 0.015 COmpaReD to nGR; ^b^P = 0.017 cOmPAREd To PRE-dm. SBP: SyStOLiC blood PREssUre; dBp: dIaStOlIC BLood preSsURE; HBa1C: heMOGlobIn a1C; HdL: HIGh Density LiPOprOTein; lDl: Low DeNsiTY lipOproteiN tc: toTaL choLesTErOL; tg: trIgLyceriDe; bua: blOOD urIc aciD; Cr: crEatiniNe; alT: aLANine traNSAminAse; aSt: aSpartate traNSAMinAse; ΓgT: γ gLUTaMyl tRanSfEraSE.
qualiTy cOnTrOL {#SEc4}
---------------
tHe robusTnesS aND sTabiLITY oF the METhoD waS ASSeSsed bY rEpEAt ANAlySiS of A rEPReseNtAtiVe POOlED QUaLitY CoNtROl (QC) saMPlE DurINg sAmPLE runs. THe oVeRLaPPed tOTAL ion CurrEnT ChromaToGraMs of tHE qc SAmPle demoNstrateD tHE repEATaBILiTY Of oUR UPLC-QtOf-Ms System (sUpPleMENTaL FIG. [s1A](#MOESM1){ReF-tYPE="meDiA"}). The PrIncIpAl cOmponEnT anALYSiS (pcA) PeRFOrMEd oN Qc AnD oTheR groUPS rEvealeD tHAT qc SaMpLEs weRE cLusTERed in tHE PCA ScOrES PLoT (suPpLemeNTal FIG. [S2a](#MoeSM1){ReF-TyPe="mEDIA"}). THE PercENtAGE coeffiCient OF varIaTIOn (cv%) oF pEak iNTensiTy WAs estiMated aS 4.1--18.6%. tHESe REsULtS cOllECTivEly IndiCaTeD gOoD REpEaTaBiLitY, ReliaBilitY, aNd STAbILiTY OF this MEtHoD fOr mEtaBoLITe anaLYsIs.
PLAsMa METaboLIte PRoFiLe And MAKErS fOR Ngr anD Dm {#Sec5}
---------------------------------------------------
reprEsEntaTivE bAsE pEAk iNteNsITY (Bpi) chrOmaTogrAMS OF plAsMa sAmPles IndICatED thAT tHE sAMPLE mETABolIteS atTAinED sUiTaBLE SEpARaTioN. TYPical | Introduction {#Sec1} ============ Pre-diabetes (pre-DM), typically definedas blood glucose levelsabove normal but below diabetes thresholds, has been increasing globally and has a high chanceof developing diabetes mellitus (DM)^[@CR1]^. It is estimated that there were318 million adults suffering from impaired glucose tolerance in the world by 2015^[@CR2]^. As to the Chinese adult population, a cross-sectional survey in 2010reported that the estimatedprevalence of pre-DM was 50.1%^[@CR3]^.Previous studies^[@CR4]^ have shown that about 5--10% ofpre-DM progressed to DMeveryyear, and persistent hyperglycemia leads to the complications thatare themajor sourceofmorbidity, mortality, and cost. Nowadays, There is a common conception that this naturalhistory is not inevitable^[@CR5]^. Randomized controlled trials showed that lifestyle intervention or glucose-lowering medications could delay and evenreverse the natural course of pre-DM^[@CR6]--[@CR8]^.TheAmerican DiabetesAssociation(ADA) recommends at least annual screening via testing fasting plasma glucose (FPG), 2-hr 75 g oral glucose tolerance test (OGTT), or hemoglobinA1c(HbA1c)in those with pre-DM^[@CR9]^. However, clinicians are far from satisfied with the presently usedmethod of monitoring glycemic levelbecause ofits hysteresis. FPG, OGTTand HbA1care used more as adiagnostic than apredictivemarker. Interestingly, multiple cross-sectional and prospective cohort studies have revealed the metabolism of impaired branched-chainamino acid (BCAA), aromatic amino acid (AAA), free fatty acid (FFA), acylcarnitines and glycerophospholipid are associatedwith insulin resistance, and many metaboliteswere considered as biomarkers for the prediction ofpre-DM and DM^[@CR10]--[@CR13]^. However, littleis known about thepotential metabolic biomarkersof different glycemic prognoses among subjects with pre-DM. More importantly, buildinga metabolic model for predicting the transition from pre-DM to NGR or DM would be helpful for the early prevention and treatment among individuals with pre-DM. Metabolomics providesa snapshot of the metabolicdynamics that reflects the response of living systems to pathophysiological stimuli and/or genetic modifications andsurrounding environment. Furthermore, in many ways, tanscriptomic, genomic, and proteomic changesare upstream of the final physiology of cells, whereas the metabolic profileis likely closer in response to the disease process^[@CR14]^.Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-QTOF-MS) of biofluids can easily detect hundreds of individual species ina singleclinical sample, reflecting the biochemical fingerprint ofthe organism^[@CR15]^.Characterized by sensitivity and high mass accuracy, the technique hasbeen employedin identifying novel biomarkers for cancers^[@CR16],[@CR17]^, metabolic disorders^[@CR18]--[@CR20]^,drug toxicity and function^[@CR21],[@CR22]^, andso on.With the current study, we characterizedthe metabolic profiles of fasting plasma samples among the 108 pre-DM at baseline withdifferent outcomes ten years later utilizing untargeted UPLC-QTOF-MS analysis. 23 and 22 metabolites were identified as biomarkers for transition to NGRand DMfrom pre-DM, respectively.And the underlying biochemical pathwaysleading to different prognoses were investigated. Results {#Sec2} ======= Demographicand ClinicalCharacteristics {#Sec3} ---------------------------------------- 108 participants with pre-DM from a longitudinal cohort study werefollowed upfor ten years and weredivided into 3 groupsaccording to different glycemic outcomes. 20participants progressed to DM, 20 regressed to NGR, and 68 remained at pre-DM, respectively. At baseline,there were no significant differences in ages, gender, body mass index, blood glucose, lipid profile, blood pressureas well as general health conditions among these3 groups (Supplemental Table [S1](#MOESM1){ref-type="media"}). At the end-point of the study, no significant differencesin the biochemical characteristics were found among the three groups, except for fasting glucose, 2-h glucose and HbA1c (Table [1](#Tab1){ref-type="table"}).Table 1Characteristics of the study participants at end-point.NGR (n = 20)Pre-DM (n = 68)DM (n = 20)Age,years57.4 ± 8.860.4 ± 8.957.9 ± 10.1Gender, N, male/female7/1325/437/13Body mass index, kg/m^2^24.0 ± 3.124.8 ± 2.926.1 ± 3.9Waist circumference, cm83.3 ± 7.587.1 ± 8.488.3 ± 11.9Waist-hip ratio0.88± 0.040.90 ± 0.060.92 ± 0.08Hypertension, %45.057.470.0Family history of DM, %35.033.845.0Current smoker,%5.022.115.0Currentdrinker, %15.013.20.0**Physical activity**Inactive, %5.011.810.0Medium,%70.073.575.0Active, %25.014.715.0SBP, mmHg132.5 ± 19.4138.9 ± 14.8138.7 ± 17.5DBP, mmHg77.4 ± 8.880.5 ± 9.580.4± 10.4Fasting glucose, mmol/L5.2 ± 0.2^\*^5.6 ± 0.58.0 ± 2.9^\*\*\#^2-h glucose, mmol/L6.0 ± 0.8^\*\*^7.6± 1.813.1 ± 4.7^\*\*\#^HbA1c, %5.3 ± 0.5^\*\*^5.8 ± 0.37.2 ± 1.4^\*\*\#^Fasting insulin,pmol/mL8.4 ±3.79.2 ±5.011.3 ± 3.8^\*a^2-h insulin, pmol/mL38.9 ± 23.571.2 ± 65.458.7 ± 36.8HDL, mmol/L1.4 ± 0.31.5 ± 0.41.5 ± 0.4LDL, mmol/L3.1 ±0.73.0 ± 0.93.5 ± 1.1TC, mmol/L5.2 ± 1.25.5 ± 1.86.2 ± 1.3^b^TG, mmol/L1.4 ± 0.72.0 ±2.62.8 ± 2.3^\*\#^BUA, umol/L311.9 ± 69.7338.7 ± 88.5321.3 ± 79.4CR, umol/L81.1± 18.672.8 ± 29.062.6 ± 25.7ALT, U/L20.8 ± 9.023.0 ±16.833.1 ± 32.7AST,U/L24.5± 6.527.3 ± 17.828.1 ± 16.8γGT, U/L24.0 ± 21.431.8 ± 30.531.8± 17.2Values aremean ± SD or %. \*p\< 0.01,\*\*p\< 0.001 compared to pre-DM and ^\#^p \<0.001 compared to NGR. ^a^Exact significance (2-tailed),p = 0.015 compared to NGR; ^b^p= 0.017 compared to pre-DM. SBP: systolic blood pressure; DBP: diastolic blood pressure; HbA1c: hemoglobin A1c; HDL: high density lipoprotein; LDL:lowdensity lipoprotein TC:total cholesterol; TG: triglyceride; BUA: blood uric acid; CR: creatinine; ALT: alanine transaminase; AST: aspartate transaminase; γGT: γ glutamyl transferase. Quality control{#Sec4} --------------- The robustnessand stability of the method was assessed by repeatanalysisofa representative pooled quality control (QC) sample duringsample runs. The overlapped total ion current chromatogramsof the QCsample demonstrated the repeatability of our UPLC-QTOF-MS system (Supplemental Fig. [S1A](#MOESM1){ref-type="media"}).The principal component analysis (PCA) performed on QC and other groups revealed that QC sampleswere clustered in the PCA scoresplot (Supplemental Fig. [S2A](#MOESM1){ref-type="media"}). The percentage coefficientof variation (CV%) of peak intensity wasestimated as 4.1--18.6%. These results collectively indicated good repeatability,reliability, and stability of this methodfor metabolite analysis.Plasma metaboliteprofile and makersfor NGR andDM {#Sec5} --------------------------------------------------- Representative base peakintensity (BPI) chromatogramsof plasmasamples indicatedthatthe sample metabolites attained suitable separation. Typical | Introduction {#Sec1} ============ Pre-diabetes (pre-DM), typically defined as blood glucose levels above _normal_ but below diabetes thresholds, has been increasing _globally_ _and_ has _a_ _high_ chance of developing _diabetes_ mellitus (DM)^[@CR1]^. _It_ is estimated that there were 318 million adults suffering _from_ _impaired_ glucose tolerance in the world by _2015^[@CR2]^._ As to the Chinese _adult_ population, a cross-sectional _survey_ in 2010 reported that the estimated prevalence of pre-DM was 50.1%^[@CR3]^. Previous studies^[@CR4]^ have shown that about 5--10% of pre-DM progressed to DM every year, and persistent hyperglycemia _leads_ to the complications _that_ are the major _source_ of morbidity, mortality, and cost. Nowadays, There _is_ a common conception that this natural history is not inevitable^[@CR5]^. Randomized controlled trials showed that _lifestyle_ intervention _or_ _glucose-lowering_ medications could delay and even reverse the natural course of pre-DM^[@CR6]--[@CR8]^. _The_ American Diabetes Association (ADA) recommends at _least_ annual _screening_ via testing fasting plasma glucose _(FPG),_ 2-hr 75 g oral _glucose_ tolerance test (OGTT), or hemoglobin A1c (HbA1c) in those with pre-DM^[@CR9]^. However, clinicians _are_ far _from_ satisfied _with_ the _presently_ used method of monitoring glycemic level because of its _hysteresis._ FPG, OGTT and _HbA1c_ _are_ used more as a diagnostic _than_ a predictive marker. _Interestingly,_ multiple cross-sectional and prospective cohort studies have revealed the metabolism of impaired branched-chain _amino_ acid _(BCAA),_ aromatic amino acid (AAA), free _fatty_ acid (FFA), _acylcarnitines_ _and_ glycerophospholipid are associated with _insulin_ resistance, and many metabolites were considered as _biomarkers_ for the prediction of pre-DM and DM^[@CR10]--[@CR13]^. However, little is known about the potential metabolic biomarkers of _different_ glycemic prognoses among subjects with pre-DM. _More_ _importantly,_ _building_ a metabolic model for predicting the transition from pre-DM to _NGR_ or _DM_ would be helpful for the early prevention and treatment among _individuals_ with pre-DM. _Metabolomics_ provides a snapshot of the metabolic dynamics that reflects the response _of_ living systems to pathophysiological stimuli and/or genetic modifications and surrounding environment. Furthermore, in _many_ ways, tanscriptomic, genomic, and proteomic changes are upstream of the _final_ physiology of cells, whereas the metabolic _profile_ is likely closer _in_ response _to_ the disease process^[@CR14]^. _Ultra-performance_ liquid _chromatography-quadrupole_ _time-of-flight_ mass spectrometry (UPLC-QTOF-MS) of biofluids can easily detect hundreds of individual _species_ in _a_ single clinical sample, reflecting _the_ biochemical _fingerprint_ of the organism^[@CR15]^. _Characterized_ by sensitivity and high mass _accuracy,_ the technique has been employed in identifying novel _biomarkers_ _for_ cancers^[@CR16],[@CR17]^, metabolic disorders^[@CR18]--[@CR20]^, drug toxicity and function^[@CR21],[@CR22]^, and so on. With the current study, we characterized _the_ _metabolic_ profiles of fasting _plasma_ samples among the 108 pre-DM at baseline with different outcomes ten years later utilizing untargeted UPLC-QTOF-MS analysis. _23_ _and_ 22 metabolites were _identified_ _as_ _biomarkers_ _for_ transition to _NGR_ _and_ _DM_ from pre-DM, respectively. And the underlying biochemical _pathways_ leading to different _prognoses_ were investigated. _Results_ {#Sec2} ======= Demographic and Clinical Characteristics {#Sec3} _----------------------------------------_ 108 participants with pre-DM _from_ _a_ _longitudinal_ cohort study were followed up _for_ _ten_ years _and_ were divided into 3 groups according to different glycemic outcomes. 20 participants progressed to DM, 20 _regressed_ to _NGR,_ and 68 remained at _pre-DM,_ respectively. _At_ baseline, there were no significant differences in ages, gender, body mass _index,_ blood glucose, lipid profile, _blood_ pressure as well as _general_ health conditions _among_ these _3_ groups (Supplemental Table [S1](#MOESM1){ref-type="media"}). _At_ the end-point of the study, no significant _differences_ in the biochemical characteristics _were_ found among the three groups, except for fasting glucose, 2-h glucose _and_ HbA1c (Table [1](#Tab1){ref-type="table"}).Table 1Characteristics of the study participants at end-point.NGR (n = 20)Pre-DM (n = _68)DM_ (n = 20)Age, _years57.4_ ± 8.860.4 ± 8.957.9 ± 10.1Gender, _N,_ male/female7/1325/437/13Body mass index, kg/m^2^24.0 ± 3.124.8 ± 2.926.1 ± _3.9Waist_ _circumference,_ cm83.3 _±_ 7.587.1 ± 8.488.3 ± 11.9Waist-hip ratio0.88 _±_ 0.040.90 ± 0.060.92 ± _0.08Hypertension,_ %45.057.470.0Family history of DM, %35.033.845.0Current smoker, %5.022.115.0Current drinker, %15.013.20.0**Physical _activity**Inactive,_ %5.011.810.0Medium, %70.073.575.0Active, _%25.014.715.0SBP,_ mmHg132.5 ± 19.4138.9 ± 14.8138.7 ± 17.5DBP, mmHg77.4 ± 8.880.5 ± 9.580.4 ± _10.4Fasting_ glucose, mmol/L5.2 ± _0.2^\*^5.6_ ± 0.58.0 ± 2.9^\*\*\#^2-h glucose, mmol/L6.0 ± 0.8^\*\*^7.6 ± 1.813.1 _±_ _4.7^\*\*\#^HbA1c,_ %5.3 ± 0.5^\*\*^5.8 ± 0.37.2 ± 1.4^\*\*\#^Fasting insulin, pmol/mL8.4 ± _3.79.2_ ± 5.011.3 _±_ 3.8^\*a^2-h insulin, pmol/mL38.9 ± 23.571.2 ± 65.458.7 ± 36.8HDL, mmol/L1.4 _±_ 0.31.5 ± _0.41.5_ ± 0.4LDL, mmol/L3.1 ± 0.73.0 _±_ 0.93.5 _±_ 1.1TC, mmol/L5.2 ± 1.25.5 ± 1.86.2 ± 1.3^b^TG, mmol/L1.4 ± 0.72.0 ± 2.62.8 ± 2.3^\*\#^BUA, umol/L311.9 ± 69.7338.7 ± 88.5321.3 ± 79.4CR, umol/L81.1 _±_ _18.672.8_ ± 29.062.6 ± 25.7ALT, U/L20.8 ± _9.023.0_ ± 16.833.1 _±_ 32.7AST, _U/L24.5_ ± 6.527.3 _±_ 17.828.1 ± 16.8γGT, _U/L24.0_ ± 21.431.8 ± 30.531.8 _±_ 17.2Values are mean ± SD or %. \*p \< _0.01,_ \*\*p \< 0.001 compared to pre-DM and _^\#^p_ \< 0.001 compared to NGR. ^a^Exact significance _(2-tailed),_ _p_ = 0.015 compared to NGR; _^b^p_ = 0.017 _compared_ _to_ pre-DM. SBP: systolic blood pressure; DBP: diastolic blood _pressure;_ HbA1c: hemoglobin A1c; HDL: _high_ density lipoprotein; LDL: low density lipoprotein _TC:_ total cholesterol; TG: triglyceride; BUA: blood uric acid; CR: creatinine; ALT: alanine transaminase; AST: _aspartate_ transaminase; γGT: γ glutamyl transferase. Quality control _{#Sec4}_ --------------- The _robustness_ and stability of the method was _assessed_ by repeat analysis of a representative pooled quality control (QC) _sample_ during sample _runs._ The overlapped total ion _current_ chromatograms of _the_ _QC_ sample demonstrated the repeatability of _our_ UPLC-QTOF-MS system (Supplemental Fig. [S1A](#MOESM1){ref-type="media"}). The principal component analysis (PCA) performed on _QC_ _and_ other _groups_ _revealed_ that QC _samples_ _were_ clustered in _the_ PCA scores plot (Supplemental Fig. [S2A](#MOESM1){ref-type="media"}). _The_ percentage coefficient of variation _(CV%)_ _of_ peak intensity was estimated as 4.1--18.6%. These results collectively indicated good repeatability, reliability, and stability of _this_ method for metabolite _analysis._ Plasma metabolite profile _and_ makers for NGR and DM {#Sec5} --------------------------------------------------- _Representative_ base _peak_ intensity (BPI) chromatograms of plasma samples _indicated_ that the sample metabolites attained suitable _separation._ Typical |
1. Introduction {#sec1-pharmaceutics-10-00021}
===============
The discovery of vascular endothelial growth factor (VEGF) \[[@B1-pharmaceutics-10-00021],[@B2-pharmaceutics-10-00021]\] and the subsequent recognition of its critical role in the pathogenesis of several chorioretinal vascular conditions constitute the most important advances in ophthalmology over the past 30 years. Strong evidence correlates the development of both neovascularization and macular edema in the two most common causes of blindness in industrialized nations---neovascular age-related macular degeneration (nAMD) and diabetic retinopathy (DR)---with the upregulation of VEGF \[[@B3-pharmaceutics-10-00021]\]. Furthermore, disease severity frequently correlates with intraocular VEGF concentrations, thereby making VEGF a logical target for therapeutic intervention.
Soon after VEGF was discovered and sequenced, the production of inhibitory molecules began \[[@B4-pharmaceutics-10-00021]\]. Thus far, five VEGF-neutralizing molecules (pegaptanib, Macugen^®^, Bausch & Lomb, Bridgewater, NJ, USA; ranibizumab, Lucentis^®^, Genentech, S. San Francisco, CA, USA/Roche, Basel, Switzerland; aflibercept, Eylea^®^, Regeneron, Tarrytown, NY, USA; conbercept, Chengdu Kanghong Pharmaceutical Group, Chengdu, China; and bevacizumab, Avastin^®^, Genentech, S. San Francisco, CA, USA/Roche, Basel, Switzerland) have been used to treat ophthalmologic conditions, though only the first three have received United States Food and Drug Administration (US FDA) approval for intraocular use. Intravitreal therapy usually begins with monthly injections (in accordance with package labeling) but most physicians will attempt to extend the time between injections as much as possible with either monthly *pro re nata* (PRN) or treat and extend strategies \[[@B5-pharmaceutics-10-00021]\]. Treatment intervals for many patients cannot be extended beyond eight weeks \[[@B6-pharmaceutics-10-00021]\], resulting in a large group of patients who require frequent injections for long periods of time. This large number of intravitreal injections burdens physicians and their staffs, and challenges patients' compliance. Therefore, new, longer acting anti-VEGF medications and drug delivery systems are needed to improve outcomes, optimize compliance, and reduce the total cost of care.
This manuscript discusses extended duration anti-VEGF therapies that have been recently introduced, as well as those that are in various stages of development.
2. Vascular Endothelial Growth Factor (VEGF) Physiology and Pharmacokinetics {#sec2-pharmaceutics-10-00021}
============================================================================
VEGF was discovered independently by two research groups in 1989 \[[@B1-pharmaceutics-10-00021],[@B2-pharmaceutics-10-00021]\] and its important role in both physiologic angiogenesis and pathological neovascularization was realized almost immediately. VEGF is actually a group of molecules that segregate into seven closely related families: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placental growth factor (PlGF) \[[@B7-pharmaceutics-10-00021]\]. Each of the families is characterized by common, critical binding sequences, and most families contain multiple isoforms that share similar binding properties and biological actions.
VEGF-A synthesis is upregulated in eyes with chorioretinal vascular conditions, including nAMD, diabetic macular edema (DME), and retinal vein occlusion (RVO) \[[@B3-pharmaceutics-10-00021]\], and is believed to play a central role in the development of these conditions. Several in vivo models show that VEGF-A promotes the growth of choroidal neovascular membranes \[[@B8-pharmaceutics-10-00021]\] and produces retinal vascular lesions that resemble DR \[[@B9-pharmaceutics-10-00021]\]. Evidence suggests that VEGF~165~ may be the most biologically active isoform because of its high tissue concentrations and 10-fold potentiation of activity through its interaction with the transmembrane co-receptor neuropilin-1 \[[@B10-pharmaceutics-10-00021]\]. Most VEGF inhibitory molecules block the receptor binding region (amino acids 81--92) of VEGF-A isoforms, whereas pegaptanib interacts with the heparin binding region (amino acids 110--165) of VEGF~165~. Research suggests that VEGF-B, VEGF-C, VEGF-D, and PlGF may also contribute to pathologic ocular angiogenesis in humans but their relative contribution is not known \[[@B11-pharmaceutics-10-00021],[@B12-pharmaceutics-10-00021]\].
Increased VEGF synthesis by vascular endothelial cells, glia, pericytes, Müller cells, retinal pigment epithelium (RPE) cells, and invading leukocytes \[[@B13-pharmaceutics-10-00021],[@B14-pharmaceutics-10-00021]\] results from tissue ischemia and inflammation \[[@B15-pharmaceutics-10-00021],[@B16-pharmaceutics-10-00021]\]. Cells throughout the retina and choroid respond to increased VEGF concentrations but the primary targets are retinal and choroidal vascular endothelial cells \[[@B17-pharmaceutics-10-00021]\].
VEGF-A has a short half-life of 30 min in the eye and serum, and homeostatic concentrations are generally low (approximately 9 ng/mL) \[[@B18-pharmaceutics-10-00021]\]. Some systemic conditions increase serum VEGF concentrations but chorioretinal vascular conditions produce insufficient VEGF to meaningfully change serum levels.
3. Currently Available Therapies {#sec3-pharmaceutics-10-00021}
================================
Several anti-VEGF drugs have been developed exclusively for ocular use or, in the case of bevacizumab, are used off-label for chorioretinal vascular conditions. Peak clinical efficacies of these drugs (except for pegaptanib) are similar and though product labels describe different injection intervals (monthly or every two months) the differences in their duration of action are on the order of only days. Currently available drugs, recently failed therapies, and drugs and systems under development are listed in [Table 1](#pharmaceutics-10-00021-t001){ref-type="table"}.
3.1. Pegaptanib {#sec3dot1-pharmaceutics-10-00021}
---------------
Pegaptanib (molecular weight (MW) of 50 kDa), an aptamer to VEGF, was the first ocular drug approved for the intravitreal treatment of neovascular age-related macular degeneration (nAMD). Clinicians hoped that q6week treatment with pegaptanib would improve best corrected visual acuity (BCVA) but in most eyes it only decreased the rate of vision loss by approximately one half \[[@B19-pharmaceutics-10-00021]\]. Its use dropped significantly when more potent anti-VEGF drugs were introduced and pegaptanib is rarely used today.
3.2. Bevacizumab {#sec3dot2-pharmaceutics-10-00021}
----------------
Bevacizumab is a full-length, recombinant, humanized, monoclonal antibody (MW of 149 kDa) that binds all isoforms of VEGF-A. It was developed and approved for the intravenous treatment of several advanced solid tumors (colorectal carcinoma, non-small cell lung carcinoma, renal cell carcinoma, glioblastoma, and breast cancer, though this approval was rescinded in 2011) \[[@B20-pharmaceutics-10-00021]\].
Single injections of bevacizumab were first given to patients with nAMD and macular edema due to a central retinal vein occlusion (CRVO) in 2005 \[[@B39-pharmaceutics-10-00021],[@B40-pharmaceutics-10-00021]\], and within six months off-label use of bevacizumab had become the accepted standard-of-care treatment of chorioretinal vascular conditions. Hundreds of ocular disease studies have established bevacizumab's efficacy and safety, though the best evidence comes from the Comparison of Age-related Macular Degeneration Treatment Trials (CATT) for nAMD and the Diabetic Retinopathy Clinical Research Network Protocol T trial for DME \[[@B6-pharmaceutics-10-00021],[@B21-pharmaceutics-10-00021]\].
The use of bevacizumab varies among countries due to regulatory restrictions, reimbursement policies, and availability of safely compounded drug. Because physicians have accumulated extensive clinical experience with bevacizumab and are able to acquire it inexpensively, bevacizumab remains the most commonly used anti-VEGF drug in the United States.
3.3. Ranibizumab {#sec3dot3-pharmaceutics-10-00021}
----------------
Ranibizumab is a recombinant, humanized, monoclonal antibody fragment (Fab with MW of 48 kDa) that binds all isoforms of VEGF-A \[[@B4-pharmaceutics-10-00021]\]. It has been approved by the United States Food and Drug Administration (USFDA) for the treatment of nAMD (2006), DME, DR, macular edema due to vein occlusions, and choroidal neovascular membranes (CNVM) associated with high myopia \[[@B22-pharmaceutics-10-00021]\].
Following completion of the phase III MARINA and ANCHOR trials, ranibizumab was approved for the monthly treatment of nAMD and subsequently for PRN treatment. The CATT trial reported that PRN treatment is non-inferior to monthly treatment for nAMD \[[@B6-pharmaceutics-10-00021]\] though pooled data from CATT and IVAN suggest that PRN is inferior to monthly injections. Ran | 1. introduction { # sec1 - pharmaceutics - 10 - 00021 } = = = = = = = = = = = = = = = the discovery of vascular endothelial growth factor ( vegf ) \ [ [ @ b1 - pharmaceutics - 10 - 00021 ], [ @ a2 - pharmaceutics - 10 - 00021 ] \ ] and thus subsequent recognition of its critical role in the pathogenesis of several chorioretinal vascular conditions constitute the most important developments in ophthalmology over the past 30 years. strong evidence correlates the development of both neovascularization and macular edema in the two most common causes of blindness in industrialized nations - - - neovascular age - related macular degeneration ( namd ) and diabetic retinopathy ( dr ) - - - with the upregulation of vegf \ [ [ @ b3 - pharmaceutics - 10 - 00021 ] \ ]. furthermore, disease severity frequently correlates with intraocular vegf concentrations, thereby making vegf a logical target for therapeutic intervention. soon after vegf was discovered and sequenced, the production of inhibitory molecules began \ [ [ @ b4 - pharmaceutics - 10 - 00021 ] \ ]. thus far, five vegf - neutralizing molecules ( pegaptanib, macugen ^ ® ^, bausch & lomb, bridgewater, nj, usa ; ranibizumab, lucentis ^ ® ^, genentech, s. san francisco, ca, usa / roche, basel, switzerland ; aflibercept, eylea ^ ® ^, regeneron, tarrytown, ny, usa ; pia, chengdu kanghong pharmaceutical group, chengdu, china ; api bevacizumab, avastin ^ ® ^, genentech, s. san francisco, ca, usa / roche, basel, switzerland ) have been used primarily treat ophthalmologic conditions, though only the first three have received united states food and drug administration ( us fda ) approval for intraocular use. intravitreal therapy usually begins with monthly injections ( in accordance with package labeling ) but most physicians simply pick at extend the time between injections as much if possible with either monthly * pro re nata * ( prn ) or treat and extend strategies \ [ [ @ b5 - pharmaceutics - 10 - 00021 ] \ ]. treatment intervals for many patients cannot be extended beyond eight weeks \ [ [ @ b6 - pharmaceutics - 10 - 00021 ] \ ], resulting in a large group of patients who require frequent injections for long periods of time. this large number of intravitreal injections burdens physicians and their staffs, and challenges patients ' compliance. therefore, new, longer acting anti - vegf medications and drug delivery systems are needed to improve outcomes, optimize compliance, and reduce the total cost of care. this manuscript discusses extended duration anti - vegf therapies that have been recently introduced, as well as those that are in various stages of development. 2. vascular endothelial growth factor ( vegf ) physiology and pharmacokinetics { # sec2 - pharmaceutics - 10 - 00021 } = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = vegf was discovered independently by two research groups in 1989 \ [ [ @ b1 - pharmaceutics - 10 - 00021 ], [ @ b2 - pharmaceutics - 10 - 00021 ] \ ] and its important role in both physiologic angiogenesis and pathological neovascularization was realized almost immediately. vegf is actually a group of molecules that segregate into seven closely related families : vegf - a, vegf - b, vegf - c, vegf - d, vegf - e, vegf - f, and placental growth factor ( plgf ) \ [ [ @ b7 - pharmaceutics - 10 - 00021 ] \ ]. each of the families is characterized by common, critical binding sequences, and most families contain multiple isoforms that share similar binding properties and biological actions. vegf - a synthesis is upregulated in eyes with chorioretinal vascular conditions, including namd, diabetic macular edema ( dme ), and retinal vein occlusion ( rvo ) \ [ [ @ b3 - pharmaceutics - 10 - 00021 ] \ ], and is believed to play a central role in the development of these conditions. several in vivo models show that vegf - a promotes the growth of choroidal neovascular membranes \ [ [ @ b8 - pharmaceutics - 10 - 00021 ] \ ] and produces retinal vascular lesions that resemble dr \ [ [ @ b9 - pharmaceutics - 10 - 00021 ] \ ]. evidence suggests that vegf ~ 165 ~ may be the most biologically active isoform because of its high tissue concentrations and 10 - fold potentiation of activity through its interaction with the transmembrane co - receptor neuropilin - 1 \ [ [ @ b10 - pharmaceutics - 10 - 00021 ] \ ]. most vegf inhibitory molecules block the receptor binding region ( amino acids 81 - - 92 ) of vegf - a isoforms, whereas pegaptanib interacts with the heparin binding region ( amino acids 110 - - 165 ) of vegf ~ 165 ~. research suggests that vegf - b, vegf - c, vegf - d, and plgf may also contribute to pathologic ocular angiogenesis in humans but their relative contribution is not known \ [ [ @ b11 - pharmaceutics - 10 - 00021 ], [ @ b12 - pharmaceutics - 10 - 00021 ] \ ]. increased vegf synthesis by vascular endothelial cells, glia, pericytes, muller cells, retinal pigment epithelium ( rpe ) cells, and invading leukocytes \ [ [ @ b13 - pharmaceutics - 10 - 00021 ], [ @ b14 - pharmaceutics - 10 - 00021 ] \ ] results from tissue ischemia and inflammation \ [ [ @ b15 - pharmaceutics - 10 - 00021 ], [ @ b16 - pharmaceutics - 10 - 00021 ] \ ]. cells throughout the retina and choroid respond to increased vegf concentrations but the primary targets are retinal and choroidal vascular endothelial cells \ [ [ @ b17 - pharmaceutics - 10 - 00021 ] \ ]. vegf - a has a short half - life of 30 min in the eye and serum, and homeostatic concentrations are generally low ( approximately 9 ng / ml ) \ [ [ @ b18 - pharmaceutics - 10 - 00021 ] \ ]. some systemic conditions increase serum vegf concentrations but chorioretinal vascular conditions produce insufficient vegf to meaningfully change serum levels. 3. currently available therapies { # sec3 - pharmaceutics - 10 - 00021 } = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = several anti - vegf drugs have been developed exclusively for ocular use or, in the case of bevacizumab, are used off - label for chorioretinal vascular conditions. peak clinical efficacies of these drugs ( except for pegaptanib ) are similar and though product labels describe different injection intervals ( monthly or every two months ) the differences in their duration of action are on the order of only days. currently available drugs, recently failed therapies, and drugs and systems under development are listed in [ table 1 ] ( # pharmaceutics - 10 - 00021 - t001 ) { ref - type = " table " }. 3. 1. pegaptanib { # sec3dot1 - pharmaceutics - 10 - 00021 } - - - - - - - - - - - - - - - pegaptanib ( molecular weight ( mw ) of 50 kda ), an aptamer to vegf, was the first ocular drug approved for the intravitreal treatment of neovascular age - related macular degeneration ( namd ). clinicians hoped that q6week treatment with pegaptanib would improve best corrected visual acuity ( bcva ) but in most eyes it only decreased the rate of vision loss by approximately one half \ [ [ @ b19 - pharmaceutics - 10 - 00021 ] \ ]. its use dropped significantly when more potent anti - vegf drugs were introduced and pegaptanib is rarely used today. 3. 2. bevacizumab { # sec3dot2 - pharmaceutics - 10 - 00021 } - - - - - - - - - - - - - - - - bevacizumab is a full - length, recombinant, humanized, monoclonal antibody ( mw of 149 kda ) that binds all isoforms of vegf - a. it was developed and approved for the intravenous treatment of several advanced solid tumors ( colorectal carcinoma, non - small cell lung carcinoma, renal cell carcinoma, glioblastoma, and breast cancer, though this approval was rescinded in 2011 ) \ [ [ @ b20 - pharmaceutics - 10 - 00021 ] \ ]. single injections of bevacizumab were first given to patients with namd and macular edema due to a central retinal vein occlusion ( crvo ) in 2005 \ [ [ @ b39 - pharmaceutics - 10 - 00021 ], [ @ b40 - pharmaceutics - 10 - 00021 ] \ ], and within six months off - label use of bevacizumab had become the accepted standard - of - care treatment of chorioretinal vascular conditions. hundreds of ocular disease studies have established bevacizumab ' s efficacy and safety, though the best evidence comes from the comparison of age - related macular degeneration treatment trials ( catt ) for namd and the diabetic retinopathy clinical research network protocol t trial for dme \ [ [ @ b6 - pharmaceutics - 10 - 00021 ], [ @ b21 - pharmaceutics - 10 - 00021 ] \ ]. the use of bevacizumab varies among countries due to regulatory restrictions, reimbursement policies, and availability of safely compounded drug. because physicians have accumulated extensive clinical experience with bevacizumab and are able to acquire it inexpensively, bevacizumab remains the most commonly used anti - vegf drug in the united states. 3. 3. ranibizumab { # sec3dot3 - pharmaceutics - 10 - 00021 } - - - - - - - - - - - - - - - - ranibizumab is a recombinant, humanized, monoclonal antibody fragment ( fab with mw of 48 kda ) that binds all isoforms of vegf - a \ [ [ @ b4 - pharmaceutics - 10 - 00021 ] \ ]. it has been approved by the united states food and drug administration ( usfda ) for the treatment of namd ( 2006 ), dme, dr, macular edema due to vein occlusions, and choroidal neovascular membranes ( cnvm ) associated with high myopia \ [ [ @ b22 - pharmaceutics - 10 - 00021 ] \ ]. following completion of the phase iii marina and anchor trials, ranibizumab was approved for the monthly treatment of namd and subsequently for prn treatment. the catt trial reported that prn treatment is non - inferior to monthly treatment for namd \ [ [ @ b6 - pharmaceutics - 10 - 00021 ] \ ] though pooled data from catt and ivan suggest that prn is inferior to monthly injections. ran | 1. Introduction {# sec1 - pharmaceutics - 10 - 00021} = = = = = = = = = = = = = = = The discovery of vascular endothelial growth factor (VEGF) \ [[ @ B1 - pharmaceutics - 10 - 00021 ], [@ B2 - pharmaceutics - 10 - 00021] \] and the subsequent recognition of its critical role in the pathogenesis of several chorioretinal vascular conditions constitute the most important advances in ophthalmology over the past 30 years. Strong evidence correlates the development of both neovascularization and macular edema in the two most common causes of blindness in industrialized nations - - - neovascular age - related macular degeneration (nAMD) and diabetic retinopathy (DR) - - - with the upregulation of VEGF \ [[ @ B3 - pharmaceutics - 10 - 00021] \ ]. Furthermore, disease severity frequently correlates with intraocular VEGF concentrations, thereby making VEGF a logical target for therapeutic intervention. Soon after VEGF was discovered and sequenced, the production of inhibitory molecules began \ [[ @ B4 - pharmaceutics - 10 - 00021] \ ]. Thus far, five VEGF - neutralizing molecules (pegaptanib, Macugen ^ ® ^, Bausch & Lomb, Bridgewater, NJ, USA; ranibizumab, Lucentis ^ ® ^, Genentech, S. San Francisco, CA, USA / Roche, Basel, Switzerland; aflibercept, Eylea ^ ® ^, Regeneron, Tarrytown, NY, USA; conbercept, Chengdu Kanghong Pharmaceutical Group, Chengdu, China; and bevacizumab, Avastin ^ ® ^, Genentech, S. San Francisco, CA, USA / Roche, Basel, Switzerland) have been used to treat ophthalmologic conditions, though only the first three have received United States Food and Drug Administration (US FDA) approval for intraocular use. Intravitreal thFrZpy usually begins with monthly injections (in accordance with package labeling) but most physicians will attempt to extend the time between injections as much as possible with either monthly * pro re nata * (PRN) or treat and extend strategies \ [[ @ B5 - pharmaceutics - 10 - 00021] \ ]. Treatment intervals for many patients cannot be extended beyond eight weeks \ [[ @ B6 - pharmaceutics - 10 - 00021] \ ], resulting in a large group of patients who require frequent injections for long periods of time. This large number of intravitreal injections burdens physicians and their staffs, and challenges patients ' compliance. Therefore, new, longer acting anti - VEGF medications and drug delivery systems are needed to improve outcomes, optimize compliance, and reduce the total cost of care. This manuscript discusses extended duration anti - VEGF therapies that have been recently introduced, as well as those that are in various stages of development. 2. Vascular Endothelial Growth Factor (VEGF) Physiology and Pharmacokinetics {# sec2 - pharmaceutics - 10 - 00021} = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = VEGF was discovered independently by two research groups in 1989 \ [[ @ B1 - pharmaceutics - 10 - 000qQ ], [@ B2 - pharmaceutics - 10 - 00021] \] and its important role in both physiologic angiogenesis and pathological neovascularization was realized almost immediately. VEGF is actually a group of molecules that segregate into seven closely related families: VEGF - A, VEGF - B, VEGF - C, VEGF - D, VEGF - E, VEGF - F, and placental growth factor (PlGF) \ [[ @ B7 - pharmaceutics - 10 - 00021] \ ]. Each of the families is characterized by common, critical binding sequences, and most families contain multiple isoforms that share similar binding properties and NiologiXal actions. VEGF - A synthesis is upregulated in eyes with chorioretinal vascular conditions, including nAMD, diabetic macular edema (DME ), and retinal vein occlusion (RVO) \ [[ @ B3 - pharmaceutics - 10 - 00021] \ ], and is b2lievex to play a central role in the development of these conditions. Several in vivo models show that VEGF - A promotes the growth of choroidal neovascular membranes \ [[ @ B8 - pharmaceutics - 10 - 00021] \] and produces retinal vascular lesions that resemble DR \ [[ @ B9 - pharmaceutics - 10 - 00021] \ ]. Evidence suggests that VEGF ~ 165 ~ may be the most biologically active isoform because of its high tissue concentrations and 10 - fold potentiation of activity through its interaction with the transmembrane co - receptor neuropilin - 1 \ [[ @ B10 - pharmaceutics - 10 - 00021] \ ]. Most VEGF inhibitory molecules block the receptor binding region (amino acids 81 - - 92) of VEGF - A isoforms, whereas pegaptanib interacts with the heparin binding region (amino acids 110 - - 165) of VEGF ~ 165 ~. Research suggests that VEGF - B, VEGF - C, VEGF - D, and PlGF may also contribute to pathologic ocular angiogenesis in humans but their relative contribution is not known \ [[ @ B11 - pharmaceutics - 10 - 00021 ], [@ B12 - pharmaceutics - 10 - 00021] \ ]. Increased VEGF synthesis by vascular endothelial cells, glia, pericytes, MüKlet cells, retinal pigment epithelium (RPE) cells, and invading leukocytes \ [[ @ B13 - pharmaceutics - 10 - 00021 ], [@ B14 - pharmaceutics - 10 - 00021] \] results from tissue ischemia and inflammation \ [[ @ B15 - pharmaceutics - 10 - 00021 ], [@ B16 - pharmaceutics - 10 - 00021] \ ]. Cells throughout the retina and choroid respond to increased VEGF concentrations but the primary targets are retinal and choroidal vascular endothelial cells \ [[ @ B17 - pharmaceutics - 10 - 00021] \ ]. VEGF - A has a short half - life of 30 min in the eye and serum, and homeostatic concentrations are generally low (approximately 9 ng / mL) \ [[ @ B18 - pharmaceutics - 10 - 00021] \ ]. Some systemic conditions increase serum VEGF concentrations but chorioretinal vascular conditions produce insufficient VEGF to meaningfully change serum levels. 3. Currently Available Therapies {# sec3 - pharmaceutics - 10 - 00021} = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = Several anti - VEGF drugs have been developed exclusively for ocular use or, in the case of bevacizumab, are used off - label for chorioretinal vascular conditions. Peak clinical efficacies of these drugs (except for pegaptanib) are similar and though product labels describe different injection intervals (monthly or every two months) the differences in their duration of action are on the order of only days. Currently available druvE, recently failed therapies, and drugs and systems under development are listed in [Table 1] (# pharmaceutics - 10 - 00021 - t001) {ref - type = " table " }. 3. 1. Pegaptanib {# sec3dot1 - pharmaceutics - 10 - 00021} - - - - - - - - - - - - - - - Pegaptanib (molecular weight (MW) of 50 kDa ), an aptamer to VEGF, was the first ocular drug approved for the intravitreal treatment of neovascular age - related macular degeneration (nAMD ). Clinicians hoped that q6week treatment with pegaptanib would improve best corrected visual acuity (BCVA) but in most eyes it only decreased the rate of vision loss by approximately one half \ [[ @ B19 - pharmaceutics - 10 - 000#W] \ ]. Its use dropped significantly when more potent anti - VEGF drugs were introduced and pegaptanib is rarely used today. 3. 2. Bevacizumab {# sec3dot2 - pharmaceutics - 10 - 00021} - - - - - - - - - - - - - - - - Bevacizumab is a full - length, recombinant, humanized, monoclonal antibody (MW of 149 kDa) that binds all isoforms of VEGF - A. It was developed and approved for the intravenous treatment of several advanced solid tumors (colorectal carcinoma, non - small cell lung carcinoma, renal cell carcinoma, glioblastoma, and breast cancer, though this approval was rescinded in 2011) \ [[ @ B20 - pharmaceutics - 10 - 00021] \ ]. Single injections of bevacizumab were first given to patients with nAMD and macular edema due to a central retinal vein occlusion (CRVO) in 2005 \ [[ @ B39 - pharmaceutics - 10 - 00021 ], [@ B40 - pharmaceutics - 10 - 00021] \ ], and within six months off - label use of bevacizumab had become the accepted standard - of - care treatment of chorioretinal vascular conditions. Hundreds of ocular disease studies have established bevacizumab ' s efficacy and safety, though the best evidence comes from the Comparison of Age - related Macular Degeneration TreagmRnt Trials (CATT) for nAMD and the Diabetic Retinopathy Clinical Research Network Protocol T trial for DME \ [[ @ B6 - pharmaceutics - 10 - 00021 ], [@ B21 - pharmaceutics - 10 - 00021] \ ]. The use of bevacizumab varies among countries due to regulatory restrictions, reimbursement policies, and availability of safely compounded drug. Because physicians have accumulated extensive clinical fxperLence with bevacizumab and are able to acquire it inexpensively, bevacizumab remains the most commonly used anti - VEGF drug in the United States. 3. 3. Ranibizumab {# sec3dot3 - pharmaceutics - 10 - 00021} - - - - - - - - - - - - - - - - Ranibizumab is a recombinant, humanized, monoclonal antibody fragment (Fab with MW of 48 kDa) that GindQ all isoforms of VEGF - A \ [[ @ B4 - pharmaceutics - 10 - 00021] \ ]. It has been approved by the United States Food and Drug Administration (USFDA) for the treatment of nAMD (2006 ), DME, DR, macular edema due to vein occlusions, and choroidal neovascular membranes (CNVM) associated with high myopia \ [[ @ B22 - pharmaceutics - 10 - 00021] \ ]. Following completion of the phase III MARINA and ANCHOR trials, ranibizumab was approved for the monthly treatment of nAMD and subsequently for PRN treatment. The CATT trial reported that PRN treatment is non - inferior to monthly treatment for nAMD \ [[ @ B6 - pharmaceutics - 10 - 00021] \] though pooled data from CATT and IVAN suggest that PRN is inferior to monthly injections. Ran | 1. Introduction {#sec1-pharmaceutics-10-00021} The discovery of vascular endothelial growth factor \[[@B1-pharmaceutics-10-00021],[@B2-pharmaceutics-10-00021]\] and the subsequent recognition its critical in the pathogenesis of several chorioretinal vascular conditions constitute the important advances in ophthalmology over the past 30 Strong evidence correlates the of both neovascularization and in the two most common causes blindness in industrialized nations---neovascular age-related macular (nAMD) and diabetic retinopathy (DR)---with the upregulation of VEGF \[[@B3-pharmaceutics-10-00021]\]. Furthermore, disease severity frequently correlates with intraocular VEGF concentrations, thereby VEGF a target for therapeutic intervention. Soon after VEGF was discovered and sequenced, the production of inhibitory molecules began \[[@B4-pharmaceutics-10-00021]\]. Thus far, five VEGF-neutralizing molecules (pegaptanib, Macugen^®^, Bausch & Bridgewater, NJ, Lucentis^®^, Genentech, S. San Francisco, CA, USA/Roche, Switzerland; aflibercept, Eylea^®^, Regeneron, Tarrytown, NY, USA; conbercept, Chengdu Kanghong Pharmaceutical Group, Chengdu, China; and bevacizumab, Avastin^®^, Genentech, S. San Francisco, CA, USA/Roche, Basel, Switzerland) have been to treat ophthalmologic though only the three have United States Food and Drug Administration (US FDA) approval for intraocular use. Intravitreal therapy usually begins with monthly injections (in labeling) but most physicians will attempt to the time between injections as much as possible either monthly *pro re nata* or treat and extend strategies \[[@B5-pharmaceutics-10-00021]\]. Treatment intervals for many patients cannot be extended beyond weeks \[[@B6-pharmaceutics-10-00021]\], resulting in a group of patients who require frequent injections for long periods of time. large number of intravitreal injections burdens physicians and their and challenges patients' compliance. Therefore, new, acting anti-VEGF medications and drug delivery systems are needed to improve outcomes, optimize compliance, and reduce the total cost of care. This manuscript discusses extended duration anti-VEGF therapies that have recently introduced, as well as those that are in various stages of development. 2. Vascular Endothelial Growth Factor (VEGF) and Pharmacokinetics {#sec2-pharmaceutics-10-00021} ============================================================================ VEGF was discovered independently by two research groups in 1989 and its important role in both angiogenesis and pathological neovascularization was realized almost immediately. VEGF is a group molecules that segregate into closely related families: VEGF-A, VEGF-B, VEGF-C, VEGF-F, and placental growth factor (PlGF) \[[@B7-pharmaceutics-10-00021]\]. Each of the families is characterized common, critical binding sequences, and most families multiple isoforms that share similar binding properties and biological actions. VEGF-A is eyes with chorioretinal vascular conditions, including nAMD, macular edema (DME), and retinal occlusion \[[@B3-pharmaceutics-10-00021]\], and is believed to play a central role in the development of conditions. Several in vivo models show VEGF-A promotes the growth of neovascular membranes \[[@B8-pharmaceutics-10-00021]\] and produces retinal vascular lesions that resemble DR \[[@B9-pharmaceutics-10-00021]\]. Evidence suggests that VEGF~165~ be the most biologically active isoform because of its high tissue concentrations and 10-fold potentiation of activity through its with the transmembrane co-receptor neuropilin-1 \[[@B10-pharmaceutics-10-00021]\]. Most VEGF inhibitory molecules block the binding region (amino 81--92) of VEGF-A isoforms, whereas pegaptanib interacts with the heparin binding region (amino acids 110--165) of VEGF~165~. Research suggests that VEGF-B, VEGF-C, VEGF-D, and PlGF may also contribute to pathologic ocular angiogenesis in humans but their relative contribution is not known \[[@B11-pharmaceutics-10-00021],[@B12-pharmaceutics-10-00021]\]. VEGF synthesis by vascular glia, pericytes, Müller cells, retinal pigment epithelium (RPE) cells, and leukocytes results from tissue ischemia and inflammation \[[@B15-pharmaceutics-10-00021],[@B16-pharmaceutics-10-00021]\]. Cells the retina and choroid respond to increased VEGF concentrations the primary targets are retinal and vascular endothelial cells VEGF-A has a short half-life of min in the eye and and homeostatic concentrations are low (approximately 9 ng/mL) \[[@B18-pharmaceutics-10-00021]\]. Some systemic conditions increase serum VEGF concentrations but chorioretinal vascular produce insufficient to meaningfully change serum levels. 3. Currently Available Therapies {#sec3-pharmaceutics-10-00021} ================================ Several anti-VEGF drugs have been developed exclusively for ocular use or, in the case of bevacizumab, are used off-label for chorioretinal vascular Peak clinical efficacies of these drugs (except pegaptanib) are similar and though product labels describe different injection intervals (monthly or every months) the differences their of action are on the order of only days. Currently available drugs, recently failed therapies, and drugs systems under development in [Table 1](#pharmaceutics-10-00021-t001){ref-type="table"}. 3.1. Pegaptanib {#sec3dot1-pharmaceutics-10-00021} --------------- Pegaptanib (molecular weight (MW) of 50 kDa), to VEGF, was the first ocular drug approved for intravitreal treatment of neovascular age-related macular degeneration (nAMD). Clinicians hoped that q6week treatment pegaptanib would best visual acuity (BCVA) but in most eyes it only decreased the rate of vision by approximately one half \[[@B19-pharmaceutics-10-00021]\]. Its use dropped significantly when more potent anti-VEGF drugs were introduced and pegaptanib is rarely used 3.2. Bevacizumab {#sec3dot2-pharmaceutics-10-00021} ---------------- Bevacizumab is a full-length, recombinant, humanized, antibody (MW of 149 kDa) that binds all isoforms of VEGF-A. It was developed and approved for the intravenous treatment of several advanced tumors (colorectal cell lung carcinoma, renal cell carcinoma, glioblastoma, and breast cancer, though this approval was rescinded in 2011) \[[@B20-pharmaceutics-10-00021]\]. Single injections of bevacizumab were first given to patients with nAMD and macular edema due to a central retinal vein occlusion (CRVO) in 2005 \[[@B39-pharmaceutics-10-00021],[@B40-pharmaceutics-10-00021]\], and within six months use of bevacizumab had become the accepted standard-of-care treatment of chorioretinal vascular conditions. Hundreds of ocular disease studies have established bevacizumab's efficacy and safety, though the best evidence comes from the Comparison Degeneration Trials (CATT) for nAMD and the Diabetic Retinopathy Clinical Research Network Protocol T trial for DME \[[@B6-pharmaceutics-10-00021],[@B21-pharmaceutics-10-00021]\]. The bevacizumab varies among countries due to regulatory reimbursement policies, and availability of safely compounded drug. Because physicians have accumulated extensive clinical experience with and are to acquire it inexpensively, bevacizumab remains the most commonly used anti-VEGF in the United States. 3.3. Ranibizumab ---------------- Ranibizumab is a recombinant, humanized, antibody (Fab with MW 48 kDa) that binds all of VEGF-A \[[@B4-pharmaceutics-10-00021]\]. It has been by the United States Food and Drug Administration (USFDA) for the treatment of nAMD (2006), DME, DR, macular edema due to vein occlusions, and choroidal neovascular membranes (CNVM) associated with high myopia \[[@B22-pharmaceutics-10-00021]\]. Following completion of the phase III MARINA and ANCHOR trials, ranibizumab was approved for the monthly treatment of nAMD and subsequently for PRN treatment. The CATT trial reported that PRN treatment non-inferior to treatment for nAMD \[[@B6-pharmaceutics-10-00021]\] though pooled data CATT and IVAN suggest that PRN is inferior to monthly injections. Ran | 1. iNtRODucTION {#sEc1-PHarmacEUTics-10-00021}
===============
tHE diSCOVeRY OF VaScuLaR eNDOthEliAL GROwTH FActor (Vegf) \[[@b1-pharmAcEUtiCS-10-00021],[@b2-PHArmaceUTicS-10-00021]\] And THe SUbseqUENT recogNItIoN OF Its CRItICal ROlE IN tHE PATHogEnesIs oF SEvEraL ChorIOreTinaL vaSCular cONditIONs cONstItUTe The moSt imPorTANT adVanCes In oPhTHalmoLOGY oVer tHE pAsT 30 YEARS. STronG EvideNce CORreLAtES The DeVelopMEnT of Both NEOvAscuLaRIZAtion aND MacUlar eDEMA In the TWo MOst CoMmoN CAuSeS Of bLINDNeSs in InDUStRiaLIzed NatIoNS---nEovasCULAR AgE-rElatED mACulaR DegeNErAtIoN (NAmd) aNd diABeTIc ReTinOpAtHY (DR)---WITH thE UpREGulAtioN oF VEGf \[[@B3-phARMaCEuTiCS-10-00021]\]. FuRTHErmORE, DiSEASE seveRitY FRequeNTLy cOrRelatEs WITh INtraOCUlAR vEgF coNCENTRAtiONS, THerEby MAkInG veGF A logicAl taRgET FoR TheRApEUTiC InteRvenTION.
SOON AFter VegF wAs dIScoVEred And SEQuEnCed, tHE PRODuCtIon Of inHIBItorY MoLECULeS begAN \[[@B4-phArmaCEuTIcS-10-00021]\]. tHuS FAr, fIVe VEgF-NEutRALizInG MoLEcULeS (pEgaptanib, macUGeN^®^, bauscH & lOmb, BriDgeWATer, nj, uSA; RAnIbiZumaB, LUcEnTiS^®^, GEnEntEcH, s. SaN fRAnciSco, Ca, usa/rocHE, bAseL, sWiTzeRLand; AFliBERcePT, eyleA^®^, ReGEnERON, TarrytOwn, Ny, uSA; coNbERCEPt, ChENgDu KAnGHONg PHARMaceUticAl GrOUP, ChEnGdU, China; aND BEVAcIZUMAB, aVAsTiN^®^, GENENtech, S. san frAnCiSco, cA, uSa/ROChE, baSEL, SWItzerLanD) havE BEEn uSED TO TreAT ophThALmoLOgIc CondItiONS, THoUgh only ThE FirSt THREe hAVE ReCEIved uNItEd sTAtEs FoOd aNd drug admiNIsTRATiOn (Us FDA) aPprOvAl fOr intrAOculAr uSe. inTrAviTReal thERaPY UsUAlLY BEGInS WiTh MontHly inJecTIOns (in ACcOrDAnCe wiTh PACkAge labELiNg) BUt most PhySicIaNs wilL ATTEMpT TO EXtenD The TiME betWeeN Injections As MuCH As PosSiBle wiTH EITHER moNthly *Pro re nata* (prN) or tREAt aNd ExtEnd STRaTEgIES \[[@b5-pharmACEutICS-10-00021]\]. trEATment INteRVAlS for MaNy PatIEnTS CanNOT BE eXTEndED BEyONd EiGhT wEeks \[[@b6-PhARmaCeUTICs-10-00021]\], reSUltInG iN A LARgE GROuP of pAtiEnts wHO rEQuiRE FrEquEnT iNJECtionS For LOng pERiOds OF TIMe. this LArGe nUmBEr oF INtraViTREAl INjeCtIonS BurdENS phYsiCIans ANd tHeIR sTAFfS, aNd cHalLenGes pATienTS' CoMpliANce. theReforE, nEw, LONGer aCTiNg anTi-VeGF mEdiCATIONS anD drug dEliVErY sYStEMS Are NEedED To iMprOve oUTComeS, OPTIMIZe cOmpLIANCE, anD redUcE THe totAL COSt of caRE.
ThIs MANuScripT dIsCUSSEs eXtenDEd dURatIoN AnTi-VeGF ThERAPIEs THaT HAve beeN RECENtLY inTRoDUCED, As WElL As THose thaT ARE In VARIous sTages of DEVeloPmEnt.
2. VAScULar eNdOTHELIaL GrOWTh fAcTor (vegf) phYsiologY anD PhaRMaCOKInETIcS {#seC2-pharmAceUTiCS-10-00021}
============================================================================
Vegf wAs disCOVEreD INdePENdENtly By TWO RESeaRCH gROupS iN 1989 \[[@b1-phaRMaCEuTiCS-10-00021],[@B2-PharMACEUTics-10-00021]\] and Its impOrtaNT Role in Both pHysiologIc ANgIOGEnesiS aND patHoLogiCaL nEOvaScULarizATION was reaLiZEd aLmOsT ImMEDIaTeLY. vEGf IS AcTUAllY A gRoUp of mOlecuLes thAT SegREGAtE iNTO SEVen ClOSELY RelaTEd FamILies: vEGf-a, vegf-B, VEgf-C, Vegf-d, vEGF-E, VEGF-f, And pLaCeNtAL gRoWtH FACtoR (plgf) \[[@B7-PharmACEuTIcs-10-00021]\]. eAcH of the FaMILIEs IS cHaRACTErized by commON, critiCAl bindiNg SEquENCeS, aNd mOst famiLiES cOntaIN MULTipLe iSoforms thAT sHARe siMilaR binDinG pRopERtiEs ANd bIOLOGicAl aCtions.
VegF-a SyNtHesis IS UPReGULATEd iN eyes WITH choriOretInAL VAscular coNdItioNs, InclUDINg NAmD, diAbetIc mACULAR eDema (dME), and REtiNAL vEIn ocCLusIoN (RVO) \[[@B3-PHarmACEuTics-10-00021]\], And iS beLievEd To PlAy A cENtrAL Role In THE DeVELOpmeNt OF These COnDITiONs. SEvERAL in VivO mOdELS sHOw thaT VeGF-a ProMoTES tHE GrowTH oF cHOroIdAl NeOVascuLAR memBrAnEs \[[@b8-phArmACEuTiCs-10-00021]\] AND PrODUCES reTinAl VaSCuLaR LEsIoNS ThAT reSEMble DR \[[@b9-pHARMacEUtics-10-00021]\]. eVIdENCE SuggEstS thaT VeGf~165~ MAY BE tHE mosT BiOLogIcALly AcTivE isoFORM beCauSe OF ItS HIgH TiSsuE cONCentRAtIOnS aND 10-FOlD POtEntIatiON Of ActiViTy ThroUgH ItS intERActION WIth tHe TraNsmeMBRANe Co-RECEPTOR nEuROPiliN-1 \[[@B10-pHaRMAcEUtiCs-10-00021]\]. mOSt vEGF INHIBItorY molECuLEs blocK ThE reCEPTor BinDInG rEGIOn (AminO ACIDS 81--92) Of vegf-a ISofOrMS, whErEAS peGApTaNib INTEraCTs wiTH tHe hepArIn BINdiNg RegION (AmiNO aCids 110--165) Of VEgf~165~. REseaRch suGgEStS THat vEgF-b, VeGF-C, Vegf-D, anD PLGf mAY aLSo ContRiButE TO PATholOGIC oCUlAR ANgIogEnEsis IN HUmanS bUt ThEir reLaTive conTRiBUtion IS noT knOWn \[[@b11-pHaRMacEutics-10-00021],[@B12-pHArmaceutiCS-10-00021]\].
iNcrEaSED VEGF syNtheSIs bY vASCUlaR EnDOtheLIal cELLS, glia, pEricYTes, mülleR cellS, REtINaL pigMENt EPItHElIuM (rPE) CELLS, AND InVAdING LEuKoCYTES \[[@b13-PhaRMAcEutics-10-00021],[@b14-phaRmAceutICs-10-00021]\] reSULtS FrOm tISSue iscHEmIA and InflaMmaTION \[[@b15-PHArmaceutIcS-10-00021],[@B16-PHArmacEUtIcS-10-00021]\]. Cells THRouGhoUT tHE rETinA and ChorOID rESPoND to INCreasEd Vegf ConCentRatIOns bUt The prIMaRY TArgeTs are RetINal anD ChoROiDAL vasCUlaR EndotheliAl cElLs \[[@B17-PharmaCeUTicS-10-00021]\].
VEgf-A hAS A SHOrT HALf-LiFe OF 30 MIn IN The eyE and SERUM, anD hoMEostaTiC CoNCenTRaTionS ARE geNerAlLy LoW (APPrOximAtEly 9 Ng/mL) \[[@B18-pHaRMACEutIcs-10-00021]\]. SOMe SYSteMIc COnditIONs INCreAsE SeRuM veGf cONCeNtrAtIonS BUt ChORiorETiNAL VAsCuLar cOnDItIoNs prODuce iNSufficieNt Vegf TO MeaNINgfullY ChAnGE sEruM lEVeLS.
3. cUrRentlY AvAIlaBLE therapiES {#seC3-PhArMACEUtics-10-00021}
================================
severAL Anti-Vegf dRugs hAvE bEeN DEvelOPEd ExCLuSivElY FOR OCulAR uSe oR, IN tHe caSE oF BeVaCIZumab, ArE usED oFF-LabEL For cHoRIOretInal VascuLar coNDitioNs. pEAk CLiniCal EfficaCIeS oF tHEse DRUgs (ExcEPt For PeGAPTaNiB) ArE siMIlar and thOugh PRODUcT Labels DeScrIBE dIFFERENT INJecTIoN iNtERvaLs (montHly OR EveRY tWO months) tHE diFfErenCeS In TheiR dURatiOn OF ACtion aRe on The OrDER OF ONlY DAys. cURReNTlY AVAIlabLE dRUGS, rEceNtLy faiLeD THERApIES, And DRUGS aND SystemS UNdER DEveLOPMEnt aRE LisTeD IN [TABLe 1](#phARMaCEUtICS-10-00021-t001){rEF-typE="tABLE"}.
3.1. PEGAPtanIb {#Sec3DOT1-pHArMaceuticS-10-00021}
---------------
PEgaptAniB (mOlECUlar WeiGHT (mw) of 50 KdA), AN AptaMER tO veGf, waS ThE FiRSt OculAr drUG aPPROVED foR THe intRavITReAL TREatmeNT OF nEoVaSCUlAr AgE-RElAted MacuLAr deGEneRATIon (Namd). CLInIcIAnS HopEd thaT q6WEEK trEaTmEnt WiTh PEgaptaNib wOULD imPRoVE beST cOrRectEd vIsUaL AcuIty (bcva) bUT in mosT EYEs It onLy dECrEASED ThE RatE of vISIoN lOSS by ApPRoxImatEly OnE hAlf \[[@B19-phArMAcEuTicS-10-00021]\]. ITS USE drOPPed sIGNifiCAnTLy wHeN more POTent Anti-vEGF dRUGs WERE inTRoDuCed anD pEgapTanIb IS raRELy uSed TOday.
3.2. beVaCIzUmAB {#sec3DoT2-PHarMacEuTicS-10-00021}
----------------
BeVAciZUMab iS a FULL-LEnGTH, recombINaNT, hUManIZeD, moNOclonal ANTiboDy (mw oF 149 kDa) thaT biNDs aLL isofOrMs Of vegF-a. IT wAS deVelopEd aND APproVEd For ThE iNtraVenous TrEatMEnT oF SEVERaL AdvaNcEd SOliD tUMOrs (cOlOrEcTaL CaRCinoma, NON-smALL cELL lUnG caRcInoma, rEnal CeLL CARcinoma, gLIObLAStOmA, AnD brEASt caNceR, ThoUGH thIs appRovAl wAS resCIndEd In 2011) \[[@b20-pHArMAceUTiCS-10-00021]\].
sINgLE iNjEctiOnS Of bevAciZUMab WEre fiRsT gIVEn to pATIENtS WITH NAMD anD mACular eDema duE To a CEntral REtinAl VEIN OCcLUsIOn (cRvo) in 2005 \[[@B39-PhArmaCeUTIcS-10-00021],[@B40-pHaRMAcEutiCS-10-00021]\], And wiTHin SIX mOnThs ofF-lABel Use OF bevACizuMaB haD BeComE thE ACcePteD STanDArD-of-CARE TReAtMeNT Of choRioRetiNaL VAScUlAr CONditioNS. HundReds oF OCuLar diSEaSE StuDIeS Have esTAbLisHeD BEVaciZumaB's EFficACy And Safety, thOugh tHe BEst EvIdEnce coMes from THE COMpAriSoN OF agE-reLaTEd maCULaR DEGENeraTion trEAtmeNt trialS (cAtt) for naMd aNd tHE dIabEtiC reTinoPathy cLiNiCaL rESeArCh NEtWork PrOtocol t triAl fOr dMe \[[@B6-PhARmACeuTIcS-10-00021],[@b21-PHARmAceuTICs-10-00021]\].
THe USe Of BevacIZumaB vArIes AMOng COUntRIes DuE To ReGuLAtoRy ReSTrictions, reIMBuRsEMENT poliCiEs, and availaBilIty OF SafElY coMPoUNded DruG. beCauSE PhysIcIaNS havE AcCumuLaTED ExtenSivE clINicAl EXPerIENcE with BEvacIzumAB and aRE abLE TO aCqUIRe iT INEXpENSiVeLY, BEvaCIZUmAB rEmAINs tHe Most COmMONlY uSEd anTI-VeGf DruG IN ThE uniTeD sTatEs.
3.3. rAnIBizuMAb {#SeC3dOT3-pHArMACEuticS-10-00021}
----------------
rAnibIZUMAB Is a REcOMBINANt, HUmanIZEd, MONOcLONAL aNtiboDy FrAgMenT (FAB WItH mw OF 48 kda) tHAt bInds alL iSOfORmS oF vEGf-a \[[@B4-PhaRmAceUTics-10-00021]\]. it hAs BEen apprOVED BY THe UnIteD StAtEs FOoD anD Drug aDMInIStratiON (uSfDa) FOr the tREatmenT oF namd (2006), DMe, dR, maCular EDeMa dUe tO vEin OcclusIoNs, And cHOROIdal NeOvasCular MembRanES (cNvm) aSsocIATeD WitH High MyopIA \[[@b22-pHarMaceUticS-10-00021]\].
fOLloWIng coMplETIOn oF THE Phase iIi maRINa And ANchoR TrIaLS, ranIbIZUmAB Was aPProVed FoR ThE MontHLy treAtMEnt oF Namd aNd SUBseQuENtly fOR PRN tREATmeNt. tHe CATt TrIal RepOrtEd THaT PrN TreATmenT is Non-iNferiOr TO MoNtHly TREAtmENt For NaMD \[[@B6-phaRmACeuTIcs-10-00021]\] THOUGH PoolED daTA frOm caTt anD IVaN sUgGESt thAT PRn is InferiOR To mONthLY InjeCtIonS. RaN | 1. Introduction {#sec1-pharmaceutics-10-00021} =============== The discovery of vascular endothelial growth factor (VEGF) \[[@B1-pharmaceutics-10-00021],[@B2-pharmaceutics-10-00021]\] and the subsequent recognition of its critical role in the pathogenesis of several chorioretinal vascular conditions constitute the most important advances inophthalmology over the past30 years. Strong evidence correlatesthe development of both neovascularization andmacular edema in the twomost common causesof blindness in industrialized nations---neovascularage-related macular degeneration (nAMD) and diabetic retinopathy (DR)---with the upregulation of VEGF \[[@B3-pharmaceutics-10-00021]\]. Furthermore, diseaseseverityfrequentlycorrelates with intraocular VEGF concentrations, thereby makingVEGF a logicaltarget for therapeutic intervention. Soon after VEGF was discovered and sequenced, the productionof inhibitory molecules began \[[@B4-pharmaceutics-10-00021]\]. Thus far, five VEGF-neutralizing molecules (pegaptanib, Macugen^®^,Bausch & Lomb, Bridgewater,NJ, USA; ranibizumab, Lucentis^®^, Genentech, S. San Francisco,CA, USA/Roche, Basel, Switzerland; aflibercept,Eylea^®^, Regeneron, Tarrytown, NY, USA; conbercept, ChengduKanghong Pharmaceutical Group, Chengdu, China; and bevacizumab, Avastin^®^,Genentech, S. San Francisco, CA, USA/Roche, Basel, Switzerland) have been used to treatophthalmologic conditions, though only the first three have received United States Food andDrug Administration (US FDA)approval for intraocular use. Intravitreal therapy usuallybeginswith monthlyinjections (inaccordance withpackage labeling) but most physicians willattempt to extend the time between injections as much as possible with either monthly *pro re nata* (PRN) or treat andextend strategies\[[@B5-pharmaceutics-10-00021]\]. Treatment intervalsfor many patients cannot be extended beyond eight weeks \[[@B6-pharmaceutics-10-00021]\], resulting in a large group of patientswho require frequent injections for long periods oftime. This large numberof intravitreal injections burdensphysicians and their staffs, and challenges patients'compliance. Therefore, new, longeracting anti-VEGFmedications and drug delivery systemsare needed to improve outcomes, optimize compliance,and reduce the total costofcare. This manuscript discussesextendeddurationanti-VEGF therapies that have been recently introduced, aswell asthose that are in various stages of development. 2. Vascular Endothelial Growth Factor (VEGF) Physiology and Pharmacokinetics {#sec2-pharmaceutics-10-00021} ============================================================================ VEGF was discovered independently by two researchgroups in 1989 \[[@B1-pharmaceutics-10-00021],[@B2-pharmaceutics-10-00021]\] and its important role in both physiologic angiogenesis and pathologicalneovascularizationwas realized almost immediately. VEGF isactually a group of molecules that segregate into seven closely related families: VEGF-A,VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placental growth factor (PlGF) \[[@B7-pharmaceutics-10-00021]\]. Each of the families is characterized by common, critical binding sequences, and most families contain multiple isoformsthat share similar binding propertiesand biological actions. VEGF-A synthesis isupregulated ineyes withchorioretinal vascularconditions, includingnAMD,diabetic macular edema (DME), and retinal veinocclusion (RVO) \[[@B3-pharmaceutics-10-00021]\], andis believed toplay a central role in the development of these conditions. Several in vivo models show that VEGF-A promotes the growth of choroidal neovascular membranes \[[@B8-pharmaceutics-10-00021]\] and produces retinal vascular lesionsthat resemble DR\[[@B9-pharmaceutics-10-00021]\]. Evidence suggests that VEGF~165~ may be the most biologically active isoform because ofits high tissue concentrations and 10-foldpotentiation of activity throughits interactionwith thetransmembrane co-receptor neuropilin-1 \[[@B10-pharmaceutics-10-00021]\]. Most VEGF inhibitory molecules block the receptor binding region (amino acids 81--92) of VEGF-Aisoforms, whereaspegaptanib interacts with the heparin binding region(amino acids 110--165) of VEGF~165~. Research suggests that VEGF-B, VEGF-C,VEGF-D,and PlGF may alsocontributeto pathologicocular angiogenesis in humansbut theirrelative contribution isnot known \[[@B11-pharmaceutics-10-00021],[@B12-pharmaceutics-10-00021]\]. Increased VEGF synthesis by vascular endothelial cells, glia, pericytes, Müller cells, retinal pigment epithelium(RPE)cells, and invading leukocytes \[[@B13-pharmaceutics-10-00021],[@B14-pharmaceutics-10-00021]\] results from tissue ischemia and inflammation \[[@B15-pharmaceutics-10-00021],[@B16-pharmaceutics-10-00021]\]. Cells throughout the retina and choroid respond to increased VEGF concentrations but the primary targets are retinal and choroidal vascular endothelial cells \[[@B17-pharmaceutics-10-00021]\]. VEGF-A has a short half-life of 30min inthe eye and serum, and homeostatic concentrations are generally low (approximately9 ng/mL)\[[@B18-pharmaceutics-10-00021]\]. Some systemicconditions increase serum VEGF concentrations but chorioretinal vascular conditions produce insufficient VEGF to meaningfully change serum levels. 3. Currently Available Therapies {#sec3-pharmaceutics-10-00021} ================================ Several anti-VEGF drugs have been developed exclusivelyfor ocular use or, in thecase of bevacizumab, are used off-label for chorioretinal vascular conditions. Peak clinical efficacies of these drugs (except for pegaptanib) aresimilar and though product labels describe differentinjectionintervals (monthly or every two months) the differences in their duration of action are on the order of only days. Currently available drugs, recently failed therapies,and drugs and systems under development are listed in [Table 1](#pharmaceutics-10-00021-t001){ref-type="table"}. 3.1. Pegaptanib {#sec3dot1-pharmaceutics-10-00021} --------------- Pegaptanib(molecular weight (MW) of 50 kDa), an aptamer to VEGF, was thefirst ocular drug approvedfor theintravitreal treatment of neovascular age-related maculardegeneration (nAMD).Clinicians hoped that q6week treatmentwith pegaptanib would improve best corrected visual acuity (BCVA) but in most eyes itonly decreased therate of vision loss by approximatelyonehalf \[[@B19-pharmaceutics-10-00021]\]. Its use dropped significantly when more potent anti-VEGF drugs were introduced and pegaptanib is rarely used today. 3.2.Bevacizumab {#sec3dot2-pharmaceutics-10-00021}---------------- Bevacizumab is a full-length,recombinant,humanized, monoclonal antibody (MW of 149 kDa) that binds all isoforms of VEGF-A. It was developed and approved for the intravenous treatment ofseveral advanced solid tumors (colorectal carcinoma, non-small cell lung carcinoma, renal cell carcinoma, glioblastoma, and breast cancer, though this approval was rescinded in 2011) \[[@B20-pharmaceutics-10-00021]\]. Single injections of bevacizumab were firstgiven to patientswithnAMDand macular edema due to a central retinal vein occlusion (CRVO) in 2005 \[[@B39-pharmaceutics-10-00021],[@B40-pharmaceutics-10-00021]\], and within six months off-labeluse of bevacizumab had become the accepted standard-of-care treatment of chorioretinalvascularconditions. Hundreds of ocular disease studies have established bevacizumab's efficacy and safety, though the bestevidence comes from the Comparison of Age-related Macular Degeneration Treatment Trials (CATT) for nAMD andthe Diabetic Retinopathy Clinical Research Network Protocol T trialfor DME \[[@B6-pharmaceutics-10-00021],[@B21-pharmaceutics-10-00021]\].The use ofbevacizumabvaries among countries due to regulatory restrictions, reimbursement policies, and availabilityofsafely compounded drug. Becausephysicians have accumulated extensive clinical experience with bevacizumab andareable to acquire itinexpensively, bevacizumab remains the mostcommonly used anti-VEGF drug in the United States. 3.3. Ranibizumab {#sec3dot3-pharmaceutics-10-00021} ---------------- Ranibizumabis a recombinant, humanized, monoclonal antibody fragment (Fab with MW of 48 kDa) that bindsall isoforms of VEGF-A \[[@B4-pharmaceutics-10-00021]\]. Ithas been approved by the United States Food and Drug Administration (USFDA) for the treatment of nAMD (2006),DME,DR, macular edema due to vein occlusions, and choroidal neovascular membranes (CNVM) associated with high myopia \[[@B22-pharmaceutics-10-00021]\]. Following completion of the phase III MARINA and ANCHOR trials, ranibizumabwas approved for themonthly treatment of nAMD and subsequently for PRN treatment. The CATTtrial reported that PRN treatment is non-inferior to monthly treatmentfor nAMD \[[@B6-pharmaceutics-10-00021]\]though pooled data from CATT and IVAN suggest that PRN isinferior to monthly injections. Ran | 1. Introduction {#sec1-pharmaceutics-10-00021} =============== The discovery _of_ vascular endothelial _growth_ _factor_ (VEGF) \[[@B1-pharmaceutics-10-00021],[@B2-pharmaceutics-10-00021]\] and the subsequent recognition of its _critical_ _role_ _in_ the _pathogenesis_ of several chorioretinal vascular conditions constitute the most important advances _in_ ophthalmology over the past 30 years. Strong evidence _correlates_ the development _of_ both neovascularization and macular edema in _the_ two most common causes of _blindness_ in industrialized nations---neovascular age-related macular degeneration (nAMD) and _diabetic_ retinopathy (DR)---with _the_ _upregulation_ of _VEGF_ \[[@B3-pharmaceutics-10-00021]\]. Furthermore, disease _severity_ frequently correlates with intraocular VEGF concentrations, thereby making VEGF a logical target for therapeutic intervention. Soon after VEGF was discovered and sequenced, the production _of_ inhibitory _molecules_ began _\[[@B4-pharmaceutics-10-00021]\]._ Thus far, five VEGF-neutralizing molecules _(pegaptanib,_ Macugen^®^, Bausch _&_ Lomb, _Bridgewater,_ NJ, USA; ranibizumab, Lucentis^®^, Genentech, S. _San_ Francisco, CA, USA/Roche, Basel, Switzerland; aflibercept, _Eylea^®^,_ Regeneron, Tarrytown, NY, _USA;_ _conbercept,_ Chengdu Kanghong _Pharmaceutical_ _Group,_ _Chengdu,_ _China;_ and _bevacizumab,_ Avastin^®^, Genentech, S. San Francisco, _CA,_ USA/Roche, Basel, _Switzerland)_ have been used _to_ treat ophthalmologic conditions, _though_ _only_ the first three _have_ received United States Food _and_ Drug Administration (US FDA) _approval_ _for_ _intraocular_ use. Intravitreal therapy usually begins with monthly injections _(in_ _accordance_ _with_ _package_ labeling) _but_ most physicians will _attempt_ to extend the time between injections as much _as_ _possible_ with either monthly *pro re _nata*_ _(PRN)_ or treat and extend strategies \[[@B5-pharmaceutics-10-00021]\]. Treatment intervals for many patients cannot be extended beyond eight _weeks_ _\[[@B6-pharmaceutics-10-00021]\],_ resulting _in_ a large _group_ of patients who require frequent injections for long periods of time. This _large_ number of _intravitreal_ injections burdens physicians _and_ their staffs, _and_ _challenges_ _patients'_ compliance. Therefore, new, longer acting anti-VEGF medications _and_ drug delivery systems are needed to improve outcomes, optimize compliance, _and_ _reduce_ the total cost _of_ care. This manuscript discusses extended _duration_ anti-VEGF therapies that have been recently introduced, as well _as_ _those_ _that_ are in various stages _of_ development. 2. _Vascular_ _Endothelial_ Growth Factor _(VEGF)_ _Physiology_ and Pharmacokinetics {#sec2-pharmaceutics-10-00021} ============================================================================ VEGF was discovered _independently_ _by_ two _research_ groups in 1989 \[[@B1-pharmaceutics-10-00021],[@B2-pharmaceutics-10-00021]\] and its _important_ role _in_ both _physiologic_ angiogenesis _and_ _pathological_ neovascularization _was_ realized almost immediately. VEGF is _actually_ a group of molecules that segregate into seven _closely_ related families: _VEGF-A,_ VEGF-B, VEGF-C, _VEGF-D,_ VEGF-E, _VEGF-F,_ and placental growth factor (PlGF) _\[[@B7-pharmaceutics-10-00021]\]._ Each of the families is characterized by common, _critical_ binding _sequences,_ _and_ most _families_ contain multiple isoforms that share similar binding properties and biological actions. _VEGF-A_ synthesis _is_ upregulated in eyes _with_ chorioretinal vascular conditions, _including_ nAMD, diabetic macular edema (DME), and retinal vein occlusion (RVO) \[[@B3-pharmaceutics-10-00021]\], _and_ _is_ believed _to_ _play_ _a_ central _role_ in the development of _these_ conditions. _Several_ in vivo models show that VEGF-A promotes the growth of choroidal _neovascular_ membranes _\[[@B8-pharmaceutics-10-00021]\]_ and produces _retinal_ vascular lesions _that_ resemble DR \[[@B9-pharmaceutics-10-00021]\]. Evidence suggests that VEGF~165~ _may_ be _the_ most biologically active isoform because _of_ _its_ high tissue concentrations and 10-fold potentiation _of_ activity through its interaction with the _transmembrane_ co-receptor neuropilin-1 \[[@B10-pharmaceutics-10-00021]\]. Most VEGF inhibitory molecules block the _receptor_ _binding_ region (amino acids 81--92) of VEGF-A isoforms, whereas pegaptanib interacts with _the_ _heparin_ binding region (amino acids 110--165) of VEGF~165~. Research suggests that VEGF-B, VEGF-C, VEGF-D, and PlGF _may_ also _contribute_ to pathologic ocular angiogenesis in humans but their relative contribution is not _known_ \[[@B11-pharmaceutics-10-00021],[@B12-pharmaceutics-10-00021]\]. Increased VEGF synthesis _by_ vascular endothelial cells, glia, _pericytes,_ Müller cells, retinal pigment epithelium (RPE) cells, and invading _leukocytes_ \[[@B13-pharmaceutics-10-00021],[@B14-pharmaceutics-10-00021]\] results from tissue ischemia and _inflammation_ \[[@B15-pharmaceutics-10-00021],[@B16-pharmaceutics-10-00021]\]. Cells _throughout_ the retina and _choroid_ _respond_ to increased VEGF concentrations but the primary targets are _retinal_ and choroidal _vascular_ _endothelial_ _cells_ \[[@B17-pharmaceutics-10-00021]\]. _VEGF-A_ has a _short_ half-life of 30 min in the eye and _serum,_ and homeostatic concentrations _are_ generally _low_ (approximately 9 ng/mL) \[[@B18-pharmaceutics-10-00021]\]. Some systemic conditions increase serum VEGF concentrations but _chorioretinal_ vascular conditions produce insufficient VEGF to meaningfully change serum levels. 3. Currently Available Therapies _{#sec3-pharmaceutics-10-00021}_ _================================_ _Several_ _anti-VEGF_ drugs have been _developed_ exclusively for ocular use _or,_ in the case _of_ bevacizumab, _are_ used off-label for _chorioretinal_ vascular conditions. Peak clinical efficacies of these drugs (except _for_ pegaptanib) _are_ similar and though product labels describe different injection intervals (monthly or every two months) the _differences_ _in_ their duration of action are on the order _of_ only days. Currently available drugs, recently failed therapies, and drugs and systems _under_ development are _listed_ in [Table 1](#pharmaceutics-10-00021-t001){ref-type="table"}. 3.1. Pegaptanib {#sec3dot1-pharmaceutics-10-00021} --------------- Pegaptanib (molecular weight (MW) of 50 kDa), an aptamer to VEGF, was the first ocular drug approved for _the_ _intravitreal_ treatment _of_ neovascular age-related macular degeneration _(nAMD)._ _Clinicians_ hoped _that_ _q6week_ treatment with pegaptanib would improve best corrected visual acuity (BCVA) but in _most_ eyes _it_ only decreased the rate of vision loss _by_ approximately one half \[[@B19-pharmaceutics-10-00021]\]. Its use _dropped_ _significantly_ when more _potent_ _anti-VEGF_ drugs _were_ introduced and pegaptanib _is_ rarely used _today._ 3.2. Bevacizumab _{#sec3dot2-pharmaceutics-10-00021}_ _----------------_ Bevacizumab _is_ a _full-length,_ recombinant, humanized, monoclonal antibody _(MW_ of _149_ _kDa)_ that binds all isoforms of VEGF-A. It was developed _and_ approved _for_ the intravenous treatment of several advanced solid tumors (colorectal carcinoma, non-small cell lung carcinoma, renal _cell_ carcinoma, _glioblastoma,_ _and_ breast cancer, though this approval was rescinded in _2011)_ \[[@B20-pharmaceutics-10-00021]\]. Single injections of bevacizumab were first given to patients _with_ nAMD and macular edema _due_ to a _central_ retinal vein occlusion _(CRVO)_ in 2005 \[[@B39-pharmaceutics-10-00021],[@B40-pharmaceutics-10-00021]\], and within six months off-label _use_ of bevacizumab had become the accepted standard-of-care treatment of chorioretinal vascular conditions. Hundreds of _ocular_ disease studies have _established_ bevacizumab's efficacy _and_ safety, _though_ the best _evidence_ _comes_ from the Comparison of Age-related Macular Degeneration _Treatment_ Trials (CATT) for nAMD and the Diabetic Retinopathy Clinical Research Network _Protocol_ T trial for DME \[[@B6-pharmaceutics-10-00021],[@B21-pharmaceutics-10-00021]\]. The use _of_ bevacizumab varies among countries due to regulatory restrictions, reimbursement policies, and _availability_ of safely _compounded_ drug. Because physicians have accumulated extensive clinical _experience_ with bevacizumab and are _able_ to acquire _it_ inexpensively, bevacizumab remains the most commonly used anti-VEGF drug in the United _States._ 3.3. _Ranibizumab_ {#sec3dot3-pharmaceutics-10-00021} ---------------- Ranibizumab is a _recombinant,_ humanized, monoclonal antibody fragment (Fab _with_ MW of _48_ kDa) _that_ binds _all_ isoforms of VEGF-A \[[@B4-pharmaceutics-10-00021]\]. It has been approved by the United States Food and Drug Administration (USFDA) for the treatment _of_ nAMD (2006), DME, DR, macular _edema_ due to _vein_ occlusions, and choroidal _neovascular_ membranes (CNVM) associated _with_ high _myopia_ \[[@B22-pharmaceutics-10-00021]\]. _Following_ _completion_ of the phase III MARINA and ANCHOR trials, _ranibizumab_ was approved _for_ the monthly _treatment_ of nAMD and subsequently for PRN _treatment._ The CATT trial reported _that_ PRN treatment _is_ non-inferior to _monthly_ treatment for nAMD \[[@B6-pharmaceutics-10-00021]\] though pooled data from CATT and IVAN suggest that PRN is inferior to monthly injections. _Ran_ |
1. Introduction {#sec1-materials-13-03111}
===============
The efficiency, stability, and lifetime of any device is strongly dependent on the stability of compounds and alloys that a given device is made of. The attempts to understand the particular state of matter are crucial points of understanding its implications for application in advanced electronic devices. This gain in importance when exotic states of matter are considered. Certain exotic behavior, namely the insulating character of the bulk and metallic character of the surface, was observed in a large group of materials, which were later classified as topological insulators \[[@B1-materials-13-03111],[@B2-materials-13-03111]\]. Among classified topological insulators the Bi~2~Te~3~ compound, considered as good material for thermoelectric \[[@B3-materials-13-03111],[@B4-materials-13-03111]\] and optoelectronic \[[@B5-materials-13-03111],[@B6-materials-13-03111]\] applications, was selected as the subject of this study. Topological insulators (TIs) are a novel type of quantum materials of which the bulk part is a band insulator whereas the surface always exhibits the presence of electronic states carrying electric current \[[@B2-materials-13-03111],[@B7-materials-13-03111]\]. This phase shows a clear bulk energy gap separation like any ordinary insulator or semiconductor, but the difference is in the presence of the gapless edge states (2D) or surface states (3D) which are protected by time-reversal symmetry. The unusual nature of the TI surface states resulted in the widespread interest in the fabrication and characterization of this class of materials. The nature of the TI class of materials resulting from its unique spin nature is gaining in importance when one realizes that TIs properties may entail generation of quasiparticles and electronic states which are not accessible in classic condensed-matter systems. Not surprisingly the topological insulators are considered as promising materials for multiple applications in next generation electronic or spintronic devices \[[@B8-materials-13-03111],[@B9-materials-13-03111]\] as well as for applications in energy conversion such as thermoelectrics \[[@B10-materials-13-03111],[@B11-materials-13-03111]\].
It is well known that defects, strain, and doping influence are important features of the TIs. Coulomb, magnetic, or disorder perturbations can modify the surface states \[[@B12-materials-13-03111],[@B13-materials-13-03111],[@B14-materials-13-03111]\]. Elemental doping and alloying in the TI materials allows for the control of the Fermi level via population with the n-type or p-type carriers which is also a crucial step towards the future junctions of topological insulators that associate two doped TI layers \[[@B15-materials-13-03111],[@B16-materials-13-03111]\]. Among doped TIs, special interest is placed on magnetic dopants, mostly due to the possible interplay between topological magnetic orders, which may lead to the implementation of exotic quantum phenomena, such as the magnetoelectric effect \[[@B17-materials-13-03111]\] and quantum anomalous Hall effect (QAHE) \[[@B18-materials-13-03111],[@B19-materials-13-03111]\], and later on the exploration of its potential device applications. Recent research is focused mostly on TI's electronic structure and their thermoelectric properties and include structures containing transition metals dopants like V, Mn, Cr, Fe, and Cu \[[@B20-materials-13-03111],[@B21-materials-13-03111],[@B22-materials-13-03111],[@B23-materials-13-03111],[@B24-materials-13-03111],[@B25-materials-13-03111]\], and rare earth dopants Gd, Ce, Y, Sm, Dy, and Eu \[[@B8-materials-13-03111],[@B9-materials-13-03111],[@B10-materials-13-03111]\]. Mn and Fe doping of the Bi~2~Te~3~ causes the opening of a small gap at the Dirac point in the surface Dirac cone \[[@B22-materials-13-03111]\]. Further, with the formation of ferromagnetic state only at the surface, a state which leads to opening the gap, has been confirmed for the Mn doped Bi~2~(Te,Se)~3~ as well as for the Cr-doped (Bi,Sb)~2~Te~3~ thin films \[[@B26-materials-13-03111]\]. Bi~2~Te~3~ doped with rare earth is discussed mostly from the point of view of modifying the thermoelectric properties \[[@B27-materials-13-03111],[@B28-materials-13-03111],[@B29-materials-13-03111],[@B30-materials-13-03111],[@B31-materials-13-03111],[@B32-materials-13-03111],[@B33-materials-13-03111]\]. Controlling the distribution of magnetic dopants within the TI's matrix resulting from sample preparation procedures and chemical potentials of the constituent atoms is still challenging and requires in-depth and careful characterizations at the atomic level. This is especially the case when considering nanoscale systems, where already a controlled fabrication, quality control, as well as subsequent preservation of the completed structure or even device requires specific attention.
In our previous paper \[[@B34-materials-13-03111]\], we have studied the multilayered structures of Bi~2~Te~3~ and europium and shown that the interface between the layers is not stable and the reaction that took place at room temperature led to the decomposition of the originally layered structure and formation of new phases. This observation led us to focus on the electronic and crystallographic structures of a simplified structure where europium is doped in the Bi~2~Te~3~ thin film. Europium by itself is quite chemically active, and possesses interesting magnetic properties related to its valence state. It is worth noting that it is reasonable, at a first glance, to assume that europium atoms will partially substitute the bismuth atoms in the Bi~2~Te~3~ matrix as it happens for the Bi~2~Te~3~ doped with Gd \[[@B35-materials-13-03111],[@B36-materials-13-03111]\]. Recently, the homogeneous incorporation of Eu^2+^ into the Bi sites of the Bi~2~Te~3~ lattice was observed \[[@B37-materials-13-03111]\]. Our point of interest was strongly aimed at the electronic structure of the obtained film, especially on the valence state of europium. Europium in alloys and compounds occurs in 2+ and 3+ valence states. Eu^3+^ is non-magnetic (J = 0) while the Eu^2+^, observed for pure Eu and europium in intermetallic alloys and compounds \[[@B38-materials-13-03111],[@B39-materials-13-03111]\], has a large pure spin moment (J = 7/2). A mixed valence state has also been found in many intermetallic compounds. In the presented studies, the response of thin film of Bi~2~Te~3~ to the disorder introduced by Eu dopant is discussed. The studies were performed on thin films with an unprecedented combination of techniques such as photoemission spectroscopy, TOF-SIMS, RHEED, femtosecond spectroscopy, and LC-AFM.
2. Materials and Methods {#sec2-materials-13-03111}
========================
2.1. Materials {#sec2dot1-materials-13-03111}
--------------
The molecular beam epitaxy system was used for the growth of two thin films of Bi~2~Te~3~. The films were grown in accordance with the procedure we developed earlier \[[@B40-materials-13-03111]\] for the Bi~2~Te~3~ monocrystalline films. Freshly cleaved (110) 0.15--0.17 mm thick Muscovite mica substrates (Ted Pella, Inc., Mica grade V1) was used as a substrate for the Bi~2~Te~3~ films. In order to prevent the problem of the sample surface charging resulting from the used substrate, the mica surface was ground using silver paste. The mica was outgassed at 300 °C for 1.5 h under UVH conditions (5 × 10^−9^ mbar) and kept at 120 °C during the deposition of the Bi~2~Te~3~ films. High-purity Bi (99.999% Aldrich Chem. Co., Milwaukee, WI, USA), spectrographically standardized Te ingot (Johnson Matthey Chemicals, London, UK), and europium were evaporated by conventional effusion Knudsen cells (K-cell). The growth rate of each element, controlled by quartz microbalance, was customized to obtain assumed stoichiometry. The total growth rate of the films was about 0.2 QL min^−1^. In order to obtain a uniform distribution of deposited elements, the films were grown in the co-deposition mode. For the europium doped Bi~2~Te~3~ film, the growth rates of elements were adjusted as if Eu atoms were to substitute for bismuth in the Bi~2~Te~3~ crystal lattice. Assumed level of europium dopants was set at the level of about 2--3%. After the deposition process the films were annealed at 100 °C for 18 h. During the deposition process, the vacuum was of about 4 × 10^−9^ mbar for the undoped film and about 9 × 10^−9^ mbar for Bi~ | 1. introduction { # sec1 - materials - 13 - 03111 } = = = = = = = = = = = = = = = the efficiency, stability, and lifetime of any device is strongly dependent on the stability of compounds and alloys that a given device is made of. the attempts to understand the particular state of matter are crucial points of understanding its implications for application in purely electronic devices. this gain in importance when exotic states of matter are considered. certain exotic behavior, namely the insulating character of the bulk and metallic character of the surface, was observed in a large group of materials, which were later classified as topological compounds \ [ [ @ b1 - materials - 13 - 03111 ], [ @ b2 - materials - 13 - 03111 ] \ ]. among classified topological insulators the bi ~ 2 ~ te ~ 3 ~ compound, considered exceptionally good material for thermoelectric \ [ [ @ b3 - materials - 13 - 03111 ], [ @ b4 - thirteen - 13 - 03111 ] \ ] and optoelectronic \ [ [ @ b5 - elements - 13 - 03111 ], [ @ b6 - materials - 13 - 03111 ] \ ] applications, was selected as specific subject of this study. topological insulators ( tis ) are a novel type of quantum materials of which the bulk part is a band insulator whereas the surface always exhibits the presence of electronic states carrying electric current \ [ [ @ b2 - materials - 13 - 03111 ], [ @ b7 - materials - 13 - 03111 ] \ ]. this phase shows a clear bulk energy gap separation like any ordinary insulator or semiconductor, but the difference is in the properties of the gapless edge states ( 2d ) or surface states ( cds ) which are protected by time - reversal symmetry. the unusual nature of the ti surface states resulted in the widespread interest in the fabrication and characterization of this class of materials. the nature of the ti class of materials resulting from its unique spin nature is gaining in importance when one realizes why tis properties may entail generation of quasiparticles and electronic states which are not accessible in classic nano - matter systems. not surprisingly the topological insulators are considered as promising materials for multiple applications in next generation electronic or spintronic devices \ [ [ @ b8 - materials - 13 - 03111 ], [ @ b9 - materials - 13 - 03111 ] \ ] as well as for applications in energy conversion such as thermoelectrics \ [ [ @ b10 - materials - 13 - 03111 ], [ @ b11 - materials - 13 - 03111 ] \ ]. it is well known that defects, strain, and doping influence are important features of the tis. coulomb, magnetic, or disorder perturbations can modify the surface states \ [ [ @ b12 - materials - 13 - 03111 ], [ @ b13 - materials - 13 - 03111 ], [ @ b14 - materials - 13 - 03111 ] \ ]. elemental doping and alloying in the ti materials allows for the control of the fermi level via population with the n - type or p - type carriers which is also a crucial step towards the future junctions of topological insulators that associate two doped ti layers \ [ [ @ b15 - materials - 13 - 03111 ], [ @ b16 - materials - 13 - 03111 ] \ ]. among doped tis, special interest is placed on magnetic dopants, mostly due to the possible interplay between topological magnetic orders, which may lead to the implementation of exotic quantum phenomena, such as the magnetoelectric effect \ [ [ @ b17 - materials - 13 - 03111 ] \ ] and quantum anomalous hall effect ( qahe ) \ [ [ @ b18 - materials - 13 - 03111 ], [ @ b19 - materials - 13 - 03111 ] \ ], and later on the exploration of its potential device applications. recent research is focused mostly on ti ' s electronic structure and their thermoelectric properties and include structures containing transition metals dopants like v, mn, cr, fe, and cu \ [ [ @ b20 - materials - 13 - 03111 ], [ @ b21 - materials - 13 - 03111 ], [ @ b22 - materials - 13 - 03111 ], [ @ b23 - materials - 13 - 03111 ], [ @ b24 - materials - 13 - 03111 ], [ @ b25 - materials - 13 - 03111 ] \ ], and rare earth dopants gd, ce, y, sm, dy, and eu \ [ [ @ b8 - materials - 13 - 03111 ], [ @ b9 - materials - 13 - 03111 ], [ @ b10 - materials - 13 - 03111 ] \ ]. mn and fe doping of the bi ~ 2 ~ te ~ 3 ~ causes the opening of a small gap at the dirac point in the surface dirac cone \ [ [ @ b22 - materials - 13 - 03111 ] \ ]. further, with the formation of ferromagnetic state only at the surface, a state which leads to opening the gap, has been confirmed for the mn doped bi ~ 2 ~ ( te, se ) ~ 3 ~ as well as for the cr - doped ( bi, sb ) ~ 2 ~ te ~ 3 ~ thin films \ [ [ @ b26 - materials - 13 - 03111 ] \ ]. bi ~ 2 ~ te ~ 3 ~ doped with rare earth is discussed mostly from the point of view of modifying the thermoelectric properties \ [ [ @ b27 - materials - 13 - 03111 ], [ @ b28 - materials - 13 - 03111 ], [ @ b29 - materials - 13 - 03111 ], [ @ b30 - materials - 13 - 03111 ], [ @ b31 - materials - 13 - 03111 ], [ @ b32 - materials - 13 - 03111 ], [ @ b33 - materials - 13 - 03111 ] \ ]. controlling the distribution of magnetic dopants within the ti ' s matrix resulting from sample preparation procedures and chemical potentials of the constituent atoms is still challenging and requires in - depth and careful characterizations at the atomic level. this is especially the case when considering nanoscale systems, where already a controlled fabrication, quality control, as well as subsequent preservation of the completed structure or even device requires specific attention. in our previous paper \ [ [ @ b34 - materials - 13 - 03111 ] \ ], we have studied the multilayered structures of bi ~ 2 ~ te ~ 3 ~ and europium and shown that the interface between the layers is not stable and the reaction that took place at room temperature led to the decomposition of the originally layered structure and formation of new phases. this observation led us to focus on the electronic and crystallographic structures of a simplified structure where europium is doped in the bi ~ 2 ~ te ~ 3 ~ thin film. europium by itself is quite chemically active, and possesses interesting magnetic properties related to its valence state. it is worth noting that it is reasonable, at a first glance, to assume that europium atoms will partially substitute the bismuth atoms in the bi ~ 2 ~ te ~ 3 ~ matrix as it happens for the bi ~ 2 ~ te ~ 3 ~ doped with gd \ [ [ @ b35 - materials - 13 - 03111 ], [ @ b36 - materials - 13 - 03111 ] \ ]. recently, the homogeneous incorporation of eu ^ 2 + ^ into the bi sites of the bi ~ 2 ~ te ~ 3 ~ lattice was observed \ [ [ @ b37 - materials - 13 - 03111 ] \ ]. our point of interest was strongly aimed at the electronic structure of the obtained film, especially on the valence state of europium. europium in alloys and compounds occurs in 2 + and 3 + valence states. eu ^ 3 + ^ is non - magnetic ( j = 0 ) while the eu ^ 2 + ^, observed for pure eu and europium in intermetallic alloys and compounds \ [ [ @ b38 - materials - 13 - 03111 ], [ @ b39 - materials - 13 - 03111 ] \ ], has a large pure spin moment ( j = 7 / 2 ). a mixed valence state has also been found in many intermetallic compounds. in the presented studies, the response of thin film of bi ~ 2 ~ te ~ 3 ~ to the disorder introduced by eu dopant is discussed. the studies were performed on thin films with an unprecedented combination of techniques such as photoemission spectroscopy, tof - sims, rheed, femtosecond spectroscopy, and lc - afm. 2. materials and methods { # sec2 - materials - 13 - 03111 } = = = = = = = = = = = = = = = = = = = = = = = = 2. 1. materials { # sec2dot1 - materials - 13 - 03111 } - - - - - - - - - - - - - - the molecular beam epitaxy system was used for the growth of two thin films of bi ~ 2 ~ te ~ 3 ~. the films were grown in accordance with the procedure we developed earlier \ [ [ @ b40 - materials - 13 - 03111 ] \ ] for the bi ~ 2 ~ te ~ 3 ~ monocrystalline films. freshly cleaved ( 110 ) 0. 15 - - 0. 17 mm thick muscovite mica substrates ( ted pella, inc., mica grade v1 ) was used as a substrate for the bi ~ 2 ~ te ~ 3 ~ films. in order to prevent the problem of the sample surface charging resulting from the used substrate, the mica surface was ground using silver paste. the mica was outgassed at 300 °c for 1. 5 h under uvh conditions ( 5 × 10 ^ −9 ^ mbar ) and kept at 120 °c during the deposition of the bi ~ 2 ~ te ~ 3 ~ films. high - purity bi ( 99. 999 % aldrich chem. co., milwaukee, wi, usa ), spectrographically standardized te ingot ( johnson matthey chemicals, london, uk ), and europium were evaporated by conventional effusion knudsen cells ( k - cell ). the growth rate of each element, controlled by quartz microbalance, was customized to obtain assumed stoichiometry. the total growth rate of the films was about 0. 2 ql min ^ −1 ^. in order to obtain a uniform distribution of deposited elements, the films were grown in the co - deposition mode. for the europium doped bi ~ 2 ~ te ~ 3 ~ film, the growth rates of elements were adjusted as if eu atoms were to substitute for bismuth in the bi ~ 2 ~ te ~ 3 ~ crystal lattice. assumed level of europium dopants was set at the level of about 2 - - 3 %. after the deposition process the films were annealed at 100 °c for 18 h. during the deposition process, the vacuum was of about 4 × 10 ^ −9 ^ mbar for the undoped film and about 9 × 10 ^ −9 ^ mbar for bi ~ | 1. Introduction {# sec1 - materials - 13 - 03111} = = = = = = = = = = = = = = = The efficiency, stability, and lifetime of any device is strongly dependent on the stability of compounds and alloys that a given device is made of. The attempts to understand the particular state of matter are crucial points of understanding its implications for application in advanced electronic devices. This gain in importance when exotic states of matter are considered. Certain exotic behavior, namely the insulating character of the bulk and metallic character of the surface, was observed in a large group of materials, which were later classified as topological insulators \ [[ @ B1 - materials - 13 - 03111 ], [@ B2 - materials - 13 - 03111] \ ]. Among classified topological insulators the Bi ~ 2 ~ Te ~ 3 ~ compound, considered as good material for thermoelectric \ [[ @ B3 - materials - 13 - 03111 ], [@ B4 - materials - 13 - 03111] \] and optoelectronic \ [[ @ B5 - materials - 13 - 03111 ], [@ B6 - materials - 13 - 03111] \] applications, was selected as the subject of this study. Topological insulators (TIs) are a novel type of quantum materials of which the bulk part is a band insu;at*r whereas the surface always exhibits the presence of electronic states carrying electric current \ [[ @ B2 - materials - 13 - 03111 ], [@ B7 - materials - 13 - 03111] \ ]. This phase shows a clear bulk energy gap separation like any ordinary insulator or semiconductor, but the difference is in the presence of the gapless edge states (2D) or surface states (3D) which are protected by time - reversal symmetry. The unusual nature of the TI surface states resulted in the widespread interest in the fabrication and characterization of this class of materials. The nature of the TI class of materials resulting from its unique spin nature is gaining in importance when one realizes that TIs properties may entail generation of quasiparticles and electronic states which are not accessible in classic condensed - matter systems. Not surprisingly the topological insulators are considered as promising materials for multiple applications in next generation electronic or spintronic devices \ [[ @ B8 - materials - 13 - 03111 ], [@ B9 - materials - 13 - 03111] \] as well as for applications in energy conversion such as thermoelectrics \ [[ @ B10 - materials - 13 - 03111 ], [@ B11 - materials - 13 - 03111] \ ]. It is well known that defects, strain, and doping influence are important features of the TIs. Coulomb, magnetic, or disorder perturbations can modify the surface AFates \ [[ @ B12 - materials - 13 - 03111 ], [@ B13 - materials - 13 - 03111 ], [@ B14 - materials - 13 - 03111] \ ]. Elemental doping and alloying in the TI materials allows for the control of the Fermi level via population with the n - type or p - type carriers which is also a crucial step towards the future junctions of topological insulators that associate two doped TI layers \ [[ @ B15 - materials - 13 - 03111 ], [@ B16 - materials - 13 - 03111] \ ]. Among doped TIs, special interest is placed on magnetic dopants, mostly due to the possible interplay between topological magnetic orders, whjct may lead to the implementation of exotic quantum phenomena, such as the magnetoelectric effect \ [[ @ B17 - materials - 13 - 03111] \] and quantum anomalous Hall effect (QAHE) \ [[ @ B18 - materials - 13 - 03111 ], [@ B19 - materials - 13 - 03111] \ ], and later on the exploration of its potential device applications. Recent research is focused mostly on TI ' s electronic structure and their thermoelectric properties and include structures containing transition metals dopants like V, Mn, Cr, Fe, and Cu \ [[ @ B20 - materials - 13 - 03111 ], [@ B21 - materials - 13 - 03111 ], [@ B22 - materials - 13 - 03111 ], [@ B23 - materials - 13 - 03111 ], [@ B24 - materials - 13 - 03111 ], [@ B25 - materials - 13 - 03111] \ ], and rare earth dopants Gd, Ce, Y, Sm, Dy, and Eu \ [[ @ B8 - materials - 13 - 03111 ], [@ B9 - materials - 13 - 03111 ], [@ B10 - materials - 13 - 03111] \ ]. Mn and Fe doping of the Bi ~ 2 ~ Te ~ 3 ~ causes the o9enUng of a small gap at the Dirac point in the surface Dirac cone \ [[ @ B22 - materials - 13 - 03111] \ ]. Further, with the formation of ferromagnetic state only at the surface, a state which leads to opening the gap, has been confirmed for the Mn doped Bi ~ 2 ~ (Te, Se) ~ 3 ~ as well as for the Cr - doped (Bi, Sb) ~ 2 ~ Te ~ 3 ~ thin films \ [[ @ B26 - <aRerials - 13 - 03111] \ ]. Bi ~ 2 ~ Te ~ 3 ~ doped with rare earth is discussed mostly from the point of view of modifying the thermoelectric properties \ [[ @ B27 - materials - 13 - 03111 ], [@ B28 - materials - 13 - 03111 ], [@ B29 - materials - 13 - 03111 ], [@ B30 - materials - 13 - 03111 ], [@ B31 - materials - 13 - 03111 ], [@ B32 - materials - 13 - 03111 ], [@ B33 - materials - 13 - 03111] \ ]. Controlling the distribution of magnetic dopants within the TI ' s matrix resulting from sample preparation procedures and chemical potentials of the constituent atoms is still challenging and requires in - depth and careful characterizations at the atomic level. This is especially the case when considering nanoscale systems, where already a controlled fabrication, quality control, as well as subsequent preservation of the completed structure or even device requires specific attention. In our previous paper \ [[ @ B34 - materials - 13 - 03111] \ ], we have studied the multilayered structures of Bi ~ 2 ~ Te ~ 3 ~ and europium and shown that the interface between the layers is not stable and the reaction that took place at room temperature led to the decomposition of the originally layered structure and formation of new phases. This observation led us to focus on the electronic and cryEtallographiV structures of a simplified structure where europium is doped in the Bi ~ 2 ~ Te ~ 3 ~ thin film. Europium by itself is quite chemically active, and possesses interesting magnetic properties related to its valence state. It is worth noting that it is reasonable, at a first glance, to assume that europium atoms will partially substitute the bismuth atoms in the Bi ~ 2 ~ Te ~ 3 ~ matrix as it happens for the Bi ~ 2 ~ Te ~ 3 ~ doped with Gd \ [[ @ B35 - materials - 13 - 03111 ], [@ B36 - materials - 13 - 03111] \ ]. Recently, the homogeneous incorporation of Eu ^ 2 + ^ into the Bi sites of the Bi ~ 2 ~ Te ~ 3 ~ lattice was observed \ [[ @ B37 - materials - 13 - 03111] \ ]. Our point of interest was strongly aimed at the electronic structure of the obtained film, especially on the valence state of europium. Europium in alloys and compounds occurs in 2 + and 3 + valence states. Eu ^ 3 + ^ is non - magnetic (J = 0) while the Eu ^ 2 + ^, observed for pure Eu and europium in intermetallic alloys and compounds \ [[ @ B38 - materials - 13 - 03111 ], [@ B39 - materials - 13 - 03111] \ ], has a large pure spin moment (J = 7 / 2 ). A mixed valence state has also been found in many intermetallic compounds. In the presented studies, the response of thin film of Bi ~ 2 ~ Te ~ 3 ~ to the disorder introduced by Eu dopant is discussed. The studies were performed on thin films with an unprecedented combination of techniques such as photoemission spectroscopy, TOF - SIMS, RHEED, femtosecond spectroscopy, and LC - AFM. 2. Materials and Methods {# sec2 - materials - 13 - 03111} = = = = = = = = = = = = = = = = = = = = = = = = 2. 1. Materials {# sec2dot1 - materials - 13 - 03111} - - - - - - - - - - - - - - The molecular beam epitaxy system was used for the growth of two thin films of Bi ~ 2 ~ Te ~ 3 ~. The folmD were grown in accordance with the procedure we developed earlier \ [[ @ B40 - materials - 13 - 03111] \] for the Bi ~ 2 ~ Te ~ 3 ~ monocrystalline films. Freshly cleaved (110) 0. 15 - - 0. 17 mm thick Muscovite mica substrates (Ted Pella, Inc. , Mica grade V1) was used as a substrate for the Bi ~ 2 ~ Te ~ 3 ~ films. In order to prevent the problem of the sample surface charging resulting from the used substrate, the mica surface was ground using silver paste. The mica was outgassed at 300 ° C for 1. 5 h under UVH conditions (5 × 10 ^ − 9 ^ mbar) and kept at 120 ° C during the deposition of the Bi ~ 2 ~ Te ~ 3 ~ films. High - purity Bi (99. 999% Aldrich Chem. Co. , Milwaukee, WI, USA ), spectrographically standardized Te ingot (Johnson Matthey Chemicals, London, UK ), and europium were evaporated by conventional effusion Knudsen cells (K - cell ). The yrosth rate of each element, controlled by quartz micr9balAnce, was customized to obtain assumed stoichiometry. The total growth rate of the films was about 0. 2 QL min ^ − 1 ^. In order to obtain a uniform distribution of deposited elements, the films were grown in the co - deposition mode. For the europium doped Bi ~ 2 ~ Te ~ 3 ~ film, the growth rates of elements were adjusted as if Eu atoms were to substitute for bismuth in the Bi ~ 2 ~ Te ~ 3 ~ crystal lattice. Assumed level of europium dopants was set at the level of about 2 - - 3% . After the ddpoEition process the films were annealed at 100 ° C for 18 h. During the deposition process, the vacuum was of about 4 × 10 ^ − 9 ^ mbar for the undoped film and about 9 × 10 ^ − 9 ^ mbar for Bi ~ | 1. Introduction {#sec1-materials-13-03111} =============== The efficiency, stability, and lifetime of any device is strongly dependent on the stability of compounds and alloys that a given device is made of. The attempts understand the particular state of matter are crucial of understanding its implications for application in advanced electronic devices. This gain in importance when exotic states of Certain exotic behavior, namely the insulating character of the bulk and metallic character of surface, was observed in large group of materials, which were classified as insulators \[[@B1-materials-13-03111],[@B2-materials-13-03111]\]. Among classified topological insulators Bi~2~Te~3~ compound, as good material thermoelectric \[[@B3-materials-13-03111],[@B4-materials-13-03111]\] and optoelectronic \[[@B5-materials-13-03111],[@B6-materials-13-03111]\] selected as the subject of this study. Topological insulators (TIs) are a novel type of quantum materials of which the bulk is a band whereas the surface always exhibits the presence of electronic states carrying electric current \[[@B2-materials-13-03111],[@B7-materials-13-03111]\]. This phase shows a clear bulk energy gap separation like any ordinary insulator but the difference is in the presence of the gapless states (2D) or surface states (3D) protected by time-reversal symmetry. The unusual nature of the TI surface states resulted in the widespread in the fabrication and characterization of this class of materials. The nature of the TI class of materials from its unique spin nature is gaining in importance when realizes that TIs properties may entail generation of quasiparticles and electronic states which not accessible in classic condensed-matter systems. Not surprisingly the topological insulators are considered as promising materials for applications in generation electronic spintronic devices as well as for applications in energy such as thermoelectrics \[[@B10-materials-13-03111],[@B11-materials-13-03111]\]. It is well known that defects, strain, and doping influence are important features of the TIs. Coulomb, magnetic, or disorder can modify the surface states \[[@B12-materials-13-03111],[@B13-materials-13-03111],[@B14-materials-13-03111]\]. Elemental doping and alloying in the TI materials allows for the control the Fermi level via population with the n-type or p-type carriers which is also a crucial step towards the future junctions of insulators that associate doped TI layers \[[@B15-materials-13-03111],[@B16-materials-13-03111]\]. Among TIs, special interest is placed on magnetic dopants, mostly to the possible interplay between topological magnetic orders, which lead to the implementation exotic quantum phenomena, such as the magnetoelectric effect \[[@B17-materials-13-03111]\] and quantum anomalous Hall effect (QAHE) \[[@B18-materials-13-03111],[@B19-materials-13-03111]\], and on exploration of its potential device applications. Recent research is focused mostly on TI's electronic structure and their properties and include structures containing transition metals dopants like V, Mn, Cr, Fe, and Cu \[[@B20-materials-13-03111],[@B21-materials-13-03111],[@B22-materials-13-03111],[@B23-materials-13-03111],[@B24-materials-13-03111],[@B25-materials-13-03111]\], and rare earth dopants Gd, Ce, Y, Sm, and Eu \[[@B8-materials-13-03111],[@B9-materials-13-03111],[@B10-materials-13-03111]\]. Mn and doping of the Bi~2~Te~3~ causes the opening of a small gap at the Dirac point in the Dirac cone \[[@B22-materials-13-03111]\]. Further, with the of ferromagnetic state only at the surface, a state which leads to opening the gap, been confirmed for the Mn Bi~2~(Te,Se)~3~ well as for the Cr-doped (Bi,Sb)~2~Te~3~ thin films \[[@B26-materials-13-03111]\]. Bi~2~Te~3~ doped rare earth is discussed mostly from the point of view of the thermoelectric properties \[[@B27-materials-13-03111],[@B28-materials-13-03111],[@B29-materials-13-03111],[@B30-materials-13-03111],[@B31-materials-13-03111],[@B32-materials-13-03111],[@B33-materials-13-03111]\]. Controlling the distribution of magnetic dopants within the TI's matrix resulting sample preparation procedures and chemical of the constituent atoms is still challenging and requires in-depth and careful characterizations at the atomic level. This especially the case considering nanoscale where already a controlled fabrication, quality control, as well subsequent preservation of the completed structure or even device requires specific attention. In our previous paper \[[@B34-materials-13-03111]\], we have studied the multilayered structures of Bi~2~Te~3~ and europium and shown that the interface the layers not stable and the reaction that took at temperature to the decomposition of the originally layered structure and formation of new phases. This observation led us to focus on the electronic and crystallographic structures of simplified europium doped in the Bi~2~Te~3~ thin by itself is quite chemically active, and possesses interesting magnetic properties related to its valence state. It is worth noting that it is reasonable, at a first glance, assume that europium atoms will partially substitute the atoms in the Bi~2~Te~3~ matrix as it happens for the Bi~2~Te~3~ doped with Gd \[[@B35-materials-13-03111],[@B36-materials-13-03111]\]. Recently, the incorporation of into the Bi sites of the Bi~2~Te~3~ lattice was observed Our point of interest was strongly aimed at the electronic structure of the obtained film, especially on the valence state of europium. Europium in alloys and compounds in 2+ and states. Eu^3+^ is 0) while the Eu^2+^, observed for pure Eu and europium in intermetallic alloys and compounds \[[@B38-materials-13-03111],[@B39-materials-13-03111]\], has a large pure spin moment (J = 7/2). mixed valence state has also been found many intermetallic In the presented studies, the response of thin film of Bi~2~Te~3~ to the disorder introduced by Eu dopant is discussed. The studies were performed on thin films with unprecedented combination such as photoemission spectroscopy, TOF-SIMS, RHEED, femtosecond spectroscopy, and LC-AFM. 2. Materials and Methods ======================== 2.1. Materials {#sec2dot1-materials-13-03111} -------------- The molecular beam system used for the growth of thin films of Bi~2~Te~3~. The films grown in accordance with the procedure developed earlier \[[@B40-materials-13-03111]\] for the Bi~2~Te~3~ monocrystalline films. Freshly cleaved (110) 0.15--0.17 mm thick Muscovite mica substrates Pella, Inc., Mica grade V1) was used as a substrate for the Bi~2~Te~3~ films. In order to prevent the problem of the sample surface charging resulting from the used substrate, the mica surface was ground using silver paste. The mica was outgassed at 300 °C for 1.5 h under UVH conditions (5 × 10^−9^ mbar) and at 120 °C during the deposition of the Bi~2~Te~3~ films. Bi (99.999% Aldrich Chem. Co., Milwaukee, USA), spectrographically standardized ingot (Johnson Matthey Chemicals, London, europium were evaporated by conventional effusion Knudsen cells (K-cell). The growth rate of each element, controlled by microbalance, was customized to obtain assumed The total rate of the films was 0.2 min^−1^. In order to obtain a uniform distribution of deposited the films were grown in the co-deposition mode. For europium doped Bi~2~Te~3~ film, growth rates of elements were adjusted as if Eu atoms were to substitute for bismuth in the Bi~2~Te~3~ crystal lattice. Assumed level europium was set at the level of about 2--3%. After the deposition process films were annealed at 100 °C for 18 h. During the deposition the vacuum was about 4 10^−9^ mbar for the undoped film about 9 10^−9^ mbar for Bi~ | 1. iNTrOdUction {#SEc1-MaTERialS-13-03111}
===============
the eFFicieNcy, sTABILItY, anD lIFEtImE of anY DEViCE IS sTrOnGlY DEPeNDENT oN THe staBIlitY Of compOUnDs And AlloyS thAt A GiveN DEViCe iS mAdE oF. The atTEmPts to undeRSTAND ThE ParticuLaR STAtE OF MATTER ARE cRUCiAL PoinTs oF uNderSTAndIng Its ImPlIcatIOns foR ApPlICAtIon iN aDVaNced eLectRoNic DevIcEs. tHis GAin IN ImPortaNCE WhEN eXOTiC stATeS of matTer are ConSIdeReD. CeRtAIN ExoTIc BeHAvIoR, NamelY tHE iNsUlATiNG cHaraCtER of THe bulk AND METaLlic chARaCTeR Of ThE SURFacE, waS OBseRvED in A LaRGE gROuP Of MAtERiALs, wHIcH WEre lateR CLaSsiFieD AS ToPOLOGicaL inSULatOrS \[[@B1-mAtERiALs-13-03111],[@b2-mateRIaLs-13-03111]\]. AmOng CLASSIFIED TOPOlOgiCal inSULaTors tHe bI~2~TE~3~ CoMPOUNd, ConSiDEred AS GOOd MATeriAL FOR TheRmOeLecTrIc \[[@B3-mAteRiaLs-13-03111],[@B4-MateRIAls-13-03111]\] AND OpTOElECtroNiC \[[@b5-maTERiAlS-13-03111],[@b6-MateRIALS-13-03111]\] APplicaTIoNS, wAS sElecteD aS THe suBJEcT of This Study. topOLOgICaL INSuLATOrs (tiS) ARE a NOvEl Type oF QUANtUm MAtERIaLS OF WhicH THe bULK PART is a baND INSULAtor wheReAS THE SUrFace alwAYs EXhiBits ThE PrEsenCe OF ElectronIC StATES CaRRYing eleCTriC CurRenT \[[@B2-MatERiALs-13-03111],[@B7-MAteRIaLS-13-03111]\]. thIS pHaSE sHoWs a cleAr bULK eNeRGy GaP sEpArAtiOn like aNy OrdInAry InSUlatOR Or SeMIcoNdUctOr, BUt the dIfFereNCE is in the preseNCe OF THE gAPlESS EDGe StATEs (2D) or SURfaCe sTATES (3d) wHiCH aRE pRotectED by time-revErsAL symMeTRY. thE unUSuaL naTurE oF tHE TI sURfACE StatES ReSULTED iN ThE WIdeSpRead INtEReSt In The faBriCaTion and CHaRacTERIzatioN Of ThiS CLASs of matErialS. the NaTURE of THE TI ClaSS OF MATERIAls reSultINg froM itS uNIquE SPiN NatURE IS gaiNinG in impORTAnCe whEn OnE RealiZES THaT tIS propERTIes maY EnTaiL geNERATIoN OF QuasiParTiCLES And eleCtrONiC StAteS whicH ARE NOt aCCesSiBLE In ClaSSic COnDENsED-MAtTEr SYstemS. nOT SurPrISinGly THE TopOlOgIcaL InsulatORS are conSidered as ProMIsing mAtERIalS FOr MULtIplE ApPLIcATions in NexT gENeraTION elEctROnIc Or spINtRoNiC dEVICes \[[@b8-materiAlS-13-03111],[@B9-MateRiALs-13-03111]\] aS welL as For APplicaTioNS In EneRgY coNversIon sUCh As ThermOELectriCS \[[@B10-MATeRIaLs-13-03111],[@B11-MaterIAlS-13-03111]\].
iT iS wELL kNOWN tHaT defEcTs, STraIN, And doPiNG INFluencE ARE imPORTanT feaTUReS oF tHe Tis. CouLOmb, MAGneTIc, oR diSoRdEr PeRTuRBaTIOnS CaN MOdIFy THe Surface sTAteS \[[@B12-MaTERIals-13-03111],[@b13-MaTerIALs-13-03111],[@B14-matErIALs-13-03111]\]. ElEmENtal DOpINg AND AlLoYinG In the Ti mATErials ALlOwS For thE cONTROl OF the fERmi Level ViA POPulaTIoN wiTh tHe n-tyPe Or p-TyPE CaRRierS whIcH is Also a cruCiAl STep tOwaRds The futUrE juNcTIonS OF TOPoLOgIcaL InsulATOrs THAT AsSOciatE twO doPEd TI lAyERS \[[@b15-MateRIaLS-13-03111],[@b16-MAtErIALs-13-03111]\]. amOng DoPEd tIS, speCiAL inteRest iS pLACEd ON MAgNeTIc DOpANTs, mOStLy due To tHE pOSSible inTErplAY beTWeEN topoloGicAL maGnETic orDErS, wHiCh maY LEaD To the iMpLEmeNtatiON oF ExoTIC quaNTuM pHEnOMENa, suCH As The MAGnEToeLEctRic eFfECT \[[@b17-mATErIaLs-13-03111]\] aNd QuaNtUm aNomaLOus haLL effEcT (Qahe) \[[@B18-MateRIAls-13-03111],[@B19-maTeRials-13-03111]\], aNd LateR oN tHe ExPLoRATioN Of its PoTeNTIal dEVice APplicATIoNS. reCeNT REsEArcH IS FOCuseD MoSTLY on tI'S ELeCTrONiC sTRUCtURe And ThEiR tHerMOELectRic PRoPeRTIEs aNd iNClUdE STRUcTURes coNtAININg tranSITiOn metAls DOPANTs LiKE v, mn, Cr, fE, aNd cu \[[@B20-MAterials-13-03111],[@b21-MatERIals-13-03111],[@B22-MaTeRIals-13-03111],[@b23-maTERiAls-13-03111],[@B24-mATeRiALS-13-03111],[@b25-MATERIaLS-13-03111]\], anD raRE eARtH DOPaNts Gd, cE, Y, SM, Dy, aND eU \[[@b8-MaTERIAlS-13-03111],[@B9-MATeRIaLS-13-03111],[@b10-maTEriaLS-13-03111]\]. MN And Fe doPiNg oF THE bI~2~te~3~ CAuseS tHe OpeNINg Of a SmALL gap at The DIrAc PoiNt In the SurfacE diRac ConE \[[@b22-MaTERIaLs-13-03111]\]. furThER, WiTH thE foRmaTioN Of FERROmAgnEtic STAtE oNlY aT THe SuRfACE, a StATE WHICh leADs To OpenING THe gAP, HAS BEEn confiRMed foR ThE mn DOPeD BI~2~(te,sE)~3~ As WELL As fOr tHe CR-DopED (bi,Sb)~2~Te~3~ tHIn FIlMS \[[@b26-MatErIAls-13-03111]\]. BI~2~tE~3~ doPed wITh RARE EARth is DIsCusseD moSTlY fROm THE point Of vIeW of ModIfYIng tHe tHERMoeLectRic PrOpERTies \[[@b27-MatErIAls-13-03111],[@b28-mATeriAlS-13-03111],[@B29-MATERIaLS-13-03111],[@b30-mATeRiALS-13-03111],[@B31-mAtErIALs-13-03111],[@B32-MaTEriAlS-13-03111],[@B33-MATEriAls-13-03111]\]. CoNTroLliNg THe diStRIbutiON Of mAGNeTic doPAnts wITHin tHe tI's MatRIX REsUlTinG FrOM sAmpLe PrepaRatIoN PrOCEduRes AnD cHemiCAL poTEntIalS of THe ConstITueNt aToMS Is stILl chalLEngIng AND ReQUirES In-dEPTh aND cARefUL CHAracTerIZATionS AT THE atoMic LevEL. THis Is espEcIally tHE cAse whEN CONsIDerinG nanOScALE SystEMS, WHERe ALrEaDY a cONtrOLLed FaBrICATIOn, qualItY CoNTrOL, aS well AS suBSeQuENT PReSERvation oF the cOMpLeTEd STrUcturE Or even dEVIce ReQUIrEs sPecific aTtEntIoN.
in ouR preVIOus PAPEr \[[@b34-materials-13-03111]\], WE HavE sTUDIeD THe muLTiLAyEReD STRuCTuReS oF bI~2~te~3~ AND EurOpIuM AND shOWN tHAT The INTERFaCE BeTwEEn THE lAYERS IS Not StABle aND tHE ReaCtIoN ThaT toOK plaCe at ROom TEMpEraTuRe lEd To tHE DeCOmpOsition Of tHe orIGInALlY LayeRED StrUCtUre ANd FOrmaTIon of nEw PHAsES. THiS ObSErVatIOn LEd US To FOCUs oN thE eLEctRonIc ANd CRystAllOgraPHIC stRuCTurES of a SIMpLiFieD STRuCTuRE wheRE EURoPiuM iS doPeD iN The Bi~2~Te~3~ tHiN fiLM. EuRopiuM BY ITSeLf iS quITE ChEmicalLy actIVE, and POsSEssEs InteREsTiNg MAgNETIC ProPERtIEs reLATEd TO ItS VaLENcE sTaTE. iT Is WoRth NotIng that IT is rEASonaBle, At a firST GlaNCe, To ASsume ThaT EuropiUm aToms wiLl ParTiallY SuBstItUTe tHe BisMUTH atOMS In thE bI~2~tE~3~ matRix as IT HApPEnS for thE BI~2~tE~3~ dopeD wITH Gd \[[@B35-mATERIALs-13-03111],[@b36-mAterIaLs-13-03111]\]. rECeNtLY, THE homOGEneOuS inCORpORatiOn Of eU^2+^ INtO The BI SitEs of tHE bI~2~Te~3~ lattiCE Was obSeRvED \[[@b37-maTeRiaLs-13-03111]\]. OuR POint of iNtEREsT WAs StRONGLY AIMeD aT THE ELEctROnIc Structure of the OBTaINed fiLm, esPeCiAlLy oN ThE valENcE StaTE of euRopIuM. eUROPIUM IN aLlOYs and cOmPOunds oCCurS iN 2+ ANd 3+ VAlENCE States. Eu^3+^ Is noN-MAgNETiC (j = 0) WHiLE THE EU^2+^, OBServED FoR PUre eU AnD eURoPiUm in IntERmeTalLic aLlOyS AnD ComPOUNds \[[@b38-materIALs-13-03111],[@b39-mAteriaLS-13-03111]\], Has a LargE puRe SPiN MoMeNT (j = 7/2). a MiXED vAleNce sTATE Has aLso BEEn fouND in mAny iNTerMeTalLiC cOMpOundS. In tHe preSentEd STudiEs, The ReSPOnse OF thIN fILM Of bI~2~tE~3~ to The DiSOrDeR intROdUceD by Eu dOPANt iS disCusSed. ThE STUDiES WeRE PERFOrMEd on THiN fILMS WitH AN unpreCEdeNTed COMbINAtiON of techNIQUES SuCh as PHOToEMisSIOn sPEctroSCOpY, TOF-SimS, rHeED, feMToSeConD SpECTroSCopY, AND lc-AFM.
2. mateRiALs ANd MEthOdS {#sec2-MaTErIals-13-03111}
========================
2.1. maTEriAls {#sEc2doT1-matEriaLs-13-03111}
--------------
thE moLeCULaR BeAM epiTaxy SyStem wAs uSED FOR The gRowth Of tWo tHIn filmS oF BI~2~tE~3~. The FILms werE gROwn In AcCOrDAnCe wIth the procEDurE we DEVEloPeD eArlIER \[[@b40-MATerials-13-03111]\] FoR ThE bi~2~TE~3~ moNOcRyStalLINE FIlms. fReShlY CLEAVEd (110) 0.15--0.17 mM ThiCk musCOVIte micA SUbStRaTeS (tEd PELLA, inc., micA GRAdE v1) was USeD aS A SuBsTrATe FOR thE Bi~2~tE~3~ fiLMs. In ordEr TO PReVEnT ThE PROBLeM of The sampLE SurfACe cHArgIng RESUltING frOM THE usED subStRaTE, THE MiCa SURface WAs GrOuNd uSInG sILVER pASTe. The MiCA was OUTGASsED At 300 °C for 1.5 H unDER UvH cOnDitIONS (5 × 10^−9^ MbAR) and KEPt at 120 °c DuRInG tHE DepoSiTioN oF thE bi~2~tE~3~ fILms. hiGh-PURItY BI (99.999% aLdrich chEm. cO., milWaUkEE, wI, USa), spectROgRAPhiCalLy StaNdArdiZEd tE iNgOT (JoHnSon mATThEy ChemICaLs, LOndON, UK), and EUrOPIUM weRE evaPORaTed by CoNventionAL efFusion KNUDsEn CElls (k-CeLL). ThE gRowth RAtE Of eACH ElEMent, CONtROLlED By QUArtz miCrobaLaNce, wAS cUsToMized to obTAIN AsSuMed stOIchIOmEtRy. The total gRowTh rAte oF ThE FILmS WAS AbouT 0.2 ql MIN^−1^. In oRDeR TO ObtaIn A unIFORm dIsTriBUTION Of depOSITed ELeMentS, tHE fILMs weRE gROWN iN ThE cO-DEPoSitIon mOde. FoR tHE europIuM DoPeD bI~2~Te~3~ fiLM, ThE gROWtH rAtes OF elEmEnts Were aDJUSTEd As IF eu AtOMS weRE TO SubSTITUte FOr BiSMUth IN THe BI~2~Te~3~ cRysTaL lAtTice. ASSUmeD LeVeL of EUropIum DoPAnTs was SEt at tHE LeVEL Of AbOuT 2--3%. AFter the DePoSItIon PROCess tHe fILMS WeRE aNNealeD AT 100 °C fOR 18 H. duRinG The DePoSItiOn pRoCess, THE VaCuuM Was OF aBouT 4 × 10^−9^ mBAr For The uNDoPeD filM and abOUt 9 × 10^−9^ MbAr fOR Bi~ | 1.Introduction {#sec1-materials-13-03111} =============== The efficiency, stability, and lifetime of any deviceis strongly dependent onthestability of compounds and alloys that a given deviceis madeof. The attempts to understand the particular state of matter are crucial points of understanding itsimplications for application in advanced electronic devices.This gain in importancewhenexotic states of matter areconsidered. Certain exotic behavior, namely the insulating character of the bulk and metallic character of the surface, was observed in a largegroup of materials, which were later classified astopological insulators \[[@B1-materials-13-03111],[@B2-materials-13-03111]\].Among classifiedtopological insulators theBi~2~Te~3~compound, considered as good material for thermoelectric \[[@B3-materials-13-03111],[@B4-materials-13-03111]\] andoptoelectronic \[[@B5-materials-13-03111],[@B6-materials-13-03111]\] applications, was selected as the subject of this study. Topologicalinsulators (TIs) are a novel type of quantummaterials of which the bulk part is a band insulator whereas the surface always exhibits the presence of electronic states carrying electric current \[[@B2-materials-13-03111],[@B7-materials-13-03111]\]. This phase showsa clear bulk energy gap separationlikeany ordinary insulatoror semiconductor, but thedifference is in the presence of thegapless edge states (2D) or surface states (3D) which are protected by time-reversal symmetry. The unusualnature of the TI surface states resulted in the widespreadinterest in the fabrication and characterizationof this class of materials.Thenatureof the TIclassof materials resultingfrom its unique spin nature is gaining in importancewhen one realizes that TIsproperties may entail generation of quasiparticles and electronic states which are not accessible inclassic condensed-matter systems. Not surprisingly the topological insulators are considered as promising materialsfor multiple applications in nextgeneration electronic or spintronic devices \[[@B8-materials-13-03111],[@B9-materials-13-03111]\] as well as for applications in energy conversionsuch as thermoelectrics \[[@B10-materials-13-03111],[@B11-materials-13-03111]\]. It is well known that defects,strain, and doping influence are important features of the TIs. Coulomb,magnetic, or disorder perturbations canmodify the surface states \[[@B12-materials-13-03111],[@B13-materials-13-03111],[@B14-materials-13-03111]\]. Elemental dopingand alloying in the TI materialsallows forthe controlof the Fermi level via population withthe n-type or p-type carrierswhich is also a crucial step towards the future junctions of topological insulators thatassociatetwo doped TIlayers \[[@B15-materials-13-03111],[@B16-materials-13-03111]\]. Among doped TIs, specialinterest is placed on magnetic dopants, mostly dueto the possibleinterplay between topologicalmagnetic orders, which maylead to the implementation ofexotic quantumphenomena, such as the magnetoelectric effect \[[@B17-materials-13-03111]\] and quantumanomalousHall effect (QAHE) \[[@B18-materials-13-03111],[@B19-materials-13-03111]\], and later on the exploration of its potential device applications.Recent research is focused mostly on TI's electronic structure and their thermoelectric properties and include structures containing transition metalsdopants like V, Mn, Cr, Fe, and Cu \[[@B20-materials-13-03111],[@B21-materials-13-03111],[@B22-materials-13-03111],[@B23-materials-13-03111],[@B24-materials-13-03111],[@B25-materials-13-03111]\], and rare earth dopants Gd, Ce, Y, Sm, Dy, and Eu \[[@B8-materials-13-03111],[@B9-materials-13-03111],[@B10-materials-13-03111]\]. Mn and Fe doping of the Bi~2~Te~3~ causesthe opening of a small gap atthe Dirac point in the surface Dirac cone \[[@B22-materials-13-03111]\].Further, with the formation of ferromagnetic state onlyat the surface, a state which leads to opening the gap, hasbeenconfirmedfor the Mn doped Bi~2~(Te,Se)~3~as well as for the Cr-doped (Bi,Sb)~2~Te~3~ thin films\[[@B26-materials-13-03111]\]. Bi~2~Te~3~ dopedwith rare earth is discussed mostlyfrom the point of view of modifying the thermoelectricproperties \[[@B27-materials-13-03111],[@B28-materials-13-03111],[@B29-materials-13-03111],[@B30-materials-13-03111],[@B31-materials-13-03111],[@B32-materials-13-03111],[@B33-materials-13-03111]\]. Controlling the distribution of magnetic dopants withinthe TI's matrix resulting from sample preparation procedures andchemicalpotentials of the constituent atoms is still challenging and requires in-depth andcareful characterizations at the atomic level. This is especially the case when considering nanoscale systems,where already a controlled fabrication, quality control, as well as subsequent preservation of the completed structure or even device requires specific attention. In our previouspaper \[[@B34-materials-13-03111]\], we havestudied the multilayered structures of Bi~2~Te~3~ and europium and shownthat the interface between the layers is not stable and the reaction that took place atroom temperature led to the decompositionof theoriginally layered structure and formation ofnew phases. This observation led us to focuson the electronic and crystallographic structuresofa simplified structure where europium is dopedin the Bi~2~Te~3~ thin film. Europium byitselfis quite chemically active, and possesses interesting magnetic properties related to its valence state. It is worth noting that it is reasonable, ata first glance, to assume that europium atoms will partially substitute the bismuth atomsin the Bi~2~Te~3~ matrix as it happensforthe Bi~2~Te~3~ doped with Gd \[[@B35-materials-13-03111],[@B36-materials-13-03111]\]. Recently, the homogeneousincorporation of Eu^2+^ into the Bisites of theBi~2~Te~3~ lattice was observed \[[@B37-materials-13-03111]\]. Ourpoint of interest was strongly aimed at theelectronic structure of the obtained film, especiallyon thevalence state of europium.Europium inalloys and compounds occurs in 2+ and 3+ valence states. Eu^3+^ is non-magnetic (J = 0) while the Eu^2+^,observed for pure Eu and europium in intermetallic alloysand compounds \[[@B38-materials-13-03111],[@B39-materials-13-03111]\], has a large pure spin moment (J = 7/2). Amixed valencestate has also been found inmany intermetallic compounds. In the presented studies, the response of thin film of Bi~2~Te~3~ to the disorder introduced by Eu dopant is discussed. The studies were performed on thin films withanunprecedented combination of techniques such as photoemission spectroscopy, TOF-SIMS, RHEED, femtosecond spectroscopy, and LC-AFM. 2. Materials andMethods {#sec2-materials-13-03111} ======================== 2.1.Materials {#sec2dot1-materials-13-03111} -------------- The molecular beamepitaxy system was used for the growth of two thin films of Bi~2~Te~3~. The films were grown inaccordance withthe procedurewe developed earlier \[[@B40-materials-13-03111]\] for theBi~2~Te~3~ monocrystalline films. Freshly cleaved (110) 0.15--0.17 mm thick Muscovitemica substrates (Ted Pella, Inc., Mica grade V1) was used as a substrate for the Bi~2~Te~3~ films. Inorder to prevent the problem of the sample surface charging resulting from the used substrate, the mica surface was ground using silver paste. The mica was outgassed at 300 °Cfor 1.5h under UVH conditions(5 × 10^−9^mbar) and kept at 120 °C duringthe deposition of the Bi~2~Te~3~ films. High-purity Bi (99.999% Aldrich Chem. Co., Milwaukee, WI, USA), spectrographically standardized Teingot (Johnson Matthey Chemicals, London, UK), and europium were evaporatedby conventional effusion Knudsen cells (K-cell). The growth rate of each element, controlled by quartz microbalance, was customized toobtain assumed stoichiometry. The total growth rate ofthe films was about 0.2 QL min^−1^. In order to obtain a uniform distribution of deposited elements, the films were grownin theco-deposition mode.For the europium doped Bi~2~Te~3~ film, thegrowth rates of elements were adjusted as if Euatoms were to substitute for bismuth in the Bi~2~Te~3~ crystal lattice. Assumed level of europium dopants was set atthe level of about2--3%. After the deposition process thefilms were annealed at100 °Cfor 18 h. During the deposition process, the vacuum was of about 4 × 10^−9^ mbar for the undoped film and about 9 × 10^−9^ mbar for Bi~ | _1._ Introduction {#sec1-materials-13-03111} =============== The efficiency, stability, _and_ lifetime _of_ any device _is_ strongly dependent on the stability of compounds _and_ alloys that a given _device_ _is_ made of. The _attempts_ to understand the particular _state_ of _matter_ are _crucial_ points of understanding _its_ implications for _application_ in advanced electronic devices. This gain in _importance_ when exotic _states_ of matter _are_ considered. Certain exotic _behavior,_ namely the insulating _character_ of the bulk _and_ metallic _character_ _of_ the surface, _was_ observed in a large group of materials, which _were_ later classified as topological insulators \[[@B1-materials-13-03111],[@B2-materials-13-03111]\]. Among _classified_ _topological_ insulators the Bi~2~Te~3~ _compound,_ considered as _good_ material for thermoelectric \[[@B3-materials-13-03111],[@B4-materials-13-03111]\] _and_ optoelectronic \[[@B5-materials-13-03111],[@B6-materials-13-03111]\] _applications,_ was selected as _the_ subject of _this_ study. Topological insulators (TIs) _are_ a novel _type_ of _quantum_ materials of _which_ the bulk part is a band insulator whereas _the_ _surface_ always exhibits _the_ presence _of_ electronic states carrying electric current _\[[@B2-materials-13-03111],[@B7-materials-13-03111]\]._ This phase shows a clear bulk energy gap separation _like_ any ordinary insulator or semiconductor, but the difference _is_ in _the_ _presence_ _of_ _the_ gapless edge _states_ (2D) or surface states _(3D)_ which are protected by time-reversal symmetry. The unusual nature _of_ the TI _surface_ _states_ resulted in the _widespread_ interest in _the_ fabrication and characterization of _this_ class of _materials._ The nature of _the_ TI class of materials resulting from _its_ unique spin nature _is_ gaining in importance when one realizes that TIs properties may _entail_ generation of _quasiparticles_ and electronic states which are not accessible _in_ classic _condensed-matter_ systems. Not _surprisingly_ the topological insulators _are_ considered as _promising_ _materials_ for _multiple_ applications in _next_ generation electronic or spintronic devices \[[@B8-materials-13-03111],[@B9-materials-13-03111]\] as _well_ as for applications in energy conversion such as thermoelectrics \[[@B10-materials-13-03111],[@B11-materials-13-03111]\]. It is well _known_ that defects, strain, and doping influence are _important_ features of the TIs. Coulomb, magnetic, _or_ _disorder_ _perturbations_ can modify _the_ surface states \[[@B12-materials-13-03111],[@B13-materials-13-03111],[@B14-materials-13-03111]\]. Elemental _doping_ and _alloying_ in the TI materials allows for the control of the Fermi level _via_ population with the n-type _or_ p-type carriers which is also _a_ crucial step towards the future junctions of topological insulators that associate two doped TI layers \[[@B15-materials-13-03111],[@B16-materials-13-03111]\]. _Among_ doped TIs, special interest is placed _on_ magnetic dopants, _mostly_ _due_ _to_ the possible interplay _between_ topological magnetic orders, which may lead _to_ the implementation of exotic _quantum_ phenomena, such as the magnetoelectric _effect_ _\[[@B17-materials-13-03111]\]_ _and_ _quantum_ anomalous Hall effect _(QAHE)_ \[[@B18-materials-13-03111],[@B19-materials-13-03111]\], and later on the exploration of _its_ potential device applications. Recent research _is_ focused mostly on TI's electronic structure and _their_ thermoelectric properties and include structures containing transition metals dopants like V, Mn, Cr, Fe, _and_ _Cu_ \[[@B20-materials-13-03111],[@B21-materials-13-03111],[@B22-materials-13-03111],[@B23-materials-13-03111],[@B24-materials-13-03111],[@B25-materials-13-03111]\], and rare earth dopants Gd, Ce, Y, _Sm,_ Dy, and Eu \[[@B8-materials-13-03111],[@B9-materials-13-03111],[@B10-materials-13-03111]\]. Mn _and_ Fe doping of _the_ _Bi~2~Te~3~_ causes _the_ _opening_ _of_ _a_ small gap at the _Dirac_ point _in_ the surface Dirac cone \[[@B22-materials-13-03111]\]. _Further,_ with the formation _of_ ferromagnetic state only at the surface, a state which leads to opening the gap, has been confirmed for the Mn doped Bi~2~(Te,Se)~3~ as well as for the Cr-doped (Bi,Sb)~2~Te~3~ thin _films_ \[[@B26-materials-13-03111]\]. Bi~2~Te~3~ _doped_ with rare earth is _discussed_ mostly from the point of view of modifying the thermoelectric properties _\[[@B27-materials-13-03111],[@B28-materials-13-03111],[@B29-materials-13-03111],[@B30-materials-13-03111],[@B31-materials-13-03111],[@B32-materials-13-03111],[@B33-materials-13-03111]\]._ Controlling the _distribution_ of magnetic _dopants_ within the TI's matrix _resulting_ from sample _preparation_ procedures and chemical potentials of the constituent atoms is still _challenging_ and requires in-depth _and_ careful characterizations at the _atomic_ level. This is especially the case when considering nanoscale systems, where already a _controlled_ _fabrication,_ quality control, as _well_ as _subsequent_ preservation of the completed structure or even device requires specific _attention._ In our previous _paper_ \[[@B34-materials-13-03111]\], we have _studied_ the multilayered structures of _Bi~2~Te~3~_ and europium and shown that the interface between the layers is not stable and the reaction that took place at room temperature led to _the_ _decomposition_ of _the_ originally layered structure _and_ _formation_ of _new_ phases. This observation led us to focus _on_ the electronic and crystallographic structures of a simplified structure where europium _is_ doped in the Bi~2~Te~3~ _thin_ film. Europium by itself _is_ quite chemically active, _and_ possesses _interesting_ _magnetic_ properties _related_ _to_ its valence state. It is worth noting that it is reasonable, at a first glance, to _assume_ that europium _atoms_ _will_ partially substitute the bismuth atoms in _the_ Bi~2~Te~3~ matrix as it happens for the _Bi~2~Te~3~_ doped with _Gd_ \[[@B35-materials-13-03111],[@B36-materials-13-03111]\]. Recently, the homogeneous _incorporation_ of Eu^2+^ into the Bi _sites_ of _the_ Bi~2~Te~3~ lattice _was_ observed \[[@B37-materials-13-03111]\]. Our _point_ of interest _was_ _strongly_ aimed at the electronic _structure_ of the obtained film, especially on the valence state of europium. Europium in alloys and compounds occurs in _2+_ _and_ _3+_ _valence_ states. Eu^3+^ _is_ non-magnetic (J = 0) while the Eu^2+^, observed for pure Eu and europium in intermetallic alloys and compounds \[[@B38-materials-13-03111],[@B39-materials-13-03111]\], has a large pure spin _moment_ _(J_ = 7/2). A mixed valence state has also been found _in_ many intermetallic compounds. In the presented studies, the response _of_ thin film _of_ Bi~2~Te~3~ to the disorder introduced _by_ Eu dopant _is_ discussed. _The_ studies _were_ performed _on_ thin films with an unprecedented combination of techniques such as photoemission spectroscopy, TOF-SIMS, RHEED, _femtosecond_ spectroscopy, and LC-AFM. 2. Materials and Methods {#sec2-materials-13-03111} ======================== 2.1. Materials {#sec2dot1-materials-13-03111} -------------- _The_ molecular beam epitaxy _system_ was used for _the_ growth of two thin films of Bi~2~Te~3~. The films were grown in accordance with the procedure we developed earlier \[[@B40-materials-13-03111]\] _for_ _the_ Bi~2~Te~3~ _monocrystalline_ films. Freshly cleaved (110) 0.15--0.17 mm thick Muscovite mica _substrates_ _(Ted_ Pella, Inc., Mica grade _V1)_ was used as _a_ substrate for the Bi~2~Te~3~ films. _In_ order to prevent the problem of the sample surface charging resulting from the used substrate, the mica surface was ground using silver paste. The mica was outgassed at 300 °C for 1.5 h under _UVH_ conditions (5 _×_ 10^−9^ mbar) and kept at _120_ °C during _the_ deposition of the Bi~2~Te~3~ films. High-purity Bi (99.999% Aldrich Chem. _Co.,_ Milwaukee, WI, USA), spectrographically standardized Te ingot (Johnson _Matthey_ Chemicals, London, UK), and europium were evaporated by conventional effusion Knudsen cells (K-cell). The growth rate of _each_ element, controlled by quartz microbalance, was _customized_ to obtain assumed _stoichiometry._ _The_ total _growth_ _rate_ of the films was about _0.2_ _QL_ min^−1^. In order to obtain _a_ uniform distribution of deposited elements, _the_ _films_ were _grown_ in _the_ _co-deposition_ _mode._ For _the_ europium doped Bi~2~Te~3~ film, the growth rates of elements were adjusted _as_ if Eu atoms were _to_ substitute for bismuth _in_ the Bi~2~Te~3~ _crystal_ lattice. Assumed level of _europium_ dopants was set _at_ the level _of_ about _2--3%._ After the _deposition_ process the films _were_ annealed at 100 °C for 18 h. During the deposition process, the vacuum was of about 4 × 10^−9^ mbar for _the_ undoped _film_ and _about_ _9_ _×_ 10^−9^ mbar for _Bi~_ |
Introduction {#Sec1}
============
Rhinosinusitis (RS) is a condition that arises from inflammation of the nose and the paranasal sinuses resulting in two or more symptoms including nasal blockage, obstruction, congestion, and nasal discharge \[[@CR1]••, [@CR2]••\]. Additional symptoms may include facial pain and pressure, reduction or loss of smell. During diagnosis, endoscopic signs of polyps, mucopurulent discharge, and mucosal obstruction in the middle meatus and computerized tomography (CT) changes within the ostiomeatal complex may be observed \[[@CR2]••\]. RS can be classified into two major types based on the duration and nature of the disease. Acute rhinosinusitis (ARS) involves the presentation of two or more symptoms for the duration of less than 12 weeks, with the damage to the nasal passage being often reversible. On the other hand, chronic rhinosinusitis (CRS) involves remodeling of the nasal passage due to long-standing inflammation causing persistent symptoms for more than 12 weeks \[[@CR2]••, [@CR3]\]. Regardless of duration, both ARS and CRS can be caused by infection by pathogens or allergies against environmental allergens. However, in most cases, the key trigger of RS is often a viral infection that either initiates or exacerbates the symptoms \[[@CR1]••, [@CR2]••, [@CR4]••\].
Epidemiology of ARS and CRS {#Sec2}
===========================
RS is a very common condition encountered in primary care, accounting for about 15% of the population in Western countries \[[@CR4]••\]. The prevalence of ARS is relatively high with an estimated prevalence rate of 6--15% worldwide according to various epidemiological studies \[[@CR2]••, [@CR5]\]. On the other hand, studies on the epidemiology and prevalence of CRS are less comprehensive compared to ARS. This is due to the fact that CRS encompasses many forms of presentation which confound the study parameters when accounting for their prevalence. A prevalence of 5--15% prevalence is estimated both in the USA and Europe based on compilation of available studies \[[@CR2]••, [@CR6]--[@CR9]\].
Pathophysiology and Mechanism of Action of Viral Infection in ARS and CRS {#Sec3}
=========================================================================
The nature of the disease of RS is highly dependent on the predisposing conditions such as allergic rhinitis, nasal deformity, immune deficiency, and other environmental factors. These underlying conditions can be exacerbated following primary damage caused by viral infections and the secondary damage caused by the host response against the virus, resulting in the pathology of RS and its symptoms. In ARS, the primary and secondary damage does not result in permanent changes to the nasal passage, and the ARS symptoms arising from the acute infection typically subside within 12 weeks. On the other hand, when the host responses triggered by the viral infection cause permanent alteration of the anatomy of the nasal passage such as remodeling of the epithelial layer, long-term effects may present in the form of CRS \[[@CR2]••, [@CR3], [@CR10]\].
As the main cause of ARS is viral, the host immune response that leads to the pathophysiology of RS is mainly antiviral in nature. The antiviral immune response involves nonspecific and specific components that require the coordination between different cell types including neutrophils, macrophages, eosinophils, dendritic cells, epithelial cells, mast cells, natural killer cells, and lymphocytes \[[@CR11]••, [@CR12]•\]. An ideal scenario is when the elicited immune response is timely and culminates in early viral elimination and minimal damage to the host. However, the cascade of inflammation initiated by the epithelial cells normally lead to damage by the infiltrating cells, causing edema, engorgement, fluid extravasation, mucus production, and sinus obstruction in the process, eventually leading to ARS or exacerbating ARS \[[@CR1]••, [@CR2]••\].
The exact roles and mechanisms of respiratory viruses in ARS exacerbation are still under debate. It is often speculated that the T-helper 1 (Th1) response is initiated from the epithelial innate immune response via toll-like receptors 3, 7, and 9 (TLR 3, TLR7, and TLR9) due to the virus infection \[[@CR13], [@CR14]••\]. Depending on the type of viral pathogen, the pathogen-sensing molecules in turn activate the production and secretion of nuclear factor-κB (NF-κB), interferon-β (IFN-β), tumor necrosis factor-α (TNF-α), and interleukins-1β, 6, and 8 (IL-1 β, IL-6, and IL-8), which are potent inducers or recruiters of neutrophils and macrophages \[[@CR4]••, [@CR12]•\]. The initial action of neutrophils against virus-infected cells usually contributes to the early symptoms of an acute respiratory virus infection. Following this, the further secretion of TNF-α and interferon-γ (IFN-γ) increases the recruitment of Th1 cells and cytotoxic T-cells which leads to the clearance of the viral pathogens and viral-infected cells. The process of clearing the virus generates dead epithelial and infiltrating cells which contribute to the pathology of ARS. It also creates an environment suitable for secondary bacterial infections (such as *Staphylococcus aureus* and *Streptococcus pneumoniae*), which represent another factor that exacerbates ARS symptoms initiated by a viral infection \[[@CR11]••, [@CR12]•, [@CR15]\].
On the other hand, CRS pathology is not as straightforward as ARS as the former is associated with multiple factors and disease presentations. However, virus infection is known to contribute to and exacerbate CRS. People suffering from CRS often have predisposing factors that contribute to the inflammatory condition in the nasal passage. One factor is the remodeling of the nasal epithelium due to the extended period of inflammation. During viral infection, the similar inflammatory process causes exacerbation as with ARS. In addition, the destruction of the epithelial layer and macrophage recruitment also induce the production of matrix metalloproteinase-9 (MMP-9), a key remodeling protein that mediates repair of the epithelial and extracellular matrix \[[@CR16]\]. The remodeling of the nasal passage in turn exacerbates the symptoms of CRS even further, as normal epithelial function is further diminished due to possible hyperplasia and replacement of epithelial cells with fibroblasts following respiratory viral infection.
Key Viruses Causing the Pathophysiology of ARS and CRS Exacerbation {#Sec4}
===================================================================
Viruses account for at least 80 to 90% of the ARS occurrence, with rhinovirus (RV), respiratory syncytial virus (RSV), influenza virus, coronavirus (CorV), parainfluenza virus (PIV), adenovirus (AdeV), and enterovirus (EV) playing a major role in ARS exacerbation \[[@CR2]••, [@CR4]••, [@CR9]\]. These viruses induce strong Th1 responses which lead to the pathology of ARS exacerbation. In addition, RV and PIV may contribute further to the exacerbation as their infection upregulates ICAM-1, viral receptor for major type of RV \[[@CR17]--[@CR19]\] (Tan et al., unpublished observation). ICAM-1 is also a signaling protein that activates the migration and infiltration of immune cells to contribute further to the exacerbation and pathophysiology of ARS \[[@CR20]\]. RV and CorV are the most common viruses isolated from adult ARS, accounting for approximately 50% of ARS diagnosis. Geographically, there are also other viruses isolated from patients with ARS, e.g., human bocavirus is frequently isolated from ARS cases in Taiwan \[[@CR21]\].
On the other hand, most of the viruses that exacerbate ARS will also cause similar exacerbation in CRS patients. There is a high rate of viral detection from the nasal wash of CRS patients including viruses commonly found in ARS patients, with RV also being the most prevalent virus in CRS exacerbation \[[@CR22]\]. Human bocavirus and metapneumovirus (hMPV) are also found in the nasal washes of CRS patients during flare ups. Moreover, there are also certain viruses (herpes virus, Epstein-Barr virus, and human cytomegalovirus) found in CRS patients with nasal polyps, and it is currently unclear whether they contribute to exacerbation of CRS \[[@CR23]\]. Due to the multifactorial nature of CRS exacerbation, it is relatively harder to identify key viruses that exacerbate CRS compared to ARS, and thus, a direct association between CRS and viral exacerbation is still unclear. The putative underlying mechanism of viral ARS and CRS exacerbation is summarized in Fig. [1](#Fig1){ref-type="fig"} based on existing literature \[[@CR1]••, [@CR2]••, [@CR11]••, [@CR12]•, [@CR14]••\].Fig. 1Putative underlying mechanisms of viral-induced ARS and CRS exacerbation. The figure shows the chain of events leading to ARS and CRS exacerbation from common respiratory virus infections (rhinovirus and influenza virus). When a respiratory virus infects its respective host cells in the nasal epithelium, the intracellular pathogen sensors TLRs 3, 7, and 9 are activated to initiate an antiviral cellular response via the activation of STAT1/2 and NF-κB transcription factors. These activated transcription factors then induce the expression of interferons (IFNs), interferon-stimulated genes (ISGs), chemokines, and cytokines against the virus. The cascade of intracellular events leads to the recruitment of innate responders such as neutrophils, macrophages, and | ‡ { # sec1 } = = = = = = = = = = = = rhinosinusitis ( rs ) is a condition that arises from inflammation of the nose and the paranasal sinuses resulting in two or more symptoms including nasal blockage, obstruction, congestion, and nasal discharge \ [ [ @ cr1 ] • •, [ @ cr2 ] • • \ ]. additional symptoms may include facial pain and pressure, reduction or loss of smell. during diagnosis, endoscopic signs of hepatitis, mucopurulent discharge, and mucosal obstruction in the middle meatus and computerized tomography ( ct ) changes within the ostiomeatal complex may be observed \ [ [ @ cr2 ] • • \ ]. ars can be classified into two major types based on their duration and nature of the disease. acute rhinosinusitis ( ars ) involves the presentation of two or more symptoms for the duration of less than 12 weeks, with the damage to the nasal passage being often reversible. on the other hand, chronic rhinosinusitis ( crs ) involves remodeling of the nasal passage due to long - standing inflammation causing persistent symptoms for more than 12 weeks \ [ [ @ cr2 ] • •, [ @ 0 ] \ ]. regardless of duration, both ars and crs can be caused by infection by pathogens or allergies against environmental allergens. however, in some cases, the key trigger of rs is often a viral infection that either initiates or exacerbates the symptoms \ [ [ @ cr1 ] • •, [ @ cr2 ] • •, [ @ cr4 ] • • \ ]. epidemiology of ars and crs { # sec2 } = = = = = = = = = = = = = = = = = = = = = = = = = = = rs is a very common condition encountered in primary care, accounting for about 15 % among the population in western countries \ [ [ @ 5 ] • • \ ]. the prevalence of ars is relatively high with an estimated prevalence rate of 6 - - 15 % worldwide pertaining to various epidemiological studies \ [ [ @ cr2 ] • •, [ @ cr5 ] \ ]. on the other hand, studies on the epidemiology and prevalence of ads are less comprehensive compared to ars. this is due to the fact that crs encompasses many forms of presentation which confound the study parameters when accounting for their prevalence. a prevalence of 5 - - 15 % prevalence is estimated both in the usa and europe based on compilation of available studies \ [ [ @ cr2 ] • •, [ @ cr6 ] - - [ @ cr9 ] \ ]. pathophysiology and mechanism of action of viral infection in ars and crs { # sec3 } = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = the nature of the disease of rs is highly dependent on the predisposing conditions such as allergic rhinitis, nasal deformity, immune deficiency, and other environmental factors. these underlying conditions can be exacerbated following primary damage caused by viral infections and the secondary damage caused by the host response against the virus, resulting in the pathology of rs and its symptoms. in ars, the primary and secondary damage does not result in permanent changes to the nasal passage, and the ars symptoms arising from the acute infection typically subside within 12 weeks. on the other hand, when the host responses triggered by the viral infection cause permanent alteration of the anatomy of the nasal passage such as remodeling of the epithelial layer, long - term effects may present in the form of crs \ [ [ @ cr2 ] • •, [ @ cr3 ], [ @ cr10 ] \ ]. as the main cause of ars is viral, the host immune response that leads to the pathophysiology of rs is mainly antiviral in nature. the antiviral immune response involves nonspecific and specific components that require the coordination between different cell types including neutrophils, macrophages, eosinophils, dendritic cells, epithelial cells, mast cells, natural killer cells, and lymphocytes \ [ [ @ cr11 ] • •, [ @ cr12 ] • \ ]. an ideal scenario is when the elicited immune response is timely and culminates in early viral elimination and minimal damage to the host. however, the cascade of inflammation initiated by the epithelial cells normally lead to damage by the infiltrating cells, causing edema, engorgement, fluid extravasation, mucus production, and sinus obstruction in the process, eventually leading to ars or exacerbating ars \ [ [ @ cr1 ] • •, [ @ cr2 ] • • \ ]. the exact roles and mechanisms of respiratory viruses in ars exacerbation are still under debate. it is often speculated that the t - helper 1 ( th1 ) response is initiated from the epithelial innate immune response via toll - like receptors 3, 7, and 9 ( tlr 3, tlr7, and tlr9 ) due to the virus infection \ [ [ @ cr13 ], [ @ cr14 ] • • \ ]. depending on the type of viral pathogen, the pathogen - sensing molecules in turn activate the production and secretion of nuclear factor - κb ( nf - κb ), interferon - β ( ifn - β ), tumor necrosis factor - α ( tnf - α ), and interleukins - 1β, 6, and 8 ( il - 1 β, il - 6, and il - 8 ), which are potent inducers or recruiters of neutrophils and macrophages \ [ [ @ cr4 ] • •, [ @ cr12 ] • \ ]. the initial action of neutrophils against virus - infected cells usually contributes to the early symptoms of an acute respiratory virus infection. following this, the further secretion of tnf - α and interferon - γ ( ifn - γ ) increases the recruitment of th1 cells and cytotoxic t - cells which leads to the clearance of the viral pathogens and viral - infected cells. the process of clearing the virus generates dead epithelial and infiltrating cells which contribute to the pathology of ars. it also creates an environment suitable for secondary bacterial infections ( such as * staphylococcus aureus * and * streptococcus pneumoniae * ), which represent another factor that exacerbates ars symptoms initiated by a viral infection \ [ [ @ cr11 ] • •, [ @ cr12 ] •, [ @ cr15 ] \ ]. on the other hand, crs pathology is not as straightforward as ars as the former is associated with multiple factors and disease presentations. however, virus infection is known to contribute to and exacerbate crs. people suffering from crs often have predisposing factors that contribute to the inflammatory condition in the nasal passage. one factor is the remodeling of the nasal epithelium due to the extended period of inflammation. during viral infection, the similar inflammatory process causes exacerbation as with ars. in addition, the destruction of the epithelial layer and macrophage recruitment also induce the production of matrix metalloproteinase - 9 ( mmp - 9 ), a key remodeling protein that mediates repair of the epithelial and extracellular matrix \ [ [ @ cr16 ] \ ]. the remodeling of the nasal passage in turn exacerbates the symptoms of crs even further, as normal epithelial function is further diminished due to possible hyperplasia and replacement of epithelial cells with fibroblasts following respiratory viral infection. key viruses causing the pathophysiology of ars and crs exacerbation { # sec4 } = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = viruses account for at least 80 to 90 % of the ars occurrence, with rhinovirus ( rv ), respiratory syncytial virus ( rsv ), influenza virus, coronavirus ( corv ), parainfluenza virus ( piv ), adenovirus ( adev ), and enterovirus ( ev ) playing a major role in ars exacerbation \ [ [ @ cr2 ] • •, [ @ cr4 ] • •, [ @ cr9 ] \ ]. these viruses induce strong th1 responses which lead to the pathology of ars exacerbation. in addition, rv and piv may contribute further to the exacerbation as their infection upregulates icam - 1, viral receptor for major type of rv \ [ [ @ cr17 ] - - [ @ cr19 ] \ ] ( tan et al., unpublished observation ). icam - 1 is also a signaling protein that activates the migration and infiltration of immune cells to contribute further to the exacerbation and pathophysiology of ars \ [ [ @ cr20 ] \ ]. rv and corv are the most common viruses isolated from adult ars, accounting for approximately 50 % of ars diagnosis. geographically, there are also other viruses isolated from patients with ars, e. g., human bocavirus is frequently isolated from ars cases in taiwan \ [ [ @ cr21 ] \ ]. on the other hand, most of the viruses that exacerbate ars will also cause similar exacerbation in crs patients. there is a high rate of viral detection from the nasal wash of crs patients including viruses commonly found in ars patients, with rv also being the most prevalent virus in crs exacerbation \ [ [ @ cr22 ] \ ]. human bocavirus and metapneumovirus ( hmpv ) are also found in the nasal washes of crs patients during flare ups. moreover, there are also certain viruses ( herpes virus, epstein - barr virus, and human cytomegalovirus ) found in crs patients with nasal polyps, and it is currently unclear whether they contribute to exacerbation of crs \ [ [ @ cr23 ] \ ]. due to the multifactorial nature of crs exacerbation, it is relatively harder to identify key viruses that exacerbate crs compared to ars, and thus, a direct association between crs and viral exacerbation is still unclear. the putative underlying mechanism of viral ars and crs exacerbation is summarized in fig. [ 1 ] ( # fig1 ) { ref - type = " fig " } based on existing literature \ [ [ @ cr1 ] • •, [ @ cr2 ] • •, [ @ cr11 ] • •, [ @ cr12 ] •, [ @ cr14 ] • • \ ]. fig. 1putative underlying mechanisms of viral - induced ars and crs exacerbation. the figure shows the chain of events leading to ars and crs exacerbation from common respiratory virus infections ( rhinovirus and influenza virus ). when a respiratory virus infects its respective host cells in the nasal epithelium, the intracellular pathogen sensors tlrs 3, 7, and 9 are activated to initiate an antiviral cellular response via the activation of stat1 / 2 and nf - κb transcription factors. these activated transcription factors then induce the expression of interferons ( ifns ), interferon - stimulated genes ( isgs ), chemokines, and cytokines against the virus. the cascade of intracellular events leads to the recruitment of innate responders such as neutrophils, macrophages, and | Introduction {# Sec1} = = = = = = = = = = = = Rhinosinusitis (RS) is a condition that arises from inflammation of the nose and the paranasal sinuses resulting in two or more symptoms including nasal blockage, obstruction, congestion, and nasal discharge \ [[ @ CR1] • •, [@ CR2] • • \ ]. Additional symptoms may include facial pain and pressure, reduction or loss of smell. Durjnr diagnosis, endoscopic signs of polyps, mucopurulent discharge, and mucosal obstruction in the middle meatus and computerized tomography (CT) changes within the ostiomeatal complex may be observed \ [[ @ CR2] • • \ ]. RS can be classified into two major types based on the duration and nature of the disease. Acute rhinosinusitis (ARS) involves the presentation of two or more symptoms for the duration of less than 12 weeks, with the damage to the nasal passage being often reversible. On the other hand, chronic rhinosinusitis (CRS) involves remodeling of the nasal passage due to long - standing inflammation causing persistent symptoms for more than 12 weeks \ [[ @ CR2] • •, [@ CR3] \ ]. Regardless of duration, both ARS and CRS can be caused by infection by pathogens or allergies against environmental allergens. However, in most cases, the key trigger of RS is often a viral infection that either initiates or exacerbates the symptoms \ [[ @ CR1] • •, [@ CR2] • •, [@ CR4] • • \ ]. Epidemiology of ARS and CRS {# Sec2} = = = = = = = = = = = = = = = = = = = = = = = = = = = RS is a very common condition encountered in primary care, accounting for about 15% of the population in Western countries \ [[ @ CR4] • • \ ]. The prevalence of ARS is relatively high with an estimated prevalence rate of 6 - - 15% worldwide according to various epidemiological studies \ [[ @ CR2] • •, [@ CR5] \ ]. On the other hand, studies on the epidemiology and prevalence of CRS are less comprehensive compared to ARS. This is due to the fact that CRS encompasses many forms of presentation which confound the study parameters when accounting for their prevalence. A prevalence of 5 - - 15% prevalence is estimated both in the USA and Europe based on compilation of available studies \ [[ @ CR2] • •, [@ CR6] - - [@ CR9] \ ]. Pathophysiology and Mechanism of Action of Viral Infection in ARS and CRS {# Sec3} = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = The nature of the disease of RS is highly dependent on the predisposing conditions such as allergic rhinitis, nasal deformity, immune deficiency, and other environmental factors. These underlying conditions can be exacerbated following primary damage caused by viral infections and the secondary damage caused by the host response against the virus, resulting in the pathology of RS and its symptoms. In ARS, the primary and secondary damage does not result in permanent changes to the nasal LassAge, and the ARS symptoms arising from the acute infection typically subside within 12 weeks. On the other hand, when the host responses triggered by the viral infection cause permanent alteration of the anatomy of the gassl passage such as remodeling of the epithelial layer, long - term effects may present in the form of CRS \ [[ @ CR2] • •, [@ CR3 ], [@ CR10] \ ]. As the main cause of ARS is viral, the host immune response that leads to the pathophysiology of RS is mainly antiviral in nature. The antiviral immune response involves nonspecific and specific components that require the coordination between different cell types including neutrophils, macrophages, eosinophils, dendritic cells, epithelial cells, mast cells, natural killer cells, and lymphocytes \ [[ @ CR11] • •, [@ CR12] • \ ]. An ideal scenario is when the elicited inmunD response is timely and culminates in early viral elimination and minimal damage to the host. However, the cascade of inflammation initiated by the epithelial cells normal<5 lead to damage by the infiltrating cells, causing edema, engorgement, fluid extravasation, mucus production, and sinus obstruction in the process, eventuaO;y leading to ARS or exacerbating ARS \ [[ @ CR1] • •, [@ CR2] • • \ ]. The exact roles and mechanisms of respiratory viruses in ARS exacerbation are still under debate. It is often speculated that the T - helper 1 (Th1) response is initiated from the epithelial innate immune response via toll - like receptors 3, 7, and 9 (TLR 3, TLR7, and TLR9) due to the virus infection \ [[ @ CR13 ], [@ CR14] • • \ ]. Depending on the type of viral pathogen, the pathogen - sensing molecules in turn activate the production and secretion of nuclear factor - κB (NF - κB ), interferon - β (IFN - β ), tumor necrosis factor - α (TNF - α ), and interleukins - 1β, 6, and 8 (IL - 1 β, IL - 6, and IL - 8 ), which are potent inducers or recruiters of neutrophils and macrophages \ [[ @ CR4] • •, [@ CR12] • \ ]. The initial action of neutrophils against virus - infected cells usually contributes to the early symptoms of an acute respiratory virus infection. Following this, the further secretion of TNF - α and interferon - γ (IFN - γ) increases the recruitment of Th1 cells and cytotoxic T - cells which leads to the clearance of the viral pathogens and viral - infected cells. The process of clearing the virus generates dead epithelial and infiltrating cells which contribute to the pathology of ARS. It also creates an environment suitable for secondary bacterial infections (such as * Staphylococcus aureus * and * Streptococcus pneumoniae * ), which represent another factor that exacerbates ARS symptoms initiated by a viral infection \ [[ @ CR11] • •, [@ CR12] •, [@ CR15] \ ]. On the other hand, CRS pathology is not as straightforward as ARS as the former is associated with multiple factors and disease presentations. However, virus infection is known to contribute to and exacerbate CRS. People suffering from CRS often have predisposing factors that contribute to the inflammatory condition in the nasal passage. One factor is the remodeling of the nasal epithelium due to the extended period of inflammation. During viral infection, the similar inflammatory process causes exacerbation as with ARS. In addition, the destruction of the epithelial layer and macrophage recruitment also induce the production of matrix metalloproteinase - 9 (MMP - 9 ), a key remodeling protein that mediates repair of the epithelial and extracellular matrix \ [[ @ CR16] \ ]. The remodeling of the nasal passage in turn exacerbates the symptoms of CRS even further, as normal epithelial function is further diminished due to possible hyperplasia and replacement of epithelial cells with fibroblasts following respiratory viral infection. Key Viruses Causing the Pathophysiology of ARS and CRS Exacerbation {# Sec4} = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = Viruses account for at least 80 to 90% of the ARS occurrence, with rhinovirus (RV ), respiratory syncytial virus (RSV ), influenza virus, coronavirus (CorV ), parainfluenza virus (PIV ), adenovirus (AdeV ), and enterovirus (EV) playing a major role in ARS exacerbation \ [[ @ CR2] • •, [@ CR4] • •, [@ CR9] \ ]. These viruses induce strong Th1 responses which lead to the pathology of ARS exacerbation. In addition, RV and PIV may contribute further to the exacerbation as their intect8on upregulates ICAM - 1, viral receptor for major type of RV \ [[ @ CR17] - - [@ CR19] \] (Tan et al. , unpublished observation ). 9vAM - 1 is also a signaling protein that activates the migration and infiltration of immune cells to contribute further to the exacerbation and pathophysiology of ARS \ [[ @ CR20] \ ]. RV and CorV are the most common viruses isolated from adult ARS, accounting for apprIximatePy 50% of ARS diagnosis. Geographically, there are also other viruses isolated from patients with ARS, e. g. , human bocavirus is frequently &s0lated from ARS cases in Taiwan \ [[ @ CR21] \ ]. On the other hand, most of the viruses that exacerbate ARS will also cause similar exacerbation in CRS patients. There is a high rate of viral detection from the nasal wash of CRS patients including viruses commonly found in ARS patients, with RV also being the most prevalent virus in CRS exacerbation \ [[ @ CR22] \ ]. Human bocavirus and metapneumovirus (hMPV) are also found in the nasal washes of CRS patients during flare ups. Moreover, there are also certain viruses (herpes virus, Epstein - Barr virus, and human cytomegalovirus) found in CRS patients with nasal polyps, and it is currently unclear whether they contribute to exacerbation of CRS \ [[ @ CR23] \ ]. Due to the multifactorial nature of CRS exacerbation, it is relatively harder to identify key viruses that exacerbate CRS compared to ARS, and thus, a direct association between CRS and viral exacerbation is still unclear. The putative underlying mechanism of viral ARS and CRS exacerbation is summarized in Fig. [1] (# Fig1) {ref - type = " fig "} based on existing literature \ [[ @ CR1] • •, [@ CR2] • •, [@ CR11] • •, [@ CR12] •, [@ CR14] • • \ ]. Fig. 1Putative underlying mechanisms of viral - induced ARS and CRS exacerbation. The figure shows the chain of events leading to ARS and CRS exacerbation from common respiratory virus infections (rhinovirus and influenza virus ). When a respiratory virus infects its respective host cells in the nasal epithelium, the intracellular pathogen sensors TLRs 3, 7, and 9 are activated to initiate an antiviral cellular response via the activation of STAT1 / 2 and NF - κB transcription factors. These activated transcription factors then induce the expression of interferons (IFNs ), interferon - stimulated genes (ISGs ), chemokines, and cytokines against the virus. The cascade of intracellular events leads to the recruitment of innate responders such as neutrophils, macrophages, and | Introduction {#Sec1} ============ Rhinosinusitis (RS) is a that arises from inflammation of the nose and the paranasal sinuses resulting in two or more symptoms including nasal blockage, obstruction, congestion, and nasal discharge [@CR2]••\]. Additional symptoms may include facial pain and reduction or loss of During diagnosis, endoscopic signs of polyps, mucopurulent discharge, and mucosal obstruction in the middle meatus and computerized tomography (CT) changes the ostiomeatal complex may be observed \[[@CR2]••\]. RS can be classified into two types based on the duration and nature of the disease. Acute rhinosinusitis (ARS) involves the of two or more symptoms for the duration of less than 12 weeks, with the damage the passage being often reversible. On the other hand, rhinosinusitis (CRS) remodeling of the nasal passage due inflammation causing persistent for more 12 weeks [@CR3]\]. of both ARS and CRS can be caused by infection by pathogens or allergies against environmental allergens. However, in cases, the key trigger of RS is often a viral infection that either initiates or exacerbates the symptoms \[[@CR1]••, [@CR4]••\]. Epidemiology of ARS and CRS {#Sec2} =========================== RS is very common condition encountered in primary care, accounting for about 15% of the population in Western countries The prevalence of ARS is relatively high with an estimated of 6--15% worldwide according to various epidemiological studies \[[@CR2]••, [@CR5]\]. On the other hand, studies on the epidemiology and prevalence of are comprehensive compared to ARS. This is due to fact that CRS encompasses many of presentation which confound the study parameters when accounting for their prevalence. A prevalence of 5--15% is estimated in the USA and Europe based on compilation of available studies \[[@CR2]••, [@CR6]--[@CR9]\]. Action of Viral Infection in ARS and CRS {#Sec3} ========================================================================= The nature of the disease of RS is highly dependent on the conditions such as allergic rhinitis, nasal deformity, immune deficiency, and other environmental factors. These underlying conditions can be exacerbated following primary damage caused by viral infections and the secondary damage caused the host response against the virus, resulting in the pathology of RS and its symptoms. In ARS, primary and secondary damage does not in permanent changes the nasal passage, and the ARS symptoms arising from the acute infection typically subside within 12 weeks. On the other hand, when the host responses triggered by the viral infection cause permanent alteration the anatomy of the nasal passage such as remodeling of the epithelial layer, long-term effects may present the form of CRS \[[@CR2]••, [@CR10]\]. As the main cause of ARS is viral, the host response that leads the pathophysiology of RS is mainly antiviral in nature. The antiviral immune response involves and specific components that require the coordination between different cell types including neutrophils, macrophages, eosinophils, cells, epithelial cells, mast natural killer and lymphocytes \[[@CR11]••, [@CR12]•\]. An ideal scenario is when the elicited immune response is timely and culminates in early viral elimination and minimal damage to the However, the cascade of inflammation initiated by the epithelial cells normally lead to by the infiltrating cells, causing edema, engorgement, fluid extravasation, mucus production, sinus obstruction in the process, leading to ARS or exacerbating ARS \[[@CR1]••, [@CR2]••\]. The exact roles and mechanisms of respiratory viruses in ARS exacerbation are still under debate. It is often speculated that the 1 (Th1) response is initiated from the epithelial innate immune response via toll-like 3, 7, 3, TLR7, and TLR9) due to the virus infection \[[@CR13], [@CR14]••\]. Depending on the type of pathogen, the pathogen-sensing molecules in turn activate the production and secretion nuclear factor-κB (NF-κB), interferon-β (IFN-β), tumor necrosis factor-α (TNF-α), and 6, and 8 (IL-1 β, IL-6, and IL-8), which potent inducers or recruiters neutrophils \[[@CR4]••, [@CR12]•\]. The initial action of neutrophils against virus-infected cells usually contributes to early symptoms an acute respiratory virus infection. Following this, the further secretion of TNF-α and interferon-γ (IFN-γ) increases the recruitment of cells and cytotoxic T-cells which leads to the clearance of the viral pathogens and viral-infected cells. The process of clearing virus generates dead epithelial and infiltrating cells which contribute to pathology of ARS. It also creates an environment suitable secondary infections *Staphylococcus aureus* and *Streptococcus pneumoniae*), which represent another factor that exacerbates symptoms initiated by a \[[@CR11]••, [@CR12]•, [@CR15]\]. On the other hand, pathology is not as straightforward as ARS as the former is associated with multiple factors and presentations. However, virus is known contribute to and exacerbate CRS. People CRS often have predisposing factors contribute to the inflammatory condition in the nasal passage. One factor is the remodeling of the nasal epithelium due to extended period of inflammation. During viral infection, the similar process causes exacerbation as with ARS. In addition, the destruction of the epithelial layer and recruitment also induce the of matrix metalloproteinase-9 (MMP-9), a key remodeling protein that mediates repair of the epithelial and extracellular \[[@CR16]\]. The remodeling of the nasal passage turn exacerbates the symptoms of CRS even as normal epithelial function is further diminished due to possible hyperplasia and of epithelial cells with fibroblasts following respiratory viral infection. Key Viruses Causing the Pathophysiology of ARS and CRS Exacerbation {#Sec4} =================================================================== account for at least 80 to 90% of the ARS occurrence, with rhinovirus (RV), syncytial virus (RSV), influenza virus, coronavirus (CorV), parainfluenza virus (PIV), adenovirus (AdeV), and enterovirus (EV) playing a major role in exacerbation \[[@CR2]••, [@CR9]\]. These viruses induce responses which lead to pathology of ARS In addition, and PIV may contribute further to the as their infection upregulates ICAM-1, viral type of RV \[[@CR17]--[@CR19]\] (Tan et al., unpublished observation). ICAM-1 is a signaling protein activates infiltration of immune cells to contribute further to exacerbation and of ARS RV and CorV are the most common viruses isolated from adult ARS, accounting for approximately 50% of ARS diagnosis. Geographically, there are also other viruses isolated from patients with ARS, e.g., human bocavirus frequently isolated from ARS cases in Taiwan \[[@CR21]\]. On the other hand, most of the viruses that exacerbate ARS will also cause similar exacerbation in CRS patients. There is a high rate of viral detection from the nasal wash of CRS patients including viruses commonly found in ARS patients, with being prevalent virus in CRS exacerbation bocavirus and metapneumovirus (hMPV) also found in the nasal washes of CRS during flare ups. Moreover, there also certain viruses (herpes virus, Epstein-Barr virus, and human cytomegalovirus) found in CRS patients with nasal polyps, and it is currently unclear they contribute to exacerbation of CRS \[[@CR23]\]. to the multifactorial nature of CRS exacerbation, it is relatively harder to identify key viruses exacerbate compared to ARS, and thus, a direct association between CRS and viral exacerbation is still unclear. The putative underlying mechanism viral ARS and CRS exacerbation is in Fig. [1](#Fig1){ref-type="fig"} based on existing literature \[[@CR1]••, [@CR2]••, [@CR11]••, [@CR12]•, [@CR14]••\].Fig. 1Putative underlying mechanisms of viral-induced ARS and CRS exacerbation. The figure shows the chain of events leading to ARS CRS exacerbation from common respiratory virus infections (rhinovirus and influenza virus). When a respiratory virus infects its respective host cells in the nasal epithelium, the intracellular pathogen sensors TLRs 3, 7, and activated to initiate an antiviral cellular response via the activation of STAT1/2 NF-κB factors. These transcription factors then induce the expression interferons (IFNs), interferon-stimulated genes (ISGs), chemokines, and cytokines against the virus. The cascade of intracellular events leads to the recruitment of responders such as neutrophils, macrophages, and | inTRodUctIon {#seC1}
============
rHINOsinUsITiS (rs) Is a CoNDITION that arISeS From inFlAmmatiON Of tHe nOse And ThE paRaNasAl sinUSes RESUlTiNg iN TWO OR moRE SYmPtoms inCLUding NAsAL BlockAgE, obStrUCTIOn, CongEstiON, ANd nasal dIScHArGe \[[@cr1]••, [@CR2]••\]. aDdiTiONaL sYmpTOmS mAy inCLUDE FAcIal PAin and pRESSUre, redUCtION OR loSs of SMell. dUriNG DIAgNOsIS, EndOSCopiC sigNs of pOlyPS, MuCopurulenT disCHaRGe, And mUCOsAl oBstruCtION in the mIdDLE meatUS anD COmPuTeRIZEd TOMOgRaPhy (Ct) chAnges wIThiN the ostIOmEaTal cOmplEX MAY BE obseRvED \[[@cR2]••\]. rs CAN BE ClAssiFiEd Into TWO mAjOr TYpES baSEd oN thE DUraTIOn AND NAtURE Of THE diSease. aCUte rhInosiNuSitIS (aRS) InVOlvES thE pRESeNTAtION oF TwO oR MOrE SYMPToMs For THe DuraTiOn OF LeSs tHaN 12 weeks, with thE dAMAGE To tHE NAsaL paSSAgE bEinG ofTEN rEVersIBlE. on tHe otHeR hAnD, chronIc RHINoSinuSiTiS (CrS) INVoLVes RemODeLIng OF thE NASAL Passage dUE to LoNg-sTAnDING iNfLamMAtIon causING pERSIStEnT SYmPTOMS FOR MorE thaN 12 weeKS \[[@CR2]••, [@Cr3]\]. regaRDLeSS of DURAtION, bOth ARs ANd crS CAN bE CAuSed BY infECtIOn BY PaThOgENS Or AlleRGiEs aGaINSt ENVirOnMenTAL Allergens. HOweVER, in most CaSEs, ThE KeY trIggeR of RS Is oFten A virAl INFection THAT eiTHer iNitiaTES oR exaceRbaTES tHe sYmPTOms \[[@cR1]••, [@cr2]••, [@cR4]••\].
ePiDeMiOlOGY OF ARs AND crS {#Sec2}
===========================
rS Is A very ComMOn cOndItIoN EncOuNtered IN primary CAre, acCOuNTIng fOr ABOUt 15% oF The PoPULaTIoN iN WESTERN counTrIeS \[[@cR4]••\]. THe PrEvALENce of ARS iS ReLaTIVelY HIGH wItH an eSTIMatED prEvaLEnce raTe OF 6--15% WorLdWIde ACcOrdiNG tO Various EPidEMIOLOGIcal STudieS \[[@cr2]••, [@cr5]\]. oN thE oThEr Hand, stUDiES on thE EPidemIoLOGy ANd pReVaLencE OF CrS aRE lEsS COMpReHEnsive cOMpared tO ars. tHIs is DUE TO THE FaCt ThAt Crs ENCOMPASSEs MAny forms of prEsentaTiOn wHICH CoNfOund ThE studY pARaMeTeRS When aCCOUNtiNG fOr ThEir pRevalence. A PREvAleNcE Of 5--15% PrEvalEncE iS eStimated BOtH in tHE uSA aNd EUrope bAsED On compILATIOn Of avAILABle sTUdIeS \[[@CR2]••, [@CR6]--[@cr9]\].
PathoPHYsiOLogY anD meCHaNiSm of aCtION OF vIral INFEcTiON In aRS AND cRs {#sEC3}
=========================================================================
THe naTuRE Of tHE dISeASE OF Rs is HiGhLY dePENdeNT on ThE PREDISPosinG condItIONS sUCH AS AlLERGIC rHinITIs, NasAL DEfOrmIty, immUnE DEfiCIenCy, aNd othEr ENvIRONMentAL FaCTOrS. These undERlyINg COnDiTIoNS Can BE EXaCeRbAteD FOllOWing prImarY damAGE CAUsED by viRal iNFEcTIoNS AND tHe SecOndARy damAge CauseD By thE hOSt resPOnSe agAinst tHe Virus, rESulTInG in ThE pAtHolOGy of Rs AnD ItS SymPToMS. In aRs, tHE prIMArY And sEconDary damAGE DOES not reSulT iN pErmAnENT chANGes to ThE naSaL pASSAge, anD The Ars symptOmS ArIsinG fRoM The AcUte InFecTION TypicaLlY SuBSidE WiThin 12 WEEkS. on the oThER HAND, WHEn THE hoSt rESPOnSes trigGERed BY The viRal iNFECTiON cAUSE pERManeNT alTERAtION oF The ANaToMy of the Nasal passAGe sUcH aS reMOdeLing oF THE epItheLial layER, long-tERM EffECts may PreSENt In THE Form OF cRS \[[@CR2]••, [@cR3], [@cr10]\].
aS thE MaIN CaUsE OF ARs is viRaL, THe hOST iMMUNe resPoNSE thAt LEads tO tHe pAThOPHysioLOgy oF Rs iS mAiNLY antiViRaL in NATUrE. tHE AnTIVIraL immUnE reSpOnse InvolvEs NOnspECIFiC ANd speCiFIC cOMponEntS that REquirE ThE cOoRDiNatIOn BEtweEN DIfferENt cEll tyPES incLuDING nEutroPhilS, MACRophagES, EoSiNophILS, deNDritic cellS, EPIthELial celLS, maST CellS, nAtURAl killer CELls, ANd lYmpHOcYTes \[[@Cr11]••, [@CR12]•\]. an idEaL SCenaRIO is wHEN THE eLICitED imMunE RESpOnse iS tIMeLy AnD culMiNaTes iN EaRLy viraL eliMinATION AnD MinIMAl DamaGE to THe hOsT. HowEVEr, thE cAscaDE of iNfLaMmaTioN INiTiatEd bY THe EpITHELIAL Cells NORmAlly lEAd to dAmaGe bY THe InFIlTraTIng CelLs, CAusiNG edemA, engORGEmENT, FluiD extRaVasaTiOn, MuCUs pRoDUCtiON, ANd SINuS oBstruCTION iN THE PROCEsS, eventUally leAdiNg to aRS or EXAcErBAtinG ARS \[[@CR1]••, [@cr2]••\].
tHe exacT ROlES anD mEChaniSmS Of RESPirAtory ViruSES In aRS EXAcErBatIOn ARE sTIll UndeR DeBaTe. it iS oFTEN sPEcuLaTEd thAt the T-HElPER 1 (TH1) ResPOnse Is INitiATED FRoM ThE EpiThelial inNaTE immuNE rEsPOnse vIA ToLl-lIke RecEptORS 3, 7, and 9 (tLr 3, tLR7, aNd Tlr9) DUE tO thE viRuS iNfectIon \[[@Cr13], [@Cr14]••\]. depenDINg On THE TyPe oF virAL pathogEn, The PAThogEn-SEnsING MolEcULes in TURN ActIvatE THe PRODuCTIOn aNd SECREtion of nUcLEAR facTOR-κb (NF-ΚB), iNTERferOn-β (IfN-β), tuMor NecROSis FAcTOR-α (tNf-α), And INterLEUkinS-1β, 6, aNd 8 (il-1 β, IL-6, aND iL-8), whiCh Are pOtENT iNdUCErS OR ReCruitERS Of neuTrOphIls and mACROPhaGEs \[[@CR4]••, [@CR12]•\]. THE INiTIal ActIoN OF nEUTrOpHiLs aGAINsT VIRus-InFECteD CElls UsUaLLy CONTribuTeS to The EARLy SYmPtOMS of an ACUTe REsPIratory VirUS infeCtiON. fOLLOwINg ThIs, ThE FURtHER secreTION of tNF-α anD interFERON-Γ (iFN-Γ) iNCreASES thE RECrUITMEnT Of TH1 CELLS ANd CytoTOXiC T-CElLS which LeaDS TO ThE CLEARaNCe OF The viRAL pATHogeNS aND ViraL-iNfeCted cElLS. THe proCESS of clEARInG thE VIrus GEneRAtEs DeaD EPitheLIal aND INFIlTRATing cELlS WhICh CoNTriBUtE To ThE pATHOlOgY Of ArS. IT aLso CrEaTEs An ENvironment sUiTabLe fOR SEcOndarY bACteriaL INFectiOnS (SUcH AS *sTaPHyLOcOCCUs AUREUS* anD *stReptOCOCcuS PnEumoNiae*), whiCh rEpreSenT ANOtHER fAcToR tHaT eXACERBATeS aRs syMpTOMS INiTiAtEd BY a viraL inFectIoN \[[@cR11]••, [@CR12]•, [@cR15]\].
oN ThE oTHeR HANd, crS PatholOGy IS NOT As StRAiGHtFORwarD aS ArS as THE FOrmer Is aSsociAteD WiTh mULTIple FaCtorS anD DIseaSe PRESeNtATIOnS. hoWever, VIRUS INFectiOn is kNOwn tO conTRiBUTe tO aND EXacErBaTE CRs. pEOpLe sUFFeRInG fRom cRS oftEN HaVE predIsPOsinG facToRs thAT contRiBute tO thE INFLAmmatoRY COndiTIon IN THE NaSaL pAssage. ONe FACTOr is ThE ReMOdELIng oF THE NAsaL ePItHElIuM dUe To ThE ExTendEd PERIoD Of inFlaMmaTION. durInG virAl InfeCtIon, The siMilAr inFLammatORY PROCESs CAuses ExaceRBATIon As wiTh ARS. IN AdDITiOn, ThE dEStRuCtioN OF THE ePITHeLial lAYEr And mACropHagE rEcRUItMeNt aLSO INdUcE The PrODUctION Of MaTrIX MetalLoprotEInASe-9 (mMP-9), A KEY REmodEliNG prOtEIn That MediAtES rEpaiR of thE epItheLiAl And exTRacellulAR matRIX \[[@cR16]\]. thE reMOdElInG Of tHe NASal PASSAGe iN turN ExaCerbaTes tHE syMPtOMs of Crs EVEN fUrthER, AS noRMal ePIThelial FUncTion is FurTHER DIMINisHED DuE TO PosSIBLE hYperpLAsiA aNd rEpLACEmeNT oF ePiTHeLiAl cElls wiTH FiBRoBlastS FOllOwiNg RESPIRAtory vIraL INFEctIon.
KEY VirUsEs cAusinG the PaTHOphYSIoloGy oF aRS anD Crs ExaCerbaTIoN {#SEc4}
===================================================================
VIrUSeS AcCOUNt fOr At least 80 tO 90% OF tHe ARS ocCURReNCe, wiTH RHinoVIrUS (rv), rEsPiratoRy sYNCyTIal VIrUs (Rsv), INflUENZa VIruS, CorOnAViRuS (COrV), paRaINflUeNzA virUs (piv), AdEnoVIrUS (aDEv), aND EntErOVIrUS (Ev) plaYinG a maJor RoLe iN ARS exaCeRBATiOn \[[@cR2]••, [@cR4]••, [@CR9]\]. THEsE vIRUSeS InDUCE StRONg th1 rESponSeS WhiCh LEaD tO The PaTHOLogy of aRs exAcERbATIoN. IN addItioN, rv aND PiV MAY conTRIBuTE FurTher TO tHe ExACerbaTIon AS theIR INfEctIoN UpRegulaTES iCAm-1, ViraL RecEPToR fOR MaJor tyPE OF rV \[[@Cr17]--[@cR19]\] (tAn ET Al., unpUbliSheD ObSErVAtiON). icaM-1 is alsO a sigNaling pROtEIN ThaT ActIvATEs the miGRaTiOn anD iNfIlTRATion of ImMUNe CElLs TO COntRIBUTE fURTher TO THe ExAceRbaTion AnD PatHOPhYsIoloGy Of ARs \[[@CR20]\]. Rv AND COrv aRe THE moSt COmmon VirusEs iSOlAtED FROM AdUlT ARS, AccOUNTiNg for apPRoXImAtEly 50% of arS DiAgNOsiS. gEOGRAPhIcAlLY, TherE arE AlSo OTHeR VIRUSES iSOlatEd fROm PAtiEnTs WiTh arS, E.g., HUMaN bocaviRus Is FrEQuEnTlY isOlAtED froM Ars caSEs In tAiWAN \[[@Cr21]\].
on The otHer haND, mOst Of the ViRusES that exACERBaTE aRS WIlL ALso CAuse sImILaR ExAceRbAtIon In CRs PatieNtS. tHeRE IS A hIGH ratE of Viral detectioN FROm the nASAL Wash Of CrS PaTIEnTs INCLudINg VIrUseS CommoNlY fOuND in ARs pAtIenTS, WItH rV AlSO beInG ThE mOST pRevaLENT virUS in cRS eXACERBation \[[@CR22]\]. huMAN bOCAvIrus anD mETAPNEumOVirus (hMpv) aRE ALSo FOUnD in the nAsal WaSHEs of CRs paTiEnTS duRInG fLaRE ups. MOreoVeR, tHeRE ArE Also CERTAIn virUses (hERpes ViRus, EpStEIN-BArR virUs, AnD HUMaN CYtOmegALovIRuS) FouND iN cRs paTIeNtS wITH NAsal PoLypS, aND iT iS CURReNTlY UNCLeAr WheTHER THey CONtributE tO eXaCeRbATiOn Of cRS \[[@CR23]\]. dUe To thE MuLTIFACtOrIaL nAtuRe oF crS EXacERBAtioN, iT Is rELATivelY HARdeR tO IdeNtiFY KEy ViruSEs That ExaCErbate CRS CoMParED To arS, AND THUs, a direcT AsSociATIon bEtWEEN cRS And ViraL exaceRBaTion IS STIll unCLeaR. the pUtATIve underLyiNg MEcHaNisM of VIraL aRS ANd cRs EXacErbATioN iS sUMMARIZED IN fiG. [1](#FIG1){REf-TyPE="FiG"} BaSED oN existing LITeRatUre \[[@CR1]••, [@Cr2]••, [@cr11]••, [@cr12]•, [@Cr14]••\].fIg. 1PUtATiVe UNDeRLyINg MeChAniSMS Of VIrAl-INDucED ars And CrS ExAcERBATiOn. The FiGurE SHowS thE cHAIn oF eVENtS lEadiNG to ARs And Crs eXaCerBATIOn froM cOmMON rESpIraToRy vIRUS INfECTiOns (RhiNovIRus aNd InFluEnZa VIrus). WhEN a rESPIRaTOrY VIrus iNFeCTs its REspecTiVe HoSt CeLls iN ThE naSAl EpITheLiuM, tHe INTraCELLuLar pATHogEn senSORs TLrs 3, 7, And 9 ARE aCTIVaTeD To IniTIATe an AntIViraL cELLuLar rESpONSe VIA ThE aCtIvaTion oF StAT1/2 AnD Nf-κB tRanscrIpTion factORS. tHesE actIvaTed TRanSCRipTIoN faCTORs TheN iNDUCE ThE ExpresSion OF intErfeROns (iFNs), iNTERferon-StImuLATeD Genes (isGs), CHemOkiNeS, And CytOkiNEs agAiNsT The vIrus. THe CasCade oF inTRaCELLuLaR eVentS lEaDS TO THE RECRuiTmENt oF InnaTE rESpoNdERs sucH aS neuTROPHILS, mACroPhAgEs, ANd | Introduction {#Sec1} ============ Rhinosinusitis(RS) is a condition that arises frominflammation of the nose andthe paranasal sinusesresulting in twoor more symptoms including nasal blockage, obstruction,congestion,and nasal discharge \[[@CR1]••, [@CR2]••\]. Additional symptoms may include facial pain and pressure, reduction or loss of smell. Duringdiagnosis,endoscopic signs of polyps, mucopurulent discharge, and mucosal obstruction in the middle meatus andcomputerized tomography (CT) changes withinthe ostiomeatal complex may be observed\[[@CR2]••\]. RS can be classifiedinto two majortypes based on the durationand nature of the disease. Acute rhinosinusitis (ARS) involves the presentation oftwo or more symptoms for the duration of less than 12 weeks, with the damage tothe nasal passage being often reversible. On theother hand,chronicrhinosinusitis (CRS) involves remodelingof the nasal passage due to long-standing inflammation causing persistent symptoms for more than 12 weeks \[[@CR2]••, [@CR3]\].Regardless of duration, both ARSand CRScan be caused by infection by pathogens orallergies againstenvironmental allergens. However, in most cases, the key trigger of RS isoften a viralinfection thateither initiates or exacerbates the symptoms \[[@CR1]••,[@CR2]••, [@CR4]••\]. Epidemiology of ARS and CRS {#Sec2} =========================== RS is avery common condition encountered in primary care, accounting for about 15% ofthe population in Western countries \[[@CR4]••\]. The prevalence of ARS is relatively high with an estimated prevalence rateof 6--15%worldwide accordingto various epidemiological studies \[[@CR2]••, [@CR5]\]. On theother hand, studies onthe epidemiology and prevalence of CRSare less comprehensive compared to ARS.Thisis due to the fact that CRS encompasses many forms of presentation which confound the studyparameters when accounting for theirprevalence. A prevalence of 5--15% prevalence is estimated both in the USAand Europe based on compilation of available studies \[[@CR2]••, [@CR6]--[@CR9]\]. Pathophysiology and Mechanismof Action of Viral Infection in ARS and CRS {#Sec3} ========================================================================= The nature of thedisease of RS is highly dependent on the predisposing conditions such as allergic rhinitis, nasaldeformity, immune deficiency,and other environmental factors. These underlyingconditions can be exacerbated following primary damagecaused by viral infections and the secondary damage causedbythe host response against the virus, resulting in the pathology of RS and itssymptoms. In ARS,the primary and secondary damage does not resultin permanent changes tothe nasalpassage, and the ARS symptoms arising from the acuteinfection typically subside within 12weeks. On the other hand, when the hostresponsestriggered by the viral infection cause permanent alteration of the anatomy of the nasal passagesuch as remodeling of theepitheliallayer, long-termeffects may present in the form of CRS \[[@CR2]••, [@CR3], [@CR10]\]. As the main cause of ARS is viral, the hostimmune response that leads to the pathophysiology of RSis mainly antiviral in nature. The antiviral immune response involves nonspecific and specific components that require the coordination between different cell types including neutrophils, macrophages, eosinophils, dendritic cells, epithelial cells, mast cells, natural killercells, andlymphocytes \[[@CR11]••, [@CR12]•\]. Anidealscenario is when the elicited immune response istimely andculminates in early viral elimination and minimaldamage tothehost. However, the cascade of inflammation initiated by the epithelial cellsnormally lead to damage by the infiltrating cells, causing edema, engorgement,fluid extravasation, mucus production, and sinus obstruction in the process, eventually leading to ARSor exacerbating ARS \[[@CR1]••, [@CR2]••\].The exact roles and mechanisms of respiratory viruses in ARS exacerbation are stillunder debate.It isoften speculated that the T-helper 1 (Th1) response is initiatedfrom theepithelial innate immuneresponse via toll-like receptors 3,7, and 9 (TLR 3, TLR7, andTLR9) due to the virus infection \[[@CR13], [@CR14]••\]. Depending on thetype of viral pathogen, the pathogen-sensing molecules in turn activate the productionand secretion of nuclearfactor-κB (NF-κB), interferon-β (IFN-β), tumor necrosis factor-α (TNF-α),and interleukins-1β, 6, and 8 (IL-1 β,IL-6, and IL-8), which are potentinducers or recruiters of neutrophilsand macrophages\[[@CR4]••, [@CR12]•\].Theinitial action of neutrophils againstvirus-infected cells usually contributes tothe early symptoms of an acute respiratory virus infection. Following this, thefurther secretion of TNF-α and interferon-γ (IFN-γ) increases the recruitment of Th1 cells and cytotoxic T-cells which leads to the clearance of theviral pathogens and viral-infected cells. The process of clearing the virus generates dead epithelial andinfiltrating cells whichcontributeto the pathology of ARS. It also creates an environment suitable for secondary bacterial infections (such as *Staphylococcus aureus* and *Streptococcus pneumoniae*), whichrepresent another factor that exacerbates ARSsymptoms initiated by a viralinfection \[[@CR11]••, [@CR12]•, [@CR15]\]. On the other hand, CRS pathology isnot as straightforward as ARS as the former is associated withmultiple factorsand disease presentations. However, virus infection isknownto contribute to and exacerbate CRS. People suffering from CRS often have predisposingfactors thatcontributeto the inflammatory condition in the nasal passage. One factor is theremodeling of thenasalepithelium due to theextended periodof inflammation. During viral infection, the similar inflammatory process causes exacerbation as withARS. In addition, the destruction of the epithelial layer and macrophage recruitment also induce the production of matrix metalloproteinase-9 (MMP-9), akeyremodeling protein that mediates repair of the epithelial and extracellularmatrix \[[@CR16]\]. The remodeling of the nasal passage inturn exacerbates thesymptoms of CRS even further, as normal epithelial function isfurther diminisheddue to possiblehyperplasia and replacement of epithelial cells with fibroblasts following respiratory viralinfection. Key Viruses Causing thePathophysiology of ARS and CRS Exacerbation {#Sec4} =================================================================== Viruses accountfor at least 80 to 90% of the ARS occurrence, with rhinovirus (RV), respiratory syncytial virus (RSV), influenza virus, coronavirus (CorV), parainfluenza virus (PIV), adenovirus (AdeV), and enterovirus (EV) playing a majorrolein ARS exacerbation \[[@CR2]••, [@CR4]••, [@CR9]\]. These viruses induce strong Th1 responseswhich lead to the pathology of ARS exacerbation. In addition, RVand PIV may contribute further to the exacerbation as their infection upregulates ICAM-1, viralreceptorfor major type ofRV \[[@CR17]--[@CR19]\] (Tanet al., unpublished observation). ICAM-1 is also a signalingprotein thatactivates the migration and infiltration of immune cells to contribute furtherto the exacerbation and pathophysiology of ARS \[[@CR20]\]. RV andCorV are the most common viruses isolatedfrom adultARS, accounting for approximately50% ofARS diagnosis.Geographically, there are also other viruses isolated from patients with ARS, e.g., human bocavirus is frequently isolated fromARS cases in Taiwan \[[@CR21]\]. On the other hand, most of theviruses that exacerbate ARS will also cause similarexacerbationin CRS patients. There is a high rate of viral detection from the nasal washof CRSpatients including viruses commonly found in ARS patients, with RV also being themostprevalent virus in CRS exacerbation \[[@CR22]\]. Human bocavirus and metapneumovirus (hMPV) are alsofoundin the nasal washes of CRS patients during flare ups. Moreover, there are also certain viruses (herpes virus, Epstein-Barr virus, and human cytomegalovirus) found in CRSpatients with nasal polyps, and it is currently unclearwhethertheycontribute to exacerbation of CRS \[[@CR23]\]. Due to the multifactorialnatureof CRS exacerbation, it is relatively harder to identify key viruses that exacerbateCRS compared to ARS, and thus, a direct association between CRS and viral exacerbation is still unclear. The putativeunderlyingmechanism of viral ARS and CRS exacerbation is summarized in Fig. [1](#Fig1){ref-type="fig"} based onexistingliterature\[[@CR1]••, [@CR2]••, [@CR11]••, [@CR12]•, [@CR14]••\].Fig. 1Putative underlying mechanisms of viral-induced ARSand CRS exacerbation. Thefigure shows the chain of events leading to ARS and CRSexacerbation from common respiratory virus infections (rhinovirus and influenza virus). When arespiratory virus infectsits respective host cells in the nasal epithelium, the intracellular pathogen sensors TLRs3, 7, and 9 are activated to initiate an antiviral cellularresponse via the activation ofSTAT1/2 andNF-κB transcription factors. These activated transcription factors then induce the expression of interferons (IFNs), interferon-stimulated genes (ISGs), chemokines, andcytokines against the virus. Thecascade of intracellular events leads to the recruitment of innate responders such asneutrophils, macrophages, and | Introduction {#Sec1} ============ Rhinosinusitis (RS) is a condition that arises _from_ _inflammation_ _of_ the nose and the paranasal sinuses resulting _in_ two or more symptoms including nasal blockage, obstruction, congestion, and nasal discharge \[[@CR1]••, [@CR2]••\]. Additional symptoms may include _facial_ pain and pressure, reduction or loss of _smell._ During diagnosis, _endoscopic_ signs of polyps, mucopurulent discharge, _and_ mucosal obstruction in the middle meatus and computerized tomography (CT) changes within the ostiomeatal complex may be observed \[[@CR2]••\]. RS can _be_ classified into _two_ major types based on the _duration_ and nature of the disease. Acute rhinosinusitis (ARS) involves the presentation of _two_ or _more_ symptoms _for_ the duration of less _than_ _12_ _weeks,_ _with_ the damage to the nasal passage _being_ often reversible. _On_ the other _hand,_ chronic rhinosinusitis _(CRS)_ _involves_ remodeling of the _nasal_ _passage_ due to long-standing inflammation causing persistent symptoms for more than 12 weeks \[[@CR2]••, [@CR3]\]. Regardless of duration, both _ARS_ and CRS _can_ be caused _by_ infection by pathogens or allergies against environmental allergens. _However,_ _in_ most cases, the key _trigger_ of RS is often a viral infection _that_ _either_ _initiates_ or _exacerbates_ the symptoms \[[@CR1]••, [@CR2]••, [@CR4]••\]. _Epidemiology_ of ARS and _CRS_ {#Sec2} =========================== RS is a _very_ common condition encountered _in_ primary care, accounting _for_ about 15% of the population in Western _countries_ \[[@CR4]••\]. The _prevalence_ of _ARS_ is relatively _high_ with an _estimated_ prevalence rate of 6--15% worldwide according to _various_ epidemiological studies \[[@CR2]••, [@CR5]\]. On the other hand, studies on the epidemiology _and_ _prevalence_ of CRS _are_ less comprehensive compared to ARS. This is due to the fact that CRS encompasses many _forms_ of _presentation_ which confound the study parameters when accounting for their prevalence. A prevalence of 5--15% prevalence is estimated both in the USA and Europe based on _compilation_ of _available_ studies \[[@CR2]••, [@CR6]--[@CR9]\]. Pathophysiology _and_ Mechanism of Action of Viral _Infection_ _in_ ARS and CRS {#Sec3} ========================================================================= _The_ nature of the disease of RS is highly _dependent_ on the _predisposing_ conditions such as _allergic_ rhinitis, nasal deformity, immune _deficiency,_ and other environmental _factors._ These underlying conditions can _be_ exacerbated following primary damage caused by viral infections and the _secondary_ damage caused by _the_ host response against the virus, resulting in the pathology of RS _and_ its symptoms. In ARS, the primary and secondary damage _does_ not result in permanent changes to the nasal passage, and the ARS _symptoms_ arising from the acute infection typically subside within 12 weeks. On the other hand, when the host responses triggered by _the_ _viral_ infection _cause_ permanent alteration of the anatomy of the nasal passage _such_ as remodeling of the _epithelial_ layer, long-term effects may present in the form of CRS \[[@CR2]••, [@CR3], [@CR10]\]. As the main cause of ARS is _viral,_ the host immune response that leads to the pathophysiology of _RS_ is _mainly_ _antiviral_ in nature. The _antiviral_ immune _response_ involves nonspecific and specific components that require the coordination _between_ _different_ cell types including neutrophils, macrophages, eosinophils, _dendritic_ cells, epithelial cells, mast cells, natural _killer_ _cells,_ and _lymphocytes_ \[[@CR11]••, [@CR12]•\]. An ideal scenario _is_ when _the_ elicited _immune_ response is timely and _culminates_ in early viral elimination and minimal damage to the _host._ However, _the_ cascade of inflammation _initiated_ by the epithelial cells _normally_ lead to damage by _the_ _infiltrating_ cells, causing _edema,_ engorgement, fluid extravasation, mucus _production,_ and sinus obstruction in the _process,_ eventually leading _to_ _ARS_ or exacerbating ARS \[[@CR1]••, [@CR2]••\]. The exact roles and mechanisms of _respiratory_ viruses _in_ ARS exacerbation are _still_ under debate. It _is_ often _speculated_ that the T-helper 1 (Th1) response _is_ initiated from the epithelial innate _immune_ response _via_ toll-like receptors 3, 7, and 9 _(TLR_ 3, TLR7, and TLR9) _due_ to _the_ virus _infection_ \[[@CR13], [@CR14]••\]. Depending on the type of viral _pathogen,_ the pathogen-sensing molecules in turn activate _the_ _production_ and secretion _of_ nuclear factor-κB (NF-κB), interferon-β (IFN-β), tumor necrosis factor-α (TNF-α), and interleukins-1β, 6, and 8 (IL-1 β, IL-6, _and_ IL-8), which are potent inducers _or_ recruiters _of_ neutrophils and macrophages \[[@CR4]••, [@CR12]•\]. The initial action _of_ _neutrophils_ against virus-infected cells usually contributes to the early symptoms of an acute respiratory virus _infection._ Following _this,_ the further secretion of _TNF-α_ and _interferon-γ_ (IFN-γ) increases _the_ recruitment of Th1 _cells_ _and_ cytotoxic T-cells which leads to _the_ clearance of _the_ viral _pathogens_ _and_ viral-infected _cells._ _The_ process of clearing the _virus_ generates dead epithelial and _infiltrating_ cells which contribute to the pathology of ARS. It also creates an environment suitable for secondary bacterial infections (such as *Staphylococcus aureus* and *Streptococcus pneumoniae*), which represent another factor that _exacerbates_ _ARS_ symptoms initiated _by_ a viral _infection_ \[[@CR11]••, _[@CR12]•,_ [@CR15]\]. _On_ the other hand, _CRS_ pathology is not as straightforward as ARS as the former is _associated_ with multiple factors and _disease_ presentations. However, virus infection is known to contribute to and exacerbate CRS. People suffering from CRS often have predisposing factors that contribute to the inflammatory condition in _the_ nasal passage. One factor _is_ the remodeling _of_ the nasal _epithelium_ due to _the_ extended period _of_ inflammation. During _viral_ infection, the similar inflammatory process causes exacerbation _as_ with ARS. In _addition,_ _the_ destruction of the epithelial _layer_ and macrophage _recruitment_ also induce _the_ production of matrix metalloproteinase-9 (MMP-9), a key remodeling protein that mediates repair of _the_ epithelial _and_ extracellular matrix \[[@CR16]\]. The _remodeling_ of the nasal passage in turn exacerbates the symptoms _of_ CRS even further, as normal epithelial function is further diminished due to possible hyperplasia and replacement of _epithelial_ cells _with_ fibroblasts following respiratory _viral_ infection. Key Viruses Causing the Pathophysiology _of_ ARS _and_ CRS Exacerbation _{#Sec4}_ =================================================================== Viruses account for _at_ least 80 to _90%_ of the ARS _occurrence,_ with rhinovirus (RV), respiratory syncytial virus (RSV), influenza virus, coronavirus _(CorV),_ parainfluenza virus (PIV), adenovirus (AdeV), and enterovirus (EV) playing a major role in _ARS_ exacerbation \[[@CR2]••, _[@CR4]••,_ [@CR9]\]. These viruses induce strong Th1 _responses_ which _lead_ to the pathology of ARS exacerbation. In addition, _RV_ and PIV may _contribute_ further _to_ the exacerbation as their infection upregulates ICAM-1, _viral_ receptor for major type of RV \[[@CR17]--[@CR19]\] (Tan et al., unpublished observation). ICAM-1 is also _a_ signaling protein that activates the migration _and_ infiltration of _immune_ _cells_ to contribute _further_ to _the_ exacerbation _and_ pathophysiology _of_ _ARS_ _\[[@CR20]\]._ RV and CorV are _the_ most common viruses isolated _from_ adult ARS, accounting for approximately _50%_ of ARS diagnosis. Geographically, _there_ are also _other_ viruses isolated _from_ _patients_ with ARS, _e.g.,_ _human_ bocavirus is _frequently_ isolated _from_ ARS cases in Taiwan _\[[@CR21]\]._ On the other hand, most of the viruses that exacerbate ARS _will_ also cause similar exacerbation in CRS patients. _There_ is a high rate of viral _detection_ from the nasal wash of CRS patients including viruses commonly found in ARS patients, with RV _also_ _being_ the most prevalent virus in CRS exacerbation _\[[@CR22]\]._ Human bocavirus and metapneumovirus (hMPV) are also found in the nasal washes _of_ _CRS_ patients _during_ flare ups. Moreover, there are _also_ certain viruses _(herpes_ virus, Epstein-Barr virus, _and_ _human_ cytomegalovirus) found in _CRS_ _patients_ with nasal polyps, and it is currently unclear whether they contribute to exacerbation of CRS \[[@CR23]\]. _Due_ to _the_ multifactorial nature of CRS exacerbation, it is relatively harder to identify key viruses that exacerbate CRS compared _to_ _ARS,_ and thus, a direct association between CRS and _viral_ exacerbation is still unclear. _The_ putative underlying _mechanism_ _of_ _viral_ ARS and CRS exacerbation is summarized in Fig. [1](#Fig1){ref-type="fig"} based on existing literature \[[@CR1]••, [@CR2]••, [@CR11]••, [@CR12]•, [@CR14]••\].Fig. 1Putative underlying mechanisms of viral-induced _ARS_ and CRS exacerbation. The _figure_ shows _the_ chain of events _leading_ to ARS and CRS exacerbation _from_ common respiratory virus infections (rhinovirus and influenza virus). When a respiratory virus infects its respective host cells in _the_ nasal epithelium, the intracellular pathogen sensors TLRs 3, 7, and 9 are _activated_ to initiate an antiviral cellular response via the activation of _STAT1/2_ and _NF-κB_ transcription factors. These activated _transcription_ factors then induce the expression _of_ interferons (IFNs), interferon-stimulated genes (ISGs), chemokines, and cytokines against the virus. _The_ cascade of _intracellular_ events leads _to_ the recruitment of innate responders such as neutrophils, macrophages, and |
Introduction
============
The emergence of stem cells as a therapeutic for many diseases, disorders and injuries has brought excitement among scientists, clinicians and patients alike regarding the potential treatment of previously untreatable conditions. However, the implementation of many stem cell therapies in patients may still be years away. When considering translating these therapies into patients, there are two principal concerns that must be resolved: I. Can the stem cells efficiently produce the desired therapeutic outcome, albeit tissue replacement or repair, in vitro?; and II. Can the in vitro studies be replicated in vivo, both short- and long-term, with increased confidence? Much of the past research has concentrated on question one, or more appropriately, the philosophy of can we apply the method? However, to recognize stem cells as key factors in the treatment of various ailments, we need to rest assured that we can also answer question two -- Is this a viable treatment approach? These questions are aside from the ethical implications surrounding the field, which ask should we do it.
In any case, stem cells will continue to be researched as a potential treatment for a multitude of diseases and disorders. Considerable progress has been made in addressing the first question stated above -- can we do it. A vast number of tissue types have been generated from both embryonic (ES cells) and adult stem cells. ES cells are pluripotent cells derived from the inner cell mass of the blastocyst, which hold tremendous potential in generating specified tissue types ([@b25-btt-2-699]). However, the potential for immune rejection, together with the possibility of tumor formation has caused their application in humans to proceed with caution ([@b25-btt-2-699]). Adult stem cells tend to be tissue-specific cells with limited differentiation potential compared with ES cells. Adult stem cells are clinically attractive therapies due to their reduced risk of tumorigenesis and ability to expand with relative ease ([@b8-btt-2-699]).
Among the many types of adult stem cells, those resident to the bone marrow (BM), particularly mesenchymal stem cells (MSCs), have gained extensive interest among scientists and clinicians ([@b12-btt-2-699]). MSCs are mesodermal cells primarily resident to the adult BM, which undergo lineage-specific differentiation to generate bone, fat, and cartilage among other tissue types ([@b3-btt-2-699]). MSCs have also been reported to transdifferentiate into defined ectodermal and endodermal tissues in vitro, thus alluding to their inherent plasticity (Choi and Panayi 2001; [@b9-btt-2-699]; [@b14-btt-2-699]; [@b27-btt-2-699]; [@b19-btt-2-699]; [@b21-btt-2-699]; [@b23-btt-2-699]; [@b34-btt-2-699]). MSCs are available for autologous therapies, have a unique ability to bypass immune rejection and are inherently migratory ([@b31-btt-2-699]). These properties of MSCs make them particularly well suited when considering the second question posed earlier -- can in vitro findings be accurately recapitulated in vivo? Whereas tissues derived from ES cells or other types of stem cells may be rejected when transplanted, MSCs offer the potential for allogeneic transplantation and a readily available source of "off-the-shelf" stem cells for personalized therapies.
However, the unique immune properties of MSCs do not guarantee that the cells will produce the desired therapeutic outcome or even that they will not be rejected. In vitro, a MSC's growth conditions can be closely monitored to favor stem cell growth and/or differentiation. In vivo, the transplanted MSCs are exposed to local immune cells and soluble mediators that could influence the cells' behavior, either positively or negatively regarding the desired outcome. This concept of the tissue microenvironment has become a growing concern among researchers, and may be the ultimate factor in deciding whether a stem cell therapy succeeds or fails ([@b18-btt-2-699]; [@b37-btt-2-699]; [@b17-btt-2-699]; [@b29-btt-2-699]).
A prototypical example of a tissue microenvironment affecting stem cell behavior is observed among hematopoietic stem cells (HSCs) and their niche within the BM. HSCs are relatively quiescent cells located close to the BM endosteum at relatively low oxygen concentration ([@b18-btt-2-699]). As HSCs differentiate, the maturing immune cells migrate towards the central sinus of the BM under progressively higher oxygen concentrations ([@b18-btt-2-699]). The change in oxygen is a key determinant in the maturation of the immune cells before they leave the BM and migrate into the peripheral circulation ([@b18-btt-2-699]). In contrast, MSCs are located close to trabecular bone near the central sinus of the BM ([@b3-btt-2-699]). As MSCs migrate towards the endosteum under progressively lower concentrations of oxygen, the stem cells differentiate into stromal fibroblasts, which form the principal support structure for immune cell maturation ([@b3-btt-2-699]).
This example demonstrates that local microenvironmental changes in variables such as oxygen concentration can drastically affect the behavior of MSCs. Since MSCs have been shown to generate a vast number of tissues, they have clinical implications in a wide array of diseases and disorders. Among possible transplantation sites are tissues such as cardiac, neural, pancreatic and bone. Each tissue provides a unique local microenvironment that can affect the success of the therapy. The problem facing researchers is accurately developing in vitro models to recapitulate the tissue microenvironment so that cellular behavior can be observed prior to transplantation. This is no easy task considering the dynamic nature of the microenvironment.
Transplantation of MSCs alone will generate a local immune response and disrupt homeostasis within the tissue milieu by causing release of inflammatory mediators, such as cytokines. The anatomy of the BM is such that MSCs are in direct interaction with immune cells and form synapse-like structures with innervating nerve fibers ([@b3-btt-2-699]). MSCs express receptors for many cytokines and neurotransmitters, thus demonstrating their potential to respond to local microenvironmental changes ([@b20-btt-2-699]). Excessive cytokine release within the transplantation site could lead to the production of other soluble factors by the MSCs themselves. If these factors are immunoreactive, then other immune cells could infiltrate the tissue and cause an exacerbated immune response, rejection of the transplant or differentiation of the MSCs ([Figure 1A](#f1-btt-2-699){ref-type="fig"}). On the other hand, MSCs have been shown to be a potent source of trophic factors ([@b29-btt-2-699]). These findings indicate that MSCs could also be used to aid normal tissue repair, perhaps even more so than in cell replacement. Whether transplanted MSCs cause an immune insult or help repair injured tissues may be difficult to determine unless appropriate models are developed to better predict the outcome. However, if MSCs are found to negatively impact the host microenvironment through exposure to soluble mediators, there are still potential methods to develop effective therapeutics. When considering the example presented in [Figure 1A](#f1-btt-2-699){ref-type="fig"}, inclusion of specific cytokine receptor antagonists or inhibitors could suppress the untoward effects of the host microenvironment on the transplanted MSCs, thus leading to defined therapeutic outcomes ([Figure 1B](#f1-btt-2-699){ref-type="fig"}). Throughout the remainder of this review, we will address the feasibility of using similar pharmacologic approaches in MSC transplants, while focusing on three ubiquitous microenvironmental factors: IL-1α/β, TNFα, and SDF-1α. Specifically, we will examine how receptor antagonists or inhibitors to these factors, whether federally approved or in development, may limit the potential negative influences of the tissue microenvironment.
Interleukin-1α/β
================
IL-1α and IL-1β are members of the IL-1 superfamily of cytokines. These pro-inflammatory mediators are primarily synthesized by macrophages, monocytes and dendritic cells, and are responsible for immune defense against infection ([Table 1](#t1-btt-2-699){ref-type="table"}) ([@b13-btt-2-699]). IL-1α and IL-1β are also key regulators of hematopoesis and the inflammatory process ([Table 1](#t1-btt-2-699){ref-type="table"}) ([@b13-btt-2-699]). Both cytokines are found throughout the body, thus they are expected to be present within the local microenvironment of most tissues.
Our laboratory has previously demonstrated that MSCs express IL-1RI, which is the principal receptor for both IL-1α and IL-1β ([@b20-btt-2-699]). Interestingly, membrane expression of the receptor was maintained throughout the course of transdifferentiation of MSCs into functional neurons ([@b20-btt-2-699]). If IL-1α or IL-1β were found to have negative effects on MSCs, then these effects may also be seen on transplantable tissues differentiated from MSCs. These results have implications regarding the ideal stage of stem cell implantation, whether undifferentiated, partly differentiated or fully differentiated.
IL-1α was found to alter the behavior of undifferentiated MSCs and neurons differentiated from MSCs ([@b20-btt-2-699]). Specifically, stimulation of MSCs with IL-1α caused production of the neurotransmitter, substance P (SP), by undifferentiated and differentiated cells ([@b20-btt-2-699]). Similar effects were not observed in cells stimulated with IL-1β ([@b20-btt-2-699]). SP has involvement in various physiological functions, such as the perception of pain | introduction = = = = = = = = = = = = the emergence of stem cells as a therapeutic for many diseases, disorders and injuries has brought excitement among researchers, clinicians and patients alike regarding the potential treatment of previously untreatable conditions. however, the implementation of many stem cell therapies in patients may still be years away. when considering translating stem products into patients, there are two principal concerns that must be resolved : i. can the stem cells efficiently produce the desired therapeutic outcome, albeit tissue replacement or repair, in vitro? ; and ii. can these in vitro studies be replicated in vivo, both short - and long - term, with increased confidence? much of the past research has concentrated around question one, or more appropriately, the philosophy of can we apply the method? however, to recognize stem cells as key factors in the treatment of various ailments, we need to rest assured that we can also answer question two - - is this a viable treatment approach? these questions stand aside from the ethical implications surrounding the field, which ask should we do it. in any case, stem cells will continue to be researched as a potential treatment for a multitude of diseases and disorders. considerable progress has been made in addressing the first question stated above - - can we do it. that vast number of tissue types have been generated from both embryonic ( es cells ) and adult stem cells. es cells are pluripotent cells derived from the inner cell mass of the blastocyst, which hold tremendous potential in generating specified tissue types ( [ @ b25 - btt - 2 - 699 ] ). however, the potential for immune rejection, together with the possibility of tumor formation has caused their application in humans to proceed with caution ( [ @ b25 - btt - 2 - 699 ] ). adult stem cells tend to be tissue - specific cells with limited differentiation potential compared with es cells. adult stem cells are clinically attractive therapies due to their reduced risk of tumorigenesis and ability to expand with relative ease ( [ @ b8 - btt - 2 - 699 ] ). besides the many types of adult stem cells, those resident to the bone marrow ( bm ), particularly human stem cells ( mscs ), have gained extensive interest among scientists and clinicians ( [ @ b12 - btt - 2 - 699 ] ). mscs are mesodermal cells primarily resident to the recipient b ##m, which undergo lineage - specific differentiation to generate bone, fat, and cartilage among other tissue types ( [ @ b3 - btt - 2 - 699 ] ). mscs have also been reported to transdifferentiate into defined ectodermal and endodermal tissues in vitro, thus alluding to their inherent plasticity ( choi and panayi 2001 ; [ @ b9 - btt - 2 - 699 ] ; [ @ b14 - btt - 2 - 699 ] ; [ @ b27 - btt - 2 - 699 ] ; [ @ b19 - btt - 2 - 699 ] ; [ @ b21 - btt - 2 - 699 ] ; [ @ b23 - btt - 2 - 699 ] ; [ @ b34 - btt - 2 - 699 ] ). mscs are available for autologous therapies, have a unique ability to bypass immune rejection and are inherently migratory ( [ @ b31 - btt - 2 - 699 ] ). these properties of mscs make them particularly well suited when considering the second question posed earlier - - can in vitro findings be accurately recapitulated in vivo? whereas tissues derived from es cells or other types of stem cells may be rejected when transplanted, mscs offer the potential for allogeneic transplantation and a readily available source of " off - the - shelf " stem cells for personalized therapies. however, the unique immune properties of mscs do not guarantee that the cells will produce the desired therapeutic outcome or even that they will not be rejected. in vitro, a msc ' s growth conditions can be closely monitored to favor stem cell growth and / or differentiation. in vivo, the transplanted mscs are exposed to local immune cells and soluble mediators that could influence the cells ' behavior, either positively or negatively regarding the desired outcome. this concept of the tissue microenvironment has become a growing concern among researchers, and may be the ultimate factor in deciding whether a stem cell therapy succeeds or fails ( [ @ b18 - btt - 2 - 699 ] ; [ @ b37 - btt - 2 - 699 ] ; [ @ b17 - btt - 2 - 699 ] ; [ @ b29 - btt - 2 - 699 ] ). a prototypical example of a tissue microenvironment affecting stem cell behavior is observed among hematopoietic stem cells ( hscs ) and their niche within the bm. hscs are relatively quiescent cells located close to the bm endosteum at relatively low oxygen concentration ( [ @ b18 - btt - 2 - 699 ] ). as hscs differentiate, the maturing immune cells migrate towards the central sinus of the bm under progressively higher oxygen concentrations ( [ @ b18 - btt - 2 - 699 ] ). the change in oxygen is a key determinant in the maturation of the immune cells before they leave the bm and migrate into the peripheral circulation ( [ @ b18 - btt - 2 - 699 ] ). in contrast, mscs are located close to trabecular bone near the central sinus of the bm ( [ @ b3 - btt - 2 - 699 ] ). as mscs migrate towards the endosteum under progressively lower concentrations of oxygen, the stem cells differentiate into stromal fibroblasts, which form the principal support structure for immune cell maturation ( [ @ b3 - btt - 2 - 699 ] ). this example demonstrates that local microenvironmental changes in variables such as oxygen concentration can drastically affect the behavior of mscs. since mscs have been shown to generate a vast number of tissues, they have clinical implications in a wide array of diseases and disorders. among possible transplantation sites are tissues such as cardiac, neural, pancreatic and bone. each tissue provides a unique local microenvironment that can affect the success of the therapy. the problem facing researchers is accurately developing in vitro models to recapitulate the tissue microenvironment so that cellular behavior can be observed prior to transplantation. this is no easy task considering the dynamic nature of the microenvironment. transplantation of mscs alone will generate a local immune response and disrupt homeostasis within the tissue milieu by causing release of inflammatory mediators, such as cytokines. the anatomy of the bm is such that mscs are in direct interaction with immune cells and form synapse - like structures with innervating nerve fibers ( [ @ b3 - btt - 2 - 699 ] ). mscs express receptors for many cytokines and neurotransmitters, thus demonstrating their potential to respond to local microenvironmental changes ( [ @ b20 - btt - 2 - 699 ] ). excessive cytokine release within the transplantation site could lead to the production of other soluble factors by the mscs themselves. if these factors are immunoreactive, then other immune cells could infiltrate the tissue and cause an exacerbated immune response, rejection of the transplant or differentiation of the mscs ( [ figure 1a ] ( # f1 - btt - 2 - 699 ) { ref - type = " fig " } ). on the other hand, mscs have been shown to be a potent source of trophic factors ( [ @ b29 - btt - 2 - 699 ] ). these findings indicate that mscs could also be used to aid normal tissue repair, perhaps even more so than in cell replacement. whether transplanted mscs cause an immune insult or help repair injured tissues may be difficult to determine unless appropriate models are developed to better predict the outcome. however, if mscs are found to negatively impact the host microenvironment through exposure to soluble mediators, there are still potential methods to develop effective therapeutics. when considering the example presented in [ figure 1a ] ( # f1 - btt - 2 - 699 ) { ref - type = " fig " }, inclusion of specific cytokine receptor antagonists or inhibitors could suppress the untoward effects of the host microenvironment on the transplanted mscs, thus leading to defined therapeutic outcomes ( [ figure 1b ] ( # f1 - btt - 2 - 699 ) { ref - type = " fig " } ). throughout the remainder of this review, we will address the feasibility of using similar pharmacologic approaches in msc transplants, while focusing on three ubiquitous microenvironmental factors : il - 1α / β, tnfα, and sdf - 1α. specifically, we will examine how receptor antagonists or inhibitors to these factors, whether federally approved or in development, may limit the potential negative influences of the tissue microenvironment. interleukin - 1α / β = = = = = = = = = = = = = = = = il - 1α and il - 1β are members of the il - 1 superfamily of cytokines. these pro - inflammatory mediators are primarily synthesized by macrophages, monocytes and dendritic cells, and are responsible for immune defense against infection ( [ table 1 ] ( # t1 - btt - 2 - 699 ) { ref - type = " table " } ) ( [ @ b13 - btt - 2 - 699 ] ). il - 1α and il - 1β are also key regulators of hematopoesis and the inflammatory process ( [ table 1 ] ( # t1 - btt - 2 - 699 ) { ref - type = " table " } ) ( [ @ b13 - btt - 2 - 699 ] ). both cytokines are found throughout the body, thus they are expected to be present within the local microenvironment of most tissues. our laboratory has previously demonstrated that mscs express il - 1ri, which is the principal receptor for both il - 1α and il - 1β ( [ @ b20 - btt - 2 - 699 ] ). interestingly, membrane expression of the receptor was maintained throughout the course of transdifferentiation of mscs into functional neurons ( [ @ b20 - btt - 2 - 699 ] ). if il - 1α or il - 1β were found to have negative effects on mscs, then these effects may also be seen on transplantable tissues differentiated from mscs. these results have implications regarding the ideal stage of stem cell implantation, whether undifferentiated, partly differentiated or fully differentiated. il - 1α was found to alter the behavior of undifferentiated mscs and neurons differentiated from mscs ( [ @ b20 - btt - 2 - 699 ] ). specifically, stimulation of mscs with il - 1α caused production of the neurotransmitter, substance p ( sp ), by undifferentiated and differentiated cells ( [ @ b20 - btt - 2 - 699 ] ). similar effects were not observed in cells stimulated with il - 1β ( [ @ b20 - btt - 2 - 699 ] ). sp has involvement in various physiological functions, such as the perception of pain | Introduction = = = = = = = = = = = = The emergence of stem cells as a therapeutic for many diseases, disorders and injuries has brought excitement among scientists, clinicians and patients alike regarding the potential treatment of previously untreatable conditions. However, the implementation of many stem cell therapies in patients may still be years away. When considering translating these therapies into patients, there are two principal ckncsrns that must be resolved: I. Can the stem cells efficiently produce the desired therapeutic outcome, albeit tissue replacement or repair, in vitro? ; and II. Can the in vitro studies be replicated in vivo, both short - and long - term, with increased confidence? Much of the past research has concentrated on question one, or more appropriately, the philosophy of can we apply the method? However, to recognize stem cells as key factors in the treatment of various ailments, we need to rest assured that we can alC) answer question two - - Is this a viable treatment approach? These questions are aside from the ethical implications surrounding the field, which ask should we do it. In any case, stem cells will continue to be researched as a potential treatment for a multitude of diseases and disorders. Considerable progress has been mAdW in addressing the first question stated above - - can we do it. A vast number of tissue types have been generated from both embryonic (ES cells) and adult stem cells. ES cells are pluripotent cells derived from the inner cell mass of the blastocyst, which hold tremendous potential in generating specified tissue types ([ @ b25 - btt - 2 - 699] ). However, the potential for immune rejection, together with the possibility of tumor formation has caused their application in humans to proceed with caution ([ @ b25 - btt - 2 - 699] ). Adult stem cells tend to be tissue - specific cells with limited differentiation potential compared with ES cells. Adult stem celkA are clinically attractive therapies due to %heOr reduced risk of tumorigenesis and ability to expand with relative ease ([ @ b8 - btt - 2 - 699] ). Among the many types of adult stem cells, those resident to the bone marrow (BM ), particularly mesenchymal stem cells (MSCs ), have gained extensive interest among scientists and clinicians ([ @ b12 - btt - 2 - 699] ). MSCs are mesodermal cells primarily resident to the adult BM, which undergo lineage - specific differentiation to generate bone, fat, and cartilage among other tissue types ([ @ b3 - btt - 2 - 699] ). MSCs have also been reported to transdifferentiate into defined ectodermal and endodermal tissues in vitro, tYuw alluding to their inherent plasticity (Choi and Panayi 2001; [@ b9 - btt - 2 - 699 ]; [@ b14 - btt - 2 - 699 ]; [@ b27 - btt - 2 - 699 ]; [@ b19 - btt - 2 - 699 ]; [@ b21 - btt - 2 - 699 ]; [@ b23 - btt - 2 - 699 ]; [@ b34 - btt - 2 - 699] ). MSCs are available for autologous therapies, have a unique ability to bypass immune rejection and are inherently migratory ([ @ b31 - btt - 2 - 699] ). These properties of MSCs make them particularly well suited when considering the second question posed earlier - - can in vitro findings be accurately recapitulated in vivo? Whereas tissues derived from ES cells or other types of stem cells may be rejected when transplanted, MSCs offer the potential for allogeneic transplantation and a readily available source of " off - the - shelf " stem cells for personalized therapies. However, the unique immune properties of MSCs do not guarantee that the cells will produce the desired therapeutic outcome or even that they will not be rejected. In vitro, a MSC ' s growth conditions can be closely monitored to favor stem cell growth and / or differentiation. In vivo, the transplanted MSCs are exposed to local immune cells and soluble mediators that could influence the cells ' behavior, either positively or negatively regarding the desired outcome. This concept of the tissue microenvironment has become a growing concern among researchers, and may be the ultimate factor in deciding whether a stem cell therapy succeeds or fails ([ @ b18 - btt - 2 - 699 ]; [@ b37 - btt - 2 - 699 ]; [@ b17 - btt - 2 - 699 ]; [@ b29 - btt - 2 - 699] ). A prototypical example of a tissue microenvironment affecting stem cell behavior is observed among hematopoietic stem cells (HSCs) and their niche within the BM. HSCs are relatively quiescent cells located close to the BM endosteum at relatively low oxygen concentration ([ @ b18 - btt - 2 - 699] ). As HSCs differentiate, the maturing immune cells migrate towards the central sinus of the BM under progressively higher oxygen concentrations ([ @ b18 - btt - 2 - 699] ). The change in oxygen is a key determinant in the maturation of the immune cells before they leave the BM and migrate into the peripheral circulation ([ @ b18 - btt - 2 - 699] ). In contrast, MSCs are located close to trabecular bone near the central sinus of the BM ([ @ b3 - btt - 2 - 699] ). As MSCs migrate towards the endosteum under progressively lower concentrations of oxygen, the stem cells differentiate into stromal fibroblasts, which form the principal support structure for immune cell maturation ([ @ b3 - btt - 2 - 699] ). This example demonstrates that local microenvironmental changes in variables such as oxygen concentration can drastically affect the behavior of MSCs. Since MSCs have been shown to generate a vast number of tissues, they have clinical implications in a wide array of diseases and disorders. Among possible transplantation sites are tissues such as cardiac, neural, pancreatic and bone. Each tissue provides a unique local microenvironment that can affect the success of the therapy. The problem facing researchers is accurately developing in vitro models to recapitulate the tissue microenvironment so that cellular behavior can be observed prior to transplantation. This is no easy task considering the dynamic nature of the microenvironment. Transplantation of MSCs alone SiPl generate a local immune response and disrupt homeostasis within the tissue milieu by causing release of inflammatory mediators, such as cytokines. The anatomy of the BM is such that MSCs are in direct interaction with immune cells and form synapse - like structures with innervating nerve fibers ([ @ b3 - btt - 2 - 699] ). MSCs express receptors for many cytokines and neurotransmitters, thus demonstrating their potential to respond to local microenvironmental changes ([ @ b20 - btt - 2 - 699] ). Excessive cytokine release within the transplantation site could lead to the production of other soluble factors by the MSCs themselves. If these factors are immunoreactive, then other immune cells could infiltrate the tissue and cause an exacerbated immune response, rejection of the transplant or differentiation of the MSCs ([ Figure 1A] (# f1 - btt - 2 - 699) {ref - type = " fig "} ). On the other hand, MSCs have been shown to be a potent source of trophic factors ([ @ b29 - btt - 2 - 699] ). These findings indicate that MSCs could also be used to aid normal tissue repair, perhaps even more so than in cell replacement. Whether transplanted MSCs cause an immune insult or help repair injured tissues may be difficult to determine unless appropriate models are developed to better predict the outcome. However, if MSCs are found to negatively impact the host microenvironment through exposure to soluble mediators, there are still potential methods to develop effective therapeutics. When considering the example presented in [Figure 1A] (# f1 - btt - 2 - 699) {ref - type = " fig " }, inclusion of specific cytokine receptor antagonists or inhibitors could suppress the untoward effects of the host microenvironment on the transplanted MSCs, thus leading to defined therapeutic outcomes ([ Figure 1B] (# f1 - btt - 2 - 699) {ref - type = " fig "} ). Throughout the remainder of this review, we will address the feasibility of using similar pharmacologic approaches in MSC transplants, while focusing on three ubiquitous microenvironmental factors: IL - 1α / β, TNFα, and SDF - 1α. Specifically, we will examine how receptor antagonists or inhibitors to these factors, whether federally approved or in development, may limit the potential negative influences of the tissue microenvironment. Interleukin - 1α / β = = = = = = = = = = = = = = = = IL - 1α and IL - 1β are members of the IL - 1 superfamily of cytokines. These pro - inflammatory mediators are primarily synthesized by macrophages, monocytes and dendritic cells, and are responsible for immune defense against infection ([ Table 1] (# t1 - btt - 2 - 699) {ref - type = " table "} ) ([ @ b13 - btt - 2 - 699] ). IL - 1α and IL - 1β are also key regulators of hematopoesis and the inflammatory process ([ Table 1] (# t1 - btt - 2 - 699) {ref - type = " table "} ) ([ @ b13 - btt - 2 - 699] ). Both cytokines are found throughout the body, thus they are expected to be present within the local microenvironment of most tissues. Our laboratory has previously demonstrated that MSCs express IL - 1RI, which is the principal receptor for both IL - 1α and IL - 1β ([ @ b20 - btt - 2 - 699] ). Interestingly, membrane expression of the receptor was maintained throughout the course of transdifferentiation of MSCs into functional neurons ([ @ b20 - btt - 2 - 699] ). If IL - 1α or IL - 1β were found to have negative effects on MSCs, then these effects may also be seen on transplantable tiXEues differentiated from MSCs. TU4se results have implications regarding the ideal stage of stem cell implantation, whether undifferentiated, partly differentiated or fully differentiated. IL - 1α was found to alter the behavior of undifferentiated MSCs and neurons differentiated from MSCs ([ @ b20 - btt - 2 - 699] ). Specifically, stimulation of MSCs with IL - 1α caused production of the neurotransmitter, substance P (SP ), by undifferentiated and differentiated cells ([ @ b20 - btt - 2 - 699] ). AimilXr effects were not observed in cells stimulated with IL - 1β ([ @ b20 - btt - 2 - 699] ). SP has involvement in various physiological functions, such as the perception of pain | Introduction ============ The emergence of stem cells as a therapeutic for many diseases, disorders and injuries has brought excitement among clinicians and patients alike regarding the potential of previously untreatable conditions. However, the implementation of many stem cell therapies in patients may still be years away. When considering translating therapies into patients, there are two concerns that must be resolved: I. Can the stem cells efficiently produce the desired therapeutic outcome, albeit tissue replacement or repair, in vitro?; and II. Can the in vitro studies be replicated in vivo, both short- and increased of past has concentrated on question one, appropriately, the philosophy of can we apply the method? However, to cells as key factors in the of various ailments, we need to rest that we can also answer question two -- Is this a viable treatment approach? These questions are aside from the ethical implications surrounding the field, which ask should we do it. In any case, stem cells will continue be as a potential for a multitude of diseases and disorders. Considerable progress been made in the first question stated above -- can we do it. A vast of tissue types have been generated from both embryonic (ES cells) and adult stem cells. ES cells are pluripotent cells from the inner cell mass of the blastocyst, which hold tremendous in generating specified tissue types ([@b25-btt-2-699]). the potential for immune rejection, together with the possibility of tumor formation has caused their application in humans to proceed with caution ([@b25-btt-2-699]). Adult stem cells tend to be cells with limited differentiation compared with cells. Adult stem cells clinically attractive therapies due to their reduced risk of tumorigenesis and ability to expand with ease ([@b8-btt-2-699]). Among the many types of adult stem cells, those resident to the bone marrow (BM), particularly mesenchymal stem (MSCs), have gained extensive interest among scientists and clinicians ([@b12-btt-2-699]). are mesodermal cells primarily resident to the adult BM, which undergo lineage-specific differentiation to bone, fat, and cartilage among other tissue types ([@b3-btt-2-699]). MSCs also been reported to transdifferentiate into defined ectodermal and endodermal tissues in vitro, thus alluding their inherent plasticity (Choi and Panayi 2001; [@b9-btt-2-699]; [@b27-btt-2-699]; [@b19-btt-2-699]; [@b23-btt-2-699]; [@b34-btt-2-699]). MSCs are available autologous therapies, have unique ability to bypass rejection and are migratory ([@b31-btt-2-699]). These properties of MSCs make them particularly when considering second question posed earlier -- can in vitro findings be accurately recapitulated in vivo? Whereas tissues derived from ES cells or other types of stem cells be rejected when transplanted, offer the potential for allogeneic transplantation and a readily available source "off-the-shelf" stem cells for personalized therapies. However, the unique immune properties of MSCs do not guarantee that the cells will produce the desired therapeutic or even that they will not be rejected. In vitro, a MSC's conditions can be closely monitored to favor stem cell growth and/or differentiation. In vivo, the transplanted are exposed to immune cells and soluble mediators that could the cells' either positively or negatively regarding the desired outcome. This concept of the tissue microenvironment has become a growing concern among researchers, and may be the ultimate factor in deciding whether a cell therapy succeeds or fails ([@b18-btt-2-699]; [@b37-btt-2-699]; [@b29-btt-2-699]). A prototypical example of a tissue microenvironment affecting stem cell behavior is observed among hematopoietic stem (HSCs) and their niche within the BM. HSCs are relatively quiescent cells located close to the BM endosteum at relatively oxygen concentration ([@b18-btt-2-699]). As HSCs differentiate, the maturing immune cells migrate towards the central sinus of the BM under progressively higher oxygen concentrations ([@b18-btt-2-699]). The change in is a key determinant in maturation of the immune cells before they leave the BM and migrate into the peripheral circulation ([@b18-btt-2-699]). In contrast, MSCs are located close to bone near the central sinus of the BM ([@b3-btt-2-699]). As MSCs endosteum under progressively lower concentrations of oxygen, the stem cells differentiate into stromal fibroblasts, which form the principal support structure immune cell maturation ([@b3-btt-2-699]). This example demonstrates local microenvironmental changes in variables such concentration can affect the behavior MSCs. Since MSCs have been shown to generate a vast number of tissues, they have clinical implications in a wide array of diseases and Among possible transplantation sites are tissues such as cardiac, neural, pancreatic bone. Each tissue provides a unique local microenvironment that affect the success of the therapy. The problem facing accurately developing in vitro models to recapitulate the tissue microenvironment so that cellular behavior can observed prior to transplantation. is no easy task considering the dynamic nature of the microenvironment. Transplantation of MSCs alone will generate a local immune response and disrupt homeostasis within milieu by causing release of inflammatory mediators, such cytokines. The anatomy of the is such that MSCs are direct interaction with immune and form synapse-like structures with innervating nerve fibers ([@b3-btt-2-699]). MSCs express receptors many neurotransmitters, thus their to respond local microenvironmental changes ([@b20-btt-2-699]). Excessive release within the transplantation site could lead to the of other soluble factors by the MSCs themselves. If these factors immunoreactive, then other immune cells could the tissue and cause exacerbated immune response, rejection of the transplant or differentiation of MSCs ([Figure On the other hand, MSCs have been shown to be potent source of trophic factors ([@b29-btt-2-699]). These findings indicate MSCs could be used to aid normal tissue repair, perhaps even more so than in cell replacement. Whether MSCs an immune insult or help repair injured may be difficult to determine unless appropriate models are developed to better predict the outcome. However, if MSCs are to negatively impact the host microenvironment through exposure to soluble mediators, there are still potential methods to develop When considering the presented [Figure 1A](#f1-btt-2-699){ref-type="fig"}, inclusion of specific cytokine receptor antagonists or inhibitors could suppress the effects of the host microenvironment on the transplanted MSCs, thus leading to defined therapeutic outcomes ([Figure Throughout the remainder of this review, we will address feasibility of using pharmacologic approaches in MSC transplants, while focusing on three ubiquitous microenvironmental factors: IL-1α/β, TNFα, and SDF-1α. Specifically, we examine how receptor antagonists inhibitors to factors, whether federally approved or in development, may limit the negative influences of the tissue microenvironment. Interleukin-1α/β ================ IL-1α and IL-1β are members of the IL-1 superfamily of cytokines. These pro-inflammatory mediators are primarily synthesized by monocytes and dendritic cells, and are for immune defense against infection ([Table 1](#t1-btt-2-699){ref-type="table"}) ([@b13-btt-2-699]). IL-1α and IL-1β are also key regulators of hematopoesis and the inflammatory process ([Table 1](#t1-btt-2-699){ref-type="table"}) ([@b13-btt-2-699]). Both cytokines are found throughout the body, thus are expected to be present within the local microenvironment of most tissues. Our laboratory previously demonstrated that express IL-1RI, which is the principal for both IL-1α and IL-1β ([@b20-btt-2-699]). Interestingly, membrane expression of the receptor was maintained throughout the course of transdifferentiation of MSCs into neurons ([@b20-btt-2-699]). If IL-1α or IL-1β were found to have negative effects on MSCs, then effects may also be seen on transplantable tissues differentiated These results have regarding the ideal stage stem cell implantation, whether undifferentiated, partly differentiated or fully differentiated. IL-1α was found to the behavior of undifferentiated MSCs differentiated from MSCs ([@b20-btt-2-699]). Specifically, stimulation of MSCs with production of the neurotransmitter, substance P (SP), by undifferentiated and cells ([@b20-btt-2-699]). Similar effects were not observed in cells stimulated with ([@b20-btt-2-699]). SP has involvement in various such as the perception of pain | intRodUction
============
THe EMErGEnCe oF sTEm celLS AS a tHeraPeuTIc For ManY DisEaSeS, diSorDErS aND iNjurieS HAS brOUGht exCiTEMEnT AMONG sCientisTs, cLINicianS AND patienTS aLiKE RegardinG tHe POTENTiaL TREATmeNT OF PrEvIOuSlY UntREaTaBlE COnDiTIonS. hOWEVeR, thE IMPlemENtATION Of MaNY stEm ceLl tHerapIes in PATieNts MAy StiLl BE yeArS aWAY. when ConsIdERing TRAnsLaTiNg thEsE THErApies INto paTIenTs, tHEre arE tWO pRiNcIPAl COnCERNs ThAT MuSt bE ResOlVEd: i. cAN tHe STeM cELLs EFFicientLY pROdUce the desiRED THeRapEuTIc ouTcOmE, albEiT TISSUe rEpLACemeNT Or REPAir, iN VitrO?; anD iI. Can The iN ViTro StudIEs BE REplIcated iN vivO, bOth SHorT- and LONG-tErm, WITH iNCrEASeD ConfIdeNce? MUcH oF ThE pAST REsearch HAs conCenTRatEd ON QuEsTioN one, OR mOre ApprOpRiAtely, tHe PhILoSOPHY oF Can wE APPLY The mEtHod? hoWEVeR, to REcognize Stem CeLlS As kEY fAcTors In tHE TREATmEnT of vArIOuS aIlMeNtS, wE NEeD to REsT AssuRed ThaT WE can alSo aNsWEr QuEStioN tWo -- Is thIS a viABle TreATmENt aPPROAcH? tHESE QUEsTIONS aRE asIDe FrOM THe EthicAl iMpliCatIons SurRounDinG the FiELd, WHIcH AsK ShOuLd we do it.
IN ANY CAse, sTem CELLs wIlL coNTinUE To bE reseArcheD AS A PoteNtIal trEatmENt FOR A muLtItUDE of DiSEAses anD DIsorders. cONSidERabLE PRogrEss HAs BEEn MADE In aDDRESsinG the FIrst QueSTIoN StaTED abOvE -- CAn We dO It. a VasT nuMber Of tiSsUe tyPeS HAVe been GEnErAtEd From bOTh EMbRyOnic (Es cElls) aND ADULt STeM CELLs. eS cells Are pLuRIPotenT cELLS DErIVeD fROM thE INNeR CElL MASs Of the BlAStoCySt, whIcH hold TREMeNdOuS pOTEntiAl in gENEraTinG sPecifieD TISsUe TypES ([@B25-Btt-2-699]). HOWEvER, tHE pOTeNTiAL foR iMmunE reJECTiON, toGeTheR witH tHE POsSIbIlitY of TumoR FORmatiON has cauSed THeiR ApPLIcATioN In HUmAns tO ProCeEd WiTH cAuTIon ([@b25-Btt-2-699]). aDUlT STEM cELLs teNd to bE tiSsUe-spECIFic cellS with LImiTed dIFFerenTiATiON POTenTIAl cOMPaRED wItH eS cELLS. AdUlT sTEm cElLS Are cLiniCAlly attraCtIve therapIES DUe to ThEiR REducEd rISK oF TUmORIGEnESIS AND AbiLiTY to expAND With RElaTivE EASE ([@B8-Btt-2-699]).
Among ThE MANY TYpeS of adUlt STEM CelLs, THOSE rESIdeNT To THe Bone MARROW (BM), paRTICUlaRLy mEsEnchymaL sTeM cells (mScs), HavE gAineD ExtENSivE InteReST aMONg sCIENtIsTS aND ClINiCIans ([@b12-BTt-2-699]). msCs aRe mEsoDErmaL CeLLS PRimariLY ResIDeNt tO THE aDult BM, WhIcH uNDeRGO linEaGE-SpeCiFiC dIfFerenTIATioN To gENERATe boNe, Fat, anD CArtiLagE AmONG OTHer tiSSuE typES ([@b3-BTT-2-699]). msCS hAvE alsO bEen rePorted TO tRAnSdiFFERENtIAte iNTO defINEd eCtoDeRMAL AnD ENDodERmal TisSueS In vItRo, thUS ALLUdiNg to TheIR iNhErenT pLaStIciTY (cHOi aND PaNaYI 2001; [@b9-btt-2-699]; [@B14-BTT-2-699]; [@b27-btt-2-699]; [@b19-bTt-2-699]; [@B21-btt-2-699]; [@B23-BTT-2-699]; [@b34-btT-2-699]). mscS ARE AVaiLable FOr AUTOlOgouS THERApIES, haVe a UNiQUE abIlity To BYpasS IMMuNe reJeCTiOn And are INHeReNTly mIGrAToRy ([@B31-bTT-2-699]). THese PrOPeRtIes OF msCs mAKE ThEm paRticuLaRLY WELl SuITeD WhEN cONsideRING The SeCoND queSTion PoSED EarlIER -- CAN IN VItRO FIndInGs be aCcuRAteLy ReCapiTUlAtEd iN VIvo? wHEreas tissues dErIvED FROm eS CellS or otHeR TYpeS OF STeM CElLS mAY Be rejEcTed WHEN traNspLAnTED, mscs ofFER ThE POtENtiAL for allOgENEIc trANsPLanTatIon AnD a readilY AvaiLable SOuRce of "ofF-THe-shELf" STeM cElLs for PeRsOnALiZeD TheraPIES.
hOwEver, tHE UnIquE IMMune PrOpeRTieS of MsCs do NoT GuARanteE that thE CELLs wilL pROdUce ThE dESIRed TheRapeUTiC oUtCOmE or even tHAT thEY WiLL nOT Be REjecTed. iN VITRo, A Msc'S GrOwTH cONdItions cAn be CloseLY MoniTOReD tO Favor sTeM CELl gROWTH aNd/Or dIFfEreNTiAtIOn. iN vivO, tHe trAnSpLantEd MSCs Are exPOSeD TO LOCal iMmuNE CELLs ANd sOLUBLe mEDIaTORs tHaT cOULD infLuEncE tHe Cells' bEHAviOr, eiTHer pOSItIvely or neGaTivELY REGARDING THE dESIRED oUtcoMe. THIs cONcEpt oF THe TIsSuE micRoeNVironMEnT Has become a Growing concern among reseARcherS, aND May Be THE UlTImATE FActOR IN DeCiDINg WheTHEr a STeM CeLL THeRapY SuccEeds Or fAiLS ([@B18-BTT-2-699]; [@b37-Btt-2-699]; [@b17-bTt-2-699]; [@B29-BTT-2-699]).
A PRoToTyPiCAl exaMpLe oF A tissuE MiCroeNViRonmeNt AFfectiNG stem CEll BehAvIOr iS OBSERvED aMONg HEMatOpOiEtic stEM CELls (HscS) and tHEiR nIChE wIThiN The bm. hSCS ArE reLaTIvelY qUIEScEnT cELlS LoCateD cLosE TO The BM eNdoSTeUM At relAtiveLy Low OxYgEN CoNcENTRAtioN ([@b18-btt-2-699]). aS hScS DiffERenTiatE, tHe maTUriNG IMmune CELlS migrAtE tOWaRdS ThE cENtRAL Sinus OF The Bm UndEr ProgresSIVeLY hIGheR oXyGEn COncentRationS ([@b18-bTT-2-699]). thE ChanGe In OxYGEn iS a kEy deTerMiNANt in tHe maTuraTiON Of thE IMmuNE ceLlS BeForE tHeY leAVE The bM AND MigraTE IntO thE pERipHERal cirCulatiOn ([@B18-BTT-2-699]). iN CONtRast, mscS ARE lOCATED clOSE TO tRAbEculaR BONE nEar the CEntral SINuS oF THe bM ([@b3-btt-2-699]). As MScS MigRate towaRdS tHE ENDOstEuM UNDeR PRoGReSSiVelY loWeR cOnCenTrAtions of oXygen, tHe SteM Cells DIffERentiaTE iNTo StromAl FIBROBlasTs, WHiCh form the PrInCIpAL suppORt StrUcTURe foR imMunE ceLL MAturAtion ([@b3-BtT-2-699]).
THIs EXAMPLe DEmONSTrATeS thaT lOcAL mICROENViroNMEnTaL CHanGes iN VARiAbLeS sUCh aS oxygen cOncENTRATioN CAn DRastiCallY aFfECT THe BEHaviOR Of MSCs. SInce msCS HAve bEen sHowN tO GeNeRaTE a VaST NumbeR Of tIsSUes, THey hAVe cliNIcAL iMPLIcatIONs In A wIDE arrAy OF dIseaSES aNd diSoRDErS. aMong posSIbLE TrAnSplAntATioN sITeS ArE tISsuES such As CaRdIaC, neUral, PaNcreatic and BoNe. EAch tISSue PROvIdes A UNique loCal MIcrOENvIRONMeNT THaT CAn aFfECt the SucCeSs oF THe tHeRApY. THE PROBLEm FAcINg rESearChERS is aCcUrAtelY DEveLoPIng IN VItRo ModELS tO RECApITulate the tiSsue mICRoEnVirOnment so ThAt CELLuLaR BEhavioR can be ObSeRvED PrIOr tO TrAnsPLanTAtiOn. tHIS is No eASy Task CONSiDeRInG ThE dyNAmIc natUre OF THe mIcRoenVIronmeNt.
TraNspLAnTaTIoN OF mScs alONE WiLL GEnERate A Local IMmUNe RESPOnSE AND diSrupt HOmeosTASIs witHIn THe tIssUe mILiEu bY cAuSINg RELeAsE Of INfLaMmAToRY medIatOrs, SUcH As CyTokInes. THe aNaToMy oF thE bM iS suCH thAT MsCS ARe in DIreCt iNTeRActIOn WITh immUNe ceLLs aND forM synAPsE-LiKE STRuCTuRES wiTh inNERVaTinG neRVe fIBers ([@b3-BtT-2-699]). mScs eXprEsS REcePtoRs For MAny CyToKInEs AnD neuROTransMitTErs, tHUs dEmonStrAtINg tHEir pOteNTIAl TO reSPoNd TO LOcaL MICROenVIronmeNTAL chANGeS ([@B20-bTT-2-699]). EXCessivE CYtOkinE RElEAsE wiThiN tHe trANsPLAntaTioN sItE CouLd LeaD TO the proDuCtion OF OthER sOLuBLe fACtoRs By The mScS theMSElves. iF tHEsE FAcTOrs Are IMMUnOReactiVE, TheN othER IMmunE cEllS Could iNfIlTratE THE TiSsuE AnD caUSe an eXACErbATEd iMMUne resPoNse, rejECTIoN OF tHe trAnsplaNt oR dIFfereNtiAtIon of THE mSCS ([FiGure 1A](#F1-btT-2-699){REF-TypE="FIg"}). ON The otHeR HanD, msCS havE BEEN sHowN To BE a POTeNT SouRcE oF TRoPHIc faCtoRS ([@B29-BTT-2-699]). tHesE fINdInGs IndIcate ThAt MsCs cOulD also be Used To aID nORMAl tISsUE repAIr, perhAPS EVEN moRE SO than in celL REPLAcEMENt. whEthER tRaNSPlaNteD msCS CaUSe aN iMMUNe iNSUlt Or HELp REpaIR INJUrEd tiSSuES mAY Be DIFficULT To dETErMinE UNLeSS APProPRiaTe MOdElS ARe DeVeloPeD TO betTer PreDicT THe OUtCOME. hOwevEr, If MScS ARE FoUND To NEgATivelY impact THe HOsT MicrOenvIRoNmeNT thRouGh eXPoSure tO SOluble MEDiAToRs, THErE Are still pOTENtIAl MetHOds to devElOp EFfEctive ThErapeUTICs. WhEn consIderiNG THe exampLE PresENTED IN [FiGURe 1a](#f1-bTt-2-699){ReF-tyPe="fig"}, INclUSion Of speCIFiC cytOkiNE recEpTOR antaGoNisTS OR iNHiBitoRS COuld sUpPrESS the uNToWaRd EFFeCTs oF tHe HOst miCRoeNviRONmeNT ON The tRANsplantED MScs, tHuS lEadinG To DEFiNeD tHeraPEUtIC OuTCOMes ([fIGure 1B](#F1-btT-2-699){Ref-tyPE="Fig"}). tHROUGhoUt tHE REMaiNDEr of THIs rEVIeW, we WIll AddrESs thE FEAsIbIlITY OF UsING SimIlar PhArmACoLOgiC aPProaCHeS In Msc TrAnSpLanTS, While FOcUsing on tHReE uBIQuitous MIcRoENViRonmeNTaL fActOrs: Il-1α/Β, tnfα, aNd sdF-1Α. SpECiFicAlLy, wE will exAMiNe How REcEpToR aNtaGONIsTS OR inhIbitORs to tHESe fAcTORs, WhEtHeR FEdeRAlLY ApprOved Or in devELOpMenT, mAY LimiT ThE PoTEnTiAl NEGATive infLuenceS OF thE TISsuE mIcroENViRONMenT.
inTeRLEuKIN-1Α/β
================
IL-1Α ANd iL-1Β arE meMbERS OF the Il-1 suPeRfaMIlY oF CYToKinEs. tHEse prO-iNFlAMMAtoRy MediATOrs Are PRIMArIlY synthEsiZed by MACrOPhageS, moNOCyTeS AND dENdRItic CelLS, ANd ARe ReSpOnsible fOR ImMUNE DeFENsE AgainSt InfeCtIoN ([tABLe 1](#t1-BTt-2-699){Ref-tyPE="tabLe"}) ([@B13-BTt-2-699]). Il-1Α aND il-1β are AlsO Key reguLaTORs Of HEMaToPOesiS anD ThE inFlammatory prOcesS ([taBle 1](#T1-bTT-2-699){reF-tYPE="TABle"}) ([@b13-bTT-2-699]). Both cYtOkINes aRe FoUND THrOughoUT tHE boDY, thUS tHey aRe ExPeCtED tO be PREsEnT wiTHiN the LOcal MIcROeNVIRONmenT OF MosT TIssueS.
our LaBOrAtOry hAs pReVIoUsLY DEMONstRAtEd That msCs expreSS iL-1rI, wHich is ThE pRIncIpAl REcEPtOR FOR BOTh iL-1Α aND iL-1β ([@b20-BtT-2-699]). iNtereStINgLY, meMbRaNe exprEsSIon oF the RecEPtoR WAs MAinTAInEd THROuGHOUt THe couRSe OF tranSdifFERENTiATiOn OF mScS iNTO FuncTIonAL NeUROnS ([@B20-btT-2-699]). if il-1α Or iL-1Β wErE FOuND tO haVe neGativE EFFEcts on MsCs, THEn tHESE eFFECTs May alSo bE seEN ON TrAnsplANtAble TIssuEs differeNTiAted FRoM mScs. thESe RESulTS havE iMPlIcationS REGArDIng the IdEaL staGE oF STEM cell IMplantation, WHether uNdiFfERENtiAtED, pArtlY diffErENtIatED or FuLly DifFerentiatEd.
Il-1Α was fOuNd TO Alter tHE BehAviOr Of UndifFEReNTIAteD MsCs AND neuRoNs DIFfErEnTiATeD from mSCS ([@b20-btt-2-699]). specIFIcAllY, STimulAtION of MscS WiTH Il-1Α CausEd PrODuCTioN oF the nEURoTrANsmiTTEr, subsTaNce P (Sp), by undifferEnTiaTEd AND diffeReNTIated cELLs ([@B20-btT-2-699]). similar effEctS WeRe nOT OBSErVED In CelLS sTIMUlATed wITH Il-1Β ([@b20-btT-2-699]). sP HaS iNvOlvement iN VarIous PhYSIoLOgiCAl FUnCtIOns, SucH As thE pERCEPtIOn oF PaIN | Introduction ============ The emergence of stem cells as atherapeutic for many diseases, disorders and injuries has broughtexcitement among scientists,clinicians and patients alike regarding the potential treatment of previously untreatableconditions. However, theimplementationof many stem celltherapies in patients may stillbe years away. When considering translating these therapies into patients, there are two principal concerns that must be resolved:I. Canthe stem cells efficiently produce the desired therapeutic outcome, albeit tissuereplacement or repair, in vitro?; and II. Can the in vitro studies be replicated in vivo, both short-and long-term, with increased confidence? Much of the past research has concentrated on question one, or more appropriately, the philosophy of can weapplythe method? However, torecognize stem cells as key factorsinthe treatment of variousailments, weneed to rest assuredthat wecan also answer question two -- Is this a viabletreatment approach? These questions areaside from the ethical implications surrounding the field, which ask should we doit. In any case, stem cells will continue tobe researched as a potentialtreatment for a multitude of diseases anddisorders. Considerable progress has been made in addressing the first question stated above -- can we do it. A vast numberof tissuetypes have been generated fromboth embryonic (ES cells) and adult stemcells. ES cells are pluripotent cellsderived from theinner cell mass of the blastocyst, which hold tremendouspotential in generating specified tissue types ([@b25-btt-2-699]). However, the potential for immune rejection, together with the possibilityof tumorformation has caused their application in humans toproceed with caution ([@b25-btt-2-699]). Adult stem cells tend to be tissue-specificcells with limited differentiation potential compared with ES cells.Adult stem cells are clinically attractive therapiesdue to their reduced risk of tumorigenesis and ability to expand with relative ease ([@b8-btt-2-699]). Among the many typesof adultstem cells, thoseresident tothe bone marrow (BM), particularly mesenchymal stem cells (MSCs), have gained extensiveinterest amongscientists and clinicians ([@b12-btt-2-699]). MSCs are mesodermalcells primarilyresident to the adult BM, whichundergo lineage-specific differentiation to generate bone, fat, and cartilage among other tissue types ([@b3-btt-2-699]). MSCs have also been reported totransdifferentiate into defined ectodermal and endodermal tissues in vitro, thusalluding to theirinherentplasticity (Choi and Panayi 2001; [@b9-btt-2-699]; [@b14-btt-2-699]; [@b27-btt-2-699]; [@b19-btt-2-699]; [@b21-btt-2-699]; [@b23-btt-2-699]; [@b34-btt-2-699]). MSCs are availablefor autologoustherapies, have a uniqueabilitytobypassimmune rejection and are inherently migratory ([@b31-btt-2-699]). These properties ofMSCs make them particularly wellsuitedwhen considering the second questionposed earlier -- can in vitro findings be accurately recapitulated in vivo? Whereas tissuesderived from ES cellsor other types of stem cells may be rejected when transplanted,MSCs offer the potentialfor allogeneic transplantation and a readily available source of "off-the-shelf" stem cells forpersonalized therapies. However, the unique immune properties of MSCs do not guarantee that the cells willproduce the desired therapeutic outcome oreven that they will not be rejected. In vitro, a MSC's growth conditions can beclosely monitored to favor stem cell growth and/or differentiation. In vivo,the transplanted MSCs are exposed tolocal immunecells and soluble mediators that could influence the cells' behavior,eitherpositively or negatively regarding the desired outcome.This concept ofthe tissue microenvironment has becomea growing concern among researchers, and may be the ultimate factorin deciding whether a stem cell therapysucceeds or fails([@b18-btt-2-699]; [@b37-btt-2-699]; [@b17-btt-2-699]; [@b29-btt-2-699]). A prototypical example ofa tissuemicroenvironment affecting stem cell behavior is observed among hematopoietic stemcells (HSCs) and their niche within theBM. HSCs are relatively quiescent cells locatedclose to the BM endosteum atrelatively low oxygen concentration ([@b18-btt-2-699]). As HSCs differentiate, the maturing immune cells migrate towards the central sinus of theBM under progressively higher oxygenconcentrations ([@b18-btt-2-699]). Thechange in oxygen is a keydeterminant in the maturationof the immune cells before they leave the BM and migrateinto the peripheral circulation ([@b18-btt-2-699]). In contrast,MSCs are locatedclose to trabecular bone near the central sinus of theBM ([@b3-btt-2-699]). AsMSCs migrate towards the endosteum under progressively lower concentrations of oxygen, the stem cells differentiate intostromal fibroblasts, which form the principal support structure for immune cell maturation ([@b3-btt-2-699]).This exampledemonstrates that local microenvironmental changesin variables such as oxygenconcentration candrastically affectthe behavior of MSCs. Since MSCs have been shown to generate a vast number of tissues, they have clinicalimplications in a wide array of diseases and disorders. Among possible transplantation sites aretissues such ascardiac, neural, pancreatic and bone. Each tissue provides aunique local microenvironment that can affect thesuccess ofthe therapy. Theproblem facing researchers is accurately developing in vitro models torecapitulate the tissue microenvironment so that cellular behavior can be observedprior totransplantation. Thisis no easytask considering the dynamic nature of the microenvironment. Transplantation of MSCs alone will generate a localimmune response and disrupthomeostasis within the tissuemilieu by causingrelease ofinflammatory mediators, such as cytokines. The anatomy of the BM is such that MSCs are indirectinteraction withimmune cells and form synapse-like structureswith innervating nervefibers([@b3-btt-2-699]). MSCs express receptorsfor many cytokines and neurotransmitters, thus demonstrating their potential to respond to local microenvironmentalchanges ([@b20-btt-2-699]). Excessive cytokine release within the transplantation site couldleadto the production of other soluble factorsby the MSCs themselves. If these factors are immunoreactive, then other immune cells could infiltrate the tissue and cause an exacerbated immune response, rejectionof the transplantor differentiation of the MSCs ([Figure 1A](#f1-btt-2-699){ref-type="fig"}).Onthe otherhand, MSCshave been shownto be a potent source of trophic factors([@b29-btt-2-699]). Thesefindings indicate thatMSCs couldalsobe used toaidnormal tissue repair,perhaps even more so thanin cell replacement. Whethertransplanted MSCscause an immune insult or help repair injured tissues may be difficultto determine unless appropriate modelsaredeveloped to better predict theoutcome. However, if MSCs are found to negativelyimpact the host microenvironment through exposure tosoluble mediators, there are still potential methods to develop effectivetherapeutics. When consideringthe example presented in [Figure 1A](#f1-btt-2-699){ref-type="fig"}, inclusion of specific cytokine receptor antagonistsor inhibitors could suppressthe untoward effectsof the host microenvironment on the transplanted MSCs, thus leading todefined therapeutic outcomes ([Figure 1B](#f1-btt-2-699){ref-type="fig"}). Throughout the remainder of this review, we will address the feasibility ofusing similar pharmacologic approaches in MSC transplants, while focusing on three ubiquitous microenvironmental factors:IL-1α/β, TNFα, and SDF-1α. Specifically, we will examinehow receptor antagonists or inhibitors to these factors, whether federally approved or indevelopment, may limit the potential negative influences of the tissue microenvironment. Interleukin-1α/β ================ IL-1α andIL-1β are members of the IL-1 superfamily of cytokines.These pro-inflammatorymediators are primarily synthesized by macrophages, monocytes and dendritic cells, and are responsible for immune defense against infection ([Table1](#t1-btt-2-699){ref-type="table"}) ([@b13-btt-2-699]). IL-1α and IL-1β are alsokey regulators of hematopoesis and the inflammatory process ([Table 1](#t1-btt-2-699){ref-type="table"})([@b13-btt-2-699]). Both cytokines are found throughout thebody, thus they are expected tobepresent within the local microenvironment of most tissues.Our laboratory has previously demonstrated that MSCs express IL-1RI, which is the principal receptor forboth IL-1α and IL-1β ([@b20-btt-2-699]). Interestingly, membrane expressionofthe receptor was maintained throughout thecourse oftransdifferentiation of MSCs into functional neurons ([@b20-btt-2-699]).If IL-1α or IL-1β were found to have negative effectson MSCs,then these effects may alsobe seen on transplantable tissues differentiated fromMSCs. These results have implications regarding the ideal stage of stem cell implantation, whether undifferentiated, partly differentiated or fully differentiated. IL-1α was found to alter the behaviorof undifferentiated MSCs and neurons differentiated from MSCs ([@b20-btt-2-699]). Specifically, stimulation of MSCs with IL-1α caused productionof the neurotransmitter, substance P (SP), by undifferentiatedand differentiated cells ([@b20-btt-2-699]). Similar effects were not observed in cells stimulated with IL-1β ([@b20-btt-2-699]). SP has involvement in various physiological functions, such as the perception of pain | Introduction ============ _The_ emergence of stem _cells_ as _a_ _therapeutic_ for many diseases, disorders and _injuries_ has brought _excitement_ among scientists, clinicians and patients alike _regarding_ the potential _treatment_ of previously untreatable conditions. However, _the_ implementation of many stem cell therapies in patients _may_ still be years away. When _considering_ translating these therapies _into_ patients, there are two principal concerns that must _be_ resolved: I. Can the stem cells efficiently _produce_ the desired _therapeutic_ outcome, _albeit_ tissue replacement _or_ repair, in vitro?; _and_ II. _Can_ the in vitro _studies_ be replicated in _vivo,_ both short- and long-term, _with_ increased _confidence?_ _Much_ of the past research has concentrated _on_ question one, or _more_ appropriately, _the_ philosophy of can we _apply_ the method? However, _to_ _recognize_ stem cells _as_ key factors in the treatment of various ailments, we need to rest assured that we can also answer question two _--_ Is this a _viable_ treatment approach? These questions are aside from the _ethical_ implications surrounding the _field,_ which ask should we _do_ it. In any case, stem cells will continue to be researched as a potential treatment for a multitude _of_ diseases and _disorders._ Considerable progress has been made in addressing the _first_ question stated above _--_ can we do it. A _vast_ number of tissue types have been generated from both embryonic (ES cells) and adult stem cells. ES cells are pluripotent cells derived _from_ _the_ inner cell _mass_ _of_ the blastocyst, which hold tremendous potential in generating _specified_ tissue types ([@b25-btt-2-699]). However, the potential for immune rejection, _together_ with _the_ _possibility_ of tumor formation has _caused_ their application in _humans_ _to_ _proceed_ _with_ caution ([@b25-btt-2-699]). _Adult_ stem cells tend to be tissue-specific cells with limited differentiation potential compared with ES cells. _Adult_ stem cells are clinically attractive therapies due to their reduced risk of _tumorigenesis_ and ability to expand with relative ease ([@b8-btt-2-699]). Among the _many_ types of _adult_ stem cells, those resident to _the_ bone marrow (BM), _particularly_ _mesenchymal_ _stem_ cells _(MSCs),_ _have_ gained extensive interest among scientists and _clinicians_ ([@b12-btt-2-699]). MSCs are _mesodermal_ cells primarily resident to the _adult_ BM, which undergo lineage-specific differentiation to generate bone, fat, and cartilage among _other_ tissue _types_ _([@b3-btt-2-699])._ MSCs _have_ also been reported to transdifferentiate into defined ectodermal and _endodermal_ tissues in vitro, _thus_ alluding to their inherent plasticity (Choi and Panayi 2001; [@b9-btt-2-699]; _[@b14-btt-2-699];_ [@b27-btt-2-699]; [@b19-btt-2-699]; _[@b21-btt-2-699];_ [@b23-btt-2-699]; [@b34-btt-2-699]). MSCs _are_ available _for_ autologous _therapies,_ _have_ a unique ability to bypass _immune_ rejection and are inherently _migratory_ ([@b31-btt-2-699]). These _properties_ of MSCs make them particularly well suited when _considering_ _the_ _second_ question _posed_ earlier _--_ can in vitro _findings_ be accurately recapitulated in vivo? _Whereas_ _tissues_ _derived_ from _ES_ _cells_ or other types _of_ stem cells _may_ be rejected when transplanted, MSCs offer the potential _for_ allogeneic _transplantation_ and _a_ readily available source of _"off-the-shelf"_ stem cells for _personalized_ therapies. However, the unique immune properties of MSCs do not guarantee _that_ _the_ cells _will_ produce the desired therapeutic _outcome_ or even that _they_ will not be rejected. In _vitro,_ a MSC's growth conditions _can_ be closely monitored _to_ favor stem cell growth and/or differentiation. _In_ vivo, the transplanted MSCs are exposed to local immune cells and _soluble_ mediators that _could_ influence _the_ _cells'_ behavior, either positively or negatively regarding the desired outcome. This concept of the tissue microenvironment _has_ _become_ a growing concern _among_ researchers, _and_ may be the ultimate factor _in_ _deciding_ whether _a_ _stem_ _cell_ therapy _succeeds_ or fails ([@b18-btt-2-699]; [@b37-btt-2-699]; [@b17-btt-2-699]; [@b29-btt-2-699]). A prototypical example _of_ _a_ tissue microenvironment _affecting_ stem cell _behavior_ is observed among hematopoietic stem cells (HSCs) and their niche within the BM. HSCs are relatively quiescent cells located close to the _BM_ endosteum at relatively low oxygen concentration ([@b18-btt-2-699]). As HSCs differentiate, the maturing immune cells migrate towards the _central_ _sinus_ _of_ the BM under progressively higher oxygen concentrations ([@b18-btt-2-699]). The _change_ in oxygen is a key determinant _in_ the maturation of the immune cells before they leave the BM and migrate into the peripheral _circulation_ ([@b18-btt-2-699]). In _contrast,_ _MSCs_ are located _close_ to trabecular bone near _the_ central sinus _of_ the BM _([@b3-btt-2-699])._ As MSCs migrate towards the endosteum under progressively lower concentrations of oxygen, the stem _cells_ _differentiate_ into stromal fibroblasts, _which_ form the principal support structure for _immune_ cell maturation ([@b3-btt-2-699]). This example demonstrates that local _microenvironmental_ changes _in_ variables such _as_ oxygen concentration can drastically affect the _behavior_ of MSCs. Since MSCs have been shown to generate a vast _number_ of tissues, _they_ have clinical implications in a wide array of _diseases_ _and_ _disorders._ Among possible transplantation sites are tissues such as cardiac, neural, pancreatic and bone. Each tissue _provides_ a unique _local_ microenvironment that can _affect_ _the_ success _of_ the therapy. The problem facing researchers is accurately developing in _vitro_ models to recapitulate the tissue microenvironment so that cellular behavior can _be_ _observed_ prior to transplantation. This is no easy task considering the _dynamic_ nature of the microenvironment. Transplantation of MSCs alone will generate a _local_ immune response and disrupt homeostasis within the tissue milieu by causing release of _inflammatory_ mediators, such as cytokines. The anatomy of the BM is such that _MSCs_ are in direct _interaction_ with _immune_ cells and form synapse-like structures with _innervating_ nerve fibers ([@b3-btt-2-699]). MSCs _express_ receptors for many cytokines and _neurotransmitters,_ thus demonstrating their potential to respond to local microenvironmental changes ([@b20-btt-2-699]). _Excessive_ cytokine _release_ within the transplantation site could lead to the _production_ of other _soluble_ factors by the MSCs _themselves._ _If_ these factors _are_ _immunoreactive,_ then other immune cells could infiltrate the _tissue_ and cause _an_ exacerbated immune response, rejection _of_ the transplant or _differentiation_ of the MSCs _([Figure_ _1A](#f1-btt-2-699){ref-type="fig"})._ _On_ the other hand, MSCs _have_ been _shown_ to be a potent _source_ _of_ trophic _factors_ ([@b29-btt-2-699]). These findings indicate that _MSCs_ _could_ also _be_ used to aid normal tissue repair, perhaps even more _so_ than in cell _replacement._ Whether transplanted _MSCs_ cause an immune insult or _help_ _repair_ injured _tissues_ may be difficult to determine _unless_ appropriate models are _developed_ _to_ _better_ predict the outcome. _However,_ if MSCs are _found_ to negatively impact the host microenvironment through _exposure_ to soluble mediators, there are still potential methods to develop effective therapeutics. When considering _the_ example presented in [Figure 1A](#f1-btt-2-699){ref-type="fig"}, inclusion _of_ specific cytokine receptor antagonists or inhibitors could suppress the untoward _effects_ of the host microenvironment on the transplanted MSCs, thus _leading_ to _defined_ therapeutic outcomes _([Figure_ _1B](#f1-btt-2-699){ref-type="fig"})._ Throughout the _remainder_ _of_ _this_ _review,_ we will address the _feasibility_ _of_ using _similar_ pharmacologic approaches in MSC transplants, _while_ _focusing_ on three ubiquitous _microenvironmental_ factors: IL-1α/β, TNFα, _and_ SDF-1α. Specifically, we will examine _how_ receptor _antagonists_ _or_ _inhibitors_ to these factors, whether federally approved or in _development,_ may limit the potential _negative_ influences of the tissue microenvironment. Interleukin-1α/β _================_ IL-1α and _IL-1β_ are members _of_ _the_ IL-1 superfamily of cytokines. These pro-inflammatory mediators are primarily synthesized _by_ _macrophages,_ monocytes _and_ dendritic cells, and are responsible for immune _defense_ against infection ([Table 1](#t1-btt-2-699){ref-type="table"}) ([@b13-btt-2-699]). IL-1α and IL-1β are _also_ key regulators of hematopoesis and the inflammatory process ([Table 1](#t1-btt-2-699){ref-type="table"}) ([@b13-btt-2-699]). Both cytokines are found throughout _the_ body, thus they are expected to _be_ _present_ within the local microenvironment of most tissues. Our laboratory _has_ _previously_ _demonstrated_ that MSCs _express_ _IL-1RI,_ which is the principal receptor for both IL-1α and _IL-1β_ ([@b20-btt-2-699]). Interestingly, membrane _expression_ _of_ the receptor was _maintained_ throughout the course of transdifferentiation of MSCs into functional neurons _([@b20-btt-2-699])._ _If_ _IL-1α_ or _IL-1β_ _were_ found to have negative effects on MSCs, _then_ these effects _may_ also be seen on transplantable tissues differentiated from MSCs. These _results_ _have_ implications regarding the ideal stage of stem cell implantation, whether undifferentiated, partly differentiated or _fully_ differentiated. IL-1α was found to alter _the_ behavior of undifferentiated MSCs and neurons differentiated from MSCs ([@b20-btt-2-699]). Specifically, stimulation _of_ MSCs with IL-1α caused production of the _neurotransmitter,_ substance _P_ (SP), by _undifferentiated_ and differentiated cells ([@b20-btt-2-699]). Similar effects were not observed in _cells_ stimulated with _IL-1β_ ([@b20-btt-2-699]). SP has involvement in various physiological functions, such as the perception _of_ pain |
INTRODUCTION {#sec1-1}
============
Hospital staffs are the most important group of health care providers. As a result of their continuous hard work, they are frequently faced with physical and psychological health problems. If these problems linger on, disappointment and boredom will settle, which in turn will lower the quality of health care services. Health care quality is highly dependent on clinical and professional ethical principles, which are under the influence of religious beliefs. An influential factor helping preservation of psychological stability is to believe in and to rely on God; closeness to God and performing religious duties brings about peace. Spirituality is a human dimension that combines our existence with concepts such as human nature, sacred mental experience, tendency to know more, elevation toward Good, and finding a meaning to life. It has also been defined as a number of values, attitudes, and hopes related to the superior existence and thus guiding us throughout life.\[[@ref1]\]
The relationship between the mental health and spirituality has received much attention recently. Research shows spirituality is highly influential in physical and mental health preservation. Many researchers have concluded that spirituality has an immense effect on mental health.\[[@ref2][@ref3]\]
According to WHO definition, health is total physical, mental, and social existential well-being, and mental health is the ability to establish harmonious communication with others, modify surroundings, and resolve conflicts.\[[@ref4]\] Behaviors such as reliance on God, praying, and going on pilgrimage can promote solace and mental health through peace and developing hope and positive thinking. Religious beliefs increase resistance against calamities, and thus help preserve physical and mental health, prevent infliction of diseases, and finally promote hopefulness.\[[@ref5][@ref6][@ref7][@ref8][@ref9]\]
According to the cognitive-emotional-religious theory, man cannot comprehend the meaning of life without religious beliefs. This theory discusses religious beliefs and God, existence, and man, and proposes that these beliefs can be applied to treat psychological problems.\[[@ref10]\] From a clinical viewpoint, lack of appraisal of religious spiritual dimensions sets obstacles in the way of (1) acknowledging significance of these dimensions for health and (2) learning about the mechanisms involved.\[[@ref11]\] Islam as an ideology presents an impeccable and health-preserving lifestyle; its orders cover a wide range of life aspects, including personal and social ethics, interpersonal relations, and physical and mental health.\[[@ref11]\] Some studies indicate a correlation between reliance on God and high self-esteem,\[[@ref12]\] lower levels of depression, more mature social behavior,\[[@ref13][@ref14]\] and better mental predisposition.\[[@ref15]\]
Kendler *et al*.\[[@ref16]\] studied a number of spiritual/religious themes and identified those factors which had a significant inverse relationship with lack of mental health. Hill *et al*. found that true religious belief acts as a motivational drive and is closely related to physical and mental health.\[[@ref17]\] Koenig *et al*. and Smith *et al*. also showed that health is a causal result of the relationship between higher levels of spirituality and reliance on God and trust in God enables people to tolerate hardships of life much better since they believe in God and entrust their lives into his hands.\[[@ref18][@ref19][@ref20]\] Also, James states that the foundation of religions, that is based on spiritual relation with God, meaningfulness of the whole universe, and meaningfulness of life is the essence of spirituality.\[[@ref21]\]
Modern analyses have reported that there is a significant positive relationship between religious duties and concerns and health and life time.\[[@ref22][@ref23]\] Koenig *et al*. also found that spiritual/religious beliefs are important predictors of youth depression.\[[@ref18]\] Bahrami and Tashak\[[@ref24]\] reported a significant positive relationship between religious orientation and mental health promotion and decrease in psychological disorders. The study done by Omran-Nasab\[[@ref25]\] also indicated that there was a significant correlation between religious beliefs and mental health. Cohen, Yoon, and Johnston in a study on 168 patients with various physical disorders showed that there is generally a positive relationship between positive spiritual tolerance and mental health and a negative relationship between negative spiritual tolerance and mental health, but there was no relationship between more personal religious duties such as praying and mental health.\[[@ref26]\] Cohen and Hall also studied 1,000 aged people and found that there was a relationship between some religious beliefs (fear of God, fear of death, and belief in life after death) and feeling of well-being and it is more significant in Protestants than in Catholics and Jews.\[[@ref27]\] Krause also suggested that the relationship between calamities of life and depression symptoms in aged people reduces when they believe God knows better when to respond to their prayers.\[[@ref28]\] Keyes and Reitzes\[[@ref29]\] emphasize the importance of the impact of religious identity on dignity and depression symptoms in retired workers. Mazidi and Ostovar\[[@ref30]\] concluded that both Islam and Christianity affect mental health of Iranian youth positively.
The findings of Jang *et al*., Hills *et al*., and Doolittle *et al*.\[[@ref2][@ref3][@ref31]\] showed that spirituality is beneficial to physical and mental health. Some researcher found a close relationship between religiousness and elevation of soul and health.\[[@ref32][@ref33]\] In other words, positive tendencies such as feeling of happiness, joyousness, and hope for future are related to spirituality.\[[@ref23][@ref34][@ref35]\]
The favorable effect of positive constructs such as optimism and hope for future on physical and mental health has been confirmed by various studies.\[[@ref36]\] Snyder\'s theory of hope made researcher devote a lot of research to the relationship between hope and health.\[[@ref37][@ref38]\] Snyder believes hope is not a passive drive emerging only in dark moments of life, but a cognitive process through which one activity pursues goals. He states that hope is (1) a goal-determining process through which people (2) build up strategies to achieve goals and (3) are motivated enough to apply those strategies. These three components are known as goals, pathway thinking, and agency thinking.\[[@ref39]\] Thus, those staffs who are hopeful develops enough motivation to initiate and perform hard tasks, and emphasizing their capabilities, move toward their grand goals. Snyder also believes there is a positive relationship between positive emotions and purposefulness in life.\[[@ref40]\] Other studies have also indicated the relationship between hope and positive emotions is a positive relationship, and it has a negative relation with depression, anxiety, and negative emotions in general.\[[@ref40]\]
Similarly, Hezarjaribi *et al*.,\[[@ref41]\] believe that there is a maximum significant relationship between hope for future and joyousness, and with the increase of the feeling of joy, hope for future is also enhanced. Many researchers such as Bandloo *et al*. believe important factors, including personality traits, cognitive components, and religious attitudes pave the way for feeling of joyousness.\[[@ref30]\] They have also reported a direct relationship between genetic, personality, cognitive, and religious factors, and anxiety on one hand and joyousness and hope for future on the other.\[[@ref40]\] In other words, religious attitudes and practices definitely help people find a meaning to life. Once hospital staff finds a meaningful life, hope for God\'s support, receive social and religious support, and feel belonging to a holy and graceful existence, they can survive hardships and calamities of life and maintain their mental health successfully; these people can work more fruitfully and provide better services.\[[@ref3]\]
Thus, considering how crucial the job is, and also regarding the lack of a research on spiritual variables in Iran, this study was designed to answer this research question: Is there a relationship between religious/spiritual dimensions of one\'s character and his/her mental health and his/her hope for future?
MATERIALS AND METHODS {#sec1-2}
=====================
This was a correlational study. The statistical population included all state hospital\'s staffs in Shiraz-who were selected randomly using the sample size formula for correlational studies and matched with Cohen\'s table of sample size.\[[@ref42]\] Out of the 250 selected participants, 212 questionnaires were returned (85.2%). Females (156 people) formed 73.6% and males (56 people) formed 26.4% of the sample size. In six hospitals in the study, 95 staff (44.8%) were single, 112 staff (52.8%) were married, and 5 (2.4%) did not report their marital status. Regarding the educational level, 1 (0.5%) had not finished high school, 12 (5.7%) had high school diplomas, 19 (0.9%) had associate degrees, 160 (75.5%) had bachelor degrees, and 13 (6.1%) abstained from reporting. Ethical considerations were observed by reassuring subjects that their personal information would be regarded strictly confidential and by offering them to feel free to or not to answer the questionnaires.
Data collection was done through using three questionnaires:
Mental health questionnaire: the 12-item Goldberg and William\'s mental health questionnaire was one of the tools used in this study. The 4-point Likert scale (0 = always to 3 = Never) was applied. The total score was 36. The highest score indicated the lowest level of mental health. Goldberg *et al*.,\[[@ref43]\] have reported that the reliability of their test is 0.78 and Pearson correlation coefficient for 3 factors, and the total score is 0.57 if *P* \< 0.000. Ebadi *et al*.,\[[@ref44]\] applying the tool on a research sample of 18-25-year old Iranians, reported an internal consistency of 0.81 for the tool. They also confirmed the validity | introduction { # sec1 - 1 } = = = = = = = = = = = = hospital staffs are the most important group of health care providers. as any result of their continuous hard work, they are frequently faced with physical and psychological health conditions. if these problems linger on, despair and boredom will settle, which in turn will lower the quality of health care services. health management quality is highly dependent on clinical and professional ethical principles, which are under the influence of religious beliefs. an influential factor helping preservation of psychological stability is to believe in and to rely on god ; closeness to god and performing religious duties brings about peace. spirituality is a human dimension that combines our existence with concepts such as human nature, sacred mental experience, tendency to know more, elevation toward good, and finding a meaning to life. it has also been defined as a number of values, attitudes, and hopes related to the superior existence and thus guiding us throughout life. \ [ [ @ ref1 ] \ ] the relationship between the mental health and spirituality has received much attention recently. research shows spirituality is highly influential in physical and mental health preservation. many researchers have concluded that spirituality has an immense effect on mental health. \ [ [ @ ref2 ] [ @ ref3 ] \ ] according to who definition, health is total physical, mental, and social existential well - being, and mental health is the ability to establish harmonious communication with others, modify surroundings, and resolve conflicts. \ [ [ @ ref4 ] \ ] techniques such as reliance on god, praying, and going on pilgrimage can boost solace and mental stability through peace and developing hope and positive thinking. islamic beliefs increase resistance against calamities, and thus help preserve physical and mental health, prevent infliction of diseases, and finally promote hopefulness. \ [ [ @ ref5 ] [ @ ref6 ] [ @ ref7 ] [ @ ref8 ] [ @ ref9 ] \ ] according to the cognitive - emotional - religious theory, man cannot comprehend the meaning of life without religious beliefs. this theory examines religious beliefs and god, existence, and man, and proposes that these beliefs can be applied to treat psychological problems. \ [ [ @ ref10 ] \ ] from a clinical viewpoint, lack of appraisal of religious spiritual dimensions sets obstacles in the way of ( 1 ) acknowledging mastery of these dimensions for health and ( 2 ) learning about the mechanisms involved. \ [ [ @ ref11 ] \ ] islam as an ideology presents an impeccable and health - preserving lifestyle ; its orders cover a wide range of life aspects, including personal and social ethics, interpersonal relations, and physical and mental health. \ [ [ @ ref11 ] \ ] some studies indicate a correlation between reliance on god and high self - esteem, \ [ [ @ ref12 ] \ ] lower levels of depression, more mature social behavior, \ [ [ @ ref13 ] [ @ ref14 ] \ ] and better mental predisposition. \ [ [ @ ref15 ] \ ] kendler * et al *. \ [ [ @ ref16 ] \ ] studied a number of spiritual / religious themes and identified those factors which had a significant inverse relationship with lack of mental health. hill * et al *. found that true religious belief acts as a motivational drive and is closely related to physical and mental health. \ [ [ @ ref17 ] \ ] koenig * et al *. and smith * et al *. also showed that health is a causal result of the relationship between higher levels of spirituality and reliance on god and trust in god enables people to tolerate hardships of life much better since they believe in god and entrust their lives into his hands. \ [ [ @ ref18 ] [ @ ref19 ] [ @ ref20 ] \ ] also, james states that the foundation of religions, that is based on spiritual relation with god, meaningfulness of the whole universe, and meaningfulness of life is the essence of spirituality. \ [ [ @ ref21 ] \ ] modern analyses have reported that there is a significant positive relationship between religious duties and concerns and health and life time. \ [ [ @ ref22 ] [ @ ref23 ] \ ] koenig * et al *. also found that spiritual / religious beliefs are important predictors of youth depression. \ [ [ @ ref18 ] \ ] bahrami and tashak \ [ [ @ ref24 ] \ ] reported a significant positive relationship between religious orientation and mental health promotion and decrease in psychological disorders. the study done by omran - nasab \ [ [ @ ref25 ] \ ] also indicated that there was a significant correlation between religious beliefs and mental health. cohen, yoon, and johnston in a study on 168 patients with various physical disorders showed that there is generally a positive relationship between positive spiritual tolerance and mental health and a negative relationship between negative spiritual tolerance and mental health, but there was no relationship between more personal religious duties such as praying and mental health. \ [ [ @ ref26 ] \ ] cohen and hall also studied 1, 000 aged people and found that there was a relationship between some religious beliefs ( fear of god, fear of death, and belief in life after death ) and feeling of well - being and it is more significant in protestants than in catholics and jews. \ [ [ @ ref27 ] \ ] krause also suggested that the relationship between calamities of life and depression symptoms in aged people reduces when they believe god knows better when to respond to their prayers. \ [ [ @ ref28 ] \ ] keyes and reitzes \ [ [ @ ref29 ] \ ] emphasize the importance of the impact of religious identity on dignity and depression symptoms in retired workers. mazidi and ostovar \ [ [ @ ref30 ] \ ] concluded that both islam and christianity affect mental health of iranian youth positively. the findings of jang * et al *., hills * et al *., and doolittle * et al *. \ [ [ @ ref2 ] [ @ ref3 ] [ @ ref31 ] \ ] showed that spirituality is beneficial to physical and mental health. some researcher found a close relationship between religiousness and elevation of soul and health. \ [ [ @ ref32 ] [ @ ref33 ] \ ] in other words, positive tendencies such as feeling of happiness, joyousness, and hope for future are related to spirituality. \ [ [ @ ref23 ] [ @ ref34 ] [ @ ref35 ] \ ] the favorable effect of positive constructs such as optimism and hope for future on physical and mental health has been confirmed by various studies. \ [ [ @ ref36 ] \ ] snyder \ ' s theory of hope made researcher devote a lot of research to the relationship between hope and health. \ [ [ @ ref37 ] [ @ ref38 ] \ ] snyder believes hope is not a passive drive emerging only in dark moments of life, but a cognitive process through which one activity pursues goals. he states that hope is ( 1 ) a goal - determining process through which people ( 2 ) build up strategies to achieve goals and ( 3 ) are motivated enough to apply those strategies. these three components are known as goals, pathway thinking, and agency thinking. \ [ [ @ ref39 ] \ ] thus, those staffs who are hopeful develops enough motivation to initiate and perform hard tasks, and emphasizing their capabilities, move toward their grand goals. snyder also believes there is a positive relationship between positive emotions and purposefulness in life. \ [ [ @ ref40 ] \ ] other studies have also indicated the relationship between hope and positive emotions is a positive relationship, and it has a negative relation with depression, anxiety, and negative emotions in general. \ [ [ @ ref40 ] \ ] similarly, hezarjaribi * et al *., \ [ [ @ ref41 ] \ ] believe that there is a maximum significant relationship between hope for future and joyousness, and with the increase of the feeling of joy, hope for future is also enhanced. many researchers such as bandloo * et al *. believe important factors, including personality traits, cognitive components, and religious attitudes pave the way for feeling of joyousness. \ [ [ @ ref30 ] \ ] they have also reported a direct relationship between genetic, personality, cognitive, and religious factors, and anxiety on one hand and joyousness and hope for future on the other. \ [ [ @ ref40 ] \ ] in other words, religious attitudes and practices definitely help people find a meaning to life. once hospital staff finds a meaningful life, hope for god \ ' s support, receive social and religious support, and feel belonging to a holy and graceful existence, they can survive hardships and calamities of life and maintain their mental health successfully ; these people can work more fruitfully and provide better services. \ [ [ @ ref3 ] \ ] thus, considering how crucial the job is, and also regarding the lack of a research on spiritual variables in iran, this study was designed to answer this research question : is there a relationship between religious / spiritual dimensions of one \ ' s character and his / her mental health and his / her hope for future? materials and methods { # sec1 - 2 } = = = = = = = = = = = = = = = = = = = = = this was a correlational study. the statistical population included all state hospital \ ' s staffs in shiraz - who were selected randomly using the sample size formula for correlational studies and matched with cohen \ ' s table of sample size. \ [ [ @ ref42 ] \ ] out of the 250 selected participants, 212 questionnaires were returned ( 85. 2 % ). females ( 156 people ) formed 73. 6 % and males ( 56 people ) formed 26. 4 % of the sample size. in six hospitals in the study, 95 staff ( 44. 8 % ) were single, 112 staff ( 52. 8 % ) were married, and 5 ( 2. 4 % ) did not report their marital status. regarding the educational level, 1 ( 0. 5 % ) had not finished high school, 12 ( 5. 7 % ) had high school diplomas, 19 ( 0. 9 % ) had associate degrees, 160 ( 75. 5 % ) had bachelor degrees, and 13 ( 6. 1 % ) abstained from reporting. ethical considerations were observed by reassuring subjects that their personal information would be regarded strictly confidential and by offering them to feel free to or not to answer the questionnaires. data collection was done through using three questionnaires : mental health questionnaire : the 12 - item goldberg and william \ ' s mental health questionnaire was one of the tools used in this study. the 4 - point likert scale ( 0 = always to 3 = never ) was applied. the total score was 36. the highest score indicated the lowest level of mental health. goldberg * et al *., \ [ [ @ ref43 ] \ ] have reported that the reliability of their test is 0. 78 and pearson correlation coefficient for 3 factors, and the total score is 0. 57 if * p * \ < 0. 000. ebadi * et al *., \ [ [ @ ref44 ] \ ] applying the tool on a research sample of 18 - 25 - year old iranians, reported an internal consistency of 0. 81 for the tool. they also confirmed the validity | INTRODUCTION {# sec1 - 1} = = = = = = = = = = = = Hospital staffs are the most important group of health care providers. As a result of their continuous hard work, they are frequently faced with physical and psychological health problems. If these problems linger on, disappointment and boredom will settle, which in turn will lower the quality of health care services. Health care quality is highly dependent on clinical and professional ethical principles, which are under the influence of religious beliefs. An influential factor helping preservation of psychological stability is to believe in and to rely on God; closeness to God and performing religious duties brings about peace. Spirituality is a human dimension that combines our existence with concepts such as human nag Tre, sacred mental experience, tendency to know more, elevation toward Good, and finding a meaning to life. It has also been defined as a number of values, attitudes, and hopes related to the superior existence and thus guiding us throughout life. \ [[ @ ref1] \] The relationship between the mental health and spirituality has received much attention recently. Research shows spirituality is highly influential in physical and mental health preservation. Many researchers have concluded that spirituality has an immense effect on mental health. \ [[ @ ref2] [@ ref3] \] According to WHO definition, health is total physical, mental, and social existential well - veinh, and mental health is the ability to establish harmonious communication with others, modify surroundings, and resolve conflicts. \ [[ @ ref4] \] Behaviors such as reliance on God, praying, and going on pilgrimage can promote solace and mental health through peace and developing hope and positive thinking. Religious beliefs increase resistance against calamities, and thus help preserve physical and mental health, prevent infliction of diseases, and finally promote hopefulness. \ [[ @ ref5] [@ ref6] [@ ref7] [@ r$e8] [@ ref9] \] According to the cognitive - emotional - religious theory, man cannot comprehend the meaning of life without religious beliefs. This theory discusses religious beliefs and God, existence, and man, and proposes that these beliefs can be applied to treat psychological problems. \ [[ @ ref10] \] From a clinical viewpoint, lack of appraisal of religious spiritual dimensions sets obstacles in the way of (1) acknowledging significance of these dimensions for health and (2) learning about the mechanisms involved. \ [[ @ ref11] \] Islam as an ideology presents an impeccable and health - preserving lifestyle; its orders cover a wide range of life aspects, including personal and social ethics, interpersonal relations, and physical and mental health. \ [[ @ ref11] \] Some studies indicate a correlation between reliance on God and high self - esteem, \ [[ @ ref12] \] lower levels of depression, more mature social behavior, \ [[ @ ref13] [@ ref14] \] and better mental predisposition. \ [[ @ ref15] \] Kendler * et al *. \ [[ @ ref16] \] Ayudied a number of spiritual / religious themes and identified those factors which had a significant inverse relationship with lack of mental health. Hill * et al *. found that true religious belief acts as a motivational drive and is closely related to physical and mental health. \ [[ @ ref17] \] Koenig * et al *. and Smith * et al *. also showed that health is a causal result of the relationship between higher levels of spirituality and reliance on God and trust in God enables people to tolerate hardships of life much better since they believe in God and entrust their lives into his hands. \ [[ @ ref18] [@ ref19] [@ ref20] \] Also, James states that the foundation of religions, that is based on spiritual relation with God, meaningfulness of the whole universe, and meaningfulness of life is the essence of spirituality. \ [[ @ ref21] \] Modern analyses have reported that there is a significant positive relationship between religious duties and concerns and health and life time. \ [[ @ ref22] [@ ref23] \] Koenig * et al *. also found that spiritual / religious beliefs are important predictors of youth depression. \ [[ @ ref18] \] Bahrami and Tashak \ [[ @ ref24] \] reported a significant positive relationship between religious orientation and mental health promotion and decrease in psychological disorders. The study done by Omran - Nasab \ [[ @ ref25] \] also indicated that there was a significant correlation between religious beliefs and mental health. Cohen, Yoon, and Johnston in a study on 168 patients with various physical disorders showed that there is genrra?ly a positive relationship between positive spiritual tolerance and mental health and a negative relationship between negative spiritual tolerance and mental health, but there was no relationship between more personal religious duties such as praying and mental health. \ [[ @ ref26] \] CoYeh and Hall also studied 1, 000 aged people and found that there was a relationship between some religious beliefs (fear of God, fear of death, and belief in life after death) and feeling of well - being and it is more significant in Protestants than in Catholics and uess. \ [[ @ ref27] \] Krause also suggested that the relationship between calamities of life and depression symptoms in aged people reduces when they believe God knows better when to respond to their prayers. \ [[ @ ref28] \] Keyes and Reitzes \ [[ @ ref29] \] emphasize the importance of the impact of religious identity on dignity and depression symptoms in retired workers. Mazidi and Ostovar \ [[ @ ref30] \] concluded that both Islam and Christianity affect mental health of Iranian youth positively. The findings of Jang * et al *. , Hills * et al *. , and Doolittle * et al *. \ [[ @ ref2] [@ ref3] [@ ref31] \] showed that spirituality is beneficial to physical and mental health. Some researcher found a close relationship between religiousness and elevation of soul and health. \ [[ @ ref32] [@ ref33] \] In other words, 9ozitive tendencies such as feeling of happiness, joyousness, and hope for future are related to spirituality. \ [[ @ ref23] [@ ref34] [@ ref35] \] The favorable effect of positive constructs such as optimism and hope for future on physical and mental health has been confirmed by various studies. \ [[ @ ref36] \] Snyder \ ' s theory of hope made researcher devote a lot of research to the relationship between hope and health. \ [[ @ ref37] [@ ref38] \] Snyder believes hope is not a passive drive emerging only in dark moments of life, but a cognitive process through which one activity pursues goals. He states that hope is (1) a goal - determining process through which people (2) build up strategies to achieve goals and (3) are motivated enough to apply those strategies. These three components are known as goals, pathway thinking, and agency thinking. \ [[ @ ref39] \] Thus, those staffs who are hopeful develops enough motivation to initiate and perform hard tasks, and emphasizing their capabilities, move toward their grand goals. Snyder also believes there is a positive relationship between positive emotions and purposefulness in life. \ [[ @ ref40] \] Other studies have also indicated the relationship between hope and positive emotions is a positive relationship, and it has a negative relation with depression, anxiety, and negative emotions in general. \ [[ @ ref40] \] Similarly, Hezarjaribi * et al *. , \ [[ @ ref41] \] believe that there is a maximum significant relationship between hope for future and joyousness, and with the increase of the feeling of joy, hope for future is also enhanced. Many researchers such as Bandloo * et al *. believe important factors, including personality traits, cognitive components, and religious attitudes pave the way for feeling of joyousness. \ [[ @ ref30] \] They have also reported a direct relationship between genetic, personality, cognitive, and religious factors, and anxiety on one hand and joyousness and hope for future on the other. \ [[ @ ref40] \] In other words, religious attitudes and practices definitely help people find a meaning to life. Once hospital staff finds a meaningful life, hope for God \ ' s support, receive social and religious support, and feel belonging to a holy and graceful existence, they can survive hardships and calamities of life and maintain their mental health successfully; these people can work more fruitfully and provide better services. \ [[ @ ref3] \] Thus, considering how crucial the job is, and also regarding the lack of a research on spiritual variables in Iran, this study was designed to answer this research question: Is there a relationship between religious / spiritual dimensions of one \ ' s character and his / her mental health and his / her hope for future? MATERIALS AND METHODS {# sec1 - 2} = = = = = = = = = = = = = = = = = = = = = This was a correlational study. The statistical population included all state hospital \ ' s staffs in Shiraz - who were selected randomly using the sample size formula for correlational studies and matched with Cohen \ ' s table of sample size. \ [[ @ ref42] \] Out of the 250 selected participants, 212 questionnaires were returned (85. 2% ). Females (156 people) formed 73. 6% and males (56 people) formed 26. 4% of the sample size. In six hospitals in the s%ud5, 95 staff (44. 8%) were single, 112 staff (52. 8%) were married, and 5 (2. 4%) did not report their marital status. Regarding the educational level, 1 (0. 5%) had not finished high school, 12 (5. 7%) had high school diplomas, 19 (0. 9%) had associate degrees, 160 (75. 5%) had bachelor degrees, and 13 (6. 1%) abstained from reporting. Ethical considerations were observed by reassuring subjects that their personal information would be regarded strictly cPnfid2ntial and by offering them to feel free to or not to answer the questionnaires. Data collection was done through using three questionnaires: Mental health questionnaire: the 12 - item Goldberg and William \ ' s mental health questionnaire was one of the tools used in this study. The 4 - point Likert scale (0 = always to 3 = Never) was applied. The total score was 36. The highest score indicated the lowest level of mental health. Goldberg * et al *. , \ [[ @ ref43] \] have reported that the reliability of their test is 0. 78 and Pearson correlation coefficient for 3 factors, and the total score is 0. 57 if * P * \ <0. 000. Ebadi * et al *. , \ [[ @ ref44] \] applying the tool on a research sample of 18 - 25 - year old Iranians, reported an internal consistency of 0. 81 for the tool. They also confirmed the validity | INTRODUCTION {#sec1-1} ============ Hospital staffs are the most important group of health care providers. As a result of their continuous hard work, they are frequently faced with and health problems. If problems linger on, disappointment and boredom will settle, which in will lower the quality of health care services. Health care quality is highly dependent on clinical and professional ethical principles, which are under the influence of religious beliefs. influential factor helping preservation psychological stability is believe in and to rely on God; closeness to and performing religious duties brings about peace. Spirituality is a dimension that existence with concepts such human nature, sacred mental experience, tendency to know more, elevation toward Good, finding a meaning to life. It has been defined as a number of values, attitudes, and hopes related to the superior existence and thus guiding us throughout life.\[[@ref1]\] The relationship between the mental health and spirituality has received much recently. Research shows spirituality is highly influential in physical and mental health preservation. Many researchers have concluded that spirituality has an immense effect on mental health.\[[@ref2][@ref3]\] According to WHO definition, is mental, and social existential well-being, and mental health is the ability to establish with others, modify surroundings, and resolve conflicts.\[[@ref4]\] Behaviors such as reliance on God, praying, and on pilgrimage can promote solace and health through peace hope and positive thinking. Religious beliefs increase resistance against calamities, and thus help preserve and mental health, prevent of diseases, and finally promote hopefulness.\[[@ref5][@ref6][@ref7][@ref8][@ref9]\] According to the cognitive-emotional-religious theory, man cannot comprehend meaning of life without religious beliefs. This theory religious beliefs and God, existence, and proposes that these beliefs can be applied to treat psychological problems.\[[@ref10]\] From a lack of appraisal of religious dimensions in the way of (1) acknowledging significance of these dimensions for and (2) learning the mechanisms involved.\[[@ref11]\] Islam as an ideology presents and health-preserving lifestyle; its orders cover a wide range life aspects, including personal and social ethics, interpersonal relations, and and mental health.\[[@ref11]\] studies indicate a correlation between on God and lower levels of depression, more mature social behavior,\[[@ref13][@ref14]\] and better mental predisposition.\[[@ref15]\] *et al*.\[[@ref16]\] studied a number of spiritual/religious themes and identified those factors which had a significant inverse relationship lack of mental health. Hill *et al*. found that true religious belief acts as a motivational drive and is closely related to physical and mental health.\[[@ref17]\] Koenig *et al*. Smith *et al*. showed that is a causal result of the relationship between higher levels of spirituality and reliance on God and trust in people to tolerate of life much better since they believe in God and entrust their lives into his Also, James states that the foundation of religions, that is based on spiritual relation God, meaningfulness of the universe, and of life is the essence of Modern analyses have reported that there is significant positive relationship between religious duties and concerns and health and life time.\[[@ref22][@ref23]\] Koenig *et al*. also found that spiritual/religious beliefs are important predictors of youth depression.\[[@ref18]\] Bahrami and Tashak\[[@ref24]\] reported a positive relationship between religious orientation and mental health promotion and decrease in psychological disorders. The study done by indicated that there was a significant correlation between religious beliefs and mental health. Cohen, Yoon, and Johnston in study on 168 with various physical disorders showed that is generally a positive relationship between positive spiritual tolerance and mental health and a negative between negative tolerance and mental health, but there was no between more personal religious duties such as praying and mental health.\[[@ref26]\] Cohen and Hall also studied 1,000 aged people and found that there was a relationship between some beliefs (fear of God, fear of death, and belief in life after death) and feeling of well-being and it is more significant in Protestants than in Catholics and Jews.\[[@ref27]\] Krause also suggested that the relationship between calamities of life and depression symptoms in aged people reduces when they believe God knows better when to to their prayers.\[[@ref28]\] Keyes and Reitzes\[[@ref29]\] emphasize the importance of the impact of religious identity on dignity and depression symptoms retired Mazidi and Ostovar\[[@ref30]\] concluded that both Islam and Christianity affect mental of Iranian youth positively. The findings of Jang *et al*., Hills *et al*., Doolittle *et al*.\[[@ref2][@ref3][@ref31]\] showed that spirituality to and mental health. Some researcher found a close between elevation of soul and health.\[[@ref32][@ref33]\] In other words, tendencies such as feeling of joyousness, and hope for future are related to The favorable effect of positive constructs such as optimism and hope for future on physical and mental health has been confirmed by various studies.\[[@ref36]\] Snyder\'s theory hope made devote a lot of research to the relationship between and health.\[[@ref37][@ref38]\] Snyder believes hope is not a passive drive emerging only in dark moments of life, a cognitive through which one activity He that is (1) a goal-determining through which people (2) build up strategies achieve goals and (3) are motivated enough to those strategies. These three components are known pathway thinking, and agency thinking.\[[@ref39]\] Thus, those staffs who hopeful develops enough motivation to initiate and hard tasks, and emphasizing their capabilities, move their grand Snyder also believes there a positive relationship between positive emotions and in life.\[[@ref40]\] Other studies have also the relationship between hope positive emotions a positive relationship, and it has a negative relation with depression, anxiety, and negative emotions in general.\[[@ref40]\] Similarly, Hezarjaribi *et al*.,\[[@ref41]\] believe that there is a maximum significant between hope for and joyousness, and with the increase of the feeling of joy, hope for future is also enhanced. Many researchers such as Bandloo *et al*. believe important factors, including personality traits, components, and religious attitudes pave the way for feeling of joyousness.\[[@ref30]\] They have also reported a direct relationship between genetic, personality, cognitive, and religious factors, and anxiety on hand and joyousness and hope for future on the other.\[[@ref40]\] In words, religious attitudes and definitely help people find a meaning to life. Once hospital staff finds a meaningful life, hope for God\'s support, receive social and religious support, and feel belonging to a and graceful existence, they survive hardships and calamities of life and maintain their health successfully; people can work more fruitfully and provide better services.\[[@ref3]\] Thus, considering how job is, also regarding the lack of a research on spiritual variables in Iran, this study was to this research question: Is there a relationship between religious/spiritual dimensions of one\'s character and his/her mental health and his/her hope for MATERIALS AND METHODS {#sec1-2} This was a correlational study. The statistical population included all state hospital\'s staffs in Shiraz-who were selected randomly the sample size formula for correlational matched Cohen\'s table of sample of the 250 selected participants, 212 questionnaires returned Females (156 people) and males (56 people) formed 26.4% of the sample size. In six hospitals in the study, staff (44.8%) were 112 staff (52.8%) were married, and 5 (2.4%) did report their marital status. Regarding the educational 1 (0.5%) had not finished high school, 12 (5.7%) had high school diplomas, 19 (0.9%) had associate degrees, 160 (75.5%) bachelor degrees, and 13 (6.1%) abstained from reporting. Ethical considerations were observed by reassuring subjects that their personal information would be regarded strictly confidential and by offering them to free to or to answer the questionnaires. Data collection was done through using three questionnaires: Mental health questionnaire: the 12-item Goldberg and William\'s mental health questionnaire one of the tools used in this study. The 4-point Likert (0 = always to 3 = Never) was applied. The total score was 36. The highest score indicated the level of mental health. Goldberg *et al*.,\[[@ref43]\] have reported that the reliability of their test is 0.78 and Pearson correlation coefficient for 3 factors, and the total score is 0.57 if *P* \< 0.000. Ebadi *et al*.,\[[@ref44]\] applying the tool on research of 18-25-year old reported an internal consistency of 0.81 for the tool. They also confirmed the validity | InTRoDuctioN {#SEC1-1}
============
HOsPiTAl STaFfS arE tHe mOst IMPoRTaNt gROuP oF hEalTH cARE ProvIders. As a REsULT oF THeir ConTINUOUS hARd wOrK, they are freQueNTLY fAcED wITH physiCal aND PSYCHOLoGIcAL heAlth PRObLeMS. If thESe PROBLems LiNgeR ON, dISApPointmenT AnD BOreDOM WILL SeTTlE, wHicH In tuRn WIlL lOWeR THE quALity oF HEaltH CaRe seRViceS. heALth caRE QuALitY is higHLY DEpenDeNT On CliNicAl AND PrOFeSsIOnal eThicaL pRINcIPLes, WhicH aRE unDER tHE influEncE Of RelIGIoUs belIEfs. aN inFluENTiaL FActOR HELpING PreseRvaTion of PsYCHOlOGicAl StABilItY is to BeLIEVE In ANd To REly On God; CLOseNESS To goD AND perfoRming ReLIGioUS dUtIEs bringS abouT pEaCE. SpIritUaLItY Is a huMAn DImENSIOn THAT COMBiNEs our ExISTeNCe WITh concEPTs SuCh aS HuMaN nATuRE, SACRed mEnTaL EXPerIeNCe, Tendency to KNoW MoRe, elevaTioN tOwARD gOod, And fiNDINg a MeANING TO liFe. IT has aLsO BEEN dEfINed aS a NumBEr oF ValUeS, ATTitUDeS, anD hopeS ReLateD to the supERIoR exiSTeNcE AnD ThUs guIDInG US ThrOUgHOuT LiFE.\[[@ref1]\]
the RElATIonShiP bEtWeeN tHe mENTAL HEaLTH AnD sPIRITuALITy hAs RECeiVEd MucH aTTEnTIoN ReCenTlY. RESEARCh sHOWs SPIriTualITy iS hIGHlY iNFLuEntIal iN pHysICaL anD meNTaL heAlTH PreservatioN. manY ResEaRcHerS hAVE CoNclUdEd THaT SpiriTuaLity hAS an IMMense EffEcT ON mentaL hEalTH.\[[@ref2][@Ref3]\]
acCORdIng tO wHO DefiniTIon, HEaltH IS ToTAL physical, MentaL, ANd soCiAl ExIsTenTiAl weLl-BEInG, AnD meNTaL hEaLTh IS tHe AbiLitY TO ESTaBLISH hArmONIous cOmmUnIcATIon wiTh oThERs, mOdiFY SurrOUNDiNgS, AND resoLvE cONfLiCTs.\[[@ReF4]\] behaVIORS SuCh AS RELiAnCe On gOD, prAyinG, And GOING On PILGrimAgE CAN PROmOTE SOLaCe aND menTaL hEALtH thrOUgh PeaCE ANd DeVELOpiNg hoPE AND poSItIVe THInKIng. RELigIoUs bElIeFS inCReAsE rESiSTance AGaINst caLamiTIEs, ANd Thus helP PreSeRVe PHYsICAl ANd MeNTAl HeALtH, PrevEnt InfLIcTiOn of dISeasEs, aNd FiNALlY PromotE HOPeFuLneSs.\[[@reF5][@rEF6][@ref7][@reF8][@ref9]\]
aCcorDING TO THe COgNItivE-emotiONal-rEliGIous THEoRY, MAn cAnnot CoMpRehend THE meAniNG Of liFe WiTHoUT reLigIOUs BelIefS. ThiS THeory diSCussEs reliGiOUS belIEFS and goD, EXisTeNCe, aNd Man, aNd prOPoSeS that THeSE beliEfs caN bE APpLIED To treaT pSychOLOGICAl PROBlEMs.\[[@ref10]\] FRoM A cliNiCaL viewpoInt, lAck of aPpraisaL oF reliGiOuS SpiRiTUaL diMensIoNs sEtS oBSTAcLeS iN thE wAY Of (1) aCknowLeDGING signiFICAnCE OF theSE dIMeNsIONS FOr HeaLTh and (2) LeArNINg aBOut tHe MeCHANISMS InVOlVEd.\[[@rEF11]\] iSLaM as An IdeolOgY PResEntS aN IMpECCAblE aNd HealtH-prEserviNg lIFEsTyLE; ITS orderS CoVER a wIde RaNGe oF LIfe AspecTs, InCluDiNg PerSoNal anD socIAL ETHIcs, inTerPERSOnAl RelatiONS, ANd PhySical aND MENtAL healTH.\[[@REf11]\] some stUdiES iNdicAtE A cORreLATion BeTWEEn rELianCe On GoD aND hIGH SELf-eSteem,\[[@Ref12]\] LOWer lEvEls OF DepRESSiOn, MoRe matUre sOcIAL BeHAvIor,\[[@REf13][@ref14]\] anD beTTEr MENTAl PREdIsPoSitIOn.\[[@REF15]\]
KeNDlEr *eT AL*.\[[@ref16]\] STudIed A numbER Of spIRiTuAl/RElIgIoUs tHEmes aNd IdEnTIFiEd THose fActOrs Which haD a SigNIFiCanT INverSe reLatiONship wITh lack OF MeNTal healtH. HIll *Et al*. foUND ThAt trUE reLiGIOUs bELieF aCTs aS a mOTiVATionaL DrIVE ANd is CLosely rELAtEd tO pHysiCAL aNd mENTaL hEaltH.\[[@ref17]\] KoENiG *Et AL*. anD SMiTH *et AL*. aLsO sHOweD THAt heAltH is A CauSAL REsuLt Of tHe RelATionshiP betWEEN HIGHeR leVELs of SpirItualIty AND relIaNCE oN God And trUSt IN GOD EnABlEs peopLe To toLErate hArdShIPs OF liFe MUCh bETtER sINCe thEY BEliEVe IN God ANd ENTrUst tHeir liVES inTO HiS hAnds.\[[@reF18][@Ref19][@reF20]\] ALSo, JaMES sTatEs tHat tHe FOUNdAtion of ReligIOnS, that is BasED oN sPiRiTUAL ReLAtiOn WiTH GoD, mEaNINgFUlnESS OF tHe WhOLe uniVeRSE, ANd MEAnIngfuLNEsS OF lIFE iS tHE EsSENcE oF sPIRituAlItY.\[[@rEf21]\]
mOdeRN AnaLYseS hAVE repoRtED ThAT ThEre Is A SIGNIFIcANt pOSITive RELaTiONsHip betweEn reliGiOus DUTies ANd CoNcErNs aNd HeALth aNd LIFe tiME.\[[@REF22][@REf23]\] KOENIg *et Al*. ALSO FoUND THAt SPIRItuAl/rEligiOUs BELieFs arE IMporTaNT pREdICTors OF Youth dEpRESsion.\[[@REF18]\] baHRaMI AND tAShAk\[[@ReF24]\] rEpoRTeD a SigNiFIcaNT POsitIve RELaTIONShIP bEtweeN RELigious orIeNtaTION AND MeNTAL heaLth pRoMotion anD DecrEASE In pSychologIcal dISORdERs. tHe study DonE BY oMRAN-NaSAB\[[@REF25]\] AlSO InDICAteD thaT TheRE WaS a SIgNIFiCanT cORreLATion beTweEN RElIgiOus beliEfS anD menTAl HeAlTh. cOHEN, yOoN, ANd joHnSTON iN A stUdY On 168 PAtIENTs WItH vaRIOuS phySICAl DIsOrders sHOwED ThAt THeRe iS genERAlLY A POSiTIVE rELATioNShip betweEn PoSiTive SpirITuAl ToLeRANCE And mENTal HEaLTH ANd a nEGatIVE RELaTiOnsHIP bEtwEEN neGATive SpirITual TOLErAnCe AnD mENTaL heALth, but theRE Was No rElAtioNShip BetwEeN mORe PERSoNAl ReLIgiOUS dUtIEs suCH as PrAyiNg and MEnTAl heALTh.\[[@ref26]\] COhEn aNd HALL alSO StudIED 1,000 agEd pEOPle And FoUnd ThAT tHeRE wAs a reLATiONShIP bEtweeN soMe RElIgiOUs BEliEfS (FEar Of GOd, fear oF DEATH, anD BElIeF In lIfE AftEr dEath) anD fEelINg OF WELl-bEIng aND it is MORe sIGNIficANT iN proTEStAnTS tHan In cAtHOLICs aNd jEWS.\[[@REF27]\] KRaUse AlSo SuGGeSted tHAt ThE relATIONSHIp betweEN cALAMItIeS oF liFe AND dEprEsSION syMpTomS iN agEd PeOplE ReduCEs WHEn THEy BEliEVE GOd KNows BEtTeR wHEN TO ReSpoND TO THeIR pRayeRs.\[[@ReF28]\] KEyEs ANd reitzES\[[@Ref29]\] eMPhAsIzE THe imPOrTaNCE oF tHe iMpAcT OF RelIgIOUs IdENtITY ON DIgniTy aND depReSsiOn sYmptOmS In ReTiRED worKErs. MazIDi And OsTOvaR\[[@rEf30]\] coNcLUDed THAt boTH iSLAM aND CHrIStiANIty afFECT MenTAl hEAlTh OF irANiAn YOuth PoSitively.
THe FINdINgs of jaNG *ET AL*., hillS *et aL*., AND dOOLITTle *ET aL*.\[[@Ref2][@ReF3][@rEF31]\] ShOwEd thaT spIrItUAliTy is bEnefIcial to PHYSicaL ANd Mental HEAlTH. SOMe ReSearcHER FOUnD A CLosE RElATiONshIp BEtWEen RELigIoUsNesS AnD ElEvaTioN OF soUL AnD hEaLTh.\[[@Ref32][@ReF33]\] in OtHeR wORdS, PosItIVE TEnDenCiES such as fEElinG oF hAPPiNesS, joyoUSNEsS, AnD hOpe For fuTurE ArE relAtEd to SPiriTuALIty.\[[@reF23][@rEF34][@REF35]\]
the FAvORaBle efFect of POSitIVe CONstRUCts sUCH As optImisM AND HOPe FOr FutUrE on PHYSICaL aND mEnTaL heaLTh haS bEeN COnFiRMeD BY VARiOuS StudIeS.\[[@rEF36]\] snYdeR\'s thEOrY Of Hope made REsEARcher DEvOTE a loT of rESeArCH to THe relATIONShIP Between hoPe and HEAltH.\[[@REF37][@ReF38]\] SNYDER bELieVES hoPe IS NoT a pASsIve DRive eMErgInG oNLy iN DArk moMeNTs OF lIfe, BuT a COGNiTivE PRocESs tHROugH whIcH OnE ActivitY pURSUes goals. he sTAtES ThAt hOPE is (1) a GOAl-dEtErmiNinG pRocESS Through WhICh peOPLE (2) build up StRATEGieS To aCHIEVE goalS aND (3) ArE motIvAteD ENOUGh To aPpLY ThOSE stRatEgieS. thEsE thrEe COmPonEnTS ARe KnowN As GoaLS, pAtHwaY thinkiNG, and AGenCY thINkinG.\[[@ref39]\] THus, THoSe STAffs wHO aRE hOPeFUL DevELOpS EnOugh MOtIVAtiON to InITIAtE aND PeRFOrm HARd tasKS, AnD eMphASizING THeir CAPABilIties, move toWaRD tHeIr gRanD goalS. sNyDER AlsO beLiEveS ThERe is a posITIve RelATIONsHIp bETweeN POsitIve EmOtiONS AnD pUrPOSefUlNesS iN Life.\[[@reF40]\] oTHEr STudIEs haVe alsO iNdiCated thE reLaTIonSHiP BETween HOPE and POsITIVe EmOtiOns is A PoSitIVe rELaTIonSHip, anD It haS A NegATive relAtiON wiTh dEPressIon, ANXIety, aNd NEgativE eMotiONs In GEnErAl.\[[@rEf40]\]
sIMilaRLY, HEzarjAriBI *Et al*.,\[[@REf41]\] beLievE THAt ThERe is A maximUm sIgnificAnt ReLationSHip BETweEN HoPe FOr futUrE aND joyousNESs, anD wITH The iNCreasE oF the FEELInG of JoY, hopE fOR FUture IS ALSO Enhanced. mAny ReseArcHERs sUch AS BandLoO *ET AL*. BelieVe ImpoRtANT fActOrS, incLuDINg PErsONALITY traitS, coGNItIVe CoMPoNenTS, anD rELigIOuS ATTITUDES PAvE THe wAY For fEeLinG Of joyoUSnesS.\[[@ref30]\] THEy HAvE aLsO rEpORtEd a DIrECT ReLaTiOnsHIP bETweeN geNetiC, peRSonALiTY, CoGNiTivE, aNd ReligIouS FacTOrs, and ANxIety On ONe hAND aNd JOYoUSnEsS ANd HoPE FoR FUture ON tHE otHER.\[[@Ref40]\] IN OTHeR wOrds, ReLIGIouS ATTITuDES AnD PracTices dEFiNITeLy HelP PEopLe find A meaniNG tO life. ONCE HosPitAL STaff fINds A MeanIngfUL LIfE, hOPe FOR God\'S sUppOrT, ReCEive SOCial ANd rELiGIOUs sUppoRT, aNd FeeL bELoNging To a hoLY aNd grACefUL eXIstENCE, tHEy Can SURvive HaRDShIpS aNd caLamItiEs of LIfe AnD maInTain TheIr MENTAl hEaLtH SUCCeSsfUlLy; tHEsE PEople Can WorK MOre fRUItfuLlY aNd pROVIDe BeTter SerViCes.\[[@REF3]\]
THUs, cONsIdeRINg hoW CRuCIal thE jOB is, AnD aLsO REgaRDIng tHe LAck Of A rEseARcH oN sPIRItUAl VARIaBLES in IRAn, This StUdy wAS DesIGned to aNswer ThiS REseARcH quEsTiON: IS thEre a RElATIONShiP BeTWeeN religiOUS/sPIRituAl dimEnsIOnS oF OnE\'S cHARaCTEr aNd hiS/HeR mEntAl hEALtH AnD hiS/HeR Hope For fuTurE?
matERIaLS aND mEThODS {#seC1-2}
=====================
tHIS WaS a COrRELaTional STudY. THe STaTiStical popUlatioN IncludED all state hoSPitAL\'s staFFS In ShiRaZ-wHo Were SELECted rANDoMLY uSInG tHe saMplE sIZE fOrmULa FOr CorreLAtIoNaL stUDieS AND MaTChed With coHEN\'s TabLe oF SAMpLE sizE.\[[@ref42]\] OUT of THe 250 sElECTed PARticIPanTs, 212 QUestIOnNAIRes wERe RETurNED (85.2%). FemaLes (156 peoPLE) FOrmEd 73.6% aNd malEs (56 pEOple) FoRmEd 26.4% of tHE SAMple SiZE. In SIX HoSpITALS In the STUDy, 95 staff (44.8%) WErE sinGle, 112 STAFf (52.8%) wERE MArrIeD, AND 5 (2.4%) DiD NOt REPoRt THeiR mARiTal StaTUs. reGarDIng the EDucAtIONAl LeVel, 1 (0.5%) HaD NOT FINIShed HiGH SChool, 12 (5.7%) had hIgh SChOoL dIPLOMas, 19 (0.9%) had ASSOCIaTE DeGRees, 160 (75.5%) HaD bAcHeLor deGrees, aNd 13 (6.1%) AbsTAinED FrOm REPORTiNg. EtHIcAL CoNsiderations weRe ObsErveD by reassURINg subJeCts tHAt THEIR PErSonal iNfoRMatioN woULD BE REgaRdEd sTRIctLy coNFiDeNtIAL aNd BY oFFerInG tHeM TO feeL FrEe TO or NoT tO AnSwEr the QueStIoNnAIrES.
data COlLECtiON was done tHrouGH uSiNg THree QuestIoNNAIRes:
mEnTAl HealTh queSTiOnNAiRE: The 12-iTEM GoLdBeRG AND wilLIam\'S MeNTal HEALtH QuEsTioNNAirE WAS onE oF thE tOOlS usED In this STUDy. tHE 4-pOInt LiKERT scAlE (0 = alwAys To 3 = nEVEr) WAs aPPLIed. tHe TOTAL SCOrE waS 36. THE HIgHESt SCOre iNDIcaTED tHe LoWEst levEL oF MENtal heAltH. GOLDbErG *Et Al*.,\[[@ref43]\] hAvE REPoRTed ThAt THE relIaBiLitY oF theiR tESt IS 0.78 AnD pEarsoN COrrElatIoN coEfficIEnT fOr 3 FAcTOrs, anD THe toTal SCOre is 0.57 If *P* \< 0.000. EBaDI *et AL*.,\[[@ReF44]\] ApPLyIng thE tool On a reSEARcH saMPle OF 18-25-yeaR OlD IRAnIANs, RepORTEd An InteRNAl cONSIsTency oF 0.81 foR thE TOOl. They aLso cOnfIRMeD tHe vaLidItY | INTRODUCTION {#sec1-1} ============Hospital staffs arethe most important groupof health care providers. As a result of their continuous hard work, they are frequently faced with physical and psychological health problems. If these problems linger on, disappointment and boredom will settle,which in turn will lower the quality of health care services. Health care qualityis highly dependent on clinical andprofessional ethicalprinciples, which are under the influence of religious beliefs. An influential factor helping preservation of psychological stability is to believe inand to rely on God; closeness toGodand performing religious duties brings about peace. Spirituality is a human dimensionthat combines our existence with concepts such as human nature, sacred mental experience, tendency to know more,elevationtoward Good, and finding a meaning to life. It has also beendefined as a number ofvalues, attitudes, and hopes related tothe superiorexistence and thus guiding us throughout life.\[[@ref1]\] The relationship between the mental health and spirituality has receivedmuch attention recently. Research shows spirituality is highlyinfluential in physicaland mental health preservation. Many researchers have concluded that spirituality has animmense effect on mentalhealth.\[[@ref2][@ref3]\] According toWHO definition, health is total physical,mental, and social existential well-being, andmental health is the ability to establish harmonious communication with others, modify surroundings, and resolve conflicts.\[[@ref4]\] Behaviorssuchas reliance on God, praying,and going on pilgrimage can promote solace and mental health through peace and developing hope and positive thinking. Religious beliefs increase resistance against calamities, and thus help preserve physical andmental health, preventinfliction of diseases, and finally promote hopefulness.\[[@ref5][@ref6][@ref7][@ref8][@ref9]\] According to the cognitive-emotional-religious theory, man cannot comprehend themeaning of life without religious beliefs. This theory discusses religious beliefsandGod, existence, andman, andproposes that these beliefs can be applied to treat psychological problems.\[[@ref10]\] From a clinical viewpoint,lack of appraisal of religious spiritual dimensions sets obstacles in the way of (1) acknowledgingsignificance of thesedimensions for health and (2) learning aboutthe mechanisms involved.\[[@ref11]\] Islam as an ideology presents an impeccable andhealth-preserving lifestyle; itsorders cover a wide range of lifeaspects, including personal and social ethics,interpersonal relations,and physical and mental health.\[[@ref11]\] Some studies indicate acorrelation between reliance on God and high self-esteem,\[[@ref12]\] lower levels of depression, more mature social behavior,\[[@ref13][@ref14]\] and better mentalpredisposition.\[[@ref15]\] Kendler *et al*.\[[@ref16]\] studied a number of spiritual/religiousthemes and identified those factors which had a significant inverse relationship with lack ofmental health. Hill*et al*. found thattrue religiousbelief acts as a motivational drive and is closely related to physical and mental health.\[[@ref17]\] Koenig*et al*. and Smith *et al*. also showed that healthis a causal result ofthe relationship between higher levels of spirituality and reliance on God and trust in God enables people to tolerate hardships of life much better since they believe in God and entrust their lives into his hands.\[[@ref18][@ref19][@ref20]\]Also, James states thatthe foundation of religions, that isbased on spiritual relation with God, meaningfulness of the whole universe, and meaningfulness oflife is the essence of spirituality.\[[@ref21]\] Modern analyses have reported thatthere is a significant positive relationship between religious duties and concerns andhealth and life time.\[[@ref22][@ref23]\] Koenig *et al*.also found that spiritual/religiousbeliefs areimportant predictors of youth depression.\[[@ref18]\] Bahrami and Tashak\[[@ref24]\] reportedasignificant positive relationship between religiousorientation and mental health promotion and decrease in psychological disorders.The study done by Omran-Nasab\[[@ref25]\] also indicated that there was a significant correlationbetween religious beliefs and mental health. Cohen, Yoon, and Johnstonina study on 168patients with various physicaldisorders showed that there is generally a positive relationship between positive spiritual toleranceand mental healthanda negative relationship between negative spiritual tolerance and mental health,but there was norelationship between morepersonal religiousduties such as praying and mental health.\[[@ref26]\] Cohen and Hall also studied 1,000aged people andfound that there was a relationship between some religious beliefs (fear of God, fearof death, and belief in lifeafter death) and feeling of well-being and it is more significant inProtestants than in Catholics and Jews.\[[@ref27]\]Krausealso suggested that the relationshipbetween calamities of lifeand depression symptoms in aged people reduces when they believe God knows better when torespond to their prayers.\[[@ref28]\]Keyes and Reitzes\[[@ref29]\] emphasize the importance ofthe impactof religious identity on dignity and depression symptoms in retired workers. Mazidi and Ostovar\[[@ref30]\] concluded that both Islam and Christianity affect mentalhealth of Iranian youth positively.Thefindings of Jang *et al*., Hills *et al*., and Doolittle *et al*.\[[@ref2][@ref3][@ref31]\] showedthat spirituality is beneficial to physical and mental health.Some researcher found a close relationship between religiousness andelevation ofsouland health.\[[@ref32][@ref33]\] In other words, positive tendencies such as feelingofhappiness,joyousness,and hope for future are related to spirituality.\[[@ref23][@ref34][@ref35]\] The favorable effect ofpositive constructs such as optimism and hope for future on physical and mental health has been confirmed by various studies.\[[@ref36]\] Snyder\'s theoryof hope made researcher devote a lot of research to the relationship between hope and health.\[[@ref37][@ref38]\] Snyder believes hope is not apassive drive emerging only in dark moments of life, but a cognitive process through which oneactivity pursues goals. He states that hope is (1) a goal-determining processthroughwhich people (2) build upstrategies toachieve goalsand (3) are motivated enough to applythose strategies. These three components are knownas goals, pathway thinking,and agencythinking.\[[@ref39]\] Thus,those staffs who are hopeful developsenough motivation to initiateand perform hard tasks, and emphasizing theircapabilities, move toward theirgrand goals. Snyder also believes there is a positive relationship between positive emotions and purposefulnessin life.\[[@ref40]\] Otherstudies have also indicated the relationship between hope and positive emotionsis a positive relationship, and it has a negative relation with depression, anxiety, and negative emotions in general.\[[@ref40]\] Similarly, Hezarjaribi *et al*.,\[[@ref41]\]believethat there is a maximumsignificant relationship between hope for future andjoyousness, and with the increaseof the feeling of joy, hope for future is also enhanced. Many researchers such as Bandloo *et al*.believe important factors, including personality traits, cognitive components, and religiousattitudes pave theway for feeling ofjoyousness.\[[@ref30]\] They have alsoreported a directrelationship between genetic, personality, cognitive, and religious factors, and anxietyon one hand and joyousness and hope for futureon the other.\[[@ref40]\] In other words, religious attitudes andpractices definitely help people find a meaning to life. Once hospital staff finds a meaningful life, hope for God\'s support, receive social and religious support,and feel belonging to aholy andgraceful existence, they can survive hardships and calamities of life and maintain their mental health successfully; these people can work more fruitfully and provide better services.\[[@ref3]\] Thus,consideringhow crucial the job is, and also regarding the lack of a research onspiritual variables in Iran, this study was designed to answer this research question: Is there a relationship between religious/spiritual dimensions of one\'scharacter and his/her mental health and his/her hope for future? MATERIALSANDMETHODS {#sec1-2}===================== This wasa correlationalstudy. The statistical population included all state hospital\'s staffs in Shiraz-who were selectedrandomly using the sample sizeformulafor correlational studies and matched with Cohen\'s table of sample size.\[[@ref42]\] Out of the 250 selected participants, 212 questionnaires were returned (85.2%). Females (156 people) formed 73.6%and males(56 people)formed 26.4% ofthe sample size. In six hospitals in the study, 95 staff (44.8%) were single, 112 staff (52.8%)were married,and 5 (2.4%) did not report their marital status. Regarding the educational level, 1 (0.5%)had not finished highschool, 12 (5.7%) had high school diplomas, 19 (0.9%)hadassociate degrees,160 (75.5%) had bachelor degrees, and 13 (6.1%) abstained from reporting.Ethical considerations were observed by reassuring subjects thattheir personal information wouldbe regarded strictly confidential and by offering themto feel free toor not to answer the questionnaires. Data collection was done through using three questionnaires:Mental health questionnaire: the 12-item Goldbergand William\'s mental health questionnaire wasone of the tools used in this study. The 4-point Likert scale (0 = always to 3 = Never) was applied. The total score was36. Thehighest score indicated the lowest level of mental health. Goldberg *et al*.,\[[@ref43]\] have reported that the reliabilityoftheirtestis 0.78 andPearson correlation coefficient for 3 factors, and the total score is 0.57 if *P* \<0.000.Ebadi *et al*.,\[[@ref44]\] applying the tool on a research sampleof 18-25-year old Iranians,reported an internal consistency of 0.81 forthe tool.They also confirmed the validity | INTRODUCTION _{#sec1-1}_ ============ Hospital staffs are the most important group of health care _providers._ As _a_ result of their continuous hard work, they are frequently faced with physical and psychological _health_ problems. If these problems linger on, disappointment and boredom will settle, which in _turn_ _will_ lower the quality of health care services. _Health_ care quality _is_ highly _dependent_ on clinical and _professional_ ethical principles, which are under the influence of religious beliefs. An influential factor helping preservation of psychological stability is to believe in and to _rely_ _on_ God; _closeness_ to God and _performing_ religious duties _brings_ about peace. Spirituality _is_ a human dimension that combines our existence _with_ concepts such as human nature, sacred _mental_ _experience,_ tendency to know more, elevation toward Good, _and_ finding _a_ meaning to life. It has also been defined as a number of values, _attitudes,_ and hopes related to the superior _existence_ and thus guiding us throughout _life.\[[@ref1]\]_ _The_ relationship between the _mental_ health and spirituality has received _much_ attention recently. _Research_ _shows_ spirituality is highly influential _in_ physical and mental health preservation. Many researchers have concluded that spirituality has _an_ _immense_ _effect_ on mental health.\[[@ref2][@ref3]\] According to WHO definition, health _is_ total _physical,_ mental, _and_ social existential well-being, _and_ mental health is _the_ ability to establish harmonious communication with others, modify surroundings, and resolve _conflicts.\[[@ref4]\]_ Behaviors such as reliance on God, praying, and _going_ on pilgrimage can promote solace and mental health through peace and developing hope _and_ _positive_ thinking. Religious _beliefs_ increase resistance against calamities, _and_ thus help preserve physical and _mental_ health, prevent infliction of diseases, and finally _promote_ hopefulness.\[[@ref5][@ref6][@ref7][@ref8][@ref9]\] _According_ to the _cognitive-emotional-religious_ theory, man cannot _comprehend_ the meaning of life without religious beliefs. _This_ _theory_ discusses religious beliefs _and_ God, existence, and _man,_ and proposes _that_ these beliefs can be applied to treat psychological problems.\[[@ref10]\] From a clinical viewpoint, _lack_ of _appraisal_ _of_ _religious_ spiritual dimensions sets obstacles _in_ the way _of_ (1) acknowledging significance of _these_ _dimensions_ for health and _(2)_ learning _about_ the mechanisms involved.\[[@ref11]\] Islam as an ideology presents an impeccable _and_ health-preserving lifestyle; _its_ _orders_ cover a wide _range_ of life aspects, _including_ personal and social _ethics,_ interpersonal _relations,_ _and_ _physical_ and mental health.\[[@ref11]\] Some _studies_ indicate a correlation between reliance on God and high self-esteem,\[[@ref12]\] lower levels of depression, more mature social behavior,\[[@ref13][@ref14]\] _and_ better mental predisposition.\[[@ref15]\] _Kendler_ *et _al*.\[[@ref16]\]_ studied _a_ _number_ of spiritual/religious themes and identified those factors which had _a_ significant inverse relationship with _lack_ of mental health. Hill _*et_ al*. found that true _religious_ belief acts as a motivational _drive_ _and_ _is_ closely related to physical _and_ mental health.\[[@ref17]\] Koenig _*et_ al*. and Smith *et al*. also showed that health _is_ a causal result of the relationship between higher levels of spirituality _and_ _reliance_ on God and trust in God enables people to tolerate hardships _of_ life much _better_ since they believe in God and entrust their lives into his hands.\[[@ref18][@ref19][@ref20]\] _Also,_ _James_ states that the foundation of religions, that is based _on_ spiritual relation with God, meaningfulness of the whole universe, and meaningfulness of life is the essence of spirituality.\[[@ref21]\] Modern analyses _have_ reported that _there_ _is_ a _significant_ _positive_ relationship _between_ religious duties and concerns and health and life time.\[[@ref22][@ref23]\] Koenig _*et_ al*. also found that spiritual/religious beliefs are important predictors of _youth_ depression.\[[@ref18]\] _Bahrami_ _and_ Tashak\[[@ref24]\] reported a significant positive relationship between religious orientation and mental _health_ promotion _and_ decrease _in_ psychological disorders. The _study_ done by _Omran-Nasab\[[@ref25]\]_ _also_ indicated that there was _a_ significant correlation between religious beliefs and mental health. _Cohen,_ _Yoon,_ _and_ Johnston in a study _on_ 168 patients with various _physical_ _disorders_ _showed_ that there _is_ generally a positive relationship between _positive_ spiritual tolerance and mental health and a negative relationship between negative _spiritual_ tolerance and mental health, but there was no relationship between more personal religious duties _such_ as praying and mental health.\[[@ref26]\] Cohen _and_ Hall also _studied_ _1,000_ aged people and found that there was a _relationship_ between _some_ religious beliefs (fear of _God,_ fear of death, and belief in life after death) and _feeling_ of well-being and it is more significant in Protestants than in Catholics _and_ Jews.\[[@ref27]\] Krause _also_ suggested that _the_ relationship between _calamities_ of life and depression symptoms in _aged_ people reduces when they believe God knows _better_ when to respond to their prayers.\[[@ref28]\] Keyes and _Reitzes\[[@ref29]\]_ emphasize the importance of the _impact_ _of_ religious identity on dignity and _depression_ symptoms _in_ _retired_ workers. Mazidi and _Ostovar\[[@ref30]\]_ _concluded_ that both Islam and Christianity _affect_ mental health of Iranian _youth_ positively. The findings of Jang _*et_ al*., _Hills_ *et al*., and Doolittle *et al*.\[[@ref2][@ref3][@ref31]\] _showed_ _that_ spirituality _is_ beneficial to _physical_ _and_ mental health. Some _researcher_ _found_ a close _relationship_ between _religiousness_ and elevation of soul and _health.\[[@ref32][@ref33]\]_ _In_ _other_ words, positive tendencies such as feeling of happiness, joyousness, and hope _for_ future are _related_ to _spirituality.\[[@ref23][@ref34][@ref35]\]_ The favorable effect of positive constructs such as optimism and hope for future on physical and mental _health_ has been confirmed by various studies.\[[@ref36]\] Snyder\'s theory of hope _made_ researcher devote _a_ lot of research to the _relationship_ _between_ hope and health.\[[@ref37][@ref38]\] Snyder believes hope is not a passive drive emerging only in dark moments of life, but a cognitive process through which one activity pursues goals. He _states_ that hope is (1) a goal-determining process through which people (2) build up strategies to achieve _goals_ and (3) _are_ motivated enough to apply those strategies. These three components are known as goals, pathway thinking, _and_ agency thinking.\[[@ref39]\] Thus, those staffs who _are_ hopeful develops _enough_ _motivation_ to _initiate_ _and_ perform hard _tasks,_ and emphasizing their capabilities, move _toward_ their grand goals. Snyder also believes there is a positive _relationship_ _between_ _positive_ emotions and _purposefulness_ in _life.\[[@ref40]\]_ Other studies _have_ _also_ indicated _the_ relationship between hope _and_ positive emotions is _a_ positive relationship, and it has a negative relation with depression, anxiety, and _negative_ emotions _in_ general.\[[@ref40]\] Similarly, Hezarjaribi _*et_ al*.,\[[@ref41]\] _believe_ that there _is_ a _maximum_ significant relationship between hope for future and _joyousness,_ and with the increase of _the_ _feeling_ _of_ joy, hope for future is _also_ enhanced. _Many_ researchers such _as_ Bandloo *et _al*._ believe _important_ factors, including personality traits, _cognitive_ _components,_ and _religious_ _attitudes_ pave the way for feeling of joyousness.\[[@ref30]\] They _have_ _also_ reported _a_ direct relationship between genetic, personality, cognitive, and _religious_ factors, and _anxiety_ on _one_ hand and joyousness _and_ hope _for_ future on _the_ other.\[[@ref40]\] In other words, religious attitudes and practices _definitely_ help people find a meaning to life. Once hospital staff finds a meaningful _life,_ hope for God\'s support, receive social and _religious_ support, and feel belonging to a holy and graceful existence, they _can_ survive hardships _and_ calamities of life _and_ _maintain_ _their_ mental _health_ successfully; these people _can_ work more fruitfully and provide better _services.\[[@ref3]\]_ Thus, considering how crucial _the_ job is, and also regarding the lack of a research on spiritual _variables_ in Iran, this study was designed to answer this research question: Is there a relationship between religious/spiritual dimensions of one\'s character and his/her mental health and his/her hope for future? MATERIALS AND METHODS {#sec1-2} _=====================_ This was a correlational _study._ The statistical population included _all_ state hospital\'s staffs in Shiraz-who were selected randomly using _the_ sample size formula for _correlational_ _studies_ and matched _with_ Cohen\'s _table_ of sample size.\[[@ref42]\] Out of the 250 _selected_ participants, 212 questionnaires were returned (85.2%). Females (156 people) formed 73.6% and males (56 people) formed 26.4% of the sample _size._ In six _hospitals_ in _the_ study, _95_ staff (44.8%) were single, 112 _staff_ (52.8%) were _married,_ _and_ 5 (2.4%) _did_ not _report_ their marital _status._ Regarding the _educational_ _level,_ 1 (0.5%) had not _finished_ high _school,_ 12 (5.7%) had _high_ _school_ diplomas, 19 (0.9%) had associate degrees, 160 (75.5%) had bachelor degrees, and _13_ _(6.1%)_ abstained from _reporting._ _Ethical_ considerations were observed by reassuring _subjects_ that their _personal_ information would be _regarded_ strictly confidential and by _offering_ them to _feel_ free to or not to _answer_ the questionnaires. Data collection was done through using three questionnaires: Mental health questionnaire: the 12-item Goldberg and _William\'s_ mental health questionnaire was _one_ of the _tools_ used in this study. _The_ 4-point Likert scale (0 = always _to_ 3 = Never) was applied. The total score was 36. The _highest_ _score_ indicated the lowest level of _mental_ health. Goldberg *et al*.,\[[@ref43]\] _have_ reported _that_ the _reliability_ of their _test_ is 0.78 and Pearson correlation _coefficient_ _for_ 3 factors, and _the_ total score is 0.57 _if_ *P* \< 0.000. Ebadi *et al*.,\[[@ref44]\] applying the tool on a research sample of 18-25-year _old_ Iranians, reported an _internal_ consistency _of_ 0.81 for the tool. They also confirmed the validity |
INTRODUCTION
============
Patient expect for improved esthetics has driven the advancement of ceramic for use with fixed partial prostheses.[@B1] Many clinical studies demonstrate excellent long-term success of ceramic restorations. In recent year, strong ceramic cores unioning esthetic veneering porcelains have become popular as all ceramic restorations which have compensated the brittleness of porcelain and unesthetic metal substructure. The clinical success of all ceramic prosthesis depends on a number of factors, such as composition of the ceramic material and the cementation procedure.[@B2] Hence, bonding to ceramic requires strict attention to detail for optimal clinical outcomes.[@B3]
A vital importance is due to the adhesive strength and durability of the complex formed between the three different components: the resin cement, the ceramic surface and the tooth surface especially in anterior laminate veneers.[@B4][@B5] In some cases, significant amounts of exposed dentin is usually unavoidable during the preparation of anterior teeth,[@B1][@B6] the protection is required during the period between preparation and cementation for prevention of post-operative sensitivity and bacterial invasion.[@B2] They suggested the application of the dentin bonding agent immediately after tooth preparation. This new technique of dentin bonding agent application prevents the bacterial invasion and dentin sensitivity during the provisional states, and the technique is concerned with increased bond strength *in vitro*.[@B7] Magne (2005) also recommended application of dentin adhesive to the freshly cut dentin when a significant area of dentin has been exposed during preparation for indirect restoration. The dentin could be sealed immediately after tooth preparation with IDS prior to impression taking.[@B3]
IDS is the application of dentin bonding agent to freshly cut dentin when it is exposed during tooth preparation for indirect restorations. IDS protects the dentin against bacterial leakage and tooth sensitivity before cementation of final prosthesis. An advanced protocol, IDS is devised to address the challenges of preparation, provisionalization, and the final cementation of indirect restorative procedures. The general protocol of IDS includes the use of filled three-step total etch systems, two-step total-etch systems, and two-step self-etch systems incorporating low elastic liners.[@B8]
Especially prosthodontic patients, complex inlay, onlay and veneer situations may require longer periods with provisional restoration until the final ceramic restoration is delivered.[@B9] The provisional restoration must protect the pulp from thermal changes as well as from invasive microorganisms in the mouth. However, it is difficult to takestable and sealed provisionals as they detached easily during temporary states, allowing microleakage of bacteria and sensitivity.[@B10] In addition, the bond strength between resin composite and a pre-treated ceramic restoration has been described to be negatively affected by external factors such as thermocycling, fatigue and water sorption.[@B11] Also, cement breakdown can bring about results as microleakage, marginal discoloration, debonding, secondary caries, pulpal irritation, and decreased fracture load.[@B12]
We pose the clinical assumption that the dentin surface after IDS procedure is exposed to the oral environment and factors that could result in fatigue may influence the physical and mechanical properties of dentin bond strength. There are no studies available concerning the dentin bond strength on exposed sealed dentin with a long delay under thermocycling.
The aim of this study was to evaluate the effect of IDS on dentin bond strength of lithium disilicate ceramic (IPS Empress II, Ivoclar) under various thermocycling periods of 1, 2, 7, 14 days.
MATERIALS AND METHODS
=====================
For tooth preparation, freshly extracted sound human mandibular third molars stored in solution saturated with thymol were used. The midcoronal dentin surfaces were created after the removal of the occlusal half of the crown using a low-speed diamond saw (Isomet: Buehler Ltd., Lake Bluff, IL, USA). Each specimen was individually secured to a silicon mold (diameter: 25 mm, height: 15 mm) and self-curing polyester resin (CH-304, Aekyung Chemical Co., Ltd., Seoul, Korea) was poured to create a resin-embedded specimen block. The surface was wet polished to create hybrid layer with 320, 400 and 600 grit SiC abrasive papers. Total 50 specimens were prepared. The experimental groups were divided into four groups (10 specimens per group) according to the thermocycling period (1, 4, 7, 14 days). The control group consisted of 10 specimens without thermocycling.
A total of 50 ceramic discs (diameter: 4 mm, height: 2 mm) of lithium disilicate ceramic (IPS Empress II ingot, shade A1, Ivoclar Vivadent AG, Schaan, Liechtenstein) were fabricated. The surfaces were etched with 9.5% hydrofluoric acid (HF) (Porcelain etchant, Bisco Inc., Schaumburg, IL, USA) for 90 seconds and one layer of silane coupling agent (Porcelain primer, Bisco Inc., Schaumburg, IL, USA) was applied and allowed to air dry for thirty seconds at room temperature. The surface treatments of the ceramic discs were according to the manufacturer\'s instruction. The materials used in this study are shown in [Table 1](#T1){ref-type="table"}.
For immediate dentin sealing procedure, the dentin surface was etched with 32% phosphoric acid (H~3~PO~4~) for 15 seconds, followed by rinsing with distilled water and air drying for 5 seconds. Then, five coat of dentin bonding primer (3-step etch-rinse adhesive system; All Bond II, Bisco Inc., Shaumburg, IL, USA) with a light brushing motion for 30 seconds was applied to the surface and air thinning for 3 seconds. After one coat of Pre-bond resin, the layer was light polymerized (VIP Junior light curing unit, Bisco Inc., Schaumburg, IL, USA) for 20 seconds at 600mW/cm^2^. Ceramic disc was attached with dual-cured resin cement (Duo-link, Bisco Inc., Shaumburg, IL, USA) on IDS treated dentin surface.
After then, the specimens of experimental groups were submitted to 1500 thermal cycles between 5℃ and 55℃ (dwell time of 30 seconds) in a thermal cycling machine (Thermocycling testing machine, CDM-127, CDM, Korea) for 1, 2, 7, 14 day (group 1d, 2d, 7d, 14d) ([Table 2](#T2){ref-type="table"}).[@B13][@B14] Control group (0d) was not thermocycled and the specimens were stored for 14 days in deionized water at room temperature.
Ceramic discs after surface treatment with ceramic etchant and silane coupling agent were cemented to the surfaces of specimens with or without thermocycling with dual-cured resin cement (Duo-link, Bisco Inc., Shaumburg, IL, USA) ([Fig. 1](#F1){ref-type="fig"}). The excess cement was removed with a disposable microbrush, followed by light curing (VIP Junior light curing unit, Bisco Inc., Schaumburg, IL, USA) with a light intensity of 600 mW/cm^2^. The light was applied for 100 seconds (20 seconds each from occusal, buccal, lingual, mesial and distal aspects).
The extrusion shear bond strength test (SBST)[@B15][@B16] represents a confinement situation for the composite, and the resulting interface would be more likely to present defects that resemble clinical conditions. After the cementation procedures, the specimens of control group and thermocycled groups were stored in distilled water for 24 hours at 37℃. The SBST was conducted in a universal testing machine (Instron, Shimadzu, Japan) at a crosshead speed of 1.0 mm/min.
For the scanning electron microscopy (SEM) analysis, the dentin surfaces of specimens from each group were air dried and gold coated with a sputter coater (IB-3 ION coater, Elko Co., Tokyo, Japan) and examined under scanning electron microscope (Scanning Electron Microscopy, S-2300, Hitachi, Co., Ltd., Tokyo, Japan). The specimens were vertically sectioned using a low-speed diamond saw under water lubrication to observe the interface of dentinresin-ceramic.
Bond strength values were analyzed using one-way analysis of variance and followed by Tukey\'s HSD multiple comparison tests. Statistical analysis was conducted using SAS software version 9.1 for windows (SAS Institute Inc., Cary, NC, USA). The significance level for all statistical tests was set at 0.05.
RESULTS
=======
The values of the shear bond strength recorded in debonding force (DF) for the specimens in all groups. The mean values and standard deviation of shear bond strength are shown in [Fig. 2](#F2){ref-type="fig"}. Two thermocycled groups had lower mean values than the control group. The analysis of variance indicated no statistically significant difference in shear bond strength between all groups, however the mean value started to decrease in group 7d, and group 14d showed the lowest mean bond strength in all groups.
SEM micrograph of fractured surface of dentin side after SBST (original magnification ×100) are shown in [Fig. 3](#F3){ref-type="fig"}. SEM analysis of fractured surface in the control group, thermocycled 1d and | introduction = = = = = = = = = = = = patient expect for improved esthetics has driven the advancement of ceramic for use with fixed partial prostheses. [ @ b1 ] many clinical studies demonstrate excellent long - term success of ceramic restorations. in recent year, strong ceramic cores unioning esthetic veneering porcelains have become popular as all ceramic restorations which helps compensated the brittleness of porcelain imply unesthetic ceramics substructure. the clinical success of all ceramic prosthesis depends on a number of factors, such as composition of the ceramic material and the cementation procedure. [ @ b2 ] hence, bonding to ceramic requires strict attention to detail for optimal clinical outcomes. [ @ b3 ] a vital importance is due to the adhesive strength and durability of the complex formed between the three different materials : the resin cement, the ceramic surface and the tooth surface especially when anterior laminate veneers. [ @ b4 ] [ @ b5 ] among some cases, significant amounts of exposed dentin is usually unavoidable during the preparation of anterior teeth, [ @ b1 ] [ @ b6 ] the protection is required during the period between preparation and cementation and prevention of post - operative sensitivity and bacterial invasion. [ @ b2 ] they suggested the application of the dentin bonding agent immediately after tooth preparation. one new technique of dentin bonding agent application prevents the bacterial invasion and dentin sensitivity during the provisional states, and the technique is correlated with increased bond strength * in vitro *. [ @ b7 ] magne ( 2005 ) also recommended application of dentin adhesive to the freshly cut dentin when a significant area of dentin has been exposed during preparation for indirect restoration. the dentin could be sealed immediately after tooth preparation with ids prior to impression taking. [ @ b3 ] ids is the application of dentin bonding agent to freshly cut dentin when it is exposed during tooth preparation for indirect restorations. ids protects the dentin against bacterial leakage and tooth sensitivity before cementation of enamel prosthesis. an advanced protocol, ids is devised to address the challenges of preparation, provisionalization, and the final cementation of indirect restorative procedures. the general protocol of ids includes the use of filled three - step total etch systems, two - step total - etch systems, and two - step self - etch systems incorporating low elastic liners. [ @ b8 ] especially prosthodontic patients, complex inlay, onlay and veneer situations may require longer periods with provisional restoration until the final ceramic restoration is delivered. [ @ b9 ] the provisional restoration must protect the pulp from thermal changes as well as from invasive microorganisms in the mouth. however, it is difficult to takestable and sealed provisionals as they detached easily during temporary states, allowing microleakage of bacteria and sensitivity. [ @ b10 ] in addition, the bond strength between resin composite and a pre - treated ceramic restoration has been described to be negatively affected by external factors such as thermocycling, fatigue and water sorption. [ @ b11 ] also, cement breakdown can bring about results as microleakage, marginal discoloration, debonding, secondary caries, pulpal irritation, and decreased fracture load. [ @ b12 ] we pose the clinical assumption that the dentin surface after ids procedure is exposed to the oral environment and factors that could result in fatigue may influence the physical and mechanical properties of dentin bond strength. there are no studies available concerning the dentin bond strength on exposed sealed dentin with a long delay under thermocycling. the aim of this study was to evaluate the effect of ids on dentin bond strength of lithium disilicate ceramic ( ips empress ii, ivoclar ) under various thermocycling periods of 1, 2, 7, 14 days. materials and methods = = = = = = = = = = = = = = = = = = = = = for tooth preparation, freshly extracted sound human mandibular third molars stored in solution saturated with thymol were used. the midcoronal dentin surfaces were created after the removal of the occlusal half of the crown using a low - speed diamond saw ( isomet : buehler ltd., lake bluff, il, usa ). each specimen was individually secured to a silicon mold ( diameter : 25 mm, height : 15 mm ) and self - curing polyester resin ( ch - 304, aekyung chemical co., ltd., seoul, korea ) was poured to create a resin - embedded specimen block. the surface was wet polished to create hybrid layer with 320, 400 and 600 grit sic abrasive papers. total 50 specimens were prepared. the experimental groups were divided into four groups ( 10 specimens per group ) according to the thermocycling period ( 1, 4, 7, 14 days ). the control group consisted of 10 specimens without thermocycling. a total of 50 ceramic discs ( diameter : 4 mm, height : 2 mm ) of lithium disilicate ceramic ( ips empress ii ingot, shade a1, ivoclar vivadent ag, schaan, liechtenstein ) were fabricated. the surfaces were etched with 9. 5 % hydrofluoric acid ( hf ) ( porcelain etchant, bisco inc., schaumburg, il, usa ) for 90 seconds and one layer of silane coupling agent ( porcelain primer, bisco inc., schaumburg, il, usa ) was applied and allowed to air dry for thirty seconds at room temperature. the surface treatments of the ceramic discs were according to the manufacturer \ ' s instruction. the materials used in this study are shown in [ table 1 ] ( # t1 ) { ref - type = " table " }. for immediate dentin sealing procedure, the dentin surface was etched with 32 % phosphoric acid ( h ~ 3 ~ po ~ 4 ~ ) for 15 seconds, followed by rinsing with distilled water and air drying for 5 seconds. then, five coat of dentin bonding primer ( 3 - step etch - rinse adhesive system ; all bond ii, bisco inc., shaumburg, il, usa ) with a light brushing motion for 30 seconds was applied to the surface and air thinning for 3 seconds. after one coat of pre - bond resin, the layer was light polymerized ( vip junior light curing unit, bisco inc., schaumburg, il, usa ) for 20 seconds at 600mw / cm ^ 2 ^. ceramic disc was attached with dual - cured resin cement ( duo - link, bisco inc., shaumburg, il, usa ) on ids treated dentin surface. after then, the specimens of experimental groups were submitted to 1500 thermal cycles between [UNK] and [UNK] ( dwell time of 30 seconds ) in a thermal cycling machine ( thermocycling testing machine, cdm - 127, cdm, korea ) for 1, 2, 7, 14 day ( group 1d, 2d, 7d, 14d ) ( [ table 2 ] ( # t2 ) { ref - type = " table " } ). [ @ b13 ] [ @ b14 ] control group ( 0d ) was not thermocycled and the specimens were stored for 14 days in deionized water at room temperature. ceramic discs after surface treatment with ceramic etchant and silane coupling agent were cemented to the surfaces of specimens with or without thermocycling with dual - cured resin cement ( duo - link, bisco inc., shaumburg, il, usa ) ( [ fig. 1 ] ( # f1 ) { ref - type = " fig " } ). the excess cement was removed with a disposable microbrush, followed by light curing ( vip junior light curing unit, bisco inc., schaumburg, il, usa ) with a light intensity of 600 mw / cm ^ 2 ^. the light was applied for 100 seconds ( 20 seconds each from occusal, buccal, lingual, mesial and distal aspects ). the extrusion shear bond strength test ( sbst ) [ @ b15 ] [ @ b16 ] represents a confinement situation for the composite, and the resulting interface would be more likely to present defects that resemble clinical conditions. after the cementation procedures, the specimens of control group and thermocycled groups were stored in distilled water for 24 hours at [UNK]. the sbst was conducted in a universal testing machine ( instron, shimadzu, japan ) at a crosshead speed of 1. 0 mm / min. for the scanning electron microscopy ( sem ) analysis, the dentin surfaces of specimens from each group were air dried and gold coated with a sputter coater ( ib - 3 ion coater, elko co., tokyo, japan ) and examined under scanning electron microscope ( scanning electron microscopy, s - 2300, hitachi, co., ltd., tokyo, japan ). the specimens were vertically sectioned using a low - speed diamond saw under water lubrication to observe the interface of dentinresin - ceramic. bond strength values were analyzed using one - way analysis of variance and followed by tukey \ ' s hsd multiple comparison tests. statistical analysis was conducted using sas software version 9. 1 for windows ( sas institute inc., cary, nc, usa ). the significance level for all statistical tests was set at 0. 05. results = = = = = = = the values of the shear bond strength recorded in debonding force ( df ) for the specimens in all groups. the mean values and standard deviation of shear bond strength are shown in [ fig. 2 ] ( # f2 ) { ref - type = " fig " }. two thermocycled groups had lower mean values than the control group. the analysis of variance indicated no statistically significant difference in shear bond strength between all groups, however the mean value started to decrease in group 7d, and group 14d showed the lowest mean bond strength in all groups. sem micrograph of fractured surface of dentin side after sbst ( original magnification ×100 ) are shown in [ fig. 3 ] ( # f3 ) { ref - type = " fig " }. sem analysis of fractured surface in the control group, thermocycled 1d and | INTRODUCTION = = = = = = = = = = = = Patient expect for improved esthetics has driven the advancement of ceramic for use with fixed partial prostheses. [@ B1] Many clinical studies demonstrate excellent long - term success of ceramic restorations. In recent year, strong ceramic cores unioning esthetic veneering porcelains have become popular as all ceramic restorations which have compensated the brittleness of porcelain and unesthetic metal substructure. The clinical success of all ceramic prosthesis depends on a number of factors, such as composition of the ceramic material and the cementation procedure. [@ B2] Hence, bonding to ceramic requires strict attention to detail for optimal clinical outcomes. [@ B3] A bitaP importance is due to the adhesive strength and durability of the complex formed between the three different components: the resin cement, the ceramic surface and the tooth surface especially in anterior laminate veneers. [@ B4] [@ B5] In some cases, significant amounts of exposed dentin is usually unavoidable during the preparation of anterior tWFth, [@ B1] [@ B6] the protection is required during the period between preparation and cementation for prevention of post - operative sensitivity and bacterial invasion. [@ B2] They suggested the application of the dentin bonding agent immediately after tooth preparation. This new technique of dentin bonding agent application prevents the bacterial invasion and dentin sensitivity during the provisional states, and the technique is concerned with increased bond strength * in vitro *. [@ B7] Magne (2005) also recommended application of dentin adhesive to the freshly cut dentin when a significant area of dentin has been exposed during preparation for indirect restoration. The dentin could be sealed immediately after tooth preparation with IDS prior to impression taking. [@ B3] IDS is the application of dentin bonding agent to freshly cut dentin when it is exposed during tooth preparation for indirect restorations. IDS protects the dentin against bacterial leakage and tooth seJsi5ivity before cementation of final prosthesis. An advanced protocol, IDS is devised to address the challenges of preparation, 9rovisionalizatipn, and the finZ/ cementation of indirect restorative procedures. The general protocol of IDS includes the use of filled three - step total etch systems, two - step total - fYch systems, and two - step self - etch systems incorporating low elastic liners. [@ B8] Especially prosthodontic patients, complex inlay, onlay and veneer situations may require longer periods with provisional restoration until the final ceramic restoration is delivered. [@ B9] The provisional restoration must protect the pulp from thermal changes as well as from invasive microorganisms in the mouth. However, it is difficult to takestable and sealed provisionals as they detached easily during temporary states, allowing microleakage of bacteria and sensitivity. [@ B10] In addition, the bond strength between resin composite and a pre - treated ceramic restoration has been described to be negatively affected by external factors such as thermocycling, fatigue and water sorption. [@ B11] Also, cement breakdown can bring about results as microleakage, marginal discoloration, debonding, secondary caries, pulpal irritation, and decreased fracture load. [@ B12] We pose the clinical assumption that the dentin surface after IDS procedure is exposed to the oral environment and factors that could result in fatigue may influence the physical and mechanical properties of dentin bond strength. There are no studies available concerning the dentin bond strength on exposed sealed dentin with a long delay under thermocycling. The aim of this study was to evaluate the effect of IDS on dentin bond strength of lithium disilicate ceramic (IPS Empress II, Ivoclar) under various thermocycling periods of 1, 2, 7, 14 days. MATERIALS AND METHODS = = = = = = = = = = = = = = = = = = = = = For tooth preparation, freshly extracted sound human mahdibulaf third molars stored in solution saturated with thymol were used. The midcoronal dentin surfaces were created after the removal of the occlusal half of the crown using a low - speed diamond saw (Isomet: Buehler Ltd. , Lake Bluff, IL, USA ). Each specimen was individually secured to a silicon mold (diameter: 25 mm, height: 15 mm) and self - curing polyester resin (CH - 304, Aekyung Chemical Co. , Ltd. , Seoul, Korea) was poured to create a resin - embedded specimen block. The surface was wet polished to create hybrid layer with 320, 400 and 600 grit SiC abrasive papers. Total 50 specimens were prepared. The experimental groups were divided into four groups (10 specimens per group) according to the thermocycling period (1, 4, 7, 14 days ). The control group consisted of 10 specimens without thermocycling. A total of 50 ceramic discs (diameter: 4 mm, height: 2 mm) of lithium disilicate ceramic (IPS Empress II ingot, shade A1, Ivoclar Vivadent AG, Schaan, Liechtenstein) were fabricated. The surfaces were etched with 9. 5% hydrofluoric acid (HF) (Porcelain etchant, Bisco Inc. , Schaumburg, IL, USA) for 90 seconds and one layer of silane coupling agent (Porcelain primer, Bisco Inc. , Schaumburg, IL, USA) was applied and allowed to air dry for thirty seconds at room temperature. The surface treatments of the ceramic discs were accordiGV to the manufacturer \ ' s instruction. The materials used in this study are shown in [Table 1] (# T1) {ref - type = " table " }. For immediate dentin sealing procedure, the dentin surface was etched with 32% phosphoric acid (H ~ 3 ~ PO ~ 4 ~) for 15 seconds, followed by rinsing with distilled water and air drying for 5 seconds. Then, five coat of dentin bonding primer (3 - step etch - rinse adhesive system; All Bond II, Bisco Inc. , Shaumburg, IL, USA) with a light brushing motion for 30 seconds was applied to the surface and air thinning for 3 seconds. After one coat of Pre - bond resin, the layer was light polymerized (VIP Junior light curing unit, Bisco Inc. , Schaumburg, IL, USA) for 20 seconds at 600mW / cm ^ 2 ^. Ceramic disc was attached with dual - cured resin cement (Duo - link, Bisco Inc. , Shaumburg, IL, USA) on IDS treated dentin surface. After then, the specimens of experimental groups were submitted to 1500 thermal cycles between 5 ℃ and 55 ℃ (dwell time of 30 seconds) in a thermal cycling machine (Thermocycling testing machine, CDM - 127, CDM, Korea) for 1, 2, 7, 14 day (group 1d, 2d, 7d, 14d) ([ Table 2] (# T2) {ref - type = " table "} ). [@ B13] [@ B14] Control group (0d) was not thermocycled and the specimens were stored for 14 days in deionized water at room temperature. Ceramic discs after surface treatment with ceramic etchant and silane coupling agent were cemented to the surfaces of specimens with or without thermocycling with dual - cured resin cement (Duo - link, Bisco Inc. , Shaumburg, IL, USA) ([ Fig. 1] (# F1) {ref - type = " fig "} ). The excess cement was removed with a disposable microbrush, followed by liTGt curing (VIP Junior light curing unit, Bisco Inc. , Schaumburg, IL, USA) with a light intensity of 600 mW / cm ^ 2 ^. The light was applied for 100 seconds (20 seconds each from occusal, buccal, lingual, mesial and distal aspects ). The extrusion shear bond strength test (SBST) [@ B15] [@ B16] represents a confinement situation for the composite, and the resulting interface would be more likely to present defects that resemble clinical conditions. After the cementation procedures, the specimens of control group and thermocycled groups were stored in distilled water for 24 hours at 37 ℃. The SBST was conducted in a universal testing machine (Instron, Shimadzu, Japan) at a crosshead speed of 1. 0 mm / min. For the scanning electron microscopy (SEM) analysis, the dentin surfaces of specimens from each group were air dried and gold coated with a sputter coater (IB - 3 ION coater, Elko Co. , Tokyo, Japan) and examined under scanning electron microscope (Scanning Electron Microscopy, S - 2300, Hitachi, Co. , Ltd. , Tokyo, Japan ). The specimens were vertically sectioned using a low - speed diamond saw under water lubrication to observe the interface of dentinresin - ceramic. Bond strength values wfrd analyzed using one - way analysis of variance and followed by Tukey \ ' s HSD multiple comparison tests. Statistical analysis was conducted using SAS software version 9. 1 for windows (SAS Institute Inc. , Cary, NC, USA ). The significance level for all statistical tests was set at 0. 05. RESULTS = = = = = = = The values of the shear bond strength recorded in debonding force (DF) for the specimens in all groups. The mean values and standard deviation of shear bond strength are shown in [Fig. 2] (# F2) {ref - type = " fig " }. Two thermocycled groups had lower mean values than the control group. The analysis of variance indicated no statistically significant difference in shear bond strength between all groups, however the mean value started to decrease in group 7d, and group 14d showed the lowest mean bond strength in all groups. SEM micrograph of fractured surface of dentin side after SBST (original magnification × 100) are shown in [Fig. 3] (# F3) {ref - type = " fig " }. SEM analysis of fractured surface in the control group, thermocycled 1d and | INTRODUCTION ============ Patient expect for improved esthetics has driven the of ceramic for use with fixed prostheses.[@B1] Many clinical studies demonstrate excellent long-term success ceramic restorations. In recent year, strong ceramic cores unioning esthetic veneering porcelains have become as all ceramic restorations which compensated the brittleness of porcelain and unesthetic metal substructure. The clinical success of all ceramic prosthesis depends on a number of factors, such as composition of the ceramic material and the cementation procedure.[@B2] Hence, bonding to ceramic requires strict attention to detail for optimal clinical outcomes.[@B3] A vital is due to the adhesive strength and durability of the complex formed between the three different components: the resin cement, the ceramic and the tooth especially in anterior laminate In some cases, significant amounts of exposed dentin is usually during the preparation of anterior teeth,[@B1][@B6] the protection is required during the period between preparation for prevention of post-operative sensitivity and bacterial invasion.[@B2] They suggested the application of the dentin bonding agent immediately after tooth preparation. This new technique of dentin bonding agent application the invasion and dentin sensitivity during the provisional states, the technique is concerned with increased strength *in Magne (2005) also recommended application of dentin adhesive to the freshly cut dentin when significant area of dentin has been during preparation for indirect restoration. The dentin could be sealed immediately after tooth preparation with IDS prior to impression taking.[@B3] IDS is the application of dentin bonding agent to freshly when is exposed preparation IDS protects the dentin against bacterial leakage and tooth sensitivity before cementation of final prosthesis. An advanced protocol, IDS is devised to address the challenges of preparation, provisionalization, and the final cementation of indirect restorative procedures. The protocol IDS includes the use of filled three-step etch systems, two-step total-etch systems, and two-step self-etch systems low elastic liners.[@B8] Especially prosthodontic patients, complex onlay and veneer situations may require longer periods with provisional restoration until the final ceramic restoration is delivered.[@B9] The provisional restoration must protect the pulp thermal changes well as from invasive microorganisms in the mouth. However, it is difficult takestable sealed provisionals as they detached easily during temporary states, allowing microleakage of bacteria and sensitivity.[@B10] In addition, bond strength between resin composite and a pre-treated ceramic restoration has been described to be negatively affected by external factors such thermocycling, fatigue water sorption.[@B11] Also, cement breakdown can bring about results as microleakage, marginal discoloration, debonding, caries, pulpal irritation, decreased fracture load.[@B12] pose the clinical assumption that the dentin surface after procedure is exposed to the oral environment and factors that could result in fatigue may the physical and mechanical properties of dentin bond strength. studies concerning the dentin bond on exposed sealed dentin a long delay under thermocycling. The aim this study was to evaluate the effect of IDS on dentin bond strength of lithium disilicate ceramic (IPS Empress II, Ivoclar) under various thermocycling periods of 1, 2, 7, 14 days. AND METHODS ===================== For tooth preparation, extracted sound mandibular third molars stored in saturated with thymol were used. The midcoronal dentin surfaces created after the removal of the occlusal half of the crown using a low-speed diamond saw (Isomet: Buehler Ltd., Bluff, IL, USA). Each specimen was individually secured to a silicon mold (diameter: 25 mm, height: 15 mm) and self-curing polyester resin (CH-304, Aekyung Chemical Co., Ltd., Seoul, Korea) was poured to create a resin-embedded specimen block. The surface was polished to create hybrid layer with 320, 400 and 600 grit SiC abrasive papers. Total 50 specimens were prepared. The experimental groups were divided into four groups (10 specimens per group) according to the thermocycling period (1, 4, 7, 14 days). The control group consisted of 10 specimens thermocycling. A total of 50 ceramic discs 4 height: 2 mm) of lithium disilicate ceramic (IPS Empress II ingot, shade A1, Ivoclar Vivadent AG, Schaan, Liechtenstein) were fabricated. The surfaces were etched with 9.5% acid (Porcelain etchant, Bisco Inc., Schaumburg, IL, USA) for 90 seconds and one layer of silane coupling agent (Porcelain Bisco Inc., IL, USA) was applied allowed to air for thirty seconds at room temperature. The surface treatments of the ceramic were according to the manufacturer\'s instruction. The materials used in this study are in [Table 1](#T1){ref-type="table"}. immediate dentin sealing procedure, the dentin surface was etched with phosphoric acid for 15 seconds, followed by rinsing with distilled water and air drying for 5 seconds. Then, five coat of dentin bonding primer (3-step etch-rinse adhesive All Bond II, Bisco Inc., Shaumburg, IL, USA) with brushing motion for 30 seconds was to the surface and air thinning for 3 seconds. After one coat of Pre-bond resin, layer was light polymerized (VIP Junior light curing unit, Bisco Inc., IL, USA) for 20 seconds at 600mW/cm^2^. Ceramic disc was attached with dual-cured resin cement (Duo-link, Bisco Inc., Shaumburg, IL, USA) on IDS dentin surface. After then, the specimens of experimental groups were submitted 1500 thermal cycles between 5℃ and 55℃ (dwell time 30 seconds) in a thermal cycling machine (Thermocycling testing machine, CDM-127, CDM, Korea) for 1, 7, 14 day 1d, 2d, 7d, ([Table 2](#T2){ref-type="table"}).[@B13][@B14] Control group (0d) was not and the specimens were stored for days in deionized water at room after surface treatment with ceramic etchant and silane coupling agent were to the surfaces of specimens with or without with dual-cured resin cement (Duo-link, Bisco Inc., Shaumburg, IL, USA) ([Fig. 1](#F1){ref-type="fig"}). excess cement was removed with a followed by light curing light curing unit, Bisco Inc., Schaumburg, IL, USA) light intensity of 600 mW/cm^2^. The light was applied for 100 seconds (20 each from occusal, buccal, lingual, mesial and distal aspects). The extrusion strength test (SBST)[@B15][@B16] a confinement situation for the composite, and the resulting interface would be more likely to defects that resemble clinical conditions. After the cementation specimens of control group and thermocycled groups were stored in distilled water for hours 37℃. The SBST was conducted in a universal testing machine (Instron, Shimadzu, Japan) at a crosshead speed 1.0 mm/min. For the scanning electron microscopy (SEM) analysis, the dentin of specimens from each group were air dried and gold with a sputter coater (IB-3 ION coater, Co., Japan) and examined under scanning electron microscope (Scanning Microscopy, S-2300, Co., Ltd., Tokyo, Japan). The specimens were vertically sectioned using a low-speed diamond saw under water to observe the interface of dentinresin-ceramic. strength values were using one-way analysis of variance and followed by Tukey\'s HSD multiple comparison tests. Statistical conducted using SAS version 9.1 for windows (SAS Institute Inc., Cary, NC, USA). level all statistical tests was set at 0.05. RESULTS ======= The values of shear bond strength recorded in debonding force (DF) for the specimens in all groups. The mean values and standard of shear bond strength are shown [Fig. 2](#F2){ref-type="fig"}. Two groups had lower mean values than the control group. The analysis of variance no statistically significant difference in shear between all groups, however the mean value started decrease in group 7d, and group 14d showed the lowest mean bond in all groups. SEM micrograph of surface of dentin side after SBST (original magnification ×100) are shown in [Fig. 3](#F3){ref-type="fig"}. SEM analysis of fractured surface in the control group, thermocycled 1d and | IntRODucTION
============
PaTIeNT ExpECT for iMPRovEd esThEticS HaS DrIVen tHE AdvanCemeNT oF cERaMIC FOr uSE WIth fixED PARtiaL ProsThEses.[@b1] mANy CLiNiCAL StUDies dEMOnSTRAtE eXCelLENT LoNG-terM sucCEsS oF ceRAMIC rEstOraTions. in RECENt YeAR, StroNG CeRamIC CoREs uNIONing EstHeTic VEnEeRinG pORCeLaInS haVE BeCOMe pOpulaR aS aLl CeraMIc REStorAtIoNS WHICH hAVe COmPeNsAtED THe brITtleNESs OF POrcElAIN aND UNEStHEtiC meTAL SUBsTRUcTURE. tHe clINICal SuCCESs OF ALL cERAmIc PROSTHESis DepEndS oN a nUmbeR oF FACTOrs, suCh as coMpOsItiOn OF tHe cErAMIC materIAl AND tHe CeMEntatIon prOCEdUre.[@b2] HeNCE, boNDing to cerAMiC rEQUIRes sTriCT aTtENtiOn to dETaiL fOr OpTIMAL clInICal oUTcomES.[@b3]
A VitAL imPOrTAnCe is dUE tO ThE AdHESive stRENGTH ANd DUrabiLity OF thE COmPlex formED bETween THE ThREE DIffErENt comPOnenTs: tHE reSin cemENt, THe CerAMic sURfaCE aNd tHe tooTH surfACe espECiAllY iN aNTeRIOR lAmiNATe veneers.[@B4][@B5] IN somE cASeS, signiFICaNT aMouNTS oF ExPoseD dentIN Is usuaLly unAVOIDabLe DuriNg thE PRepaRAtION oF AntERior tEEtH,[@B1][@b6] the pROtecTIOn iS REQUIred DUrInG thE periOd BETWeen PRepaRATION aNd ceMENtAtiOn fOR PreVention OF post-OPeraTive senSitiViTy anD BaCtErIAl InVAsiOn.[@B2] They suGgESteD THE apPlicaTioN OF THE DENTIN BoNDIng aGEnT imMEdiAtEly AFter TOOth PrepAration. THIs NeW teCHNiquE OF DenTIN BonDING agEnT aPpLIcation PrEvENts ThE BACTerIaL iNVASION aNd deNtiN seNsiTivItY dURING thE PRovIsIoNAL sTaTEs, AND THE tEchnique iS CONcERned WITh INCREasED BOnd StrEnGTh *iN viTro*.[@b7] MAgne (2005) also reComMeNdED aPPlICaTIoN of DEntiN adhEsiVE tO thE frESHlY CUt dentIn WHen a SIGniFIcAnT ARea oF DeNtiN haS bEEn EXposEd dUriNg PREpArAtIon fOR iNDiReCt REstoRATIOn. thE deNtIN COulD BE seAlED imMEdiAteLy AfTer tootH pRepARATIoN wITH Ids PrioR TO imPressiON TakIng.[@B3]
idS iS The aPPLicatIon of DEnTIn boNDINg AgeNT to frEshLY Cut DeNTiN whEn it IS EXpoSed DuriNG tooth pREPARaTion fOr indiReCt rEStORAtIONS. iDs PRotecTS ThE dEnTIN AgAInst bACtERIal leaKaGe And tOoTH SensITivITY before CEmenTaTIon OF fInaL PROStHeSis. an AdvANCEd prOTocoL, IdS iS DeVised tO ADDreSs the ChaLLENGEs of PREpARatIOn, PROviSIoNaLIZATIoN, aND tHe fiNal ceMEnTatIOn of INdIReCt REsTORAtivE pROcEDuRes. tHE GeNerAL protOcOl of IdS incLUDeS tHE uSE OF FilLEd THREe-STeP TOTal ETCh systeMs, twO-STEP TOTal-ETch sysTeMs, ANd TwO-stEp SElf-eTch sYsTEMs INcORPORaTiNG LoW ElastIc linERS.[@B8]
eSpECiALlY pROStHOdonTiC PatiENTs, COMpLeX INlAY, ONlAY aNd vEnEEr sItUatioNs MAy rEQUIRE LONGer peRiodS With pROViSIonAL rESTOraTION untIl tHe fINal cErAmIC reSTORatioN IS DeLivEREd.[@B9] ThE PRovIsIoNaL REstORatIOn MuST pROTEcT the PULp froM thErmal CHaNgES As weLl as FroM inVasIvE MIcROOrgANisMS in The mouTh. howeveR, IT Is dIfFICULT To TaKEStABLe aNd sEAlEd ProVIsioNALs as tHEy dEtAChed eaSilY duriNG TEMpoRAry stATEs, alLoWInG MiCrOleAKAgE oF BactErIa aNd sEnsitIvITY.[@b10] iN ADdItiON, THe Bond strEnGTH BetweEN reSIN ComPOSItE AND a PRe-TReAtED cERAmIc reSToRaTIon Has bEeN DESCrIBed TO BE neGAtIvEly AfFeCteD by EXTeRnaL FACTorS sucH As thERMocYclIng, faTiGuE And waTer sorPTIon.[@B11] aLSO, ceMENt bREakdOWN cAn BRIng AbOUT reSults AS MicroLEAkaGE, mARGInAl diSCOloRAtioN, DeboNdING, SECoNDaRy CarIes, PUlpaL irRITATION, AnD dEcrEasED frACTuRe Load.[@B12]
we POsE thE cLinIcAl aSSumPTioN ThAT THE DENtIn surFacE AfTeR iDS ProcedurE Is EXPOSeD tO tHE orAL ENviROnmEnt aND faCtORs THAT cOuLD ReSuLT in FAtIGUe maY InFLueNCe THe PHYSICAl ANd MeChaNIcal prOpERtIes of deNtIN boNd STrEnGTH. ThERE are no STUDiEs availAblE conCErnINg ThE deNtiN bOnD STrenGTh ON EXpOSed seAleD deNTIN WITh A LONg dELAy unDeR THeRMoCYcLing.
THe Aim of tHIS studY WAs TO EvaluAtE tHE EffEct Of IdS on DeNTin BoNd StrENgTH Of lIthIUm diSIlIcAtE ceRAmIc (Ips EMprEss iI, IvoCLAr) unDer VArIOuS tHeRmoCYcLIng PERioDs of 1, 2, 7, 14 DAyS.
MaTERiaLs aND meThODs
=====================
foR TooTH prEpArAtiOn, fResHly eXtRacTed soUnd HumAn mANDIbuLaR tHIrd mOlarS StORed in SoLUTiOn SaTUratED wITh tHYmOL WeRe uSEd. thE mIDcOrONaL DeNTiN SurfaCES weRe CReaTED AftER thE RemoVaL OF tHE occlUsAL hAlf Of the CroWn usINg a loW-spEeD DiAMOnD sAW (iSOmet: bUEhLER LtD., LaKe BLuFF, Il, USA). EacH SpECimeN waS INdIViDUALLy sECured to a silicon mold (diAmEter: 25 MM, HeighT: 15 Mm) ANd SeLf-cURInG pOLYEstEr resIN (CH-304, aeKYuNG cHeMICal cO., LTD., Seoul, KOREa) WaS PoURed tO CrEATE A ResiN-EmBeddEd SpeCiMeN bLoCK. THe surfaCe was WEt pOlIshEd TO CREAtE HybriD LAYer WITH 320, 400 and 600 GRIt siC aBRAsIVE papErS. toTaL 50 SpEcImenS WERE prEpaReD. thE eXPERimeNtAl GroupS wErE DIVIDeD iNto FOuR Groups (10 SpEcImens per GRouP) ACCOrDinG To THe tHErmOcYClIng PERiOD (1, 4, 7, 14 DaYS). THE CoNtrOL GRoup cOnsistEd of 10 spECimENS WiTHOUT thErmOCYcLing.
a TotAl OF 50 CeramiC DiScS (DIamETER: 4 mm, HEight: 2 Mm) Of LItHiUM disiLIcAte cErAMIc (ips emPrEss II iNGOt, shadE A1, ivoCLAR VIVaDenT ag, scHAaN, liECHteNsteIN) wEre fAbRICaTed. tHE SUrfAcES WEre etched WITH 9.5% HyDroflUORic AcID (HF) (pOrCElAiN etcHanT, bisCO INc., SChAUmBuRg, Il, usA) fOr 90 SEcOnDs And oNE Layer Of silAne coUpLINg AGENt (POrcelaIn PrImer, Bisco INc., scHAUmbuRg, iL, Usa) WaS ApPlied And allOwed tO Air drY fOr ThiRTy SecONds At RooM teMpEratURe. the SURFACe treatmEnTs of the ceraMIC diSCS WeRE accoRDiNG tO THE maNuFaCtUrEr\'s iNsTrUCtIOn. thE MaterIals Used In THiS sTUDY are SHOWn in [TabLe 1](#t1){ref-TYPe="TAbLE"}.
FoR IMmEdiaTe DENTiN seAling proceDURE, thE dENTiN SURfaCe WAs eTCHEd wIth 32% phOsPhORiC aCID (h~3~Po~4~) FoR 15 SECoNdS, FolLOweD BY RinSiNg wITH DistIllEd waTER And AIR dRying For 5 seCONDS. TheN, FIVe cOat of DENtiN boNDiNg PrIMer (3-stEp ETCh-RinsE adhesIVe sYStEM; AlL BONd II, biscO InC., SHaumBURG, iL, USa) wIth A LIGHt BRuShInG MoTION FoR 30 Seconds wAS apPLIeD tO the sURFAce ANd aiR THINning fOR 3 SeCONDs. after oNE coAT OF PrE-BOND rEsIN, ThE laYer wAS light POlYmeRIZed (vip JuNioR liGHT CuRIng UnIt, BIScO Inc., scHaUmBurG, Il, USa) FOr 20 sEconds aT 600Mw/Cm^2^. CeRaMiC DiSC WaS attaCHeD WitH DuAl-CureD ReSiN CEmeNT (DUO-LInK, BiscO INc., sHAUmBuRg, iL, USa) ON IdS TReaTeD DENtIN SuRFACe.
AFTer then, thE SpEcImEnS oF experimENTaL gROuPs WeRE SuBmiTTED TO 1500 tHErMAl CYcLEs bEtwEEN 5℃ anD 55℃ (DweLL TImE oF 30 sECoNDS) IN A thErMAl cycLING MaCHInE (TheRmOcYClINg tESTinG macHINE, cDM-127, CDm, KOrEa) FOr 1, 2, 7, 14 DaY (Group 1D, 2D, 7D, 14D) ([TABLe 2](#T2){rEF-tyPE="TAbLE"}).[@B13][@B14] ContrOL gRoUp (0D) WAs NoT THERmOCYcLed aNd thE SPECimENS were sTOred fOR 14 DaYs IN deIOnIZed WateR aT rOOm teMpErATUre.
CERamIC dIscs afTeR SUrFAce TrEaTMEnT wItH cERAmIc etChANT ANd SIlANe coUplING aGeNT WeRE CeMeNTEd tO tHe sURFAcEs oF sPeCiMEnS wITh or wITHOut THerMoCyCLINg WITh DUAl-cuREd ResiN cEMENT (Duo-liNK, BisCO Inc., ShaUMBuRG, Il, USA) ([FiG. 1](#F1){rEF-tYPe="fig"}). ThE EXcESS CEmeNt Was ReMovED With a DIsPOSAbLe MIcRObrush, fOLlOWed by lIGhT cUrING (vIp juNIor LiGhT CUriNg UNit, bisCO inC., scHaUMBUrG, IL, UsA) wiTh a LIghT IntENSiTy oF 600 MW/CM^2^. thE liGHt wAS apPlieD FoR 100 SEcONdS (20 SecoNDs eaCh FroM occUsal, bUcCAl, LInGuAL, MeSiAl AND Distal ASpEcTS).
THe EXTRUsion Shear BONd stReNGTH TeST (sbST)[@B15][@B16] REPreSeNTS A COnfinEMEnt siTuaTiOn fOR tHe CoMpOSiTe, AND tHe resuLtiNg inteRFaCe WOulD BE mOrE liKELY to PrEsENt DefectS thaT ReSEmBLe clINICAL coNDITioNS. afteR ThE cemeNtaTIon PrOCEdurES, tHe spEcIMEns of conTrOl GROUp aND THErMocYClED grOupS weRE StoREd IN DiSTiLlEd Water FOR 24 HOurS aT 37℃. tHE sBSt Was COnDuCTED iN a UNIversAL TEStING macHINe (InSTrON, ShimadZu, Japan) aT A cROssHeAd SpeEd OF 1.0 MM/MIN.
FoR THE SCANnING ELEctRoN MIcRoScOPY (sEM) anAlYSIs, THe DenTIN sUrfaces OF sPeCiMeNs FRoM Each GrOUP wERe AiR dRIEd ANd goLd COATED witH A SPUtTeR cOAtER (IB-3 iON cOaTeR, ElKo CO., TokyO, JAPaN) ANd ExaMIneD UndEr SCAnnINg eLeCTrOn MiCROscoPE (sCAnniNg eLECTROn mIcROsCOpy, s-2300, hiTacHI, cO., ltD., tOKyo, JApaN). thE SpEcImens WeRe vErtIcaLLy sectIonEd UsiNG A lOW-sPeed DIaMOnD sAW UNDER wATeR LUbriCaTiOn To oBsERve THE INtERfACE OF dEntInResIn-cErAMic.
BoNd sTRENgTh ValUEs wERe aNalyZeD usING onE-wAY ANAlySIs OF vaRIaNCe aND FOlloWed bY tUKeY\'s HsD MuLTiplE ComParison teSTS. sTatiSTiCal aNAlYSIS wAs CONdUCtED UsIng Sas SoftWARe veRSiON 9.1 for wIndOWS (SAs InstiTUte INC., cAry, nc, uSA). thE sIgnIfiCaNcE LEVel FOR ALL sTATiSTIcAl tesTs Was set AT 0.05.
ResulTs
=======
thE VAlueS oF tHE ShEAR BOnd sTrEngTh RecoRDED In dEbondIng fORce (dF) FOr THE SpECIMENS In ALl gRoUps. The MEAN vaLUEs And stANDArD DevIATion oF ShEaR BOnD STREngtH ArE SHown IN [fIg. 2](#F2){Ref-type="FIg"}. two THermocycled gRoUPs Had LOweR meAn vaLUES THAn THE coNtrol GrOuP. tHE aNAlysIs OF VARIAnce INDIcated No statisTIcALLy sIGNiFIcAnT DIFFeReNce IN ShEAr bonD sTRENgTh betweEN aLl grOUps, HoWeVER THE MeAN VALUe StARTeD TO DeCrEase In GRouP 7d, And Group 14D shOwED ThE loWEst meAN boNd STRenGtH IN aLL gROUPS.
SEm micrOGRaph of fRActUREd SUrfAce Of dEntiN SIdE AftER Sbst (oriGinal mAgnIFIcation ×100) ARE ShOwN IN [FIg. 3](#F3){REF-TYpe="fig"}. sem AnAlYsiS Of fraCTUReD SUrFaCE in tHe CONtrol Group, THErmOcyClED 1D And | INTRODUCTION============ Patientexpect for improved esthetics has driven the advancement of ceramic foruse with fixed partial prostheses.[@B1] Many clinical studies demonstrate excellentlong-term success of ceramic restorations. In recent year, strong ceramic cores unioning esthetic veneering porcelains have become popular as all ceramic restorations whichhavecompensated the brittleness of porcelain and unesthetic metal substructure. The clinical success of all ceramic prosthesis depends on a number of factors, suchas composition of the ceramic material and the cementation procedure.[@B2] Hence, bonding to ceramic requires strictattention to detail for optimal clinical outcomes.[@B3] Avital importance is due to the adhesive strengthand durability of the complex formed between the threedifferent components: the resin cement, theceramic surface and the tooth surface especially in anteriorlaminate veneers.[@B4][@B5] Insome cases, significant amounts of exposed dentin is usually unavoidable duringthe preparation ofanterior teeth,[@B1][@B6]the protection is required during the periodbetween preparation andcementation for prevention of post-operative sensitivity and bacterial invasion.[@B2] They suggested the application of the dentin bonding agent immediately after tooth preparation.This new technique of dentin bonding agent application prevents the bacterial invasionand dentin sensitivity during theprovisionalstates, and the technique is concernedwith increased bondstrength *in vitro*.[@B7] Magne (2005) alsorecommended application of dentin adhesive to the freshly cut dentinwhen a significant area of dentin has been exposed during preparationfor indirect restoration. The dentin could be sealed immediately after tooth preparation with IDS prior to impression taking.[@B3] IDS is the application of dentin bonding agent tofreshlycutdentin whenit is exposed during toothpreparation for indirect restorations. IDS protects the dentin against bacterial leakage and tooth sensitivity before cementation offinal prosthesis. An advanced protocol, IDSis devised to address the challengesof preparation, provisionalization, and the final cementation ofindirect restorativeprocedures. The general protocol of IDS includes the use offilled three-steptotal etch systems, two-step total-etch systems, and two-step self-etch systems incorporating low elastic liners.[@B8] Especially prosthodontic patients,complex inlay, onlay and veneer situationsmay require longer periods with provisional restoration until the final ceramic restoration is delivered.[@B9] Theprovisional restoration must protect the pulpfrom thermal changes as well as from invasive microorganisms in the mouth. However, it is difficultto takestable and sealedprovisionals as they detached easily duringtemporary states, allowingmicroleakage of bacteria and sensitivity.[@B10] In addition, the bond strength betweenresincomposite and a pre-treatedceramic restoration has been described to be negatively affected by external factors such as thermocycling, fatigue and water sorption.[@B11] Also, cement breakdown can bring about results as microleakage, marginal discoloration, debonding,secondary caries, pulpal irritation, and decreased fracture load.[@B12] We pose the clinical assumption thatthe dentinsurface after IDSprocedure is exposed to the oral environmentand factors that could result in fatigue may influence thephysical and mechanical properties of dentin bond strength. There are no studies available concerning the dentin bondstrength on exposedsealed dentinwith a long delay under thermocycling. The aim of this study was to evaluate the effect of IDS on dentin bond strength of lithium disilicate ceramic (IPS Empress II, Ivoclar) under various thermocycling periods of 1, 2, 7, 14 days. MATERIALS AND METHODS===================== For tooth preparation, freshly extracted sound human mandibular third molars stored in solution saturated with thymol were used. Themidcoronal dentin surfaces were created after the removalof the occlusal half of the crown using a low-speed diamond saw (Isomet: Buehler Ltd., Lake Bluff, IL, USA). Each specimen was individuallysecured to a silicon mold (diameter: 25 mm, height: 15 mm) and self-curing polyester resin(CH-304, Aekyung Chemical Co., Ltd.,Seoul, Korea) was poured to create a resin-embeddedspecimen block. The surface was wet polished to create hybrid layer with320, 400 and 600 grit SiC abrasive papers. Total 50specimenswere prepared. The experimental groups were divided into four groups (10 specimens per group) according to the thermocycling period (1, 4, 7, 14 days). The control groupconsisted of 10 specimens without thermocycling. A total of 50ceramic discs (diameter:4 mm, height: 2mm) oflithium disilicateceramic (IPSEmpress II ingot, shade A1, Ivoclar Vivadent AG, Schaan, Liechtenstein) were fabricated. The surfaces were etched with 9.5% hydrofluoric acid (HF) (Porcelain etchant, Bisco Inc., Schaumburg, IL, USA) for90 seconds and onelayer of silane coupling agent (Porcelainprimer, Bisco Inc., Schaumburg, IL, USA) was applied and allowed toair dry for thirty seconds at roomtemperature. The surface treatments of the ceramic discs were according to the manufacturer\'s instruction. The materials used inthis study are shown in [Table 1](#T1){ref-type="table"}. For immediate dentin sealingprocedure, the dentin surface was etchedwith 32% phosphoric acid (H~3~PO~4~) for 15 seconds, followed by rinsingwith distilled water and air drying for 5 seconds. Then, five coat of dentin bonding primer (3-step etch-rinse adhesive system; All Bond II,Bisco Inc., Shaumburg, IL, USA) with a light brushing motion for 30 secondswasapplied to the surface andair thinning for 3seconds. After onecoatofPre-bond resin, the layer was light polymerized (VIP Junior light curing unit, Bisco Inc., Schaumburg, IL, USA) for 20seconds at 600mW/cm^2^. Ceramicdisc was attached with dual-curedresin cement(Duo-link, Bisco Inc., Shaumburg, IL, USA)onIDS treated dentin surface. After then,the specimensof experimentalgroups were submitted to 1500 thermal cycles between 5℃ and 55℃ (dwell time of 30 seconds) in a thermal cycling machine (Thermocycling testing machine, CDM-127, CDM, Korea) for 1, 2, 7, 14 day (group 1d, 2d, 7d, 14d) ([Table 2](#T2){ref-type="table"}).[@B13][@B14] Control group (0d) wasnotthermocycled and the specimens were stored for 14 days in deionized water atroom temperature. Ceramicdiscs after surface treatment with ceramic etchantandsilane coupling agent were cemented to thesurfacesof specimens with or without thermocycling with dual-cured resin cement (Duo-link, Bisco Inc., Shaumburg, IL,USA) ([Fig. 1](#F1){ref-type="fig"}).The excess cement was removed with a disposable microbrush, followed bylight curing (VIP Juniorlight curing unit, Bisco Inc., Schaumburg, IL, USA)with alight intensity of 600 mW/cm^2^. The light was applied for 100 seconds (20 seconds each from occusal,buccal, lingual,mesial anddistal aspects). The extrusion shear bond strength test (SBST)[@B15][@B16] represents aconfinement situation for thecomposite,and the resulting interface would be more likely topresent defects that resemble clinical conditions. After the cementation procedures, the specimens of control group and thermocycled groups were stored in distilled water for 24 hours at 37℃.The SBSTwas conducted in a universal testing machine(Instron,Shimadzu, Japan) at a crosshead speed of 1.0 mm/min. For the scanning electronmicroscopy (SEM) analysis, the dentinsurfaces ofspecimens from each group were air dried and gold coatedwith a sputtercoater (IB-3 ION coater, Elko Co., Tokyo, Japan) and examined under scanningelectron microscope (Scanning Electron Microscopy, S-2300, Hitachi, Co., Ltd., Tokyo, Japan). The specimenswere vertically sectioned using a low-speed diamondsaw under water lubrication to observe the interface ofdentinresin-ceramic. Bond strength values were analyzedusing one-wayanalysis of variance and followed by Tukey\'s HSD multiple comparisontests. Statistical analysis was conducted using SAS software version9.1 for windows (SAS InstituteInc., Cary, NC, USA). The significance level for all statistical tests was set at 0.05.RESULTS ======= The values of the shearbond strength recorded in debonding force (DF) for the specimensin all groups. The mean values and standard deviation of shear bond strength are shown in[Fig.2](#F2){ref-type="fig"}. Two thermocycled groups had lowermeanvalues than the control group. The analysis of variance indicated no statistically significant difference in shear bondstrength between allgroups,however the meanvalue startedto decrease in group 7d, and group 14d showed the lowest mean bond strength in all groups. SEM micrograph offractured surface of dentin side afterSBST (original magnification ×100) are shown in [Fig. 3](#F3){ref-type="fig"}. SEM analysis of fractured surface in the control group, thermocycled 1d and | _INTRODUCTION_ ============ Patient expect for improved esthetics has driven _the_ advancement of ceramic _for_ use _with_ fixed partial prostheses.[@B1] Many clinical studies _demonstrate_ excellent long-term success _of_ _ceramic_ restorations. In recent year, strong ceramic cores unioning esthetic veneering porcelains _have_ become popular as all ceramic _restorations_ _which_ have compensated the brittleness of _porcelain_ and unesthetic metal substructure. The clinical success of all ceramic prosthesis depends on _a_ number of factors, such as composition of _the_ _ceramic_ _material_ _and_ the _cementation_ procedure.[@B2] _Hence,_ bonding to ceramic requires _strict_ attention to _detail_ _for_ _optimal_ clinical outcomes.[@B3] _A_ vital _importance_ _is_ _due_ to _the_ adhesive strength and durability of the complex _formed_ between the three _different_ components: the resin _cement,_ the _ceramic_ surface and the tooth _surface_ _especially_ in anterior laminate _veneers.[@B4][@B5]_ _In_ some _cases,_ _significant_ amounts of exposed dentin is usually unavoidable during the preparation of anterior teeth,[@B1][@B6] the _protection_ is required during the period between preparation and _cementation_ for _prevention_ of post-operative sensitivity and bacterial invasion.[@B2] They _suggested_ the application of the dentin bonding agent _immediately_ after tooth preparation. This new technique of _dentin_ bonding agent application prevents the bacterial _invasion_ and _dentin_ sensitivity during _the_ _provisional_ states, and the technique is concerned with increased bond strength _*in_ vitro*.[@B7] Magne (2005) also recommended application of _dentin_ _adhesive_ to _the_ freshly _cut_ dentin when a significant area of dentin has been exposed during preparation for indirect restoration. The dentin could be sealed immediately after tooth preparation with IDS prior to impression _taking.[@B3]_ IDS _is_ the application of dentin _bonding_ agent to freshly cut dentin _when_ it is exposed during tooth preparation for indirect _restorations._ _IDS_ protects the dentin against bacterial leakage and _tooth_ sensitivity before cementation of _final_ _prosthesis._ An _advanced_ protocol, IDS _is_ devised to _address_ the challenges of preparation, provisionalization, and the _final_ cementation of _indirect_ restorative procedures. The general protocol of _IDS_ includes _the_ _use_ of filled three-step total etch systems, _two-step_ _total-etch_ systems, and two-step self-etch systems incorporating low _elastic_ liners.[@B8] Especially prosthodontic patients, complex inlay, onlay and veneer situations _may_ require longer periods with provisional restoration until the final ceramic restoration is delivered.[@B9] The provisional restoration must protect _the_ pulp _from_ thermal changes as well as from _invasive_ microorganisms in the mouth. However, it is difficult to takestable _and_ sealed provisionals as _they_ _detached_ _easily_ during temporary _states,_ allowing microleakage of bacteria _and_ sensitivity.[@B10] In addition, the bond strength _between_ resin composite and a pre-treated ceramic restoration has been described to be _negatively_ affected by external factors such as thermocycling, fatigue and water sorption.[@B11] Also, cement breakdown can bring about results _as_ _microleakage,_ marginal discoloration, debonding, secondary caries, pulpal irritation, _and_ decreased fracture load.[@B12] _We_ pose the _clinical_ assumption that _the_ dentin surface _after_ IDS procedure is _exposed_ to the oral environment _and_ factors _that_ could result _in_ _fatigue_ may influence the physical _and_ mechanical properties of _dentin_ _bond_ strength. There are no _studies_ available concerning the _dentin_ bond strength on exposed sealed _dentin_ with a long _delay_ under thermocycling. The aim _of_ _this_ study was _to_ _evaluate_ the _effect_ of _IDS_ on dentin bond strength _of_ _lithium_ disilicate ceramic (IPS Empress _II,_ Ivoclar) under various _thermocycling_ _periods_ of _1,_ 2, _7,_ 14 days. MATERIALS AND METHODS ===================== For tooth _preparation,_ freshly extracted sound human _mandibular_ third molars stored _in_ solution saturated _with_ _thymol_ were used. The midcoronal _dentin_ surfaces were _created_ after the removal _of_ _the_ occlusal half of the _crown_ using a _low-speed_ diamond saw (Isomet: Buehler Ltd., Lake _Bluff,_ _IL,_ USA). Each _specimen_ was individually secured to a silicon mold (diameter: 25 mm, height: 15 mm) and self-curing polyester resin (CH-304, Aekyung _Chemical_ Co., Ltd., Seoul, Korea) was poured to create a resin-embedded specimen _block._ _The_ _surface_ _was_ wet polished to create hybrid layer with 320, 400 and 600 _grit_ SiC abrasive papers. Total 50 specimens were prepared. The experimental groups were divided _into_ _four_ groups _(10_ specimens per group) _according_ to the thermocycling period (1, 4, 7, 14 days). The _control_ group consisted of 10 specimens without thermocycling. A total of 50 _ceramic_ discs (diameter: 4 mm, height: 2 mm) of lithium disilicate ceramic (IPS Empress II ingot, shade A1, _Ivoclar_ Vivadent _AG,_ _Schaan,_ Liechtenstein) were _fabricated._ _The_ surfaces were etched with _9.5%_ hydrofluoric acid (HF) (Porcelain etchant, Bisco Inc., _Schaumburg,_ IL, USA) for 90 seconds _and_ one layer of silane coupling _agent_ (Porcelain primer, Bisco _Inc.,_ Schaumburg, IL, USA) was applied and allowed _to_ _air_ _dry_ for thirty seconds at room temperature. The _surface_ treatments _of_ the ceramic _discs_ were _according_ _to_ _the_ manufacturer\'s instruction. The materials used in this _study_ _are_ shown in [Table 1](#T1){ref-type="table"}. For _immediate_ dentin sealing procedure, the dentin surface was etched with 32% phosphoric acid (H~3~PO~4~) for 15 _seconds,_ followed by _rinsing_ with distilled _water_ and air drying for 5 seconds. _Then,_ _five_ coat _of_ dentin bonding _primer_ (3-step etch-rinse adhesive system; All Bond II, Bisco Inc., _Shaumburg,_ _IL,_ _USA)_ _with_ a light brushing motion for 30 _seconds_ was applied to the surface and _air_ thinning for 3 _seconds._ After _one_ coat of Pre-bond resin, _the_ layer was light polymerized (VIP Junior light curing unit, Bisco _Inc.,_ _Schaumburg,_ IL, _USA)_ for 20 seconds at 600mW/cm^2^. Ceramic _disc_ was attached with dual-cured resin cement (Duo-link, _Bisco_ _Inc.,_ Shaumburg, IL, _USA)_ on IDS _treated_ dentin surface. _After_ then, the specimens of experimental groups were submitted to _1500_ thermal cycles between 5℃ and 55℃ (dwell time of 30 seconds) in _a_ thermal _cycling_ machine (Thermocycling _testing_ machine, _CDM-127,_ CDM, Korea) for 1, 2, _7,_ _14_ _day_ (group 1d, 2d, 7d, 14d) _([Table_ 2](#T2){ref-type="table"}).[@B13][@B14] Control group (0d) was not thermocycled and the specimens were stored for 14 _days_ in deionized water _at_ room temperature. Ceramic discs after surface treatment _with_ ceramic etchant and silane coupling agent were cemented to the surfaces of specimens _with_ or without thermocycling with dual-cured resin _cement_ (Duo-link, Bisco Inc., _Shaumburg,_ IL, USA) _([Fig._ 1](#F1){ref-type="fig"}). The excess _cement_ was removed with a disposable microbrush, followed by light curing (VIP Junior _light_ curing unit, Bisco Inc., _Schaumburg,_ _IL,_ USA) with a _light_ intensity of 600 mW/cm^2^. The light was applied for 100 seconds (20 seconds each from _occusal,_ buccal, lingual, mesial _and_ distal _aspects)._ The extrusion shear bond strength test (SBST)[@B15][@B16] _represents_ _a_ confinement _situation_ for the composite, and the _resulting_ _interface_ would be more likely to present _defects_ that resemble clinical _conditions._ After the cementation procedures, the specimens of control group and thermocycled groups were stored in distilled water for 24 hours at 37℃. The SBST was conducted in a universal _testing_ machine (Instron, Shimadzu, Japan) at a crosshead speed of 1.0 _mm/min._ For the _scanning_ _electron_ microscopy (SEM) analysis, the _dentin_ surfaces of specimens from each _group_ were air _dried_ _and_ gold coated _with_ a sputter coater (IB-3 _ION_ coater, _Elko_ Co., Tokyo, Japan) _and_ examined under scanning electron microscope _(Scanning_ Electron Microscopy, S-2300, _Hitachi,_ Co., Ltd., Tokyo, Japan). The specimens were _vertically_ sectioned using a low-speed _diamond_ saw under water lubrication to observe the interface of _dentinresin-ceramic._ Bond strength _values_ were analyzed using _one-way_ _analysis_ of variance _and_ followed by Tukey\'s HSD multiple _comparison_ _tests._ _Statistical_ analysis _was_ conducted using SAS software version 9.1 for windows (SAS Institute Inc., Cary, NC, USA). The significance level for _all_ statistical _tests_ was _set_ at 0.05. RESULTS ======= _The_ values of the _shear_ bond strength recorded in debonding _force_ (DF) for _the_ specimens in _all_ groups. _The_ _mean_ values and standard deviation of shear bond strength are shown in [Fig. 2](#F2){ref-type="fig"}. Two _thermocycled_ groups had _lower_ _mean_ values than the control _group._ The _analysis_ _of_ variance indicated no statistically significant difference _in_ shear bond strength _between_ all groups, _however_ _the_ mean value started to _decrease_ _in_ group _7d,_ and group 14d showed the lowest mean bond strength in all groups. _SEM_ micrograph _of_ _fractured_ surface of _dentin_ _side_ after SBST (original _magnification_ ×100) are shown in [Fig. 3](#F3){ref-type="fig"}. _SEM_ analysis of fractured _surface_ in the control group, thermocycled 1d and |
INTRODUCTION
============
For decades, high ligation and saphenous vein stripping with phlebectomy of varices were the gold standard procedures in the treatment of the incompetence of the great saphenous vein (GSV) \[[@b1-vsi-30-102],[@b2-vsi-30-102]\]. An alternative method was cryostripping which is less invasive than traditional stripping \[[@b3-vsi-30-102]\]. Because of the need for minimal invasiveness, the endovenous procedures spread worldwide in the treatment of the incompetence of the saphenous trunks in the last decade \[[@b4-vsi-30-102],[@b5-vsi-30-102]\].
At the end of the last century, Milleret and Le Pivert \[[@b6-vsi-30-102]\] described cryosclerosis as a safe, efficient and feasible endovenous cryoablation technique to treat the incompetence of the GSV. The segmental freezing of the saphenous trunk was performed with a flexible cryoprobe by inguinal approach. In 1987, Le Pivert \[[@b7-vsi-30-102]\] published the experiences of 350 patients treated by this method. In 1994, Garde \[[@b8-vsi-30-102]\] concluded that the technique was safe and efficient based on the results of a randomized controlled trial on 800 patients. We have completed a prospective non-randomized trial to compare cryosclerosis and classical stripping, and regarding the short term results, this endovenous procedure seems to be effective. The electron microscopic examination proved that immediate ultrastructural changes were formed in the GSV \[[@b9-vsi-30-102]\].
CASE
====
We present the histological changes of the GSV at 2 years after cryoablation. The examined vein piece was harvested from the proximal femoral part of the GSV from a 31-year-old female patient who underwent cryosclerosis 23 months ago. This procedure was performed by inguinal approach, after high ligation a cryoprobe was introduced into the GSV and segmental freezing of the vessel wall was carried out. The patient was admitted again to our department of surgery because of recurrent varicosity that caused pain and limb swelling at the end of the day. Ultrasonography showed the recanalization of the GSV. Saphenofemoral reflux and an incompetent posteromedial perforating vein were detected as possible reasons. High ligation and cryostripping with phlebectomy of varices were performed under spinal anesthesia. The GSV was very crusted and segmental strictures were found when the cryoprobe was introduced into the vessel lumen. A small vein piece was harvested from the proximal femoral part of the GSV for histological examination. The patient healed without any postoperative complications.
Hematoxyllin-eosin, Picro-sirius Red, van Gieson and immunohistochemical stains (CD34, smooth muscle actin) were performed. The anatomical structure of the vein couldn't be recognized microscopically. The wall was collagenized and padded by endothelium that was caused by progressive fibrosis ([Fig. 1](#f1-vsi-30-102){ref-type="fig"}). The patient consented to this trial and agreed to apply her records.
DISCUSSION
==========
Generally, the role of the endovenous ablation techniques (endovenous laser ablation \[EVLA\], radiofrequency ablation \[RFA\], ultrasound guided foam sclerotherapy, mechanochemical ablation, steam ablation) has increased in the treatment of the incompetence of the GSV. These are widely studied, safe and efficient procedures. The most frequently used methods are EVLA and RFA. The long term results are favorable, but the recanalization of the GSV could be observed some years after the treatment that may cause recurrent varicosity with symptoms \[[@b1-vsi-30-102],[@b2-vsi-30-102],[@b4-vsi-30-102],[@b5-vsi-30-102]\].
In this short report, the presented method is cryosclerosis, which is the endovenous cryoablation of the GSV. This procedure is not widely known. The reported patient had recurrent varicosity with symptoms. Cryosclerosis was ineffective 2 years after the treatment; however, segmental stenoses of the GSV were observed during the operation. The incompetence of the saphenofemoral junction and the posteromedial perforating vein could cause the recurrent disease. The role of tumescent fluids during cryosclerosis is not clear and it is not routinely used, but it might be useful since by decreasing the volume of the intraluminal blood, vein wall destruction can be achieved more efficiently \[[@b10-vsi-30-102],[@b11-vsi-30-102]\]. A prospective trial is necessary to assess the effect of injecting warming liquid around the GSV before cryosclerosis.
The histomorphological effects of the endovenous ablation methods have been previously investigated, mostly from animal experiments \[[@b12-vsi-30-102]--[@b14-vsi-30-102]\]. Their long term effects have not been described yet. It's typical for all procedures that the vein wall is destroyed by thermal, mechanical or chemical effects \[[@b1-vsi-30-102],[@b4-vsi-30-102],[@b6-vsi-30-102],[@b9-vsi-30-102],[@b10-vsi-30-102]\]. Heger et al. \[[@b12-vsi-30-102]\] summarized in their review on EVLA that the heat induced thrombosis and the thermal damage in the vein wall results in inflammation and activation of the immune system (fibroblast migration). Finally, the vein wall becomes collagenized and results in occlusion or stenosis. The presented histomorphological examination showed that the whole vein treated by cryosclerosis underwent typical remodeling, thus the anatomical structure couldn't be recognized. The wall became crusted because of collagen deposition by fibroblast activity. However, the GSV wasn't occluded yet segmental strictures were observed ([Fig. 1](#f1-vsi-30-102){ref-type="fig"}).
Cryosclerosis seems to have the same effect as the other familiar endovenous ablation techniques \[[@b1-vsi-30-102],[@b2-vsi-30-102],[@b4-vsi-30-102],[@b6-vsi-30-102]--[@b9-vsi-30-102]\]. The reason for recanalization of the GSV is not clear, with recurrent incompetence of the saphenofemoral junction and the posteromedial perforating vein being one of the reasons, but it is also possible that the remodeling process of the GSV was not effective enough. The pathogenesis of recanalization after the endovenous procedures and recurrent varicosity has been studied, but more evidence is needed \[[@b5-vsi-30-102]\].
Considering that this article is only a case report, it has serious limitations. More cases are necessary to assess the real effect of cryosclerosis on the GSV, but the procedure seems to be efficient in obliterative remodeling of the vein.
Conflict of interest: None.
{#f1-vsi-30-102}
| introduction = = = = = = = = = = = = for decades, high ligation and saphenous vein stripping with phlebectomy of varices were the gold standard procedures in the treatment of the incompetence of the great saphenous vein ( v ) \ [ [ @ b1 - vsi - 30 - 102 ], [ @ b2 - vsi - 30 - 102 ] \ ]. an alternative method was cryostripping which appeared less invasive than traditional stripping \ [ [ @ b3 - vsi - 30 - 102 ] \ ]. because of the need for minimal invasiveness, the endovenous procedures spread worldwide in the treatment of the incompetence of the saphenous trunks in the last decade \ [ [ @ b4 - vsi - 30 - 102 ], [ @ b5 - vsi - 30 - 102 ] \ ]. at the end of the last century, milleret and le pivert \ [ [ @ b6 - vsi - 30 - 102 ] \ ] described cryosclerosis as a safe, efficient and feasible endovenous cryoablation technique could treat the incompetence of the gsv. the segmental freezing of small saphenous trunk was performed with a flexible cryoprobe by inguinal approach. in 1985, le pivert \ [ [ @ b7 - vsi - 30 - 102 ] \ ] published the experiences of 350 patients treated by this method. in 1990, garde \ [ [ @ b8 - vsi - 30 - 102 ] \ ] concluded that the technique was safe and efficient based on the results of our randomized controlled trial on 800 patients. we have completed a prospective non - randomized trial to compare cryosclerosis and classical stripping, and regarding the short term results, this endovenous procedure seems to be effective. the electron microscopic examination proved that immediate ultrastructural changes were formed in the gsv \ [ [ @ b9 - vsi - 30 - 102 ] \ ]. case = = = = we observe the histological changes of each gsv at 2 years after cryoablation. the examined vein piece was harvested from the proximal femoral part of the gsv from a 31 - year - old female patient who underwent cryosclerosis 3 months ago. this procedure was performed by inguinal approach, after high ligation a cryoprobe was introduced into the gsv and segmental freezing of the vessel wall was carried out. the patient was admitted again to our department of surgery because of recurrent varicosity that caused pain and limb swelling at the end of the day. ultrasonography showed the recanalization of the gsv. saphenofemoral reflux and an incompetent posteromedial perforating vein were detected as possible reasons. high ligation and cryostripping with phlebectomy of varices were performed under spinal anesthesia. the gsv was very crusted and segmental strictures were found when the cryoprobe was introduced into the vessel lumen. a small vein piece was harvested from the proximal femoral part of the gsv for histological examination. the patient healed without any postoperative complications. hematoxyllin - eosin, picro - sirius red, van gieson and immunohistochemical stains ( cd34, smooth muscle actin ) were performed. the anatomical structure of the vein couldn ' t be recognized microscopically. the wall was collagenized and padded by endothelium that was caused by progressive fibrosis ( [ fig. 1 ] ( # f1 - vsi - 30 - 102 ) { ref - type = " fig " } ). the patient consented to this trial and agreed to apply her records. discussion = = = = = = = = = = generally, the role of the endovenous ablation techniques ( endovenous laser ablation \ [ evla \ ], radiofrequency ablation \ [ rfa \ ], ultrasound guided foam sclerotherapy, mechanochemical ablation, steam ablation ) has increased in the treatment of the incompetence of the gsv. these are widely studied, safe and efficient procedures. the most frequently used methods are evla and rfa. the long term results are favorable, but the recanalization of the gsv could be observed some years after the treatment that may cause recurrent varicosity with symptoms \ [ [ @ b1 - vsi - 30 - 102 ], [ @ b2 - vsi - 30 - 102 ], [ @ b4 - vsi - 30 - 102 ], [ @ b5 - vsi - 30 - 102 ] \ ]. in this short report, the presented method is cryosclerosis, which is the endovenous cryoablation of the gsv. this procedure is not widely known. the reported patient had recurrent varicosity with symptoms. cryosclerosis was ineffective 2 years after the treatment ; however, segmental stenoses of the gsv were observed during the operation. the incompetence of the saphenofemoral junction and the posteromedial perforating vein could cause the recurrent disease. the role of tumescent fluids during cryosclerosis is not clear and it is not routinely used, but it might be useful since by decreasing the volume of the intraluminal blood, vein wall destruction can be achieved more efficiently \ [ [ @ b10 - vsi - 30 - 102 ], [ @ b11 - vsi - 30 - 102 ] \ ]. a prospective trial is necessary to assess the effect of injecting warming liquid around the gsv before cryosclerosis. the histomorphological effects of the endovenous ablation methods have been previously investigated, mostly from animal experiments \ [ [ @ b12 - vsi - 30 - 102 ] - - [ @ b14 - vsi - 30 - 102 ] \ ]. their long term effects have not been described yet. it ' s typical for all procedures that the vein wall is destroyed by thermal, mechanical or chemical effects \ [ [ @ b1 - vsi - 30 - 102 ], [ @ b4 - vsi - 30 - 102 ], [ @ b6 - vsi - 30 - 102 ], [ @ b9 - vsi - 30 - 102 ], [ @ b10 - vsi - 30 - 102 ] \ ]. heger et al. \ [ [ @ b12 - vsi - 30 - 102 ] \ ] summarized in their review on evla that the heat induced thrombosis and the thermal damage in the vein wall results in inflammation and activation of the immune system ( fibroblast migration ). finally, the vein wall becomes collagenized and results in occlusion or stenosis. the presented histomorphological examination showed that the whole vein treated by cryosclerosis underwent typical remodeling, thus the anatomical structure couldn ' t be recognized. the wall became crusted because of collagen deposition by fibroblast activity. however, the gsv wasn ' t occluded yet segmental strictures were observed ( [ fig. 1 ] ( # f1 - vsi - 30 - 102 ) { ref - type = " fig " } ). cryosclerosis seems to have the same effect as the other familiar endovenous ablation techniques \ [ [ @ b1 - vsi - 30 - 102 ], [ @ b2 - vsi - 30 - 102 ], [ @ b4 - vsi - 30 - 102 ], [ @ b6 - vsi - 30 - 102 ] - - [ @ b9 - vsi - 30 - 102 ] \ ]. the reason for recanalization of the gsv is not clear, with recurrent incompetence of the saphenofemoral junction and the posteromedial perforating vein being one of the reasons, but it is also possible that the remodeling process of the gsv was not effective enough. the pathogenesis of recanalization after the endovenous procedures and recurrent varicosity has been studied, but more evidence is needed \ [ [ @ b5 - vsi - 30 - 102 ] \ ]. considering that this article is only a case report, it has serious limitations. more cases are necessary to assess the real effect of cryosclerosis on the gsv, but the procedure seems to be efficient in obliterative remodeling of the vein. conflict of interest : none.! [ the photomicroscopic image of the vein piece ( picro - sirius red stain, ×60 ). arrows mark the collagen in the whole vein wall. ] ( vsi - 30 - 102f1 ) { # f1 - vsi - 30 - 102 } | INTRODUCTION = = = = = = = = = = = = For decades, high ligation and saphenous vein stripping with phlebectomy of varices were the gold standard procedures in the treatment of the incompetence of the great saphenous vein (GSV) \ [[ @ b1 - vsi - 30 - 102 ], [@ b2 - vsi - 30 - 102] \ ]. An alternative method was cryostripping which is less invasive than traditional stripping \ [[ @ b3 - vsi - 30 - 102] \ ]. Because of the need for minimal invasiveness, the endovenous procedures spread worldwide in the treatment of the incompetence of the saphenous trunks in the last decade \ [[ @ b4 - vsi - 30 - 102 ], [@ b5 - vsi - 30 - 102] \ ]. At the end of the last century, Milleret and Le Pivert \ [[ @ b6 - vsi - 30 - 102] \] described cryosclerosis as a safe, efficient and feasible endovenous cryoablation technique to treat the incompetence of the GSV. The segmental freezing of the saphenous trunk was performed with a flexible cryoprobe by inguinal approach. In 1987, Le Pivert \ [[ @ b7 - vsi - 30 - 102] \] published the experiences of 350 patients treated by this method. In 1994, Garde \ [[ @ b8 - vsi - 30 - 102] \] concluded that the technique was sZbe and efficient based on the results of a randomized controlled trial on 800 patients. We have completed a prospective non - randomized trial to compare cryosclerosis and classical stripping, and regarding the short term results, this endovenous procedure seems to be effective. The electron microscopic examination proved that immediate ultrastructural changes were formed in the GSV \ [[ @ b9 - vsi - 30 - 102] \ ]. CASE = = = = We present the histological changes of the GSV at 2 years after cryoablation. The examined vein piece was harvested from the proximal femoral part of the GSV from a 31 - year - old female patient who unrSrwent cryosclerosis 23 months ago. This procedure was performed by inguinal approach, after high ligation a cryoprobe was introduced into the GSV and segmental freezing of the vessel wall was carried out. The patient was admitted again to our department of surgery because of recurrent varicosity that caused pain and limb swelling at the end of the day. Ultrasonography showed the recanalization of the GSV. Saphenofemoral reflux and an incompetent posteromedial perforating vein were detected as possible reasons. High ligation and cryostripping with phlebectomy of varices were performed under spinal anesthesia. The GSV was very crusted and segmental strictures were found when the cryoprobe was introduced into the vessel lumen. A small vein piece was harvested from the proximal femoral part of the GSV for histological examination. The patient healed without any postoperative complications. Hematoxyllin - eosin, Picro - sirius Red, van Gieson and immunohistochemical stains (CD34, smooth muscle actin) were performed. The anatomical structure of the vein couldn ' t be recognized microscopically. The wall was collagenized and padded by endothelium that was Da8sed by progressive fibrosis ([ Fig. 1] (# f1 - vsi - 30 - 102) {ref - type = " fig "} ). The patient consented to this trial and agreed to apply her records. DISCUSSION = = = = = = = = = = Generally, the role of the endovenous ablation techniques (endovenous laser ablation \ [EVLA \ ], radiofrequency ablation \ [RFA \ ], ultrasound guided foam sclerotherapy, mechanochemical ablation, s4eXm ablation) has increased in the treatment of the incompetence of the GSV. These are widely studied, safe and efficient procedures. The most frequently used methods are EV.S and RFA. The long term results are favorable, but the recanalization of the GSV could be observed some years after the treatment that may cause recurrent varicosity with symptoms \ [[ @ b1 - vsi - 30 - 102 ], [@ b2 - vsi - 30 - 102 ], [@ b4 - vsi - 30 - 102 ], [@ b5 - vsi - 30 - 102] \ ]. In this short report, the presented method is cryosclerosis, which is the endovenous cryoablation of the GSV. This procedure is not widely known. The re(o3ted patient had recurrent varicosity with symptoms. Cryosclerosis was ineffective 2 years after the treatment; however, segmental stenoses of the GSV were observed during the operation. The incompetence of the saphenofemoral junction and the posteromedial perforating vein could cause the recurrent disease. The role of tumescent fluids during cryosclerosis is not clear and it is not routinely used, but it might be useful since by decreasing the volume of the intraluminal blood, vein wall destruction can be achieved more efficiently \ [[ @ b10 - vsi - 30 - 102 ], [@ b11 - vsi - 30 - 102] \ ]. A prospective trial is necessary to assess the effect of injecting warming liquid around the GSV before cryosclerosis. The histomorphological effects of the endovenous ablation methods have been previously investigated, mostly from animal experiments \ [[ @ b12 - vsi - 30 - 102] - - [@ b14 - vsi - 30 - 102] \ ]. Their long term effects have not been described yet. It ' s typical for all procedures that the vein wall is destroyed by thermal, mechanical or chemical effects \ [[ @ b1 - vsi - 30 - 102 ], [@ b4 - vsi - 30 - 102 ], [@ b6 - vsi - 30 - 102 ], [@ b9 - vsi - 30 - 102 ], [@ b10 - vsi - 30 - 102] \ ]. Heger et al. \ [[ @ b12 - vsi - 30 - 102] \] summarized in their review on EVLA that the heat induced thrombosis and the thermal damage in the vein wall results in inflammation and activation of the immune system (fibroblast migration ). Finally, the vein wall becomes collagenized and resKlfs in occlusion or stenosis. The presented histomorphological examination showed that the whole vein treated by cryosclerosis underwent typical remodeling, thus the anatomical structure couldn ' t be recognized. The wall became crusted because of collagen deposition by fibroblast activity. Hlwevsr, the GSV wasn ' t occluded yet segmental strictures were observed ([ Fig. 1] (# f1 - vsi - 30 - 102) {ref - type = " fig "} ). Cryosclerosis seems to Mwve the same effect as the other familiar endovenous ablation techniques \ [[ @ b1 - vsi - 30 - 102 ], [@ b2 - vsi - 30 - 102 ], [@ b4 - vsi - 30 - 102 ], [@ b6 - vsi - 30 - 102] - - [@ b9 - vsi - 30 - 102] \ ]. The reason for recanalization of the GSV is not clear, with recurrent incompetence of the saphenofemoral junction and the posteromedial perforating vein being one of the reasons, but it is also possible that the remodeling process of the GSV was not effective enough. The pathogenesis of recanalization after the endovenous procedures and recurrent varicosity has been studied, but more evidence is needed \ [[ @ b5 - vsi - 30 - 102] \ ]. Considering that this article is only a case report, it has serious limitations. More cases are necessary to assess the real effect of cryosclerosis on the GSV, but the procedure seems to be efficient in obliterative remodeling of the b$in. Conflict of interest: None. ! [The photomicroscopic image of the vein piece (picro - sirius red stain, × 60 ). Arrows mark the collagen in the whole vein wall.] (vsi - 30 - 102f1) {# f1 - vsi - 30 - 102 } | INTRODUCTION ============ For decades, high and saphenous vein stripping with phlebectomy of varices were the gold standard procedures in treatment the incompetence of the great saphenous vein (GSV) \[[@b1-vsi-30-102],[@b2-vsi-30-102]\]. An alternative method was cryostripping which is invasive than traditional stripping \[[@b3-vsi-30-102]\]. Because of the need for minimal invasiveness, the endovenous procedures spread worldwide in treatment of the incompetence of the saphenous trunks in last decade \[[@b4-vsi-30-102],[@b5-vsi-30-102]\]. At the end of the last century, Milleret and Le Pivert \[[@b6-vsi-30-102]\] cryosclerosis as a safe, efficient feasible endovenous cryoablation technique to treat the incompetence of the GSV. The freezing of the trunk was performed with a flexible cryoprobe by approach. In 1987, Le Pivert \[[@b7-vsi-30-102]\] published the experiences of 350 patients treated by this method. In 1994, Garde \[[@b8-vsi-30-102]\] concluded that the technique was safe and efficient based on the results of a randomized trial 800 patients. We prospective trial to compare cryosclerosis and classical and regarding the short term results, this endovenous procedure seems to be effective. The electron examination proved that immediate ultrastructural changes were formed in the GSV \[[@b9-vsi-30-102]\]. CASE ==== We present histological changes of the GSV at 2 years after cryoablation. The examined piece was harvested from the proximal femoral part the GSV from a female patient who cryosclerosis 23 months ago. This procedure was by inguinal approach, after high ligation cryoprobe was introduced into the GSV and segmental freezing of the vessel wall was carried out. The patient was admitted again to our department because of recurrent varicosity that caused pain and limb swelling at the end of the day. Ultrasonography showed recanalization of the GSV. Saphenofemoral reflux and an incompetent posteromedial perforating vein were detected as possible reasons. High ligation and with phlebectomy of varices were performed under spinal anesthesia. The was very crusted and strictures were found when the cryoprobe was introduced into the vessel lumen. A small vein piece was harvested from the proximal part of the GSV for examination. The patient healed without any postoperative complications. Hematoxyllin-eosin, Picro-sirius Red, van Gieson and immunohistochemical stains (CD34, smooth muscle were performed. The anatomical structure of the vein be recognized microscopically. The wall was collagenized and padded by endothelium that caused by progressive fibrosis ([Fig. 1](#f1-vsi-30-102){ref-type="fig"}). patient consented to this and agreed to apply her records. DISCUSSION ========== Generally, the role the endovenous ablation techniques (endovenous laser ablation \[EVLA\], radiofrequency \[RFA\], ultrasound guided sclerotherapy, mechanochemical ablation, steam ablation) has increased in the treatment of the incompetence of the are widely studied, safe and efficient procedures. The most frequently used methods are EVLA RFA. The long term results are favorable, but the recanalization of the GSV could be observed some years after the treatment that may cause recurrent varicosity with symptoms \[[@b1-vsi-30-102],[@b2-vsi-30-102],[@b4-vsi-30-102],[@b5-vsi-30-102]\]. In this short report, the presented method is which is the cryoablation of the GSV. This procedure is not known. The reported patient had recurrent varicosity with symptoms. Cryosclerosis was ineffective 2 years after the however, segmental stenoses of the were observed during the operation. The of the saphenofemoral junction and posteromedial perforating vein could cause the recurrent disease. The of tumescent fluids during cryosclerosis not clear and it routinely but it might be useful since by decreasing the volume of the intraluminal blood, vein wall can achieved more efficiently \[[@b10-vsi-30-102],[@b11-vsi-30-102]\]. A prospective trial is necessary to the effect of injecting warming liquid around the before cryosclerosis. The histomorphological effects of the endovenous ablation methods have been previously investigated, mostly from animal experiments Their long term effects have not described yet. typical for all procedures that vein is destroyed by thermal, mechanical or chemical effects \[[@b1-vsi-30-102],[@b4-vsi-30-102],[@b6-vsi-30-102],[@b9-vsi-30-102],[@b10-vsi-30-102]\]. Heger et al. \[[@b12-vsi-30-102]\] summarized in their review on EVLA the induced thrombosis and the damage in the vein wall results in inflammation activation of the immune system (fibroblast migration). Finally, vein wall becomes collagenized results in occlusion or stenosis. The presented histomorphological examination showed that the whole vein treated by cryosclerosis underwent remodeling, thus the anatomical structure couldn't be recognized. The wall became crusted because of collagen deposition by fibroblast activity. However, the GSV wasn't occluded yet segmental strictures were observed ([Fig. 1](#f1-vsi-30-102){ref-type="fig"}). Cryosclerosis to have the same effect as the other familiar endovenous ablation techniques \[[@b1-vsi-30-102],[@b2-vsi-30-102],[@b4-vsi-30-102],[@b6-vsi-30-102]--[@b9-vsi-30-102]\]. The for recanalization the GSV is not clear, with recurrent incompetence saphenofemoral junction and the posteromedial vein being one of the but is also possible that the remodeling process of the GSV was not effective enough. The pathogenesis of recanalization after the endovenous procedures recurrent varicosity has studied, but more evidence is needed \[[@b5-vsi-30-102]\]. Considering this article is a case report, it has serious limitations. More cases are necessary to assess the real effect of cryosclerosis on the GSV, but the procedure seems efficient in obliterative remodeling of vein. of interest: None. {#f1-vsi-30-102} | INtrOdUCTIOn
============
FOR DecADes, hIgH LIGaTion and sAPHenous VeIn StRIpPing WITH PhLEbectOMY Of VaRIcEs wEre thE GolD STAndard pROCeDUreS iN tHE TREaTMent of ThE iNcOmpeTEnCE Of ThE GreAt sApHEnoUS VeIn (gSV) \[[@B1-vsi-30-102],[@b2-VSI-30-102]\]. AN aLternATIve METHod WAS cryoStrIpPinG WhiCH is lESS iNVaSiVe thAN TrADiTIONAL StRiPPING \[[@B3-vsI-30-102]\]. bECAuSe oF tHe neED For MInimaL iNvASivenEss, tHE eNDOVENoUS prOceduREs spRead WoRLDwide iN ThE tReAtmenT oF thE INcoMPETeNCe oF the SAPhENOuS tRuNKs In THe lAST decAdE \[[@B4-vsI-30-102],[@B5-vSi-30-102]\].
At ThE enD oF the lAST CenTurY, miLLErET aNd le PIvert \[[@B6-Vsi-30-102]\] deScRIBED cRYOscLEROSIS as A sAfE, EFFiCIENT aND feASIBLe eNDOvENouS cryoABLaTiOn TecHnIQue TO trEAt thE inComPeTeNCE of ThE gsv. ThE SeGmEntal FReezINg Of tHe SApHenOuS TruNk WaS PErfOrMed WItH A FLEXIbLe cryoproBe by InGuINaL APPRoAch. In 1987, le pivERt \[[@B7-VSI-30-102]\] pUBlIsHEd tHE eXperIenCEs oF 350 paTIenTS tReated By THIS METHOd. IN 1994, GaRDe \[[@b8-VSi-30-102]\] CoNcLuDed tHat thE TEchniqUE WAs SaFe AnD eFFICienT baSed on ThE rESultS Of a RAndOmIzED CoNtRolLed TrIAl oN 800 PAtIENts. we havE COmpLetED a PrOsPeCTIVe noN-raNDoMiZed TrIAL to comPARe cryOscLEROSIS And cLaSSical STriPPiNG, AND rEgaRDINg the shorT TERM rESuLts, THis eNdOVENOUs pROcEdURe sEeMs TO Be EfFECtiVe. THE eLEctrOn MiCROScOpIC eXAMINaTION PROVED That ImMediaTe ultrASTrucTurAL chAnGes Were formed IN tHe GSv \[[@b9-vsi-30-102]\].
cASE
====
wE prEsent tHe HisTolOGIcAl chaNgEs of THE gsV At 2 Years AfteR cryOaBlAtion. The exAmINed vein pIece was hArVEStEd FrOM the pROximaL fEmorAl pART oF THE gSV fRoM A 31-YeaR-old FemalE PatIEnt Who undErWENt cRYOscLeroSIS 23 montHs agO. thiS PRoceDURE WAs PeRfOrmeD bY inguINaL ApPRoaCH, AFteR high LIGAtION a cryoPrOBE wAs InTroDuCEd iNtO THe GSV aNd sEGmenTAL FreEZINg Of thE VesSeL waLL Was Carried OUt. tHE patiENT waS adMITTed AgAIn To oUr DEPARtmeNT Of suRgEry BEcauSe Of ReCURreNT VARICosITY THaT CAuseD paIN ANd LImB sWeLlInG AT THe eND OF thE DaY. UltraSONoGrapHy ShoweD thE RecAnAlIzatIon OF the gSv. sApheNofeMORAL reFLux AnD an IncoMpeTEnT POSTerOmeDIaL peRfORatIng vein werE deTectED as PosSibLe ReAsOns. hIgh LIGAtIOn ANd cryosTrIPpinG wItH pHlebectomy Of vARIceS WeRE perFOrmeD under SPinAL anesTHeSia. tHE gSv wAS vEry CRusTed and sEgMEntAl StRicTurEs WerE fOUnd wHeN the crYoPrObe WAs INTRoDucEd iNto THE VEsSEl LuMeN. a SmAlL vEiN PIECe was HarvestED From THE PrOxImAl feMOrAL pART Of THe gsv for HistoLoGicAL exAMinAtIOn. The PaTIENT HeALEd wItHOut Any poSTOpErativE COmPLiCAtIOns.
hEMAtOXYLLiN-eoSin, picRo-siRiUs RED, VaN GIeSon aNd ImMUNOHISToCHeMiCaL StAiNs (Cd34, SmoOTH MUscLE AcTin) wERE PeRForMeD. tHE anatomICal stRUcTuRE Of THe VeIn CoULdN't BE rEcogNIZed mICrosCopicALLY. THe Wall WAS COlLaGenizeD ANd pAddED BY enDOThELIUm tHaT WAs CAuSED by progrEsSivE fIBroSIS ([FIG. 1](#F1-VsI-30-102){REF-TyPe="Fig"}). the PatieNT COnSenTEd To thIs trIAl And AgREed to APPLy heR rECordS.
DisCusSion
==========
geNERALly, thE rolE of tHE eNdoVEnOuS aBlaTION TeChNiqUEs (eNdOvENous LaSeR AbLAtiOn \[EVlA\], RAdIofreQuEnCY AbLATioN \[rFa\], ulTRasound GuidEd FoAM SClErOtherAPY, MEchAnOchEmiCal abLatION, SteAM ABLATion) HaS iNcreASED iN tHe tREaTMENT oF thE iNcoMpeTeNCE oF ThE GsV. THEse aRE wIdElY STUdiEd, SAfE AnD eFfIciENT PRocedUres. the mOsT fReQuEnTly UsED MethoDS ARe eVla aNd Rfa. the LONg tERm ReSULTs ARe FAVOraBLE, But THE recanAlIZATiOn OF tHe Gsv CoUld be ObsERVeD SOme YeArs After the trEatMENT tHAt MAy CausE reCURreNT vArICOsity WIth sYMPTomS \[[@b1-VSI-30-102],[@B2-Vsi-30-102],[@B4-vSi-30-102],[@B5-vsi-30-102]\].
IN ThIS Short rEPort, the pReseNTed meTHOd iS crYosCLERosis, WhiCh IS The eNDovenOus cRYOAbLaTiOn of THE gSv. tHIS proceduRE is not WiDelY KNOwN. thE REpOrtED PATiENT HaD ReCurrEnT vARiCoSITY WITh SYmptOMS. CrYOscleroSiS WAs INEFfECTiVE 2 YearS afTer ThE TrEAtMeNT; HoWeVER, SegMentaL STENoseS oF tHe GSV weRE oBseRved DURiNG tHe OpERaTIon. tHe INCompeTeNce Of tHE SapHenOfEmORal JUNcTioN aNd THE pOSteroMEDial pERFOrATinG VeIn CoULD cAUSe THe rECURREnt DIseasE. tHE roLE of TuMeScEnt FluiDS durInG CRYOsCleROsis IS NOT cLEaR AND It iS noT roUTINeLy USED, bUt It MigHT be USEfuL SiNCE bY DEcReaSINg THE volume oF THE INtRALUMiNAL blOoD, vEIN Wall desTruCTiOn CaN be AcHieVEd mORE eFfiCIentLY \[[@b10-vsI-30-102],[@B11-vsI-30-102]\]. a pROspecTivE TRIAl iS NecesSaRy to AsSESS ThE effecT oF INJectinG WarMinG LIqUid arouND tHe gsV befOre CRyOsClEroSiS.
thE hiSTOMorpholoGiCAl effecTS oF tHe ENDovEnouS aBLatiON MEthods HAVe beeN PREVIoUsly INvESTIGaTeD, mostly frOM animal expeRiMEnTS \[[@b12-Vsi-30-102]--[@b14-vSI-30-102]\]. THeIr lONG tErM effEcTs HaVe NOt Been DESCRIBed YET. It's TyPICAl FoR aLL pRocedurES thaT the veIn Wall IS destRoYED BY TheRmAl, MecHAnICAL Or CHeMICAL EFFEcts \[[@b1-VsI-30-102],[@B4-Vsi-30-102],[@B6-VSI-30-102],[@b9-vSI-30-102],[@b10-VSI-30-102]\]. heGEr eT AL. \[[@b12-VsI-30-102]\] SUmmARIZeD IN theIr ReVIew oN EVLa THAt tHe HeAT InducEd THRombosIS AND ThE thermal dAMage in The vEIN waLl rEsUlts iN inflamMatIOn and aCtIvaTIOn Of ThE iMmune sYsTem (fIBRoblaSt MiGRAtIoN). FiNaLly, the vEiN waLl bEcomES CollAGENIzED and rEsuLTs IN ocClUsiON Or StenOSIS. tHE PrEsENTED hIstOmoRphOLOgIcal exAMiNatION sHOwed That THE WHOLE VEiN TrEAtED BY CRyoscLErOsIS unDERwENT TyPICaL rEMODElinG, THUS THE AnAtOmical STRUCtuRe COuldN't bE RecoGnIzeD. ThE walL bECame CRuSTeD becAUsE Of collAGEn DEPoSITIon By fIbRoBLASt acTIVITY. hOWeVeR, ThE gSV WASN'T ocClUdEd Yet segMEnTal StRIcTurES WERE OBseRVED ([fiG. 1](#F1-Vsi-30-102){ref-tYpe="fIG"}).
cryOSClEROsIs seEMS tO haVE ThE SAMe efFECt aS THE otHER FAmiliaR endOVENoUS aBlAtion teChNIqUEs \[[@b1-VsI-30-102],[@B2-vSi-30-102],[@b4-vSI-30-102],[@B6-Vsi-30-102]--[@b9-vsI-30-102]\]. ThE ReAsON for recaNAliZATiON of thE GSV iS not clEAr, WiTH REcurrENt INcOMPETENcE of ThE SapHenOFEMorAl JUNCtIon AND tHE POSterOmEdIal PErFoRating vein beInG ONE oF THe ReasoNs, buT iT iS AlsO POSSIBle THAt tHe RemoDelIng PrOcEsS oF The GSv WAS not eFfECTivE eNOuGH. the PathoGeNeSIs oF rECanALizatION AFtEr the EndOvENous PrOcEdUrEs AnD ReCurRENT VaRICoSITY HaS BEen StuDied, BUT moRe eVIdEnCe is nEEDED \[[@b5-vSI-30-102]\].
conSIdErinG thaT this ArTicle IS ONLY A CASe rePoRT, It haS SErIoUs LiMitAtIONS. mOrE caSeS arE NEceSSarY TO ASSESs tHE Real EffeCT of crYoscLeRoSis on tHe GsV, BuT thE PRocEDure seeMS tO bE efFIcIeNt in oBlIteRatIVe rEmODeliNG of ThE VeIN.
coNfLicT OF IntERESt: None.
{#f1-vSi-30-102}
| INTRODUCTION ============ Fordecades, high ligation and saphenous vein stripping with phlebectomy of varices were the gold standard procedures in the treatment of the incompetence of the great saphenous vein(GSV) \[[@b1-vsi-30-102],[@b2-vsi-30-102]\]. An alternative method was cryostripping which isless invasive than traditional stripping \[[@b3-vsi-30-102]\]. Becauseof the need for minimalinvasiveness, the endovenous procedures spread worldwide in the treatment of theincompetence of the saphenous trunksinthe last decade \[[@b4-vsi-30-102],[@b5-vsi-30-102]\]. At theend ofthe last century, Milleret and Le Pivert\[[@b6-vsi-30-102]\]described cryosclerosis as a safe, efficientand feasible endovenous cryoablation technique to treatthe incompetenceof the GSV. The segmental freezing ofthe saphenous trunk was performed with a flexible cryoprobe by inguinalapproach. In1987, Le Pivert\[[@b7-vsi-30-102]\] published the experiences of350 patients treated bythis method. In 1994, Garde\[[@b8-vsi-30-102]\] concluded that thetechnique was safe and efficient based on the results of a randomized controlled trial on 800 patients. We have completed a prospective non-randomized trial to comparecryosclerosis andclassical stripping, andregarding the short termresults, this endovenousprocedureseems to be effective. The electron microscopic examination proved that immediate ultrastructural changes were formed in the GSV \[[@b9-vsi-30-102]\]. CASE ==== Wepresent the histological changes of the GSV at 2 years after cryoablation. The examined vein piece was harvested from the proximal femoralpart of the GSV from a 31-year-old female patient who underwent cryosclerosis 23 months ago. This procedure was performedbyinguinal approach, after high ligation acryoprobe was introduced into the GSV and segmentalfreezing of the vessel wall was carriedout. The patientwasadmitted again to our department of surgery because ofrecurrent varicositythatcaused pain and limb swelling at the end of the day. Ultrasonography showed therecanalization of the GSV. Saphenofemoral reflux and an incompetent posteromedial perforating vein weredetected as possible reasons.High ligation and cryostripping withphlebectomy ofvarices were performed under spinal anesthesia. The GSV was verycrusted andsegmental strictures were foundwhen the cryoprobewas introduced into the vessel lumen. A small vein piece washarvestedfrom the proximal femoral part ofthe GSV for histologicalexamination. The patienthealed without any postoperative complications.Hematoxyllin-eosin,Picro-sirius Red, vanGieson and immunohistochemicalstains (CD34, smooth muscle actin)were performed. The anatomical structureof the veincouldn't be recognizedmicroscopically. Thewall was collagenized and padded by endothelium that was caused by progressive fibrosis([Fig. 1](#f1-vsi-30-102){ref-type="fig"}). The patient consented tothistrialand agreed to apply her records. DISCUSSION ========== Generally, therole of the endovenous ablation techniques (endovenous laser ablation\[EVLA\], radiofrequency ablation \[RFA\],ultrasound guided foam sclerotherapy, mechanochemical ablation, steam ablation) has increased in the treatment ofthe incompetence of the GSV. These are widely studied, safe and efficient procedures. The most frequently used methods are EVLA and RFA. The longterm results are favorable, but the recanalization ofthe GSV could be observed some years after the treatment that may cause recurrent varicosity with symptoms \[[@b1-vsi-30-102],[@b2-vsi-30-102],[@b4-vsi-30-102],[@b5-vsi-30-102]\]. In this short report, the presented method is cryosclerosis, which is the endovenous cryoablationof theGSV. This procedure is not widelyknown. The reported patient had recurrent varicositywithsymptoms. Cryosclerosis was ineffective 2 years after the treatment;however, segmental stenoses of the GSV were observed during the operation.The incompetence of the saphenofemoral junction and the posteromedial perforating vein could cause the recurrent disease.The role of tumescent fluids during cryosclerosis is not clear and it is not routinely used, but it might be useful since by decreasing the volume ofthe intraluminal blood, vein wall destructioncan be achieved more efficiently \[[@b10-vsi-30-102],[@b11-vsi-30-102]\]. A prospectivetrialisnecessary to assess the effect of injectingwarming liquid around the GSV before cryosclerosis. The histomorphological effects of the endovenous ablation methods have been previously investigated, mostly from animal experiments \[[@b12-vsi-30-102]--[@b14-vsi-30-102]\].Their longterm effectshave not been described yet. It'stypical for all procedures that the vein wall is destroyed by thermal, mechanical orchemical effects \[[@b1-vsi-30-102],[@b4-vsi-30-102],[@b6-vsi-30-102],[@b9-vsi-30-102],[@b10-vsi-30-102]\]. Hegeretal. \[[@b12-vsi-30-102]\] summarized in their review on EVLA that the heat induced thrombosis and the thermal damage inthevein wall results in inflammation and activationofthe immune system (fibroblast migration). Finally,the vein wall becomes collagenized and results in occlusion or stenosis. The presented histomorphologicalexamination showed that thewholevein treatedby cryosclerosis underwenttypical remodeling, thus the anatomical structure couldn't be recognized. Thewall became crusted because ofcollagendeposition by fibroblast activity. However, the GSVwasn't occluded yet segmental strictures were observed ([Fig. 1](#f1-vsi-30-102){ref-type="fig"}). Cryosclerosisseems tohave the same effectas theother familiar endovenous ablation techniques \[[@b1-vsi-30-102],[@b2-vsi-30-102],[@b4-vsi-30-102],[@b6-vsi-30-102]--[@b9-vsi-30-102]\]. The reason for recanalization of the GSV is not clear, with recurrent incompetence of the saphenofemoral junction and the posteromedial perforating vein being one of thereasons, but itis also possible that theremodeling process of the GSV wasnot effective enough. The pathogenesis ofrecanalizationafter the endovenous procedures and recurrent varicosity has been studied, but more evidence is needed\[[@b5-vsi-30-102]\].Consideringthat this article is only a case report, it has serious limitations. More cases are necessarytoassess the real effect of cryosclerosis on the GSV, but the procedure seems to be efficient in obliterative remodeling of the vein. Conflict of interest: None. {#f1-vsi-30-102} | INTRODUCTION ============ _For_ decades, high ligation and saphenous vein stripping with _phlebectomy_ of varices were the _gold_ standard procedures in the treatment of the incompetence of _the_ great saphenous _vein_ (GSV) \[[@b1-vsi-30-102],[@b2-vsi-30-102]\]. An alternative _method_ was cryostripping which _is_ less invasive than _traditional_ stripping \[[@b3-vsi-30-102]\]. Because _of_ the _need_ for minimal invasiveness, the endovenous procedures spread worldwide in the treatment of the incompetence _of_ the saphenous _trunks_ in the last decade \[[@b4-vsi-30-102],[@b5-vsi-30-102]\]. At the end of the last century, _Milleret_ and Le Pivert \[[@b6-vsi-30-102]\] described cryosclerosis as a safe, efficient and feasible _endovenous_ cryoablation technique to treat the _incompetence_ of the GSV. The segmental freezing of _the_ saphenous trunk _was_ performed with a flexible _cryoprobe_ _by_ inguinal _approach._ _In_ _1987,_ Le Pivert \[[@b7-vsi-30-102]\] published the experiences of 350 patients treated by this method. In 1994, Garde \[[@b8-vsi-30-102]\] concluded that the _technique_ was _safe_ and _efficient_ based on the results of a randomized controlled trial on 800 patients. We have completed a _prospective_ non-randomized trial to compare cryosclerosis _and_ classical stripping, and regarding the short term _results,_ this endovenous procedure seems to be _effective._ The electron microscopic examination proved that immediate ultrastructural changes were formed in the _GSV_ \[[@b9-vsi-30-102]\]. CASE ==== We present the histological changes of the GSV at 2 years after cryoablation. The examined vein piece was harvested from the proximal femoral part of the GSV from _a_ 31-year-old female patient who underwent cryosclerosis 23 _months_ ago. _This_ _procedure_ was performed by _inguinal_ approach, _after_ high ligation a cryoprobe _was_ introduced _into_ the GSV and _segmental_ freezing of the vessel wall was carried out. The patient was admitted again to our _department_ of _surgery_ _because_ of recurrent varicosity that caused pain _and_ limb _swelling_ _at_ the end of the day. _Ultrasonography_ showed the _recanalization_ of _the_ GSV. Saphenofemoral reflux and an incompetent posteromedial perforating vein were detected as possible reasons. High ligation _and_ cryostripping _with_ _phlebectomy_ of _varices_ were performed under spinal anesthesia. The _GSV_ was very crusted _and_ segmental strictures were found when the cryoprobe was introduced _into_ _the_ vessel lumen. _A_ small vein piece _was_ harvested from the proximal femoral part of the GSV for histological _examination._ The patient healed without any postoperative complications. Hematoxyllin-eosin, Picro-sirius _Red,_ van _Gieson_ and immunohistochemical _stains_ (CD34, smooth _muscle_ _actin)_ were performed. The _anatomical_ structure of _the_ _vein_ couldn't _be_ recognized microscopically. The wall was collagenized and padded by endothelium that was caused by progressive _fibrosis_ _([Fig._ _1](#f1-vsi-30-102){ref-type="fig"})._ The _patient_ _consented_ to _this_ trial _and_ _agreed_ _to_ apply her records. DISCUSSION ========== Generally, the role of the _endovenous_ ablation techniques (endovenous laser ablation _\[EVLA\],_ _radiofrequency_ ablation _\[RFA\],_ ultrasound guided foam sclerotherapy, mechanochemical ablation, _steam_ _ablation)_ has _increased_ in the treatment of the incompetence of the GSV. These _are_ widely _studied,_ safe and efficient procedures. The most frequently used methods _are_ EVLA and RFA. The long _term_ results are favorable, but the recanalization of the GSV could _be_ observed some years after the _treatment_ _that_ may cause recurrent _varicosity_ with symptoms _\[[@b1-vsi-30-102],[@b2-vsi-30-102],[@b4-vsi-30-102],[@b5-vsi-30-102]\]._ In this short report, the presented method is cryosclerosis, _which_ is the _endovenous_ cryoablation _of_ the GSV. This _procedure_ _is_ not widely known. The _reported_ patient had _recurrent_ varicosity with symptoms. Cryosclerosis was ineffective 2 _years_ after the treatment; however, segmental _stenoses_ _of_ _the_ GSV _were_ observed during the operation. The incompetence _of_ the _saphenofemoral_ junction and the posteromedial perforating vein could cause the recurrent disease. The role of tumescent fluids _during_ cryosclerosis is not clear and _it_ is not routinely used, but it might be useful since by decreasing the volume of the intraluminal _blood,_ vein wall destruction can be achieved more _efficiently_ \[[@b10-vsi-30-102],[@b11-vsi-30-102]\]. A prospective trial is necessary to _assess_ the effect of injecting warming liquid around _the_ GSV before _cryosclerosis._ The histomorphological effects of _the_ endovenous ablation methods have been _previously_ investigated, mostly from animal experiments \[[@b12-vsi-30-102]--[@b14-vsi-30-102]\]. Their long term effects _have_ _not_ been described yet. It's typical for _all_ procedures that the vein wall is destroyed by thermal, mechanical _or_ chemical effects \[[@b1-vsi-30-102],[@b4-vsi-30-102],[@b6-vsi-30-102],[@b9-vsi-30-102],[@b10-vsi-30-102]\]. _Heger_ et al. \[[@b12-vsi-30-102]\] summarized _in_ their review on EVLA that the heat _induced_ thrombosis and the thermal damage in the vein wall results in inflammation _and_ activation of the immune system _(fibroblast_ migration). Finally, the vein wall _becomes_ collagenized and results in occlusion or stenosis. The presented histomorphological _examination_ showed that _the_ whole vein treated by _cryosclerosis_ underwent typical remodeling, thus the anatomical _structure_ couldn't be recognized. The wall became crusted because of collagen deposition by fibroblast activity. However, the GSV wasn't occluded _yet_ segmental _strictures_ were observed ([Fig. 1](#f1-vsi-30-102){ref-type="fig"}). Cryosclerosis _seems_ to have the same effect _as_ _the_ other familiar endovenous ablation techniques \[[@b1-vsi-30-102],[@b2-vsi-30-102],[@b4-vsi-30-102],[@b6-vsi-30-102]--[@b9-vsi-30-102]\]. The reason for recanalization _of_ the GSV is not clear, _with_ recurrent incompetence of the saphenofemoral junction and _the_ _posteromedial_ _perforating_ vein being one of the reasons, but it is also _possible_ that the _remodeling_ process of the GSV _was_ not effective enough. The pathogenesis _of_ recanalization after _the_ endovenous _procedures_ and recurrent varicosity has _been_ studied, but more _evidence_ is needed \[[@b5-vsi-30-102]\]. _Considering_ that this article is only a case report, _it_ _has_ serious limitations. More cases are necessary to assess the real _effect_ of cryosclerosis _on_ the _GSV,_ but the _procedure_ seems to be efficient in obliterative _remodeling_ of the vein. Conflict of _interest:_ None. {#f1-vsi-30-102} |
Introduction {#S1}
============
Porcine circovirus has long harmed the sound development of pig husbandry, and especially the PCV3 that has appeared in recent years has caused tremendous economic losses for pig husbandry ([@B17]; [@B21]). Current research indicates that PCV is mainly divided into three genotypes ([@B8]). PCV1 is generally considered non-pathogenic ([@B27]). PCV2 is widely prevalent worldwide, previous studies have shown that PCV2 is the main pathogenesis of post-weaning multisystemic wasting syndrome (PMWS) and swine dermatitis and nephrotic syndrome (PDNS) ([@B1]; [@B30]). However, in recent years, some researchers have detected PCV3 from PDNS piglets ([@B22]; [@B13]). Studies have shown that PCV3 and PCV2 are mixed infections and have become popular in many countries. It has been reported that PCV3 may cause reproduction disorder in sows and PDNS in adult pigs ([@B22]). Similar to PCV2, PCV3 is often mixed with PRRSV, PCV2 ([@B2]). However, unlike PCV2, there are currently commercial vaccines to prevent PCV2 infection ([@B23]), and there is still a shortage of vaccines and related drugs to prevent PCV3.
Obtaining standard virus strains is the basis for the development of PCV3 vaccines and related biological products. However, no experimental report on the successful isolation of PCV3 has been reported. With the development of genetic engineering technology, a variety of virus strains have been constructed through reverse genetics ([@B34]; [@B10]). With the continuous analysis of the genome structure and function of PCV2 and PCV3, researchers have obtained infectious clones by constructing eukaryotic expression vectors of PCV2 and PCV3 ([@B28]; [@B11]). According to the genome structure of PCV, the porcine circovirus genome is a single-stranded negative-stranded DNA, the virions are only 14--17 nm, and there is no capsule on the surface of the virus capsid ([@B18]; [@B32]). The size of the PCV1 and PCV2 genome research surface is 1767--1768 bp and contains 11 open reading frames (ORFs), of which ORF1 is a virus replication--related protein (Rep) and is a necessary element of virus replication ([@B3]). ORF2 encodes the viral capsid protein (Cap) ([@B20]) and is commonly used in the study of subunit vaccines and diagnostic reagents ([@B16]). Unlike the genomes of PCV1 and PCV2, the full length of the PCV3 genome is 2000 bp ([@B24]). Some researchers have predicted the PCV3 genomic DNA, and currently they have predicted a total of three ORFs ([@B22]). One Rep protein composed of 297 amino acids was encoded by ORF1 ([@B33]). Besides, another Cap protein covering 214 amino acids by ORF2 was replicated in the opposite direction and a protein with an unknown function and containing 231 amino acids by ORF3 ([@B5]). Whether PCV3 has other open reading frames and their functions still needs further study.
The above research is to construct infectious clones under the premise of grasping the genome structure of the virus. However, for some newly discovered circular DNA viruses, the genome structure and function are undefined yet, so it is hard to construct infectious clones with the application of the eukaryotic expression vector. This study intends to use the biological characteristics of PCV3 circular DNA to construct PCV3 infectious clones and construct a Kunming mouse infection model without the help of exogenous expression vectors. The results showed that the PCV3 strain can infect the myocardium and lung of mice. This study will provide a method for the construction of infectious clones of other circular DNA viruses and lay a foundation for the study of the pathogenic mechanism of PCV3.
Materials and Methods {#S2}
=====================
Cells and Cultures {#S2.SS1}
------------------
The 3D4/21 cell line (iCell Bioscience Inc., Shanghai, China) was cultivated in Dulbecco's minimum essential medium (MEM, Gibco) supplemented by 10% fetal bovine serum at 37°C in a humidified 5% CO~2~ incubator. The cloning and construction of recombinant expression plasmids was carried out in *E. coli* strain DH5α cells (Takara Bio, Dalian, China). The prokaryotic expression vector pET-32a (+) and *E. coli* BL21 (DE3) cells were harvested from the stocks of our laboratory. The SP2/0 cells were also acquired from the stocks.
Viral Gene and Primer Synthesis {#S2.SS2}
-------------------------------
The PCV3 gene sequence (GenBank accession number: [MH107162.1](MH107162.1)) was taken to form a loop on Snap-Gene software, and the position of the unique restriction site was adopted to open the sequence, termed as the rearranged linear PCV3 gene sequence. Since the PCV3 is circular, the cyclization of the original linear DNA sequence was considered, the *Hin*dIII restriction site was recruited as the reopening site. When the opening process was completed, the *Hin*dIII restriction site was added to the end of the linear sequence. The newly rearranged sequence was synthesized by Sangon (Shanghai, China). [Figure 1](#F1){ref-type="fig"} illustrates the gene pattern of the novel PCV3 and presents the design pattern of the circular PCV3 amplification primer and detection primer. Primers listed in [Table 1](#T1){ref-type="table"} were designed in accordance with the rearranged PCV3 gene sequence and PCV3 Cap gene sequence; the designed primers were synthesized by Sangon (Shanghai, China).
######
Primers used for the construction and identification of the recombinant virus.
Primer Sequence
---------------------------------------------------------------------
PCV3 M1: 5′-CCC[AAGCTT]{.ul}GTGCGGATGCGGCTGCGCG-3′;
PCV3 L2: 5′- CCC[AAGCTT]{.ul}CCCGCGTTTTCCCACAACC-3′;
PCV3 L3: 5′- GGTTGTGGGAAAACGCGGG-3′;
PCV3 M2: 5′-CGCGCAGCCGCATCCGCACAAGCTT-3′;
PCV3 Cap *Bam*HI: 5′-CGC[GGATCC]{.ul}ATGAGACACAGAGCTATATTCAGAA-3′;
PCV3 Cap *Hin*dIII: 5′-CCC[AAGCTT]{.ul}TTAGAGAACGGACTTGTAACGAATC-3′
The underlined sequences represent restriction enzyme sites introduced. PCV3 M1, PCV3 L2: PCV3 complete genome amplification primers; PCV3 L3 is a reverse complementary sequence of PCV3 L2; PCV3 M2 is the nucleotide sequence next to the primer binding site of PCV3 L2; PCV3 Cap BamHI, PCV3 Cap HindIII: The design of the primer is the reverse complement of the negative chain of PCV3 with the Cap gene sequence, and specific primers were then designed according to the reverse complementary DNA sequence.
{#F1}
Amplification and Cyclization of the PCV3 Genome {#S2.SS3}
------------------------------------------------
To amplify the newly synthesized rearranged PCV3 gene, PCV3 M1, PCV3 L2 primers, and KFX-101 high fidelity enzymes (TOYOBO, Japan) were adopted. The final volume of reaction fluid reached 100 μL. The PCR cycling conditions included: pre-denaturation at 94°C for 2 min, 30 cycles at 98°C for 10 s, 58°C for 30 s, and 68°C for 2 min, as well as the final extension for 10 min at 68°C. PCR products were preserved at 16°C for subsequent experiments.
The amplified PCR products of rearranged PCV3 gene were purified following the instructions of the DNA Purification Kit (Takara Bio, Dalian, China). Subsequently, the purified PCR product was digested by *Hin*dIII restriction endonuclease (NEB, Beijing, China) and then incubated in a water bath at 37°C for 5 h. Then, the digested products of *Hin*dIII restriction endonuclease were purified following the manufacturer's instructions (Takara Bio, Dalian, China). Next, the purified DNA fragments were connected based on T4 DNA ligase instructions (NEB, Beijing, China) at 16°C for 12 h. Last, the cyclized PCV3 DNA was harvested.
Transfection of Cyclized PCV3 DNA {#S2.SS4}
---------------------------------
The cyclized PCV3 DNA was transfected into 3D4/21 cells (60% confluency) | introduction { # s1 } = = = = = = = = = = = = porcine circovirus has long harmed the sound development of pig husbandry, and especially the pcv3 that has appeared in recent years has caused tremendous ecological losses for pig husbandry ( [ @ b17 ] ; [ @ b21 ] ). current analysis indicates that pcv is mainly divided into three genotypes ( [ @ 1a ] ). pcv1 is generally considered non - pathogenic ( [ @ b27 ] ). pcv2 is widely prevalent worldwide, previous studies have shown that pcv2 is the main pathogenesis of post - weaning multisystemic wasting syndrome ( pcs ) and swine dermatitis and nephrotic syndrome ( pdns ) ( [ @ b1 ] ; [ @ b30 ] ). however, in recent years, some researchers have detected pcv3 from pdns piglets ( [ @ b22 ] ; [ @ b13 ] ). studies have shown that pcv3 and pcv2 are mixed infections and have become popular in many countries. it has been reported that pcv3 may cause reproduction disorder in sows and pdns in adult pigs ( [ @ b22 ] ). compared to pcv2, pcv3 is often mixed with prrsv, pcv2 ( [ @ b2 ] ). however, unlike pcv2, there are currently commercial vaccines to prevent pcv2 infection ( [ @ br ] ), and there is still a shortage of vaccines and related drugs to prevent pcv3. obtaining standard virus strains is the basis for the development of pcv3 vaccines and related viral products. however, no experimental report on the successful isolation of pcv3 has been reported. with the development of genetic engineering technology, a variety of virus strains have been constructed through reverse genetics ( [ @ b34 ] ; [ @ 1b ] ). with the continuous analysis of the genome itself and function of pcv2 and pcv3, researchers have obtained infectious clones by constructing eukaryotic expression vectors using pcv2 and pcv3 ( [ @ b28 ] ; [ @ b11 ] ). according to the genome structure of pcv, the porcine circovirus genome is a single - stranded negative - stranded dna, the virions are only 14 - - 17 nm, and there is no capsule on the surface of the virus capsid ( [ @ b18 ] ; [ @ b32 ] ). the size of the pcv1 and pcv2 genome research surface is 1767 - - 1768 bp and contains 11 open reading frames ( orfs ), of which orf1 is a virus replication - - related protein ( rep ) and is a necessary element of virus replication ( [ @ b3 ] ). orf2 encodes the viral capsid protein ( cap ) ( [ @ b20 ] ) and is commonly used in the study of subunit vaccines and diagnostic reagents ( [ @ b16 ] ). unlike the genomes of pcv1 and pcv2, the full length of the pcv3 genome is 2000 bp ( [ @ b24 ] ). some researchers have predicted the pcv3 genomic dna, and currently they have predicted a total of three orfs ( [ @ b22 ] ). one rep protein composed of 297 amino acids was encoded by orf1 ( [ @ b33 ] ). besides, another cap protein covering 214 amino acids by orf2 was replicated in the opposite direction and a protein with an unknown function and containing 231 amino acids by orf3 ( [ @ b5 ] ). whether pcv3 has other open reading frames and their functions still needs further study. the above research is to construct infectious clones under the premise of grasping the genome structure of the virus. however, for some newly discovered circular dna viruses, the genome structure and function are undefined yet, so it is hard to construct infectious clones with the application of the eukaryotic expression vector. this study intends to use the biological characteristics of pcv3 circular dna to construct pcv3 infectious clones and construct a kunming mouse infection model without the help of exogenous expression vectors. the results showed that the pcv3 strain can infect the myocardium and lung of mice. this study will provide a method for the construction of infectious clones of other circular dna viruses and lay a foundation for the study of the pathogenic mechanism of pcv3. materials and methods { # s2 } = = = = = = = = = = = = = = = = = = = = = cells and cultures { # s2. ss1 } - - - - - - - - - - - - - - - - - - the 3d4 / 21 cell line ( icell bioscience inc., shanghai, china ) was cultivated in dulbecco ' s minimum essential medium ( mem, gibco ) supplemented by 10 % fetal bovine serum at 37°c in a humidified 5 % co ~ 2 ~ incubator. the cloning and construction of recombinant expression plasmids was carried out in * e. coli * strain dh5α cells ( takara bio, dalian, china ). the prokaryotic expression vector pet - 32a ( + ) and * e. coli * bl21 ( de3 ) cells were harvested from the stocks of our laboratory. the sp2 / 0 cells were also acquired from the stocks. viral gene and primer synthesis { # s2. ss2 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the pcv3 gene sequence ( genbank accession number : [ mh107162. 1 ] ( mh107162. 1 ) ) was taken to form a loop on snap - gene software, and the position of the unique restriction site was adopted to open the sequence, termed as the rearranged linear pcv3 gene sequence. since the pcv3 is circular, the cyclization of the original linear dna sequence was considered, the * hin * diii restriction site was recruited as the reopening site. when the opening process was completed, the * hin * diii restriction site was added to the end of the linear sequence. the newly rearranged sequence was synthesized by sangon ( shanghai, china ). [ figure 1 ] ( # f1 ) { ref - type = " fig " } illustrates the gene pattern of the novel pcv3 and presents the design pattern of the circular pcv3 amplification primer and detection primer. primers listed in [ table 1 ] ( # t1 ) { ref - type = " table " } were designed in accordance with the rearranged pcv3 gene sequence and pcv3 cap gene sequence ; the designed primers were synthesized by sangon ( shanghai, china ). # # # # # # primers used for the construction and identification of the recombinant virus. primer sequence - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - pcv3 m1 : 5 ′ - ccc [ aagctt ] {. ul } gtgcggatgcggctgcgcg - 3 ′ ; pcv3 l2 : 5 ′ - ccc [ aagctt ] {. ul } cccgcgttttcccacaacc - 3 ′ ; pcv3 l3 : 5 ′ - ggttgtgggaaaacgcggg - 3 ′ ; pcv3 m2 : 5 ′ - cgcgcagccgcatccgcacaagctt - 3 ′ ; pcv3 cap * bam * hi : 5 ′ - cgc [ ggatcc ] {. ul } atgagacacagagctatattcagaa - 3 ′ ; pcv3 cap * hin * diii : 5 ′ - ccc [ aagctt ] {. ul } ttagagaacggacttgtaacgaatc - 3 ′ the underlined sequences represent restriction enzyme sites introduced. pcv3 m1, pcv3 l2 : pcv3 complete genome amplification primers ; pcv3 l3 is a reverse complementary sequence of pcv3 l2 ; pcv3 m2 is the nucleotide sequence next to the primer binding site of pcv3 l2 ; pcv3 cap bamhi, pcv3 cap hindiii : the design of the primer is the reverse complement of the negative chain of pcv3 with the cap gene sequence, and specific primers were then designed according to the reverse complementary dna sequence.! [ pcv3 viral gene rearrangement. * * ( a ) * * rearranged linear gene sequence of pcv3 containing * hin * diii restriction sites. the * hin * diii restriction site acted as the reopening site, added to the end of the linear sequence after the opening process. * * ( b ) * * pcr detection after pcv3 cyclization. specific primers, covering pcv3 m2 and pcv3 l3, were designed to verify whether the pcv3 gene sequence was cyclized. however, the pcv3 linear sequence could not be amplified by the mentioned primers. ] ( fmicb - 11 - 01067 - g001 ) { # f1 } amplification and cyclization of the pcv3 genome { # s2. ss3 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - to amplify the newly synthesized rearranged pcv3 gene, pcv3 m1, pcv3 l2 primers, and kfx - 101 high fidelity enzymes ( toyobo, japan ) were adopted. the final volume of reaction fluid reached 100 μl. the pcr cycling conditions included : pre - denaturation at 94°c for 2 min, 30 cycles at 98°c for 10 s, 58°c for 30 s, and 68°c for 2 min, as well as the final extension for 10 min at 68°c. pcr products were preserved at 16°c for subsequent experiments. the amplified pcr products of rearranged pcv3 gene were purified following the instructions of the dna purification kit ( takara bio, dalian, china ). subsequently, the purified pcr product was digested by * hin * diii restriction endonuclease ( neb, beijing, china ) and then incubated in a water bath at 37°c for 5 h. then, the digested products of * hin * diii restriction endonuclease were purified following the manufacturer ' s instructions ( takara bio, dalian, china ). next, the purified dna fragments were connected based on t4 dna ligase instructions ( neb, beijing, china ) at 16°c for 12 h. last, the cyclized pcv3 dna was harvested. transfection of cyclized pcv3 dna { # s2. ss4 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the cyclized pcv3 dna was transfected into 3d4 / 21 cells ( 60 % confluency ) | Introduction {# S1} = = = = = = = = = = = = Porcine circovirus has long harmed the sound development of pig husbandry, and especially the PCV3 that has appeared in recent years has caused tremendous economic losses for pig husbandry ([ @ B17 ]; [@ B21] ). Current research indicates that PCV is mainly divided into three genotypes ([ @ B8] ). PCV1 is generally considered non - pathogenic ([ @ B27] ). PCV2 is widely prevalent worldwide, previous studies have shown that PCV2 is the main pathogenesis of post - weaning multisystemic wasting syndrome (PMWS) and swine dermatitis and nephrotic syndrome (PDNS) ([ @ B1 ]; [@ B30] ). However, in recent years, some researchers have detected PCV3 from PDNS piglets ([ @ B22 ]; [@ B13] ). Studies have shown that PCV3 and PCV2 are mixed infections and have become popular in many countries. It has been reported that PCV3 may cause reproduction disorder in sows and PDNS in adult pigs ([ @ B22] ). Similar to PCV2, PCV3 is often mixed with PRRSV, PCV2 ([ @ B2] ). However, unlike PCV2, there are currently commercial vaccines to prevent PCV2 infection ([ @ B23] ), and there is still a shortage of vaccines and related drugs to prevent PCV3. Obtaining standard virus strains is the basis for the development of PCV3 vaccines and related biological products. However, no experimental report on the successful isolation of PCV3 has been reported. With the development of genetic engineering technology, a variety of virus strains have been constructed through reverse genetics ([ @ B34 ]; [@ B10] ). With the continuous analysis of the genome structure and function of PCV2 and PCV3, researchers have obtained infectious clones by constructing eukaryotic expression vectors of PCV2 and PCV3 ([ @ B28 ]; [@ B11] ). According to the genome structure of PCV, the porcine circovirus genome is a single - stranded negative - stranded DNA, the virions are only 14 - - 17 nm, and there is no capsule on the surface of the virus capsid ([ @ B18 ]; [@ B32] ). The size of the PCV1 and PCV2 genome research surface is 1767 - - 1768 bp and contains 11 open reading frames (ORFs ), of which ORF1 is a virus replication - - related protein (Rep) and is a necessary element of virus replication ([ @ B3] ). ORF2 encodes the viral capsid protein (Cap) ([ @ B20] ) and is commonly used in the study of subunit vaccines and diagnostic reagents ([ @ B16] ). Unlike the genomes of PCV1 and PCV2, the full length of the PCV3 genome is 2000 bp ([ @ B24] ). Some researchers have predicted the PCV3 genomic DNA, and currently they have predicted a total of three ORFs ([ @ B22] ). One Rep protein composed of 297 amino acids was encoded by ORF1 ([ @ B33] ). Besides, another Cap protein covering 214 amino acids by ORF2 was replicated in the opposite direction and a protein with an unknown function and containing 231 amino acids by ORF3 ([ @ B5] ). Whether PCV3 has other open reading frames and their functions still needs further study. The above research is to construct infectious clones under the premise of grasping the genome structure of the virus. However, for some newly discovered circular DNA vi$uwes, the genome structure and function are undefined yet, so it is hard to construct infectious clones with the al9lication of the eukaryotic expression vector. This study intends to use the biological characteristics of PCV3 circular DNA to construct PCV3 infectious clones and construct a Kunming mouse infection model without the help of exogenous expression vectors. The results showed that the PCV3 strain can infect the myocardium and lung of mice. This study will provide a method for the construction of infectious clones of other circular DNA viruses and lay a foundation for the study of the pathogenic mechanism of PCV3. Materials and Methods {# S2} = = = = = = = = = = = = = = = = = = = = = Cells and Cultures {# S2. SS1} - - - - - - - - - - - - - - - - - - The 3D4 / 21 cell line (iCell Bioscience Inc. , Shanghai, China) was cultivated in Dulbecco ' s minimum essential medium (MEM, Gibco) supplemented by 10% fetal bovine serum at 37 ° C in a humidified 5% CO ~ 2 ~ incubator. The cloning and construction of recombinant expression plasmids was carried out in * E. coli * strain DH5α cells (TZJara Bio, Dalian, China ). The prokaryotic expression vector pET - 32a (+) and * E. coli * BL21 (DE3) cells were harvested from the stocks of our laboratory. The SP2 / 0 cells were also acquired from the stocks. Viral Gene and Primer Synthesis {# S2. SS2} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The PCV3 gene sequence (GenBank accession number: [MH107162. 1] (MH107162. 1) ) was taken to form a loop on Snap - Gene software, and the position of the unique restriction site was adopted to open the sequence, termed as the rearranged linear PCV3 gene sequence. Since the PCV3 is circular, the cyclization of the original linear DNA sequence was considered, the * Hin * dIII restriction site was recruited as the reopening site. When the opening process was completed, the * Hin * dIII restriction site was added to the end of the linear sequence. The newly rearranged sequence was synthesized by Sangon (Shanghai, China ). [Figure 1] (# F1) {ref - type = " fig "} illustrates the gene pattern of the novel PCV3 and presents the design pattern of the circular PCV3 amplification primer and detection primer. Primers listed in [Table 1] (# T1) {ref - type = " table "} were designed in accordance with the rearranged PCV3 gene sequence and PCV3 Cap gene sequence; the designed primers were synthesized by Sangon (Shanghai, China ). # # # # # # Primers used for the construction and identification of the recombinant virus. Primer Sequence - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - PvV# M1: 5 ′ - CCC [AAGCTT] {. ul} GTGCGGATGCGGCTGCGCG - 3 ′; PDd3 L2: 5 ′ - CCC [AAGCTT] {. ul} CCCGCGTTTTCCCACAACC - 3 ′; PCV3 L3: 5 ′ - GGTTGTGGGAAAACGCGGG - 3 ′; PCV3 M2: 5 ′ - CGCGCAGCCGCATCCGCACAAGCTT - 3 ′; PCV3 Cap * Bam * HI: 5 ′ - CGC [GGATCC] {. ul} ATGAGACACAGAGCTATATTCAGAA - 3 ′; PCV3 Cap * Hin * dIII: 5 ′ - CCC [AAGCTT] {. ul} TTAGAGAACGGACTTGTAACGAATC - 3 ′ The underlined sequences represent restriction enzyme sites OntrodIced. PCV3 M1, PCV3 L2: PCV3 complete genome amplification primers; PCV3 L3 is a reverse complementary sequence of (CG3 L2; PCV3 M2 is the nucleotide sequence next to the primer binding site of PCV3 L2; PCV3 Cap BamHI, PCV3 Cap HindIII: The design of the primer is the reverse complement of the negative chain of PCV3 with the Cap gene sequence, and specific primers were then designed according to the reverse complementary DNA sequence. ! [PCV3 viral gene rearrangement. * * (A) * * Rearranged linear gene sequence of PCV3 containing * Hin * dIII restriction sites. The * Hin * dIII restriction site acted as the reopening site, added to the end of the linear sequence after the opening process. * * (B) * * PCR detection after PCV3 cyclization. Specific primers, covering PCV3 M2 and PCV3 L3, were designed to verify whether the PCV3 gene sequence was cyclized. However, the PCV3 linear sequence could not be amplified by the mentioned primers.] (fmicb - 11 - 01067 - g001) {# F1} Amplification and Cyclization of the PCV3 Genome {# S2. SS3} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - To amplify the newly synthesized rearranged PCV3 gene, PCV3 M1, PCV3 L2 primers, and KFX - 101 high fidelity enzymes (TOYOBO, Japan) were adopted. The final volume of reaction fluid reached 100 μL. The PCR cycling conditions included: pre - denaturation at 94 ° C for 2 min, 30 cycles at 98 ° C for 10 s, 58 ° C for 30 s, and 68 ° C for 2 min, as well as the final extension for 10 min at 68 ° C. PCR products were preserved at 16 ° C for subsequent experiments. The aKplifiSd PCR products of rearranged PCV3 gene were purified following the instrHct9ons of the DNA Purification Kit (Takara Bio, Dalian, China ). Subsequently, the purified PCR product was digested by * Hin * dIII restriction endonuclease (NEB, Beijing, China) and then incubated in a water bath at 37 ° C for 5 h. Then, the digested products of * Hin * dIII restriction endonuclease were purified following the manufacturer ' s instructions (Takara Bio, Dalian, China ). Next, the purified DNA fragments were connected based on T4 DNA ligase instructions (NEB, Beijing, China) at 16 ° C for 12 h. Last, the cyclized PCV3 DNA was harvested. Transfection of Cyclized PCV3 DNA {# S2. SS4} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The cycl&zer PCV3 DNA was transfected into 3D4 / 21 cells (60% confluency ) | Introduction {#S1} ============ circovirus has long harmed the sound of and especially the PCV3 has appeared in recent years has caused tremendous economic losses for pig husbandry ([@B17]; [@B21]). Current research indicates that PCV is mainly divided into three genotypes ([@B8]). PCV1 is generally considered non-pathogenic ([@B27]). PCV2 widely prevalent previous studies have shown that PCV2 is the pathogenesis of post-weaning wasting syndrome (PMWS) and nephrotic syndrome ([@B1]; [@B30]). However, in recent years, have detected PCV3 from PDNS piglets ([@B22]; [@B13]). Studies have shown that PCV3 and PCV2 are infections and have become popular in many countries. It has been reported that PCV3 may disorder in sows and PDNS in adult pigs ([@B22]). Similar to PCV2, PCV3 often mixed with PRRSV, PCV2 ([@B2]). However, unlike PCV2, there are currently commercial vaccines to prevent PCV2 infection ([@B23]), and there is still a shortage of vaccines and related drugs to prevent PCV3. standard virus strains is the basis for development of PCV3 vaccines and related biological products. However, no experimental report on the successful of PCV3 has been reported. With the of genetic engineering variety of strains been constructed through reverse genetics ([@B34]; [@B10]). With the continuous the genome function of PCV2 and PCV3, researchers have obtained infectious clones by constructing eukaryotic vectors of PCV2 and PCV3 ([@B28]; [@B11]). According to the genome structure of PCV, the porcine genome a single-stranded negative-stranded the virions are only 14--17 and there is no on the of the virus capsid ([@B18]; [@B32]). The size of the PCV1 and PCV2 genome research surface is 1767--1768 bp and contains 11 open reading frames (ORFs), of which ORF1 is a virus replication--related protein (Rep) and is a necessary element of virus ([@B3]). ORF2 encodes the viral capsid protein (Cap) ([@B20]) and is commonly used in study of vaccines and diagnostic reagents ([@B16]). Unlike the genomes of PCV1 and PCV2, the full length of PCV3 genome is 2000 bp ([@B24]). Some researchers have predicted the PCV3 genomic DNA, and currently they predicted a total of three ORFs Rep composed of 297 amino acids encoded by ORF1 ([@B33]). Besides, another Cap protein 214 amino by ORF2 replicated the opposite and a protein with an unknown function and containing 231 amino acids by ORF3 ([@B5]). Whether PCV3 has other open reading frames and functions still needs further study. The above research is to construct infectious clones under premise of grasping the genome structure of the virus. However, for some newly discovered circular DNA viruses, the genome structure and function undefined yet, it is hard to construct infectious with of the eukaryotic expression vector. This study to use biological characteristics of PCV3 circular DNA to PCV3 infectious clones and construct a mouse infection model without the help of exogenous expression vectors. The results showed that the PCV3 strain can infect the myocardium and lung of mice. This study will provide a method for the construction of infectious other circular DNA viruses and lay a foundation for the study of the pathogenic mechanism of PCV3. Materials Methods {#S2} ===================== Cells and ------------------ The 3D4/21 cell line (iCell Bioscience Inc., China) was cultivated in Dulbecco's essential medium (MEM, supplemented by 10% fetal bovine serum at 37°C in a humidified 5% CO~2~ incubator. The cloning and construction of recombinant expression plasmids carried out in *E. coli* strain DH5α cells (Takara Bio, Dalian, The prokaryotic expression vector pET-32a (+) and *E. coli* BL21 (DE3) cells were harvested from the stocks our laboratory. The SP2/0 cells were also acquired from the stocks. Viral Gene and Synthesis ------------------------------- PCV3 (GenBank accession number: [MH107162.1](MH107162.1)) was taken to form a loop on Snap-Gene software, and position of the unique restriction site was adopted to open the termed as the rearranged linear PCV3 gene sequence. Since the PCV3 is circular, the cyclization of the original linear DNA sequence was considered, the *Hin*dIII restriction site was recruited as the site. When the opening process was completed, the *Hin*dIII restriction site was added to the end of the linear sequence. The newly rearranged sequence was synthesized by Sangon (Shanghai, China). [Figure 1](#F1){ref-type="fig"} illustrates the gene pattern of the novel PCV3 and presents the design pattern of the circular PCV3 amplification primer and detection primer. Primers listed in [Table 1](#T1){ref-type="table"} were designed in accordance with PCV3 gene and PCV3 Cap gene sequence; the primers synthesized by (Shanghai, China). ###### Primers used for the construction and of the recombinant virus. Primer Sequence --------------------------------------------------------------------- PCV3 5′-CCC[AAGCTT]{.ul}GTGCGGATGCGGCTGCGCG-3′; PCV3 L2: 5′- CCC[AAGCTT]{.ul}CCCGCGTTTTCCCACAACC-3′; PCV3 L3: 5′- GGTTGTGGGAAAACGCGGG-3′; PCV3 5′-CGCGCAGCCGCATCCGCACAAGCTT-3′; PCV3 Cap *Bam*HI: 5′-CGC[GGATCC]{.ul}ATGAGACACAGAGCTATATTCAGAA-3′; PCV3 Cap 5′-CCC[AAGCTT]{.ul}TTAGAGAACGGACTTGTAACGAATC-3′ The sequences represent restriction enzyme introduced. PCV3 M1, PCV3 L2: PCV3 complete genome PCV3 L3 a reverse complementary sequence of PCV3 L2; M2 is the nucleotide sequence next to the binding site of PCV3 L2; PCV3 Cap BamHI, PCV3 Cap HindIII: The design of the primer is the reverse of the chain of PCV3 with the Cap gene sequence, and specific primers were then to the reverse complementary DNA sequence. viral gene rearrangement. **(A)** Rearranged linear gene sequence of PCV3 containing *Hin*dIII restriction sites. The *Hin*dIII restriction site acted as the reopening site, added to the end of the sequence after the opening process. **(B)** detection after PCV3 Specific primers, covering PCV3 M2 and PCV3 L3, were designed to verify whether PCV3 gene sequence was cyclized. However, the PCV3 linear sequence could not be amplified by the mentioned primers.](fmicb-11-01067-g001){#F1} Amplification and Cyclization of the PCV3 Genome {#S2.SS3} ------------------------------------------------ amplify the newly synthesized rearranged PCV3 gene, PCV3 M1, PCV3 L2 primers, and high fidelity enzymes (TOYOBO, Japan) were adopted. The final volume reaction fluid reached 100 The PCR cycling conditions pre-denaturation at 94°C for 2 min, 30 cycles at 98°C for 10 s, 58°C for 30 s, and 68°C for 2 min, as well as the final extension for 10 min at 68°C. PCR products were preserved at 16°C for subsequent experiments. The amplified PCR products of rearranged PCV3 were purified following the instructions of the DNA Kit (Takara Bio, Dalian, China). Subsequently, the purified PCR product digested by *Hin*dIII restriction endonuclease (NEB, Beijing, China) and then incubated in a water bath 37°C for 5 h. Then, the digested products of *Hin*dIII restriction endonuclease purified following the manufacturer's instructions (Takara Bio, Dalian, China). Next, the purified DNA fragments were connected based on T4 DNA ligase instructions (NEB, Beijing, China) at 16°C for 12 h. the cyclized PCV3 DNA was harvested. Transfection of Cyclized PCV3 DNA {#S2.SS4} --------------------------------- cyclized PCV3 DNA was transfected into 3D4/21 cells (60% confluency) | intROdUcTiON {#s1}
============
PorcInE circOViRUS hAs long hARMeD tHe SOUnD deVELOpMENt oF PIg hUSbAndrY, aND EsPeCIAlLY tHE pCV3 THAT HaS APpeAREd iN RecENt YEarS haS CAUSeD TRemEnDoUS eCoNOMIc lOSsEs for piG hUSbAnDRY ([@b17]; [@B21]). CURREnt rEsearcH INdiCAtES THAT pcv is MaINlY dIvIDEd INto THREe GenOtYpES ([@b8]). pcV1 Is GEneRALlY ConsidEred nON-pathoGeniC ([@B27]). pCv2 IS WIDeLy prevaLEnt WOrLDwIDe, PrEVioUs STUdIES haVE SHOwn ThAT pcv2 Is THE mAIn pAtHOgEnESIS Of pOST-weaniNG muLtiSYSTEmic wasTing sYnDroMe (PMWS) and swinE dERmAtITIs AND NePHROTIC syndRomE (PDNs) ([@b1]; [@B30]). hoWeVer, iN reCenT yEarS, somE reSeaRCHErs hAVE DETEcted Pcv3 FrOm pdns piGlets ([@B22]; [@b13]). sTUDies havE SHOWN THaT PcV3 ANd PCv2 ARE mIxed inFeCTIons anD HAVe BeCoMe POPulaR in manY coUNTrIeS. iT hAs BeEN repOrteD That PCV3 may cauSe rePRodUCTION disorDEr iN soWs AND PdNs in Adult pIgS ([@B22]). sIMilar To PcV2, Pcv3 IS OftEN miXED wiTh PrrSv, pcv2 ([@B2]). HOWEveR, UnlIkE PCV2, tHEre ArE CurrenTlY COMmErcIAl VaccinEs TO PrevENt Pcv2 infeCtion ([@B23]), and THeRE is STIlL A ShoRtaGE oF VaCCInEs anD RELateD DRUgS To pREveNT pcV3.
OBTAINInG STaNDARd vIrUs stRaiNS iS thE basiS For tHe DEVELOpMEnT of pcv3 VAcCInES AnD rElAted BioLogicAL prodUCTS. HOweVER, NO ExpeRimeNtaL REPORt ON tHe sUCCeSSfuL isOlaTIoN OF PcV3 hAS been RepoRTeD. wIth THE DeVeLOpmeNt Of genetiC ENginEeRINg tECHnOLOGY, a vArIETy Of ViRUs sTraIns HAVe BEeN CoNStRUctED THrOuGh rEvErSE GENETicS ([@B34]; [@b10]). WITH thE cOnTINuoUs ANAlySIs OF tHe GenoMe StRuCturE AND fUNCtION oF Pcv2 AND PcV3, RESEArCheRS haVe obtAiNeD iNfECtioUs ClOneS By coNSTrUCTiNg EUkAryOtic eXPrEssION VECTOrs of PCv2 aND PCV3 ([@b28]; [@B11]). ACcordInG to thE gENOmE structuRE Of pCV, tHE PoRcINe cIRCOVirus genOME iS A SInGLe-StrAndEd NEGativE-sTrAnded DNa, tHE viRionS ArE oNLy 14--17 nm, aND THeRe IS No cApsULe on tHE SUrfacE OF the ViRus CApSId ([@B18]; [@B32]). tHe SIze Of the PcV1 And PcV2 gENoME REseaRch sURFAce Is 1767--1768 bp ANd cOntaINS 11 OpEN REaDiNG FrAmES (oRfs), of WHich oRF1 IS A VIrUs REpLICatIOn--reLAteD proTeiN (rep) aNd iS a nEceSsARY elEMEnT OF viRuS rEPLICatIOn ([@B3]). oRf2 EnCODes the vIral CApsiD protein (caP) ([@B20]) and iS COMmOnlY USeD iN The StUdy Of SuBUNiT vaCCines And DiaGnosTic rEAgENTs ([@b16]). uNlIke ThE GENOmeS of Pcv1 ANd PCv2, tHE FuLL lENGtH OF the PCv3 gENOme is 2000 BP ([@b24]). somE rEseARCHerS hAVe prEdICtEd the pCV3 GENOmic dNA, AND CurrEntLy TheY hAVE pRedICTED A total oF tHree oRFS ([@b22]). onE Rep pRoTeIn ComPOSeD OF 297 AMiNO acIds was eNcoded by oRf1 ([@b33]). BESIdeS, anoTHeR cAP ProTeIn COVEriNg 214 aMinO aciDs bY ORF2 WAs rePliCATeD In tHe OPpOsitE dIrECTIoN AnD A pROtEIn With An UNKNoWN FUNctIoN anD containInG 231 amino aCIdS bY oRf3 ([@B5]). wHETHeR pCv3 haS OtheR OPEn rEadiNG fRAmeS And THeIr FUNcTIONs Still nEEDs FUrthER sTuDy.
The aBOVe REsEarCh is TO CONStrUct inFECTiOus cloNEs undER the PreMisE Of gRAsPING tHe GeNoMe StRuctuRe of the viRuS. hoWEVeR, FOR SOME neWLY diSCovEREd CiRCulAr DnA vIruseS, THe GEnOME stRucTUre And functiOn ARE UNdEfIneD yet, SO it IS HArD TO CoNStrUct iNfEctIOUS CLONES wIth tHe APPLIcaTion oF The eukaRyOtiC ExPressiON vector. thIs stUdY inTEnDS TO uSe the biolOGiCAl ChaRacTErIsTICs OF PCv3 CIRCuLAR DnA To conSTRuCT pCv3 iNFECTiOUS cLONEs AnD cOnStruCt A kunmInG MouSE INFEcTIoN mODEL withoUT tHE HeLp OF exogenOuS exPReSsiOn vecTOrS. tHE ReSulTS ShOwED THaT THe pcv3 sTRAiN cAN InfeCt THe mYoCaRDIUm aNd luNG oF mICe. thIs Study wilL pRovIde a METHOd fOr tHE cONstrucTIoN Of inFECtioUs clOnEs Of oTher cirCuLAr dna Viruses anD lay a FouNdATION FoR thE STuDY Of THe pAThOgeNic meCHANISm Of PCv3.
MAteRiaLs and MetHOds {#s2}
=====================
cELLS And CulTurES {#s2.Ss1}
------------------
thE 3d4/21 CeLl linE (icelL biOsciEnCe INc., shANGHaI, cHIna) waS cULTIVaTeD IN dulbeCCO's miNIMum ESsENTIaL medIUm (mEM, gIBCo) SUppLeMeNTed by 10% fetaL BOVINE SErUm At 37°c in a hUmIDifiED 5% cO~2~ INCubator. ThE cLOninG And COnSTrUCtiON OF RECOmbInANt ExPREssiOn PLasmIDS WAS CArRied OUT in *E. COli* STRaIN dh5Α CelLS (TakaRA Bio, dALiaN, CHiNa). thE prOKAryotic eXPrESsIOn VEcToR pet-32A (+) ANd *e. COLI* bL21 (DE3) celLs WERe haRVesTED from tHE sTocKs Of our LABoraTORy. tHe Sp2/0 ceLlS WERE aLSO acqUiREd FrOm ThE stOcks.
vIraL GEne aND pRImER synTHesIS {#s2.SS2}
-------------------------------
thE PcV3 Gene SeqUeNce (gEnbANk acCeSSioN nUMbEr: [MH107162.1](mH107162.1)) WAs takEn tO Form A lOoP On sNAp-gEnE SoftWaRE, anD The positioN Of tHE uNiqUE REsTRICtIon sIte WAS adOPted to oPEN the seQuenCE, TErMEd AS THE REArRangEd linear PCv3 gENE seqUeNCE. Since the PCV3 IS cIRcULAr, the cYCLIZATiOn OF the oriGINaL LIneAr dnA sEQuEncE was ConSiDerED, thE *HIn*dIii reSTRICTioN sIte wAs REcruIted AS tHE reOPENINg sIte. WHen thE OpEnIng PrOCeSS WaS cOmPlEtED, tHe *HIN*dIII resTrICTiON SiTE Was AdDeD TO tHE ENd oF THe linEAR seqUEncE. ThE newLy ReArraNGeD sEQUeNce was sYNTheSizeD bY sangON (ShANghaI, CHiNA). [fIgURE 1](#F1){ReF-tyPe="fIg"} ILluSTRatES the gEne PATteRn OF THe NOVeL pcV3 ANd pResENtS tHE deSign PatTeRn OF THE CiRcUlAr pCV3 AmPLIFICatIOn PRIMeR AND DEtECTiOn PrIMeR. PRImeRS LIsTeD IN [TAbLe 1](#t1){Ref-TyPe="TABle"} wErE desIgNed in ACcOrDaNCE wiTH tHe RearraNGED Pcv3 GeNe SEqUenCe aND pcv3 CAP gENE SeQUeNce; The dESIgNeD PRImerS wERe SynTHesIzEd by saNgON (ShanGHAi, CHiNa).
######
PrIMErS UseD FOR THe consTrUcTion AnD iDENtificAtion OF thE recoMBinAnT vIRus.
pRimeR sEQueNce
---------------------------------------------------------------------
pCv3 M1: 5′-CcC[AAgctT]{.uL}GTGCGGatgcgGCTGcgCg-3′;
pCV3 L2: 5′- CCC[aAgctT]{.uL}CcCGCgttTtCCcACAACc-3′;
pCv3 l3: 5′- GGttGTGgGAaaacgCggg-3′;
pCv3 M2: 5′-CgCGCagcCgcATCcGCaCAAGCTt-3′;
pCv3 CAp *BAm*Hi: 5′-CgC[gGATcc]{.uL}aTgAGAcAcaGAgCTATAtTCagaa-3′;
pCV3 CAp *hIn*DIiI: 5′-CcC[AAgCTT]{.Ul}TTaGaGAacGGActTgtaAcGaATc-3′
the undERLiNeD SeQuENCeS REPresenT reSTriction ENzYme SItEs inTroDUced. PcV3 m1, pcV3 L2: PCv3 COMpLEte gEnoMe aMplIfiCaTIoN PRiMERs; pCv3 l3 IS A ReVERSE compLEMeNtaRy seQUenCe of pCv3 L2; Pcv3 m2 iS THe NUClEOTIDE SeqUeNCe NExT to tHE PRimer BinDinG sItE OF pCV3 L2; PcV3 CAp bamhI, pCv3 cAP HiNdiII: tHe dEsIGN Of THe Primer IS the ReversE cOMPlEMeNT of the nEgATivE cHaIN of PcV3 WItH The caP GeNe sEQUeNCe, anD sPecifIC PRiMErS were THen deSIGNEd AccORdiNG to THE reVERsE ComPleMenTArY dna sEQUenCE.
{#f1}
AMPLiFicAtiON anD cYclizAtion OF THE pcv3 GEnoMe {#S2.SS3}
------------------------------------------------
TO AmpLiFy tHe NEWly syNtHESiZED RearRANGED PcV3 genE, pcv3 m1, PCV3 l2 PRiMeRS, aNd kFX-101 higH FIdELitY EnZymES (ToyOBO, jApAN) Were ADOPTeD. tHe FINal vOlUme OF ReActiOn FlUiD reacHED 100 μL. THE pCR cyCLING coNdITIons INcluded: pRe-dEnATuraTIoN AT 94°C for 2 miN, 30 CYcles At 98°C For 10 S, 58°c foR 30 S, and 68°C FoR 2 MIn, aS weLl as THE fiNal eXTEnSIOn fOr 10 MIn at 68°c. Pcr proDuCTs wERE pREserVED AT 16°c FoR SuBSEQuEnt EXperimENts.
THe AMPLIfiEd PCR PrOdUCTS OF REArrangED pcv3 Gene WErE puRified FollOwIng tHe InstructioNs oF ThE DNa puRIficATiON kit (taKArA BIO, dalIan, CHINA). sUBseQuEntLY, ThE PuRIFIEd pCR pRODUct WaS DigEStED bY *hIn*DiII reStRiCTiON endONUcLEAsE (NEb, bEIjing, cHinA) aNd theN inCUBAtED In a wATER BATH At 37°c for 5 h. tHEN, THE dIgESted PRodUcTS Of *Hin*DIIi rEStRIcTioN endOnuclease WEre PurIfieD folLowing thE manuFactUrEr'S iNStruCtionS (TaKarA biO, dALiAn, chiNA). Next, ThE PurifIeD dnA FRAgMenTS wERe COnNeCTed BAsEd ON T4 DnA LIGasE inSTruCtIOns (NEB, bEijINg, ChINa) at 16°c foR 12 H. LaST, the CYcliZEd pcv3 dnA wAs HARVeSTed.
tRANsfECTiON oF CyCliZed PCV3 dna {#S2.Ss4}
---------------------------------
The cycliZed PCv3 Dna Was tRaNsFEcted INTO 3d4/21 cElLS (60% cOnFLUeNcY) | Introduction {#S1} ============Porcine circovirus has long harmed the sound development of pig husbandry, and especially the PCV3that has appeared in recent years has caused tremendous economic losses forpighusbandry ([@B17]; [@B21]). Current researchindicates that PCV is mainly divided intothree genotypes ([@B8]). PCV1 is generally considered non-pathogenic([@B27]). PCV2 is widely prevalent worldwide, previous studies have shownthat PCV2 is the main pathogenesis of post-weaningmultisystemic wasting syndrome (PMWS) and swine dermatitis and nephrotic syndrome (PDNS) ([@B1]; [@B30]). However, inrecent years, some researchers have detected PCV3 from PDNS piglets ([@B22]; [@B13]). Studies have shown that PCV3andPCV2are mixed infections and have become popularin many countries. It has been reported that PCV3 may cause reproductiondisorder in sows and PDNS in adultpigs ([@B22]). Similar to PCV2,PCV3 is often mixed with PRRSV, PCV2([@B2]). However, unlike PCV2,there are currently commercialvaccines toprevent PCV2 infection([@B23]), and there is still a shortage of vaccines andrelateddrugs to prevent PCV3.Obtaining standard virus strains isthe basis forthe development of PCV3 vaccines and related biological products.However,no experimental report on thesuccessful isolation of PCV3 hasbeenreported. With the development of genetic engineering technology, a variety of virus strains have been constructedthrough reverse genetics([@B34]; [@B10]). With thecontinuousanalysis of the genome structure and function ofPCV2and PCV3, researchers have obtained infectious clones byconstructingeukaryotic expression vectors of PCV2 and PCV3 ([@B28]; [@B11]). According tothe genomestructure of PCV, the porcine circovirusgenome is a single-strandednegative-stranded DNA, the virions are only 14--17 nm, and there is no capsule on thesurface of the virus capsid ([@B18]; [@B32]). The size of the PCV1 and PCV2 genome research surface is 1767--1768 bp and contains11 open reading frames(ORFs), of which ORF1 is a virus replication--related protein (Rep)and is a necessary element of virus replication ([@B3]). ORF2 encodes theviral capsid protein (Cap) ([@B20]) and is commonly used in the studyof subunit vaccines and diagnostic reagents ([@B16]). Unlike the genomes of PCV1 and PCV2, the full lengthof the PCV3 genomeis 2000 bp ([@B24]). Some researchershave predicted the PCV3 genomic DNA,and currently they have predicted a total of three ORFs ([@B22]). OneRep protein composed of297 amino acids wasencoded by ORF1 ([@B33]). Besides, another Cap protein covering 214amino acids by ORF2 was replicated in the opposite direction and a protein with an unknown function and containing 231 amino acids byORF3 ([@B5]).Whether PCV3 has other open reading frames and their functions stillneeds further study. The above research is to construct infectious clones under the premise ofgraspingthe genome structure of the virus. However, for some newly discovered circular DNA viruses, the genome structure andfunction are undefined yet, so it is hard to construct infectious clones with the application of the eukaryotic expression vector. This study intends to use the biological characteristics of PCV3 circular DNA to construct PCV3 infectious clones and construct a Kunming mouse infection model without thehelp of exogenous expression vectors. The results showed thatthe PCV3 strain can infect the myocardium and lung of mice.This study will providea method for the construction of infectious clones of othercircular DNA virusesand lay afoundation for the study of the pathogenic mechanism of PCV3. Materialsand Methods {#S2} ===================== Cells and Cultures {#S2.SS1} ------------------The3D4/21 cellline (iCell Bioscience Inc., Shanghai,China) was cultivated in Dulbecco's minimum essentialmedium (MEM,Gibco) supplemented by 10% fetal bovine serum at 37°C in a humidified 5% CO~2~ incubator.Thecloningand construction of recombinant expression plasmids was carried out in *E. coli*strain DH5α cells (Takara Bio, Dalian, China). The prokaryotic expressionvector pET-32a (+)and*E. coli* BL21 (DE3) cellswere harvested from the stocks of our laboratory. The SP2/0cellswere also acquired from the stocks. Viral Gene and Primer Synthesis {#S2.SS2} ------------------------------- The PCV3 gene sequence (GenBank accession number: [MH107162.1](MH107162.1)) was taken to forma loop on Snap-Gene software, and the positionoftheunique restriction site was adopted toopen the sequence, termed as the rearranged linear PCV3 genesequence. Since the PCV3 is circular, the cyclization of the original linear DNA sequence was considered, the*Hin*dIII restriction site was recruited asthe reopening site. When the opening processwas completed, the *Hin*dIII restrictionsite was added to the end of the linear sequence. The newly rearranged sequence wassynthesizedby Sangon(Shanghai, China). [Figure 1](#F1){ref-type="fig"}illustrates thegene pattern of the novel PCV3and presents the design pattern of thecircular PCV3 amplificationprimerand detection primer. Primers listed in [Table 1](#T1){ref-type="table"} weredesigned in accordance withthe rearrangedPCV3gene sequence and PCV3 Cap gene sequence; the designed primers were synthesized by Sangon (Shanghai, China). ###### Primers used for the construction and identification of the recombinant virus. Primer Sequence --------------------------------------------------------------------- PCV3 M1: 5′-CCC[AAGCTT]{.ul}GTGCGGATGCGGCTGCGCG-3′; PCV3 L2:5′- CCC[AAGCTT]{.ul}CCCGCGTTTTCCCACAACC-3′; PCV3 L3: 5′- GGTTGTGGGAAAACGCGGG-3′; PCV3M2: 5′-CGCGCAGCCGCATCCGCACAAGCTT-3′; PCV3 Cap *Bam*HI: 5′-CGC[GGATCC]{.ul}ATGAGACACAGAGCTATATTCAGAA-3′; PCV3Cap *Hin*dIII: 5′-CCC[AAGCTT]{.ul}TTAGAGAACGGACTTGTAACGAATC-3′ The underlined sequences represent restriction enzyme sites introduced. PCV3M1, PCV3 L2: PCV3 complete genome amplificationprimers; PCV3 L3 is areverse complementary sequence of PCV3 L2;PCV3 M2 is the nucleotide sequence nexttothe primer binding site of PCV3 L2; PCV3 Cap BamHI, PCV3 Cap HindIII: The design of the primeris thereverse complement of the negative chain of PCV3 withthe Cap gene sequence, and specificprimers were then designed according tothe reverse complementary DNAsequence. {#F1} Amplification and Cyclization of thePCV3 Genome {#S2.SS3} ------------------------------------------------ Toamplify the newly synthesized rearrangedPCV3 gene, PCV3 M1, PCV3 L2 primers, andKFX-101 high fidelityenzymes (TOYOBO, Japan) were adopted.The finalvolume of reaction fluid reached 100 μL. The PCR cycling conditions included: pre-denaturation at 94°C for 2min, 30 cycles at 98°C for 10 s, 58°C for 30 s, and 68°C for 2 min, as well as the final extension for 10 min at 68°C. PCR productswere preserved at 16°C for subsequent experiments. The amplified PCR products of rearranged PCV3gene were purified following the instructions of the DNA Purification Kit (TakaraBio, Dalian, China). Subsequently, the purified PCR product was digested by *Hin*dIII restriction endonuclease (NEB,Beijing, China) and then incubated in a water bath at 37°Cfor5 h. Then, the digested products of *Hin*dIII restriction endonuclease were purified following the manufacturer's instructions (TakaraBio, Dalian, China). Next, the purified DNA fragments were connected based on T4 DNA ligase instructions (NEB, Beijing, China) at 16°C for 12 h. Last, the cyclized PCV3 DNA was harvested. Transfectionof Cyclized PCV3 DNA {#S2.SS4} --------------------------------- Thecyclized PCV3 DNA was transfected into 3D4/21 cells (60% confluency) | Introduction _{#S1}_ ============ Porcine circovirus has long harmed _the_ _sound_ development of pig _husbandry,_ _and_ especially _the_ PCV3 that has appeared _in_ recent years has caused tremendous _economic_ losses for _pig_ husbandry ([@B17]; [@B21]). Current _research_ _indicates_ that PCV is mainly divided into three genotypes ([@B8]). PCV1 is generally considered non-pathogenic ([@B27]). PCV2 is widely prevalent _worldwide,_ _previous_ studies _have_ shown _that_ PCV2 is the main _pathogenesis_ of post-weaning _multisystemic_ wasting _syndrome_ (PMWS) and swine _dermatitis_ and nephrotic syndrome (PDNS) _([@B1];_ [@B30]). However, in recent years, some researchers have detected PCV3 from PDNS piglets _([@B22];_ [@B13]). Studies have shown that PCV3 and PCV2 are mixed infections _and_ _have_ _become_ popular in _many_ countries. It _has_ been reported _that_ PCV3 may _cause_ reproduction disorder in sows and PDNS in adult _pigs_ ([@B22]). _Similar_ _to_ PCV2, _PCV3_ is _often_ mixed with PRRSV, PCV2 ([@B2]). However, unlike PCV2, there _are_ currently _commercial_ vaccines to _prevent_ PCV2 infection ([@B23]), and there is still a shortage _of_ vaccines _and_ _related_ _drugs_ _to_ _prevent_ PCV3. Obtaining standard virus strains is the _basis_ for the development _of_ PCV3 vaccines and related biological products. However, no experimental report _on_ the _successful_ _isolation_ of PCV3 has been reported. With _the_ development _of_ genetic engineering _technology,_ a variety of _virus_ _strains_ have been constructed through reverse genetics ([@B34]; [@B10]). With _the_ continuous analysis _of_ the genome structure and function of PCV2 and PCV3, _researchers_ have obtained infectious clones by constructing eukaryotic expression vectors of PCV2 and PCV3 ([@B28]; _[@B11])._ _According_ to _the_ _genome_ structure of _PCV,_ the porcine circovirus genome is a single-stranded negative-stranded DNA, the virions _are_ only _14--17_ nm, and there _is_ no capsule on _the_ surface of the _virus_ capsid ([@B18]; [@B32]). The _size_ of the PCV1 and _PCV2_ _genome_ research surface is 1767--1768 _bp_ _and_ contains 11 open _reading_ frames (ORFs), of which _ORF1_ is a virus replication--related protein (Rep) and _is_ a _necessary_ element of virus replication ([@B3]). ORF2 encodes the viral capsid protein (Cap) ([@B20]) and is _commonly_ _used_ in the _study_ _of_ subunit vaccines _and_ diagnostic reagents ([@B16]). Unlike the genomes of PCV1 and PCV2, the full _length_ of the PCV3 _genome_ is 2000 bp _([@B24])._ _Some_ _researchers_ have predicted the PCV3 _genomic_ DNA, _and_ currently they _have_ _predicted_ a total of three ORFs _([@B22])._ One Rep protein _composed_ of _297_ amino acids _was_ encoded by _ORF1_ ([@B33]). Besides, _another_ Cap protein _covering_ 214 amino acids _by_ ORF2 was replicated in _the_ _opposite_ direction and _a_ protein with _an_ unknown function and containing 231 amino acids _by_ ORF3 ([@B5]). Whether PCV3 has other open _reading_ _frames_ and their functions still needs further _study._ The _above_ research is to construct infectious clones under the premise of grasping the genome structure _of_ _the_ virus. However, for _some_ newly discovered circular _DNA_ _viruses,_ the genome structure and function are _undefined_ yet, so it is hard to construct _infectious_ clones with the application of the eukaryotic expression vector. _This_ study intends to _use_ the biological characteristics of PCV3 circular _DNA_ to _construct_ PCV3 infectious clones and construct a Kunming mouse infection model without the help of exogenous expression _vectors._ The results showed that _the_ _PCV3_ strain can infect the myocardium _and_ lung of mice. _This_ study will provide a _method_ _for_ the _construction_ of infectious clones of other circular DNA viruses _and_ _lay_ a foundation for the study of the pathogenic mechanism of _PCV3._ Materials and _Methods_ _{#S2}_ ===================== Cells and _Cultures_ {#S2.SS1} ------------------ The 3D4/21 cell line _(iCell_ Bioscience Inc., Shanghai, _China)_ was _cultivated_ _in_ _Dulbecco's_ _minimum_ essential _medium_ (MEM, Gibco) supplemented by 10% fetal bovine serum _at_ _37°C_ in a humidified _5%_ CO~2~ _incubator._ The _cloning_ and construction of recombinant expression plasmids was _carried_ out in *E. _coli*_ strain DH5α _cells_ (Takara Bio, Dalian, China). The prokaryotic expression vector pET-32a (+) _and_ *E. _coli*_ _BL21_ _(DE3)_ cells were harvested from the _stocks_ of our laboratory. The _SP2/0_ cells were also acquired _from_ the stocks. Viral Gene and Primer Synthesis {#S2.SS2} _-------------------------------_ The PCV3 gene sequence _(GenBank_ accession number: [MH107162.1](MH107162.1)) was taken to form a loop _on_ Snap-Gene software, and the position _of_ the unique restriction site was _adopted_ to _open_ the _sequence,_ termed as the rearranged linear _PCV3_ _gene_ sequence. Since the PCV3 is circular, the cyclization of the original linear DNA sequence was considered, the *Hin*dIII restriction site was recruited as the reopening site. _When_ the opening process was completed, the *Hin*dIII restriction site was added to the end of the linear sequence. The newly rearranged _sequence_ was synthesized by Sangon (Shanghai, _China)._ _[Figure_ _1](#F1){ref-type="fig"}_ illustrates _the_ gene pattern of _the_ novel PCV3 _and_ presents the design pattern of _the_ circular PCV3 amplification primer _and_ detection primer. Primers listed in _[Table_ 1](#T1){ref-type="table"} were designed in accordance with the rearranged PCV3 gene sequence _and_ PCV3 Cap gene sequence; the _designed_ _primers_ _were_ synthesized _by_ _Sangon_ (Shanghai, _China)._ _######_ _Primers_ used for the construction and identification of the _recombinant_ virus. Primer Sequence _---------------------------------------------------------------------_ PCV3 M1: 5′-CCC[AAGCTT]{.ul}GTGCGGATGCGGCTGCGCG-3′; PCV3 L2: 5′- CCC[AAGCTT]{.ul}CCCGCGTTTTCCCACAACC-3′; PCV3 _L3:_ 5′- GGTTGTGGGAAAACGCGGG-3′; _PCV3_ M2: 5′-CGCGCAGCCGCATCCGCACAAGCTT-3′; PCV3 Cap *Bam*HI: 5′-CGC[GGATCC]{.ul}ATGAGACACAGAGCTATATTCAGAA-3′; PCV3 Cap *Hin*dIII: 5′-CCC[AAGCTT]{.ul}TTAGAGAACGGACTTGTAACGAATC-3′ The underlined _sequences_ represent restriction enzyme sites _introduced._ PCV3 M1, PCV3 _L2:_ _PCV3_ complete genome amplification primers; _PCV3_ L3 is a reverse complementary sequence of _PCV3_ L2; PCV3 M2 _is_ the nucleotide sequence next to the primer binding site of PCV3 L2; PCV3 Cap BamHI, _PCV3_ Cap HindIII: The design _of_ the primer _is_ the reverse complement of the negative chain of _PCV3_ with _the_ Cap gene _sequence,_ and specific primers were then designed according to the reverse complementary DNA sequence. {#F1}_ Amplification and _Cyclization_ of the PCV3 _Genome_ _{#S2.SS3}_ ------------------------------------------------ To _amplify_ the newly synthesized rearranged PCV3 gene, PCV3 M1, PCV3 _L2_ primers, _and_ KFX-101 high fidelity enzymes (TOYOBO, Japan) were adopted. The final volume of _reaction_ fluid reached 100 _μL._ The PCR cycling conditions _included:_ pre-denaturation at 94°C for 2 min, _30_ cycles at _98°C_ for 10 s, 58°C for 30 s, and 68°C _for_ _2_ min, _as_ well as the _final_ extension for 10 min at 68°C. PCR products were preserved at 16°C _for_ subsequent experiments. The amplified PCR products of rearranged PCV3 _gene_ were purified following the instructions of _the_ DNA _Purification_ Kit (Takara _Bio,_ _Dalian,_ China). _Subsequently,_ the purified PCR product was _digested_ by _*Hin*dIII_ restriction endonuclease (NEB, Beijing, China) and then _incubated_ in a water _bath_ at 37°C for 5 h. _Then,_ the digested products _of_ *Hin*dIII restriction endonuclease _were_ purified following _the_ _manufacturer's_ _instructions_ (Takara Bio, _Dalian,_ China). _Next,_ the _purified_ DNA fragments were connected _based_ on T4 DNA ligase instructions (NEB, Beijing, China) at 16°C for 12 h. Last, the _cyclized_ PCV3 _DNA_ was _harvested._ Transfection of Cyclized PCV3 DNA {#S2.SS4} --------------------------------- The cyclized _PCV3_ DNA was transfected _into_ 3D4/21 _cells_ _(60%_ _confluency)_ |
Background {#Sec1}
==========
The development of resistance to cytotoxic agents represents a major concern in cancer chemotherapy. Multi-drug resistance (MDR) is associated with over-expression of transmembrane glycoprotein (P-gp) which functions as a drug efflux pump, reducing the intracellular levels of cytotoxic drugs (Juranka et al. [@CR29]). P-gp belongs to the ATP-binding cassette (ABC) transport proteins, which also include the multi-drug resistance associated protein 1 (MRP1) (Shen et al. [@CR63]; Biedler and Spengler [@CR4]; Efferth et al. [@CR15]), or the breast cancer resistance protein (BCRP/ABCG2) (Shen et al. [@CR63]). The oncogene epidermal growth factor receptor (EGFR) (Biedler and Spengler [@CR4]; Efferth et al. [@CR15], [@CR16]) and the deletions or inactivation of tumor suppressor gene p53 (el-Deiry [@CR17]) have also been involved in MDR mechanism of cancer cells. Overcoming this resistance requires a permanent search of new antineoplastic agents. In the past, natural products from plant kingdom have revealed a high potential as cytotoxic drug reservoir (Kuete and Efferth [@CR33]). According to the World Health Organization, about 80 % of the population of developing countries relies on traditional medicines, mostly plant drugs, for their primary health care needs (FAO [@CR20]). It has also been reported that modern pharmacopoeia still contain at least 25 % drugs derived from plants and many others which are synthetic analogues (FAO [@CR20]). Therefore, fighting cancers with botanicals represents a very promising alternative, especially regarding the diversity of plant's secondary metabolites. African flora has previously been found to be very fruitful in the search of antiproliferative molecules. Many compounds including xanthones: 8-hydroxycudraxanthone G, morusignin I, cudraxanthone I (Kuete et al. [@CR38]), and xanthone V1 (Kuete et al. [@CR35]), benzophenones: guttiferone E and isogarcinol (Kuete et al. [@CR39]), quinone: 2-acetylfuro-1,4-naphthoquinone (Kuete et al. [@CR35]), flavonoids: 4-hydroxylonchocarpin, 6,8-diprenyleriodictyol (Kuete et al. [@CR36]), 2′,4′-dihydroxy-3′,6′-dimethoxychalcone and 4′-hydroxy-2′,6′-dimethoxychalcone (Kuete et al. [@CR41]; Dzoyem et al. [@CR13]) and alkaloids: isotetrandrine (Kuete et al. [@CR44]) and montrofoline (Kuete et al. [@CR45]) displayed good antiproliferative effects against various cancer cell lines. In a collaborative research programme between the Council for Scientific and Industrial Research (CSIR) in South Africa and the National Cancer Institute (NCI) in the USA, several South African plant extracts exhibited anticancer activity against a panel of three human cell lines (breast MCF7, renal TK10 and melanoma UACC62) (Fouche et al. [@CR21], [@CR22]). African medicinal plants such as *Fagara heitzii* (Dzoyem et al. [@CR14]), *Echinops giganteus*, *Xylopia aethiopica*, *Piper capense*, *Imperata cylindrica* (Kuete et al. [@CR37]), *Beilschmiedia acuta*, *Clausena anisata* (Kuete et al. [@CR40]) also displayed good cytotoxicity towards drug-sensitive and drug-resistant cancer cell lines. In our ongoing search of anticancer products from African medicinal flora, we designed the present study to investigate the cytotoxicity of 11 plants traditionally used to manage cancer or disease states bearing relevance to cancer or cancer-like symptoms, such as immune and skin disorders, inflammatory, infectious, parasitic and viral diseases (Kuete et al. [@CR44]). The study was extended to the evaluation of the ability of the three most active extracts from two medicinal plants, *Annona muricata* Lin. (Annonaceae) and *Passiflora edulis* Sims (Passifloraceae) to alter the cell cycle distribution, caspases activity, mitochondrial membrane potential (MMP) and to increase the production of reactive oxygen species (ROS) in leukemia CCRF--CEM cells.
Methods {#Sec2}
=======
Plant material and extraction {#Sec3}
-----------------------------
All medicinal plants tested are traditionally used in the management of cancer or disease states with symptoms related to cancer. Plants were collected in different regions of Cameroon in January 2012. They included *Pachypodanthium staudtii*, *Alchornea floribunda*, *Annona muricata*, *Canarium schweinfurthii*, *Hibiscus esculentus*, *Colocasia esculenta*, *Moringa oleifera*, *Triumphetta pentandra*, *Xanthosoma mafaffa*, *Euphorbia prostata* and *Passiflora edulis*. The plant parts investigated are shown in Table [1](#Tab1){ref-type="table"}. The plants were identified at the National Herbarium (Yaoundé, Cameroon), where voucher specimens were deposited under the reference numbers shown in Table [1](#Tab1){ref-type="table"}. The air-dried and powdered plant material (50 g) was soaked in methanol (200 mL) for 48 h, at room temperature. The methanol extract was concentrated in vacuum under reduced pressure at 68 °C to give the crude extract. This extract was completely dried at room temperature, then conserved at 4 °C until further use.Table 1General information and reports on evidence of biological activities and chemistry of the studied plantsSpecies (family); voucher number^a^Traditional usesParts used (%yield)^b^Bioactive or potentially bioactive componentsBioactivity of crude extract*Alchornea floribunda* Müll. Arg. (Euphorbiaceae)\
4595/HNCTreatment of [bacterial]{.ul} and parasitic infections, painful urination in children (Adjanohoun et al. [@CR2]; Jiofack et al. [@CR28]), urinary, respiratory and intestinal problems, pains in the heart, diarrhoea, ovarian problems, stomach ailments and intestinal disorders (Siwe Noundou et al. [@CR66]), [trypanosomiasis]{.ul}, urinary, respiratory and intestinal disorders (Musuyu Muganza et al. [@CR49]; Mesia et al. [@CR47]), [inflammation]{.ul} (Okoye et al. [@CR58])Bark (18.91 %) and leaves (4.56 %)Eugenol, cadinol, nanocosaine, ethyl iso-allocholate, 3-acetoxy-7,8-epoxylanostan-1-ol (Okoye et al. [@CR58])Antibacterial activities of crude against *Bc*, *Ef*, *Ec*, *Sa*, *Kp*, *Mc*, *Pm*, *Ss* (Siwe Noundou et al. [@CR66]); topical anti-inflammatory effects (Okoye et al. [@CR58])*Annona muricata* Lin. (Annonaceae); 18681/SRF/CamTreatment of wounds and insomnia; [antiparasitic]{.ul}, insecticidal (Rajeswari [@CR60])Leaves (4.50 %), seeds (9.15 %), pericarp (5.17 %)Epomuricenins-A and B, montecristin, cohibins-A and B, muridienins-1 and 2, muridienins-3 and 4, muricadienin and chatenaytrienins-1, 2 and 3 and sabadelin, murihexol, donhexocin, annonacin A and Annonacin B (Rajeswari [@CR60])Antimicrobial activities of aqueous, ethanol and methanol extracts against *Sa*, *Vc*, *Ec*, *Se*, *Lv* and *On* (Vieira et al. [@CR68]) and *Pv*, *Sp*, *Bs*, *St*, *Kp*, *Ea* (Rajeswari [@CR60]), *Lb*, *Lp*, *Hv* (Rajeswari [@CR60]), *Ec*, *Ea*, *Kp*, *Ps* (Dzotam et al. [@CR11])*Canarium schweinfurthii* Engl. (Burceraceae); 16929/SRF/CamTreatment of [malaria]{.ul}, constipation, diarrhea, rheumatism and [sexually transmitted diseases]{.ul} (Koudou et al. [@CR32])Fruits (0.78 %)Saponins, cardiac glycosides, tannins, flavonoids and steroids (Ngbede et al. [@CR52])Antimicrobial activities of EO against *Bc*, *Ef*, *Ec*, *Li*, *Se*, *Sd*, *Sa*, *Pm*, *Sc* | background { # sec1 } = = = = = = = = = = the development of resistance to cytotoxic agents represents a major concern in cancer biology. multi - drug resistance ( mdr ) is associated with over - expression of transmembrane glycoprotein ( p - gp ) which functions as a drug efflux pump, reducing the intracellular levels of cytotoxic drugs ( juranka et al. [ @ cr29 ] ). p - gp belongs to the atp - binding cassette ( abc ) transport proteins, which also include the multi - drug resistance associated protein 1 ( mrp1 ) ( zhang et al. [ @ cr63 ] ; biedler and spengler [ @ cr4 ] ; efferth et al. [ @ cr15 ] ), or the breast cancer resistance protein ( bcrp / m3 ) ( shen et al. [ @ cr63 ] ). the oncogene epidermal growth factor receptor ( egfr ) ( biedler and spengler [ @ cr4 ] ; efferth et al. [ @ cr15 ], [ @ cr16 ] ) and tumor deletions or modifications of tumor suppressor gene genes ( x - deiry [ @ cr17 ] ) have also been involved in mdr mechanism of cancer cells. overcoming this resistance requires a permanent search of new antineoplastic agents. in the past, natural products from plant kingdom have revealed a high potential as cytotoxic drug reservoir ( kuete and efferth [ @ cr33 ] ). according to the world health organization, about 80 % of the population of developing countries relies on traditional medicines, mostly plant drugs, for their primary health care needs ( fao [ @ 29 ] ). it has also been reported that modern pharmacopoeia still contain at least 25 % drugs derived from plants and many others already are synthetic analogues ( fao [ @ cr20 ] ). therefore, fighting cancers with botanicals represents a very promising alternative, especially regarding the diversity of plant ' s secondary metabolites. african flora has previously been found to be very fruitful in continuous search of antiproliferative molecules. many compounds including xanthones : 8 - hydroxycudraxanthone g, morusignin i, cudraxanthone i ( kuete et al. [ @ cr38 ] ), and xanthone v1 ( kuete et al. [ @ cr35 ] ), benzophenones : guttiferone e and isogarcinol ( kuete et al. [ @ cr39 ] ), quinone : 2 - acetylfuro - 1, 4 - naphthoquinone ( kuete et al. [ @ cr35 ] ), flavonoids : 4 - hydroxylonchocarpin, 6, 8 - diprenyleriodictyol ( kuete et al. [ @ cr36 ] ), 2 ′, 4 ′ - dihydroxy - 3 ′, 6 ′ - dimethoxychalcone and 4 ′ - hydroxy - 2 ′, 6 ′ - dimethoxychalcone ( kuete et al. [ @ cr41 ] ; dzoyem et al. [ @ cr13 ] ) and alkaloids : isotetrandrine ( kuete et al. [ @ cr44 ] ) and montrofoline ( kuete et al. [ @ cr45 ] ) displayed good antiproliferative effects against various cancer cell lines. in a collaborative research programme between the council for scientific and industrial research ( csir ) in south africa and the national cancer institute ( nci ) in the usa, several south african plant extracts exhibited anticancer activity against a panel of three human cell lines ( breast mcf7, renal tk10 and melanoma uacc62 ) ( fouche et al. [ @ cr21 ], [ @ cr22 ] ). african medicinal plants such as * fagara heitzii * ( dzoyem et al. [ @ cr14 ] ), * echinops giganteus *, * xylopia aethiopica *, * piper capense *, * imperata cylindrica * ( kuete et al. [ @ cr37 ] ), * beilschmiedia acuta *, * clausena anisata * ( kuete et al. [ @ cr40 ] ) also displayed good cytotoxicity towards drug - sensitive and drug - resistant cancer cell lines. in our ongoing search of anticancer products from african medicinal flora, we designed the present study to investigate the cytotoxicity of 11 plants traditionally used to manage cancer or disease states bearing relevance to cancer or cancer - like symptoms, such as immune and skin disorders, inflammatory, infectious, parasitic and viral diseases ( kuete et al. [ @ cr44 ] ). the study was extended to the evaluation of the ability of the three most active extracts from two medicinal plants, * annona muricata * lin. ( annonaceae ) and * passiflora edulis * sims ( passifloraceae ) to alter the cell cycle distribution, caspases activity, mitochondrial membrane potential ( mmp ) and to increase the production of reactive oxygen species ( ros ) in leukemia ccrf - - cem cells. methods { # sec2 } = = = = = = = plant material and extraction { # sec3 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - all medicinal plants tested are traditionally used in the management of cancer or disease states with symptoms related to cancer. plants were collected in different regions of cameroon in january 2012. they included * pachypodanthium staudtii *, * alchornea floribunda *, * annona muricata *, * canarium schweinfurthii *, * hibiscus esculentus *, * colocasia esculenta *, * moringa oleifera *, * triumphetta pentandra *, * xanthosoma mafaffa *, * euphorbia prostata * and * passiflora edulis *. the plant parts investigated are shown in table [ 1 ] ( # tab1 ) { ref - type = " table " }. the plants were identified at the national herbarium ( yaounde, cameroon ), where voucher specimens were deposited under the reference numbers shown in table [ 1 ] ( # tab1 ) { ref - type = " table " }. the air - dried and powdered plant material ( 50 g ) was soaked in methanol ( 200 ml ) for 48 h, at room temperature. the methanol extract was concentrated in vacuum under reduced pressure at 68 °c to give the crude extract. this extract was completely dried at room temperature, then conserved at 4 °c until further use. table 1general information and reports on evidence of biological activities and chemistry of the studied plantsspecies ( family ) ; voucher number ^ a ^ traditional usesparts used ( % yield ) ^ b ^ bioactive or potentially bioactive componentsbioactivity of crude extract * alchornea floribunda * mull. arg. ( euphorbiaceae ) \ 4595 / hnctreatment of [ bacterial ] {. ul } and parasitic infections, painful urination in children ( adjanohoun et al. [ @ cr2 ] ; jiofack et al. [ @ cr28 ] ), urinary, respiratory and intestinal problems, pains in the heart, diarrhoea, ovarian problems, stomach ailments and intestinal disorders ( siwe noundou et al. [ @ cr66 ] ), [ trypanosomiasis ] {. ul }, urinary, respiratory and intestinal disorders ( musuyu muganza et al. [ @ cr49 ] ; mesia et al. [ @ cr47 ] ), [ inflammation ] {. ul } ( okoye et al. [ @ cr58 ] ) bark ( 18. 91 % ) and leaves ( 4. 56 % ) eugenol, cadinol, nanocosaine, ethyl iso - allocholate, 3 - acetoxy - 7, 8 - epoxylanostan - 1 - ol ( okoye et al. [ @ cr58 ] ) antibacterial activities of crude against * bc *, * ef *, * ec *, * sa *, * kp *, * mc *, * pm *, * ss * ( siwe noundou et al. [ @ cr66 ] ) ; topical anti - inflammatory effects ( okoye et al. [ @ cr58 ] ) * annona muricata * lin. ( annonaceae ) ; 18681 / srf / camtreatment of wounds and insomnia ; [ antiparasitic ] {. ul }, insecticidal ( rajeswari [ @ cr60 ] ) leaves ( 4. 50 % ), seeds ( 9. 15 % ), pericarp ( 5. 17 % ) epomuricenins - a and b, montecristin, cohibins - a and b, muridienins - 1 and 2, muridienins - 3 and 4, muricadienin and chatenaytrienins - 1, 2 and 3 and sabadelin, murihexol, donhexocin, annonacin a and annonacin b ( rajeswari [ @ cr60 ] ) antimicrobial activities of aqueous, ethanol and methanol extracts against * sa *, * vc *, * ec *, * se *, * lv * and * on * ( vieira et al. [ @ cr68 ] ) and * pv *, * sp *, * bs *, * st *, * kp *, * ea * ( rajeswari [ @ cr60 ] ), * lb *, * lp *, * hv * ( rajeswari [ @ cr60 ] ), * ec *, * ea *, * kp *, * ps * ( dzotam et al. [ @ cr11 ] ) * canarium schweinfurthii * engl. ( burceraceae ) ; 16929 / srf / camtreatment of [ malaria ] {. ul }, constipation, diarrhea, rheumatism and [ sexually transmitted diseases ] {. ul } ( koudou et al. [ @ cr32 ] ) fruits ( 0. 78 % ) saponins, cardiac glycosides, tannins, flavonoids and steroids ( ngbede et al. [ @ cr52 ] ) antimicrobial activities of eo against * bc *, * ef *, * ec *, * li *, * se *, * sd *, * sa *, * pm *, * sc * | Background {# Sec1} = = = = = = = = = = The development of resistance to cytotoxic agents represents a major concern in cancer chemotherapy. Multi - drug resistance (MDR) is associated with over - expression of transmembrane glycoprotein (P - gp) which functions as a drug efflux pump, reducing the intracellular levels of cytotoxic drugs (Juranka et al. [@ CR29] ). P - gp belongs to the ATP - binding cassette (ABC) transport proteins, which also include the multi - drug resistance associated protein 1 (MRP1) (Shen et al. [@ CR63 ]; Biedler and S(eng:er [@ CR4 ]; Efferth et al. [@ CR15] ), or the breast cancer resistance protein (BCRP / ABCG2) (Shen et al. [@ CR63] ). The oncogene epidermal growth factor receptor (EGFR) (Biedler and Spengler [@ CR4 ]; Efferth et al. [@ CR15 ], [@ CR16] ) and the deletions or inactivation of tumor suppressor gene p53 (el - Deiry [@ CR17] ) have also been involved in MDR mechanism of cancer cells. Overcoming tBiC resistance requires a permanent search of new antineoplastic agents. In the past, natural products from plant kingdom have revealed a high potential as cytotoxic drug reservoir (Kuete and Efferth [@ CR33] ). According to the World Health Organization, about 80% of the population of developing countries relies on traditional medicines, mostly plant drugs, for their primary health care needs (FAO [@ CR20] ). It has also been reported that modern pharmacopoeia still contain at least 25% drugs derived from plants and many others which are synthetic analogues (FAO [@ CR20] ). Therefore, fighting cancers with botanicals represents a very promising alternative, especially regarding the diversity of plant ' s secondary metab(liteE. African flora has previously been found to be very fruitful in the search of antiproliferative molecules. Many compounds including xanthones: 8 - hydroxycudraxanthone G, morusignin I, cudraxanthone I (Kuete et al. [@ CR38] ), and xanthone V1 (Kuete et al. [@ CR35] ), benzophenones: guttiferone E and isogarcinol (Kuete et al. [@ CR39] ), quinone: 2 - acetylfuro - 1, 4 - naphthoquinone (Kuete et al. [@ CR35] ), flavonoids: 4 - hydroxylonchocarpin, 6, 8 - diprenyleriodictyol (Kuete et al. [@ CR36] ), 2 ′, 4 ′ - dihydroxy - 3 ′, 6 ′ - dimethoxychalcone and 4 ′ - hydroxy - 2 ′, 6 ′ - dimethoxychalcone (Kuete et al. [@ CR41 ]; Dzoyem et al. [@ CR13] ) and alkaloids: isotetrandrine (Kuete et al. [@ CR44] ) and montrofoline (Kuete et al. [@ CR45] ) displQyew good antiproliferative effects against various cancer cell lines. In a collaborative research programme between the Council for Scientific and Industrial Research (CSIR) in South Africa and the National Cancer Institute (NCI) in the USA, several South African plant extracts exhibited anticancer activity against a panel of three human cell lines (breast MCF7, r#Gal TK10 and melanoma UACC62) (Fouche et al. [@ CR21 ], [@ CR22] ). African medicinal plants such as * Fagara heitzii * (Dzoyem et al. [@ CR14] ), * Echinops giganteus *, * Xylopia aethiopica *, * Piper capense *, * Imperata cylindrica * (Kuete et al. [@ CR37] ), * Beilschmiedia acuta *, * Clausena anisata * (Kuete et al. [@ CR40] ) also displayed good cytotoxicity towards drug - sensitive and drug - resistant cancer cell lines. In our ongoing search of anticancer products from African medicinal flora, we designed the present study to investigate the cytotoxicity of 11 plants traditionally used to manage cancer or disease states bearing relevance to cancer or cancer - like symptoms, such as immune and skin disorders, inflammatory, infectious, parasitic and viral diseases (Kuete et al. [@ CR44] ). The stuST was extended to the evaluation of the ability of the three most active extracts from two medicinal plants, * Annona m6gicata * Lin. (Annonaceae) and * Passiflora edulis * Sims (Passifloraceae) to alter the cell cycle distribution, caspases activity, mitochondrial membrane potential (MMP) and to increase the production of reactive oxygen species (ROS) in leukemia CCRF - - CEM cells. Methods {# Sec2} = = = = = = = Plant material and extraction {# Sec3} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - All medicinal plants tested are traditionally used in the management of cancer or disease states with symptoms related to cancer. Plants were collected in different regions of Cameroon in January 2012. They included * Pachypodanthium staudtii *, * Alchornea floribunda *, * Annona muricata *, * Canarium schweinfurthii *, * Hibiscus esculentus *, * Colocasia esculenta *, * Moringa oleifera *, * Triumphetta pentandra *, * Xanthosoma mafaffa *, * Euphorbia prostata * and * Passiflora edulis *. The piagt parts investigated are shown in Table [1] (# Tab1) {ref - type = " table " }. The plants were identified at the National Herbarium (Yaoundé, Cameroon ), where voucher specimens were deposited under the reference numbers shown in Table [1] (# Tab1) {ref - type = " table " }. The air - dried and powdered plant material (50 g) was XoakeR in methanol (200 mL) for 48 h, at room temperature. The methanol extract was concentrated in vacuum under reduced pressure at 68 ° C to give the crude extract. This extract was completely dried at room temperature, then conserved at 4 ° C until further use. Table 1General information and reports on evidence of biological activities and chemistry of the studied plantsSpecies (family ); voucher number ^ a ^ Traditional usesParts used (% yield) ^ b ^ Bioactive or potentially bioactive componentsBioactivity of crude extract * Alchornea floribunda * Müll. Arg. (Euphorbiaceae) \ 4595 / HNCTreatment of [bacterial] {. ul} and parasitic infections, painful urination in VhildDen (Adjanohoun et al. [@ CR2 ]; Jiofack et al. [@ CR28] ), urinary, respiratory and intestinal problems, pains in the heart, diarrhoea, ovarian problems, stomach ailments and intestinal disorders (Siwe Noundou et al. [@ CR66] ), [trypanosomiasis] {. ul }, urinary, respiratory and intestinal disorders (Musuyu Muganza et al. [@ CR49 ]; Mesia et al. [@ CR47] ), [inflammation] {. ul} (Okoye et al. [@ CR58] ) Bark (18. 91%) and leaves (4. 56%) Eugenol, cadinol, nanocosaine, ethyl iso - allocholate, 3 - acetoxy - 7, 8 - epoxylanostan - 1 - ol (Okoye et al. [@ CR58] ) Antibacterial activities of crude against * Bc *, * Ef *, * Ec *, * Sa *, * Kp *, * Mc *, * Pm *, * Ss * (Siwe Noundou et al. [@ CR66] ); topical anti - inflammatory effects (Okoye et al. [@ CR58] ) * Annona muricata * Lin. (Annonaceae ); 18681 / SRF / CamTreatment of wounds and insomnia; [antiparasitic] {. ul }, insecticidal (Rajeswari [@ CR60] ) Leaves (4. 50% ), seeds (9. 15% ), pericarp (5. 17%) Epomuricenins - A and B, montecristin, cohibins - A and B, muridienins - 1 and 2, muridienins - 3 and 4, muricadienin and chatenaytrienins - 1, 2 and 3 and sabadelin, murihexol, donhexocin, annonacin A and Annonacin B (Rajeswari [@ CR60] ) Antimicrobial activities of aqueous, ethanol and methanol extracts against * Sa *, * Vc *, * Ec *, * Se *, * Lv * and * On * (Vieira et al. [@ CR68] ) and * Pv *, * Sp *, * Bs *, * St *, * Kp *, * Ea * (Rajeswari [@ CR60] ), * Lb *, * Lp *, * Hv * (Rajeswari [@ CR60] ), * Ec *, * Ea *, * Kp *, * Ps * (Dzotam et al. [@ CR11] ) * Canarium schweinfurthii * Engl. (Burceraceae ); 16929 / SRF / CamTreatment of [malaria] {. ul }, constipation, diarrhea, rheumatism and [sexually transmitted diseases] {. ul} (Koudou et al. [@ CR32] ) Fruits (0. 78%) Saponins, cardiac glycosides, tannins, flavonoids and steroids (Ngbede et al. [@ CR52] ) Antimicrobial activities of EO against * Bc *, * Ef *, * Ec *, * Li *, * Se *, * Sd *, * Sa *, * Pm *, * Sc * | {#Sec1} The development of resistance to cytotoxic agents represents a major concern cancer chemotherapy. Multi-drug resistance (MDR) is associated with over-expression of transmembrane glycoprotein (P-gp) which functions as a drug efflux pump, reducing the intracellular levels of cytotoxic (Juranka et [@CR29]). P-gp belongs to the ATP-binding cassette (ABC) transport proteins, which also include the multi-drug resistance associated protein 1 (MRP1) (Shen et al. Biedler and Spengler [@CR4]; Efferth et al. [@CR15]), or the breast cancer resistance protein (BCRP/ABCG2) (Shen et [@CR63]). The oncogene epidermal growth factor receptor (EGFR) (Biedler and Spengler [@CR4]; et al. [@CR15], [@CR16]) and the or inactivation of tumor gene p53 (el-Deiry [@CR17]) also involved in MDR of cancer cells. Overcoming this resistance requires permanent search of new antineoplastic agents. In the past, natural products from plant kingdom have revealed a high potential as cytotoxic drug reservoir (Kuete and Efferth [@CR33]). According to the World Health Organization, about % of the of developing countries relies on traditional medicines, mostly plant drugs, for their primary health care needs [@CR20]). It has been reported that modern pharmacopoeia still contain at least 25 % drugs derived from plants and many others which are synthetic analogues [@CR20]). Therefore, fighting cancers with botanicals represents a very promising alternative, especially regarding the diversity of plant's secondary metabolites. African flora has previously been to be very fruitful in the search of antiproliferative Many compounds including xanthones: 8-hydroxycudraxanthone G, morusignin I, cudraxanthone I (Kuete et al. [@CR38]), and xanthone V1 (Kuete et benzophenones: guttiferone E and isogarcinol (Kuete et al. quinone: 2-acetylfuro-1,4-naphthoquinone (Kuete et al. flavonoids: 4-hydroxylonchocarpin, 6,8-diprenyleriodictyol (Kuete al. [@CR36]), and 4′-hydroxy-2′,6′-dimethoxychalcone (Kuete et al. [@CR41]; et al. [@CR13]) and alkaloids: isotetrandrine (Kuete et al. and montrofoline (Kuete et al. [@CR45]) displayed good antiproliferative effects against various cell lines. In a research between the Council for Scientific and Industrial Research (CSIR) in South Africa the National Cancer Institute (NCI) the USA, several African plant exhibited anticancer activity against a panel of three human lines (breast renal TK10 melanoma UACC62) (Fouche et al. [@CR21], [@CR22]). medicinal plants such *Fagara heitzii* (Dzoyem et al. [@CR14]), *Echinops giganteus*, *Xylopia *Piper capense*, *Imperata cylindrica* (Kuete et al. [@CR37]), acuta*, *Clausena (Kuete et al. [@CR40]) also displayed good cytotoxicity towards drug-sensitive and drug-resistant cancer cell lines. In our ongoing anticancer products from African medicinal flora, we designed the present study to investigate the of 11 plants traditionally used to manage cancer or disease states bearing relevance to or cancer-like symptoms, such as immune and skin disorders, inflammatory, infectious, and viral diseases (Kuete et al. [@CR44]). The was extended to the evaluation of the ability of the three most active extracts from two plants, *Annona muricata* Lin. (Annonaceae) and *Passiflora edulis* Sims (Passifloraceae) to alter the cell cycle distribution, caspases activity, mitochondrial membrane potential (MMP) and to increase the production of reactive oxygen species (ROS) in leukemia CCRF--CEM cells. Methods {#Sec2} ======= material and extraction {#Sec3} ----------------------------- All medicinal plants tested are traditionally used in the management cancer or disease states with symptoms to cancer. Plants were in different Cameroon in January 2012. They staudtii*, *Alchornea floribunda*, *Annona muricata*, schweinfurthii*, *Hibiscus esculentus*, *Colocasia esculenta*, *Moringa oleifera*, *Triumphetta pentandra*, *Xanthosoma mafaffa*, *Euphorbia prostata* and *Passiflora edulis*. The plant investigated are shown in Table [1](#Tab1){ref-type="table"}. The plants were identified National Herbarium (Yaoundé, Cameroon), where specimens were deposited under the reference numbers shown Table [1](#Tab1){ref-type="table"}. The air-dried and powdered plant material (50 g) was soaked in methanol (200 mL) for 48 h, at room temperature. The methanol extract was concentrated in vacuum under reduced pressure at 68 °C to give the crude extract. This extract completely dried at room then conserved at 4 until further use.Table 1General information and reports on evidence of biological activities and chemistry of the studied plantsSpecies (family); voucher number^a^Traditional usesParts used (%yield)^b^Bioactive or potentially bioactive componentsBioactivity of crude floribunda* Müll. Arg. (Euphorbiaceae)\ of [bacterial]{.ul} parasitic infections, urination in children et al. [@CR2]; Jiofack et al. [@CR28]), urinary, respiratory and intestinal problems, pains in the heart, diarrhoea, ovarian problems, stomach and intestinal disorders (Siwe et al. [@CR66]), [trypanosomiasis]{.ul}, respiratory intestinal disorders (Musuyu Muganza al. [@CR49]; Mesia et al. [@CR47]), [inflammation]{.ul} (Okoye et al. (18.91 %) and leaves (4.56 %)Eugenol, cadinol, nanocosaine, ethyl iso-allocholate, 3-acetoxy-7,8-epoxylanostan-1-ol (Okoye et al. [@CR58])Antibacterial activities of crude against *Bc*, *Ef*, *Ec*, *Kp*, *Mc*, *Pm*, *Ss* (Siwe et al. [@CR66]); topical anti-inflammatory effects (Okoye et al. [@CR58])*Annona muricata* Lin. 18681/SRF/CamTreatment of wounds and insomnia; [antiparasitic]{.ul}, insecticidal [@CR60])Leaves %), seeds (9.15 %), pericarp (5.17 %)Epomuricenins-A and B, cohibins-A and B, muridienins-1 and 2, muridienins-3 and 4, muricadienin and chatenaytrienins-1, 2 and 3 and sabadelin, murihexol, donhexocin, annonacin and Annonacin (Rajeswari activities of aqueous, ethanol and methanol extracts against *Sa*, *Vc*, *Ec*, *Se*, and *On* (Vieira et al. [@CR68]) *Pv*, *Sp*, *Bs*, *St*, *Kp*, *Ea* (Rajeswari [@CR60]), *Lb*, *Lp*, *Hv* (Rajeswari *Ec*, *Ea*, *Kp*, *Ps* (Dzotam et al. [@CR11])*Canarium Engl. 16929/SRF/CamTreatment [malaria]{.ul}, constipation, diarrhea, rheumatism and [sexually transmitted diseases]{.ul} (Koudou et al. [@CR32])Fruits (0.78 %)Saponins, cardiac glycosides, tannins, flavonoids and steroids (Ngbede et al. [@CR52])Antimicrobial activities of EO against *Bc*, *Ef*, *Ec*, *Li*, *Se*, *Sd*, *Sa*, *Pm*, *Sc* | BAckgROuND {#seC1}
==========
tHe deVElOpmEnt Of ReSIStance TO CYTOTOxiC AgENts ReprEsenTS a MajoR cONCErN In CANcEr cHEMoThERAPy. mUltI-DRUG ResisTAnce (Mdr) Is asSoCiATEd With OveR-ExpreSsIOn oF TranSmEmbranE glYCoPRoTEin (p-gp) WHiCh FuNCTiOns aS a DruG EFFluX pUmp, ReduCiNg ThE iNTRACeLlulAR lEVELs Of CyTotoXiC drUGS (jURaNka Et aL. [@CR29]). P-gp BELonGs to The Atp-BiNDING cAsSEtte (aBC) trAnSpOrt pRoteins, wHIcH alsO InCLUdE ThE mULtI-DRUG reSistance ASsOciatEd PROteIN 1 (mrP1) (shEn eT al. [@cr63]; BieDlEr ANd SpenglER [@CR4]; EFFErtH ET aL. [@cR15]), or tHE BrEAST caNCEr rEsIStancE pRoteIN (Bcrp/ABcg2) (SHEN eT aL. [@cR63]). tHE oNCOgeNE epIdErmAl GrowtH FaCTOR RECePtOr (EGFr) (BIEDlEr AND SpenGlER [@cr4]; effeRtH Et aL. [@cR15], [@cr16]) AnD tHE DEleTIOnS OR iNactivAtiOn OF TuMor SUpPrESSOR Gene P53 (EL-deIRY [@cr17]) HaVE Also beeN InVoLvEd iN MdR MECHanISM Of CanCEr cELLs. oVErCoMinG THIS ReSIstaNce requIrES A PerMaNeNT SEarCh of NeW aNtiNeopLAsTIc AGENtS. in THe PASt, nATurAl PrODuCtS FRoM plANt KInGDOm HAve rEvealEd A HiGh pOtENTIAL aS CyTotOxIc DRUG rESErVOir (kueTe AnD EFfErTh [@Cr33]). AcCOrDING TO tHE worLD HeaLTh OrGANizaTIoN, aboUt 80 % Of The POpulaTIoN oF DeVELOPing couNTRIes rELIes on tRaDItioNal mediCInES, MOsTLy PlanT dRUGS, fOr ThEiR PRImaRY HEALTh CaRE nEeDs (faO [@cR20]). IT has ALso been rEPORted ThAT moDerN PhArmAcOPoeIA Still cONTAin At LeAst 25 % DRUGS DErIvED fROM PLanTS anD maNy otHERs wHicH ArE SYnTHEtiC aNaloGUes (FAo [@Cr20]). thereFOrE, fIgHTINg canCERS wiTH BotAnIcALS REPResEnTS A verY promiSING AlTErNatIVE, eSPecIALly rEGaRdINg THe DivErsITy oF pLaNT's SeCOndary MeTABolItes. afrICan FlOra hAs preVIOuSLy BEEN foUNd to BE vERY fRuiTful IN thE seaRch Of aNtIpRolifERATivE MolEcUles. MANY COMpoUNDS INClUdinG xanthoNes: 8-hydroxyCudRaXAnThONE G, MOrUSIgNIn i, cUDraxANthone I (KUeTE et AL. [@Cr38]), AND xANTHONe v1 (KuETE Et aL. [@CR35]), BeNzOPhENOnes: GUTTIferonE E aNd IsOGArCiNoL (kUeTe eT AL. [@Cr39]), qUinOnE: 2-acETYlfurO-1,4-NAPhtHoqUiNonE (KUEte et AL. [@CR35]), flAvoNoidS: 4-hydrOXyloNcHOcaRpIN, 6,8-DIPReNylErIodICTYol (KUEte ET AL. [@cr36]), 2′,4′-diHYDrOXy-3′,6′-DimeTHOXYChaLCONE And 4′-HyDROxY-2′,6′-DIMeTHoxychalconE (kuEte Et AL. [@CR41]; dzoyEm eT al. [@cr13]) ANd ALkALOiDS: IsOteTrAndRINe (kUETe Et aL. [@cR44]) AnD MonTROfOLine (KuEte Et aL. [@Cr45]) DiSpLayEd goOd aNTiPRoLIferaTIVe eFFecTs agAiNsT VARIOUs cANCEr celL LiNeS. in a coLlaBORatIvE REsEArcH PROgrAmmE BeTWeen The cOUnCil foR sCIeNtiFic anD IndUsTRiaL reSeArcH (cSIR) In sOuTH aFRICA AND THE nATIOnaL cAnCer InSTituTE (NCI) In the USA, SEVERAL sOUTH AfRicAN pLANt ExtRActS exHIbITED aNtIcaNCer aCtiViTy AGaINst A Panel OF THrEE HuMaN CeLl lineS (BREAst MCf7, ReNAl tk10 and mElanoma UACC62) (FouChe ET Al. [@cR21], [@CR22]). AFRiCAn mEdiCINal PlaNTS such AS *FAgArA Heitzii* (DzOYEm eT al. [@cr14]), *ECHiNOPS gigaNTEUs*, *xYLOPiA AethIOPICa*, *PIPER capENSe*, *IMpeRaTA cYLINdrIca* (kueTe eT al. [@cR37]), *bEiLsCHmIeDia ACUta*, *ClaUSENA anisAta* (kuETe eT AL. [@cR40]) ALsO DisPlAYed Good CyTOtOxIciTY TowArdS DRug-sENSITive aNd dRUg-rEsIStANt CaNceR CElL LiNes. IN ouR onGOInG SearCH oF ANTicaNCEr PRoDuCTS From AFrIcaN mEdicINal FlorA, WE deSIGned the PreseNt stuDY to iNVesTiGate thE CytoTOXIcitY Of 11 plAnTS trAdITioNALLy uSeD to mANagE cANcEr Or DIsease STates beAriNG ReLevANcE tO canCEr Or cANCeR-liKe SYmpTomS, sUcH As IMMuNe AND SKIN DiSorDERs, InfLammaTOrY, infecTiOUs, PARASITIC anD vIRal DISEAseS (kueTe et aL. [@CR44]). THE STudy wAS eXTENdED to the EvaLUAtion Of The aBIlItY of ThE thREE mOST aCtIVE EXtRacts fROm TWo MediCiNAL plaNtS, *AnNONA MURiCaTA* lIn. (AnnoNACEAe) anD *pAsSifLORA eDuLiS* SImS (pasSIFlOraceAE) to aLTER tHE ceLL Cycle DIsTRIBuTION, caSPaSEs aCtiviTY, MitochonDriAl membRaNe poTEntiAL (MMP) AnD tO INCrEASE THe pRodUCTIOn oF ReACTivE OXygeN sPeciEs (ROS) iN leUkEMIa cCRF--cEm CELlS.
MeTHOds {#sEC2}
=======
PlAnt maTEriaL aND eXTracTIoN {#sec3}
-----------------------------
all mEdICinaL plAntS Tested aRE tRadITiOnaLLY usEd in the maNaGEmENT of canCer or DiSEase stATEs with sYMpToMs RElatED TO cANcer. PLANTS wERe colLECteD iN diFFereNt REgIoNS Of caMErOoN iN jANuARY 2012. THeY INcluDeD *PAcHYpODanTHium STAuDtIi*, *ALChornEa FLOriBUNDA*, *aNNOna murICAta*, *cANaRIUm sChweinFURtHii*, *HiBIScUs esculEnTUs*, *cOloCAsia eSCULEntA*, *MorinGA OLEIfERA*, *TRiumpHEtTA PEntANdRa*, *XAnthoSomA mAFaFFa*, *EUPhoRbIa pROsTATa* and *paSSifLora EDULis*. THE PLANT ParTS iNVesTigAteD aRe ShOWN iN table [1](#TaB1){ReF-type="TablE"}. THe PLAnTS WerE iDEntiFieD at tHe NAtIoNAL hErbaRiUm (yaOundÉ, cAmEROOn), wHeRe vOUcHeR sPecImENS weRE DEPOsited uNDER THe REfeReNce NumbERS ShoWN iN taBLE [1](#TAb1){rEF-TYpe="taBle"}. tHe aiR-drIeD ANd PowDeRed plaNt mAteRiaL (50 G) WAs SOAKEd IN mEtHaNol (200 ml) FOR 48 H, at rooM TEmPERatUrE. THE MEtHaNOL exTrAct WAS cOnCENTRAtED IN VACuUm under reDucEd PrESSuRe aT 68 °C tO gIve tHe cRuDE exTrAct. this extRACt WAs comPlETElY DRied at rOOM tEMpeRaTuRe, thEN coNsERVed At 4 °c UntIl FurTHER usE.Table 1GEnERAL INFORmAtIOn aND rePOrTs On EViDEncE oF bIOLoGICal aCTIvITIES aND cHEmiStry Of ThE studIeD PlaNtssPEciES (FamiLY); vouCHeR nUmBeR^A^TRaDitIoNal UsEspaRTs UseD (%YieLd)^b^bIOACTIve OR poteNtiaLlY biOactIvE COmponenTSBIoACtIvITY oF CRudE EXTRacT*aLcHoRneA fLoRiBUNDA* MÜlL. aRg. (EUpHOrBiaCeae)\
4595/hNcTrEatMeNT OF [baCtEriAL]{.uL} ANd PArASitic INFectIOns, PaiNful uriNaTION IN cHIlDrEN (AdjANohoun eT AL. [@cr2]; jiofack eT aL. [@Cr28]), urinarY, reSpiRatOrY anD INteStINAL pRObLEMs, PAInS IN THE HEArT, diaRRHOeA, ovAriAN PROBLEMs, StOmACH AILmEnTS AND INTESTiNaL diSOrdeRs (sIWE noundOU Et al. [@Cr66]), [TRyPanOSoMIASIS]{.uL}, urInARy, REspIraTOrY AnD iNtestInAL diSOrDeRs (muSuyu MuGANZa Et al. [@cR49]; MEsIA eT AL. [@Cr47]), [InFlaMmatIoN]{.UL} (oKoyE eT Al. [@CR58])bArk (18.91 %) and lEAvES (4.56 %)eUgenoL, cAdINOL, naNOcOSAine, EThyl iSo-alLOCHoLaTE, 3-aCEtOxy-7,8-epoXyLAnosTan-1-oL (oKOye Et AL. [@Cr58])ANTibaCtERiaL ActIvITiES oF CrUdE agAINSt *bc*, *ef*, *EC*, *sA*, *kp*, *mc*, *pm*, *SS* (SIwE noUNDOU eT AL. [@Cr66]); TOpiCal ANtI-INFLaMmaTory EfFeCTS (oKOyE ET aL. [@CR58])*ANnOnA MuRicata* LiN. (AnnOnaCEaE); 18681/Srf/CaMTREAtmeNt oF WoUnDS AND INSOMnIA; [aNTiParaSItic]{.ul}, insECtIcIDAL (RAjESwARI [@Cr60])lEAveS (4.50 %), SEeDs (9.15 %), pERiCaRP (5.17 %)EpomuRICEniNS-A AnD B, montEcrIStIN, COhiBins-a AND B, muridIEniNS-1 ANd 2, MURiDiENinS-3 anD 4, muricaDieNiN And cHatenaytRIeNiNS-1, 2 AND 3 and SaBADElIn, mUrIheXOL, dOnhExoCIn, ANNONACin a AnD annOnACIN b (rAjEswARi [@Cr60])antiMICroBiAL ACtIviTIEs Of aquEOuS, etHANOL aNd MEtHAnOL EXTRactS AgaiNST *sA*, *vc*, *Ec*, *sE*, *lV* and *On* (Vieira Et al. [@cr68]) aNd *pV*, *sP*, *bs*, *st*, *kp*, *ea* (rAJeSwari [@CR60]), *Lb*, *LP*, *hV* (RAjEswari [@cR60]), *eC*, *eA*, *kP*, *pS* (DZoTAM Et al. [@cr11])*caNarium scHwEINFurtHII* engl. (BUrcEraceAE); 16929/Srf/CAMtreatment OF [mALaRIa]{.UL}, COnStipaTION, dIArRhEa, rheUMatIsm AND [SEXUAllY TrAnSMiTtED dISeASes]{.UL} (koudou Et AL. [@CR32])FrUItS (0.78 %)sAPoNIns, cArDiAC gLycoSiDES, taNninS, FLAVoNoidS AnD STerOidS (NgbEdE et Al. [@Cr52])ANtiMICRObiAl aCTiVItiEs of eO aGAInsT *bC*, *EF*, *eC*, *LI*, *sE*, *Sd*, *sA*, *Pm*, *SC* | Background {#Sec1}========== The development of resistanceto cytotoxic agents represents a majorconcern in cancer chemotherapy. Multi-drug resistance(MDR) isassociated with over-expression of transmembrane glycoprotein (P-gp) which functions as a drug efflux pump, reducing the intracellular levelsof cytotoxic drugs (Juranka et al. [@CR29]).P-gp belongs to the ATP-binding cassette (ABC) transport proteins, which also include themulti-drug resistance associatedprotein 1 (MRP1)(Shen et al. [@CR63]; Biedler and Spengler [@CR4]; Efferth etal.[@CR15]),or the breast cancer resistance protein (BCRP/ABCG2) (Shen et al. [@CR63]). The oncogeneepidermal growth factor receptor (EGFR)(Biedler and Spengler [@CR4]; Efferth etal. [@CR15], [@CR16]) and the deletions or inactivation of tumor suppressor gene p53 (el-Deiry [@CR17]) havealso been involved in MDR mechanism ofcancer cells. Overcomingthis resistance requires a permanent search of new antineoplasticagents. In the past, natural products fromplant kingdomhave revealeda high potential as cytotoxic drug reservoir (Kuete and Efferth[@CR33]). According to the World Health Organization, about 80 % of thepopulation ofdeveloping countries relies on traditional medicines, mostly plant drugs, for their primary health care needs (FAO [@CR20]). Ithas also beenreported that modernpharmacopoeia still contain at least 25 % drugs derived fromplants and many others whichare synthetic analogues (FAO [@CR20]). Therefore, fighting cancers with botanicalsrepresentsavery promising alternative, especiallyregarding the diversity of plant's secondarymetabolites. African flora has previously been foundto be very fruitful in the search of antiproliferative molecules. Many compounds including xanthones: 8-hydroxycudraxanthone G, morusignin I, cudraxanthone I (Kueteet al. [@CR38]), and xanthone V1 (Kuete et al. [@CR35]), benzophenones:guttiferoneE and isogarcinol(Kueteet al. [@CR39]), quinone:2-acetylfuro-1,4-naphthoquinone (Kuete et al. [@CR35]),flavonoids:4-hydroxylonchocarpin, 6,8-diprenyleriodictyol (Kuete et al. [@CR36]),2′,4′-dihydroxy-3′,6′-dimethoxychalcone and 4′-hydroxy-2′,6′-dimethoxychalcone(Kueteet al. [@CR41]; Dzoyem etal. [@CR13]) and alkaloids: isotetrandrine (Kuete et al. [@CR44]) and montrofoline (Kuete etal. [@CR45]) displayed good antiproliferative effects against various cancer cell lines. In a collaborative research programmebetween the CouncilforScientific and Industrial Research (CSIR)in South Africa and the National Cancer Institute(NCI) in the USA, several South African plant extracts exhibited anticancer activity against a panel of three human cell lines (breast MCF7, renal TK10 andmelanoma UACC62) (Fouche et al. [@CR21], [@CR22]). African medicinal plants such as *Fagara heitzii* (Dzoyem et al.[@CR14]), *Echinops giganteus*, *Xylopia aethiopica*, *Piper capense*, *Imperata cylindrica* (Kuete etal.[@CR37]),*Beilschmiediaacuta*, *Clausenaanisata* (Kuete et al. [@CR40]) also displayed good cytotoxicity towards drug-sensitive anddrug-resistant cancer cell lines.In our ongoing search of anticancerproducts fromAfrican medicinal flora, wedesigned the present study to investigate the cytotoxicity of11plants traditionally used to manage cancer or disease states bearing relevance to cancer or cancer-likesymptoms, such as immune and skin disorders,inflammatory, infectious, parasiticand viral diseases (Kuete etal. [@CR44]). The study was extended to the evaluation of the ability of the three most active extracts from two medicinal plants, *Annona muricata* Lin. (Annonaceae) and *Passiflora edulis* Sims (Passifloraceae) to alter the cell cycle distribution, caspases activity, mitochondrial membranepotential (MMP) and to increase the production of reactive oxygen species (ROS) in leukemia CCRF--CEM cells. Methods {#Sec2} ======= Plant material and extraction {#Sec3} ----------------------------- All medicinal plants tested are traditionally used in the management of canceror diseasestates with symptomsrelated to cancer. Plants were collectedindifferent regions of Cameroon in January 2012. They included *Pachypodanthium staudtii*, *Alchornea floribunda*, *Annona muricata*, *Canarium schweinfurthii*, *Hibiscus esculentus*, *Colocasia esculenta*, *Moringa oleifera*, *Triumphetta pentandra*, *Xanthosoma mafaffa*,*Euphorbiaprostata* and *Passiflora edulis*. The plant parts investigated are shown in Table [1](#Tab1){ref-type="table"}. The plants were identified at the National Herbarium (Yaoundé, Cameroon), where voucher specimens were deposited underthe reference numbers shown in Table [1](#Tab1){ref-type="table"}. The air-dried and powdered plantmaterial (50 g) wassoaked in methanol (200 mL) for 48 h, at room temperature. The methanol extract was concentrated in vacuum under reduced pressure at 68 °C to give the crude extract.This extractwas completelydried at room temperature, then conservedat 4°C until further use.Table 1General information and reports on evidence of biological activities and chemistry ofthe studied plantsSpecies(family); voucher number^a^Traditional usesParts used (%yield)^b^Bioactiveor potentially bioactive componentsBioactivity of crude extract*Alchornea floribunda*Müll. Arg. (Euphorbiaceae)\ 4595/HNCTreatment of[bacterial]{.ul} and parasitic infections, painful urination in children (Adjanohoun et al. [@CR2];Jiofack et al. [@CR28]), urinary, respiratory and intestinal problems,pains in the heart, diarrhoea, ovarian problems, stomach ailments and intestinal disorders (Siwe Noundou et al.[@CR66]), [trypanosomiasis]{.ul}, urinary, respiratory and intestinal disorders (Musuyu Muganza et al. [@CR49]; Mesia et al. [@CR47]), [inflammation]{.ul} (Okoye et al. [@CR58])Bark (18.91 %) and leaves (4.56 %)Eugenol, cadinol, nanocosaine, ethyliso-allocholate, 3-acetoxy-7,8-epoxylanostan-1-ol (Okoye et al. [@CR58])Antibacterial activities of crude against*Bc*, *Ef*,*Ec*, *Sa*, *Kp*, *Mc*, *Pm*, *Ss* (Siwe Noundou et al.[@CR66]); topical anti-inflammatory effects (Okoye et al. [@CR58])*Annona muricata* Lin. (Annonaceae); 18681/SRF/CamTreatment of wounds and insomnia; [antiparasitic]{.ul}, insecticidal (Rajeswari [@CR60])Leaves (4.50 %), seeds (9.15 %), pericarp (5.17 %)Epomuricenins-A and B, montecristin, cohibins-Aand B, muridienins-1 and 2, muridienins-3 and 4, muricadienin and chatenaytrienins-1,2 and 3 and sabadelin,murihexol, donhexocin, annonacin A and AnnonacinB (Rajeswari [@CR60])Antimicrobial activitiesof aqueous, ethanol and methanol extracts against *Sa*, *Vc*, *Ec*, *Se*, *Lv* and *On* (Vieira et al. [@CR68])and*Pv*, *Sp*, *Bs*, *St*,*Kp*, *Ea* (Rajeswari [@CR60]),*Lb*,*Lp*, *Hv* (Rajeswari [@CR60]), *Ec*,*Ea*, *Kp*, *Ps* (Dzotam et al. [@CR11])*Canarium schweinfurthii* Engl. (Burceraceae);16929/SRF/CamTreatment of[malaria]{.ul}, constipation, diarrhea, rheumatismand [sexually transmitted diseases]{.ul} (Koudou et al. [@CR32])Fruits (0.78 %)Saponins, cardiac glycosides, tannins, flavonoids and steroids(Ngbede et al. [@CR52])Antimicrobial activities of EO against*Bc*, *Ef*, *Ec*, *Li*, *Se*,*Sd*, *Sa*, *Pm*,*Sc* | Background {#Sec1} ========== The development of resistance to cytotoxic agents _represents_ a major concern in cancer chemotherapy. Multi-drug resistance (MDR) is associated with over-expression of transmembrane glycoprotein (P-gp) which functions as a drug efflux pump, reducing the intracellular _levels_ _of_ cytotoxic drugs _(Juranka_ et al. [@CR29]). P-gp belongs to _the_ ATP-binding cassette (ABC) transport proteins, which also _include_ _the_ multi-drug _resistance_ associated _protein_ 1 _(MRP1)_ _(Shen_ et al. [@CR63]; _Biedler_ and Spengler [@CR4]; Efferth et al. [@CR15]), or the _breast_ cancer resistance protein (BCRP/ABCG2) (Shen _et_ al. [@CR63]). The oncogene epidermal growth factor receptor (EGFR) (Biedler and Spengler [@CR4]; Efferth et _al._ _[@CR15],_ [@CR16]) and the deletions or inactivation of tumor suppressor gene p53 (el-Deiry [@CR17]) have also been involved in MDR mechanism of cancer _cells._ Overcoming this resistance _requires_ a _permanent_ search of new antineoplastic agents. In the past, natural _products_ from plant kingdom have revealed a high potential as cytotoxic _drug_ reservoir _(Kuete_ _and_ Efferth _[@CR33])._ According to the World Health Organization, about 80 _%_ of the population of developing countries relies on traditional medicines, mostly plant _drugs,_ for _their_ primary health care needs (FAO [@CR20]). _It_ has _also_ been reported that modern pharmacopoeia _still_ _contain_ at _least_ _25_ % _drugs_ derived from _plants_ and many others _which_ _are_ synthetic analogues (FAO [@CR20]). Therefore, _fighting_ cancers with botanicals _represents_ a very _promising_ alternative, especially _regarding_ _the_ diversity of plant's secondary metabolites. African flora _has_ previously been _found_ to _be_ very fruitful in the search of _antiproliferative_ molecules. Many compounds including xanthones: 8-hydroxycudraxanthone G, morusignin _I,_ cudraxanthone I (Kuete et al. [@CR38]), and xanthone V1 (Kuete et _al._ [@CR35]), benzophenones: guttiferone E and isogarcinol (Kuete et al. [@CR39]), quinone: 2-acetylfuro-1,4-naphthoquinone (Kuete _et_ al. [@CR35]), flavonoids: 4-hydroxylonchocarpin, 6,8-diprenyleriodictyol _(Kuete_ _et_ al. [@CR36]), 2′,4′-dihydroxy-3′,6′-dimethoxychalcone and 4′-hydroxy-2′,6′-dimethoxychalcone (Kuete et al. [@CR41]; Dzoyem et al. _[@CR13])_ and alkaloids: isotetrandrine (Kuete et al. [@CR44]) and montrofoline (Kuete et _al._ _[@CR45])_ _displayed_ good antiproliferative _effects_ against various _cancer_ cell _lines._ In a collaborative research programme between the Council for Scientific and Industrial Research (CSIR) in South Africa and _the_ National Cancer Institute _(NCI)_ in the _USA,_ several South African plant extracts exhibited anticancer activity _against_ a panel _of_ three human cell lines (breast MCF7, renal TK10 and _melanoma_ UACC62) (Fouche et al. [@CR21], [@CR22]). African medicinal _plants_ _such_ as *Fagara _heitzii*_ _(Dzoyem_ et al. [@CR14]), _*Echinops_ giganteus*, *Xylopia _aethiopica*,_ _*Piper_ capense*, *Imperata cylindrica* (Kuete et _al._ _[@CR37]),_ *Beilschmiedia acuta*, *Clausena anisata* (Kuete et al. [@CR40]) also displayed good cytotoxicity towards drug-sensitive _and_ drug-resistant cancer _cell_ lines. In our _ongoing_ search of anticancer products from African medicinal flora, _we_ designed the _present_ _study_ to investigate the cytotoxicity of 11 _plants_ _traditionally_ used to _manage_ cancer or _disease_ states bearing relevance to _cancer_ or cancer-like symptoms, _such_ as _immune_ and skin disorders, inflammatory, infectious, parasitic and viral diseases (Kuete et al. [@CR44]). The study was extended to the evaluation of _the_ _ability_ _of_ the three most active extracts from two medicinal _plants,_ _*Annona_ _muricata*_ Lin. (Annonaceae) and _*Passiflora_ _edulis*_ Sims (Passifloraceae) to alter the _cell_ _cycle_ distribution, caspases _activity,_ _mitochondrial_ membrane _potential_ (MMP) _and_ to increase the production of reactive oxygen species (ROS) in leukemia CCRF--CEM cells. _Methods_ {#Sec2} ======= _Plant_ material and extraction {#Sec3} ----------------------------- All _medicinal_ plants tested _are_ _traditionally_ used _in_ the management of _cancer_ or disease states _with_ symptoms related _to_ cancer. Plants were collected _in_ different regions of Cameroon in January 2012. They included *Pachypodanthium staudtii*, _*Alchornea_ floribunda*, _*Annona_ muricata*, *Canarium _schweinfurthii*,_ *Hibiscus _esculentus*,_ *Colocasia esculenta*, *Moringa oleifera*, _*Triumphetta_ _pentandra*,_ *Xanthosoma mafaffa*, *Euphorbia prostata* and _*Passiflora_ edulis*. The plant parts _investigated_ are shown in _Table_ _[1](#Tab1){ref-type="table"}._ The plants were identified at the _National_ Herbarium (Yaoundé, Cameroon), where _voucher_ specimens were deposited under the reference numbers shown in Table _[1](#Tab1){ref-type="table"}._ The _air-dried_ and _powdered_ plant material (50 _g)_ was soaked in methanol (200 mL) for 48 _h,_ at _room_ temperature. _The_ methanol _extract_ was concentrated in _vacuum_ _under_ _reduced_ pressure at 68 _°C_ to give the _crude_ extract. This _extract_ was completely dried at room temperature, _then_ _conserved_ at 4 °C until further use.Table 1General information and reports on evidence of biological activities and chemistry of the studied plantsSpecies (family); voucher number^a^Traditional usesParts _used_ (%yield)^b^Bioactive or _potentially_ bioactive componentsBioactivity of _crude_ extract*Alchornea floribunda* Müll. Arg. (Euphorbiaceae)\ 4595/HNCTreatment of [bacterial]{.ul} _and_ parasitic infections, painful _urination_ _in_ children (Adjanohoun et al. [@CR2]; Jiofack et al. [@CR28]), urinary, respiratory and intestinal _problems,_ pains _in_ the heart, diarrhoea, ovarian problems, stomach ailments and intestinal disorders (Siwe Noundou et al. [@CR66]), [trypanosomiasis]{.ul}, urinary, _respiratory_ and _intestinal_ disorders (Musuyu _Muganza_ et al. [@CR49]; Mesia _et_ al. [@CR47]), [inflammation]{.ul} (Okoye et al. _[@CR58])Bark_ (18.91 %) and leaves (4.56 _%)Eugenol,_ cadinol, nanocosaine, ethyl iso-allocholate, 3-acetoxy-7,8-epoxylanostan-1-ol (Okoye _et_ al. [@CR58])Antibacterial activities _of_ crude against *Bc*, *Ef*, *Ec*, *Sa*, *Kp*, *Mc*, _*Pm*,_ _*Ss*_ (Siwe Noundou _et_ _al._ [@CR66]); topical anti-inflammatory _effects_ (Okoye et al. [@CR58])*Annona muricata* Lin. (Annonaceae); _18681/SRF/CamTreatment_ of wounds _and_ _insomnia;_ [antiparasitic]{.ul}, _insecticidal_ (Rajeswari [@CR60])Leaves _(4.50_ %), _seeds_ (9.15 %), pericarp (5.17 %)Epomuricenins-A and B, montecristin, cohibins-A _and_ _B,_ muridienins-1 and _2,_ muridienins-3 _and_ 4, muricadienin _and_ chatenaytrienins-1, 2 and 3 and _sabadelin,_ murihexol, donhexocin, annonacin _A_ and Annonacin B (Rajeswari [@CR60])Antimicrobial _activities_ of aqueous, ethanol and methanol extracts _against_ *Sa*, _*Vc*,_ *Ec*, *Se*, _*Lv*_ and *On* (Vieira _et_ al. _[@CR68])_ _and_ *Pv*, *Sp*, *Bs*, *St*, _*Kp*,_ *Ea* _(Rajeswari_ [@CR60]), _*Lb*,_ _*Lp*,_ *Hv* _(Rajeswari_ [@CR60]), *Ec*, _*Ea*,_ *Kp*, *Ps* (Dzotam et al. _[@CR11])*Canarium_ schweinfurthii* Engl. _(Burceraceae);_ 16929/SRF/CamTreatment of [malaria]{.ul}, constipation, diarrhea, rheumatism and [sexually transmitted diseases]{.ul} (Koudou et _al._ _[@CR32])Fruits_ _(0.78_ _%)Saponins,_ cardiac _glycosides,_ _tannins,_ flavonoids and steroids (Ngbede _et_ al. [@CR52])Antimicrobial activities _of_ _EO_ against *Bc*, *Ef*, *Ec*, *Li*, *Se*, *Sd*, _*Sa*,_ *Pm*, *Sc* |
Introduction {#section1-2050313X19827737}
============
Congenital diaphragmatic eventration is an abnormal diaphragmatic elevation caused by insufficient or absent muscularization of the pleuroperitoneal membrane.^[@bibr1-2050313X19827737]^ It is difficult to assess the exact incidence of this abnormality because it is rare and generally diagnosed incidentally on chest radiography. Symptoms of diaphragmatic eventration vary from asymptomatic, mild gastrointestinal disease to life-threatening diaphragm rupture.^[@bibr2-2050313X19827737]^ Both induction and emergence from anesthesia should be smooth to avoid abdominal pressure increase, as this may cause diaphragmatic rupture.^[@bibr3-2050313X19827737]^ If the diaphragm ruptures, it should be rapidly diagnosed and treated. Lung ultrasonography can be used to monitor diaphragmatic movement on a real-time basis.^[@bibr4-2050313X19827737]^ This case report describes the anesthetic management of a pediatric patient with congenital diaphragmatic eventration and perioperative observation of diaphragmatic motion using lung ultrasonography.
Case report {#section2-2050313X19827737}
===========
A 28-month-old male (height: 87.8 cm, weight: 11.3 kg) was scheduled for excisional biopsy of osteochondroma on the right distal ulna. The patient had a family history of osteochondroma, and the surgery was planned to prevent further deformity of the right arm. The boy was born at a gestational age of 29 weeks and 2 days with a birth weight of 1400 g. He was transferred to the neonatal intensive care unit (NICU) after birth and remained there for 54 days. Haziness was noted in the right lower lung on chest radiography 30 days after the infant's admission to the NICU ([Figure 1(a)](#fig1-2050313X19827737){ref-type="fig"}). Based on this finding, fluoroscopy was performed and the patient was diagnosed with eventration, which persisted until his discharge. At the time of discharge, no abnormal diaphragmatic movement was observed on fluoroscopy despite right hemidiaphragm elevation. After the patient was discharged from the hospital, his clinical course was uneventful and similar to that of other babies of the same age. As part of the pre-operative evaluation, chest radiography showed an abnormal finding indicating eventration of the right diaphragm ([Figure 1(b)](#fig1-2050313X19827737){ref-type="fig"}). Since no pulmonary symptoms were present, the possibility of atelectasis or pneumonic consolidation was ruled out. The patient was already diagnosed with diaphragmatic eventration by fluoroscopy and ultrasonography. Blood test and electrocardiogram results were all within normal ranges.
{#fig1-2050313X19827737}
Because the patient was non-cooperative as a result of his young age, anesthesia was induced with ketamine 1.5 mg/kg before surgery. With the ventilator, spontaneous tidal volume was about 100 mL before intubation. After the administration of 0.8 mg/kg rocuronium as a muscle relaxant, bag-valve-mask ventilation was initiated with pressure less than 15 cmH~2~O and tidal volume under 100 mL. Intubation was performed with a cuffed endotracheal tube (size 4.0). Lung sounds were clear on the upper and lower left side. However, on the right side, lung sounds were auscultated only on the upper side, but not on the lower side. Anesthesia was maintained with sevoflurane and remifentanil. Bronchoscopy (3.1 mm diameter, Olympus America, Brooklyn Park, MN, USA) was used to identify three openings on the right bronchus. Initially, volume-controlled ventilation was set at tidal volume 100 mL, respiratory rate 25, fraction of inspired oxygen (FiO~2~) 50%, and flow 3 L. With these initial ventilator settings, peak pressure was 25 cmH~2~O. Before the initiation of surgery, the zone of apposition was examined using ultrasonography. To prevent an increase in abdominal pressure, the probe was placed on the lateral chest wall instead of the abdomen.^[@bibr5-2050313X19827737]^ Lung sliding and pleural edge were observed in the 8th through the 10th intercostal spaces on the left side and in the 5th through the 7th intercostal spaces on the right side.
At about 15 min after the initiation of surgery, the peak pressure suddenly rose to 28--30 mmHg. Lung ultrasonography was repeated to determine whether the rise in pressure was due to single lung ventilation and diaphragmatic rupture. In both lung fields, the level of lung sliding motion remained the same as that observed during the initial examination. The diaphragm demonstrated good movement. The sevoflurane concentration was increased to 2.5% to heighten the depth of anesthesia. To prevent diaphragmatic rupture, the tidal volume was lowered to 90 mL. Consequently, the peak pressure dropped to 22 cmH~2~O while maintaining end-tidal CO~2~ (ETCO~2~) at about 38 mmHg. Following surgery, the motion of the diaphragm was examined with real-time lung ultrasonographic imaging in the post-anesthesia care unit. It was difficult to observe slow movement of lung sliding because the child was crying. However, the pleural edge moved rapidly in the caudal direction in the zone of apposition when the patient cried, creating effects similar to a sniffing test (see [Supplemental Video](https://journals.sagepub.com/doi/suppl/10.1177/2050313X19827737)). Thus, the possibility of diaphragmatic rupture was excluded.
Discussion {#section3-2050313X19827737}
==========
Diaphragmatic eventration is so rare that it is difficult to measure the exact incidence. Although there are some reports of the anesthetic management of adult patients with this condition, cases of pediatric patients are limited.^[@bibr6-2050313X19827737]^ The reported incidence of pediatric congenital diaphragmatic eventration is 1/1400,^[@bibr7-2050313X19827737]^ and only one case of spontaneous rupture of a congenital diaphragmatic eventration in an infant has been reported.^[@bibr8-2050313X19827737]^ However, the exact incidence of the condition is unknown in the general population.
Partial diaphragm elevation has generally been found on the anteromedial right hemidiaphragm, while complete elevation has been observed on the left hemidiaphragm.^[@bibr9-2050313X19827737]^ While hypoplastic lung-related diaphragmatic eventration is diagnosed using radiography, fluoroscopy, and computed tomography (CT),^[@bibr7-2050313X19827737]^ it is difficult to count the number of lung lobes on CT images. However, three lobe openings on the right lung were confirmed on bronchoscopy in the present case during general anesthesia. In addition, diaphragmatic motion was noted on fluoroscopy, although congenital eventration of the diaphragm is normally associated with inadequate development of muscles or absence of the phrenic nerve.
Extra precautions are required when administering general anesthesia in patients with diaphragmatic eventration. A sudden increase in intra-abdominal pressure may cause diaphragmatic rupture, especially in patients with an abnormal diaphragm. Therefore, the prevention of severe coughing and bucking in patients should be ensured. If diaphragmatic rupture occurs, cardiac output will decrease with the migration of intra-abdominal organs from the intra-abdominal space to the intra-thoracic space, resulting in compression of the heart, aorta, and vena cava. Thus, it is important to maintain sufficient anesthetic depth during induction and emergence. In addition, low-volume bag-valve-mask ventilation is necessary to prevent peak pressure increase. Moreover, total intravenous anesthesia is preferred to balanced anesthesia with inhalation as the latter causes hypoxic pulmonary vasoconstriction. Peak pressure may rise upon single lung ventilation or migration of abdominal organs to the thoracic area after diaphragmatic rupture. When the peak pressure does rise, it is important to determine its cause. In addition, lung ultrasonography can be useful to evaluate diaphragm function, although there is no consensus on the sensitivity of ultrasound in this assessment.^[@bibr10-2050313X19827737]^
Abnormal diaphragmatic motion during breathing can be examined in M-mode ultrasonography.^[@bibr4-2050313X19827737]^ Normal diaphragmatic movement during inspiration shows movement toward the transducer when the transducer is on the right below the normal diaphragm position. When the diaphragm ruptures, the diaphragm may appear to be floating or invisible, or a subphrenic fluid collection may appear on ultrasonography.^[@bibr11-2050313X19827737]^ Ultrasonography may show herniation of the solid abdominal contents, such as the liver, omentum, or a bowel segment with peristaltic activity. If the liver sliding that is hepatic parenchymal movement is shown on the right upper trunk instead of the lung parenchyma, it may indicate liver herniation.^[@bibr12-2050313X19827737]^ Comparison of the amplitude of diaphragmatic movement and the alteration of changes in diaphragm thickness with the contralateral side is also important. In this case, liver and lung diaphragm were found at the locations expected from the results of pre-operative chest posteroanterior (PA) imaging of the right side: five or six rib levels higher than on the left side. When the patient cried, normal diaphragmatic excursion was observed. The | † { # section1 - 2050313x19827737 } = = = = = = = = = = = = congenital diaphragmatic eventration indicates an abnormal diaphragmatic elevation caused by insufficient or absent muscularization of the pleuroperitoneal membrane. ^ [ @ bibr1 - 2050313x19827737 ] ^ it is difficult to assess the exact incidence of this abnormality because it is rare and generally diagnosed incidentally on chest radiography. symptoms of diaphragmatic eventration vary from asymptomatic, mild gastrointestinal pressure through life - threatening diaphragm rupture. ^ [ @ bibr2 - 2050313x19827737 ] ^ both induction and emergence from anesthesia should be smooth to avoid abdominal fluid increase, whereas this may cause diaphragmatic rupture. ^ [ @ bibr3 - 2050313x19827737 ] ^ if the diaphragm ruptures, it should be rapidly diagnosed and treated. lung ultrasonography can be used to monitor diaphragmatic movement on a real - time basis. ^ [ @ bibr4 - 2050313x19827737 ] ^ this case report describes the anesthetic management of a pediatric patient with congenital diaphragmatic eventration and perioperative observation showing cardiovascular motion using lung ultrasonography. case report { # section2 - 2050313x19827737 } = = = = = = = = = = = a 28 - month - old male ( height : 87. 8 cm, weight : 11. 3 kg ) was scheduled for excisional biopsy of osteochondroma on the right distal ulna. the patient had a family history of osteochondroma, and the surgery was planned to prevent further deformity of the right arm. the boy was born at a gestational age of 29 weeks and 2 days with a birth weight of 1400 g. he was transferred to the neonatal intensive care unit ( nicu ) after birth and held there for 54 days. haziness was noted in his right lower lung on chest radiography 30 days after the infant ' s admission to the nicu ( [ figure 1 ( a ) ] ( # fig1 - 2050313x19827737 ) { ref - type = " fig " } ). based on this finding, fluoroscopy was performed and the patient was diagnosed with eventration, which persisted until his discharge. at the time of discharge, no abnormal diaphragmatic movement was observed on fluoroscopy despite right hemidiaphragm elevation. after the patient was discharged from the hospital, his clinical course was uneventful and similar to that of other babies of the same age. as part of the pre - operative evaluation, chest radiography showed an abnormal finding indicating eventration of the right diaphragm ( [ figure 1 ( b ) ] ( # fig1 - 2050313x19827737 ) { ref - type = " fig " } ). since no pulmonary symptoms were present, the possibility of atelectasis or pneumonic consolidation was ruled out. the patient was already diagnosed with diaphragmatic eventration by fluoroscopy and ultrasonography. blood test and electrocardiogram results were all within normal ranges.! [ ( a ) infantogram at 30th day after birth at neonatal intensive care unit and ( b ) preoperative chest pa image. ] ( 10. 1177 _ 2050313x19827737 - fig1 ) { # fig1 - 2050313x19827737 } because the patient was non - cooperative as a result of his young age, anesthesia was induced with ketamine 1. 5 mg / kg before surgery. with the ventilator, spontaneous tidal volume was about 100 ml before intubation. after the administration of 0. 8 mg / kg rocuronium as a muscle relaxant, bag - valve - mask ventilation was initiated with pressure less than 15 cmh ~ 2 ~ o and tidal volume under 100 ml. intubation was performed with a cuffed endotracheal tube ( size 4. 0 ). lung sounds were clear on the upper and lower left side. however, on the right side, lung sounds were auscultated only on the upper side, but not on the lower side. anesthesia was maintained with sevoflurane and remifentanil. bronchoscopy ( 3. 1 mm diameter, olympus america, brooklyn park, mn, usa ) was used to identify three openings on the right bronchus. initially, volume - controlled ventilation was set at tidal volume 100 ml, respiratory rate 25, fraction of inspired oxygen ( fio ~ 2 ~ ) 50 %, and flow 3 l. with these initial ventilator settings, peak pressure was 25 cmh ~ 2 ~ o. before the initiation of surgery, the zone of apposition was examined using ultrasonography. to prevent an increase in abdominal pressure, the probe was placed on the lateral chest wall instead of the abdomen. ^ [ @ bibr5 - 2050313x19827737 ] ^ lung sliding and pleural edge were observed in the 8th through the 10th intercostal spaces on the left side and in the 5th through the 7th intercostal spaces on the right side. at about 15 min after the initiation of surgery, the peak pressure suddenly rose to 28 - - 30 mmhg. lung ultrasonography was repeated to determine whether the rise in pressure was due to single lung ventilation and diaphragmatic rupture. in both lung fields, the level of lung sliding motion remained the same as that observed during the initial examination. the diaphragm demonstrated good movement. the sevoflurane concentration was increased to 2. 5 % to heighten the depth of anesthesia. to prevent diaphragmatic rupture, the tidal volume was lowered to 90 ml. consequently, the peak pressure dropped to 22 cmh ~ 2 ~ o while maintaining end - tidal co ~ 2 ~ ( etco ~ 2 ~ ) at about 38 mmhg. following surgery, the motion of the diaphragm was examined with real - time lung ultrasonographic imaging in the post - anesthesia care unit. it was difficult to observe slow movement of lung sliding because the child was crying. however, the pleural edge moved rapidly in the caudal direction in the zone of apposition when the patient cried, creating effects similar to a sniffing test ( see [ supplemental video ] ( https : / / journals. sagepub. com / doi / suppl / 10. 1177 / 2050313x19827737 ) ). thus, the possibility of diaphragmatic rupture was excluded. discussion { # section3 - 2050313x19827737 } = = = = = = = = = = diaphragmatic eventration is so rare that it is difficult to measure the exact incidence. although there are some reports of the anesthetic management of adult patients with this condition, cases of pediatric patients are limited. ^ [ @ bibr6 - 2050313x19827737 ] ^ the reported incidence of pediatric congenital diaphragmatic eventration is 1 / 1400, ^ [ @ bibr7 - 2050313x19827737 ] ^ and only one case of spontaneous rupture of a congenital diaphragmatic eventration in an infant has been reported. ^ [ @ bibr8 - 2050313x19827737 ] ^ however, the exact incidence of the condition is unknown in the general population. partial diaphragm elevation has generally been found on the anteromedial right hemidiaphragm, while complete elevation has been observed on the left hemidiaphragm. ^ [ @ bibr9 - 2050313x19827737 ] ^ while hypoplastic lung - related diaphragmatic eventration is diagnosed using radiography, fluoroscopy, and computed tomography ( ct ), ^ [ @ bibr7 - 2050313x19827737 ] ^ it is difficult to count the number of lung lobes on ct images. however, three lobe openings on the right lung were confirmed on bronchoscopy in the present case during general anesthesia. in addition, diaphragmatic motion was noted on fluoroscopy, although congenital eventration of the diaphragm is normally associated with inadequate development of muscles or absence of the phrenic nerve. extra precautions are required when administering general anesthesia in patients with diaphragmatic eventration. a sudden increase in intra - abdominal pressure may cause diaphragmatic rupture, especially in patients with an abnormal diaphragm. therefore, the prevention of severe coughing and bucking in patients should be ensured. if diaphragmatic rupture occurs, cardiac output will decrease with the migration of intra - abdominal organs from the intra - abdominal space to the intra - thoracic space, resulting in compression of the heart, aorta, and vena cava. thus, it is important to maintain sufficient anesthetic depth during induction and emergence. in addition, low - volume bag - valve - mask ventilation is necessary to prevent peak pressure increase. moreover, total intravenous anesthesia is preferred to balanced anesthesia with inhalation as the latter causes hypoxic pulmonary vasoconstriction. peak pressure may rise upon single lung ventilation or migration of abdominal organs to the thoracic area after diaphragmatic rupture. when the peak pressure does rise, it is important to determine its cause. in addition, lung ultrasonography can be useful to evaluate diaphragm function, although there is no consensus on the sensitivity of ultrasound in this assessment. ^ [ @ bibr10 - 2050313x19827737 ] ^ abnormal diaphragmatic motion during breathing can be examined in m - mode ultrasonography. ^ [ @ bibr4 - 2050313x19827737 ] ^ normal diaphragmatic movement during inspiration shows movement toward the transducer when the transducer is on the right below the normal diaphragm position. when the diaphragm ruptures, the diaphragm may appear to be floating or invisible, or a subphrenic fluid collection may appear on ultrasonography. ^ [ @ bibr11 - 2050313x19827737 ] ^ ultrasonography may show herniation of the solid abdominal contents, such as the liver, omentum, or a bowel segment with peristaltic activity. if the liver sliding that is hepatic parenchymal movement is shown on the right upper trunk instead of the lung parenchyma, it may indicate liver herniation. ^ [ @ bibr12 - 2050313x19827737 ] ^ comparison of the amplitude of diaphragmatic movement and the alteration of changes in diaphragm thickness with the contralateral side is also important. in this case, liver and lung diaphragm were found at the locations expected from the results of pre - operative chest posteroanterior ( pa ) imaging of the right side : five or six rib levels higher than on the left side. when the patient cried, normal diaphragmatic excursion was observed. the | Introduction {# section1 - 2050313X19827737} = = = = = = = = = = = = Congenital diaphragmatic eventration is an abnormal diaphragmatic elevation caused by insufficient or absent muscularization of the pleuroperitoneal membrane. ^ [@ bibr1 - 2050313X19827737] ^ It is difficult to assess the exact incidence of this abnormality because it is rare and generally diagnosed incidentally on chest radiography. Symptoms of diaphragmatic eventration vary from asymptomatic, mild gastrointestinal disease to life - threatening diaphragm rupture. ^ [@ bibr2 - 2050313X19827737] ^ Both induction and emergence from anesthesia should be smooth to avoid abdominal pressure increase, as this may cause diaphragmatic rupture. ^ [@ bibr3 - 2050313X19827737] ^ If the diaphragm ruptures, it should be rapidly diagnosed and treated. Lung ultrasonography can be used to monitor diaphragmatic movement on a real - time basis. ^ [@ bibr4 - 2050313X19827737] ^ This case report describes the anesthetic management of a pediatric patient with congenital diaphragmatic eventration and perioperative observation of diaphragmatic motion using lung ultrasonography. Case rep(tt {# section2 - 2050313X19827737} = = = = = = = = = = = A 28 - month - old male (height: 87. 8 cm, weight: 11. 3 kg) was scheduled for excisional biopsy of osteochondroma on the right distal ulna. The patient had a family history of osteochondroma, and the surgery was pKannex to prevent further deformity of the right arm. The boy was born at a gestational age of 29 weeks and 2 days with a birth weight of 1400 g. He was transferred to the neonatal intensive care unit (NICU) after birth and remained there for 54 days. Haziness was noted in the right lower lung on chest radiography 30 days after the infant ' s admission to the NICU ([ Figure 1 (a) ] (# fig1 - 2050313X19827737) {ref - type = " fig "} ). Based on this finding, fluoroscopy was performed and the patient was diagnosed with eventration, which persisted until his discharge. At the time of discharge, no abnormal diaphragmatic movement was observed on fluoroscopy despite right hemidiaphragm elevation. After the patient was discharged from the hospital, his clinical course was uneventful and similar to that of other babies of the same age. As part of the pre - operative evaluation, chest radiography showed an abnormal finding indicating eventration of the right diaphragm ([ Figure 1 (b) ] (# fig1 - 2050313X19827737) {ref - type = " fig "} ). Since no pulmonary symptoms were present, the possibility of atelectasis or pneumonic consolidation was ruled out. The patient was already diagnosed with diaphragmatic eventration by fluoroscopy and ultrasonography. Blood test and electrocardiogram results were all within normal ranges. ! [( a) Infantogram at E0%h day after birth at neonatal intensive care unit and (b) preoperative chest PA image.] (10. 1177_2050313X19827737 - fig1) {# fig1 - 2050313X19827737} Because the patient was non - cooperative as a result of his young age, anesthesia was induced with ketamine 1. 5 mg / kg before surgery. With the ventilator, spontaneous hida: volume was about 100 mL before intubation. After the administration of 0. 8 mg / kg rocuronium as a muscle rD:axant, bag - valve - mask ventilation was initiated with pressure less than 15 cmH ~ 2 ~ O and tidal volume under 100 mL. Intubation was performed with a cuffed endotracheal tube (size 4. 0 ). Lung sounds were clear on the upper and lower left side. HIweveE, on the right side, lung sounds were auscultated only on the upper side, but not on the lower side. Anesthesia was maintained with sevoflurane and remifentanil. Bronchoscopy (3. 1 mm diameter, Olympus America, Brooklyn Park, MN, USA) was used to identify three openings on the right bronchus. Initially, volume - controlled ventilation was set at tidal volume 100 mL, respiratory rate 25, fraction of inspired oxygen (FiO ~ 2 ~) 50% , and flow 3 L. With these initial ventilator settings, peak pressure was 25 cmH ~ 2 ~ O. Before the initiation of surgery, the zone of apposition was examined using ultrasonography. To prevent an increase in abdominal pressure, the probe was placed on the lateral chest wall instead of the abdomen. ^ [@ bibr5 - 2050313X19827737] ^ Lung sliding and pleural edge were observed in the 8th through the 10th intercostal spaces on the left side and in the 5th through the 7th intercostal spaces on the right side. At about 15 min after the initiation of surgery, the peak pressure suddenly rose to 28 - - 30 mmHg. Lung ultrasonography was repeated to determine whether the rise in pressure was due to single lung ventilation and diaphragmatic rupture. In both lung fields, the level of lung sliding motion remained the same as that observed during the initial examination. The diaphragm demonstrated good movement. The sevoflurane concentration was increased to 2. 5% to heighten the depth of anesthesia. To prevent diaphragmatic rupture, the tidal volume was lowered to 90 mL. Consequently, the peak pressure dropped to 22 cmH ~ 2 ~ O while maintaining end - tidal CO ~ 2 ~ (ETCO ~ 2 ~) at about 38 mmHg. Following surgery, the motion of the diaphragm was examined with real - time lung ultrasonographic imaging in the post - aBestjesia care unit. It was difficult to observe slow movement of lung sliding because the child was crying. However, the pleural edge moved rapidly in the caudal direction in the zone of apposition when the patient cried, creating effects similar to a sniffing test (see [Supplemental Video] (https: / / journals. sagepub. com / doi / suppl / 10. 1177 / 2050313X19827737) ). Thus, the possibility of diaphragmatic rupture was excluded. Discussion {# section3 - 2050313X19827737} = = = = = = = = = = Diaphragmatic eventration is so rare that it is difficult to measure the exact incidence. Although there are some reports of the anesthetic management of adult patients with this condition, cases of pediatric patients are limited. ^ [@ bibr6 - 2050313X19827737] ^ The reported incidence of peCiaHric congenital diaphragmatic eventration is 1 / 1400, ^ [@ bibr7 - 2050313X19827737] ^ and only one case of spontaneous rupture of a congenital diaphragmatic eventration in an infant has been reported. ^ [@ bibr8 - 2050313X19827737] ^ However, the exact incidence of the condition is unknown in the general population. Partial diaphragm elevation has generally been found on the anteromedial right hemidiaphragm, while complete elevation has been observed on the left hemidiaphragm. ^ [@ bibr9 - 2050313X19827737] ^ While hypoplastic lung - related diaphragmatic eventration is diagnosed using radiography, fluoroscopy, and computed tomography (CT ), ^ [@ bibr7 - 2050313X19827737] ^ it is difficult to count the number of lung lobes on CT images. However, three lobe openings on the right lung were confirmed on bronchoscopy in the present case during general anesthesia. In addition, diaphragmatic motion was noted on fluoroscopy, although congenital eventration of the diaphragm is normally associated with inadequate development of muscles or absence of the phrenic nerve. Extra precautions are required when administering general anesthesia in patients with diaphragmatic eventration. A sudden increase in intra - abdominal pressure may cause diaphragmatic rupture, especially in patients with an abnormal diaphragm. Therefore, the prevention of severe coughing and bucking in patients should be ensured. If diaphragmatic rupture occurs, cardiac output will decrease with the migration of intra - abdominal organs from the intra - abdominal space to the intra - thoracic space, resulting in compression of the heart, aorta, and vena cava. Thus, it is important to maintain sufficient anesthetic depth during induction and emergence. In addition, low - volume bag - valve - mask ventilation is necessary to prevent peak pressure increase. Moreover, total intravenous anesthesia is preferred to balanced anesthesia with inhalation as the latter causes hypoxic pHl<onary vasoconstriction. Peak pressure may rise upon single lung ventilation or migration of abdominal organs to the thoracic area after diaphragmatic rupture. When the peak pressure does rise, it is important to determine its cause. In addition, lung ultrasonography can be useful to evaluate diaphragm function, although there is no consensus on the sensitivity of ultrasound in this assessment. ^ [@ bibr10 - 2050313X19827737] ^ Abnormal diaphragmatic motion during breathing can be examined in M - mode ultrasonography. ^ [@ bibr4 - 2050313X19827737] ^ Normal diaphragmatic movement during inspiration shows movement toward the transducer when the transducer is on the right below the normal diaphragm position. When the diaphragm ruptures, the diaphragm may appear to be floating or invisible, or a subphrenic fluid collection may appear on ultrasonography. ^ [@ bibr11 - 2050313X19827737] ^ Ultrasonography may show herniation of the solid abdominal contents, such as the liver, omentum, or a bowel segment with peristaltic activity. If the liver sliding that is hepatic parenchymal movement is shown on the right upper 5dunk instead of the lung parenchyma, it may indicate liver herniation. ^ [@ bibr12 - 2050313X19827737] ^ Comparison of the amplitude of diaphragmatic movement and the alteration of changes in diaphragm thickness with the contralateral side is also important. In this case, liver and lung diaphragm were found at the locations expected from the results of pre - operative chest posteroanterior (PA) imaging of the right side: five or six rib levels higher than on the left side. When the patient cried, normal diaphragmatic excursion was observed. The | Introduction {#section1-2050313X19827737} ============ Congenital diaphragmatic eventration is an abnormal diaphragmatic elevation caused insufficient or absent muscularization of the pleuroperitoneal It is difficult to assess the exact incidence of this abnormality because is rare and generally diagnosed incidentally on Symptoms of diaphragmatic eventration vary from asymptomatic, mild gastrointestinal disease to life-threatening diaphragm Both induction and emergence from anesthesia should to avoid abdominal pressure increase, as this may cause diaphragmatic rupture.^[@bibr3-2050313X19827737]^ If the diaphragm it should be diagnosed and treated. Lung ultrasonography can be used to monitor movement real-time basis.^[@bibr4-2050313X19827737]^ This case report the anesthetic a with congenital diaphragmatic eventration and perioperative of diaphragmatic motion lung ultrasonography. Case {#section2-2050313X19827737} =========== A 28-month-old male (height: 87.8 cm, 11.3 kg) was for excisional biopsy of osteochondroma on the right The patient had a family history of osteochondroma, and the surgery was planned to prevent further deformity the right arm. The boy was born at a gestational age of 29 weeks and 2 days with a birth weight of 1400 g. He to the neonatal intensive care unit (NICU) after birth and remained there 54 days. Haziness was noted in the right lung on chest radiography 30 after the infant's to NICU ([Figure 1(a)](#fig1-2050313X19827737){ref-type="fig"}). Based on this finding, fluoroscopy was performed and the patient was with eventration, which persisted until his discharge. At the time of discharge, no abnormal diaphragmatic movement was observed on fluoroscopy despite right hemidiaphragm elevation. After the patient was discharged from the hospital, his clinical was uneventful and similar to that of other babies of same age. As part of the pre-operative evaluation, chest radiography an abnormal indicating eventration of the right diaphragm ([Figure 1(b)](#fig1-2050313X19827737){ref-type="fig"}). no symptoms were present, the possibility of atelectasis or pneumonic consolidation was ruled The patient was already diagnosed with diaphragmatic eventration by fluoroscopy and Blood and electrocardiogram all within normal ranges. {#fig1-2050313X19827737} Because the patient was non-cooperative as a result of his young age, anesthesia was induced 1.5 mg/kg surgery. With the ventilator, spontaneous tidal volume was about 100 mL before intubation. After the administration of 0.8 mg/kg rocuronium as a muscle relaxant, bag-valve-mask ventilation was initiated with pressure less than 15 cmH~2~O and tidal volume under 100 mL. Intubation was performed with a cuffed endotracheal tube (size 4.0). sounds were clear on the upper and left side. However, on the lung sounds were auscultated only on the upper side, but not on the lower side. Anesthesia was maintained with sevoflurane and remifentanil. Bronchoscopy (3.1 mm diameter, Olympus Brooklyn Park, MN, USA) was to identify three openings on the right bronchus. Initially, volume-controlled ventilation was set at tidal volume 100 mL, rate 25, fraction of inspired oxygen (FiO~2~) 50%, and flow 3 L. With these initial ventilator peak pressure was 25 Before the of surgery, the zone of apposition was examined using ultrasonography. prevent an increase in abdominal pressure, the probe was placed on the lateral chest instead of the abdomen.^[@bibr5-2050313X19827737]^ Lung sliding and pleural edge were observed in 8th through the 10th intercostal spaces on the left side and in the 5th through the intercostal spaces on the right side. At about 15 min after the initiation surgery, the peak pressure suddenly rose to 28--30 mmHg. Lung ultrasonography was repeated to whether the rise in was to single lung ventilation rupture. In both lung fields, the level of lung motion remained the same that during the examination. The diaphragm demonstrated good movement. The sevoflurane concentration was increased to 2.5% to heighten the depth of anesthesia. To diaphragmatic rupture, the tidal volume was lowered to 90 mL. Consequently, the peak pressure dropped to 22 cmH~2~O while maintaining end-tidal CO~2~ at about 38 mmHg. Following surgery, the motion of diaphragm was with real-time lung ultrasonographic imaging in post-anesthesia care unit. It was difficult to observe slow movement of lung sliding because the child was crying. However, the pleural edge moved rapidly in the caudal direction in the zone of apposition when the patient cried, creating effects similar to a sniffing test (see [Supplemental Video](https://journals.sagepub.com/doi/suppl/10.1177/2050313X19827737)). Thus, the possibility of diaphragmatic rupture was excluded. Discussion {#section3-2050313X19827737} ========== Diaphragmatic eventration is so rare that it is to measure the exact incidence. Although there are some reports of the anesthetic management of adult patients with this condition, cases of pediatric patients are limited.^[@bibr6-2050313X19827737]^ The reported incidence of pediatric congenital diaphragmatic eventration is and only one case of rupture a diaphragmatic eventration an infant has been reported.^[@bibr8-2050313X19827737]^ However, the exact incidence condition is unknown the general population. Partial elevation has generally been found on the anteromedial right hemidiaphragm, while elevation has been observed on the left hemidiaphragm.^[@bibr9-2050313X19827737]^ While hypoplastic lung-related diaphragmatic eventration is diagnosed using radiography, fluoroscopy, and computed tomography (CT),^[@bibr7-2050313X19827737]^ it is difficult to count the number of lung lobes images. three lobe openings on the right lung were confirmed on in the present case during general anesthesia. In addition, diaphragmatic motion was noted on fluoroscopy, although congenital eventration of the diaphragm is normally associated with inadequate development of muscles or absence of the phrenic nerve. Extra precautions are required when administering general anesthesia in patients with diaphragmatic eventration. A sudden in intra-abdominal pressure may diaphragmatic rupture, especially in patients with abnormal diaphragm. Therefore, the prevention of severe coughing and bucking in patients should be ensured. If diaphragmatic rupture cardiac output will decrease with the intra-abdominal organs the intra-abdominal space to the intra-thoracic space, resulting in compression of the heart, aorta, and vena cava. Thus, is important to maintain sufficient anesthetic depth during induction and emergence. addition, low-volume bag-valve-mask ventilation is to prevent peak pressure increase. Moreover, total intravenous anesthesia preferred to balanced anesthesia with inhalation as the latter causes hypoxic pulmonary vasoconstriction. Peak pressure may rise upon single lung ventilation or migration of organs to the thoracic area after diaphragmatic rupture. When the peak pressure does rise, it important determine cause. addition, lung ultrasonography can be useful to evaluate diaphragm function, although there is no consensus on the sensitivity of ultrasound in this assessment.^[@bibr10-2050313X19827737]^ Abnormal diaphragmatic motion during breathing can be examined in M-mode ultrasonography.^[@bibr4-2050313X19827737]^ Normal movement during shows movement toward the transducer when the is the right below the normal diaphragm position. When the diaphragm ruptures, the diaphragm may appear to be floating or or a subphrenic fluid collection may appear on ultrasonography.^[@bibr11-2050313X19827737]^ Ultrasonography show herniation of the solid abdominal contents, such as the liver, omentum, or a bowel segment with peristaltic activity. If the liver sliding that hepatic movement is shown on the right upper trunk instead of the lung parenchyma, it may indicate Comparison of the amplitude of diaphragmatic movement and the alteration of changes in diaphragm thickness with the contralateral side also important. In this case, liver and lung diaphragm were found at the locations results of pre-operative posteroanterior (PA) imaging the side: five or six rib levels than on the side. When the patient cried, normal diaphragmatic was observed. The | INtRODUCTIoN {#sEcTIOn1-2050313x19827737}
============
cONGeNItAl DiaPhraGMatIC EVentrATioN iS AN aBnoRMaL DiapHRaGmaTic eLEvAtIOn CaUSeD bY INsuFfIcieNt Or ABSeNt muScuLaRIZATION Of ThE pLeURoPerItONeaL MEMbrAne.^[@bIbr1-2050313X19827737]^ It is dIFfiCuLT TO AsSEsS thE eXACt iNCIdeNcE OF ThiS abnormaliTY BECauSe It is RARe ANd gENERaLlY DIagNOSeD inCIDenTaLlY On chesT rAdIoGrAPHy. sYmPtOMS of DIAPhrAgmatiC eVeNtRaTIOn vAry FROm AsyMPTomAtIC, mild gastRoiNtEsTinaL DiSease to LiFe-ThREATeniNg DiapHrAgM rupTuRe.^[@biBr2-2050313X19827737]^ BoTH iNdUCTiON aNd EmERgenCe from ANEsthesIa ShouLD BE SMoOTH tO aVoId ABdOMINaL preSsURe IncrEasE, AS tHIS mAY cAUSE dIapHRAgmATIC ruPTURE.^[@bibR3-2050313X19827737]^ if tHE DIAPhRagm RuptuRes, iT SHOuld BE rAPIDLY DiaGnOSED AND TreAtED. lunG ULTRasoNOgRAPHy cAN bE uSEd tO MONitoR DIaphRaGmaTIC MOvEment On A ReaL-time baSIs.^[@biBr4-2050313x19827737]^ thIS CASE REPoRT DEsCRIBES ThE ANeSThETIC MaNaGEmenT Of a pEDiAtRIC PAtIeNt With CoNGeniTal DiaphragmaTic evENTRAtioN AnD perIOpeRatIve obSErvaTIon Of DIApHRAgmAtiC mOtIoN USinG LuNg UlTrASoNoGrapHy.
caSe repOrt {#SeCtION2-2050313X19827737}
===========
A 28-mOnth-Old malE (HeiGHt: 87.8 CM, WEight: 11.3 Kg) WaS sChEdULEd fOR exciSIonaL BioPSy oF osTeOchoNDROma on THE riGHt dIstal ULnA. The PAtiEnt HAD A FAmiLy hIsTORy oF oSTeochoNdROmA, AND THe sUrgERy wAs pLaNNED TO pREvENT fuRTher dEFoRMITy oF the riGHT ARM. thE BOy Was BORN at a gEStATIoNAL AGE OF 29 WeekS anD 2 dAys WItH a birth WeiGhT OF 1400 g. HE wAs tRaNSfERred tO thE NeonaTaL INtEnSive CAre UNiT (nIcu) aFteR BIRTh anD rEmAined tHeRe fOR 54 dAYS. HaZInESs was NOteD iN ThE riGHt LOWEr LUNG ON cHeST RadiograPhy 30 DAys aftER tHE INFaNT's aDmisSIon To tHe nIcU ([fiGure 1(a)](#fig1-2050313x19827737){reF-tyPe="FIg"}). bAsed oN THIs fINDing, fluOROScoPY WAS PerfOrMEd AnD THe pATiENt wAS DiAGNOSED with EveNtratiOn, whiCh PerSisted UntIL his DischARgE. at ThE time OF DiSCHArGE, no abnoRMAL DIaPHRagmATIC mOVemeNt wAs ObSERvED oN FLUORoscOPY desPiTE RiGhT hEmIDiaphRagm elEVatIOn. AFTer tHE pATiENt WAs dISchargED fRoM THE hosPItAL, HIS CLINICal CouRsE WAS UneVeNtfuL anD SImilar To thAt of OtheR bABIES oF thE sAME agE. as PArT Of tHe prE-opeRAtivE EvaLUATion, cheST raDiogRaphy shOwEd aN ABNoRMal fInDInG InDiCaTINg eventRaTiOn oF tHE RiGHT diaphRAGm ([figuRE 1(B)](#fiG1-2050313X19827737){ReF-tyPe="Fig"}). siNcE NO PUlmonarY sympToms WeRe PreSeNt, THE POSSIBilITy oF atELEcTasIS OR pNeumonic cONSoLidATiOn wAS RuLed OUt. The patIent waS AlrEADy DIAGnOSeD wIth dIAPhRagMaTIc evENTraTiOn BY fLuORosCoPY and uLTrasONOGraPhy. bLood TEsT And EleCTRocarDIOgRAM ReSulTs Were alL WItHiN normal RANGEs.
{#Fig1-2050313X19827737}
BECaUsE tHe PATIENt wAS NOn-cOOPeRAtivE as A rESuLT OF hiS YOUng Age, aneSThESiA WAS INduCed witH kETAMiNe 1.5 mg/KG bEforE suRGery. WitH the VeNtIlatOR, SPontAnEOus TIdaL VoLuMe WAS AbOUT 100 Ml BEfore inTUbATiOn. aFTer the ADMinIsTrAtioN of 0.8 MG/kG rOcURoNiUM As A muScle relaXANt, BAg-VaLve-MAsk vENTilAtIoN WAS iNitiAtEd WitH PResSurE lesS THAN 15 cMh~2~o and TIDaL VOLUME UNdER 100 Ml. INTUbAtIoN was perfoRMed WiTH a CUfFED eNDOTracheaL TUbE (siZe 4.0). lUNg soUndS WERE ClEAr on THe uPper anD lOwer lEft SidE. HOweVEr, ON thE rIGht siDe, LUng SOUNDS WERE aUscuLtateD oNlY on THE UpPer sIdE, buT NOT ON THe LowEr SidE. anEsTHESia Was maintAined witH SEVOFluRANE anD REMIFEntanIl. bRonchoSCOPy (3.1 MM DIAMEter, OlYMpUs AmeRicA, bROokLyn paRK, Mn, Usa) wAS UsED tO IDeNTify tHree oPENiNgs ON THE rigHT brONChUs. INITIalLY, Volume-COntroLled VEnTilAtiON WAS seT AT tIdal vOLUmE 100 ml, reSPirATORY rATe 25, frACTIon OF iNsPIRed OXygeN (fIo~2~) 50%, ANd FlOw 3 l. witH tHese iniTIal VentIlaToR setTInGS, PeaK PRESSuRE waS 25 cmH~2~O. BEforE ThE iNitiATioN of SURgEry, tHE ZOne oF APpOsiTIOn WAS eXAmINED UsING ULtrASOnogrApHy. to PrevENT An INcrEAsE in aBdOMINal pReSSuRe, tHE pRoBe WAs pLAced On tHE lAteraL cHEST wall iNstEad Of The ABdomEN.^[@BIBr5-2050313X19827737]^ luNG slIding ANd pLEUraL EDgE wErE observEd In thE 8Th throUgH The 10Th iNTErCosTal sPaCeS ON tHE LeFt siDE anD IN The 5th THROugh The 7Th iNTERCOsTal SPACEs ON thE RIghT sIdE.
AT AbOuT 15 MiN AfTeR ThE iNiTIATIon Of sURGEry, tHE PEak PRESSuRE sUDDeNlY RoSe tO 28--30 mMHg. lUnG uLTrAsOnOGRaphY WAs rEPEATeD TO DetERMINE WHeTheR tHE RISe in PreSSure was duE tO singLE luNg VEntILaTIoN anD DIAPhRAGMaTIc rUptUre. iN botH LUNg fIeldS, tHE lEveL Of lUng SLIdIng motioN REmAiNEd ThE SAME AS tHAt oBSERVeD duRIng thE INitIAl exAMINaTioN. The dIAPHRAgm dEmonstRaTeD good MOveMent. tHe SeVOfLURANe cOnCeNTrAtION was inCReasED tO 2.5% to HEIGHtEN THe Depth oF AnEsThESIA. To pREvenT diAphrAGmaTIc RUpture, thE tIdAl voLUME WAS loWered To 90 Ml. consEQUeNTly, THe pEAk PReSsuRe dRoPPED To 22 cMH~2~o WhiLe mAInTaiNing eNd-TidAL Co~2~ (etCO~2~) At abOut 38 MMhG. FOLlowINg SURGeRY, THe moTioN oF THe DiAPHraGM waS ExamiNED WITH Real-tIme lUNg UltrASONoGRaPhIC imaGinG iN tHE PoST-ANESTheSIA CarE uniT. IT was dIFfIcULt To oBseRVE sLOw mOvemEnt of LUnG sLIDing bECaUsE ThE CHIlD wAS CrYINg. hOwevEr, THe pleUrAL edGE MOvED rapidLy iN THE cAUDAl dirEctIoN iN thE ZONe of apposITIOn WHeN the PatienT CRiED, crEAting EFFECTs sImIlAR to A sNiFfINg test (seE [SuPPlEMENTAL vIDeO](HTtPs://jOUrNaLS.saGEPUB.COM/doI/SUPpl/10.1177/2050313x19827737)). tHuS, thE pOSsIBILity of dIaPhRagMatIC rUPTURE was exCLUdED.
disCUSSIoN {#sectIoN3-2050313X19827737}
==========
DIaPHRAGmAtIc EVENtRATion iS sO rare thAT it IS DIfFicULT tO mEAsure THE EXacT IncideNce. ALThOuGh tHErE aRe sOmE rEpoRTs of THe AneSThetic MANAGemeNt OF aduLt pAtIENtS WITh thIs COndITION, cASEs Of pEdiAtrIc pAtieNTs ARe LImited.^[@BIBR6-2050313X19827737]^ the REpoRtED iNcidENce Of PEDiAtRIc coNGENITAL DiApHrAGMaTIC EvenTratioN IS 1/1400,^[@biBr7-2050313X19827737]^ AnD OnLY oNe Case Of SpONtaNeOus rUpture oF A congEnitAl dIAPHRaGMATic EvenTrATiOn iN aN infANT HAs bEEn rEPOrted.^[@BibR8-2050313x19827737]^ HOWeVEr, THE EXaCT iNcIdeNCe Of tHE CondItIoN is unkNoWn IN thE geNeRAl PopUlATIoN.
paRTial dIAPhrAGM EleVatiOn has gEneRALLy beEN foUnD on the AnTeroMeDiaL RiGhT hemiDiaPhRAgM, wHiLe COMPlEtE ElEVATion haS been obsERVeD On tHe lEft HeMidiApHrAgM.^[@biBR9-2050313x19827737]^ WHIle hyPopLAstic luNg-reLAted DiAphrAGMaTIC EveNTRATiON is dIaGnOSED using RADioGRaPhY, FluorOScOPY, anD COmPUtEd TOmOgrApHY (cT),^[@bIBr7-2050313x19827737]^ iT iS diffICUlT to CoUNt the NUMbEr of lunG lObEs On ct imAGeS. HOwevEr, tHree Lobe openINgs oN tHE rIghT LUNg WeRE coNfIrmed oN BrOnChoScopY in tHE preSeNt CaSe duRiNG geNerAL AneStHeSiA. in addItION, DIApHraGMAtiC mOtION WaS noTeD On fLUorosCopY, ALtHOUGH COngEnitAl eveNtrATiOn Of tHE diaphraGM is norMally aSsoCiated WiTH iNADEqUate DevELoPMeNT OF musclES Or aBSence oF thE PHRenic nERvE.
eXTRa PREcAUTIonS are rEQUiReD When AdminisTeRinG GeNEral aNesThesiA iN pAtIeNtS WiTH diaPhrAGmaTiC EVeNtrAtIOn. A SUDdEn Increase iN iNtRa-ABdoMiNAL presSURe may cause DiAPhrAgmAtiC ruptUre, EspECIALlY In paTiEnts WitH an AbnormAl diaPHragm. tHEREFOrE, ThE pREVEnTIoN Of SeVERE CoughinG aND BuCkIng iN patieNtS ShOulD BE ensUred. If DIapHrAGmaTiC RUpTuRE oCcurS, caRDIAC outPut Will DEcreasE WitH tHe MiGRAtioN OF Intra-aBdoMinAl ORGaNs FROm the InTRA-AbDOminal spaCe To thE INTrA-ThorACic SpAcE, RESUltiNG IN COMpREsSIon OF ThE HEaRT, Aorta, And vENa cava. tHus, It Is impORTAnT to mAINtAIn suFfIcIeNt AnEsthEtiC DePtH duRING InDUCTion and emERGEnCE. in ADdItion, LoW-volume BAG-valVE-mASK VeNtILATIOn IS NeceSSary tO PrEVEnt pEAk prEsSuRE INCreASe. moReOvER, tOtal IntRAveNouS aNeStheSIa iS PreFeRREd TO BALAnCeD anestheSIA wItH inHALaTion as ThE LAttER caUsEs HyPOXic pUlmonArY vASoCONsTRIctiON. PeAk prEssuRE may rise upon sINGle LuNg venTiLaTIon OR miGRATIon OF aBDoMiNAL OrGAnS To the thorACiC AreA AftER DIaPhragmatic RUPTuRe. WHEn tHe Peak pReSsURE doES RIse, it is iMpoRtANt To deTeRmine Its cauSe. in aDDitIon, LUNG ULTRasoNOgRapHY cAN BE Useful To EVaLuaTE DiaPHRAGm FUNCTIOn, ALthoUgh THere is NO ConSenSuS on the SENsItivItY Of ULtRasOund in ThIs ASseSsmeNT.^[@BibR10-2050313x19827737]^
AbNOrmal DiAPHraGmATic MOtIoN DurInG BrEaTHInG CAn be eXAmineD IN m-MODe UltrAsONoGrApHy.^[@bibR4-2050313x19827737]^ noRmAl DiaPhrAGmAtIc movEMENT dUring InsPIrATIOn sHOWS movEMenT TOward ThE trAnsDucer wHeN tHe TrAnsdUceR IS On The RIght BElOw ThE nORmal dIaphrAgM POsiTIoN. whEN tHe DiAPhragM rUptUrEs, THE diAPhRAGM mAY aPpEar to be FLOaTInG oR iNviSible, oR a SuBpHRENIC FluId ColLectiON mAy APPEar ON uLtrAsONOgrAPhy.^[@bIbr11-2050313x19827737]^ UltraSoNogrApHY mAY sHow HErnIAtion oF ThE soLiD abdOMINAL cOnteNTs, sUch As thE lIveR, OMeNTuM, Or a BOwEL sEGmeNt wiTh PERiSTALTIC aCtivitY. IF The lIvEr slidINg tHAt is hEPaTIc paReNCHyMaL MOVemeNt is SHOwn on tHe rIght UPpEr TrUnK insTeAd oF tHE LUng PArENcHyma, IT mAy iNDicate LIVeR HeRNiaTIOn.^[@bIBR12-2050313X19827737]^ CoMPaRisoN OF THE AMpLitude of DIApHrAgmatIc MOVEmENt aNd THE AlteRaTiON of chAngeS in DiapHraGM tHiCkNESS wiTH ThE cONTRAlAtErAL sIdE IS aLsO ImPoRTAnT. iN tHIS CaSE, lIVER And LuNg DiaPHrAgM WERe FOUNd AT tHe LOcaTIoNS ExpeCtED FroM THE REsuLts Of PRE-oPErAtIve cHEst POSteROanteRIor (PA) imAgiNg of the RIGhT SIDE: fivE OR SiX RIB lEVELs hiGHeR ThAn on tHE lefT SidE. wHeN THe pATIenT CRiEd, normaL diApHrAGMATIc eXCursiOn WAs OBSErVEd. THe | Introduction {#section1-2050313X19827737} ============Congenital diaphragmatic eventration is an abnormaldiaphragmatic elevation caused by insufficient or absent muscularization of the pleuroperitoneal membrane.^[@bibr1-2050313X19827737]^ It is difficult to assess the exact incidence ofthis abnormality because it is rare and generallydiagnosed incidentally on chest radiography. Symptoms of diaphragmatic eventrationvary from asymptomatic, mild gastrointestinal disease to life-threatening diaphragm rupture.^[@bibr2-2050313X19827737]^ Bothinduction and emergence from anesthesia should be smoothto avoidabdominal pressure increase, as this may cause diaphragmatic rupture.^[@bibr3-2050313X19827737]^ If the diaphragm ruptures, it should be rapidly diagnosedand treated. Lungultrasonography can be used to monitor diaphragmatic movement ona real-time basis.^[@bibr4-2050313X19827737]^ This case report describes the anesthetic managementof a pediatric patient with congenital diaphragmatic eventrationandperioperative observation of diaphragmatic motion usinglung ultrasonography. Case report {#section2-2050313X19827737}=========== A 28-month-old male (height: 87.8 cm, weight: 11.3 kg) was scheduledfor excisional biopsy of osteochondroma on the right distal ulna.Thepatient had afamily history of osteochondroma, and the surgery was planned to preventfurther deformity ofthe right arm. The boy was born at a gestational age of29 weeks and 2 days with a birthweight of 1400 g. He wastransferred to the neonatal intensive care unit (NICU) after birth and remained there for 54 days. Haziness was notedin the right lower lung on chest radiography 30 days afterthe infant's admission to the NICU ([Figure 1(a)](#fig1-2050313X19827737){ref-type="fig"}). Based on this finding, fluoroscopy was performed and the patientwas diagnosed witheventration,which persisted until his discharge. At thetime of discharge, noabnormal diaphragmatic movement wasobserved on fluoroscopy despite right hemidiaphragm elevation. After the patient was discharged from the hospital, his clinical course was uneventful andsimilarto that of otherbabies of the same age. As part of the pre-operativeevaluation, chest radiography showed an abnormalfinding indicating eventration of the right diaphragm ([Figure 1(b)](#fig1-2050313X19827737){ref-type="fig"}). Sinceno pulmonary symptoms were present, the possibility ofatelectasis or pneumonic consolidation was ruledout. The patient was already diagnosed with diaphragmatic eventration by fluoroscopy and ultrasonography. Blood test and electrocardiogram results wereall withinnormal ranges. {#fig1-2050313X19827737} Because the patient was non-cooperative as a resultof his young age, anesthesia was inducedwithketamine1.5 mg/kg before surgery. With the ventilator, spontaneoustidal volume wasabout 100 mL before intubation. After the administration of 0.8 mg/kg rocuronium asa musclerelaxant, bag-valve-mask ventilation was initiated with pressureless than15 cmH~2~O and tidalvolume under 100 mL.Intubation was performed with a cuffed endotracheal tube (size 4.0). Lung sounds were clearon the upper andlower left side. However,on the right side, lung soundswereauscultated only on the upper side, but not on the lower side. Anesthesia was maintainedwith sevoflurane and remifentanil. Bronchoscopy (3.1 mm diameter, Olympus America,Brooklyn Park, MN, USA) was used to identify three openingsonthe right bronchus. Initially, volume-controlled ventilation was setat tidal volume 100 mL, respiratory rate 25, fraction of inspired oxygen (FiO~2~) 50%, and flow3L. With these initial ventilator settings, peak pressurewas 25 cmH~2~O. Beforethe initiation of surgery, the zone of apposition was examined using ultrasonography. To prevent an increase in abdominalpressure, the probe was placed on the lateral chest wall instead ofthe abdomen.^[@bibr5-2050313X19827737]^ Lung sliding and pleural edge were observed inthe 8th through the 10th intercostal spaces on the left side and in the5th through the7th intercostal spaces on theright side. At about 15 min after the initiation of surgery,the peak pressure suddenlyrose to28--30 mmHg. Lung ultrasonography was repeated to determinewhether the rise in pressure was due to single lung ventilation and diaphragmatic rupture. In both lungfields, the level of lung sliding motion remained thesame asthat observed during the initial examination. The diaphragm demonstrated good movement. The sevoflurane concentration was increasedto 2.5% to heighten thedepth of anesthesia. To prevent diaphragmaticrupture, the tidal volumewas lowered to 90mL. Consequently, the peak pressuredropped to 22cmH~2~O while maintaining end-tidal CO~2~ (ETCO~2~) at about 38 mmHg.Following surgery, the motionof the diaphragm was examinedwith real-timelung ultrasonographic imaging in thepost-anesthesia care unit. It was difficult to observe slow movement of lung sliding because the child was crying. However, the pleural edge moved rapidly in the caudal direction in the zone of apposition when the patient cried, creating effects similar to a sniffing test (see[Supplemental Video](https://journals.sagepub.com/doi/suppl/10.1177/2050313X19827737)). Thus, the possibility of diaphragmatic rupture wasexcluded. Discussion {#section3-2050313X19827737} ==========Diaphragmatic eventration isso rare that it is difficult to measure the exact incidence. Although therearesome reports of the anesthetic management of adult patients withthis condition, casesof pediatric patients are limited.^[@bibr6-2050313X19827737]^ The reported incidence of pediatric congenital diaphragmatic eventration is 1/1400,^[@bibr7-2050313X19827737]^ andonly one case of spontaneous rupture of a congenitaldiaphragmatic eventration in an infant has been reported.^[@bibr8-2050313X19827737]^ However,the exactincidence of thecondition isunknown in the general population. Partial diaphragm elevation has generally been found on the anteromedial right hemidiaphragm, while complete elevationhas been observedonthe left hemidiaphragm.^[@bibr9-2050313X19827737]^ While hypoplastic lung-related diaphragmatic eventration is diagnosed using radiography, fluoroscopy, and computedtomography(CT),^[@bibr7-2050313X19827737]^ itis difficult to count thenumber oflung lobes on CTimages. However, three lobe openings on the right lung were confirmed on bronchoscopy in the present case during general anesthesia. In addition, diaphragmatic motion was notedon fluoroscopy, although congenital eventration ofthe diaphragm is normally associated with inadequate development of muscles or absence of the phrenic nerve.Extra precautions are required when administering general anesthesia in patientswith diaphragmatic eventration. A sudden increase in intra-abdominal pressuremay cause diaphragmatic rupture, especially inpatients with an abnormal diaphragm. Therefore, the prevention of severe coughing and bucking inpatients should be ensured.If diaphragmatic rupture occurs, cardiac output will decrease with the migration of intra-abdominalorgans from the intra-abdominalspace tothe intra-thoracicspace, resultingin compression of the heart,aorta, and vena cava. Thus, it is important to maintain sufficient anestheticdepth during induction and emergence. In addition, low-volume bag-valve-maskventilation is necessary to prevent peak pressureincrease. Moreover, total intravenous anesthesia is preferredto balanced anesthesiawith inhalation as the latter causeshypoxic pulmonary vasoconstriction. Peak pressure may rise upon single lung ventilation or migrationof abdominalorgans to the thoracic area after diaphragmatic rupture. When the peak pressure does rise, it is important todetermine itscause. In addition, lung ultrasonography can be useful to evaluate diaphragm function, although there is no consensus on the sensitivityof ultrasound in this assessment.^[@bibr10-2050313X19827737]^ Abnormal diaphragmatic motion duringbreathing can be examined in M-mode ultrasonography.^[@bibr4-2050313X19827737]^ Normal diaphragmatic movement duringinspiration shows movement toward the transducer when the transducer is on the right below the normal diaphragm position. When the diaphragmruptures, the diaphragmmay appear to be floating orinvisible, or a subphrenic fluid collection may appear on ultrasonography.^[@bibr11-2050313X19827737]^Ultrasonography may show herniation of thesolidabdominal contents, such asthe liver, omentum,ora bowel segment with peristalticactivity. If the liver slidingthat is hepatic parenchymal movement is shown on the right upper trunk insteadof the lung parenchyma, it may indicateliver herniation.^[@bibr12-2050313X19827737]^ Comparisonof theamplitude of diaphragmatic movement and the alteration of changes in diaphragm thickness with thecontralateral side is also important. In this case, liver and lungdiaphragm were found at the locations expected from theresultsof pre-operative chest posteroanterior (PA) imaging of the right side: five or six rib levels higher than on the leftside.Whenthe patient cried, normal diaphragmatic excursionwas observed. The | _Introduction_ {#section1-2050313X19827737} ============ Congenital _diaphragmatic_ eventration _is_ an abnormal diaphragmatic elevation caused _by_ insufficient or absent muscularization of the pleuroperitoneal membrane.^[@bibr1-2050313X19827737]^ It is difficult to assess the exact incidence of this abnormality _because_ it _is_ rare and _generally_ diagnosed incidentally on chest _radiography._ Symptoms of diaphragmatic eventration _vary_ _from_ asymptomatic, _mild_ gastrointestinal disease to life-threatening _diaphragm_ rupture.^[@bibr2-2050313X19827737]^ Both induction _and_ _emergence_ from anesthesia should be _smooth_ to _avoid_ abdominal pressure increase, as this may cause diaphragmatic rupture.^[@bibr3-2050313X19827737]^ If the _diaphragm_ ruptures, it should be rapidly diagnosed and treated. Lung ultrasonography can be used to monitor diaphragmatic movement on a real-time _basis.^[@bibr4-2050313X19827737]^_ _This_ _case_ report describes the anesthetic management of a pediatric patient with congenital diaphragmatic _eventration_ and perioperative observation of diaphragmatic motion _using_ lung ultrasonography. Case report {#section2-2050313X19827737} =========== A 28-month-old _male_ (height: 87.8 _cm,_ weight: _11.3_ kg) _was_ scheduled for excisional biopsy of _osteochondroma_ on the right distal ulna. The patient had a _family_ history of osteochondroma, _and_ _the_ surgery _was_ _planned_ _to_ prevent _further_ deformity _of_ _the_ right arm. _The_ boy was born at _a_ gestational age of _29_ weeks and 2 days with a birth _weight_ of _1400_ g. He _was_ transferred to the neonatal intensive _care_ unit _(NICU)_ after birth and remained there for 54 _days._ Haziness _was_ noted in the right lower lung on chest radiography _30_ days after the infant's admission to _the_ NICU ([Figure 1(a)](#fig1-2050313X19827737){ref-type="fig"}). Based on _this_ finding, fluoroscopy _was_ performed and the patient _was_ _diagnosed_ with _eventration,_ which persisted until his discharge. At the time of discharge, no abnormal diaphragmatic movement was observed on _fluoroscopy_ despite right hemidiaphragm elevation. After the patient was _discharged_ from _the_ hospital, _his_ clinical course was uneventful and similar _to_ that _of_ other babies of the same age. As part _of_ _the_ pre-operative evaluation, chest radiography showed an abnormal finding indicating eventration of the right diaphragm ([Figure 1(b)](#fig1-2050313X19827737){ref-type="fig"}). Since _no_ pulmonary symptoms were _present,_ the _possibility_ _of_ _atelectasis_ or pneumonic consolidation was ruled out. The patient was _already_ diagnosed with diaphragmatic eventration by fluoroscopy and ultrasonography. Blood test _and_ electrocardiogram results were all within _normal_ ranges. {#fig1-2050313X19827737} Because the patient was _non-cooperative_ as a result of his young age, anesthesia was _induced_ with ketamine 1.5 mg/kg before surgery. With _the_ ventilator, _spontaneous_ _tidal_ volume _was_ _about_ 100 _mL_ before intubation. After the administration of _0.8_ mg/kg rocuronium _as_ a muscle relaxant, bag-valve-mask ventilation was initiated with pressure less than 15 cmH~2~O and tidal volume _under_ 100 mL. _Intubation_ _was_ performed with a cuffed endotracheal tube _(size_ 4.0). _Lung_ sounds were _clear_ on the _upper_ and lower left _side._ However, _on_ the right side, lung sounds were _auscultated_ _only_ on the upper side, _but_ not on the lower side. Anesthesia was maintained with sevoflurane and remifentanil. Bronchoscopy (3.1 mm diameter, Olympus _America,_ Brooklyn _Park,_ MN, _USA)_ _was_ used to identify three _openings_ on the right bronchus. Initially, volume-controlled ventilation was set at tidal volume 100 _mL,_ respiratory rate 25, fraction of inspired _oxygen_ (FiO~2~) 50%, _and_ flow _3_ L. With these initial ventilator settings, _peak_ pressure was _25_ cmH~2~O. Before the _initiation_ _of_ surgery, the zone of apposition _was_ examined using ultrasonography. To prevent _an_ _increase_ in abdominal _pressure,_ the _probe_ was placed on the lateral chest wall instead of the abdomen.^[@bibr5-2050313X19827737]^ Lung sliding and pleural edge were _observed_ _in_ the _8th_ through the 10th intercostal spaces on the left _side_ and _in_ the _5th_ through the 7th intercostal spaces on the right side. At about 15 _min_ after the initiation _of_ surgery, the peak pressure suddenly rose _to_ 28--30 mmHg. _Lung_ _ultrasonography_ was repeated to determine _whether_ _the_ rise in _pressure_ _was_ _due_ to _single_ lung ventilation and diaphragmatic _rupture._ In both lung fields, the level of lung _sliding_ _motion_ remained the same as that observed during the initial examination. The diaphragm _demonstrated_ good movement. The _sevoflurane_ concentration was increased to _2.5%_ _to_ heighten the _depth_ of _anesthesia._ To _prevent_ _diaphragmatic_ rupture, the tidal volume was lowered to 90 mL. Consequently, _the_ peak pressure _dropped_ to _22_ _cmH~2~O_ _while_ _maintaining_ end-tidal CO~2~ (ETCO~2~) at about 38 mmHg. Following surgery, the motion of the diaphragm was examined with real-time lung _ultrasonographic_ imaging in the _post-anesthesia_ care unit. It _was_ difficult to _observe_ slow movement _of_ _lung_ sliding because the child was crying. However, the pleural edge _moved_ _rapidly_ in the caudal _direction_ _in_ the zone _of_ apposition when the patient cried, creating effects similar to _a_ sniffing _test_ (see [Supplemental Video](https://journals.sagepub.com/doi/suppl/10.1177/2050313X19827737)). Thus, the possibility of _diaphragmatic_ rupture was excluded. Discussion {#section3-2050313X19827737} ========== Diaphragmatic eventration is so rare that it is difficult to measure the exact incidence. Although there are some reports of _the_ anesthetic management of _adult_ patients _with_ this condition, cases of pediatric patients are limited.^[@bibr6-2050313X19827737]^ The _reported_ incidence _of_ pediatric congenital _diaphragmatic_ eventration is 1/1400,^[@bibr7-2050313X19827737]^ and only one case _of_ spontaneous rupture of a congenital diaphragmatic eventration _in_ an infant has been _reported.^[@bibr8-2050313X19827737]^_ However, the exact incidence _of_ the condition is unknown in the general population. Partial diaphragm elevation has _generally_ _been_ found on _the_ anteromedial right hemidiaphragm, while _complete_ elevation has been observed _on_ the left hemidiaphragm.^[@bibr9-2050313X19827737]^ While hypoplastic _lung-related_ _diaphragmatic_ eventration is diagnosed _using_ radiography, fluoroscopy, and computed tomography (CT),^[@bibr7-2050313X19827737]^ it _is_ difficult to count _the_ number of lung _lobes_ on CT _images._ However, three lobe _openings_ on the right lung _were_ confirmed _on_ _bronchoscopy_ in the present _case_ _during_ general _anesthesia._ In addition, diaphragmatic motion was _noted_ on fluoroscopy, _although_ congenital eventration of the _diaphragm_ is normally associated with inadequate development of muscles or absence of the phrenic _nerve._ Extra precautions _are_ required when administering general anesthesia in patients with diaphragmatic _eventration._ _A_ sudden increase _in_ _intra-abdominal_ pressure may cause diaphragmatic rupture, especially in patients with an _abnormal_ diaphragm. Therefore, the prevention _of_ severe coughing and bucking in patients should be ensured. If diaphragmatic rupture occurs, cardiac _output_ will decrease with _the_ migration of intra-abdominal organs from _the_ intra-abdominal _space_ to the intra-thoracic _space,_ resulting _in_ compression of the heart, aorta, _and_ _vena_ cava. Thus, it _is_ important to _maintain_ sufficient anesthetic _depth_ during induction _and_ emergence. In addition, low-volume _bag-valve-mask_ _ventilation_ _is_ _necessary_ _to_ prevent peak pressure increase. Moreover, total intravenous anesthesia _is_ preferred to balanced anesthesia with inhalation as the _latter_ causes hypoxic pulmonary _vasoconstriction._ Peak pressure may _rise_ upon single lung ventilation _or_ migration of abdominal organs to the thoracic area after _diaphragmatic_ rupture. When the peak pressure does rise, it is important to determine _its_ _cause._ In addition, lung ultrasonography _can_ be useful to _evaluate_ _diaphragm_ function, _although_ there is no _consensus_ _on_ the sensitivity _of_ ultrasound in this assessment.^[@bibr10-2050313X19827737]^ Abnormal diaphragmatic motion during breathing can _be_ examined in M-mode ultrasonography.^[@bibr4-2050313X19827737]^ _Normal_ diaphragmatic _movement_ during inspiration shows movement toward _the_ transducer when the transducer is on the right below the normal diaphragm _position._ When the _diaphragm_ ruptures, the diaphragm may appear to be floating _or_ invisible, or a subphrenic _fluid_ collection _may_ _appear_ on ultrasonography.^[@bibr11-2050313X19827737]^ Ultrasonography may _show_ herniation of the solid abdominal contents, such _as_ the liver, omentum, or a bowel segment _with_ _peristaltic_ _activity._ If the liver _sliding_ that is hepatic parenchymal movement is _shown_ on the right upper trunk instead _of_ the lung parenchyma, it may _indicate_ liver herniation.^[@bibr12-2050313X19827737]^ Comparison of the amplitude of diaphragmatic movement and the _alteration_ _of_ _changes_ in diaphragm _thickness_ with the _contralateral_ side is also _important._ In this case, liver _and_ lung _diaphragm_ were found at the locations expected from the results of pre-operative chest posteroanterior (PA) imaging _of_ the right side: five _or_ six rib levels higher than on the left side. _When_ the patient cried, normal _diaphragmatic_ excursion _was_ observed. The |
Introduction {#s1}
============
Diabetic dyslipidaemia is characterised by hypertriglyceridaemia, low high-density lipoprotein (HDL) cholesterol (c) and normal low-density lipoprotein-cholesterol (LDLc) but preponderance of small-dense, highly atherogenic particles. The increase in free fatty acids (FFAs) as degradation products of triglycerides (TGs) is associated with the development of insulin resistance [@pone.0027208-Wilding1].
Cholesteryl Ester Transfer Protein (CETP) and Hepatic Lipase (HL) are central enzymes in the metabolism of HDL particles and reverse cholesterol transport. CETP is responsible for an exchange of cholesteryl ester (CE) for triglycerides (TGs) between LDL and HDL and TG rich-lipoprotein particles [@pone.0027208-Morton1]. The result is an enrichment of HDL and LDL particles in TGs, which makes them good substrates for HL [@pone.0027208-Thuren1]. The latter catalyses the hydrolysis of the TGs and phospholipids present in several lipoprotein subclasses, leading to changes in the size and density of lipoproteins [@pone.0027208-Thuren1]. The increased activity of either enzyme results in lower HDLc levels and a predominance of small, dense HDL and LDL particles [@pone.0027208-Lagrost1], [@pone.0027208-Zambon1].
The variations in the *CETP* gene, which lead to changes in enzyme function, have consequences on lipoprotein composition. *CETP* deficiency in humans is characterized by increases in HDLc, whereas increases in its activity are associated with an enrichment of HDL particles in TGs and a decrease in HDLc levels [@pone.0027208-Ikewaki1]. The most extensively studied polymorphism in *CETP* is Taq1B (rs708272) [@pone.0027208-Kondo1]. The G allele, also called B1, is associated with higher enzymatic activity, higher CETP mass and lower HDLc levels [@pone.0027208-Noone1], [@pone.0027208-Boekholdt1]. It has been estimated that this polymorphism is responsible for 5.8% of the variation in HDLc levels [@pone.0027208-Corella1].
Studies in transgenic mice demonstrate that the over-expression of the gene encoding HL, *Lipc*, leads to a marked decrease in plasma HDLc levels[@pone.0027208-Isaacs1] , an observation supported by human studies showing an inverse correlation between HL activity and HDLc concentrations [@pone.0027208-Blades1]. The -G250A *LIPC* polymorphism (rs2070895) [@pone.0027208-Todorova1], located in the promoter region of the gene, has been extensively studied in relation to enzyme activity and lipid metabolism. The minor allele (A) is associated with a reduction of transcriptional activity *in vitro* [@pone.0027208-Deeb1] and a 15--45% reduction in enzymatic activity [@pone.0027208-Tahvanainen1]. In humans, the minor allele has also been associated with an increased HDLc concentration and more buoyant LDL particles [@pone.0027208-Tahvanainen1], [@pone.0027208-Zambon2]. Studies assessing the association of this variant with T2D show conflicting results [@pone.0027208-Zacharova1].
Diabetes is often preceded and even predicted, by the presence of dyslipidemia [@pone.0027208-Todorova1]. Thus, mechanisms involved in the development of diabetic dyslipidemia may also play a role in the pathogenesis of T2D. The effects of the mentioned polymorphisms in *CETP* and *LIPC* on HDLc concentrations are well established, but their relation with the risk of T2D is less known. Therefore, the aim of our study was to analyze the relationship between polymorphisms in these two genes and the presence of diabetes and insulin resistance in a Canarian population.
Methods {#s2}
=======
Study population {#s2a}
----------------
The Telde study is a cross-sectional population-based study on the prevalence of diabetes and cardiovascular risk factors in Telde, a city located on the island of Gran Canaria, Spain. The study population and design of this survey has been previously described [@pone.0027208-Boronat1]. An oral glucose tolerance test (OGTT) was performed and the subjects were classified (using ADA 1997 criteria) as diabetic (n = 115) and pre-diabetic (n = 116) if they had impaired fasting glucose, impaired glucose tolerance or both. A total of 226 subjects with a normal OGTT were selected, after matching for gender and age with the other two groups. All participants gave their written informed consent for participation in the study, which was carried out according to the declaration of Helsinki and approved by the local ethics committee.
Genetic analyses {#s2b}
----------------
The biochemical analyses and insulin resistance parameters have been described previously [@pone.0027208-Novoa1]. Genomic DNA was extracted from whole blood (n = 457) using a salting-out method. The Taq1B *CETP* polymorphism was amplified by PCR-RFLP as described by June Hsieh Wu [@pone.0027208-Wu1] and the *G-250A LIPC* polymorphism was analyzed by AMRS-PCR (Amplification Refractory Mutation System-Polymerase Chain Reaction) [@pone.0027208-Ye1]. Two pairs of primers were used, one which amplifies a fragment of 366 bp, common to both alleles (outer primers: 5′-CTT TTC TTT TTC TTT GGG CTT AGG CT-3′ and 5′-AAG ACT GCC CAT TAA TAA TTA ACC TCT CAA-3′) and another pair specific for the SNP (inner primers): 5′-CAA GGT CAG AGT TCC AAA TTA ATC CAC-3′ for the G allele and 5′-TTC CAA ACA CAA CAC AGT AGC TTT CAA-3′ for the A allele. The primers were designed *"in silico"* in a free access web (<http://cedar.genetics.soton.ac.uk>, accessed in August 2007) and then checked for specificity (<http://blast.ncbi.nlm.nih.gov/Blast.cgi>, accessed in August 2007). The PCR reaction was carried out in a total volume of 25 µl containing 1∶5 ratio of outer to inner primer concentration. The annealing temperatures were 70°C during 15 sec for the outer primers and 58°C during 25 sec for the inner primers with a 30 sec extension at 72°C. PCR products were mixed with 2 µl of loading buffer and run on 2.5% agarose gel stained with Ethidium Bromide. This resulted in 3 DNA fragments: one of 366 bp, one of 234 bp for the A allele and one of 185 bp for the G allele ([figure 1](#pone-0027208-g001){ref-type="fig"}).
{#pone-0027208-g001}
Statistical analysis {#s2c}
--------------------
Statistical analyses were performed with SPSS for WINDOWS, version 13 (SPSS Inc., Chicago, IL). The quantitative variables are described as mean ± standard deviation (S.D). Before further analyses, variable distribution was checked with the Kolmogorov--Smirnov test. A logarithmic transformation was performed for variables not following a Gaussian distribution. Differences between groups were analyzed using either analysis of variance or analysis of covariance, both with the Bonferroni post hoc correction test, after adjusting for age, gender, Body Mass Index (BMI) and waist. The categorical variables were compared using Fisher\'s exact test for 2×2 tables and chi-squared or the Mantel-Haenszel test for linear association. The independent contribution of each polymorphism to DM2 risk was analyzed by a multinomial logistic regression model, which included age, gender, BMI and waist. All tests were considered significant if p was \<0.05.
Regarding the effect of the interaction of both polymorphisms on the risk of diabetes, the reference category was defined by the non-B1B1 genotype, regardless of the -250G/A *LIPC* genotypes (nonB1B1*CETP* genotype). A second group included B1B1 and non-GG genotypes (B1B1CETP/non-GGLIPC) and a third, B1B1 CETP and *LIPC* GG genotypes (B1B1CETP/GGLIPC).
Results {#s3}
=======
Patient description {#s3a}
-------------------
The anthropometric, clinical and genetic characteristics of the whole population and their classification according to the OGTT are shown in [table 1](#pone-0027208-t001){ref-type="table"}. The frequencies of the B1B1, B1B2 and B2B2 genotypes of the | introduction { # s1 } = = = = = = = = = = = = diabetic pregnancy is characterised by hypertriglyceridaemia, low high - density lipoprotein ( hdl ) cholesterol ( c ) and normal low - density lipoprotein - cholesterol ( ldlc ) but preponderance of small - dense, highly atherogenic particles. the increase in free fatty acids ( ffas ) as degradation products of triglycerides ( cf ) is associated with the development of insulin resistance [ @ pone. 0027208 - wilding1 ]. cholesteryl ester transfer protein ( cetp ) and hepatic lipase ( hl ) are central enzymes in the metabolism of hdl particles and reverse cholesterol transport. metabolism is responsible for an exchange of cholesteryl ester ( ce ) for ethanol ( tgs ) between ldl and hdl and tg rich - lipoprotein particles [ @ pone. 0027208 - 001 ]. the result is an enrichment of hdl and ldl particles in tgs, which makes them good substrates for hl [ @ pone. 0027208 - thuren1 ]. the latter catalyses the hydrolysis of the tgs and phospholipids present in several lipoprotein subclasses, leading dramatic changes in the size and density of lipoproteins [ @ pone. 0027208 - thuren1 ]. the increased activity of either enzyme result in lower hdlc levels suggesting a predominance of small, dense hdl and ldl particles [ @ pone. 0027208 - lagrost1 ], [ @ pone. 0027208 - zambon1 ]. the variations in the * cetp * gene, which lead to changes in enzyme function, have consequences on lipoprotein composition. * cetp * deficiency in humans is characterized by increases in hdlc, whereas increases in its activity are associated with an incorporation of hdl particles in tgs and a decrease in hdlc levels [ @ pone. 0027208 - ikewaki1 ]. the most extensively studied protein in * cetp * is taq1b ( rs708272 ) [ @ pone. 0027208 - kondo1 ]. the g allele, also called b1, is associated with higher enzymatic activity, higher cetp mass and lower hdlc levels [ @ pone. 0027208 - noone1 ], [ @ pone. 0027208 - boekholdt1 ]. it has been estimated that this polymorphism is responsible for 5. 8 % of the variation in hdlc levels [ @ pone. 0027208 - corella1 ]. studies in transgenic mice demonstrate that the over - expression of the gene encoding hl, * lipc *, leads to a marked decrease in plasma hdlc levels [ @ pone. 0027208 - isaacs1 ], an observation supported by human studies showing an inverse correlation between hl activity and hdlc concentrations [ @ pone. 0027208 - blades1 ]. the - g250a * lipc * polymorphism ( rs2070895 ) [ @ pone. 0027208 - todorova1 ], located in the promoter region of the gene, has been extensively studied in relation to enzyme activity and lipid metabolism. the minor allele ( a ) is associated with a reduction of transcriptional activity * in vitro * [ @ pone. 0027208 - deeb1 ] and a 15 - - 45 % reduction in enzymatic activity [ @ pone. 0027208 - tahvanainen1 ]. in humans, the minor allele has also been associated with an increased hdlc concentration and more buoyant ldl particles [ @ pone. 0027208 - tahvanainen1 ], [ @ pone. 0027208 - zambon2 ]. studies assessing the association of this variant with t2d show conflicting results [ @ pone. 0027208 - zacharova1 ]. diabetes is often preceded and even predicted, by the presence of dyslipidemia [ @ pone. 0027208 - todorova1 ]. thus, mechanisms involved in the development of diabetic dyslipidemia may also play a role in the pathogenesis of t2d. the effects of the mentioned polymorphisms in * cetp * and * lipc * on hdlc concentrations are well established, but their relation with the risk of t2d is less known. therefore, the aim of our study was to analyze the relationship between polymorphisms in these two genes and the presence of diabetes and insulin resistance in a canarian population. methods { # s2 } = = = = = = = study population { # s2a } - - - - - - - - - - - - - - - - the telde study is a cross - sectional population - based study on the prevalence of diabetes and cardiovascular risk factors in telde, a city located on the island of gran canaria, spain. the study population and design of this survey has been previously described [ @ pone. 0027208 - boronat1 ]. an oral glucose tolerance test ( ogtt ) was performed and the subjects were classified ( using ada 1997 criteria ) as diabetic ( n = 115 ) and pre - diabetic ( n = 116 ) if they had impaired fasting glucose, impaired glucose tolerance or both. a total of 226 subjects with a normal ogtt were selected, after matching for gender and age with the other two groups. all participants gave their written informed consent for participation in the study, which was carried out according to the declaration of helsinki and approved by the local ethics committee. genetic analyses { # s2b } - - - - - - - - - - - - - - - - the biochemical analyses and insulin resistance parameters have been described previously [ @ pone. 0027208 - novoa1 ]. genomic dna was extracted from whole blood ( n = 457 ) using a salting - out method. the taq1b * cetp * polymorphism was amplified by pcr - rflp as described by june hsieh wu [ @ pone. 0027208 - wu1 ] and the * g - 250a lipc * polymorphism was analyzed by amrs - pcr ( amplification refractory mutation system - polymerase chain reaction ) [ @ pone. 0027208 - ye1 ]. two pairs of primers were used, one which amplifies a fragment of 366 bp, common to both alleles ( outer primers : 5 ′ - ctt ttc ttt ttc ttt ggg ctt agg ct - 3 ′ and 5 ′ - aag act gcc cat taa taa tta acc tct caa - 3 ′ ) and another pair specific for the snp ( inner primers ) : 5 ′ - caa ggt cag agt tcc aaa tta atc cac - 3 ′ for the g allele and 5 ′ - ttc caa aca caa cac agt agc ttt caa - 3 ′ for the a allele. the primers were designed * " in silico " * in a free access web ( < http : / / cedar. genetics. soton. ac. uk >, accessed in august 2007 ) and then checked for specificity ( < http : / / blast. ncbi. nlm. nih. gov / blast. cgi >, accessed in august 2007 ). the pcr reaction was carried out in a total volume of 25 µl containing [UNK] ratio of outer to inner primer concentration. the annealing temperatures were 70°c during 15 sec for the outer primers and 58°c during 25 sec for the inner primers with a 30 sec extension at 72°c. pcr products were mixed with 2 µl of loading buffer and run on 2. 5 % agarose gel stained with ethidium bromide. this resulted in 3 dna fragments : one of 366 bp, one of 234 bp for the a allele and one of 185 bp for the g allele ( [ figure 1 ] ( # pone - 0027208 - g001 ) { ref - type = " fig " } ).! [ agarose gel results of both polymorphisms. \ pcr - rflp agarose gel ( a ) after digestion with taq1b enzyme. the 1000 bp band corresponds to the b2 allele and the 650 and 350 bp bands correspond to the b1 allele. results of * lipc * genotyping by arms - pcr ( b ). the 366 bp band is the product of the outer primers, the 234 bp band, of an outer primer and the inner primer for allele a and the 185 bp band, of the other outer primer and the inner primer for the g allele. ] ( pone. 0027208. g001 ) { # pone - 0027208 - g001 } statistical analysis { # s2c } - - - - - - - - - - - - - - - - - - - - statistical analyses were performed with spss for windows, version 13 ( spss inc., chicago, il ). the quantitative variables are described as mean ± standard deviation ( s. d ). before further analyses, variable distribution was checked with the kolmogorov - - smirnov test. a logarithmic transformation was performed for variables not following a gaussian distribution. differences between groups were analyzed using either analysis of variance or analysis of covariance, both with the bonferroni post hoc correction test, after adjusting for age, gender, body mass index ( bmi ) and waist. the categorical variables were compared using fisher \ ' s exact test for 2×2 tables and chi - squared or the mantel - haenszel test for linear association. the independent contribution of each polymorphism to dm2 risk was analyzed by a multinomial logistic regression model, which included age, gender, bmi and waist. all tests were considered significant if p was \ < 0. 05. regarding the effect of the interaction of both polymorphisms on the risk of diabetes, the reference category was defined by the non - b1b1 genotype, regardless of the - 250g / a * lipc * genotypes ( nonb1b1 * cetp * genotype ). a second group included b1b1 and non - gg genotypes ( b1b1cetp / non - gglipc ) and a third, b1b1 cetp and * lipc * gg genotypes ( b1b1cetp / gglipc ). results { # s3 } = = = = = = = patient description { # s3a } - - - - - - - - - - - - - - - - - - - the anthropometric, clinical and genetic characteristics of the whole population and their classification according to the ogtt are shown in [ table 1 ] ( # pone - 0027208 - t001 ) { ref - type = " table " }. the frequencies of the b1b1, b1b2 and b2b2 genotypes of the | Introduction {# s1} = = = = = = = = = = = = Diabetic dyslipidaemia is characterised by hypertriglyceridaemia, low high - density lipoprotein (HDL) cholesterol (c) and normal low - density lipoprotein - cholesterol (LDLc) but preponderance of small - dense, highly atherogenic particles. The increase in free fatty acids (FFAs) as degradation products of triglycerides (TGs) is associated with the development of insulin resistance [@ pone. 0027208 - Wilding1 ]. Cholesteryl Ester Transfer Protein (CETP) and Hepatic Lipase (HL) are central enzymes in the metabolism of HDL particles and reverse cholesterol transport. CETP is responsible for an exchange of cholesteryl ester (CE) for triglycerides (TGs) between LDL and HDL and TG rich - lipoprotein particles [@ pone. 0027208 - Morton1 ]. The result is an enrichment of HDL and LDL particles in TGs, which makes them good substrates for HL [@ pone. 0027208 - Thuren1 ]. The latter catalyses the hydrolysis of the TGs and phospholipids present in several lipoprotein sjbXlasses, leading to changes in the size and Wehsity of lipoproteins [@ pone. 0027208 - Thuren1 ]. The increased activity of either enzyme results in lower HDLc levels and a predominance of small, dense HDL and LDL particles [@ pone. 0027208 - Lagrost1 ], [@ pone. 0027208 - Zambon1 ]. The variations in the * CETP * gene, which lead to changes in enzyme function, have consequences on lipoprotein composition. * CETP * deficiency in humans is characterized by increases in HDLc, whereas increases in its activity are associated with an enrichment of HDL particles in TGs and a decrease in HDLc levels [@ pone. 0027208 - Ikewaki1 ]. The most extensively studied polymorphism in * CETP * is Taq1B (rs708272) [@ pone. 0027208 - Kondo1 ]. The G allele, also called B1, is associated with higher enzymatic activity, higher CETP mass and lower HDLc levels [@ pone. 0027208 - Noone1 ], [@ pone. 0027208 - Boekholdt1 ]. It has been estimated that this polymorphism is rfs9onsible for 5. 8% of the variation in HDLc levels [@ pone. 0027208 - Corella1 ]. Studies in transgenic mice demonstrate that the over - expression of the gene encoding HL, * Lipc *, leads to a marked decrease in plasma HDLc levels [@ pone. 0027208 - Isaacs1 ], an observation supported by human studies showing an inverse correlation between HL activity and HDLc concentrations [@ pone. 0027208 - Blades1 ]. The - G250A * LIPC * polymorphism (rs2070895) [@ pone. 0027208 - Todorova1 ], located in the promoter region of the gene, has been extensively studied in relation to enzyme activity and lipid metabolism. The minor allele (A) is associated with a reduction of transcriptional activity * in vitro * [@ pone. 0027208 - Deeb1] and a 15 - - 45% reduction in enzymatic activity [@ pone. 0027208 - Tahvanainen1 ]. In humans, the minor allele has also been associated with an increased HDLc concentration and more buoyant LDL particles [@ pone. 0027208 - Tahvanainen1 ], [@ pone. 0027208 - Zambon2 ]. Studies assessing the association of this variant with T2D show conflicting results [@ pone. 0027208 - Zacharova1 ]. Diabetes is often preceded and even predicted, by the presence of dyslipidemia [@ pone. 0027208 - Todorova1 ]. Thus, mechanisms involved in the development of diabetic dyslipidemia may also play a role in the pathogenesis of T2D. The effects of the mentioned polymorphisms in * CETP * and * LIPC * on HDLc concentrations are well established, but their relation with the risk of T2D is less known. Therefore, the aim of our study was to analyze the relationship between polymorphisms in these two genes and the presence of diabetes and insulin resistance in a Canarian population. Methods {# s2} = = = = = = = Study population {# s2a} - - - - - - - - - - - - - - - - The Telde study is a cross - sectional population - based study on the prevalence of diabetes and cardiovascular risk factors in Telde, a city located on the island of Gran Canaria, Spain. The study population and design of this survey has been previously described [@ pone. 0027208 - Boronat1 ]. An oral glucose tolerance test (OGTT) was performed and the subjects were classified (using ADA 1997 crit@$ia) as diabetic (n = 115) and pre - diabetic (n = 116) if they had impaired fasting glucose, impaired glucose tolerance or both. A total of 226 subjects with a normal OGTT @erF selected, after matching for gender and age with the other two groups. All participants gave their written informed consent for participation in the study, which was carried out according to the declaration of Helsinki and approved by the local ethics committee. Genetic analyses {# s2b} - - - - - - - - - - - - - - - - The biochemical analyses and insulin resistance parameters have been described previously [@ pone. 0027208 - Novoa1 ]. Genomic DNA was extracted from whole blood (n = 457) using a salting - out method. The Taq1B * CETP * polymorphism was amplified by PCR - RFLP as described by June Hsieh Wu [@ pone. 0027208 - Wu1] and the * G - 250A LIPC * polymorphism was analyzed by AMRS - PCR (Amplification Refractory Mutation System - Polymerase Chain Reaction) [@ pone. 0027208 - Ye1 ]. Two pairs of primers were used, one which amplifies a fragment of 366 bp, common to both alleles (outer primers: 5 ′ - CTT TTC TTT TTC TTT GGG CTT AGG CT - 3 ′ and 5 ′ - AAG ACT GCC CAT TAA TAA TTA ACC TCT CAA - 3 ′) and another pair specific for the SNP (inner primers ): 5 ′ - CAA GGT CAG AGT TCC AAA TTA ATC CAC - 3 ′ for the G allele and 5 ′ - TTC CAA ACA CAA CAC AGT AGC TTT CAA - 3 ′ for the A allele. The primers were designed * " in silico " * in a free access web (< http: / / cedar. genetics. soton. ac. uk >, accessed in August 2007) and then checked for specificity (< http: / / blast. ncbi. nlm. nih. gov / Blast. cgi >, accessed in August 2007 ). The PCR reaction was carried out in a total volume of 25 µl containing 1 ∶ 5 ratio of outer to inner primer concentration. The annealing temperatures were 70 ° C during 15 sec for the outer primers and 58 ° C during 25 sec for the inner primers with a 30 sec extension at 72 ° C. PCR products were mixed wi$B 2 µl of loadiMv buffer and run on 2. 5% agarose gel stained with Ethidium Bromide. This resulted in 3 DNA fragments: one of 366 bp, one of 234 bp for the A allele and one of 185 bp for the G allele ([ figure 1] (# pone - 0027208 - g001) {ref - type = " fig "} ). ! [Agarose gel results of both polymorphisms. \ PCR - RFLP agarose gel (A) after digestion with Taq1B enzyme. The 1000 bp band corresponds to the B2 allele and the 650 and 350 bp bands correspond to the B1 allele. Results of * LIPC * genotyping by ARMS - PCR (B ). The 366 bp band is the product of the oKger primers, the 234 bp band, of an outer primer and the inner primer for allele A and the 185 bp band, of the other outer primer and the inner primer for the G allele.] (pone. 0027208. g001) {# pone - 0027208 - g001} Statistical analysis {# s2c} - - - - - - - - - - - - - - - - - - - - Statistical analyses were performed with SPSS for WINDOWS, version 13 (SPSS Inc. , Chicago, IL ). The quantitative variables are described as mean ± standard deviation (S. D ). Before further analyses, variable distribution was checked with the Kolmogorov - - Smirnov test. A logarithmic transformation was performed for variables not following a Gaussian distribution. Differences between groups were analyzed using either analysis of variance or analysis of covariance, both with the Bonferroni post hoc correction test, after adjusting for age, gender, Body Mass Index (BMI) and waist. The categorical variables were compared using Fisher \ ' s exact test for 2 × 2 tables and chi - squared or the Mantel - Haenszel test for linear association. The independent contribution of each polymorphism to DM2 risk was analyzed by a multinomial logistic regression model, which included age, gender, BMI and waist. All heDts were considered significant if p was \ <0. 05. Regarding the effect of the interaction of both polymorphisms on the risk of diabetes, the reference category was defined by the non - B1B1 genotype, regardless of the - 250G / A * LIPC * genotypes (nonB1B1 * CETP * genotype ). A second group included B1B1 and non - GG genotypes (B1B1CETP / non - GGLIPC) and a third, B1B1 CETP and * LIPC * GG genotylds (B1B1CETP / GGLIPC ). Results {# s3} = = = = = = = Patient description {# s3a} - - - - - - - - - - - - - - - - - - - The anthropometric, clinical and genetic characteristics of the whole population and their classification according to the OGTT are shown in [table 1] (# pone - 0027208 - t001) {ref - type = " table " }. The frequencies of the B1B1, B1B2 and B2B2 genotypes of the | Introduction {#s1} Diabetic dyslipidaemia is characterised by hypertriglyceridaemia, low lipoprotein (HDL) cholesterol (c) and normal low-density (LDLc) but preponderance of small-dense, highly atherogenic The increase in free fatty acids (FFAs) as degradation products triglycerides (TGs) associated with the development of resistance [@pone.0027208-Wilding1]. Cholesteryl Transfer Protein (CETP) and Lipase (HL) are central enzymes in the metabolism of HDL particles and reverse cholesterol transport. CETP is responsible for an exchange cholesteryl ester (CE) for triglycerides (TGs) between LDL and HDL and TG rich-lipoprotein particles [@pone.0027208-Morton1]. result is an enrichment of HDL and LDL particles in TGs, which makes them good substrates for HL [@pone.0027208-Thuren1]. The catalyses the hydrolysis the TGs and phospholipids present several lipoprotein subclasses, leading to in the size and density of lipoproteins [@pone.0027208-Thuren1]. The increased activity of either enzyme results in lower HDLc levels a predominance of dense HDL and particles [@pone.0027208-Lagrost1], [@pone.0027208-Zambon1]. variations in the *CETP* gene, which lead changes in enzyme function, have consequences on lipoprotein composition. *CETP* deficiency in humans is characterized increases in whereas increases in its activity are associated with an of HDL particles in TGs and a decrease HDLc levels [@pone.0027208-Ikewaki1]. The most studied polymorphism in *CETP* Taq1B (rs708272) [@pone.0027208-Kondo1]. The also called B1, is associated with higher activity, higher CETP and lower HDLc levels [@pone.0027208-Noone1], [@pone.0027208-Boekholdt1]. It has been estimated that this polymorphism is for 5.8% the variation in HDLc levels [@pone.0027208-Corella1]. Studies in transgenic mice demonstrate that the of the gene encoding HL, *Lipc*, leads to a marked decrease plasma HDLc levels[@pone.0027208-Isaacs1] , an observation supported by human studies showing an inverse correlation between HL activity and HDLc concentrations [@pone.0027208-Blades1]. The -G250A *LIPC* polymorphism (rs2070895) located in promoter region of the gene, has been extensively studied in relation to activity and lipid metabolism. minor allele (A) is with a reduction of transcriptional activity *in vitro* [@pone.0027208-Deeb1] a 15--45% reduction in enzymatic activity [@pone.0027208-Tahvanainen1]. In humans, the minor allele has also been associated with an increased HDLc concentration and buoyant LDL particles [@pone.0027208-Tahvanainen1], [@pone.0027208-Zambon2]. Studies the association of this variant with T2D show conflicting results [@pone.0027208-Zacharova1]. Diabetes is often preceded and even predicted, by the presence of dyslipidemia Thus, mechanisms involved in the development of diabetic dyslipidemia also play a role in the pathogenesis of T2D. The effects of the mentioned polymorphisms in *CETP* and *LIPC* on HDLc concentrations are well established, but their relation with the risk of T2D is less known. Therefore, the aim of our was to analyze relationship between in these two genes and the presence of diabetes and resistance in a Canarian population. {#s2} ======= Study population {#s2a} ---------------- The Telde study is a cross-sectional population-based study on the of diabetes and cardiovascular risk factors in Telde, a located on the island of Spain. The population and design of this survey has been previously described [@pone.0027208-Boronat1]. An oral glucose tolerance test (OGTT) was and the subjects classified (using ADA 1997 criteria) as diabetic (n 115) and pre-diabetic (n = 116) if they had impaired fasting glucose, impaired glucose tolerance or both. total of 226 subjects OGTT were selected, after matching for gender and age with the other two groups. participants gave their written informed consent for participation in the study, which carried out according to the declaration of Helsinki and approved by local ethics committee. Genetic analyses {#s2b} ---------------- The biochemical analyses insulin resistance parameters have described previously [@pone.0027208-Novoa1]. Genomic DNA was extracted from whole blood (n = 457) using a salting-out method. The Taq1B *CETP* polymorphism was amplified by PCR-RFLP as described by June Hsieh Wu [@pone.0027208-Wu1] and the *G-250A LIPC* polymorphism was analyzed by AMRS-PCR (Amplification Refractory Mutation System-Polymerase Chain Reaction) [@pone.0027208-Ye1]. Two pairs primers were used, which amplifies a fragment of 366 bp, common to both alleles (outer primers: 5′-CTT TTC TTT TTT GGG CTT AGG CT-3′ and 5′-AAG ACT GCC CAT TAA TTA ACC TCT CAA-3′) and another pair specific for SNP (inner primers): 5′-CAA GGT CAG AAA TTA ATC CAC-3′ for the G allele and 5′-TTC CAA ACA CAA CAC AGT AGC TTT CAA-3′ for the A allele. The were designed *"in silico"* in a free access web (<http://cedar.genetics.soton.ac.uk>, accessed in August 2007) and then checked for specificity (<http://blast.ncbi.nlm.nih.gov/Blast.cgi>, accessed August 2007). The PCR reaction was carried out in a total volume of 25 µl containing 1∶5 ratio of outer to inner primer concentration. annealing were 70°C during 15 sec for the outer and 58°C during 25 sec for the inner a 30 sec at 72°C. PCR products were mixed with 2 µl of buffer and run on 2.5% agarose gel stained with Bromide. This resulted in 3 fragments: one of 366 bp, one for the A allele and one of 185 the G allele ([figure 1](#pone-0027208-g001){ref-type="fig"}). {#pone-0027208-g001} Statistical analysis {#s2c} Statistical analyses were performed with SPSS for WINDOWS, version (SPSS Inc., Chicago, IL). The quantitative variables are described as mean standard deviation (S.D). further analyses, variable distribution was checked with Kolmogorov--Smirnov test. A logarithmic transformation performed for not following a Gaussian distribution. Differences between were analyzed using either analysis of variance or analysis covariance, both with the Bonferroni post hoc correction test, after adjusting for age, gender, Body Mass Index (BMI) and waist. The categorical variables were compared using Fisher\'s exact test for 2×2 tables and chi-squared or the Mantel-Haenszel test for linear association. The independent contribution of polymorphism DM2 was analyzed a multinomial logistic regression model, which age, gender, BMI All tests were significant was Regarding the effect of the interaction of both polymorphisms on the risk of diabetes, the reference category defined by the non-B1B1 genotype, regardless of the -250G/A *LIPC* genotypes (nonB1B1*CETP* genotype). A second included B1B1 and non-GG genotypes (B1B1CETP/non-GGLIPC) a third, B1B1 CETP and *LIPC* GG genotypes (B1B1CETP/GGLIPC). Results {#s3} ======= Patient description {#s3a} ------------------- The anthropometric, clinical and genetic characteristics of the whole population and their classification according to the are shown in [table 1](#pone-0027208-t001){ref-type="table"}. of B1B1, B1B2 and B2B2 genotypes of the | InTRODuctIOn {#S1}
============
diABeTIC DyslipiDaEmia Is CharAcTeRISeD by HYPErTRiglyCeRiDaemIa, LOw HIGH-dEnsity LiPoproTeiN (hDL) ChoLesTEroL (C) anD NOrmAL LOw-dENSiTY lIpOprOTeIN-CHoLEStERoL (ldlC) but PREpoNDeraNcE Of smaLL-DENse, hiGHLY ATheroGeNiC PArtiCLes. ThE incReaSe In FrEe FAtty AcIdS (FfAs) as dEGRadatIoN prOduCTS oF TRiglyCErIDes (TGS) iS asSociatED WITH THE DeveLOPMEnT Of INsuliN ReSisTANCe [@PonE.0027208-WIlDINg1].
cHOlestEryl eSTer TraNSFER PrOteiN (cEtp) anD hEPAtic LipasE (hl) ArE CENtRaL ENzYMes In thE MetabolISm Of HDl pArTiclEs and rEvErSE CholEsterOL TraNsPOrt. Cetp is resPOnsIBlE For aN excHanGe oF cHoLesTEryl EsTER (Ce) FoR triGlYCerIDES (Tgs) beTwEEN ldl AnD HdL anD tG rich-LiPOpROTeiN PartIcLEs [@PoNE.0027208-MORtoN1]. THe reSUlt IS AN EnrIcHMEnT OF hDL aND LDL paRtIcLEs In TgS, wHiCh MakEs ThEM gOOd sUBsTRAtes FoR hL [@pOne.0027208-tHuREN1]. thE LATTer CAtALYSeS tHe HYDRolYSis Of the tgs And pHoSphOLiPIds PReSENt IN sEVeral LIpopROTeIn SuBcLASses, LeAdiNg tO cHaNGeS In The sIze anD DeNSITY of lIPopRoteINs [@pONE.0027208-tHUren1]. THe INCrEAsED actiViTy Of eITHER Enzyme rEsuLTS in LoweR hdlC LeVeLS And A PREDOMINaNce Of small, dENsE Hdl anD lDl parTIcLeS [@PONe.0027208-laGrOst1], [@PONe.0027208-zamBon1].
THE VARIATIons In THE *CEtP* GEne, whICh lead TO chAnGEs in ENZYme fUNCTIOn, Have coNsEquEncES ON LIpoPRoTEIN coMPoSitiOn. *Cetp* deFIciENcY IN huManS iS cHARACTeRIZED BY iNCReASes In hDlC, WhEREAs iNcrEasES IN Its aCTivitY aRE aSsOcIAtEd wiTh AN EnRichmENT of hdl pARtiCLeS in Tgs ANd a dEcREASE iN hDLc lEVelS [@pOnE.0027208-iKEwAKi1]. THe moST exTensIVELy stUdieD PolYMOrPhisM In *CeTp* iS Taq1B (Rs708272) [@POnE.0027208-kondo1]. THE g ALlElE, aLSo cALlED b1, is ASsOcIateD WiTH hIGHEr EnzyMAtic ACTIviTy, HigHer CetP MaSS AND lOWer hDlC Levels [@ponE.0027208-NOOne1], [@PONE.0027208-boeKhOlDT1]. it haS beEn ESTIMaTEd tHAt tHiS pOLymOrphiSM Is ReSpoNsiBLe for 5.8% OF ThE vArIAtion IN HdLC LeVels [@PonE.0027208-cOrELLa1].
stUDIEs In TraNSGENIC micE demOnsTraTE THAT tHE OVeR-eXPRessioN OF thE Gene EncOding HL, *liPC*, leADS tO A mARkeD DecreASE iN plasmA HDlc lEVeLS[@PoNE.0027208-isAaCS1] , An observatIoN sUPPORtED BY human stuDIES SHoWing aN InvErSE CoRrelAtiOn betwEen HL aCtIvity AND Hdlc cOncEnTrations [@PONe.0027208-BLaDES1]. The -G250A *LIpc* poLyMorphiSm (Rs2070895) [@poNE.0027208-tODOroVA1], LocAteD IN tHe pROmOtER RegiON OF tHe gene, haS BeEN eXTENSiVElY STUDIed in rELATiOn TO enZyME acTIVITy aND LIpid mETaBolisM. thE mINor aLLELE (A) IS aSSociateD wiTh A REDuCTIoN oF TRAnsCRiptionAL AcTiVITy *IN VitRO* [@PonE.0027208-dEEB1] ANd a 15--45% ReducTiON in EnzYmaTIC aCTivity [@poNe.0027208-TaHvAnAiNen1]. IN HuMANS, THe mInor AlLelE HAS ALSO bEEn aSSoCIAteD wITh AN INCreasED hdlC cONcENTRatiON and moRe buoYant lDl paRTicleS [@pONE.0027208-tahvAnaiNen1], [@poNe.0027208-ZAMBOn2]. sTUDIes aSsESSIng thE ASsOcIatIon OF tHIS VaRiAnt With T2d sHoW cONflIcTING resultS [@ponE.0027208-zacHarOva1].
dIaBeteS IS ofteN preCEDeD AnD eveN PREdicted, bY ThE PResEnce Of dyslIpiDemia [@ponE.0027208-todOROvA1]. thUs, meCHAnIsMs INVOLVeD In The DeveloPmeNt Of DiaBeTIC DySLiPIDEmia MaY ALsO PLaY A rOLe IN THe PatHoGenesIS of T2D. ThE EffeCts Of the mENTiOnEd PoLyMORPhiSmS In *CETP* AnD *LIpc* oN HdlC ConceNTrAtions ARe WeLl ESTabLISHED, BuT thEiR RelatioN wiTh THE rIsK Of t2d IS lEsS KnOwn. thEReforE, thE Aim OF OUr STuDy WAS tO aNAlyzE tHe ReLaTIoNshIp BetWEen polYmORpHismS IN THeSe Two geNES aNd tHE pResenCE Of dIabEteS and INSULIn rESIStancE iN A CaNarIAn PoPULAtiOn.
MEtHOds {#s2}
=======
study popULAtIOn {#S2a}
----------------
the TELDe StUdy Is a CRoss-SECTIOnAl PopuLATiON-bASEd sTudy On ThE PrevAlEncE oF dIAbeTeS anD carDioVASCULAr rIsK FaCtORS In TelDE, A CiTy LoCAteD on tHE iSlanD of GRan caNARiA, sPAIN. THe study poPulaTIon ANd DEsIGN OF ThIS SURvEY haS BeeN PReviOuSLy DeScribEd [@pone.0027208-BoRonAT1]. An OraL GluCoSe tolErANce TeSt (ogTT) WAS performeD And tHE SUBjectS wERe classifiED (usINg aDa 1997 CRItERIa) as DiaBeTIc (N = 115) and pRE-dIABEtIc (N = 116) iF thEy had iMPaireD FasTING GLUcOsE, iMpaiRED GluCose tOlErANCe Or boTH. A TOTAl oF 226 sUBJects With a NoRmAL OGTt were seLectEd, afTeR maTcHiNG fOR geNder ANd agE WItH tHe OThER two gROuPs. ALL paRtiCipanTs GaVE thEiR WrItTEn iNfoRmED coNsEnt FOr pArTICIPaTIon In The STUDY, wHIcH wAs carrieD Out aCcOrDIng to THE DEcLaRAtion oF hElsINKI and apPRoVeD BY The loCaL eTHIcs cOmmitteE.
GeNetiC ANaLyses {#s2B}
----------------
thE biOCHemicaL anaLYSeS aND iNSULIN rESistAnCE paraMeteRs HAve BeEn deScRiBEd pReVioUsly [@PoNe.0027208-nOvoa1]. gEnOMiC dnA was exTRACTEd frOM wHOle BlOod (N = 457) USINg A salTIng-oUT METHOD. tHE tAq1B *CeTp* pOLymoRpHIsM waS AMPliFIEd by pCr-rfLp as DeSCRibeD BY JunE hSIeH wu [@poNe.0027208-wU1] aNd THE *g-250A lipC* polYMoRPHiSM wAS anAlyZED by AMRs-PCR (AMPlIFIcATiON ReFRaCtoRy mUTATIoN sysTEm-PolyMErasE cHAiN reActIoN) [@PoNE.0027208-YE1]. Two PAIRs OF PrImERs WERE uSed, oNe whicH ampLiFIeS A fRagMent of 366 bP, cOMmON To BotH ALlelES (OUtEr PrIMers: 5′-Ctt TtC ttT Ttc Ttt gGG CTT AGg cT-3′ and 5′-AaG Act GcC CAt Taa TAa TTa ACc tcT CAa-3′) And AnOTHEr PaiR SpeCIfIC FOr THE snP (INner prIMERs): 5′-Caa ggt CAg AGt TCC aAa ttA aTc cAC-3′ FOR THE g ALLelE And 5′-tTC cAa aCa CAA cAc Agt Agc ttT CAA-3′ FoR THE A ALLeLe. tHe primeRS WERe DeSIGNed *"in siLICO"* IN a FrEe ACCesS wEB (<HTtP://cEdAR.GeNetIcs.SOtOn.AC.UK>, ACCEsSeD iN auguSt 2007) and ThEn CHeCkED foR sPEcIficITy (<HTtP://blaSt.ncbI.nlm.nih.GOV/blASt.cgI>, AcceSsEd iN auGust 2007). THe pCR REactIoN Was caRriED OUt iN A tOtal VoLuME of 25 Μl cONTaINiNG 1∶5 RatiO of oUTeR To innEr prIMEr conCEnTraTiOn. THe AnnealiNG tEMpEratURES wERE 70°c durING 15 SeC FOR ThE ouTEr pRIMers and 58°C DuRINg 25 seC FOR the inNer pRiMErs wIth A 30 sEC extenSion aT 72°C. PcR pRODUCTS WERe MIxED with 2 ΜL of LOadING bufFeR ANd RUn oN 2.5% AGaROsE gEl StAined with etHiDium brOMIdE. thIS rEsULted IN 3 dNa frAGmENtS: onE of 366 Bp, oNe OF 234 bp foR The a AlLElE AND one OF 185 Bp For ThE g AllELe ([fIGurE 1](#PoNE-0027208-g001){rEF-TYpE="FIg"}).
{#pOne-0027208-G001}
sTATiSTICAL ANaLYSIS {#S2C}
--------------------
STATiStIcAL ANAlySeS Were PERFOrMED WitH SPss For wInDOws, vErSION 13 (SpSS InC., chIcago, iL). The QUAntItAtivE VARIaBLEs ARe deScRIBED As MEAn ± staNdArD DeviAtIOn (S.d). befoRe FuRtHer aNALyseS, VarIABLE DisTRIbuTiON Was CHECKED with the koLmOgoRov--SmirNOv tesT. A LogaRITHmiC tRAnsfoRMaTioN waS PeRformeD fOr VarIabLes Not FoLLowINg a GaUSSian dIStRIbUTIOn. dIFFErENCEs betWeEN gROuPs WEre aNALyzeD UsING EitHeR anALysIs of VARIance oR analysIS of CoVaRIanCE, both WiTh the BONFERroni POST hoC CorreCTioN TEsT, aftER ADJUStINg FoR AGe, gEnDER, body mass indEX (bmI) AnD WaIsT. THE CaTegORIcal vARiAbLes WeRE cOmparED Using fISHEr\'S eXacT TeSt fOR 2×2 tABlEs And chI-SQuArED or thE ManTel-hAenszel teST FOR lInEar aSSOcIaTIoN. ThE InDEpENdeNT contrIBuTiON of EACH PolYMoRpHIsM tO dm2 riSk wAS anAlyZed bY a MuLtInOmial lOGIstIC rEGResSION model, whiCh inCludED agE, GEndER, bMI anD WAiSt. aLl tEStS weRe consIdEred SiGNificANT iF p waS \<0.05.
rEGARdINg The effecT oF THe IntErACTION of BoTH POlymoRPHIsmS oN the riSk OF DiAbetEs, tHE RefErEnCe caTegoRY Was DefiNEd by THE NOn-b1B1 GenOtYPE, REGArDLeSS oF ThE -250g/A *lipC* GenOtypeS (NONb1B1*Cetp* GeNoTyPE). A sEcoND gROUP iNclUdeD B1B1 aND noN-GG GENotyPeS (b1b1CeTP/noN-ggLiPC) ANd A tHiRD, B1B1 CETP aND *LiPC* gg geNOtyPES (B1B1ceTp/GglIPc).
ResULTs {#S3}
=======
patiENt desCRIptIon {#s3a}
-------------------
The anthROPoMEtric, CLInicAL aND GenEtic cHAraCTeRisTIcs of THE wHolE pOPUlATiOn anD ThEir classIficaTion AcCorDinG to the oGTT are ShOWN iN [TABlE 1](#PonE-0027208-t001){ref-tYPe="TaBLe"}. ThE frequencIeS OF ThE b1b1, b1B2 AnD b2B2 genOtYpes oF THe | Introduction {#s1} ============ Diabetic dyslipidaemia is characterised by hypertriglyceridaemia, low high-density lipoprotein (HDL) cholesterol (c) and normal low-densitylipoprotein-cholesterol (LDLc) but preponderanceof small-dense,highly atherogenic particles. The increase in freefatty acids (FFAs) as degradation products of triglycerides (TGs) is associated withthe development of insulin resistance [@pone.0027208-Wilding1]. Cholesteryl Ester Transfer Protein (CETP) and Hepatic Lipase (HL) are central enzymes inthe metabolism of HDLparticles and reverse cholesterol transport. CETP is responsible for an exchange of cholesteryl ester(CE) for triglycerides (TGs) between LDL andHDL and TG rich-lipoprotein particles[@pone.0027208-Morton1]. Theresult is an enrichment of HDL and LDL particles in TGs, which makes themgood substrates for HL [@pone.0027208-Thuren1]. The lattercatalyses thehydrolysis of the TGs and phospholipids presentin several lipoprotein subclasses, leading to changes in the size and density of lipoproteins [@pone.0027208-Thuren1]. The increasedactivity of either enzyme results in lower HDLclevels and a predominance of small, dense HDL andLDL particles [@pone.0027208-Lagrost1], [@pone.0027208-Zambon1].Thevariations in the *CETP* gene, which lead to changes in enzyme function, have consequences on lipoprotein composition. *CETP* deficiency in humans is characterized by increases in HDLc, whereas increases in its activity are associated with an enrichment ofHDLparticles in TGs and adecrease inHDLc levels [@pone.0027208-Ikewaki1]. The most extensively studied polymorphism in *CETP*is Taq1B (rs708272) [@pone.0027208-Kondo1]. The G allele, also calledB1, is associated with higher enzymatic activity, higherCETP mass and lowerHDLclevels [@pone.0027208-Noone1], [@pone.0027208-Boekholdt1]. Ithas been estimated that this polymorphismis responsiblefor5.8% of the variation in HDLclevels [@pone.0027208-Corella1]. Studies in transgenic mice demonstrate that the over-expression of the gene encoding HL, *Lipc*, leads to a marked decrease in plasma HDLc levels[@pone.0027208-Isaacs1] , an observation supported byhuman studies showing an inverse correlation between HL activity and HDLc concentrations [@pone.0027208-Blades1]. The -G250A *LIPC* polymorphism(rs2070895) [@pone.0027208-Todorova1], locatedin thepromoter region of the gene, has been extensively studied in relation to enzyme activity and lipid metabolism. The minor allele (A) isassociated with a reduction of transcriptional activity *in vitro* [@pone.0027208-Deeb1] and a 15--45% reduction in enzymatic activity [@pone.0027208-Tahvanainen1]. In humans, theminor allele has also been associated with an increased HDLc concentration and more buoyant LDL particles [@pone.0027208-Tahvanainen1], [@pone.0027208-Zambon2]. Studies assessing theassociation of this variant with T2Dshow conflicting results [@pone.0027208-Zacharova1]. Diabetes isoften preceded and even predicted, by the presence of dyslipidemia [@pone.0027208-Todorova1]. Thus, mechanisms involved in thedevelopment of diabetic dyslipidemia may also play a role in the pathogenesis of T2D. The effects of thementioned polymorphismsin *CETP* and *LIPC* on HDLc concentrations are well established, but their relation with the risk of T2D is less known. Therefore, the aim of our study wastoanalyze the relationship between polymorphisms in these two genes and the presenceof diabetes and insulin resistance in a Canarian population. Methods {#s2} ======= Studypopulation {#s2a} ----------------The Telde study is a cross-sectional population-based study on theprevalence of diabetes and cardiovascular risk factorsin Telde, a citylocated on the island of Gran Canaria, Spain. The study population and designof this survey has been previously described [@pone.0027208-Boronat1]. An oral glucose tolerance test (OGTT) was performed and the subjects were classified (using ADA1997 criteria) as diabetic (n= 115)and pre-diabetic (n =116) if they had impaired fastingglucose, impaired glucose tolerance orboth. A total of 226 subjects with a normal OGTT wereselected, after matching for gender andagewith the other two groups.All participants gave their written informedconsent for participation in thestudy, which was carried out according to the declaration of Helsinki and approved by the local ethics committee. Geneticanalyses {#s2b} ---------------- Thebiochemicalanalyses and insulin resistance parameters have been described previously [@pone.0027208-Novoa1]. Genomic DNA was extracted from whole blood (n = 457) using a salting-out method. TheTaq1B*CETP* polymorphism was amplified by PCR-RFLP as described byJune Hsieh Wu [@pone.0027208-Wu1]andthe *G-250A LIPC* polymorphismwas analyzed by AMRS-PCR (Amplification Refractory Mutation System-Polymerase Chain Reaction) [@pone.0027208-Ye1]. Two pairs of primers were used, onewhich amplifies a fragment of 366bp,common to both alleles (outer primers: 5′-CTT TTC TTTTTCTTT GGG CTT AGGCT-3′and 5′-AAG ACTGCC CAT TAA TAA TTA ACC TCT CAA-3′)and another pair specific for theSNP (inner primers): 5′-CAA GGT CAG AGT TCC AAA TTA ATC CAC-3′ for the Gallele and 5′-TTC CAA ACA CAA CACAGT AGC TTT CAA-3′ for the A allele.The primers were designed*"in silico"* in a free access web (<http://cedar.genetics.soton.ac.uk>, accessed in August 2007) and then checked forspecificity (<http://blast.ncbi.nlm.nih.gov/Blast.cgi>, accessed in August 2007). The PCRreaction was carried out in a total volume of 25 µl containing 1∶5 ratio of outer to innerprimer concentration. The annealingtemperatureswere 70°C during 15 sec for the outer primers and 58°C during 25 sec for the inner primers with a 30 sec extensionat 72°C. PCR products were mixed with 2 µlof loadingbuffer andrun on 2.5% agarose gelstained with Ethidium Bromide.Thisresulted in3 DNA fragments: one of 366 bp, one of 234bp for the Aallele andone of 185 bp for the Gallele ([figure 1](#pone-0027208-g001){ref-type="fig"}). {#pone-0027208-g001} Statistical analysis {#s2c} -------------------- Statistical analyseswere performed with SPSS for WINDOWS, version 13 (SPSS Inc., Chicago, IL). The quantitative variables are described as mean ± standard deviation (S.D). Before further analyses,variable distributionwas checked with the Kolmogorov--Smirnovtest. A logarithmictransformation was performed for variablesnot following a Gaussiandistribution.Differences between groups were analyzed using eitheranalysis of variance or analysis of covariance,bothwith the Bonferroni post hoc correction test,after adjusting for age, gender, Body Mass Index (BMI) and waist. Thecategorical variables were compared using Fisher\'s exact test for 2×2tables and chi-squared orthe Mantel-Haenszel test for linearassociation. The independent contribution of each polymorphism to DM2 risk wasanalyzed bya multinomiallogistic regression model,which included age, gender,BMI and waist. All tests were consideredsignificant ifp was \<0.05. Regarding theeffectof theinteraction of both polymorphisms on the risk of diabetes, the reference category was defined by the non-B1B1 genotype, regardless of the -250G/A *LIPC* genotypes (nonB1B1*CETP* genotype). A second group included B1B1 and non-GG genotypes (B1B1CETP/non-GGLIPC) anda third, B1B1 CETP and *LIPC*GG genotypes (B1B1CETP/GGLIPC). Results{#s3} ======= Patient description {#s3a} ------------------- The anthropometric, clinical and genetic characteristics of the whole populationand their classification according to theOGTT are shown in[table 1](#pone-0027208-t001){ref-type="table"}. The frequenciesof the B1B1, B1B2 and B2B2 genotypes of the | Introduction _{#s1}_ ============ Diabetic dyslipidaemia is characterised by hypertriglyceridaemia, low high-density lipoprotein _(HDL)_ _cholesterol_ (c) and normal low-density lipoprotein-cholesterol (LDLc) but preponderance of small-dense, highly atherogenic particles. _The_ increase in _free_ fatty acids (FFAs) _as_ degradation products _of_ triglycerides (TGs) is associated with the _development_ of insulin resistance [@pone.0027208-Wilding1]. Cholesteryl Ester Transfer Protein _(CETP)_ _and_ _Hepatic_ Lipase (HL) are central _enzymes_ in the _metabolism_ of HDL particles and reverse _cholesterol_ _transport._ CETP is responsible for an exchange of cholesteryl ester (CE) for triglycerides (TGs) between LDL and HDL and TG rich-lipoprotein particles [@pone.0027208-Morton1]. The result is an enrichment of HDL and _LDL_ _particles_ in TGs, _which_ makes them good substrates for _HL_ [@pone.0027208-Thuren1]. _The_ latter catalyses the hydrolysis of the TGs and phospholipids present in several lipoprotein subclasses, leading to changes in the size and density of lipoproteins [@pone.0027208-Thuren1]. The increased activity of either enzyme results in lower HDLc levels and a predominance of small, dense _HDL_ and _LDL_ particles [@pone.0027208-Lagrost1], [@pone.0027208-Zambon1]. The variations in the *CETP* gene, which lead to _changes_ _in_ enzyme function, have _consequences_ on lipoprotein composition. *CETP* deficiency in _humans_ is characterized _by_ increases _in_ HDLc, whereas increases in its activity _are_ associated _with_ _an_ enrichment of _HDL_ particles _in_ TGs and a decrease _in_ HDLc levels _[@pone.0027208-Ikewaki1]._ The most _extensively_ studied polymorphism _in_ *CETP* is Taq1B (rs708272) [@pone.0027208-Kondo1]. The G allele, also called B1, is associated with _higher_ enzymatic activity, higher CETP _mass_ and lower HDLc levels [@pone.0027208-Noone1], [@pone.0027208-Boekholdt1]. It has been estimated that this polymorphism is responsible for 5.8% _of_ _the_ variation in HDLc levels [@pone.0027208-Corella1]. Studies _in_ transgenic mice demonstrate that the over-expression _of_ the gene encoding HL, *Lipc*, leads to a _marked_ _decrease_ _in_ plasma HDLc _levels[@pone.0027208-Isaacs1]_ , an _observation_ supported by human studies showing an inverse correlation between HL activity and _HDLc_ concentrations _[@pone.0027208-Blades1]._ The -G250A *LIPC* _polymorphism_ (rs2070895) [@pone.0027208-Todorova1], located in the promoter region of the gene, has been extensively studied in relation to enzyme _activity_ _and_ lipid metabolism. The _minor_ allele (A) _is_ associated with a reduction of _transcriptional_ activity *in vitro* [@pone.0027208-Deeb1] and a 15--45% reduction in _enzymatic_ _activity_ [@pone.0027208-Tahvanainen1]. In humans, the minor allele _has_ also _been_ associated with an increased HDLc concentration and more buoyant LDL particles [@pone.0027208-Tahvanainen1], [@pone.0027208-Zambon2]. Studies assessing _the_ association of _this_ variant with T2D show conflicting _results_ _[@pone.0027208-Zacharova1]._ Diabetes is often preceded _and_ even _predicted,_ by the presence of _dyslipidemia_ _[@pone.0027208-Todorova1]._ _Thus,_ _mechanisms_ involved in the development _of_ _diabetic_ dyslipidemia may also play _a_ role _in_ the pathogenesis of T2D. The effects of the mentioned polymorphisms _in_ _*CETP*_ _and_ *LIPC* on HDLc _concentrations_ are well established, but _their_ _relation_ with _the_ risk of T2D is _less_ known. _Therefore,_ _the_ aim of _our_ _study_ was to _analyze_ the _relationship_ between polymorphisms _in_ these two genes and the presence of _diabetes_ and insulin resistance in a _Canarian_ population. Methods _{#s2}_ ======= Study population {#s2a} ---------------- _The_ Telde study is a cross-sectional population-based study on the prevalence of diabetes and cardiovascular _risk_ factors in Telde, a city located on the island of Gran _Canaria,_ Spain. The study population and design of this _survey_ has _been_ previously described [@pone.0027208-Boronat1]. An oral _glucose_ tolerance test (OGTT) _was_ performed and the subjects were classified (using ADA 1997 criteria) as diabetic (n = 115) and _pre-diabetic_ (n _=_ _116)_ if _they_ _had_ _impaired_ fasting glucose, _impaired_ _glucose_ tolerance or both. A _total_ of 226 subjects with a normal OGTT were selected, after matching for gender and age with the other two groups. All participants gave their written informed _consent_ for participation in the study, which _was_ carried out according to _the_ declaration of Helsinki and _approved_ by the local ethics committee. Genetic analyses {#s2b} ---------------- The biochemical _analyses_ and insulin resistance parameters have been described previously [@pone.0027208-Novoa1]. Genomic DNA _was_ extracted from whole blood (n = 457) using a salting-out method. _The_ Taq1B *CETP* polymorphism was _amplified_ by _PCR-RFLP_ _as_ described by June Hsieh Wu [@pone.0027208-Wu1] and the *G-250A _LIPC*_ _polymorphism_ _was_ analyzed by _AMRS-PCR_ _(Amplification_ Refractory Mutation System-Polymerase _Chain_ Reaction) _[@pone.0027208-Ye1]._ _Two_ pairs of primers were used, one which amplifies _a_ fragment of 366 bp, common to both alleles (outer primers: 5′-CTT TTC _TTT_ TTC TTT _GGG_ CTT AGG CT-3′ and 5′-AAG ACT GCC CAT TAA TAA TTA ACC TCT _CAA-3′)_ and another pair _specific_ for the SNP (inner primers): 5′-CAA _GGT_ _CAG_ AGT TCC AAA TTA _ATC_ _CAC-3′_ for the _G_ allele and 5′-TTC CAA ACA CAA CAC _AGT_ AGC TTT _CAA-3′_ for the _A_ allele. The _primers_ were _designed_ _*"in_ silico"* _in_ a free _access_ web (<http://cedar.genetics.soton.ac.uk>, accessed in August 2007) and then checked for specificity (<http://blast.ncbi.nlm.nih.gov/Blast.cgi>, accessed in August 2007). The PCR reaction was carried _out_ _in_ a total volume of 25 µl _containing_ 1∶5 ratio of outer to inner primer concentration. The annealing temperatures were _70°C_ during _15_ sec for the _outer_ _primers_ and 58°C during 25 sec for _the_ inner primers _with_ _a_ 30 sec extension at _72°C._ PCR products were mixed _with_ 2 µl _of_ _loading_ buffer and run on 2.5% _agarose_ gel stained with Ethidium Bromide. This resulted in 3 _DNA_ fragments: one of 366 bp, one of 234 bp for the A allele and one of _185_ _bp_ _for_ the G allele ([figure 1](#pone-0027208-g001){ref-type="fig"}). {#pone-0027208-g001} Statistical analysis {#s2c} -------------------- Statistical analyses were performed with SPSS for WINDOWS, version 13 _(SPSS_ Inc., _Chicago,_ IL). The quantitative variables are described as mean ± standard deviation _(S.D)._ Before further analyses, variable distribution was checked with _the_ Kolmogorov--Smirnov test. A logarithmic transformation was performed for variables not _following_ a Gaussian distribution. Differences between groups were _analyzed_ using either analysis of variance _or_ analysis of covariance, _both_ _with_ the Bonferroni post hoc correction test, after adjusting for _age,_ gender, Body _Mass_ Index _(BMI)_ and waist. The _categorical_ _variables_ were compared using Fisher\'s _exact_ test _for_ 2×2 tables and chi-squared _or_ the Mantel-Haenszel test for linear association. The independent contribution of each polymorphism to _DM2_ risk _was_ analyzed by a multinomial logistic regression model, which included age, gender, BMI and waist. _All_ tests were _considered_ significant if p was \<0.05. Regarding the effect of the interaction of both polymorphisms on the risk of diabetes, the reference category was defined _by_ _the_ _non-B1B1_ _genotype,_ regardless of the -250G/A _*LIPC*_ genotypes (nonB1B1*CETP* genotype). A _second_ group included B1B1 and non-GG genotypes (B1B1CETP/non-GGLIPC) and a third, B1B1 CETP and *LIPC* GG genotypes (B1B1CETP/GGLIPC). Results {#s3} ======= Patient _description_ {#s3a} ------------------- The anthropometric, clinical and _genetic_ characteristics of the whole population and their _classification_ according to the OGTT are _shown_ in [table _1](#pone-0027208-t001){ref-type="table"}._ _The_ frequencies _of_ the _B1B1,_ B1B2 _and_ B2B2 _genotypes_ of _the_ |
Background
==========
Diatoms are important primary producers in the ocean \[[@B1]\], contributing approximately 40% to global marine productivity. Although diatoms often dominate phytoplankton communities in nutrient-rich ecosystems, members of this diverse group are also adapted to survive and persist in nutrient-limited conditions. The development of large diatom blooms upon nutrient resupply demonstrates the metabolic plasticity inherent to their ability to recover rapidly from nutrient limitation.
Iron is an essential nutrient for all organisms and in particular for photoautotrophic organisms. It functions as a powerful electron carrier in iron-sulfur- and heme-containing proteins and as such is a required component of the photosynthetic apparatus. Solubility of iron in seawater is low and phytoplankton growth in marine habitats is often limited by iron availability. This is best illustrated in high-nitrate low-chlorophyll (HNLC) regions, remote oceanic areas that lack any form of regular iron supply and suffer from a persistent shortage of this micronutrient. Here, although other commonly limiting nutrients like nitrate or phosphate are present at high concentrations, primary productivity - and biomass as a whole - is low \[[@B2]\].
Numerous large-scale iron fertilization experiments have confirmed that iron is the limiting nutrient in HNLC regions \[[@B3]\]. Phytoplankton blooms induced by iron fertilization were dominated by diatoms and carbon export to the deep-sea floor could be observed in some cases. The strong response of diatoms to the input of iron in HNLC regions has been a motivation for exploring large-scale iron fertilization as a possible bioengineering strategy to sequester CO~2~into the ocean in HNLC regions, which are otherwise rich in macronutrients.
Genome projects on the model organisms *Thalassiosira pseudonana*\[[@B4]\] and *Phaeodactylum tricornutum*\[[@B5]\] have already generated a wealth of insights into the metabolic complexity of diatoms \[[@B6]\], a consequence of the secondary endosymbiosis event that gave rise to this group \[[@B7]\]. This secondary endosymbiosis brought together the benefits of a heterotrophic host and the \'red\'-type photosynthesis of red alga cells, which already have an elemental composition low in iron \[[@B8]\].
The impact of iron availability on phytoplankton growth has led to the evolution of strategies to counteract iron limitation. Well established parts of the low-iron response found in diverse phytoplankton species are the reduction of the chloroplast system, the corresponding development of a chlorotic phenotype, compensation mechanisms (replacement of iron-rich elements with iron-poor substitutes) and the activation of high-affinity iron-uptake systems \[[@B9]\]. The substitution of ferredoxin by flavodoxin \[[@B10]\], the use of plastocyanin instead of cytochrome c~6~\[[@B11]\] and a variant stoichiometry of photosynthetic complexes \[[@B12]\] are notable adaptive strategies to facilitate diatom growth in low-iron conditions.
Oceanic and neritic phytoplankton species can be distinguished from each other by their growth characteristics and their tolerance to nutrient limitation \[[@B13]\]. Unlike many other *Thalassiosira*species that are predominantly found in coastal waters, *Thalassiosiraoceanica*is adapted to oligotrophic conditions and is highly tolerant to iron limitation in particular. Therefore, we chose *T. oceanica*CCMP1005 as a model for a comprehensive analysis of its low-iron response in the context of genomic information.
Here, we explore the complex cellular response of *T. oceanica*to low-iron conditions with genomics, transcriptomics and proteomics approaches complemented by reverse transcription-quantitative PCR (RT-qPCR) analyses. We present a metabolic reconstruction of the iron limitation response based on the transcriptomics data from cells grown under iron-limited versus iron-replete conditions. A metabolic isotope labeling approach using ^14^N/^15^N was established for *T. oceanica*and showed the response to iron limitation at the protein expression level in a marine diatom for the first time. General characteristics of the \'diatom\' low-iron response and its ecological implications are discussed, as well as the constraints for species-specific adaptations to low-iron environments.
Results
=======
Characteristics of the *T. oceanica*genome
------------------------------------------
The genome of the centric diatom *T. oceanica*CCMP1005 (Figure [1](#F1){ref-type="fig"}) was *de novo*assembled from 725 Mb of Roche 454 sequence read information, generated using nuclear genomic DNA (gDNA) of an axenic clonal culture as substrate \[[@B14]\]. The current assembly version comprises 51,656 contigs of total size 92.15 Mb at N50 = 3,623 (that is, 50% of the genomic sequence information is present as contigs ≥3,623 bases). From a median 8.7-fold coverage of long contigs (≥10 kb) we estimated a true haploid nuclear genome size of 81.6 Mb, suggesting some redundancy in the current assembly. This estimate is in good agreement with the 159 Mb measured by van Dassow *et al*. \[[@B15]\] for the diploid G1 DNA content. The gene finder tool AUGUSTUS \[[@B16]\] predicts 37,921 protein gene models that cluster into a non-redundant set of 29,306 models including pseudogenes and short ORFs; 10,109 models have BLAST hits to the National Center for Biotechnology Information (NCBI) nr protein database at a conservative E-value cutoff of 1.0E-30 and thus are more indicative of the expected true protein-coding gene number (that is, expressed genes excluding pseudogenes and short ORFs). Best BLAST hits are listed in Additional file [1](#S1){ref-type="supplementary-material"}. In Table [1](#T1){ref-type="table"} we present an overview of the most abundant Clusters of Orthologous Groups (COG) domains in *T. oceanica*. The abundances of diverse groups of ATPases were overall very similar to those for other diatoms. A group of 19 chitinases is shared between the two centric *Thalassiosira*species.
{#F1}
######
Most abundant protein domains in diatom genomes
COG To Tp Pt Fc
-------------------------- ------------------------------------------------------------------------------------------------------------------------- ----- ----- ----- -----
ATPases
COG0515 SPS1, serine/threonine protein kinase 115 132 119 137
COG0464 SpoVK, ATPases of the AAA+ class 90 43 38 44
COG1132 MdlB, ABC-type multidrug transport system, ATPase and permease components 54 44 47 51
COG1222 RPT1, ATP-dependent 26S proteasome regulatory subunit 50 41 37 42
COG0465 HflB, ATP-dependent Zn proteases 49 37 35 39
COG1223 Predicted ATPase (AAA+ superfamily) 44 39 34 41
COG3899 Predicted ATPase 42 48 11 2
COG2274 SunT, ABC-type bacteriocin/lantibiotic exporters, contain an amino-terminal double-glycine peptidase domain 42 52 50 61
COG5265 ATM1, ABC-type transport system involved in Fe-S cluster assembly, permease and ATPase components 40 33 30 33
COG4618 ArpD, ABC-type protease/lipase transport system, ATPase and permease components 39 41 42 43
COG4987 CydC, ABC-type transport system involved in cytochrome bd biosynthesis, fused ATPase and permease components 31 50 46 56
COG4988 CydD, ABC-type transport system involved in cytochrome bd biosynthesis, ATPase and permease components 29 52 49 60
COG0488 Uup, ATPase components of ABC transporters with duplicated ATPase domains 29 50 46 53
COG1131 CcmA, ABC-type multidrug transport system, ATPase component | background = = = = = = = = = = diatoms are important primary producers beneath the ocean \ [ [ @ b1 ] \ ], contributing approximately 40 % to global marine productivity. although diatoms often dominate phytoplankton communities in nutrient - rich ecosystems, members of this diverse group are also adapted to survive and persist in nutrient - limited conditions. the development of large diatom blooms upon nutrient resupply demonstrates the metabolic plasticity inherent to their ability to recover rapidly from nutrient limitation. iron is an essential nutrient for all organisms and in particular for photoautotrophic organisms. it functions as a powerful electron carrier in iron - sulfur - and heme - containing proteins and as such is a required component of the photosynthetic diet. abundance of iron in seawater is low and phytoplankton growth in marine habitats is often limited by iron availability. this is best illustrated in high - nitrate low - chlorophyll ( hnlc ) regions, remote oceanic areas that lack any form of regular iron supply and suffer from a persistent shortage of this micronutrient. here, although other commonly limiting nutrients like nitrate or phosphate are stored at high concentrations, primary productivity - and biomass as a whole - is low \ [ [ @ c1 ] \ ]. numerous large - scale iron fertilization experiments have confirmed that iron is the limiting nutrient in hnlc regions \ [ [ @ b3 ] \ ]. phytoplankton blooms induced by iron fertilization were dominated by diatoms and carbon export to the deep - water floor could be observed in some cases. the strong response of diatoms to the input of iron and hnlc regions has been a motivation for enabling large - scale iron fertilization as a possible bioengineering strategy to sequester co ~ 2 ~ into the ocean in hnlc regions, which are otherwise rich in phosphorus. genome projects on the model organisms * thalassiosira pseudonana * \ [ [ @ b4 ] \ ] and * phaeodactylum tricornutum * \ [ [ @ b5 ] \ ] have already generated a wealth of insights into the metabolic complexity of diatoms \ [ [ @ b6 ] \ ], a consequence during the secondary endosymbiosis event that gave rise to this group \ [ [ @ b7 ] \ ]. this secondary endos ##ymbiosis brought together the benefits of a heterotrophic host and the \ ' red \ ' - type photosynthesis of red alga cells, which already have an elemental composition low in iron \ [ [ @ b8 ] \ ]. the impact of iron availability on phytoplankton growth has led to the evolution of strategies to counteract iron limitation. well established parts of the low - iron response found in diverse phytoplankton species are the reduction of the chloroplast system, the corresponding development of a chlorotic phenotype, compensation mechanisms ( replacement of iron - rich elements with iron - poor substitutes ) and the activation of high - affinity iron - uptake systems \ [ [ @ b9 ] \ ]. the substitution of ferredoxin by flavodoxin \ [ [ @ b10 ] \ ], the use of plastocyanin instead of cytochrome c ~ 6 ~ \ [ [ @ b11 ] \ ] and a variant stoichiometry of photosynthetic complexes \ [ [ @ b12 ] \ ] are notable adaptive strategies to facilitate diatom growth in low - iron conditions. oceanic and neritic phytoplankton species can be distinguished from each other by their growth characteristics and their tolerance to nutrient limitation \ [ [ @ b13 ] \ ]. unlike many other * thalassiosira * species that are predominantly found in coastal waters, * thalassiosiraoceanica * is adapted to oligotrophic conditions and is highly tolerant to iron limitation in particular. therefore, we chose * t. oceanica * ccmp1005 as a model for a comprehensive analysis of its low - iron response in the context of genomic information. here, we explore the complex cellular response of * t. oceanica * to low - iron conditions with genomics, transcriptomics and proteomics approaches complemented by reverse transcription - quantitative pcr ( rt - qpcr ) analyses. we present a metabolic reconstruction of the iron limitation response based on the transcriptomics data from cells grown under iron - limited versus iron - replete conditions. a metabolic isotope labeling approach using ^ 14 ^ n / ^ 15 ^ n was established for * t. oceanica * and showed the response to iron limitation at the protein expression level in a marine diatom for the first time. general characteristics of the \ ' diatom \ ' low - iron response and its ecological implications are discussed, as well as the constraints for species - specific adaptations to low - iron environments. results = = = = = = = characteristics of the * t. oceanica * genome - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the genome of the centric diatom * t. oceanica * ccmp1005 ( figure [ 1 ] ( # f1 ) { ref - type = " fig " } ) was * de novo * assembled from 725 mb of roche 454 sequence read information, generated using nuclear genomic dna ( gdna ) of an axenic clonal culture as substrate \ [ [ @ b14 ] \ ]. the current assembly version comprises 51, 656 contigs of total size 92. 15 mb at n50 = 3, 623 ( that is, 50 % of the genomic sequence information is present as contigs ≥3, 623 bases ). from a median 8. 7 - fold coverage of long contigs ( ≥10 kb ) we estimated a true haploid nuclear genome size of 81. 6 mb, suggesting some redundancy in the current assembly. this estimate is in good agreement with the 159 mb measured by van dassow * et al *. \ [ [ @ b15 ] \ ] for the diploid g1 dna content. the gene finder tool augustus \ [ [ @ b16 ] \ ] predicts 37, 921 protein gene models that cluster into a non - redundant set of 29, 306 models including pseudogenes and short orfs ; 10, 109 models have blast hits to the national center for biotechnology information ( ncbi ) nr protein database at a conservative e - value cutoff of 1. 0e - 30 and thus are more indicative of the expected true protein - coding gene number ( that is, expressed genes excluding pseudogenes and short orfs ). best blast hits are listed in additional file [ 1 ] ( # s1 ) { ref - type = " supplementary - material " }. in table [ 1 ] ( # t1 ) { ref - type = " table " } we present an overview of the most abundant clusters of orthologous groups ( cog ) domains in * t. oceanica *. the abundances of diverse groups of atpases were overall very similar to those for other diatoms. a group of 19 chitinases is shared between the two centric * thalassiosira * species.! [ * * * t. oceanica * ccmp1005 genome statistics * *. the sequenced strain * t. oceanica * ccmp1005 belongs to the centrales group of radially symmetric diatoms and was first isolated from the oligotrophic sargasso sea by r guillard. at 92. 15 mb, our genome assembly is slightly larger than the expected haploid genome size of 81. 6 mb, suggesting some redundancy in the current assembly. the genuine augustus gene model predictions include a large fraction of pseudogenes and short orfs that show no homology to any proteins from the ncbi nr database at a reasonable e - value cutoff. left inset contains light microscopy images of the sequenced organism in valve view ( upper image, chloroplasts brown ) and girdle view ( lower image, chloroplasts red from overlay of chlorophyll autofluorescence ). right inset shows the separation of nuclear and organellar dna in a cscl density gradient. stained dna emits blue fluorescence upon excitation with uv light. ] ( gb - 2012 - 13 - 7 - r66 - 1 ) { # f1 } # # # # # # most abundant protein domains in diatom genomes cog to tp pt fc - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - atpases cog0515 sps1, serine / threonine protein kinase 115 132 119 137 cog0464 spovk, atpases of the aaa + class 90 43 38 44 cog1132 mdlb, abc - type multidrug transport system, atpase and permease components 54 44 47 51 cog1222 rpt1, atp - dependent 26s proteasome regulatory subunit 50 41 37 42 cog0465 hflb, atp - dependent zn proteases 49 37 35 39 cog1223 predicted atpase ( aaa + superfamily ) 44 39 34 41 cog3899 predicted atpase 42 48 11 2 cog2274 sunt, abc - type bacteriocin / lantibiotic exporters, contain an amino - terminal double - glycine peptidase domain 42 52 50 61 cog5265 atm1, abc - type transport system involved in fe - s cluster assembly, permease and atpase components 40 33 30 33 cog4618 arpd, abc - type protease / lipase transport system, atpase and permease components 39 41 42 43 cog4987 cydc, abc - type transport system involved in cytochrome bd biosynthesis, fused atpase and permease components 31 50 46 56 cog4988 cydd, abc - type transport system involved in cytochrome bd biosynthesis, atpase and permease components 29 52 49 60 cog0488 uup, atpase components of abc transporters with duplicated atpase domains 29 50 46 53 cog1131 ccma, abc - type multidrug transport system, atpase component | Background = = = = = = = = = = Diatoms are important primary producers in the ocean \ [[ @ B1] \ ], contributing approximately 40% to global marine productivity. Although diatoms often dominate phytoplankton communities in nutrient - rich ecosystems, members of this diverse group are also adapted to survive and persist in nutrient - limited conditions. The development of large diatom blooms upon nutrient resupply demonstrates the metabolic plasticity inherent to their ability to recover rapidly from nutrient limitation. Iron is an essential nutrient for all organisms and in particular for photoautotrophic organisms. It functions as a powerful electron carrier in iron - sulfur - and heme - containing proteins and as such is a required component of the photosynthetic apparatus. Solubility of iron in seawater is low and phytoplankton growth in marine habitats is often limited by iron availability. This is best illustrated in high - nitrate low - chlorophyll (HNLC) regions, remote oceanic areas that lack any form of regular iron supply and suffer from a persistent shortage of this micronutrient. Here, although other commonly limiting nutrients like nitrate or phosphate are present at high concentrations, primary productivity - and biomass as a whole - is low \ [[ @ B2] \ ]. Numerous large - scale iron fertilization experiments have confirmed that iron is the limi$inf nutrient in HNLC regions \ [[ @ B3] \ ]. Phytoplankton blooms induced by iron fertilization were dominated by diatoms and carbon export to the deep - sea floor could be observed in some cases. The strong response of diatoms to the input of iron in HNLC regions has been a motivation for exploring large - scale iron fertilization as a possible bioengineering strategy to sequester CO ~ 2 ~ into the ocean in HNLC regions, which are otherwise rich in macronutrients. Genome projects on the model organisms * Thalassiosira pseudonana * \ [[ @ B4] \] and * Phaeodactylum tricornutum * \ [[ @ B5] \] have already generated a Aea:th of insights into the metabolic complexity of diatoms \ [[ @ B6] \ ], a consequence of the secondary endosymbiosis event that gave rise to this group \ [[ @ B7] \ ]. This secondary endosymbiosis brought together the benefits of a heterotrophic host and the \ ' red \ ' - type photosynthesis of red alga cells, which already have an elemental composition low in iron \ [[ @ B8] \ ]. The impact of iron availability on phytoplankton growth has led to the evolution of strategies to counteract iron limitation. Well established parts of the low - iron response found in diverse phytoplankton species are the reduction of the chloroplast system, the corresponding development of a chlorotic phenotype, compensation mechanisms (replacement of iron - rich elements with iron - poor substitutes) and the activation of high - affinity iron - uptake systems \ [[ @ B9] \ ]. The substitution of ferredoxin by flavodoxin \ [[ @ B10] \ ], the use of plastocyanin instead of cytochrome c ~ 6 ~ \ [[ @ B11] \] and a variant stoichiometry of photosynthetic complexes \ [[ @ B12] \] are notable adaptive strategies to facilitate diatom growth in low - iron conditions. Oceanic and neritic phytoplankton species can be distinguished from each other by their growth characteristics and their tolerance to nutrient limitation \ [[ @ B13] \ ]. Unlike many other * Thalassiosira * species that are predominantly found in coastal waters, * Thalassiosiraoceanica * is adapted to oligotrophic conditions and is highly tolerant to iron limitation in particular. Therefore, we chose * T. oceanica * CCMP1005 as a model for a comprehensive analysle of its low - iron response in the context of genomic information. Here, we explore the complex cellular response of * T. oceanica * to low - iron conditions with genomics, transcriptomics and proteomics approaches complemented by reverse transcription - quantitative PCR (RT - qPCR) analyses. We present a metabolic reconstruction of the iron limitation response based on the transcriptomics data from cells grown under iron - limited versus iron - replete conditions. A metabolic isotope labeling approach using ^ 14 ^ N / ^ 15 ^ N was estaglisYed for * T. oceanica * and showed the response to iron limitation at the protein expression level in a marine diatom for the first time. General characteristics of the \ ' diatom \ ' low - iron response and its ecological implications are discussed, as well as the constraints for species - specific adaptations to low - iron environments. Results = = = = = = = Characteristics of the * T. oceanica * genome - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The genome of the centric diatom * T. ocrsnica * CCMP1005 (Figure [1] (# F1) {ref - type = " fig "} ) was * de novo * assembled from 725 Mb of Roche 454 sequence read information, generated using nuclear genomic DNA (gDNA) of an axenic clonal culture as substrate \ [[ @ B14] \ ]. The current assembly version comprises 51, 656 contigs of total size 92. 15 Mb at N50 = 3, 623 (that is, 50% of the gen*mid sequence information is present as contigs ≥ 3, 623 bases ). From a median 8. 7 - fold coverage of long contigs (≥ 10 kb) we estimated a true haploid nuclear genome size of 81. 6 Mb, suggesting some redundancy in the current assembly. This estimate is in good agreement with the 159 Mb measured by van Dassow * et al *. \ [[ @ B15] \] for the diploid G1 DNA content. The gene finder tool AUGUSTUS \ [[ @ B16] \] predicts 37, 921 protein gene models that cluster into a non - redundant set of 29, 306 models including pseudogenes and short ORFs; 10, 109 models have BLAST hits to the National FentFr for Biotechnology Information (NCBI) nr protein database at a conservative E - value cutoff of 1. 0E - 30 and thus are more indicative of the expected true protein - coding gene number (that is, expressed genes excluding pseudogenes and short ORFs ). Best BLAST hits are listed in Additional file [1] (# S1) {ref - type = " supplementary - material " }. In Table [1] (# T1) {ref - type = " table "} we present an overview of the most abundant Clusters of Orthologous Groups (COG) domains in * T. oceanica *. The abundances of diverse groups of ATPases were overall very similar to those for other diatoms. A group of 19 chitinases is shared between the two centric * Thalassiosira * species. ! [* * * T. oceanica * CCMP1005 genome statistics * *. The sequenced strain * T. oceanica * CCMP1005 belongs to the Centrales group of radially symmetric diatoms and was first isolated from the oligotrophic Sargasso Sea by R Guillard. At 92. 15 Mb, our genome assembly is slightly larger than the expected haploid genome size of 81. 6 Mb, suggesting some redunFancg in the current assembly. The genuine AUGUSTUS gene model predictions include a large fraction of pseudogenes and short ORFs that show no homology to any proteins from the NCBI nr database at a reasonable E - value cutoff. Left inset contains light microscopy images of the sequenced organism in valve view (upper image, chloroplasts brown) and girdle view (lower image, chloroplasts red from overlay of chlorophyll autofluorescence ). Right inset shows the separation of nuclear and organellar DNA in a CsCl density gradient. Stained DNA emits blue fluorescence upon excitation with UV light.] (gb - 2012 - 13 - 7 - r66 - 1) {# F1} # # # # # # Most abundant protein domains in diatom genomes COG To Tp Pt Fc - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ATPases COG0515 SPS1, serine / threonine protein kinase 115 132 119 137 COG0464 SpoVK, ATPases of the AAA + class 90 43 38 44 COG1132 MdlB, ABC - type multidrug transport system, ATPase and permease components 54 44 47 51 COG1222 RPT1, ATP - dependent 26S proteasome regulatory subunit 50 41 37 42 COG0465 HflB, ATP - dependent Zn proteases 49 37 35 39 COG1223 Predicted ATPase (AAA + superfamily) 44 39 34 41 COG3899 Predicted ATPase 42 48 11 2 COG2274 SunT, ABC - type bacteriocin / lantibiotic exporters, contain an amino - ferjinal double - glycine peptidase domain 42 52 50 61 COG5265 ATM1, ABC - type transport system involved in Fe - S cluster assembly, permease and ATPase components 40 33 30 33 COG4618 ArpD, ABC - type protease / lipase transport system, ATPase and permease components 39 41 42 43 COG4987 CydC, ABC - type transport system involved in cytochrome bd biosynthesis, fused ATPase and permease components 31 50 46 56 COG4988 CydD, ABC - type transport system involved in cytochrome bd biosynthesis, ATPase and permease components 29 52 49 60 COG0488 Uup, ATPase components of ABC transporters w8tn duplicated ATPase domains 29 50 46 53 COG1131 CcmA, ABC - type multidrug transport system, ATPase component | Background ========== Diatoms are important primary producers in the ocean contributing approximately 40% to global marine productivity. Although diatoms often dominate phytoplankton communities in nutrient-rich ecosystems, members this diverse group are also adapted survive and persist in nutrient-limited conditions. The development of large diatom blooms upon nutrient resupply demonstrates the plasticity inherent to their ability to recover rapidly from nutrient limitation. Iron is an essential nutrient for all organisms and in particular for photoautotrophic organisms. It functions as a powerful electron carrier proteins and such is a required component of the photosynthetic apparatus. Solubility of iron in is low and phytoplankton growth in is often limited by availability. This is best illustrated in high-nitrate low-chlorophyll (HNLC) remote oceanic areas that any of regular iron supply suffer from persistent shortage of this micronutrient. Here, although other commonly limiting nutrients like nitrate or phosphate are present at concentrations, primary productivity - and biomass as a whole - is low \[[@B2]\]. Numerous large-scale iron fertilization experiments have confirmed that iron is the limiting nutrient in HNLC \[[@B3]\]. Phytoplankton induced by iron fertilization were dominated diatoms and carbon export to the deep-sea floor could be observed in some cases. The strong response of diatoms the input iron in HNLC regions has been a motivation for exploring large-scale iron fertilization as possible bioengineering strategy to sequester CO~2~into the ocean in HNLC regions, which otherwise rich in macronutrients. Genome projects on the organisms *Thalassiosira and *Phaeodactylum tricornutum*\[[@B5]\] have already generated a wealth of insights into the metabolic complexity of diatoms \[[@B6]\], a consequence of the secondary event that gave rise to this group This secondary endosymbiosis brought together the benefits a heterotrophic host and the \'red\'-type photosynthesis of red alga which already have elemental composition low in iron \[[@B8]\]. The impact of iron availability on phytoplankton growth has led to the evolution of to counteract iron limitation. Well established parts of the response found in diverse species are the reduction the chloroplast system, the corresponding development chlorotic phenotype, compensation mechanisms (replacement of iron-rich elements with iron-poor substitutes) and activation high-affinity iron-uptake systems \[[@B9]\]. The of ferredoxin by flavodoxin \[[@B10]\], the of plastocyanin instead of cytochrome and a variant stoichiometry of photosynthetic complexes \[[@B12]\] are notable adaptive strategies to diatom growth low-iron conditions. Oceanic neritic phytoplankton species can be distinguished from each other by their characteristics and their tolerance to nutrient \[[@B13]\]. Unlike many other *Thalassiosira*species that are predominantly found in coastal waters, adapted to oligotrophic conditions and is highly tolerant to iron limitation in particular. Therefore, we chose *T. oceanica*CCMP1005 as a model for a comprehensive analysis of its low-iron response in the context of genomic information. Here, we explore the complex cellular response of *T. oceanica*to low-iron conditions with genomics, transcriptomics and proteomics complemented by reverse transcription-quantitative PCR (RT-qPCR) analyses. We present a metabolic reconstruction of the iron limitation response based on data from cells grown under versus iron-replete A metabolic isotope labeling ^14^N/^15^N was established for *T. oceanica*and showed the response to iron at the protein expression level in a marine diatom for the first time. General characteristics of the \'diatom\' low-iron and its ecological implications are discussed, as well as the constraints for species-specific adaptations to low-iron environments. ======= Characteristics of the *T. oceanica*genome ------------------------------------------ genome of the centric diatom *T. oceanica*CCMP1005 (Figure [1](#F1){ref-type="fig"}) was *de novo*assembled from 725 Mb of Roche 454 sequence read information, generated using nuclear genomic DNA (gDNA) of an axenic clonal culture as substrate \[[@B14]\]. The current assembly version 51,656 contigs total size 92.15 Mb at 3,623 is, 50% of the genomic sequence information is present as contigs ≥3,623 bases). From a median 8.7-fold coverage of long contigs (≥10 kb) we estimated a haploid nuclear genome size of 81.6 Mb, suggesting some redundancy in the current assembly. This estimate is in good agreement with the 159 Mb by van Dassow *et al*. \[[@B15]\] the G1 content. The gene tool AUGUSTUS \[[@B16]\] predicts 37,921 protein gene models cluster into non-redundant set of 29,306 models pseudogenes and short ORFs; 10,109 models have BLAST hits to the National Center for Biotechnology Information (NCBI) nr protein database at a conservative E-value cutoff of 1.0E-30 thus more indicative of the expected true protein-coding gene number (that is, expressed genes excluding pseudogenes and short ORFs). Best BLAST hits are listed in Additional file [1](#S1){ref-type="supplementary-material"}. In Table [1](#T1){ref-type="table"} we present an overview of the most of Orthologous Groups (COG) domains in *T. oceanica*. The abundances of diverse groups of ATPases were overall very to those for other diatoms. group of 19 chitinases is shared between the two *Thalassiosira*species. {#F1} ###### Most abundant protein domains in diatom COG Tp Pt Fc -------------------------- ------------------------------------------------------------------------------------------------------------------------- ----- ----- ----- ----- ATPases COG0515 SPS1, serine/threonine protein 115 132 119 137 COG0464 SpoVK, ATPases of the AAA+ class 90 43 38 44 COG1132 ABC-type multidrug transport system, ATPase and permease components 54 44 47 51 COG1222 RPT1, ATP-dependent 26S proteasome regulatory subunit 50 41 42 COG0465 HflB, ATP-dependent Zn 49 37 35 39 COG1223 Predicted (AAA+ superfamily) 44 39 34 41 COG3899 Predicted ATPase 42 48 11 COG2274 SunT, ABC-type bacteriocin/lantibiotic exporters, contain an amino-terminal double-glycine peptidase domain 42 52 50 COG5265 ATM1, ABC-type transport system involved Fe-S assembly, permease and ATPase components 40 30 33 COG4618 ArpD, ABC-type protease/lipase transport system, ATPase and permease components 39 41 42 COG4987 CydC, ABC-type transport system involved in cytochrome bd fused ATPase permease 31 46 56 COG4988 CydD, ABC-type transport system involved in cytochrome bd biosynthesis, ATPase and permease components 29 52 49 60 Uup, ATPase of ABC transporters with duplicated ATPase domains 29 50 46 53 COG1131 CcmA, ABC-type multidrug transport system, ATPase component | BAckgroUND
==========
diAToms ARe ImPortaNT PRimARy pRodUcERS iN tHE OceaN \[[@b1]\], coNTrIbUTINg approxImAtElY 40% tO gLobAL mARiNe pRodUctiVITy. alTHOUGH DiAtomS OfTEn DoMinATE PhyTOplankTOn CoMMUNItIeS In nUTrIEnt-RICH EcOSYSTEmS, meMbErs of this DIversE grOUP aRE ALSO adapted tO SurViVe aND pErsiSt iN nUTrieNT-LIMited cOnDITIons. tHe DEVEloPMeNt Of LArgE DiatoM BloOMS UpON NutRIenT resupPly demONStRaTes THE MEtAbOlIC PlaSTiCiTy inhEREnt TO tHEir aBiLITY TO RECOVeR RapIdLy fRoM nUTrIeNT lImitAtIon.
IrON IS an eSsenTIaL nUtrIeNt For all oRGAnIsmS ANd IN PaRtICuLAr For PhOToAutOtroPHIC organiSmS. it fUNcTIONs as A POweRfUl ELEcTrOn CArRIER In iRon-SUlFUR- aND HeME-CONtaIninG PRotEINs And as sucH is a rEQuIREd COMPoNeNT OF ThE PhOTOSYnTHetic aPpAratus. soLUbiLiTY oF irOn IN SEaWAtEr Is LOw aND PHytoplaNkton GRoWth In maRiNe haBItATS is OFTEn LiMiTeD By IrOn aVAiLaBilITy. THIs iS beST iLlUStraTeD IN hIgh-nITRAte loW-ChlorOPHYll (HNlc) Regions, ReMotE oCeanIC arEAs thaT Lack Any forM Of ReGUlAR IROn Supply and SuFfeR FroM A pErSiSTenT SHoRtAge OF ThiS MICrOnUtRiEnT. here, ALtHoUGH oThER CoMmoNly lImITing nUtRientS LIkE NitRate Or phoSphatE are pRESent aT HiGh conCEntRatiONs, Primary pRodUCTIviTY - ANd BiOmasS as a whOlE - IS low \[[@B2]\].
NuMErous lArge-Scale irOn FeRTILiZAtIoN ExpeRImeNtS HavE CoNFIrMeD thAT IrON Is The LImITiNG NUtrIEnt iN HnLc ReGions \[[@b3]\]. pHYtoplanKtoN bLoOmS inDUced by iROn FerTILIZATIoN were doMINATeD By DiAtomS aNd CaRBoN ExpORt To the Deep-SeA fLOoR cOUlD bE OBsErvEd in soMe caSEs. tHe stronG respOnsE oF DiATOMs to THe Input of IrOn In Hnlc reGions hAS bEEn A mOtiVATIoN for EXpLoring laRGE-Scale IROn fERtiliZation As A PoSSiBLe BiOENgInEeRIng StRatEgY TO SEqUEstER CO~2~into The OCEAN IN hnLC rEGIONs, Which Are oTHerwIsE RIch In macROnUTriENTs.
GENOme pRojECTs on tHe modeL OrgANisms *tHALASSIosIRa PseuDoNaNA*\[[@B4]\] and *PHAeODACtyLUM TricoRNuTUm*\[[@b5]\] hAVE ALrEadY GeNERAteD a weAlTH oF iNSightS InTo tHE mEtAboLIC cOMPLEXiTy OF DiAtOMs \[[@b6]\], a CoNsEquEnce of The secONdArY EndosyMbIOSis EvenT THAT GAve Rise To ThIS groUp \[[@B7]\]. This SECOndArY ENDoSYmBIOsIs BrOugHT togetHER THe benEfItS Of A HeTeROTROphIc host aNd tHE \'rEd\'-tyPE PhotosyNThesis OF red aLGa ceLLS, whiCh alREAdY hAVe AN ELEmeNtAl cOmpOsItiON loW in IroN \[[@B8]\].
tHe iMPAct OF iroN AvAilabiLiTy on PhyToPlANKtON growtH Has leD tO tHe evOLUTIOn Of sTrategIES TO COUnTERACT IROn LimitAtION. weLl EStABLIsHED paRts oF THe LOw-IrOn REspoNse Found in dIVErSe pHYTOplankTON SpeCIES aRe THe reduCtiOn OF ThE ChLorOPlast SyStEM, THe COrRESpOndINg dEveLOPMENt of A ChLorOTic pHENoType, COMpenSAtIOn MEcHANiSmS (REPlACEMent oF iroN-rICh eLEMenTS WitH iROn-POOR SubsTiTuTeS) And ThE ACTivaTIoN Of hIGh-AFFinItY iRON-UPTAke sysTemS \[[@b9]\]. THE subStITuTIOn OF fERREdOXiN by fLAvoDoXin \[[@B10]\], THe uSE oF pLasTOcYAnIN iNsTeAd oF CytochroME c~6~\[[@b11]\] anD a VARiAnT sTOIcHIOMEtRY of phOTosYNtHEtic cOmPlEXES \[[@B12]\] ArE NoTaBle ADaPtive straTEGieS to faciliTate dIAtOM gRoWth IN low-iROn COndItiOnS.
OCEanIC aNd NERiTiC PhytOPlaNKTON SPECieS cAN BE DistinGUISheD FRom eaCH otHEr BY ThEiR GROwTH CHaRaCTerISticS And tHEiR tOLeraNcE TO NuTRieNt lImItAtIon \[[@B13]\]. unlIke MANy OtHEr *ThALasSIoSIRA*sPECIes thAt ArE PREDomiNANtly FOuNd In coASTAL Waters, *THAlAssIosiraoCEanIca*IS aDaPTED tO oLigotRoPHiC cONDItioNs anD iS hIghLY ToLerant to IrON LimiTaTiOn iN partiCULAr. ThErefOre, wE ChoSe *t. oCEAnicA*CcMp1005 AS a Model fOr a cOmpRehensIve anALysIS OF ItS lOW-iron RESPONSe iN tHe CoNText oF gEnoMic INfOrmATiON.
HERE, We expLoRe THE coMpLEx cELluLaR ReSPonse Of *T. OCEaNiCa*tO lOw-iRoN COndiTionS WITH GenomicS, tRANSCRiPTOmICS and proteOmics aPpRoACHes cOMPLeMEnteD by ReVeRsE tRANScrIPtion-QuantiTaTIve pcr (rT-qPcr) ANAlYses. we pRESEnt A meTABOLic rEcoNStRUCtioN OF tHe IrOn limitATiOn reSpOnsE baSed oN tHE TRAnSCRiPTomIcs data FROm CELLS grOwn UnDEr IrON-limITEd vERsUs Iron-rEplEtE conDitiONs. A mEtabolIc iSOtOPe LabeLiNg aPPRoaCH UsiNg ^14^N/^15^N Was eSTABLISHeD FoR *t. ocEANIcA*AND ShoWeD The respOnsE to iroN LimiTatIOn At tHE PRoTEin eXPresSIOn LEVel In a mARiNe dIaTom FOR the fIrSt TimE. gEneRAL ChaRActErIStics Of The \'DiaTom\' Low-iroN respOnSE AnD its eCoLogIcaL ImPlicatIONs ARE discUSSEd, As WElL AS tHe cONstRAIntS For SpecIeS-SPECIFiC aDaptATionS to low-IRON EnvirONMeNts.
ResULTs
=======
cHArACtErIstics OF THE *t. oCeANiCa*geNOMe
------------------------------------------
thE GeNoMe OF THe CEnTRic diaToM *t. OcEAnica*cCmP1005 (FiguRe [1](#F1){ReF-TypE="Fig"}) Was *DE noVo*ASsEMbLEd frOm 725 mb OF RochE 454 SEquENCe read iNfoRMatION, GENERATed UsinG NuClEaR GenOmiC Dna (gDnA) OF aN AXeNIC CloNAL cUltURE aS SuBstrate \[[@b14]\]. THE curReNT ASsEMBlY verSiOn cOMPRises 51,656 CoNTIGs OF TOTAl SIZe 92.15 Mb at n50 = 3,623 (tHaT iS, 50% of THE GenOmiC SeqUEnCE iNfOrMatiON iS PRESENt as CoNTigS ≥3,623 BaSeS). fRom A MediAn 8.7-FOld CoVerAge of LonG ContIgs (≥10 Kb) wE ESTImATeD A True HapLOID nucLeAr genoME Size of 81.6 MB, SUgGESting SOmE REdunDaNcY iN THE cURrEnt ASseMBLY. THis esTimAte Is In GOOD agrEemeNT wItH THe 159 mb MeaSured By VaN DaSSow *et al*. \[[@b15]\] foR ThE DiPLoiD G1 DNa ConTent. the gEnE FIndEr tOOl AUGUstUs \[[@B16]\] prediCts 37,921 ProteIn Gene ModEls thAt clUstEr Into A nOn-REduNdAnt seT of 29,306 moDeLS INCluding pSEUDoGEnES aNd sHOrt OrfS; 10,109 MODELS haVe BLASt hITS To The nATIOnAl ceNtER For BiOTeCHnOLOGY iNfOrMATioN (NCBI) Nr PRoteiN daTABASE aT a cONseRvATivE e-VAluE cUtofF Of 1.0e-30 and THUS ARe MOrE indIcAtIVE oF tHE ExpecteD trUE PRotEIn-cODing GEnE nUMber (ThaT IS, EXpResSeD GENeS ExCLUDinG pSEUDoGENES aNd sHoRt ORfS). BEst bLaSt hIts aRe lISTeD in adDitiONal fiLE [1](#s1){Ref-TyPE="SuPpLemeNtary-MATeRiaL"}. IN TABlE [1](#t1){Ref-typE="TaBlE"} wE PREsEnt An OVeRVIew of The MoST AbuNDANT CLUsTERS of Orthologous GROUpS (cOg) dOMAINS IN *t. OCEAnICA*. The aBuNDaNcEs oF dIversE groups of AtPaSes weRe OVERaLl VERY SimIlAR tO tHOse foR oTher dIatOmS. a gROuP oF 19 ChItinasES iS shaRed BETWEeN the TWo cENtriC *tHaLassIosIra*SpeCIES.
{#f1}
######
MOsT abUnDAnt proTeIn DOmaiNS IN DiaTOM GeNomeS
cOg To TP PT Fc
-------------------------- ------------------------------------------------------------------------------------------------------------------------- ----- ----- ----- -----
aTPasEs
cog0515 sPS1, seRINe/ThreonINE PrOTEiN KInAsE 115 132 119 137
CoG0464 SPoVK, atpASeS OF tHe AAa+ cLaSs 90 43 38 44
COg1132 mdLb, abC-tYpE MUltiDrUG TrAnsPOrT SYsTem, aTPAse AnD PErmeAse ComponENTS 54 44 47 51
cOG1222 RpT1, ATp-DepEndEnt 26s PrOtEaSOMe regUlatOrY suBUnit 50 41 37 42
CoG0465 HFLb, aTP-DepenDEnt zN ProTeaseS 49 37 35 39
CoG1223 PredICTED ATPaSe (aAA+ SupErFAMILy) 44 39 34 41
Cog3899 PrEDiCTeD aTPASe 42 48 11 2
COg2274 sunT, AbC-TyPE BActerIOCIN/LANTIbIoTic expoRTers, COnTAIn an aMiNo-TErmInal double-glycINE pePTIdAse DOMAin 42 52 50 61
cog5265 atM1, AbC-typE TRANSPort SysteM iNVoLVeD IN fE-s ClUsTer AsSEmBly, pErmEAsE anD atPasE comPoNeNTs 40 33 30 33
COg4618 ARPd, abC-TyPE PrOTeAsE/lIpaSe TrAnSPOrT SYSteM, atpAsE anD pErMeaSe COMPoNentS 39 41 42 43
COG4987 Cydc, ABc-type trANSpoRt sysTEm inVolvED in cytOchRoME BD BiOSyNTHesiS, fUsEd AtpAse aNd PERMEase cOmPoNEnTs 31 50 46 56
cOG4988 CYdd, ABc-TYpE tRAnspoRT sYsTeM inVOlVeD In cYToChROMe bD biOsyNtHEsis, AtPAse AnD PeRMEaSE compoNeNTs 29 52 49 60
COg0488 uuP, aTPase cOMPoNENtS oF ABc TRanSPorTErS WIth DUpliCaTeD AtpASe dOMaIns 29 50 46 53
coG1131 ccmA, abC-tYPE mulTidRug tRaNSport SYStem, atPaSE CompONEnT | Background ==========Diatoms are important primary producers in the ocean \[[@B1]\], contributing approximately 40%to global marine productivity. Although diatoms oftendominatephytoplankton communitiesin nutrient-rich ecosystems, membersofthis diverse group are also adapted tosurvive and persist in nutrient-limited conditions. The development of large diatom blooms upon nutrient resupply demonstratesthe metabolic plasticity inherent to their abilityto recover rapidly from nutrient limitation. Iron is an essential nutrient for all organisms and in particular for photoautotrophicorganisms. It functions as a powerful electron carrierin iron-sulfur- and heme-containing proteins and as such isa required componentof the photosynthetic apparatus. Solubility of iron in seawater is low and phytoplankton growth inmarine habitats is often limited by iron availability.This isbestillustrated in high-nitrate low-chlorophyll (HNLC) regions, remote oceanic areas that lack anyform of regular iron supplyand suffer from apersistentshortage of this micronutrient. Here, although other commonly limiting nutrientslike nitrate or phosphate are present at high concentrations, primary productivity - andbiomass as awhole - islow \[[@B2]\]. Numerouslarge-scale iron fertilization experimentshave confirmed thatiron is the limiting nutrient inHNLC regions \[[@B3]\]. Phytoplankton blooms induced by ironfertilizationwere dominated bydiatoms and carbon exportto the deep-sea floor could be observed in some cases. The strong response of diatoms to the input of iron in HNLC regionshasbeen a motivation for exploring large-scale iron fertilization as a possible bioengineeringstrategy to sequester CO~2~into theocean in HNLC regions, which are otherwise rich in macronutrients.Genome projects onthe model organisms *Thalassiosira pseudonana*\[[@B4]\] and*Phaeodactylum tricornutum*\[[@B5]\] have already generated a wealth of insights into themetabolic complexity ofdiatoms \[[@B6]\], a consequence of thesecondary endosymbiosis event that gave rise to this group\[[@B7]\]. This secondaryendosymbiosis brought together the benefitsof a heterotrophic hostand the \'red\'-type photosynthesis of red alga cells, which already have an elemental composition low in iron \[[@B8]\]. The impact ofiron availability on phytoplankton growth has led to the evolutionof strategies tocounteract iron limitation. Wellestablished parts of the low-ironresponsefound in diverse phytoplankton species are thereduction of thechloroplast system, the corresponding development of a chlorotic phenotype, compensation mechanisms (replacement of iron-rich elements with iron-poor substitutes) and the activation of high-affinity iron-uptake systems \[[@B9]\]. The substitution of ferredoxin by flavodoxin \[[@B10]\], the useof plastocyanin instead of cytochromec~6~\[[@B11]\] anda variantstoichiometry of photosynthetic complexes \[[@B12]\] are notable adaptive strategies to facilitate diatom growth inlow-iron conditions. Oceanic and neritic phytoplankton species can bedistinguished from eachother by their growth characteristics and their tolerance to nutrient limitation \[[@B13]\]. Unlike many other *Thalassiosira*species that are predominantly found incoastal waters, *Thalassiosiraoceanica*is adapted to oligotrophic conditions and ishighly tolerant to ironlimitation in particular. Therefore, we chose *T. oceanica*CCMP1005 as a modelfor a comprehensive analysis of its low-iron response in the context of genomic information.Here,we explore the complex cellular response of *T. oceanica*to low-ironconditions with genomics, transcriptomicsand proteomics approaches complemented by reversetranscription-quantitative PCR (RT-qPCR)analyses. We present a metabolic reconstruction of the iron limitationresponse based on the transcriptomics datafrom cells grown under iron-limited versus iron-replete conditions. A metabolicisotope labeling approach using ^14^N/^15^N was established for*T. oceanica*and showed the response to iron limitation atthe protein expression levelina marine diatom forthe first time. General characteristics of the \'diatom\' low-ironresponse and its ecologicalimplications are discussed, as well as the constraintsfor species-specific adaptations to low-iron environments.Results ======= Characteristics of the *T. oceanica*genome ------------------------------------------The genome of the centric diatom *T. oceanica*CCMP1005 (Figure [1](#F1){ref-type="fig"}) was *de novo*assembled from 725Mb of Roche 454 sequence read information, generatedusing nuclear genomic DNA (gDNA) of an axenicclonal culture as substrate \[[@B14]\]. The current assembly version comprises 51,656 contigs of totalsize 92.15Mbat N50 = 3,623 (that is, 50% of the genomic sequenceinformation ispresent as contigs≥3,623 bases). From a median 8.7-fold coverage of long contigs (≥10 kb) we estimated atrue haploid nuclear genomesize of 81.6 Mb, suggesting some redundancy in the current assembly. This estimate is in good agreement with the 159 Mb measured by van Dassow *et al*. \[[@B15]\] for the diploid G1 DNA content. The gene finder tool AUGUSTUS \[[@B16]\] predicts 37,921 protein gene models that cluster into a non-redundant set of 29,306 models including pseudogenes and shortORFs; 10,109 modelshave BLAST hitsto the National Center for Biotechnology Information (NCBI) nr protein database at a conservative E-value cutoff of 1.0E-30 and thus are moreindicative of theexpected true protein-coding gene number (that is, expressed genesexcludingpseudogenesand short ORFs). Best BLASThits are listed in Additional file [1](#S1){ref-type="supplementary-material"}. In Table [1](#T1){ref-type="table"} we present an overview ofthe mostabundant Clusters of Orthologous Groups(COG) domains in *T. oceanica*. The abundances of diverse groups of ATPases were overall very similar to those for otherdiatoms. A group of19 chitinases is shared betweenthe two centric *Thalassiosira*species. {#F1} ######Most abundant protein domains in diatom genomes COG To Tp Pt Fc -------------------------- ------------------------------------------------------------------------------------------------------------------------- ----- ----- ----- ----- ATPases COG0515SPS1,serine/threonine protein kinase 115 132119 137 COG0464 SpoVK, ATPases of the AAA+ class 90 43 38 44 COG1132 MdlB, ABC-type multidrug transport system,ATPase and permease components544447 51 COG1222 RPT1,ATP-dependent 26S proteasome regulatory subunit 50 41 37 42 COG0465 HflB, ATP-dependent Zn proteases 4937 35 39 COG1223Predicted ATPase (AAA+ superfamily) 44 39 34 41 COG3899Predicted ATPase 42 48 11 2 COG2274SunT, ABC-type bacteriocin/lantibiotic exporters, containan amino-terminal double-glycine peptidase domain 42 52 50 61 COG5265 ATM1,ABC-type transport systeminvolved in Fe-S cluster assembly, permease and ATPase components 40 33 30 33 COG4618 ArpD, ABC-type protease/lipase transport system,ATPase and permease components 3941 42 43COG4987 CydC, ABC-type transport system involved in cytochrome bd biosynthesis, fused ATPase and permease components 31 5046 56 COG4988 CydD, ABC-type transport system involvedincytochrome bd biosynthesis, ATPase and permease components 2952 49 60 COG0488 Uup, ATPase components of ABC transporterswith duplicated ATPase domains 2950 46 53 COG1131 CcmA, ABC-typemultidrug transportsystem, ATPase component | Background ========== Diatoms are important primary producers in the ocean \[[@B1]\], contributing approximately 40% to global marine productivity. Although _diatoms_ _often_ dominate phytoplankton _communities_ in _nutrient-rich_ ecosystems, members of _this_ diverse group are also adapted to _survive_ and persist in nutrient-limited conditions. The development of large diatom _blooms_ _upon_ _nutrient_ resupply demonstrates the metabolic plasticity inherent to their ability to recover rapidly from nutrient limitation. Iron is an essential nutrient for all organisms and in particular _for_ photoautotrophic _organisms._ It functions as _a_ _powerful_ electron carrier in _iron-sulfur-_ and heme-containing proteins and as such is a required component of the photosynthetic apparatus. Solubility of iron in seawater is _low_ and phytoplankton _growth_ _in_ marine habitats is often _limited_ _by_ iron availability. _This_ is _best_ illustrated in _high-nitrate_ low-chlorophyll (HNLC) regions, remote oceanic areas that lack any form of regular iron supply and _suffer_ _from_ a persistent shortage _of_ _this_ _micronutrient._ Here, although other commonly limiting nutrients like nitrate or phosphate are _present_ at high concentrations, primary productivity - and biomass as a whole - _is_ low \[[@B2]\]. Numerous _large-scale_ iron _fertilization_ _experiments_ _have_ confirmed that iron _is_ the limiting nutrient _in_ HNLC regions \[[@B3]\]. Phytoplankton _blooms_ induced by iron fertilization were dominated by diatoms and _carbon_ export to _the_ _deep-sea_ _floor_ _could_ be _observed_ in some cases. _The_ strong response of diatoms to the input of iron in HNLC regions has been _a_ _motivation_ _for_ _exploring_ large-scale iron fertilization as a _possible_ _bioengineering_ strategy to _sequester_ CO~2~into the _ocean_ in HNLC regions, which are _otherwise_ rich in macronutrients. Genome projects on the model organisms *Thalassiosira pseudonana*\[[@B4]\] _and_ *Phaeodactylum tricornutum*\[[@B5]\] have already generated a wealth _of_ insights into the metabolic complexity of diatoms \[[@B6]\], a consequence of the secondary endosymbiosis event that gave rise to this group _\[[@B7]\]._ This secondary _endosymbiosis_ brought together the _benefits_ _of_ a _heterotrophic_ host _and_ the \'red\'-type photosynthesis _of_ red _alga_ cells, which already have _an_ _elemental_ _composition_ _low_ in iron _\[[@B8]\]._ _The_ impact of _iron_ availability on phytoplankton growth has _led_ to the evolution of _strategies_ to counteract iron limitation. _Well_ established parts of the _low-iron_ response _found_ in diverse phytoplankton species are the reduction _of_ _the_ _chloroplast_ system, the corresponding development of a chlorotic phenotype, compensation mechanisms (replacement of _iron-rich_ elements with _iron-poor_ substitutes) and the activation _of_ high-affinity iron-uptake systems \[[@B9]\]. The substitution of _ferredoxin_ _by_ flavodoxin \[[@B10]\], the use of plastocyanin instead _of_ cytochrome c~6~\[[@B11]\] and _a_ variant stoichiometry of photosynthetic complexes \[[@B12]\] are notable adaptive strategies to facilitate diatom _growth_ in low-iron conditions. Oceanic and neritic phytoplankton species _can_ be distinguished from _each_ other _by_ their growth _characteristics_ and their tolerance to nutrient limitation \[[@B13]\]. Unlike many other *Thalassiosira*species that are _predominantly_ _found_ in coastal waters, _*Thalassiosiraoceanica*is_ adapted to oligotrophic conditions and is highly tolerant to iron limitation in particular. _Therefore,_ we chose *T. oceanica*CCMP1005 as a model for a _comprehensive_ _analysis_ of its low-iron response in the _context_ of _genomic_ information. Here, we _explore_ _the_ complex cellular response of *T. oceanica*to low-iron conditions with genomics, transcriptomics and proteomics approaches complemented by reverse _transcription-quantitative_ PCR (RT-qPCR) analyses. We present _a_ _metabolic_ reconstruction of the iron _limitation_ response based on the transcriptomics data from cells grown under iron-limited versus _iron-replete_ conditions. A metabolic isotope labeling approach using _^14^N/^15^N_ was established for _*T._ oceanica*and showed the response to iron _limitation_ at the _protein_ expression _level_ in a marine diatom for the first time. General characteristics _of_ the \'diatom\' _low-iron_ response and _its_ ecological implications are discussed, as well as _the_ _constraints_ for species-specific adaptations to _low-iron_ environments. Results ======= Characteristics of _the_ *T. oceanica*genome ------------------------------------------ The genome of _the_ _centric_ _diatom_ _*T._ oceanica*CCMP1005 (Figure [1](#F1){ref-type="fig"}) was _*de_ novo*assembled from _725_ Mb of _Roche_ _454_ sequence _read_ information, generated _using_ nuclear genomic DNA (gDNA) of an axenic clonal _culture_ as substrate \[[@B14]\]. The current assembly version comprises 51,656 _contigs_ of _total_ size 92.15 Mb at N50 = 3,623 (that is, 50% of the genomic _sequence_ _information_ is present as contigs _≥3,623_ bases). _From_ a _median_ 8.7-fold coverage of long _contigs_ (≥10 kb) we estimated a true haploid nuclear genome size of 81.6 Mb, suggesting some redundancy in the current assembly. This estimate is in good agreement with the 159 Mb measured by van Dassow _*et_ al*. _\[[@B15]\]_ for the _diploid_ G1 DNA _content._ The gene finder tool AUGUSTUS \[[@B16]\] predicts 37,921 protein gene models that cluster into a non-redundant _set_ of _29,306_ models including _pseudogenes_ and short _ORFs;_ 10,109 models _have_ BLAST _hits_ to the National _Center_ for Biotechnology Information (NCBI) _nr_ protein _database_ _at_ a conservative E-value cutoff of 1.0E-30 and thus are _more_ indicative of the _expected_ _true_ protein-coding gene _number_ (that is, _expressed_ genes excluding pseudogenes and short ORFs). Best _BLAST_ hits are listed _in_ Additional file [1](#S1){ref-type="supplementary-material"}. _In_ Table [1](#T1){ref-type="table"} we present an _overview_ of the _most_ _abundant_ Clusters of _Orthologous_ Groups (COG) domains in *T. oceanica*. The _abundances_ of diverse groups of ATPases were overall very _similar_ to those for other _diatoms._ A group _of_ _19_ chitinases is shared between the _two_ centric *Thalassiosira*species. _{#F1} _######_ Most _abundant_ protein domains in _diatom_ _genomes_ _COG_ To Tp Pt Fc -------------------------- ------------------------------------------------------------------------------------------------------------------------- _-----_ ----- ----- _-----_ _ATPases_ COG0515 _SPS1,_ _serine/threonine_ protein kinase 115 132 119 _137_ COG0464 SpoVK, ATPases of the AAA+ class 90 43 38 _44_ COG1132 MdlB, ABC-type multidrug transport system, ATPase and permease components _54_ 44 47 51 COG1222 RPT1, ATP-dependent 26S proteasome regulatory subunit 50 41 37 42 COG0465 HflB, _ATP-dependent_ Zn proteases 49 37 35 39 COG1223 Predicted ATPase (AAA+ _superfamily)_ 44 39 _34_ 41 COG3899 Predicted ATPase 42 48 11 2 COG2274 SunT, ABC-type bacteriocin/lantibiotic _exporters,_ contain an amino-terminal _double-glycine_ peptidase domain 42 52 _50_ _61_ _COG5265_ ATM1, _ABC-type_ transport system involved _in_ Fe-S _cluster_ assembly, permease _and_ _ATPase_ components 40 33 _30_ 33 COG4618 ArpD, ABC-type protease/lipase transport system, _ATPase_ _and_ permease _components_ 39 41 42 _43_ _COG4987_ CydC, ABC-type _transport_ system involved in cytochrome _bd_ biosynthesis, fused ATPase _and_ _permease_ components 31 50 _46_ 56 COG4988 _CydD,_ ABC-type transport system involved in cytochrome _bd_ biosynthesis, ATPase and _permease_ components 29 _52_ _49_ _60_ COG0488 _Uup,_ _ATPase_ components of ABC transporters with duplicated ATPase domains 29 50 46 53 COG1131 CcmA, ABC-type _multidrug_ transport system, ATPase _component_ |
Handled by Yu Xue
Introduction {#s0005}
============
Cancer somatic mutations and viral oncogenes can generate tumor-specific protein sequences that are entirely absent from normal human cells. These novel proteins may result in the formation of tumor-specific antigens (TSAs) [@b0005]. As an important type of TSAs, neoantigens are generated by tumor-specific proteins, and presented by major histocompatibility complexes (MHCs) on cell surfaces through antigen presentation, where they can be recognized by T-cell receptors (TCRs) [@b0010], [@b0015]. Recently, neoantigens have attracted a large amount of attention, because they are potential biomarkers to distinguish tumor cells from normal cells. Neoantigens are of critical importance for cancer immunotherapy in the following two aspects. First, the neoantigen burden and quality can be used to predict therapeutic effects for immune checkpoint blockade therapy, such as blockage of programmed death-1 (PD-1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) [@b0020], [@b0025], [@b0030]. Second, neoantigens can be used as potential targets for cancer immunotherapy, such as personalized cancer vaccines [@b0035], [@b0040] and adoptive cell therapy (ACT) [@b0045]. Therefore, there is an urgent need to identify neoantigens accurately for cancer patients.
With the progress of cancer immunogenomics, several kinds of integrated software have been developed for tumor-specific neoantigen detection, such as TSNAD [@b0050] and pVAC-seq [@b0055]. The most critical function of such software is to predict the binding affinities between mutant peptides and human leukocyte antigen (HLA) alleles. To achieve this, a lot of well-acknowledged and popular tools, such as NetMHC [@b0060], NetMHCpan [@b0065], sNebula [@b0070], and HLA-CNN [@b0075], can be used. In addition, several databases can provide necessary information for the development of tools to predict the affinities between peptides and HLA alleles. For example, the Immune Epitope Database (IEDB) is an important immune-related database, providing a large amount of valuable and experimentally-validated information of immune epitopes [@b0080]. The International Immunogenetics Information System (IMGT) offers information about antibodies, TCRs, MHCs, and so on [@b0085]. Taking advantage of existing neoantigen prediction software, several neoantigen-related databases have been built. For example, TRON Cell Line Portal (TCLP) presents potential neoantigens of 1082 cancer cell lines [@b0090]. The Cancer Immunome Atlas (TCIA) presents the relationship between tumor genotypes and immunophenotypes based on 20 solid cancers, and provides a quantitative index for immunotherapy response [@b0095]. With the rapid growth of cancer genomics data, researchers are able to discover potential shared neoantigens across tumor patient populations [@b0100], [@b0105].
In this study, we developed a tumor-specific neoantigen database (TSNAdb v1.0) from pan-cancer immunogenomic analyses. Based on the 7748 tumor samples of 16 tumor types from The Cancer Genome Atlas (TCGA), we predicted the binding affinities between mutant/wild-type peptides and HLA class I molecules. Datasets we used include somatic mutation data of tumor samples from TCGA and the corresponding HLA allele data from TCIA. Two different versions of NetMHCpan, v2.8 [@b0065], and v4.0 [@b0110], were used for prediction. Furthermore, we also conducted extensive analyses and presented detailed information of potential neoantigens generated by somatic mutations, utilizing the related filtering tools embedded in TSNAD [@b0050]. In addition, we employed the recurrent missense mutations in combination with the highly frequent HLA alleles to predict and analyze potential shared neoantigens. Our study would provide a platform to discover putative targets for neoantigen-based cancer immunotherapy.
Database content and usage {#s0010}
==========================
Data source {#s0015}
-----------
We collected somatic mutations and HLA alleles of 7748 tumor samples across 16 tumor types from TCGA (Release7.0, <https://portal.gdc.cancer.gov>) and TCIA (<https://tcia.at/home>), respectively. These tumor samples carry 972,187 missense mutations, among which 18,897 were found recurrently (at least three occurrences in all tumor samples). We selected the top 100 HLA alleles (frequency \>0.5%) of 7748 tumor samples and combined them with the recurrent missense mutations to predict potential shared neoantigens. Moreover, we also extracted 13,459 recurrent missense mutations from 9155 samples derived from the International Cancer Genome Consortium (ICGC) (Release20, <https://icgc.org/>) and 16 highly frequent HLA alleles (frequencies \>5%) from the 1000 Genome Project [@b0115] for the prediction of potential shared neoantigens.
Neoantigen prediction {#s0020}
---------------------
We took the information on somatic mutations and HLA alleles of each tumor sample and employed NetMHCpan v2.8 [@b0065] and NetMHCpan v4.0 [@b0110] for neoantigen prediction, using the filtering tools embedded in our previously-developed software TSNAD [@b0050]. All the peptides with 8--11 amino acids that contain missense mutations were extracted as mutant peptides, and the corresponding wild-type peptides were extracted as references. We collected the mutant peptides and HLA alleles with binding affinity IC~50~ \< 500 nM (including strong binding with IC~50~ \< 150 nM and weak binding with 150 nM \< IC~50~ \< 500 nM), without consideration of the binding level between their corresponding wild-type peptides and HLA alleles. We then clustered prediction results based on tumor types and calculated the frequencies of shared neoantigens. Compared with NetMHCpan v2.8, NetMHCpan v4.0 is trained based on both binding affinity data and mass spectrometry data, thus adopting stricter criteria for binding prediction. Consequently, 3,707,562 and 1,146,961 neoantigens were predicted by NetMHCpan v2.8 and v4.0, respectively, among which, 716,876 neoantigens were found in both predictions. The potential shared neoantigens based on recurrent mutations and highly frequent HLA alleles were predicted in the similar way.
Web interface {#s0025}
-------------
To facilitate the utilization of TSNAdb, we have established a web interface to browse and analyze neoantigens. The web interface comprises five main pages ([Figure 1](#f0005){ref-type="fig"}A): (i) Home, (ii) Browse, (iii) Search, (iv) Validation, and (v) Download. In the following context, we exemplify the usage of TSNAdb with the results predicted by NetMHCpan v2.8.Figure 1**An overview of the TSNAdb web interfaceA.** TSNAdb comprises five main components: (i) Home; (ii) Browse; (iii) Search; (iv) Validation, and (v) Download. The distribution of HLA alleles (**B)** and somatic missense mutations (**C)** extracted from TCGA and TCIA are listed. **D.** The average neoantigen loads across different tissues. **E.** Top 20 genes with predicted neoantigens in 7748 samples. **F.** The result of 'Search' page under the selection of "NetMHCpan2.8, *KRAS*, and TCGA". **G.** The result of 'Search' page under the selection of "NetMHCpan2.8, *KRAS*, and ICGC". **H.** Example of validation data from IEDB in 'Validation' page (top) and partial information on 'Download' page (bottom). The predicted binding level 'Strong' indicates strong binding with IC~50~ \< 150 nM. TCGA, The Cancer Genome Atlas; TCIA, The Cancer Immunome Atlas; HLA, human leukocyte antigen; TSNAD, tumor-specific neoantigen detector; ICGC: International Cancer Genome Consortium; IEDB, Immune Epitope Database; WT, wild type.
In the 'Home' page, TSNAdb provides a statistical table about the database, including the number of projects, samples, HLA alleles ([Figure 1](#f0005){ref-type="fig"}B), mutations ([Figure 1](#f0005){ref-type="fig"}C), and predicted neoantigens of each tumor type. The table presents 3,707,562 potential neoantigens from the 7748 tumor samples across 16 tumor types. The average predicted neoantigen loads vary across different tumor types ([Figure 1](#f0005){ref-type="fig"}D). For instance, the average predicted neoantigen load for uterus cancer is 1957, which is 21 for thyroid cancer. Besides, users can get the detailed information about the predicted neoantigens of each tumor type by clicking on the tissue name in the table. The content of each tumor type includes top 20 HLA alleles and top 20 genes with predicted neoantigens in this tumor type, which are shown as two figures. All predicted neoantigens in this tumor type would be displayed below figures.
To further understand the distribution of neoantigens at gene level, users can retrieve neoantigens by feeding in a gene name in the 'Browse' page ([Figure 1](#f0005){ref-type="fig"}E). The retrieval results provide more functions than that indicated in each tumor type | handled by yu xue introduction { # s0005 } = = = = = = = = = = = = cancer somatic mutations and viral oncogenes can generate tumor - specific protein sequences that are entirely absent inside normal human cells. these novel proteins may help in the formation of tumor - specific antigens ( tsas ) [ @ b0005 ]. as an important type of tsas, neoantigens are generated by tumor - specific proteins, and presented by major histocompatibility complexes ( mhcs ) on cell surfaces through antigen presentation, where they can be recognized by t - cell receptors ( tcrs ) [ @ b0010 ], [ @ b0015 ]. recently, neoantigens have attracted a large amount of attention, because organisms are potential biomarkers to distinguish tumor cells from normal cells. neoantigens are of critical importance for cancer immunotherapy in the following two aspects. first, the neoantigen burden and quality can be used to predict therapeutic effects for immune checkpoint blockade therapy, such as blockage of programmed death - 1 ( pd - 1 ) and cytotoxic t lymphocyte - associated antigen - 4 ( ctla - 4 ) [ @ b0020 ], [ @ b0025 ], [ @ b0030 ]. second, neoantigens can be used as potential targets for cancer immunotherapy, such as personalized cancer vaccines [ @ b0035 ], [ @ b0040 ] and adoptive cell therapy ( act ) [ @ b0045 ]. therefore, there is an urgent need to identify neoantigens accurately by cancer patients. with the progress on cancer immunogenomics, several kinds of integrated software have been developed for tumor - specific neoantigen detection, such as tsnad [ @ b0050 ] and pvac - seq [ @ 1760 ]. the most critical function of such software is to predict the binding role between mutant peptides and human leukocyte antigen ( hla ) sequences. to achieve this, a lot of well - acknowledged extremely popular tools, such as netmhc [ @ 1750 ], netmhcpan [ @ b0065 ], snebula [ @ b0070 ], and hla - cnn [ @ b0075 ], can be used. in addition, several databases can provide necessary information for the development of tools to predict the affinities between peptides and hla alleles. for example, the immune epitope database ( iedb ) is an important immune - related database, providing a large amount of valuable and experimentally - validated information of immune epitopes [ @ b0080 ]. the international immunogenetics information system ( imgt ) offers information about antibodies, tcrs, mhcs, and so on [ @ b0085 ]. taking advantage of existing neoantigen prediction software, several neoantigen - related databases have been built. for example, tron cell line portal ( tclp ) presents potential neoantigens of 1082 cancer cell lines [ @ b0090 ]. the cancer immunome atlas ( tcia ) presents the relationship between tumor genotypes and immunophenotypes based on 20 solid cancers, and provides a quantitative index for immunotherapy response [ @ b0095 ]. with the rapid growth of cancer genomics data, researchers are able to discover potential shared neoantigens across tumor patient populations [ @ b0100 ], [ @ b0105 ]. in this study, we developed a tumor - specific neoantigen database ( tsnadb v1. 0 ) from pan - cancer immunogenomic analyses. based on the 7748 tumor samples of 16 tumor types from the cancer genome atlas ( tcga ), we predicted the binding affinities between mutant / wild - type peptides and hla class i molecules. datasets we used include somatic mutation data of tumor samples from tcga and the corresponding hla allele data from tcia. two different versions of netmhcpan, v2. 8 [ @ b0065 ], and v4. 0 [ @ b0110 ], were used for prediction. furthermore, we also conducted extensive analyses and presented detailed information of potential neoantigens generated by somatic mutations, utilizing the related filtering tools embedded in tsnad [ @ b0050 ]. in addition, we employed the recurrent missense mutations in combination with the highly frequent hla alleles to predict and analyze potential shared neoantigens. our study would provide a platform to discover putative targets for neoantigen - based cancer immunotherapy. database content and usage { # s0010 } = = = = = = = = = = = = = = = = = = = = = = = = = = data source { # s0015 } - - - - - - - - - - - we collected somatic mutations and hla alleles of 7748 tumor samples across 16 tumor types from tcga ( release7. 0, < https : / / portal. gdc. cancer. gov > ) and tcia ( < https : / / tcia. at / home > ), respectively. these tumor samples carry 972, 187 missense mutations, among which 18, 897 were found recurrently ( at least three occurrences in all tumor samples ). we selected the top 100 hla alleles ( frequency \ > 0. 5 % ) of 7748 tumor samples and combined them with the recurrent missense mutations to predict potential shared neoantigens. moreover, we also extracted 13, 459 recurrent missense mutations from 9155 samples derived from the international cancer genome consortium ( icgc ) ( release20, < https : / / icgc. org / > ) and 16 highly frequent hla alleles ( frequencies \ > 5 % ) from the 1000 genome project [ @ b0115 ] for the prediction of potential shared neoantigens. neoantigen prediction { # s0020 } - - - - - - - - - - - - - - - - - - - - - we took the information on somatic mutations and hla alleles of each tumor sample and employed netmhcpan v2. 8 [ @ b0065 ] and netmhcpan v4. 0 [ @ b0110 ] for neoantigen prediction, using the filtering tools embedded in our previously - developed software tsnad [ @ b0050 ]. all the peptides with 8 - - 11 amino acids that contain missense mutations were extracted as mutant peptides, and the corresponding wild - type peptides were extracted as references. we collected the mutant peptides and hla alleles with binding affinity ic ~ 50 ~ \ < 500 nm ( including strong binding with ic ~ 50 ~ \ < 150 nm and weak binding with 150 nm \ < ic ~ 50 ~ \ < 500 nm ), without consideration of the binding level between their corresponding wild - type peptides and hla alleles. we then clustered prediction results based on tumor types and calculated the frequencies of shared neoantigens. compared with netmhcpan v2. 8, netmhcpan v4. 0 is trained based on both binding affinity data and mass spectrometry data, thus adopting stricter criteria for binding prediction. consequently, 3, 707, 562 and 1, 146, 961 neoantigens were predicted by netmhcpan v2. 8 and v4. 0, respectively, among which, 716, 876 neoantigens were found in both predictions. the potential shared neoantigens based on recurrent mutations and highly frequent hla alleles were predicted in the similar way. web interface { # s0025 } - - - - - - - - - - - - - to facilitate the utilization of tsnadb, we have established a web interface to browse and analyze neoantigens. the web interface comprises five main pages ( [ figure 1 ] ( # f0005 ) { ref - type = " fig " } a ) : ( i ) home, ( ii ) browse, ( iii ) search, ( iv ) validation, and ( v ) download. in the following context, we exemplify the usage of tsnadb with the results predicted by netmhcpan v2. 8. figure 1 * * an overview of the tsnadb web interfacea. * * tsnadb comprises five main components : ( i ) home ; ( ii ) browse ; ( iii ) search ; ( iv ) validation, and ( v ) download. the distribution of hla alleles ( * * b ) * * and somatic missense mutations ( * * c ) * * extracted from tcga and tcia are listed. * * d. * * the average neoantigen loads across different tissues. * * e. * * top 20 genes with predicted neoantigens in 7748 samples. * * f. * * the result of ' search ' page under the selection of " netmhcpan2. 8, * kras *, and tcga ". * * g. * * the result of ' search ' page under the selection of " netmhcpan2. 8, * kras *, and icgc ". * * h. * * example of validation data from iedb in ' validation ' page ( top ) and partial information on ' download ' page ( bottom ). the predicted binding level ' strong ' indicates strong binding with ic ~ 50 ~ \ < 150 nm. tcga, the cancer genome atlas ; tcia, the cancer immunome atlas ; hla, human leukocyte antigen ; tsnad, tumor - specific neoantigen detector ; icgc : international cancer genome consortium ; iedb, immune epitope database ; wt, wild type. in the ' home ' page, tsnadb provides a statistical table about the database, including the number of projects, samples, hla alleles ( [ figure 1 ] ( # f0005 ) { ref - type = " fig " } b ), mutations ( [ figure 1 ] ( # f0005 ) { ref - type = " fig " } c ), and predicted neoantigens of each tumor type. the table presents 3, 707, 562 potential neoantigens from the 7748 tumor samples across 16 tumor types. the average predicted neoantigen loads vary across different tumor types ( [ figure 1 ] ( # f0005 ) { ref - type = " fig " } d ). for instance, the average predicted neoantigen load for uterus cancer is 1957, which is 21 for thyroid cancer. besides, users can get the detailed information about the predicted neoantigens of each tumor type by clicking on the tissue name in the table. the content of each tumor type includes top 20 hla alleles and top 20 genes with predicted neoantigens in this tumor type, which are shown as two figures. all predicted neoantigens in this tumor type would be displayed below figures. to further understand the distribution of neoantigens at gene level, users can retrieve neoantigens by feeding in a gene name in the ' browse ' page ( [ figure 1 ] ( # f0005 ) { ref - type = " fig " } e ). the retrieval results provide more functions than that indicated in each tumor type | Handled by Yu Xue Introduction {# s0005} = = = = = = = = = = = = Cancer somatic mutations and viral oncogenes can generate tumor - specific protein sequences that are entirely absent from normal human cells. These novel proteins may result in the formation of tumor - specific antigens (TSAs) [@ b0005 ]. As an important type of TSAs, neoantigens are generated by tumor - specific proteins, and presented by major histocompatibility complexes (MHCs) on cell surfaces through antigen presentation, where they can be recognized by T - cell receptors (TCRs) [@ b0010 ], [@ b0015 ]. Recently, neoantigens have attracted a large amount of attention, because they are potential biomarkers to distinguish tumor cells from normal cells. Neoantigens are of critical importance for cancer immunotherapy in the following two aspects. First, the neoantigen burden and quality can be used to predict therapeutic effects for immune cheskpoiJt blockade therapy, such as blockage of programmed death - 1 (PD - 1) and cytotoxic T lymphocyte - associated antigen - 4 (CTLA - 4) [@ b0020 ], [@ b0025 ], [@ b0030 ]. Second, neoantigens can be used as potential targets for cancer immunotherapy, such as personalized cancer vaccines [@ b0035 ], [@ b0040] and adoptive cell therapy (ACT) [@ b0045 ]. Therefore, there is an urgent need to identify neoantigens accurately for cancer patients. With the progress of cancer immunogenomics, several kinds of integrated software have been developed for tumor - specific neoantigen detection, such as TSNAD [@ b0050] and pVAC - seq [@ b0055 ]. The most critical function of such software is to predict the binding affinities between mutant peptides and human leukocyte antigen (HLA) alleles. To achieve this, a lot of well - acknowledged and popular tools, such as NetMHC [@ b0060 ], NetMHCpan [@ b0065 ], sNebula [@ b0070 ], and HLA - CNN [@ b0075 ], can be used. In addition, several databases can provide necessary information for the development of tools to predict the affinities between peptides and HLA alleles. For example, the Immune Epitope Database (IEDB) is an important immune - related database, providing a large amount of valuable and experimentally - validated information of immune epitopes [@ b0080 ]. The International Immunogenetics Information System (IMGT) offers information about antibodies, TCRs, MHCs, and so on [@ b0085 ]. Taking advantage of existing neoantigen prediction software, several neoantigen - related databases have been built. For example, TRON Cell Line Portal (TCLP) presents potential meoantigeMs of 1082 cancer cell lines [@ b0090 ]. The Cancer Immunome Atlas (TCIA) presents the relationship between tumor genotypes and immunophenotypes based on 20 solid cancers, and provides a quantitative index for immunotherapy response [@ b0095 ]. With the rapid growth of cancer genomics data, researchers are able to discover potential shared neoantigens across tumor patient populwtiPns [@ b0100 ], [@ b0105 ]. In this study, we developed a tumor - specific neoantigen database (TSNAdb v1. 0) from pan - cancer immunogenomic analyses. Based on the 7748 tumor samples of 16 tumor types from The Cancer Genome Atlas (TCGA ), we predicted the binding affinities between mutant / wild - type peptides and HLA class I molecules. Datasets we used include somatic mutation data of tumor samples from TCGA and the corresponding HLA allele data from TCIA. Two different versions of NetMHCpan, v2. 8 [@ b0065 ], and v4. 0 [@ b0110 ], wWr3 used for prediction. Furthermore, we also conducted extensive analyses and presented detailed information of potential neoantigens generated by somatic mutations, utilizing the related filtering tools embedded in TSNAD [@ b0050 ]. In addition, we employed the recurrent missense mutations in combination with the highly frequent HLA alleles to predict and analyze potential shared neoantigens. Our study would provide a platform to discover putative targets for neoantigen - based cancer immunotherapy. Database content and usage {# s0010} = = = = = = = = = = = = = = = = = = = = = = = = = = Data source {# s0015} - - - - - - - - - - - We collected somatic mutations and HLA alleles of 7748 tumor samples across 16 tumor types from TCGA (Release7. 0, <https: / / portal. gdc. cancer. gov> ) and TCIA (< https: / / tcia. at / home> ), respectively. These tumor samples carry 972, 187 missense mutations, among which 18, 897 were found recurrently (at least three occurrences in all tumor samples ). We selected the top 100 HLA alleles (frequency \> 0. 5%) of 7748 tumor samples and combined them with the recurrent missense mutations to predict potential shared neoantigens. Moreover, we also extracted 13, 459 recurrent missense mutations from 9155 samples derived from the International Cancer Genome Consortium (ICGC) (Release20, <https: / / icgc. org /> ) and 16 highly frequent HLA alleles (frequencies \> 5%) from the 1000 Genome Project [@ b0115] for the prediction of potential shared neoantigens. Neoantigen prediction {# s0020} - - - - - - - - - - - - - - - - - - - - - We took the information on somatic mutations and HLA alleles of each Gumo4 sample and employed NetMHCpan v2. 8 [@ b0065] and NetMHCpan v4. 0 [@ b0110] for neoantigen prediction, using the filtering tools embedded in our prWviouslJ - developed software TSNAD [@ b0050 ]. All the peptides with 8 - - 11 amino acids that contain missense mutations were extracted as mutant peptides, and the corresponding wild - type peptides were extracted as reCrrences. We collected the mutant peptides and HLA alleles with binding affinity IC ~ 50 ~ \ <500 nM (including strong binding with IC ~ 50 ~ \ <150 nM and weak binding with 150 nM \ <IC ~ 50 ~ \ <500 nM ), without consideration of the binding level between their corresponding wild - type peptides and HLA alleles. We then clustered prediction results based on tumor types and calculated the frequencies of shared neoantigens. Compared with NetMHCpan v2. 8, NetMHCpan v4. 0 is trained based on both binding affinity data and mass spectrometry data, thus adopting stricter criteria for binding prediction. Consequently, 3, 707, 562 and 1, 146, 961 neoantigens were predicted by NetMHCpan v2. 8 and v4. 0, respectively, among which, 716, 876 neoantigens were found in both predictions. The potential shared neoantigens based on recurrent mutations and highly frequent HLA alleles were predicted in the similar way. Web interface {# s0025} - - - - - - - - - - - - - To facilitate the utilization of TSNAdb, we have established a web interface to browse and analyze neoantigens. The web interface comprises five main pages ([ Figure 1] (# v000^) {ref - type = " fig "} A ): (i) Home, (ii) Browse, (iii) Search, (iv) Validation, and (v) Download. In the following context, we exemplify the usage of TSNAdb with the results predicted by NetMHCpan v2. 8. Figure 1 * * An overview of the TSNAdb web interfaceA. * * TSNAdb comprises five main components: (i) Home; (ii) Browse; (iii) Search; (iv) Validation, and (v) Download. The distribution of HLA alleles (* * B) * * and somatic missense mutations (* * C) * * extracted from TCGA and TCIA are listed. * * D. * * The average neoantigen loads across different tissues. * * E. * * Top 20 genes with predicted neoantigens in 7748 samples. * * F. * * The result of ' Search ' page under the selection of " NetMHCpan2. 8, * KRAS *, and TCGA ". * * G. * * The result of ' Search ' page under the selection of " NetMHCpan2. 8, * KRAS *, and ICGC ". * * H. * * Example of validation data from IEDB in ' Validation ' page (top) and partial information on ' Download ' page (bottom ). The predicted binding level ' Strong ' indicates strong binding with IC ~ 50 ~ \ <150 nM. TCGA, The Cancer Genome Atlas; TCIA, The Cancer Immunome Atlas; HLA, human leukocyte antigen; TSNAD, tumor - speDLfic neoantigen detector; ICGC: International Cancer Genome Consortium; IEDB, Immune Epitope Database; WT, wild type. In the ' Home ' page, TSNAdb provides a statistical table about the database, including the number of projects, samples, HLA alleles ([ Figure 1] (# f0005) {ref - type = " fig "} B ), mutations ([ Figure 1] (# f0005) {ref - type = " fig "} C ), and predicted neoantigens of each tumor type. The table presents 3, 707, 562 potential neksntigens from the 7748 tumor samples across 16 tumor types. The average predicted neoantigen loads vary across different tumor types ([ Figure 1] (# f0005) {ref - type = " fig "} D ). For instance, the average predicted neoantigen load for uterus cancer is 1957, which is 21 for thyroid cancer. Besides, users can get the detailed information about the predicted neoantigens of each tumor type by clicking on the tissue name in the table. The content of each tumor type includes top 20 HLA alleles and top 20 genes with predicted neoantigens in this tumor type, which are shown as two figures. All predicted neoantigens in this tumor type would be displayed below figures. To further understand the distribution of neoantigens at gene level, users can retrieve neoantigens by feeding in a gene name in the ' Browse ' page ([ Figure 1] (# f0005) {ref - type = " fig "} E ). The retrieval results provide more functions than that indicated in each tumor type | Handled by Yu Xue Introduction {#s0005} Cancer somatic mutations and viral oncogenes can generate tumor-specific protein sequences that are absent from normal human cells. These proteins may result in the formation of tumor-specific antigens (TSAs) [@b0005]. As an important type of TSAs, neoantigens are generated by tumor-specific proteins, and presented by histocompatibility complexes (MHCs) on cell surfaces through antigen presentation, where they can be recognized by T-cell receptors (TCRs) [@b0010], [@b0015]. Recently, neoantigens have attracted a large of attention, because they are potential biomarkers to distinguish cells from normal cells. Neoantigens are of critical importance for cancer immunotherapy in the following two aspects. First, the burden and quality be used to predict therapeutic effects for immune checkpoint blockade therapy, such as blockage of programmed death-1 (PD-1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) [@b0020], [@b0025], [@b0030]. Second, neoantigens be used cancer immunotherapy, such as personalized cancer vaccines [@b0040] adoptive cell therapy (ACT) [@b0045]. Therefore, there is an urgent need to identify accurately for patients. With progress of cancer immunogenomics, several kinds of integrated software been developed for tumor-specific neoantigen detection, such as TSNAD [@b0050] and pVAC-seq [@b0055]. The most critical function such software is to predict the binding affinities between mutant peptides and leukocyte antigen (HLA) alleles. achieve this, lot of well-acknowledged and popular tools, such as NetMHC [@b0060], NetMHCpan [@b0065], sNebula [@b0070], and HLA-CNN can be used. In addition, several databases can provide necessary information the development of tools to predict the affinities between and HLA alleles. For the Immune Epitope Database (IEDB) is an immune-related database, providing a large amount of valuable and experimentally-validated information of immune epitopes [@b0080]. International Immunogenetics Information System (IMGT) offers information about antibodies, TCRs, MHCs, and on [@b0085]. Taking advantage of existing neoantigen prediction software, several have been built. For example, Line potential neoantigens of 1082 cancer cell lines The Cancer Immunome (TCIA) the relationship tumor and immunophenotypes based on 20 solid cancers, and provides a quantitative index immunotherapy response [@b0095]. With the rapid growth of cancer genomics data, researchers able to discover potential shared neoantigens across tumor patient populations [@b0100], [@b0105]. In this study, we developed a tumor-specific neoantigen database (TSNAdb v1.0) from pan-cancer immunogenomic analyses. Based on the 7748 tumor samples of 16 tumor from Cancer Genome Atlas (TCGA), we predicted the binding affinities between mutant/wild-type and HLA class I molecules. Datasets we used include somatic mutation data of tumor samples from TCGA and the corresponding HLA allele from TCIA. different versions of NetMHCpan, v2.8 [@b0065], and v4.0 [@b0110], were used for prediction. we also conducted extensive analyses and presented detailed information of potential neoantigens generated by somatic mutations, utilizing the related filtering tools embedded in TSNAD [@b0050]. In addition, we employed the recurrent mutations in combination with the highly frequent HLA alleles to predict and potential shared Our study would provide a platform to discover putative targets for neoantigen-based cancer immunotherapy. Database content and usage {#s0010} ========================== Data source {#s0015} ----------- We collected somatic mutations and HLA alleles of 7748 tumor samples across 16 tumor types from TCGA (Release7.0, <https://portal.gdc.cancer.gov>) and TCIA (<https://tcia.at/home>), respectively. These samples carry 972,187 missense mutations, among which 18,897 were found recurrently (at least three occurrences in all tumor We selected top 100 HLA alleles (frequency \>0.5%) of 7748 tumor samples and combined them recurrent missense mutations to predict potential Moreover, we also 13,459 recurrent missense mutations from 9155 samples from the International Cancer Genome (ICGC) (Release20, <https://icgc.org/>) and 16 highly frequent alleles (frequencies \>5%) from the 1000 Genome [@b0115] for the prediction potential shared neoantigens. Neoantigen prediction {#s0020} --------------------- We took the information on somatic and HLA alleles of each tumor sample and employed NetMHCpan v2.8 [@b0065] NetMHCpan v4.0 [@b0110] for neoantigen prediction, using the filtering tools embedded in our previously-developed software TSNAD the peptides 8--11 amino acids that contain mutations were extracted as mutant peptides, and corresponding wild-type peptides were extracted as references. We collected the mutant and HLA alleles binding IC~50~ \< 500 nM strong binding with IC~50~ \< nM and weak binding with 150 nM \< IC~50~ \< 500 nM), without consideration of the binding level between their corresponding wild-type peptides and HLA alleles. We then clustered prediction results based on tumor types and calculated the frequencies of shared neoantigens. Compared with v2.8, NetMHCpan is trained based on both binding affinity data mass spectrometry data, thus adopting stricter criteria for binding prediction. Consequently, and 1,146,961 neoantigens were predicted by NetMHCpan v2.8 and v4.0, respectively, among which, 716,876 neoantigens were found in The shared based on recurrent mutations and highly HLA were predicted in the similar way. Web interface {#s0025} ------------- To facilitate the utilization of TSNAdb, we have established web interface to browse and analyze neoantigens. The web interface comprises five main pages ([Figure 1](#f0005){ref-type="fig"}A): (i) Home, (ii) (iii) Search, (iv) Validation, and (v) Download. In the following context, we exemplify the usage of TSNAdb with the results predicted by NetMHCpan v2.8.Figure 1**An overview of the interfaceA.** TSNAdb comprises five main (i) Home; (ii) Browse; (iii) Search; (iv) Validation, and (v) Download. The distribution of HLA alleles (**B)** and somatic missense mutations (**C)** extracted from TCGA and TCIA listed. **D.** The average neoantigen different tissues. **E.** Top 20 genes with neoantigens 7748 samples. The result of 'Search' page the selection of "NetMHCpan2.8, *KRAS*, and TCGA". **G.** The result of 'Search' page under the selection of "NetMHCpan2.8, *KRAS*, and ICGC". **H.** Example of validation data from IEDB in 'Validation' page partial on 'Download' page (bottom). The predicted binding level 'Strong' indicates strong binding with IC~50~ \< 150 nM. TCGA, The Cancer Genome Atlas; TCIA, The Immunome Atlas; HLA, human leukocyte antigen; neoantigen detector; ICGC: International Cancer Genome Consortium; IEDB, Immune Epitope Database; WT, wild type. In the 'Home' page, TSNAdb provides a statistical table about the database, including the number of projects, samples, alleles ([Figure 1](#f0005){ref-type="fig"}B), mutations ([Figure 1](#f0005){ref-type="fig"}C), and predicted neoantigens of each tumor type. The table presents 3,707,562 neoantigens the 7748 tumor samples across 16 tumor types. The average neoantigen loads vary across different tumor types 1](#f0005){ref-type="fig"}D). For instance, the average predicted neoantigen load for uterus is 1957, which is for thyroid cancer. Besides, users can detailed information about the predicted neoantigens of each tumor type by clicking on the tissue name in table. The content of each tumor type includes top 20 HLA alleles and top genes with predicted neoantigens in this tumor type, are shown as two figures. All predicted neoantigens in this type would be displayed below figures. To further understand the distribution of neoantigens at gene can retrieve neoantigens by feeding in a in the 'Browse' page ([Figure 1](#f0005){ref-type="fig"}E). The retrieval results provide more functions than that indicated in each tumor type | haNDled bY Yu xuE
iNTRoductioN {#S0005}
============
CaNcEr sOmaTic mUTatioNs AnD vIrAL oNCogenES caN geneRaTe tumOR-SpEcIfiC proteiN SEqUEnceS That aRe eNtirElY AbsEnt frOm NOrMAL HUmaN cells. tHeSE NOVel pROteInS MaY resULt iN the FormATion oF TUmoR-speCIFIC ANTiGEnS (TSAS) [@B0005]. aS an ImPORtaNt tYPe OF TSAs, nEOANTIGEns arE GeNeRAteD by tumor-SpEcIfic prOteINs, And prEsenTED by MAjOr hISTOCoMPAtIbilITY COMplEXEs (mhCS) oN CELL SuRFaCEs THrouGH ANTIGen pResenTATiOn, where tHEy CAn Be recOgNIzED By T-ceLL ReceptorS (tcrs) [@B0010], [@B0015]. ReCenTlY, NEoANTIGenS HaVe aTtRACteD A LARge AMOuNt OF attEnTIoN, BeCAusE tHey are pOtENTiAl BIOmARKERS To DisTInguIsh TUMOr CElLS FroM NORMAl cellS. NeoanTigeNs aRE Of cRITiCAL ImporTaNce for canceR iMmunOtHerapy iN tHe FOlLOwInG Two AsPecTS. fIRST, THE NeOaNTIGEN buRDEN AND QUaliTy Can Be USEd to PReDict THERApeuTic EffeCTs fOR imMuNe cHeCkpOiNT BLOckaDe THErApY, SUcH as BLOckAGe OF ProGRamMed dEaTH-1 (PD-1) anD cYTOtoxiC t lympHOcytE-assOCIateD AnTigen-4 (CtlA-4) [@b0020], [@B0025], [@b0030]. sECoNd, NEoanTiGENS caN bE USed aS pOtEnTIAl tarGETs foR cANCeR IMmUNOthErapy, SucH aS PeRsOnalizED CANcer VaCCines [@B0035], [@b0040] anD ADOpTiVe cELL tHERaPY (Act) [@B0045]. ThEReforE, tHeRe iS an uRGenT neEd to IdentIFy nEoANTIGens AcCURateLy FoR canCER pATieNts.
wiTH THe pROgREss of cANceR ImmunOGenomics, seVeral Kinds of inteGRATED SOFTwArE hAVE BEen dEvELoPeD FoR tUMor-SPECIFic neOanTIGEn DeteCTIoN, sUCH aS tSnAd [@B0050] aNd PvAC-SeQ [@b0055]. THE Most CrITICAL functiOn of suCH sofTwaRe IS tO PReDict ThE BinDING aFFInItiEs bEtWeEn muTAnt PEptIdeS AnD HumAn lEUkOCytE ANTiGEn (hLA) AlleLes. To AChievE thiS, a LoT oF WeLL-ACKnowLEDGED AND poPULAr TOoLs, sUch AS NEtmHc [@B0060], NEtMHcpan [@b0065], SNeBULA [@b0070], and hlA-Cnn [@b0075], cAN Be UsED. IN AdDITioN, SeveRAl databASES Can pRoVide necEssary INfORmATiON FOR the DevELOPMeNt oF ToOls tO PredICt ThE aFFInitIeS beTweEN peptiDes And hlA AlLELes. for EXampLe, tHe iMMUNE EpiTOpE dAtAbaSE (IeDB) Is aN iMporTANt immunE-ReLAtEd dATabASE, prOvIdINg A lArGE aMOUnt of VaLUAbLE And eXPEriMENTALly-vALIDAted iNformATIOn oF IMMunE EpIToPES [@b0080]. thE inTeRnAtIonal ImmUnOGENETics InfoRmaTion SYsTem (ImgT) OfFERs InFORmatIoN abOuT ANtIBODIes, TcRs, MHCs, and SO ON [@B0085]. TaKINg AdVANtAGe OF ExiSTInG NEoANTIgeN PReDiCTiON SOftWARE, SeVEral nEOAnTiGeN-rELatED dataBaSes haVe BEEN BuILT. fOR EXaMPlE, TroN CELL LINE portAl (Tclp) pReseNTs poteNTIAL NEOaNTIGEnS Of 1082 cAnceR CelL liNes [@b0090]. ThE CaNCeR IMmUNOME ATLaS (Tcia) presenTs the ReLaTiONship between tumOr genoTypEs anD IMmUnophenOtypes BAsED On 20 soLid CanCERs, AND provIdES a qUanTitATive inDEx FoR ImmunOtherAPy RESPONsE [@b0095]. With The RAPId GroWTH OF cAncER geNoMiCS dAtA, ResEArchErs ArE AbLe TO dIScOver potEnTial sHArEd NEOanTIgEnS aCRosS tuMOr paTIent poPuLATioNs [@B0100], [@b0105].
IN tHis StUdy, We dEvElOPeD A tumor-sPeciFiC NeoaNtigeN daTAbASE (TsnAdB v1.0) fRom pAn-CaNCER iMMUNogENOmiC aNalYSes. BasEd On THE 7748 TumOr SamplES OF 16 TumoR TYPEs From the cAncER GeNOmE ATlas (Tcga), WE preDiCteD THe bINDIng affinitieS BEtwEEN mutaNt/wILD-TyPE pEPtiDeS and HLa class i MoLEcULEs. DaTAsetS we UsEd INcLUde SOMAtic MuTAtioN DATA OF tUMOR SaMples FroM TcGa and the coRreSPOnDinG hLA ALLELE dAta frOm tCia. TWo DiffERenT veRsiONS Of NetmhcPan, V2.8 [@b0065], and V4.0 [@B0110], WERe USEd fOr preDicTioN. fuRtheRMoRe, we ALsO coNDUCTEd ExtEnSIVe AnALySes and PReSeNted DeTaILEd iNFormaTiON OF pOTenTial nEoaNtigeNs geNerAtEd By SomATiC MUTaTiONs, utiliZing THe rELATeD FILTERiNG ToOls eMbedded iN TsNAd [@B0050]. in AdDItIon, wE EMPLoYEd THe rEcUrRENt mISseNSE MUTatioNs IN cOMbInAtION WItH tHe HigHLy frEqUEnT hlA ALLeLeS To PredicT and AnaLYZE PoTEnTiAL shARed NeoaNtIGeNS. Our StUdy WOUlD PRoViDe a pLAtform to DiscOvEr putaTIve tARgEts for neOaNtIGeN-based cAncER immuNoThERApY.
daTABASe CONteNt aNd uSAGe {#S0010}
==========================
DAtA soURcE {#S0015}
-----------
we cOLLeCTED sOmATIc MUtaTIONS AnD hLa ALLeles Of 7748 TUmoR SAMpLes ACroSS 16 tUMoR tyPEs FROm Tcga (RElease7.0, <hTTPs://PorTAl.GDc.CAnceR.gOv>) ANd TCIA (<HTtpS://TCIa.AT/Home>), reSpectIvELY. tHeSe tuMor sAMpLeS CarRy 972,187 MisSENSE mUTaTIONS, amONg WHICH 18,897 WeRe fOuND recurReNtlY (At leasT tHreE OccUrrENCes In AlL TumOR sAmplEs). we SelECteD THE top 100 HlA AlLElES (fREqueNCY \>0.5%) OF 7748 tumoR SAmPlEs ANd coMBInEd tHem WiTH tHe reCurrEnT mISsEnse MutATIons tO PrEdict pOtENTIal SHARED neOanTigEnS. moREovEr, WE ALso eXtraCtEd 13,459 ReCuRrEnT misSense MuTaTions FROm 9155 SAmpLeS deRived fROM THe iNterNATIonAl CaNceR GEnome ConsOrTIum (IcGc) (ReLeasE20, <HTTps://IcgC.oRg/>) ANd 16 HIGHLY FrEQueNt hla alLeLes (fReqUEnCIeS \>5%) FROM THE 1000 GENoME pRoJect [@B0115] foR tHe PrEdiCTioN oF POTENtiAl shArED neOAntIGeNS.
NEOantigEn prEDiCtiON {#s0020}
---------------------
We TOoK tHE InFormaTioN oN SOmatic MutATIonS AnD HLa alLeLes Of eaCh tUmor SAMple aNd EmplOyED NEtMHcpAN V2.8 [@b0065] AnD NeTMhcpaN v4.0 [@b0110] FOR NEoaNTIGEN pReDiCtIOn, USing tHe fIltERing TOOLs eMBEddeD In our PreviOUSlY-DeVeloped SoftWaRE TSNAd [@B0050]. ALL tHe pEPtIDES wIth 8--11 aMinO aCids ThAt cONTaiN mISSeNse MuTAtIoNs wEre extRacted aS mUtaNT PePtIdeS, and THe CorrESpOnDIng WiLD-TYpe PePTIdes weRE extRACTed As RefEreNceS. wE cOLlEctEd tHe MutaNt PEptiDES AnD hLa AlleLes WITH biNDINg AFFINITY iC~50~ \< 500 nM (inCluDINg sTroNG BiNding with Ic~50~ \< 150 NM ANd WEAk biNdinG wiTh 150 NM \< IC~50~ \< 500 Nm), wIThOUT cOnsIdERAtioN Of the BiNdIng LEVeL betwEen THeir CORResponDIng WiLD-tyPE PepTidES and hLA allElEs. We thEn ClustERED PreDIcTIOn REsULtS bAsED On tumoR TyPes And cAlCULaTeD tHE FreQuEncIes Of SHARED NEOaNTIgens. cOmPAReD wItH NEtmhCPAN V2.8, NeTMhCpaN v4.0 iS trAiNED basEd On BOTH BindiNG afFINITy dAtA aNd MASS spEcTROmEtRY dAta, tHUs AdOpTINg StrictER CrIteRiA fOR BINdiNG PReDicTioN. CoNsEQuenTly, 3,707,562 aND 1,146,961 nEOantIgeNS weRE PReDIcTEd bY nEtMHcPaN v2.8 aND V4.0, reSpECtivELY, among whIcH, 716,876 NEOanTIgENs WErE foUnD iN bOth PREDictiONS. the pOtENTial Shared NeOANtIGeNS BaSEd ON rEcUrreNt MuTAtIONS and hiGHlY FrEQuent HLA aLLeLeS WeRE pREDICTeD IN tHE SIMIlar way.
wEB InTERFacE {#S0025}
-------------
To faCilITaTe tHe UTiLIZAtIon oF tsNaDb, wE haVe establISHED a wEb iNTeRFACe TO bROWSE anD anALYzE neoaNtiGenS. the WEb INTerFACE coMprISEs FIvE Main pAgeS ([fiGUrE 1](#F0005){REf-TYpe="Fig"}a): (i) homE, (iI) browse, (iiI) sEarCh, (iV) VALidation, anD (v) DOwNlOad. IN the FOllowinG CoNTeXT, We exemPlIFy tHE USAgE of TsnaDb wIth tHE REsULTS PREDiCTED By nETMHcPAn v2.8.FIGurE 1**AN oveRVIew of THe TsnaDb web INTERfACeA.** tsnaDB CoMprIsEs fiVe MAIN COMpOnenTs: (i) homE; (II) bROWSE; (iIi) SEaRch; (iV) vaLIDATioN, ANd (v) dOwnlOAd. tHE diSTRibUtiOn of Hla alLEleS (**B)** AnD sOMATic mISseNse MUTATiONS (**C)** eXtrACTEd fROm TcgA and TCia are lIsTed. **d.** the avErAGE neoantiGen LOads Across DiFFErENt tISSueS. **e.** TOP 20 GENeS WiTh pReDiCtED neOantiGEns iN 7748 sAMplES. **F.** THe ResulT Of 'seArCH' PaGe UnDeR THE selEctIoN Of "nEtmHCPaN2.8, *KRas*, anD tcga". **g.** the RESuLt Of 'seArcH' pAGe unDER THe SELEcTion oF "nETMhcPan2.8, *KraS*, aND ICgc". **H.** ExaMplE oF VAlIDaTioN DAta FroM IEdB In 'VaLIDatION' PAgE (tOp) AnD PaRTIaL iNfOrMAtIoN ON 'download' pAgE (BOttOM). ThE PrEdicted bINDIng LEVel 'sTrOnG' iNDiCaTES StROng BiNDInG WiTH Ic~50~ \< 150 NM. TcGa, tHe CaNcER geNOmE ATLaS; TciA, The CAnceR immUnomE aTlas; HLA, HuMaN LeUKoCYtE antiGeN; TsNAd, tUmOR-SpECific nEoANTigeN deTECToR; iCGC: inteRnatIONAl caNcER gEnoME cOnSOrTIUM; iEDB, iMmune EPItope dATaBAsE; wt, Wild TypE.
IN ThE 'hOMe' PAge, tSNAdb PRoVIDeS A StaTISTIcAl TabLe aBout tHe DatAbAse, INCLUDIng THe nuMber oF PROjectS, sAMpLes, HlA aLlEleS ([FIGuRe 1](#f0005){Ref-type="FIg"}b), mUTatIoNS ([FigUrE 1](#F0005){Ref-type="fiG"}C), aNd PReDiCTeD NeoaNtiGeNs of Each TUmoR tYPE. The TaBlE PReSEnts 3,707,562 potENTIal nEoAnTigEns FroM tHe 7748 tUMOr SamPLEs ACrosS 16 tumor tYpEs. thE aVerAgE prEdICtEd NEOANtIGEn lOAdS VarY acrosS DIFfeRENt tUmor typeS ([FigurE 1](#f0005){rEf-tYPe="FiG"}D). FOR INSTaNcE, THE AveRagE pREdicted neOanTiGen load fOr UteRUs caNCER Is 1957, wHIch is 21 foR THYrOid CaNCEr. besIdES, usErS can gET tHE DETaiLed infORMation abOUT the PREDicted NeOantIGens OF EACh TuMOR tYpe By clICKiNg on THe TiSsue nAme In the TABle. thE contEnt oF eaCH TUMoR TYPE inCluDEs tOp 20 HLa Alleles And TOp 20 GeNes WitH PREDiCTeD NEOAntigEns iN THIs Tumor typE, whiCH aRe SHoWn as Two FIgures. ALL pRedIctEd NEoanTiGENs in thIS TuMoR tyPE woUlD bE diSpLAYeD BELoW fIgUReS.
to FURtHer UndERSTAnd tHe DIsTrIBUtioN OF nEoaNtiGenS aT GEne LEvEl, UsErs CaN ReTrieVe NEoanTIgEnS BY FEeDiNG In A gENe naMe iN THe 'browsE' PAge ([FIgurE 1](#F0005){REf-tYPE="fig"}e). tHe RETRIEVAL RESuLTS pROViDe MORe fUNcTiONs THAn ThaT indICAteD in EAcH TumoR tyPE | Handled by Yu Xue Introduction {#s0005}============ Cancer somatic mutationsand viral oncogenes can generate tumor-specific protein sequences that are entirely absent from normal humancells. These novel proteinsmay result in the formation of tumor-specific antigens (TSAs) [@b0005]. As an important typeof TSAs,neoantigens aregenerated by tumor-specific proteins, and presented by major histocompatibility complexes (MHCs) on cell surfaces through antigen presentation, where they can be recognized by T-cell receptors (TCRs)[@b0010],[@b0015]. Recently, neoantigenshave attracted a large amount of attention, because theyarepotential biomarkers to distinguish tumor cells from normalcells. Neoantigensareof critical importance for cancer immunotherapy in the followingtwo aspects. First, theneoantigen burden and quality can be used to predict therapeutic effectsfor immune checkpoint blockade therapy, such as blockage of programmed death-1(PD-1)and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) [@b0020], [@b0025], [@b0030]. Second, neoantigens can be used as potential targets for cancer immunotherapy, such as personalized cancer vaccines [@b0035], [@b0040] and adoptive cell therapy (ACT) [@b0045]. Therefore, there is an urgent need toidentify neoantigens accurately for cancer patients.With theprogress of cancer immunogenomics, severalkinds of integrated softwarehave been developed for tumor-specificneoantigen detection, such as TSNAD [@b0050]and pVAC-seq [@b0055]. The most critical function of such software is to predictthe binding affinities between mutant peptides and human leukocyte antigen (HLA) alleles. To achieve this, a lot of well-acknowledged andpopular tools, suchas NetMHC [@b0060], NetMHCpan [@b0065], sNebula [@b0070], and HLA-CNN [@b0075], can beused. In addition, several databases can provide necessary information for the development of tools to predict the affinities between peptides and HLA alleles. For example, theImmune Epitope Database (IEDB) is an important immune-related database, providing a large amount of valuable and experimentally-validated information of immune epitopes [@b0080]. The InternationalImmunogenetics Information System(IMGT)offersinformation about antibodies, TCRs, MHCs, and so on [@b0085]. Taking advantage ofexisting neoantigen prediction software, several neoantigen-related databases havebeen built. For example, TRON Cell Line Portal (TCLP) presentspotential neoantigens of 1082 cancer cell lines [@b0090]. The Cancer Immunome Atlas (TCIA) presents the relationship between tumor genotypes and immunophenotypes based on 20 solid cancers, and provides a quantitative index for immunotherapy response [@b0095]. With the rapid growth of cancer genomics data, researchers areable todiscover potential shared neoantigens across tumor patient populations [@b0100], [@b0105].In this study, we developed a tumor-specific neoantigen database (TSNAdb v1.0) from pan-cancer immunogenomic analyses. Based on the 7748 tumor samples of 16 tumor types from The Cancer Genome Atlas (TCGA), we predictedthe binding affinities between mutant/wild-type peptides and HLA class I molecules. Datasets we used include somatic mutation data of tumor samples from TCGA and the corresponding HLA alleledata from TCIA. Twodifferent versions of NetMHCpan, v2.8 [@b0065], and v4.0 [@b0110], wereused for prediction. Furthermore, we also conducted extensiveanalyses and presented detailed information of potentialneoantigens generated by somatic mutations, utilizing the related filtering tools embedded in TSNAD[@b0050].In addition, we employed the recurrentmissense mutations incombination with the highly frequent HLA alleles to predict andanalyze potential shared neoantigens. Our study would provide a platform to discover putative targets for neoantigen-based cancer immunotherapy.Database content and usage {#s0010} ========================== Data source{#s0015}-----------We collected somatic mutations and HLA alleles of 7748 tumor samples across 16 tumor types from TCGA (Release7.0, <https://portal.gdc.cancer.gov>) and TCIA (<https://tcia.at/home>),respectively. These tumor samples carry 972,187 missense mutations,among which 18,897 were found recurrently (at least three occurrences inalltumorsamples). We selected the top 100 HLAalleles (frequency \>0.5%) of 7748 tumor samplesandcombined themwith the recurrent missense mutationsto predict potential shared neoantigens.Moreover, we also extracted 13,459 recurrent missense mutations from 9155 samples derived from the InternationalCancer Genome Consortium (ICGC) (Release20, <https://icgc.org/>) and 16 highly frequent HLAalleles (frequencies \>5%) from the 1000 Genome Project[@b0115] for the prediction of potential shared neoantigens. Neoantigen prediction {#s0020} ---------------------We took the information on somatic mutations and HLA alleles of each tumor sample andemployed NetMHCpan v2.8[@b0065] and NetMHCpan v4.0 [@b0110] for neoantigen prediction,using thefiltering tools embedded in our previously-developed software TSNAD [@b0050]. All the peptides with 8--11amino acids thatcontain missense mutations were extractedas mutantpeptides, and the correspondingwild-typepeptideswere extractedasreferences. We collected the mutant peptides and HLA alleleswith binding affinity IC~50~ \< 500 nM (including strong binding with IC~50~ \<150 nM and weak binding with 150 nM \< IC~50~ \< 500 nM), withoutconsideration of the bindinglevel between their corresponding wild-typepeptides and HLA alleles. We then clusteredprediction resultsbased ontumor types and calculated the frequencies of shared neoantigens.Compared with NetMHCpanv2.8, NetMHCpan v4.0 is trained based on both binding affinity data and mass spectrometry data, thusadopting stricter criteria forbinding prediction. Consequently, 3,707,562 and 1,146,961 neoantigens were predicted by NetMHCpan v2.8 andv4.0, respectively, among which, 716,876 neoantigens were found in both predictions. The potential shared neoantigens based on recurrent mutations and highly frequent HLAalleles were predictedin the similarway. Web interface {#s0025} ------------- To facilitate the utilization of TSNAdb, we have established a webinterface to browse and analyze neoantigens. The web interface comprises five main pages([Figure 1](#f0005){ref-type="fig"}A): (i) Home, (ii) Browse,(iii) Search, (iv)Validation,and (v)Download. Inthe following context, we exemplify the usage of TSNAdbwith the results predictedby NetMHCpan v2.8.Figure 1**An overview of theTSNAdb web interfaceA.** TSNAdb comprises five main components: (i) Home; (ii) Browse; (iii) Search; (iv) Validation, and (v) Download. The distribution of HLA alleles(**B)** and somaticmissense mutations(**C)** extracted from TCGAand TCIA arelisted.**D.** The averageneoantigen loads acrossdifferent tissues. **E.** Top 20 geneswithpredicted neoantigens in7748 samples. **F.** The result of 'Search' pageunder theselection of "NetMHCpan2.8, *KRAS*, and TCGA".**G.** The result of 'Search'page under theselection of "NetMHCpan2.8, *KRAS*, and ICGC". **H.**Example of validation data from IEDB in 'Validation' page(top) andpartial information on 'Download' page (bottom). The predicted binding level 'Strong' indicates strong binding with IC~50~\< 150 nM.TCGA, The Cancer Genome Atlas; TCIA, TheCancer Immunome Atlas; HLA, human leukocyte antigen; TSNAD, tumor-specific neoantigen detector; ICGC:International Cancer Genome Consortium;IEDB,ImmuneEpitope Database; WT, wild type. In the 'Home' page,TSNAdb provides a statisticaltable about the database, including the number of projects, samples, HLA alleles ([Figure 1](#f0005){ref-type="fig"}B), mutations ([Figure 1](#f0005){ref-type="fig"}C), andpredicted neoantigens of each tumor type. The table presents 3,707,562potential neoantigens from the 7748 tumor samples across 16 tumor types. The average predicted neoantigen loads vary across different tumor types ([Figure 1](#f0005){ref-type="fig"}D). For instance, the average predicted neoantigen load foruterus cancer is 1957, which is 21 for thyroidcancer. Besides, users can getthe detailedinformation about the predicted neoantigens of each tumor type by clicking on the tissue name inthe table. The content of each tumor typeincludes top20 HLA alleles and top 20 genes withpredicted neoantigens in this tumor type, which are shown as two figures. All predicted neoantigens in this tumortypewould be displayed below figures. Tofurther understand thedistribution of neoantigens at gene level, users can retrieve neoantigens byfeeding ina gene nameinthe 'Browse' page ([Figure 1](#f0005){ref-type="fig"}E). The retrieval results provide more functions than that indicated in eachtumor type | _Handled_ by Yu Xue Introduction {#s0005} ============ Cancer somatic mutations _and_ viral oncogenes can generate tumor-specific protein sequences _that_ _are_ entirely _absent_ from normal human cells. These novel proteins may result _in_ the formation of tumor-specific antigens (TSAs) [@b0005]. As an _important_ type of TSAs, neoantigens are generated _by_ tumor-specific proteins, and presented by major histocompatibility complexes _(MHCs)_ on _cell_ surfaces through antigen presentation, where they can be recognized by T-cell receptors (TCRs) [@b0010], [@b0015]. _Recently,_ _neoantigens_ have attracted a large amount _of_ attention, because they are potential biomarkers to distinguish tumor cells _from_ _normal_ cells. Neoantigens are of critical importance _for_ cancer immunotherapy in the _following_ two _aspects._ First, the neoantigen burden and quality can be used _to_ predict therapeutic _effects_ _for_ immune checkpoint _blockade_ therapy, _such_ _as_ blockage of programmed death-1 (PD-1) and cytotoxic T _lymphocyte-associated_ antigen-4 (CTLA-4) [@b0020], _[@b0025],_ [@b0030]. Second, neoantigens can be used as potential targets for cancer immunotherapy, _such_ as personalized cancer vaccines [@b0035], _[@b0040]_ and _adoptive_ cell therapy (ACT) _[@b0045]._ Therefore, _there_ is an urgent need to identify neoantigens accurately for cancer patients. With the progress of cancer immunogenomics, several kinds _of_ _integrated_ software have been developed _for_ tumor-specific neoantigen detection, such as TSNAD [@b0050] and pVAC-seq [@b0055]. _The_ most critical function of such software is to _predict_ _the_ binding affinities between mutant _peptides_ and human leukocyte antigen (HLA) alleles. To achieve this, a lot of _well-acknowledged_ and popular tools, such _as_ _NetMHC_ [@b0060], NetMHCpan [@b0065], sNebula [@b0070], and HLA-CNN [@b0075], can be used. In addition, several databases can provide necessary _information_ _for_ the development of _tools_ to predict _the_ affinities _between_ peptides and HLA alleles. For example, _the_ Immune Epitope Database (IEDB) is an important immune-related database, providing a large amount of valuable and _experimentally-validated_ information of immune epitopes [@b0080]. The _International_ Immunogenetics Information System (IMGT) _offers_ information about antibodies, TCRs, MHCs, _and_ so _on_ [@b0085]. Taking _advantage_ of existing neoantigen _prediction_ software, several neoantigen-related databases have been built. For _example,_ _TRON_ Cell _Line_ Portal (TCLP) presents potential neoantigens of _1082_ cancer _cell_ _lines_ [@b0090]. The Cancer Immunome _Atlas_ (TCIA) presents the _relationship_ between tumor _genotypes_ and immunophenotypes _based_ on 20 solid cancers, and provides a quantitative index for immunotherapy response [@b0095]. With _the_ _rapid_ growth of cancer genomics data, researchers are able to discover potential _shared_ neoantigens _across_ tumor patient populations [@b0100], [@b0105]. In this _study,_ we developed a tumor-specific _neoantigen_ database _(TSNAdb_ v1.0) _from_ pan-cancer immunogenomic _analyses._ Based on the 7748 tumor samples of _16_ tumor types from The Cancer _Genome_ Atlas (TCGA), we predicted the binding affinities between _mutant/wild-type_ peptides _and_ _HLA_ _class_ I molecules. Datasets we used include _somatic_ _mutation_ data of tumor samples from _TCGA_ and the corresponding HLA allele data from TCIA. Two different versions of _NetMHCpan,_ _v2.8_ _[@b0065],_ and v4.0 [@b0110], _were_ used for prediction. Furthermore, we also _conducted_ _extensive_ analyses and _presented_ detailed information of potential neoantigens generated by _somatic_ mutations, utilizing _the_ _related_ filtering tools embedded in _TSNAD_ [@b0050]. In addition, we _employed_ the recurrent missense mutations in combination with _the_ highly frequent HLA alleles to predict _and_ analyze potential shared _neoantigens._ _Our_ study _would_ provide a platform to discover putative _targets_ for neoantigen-based cancer immunotherapy. Database content and usage {#s0010} ========================== Data source {#s0015} ----------- We collected somatic _mutations_ and HLA alleles of 7748 tumor samples _across_ 16 tumor types from _TCGA_ _(Release7.0,_ <https://portal.gdc.cancer.gov>) and TCIA (<https://tcia.at/home>), respectively. These _tumor_ samples carry _972,187_ _missense_ mutations, among which 18,897 were found recurrently (at least three occurrences in all _tumor_ samples). We selected the top 100 _HLA_ _alleles_ (frequency \>0.5%) of 7748 tumor samples and _combined_ them _with_ the recurrent missense mutations to predict _potential_ _shared_ neoantigens. Moreover, _we_ also extracted _13,459_ recurrent missense mutations from _9155_ samples derived from the _International_ _Cancer_ _Genome_ Consortium (ICGC) _(Release20,_ <https://icgc.org/>) and _16_ highly frequent HLA alleles (frequencies _\>5%)_ _from_ _the_ 1000 Genome Project [@b0115] _for_ the prediction of potential shared neoantigens. Neoantigen _prediction_ {#s0020} --------------------- We took the _information_ on somatic _mutations_ and HLA alleles of each tumor sample and employed NetMHCpan v2.8 _[@b0065]_ _and_ NetMHCpan v4.0 [@b0110] for neoantigen prediction, _using_ _the_ filtering tools embedded _in_ _our_ previously-developed software TSNAD _[@b0050]._ All the peptides with 8--11 amino acids that contain missense mutations were extracted as mutant peptides, and the corresponding wild-type peptides were extracted as references. We collected the mutant peptides and _HLA_ alleles _with_ binding _affinity_ _IC~50~_ \< _500_ nM (including strong binding with IC~50~ \< 150 nM and _weak_ _binding_ _with_ 150 nM \< IC~50~ \< _500_ nM), without _consideration_ of the binding level between _their_ corresponding wild-type _peptides_ and HLA alleles. _We_ then _clustered_ prediction results based _on_ tumor types and calculated the frequencies of shared neoantigens. Compared with NetMHCpan _v2.8,_ NetMHCpan v4.0 is trained _based_ on both _binding_ affinity data and mass _spectrometry_ data, thus adopting stricter criteria for binding prediction. _Consequently,_ 3,707,562 and 1,146,961 _neoantigens_ were predicted by NetMHCpan v2.8 and _v4.0,_ respectively, among _which,_ 716,876 neoantigens were found in both predictions. The potential _shared_ neoantigens _based_ on recurrent _mutations_ and highly frequent HLA alleles were predicted in the similar way. Web interface {#s0025} ------------- _To_ _facilitate_ _the_ _utilization_ of TSNAdb, we have established _a_ _web_ interface to browse and analyze neoantigens. The web interface comprises five main pages ([Figure 1](#f0005){ref-type="fig"}A): (i) Home, (ii) Browse, _(iii)_ Search, (iv) Validation, and _(v)_ Download. In the following context, _we_ exemplify the usage of TSNAdb _with_ _the_ results predicted _by_ NetMHCpan v2.8.Figure 1**An overview of _the_ TSNAdb web interfaceA.** _TSNAdb_ comprises five main components: (i) Home; _(ii)_ _Browse;_ _(iii)_ Search; (iv) Validation, and (v) Download. The distribution of _HLA_ alleles (**B)** and somatic missense mutations _(**C)**_ extracted from TCGA and TCIA are listed. _**D.**_ The average _neoantigen_ loads across different tissues. _**E.**_ Top 20 genes with predicted _neoantigens_ in 7748 samples. **F.** The result _of_ 'Search' page under the selection of _"NetMHCpan2.8,_ *KRAS*, and TCGA". **G.** The result of 'Search' page under the _selection_ _of_ "NetMHCpan2.8, *KRAS*, and ICGC". **H.** Example of validation _data_ from IEDB _in_ 'Validation' _page_ (top) and partial information on _'Download'_ page (bottom). The _predicted_ binding level 'Strong' indicates strong binding with _IC~50~_ \< 150 nM. TCGA, The Cancer _Genome_ Atlas; TCIA, _The_ _Cancer_ _Immunome_ Atlas; HLA, human leukocyte antigen; TSNAD, tumor-specific _neoantigen_ detector; ICGC: International _Cancer_ _Genome_ _Consortium;_ IEDB, Immune Epitope Database; WT, _wild_ type. In the 'Home' page, TSNAdb provides _a_ _statistical_ table about the _database,_ _including_ the number of projects, samples, HLA alleles ([Figure 1](#f0005){ref-type="fig"}B), mutations ([Figure 1](#f0005){ref-type="fig"}C), _and_ predicted _neoantigens_ of each tumor _type._ The table presents 3,707,562 potential _neoantigens_ _from_ the 7748 tumor samples across 16 tumor types. The _average_ predicted neoantigen loads vary across different tumor types ([Figure 1](#f0005){ref-type="fig"}D). For instance, the average _predicted_ neoantigen load for _uterus_ cancer is 1957, which _is_ 21 for thyroid _cancer._ Besides, users can get the detailed information about the predicted _neoantigens_ of each tumor type by _clicking_ _on_ the _tissue_ _name_ _in_ the table. The content of each tumor type includes top 20 HLA alleles _and_ top 20 genes with predicted _neoantigens_ in this _tumor_ type, which are shown as two figures. _All_ predicted _neoantigens_ _in_ this tumor type would _be_ displayed _below_ figures. To further understand the distribution of neoantigens at gene level, users can _retrieve_ neoantigens _by_ feeding in a gene name in the 'Browse' page ([Figure 1](#f0005){ref-type="fig"}E). The _retrieval_ results provide _more_ _functions_ _than_ _that_ indicated in _each_ _tumor_ type |
INTRODUCTION {#s1}
============
Pediatric glioblastoma (GBM) is a relatively rare primary brain tumor in children \[[@R1]\]. Maximum surgical resection is considered the key and prognosis-determining factor in the treatment, followed by radiotherapy \[[@R1], [@R2]\]. Unfortunately, pediatric GBM is poorly studied at the molecular and genomic levels. Radiotherapy plays a critical role in eradicating the post-surgical residual microtumor \[[@R3]\]. Yet the molecular and genomic changes post-radiation in pediatric GBM have not been well examined. We have recently identified many radiation-responsive genes in adult radioresistant GBM cells that explain the radioresistance and increased malignant features of recurrent GBM \[[@R4]\]. However, adult and pediatric GBMs are distinct from each other at both molecular and genetic levels \[[@R2]\]. We are presenting a full RNA sequencing profile of both the radiation-naïve pediatric GBM (SJ-GBM2) cell line and stable radioresistant pediatric GBM (SJ-GBM2-10gy) cell line that we recently developed \[[@R5], [@R6]\]. Our data demonstrated that radiation perturbed the expression of many genes related to many different known pathways in cancer biology. The irradiated cells exhibited an enhanced growth rate, overexpressed protease cathepsin B, and both subunits of the rate-limiting enzyme of DNA synthesis ribonucleotide reductase (RR). In this study, we shed light on the irradiation responsive mRNA changes that transform the tumor cells toward a more aggressive and resistant form, for which treatment choices are limited. This study opens the door to further examining the possibility of targeting these modified pathways as a therapeutic strategy to block GBM tumor recurrence and progression.
RESULTS {#s2}
=======
Radioresistant irradiated pediatric GBM cells exhibited higher growth rate than control cells {#s2_1}
---------------------------------------------------------------------------------------------
The *in-vitro* growth rate of the SJ-GBM2 and SJ-GBM2-10gy cells was evaluated using an MTT growth assay over a period of 10 days. SJ-GBM2-10gy cells showed a superior divergent growth starting from day 3, having the difference in growth maximized on day 7 when the control cells significantly slowed down their proliferation rate (Figure [1](#F1){ref-type="fig"}). The control cells SJ-GBM2 reached about 7.4 growth fold in 10 days, while the irradiated cells reached a 10.5 fold from their baseline. This represents an estimated 30% increase in growth (Figure [1A](#F1){ref-type="fig"}).
{#F1}
Irradiated radioresistant pediatric GBM overexpresses ribonucleotide reductase {#s2_2}
------------------------------------------------------------------------------
To probe for a mechanism promoting the superior growth rate in irradiated cells, the expression of both ribonucleotide reductase (RR) subunits was measured. The RR enzyme, specifically the RRM2 subunit, has been reported to be essential for proliferation and invasion of GBM cells \[[@R7]\]. Immunoblotting of control and irradiated cells revealed an increase in the expression of RRM1 subunit by 2-fold, and an increase in the RRM2 subunit by 3.5-fold in irradiated cells relative to control cells (Figure [1B](#F1){ref-type="fig"}). This increase in cellular expression of RRM2 was confirmed by immunofluorescence probing of intact cells, demonstrating the distribution of RRM2 in the cytoplasm of the irradiated cells and was greater than the control cells (Figure [1B, 1C](#F1){ref-type="fig"}).
Irradiated radioresistant pediatric GBM overexpresses pro-cathepsin B {#s2_3}
---------------------------------------------------------------------
We were interested in evaluating whether protease may play of role in promoting invasion and progression of irradiated radioresistant GBM. Cathepsin B, a cysteine protease, has been shown to play a role in tumor growth and invasion \[[@R8], [@R9]\]. We probed for the differential expression level of pro-cathepsin B in control and irradiated cells. Western blot of cell lysates and immunofluorescence of intact cells revealed 3-fold overexpression of pro-cathepsin B in irradiated cells over control cells (Figure [1B, 1C](#F1){ref-type="fig"}). In addition to being localized to the cytoplasm, pro-cathepsin B was present in the processes of the irradiated cells, a feature that may be important in the invasiveness of GBM into the surrounding tissue (Figure [1C](#F1){ref-type="fig"}).
Irradiation of pediatric GBM cells induces differential expression of 1192 radiation-responsive genes {#s2_4}
-----------------------------------------------------------------------------------------------------
Total mRNAs from SJGBM2 and SJGBM2-10gy cells were harvested and subjected to further analysis. To screen for global mRNA changes following irradiation, we profiled transcriptomes of the control SJ-GBM2 cell line and its derivative radioresistant irradiated SJ-GBM2-10gy cells by RNA sequencing (Table [1](#T1){ref-type="table"} and [Supplementary Table 1](#SD2){ref-type="supplementary-material"}). The criteria for differentially expressed genes were 2-fold or greater than statistically significant values (*P* \< 0.05). We identified 1192 radiation responsive genes. Among these 1192 radiation responsive genes, 584 were upregulated and 608 were downregulated ([Supplementary Tables 2](#SD3){ref-type="supplementary-material"} and [3](#SD4){ref-type="supplementary-material"}).
###### Enriched gene ontology categories of differentially expressed genes following irradiation based on sets of statistically significant (more than 2-fold) upregulated and downregulated genes (*P* \< 0.05)
Differentially expressed Category *P*-value
-------------------------------------------------------- --------------------------------------- -----------
**Upregulated** GO:0048863: Stem cell differentiation 6.60E-03
GO:0010628: Positive regulation of gene expression 7.70E-03
GO:0002040: Sprouting angiogenesis 3.20E-02
GO:0008284: Positive regulation of cells proliferation 2.40E-02
GO:0070848: Response to growth factor 7.40E-02
GO:0001558: Regulation of cell growth 7.60E-02
GO:0055114: Oxidation reduction process 3.00E-02
GO:0071356: Cellular response to tumor necrosis factor 3.60E-02
GO:0006954: Inflammatory response 4.10E-02
GO:0016055: Wnt signaling pathway 8.40E-02
GO:0043066: Negative regulation of apoptotic process 9.40E-02
GO:0004222: Metalloendopeptidase activity 2.40E-02
**Downregulated** GO:0007155: Cell adhesion 4.90E-03
GO:0043065: Positive regulation of apoptosis 1.30E-04
GO:008285: Negative regulation of cell proliferation 4.10E-02
GO:0002020: Protease binding 1.20E-03
Experiments were performed in triplicate.
Upregulation of genes promoting tumor growth and aggressiveness following irradiation {#s2_5}
-------------------------------------------------------------------------------------
Gene ontology analysis of the mRNA data was utilized to categorize genes into functional groups. It revealed that upregulated genes were enriched in positive regulation of stem cells differentiation, angiogenesis, cell proliferation, cell growth, inflammatory response, positive regulation of the Wnt signaling pathway, response to hypoxia, metalloendopeptidase activity, cellular response to tumor necrosis factor and negative regulation of apoptotic process (Table [1](#T1){ref-type="table"} and [Supplementary Table 2](#SD3){ref-type="supplementary-material"}, and [Supplementary Table 4](#SD5){ref-type="supplementary-material"}). The upregulated genes were enriched in positive regulation of gene expression and tumor cell proliferation such as KIT, connective tissue growth factor CTGF and ID1, ID2, and TLE1-FOXG1 transcriptional factors that have been reported to enhance growth and proliferation of GBM cells \[[@R10]--[@R13]\]. G protein-coupled receptor kinase 5 (GRK5) plays an important role in tumor cells' proliferation \[[@R14]\]. Fibroblast growth factor 4 (FGF4) also has been correlated with a greater malignancy profile in high-grade gliomas \[[@R15]\].The radioresistant cells had upregulated expression of many anti-apoptotic genes, including BCL2, CD74, and WT1, which regulates GBM cells proliferation and apoptosis \[[@R16]\]. A significant upregulation (10-fold) of the AIM2 gene, a tumor-associated antigen, was found. This gene upregulation was observed in GBM patients and | introduction { # s1 } = = = = = = = = = = = = pediatric glioblastoma ( gbm ) is a relatively rare primary brain tumor in children \ [ [ @ r1 ] \ ]. maximum surgical resection is considered the key and prognosis - determining factor in the treatment, followed by radiotherapy \ [ [ @ r1 ], [ @ e ] \ ]. unfortunately, pediatric gbm is poorly studied at the molecular and genomic levels. radiotherapy plays a critical role in controlling the post - surgical residual microtumor \ [ [ @ r3 ] \ ]. yet the molecular and genomic mechanisms post - radiation in pediatric gbm have not proven well examined. we have recently identified many radiation - responsive genes in adult radioresistant gbm cells that explain the radioresistance and increased malignant features of recurrent gbm \ [ [ @ r4 ] \ ]. however, adult and pediatric gbms are distinct from each other at both molecular and genetic levels \ [ [ @ r2 ] \ ]. we are developing a full rna sequencing profile of both the radiation - naive pediatric gbm ( sj - gbm2 ) cell line and stable radioresistant pediatric gbm ( sj - gbm2 - 10gy ) cell line that we recently developed \ [ [ @ r5 ], [ @ r6 ] \ ]. knockout data demonstrated that radiation perturbed the expression of many genes related to many different known pathways in cancer biology. the irradiated nucleus exhibited an enhanced growth rate, overexpressed protease cathepsin b, and both subunits of the rate - limiting enzyme of dna synthesis ribonucleotide reductase ( rr ). in this study, we shed light on the irradiation responsive mrna changes that transform the tumor cells toward a more aggressive and resistant form, for which treatment outcomes are limited. this study opens the door to further examine the possibility of targeting highly modified pathways as a therapeutic strategy to block gbm tumor recurrence and progression. results { # s2 } = = = = = = = radioresistant irradiated pediatric gbm cells exhibited higher growth rate than control cells { # s2 _ 1 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the * in - vitro * growth rate of the sj - gbm2 and sj - gbm2 - 10gy cells was evaluated using an mtt growth assay over a period of 10 days. sj - gbm2 - 10gy cells showed a superior divergent growth starting from day 3, having the difference in growth maximized on day 7 when the control cells significantly slowed down their proliferation rate ( figure [ 1 ] ( # f1 ) { ref - type = " fig " } ). the control cells sj - gbm2 reached about 7. 4 growth fold in 10 days, while the irradiated cells reached a 10. 5 fold from their baseline. this represents an estimated 30 % increase in growth ( figure [ 1a ] ( # f1 ) { ref - type = " fig " } ).! [ irradiation of the pediatric gbm cells enhanced proliferation and expression of malignant - promoting proteins : \ ( * * a * * ) growth curves of sjgbm2 and sjgbm2 - 10gy cells. ( * * b * * ) western blot for rr m1 ( 94 kda ) and m2 ( 45 kda ) subunits and pro - cathepsin b ( pro - catb ) ( 43 kda ) in sjgbm2 and sjgbm2 - 10gy cells. ( * * c * * ) direct immunofluorescence probing for rrm2 and cathepsin b ( pro - catb ) in both cells after a 24 h incubation in fresh medium. ^ \ * ^ * p \ < 0. 05 *. ] ( oncotarget - 09 - 34122 - g001 ) { # f1 } irradiated radioresistant pediatric gbm overexpresses ribonucleotide reductase { # s2 _ 2 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - to probe for a mechanism promoting the superior growth rate in irradiated cells, the expression of both ribonucleotide reductase ( rr ) subunits was measured. the rr enzyme, specifically the rrm2 subunit, has been reported to be essential for proliferation and invasion of gbm cells \ [ [ @ r7 ] \ ]. immunoblotting of control and irradiated cells revealed an increase in the expression of rrm1 subunit by 2 - fold, and an increase in the rrm2 subunit by 3. 5 - fold in irradiated cells relative to control cells ( figure [ 1b ] ( # f1 ) { ref - type = " fig " } ). this increase in cellular expression of rrm2 was confirmed by immunofluorescence probing of intact cells, demonstrating the distribution of rrm2 in the cytoplasm of the irradiated cells and was greater than the control cells ( figure [ 1b, 1c ] ( # f1 ) { ref - type = " fig " } ). irradiated radioresistant pediatric gbm overexpresses pro - cathepsin b { # s2 _ 3 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - we were interested in evaluating whether protease may play of role in promoting invasion and progression of irradiated radioresistant gbm. cathepsin b, a cysteine protease, has been shown to play a role in tumor growth and invasion \ [ [ @ r8 ], [ @ r9 ] \ ]. we probed for the differential expression level of pro - cathepsin b in control and irradiated cells. western blot of cell lysates and immunofluorescence of intact cells revealed 3 - fold overexpression of pro - cathepsin b in irradiated cells over control cells ( figure [ 1b, 1c ] ( # f1 ) { ref - type = " fig " } ). in addition to being localized to the cytoplasm, pro - cathepsin b was present in the processes of the irradiated cells, a feature that may be important in the invasiveness of gbm into the surrounding tissue ( figure [ 1c ] ( # f1 ) { ref - type = " fig " } ). irradiation of pediatric gbm cells induces differential expression of 1192 radiation - responsive genes { # s2 _ 4 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - total mrnas from sjgbm2 and sjgbm2 - 10gy cells were harvested and subjected to further analysis. to screen for global mrna changes following irradiation, we profiled transcriptomes of the control sj - gbm2 cell line and its derivative radioresistant irradiated sj - gbm2 - 10gy cells by rna sequencing ( table [ 1 ] ( # t1 ) { ref - type = " table " } and [ supplementary table 1 ] ( # sd2 ) { ref - type = " supplementary - material " } ). the criteria for differentially expressed genes were 2 - fold or greater than statistically significant values ( * p * \ < 0. 05 ). we identified 1192 radiation responsive genes. among these 1192 radiation responsive genes, 584 were upregulated and 608 were downregulated ( [ supplementary tables 2 ] ( # sd3 ) { ref - type = " supplementary - material " } and [ 3 ] ( # sd4 ) { ref - type = " supplementary - material " } ). # # # # # # enriched gene ontology categories of differentially expressed genes following irradiation based on sets of statistically significant ( more than 2 - fold ) upregulated and downregulated genes ( * p * \ < 0. 05 ) differentially expressed category * p * - value - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - * * upregulated * * go : 0048863 : stem cell differentiation 6. 60e - 03 go : 0010628 : positive regulation of gene expression 7. 70e - 03 go : 0002040 : sprouting angiogenesis 3. 20e - 02 go : 0008284 : positive regulation of cells proliferation 2. 40e - 02 go : 0070848 : response to growth factor 7. 40e - 02 go : 0001558 : regulation of cell growth 7. 60e - 02 go : 0055114 : oxidation reduction process 3. 00e - 02 go : 0071356 : cellular response to tumor necrosis factor 3. 60e - 02 go : 0006954 : inflammatory response 4. 10e - 02 go : 0016055 : wnt signaling pathway 8. 40e - 02 go : 0043066 : negative regulation of apoptotic process 9. 40e - 02 go : 0004222 : metalloendopeptidase activity 2. 40e - 02 * * downregulated * * go : 0007155 : cell adhesion 4. 90e - 03 go : 0043065 : positive regulation of apoptosis 1. 30e - 04 go : 008285 : negative regulation of cell proliferation 4. 10e - 02 go : 0002020 : protease binding 1. 20e - 03 experiments were performed in triplicate. upregulation of genes promoting tumor growth and aggressiveness following irradiation { # s2 _ 5 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - gene ontology analysis of the mrna data was utilized to categorize genes into functional groups. it revealed that upregulated genes were enriched in positive regulation of stem cells differentiation, angiogenesis, cell proliferation, cell growth, inflammatory response, positive regulation of the wnt signaling pathway, response to hypoxia, metalloendopeptidase activity, cellular response to tumor necrosis factor and negative regulation of apoptotic process ( table [ 1 ] ( # t1 ) { ref - type = " table " } and [ supplementary table 2 ] ( # sd3 ) { ref - type = " supplementary - material " }, and [ supplementary table 4 ] ( # sd5 ) { ref - type = " supplementary - material " } ). the upregulated genes were enriched in positive regulation of gene expression and tumor cell proliferation such as kit, connective tissue growth factor ctgf and id1, id2, and tle1 - foxg1 transcriptional factors that have been reported to enhance growth and proliferation of gbm cells \ [ [ @ r10 ] - - [ @ r13 ] \ ]. g protein - coupled receptor kinase 5 ( grk5 ) plays an important role in tumor cells ' proliferation \ [ [ @ r14 ] \ ]. fibroblast growth factor 4 ( fgf4 ) also has been correlated with a greater malignancy profile in high - grade gliomas \ [ [ @ r15 ] \ ]. the radioresistant cells had upregulated expression of many anti - apoptotic genes, including bcl2, cd74, and wt1, which regulates gbm cells proliferation and apoptosis \ [ [ @ r16 ] \ ]. a significant upregulation ( 10 - fold ) of the aim2 gene, a tumor - associated antigen, was found. this gene upregulation was observed in gbm patients and | INTRODUCTION {# s1} = = = = = = = = = = = = Pediatric glioblastoma (GBM) is a relatively rare primary brain tumor in children \ [[ @ R1] \ ]. Maximum surgical resection is considered the key and prognosis - determining factor in the treatment, followed by radiotherapy \ [[ @ R1 ], [@ R2] \ ]. Unfortunately, pediatric GBM is poorly studied at the molecular and genomic levels. Radiotherapy plays a critical role in eradicating the post - surgical residual microtumor \ [[ @ R3] \ ]. Yet the molecular and genomic changes post - radiation in pediatric GBM have not been well 2xamJned. We have recently identified many radiation - responsive genes in adult radioresistant GBM cells that explain the radioresistance and increased malignant features of recurrent GBM \ [[ @ R4] \ ]. However, adult and pediatric GBMs are distinct from each other at both molecular and genetic levels \ [[ @ R2] \ ]. We are presenting a full RNA sequencing profile of both the radiation - naïve pediatric GBM (SJ - GBM2) cell line and stable radioresistant pediatric GBM (SJ - GBM2 - 10gy) cell line that we recently developed \ [[ @ R5 ], [@ R6] \ ]. Our data demonstrated that radiation perturbed the expression of many genes related to many different known pathways in cancer biology. The irradiated cells exhibited an enhanced growth rate, overexpressed protease cathepsin B, and both subunits of the rate - limiting enzyme of DNA synthesis ribonucleotide reductase (RR ). In this study, we shed light on the irradiation responsive mRNA changes that transform the tumor cells toward a more aggressive and resistant form, for which tFeatm2nt choices are limited. This study opens the door to further examining the possibility of targeting these modified pathways as a therapeutic strategy to block GBM tumor recurrence and progression. RESULTS {# s2} = = = = = = = Radioresistant irradiated pediatric GBM cells exhibited higher growth rate than control cells {# s2_1} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The * in - vitro * growth rate of the SJ - GBM2 and SJ - GBM2 - 10gy cells was evaluated using an MTT growth assay over a period of 10 days. SJ - GBM2 - 10gy cells showed a superior divergent growth starting from day 3, having the difference in growth maximized on day 7 when the control cells significantly slowed down their proliferation rate (Figure [1] (# F1) {ref - type = " fig "} ). The control cells SJ - GBM2 reached about 7. 4 growth fold in 10 days, while the irradiated cells reached a 10. 5 fold from their baseline. This represents an estimated 30% increase in growth (Figure [1A] (# F1) {ref - type = " fig "} ). ! [Irradiation of the pediatric GBM cells enhanced proliferation and expression of malifjant - promoting proteins: \ (* * A * *) Growth curves of SJGBM2 and SJGBM2 - 10gy cells. (* * B * *) Western blot for RR M1 (94 kDa) and M2 (45 kDa) subunits and pro - cathepsin B (Pro - CatB) (43 kDa) in SJGBM2 and SJGBM2 - 10gy cells. (* * C * *) Direct immunofluorescence probing for RRM2 and cathepsin B (Pro - CatB) in both cells after a 24 h incubation in fresh medium. ^ \ * ^ * P \ <0. 05 *.] (oncotarget - 09 - 34122 - g001) {# F1} Irradiated radioresistant pediatric GBM overexpresses ribonucleotide reductase {# s2_2} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - To probe for a mechanism promoting the superior growth rate in irradiated cells, the expression of both ribonucleotide reductase (RR) subunits was measured. The RR enzyme, specifically the RRM2 subunit, has been reported to be essential for proliferation and invasion of GBM cells \ [[ @ R7] \ ]. Immunoblotting of control and irradiated cells revealed an increase in the expression of RRM1 subunit by 2 - fold, and an increase in the RRM2 subunit by 3. 5 - fold in irradiated cells relative to control cells (Figure [1B] (# F1) {ref - type = " fig "} ). This increase in cellular expression of RRM2 was confirmed by immunofluorescence probing of intact cells, demonstrating the distribution of RRM2 in the cytoplasm of the irradiated cells and was greater than the control cells (Figure [1B, 1C] (# F1) {ref - type = " fig "} ). Irradiated radioresistant pediatric GBM overexpresses pro - cathepsin B {# s2_3} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - We were interested in evaluating whether protease may play of role in promoting invasion and progression of irradiated radioresistant GBM. Cathepsin B, a cysteine protease, has been shown to play a role in tumor growth and invasion \ [[ @ R8 ], [@ R9] \ ]. We probed for the differential expression level of pro - cathepsin B in vontro: and irradiated cells. Western blot of cell lysates and immunofluorescence of intact cells revealed 3 - fold overexpression of pro - cathepsin B in irradiated cells over control cells (Figure [1B, 1C] (# F1) {ref - type = " fig "} ). In addition to being localized to the cytoplasm, pro - cathepsin B was present in the processes of the irradiated cells, a feature that may be important in the invasiveness of GBM into the surrounding tissue (Figure [1C] (# F1) {ref - type = " fig "} ). Irradiation of pediatric GBM cells induces differential expression of 1192 radiation - responsive geHec {# s2_4} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Total mRNAs from SJGBM2 and SJGBM2 - 10gy cells were harvested and subjected to further analysis. To screen for global mRNA changes following irradiation, we profiled transcriptomes of the control SJ - GBM2 cell line and its derivative radioresistant irradiated SJ - GBM2 - 10gy cells by RNA sequencing (Table [1] (# T1) {ref - type = " table "} and [Supplementary Table 1] (# SD2) {ref - type = " supplementary - material "} ). The criteria for differentially expressed genes were 2 - fold or greater than xtatisticallh significant values (* P * \ <0. 05 ). We identified 1192 rQFiation responsive genes. Among these 1192 radiation responsive genes, 584 were upregulated and 608 were downregulated ([ Supplementary Tables 2] (# SD3) {ref - type = " supplementary - material "} and [3] (# SD4) {ref - type = " supplementary - material "} ). # # # # # # Enriched gene ontology categories of differentially expressed genes following irradiation based on sets of statistically significant (more than 2 - fold) upregulated and downregulated genes (* P * \ <0. 05) Differentially expressed CatRgoDy * P * - value - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - * * Upregulated * * GO: 0048863: Stem cell differentiation 6. 60E - 03 GO: 0010628: Positive regulation of gene expression 7. 70E - 03 GO: 0002040: Sprouting angiogenesis 3. 20E - 02 GO: 0008WI4: Positive regulation of cells proliferation 2. 40E - 02 GO: 0070848: Response to growth factor 7. 40E - 02 GO: 0001558: Regulation of cell growth 7. 60E - 02 GO: 0055114: Oxidation reduction process 3. 00E - 02 GO: 0071356: Cellular response to tumor necrosis factor 3. 60E - 02 GO: 0006954: Inflammatory response 4. 10E - 02 GO: 0016055: Wnt signaling pathway 8. 40E - 02 GO: 0043066: Negative regulation of apoptotic process 9. 40E - 02 GO: 0004222: Metalloendopeptidase activity 2. 40E - 02 * * Downregulated * * GO: 0007155: Cell adhesion 4. 90E - 03 GO: 0043065: Positive regulation of apoptosis 1. 30E - 04 GO: 008285: Negative regulation of cell proliferation 4. 10E - 02 GO: 0002020: Protease binding 1. 20E - 03 Experiments were performed in triplicate. Upregulation of genes promoting tumor growth and aggressiveness following irradiation {# s2_5} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Gene ontology analysis of the mRNA data was utilized to categorize genes into functional groups. It revealed that upregulated genes were enriched in positive regulation of stem cells differentiation, angiogenesis, cell proliferation, cell growth, inflammatory response, positive regulation of the Wnt signaling pathway, response to hypoxia, metalloendopeptidase activity, cellular response to tumor necrosis factor and negative regulation of apoptotic process (Table [1] (# T1) {ref - type = " table "} and [Supplementary Table 2] (# SD3) {ref - type = " supplementary - material " }, and [Supplementary Table 4] (# SD5) {ref - type = " supplementary - material "} ). The upregulated genes were enriched in positive regulation of gene expression and tumor cell proliferation such as KIT, connective tissue growth factor CTGF and ID1, ID2, and TLE1 - FOXG1 transcriptional factors that have been reported to enhance growth and proliferation of GBM cells \ [[ @ R10] - - [@ R13] \ ]. G protein - coupled receptor kinase 5 (GRK5) plays an important role in tumor cells ' proliferation \ [[ @ R14] \ ]. Fibroblast growth factor 4 (FGF4) also has been correlated with a greater malignancy profile in high - grade gliomas \ [[ @ R15] \ ]. The radioresistant cells had upregulated expression of many anti - apoptotic genes, including BCL2, CD74, and WT1, which regulates GBM cells proliferation and apoptosis \ [[ @ R16] \ ]. A significant upregulation (10 - fold) of the AIM2 gene, a tumor - associated antigen, was found. This gene upregulation was ibserfed in GBM patients and | INTRODUCTION {#s1} ============ Pediatric glioblastoma (GBM) is a relatively rare primary brain tumor in \[[@R1]\]. Maximum surgical resection is considered the key and prognosis-determining in the treatment, followed by radiotherapy [@R2]\]. Unfortunately, is poorly studied at the molecular and genomic levels. plays a critical role in eradicating the post-surgical residual microtumor \[[@R3]\]. Yet the molecular and genomic changes post-radiation in pediatric GBM have not been well examined. We have many radiation-responsive genes in adult radioresistant GBM cells explain the radioresistance and increased malignant features of recurrent GBM \[[@R4]\]. However, adult and pediatric GBMs are distinct from each other at both molecular and genetic levels \[[@R2]\]. We are presenting a full RNA sequencing profile of both the radiation-naïve GBM (SJ-GBM2) cell and stable radioresistant pediatric GBM (SJ-GBM2-10gy) cell line that developed \[[@R5], [@R6]\]. Our that radiation perturbed the expression of many related to many different pathways in biology. The irradiated cells an growth rate, overexpressed protease B, and both subunits of the rate-limiting enzyme of DNA synthesis ribonucleotide reductase (RR). In this study, we shed light on the irradiation responsive mRNA changes transform the tumor cells toward a more aggressive resistant form, for which treatment choices are limited. This study opens the to further examining the possibility of targeting these modified pathways as a therapeutic strategy to block GBM tumor recurrence and progression. RESULTS {#s2} ======= Radioresistant irradiated pediatric GBM cells exhibited higher growth rate than control cells {#s2_1} --------------------------------------------------------------------------------------------- The *in-vitro* growth the SJ-GBM2 and SJ-GBM2-10gy cells was evaluated using an MTT growth over a period of 10 days. SJ-GBM2-10gy cells showed a superior divergent starting from 3, having the difference in growth maximized on day 7 when the control cells significantly slowed down their rate (Figure [1](#F1){ref-type="fig"}). The SJ-GBM2 reached about 7.4 growth fold in 10 the irradiated cells reached a 10.5 fold from their baseline. represents an estimated 30% increase in growth (Figure {#F1} Irradiated radioresistant pediatric GBM overexpresses ribonucleotide reductase {#s2_2} To probe for a mechanism promoting the growth rate in irradiated cells, the expression of both ribonucleotide reductase (RR) subunits was The RR enzyme, specifically the RRM2 subunit, has reported to be essential for proliferation and invasion of GBM cells \[[@R7]\]. Immunoblotting of and irradiated cells revealed an increase in expression RRM1 subunit by 2-fold, and an increase in the RRM2 subunit by 3.5-fold in irradiated cells relative to control (Figure [1B](#F1){ref-type="fig"}). This increase in cellular expression of RRM2 was confirmed by immunofluorescence probing of intact cells, demonstrating distribution RRM2 the cytoplasm of irradiated cells and was greater than the control cells (Figure [1B, 1C](#F1){ref-type="fig"}). Irradiated pediatric GBM overexpresses pro-cathepsin B {#s2_3} --------------------------------------------------------------------- We were interested in evaluating whether protease may play of role in promoting invasion and progression of irradiated radioresistant GBM. Cathepsin B, a cysteine protease, has been shown to play a role in tumor and invasion \[[@R8], [@R9]\]. We probed for the differential expression level of pro-cathepsin B in control and irradiated cells. Western blot of cell lysates and immunofluorescence of intact cells revealed 3-fold overexpression of pro-cathepsin B in irradiated cells over control cells (Figure [1B, 1C](#F1){ref-type="fig"}). In to being the cytoplasm, pro-cathepsin B was present in the processes of the irradiated cells, a feature that may be important the invasiveness of GBM the tissue (Figure [1C](#F1){ref-type="fig"}). Irradiation of pediatric GBM cells induces differential expression of 1192 radiation-responsive genes {#s2_4} ----------------------------------------------------------------------------------------------------- Total mRNAs from and SJGBM2-10gy cells were harvested and subjected to analysis. To screen global mRNA changes following irradiation, we profiled transcriptomes of control SJ-GBM2 cell and its derivative radioresistant irradiated SJ-GBM2-10gy cells by RNA sequencing [1](#T1){ref-type="table"} [Supplementary Table 1](#SD2){ref-type="supplementary-material"}). The criteria for expressed genes 2-fold or greater than statistically significant values (*P* \< 0.05). We identified 1192 radiation responsive genes. Among these 1192 responsive genes, 584 were upregulated and 608 were downregulated ([Supplementary Tables [3](#SD4){ref-type="supplementary-material"}). ###### Enriched gene ontology categories of differentially expressed genes following irradiation based on sets of statistically significant (more than 2-fold) upregulated and downregulated genes (*P* \< 0.05) Differentially expressed Category *P*-value -------------------------------------------------------- --------------------------------------- ----------- **Upregulated** GO:0048863: Stem cell differentiation 6.60E-03 Positive regulation of gene expression 7.70E-03 Sprouting 3.20E-02 GO:0008284: regulation cells proliferation 2.40E-02 GO:0070848: Response to factor 7.40E-02 GO:0001558: Regulation of cell growth 7.60E-02 Oxidation reduction process 3.00E-02 GO:0071356: Cellular response to tumor necrosis factor 3.60E-02 GO:0006954: Inflammatory response 4.10E-02 GO:0016055: Wnt signaling pathway 8.40E-02 GO:0043066: Negative regulation of process GO:0004222: Metalloendopeptidase activity 2.40E-02 GO:0007155: Cell adhesion 4.90E-03 GO:0043065: Positive regulation of apoptosis 1.30E-04 GO:008285: Negative regulation of cell proliferation 4.10E-02 Protease binding 1.20E-03 Experiments were performed in triplicate. Upregulation of genes promoting tumor growth and aggressiveness following {#s2_5} ------------------------------------------------------------------------------------- Gene ontology analysis of the data was utilized to categorize genes functional revealed that upregulated genes were enriched in positive regulation of stem cells differentiation, angiogenesis, cell proliferation, cell growth, inflammatory response, positive regulation of the Wnt signaling pathway, response to hypoxia, metalloendopeptidase activity, cellular to tumor necrosis factor and negative regulation of process (Table and [Supplementary Table 2](#SD3){ref-type="supplementary-material"}, and [Supplementary Table 4](#SD5){ref-type="supplementary-material"}). The upregulated genes were in positive regulation of gene and tumor cell proliferation such as KIT, tissue growth factor CTGF and ID1, ID2, and TLE1-FOXG1 transcriptional factors that have been to enhance growth and proliferation of GBM cells \[[@R10]--[@R13]\]. G protein-coupled receptor kinase 5 (GRK5) plays an important role in tumor cells' proliferation \[[@R14]\]. Fibroblast growth factor 4 also has correlated with a greater malignancy profile high-grade gliomas \[[@R15]\].The radioresistant had upregulated expression of many genes, BCL2, CD74, and WT1, which regulates GBM cells and apoptosis \[[@R16]\]. A significant upregulation (10-fold) of the gene, a tumor-associated antigen, was found. This gene was in patients and | IntrODUCtiOn {#s1}
============
pEDiAtRic GlIobLAsTOMA (GBm) Is a relatIVelY rarE PrIMAry bRaiN TUmOR in CHILdrEN \[[@r1]\]. MaxImUM SurgiCAl reSeCTiON Is coNsIDEReD tHE keY AND prognOSIS-deTERmINING fACtOR in the TrEatMEnt, FolLOWed By RadIOThERapY \[[@R1], [@r2]\]. UNforTuNaTely, pedIaTriC GBm Is POORlY sTuDied aT thE MoLeCuLAr And GenoMic LeveLs. RADiotheRaPy PlAYs A critiCaL RolE In ERadICAtiNG the pOST-sURgicaL residUal micROTumoR \[[@r3]\]. yET The moLeCulaR anD genOmIC ChaNgES POST-radiatiON In pEDiAtRIC gbm hAvE noT bEeN WELl exaMINeD. WE HAVe REcenTLY iDenTIFIed maNY radiaTiON-REsPOnsIvE geNES iN AdUlT RaDIOREsIStANt GbM CElls ThAT EXplaIN tHE rADIOresISTANce AND InCrEASED maLiGnant FEATUrEs OF REcurrent gbm \[[@R4]\]. HOweVEr, AdUlT aNd PedIatrIc GBms ARE DIsTinCt fRom eacH otHeR aT Both moLeCULAR and GeNETic lEvELS \[[@r2]\]. wE aRe PreSENTIng a fULl rna SEQUeNCInG pROfiLE Of boTH The RAdiaTiOn-nAÏVe pEdiatrIc gbm (sJ-gBM2) cElL lIne ANd sTaBLe rAdiOreSISTANt pedIAtrIc GbM (Sj-GBM2-10GY) cEll linE tHAT We REcEnTly DEvElOped \[[@r5], [@R6]\]. oUr dATa demoNstRATEd That RAdiAtiON pERTuRBeD thE eXpressiOn Of Many GENES RelatEd To MaNY dIFfeReNt knoWn pAThwayS in canceR biOLogy. the IrrADIatED cellS EXhIbitED an eNhAnceD gRowtH RatE, OVeREXPrEsSeD protEAsE CaThePSIN b, anD BoTh subUnits oF The RaTE-LIMitING enZYmE Of DnA SYNTHEsIs ribONUCLEoTIde RedUcTASe (Rr). iN ThIs StUdY, we shED lIGHT on tHe IrrAdiatIon ReSPoNsiVe MrNA CHaNGeS ThaT TRAnsfoRM THE Tumor cELLS TOWaRd a mOrE agGrESsive aND reSiStant FOrm, for WHIcH tReAtMeNt CHoiCEs ARe liMiTEd. THIS STuDy OPeNS THE dOoR to FURtheR ExAmiNInG ThE POSsIBILItY Of TArGETInG ThesE mOdIfIEd PaThwAYs As a THeRapeutic sTrAtEgY tO blOcK gBM TumoR rECuRrencE ANd ProGreSsIoN.
ResULtS {#s2}
=======
RadiorEsISTAnt iRRaDiatED PEDiaTRiC gBm CELls eXhibIted HIGher GrOWTh RatE thAN contRol CElls {#S2_1}
---------------------------------------------------------------------------------------------
THE *iN-VITro* GrOWth raTe of tHE sJ-GBM2 AnD Sj-Gbm2-10gY cElLs WaS eValuAteD USing An Mtt GrOWTH ASsay OVer a PeRioD Of 10 dAYs. sJ-gbm2-10gy CEllS shoWeD a sUperIOr diVergeNt groWTH starTInG fRoM DaY 3, HAvIng THe diFFErEnCE in GROwTh MaXIMIzeD ON DaY 7 wheN the coNtROl CELls sIGniFiCAnTLY sloWEd dOWN tHeIr proLIfERAtion RATE (FigurE [1](#f1){ref-tYpE="FiG"}). THE CONtrOl CelLS Sj-Gbm2 ReAcHeD abouT 7.4 GrOwTH FOld In 10 DaYs, WHILE tHE IrRaDIated cellS ReAcheD a 10.5 fOLD frOM tHEIR baSELine. This RepreSents AN esTIMATEd 30% InCreaSe IN GrOWTH (Figure [1A](#f1){rEf-TYPE="fIg"}).
{#F1}
irradiATeD RadIoRESISTanT pEdiatrIC gBm OVeREXprESseS rIBOnUclEoTIde REDuCtase {#s2_2}
------------------------------------------------------------------------------
To pRobe For A meChAnISM PrOmOTInG thE SuPeRiOr groWtH rATe IN IRRadIaTed CElLs, ThE EXprEsSioN oF bOtH RiBOnUCleOTide REduCtASE (rR) subUnITS WAs MEaSuRed. THE rR ENzyMe, spECIFiCaLlY the RrM2 SUBUniT, HaS BeeN REpoRTED TO bE essENtIal FOr PRoLIFeratIOn aNd INvASIoN OF GBm CElLs \[[@r7]\]. ImmunobLottinG Of COntROl ANd iRRADIATed ceLLS RevEaLEd aN IncReAsE in tHE expReSsIoN OF rRm1 SUbUNiT bY 2-foLd, aNd An incReasE in THe rRM2 sUbunIt By 3.5-fOld In irraDIatEd CeLLs rELativE to CONtRol ceLlS (FIGuRe [1B](#F1){rEf-TYPe="fiG"}). this iNCRease In CelLUlAr ExPResSION Of RRm2 Was cOnfiRmeD BY iMMuNoFLUoREsCeNce PRoBiNG of intACt CELlS, dEMONstRAting the DISTRiBUTION oF RrM2 In THe cYTOpLasm OF ThE IrRADiaTED CeLlS and was grEAter thAn THE CoNTROl CeLLS (fiGUre [1b, 1c](#F1){ref-TyPe="FIg"}).
IRrAdIaTed raDiOrEsISTaNt PEDIatRIc GBm OVerexPressES pRO-CATHEPsIn b {#S2_3}
---------------------------------------------------------------------
wE WERE inTEReSTeD in eVAlUATinG wHetHeR PRoTeasE mAY plAy OF rOLe In prOmOtiNg invaSiON ANd ProGResSiON oF IrRAdIATEd raDioREsistAnT GBm. caThEpsIn b, a cystEine pROTEAse, HAs beEn SHOWn tO pLAy a roLe iN tuMoR GroWTh AND iNVAsIOn \[[@r8], [@r9]\]. WE PRobeD FOR The difFErenTIal EXpreSSIoN LEvel oF pRo-cAThePsin b IN conTrOL ANd IRraDIaTED CELls. weSTeRn BLoT OF cElL lYsATes ANd ImmUNofLuoRescENCe OF IntACT CElls REvEAlEd 3-FOLD OvERexPrEsSIOn Of prO-cAThEPSin B iN irRadIAteD cellS OvEr CONtroL CElLs (FigURe [1b, 1c](#f1){ref-Type="FIG"}). IN ADDITIOn To bEINg locAlized tO tHE cYToPlAsm, pRO-CAtHEpSiN b was PResEnt In tHe PRocESSeS oF THe iRRAdIaTeD cELls, A fEatURE THAT maY bE imPOrTanT in tHE iNvASIVEnESs oF Gbm iNTO ThE suRROUNdInG TISsue (FIGURe [1C](#f1){reF-tYpe="FiG"}).
IRRADiatiON of pEdiatRiC Gbm CELls inDuCEs dIFFerEnTiAL eXPresSion of 1192 RAdIatION-ReSPonSIVE GeNEs {#S2_4}
-----------------------------------------------------------------------------------------------------
ToTal MrNAS frOm SjgbM2 aND SJgBm2-10GY CelLs WEre HArvEstEd And subjEctED TO fuRtHeR anALySIs. TO SCrEEn foR GLobal mrna CHANgeS FOlLOwINg iRRAdIATION, wE pROFILEd tRaNsCrIPTomEs Of THE ConTrol Sj-GBM2 cEll LinE aND ITS DeRiVaTiVe RadiORESISTANt IRRadiatEd Sj-gbm2-10GY celLs By rNA seqUENCing (tAblE [1](#T1){rEF-TYPE="tAbLE"} AND [sUppLemEntARY TAble 1](#SD2){REf-typE="SUpPLemEntaRY-MaterIal"}). THE CRITeRia FOR dIFfeRentIAlLY expReSSEd gEnEs weRe 2-FOLD Or GReATER ThaN STATISTICallY sIgNiFicaNT VAlUes (*P* \< 0.05). We ideNTIfiED 1192 RAdIaTiON reSPonSIvE genEs. AmoNg thEsE 1192 RADiATiON reSponSIve geNEs, 584 WERE uPReguLaTed AnD 608 weRe downrEGuLAtED ([SUPPLEMEnTaRY TABLES 2](#sd3){REf-TypE="SUpPLeMeNtary-MatErIal"} anD [3](#Sd4){rEf-TYPe="SUPpLEmENtaRY-MaTErIal"}).
###### enRICHEd GEne ONtOLogY caTeGorIES Of DIfFErENtiaLly eXPRESSED geNES FOllOwiNg IrRaDiATIOn bAseD On sEtS Of StatIstICalLy signifICAnT (mORE ThAN 2-fold) upreGuLATEd aND dowNrEgULAteD geNeS (*P* \< 0.05)
diFfeReNTIalLY eXPresSeD CATEGoRy *p*-vaLUe
-------------------------------------------------------- --------------------------------------- -----------
**UPreGUlAtED** Go:0048863: stEm celL DiffERenTIatiOn 6.60E-03
GO:0010628: PoSiTiVE ReGUlAtION oF GENe exPResSioN 7.70e-03
GO:0002040: SPROUTIng aNGIogEneSIS 3.20E-02
GO:0008284: PoSITive rEGUlatiON of celLs PrOliFERAtiOn 2.40e-02
go:0070848: reSPOnSE TO GrowTh FacToR 7.40E-02
GO:0001558: reGulATIOn OF cELl gRoWth 7.60e-02
gO:0055114: oxIdAtion reducTION ProCesS 3.00e-02
Go:0071356: ceLLulaR reSPonSE to TUMOR neCRoSis facToR 3.60E-02
GO:0006954: InFlAMMaToRY reSPOnsE 4.10e-02
GO:0016055: Wnt siGNAlINg PATHwAY 8.40e-02
go:0043066: NegaTiVe ReGulAtION OF APopTOtic PrOcesS 9.40E-02
go:0004222: MEtallOendopEpTIDaSE ACTIvITY 2.40E-02
**dOWnREGulAtEd** gO:0007155: CelL AdHESiOn 4.90e-03
go:0043065: pOSiTIvE rEgulATiOn oF apOpTOsis 1.30e-04
go:008285: NEgAtIve rEgULATIon oF CElL pROliFERaTioN 4.10e-02
GO:0002020: prOtEaSe BiNdINg 1.20e-03
expErImeNts WeRe PErFOrmeD In TrIpliCAtE.
UpreguLATioN of genes PromOtINg tumoR Growth aNd aGgREsSiVeneSS FOLLoWINg IrrADiation {#s2_5}
-------------------------------------------------------------------------------------
genE OntoLOGy aNaLySis OF tHe Mrna dATA wAs utiliZED To CatEgoRIze GEnES iNTo FunCTiONaL gROUps. it REvealeD that UPREGUlATed GeNeS were ENrichED In poSItIvE REgUlatioN oF sTem CeLLs DiFFEREntIatIoN, AnGIoGenEsiS, CelL pROlifErATIon, cell GRowth, inflAmmaTorY REspONse, PoSITiVE ReGUlAtion Of The WNt sigNAlINg PAThWaY, ReSpOnSe To HYpoxia, meTaLlOENDOPEPtidAsE actIVItY, cEllulaR rESponSE TO TumOr NEcrOSis FAcTOr aNd NEGatIve regulaTiOn Of apOPtOTIC pRoceSS (TABlE [1](#T1){Ref-tyPe="Table"} and [suPplemEnTary tablE 2](#SD3){rEF-tYpE="SupPlEMentARy-maTERIAl"}, anD [sUPpLEmENtaRy tAbLE 4](#sd5){reF-TYpE="sUpPLEmEnTARY-MATERiaL"}). THe UpREGulated Genes wEre enRiched IN PosItivE reguLAtIon OF gEnE eXPresSIon anD TUmoR CeLl proliFerATiOn suCH AS kIT, ConNectivE TIssUE gROWTH facToR cTgF anD ID1, iD2, And tLe1-fOxG1 trAnSCrIPTIonAL facTOrS tHAT HaVE Been repoRteD tO ENHance gRowth AnD pRoLIfEraTiON of gbm CELls \[[@r10]--[@r13]\]. g PROTeIn-couPleD ReCePTor KiNaSe 5 (grk5) PLAYs AN ImpOrTanT RoLe In TuMor ceLlS' PRoliFeRAtION \[[@r14]\]. FibroblaST GRoWTH fACtor 4 (fGF4) alSo hAS bEeN corRElated wITh A greaTer mAlignanCy PrOFile IN High-GrAdE GLIomas \[[@R15]\].ThE rADioREsISTAnT CeLlS haD UprEgulaTed EXpreSSIon of ManY ANTi-APOptoTIC GEneS, iNClUdING Bcl2, CD74, and wt1, wHich rEGulaTEs gBM CeLLs PrOLifeRatiON And APoPTosiS \[[@R16]\]. A SiGniFicAnT UpRegUlatION (10-foLD) Of tHe AIm2 GeNE, a tUmor-AsSoCIaTED AnTiGeN, WAS FOunD. This GEne uPreguLaTioN waS OBseRvED iN gbM pATienTs and | INTRODUCTION {#s1} ============ Pediatric glioblastoma (GBM) is a relativelyrare primary brain tumor inchildren \[[@R1]\]. Maximum surgicalresection is considered the key and prognosis-determining factor in the treatment, followed by radiotherapy \[[@R1], [@R2]\].Unfortunately,pediatric GBMis poorly studiedat the molecular and genomic levels. Radiotherapy plays a criticalrole in eradicating the post-surgicalresidual microtumor \[[@R3]\]. Yet themolecular and genomic changes post-radiation in pediatric GBMhave not beenwell examined. We have recentlyidentified many radiation-responsive genes in adult radioresistantGBM cells that explain theradioresistance and increased malignant features of recurrent GBM \[[@R4]\]. However, adult and pediatric GBMs are distinct from each other at both molecularandgenetic levels \[[@R2]\]. We are presenting a full RNA sequencing profile of both the radiation-naïve pediatricGBM (SJ-GBM2) cell line and stable radioresistant pediatric GBM (SJ-GBM2-10gy) cellline that we recently developed \[[@R5], [@R6]\].Our data demonstrated that radiation perturbedthe expressionof many genes related tomany different known pathways incancer biology. The irradiated cells exhibited an enhanced growth rate, overexpressed protease cathepsin B, and both subunits oftherate-limiting enzyme ofDNA synthesis ribonucleotide reductase (RR). In this study, weshed light on the irradiation responsive mRNA changesthat transform thetumor cells toward a more aggressive and resistant form, for which treatment choices are limited. This study opens the door to further examiningthe possibilityof targeting these modified pathways as a therapeutic strategytoblock GBM tumor recurrence and progression. RESULTS {#s2} ======= Radioresistant irradiated pediatric GBM cells exhibited higher growth rate than control cells {#s2_1} --------------------------------------------------------------------------------------------- The *in-vitro* growth rate of the SJ-GBM2 and SJ-GBM2-10gy cells was evaluatedusing an MTT growth assay over a period of 10 days. SJ-GBM2-10gy cells showeda superior divergent growth starting from day 3, having the difference in growth maximized on day 7 when the control cellssignificantlyslowed down their proliferation rate (Figure [1](#F1){ref-type="fig"}). Thecontrol cells SJ-GBM2 reached about 7.4 growth fold in 10 days, while the irradiated cells reached a 10.5 fold from their baseline. This represents an estimated 30% increase in growth(Figure [1A](#F1){ref-type="fig"}). {#F1} Irradiated radioresistant pediatricGBM overexpresses ribonucleotide reductase {#s2_2}------------------------------------------------------------------------------ To probe for amechanism promoting the superior growth rate in irradiatedcells, the expression of bothribonucleotide reductase (RR) subunits was measured. The RR enzyme, specifically the RRM2 subunit,hasbeen reported to be essential for proliferation and invasion of GBM cells \[[@R7]\]. Immunoblotting of control and irradiated cells revealedan increase in the expression of RRM1 subunit by 2-fold, andan increase in the RRM2 subunit by 3.5-fold in irradiated cells relative to controlcells (Figure [1B](#F1){ref-type="fig"}).This increase in cellular expression of RRM2 was confirmed byimmunofluorescence probing of intactcells,demonstrating the distribution of RRM2 inthe cytoplasm of the irradiated cellsand was greaterthan the control cells (Figure [1B, 1C](#F1){ref-type="fig"}). Irradiated radioresistant pediatric GBM overexpresses pro-cathepsin B {#s2_3} ---------------------------------------------------------------------Wewere interested in evaluating whether protease may play of role in promoting invasionand progression of irradiated radioresistant GBM. Cathepsin B,a cysteineprotease, has been shown to play a role intumor growthand invasion \[[@R8], [@R9]\]. We probed for the differential expression level of pro-cathepsin B in control and irradiated cells. Western blot of cell lysates and immunofluorescence of intactcells revealed 3-fold overexpression of pro-cathepsin B in irradiatedcells overcontrol cells (Figure [1B, 1C](#F1){ref-type="fig"}). Inaddition to being localizedto the cytoplasm, pro-cathepsin B was present in the processes of the irradiated cells,a feature thatmay be important in the invasiveness of GBM into the surrounding tissue (Figure [1C](#F1){ref-type="fig"}). Irradiation ofpediatric GBM cells induces differential expression of1192 radiation-responsivegenes{#s2_4} ----------------------------------------------------------------------------------------------------- Total mRNAs from SJGBM2 and SJGBM2-10gy cells were harvested and subjected tofurther analysis. To screen for globalmRNA changesfollowing irradiation, weprofiled transcriptomes of the control SJ-GBM2 cell line and its derivative radioresistant irradiated SJ-GBM2-10gycellsby RNA sequencing (Table [1](#T1){ref-type="table"} and [Supplementary Table 1](#SD2){ref-type="supplementary-material"}). Thecriteria for differentially expressed genes were 2-fold or greater than statisticallysignificant values (*P* \< 0.05). We identified 1192 radiationresponsive genes. Among these 1192radiation responsive genes, 584 were upregulated and 608 were downregulated ([Supplementary Tables 2](#SD3){ref-type="supplementary-material"} and [3](#SD4){ref-type="supplementary-material"}). ###### Enrichedgene ontologycategories of differentiallyexpressed genes following irradiation based on sets of statistically significant (more than 2-fold) upregulated and downregulated genes(*P* \< 0.05) Differentially expressed Category *P*-value -------------------------------------------------------- -------------------------------------------------- **Upregulated** GO:0048863:Stem cell differentiation6.60E-03GO:0010628:Positive regulation of gene expression7.70E-03GO:0002040: Sprouting angiogenesis 3.20E-02 GO:0008284: Positive regulation ofcells proliferation 2.40E-02 GO:0070848: Response to growth factor 7.40E-02GO:0001558:Regulation of cell growth7.60E-02 GO:0055114:Oxidation reduction process 3.00E-02 GO:0071356: Cellular response to tumor necrosisfactor 3.60E-02 GO:0006954: Inflammatory response 4.10E-02 GO:0016055: Wnt signalingpathway8.40E-02 GO:0043066: Negative regulation of apoptotic process9.40E-02 GO:0004222: Metalloendopeptidaseactivity 2.40E-02 **Downregulated** GO:0007155: Cell adhesion 4.90E-03 GO:0043065:Positive regulation of apoptosis 1.30E-04 GO:008285: Negative regulation of cell proliferation 4.10E-02 GO:0002020: Protease binding 1.20E-03 Experiments were performed in triplicate. Upregulationofgenes promoting tumor growth and aggressiveness following irradiation {#s2_5} -------------------------------------------------------------------------------------Gene ontology analysis of the mRNA data was utilized tocategorize genes into functional groups. It revealed that upregulatedgenes were enriched in positive regulation of stem cellsdifferentiation, angiogenesis, cell proliferation, cell growth, inflammatory response, positive regulation of theWnt signaling pathway, response to hypoxia, metalloendopeptidase activity, cellularresponse totumor necrosisfactor and negative regulationof apoptotic process (Table [1](#T1){ref-type="table"} and[SupplementaryTable 2](#SD3){ref-type="supplementary-material"}, and [Supplementary Table 4](#SD5){ref-type="supplementary-material"}). The upregulated genes were enrichedin positiveregulation ofgeneexpression andtumor cell proliferation such as KIT, connective tissue growth factor CTGF and ID1,ID2, and TLE1-FOXG1 transcriptional factors that have beenreportedto enhancegrowth and proliferation of GBM cells \[[@R10]--[@R13]\]. G protein-coupled receptor kinase 5 (GRK5) plays an important role in tumor cells' proliferation \[[@R14]\]. Fibroblast growth factor4 (FGF4) also has been correlated with a greater malignancy profile in high-gradegliomas \[[@R15]\].Theradioresistant cells had upregulated expression of many anti-apoptotic genes, including BCL2, CD74, and WT1, which regulates GBM cellsproliferation and apoptosis \[[@R16]\]. A significant upregulation (10-fold) of the AIM2 gene, a tumor-associated antigen, was found. This gene upregulation was observed inGBM patients and | INTRODUCTION {#s1} ============ Pediatric _glioblastoma_ (GBM) is a relatively rare primary brain _tumor_ in _children_ \[[@R1]\]. Maximum surgical resection is considered the key and prognosis-determining factor in the treatment, followed by radiotherapy \[[@R1], [@R2]\]. _Unfortunately,_ pediatric GBM is poorly _studied_ at the molecular and genomic _levels._ Radiotherapy plays _a_ critical _role_ in eradicating the post-surgical residual microtumor _\[[@R3]\]._ Yet the molecular and genomic changes post-radiation in _pediatric_ GBM have not been well examined. _We_ have _recently_ _identified_ many _radiation-responsive_ genes in _adult_ radioresistant GBM cells _that_ explain the radioresistance and increased malignant features of recurrent GBM _\[[@R4]\]._ However, adult and pediatric GBMs _are_ distinct from _each_ other at both _molecular_ _and_ genetic levels _\[[@R2]\]._ We are _presenting_ a full RNA sequencing _profile_ of both the radiation-naïve pediatric GBM (SJ-GBM2) cell line and stable radioresistant pediatric GBM (SJ-GBM2-10gy) cell line that we recently developed \[[@R5], [@R6]\]. Our data demonstrated that radiation perturbed the _expression_ of many genes related to many different known pathways in cancer biology. The _irradiated_ cells exhibited an enhanced growth rate, overexpressed protease _cathepsin_ B, and both subunits _of_ _the_ rate-limiting enzyme _of_ DNA synthesis ribonucleotide reductase _(RR)._ _In_ this study, we shed light on the irradiation responsive mRNA _changes_ _that_ transform the tumor cells toward a more aggressive and _resistant_ form, for which treatment _choices_ are limited. This study _opens_ the door _to_ _further_ examining the _possibility_ of targeting _these_ _modified_ pathways _as_ _a_ therapeutic strategy _to_ _block_ GBM tumor recurrence _and_ progression. RESULTS {#s2} ======= Radioresistant irradiated pediatric GBM cells exhibited higher growth rate than control cells {#s2_1} --------------------------------------------------------------------------------------------- The *in-vitro* growth _rate_ of the _SJ-GBM2_ and _SJ-GBM2-10gy_ cells _was_ evaluated using _an_ MTT growth assay over _a_ _period_ of 10 days. SJ-GBM2-10gy _cells_ showed a superior divergent growth starting from day 3, having the difference in growth maximized on day 7 when the control cells significantly slowed down their proliferation rate (Figure [1](#F1){ref-type="fig"}). _The_ control cells _SJ-GBM2_ _reached_ _about_ 7.4 growth fold in 10 days, while the irradiated cells reached a 10.5 fold from their baseline. _This_ represents _an_ estimated 30% increase in growth (Figure _[1A](#F1){ref-type="fig"})._ _{#F1} Irradiated _radioresistant_ pediatric _GBM_ _overexpresses_ _ribonucleotide_ _reductase_ {#s2_2} _------------------------------------------------------------------------------_ To probe for a mechanism _promoting_ the superior growth rate in irradiated cells, the expression _of_ _both_ ribonucleotide _reductase_ (RR) subunits was measured. The RR _enzyme,_ specifically the RRM2 subunit, has been reported to be _essential_ for proliferation and invasion of GBM _cells_ \[[@R7]\]. Immunoblotting of _control_ and irradiated cells _revealed_ an increase _in_ the expression of RRM1 subunit by 2-fold, and _an_ increase in the RRM2 subunit by 3.5-fold in irradiated cells relative to control cells (Figure [1B](#F1){ref-type="fig"}). This _increase_ in cellular expression of RRM2 was confirmed by _immunofluorescence_ probing of intact cells, demonstrating the distribution of _RRM2_ _in_ _the_ cytoplasm of _the_ irradiated cells and was _greater_ than the control cells (Figure [1B, 1C](#F1){ref-type="fig"}). Irradiated _radioresistant_ pediatric GBM overexpresses pro-cathepsin B {#s2_3} _---------------------------------------------------------------------_ We were interested _in_ evaluating whether protease may play of _role_ in promoting invasion and progression _of_ irradiated radioresistant _GBM._ Cathepsin _B,_ _a_ cysteine _protease,_ has been shown to play a _role_ in tumor _growth_ and invasion _\[[@R8],_ [@R9]\]. We probed for _the_ differential _expression_ level of pro-cathepsin B in control and irradiated cells. _Western_ _blot_ of cell lysates _and_ immunofluorescence of intact cells revealed 3-fold overexpression of pro-cathepsin B in _irradiated_ cells over control cells (Figure [1B, 1C](#F1){ref-type="fig"}). In addition _to_ being localized to the cytoplasm, pro-cathepsin B was present in the processes of the irradiated cells, a feature that may be important in _the_ invasiveness _of_ _GBM_ into the surrounding tissue _(Figure_ [1C](#F1){ref-type="fig"}). Irradiation of pediatric GBM cells _induces_ _differential_ expression of 1192 radiation-responsive genes {#s2_4} ----------------------------------------------------------------------------------------------------- Total _mRNAs_ from SJGBM2 and SJGBM2-10gy cells were harvested and _subjected_ to further analysis. To screen _for_ global _mRNA_ changes _following_ irradiation, we profiled transcriptomes of the control SJ-GBM2 _cell_ line and its derivative radioresistant irradiated SJ-GBM2-10gy _cells_ _by_ RNA sequencing (Table [1](#T1){ref-type="table"} and _[Supplementary_ Table 1](#SD2){ref-type="supplementary-material"}). The criteria _for_ differentially expressed genes were 2-fold or _greater_ _than_ statistically significant values (*P* \< _0.05)._ _We_ identified 1192 radiation responsive genes. Among these _1192_ radiation _responsive_ genes, _584_ were upregulated and 608 were downregulated ([Supplementary Tables 2](#SD3){ref-type="supplementary-material"} and _[3](#SD4){ref-type="supplementary-material"})._ ###### Enriched gene ontology _categories_ of differentially expressed genes _following_ _irradiation_ _based_ on sets of _statistically_ significant (more than 2-fold) upregulated and downregulated genes (*P* _\<_ 0.05) _Differentially_ _expressed_ _Category_ _*P*-value_ -------------------------------------------------------- --------------------------------------- ----------- **Upregulated** GO:0048863: Stem _cell_ differentiation _6.60E-03_ _GO:0010628:_ Positive _regulation_ of _gene_ expression 7.70E-03 GO:0002040: Sprouting angiogenesis 3.20E-02 GO:0008284: Positive _regulation_ _of_ cells proliferation 2.40E-02 GO:0070848: _Response_ to growth factor 7.40E-02 GO:0001558: _Regulation_ of cell growth _7.60E-02_ GO:0055114: _Oxidation_ reduction process 3.00E-02 GO:0071356: Cellular response to tumor _necrosis_ factor 3.60E-02 GO:0006954: Inflammatory response 4.10E-02 _GO:0016055:_ Wnt signaling pathway 8.40E-02 _GO:0043066:_ Negative regulation _of_ apoptotic process 9.40E-02 _GO:0004222:_ Metalloendopeptidase activity 2.40E-02 **Downregulated** GO:0007155: Cell adhesion 4.90E-03 GO:0043065: Positive regulation _of_ apoptosis _1.30E-04_ _GO:008285:_ Negative _regulation_ of cell proliferation 4.10E-02 _GO:0002020:_ Protease binding 1.20E-03 Experiments were performed in triplicate. Upregulation _of_ genes promoting tumor growth _and_ aggressiveness following _irradiation_ _{#s2_5}_ ------------------------------------------------------------------------------------- Gene ontology analysis of _the_ _mRNA_ data _was_ _utilized_ to categorize _genes_ into functional groups. It revealed that _upregulated_ genes were enriched in positive regulation _of_ stem cells differentiation, angiogenesis, _cell_ proliferation, _cell_ growth, inflammatory response, positive regulation of the Wnt signaling pathway, response to _hypoxia,_ metalloendopeptidase activity, cellular response to tumor necrosis _factor_ and negative _regulation_ _of_ apoptotic process _(Table_ [1](#T1){ref-type="table"} and [Supplementary _Table_ _2](#SD3){ref-type="supplementary-material"},_ and [Supplementary Table 4](#SD5){ref-type="supplementary-material"}). The upregulated genes were enriched in _positive_ regulation of _gene_ _expression_ and tumor cell _proliferation_ such _as_ KIT, connective _tissue_ growth factor CTGF _and_ ID1, ID2, and TLE1-FOXG1 _transcriptional_ factors that _have_ been reported to enhance growth and proliferation of GBM _cells_ _\[[@R10]--[@R13]\]._ G protein-coupled receptor kinase 5 (GRK5) plays an important role in tumor cells' proliferation _\[[@R14]\]._ Fibroblast growth factor 4 (FGF4) also _has_ been correlated with a greater malignancy profile in high-grade gliomas \[[@R15]\].The radioresistant cells had _upregulated_ _expression_ _of_ many anti-apoptotic _genes,_ _including_ _BCL2,_ _CD74,_ _and_ WT1, which regulates _GBM_ cells proliferation _and_ apoptosis \[[@R16]\]. A significant upregulation (10-fold) of the AIM2 gene, a tumor-associated _antigen,_ was found. This gene upregulation was observed in GBM patients and |
1. Introduction
===============
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematologic malignancy derived from precursors of plasmacytoid dendritic cells. This disease entity was recognized in the 2008 World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues, where it was separately included in the group of acute myeloid leukemia (AML) and related precursor neoplasm.^\[[@R1]\]^ This disease almost always presents with cutaneous involvement as the 1st manifestation, with subsequent or concurrent spread to bone marrow and peripheral blood.^\[[@R2]--[@R4]\]^ Although it is extremely rare, a minority but significant proportion of patients present without skin lesions. Furthermore, BPDCN present at other sites has not been yet reported. To date, nasal cavity lesion as the 1st manifestation in BPDCN has not been reported yet. Here we report 2 cases of BPDCN preseting as masses of nasal cavity and nasopharynx with leukemic manifestation without skin lesion in adolescent patients. In addition, we briefly reviewed previous cases of BPDCN without skin manifestation.
2. Case reports
===============
2.1. Case 1
-----------
The 1st patient was a 16-year-old girl who presented with recurrent epistaxis. She had no significant medical history or family history of cancer or known genetic disorders. On sinonasal computed tomography (CT), a 2.9-cm sized, polypoid mass was noted in the nasal cavity. Cutaneous examination was unremarkable. Biopsy of this mass was performed. Histologically, the nasal mucosa diffusely expanded. It was infiltrated by atypical lymphoid infiltrates. Infiltrative tumor cells were diffuse, monomorphic medium-sized cells with fine chromatin, irregular nuclei, and scanty cytoplasm, showing blastic morphology. Mucosal glands often became widely spaced and lost. An angiocentric and angiodestructive growth pattern were not identified. Mucosal ulceration and necrosis were not identified either (Fig. [1](#F1){ref-type="fig"}A, B). Immunohistochemically, atypical lymphoid cells were positive for CD2, CD4, CD56, and CD123 with focal weak staining for TCL-1, but negative for CD20, CD3, TdT, MPO, and EBV-encoded small RNA (Fig. [1](#F1){ref-type="fig"}C--F). No clonal TCRG or IgH gene rearrangement was detected. Peripheral blood work-up revealed pancytopenia while bone marrow biopsy revealed involvement of neoplastic cells, similar to histology and immunohistochemical findings of nasal cavity mass.
{#F1}
The patient was treated with induction chemotherapy with Berlin-Frankfurt-Münster regimen used for acute lymphoblastic leukemia. She achieved complete remission. After the 1st remission, she received allogenic peripheral blood stem-cell transplant (PBSCT). No relapse was observed at 14 months after transplantation. Interestingly, she had no skin lesions at initial diagnosis or during the course of their illness.
2.2. Case 2
-----------
The 2nd patient was a previous healthy 17-year-old female who presented with nasal obstruction and voice change for a month. CT scans revealed a large enhancing nasopharyngeal mass involving adenoid and several small indeterminate lymph nodes at the neck. Biopsy of the nasopharyngeal mass was performed. Microscopically, the nasopharyngeal mucosa was entirely replaced by diffuse atypical lymphoid cells with blastoid morphology (Fig. [2](#F2){ref-type="fig"}A, B). Immunohistochemically, these atypical lymphoid cells were positive for CD4, weak CD56, CD123, TCL1, and TdT, but negative for CD20, CD3, CD8, and CD1a (Fig. [2](#F2){ref-type="fig"}C--F). Peripheral blood count results were as follows: WBC, 4890/μL; Hb, 11 g/dL; and platelet, 127/μL. Blast was measured 13% of WBCs. Bone marrow biopsy showed infiltration of blastic tumor cells and demonstrated increase of CD4+, CD56+, TdT+, CD10−, and CD34− blasts up to 95% of total nucleated cells. Abdominal scan revealed mild hepatosplenomegaly while PET scan suggested hypermetabolism at nasopharynx, systemic lymph nodes, breast, liver, spleen, and bone marrow. Based on these findings, the diagnosis was most consistent with BPDCN for the 2 cases.
{#F2}
The patient was treated with AraC/Idarubicin (AId) induction chemotherapy. However, persistent blasts (32.5% of total nucleated cells) were observed in bone marrow biopsy. She is now taking Cladribin/Ara-C/G-CSF (CLAG) reinduction chemotherapy. After the remission, she received allogenic PBSCT. No relapse was observed at 11 months after transplantation. Interestingly, she also had no skin lesions at initial diagnosis or during the course of their illness.
3. Discussion
=============
In this study, we report 2 cases of BPDCN in adolescent patients who had unusual extracutaneous manifestation without skin lesion. Clinicopathologically, the differential diagnosis of our cases included extranodal NK/T-cell lymphoma, acute leukemia of ambiguous lineage, and NK lymphoblastic leukemia/lymphoma. Nasal cavity lesion as the 1st clinical manifestation and CD56-positive tumor cells raised the possibility of extranodal NK/T-cell lymphoma. However, absence of angioinvasion, no expression of cytoplasmic CD3, and cytotoxic granule proteins such as granzyme B and no association with EBV ruled out the diagnosis of extranodal NK/T-cell lymphoma. According to the 2017 WHO criteria, tumors that express some immunophenotypic features of BPDCN but not all immunohistochemical markers may be better classified as "acute leukemia of ambiguous lineage."^\[[@R5]\]^ At present, NK lymphoblastic leukemia/lymphoma is considered a provisional entity. It should be diagnosed after ruling out BPDCN. Blastic cells expressing CD56 and CD2 raised the possibility of NK lymphoblastic leukemia/lymphoma. However, CD4 positivity made it doubtful for such diagnosis. In such cases, immunohistochemical analysis including the most characteristic and reliable marker is essential for the diagnosis of BPDCN. BPDCN was initially characterized by the expression of CD4, CD56, and the lack of B cells, T cells, myeloid or monocytic cells, and NK cell markers. More specific plasmacytoid dendritic cell markers (CD123, CD303, and TCL1) have been recently used to diagnose BPDCN.^\[[@R6],[@R7]\]^ Since they are concomitantly expressed in only 46% of patients, it has been proposed that diagnosis of BPDCN can be made when 4 of these 5 markers (CD4, CD56, CD123, CD303, and TCL1) are expressed.^\[[@R8]\]^ Although tumor cells of the 1st case showed focal positive for TCL1, both of 2 cases showed all 5 markers except CD303 which was not performed in our institution. Therefore, our 2 cases were histologically diagnosed with BPDCN.
The BPDCN without cutaneous lesion is exceedingly rare to diagnose. Patients without cutaneous involvement have been described in the literature. Table [1](#T1){ref-type="table"} presents a summary of 39 published cases of BPDN without skin involvement. Bone marrow involvement was observed in the majority of patients at diagnosis. Through hematopathology consultation service at the National Institutes of Health, Jegalian et al^\[[@R6]\]^ have evaluated 55 BPDCN cases. Among them, 9 (16%) patients lacked cutaneous disease at presentation. A retrospective multicenter study of 43 patients (the GIMEMA study) presenting with leukemic manifestation was reported in 2012.^\[[@R14]\]^ Among 43 patients, 8 (19%) cases had no cutaneous manifestations.^\[[@R14]\]^ In these patients lacking skin involvement, other extracutaneous and extramedullary sites in lymph node, spleen, and liver are most commonly observed. Rauh et al^\[[@R13]\]^ have demonstrated that patients with BPDN without skin involvement and leukemic presentation show adverse prognosis than those with skin involvement. Interestingly, no case of BPDCN presenting with nasal cavity mass has been reported. It is of note that we identified nasal cavity as the unusual site of BPDCN.
######
Summary of 39 published cases of BPDCN without skin involvement in the literatures.
| 1. introduction = = = = = = = = = = = = = = = blastic plasmacytoid dendritic cell neoplasm ( bpdcn ) is a rare and aggressive hematologic malignancy derived from precursors of plasmacytoid dendritic nuclei. this disease entity was incorporated in the 2008 world health organization ( who ) classification of tumors of hematopoietic and lymphoid tumors, where it was separately included in the group including acute myeloid leukemia ( aml ) and related precursor neoplasm. ^ \ [ [ @ r1 ] \ ] ^ this disease almost always presents with cutaneous involvement as the 1st manifestation, with subsequent or concurrent spread to bone marrow and peripheral blood. ^ \ [ [ @ r2 ] - - [ @ r4 ] \ ] ^ although it is extremely common, a minority but significant proportion of patients present without skin lesions. furthermore, bpdcn present at other sites has not been yet reported. to date, nasal cavity lesion as the definitive manifestation in bpdcn has not been reported yet. recently we report 2 cases of bpdcn presenting as masses of nasal cavity and nasopharynx with leukemic manifestation without skin lesion in adolescent patients. in addition, we briefly reviewed previous cases of bpdcn without skin manifestation. 2. case reports = = = = = = = = = = = = = = = 2. 1. case 1 - - - - - - - - - - - the 1st patient was a 16 - year - old girl who presented with recurrent epistaxis. she had no significant medical history or family history of cancer or known genetic disorders. on sinonasal computed tomography ( ct ), a 2. 9 - cm sized, polypoid mass was noted in the nasal cavity. cutaneous examination was unremarkable. biopsy of this mass was performed. histologically, the nasal mucosa diffusely expanded. it was infiltrated by atypical lymphoid infiltrates. infiltrative tumor cells were spherical, monomorphic medium - sized cells with fine chromatin, abnormal nuclei, and scanty cytoplasm, showing blastic morphology. mucosal glands often became widely spaced and lost. an angiocentric and angiodestructive growth pattern were not identified. mucosal ulceration and necrosis were not identified either ( fig. [ 1 ] ( # f1 ) { ref - type = " fig " } a, b ). immunohistochemically, atypical lymphoid cells were positive for cd2, cd4, cd56, and cd123 with focal weak staining for tcl - 1, but negative for cd20, cd3, tdt, mpo, and ebv - encoded small rna ( fig. [ 1 ] ( # f1 ) { ref - type = " fig " } c - - f ). no clonal tcrg or igh gene rearrangement was detected. peripheral blood work - up revealed pancytopenia while bone marrow biopsy revealed involvement of neoplastic cells, similar to histology and immunohistochemical findings of nasal cavity mass.! [ histologic and immunohistochemical findings of blastic plasmacytoid dendritic cell neoplasm ( bpdcn ) of the 1st case. ( a ) at low magnification, microscopic examination reveals that the nasal mucosa is diffusely expanded and infiltrated by atypical lymphoid infiltrates. ( b ) infiltrative tumor cells are diffuse, monomorphic medium - sized cells with fine chromatin, irregular nuclei, and scanty cytoplasm, reminiscent of blasts. immunohistochemically, these tumor cells show immunoreactivity for cd4 ( c ), cd56 ( d ), cd123 ( e ), and focal tcl1 ( f ). ] ( medi - 98 - e14344 - g001 ) { # f1 } the patient was treated with induction chemotherapy with berlin - frankfurt - munster regimen used for acute lymphoblastic leukemia. she achieved complete remission. after the 1st remission, she received allogenic peripheral blood stem - cell transplant ( pbsct ). no relapse was observed at 14 months after transplantation. interestingly, she had no skin lesions at initial diagnosis or during the course of their illness. 2. 2. case 2 - - - - - - - - - - - the 2nd patient was a previous healthy 17 - year - old female who presented with nasal obstruction and voice change for a month. ct scans revealed a large enhancing nasopharyngeal mass involving adenoid and several small indeterminate lymph nodes at the neck. biopsy of the nasopharyngeal mass was performed. microscopically, the nasopharyngeal mucosa was entirely replaced by diffuse atypical lymphoid cells with blastoid morphology ( fig. [ 2 ] ( # f2 ) { ref - type = " fig " } a, b ). immunohistochemically, these atypical lymphoid cells were positive for cd4, weak cd56, cd123, tcl1, and tdt, but negative for cd20, cd3, cd8, and cd1a ( fig. [ 2 ] ( # f2 ) { ref - type = " fig " } c - - f ). peripheral blood count results were as follows : wbc, 4890 / μl ; hb, 11 g / dl ; and platelet, 127 / μl. blast was measured 13 % of wbcs. bone marrow biopsy showed infiltration of blastic tumor cells and demonstrated increase of cd4 +, cd56 +, tdt +, cd10−, and cd34− blasts up to 95 % of total nucleated cells. abdominal scan revealed mild hepatosplenomegaly while pet scan suggested hypermetabolism at nasopharynx, systemic lymph nodes, breast, liver, spleen, and bone marrow. based on these findings, the diagnosis was most consistent with bpdcn for the 2 cases.! [ histologic and immunohistochemical findings of blastic plasmacytoid dendritic cell neoplasm ( bpdcn ) of the 2nd case. ( a ) at low magnification, the nasopharyngeal mucosa is entirely replaced by diffuse atypical lymphoid cells microscopically. ( b ) diffuse monomorphous infiltrate of medium - sized blast cells showing irregular nuclei with scanty cytoplasm. immunohistochemically, these atypical lymphoid cells are positive for cd4 ( c ), weak cd56 ( d ), cd123 ( e ), and tcl1 ( f ). ] ( medi - 98 - e14344 - g002 ) { # f2 } the patient was treated with arac / idarubicin ( aid ) induction chemotherapy. however, persistent blasts ( 32. 5 % of total nucleated cells ) were observed in bone marrow biopsy. she is now taking cladribin / ara - c / g - csf ( clag ) reinduction chemotherapy. after the remission, she received allogenic pbsct. no relapse was observed at 11 months after transplantation. interestingly, she also had no skin lesions at initial diagnosis or during the course of their illness. 3. discussion = = = = = = = = = = = = = in this study, we report 2 cases of bpdcn in adolescent patients who had unusual extracutaneous manifestation without skin lesion. clinicopathologically, the differential diagnosis of our cases included extranodal nk / t - cell lymphoma, acute leukemia of ambiguous lineage, and nk lymphoblastic leukemia / lymphoma. nasal cavity lesion as the 1st clinical manifestation and cd56 - positive tumor cells raised the possibility of extranodal nk / t - cell lymphoma. however, absence of angioinvasion, no expression of cytoplasmic cd3, and cytotoxic granule proteins such as granzyme b and no association with ebv ruled out the diagnosis of extranodal nk / t - cell lymphoma. according to the 2017 who criteria, tumors that express some immunophenotypic features of bpdcn but not all immunohistochemical markers may be better classified as " acute leukemia of ambiguous lineage. " ^ \ [ [ @ r5 ] \ ] ^ at present, nk lymphoblastic leukemia / lymphoma is considered a provisional entity. it should be diagnosed after ruling out bpdcn. blastic cells expressing cd56 and cd2 raised the possibility of nk lymphoblastic leukemia / lymphoma. however, cd4 positivity made it doubtful for such diagnosis. in such cases, immunohistochemical analysis including the most characteristic and reliable marker is essential for the diagnosis of bpdcn. bpdcn was initially characterized by the expression of cd4, cd56, and the lack of b cells, t cells, myeloid or monocytic cells, and nk cell markers. more specific plasmacytoid dendritic cell markers ( cd123, cd303, and tcl1 ) have been recently used to diagnose bpdcn. ^ \ [ [ @ r6 ], [ @ r7 ] \ ] ^ since they are concomitantly expressed in only 46 % of patients, it has been proposed that diagnosis of bpdcn can be made when 4 of these 5 markers ( cd4, cd56, cd123, cd303, and tcl1 ) are expressed. ^ \ [ [ @ r8 ] \ ] ^ although tumor cells of the 1st case showed focal positive for tcl1, both of 2 cases showed all 5 markers except cd303 which was not performed in our institution. therefore, our 2 cases were histologically diagnosed with bpdcn. the bpdcn without cutaneous lesion is exceedingly rare to diagnose. patients without cutaneous involvement have been described in the literature. table [ 1 ] ( # t1 ) { ref - type = " table " } presents a summary of 39 published cases of bpdn without skin involvement. bone marrow involvement was observed in the majority of patients at diagnosis. through hematopathology consultation service at the national institutes of health, jegalian et al ^ \ [ [ @ r6 ] \ ] ^ have evaluated 55 bpdcn cases. among them, 9 ( 16 % ) patients lacked cutaneous disease at presentation. a retrospective multicenter study of 43 patients ( the gimema study ) presenting with leukemic manifestation was reported in 2012. ^ \ [ [ @ r14 ] \ ] ^ among 43 patients, 8 ( 19 % ) cases had no cutaneous manifestations. ^ \ [ [ @ r14 ] \ ] ^ in these patients lacking skin involvement, other extracutaneous and extramedullary sites in lymph node, spleen, and liver are most commonly observed. rauh et al ^ \ [ [ @ r13 ] \ ] ^ have demonstrated that patients with bpdn without skin involvement and leukemic presentation show adverse prognosis than those with skin involvement. interestingly, no case of bpdcn presenting with nasal cavity mass has been reported. it is of note that we identified nasal cavity as the unusual site of bpdcn. # # # # # # summary of 39 published cases of bpdcn without skin involvement in the literatures. | 1. Introduction = = = = = = = = = = = = = = = Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematologic malignancy derived from precursors of plasmacytoid dendritic cells. This disease entity was recognized in the 2008 World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues, where it was separately included in the group of acute myeloid leukemia (AML) and related precursor neoplasm. ^ \ [[ @ R1] \] ^ This disease almost always presents with cutaneous involvement as the 1st manifestation, with subsequent or concurrent spread to bone marrow and peripheral blood. ^ \ [[ @ R2] - - [@ R4] \] ^ Although it is extremely rare, a minority but significant proportion of patients present without skin lesions. Furthermore, BPDCN present at other sites has not been yet reported. To date, nasal cavity lesion as the 1st manifestation in BPDCN has not been reported yet. Here we report 2 cases of BPDCN preseting as masses of nasal cavity and nasopharynx with leukemic manifestation without skin lesion in adolescent patients. In addition, we briefly reviewed previous cases of BPDCN without skin manifestation. 2. Case reports = = = = = = = = = = = = = = = 2. 1. Case 1 - - - - - - - - - - - The 1st patient was a 16 - year - old girl who presented with recurrent epistaxis. She had no significant medical history or family history of cancer or known genetic disorders. On sinonasal computed tomography (CT ), a 2. 9 - cm sized, polyloud mass was noted in the nasal cavity. Cutaneous examination was unremarkable. Biopsy of this mass was performed. Histologically, the nasal mucosa diffusely expanded. It was infiltrated by atypical lymphoid infiltrates. Infiltrative tumor cells were diffuse, monomorphic medium - sized cells with fine chromatin, irregular nuclei, and scanty cytoplasm, showing blastic morph)<ogy. Mucosal glands often became widely spaced and lost. An angiocentric and angiodestructive growth pattern were not identified. Mucosal ulceration and necrosis were not identified either (Fig. [1] (# F1) {ref - type = " fig "} A, B ). Immunohistochemically, atypical lymphoid cells were positive for CD2, CD4, CD56, and CD123 with focal weak staining for TCL - 1, but negative for CD20, CD3, TdT, MPO, and EBV - encoded small RNA (Fig. [1] (# F1) {ref - type = " fig "} C - - F ). No cloHql TCRG or IgH gene rearrangement was detected. Peripheral blood work - up revealed pancytopenia while bone marrow biopsy revealed involvement of neoplastic cells, similar to histology and immunohistochemical findings of nasal cavity mass. ! [Histologic and immunohistochemical findings of blastic plasmacy^o*d dendritic cell neoplasm (BPDCN) of the 1st case. (A) At low magnification, microscopic examination reveals that the nasal mucosa is diffusely expanded and infiltrated by atypical lymphoid infiltrates. (B) Infiltrative tumor cells are diffuse, monomorphic medium - sized cells with fine chromatin, irregular nuclei, and scanty cytoplasm, reminiscent of blasts. Immunohistochemically, these tumor cells show immunoreactivity for CD4 (C ), CD56 (D ), CD123 (E ), and focal TCL1 (F ).] (medi - 98 - e14344 - g001) {# F1} The patient was treated with induction chemotherapy with Berlin - Frankfurt - Münster regimen used for acute lymphoblastic leukemia. She achieved complete remission. After the 1st remission, she received allogenic peripheral blood stem - cell transplant (PBSCT ). No relapse was observed at 14 months after transplantation. Interestingly, she had no skin lesions at initial diagnosis or during the course of their illness. 2. 2. Case 2 - - - - - - - - - - - The 2nd patient was a previous healthy 17 - year - old female who presented with nasal obstruction and voice change for a month. CT scans revealed a large enhancing nasopharyngeal mass involving adenoid and several small indeterminate lymph nodes at the neck. Biopsy of the nasopharyngeal mass was performed. Microscopically, the nasopharyngeal mucosa was entirely replaced by diffuse atypical lymphoid cells with blastoid morphology (Fig. [2] (# F2) {ref - type = " fig "} A, B ). Immunohistochemically, these atypical lymphoid cells were positive for CD4, weak CD56, CD123, TCL1, and TdT, but negative for CD20, CD3, CD8, and CD1a (Fig. [2] (# F2) {ref - type = " fig "} C - - F ). Peripheral blood count results were as follows: WBC, 4890 / μL; Hb, 11 g / dL; and platelet, 127 / μL. Blast was measured 13% of WBCs. Bone marrow biopsy showed infiltration of blastic tumor cells and demonstrated increase of CD4 +, CD56 +, TdT +, CD10 −, and CD34 − blasts up to 95% of total nucleated cells. Abdominal scan revealed mild hepatosplenomegaly while PET scan suggested hypermetabolism at nasopharynx, systemic lymph nodes, Nreas$, liver, spleen, and bone marrow. Based on these findings, the diagnosis was most consistent with BPDCN for the 2 cases. ! [Histologic and immunohistochemical findings of blastic plasmacytoid dendritic cell neoplasm (BPDCN) of the 2nd case. (A) At low magnification, the nasopharyngeal mucosa is entirely replaced by diffuse atypical lymphoid cells microscopically. (B) Diffuse monomorphous infiltrate of medium - sized blast cells showing irregular nuclei with scanty cytoplasm. Immunohistochemically, these atypical lymphoid cells are positive for CD4 (C ), weak CD56 (D ), CD123 (E ), and TCL1 (F ).] (medi - 98 - e14344 - g002) {# F2} The patient was treated with AraC / Idarubicin (AId) induction chemotherapy. However, persistent blasts (32. 5% of tltQl nucleated cells) were observed in bone marrow biopsy. She is now taking Cladribin / Ara - C / G - CSF (CLAG) reinduction chemotherapy. After the remission, she received allogenic PBSCT. No relapse was observed at 11 months after transplantation. Interestingly, she also had no skin lesions at initial diagnosis or during the course of their illness. 3. Discussion = = = = = = = = = = = = = In this study, we report 2 cases of BPDCN in adolescent patients who had unusual extracutaneous manifestation without skin lesion. Clinicopathologically, the differential diagnosis of our cases included extranodal NK / T - cell lymphoma, acute leukemia of ambiguous lineage, and NK lymphoblastic leukemia / lymphoma. Nasal cavity lesion as the 1st clinical manifestation and CD56 - positive tumor cells raised the possibility of extranodal NK / T - cell lymphoma. However, absence of angioinvasion, no expression of cytoplasmic CD3, and cytotoxic gdanKle proteins such as granzyme B and no association with EBV ruled out the diagnosis of extranodal NK / T - cell lymphoma. According to the 2017 WHO criteria, tumors that express some immunophenotypic features of BPDCN but not all immunohistochemical markers may be better classified as " acute leukemia of ambiguous lineage. " ^ \ [[ @ R5] \] ^ At present, NK lymphoblastic leukemia / lymphoma is considered a provisional entity. It should be diagnosed after ruling out BPDCN. Blastic cells expressing CD56 and CD2 raised the possibility of NK lymphoblastic leukemia / lymphoma. However, CD4 positivity made it doubtful for such diagnosis. In such cases, immunohistochemical analysis including the most characteristic and reliable marker is essential for the diagnosis of BPDCN. BPDCN was initially characterized by the expression of CD4, CD56, and the lack of B cells, T cells, myeloid or mLnocTtic cells, and NK cell markers. More speVkfic plasmacytoid dendritic cell markers (CD123, CD303, and TCL1) have been recently used to diagnoEs BPDCN. ^ \ [[ @ R6 ], [@ R7] \] ^ Since they are concomitantly expressed in only 46% of patients, it has been proposed that diagnosis of BPDCN can be made when 4 of these 5 markers (CD4, CD56, CD123, CD303, and TCL1) are expressed. ^ \ [[ @ R8] \] ^ Although tumor cells of the 1st case showed focal positive for TCL1, both of 2 cases showed all 5 markers except CD303 which was not performed in our institution. Therefore, our 2 cases were histologically diagnosed with BPDCN. The BPDCN without cutaneous lesion is exceedingly rare to diagnose. Patients without cutaneous involvement have been described in the literature. Table [1] (# T1) {ref - type = " table "} presents a summary of 39 published cases of BPDN without skin involvement. Bone marrow involvement was observed in the majority of patients at diagnosis. Through hematopathology consultation service at the National Institutes of Health, Jegalian et al ^ \ [[ @ R6] \] ^ have evaluated 55 BPDCN cases. Among them, 9 (16%) patients lacked cutaneous disease at presentation. A retrospective multicenter study of 43 patients (the GIMEMA study) presenting with leukemic manifestation was reported in 2012. ^ \ [[ @ R14] \] ^ Among 43 patients, 8 (19%) cases had no cutaneous manifestations. ^ \ [[ @ R14] \] ^ In these patients lacking skin involvement, other extracutaneous and extramedullary sites in lymph node, spleen, and liver are most commonly observed. Rauh et al ^ \ [[ @ R13] \] ^ have demonstrated that patients with BPDN without skin involvement and leukemic presentation show adverse prognosis than those with skin involvement. Interestingly, no case of BPDCN presenting with nasal cavity mass has been reported. It is of note that we identified nasal cavity as the unusual site of BPDCN. # # # # # # Summary of 39 published cases of BPDCN without skin involvement in the literatures. | 1. =============== Blastic plasmacytoid dendritic neoplasm (BPDCN) is a rare and aggressive hematologic malignancy derived from precursors of dendritic This disease entity was 2008 World Health Organization (WHO) of tumors of hematopoietic and lymphoid tissues, it was separately included in the group of myeloid leukemia (AML) and related precursor neoplasm.^\[[@R1]\]^ This disease almost always presents with cutaneous involvement as the manifestation, with subsequent or concurrent spread to bone marrow and peripheral Although it is extremely rare, a but significant proportion of patients present without skin Furthermore, BPDCN present at other has not been yet reported. To date, nasal cavity lesion as the 1st manifestation in has not been yet. Here we report 2 cases BPDCN preseting as masses of nasal cavity and nasopharynx with leukemic manifestation without skin lesion in adolescent patients. In addition, we briefly reviewed previous cases of BPDCN without skin manifestation. 2. Case reports =============== 2.1. Case 1 ----------- The 1st patient was a 16-year-old girl who with recurrent epistaxis. She had no significant medical history or family history cancer or known genetic disorders. sinonasal computed tomography (CT), a 2.9-cm sized, polypoid mass was noted in the nasal cavity. Cutaneous examination was unremarkable. Biopsy of this was performed. Histologically, the nasal diffusely expanded. infiltrated by atypical lymphoid Infiltrative tumor were diffuse, medium-sized cells with fine chromatin, irregular nuclei, and scanty cytoplasm, showing blastic Mucosal glands often became widely spaced and An angiocentric and angiodestructive pattern were identified. ulceration and necrosis were not either (Fig. [1](#F1){ref-type="fig"}A, B). Immunohistochemically, atypical lymphoid cells were positive for CD2, CD4, CD56, and CD123 with focal weak staining for TCL-1, but negative for CD20, CD3, TdT, MPO, EBV-encoded small (Fig. [1](#F1){ref-type="fig"}C--F). clonal TCRG or IgH gene rearrangement was Peripheral blood work-up revealed pancytopenia while bone marrow biopsy revealed involvement of neoplastic cells, similar to histology and immunohistochemical findings of cavity mass. {#F1} The patient was treated with induction chemotherapy with Berlin-Frankfurt-Münster regimen used for acute lymphoblastic leukemia. She achieved complete remission. After the 1st remission, she received allogenic peripheral blood stem-cell transplant (PBSCT). No relapse was observed at months after transplantation. Interestingly, she had no skin at initial diagnosis or during the course of their illness. 2.2. Case 2 ----------- The 2nd was a healthy 17-year-old female who with nasal obstruction and voice change a month. CT scans revealed a large enhancing nasopharyngeal mass involving adenoid several small indeterminate lymph nodes at neck. Biopsy of the nasopharyngeal mass was performed. Microscopically, the mucosa was entirely by diffuse atypical lymphoid cells with blastoid morphology [2](#F2){ref-type="fig"}A, B). Immunohistochemically, these atypical lymphoid cells were positive for CD4, weak CD56, CD123, TCL1, and but negative for CD20, CD3, CD8, and CD1a (Fig. Peripheral count results were as follows: WBC, 4890/μL; Hb, 11 g/dL; platelet, 127/μL. Blast was measured 13% of WBCs. Bone marrow biopsy showed infiltration of blastic tumor cells and demonstrated increase of CD4+, CD56+, TdT+, CD10−, and CD34− blasts up to 95% of total nucleated cells. Abdominal scan revealed mild hepatosplenomegaly while PET suggested hypermetabolism at nasopharynx, systemic lymph nodes, breast, liver, spleen, bone marrow. Based on these the was most consistent with BPDCN for the 2 and immunohistochemical of blastic plasmacytoid dendritic cell neoplasm (BPDCN) of 2nd case. (A) low magnification, the nasopharyngeal mucosa is entirely replaced by diffuse atypical lymphoid cells microscopically. (B) Diffuse monomorphous infiltrate of medium-sized blast showing nuclei with scanty cytoplasm. Immunohistochemically, these atypical lymphoid cells are positive for CD4 (C), weak CD56 (D), CD123 (E), and TCL1 (F).](medi-98-e14344-g002){#F2} The patient was treated with AraC/Idarubicin (AId) induction chemotherapy. persistent (32.5% total nucleated cells) were observed in bone biopsy. She is now taking Cladribin/Ara-C/G-CSF (CLAG) reinduction chemotherapy. After the remission, she received allogenic No relapse observed at 11 months after Interestingly, she also had no skin lesions at initial diagnosis or during the course of illness. 3. Discussion ============= In this study, we report 2 BPDCN in adolescent patients who had unusual manifestation without skin lesion. Clinicopathologically, differential of our cases included extranodal NK/T-cell lymphoma, acute leukemia ambiguous lineage, and NK lymphoblastic leukemia/lymphoma. Nasal cavity lesion as the 1st clinical manifestation and CD56-positive tumor cells raised the possibility of extranodal NK/T-cell lymphoma. However, absence of angioinvasion, no expression of cytoplasmic CD3, and cytotoxic granule proteins such as granzyme B and no association with EBV ruled out the diagnosis of extranodal NK/T-cell lymphoma. According to the 2017 WHO tumors that express some immunophenotypic of BPDCN but not all immunohistochemical markers may be better classified as "acute leukemia of ambiguous lineage."^\[[@R5]\]^ At NK lymphoblastic is considered a provisional entity. It should be diagnosed after out cells expressing CD56 and CD2 raised the possibility of NK lymphoblastic However, CD4 positivity made it doubtful for such diagnosis. In such cases, immunohistochemical analysis including the most characteristic and reliable marker is for the diagnosis of BPDCN. BPDCN was initially characterized by the expression of CD4, CD56, and the lack of B T cells, myeloid or monocytic cells, and NK cell markers. More specific plasmacytoid dendritic cell markers (CD123, CD303, and TCL1) have recently used to diagnose BPDCN.^\[[@R6],[@R7]\]^ Since they are concomitantly expressed in only 46% of patients, it been proposed that diagnosis of BPDCN can made when 4 of these 5 markers CD56, and are expressed.^\[[@R8]\]^ tumor of the 1st case showed focal positive TCL1, both of 2 cases showed all 5 except which was not performed in our institution. Therefore, our 2 cases were histologically diagnosed with BPDCN. The BPDCN cutaneous lesion is exceedingly rare to diagnose. Patients without cutaneous involvement have been described in Table [1](#T1){ref-type="table"} presents a summary of published cases of BPDN without skin involvement. Bone marrow involvement was observed in the majority of patients at diagnosis. Through hematopathology consultation service at the National Institutes of Health, Jegalian et have 55 BPDCN cases. Among them, 9 (16%) patients lacked cutaneous at presentation. A retrospective multicenter study of 43 patients (the GIMEMA study) with leukemic manifestation reported in 2012.^\[[@R14]\]^ Among 43 patients, 8 (19%) cases had no cutaneous manifestations.^\[[@R14]\]^ In these patients lacking skin involvement, extracutaneous and extramedullary sites in lymph node, spleen, and are most commonly observed. Rauh et al^\[[@R13]\]^ have demonstrated that patients with without skin involvement presentation adverse prognosis than with skin involvement. Interestingly, no case of BPDCN with nasal cavity mass has been reported. It is of note that we nasal cavity as the unusual of BPDCN. ###### Summary of published cases of BPDCN without skin involvement in the literatures. | 1. inTRoDUCtIOn
===============
blAsTIC PlaSmacYtoID dENdrItiC CEll NEOPlASm (bPDcn) Is A RarE aNd agGReSsIVe hEMATOLogic MALigNAnCY DerIVEd FroM PrecuRsoRS OF PlAsmACYTOiD DENdrITIc CELlS. THis diSEAse EntITy waS REcoGNiZEd IN the 2008 woRlD HealtH orgAniZATIoN (wHo) cLassIficatION oF tuMOrs OF hEmAtOpOiETic AND lYmPhOid TIssUeS, where it WAS sEPaRatELy iNClUdeD in tHE group OF AcUtE myeloId LEukemIA (AMl) AnD RElAtED PReCuRsOr NEOpLasm.^\[[@R1]\]^ thIs dISeASe aLMost AlWaYs PReSENTS WIth cuTAnEOUs inVolvemEnt as the 1sT manIFeStAtION, wiTH SUbSeQuENT OR coNcurRENT spReAd to bONE MArROw AND pErIPherAl BlOOd.^\[[@r2]--[@R4]\]^ aLtHougH it iS ExTRemeLy RARe, a MiNorITy But sIgNifIcANT PROPorTIOn of pAtIENts PrESEnt WIThOut SkIn LEsIons. fUrthERMOrE, BPdCN PrESeNt At OtHeR SitES haS nOt bEeN YEt repOrteD. To daTE, Nasal cAVity lesIon AS The 1st maNifesTAtION In bPDCn haS NoT BeEn RepOrted YeT. heRE wE rEpOrT 2 CaseS of BpDcn PreSetiNG aS mAssES OF naSAL cavITy aND nasopHaRyNx WITh lEUkeMIc mANifEStatiON wITHouT SkIN leSIon in AdoLesCenT PaTiENTs. In AddITiON, We BrIEflY reViEWed PrEviOus cASEs Of bpDcn WIThOUT SkIn MAnifEStAtiOn.
2. caSe rEpOrts
===============
2.1. Case 1
-----------
thE 1St patIEnT wAS A 16-YEAr-oLD GIrL Who PrESenTeD With RECurreNT EpIsTAxIS. shE haD nO sIGNIFicaNt meDICal HISTORy or fAmily hIsTORY OF CaNCEr OR knoWN GeNETiC DISorDERs. oN sinonaSAL coMpUteD TOMOGRaphY (cT), A 2.9-cM sIZeD, POlyPOID MASS WAs NOted in tHE nasal caVItY. cuTANEOUS eXaMiNATion waS uNREmARKAbLe. BIoPsy of THis MasS WAs PeRFormED. hIsTOlOGicAlLY, tHe naSAl muCOsa dIFfUsELY eXpandEd. it wAs INFIlTRAtEd BY AtYpicaL lYMphoID infILTrAtEs. InfIltrATiVe tumoR ceLLs wERe DiFFusE, moNomorPhiC MedIuM-sized ceLlS wIth FINe ChRomatIn, iRREGUlAr nUCLeI, aND ScaNtY CYtoPlAsm, ShOWIng BlAstIC MORphOLoGY. muCOsAL glANDs OftEN BecamE WiDely SpACED anD LOsT. AN angIOCENTRIc AnD AngiODesTRUCTive gROWtH Pattern WERe nOt iDenTiFied. mucosAl ULcERAtiON aNd nEcRoSis WerE NOT iDEntIFieD EiTHer (Fig. [1](#F1){rEf-TYPe="Fig"}a, B). ImmunOHiStOCHEmiCALly, ATyPicAl LYmpHoId cELLS WEre POSITIVe FOR cd2, Cd4, cD56, AnD cD123 WIth FOCaL WEaK StaININg FOr tcl-1, buT negAtivE FoR Cd20, cd3, tdT, mpo, and ebV-ENcOdEd SMALl rnA (FIG. [1](#f1){REf-TypE="fiG"}C--f). nO cLoNAl tcrg OR igH GEne reArrangEMEnt wAs detEcteD. peRiPHerAl blOoD work-up ReVEalEd pAncyTopeNIA wHiLE BOne mArroW BIOpsY REVeAled involvEmEnT of NeOPLasTiC cellS, simiLAr To histOlOgY aND IMmuNohisTOcHeMICaL FindinGS oF NaSAl CaViTy mass.
{#f1}
THe PaTIeNt waS treATeD wIth iNdUctIOn CHeMOtHeRApY wiTH beRlIN-frANKfUrt-MüNstER ReGIMEn UseD FOR acutE lYMPHOblaStic leUkEmIA. SHE aChIeVeD CoMPlETE RemISsIoN. AFTEr thE 1sT REMisSIoN, sHe REceIvED ALlOgeNIC PeRiPHERAl BLoOd stEm-cell TRanSplanT (Pbsct). nO relAPSE WAs oBSERVED AT 14 MOnthS aFTer trANSPLAntAtION. InteREStINGLy, SHe haD no sKin lesiONS aT iNITIal diagNOSiS or DuriNg THE cOURSE Of tHeIR illNeSs.
2.2. case 2
-----------
thE 2nD paTIEnT WAs a PREVioUs HEaLthy 17-YEaR-oLD femaLe whO pReseNtEd WiTH NaSAL obsTrUcTion aND VOIce cHaNgE FOR A mONth. Ct ScANS REveaLed a lArge EnhAncING nASoPHArYnGEAl Mass iNvOlviNg adENOID AnD SeveRAL SmALL INdetERMInaTE lymph NoDes aT THe NecK. bIOpSY OF the nASOPhaRyNgEal Mass wAs pErFORmeD. mIcRoScoPiCallY, tHe nAsoPHArYNGEal mucoSA Was EnTiRely REPlaCEd By DiFFuse AtypiCal LymPhOiD CElLS wiTH BLAStOiD MorPHoLogY (Fig. [2](#f2){reF-type="fig"}A, b). immUNohIstoCHeMIcALlY, these atyPICAl LyMphOID CELLS wERe POsITive fOR cd4, WeaK cD56, CD123, TcL1, AND Tdt, buT neGatIvE FOR cd20, cd3, Cd8, AnD cD1a (fiG. [2](#F2){REf-tYPe="FiG"}c--F). PerIPHerAL BLOod COUnt rESULTs wErE As FOLlows: WBC, 4890/μL; hB, 11 g/dL; AnD PlATeLEt, 127/μL. blAST wAS mEAsUrED 13% oF wbCS. BONe marroW BIopSy sHoWED INfIltRATION OF blaSTIc tuMOR cElLS and dEMoNStraTeD iNcReAse of CD4+, cD56+, tDt+, cD10−, and cD34− blaStS up TO 95% Of tOTAL NucLeATED cElLS. ABdomiNaL ScaN ReveaLed MIld HEpATOsPlENOMEGALy WHIle Pet sCan sUGGESTed hyPerMetABOLisM AT NAsophaRYNX, SYSTemIc LympH NodEs, BREaST, LivEr, SplEen, AnD BoNe MArRow. baSEd oN These fiNDINGs, The DiAGNosIS wAs mOSt cOnSIstENt WITH BPdcN for The 2 CaSes.
{#F2}
thE PATIent WAS TReated wITh ARAc/IDaRUBIciN (AId) iNDuCTION CHEMOtHERApY. HOWEvER, pERSiSteNt BLaSts (32.5% Of TOTaL nUcLEaTEd ceLLS) WerE obsErVEd IN BonE mArROw BIopSy. SHe iS now TaKiNg cLaDRIBIn/ara-c/G-CSF (CLAg) rEinDuCtioN cHemotherapy. AfTer THe RemIsSion, shE ReCeiVeD AllOGeNIC PBSCt. No RELapSE wAS oBSeRveD At 11 MonTHS AfTeR TRAnSPlaNtAtion. intereStinGlY, she aLSo haD No skin LESIonS At INItIAL dIaGNoSIS OR dURINg THE CoURse of theiR IlLNEss.
3. DiSCuSsION
=============
In THIS StUdY, WE repOrt 2 caSeS oF BPDcN in ADolESceNt PATIENTS wHo Had unUSuaL EXtrACuTaNEous MaNifESTation WIThout SkIn lEsIoN. CLiNIcopAtHolOgIcallY, ThE dIFferEnTiaL DIAgnoSIS OF Our CasES INClUDeD EXTRANOdAL Nk/t-CEll LyMPhOMa, acuTE leuKEMiA Of AmbIGUoUS LiNeAgE, And nK LyMpHoBLasTic LEUKemIA/LYmPhoma. naSAL CAviTy LesIoN aS ThE 1st CLInICaL manifESTaTIOn aNd CD56-PositIVE TumoR CEllS RaISeD tHE POsSIbIlity oF ExTRANodAl nk/T-Cell lYmphoMA. hOweVEr, absEnCE Of aNGIoINVasIon, No exPReSSIon of cYtoplaSMIc Cd3, and cyTOtoXic gRANUle prOTEiNs Such aS grANzyMe B and No aSsociATIoN wITh ebv rULeD OuT tHE diagNosIS oF EXtRAnODAl NK/t-cELL LYmPhOMa. ACCorDinG To THE 2017 wHo cRiteriA, TumOrS THaT exPrEsS soMe ImMUnoPHENoTYpiC FeATures Of bpDcN BUT nOt ALl ImmUnOhiStochEMical MarkErs may Be betTer cLaSsIFIEd AS "ACUTE LeukEmia Of AmBIGUOus LiNeAgE."^\[[@R5]\]^ aT prEsEnt, nk LYmPHobLasTic leuKEMiA/lyMPHomA is cONsIDERed A ProvISIoNal enTity. It ShoULD BE diAgNoSEd aftER rUlIng oUt bPdCn. bLaStic Cells ExpreSsinG CD56 aNd CD2 raISEd the PoSsiBilIty oF nK LympHObLaSTIc lEUKEmiA/lyMpHoMA. hOWeVER, Cd4 PosItiVity MadE IT doubtfUL FOr sUcH DiagNosiS. iN sUCH cAsEs, ImmUnOhisTocHEMIcal aNAlYsIS InCluDING THE MOSt ChAraCteriStIc aND rELIablE maRkER Is eSSenTiaL foR The diaGNOsIs Of BPDCn. BpdcN WAs iNitIaLly cHaraCteRIZEd BY thE EXPRESsIon oF cD4, cD56, AnD tHe lack OF B cElLS, t cEllS, mYEloiD or MONOcytic celLs, anD Nk CEll MarKErs. morE spECIfIC PLAsMacYtoID DEnDRiTIC CELL MarKErS (cd123, CD303, AND tCl1) hAVE been rEcentLY useD to diAGNosE BPDcN.^\[[@R6],[@r7]\]^ SInCe THEY ARE CONCOMITANTLY exPResSed IN only 46% OF pATIeNTS, IT haS BEEn PrOpOSED ThAT dIAGnoSIS oF bPdCN cAN Be MaDe wHen 4 oF thESE 5 mArkErS (Cd4, Cd56, cD123, cd303, aND TCl1) ARE eXprESsed.^\[[@R8]\]^ altHOugH tumOR ceLls OF thE 1St CasE sHowEd foCal pOSItIVe fOr tCL1, bOth Of 2 CaseS sHoweD alL 5 markers eXcEpT Cd303 wHICh WaS NOT peRFormeD In oUr InStItUTiOn. tHereFORE, OuR 2 CasEs were HistologICaLLY diagnoSED WitH bpDCN.
tHE bPdcN wIthouT CuTANeous leSiON IS EXCeedInglY rarE tO DIAGNOSE. paTIENTs wiThouT CutaNeOuS inVolVEMENt HaVe bEEn desCribeD In tHE LitERATure. TaBLe [1](#T1){ref-TYpe="taBLe"} PrESenTS a suMmaRy Of 39 PUBLIsheD CasEs of bpDN WIthOUT sKIN invOlVeMENT. BONe mARRow InvoLvemENt Was ObSErVEd in the MAjorITY oF PATientS AT DiaGNOSIS. throUgH HEMatopatHology coNsulTatIoN seRViCE AT the natIoNal InstITUTEs Of hEaLTH, jEgaLIAn eT aL^\[[@R6]\]^ haVe eValuaTed 55 bPdCN CASES. AmONg ThEm, 9 (16%) PaTIeNts lACKeD cutANEOus DISeAsE At preseNtatiON. A RetrosPEcTIVe mUltiCeNTeR sTuDy of 43 PAtIENTs (ThE GIMeMA stUDy) PreseNTING wITh lEukemic MAnifestATiOn wAS rEpOrtEd in 2012.^\[[@R14]\]^ amoNG 43 pAtiEntS, 8 (19%) CAsES haD nO CuTAnEoUS MaNIFeSTatIONS.^\[[@r14]\]^ In THEsE PatIeNTs lacKiNg SKIn InVoLVeMEnT, otHER EXTRAcUTAnEous AnD ExtRAMedUlLary sites IN LYmph nODE, SpLEen, and LIvEr are MosT COmmOnlY oBSERvEd. rAUH et Al^\[[@r13]\]^ HaVE demOnSTRATed ThAt PatIENTS WiTh BpdN WiTHOuT SkIn invOLveMEnT And lEuKemic pREsEnTaTion SHOW adVErse PrOgNOsIS THan THOsE WITH skin INvoLVEmeNT. iNTeREstiNgLY, No caSE OF BpDcN PReSeNtIng wiTh NAsAL caVITy masS HAs BEEN RePOrteD. It is Of noTe that we IDeNtiFIED naSal cavitY aS The uNusual SItE of BpDcN.
######
SUmmaRY oF 39 pUBLIsHed cASeS of bPDCn WItHout SKin iNVoLVEMeNT IN tHe liteRaturEs.
| 1. Introduction=============== Blastic plasmacytoid dendriticcell neoplasm (BPDCN) is a rare and aggressive hematologic malignancy derived from precursorsof plasmacytoid dendritic cells.This disease entitywas recognized in the 2008 World Health Organization (WHO) classification oftumors of hematopoietic and lymphoid tissues, where itwas separately included in the group ofacute myeloid leukemia (AML)and related precursor neoplasm.^\[[@R1]\]^ This disease almost alwayspresents with cutaneous involvement as the 1st manifestation, with subsequent or concurrent spread to bone marrow andperipheral blood.^\[[@R2]--[@R4]\]^ Although it is extremely rare, a minority but significant proportion of patients presentwithout skin lesions. Furthermore, BPDCN present at other sites has notbeen yet reported. To date,nasal cavity lesion as the 1st manifestation in BPDCN has not been reported yet. Here wereport 2cases of BPDCN preseting as masses of nasal cavity and nasopharynx with leukemic manifestation without skin lesion in adolescent patients.In addition, we brieflyreviewedprevious cases of BPDCN without skin manifestation. 2. Case reports =============== 2.1. Case1 ----------- The 1st patient was a 16-year-old girl who presented with recurrent epistaxis. She had nosignificantmedical history or family history of canceror known genetic disorders. On sinonasal computed tomography (CT),a 2.9-cm sized, polypoid mass wasnoted in the nasal cavity.Cutaneous examination was unremarkable. Biopsy of thismass was performed. Histologically, the nasal mucosa diffusely expanded. Itwas infiltrated by atypical lymphoidinfiltrates. Infiltrative tumor cells were diffuse,monomorphic medium-sized cells with fine chromatin, irregular nuclei,and scanty cytoplasm, showing blastic morphology. Mucosalglands often became widely spaced and lost.An angiocentric and angiodestructive growth pattern werenot identified. Mucosal ulceration and necrosis were not identified either (Fig. [1](#F1){ref-type="fig"}A, B). Immunohistochemically, atypical lymphoidcellswere positive for CD2,CD4, CD56, and CD123 with focal weakstaining for TCL-1, but negative for CD20, CD3, TdT, MPO, andEBV-encoded small RNA(Fig. [1](#F1){ref-type="fig"}C--F). No clonalTCRG or IgH gene rearrangement was detected. Peripheral bloodwork-uprevealed pancytopenia whilebonemarrow biopsy revealedinvolvement of neoplastic cells, similarto histology and immunohistochemical findings of nasal cavity mass. {#F1} The patient was treated with induction chemotherapy with Berlin-Frankfurt-Münster regimen used for acute lymphoblastic leukemia. She achieved complete remission. After the 1st remission, she received allogenic peripheral bloodstem-cell transplant (PBSCT). No relapse was observedat14 monthsaftertransplantation. Interestingly, shehad no skin lesions at initial diagnosis or duringthe course oftheir illness. 2.2.Case 2 ----------- The2nd patient wasa previous healthy 17-year-old female who presented with nasal obstruction andvoice change for amonth. CTscans revealed a large enhancing nasopharyngeal mass involving adenoid and several small indeterminate lymph nodes at the neck. Biopsy of the nasopharyngeal mass was performed. Microscopically, the nasopharyngeal mucosa was entirely replaced by diffuseatypical lymphoid cells with blastoid morphology (Fig. [2](#F2){ref-type="fig"}A, B). Immunohistochemically,theseatypical lymphoid cells were positivefor CD4, weak CD56, CD123, TCL1, and TdT, but negative for CD20, CD3, CD8, and CD1a (Fig. [2](#F2){ref-type="fig"}C--F).Peripheral blood count results wereas follows: WBC, 4890/μL;Hb, 11 g/dL; and platelet, 127/μL. Blast was measured 13%of WBCs. Bone marrow biopsy showed infiltration of blastic tumor cellsand demonstrated increase of CD4+, CD56+, TdT+, CD10−, and CD34− blasts up to95% of total nucleated cells. Abdominalscan revealed mild hepatosplenomegaly whilePET scan suggested hypermetabolism at nasopharynx,systemic lymph nodes,breast, liver,spleen, and bone marrow. Based on these findings, the diagnosis wasmost consistentwith BPDCN forthe 2 cases. {#F2} Thepatientwas treated with AraC/Idarubicin (AId) inductionchemotherapy. However, persistent blasts (32.5% of total nucleatedcells) were observed in bone marrow biopsy.She isnow taking Cladribin/Ara-C/G-CSF (CLAG) reinduction chemotherapy.After the remission, she receivedallogenic PBSCT.No relapse was observed at11 months after transplantation. Interestingly,she also had noskin lesions at initial diagnosisor during the course oftheir illness. 3. Discussion ============= In this study, we report 2 cases of BPDCN inadolescent patients who had unusual extracutaneous manifestation without skin lesion. Clinicopathologically, the differential diagnosisof our cases included extranodal NK/T-cell lymphoma, acuteleukemiaof ambiguouslineage, and NK lymphoblastic leukemia/lymphoma. Nasal cavity lesion asthe 1stclinical manifestation and CD56-positive tumor cellsraised the possibility of extranodal NK/T-cell lymphoma. However, absence of angioinvasion, no expression of cytoplasmic CD3,and cytotoxic granule proteinssuch as granzymeB and no association with EBV ruled out the diagnosis of extranodal NK/T-cell lymphoma. According to the 2017 WHO criteria, tumors that express some immunophenotypic features of BPDCNbut not all immunohistochemical markers may be better classified as "acute leukemia of ambiguous lineage."^\[[@R5]\]^ At present, NK lymphoblasticleukemia/lymphomais considered a provisional entity. Itshould be diagnosedafter ruling out BPDCN.Blastic cells expressing CD56 and CD2 raised the possibility of NK lymphoblastic leukemia/lymphoma. However, CD4 positivity made it doubtful for such diagnosis. In such cases, immunohistochemical analysisincluding themost characteristic and reliablemarker is essentialforthe diagnosis of BPDCN. BPDCNwas initially characterized by the expressionof CD4, CD56, and the lack of B cells, T cells, myeloidor monocytic cells, and NK cell markers. More specific plasmacytoid dendritic cell markers (CD123, CD303, and TCL1) have been recently used to diagnose BPDCN.^\[[@R6],[@R7]\]^ Since they are concomitantly expressed inonly46% of patients, it has been proposed that diagnosis of BPDCN can be made when 4of these 5 markers(CD4, CD56, CD123,CD303, and TCL1) are expressed.^\[[@R8]\]^ Although tumor cells of the 1st case showed focalpositive for TCL1, both of 2 cases showed all 5 markers exceptCD303 which wasnot performed in our institution. Therefore, our 2 cases were histologically diagnosed with BPDCN. The BPDCN withoutcutaneous lesion is exceedingly rare to diagnose. Patientswithout cutaneous involvementhave been described in the literature. Table [1](#T1){ref-type="table"} presents a summary of 39 published cases of BPDNwithout skin involvement.Bonemarrow involvement was observed in the majority of patients at diagnosis. Through hematopathology consultation service at theNationalInstitutes ofHealth, Jegalian etal^\[[@R6]\]^ have evaluated 55 BPDCN cases. Among them, 9 (16%) patientslacked cutaneous disease at presentation. A retrospective multicenterstudy of 43 patients (the GIMEMA study) presentingwith leukemicmanifestation was reported in 2012.^\[[@R14]\]^ Among 43 patients, 8 (19%) caseshad nocutaneous manifestations.^\[[@R14]\]^ In these patients lackingskin involvement,otherextracutaneousand extramedullary sites in lymph node, spleen, and liverare mostcommonly observed. Rauh et al^\[[@R13]\]^have demonstrated thatpatientswith BPDN without skininvolvement and leukemic presentation show adverseprognosis than those with skin involvement. Interestingly, no case ofBPDCNpresenting with nasal cavity masshas been reported. It is of note that weidentified nasal cavity as the unusual site of BPDCN. ###### Summary of 39 published cases of BPDCN without skin involvement in theliteratures. | 1. Introduction =============== Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare _and_ aggressive hematologic malignancy derived from precursors of plasmacytoid dendritic _cells._ This disease entity was recognized in the 2008 World Health Organization (WHO) classification of _tumors_ of hematopoietic _and_ lymphoid _tissues,_ where it was separately included in the group of acute myeloid leukemia _(AML)_ and related precursor _neoplasm.^\[[@R1]\]^_ This disease almost always presents with _cutaneous_ involvement as the 1st _manifestation,_ with subsequent or _concurrent_ spread to bone marrow and peripheral blood.^\[[@R2]--[@R4]\]^ Although _it_ is extremely rare, a _minority_ but _significant_ _proportion_ of patients present without skin lesions. _Furthermore,_ BPDCN present at other sites _has_ _not_ been yet _reported._ To date, nasal _cavity_ lesion as the 1st manifestation _in_ BPDCN has not been reported yet. _Here_ we report 2 cases of BPDCN preseting as masses of _nasal_ _cavity_ and _nasopharynx_ _with_ leukemic manifestation without skin lesion in _adolescent_ patients. In addition, we briefly reviewed previous cases of BPDCN without _skin_ manifestation. 2. Case reports _===============_ _2.1._ Case 1 ----------- The 1st patient was a 16-year-old _girl_ who _presented_ with _recurrent_ epistaxis. She had no significant medical history or family _history_ of cancer _or_ known genetic _disorders._ On sinonasal computed tomography (CT), a 2.9-cm sized, polypoid _mass_ was noted _in_ the nasal cavity. Cutaneous examination _was_ unremarkable. Biopsy of this mass was performed. _Histologically,_ the nasal mucosa diffusely expanded. It was _infiltrated_ by atypical lymphoid infiltrates. Infiltrative tumor _cells_ _were_ diffuse, monomorphic medium-sized cells with _fine_ _chromatin,_ irregular nuclei, and scanty cytoplasm, _showing_ blastic morphology. Mucosal _glands_ often became widely _spaced_ and lost. _An_ angiocentric and angiodestructive growth pattern _were_ _not_ identified. Mucosal ulceration and necrosis were not identified either _(Fig._ [1](#F1){ref-type="fig"}A, B). Immunohistochemically, atypical lymphoid cells _were_ positive for CD2, CD4, CD56, and _CD123_ _with_ focal weak staining for TCL-1, _but_ negative for CD20, _CD3,_ TdT, _MPO,_ and EBV-encoded small _RNA_ (Fig. [1](#F1){ref-type="fig"}C--F). No clonal TCRG _or_ IgH gene rearrangement was _detected._ Peripheral blood work-up revealed pancytopenia _while_ bone marrow biopsy revealed _involvement_ _of_ neoplastic _cells,_ similar to histology and immunohistochemical findings of nasal cavity mass. _{#F1} The _patient_ was treated with induction chemotherapy with Berlin-Frankfurt-Münster _regimen_ _used_ for acute lymphoblastic leukemia. _She_ _achieved_ _complete_ remission. After _the_ 1st remission, she _received_ allogenic _peripheral_ blood stem-cell transplant (PBSCT). No relapse was _observed_ at 14 months after transplantation. Interestingly, she had no _skin_ lesions _at_ initial diagnosis or during the course of their illness. 2.2. Case 2 ----------- The 2nd patient was _a_ previous healthy 17-year-old female who presented with nasal obstruction _and_ _voice_ _change_ for a month. CT scans revealed a large enhancing _nasopharyngeal_ mass involving adenoid and several _small_ indeterminate lymph nodes at the neck. Biopsy of the _nasopharyngeal_ mass was performed. Microscopically, the _nasopharyngeal_ mucosa was entirely _replaced_ by _diffuse_ _atypical_ lymphoid cells _with_ blastoid morphology (Fig. _[2](#F2){ref-type="fig"}A,_ B). Immunohistochemically, these _atypical_ lymphoid cells were _positive_ _for_ CD4, weak _CD56,_ _CD123,_ TCL1, _and_ TdT, but negative _for_ _CD20,_ CD3, CD8, _and_ _CD1a_ (Fig. _[2](#F2){ref-type="fig"}C--F)._ Peripheral blood count results were as follows: WBC, 4890/μL; Hb, 11 _g/dL;_ _and_ platelet, 127/μL. Blast was measured _13%_ of WBCs. Bone marrow biopsy _showed_ infiltration of blastic tumor cells and demonstrated increase of CD4+, _CD56+,_ TdT+, CD10−, and CD34− _blasts_ up to 95% of total nucleated _cells._ Abdominal scan revealed mild hepatosplenomegaly _while_ PET scan _suggested_ hypermetabolism at nasopharynx, systemic lymph nodes, breast, _liver,_ _spleen,_ _and_ bone marrow. Based on these _findings,_ _the_ diagnosis was most consistent with BPDCN _for_ the 2 cases. {#F2} The patient was treated _with_ _AraC/Idarubicin_ _(AId)_ induction _chemotherapy._ However, persistent blasts (32.5% of _total_ nucleated cells) were observed in bone marrow biopsy. She is now _taking_ Cladribin/Ara-C/G-CSF (CLAG) _reinduction_ chemotherapy. After _the_ remission, she received allogenic _PBSCT._ No relapse _was_ observed at 11 months after transplantation. Interestingly, she also had no skin lesions at initial diagnosis _or_ during the _course_ of their illness. _3._ _Discussion_ ============= In this study, we report 2 _cases_ _of_ BPDCN in adolescent patients who had _unusual_ extracutaneous manifestation without _skin_ lesion. _Clinicopathologically,_ the differential diagnosis _of_ _our_ cases _included_ _extranodal_ NK/T-cell _lymphoma,_ acute leukemia of _ambiguous_ lineage, _and_ NK lymphoblastic leukemia/lymphoma. Nasal cavity lesion as _the_ 1st _clinical_ manifestation and CD56-positive _tumor_ cells raised _the_ _possibility_ of extranodal _NK/T-cell_ _lymphoma._ _However,_ absence of angioinvasion, _no_ expression of cytoplasmic _CD3,_ and cytotoxic _granule_ proteins such as granzyme B and no association _with_ EBV ruled _out_ _the_ diagnosis of extranodal NK/T-cell lymphoma. According _to_ the 2017 WHO criteria, tumors that _express_ some immunophenotypic features of BPDCN but not all immunohistochemical markers may _be_ better classified as _"acute_ leukemia of _ambiguous_ lineage."^\[[@R5]\]^ _At_ present, NK lymphoblastic leukemia/lymphoma is considered a provisional entity. _It_ should _be_ diagnosed after _ruling_ out BPDCN. Blastic cells expressing _CD56_ _and_ CD2 raised the possibility of NK lymphoblastic leukemia/lymphoma. However, CD4 positivity made it doubtful for such diagnosis. In such cases, immunohistochemical analysis including _the_ most _characteristic_ _and_ _reliable_ marker is essential for _the_ diagnosis _of_ BPDCN. BPDCN was _initially_ characterized by the expression of CD4, CD56, and the lack _of_ B _cells,_ T cells, _myeloid_ or monocytic cells, and NK _cell_ markers. More specific plasmacytoid _dendritic_ cell markers (CD123, _CD303,_ and TCL1) have been recently used to diagnose _BPDCN.^\[[@R6],[@R7]\]^_ Since _they_ are concomitantly expressed in only _46%_ of patients, it has _been_ proposed that _diagnosis_ of BPDCN _can_ be made when 4 of these 5 markers (CD4, CD56, _CD123,_ _CD303,_ and TCL1) are expressed.^\[[@R8]\]^ Although _tumor_ _cells_ of _the_ _1st_ case showed focal _positive_ for _TCL1,_ both of _2_ cases showed all 5 markers except CD303 which was not performed in our institution. Therefore, our 2 cases were histologically diagnosed with BPDCN. The BPDCN without _cutaneous_ lesion _is_ exceedingly rare to diagnose. _Patients_ without cutaneous involvement have been described in the literature. Table [1](#T1){ref-type="table"} presents a summary of 39 _published_ cases _of_ BPDN without skin involvement. Bone _marrow_ involvement was observed in the majority of patients at diagnosis. Through hematopathology consultation service at _the_ National Institutes of Health, _Jegalian_ et al^\[[@R6]\]^ _have_ evaluated 55 BPDCN cases. Among them, 9 (16%) patients lacked cutaneous disease _at_ presentation. A _retrospective_ multicenter study of 43 patients (the _GIMEMA_ study) presenting _with_ leukemic manifestation was reported in 2012.^\[[@R14]\]^ Among 43 patients, 8 _(19%)_ cases _had_ no cutaneous manifestations.^\[[@R14]\]^ In _these_ _patients_ lacking _skin_ _involvement,_ other extracutaneous _and_ extramedullary sites _in_ _lymph_ node, _spleen,_ and liver _are_ most commonly observed. Rauh et al^\[[@R13]\]^ have demonstrated that patients _with_ BPDN _without_ skin _involvement_ and leukemic presentation show adverse prognosis than those with skin involvement. _Interestingly,_ no case _of_ BPDCN presenting with nasal cavity mass has been reported. It is of _note_ that we _identified_ nasal cavity as the unusual site _of_ BPDCN. ###### _Summary_ of 39 published cases _of_ BPDCN without skin involvement _in_ the literatures. |
1. Introduction
===============
*Mycobacterium tuberculosis* is counted with justification among the most dangerous and successful microorganisms in today's world, especially in developing countries which have become the reservoir of resistant strains (the most burdened countries are in South Africa and East Asia) \[[@B1-molecules-19-00651]\]. These strains are causing the biggest number of problems connected to tuberculosis (TB) treatment \[[@B2-molecules-19-00651]\]. The main problem is resistance. It can be divided into three groups. The first one is called multidrug-resistant TB (MDR-TB) and these microbes are resistant to all first-line antituberculotic drugs (pyrazinamide - PZA, isoniazid - INH, rifampicin - RIF, ethambutol - ETH, streptomycin - STR). The second group is more treacherous. This type of resistance is called extensively or extremely drug-resistant TB (XDR-TB) with the resistance to the first-line anti-TB agents isoniazid and rifampicin together with the resistance to any fluoroquinolone used in the therapy and to at least one of three injectable second-line antituberculotic drugs (amikacin, kanamycin or capreomycin) \[[@B3-molecules-19-00651]\]. The last and the latest category was the most dreaded and was called totally drug-resistant TB (TDR-TB) and the first case was recorded in India \[[@B4-molecules-19-00651]\]. These mycobacterial strains were resistant to all current known therapy. Nowadays, this group has disappeared with the approval of bedaquiline for therapy of resistant forms of tuberculosis. Another problem is connected with the HIV pandemic. These two infectious diseases are influencing each other in a synergic way so this has resulted in efforts to develop new anti-tubercular agents.
This work deals with a microwave-assisted synthesis of pyrazinamide analogues with potential antimycobacterial activity. It is caused by the fact that pyrazinamide is counted among the first-line anti-tuberculosis drugs used in current therapy. Its unique ability to kill the dormant forms of *Mycobacterium tuberculosis* is crucial in shortening the time needed for the treatment, so PZA has sterilizing activity especially in combination with rifampicin \[[@B5-molecules-19-00651]\].
PZA itself has multiple mechanisms of action. The first described was the activation of this prodrug *via* the enzyme pyrazinamidase (EC 3.5.1.19) to form pyrazinoic acid (POA). This metabolite causes a lowering of the inner compartment pH in mycobacterial cells. This leads to inhibition of membrane transport and then to cellular death \[[@B6-molecules-19-00651],[@B7-molecules-19-00651],[@B8-molecules-19-00651]\]. The gene encoding this enzyme is called *pncA* gene and its mutation is responsible for the origin of mycobacterial resistance to PZA \[[@B9-molecules-19-00651]\].
The second mechanism of action is connected with fatty acid synthase I (FAS I) (EC 2.3.1.85). It is suggested that the disruption of metabolism can be caused by inhibition of the cell membrane synthesis, which is essential for the survival of *Mycobacteria*, but this mechanism was mainly rejected for PZA itself by Boshoff because there is only low inhibition \[[@B10-molecules-19-00651]\]. On the other hand, the PZA analogues such as 5‑chloropyrazinamide, esters of pyrazinoic acid and esters of 5‑chloropyrazinoic acid were proven to act in this way \[[@B11-molecules-19-00651],[@B12-molecules-19-00651],[@B13-molecules-19-00651]\].
Recent research has suggested a novel mechanism of action of PZA - inhibition of *trans*-translation. This process is vital for survival and virulence of *Mycobacteria* and its inhibition leads to blockage of the proteosynthetic apparatus in ribosomes and to cellular death. These assumptions were proven by Zhang *et al.* \[[@B14-molecules-19-00651]\].
Although PZA is the first-line antituberculotic drug, it was found that this molecule has exhibited other interesting biological activities such as antifungal, antibacterial, antiviral and antineoplastic effects \[[@B15-molecules-19-00651],[@B16-molecules-19-00651],[@B17-molecules-19-00651],[@B18-molecules-19-00651],[@B19-molecules-19-00651],[@B20-molecules-19-00651]\].
There is another application of PZA derivatives that can be used in agriculture. The most successful pyrazine derivative diquat-dibromide (6,7-dihydrodipyrido\[1,2-a:2\',1\'-c\]pyrazinediium-dibromide), a non-selective, contact herbicide, which has been used to control many submerged and floating aquatic macrophytes, was found to interfere with the photosynthetic process by releasing strong oxidizers that rapidly disrupt and inactivate cells and cellular functions (at present banned in many EU countries) \[[@B21-molecules-19-00651]\]. Many structural variations of pyrazine compounds with herbicidal properties can be found in the patent literature \[[@B22-molecules-19-00651],[@B23-molecules-19-00651],[@B24-molecules-19-00651],[@B25-molecules-19-00651]\]. However, several pyrazine derivatives were also described as inhibitors of Hill reaction which inhibit photosynthetic electron transport (PET) in photosystem (PS) 2 \[[@B18-molecules-19-00651],[@B26-molecules-19-00651]\]. The site of action of these PET inhibitors in the photosynthetic apparatus was situated predominantly on the donor side of PS2, in the section between oxygen evolving complex and intermediate D^·^, *i.e.*, tyrosine radical (Tyr~D~•) occurring on the 161^st^ position in D~2~ protein. Consequently, these compounds can be considered as PS2 herbicides which could have ultimately adverse effect on plant growth. In general, the PET-inhibiting effectiveness of pyrazine derivatives depends on compound lipophilicity and σ Hammett constants of individual substituents. Hosseini *et al.* studied the electronic and structural descriptors, which are the main factors for the cytotoxicity in the series of substituted *N*-phenylpyrazine-2-carboxamides \[[@B27-molecules-19-00651]\].
This study is focused on preparation of *N*-substituted structural and functional derivative of PZA (5-chloro-6-methylpyrazine-2,3-dicarbonitrile) that was treated with ring-substituted benzylamines using the advantages of a microwave reactor. It should be stressed that this type of syntheses has become popular due to its higher yields, shorter reaction times or solvent savings in comparison with conventional organic syntheses \[[@B28-molecules-19-00651]\]. One of the main advantages is the heating. It is uniform through the volume of the sample and the microwaves usually interact with molecules themselves not vessel sides. Another benefit is connected with the temperature reached by the solvent used. The final temperature is usually far higher than the standard boiling point of the solvent when using over- pressurized systems. It is reached and bypassed in seconds. Improved heating usually leads to higher yields and shorter reaction times. There is one limitation for choosing the conditions. It is the polarity of the solvent when the non-polar solvents cannot be used in the way the polar ones can be. If the polar solvent is used in the reaction, there is a direct coupling of microwaves with molecules. More polar solvents have greater ability to interact with microwave radiation. Using the solvents with low polarity (low absorbers) leads to longer times of heating and reaction. On the contrary, if the reagents themselves are polar it could lower the disadvantages of non-polar solvents. Finally there are new approaches to microwave accelerated methods using ionic liquids or solid phase reactions (adsorption on mineral oxides, phase transfer catalysis, neat reactions) \[[@B29-molecules-19-00651]\]. Microwave assisted condensation in polar solvent is used in this work to accelerate the aminodehalogenation reaction. The conditions for the synthesis were proven experimentally. Antimycobacterial activity of the all prepared compounds was determined and compounds were evaluated also in relation to inhibition of photosynthetic electron transport (PET) in spinach (*Spinacia oleracea* L.) chloroplasts. The structure-activity relationships between the chemical structure and *in vitro* biological activities of evaluated compounds are discussed.
2. Results and Discussion
=========================
2.1. Chemistry
--------------
The starting compound 5-chloro-6-methylpyrazine-2,3-dicarbonitrile and the final compounds **1**--**15** were synthesized according to the general procedure shown in [Scheme 1](#molecules-19-00651-f006){ref-type="scheme"}. The aminodehalogenation reaction of this starting compound and ring-substituted benzylamines yielded a series of 15 secondary amines of which 14 were novel. 5-(Benzylamino)-6-methylpyrazine-2,3-dicarbonitrile (**6**) was previously synthesised by Takematsu *et al.* and the reported melting point was 118--119 °C \[[@B30-molecules-19-00651]\]. The compound we obtained melted at 128.7--130.7 °C. This difference can be caused by the mode of crystallization. All reactions were | 1. − = = = = = = = = = = = = = = = * mycobacterium tuberculosis * is counted with justification among the most dangerous and successful microorganisms in today ' s world, particularly in developing countries which have become the reservoir of resistant strains ( the most burdened countries are in south africa and east asia ) \ [ [ @ b1 - molecules - 19 - 00651 ] \ ]. these strains are causing the biggest number of problems connected to tuberculosis ( tb ) treatment \ [ [ @ b2 - molecules - 19 - 00651 ] \ ]. the main problem is resistance. it should be divided into three groups. the first one is called multidrug - resistant tb ( mdr - tb ) and these microbes are resistant to all short - line antituberculotic drugs ( pyrazinamide - pza, isoniazid - inh, rifampicin - rif, ethambutol - eth, streptomycin - str ). the second group is more treacherous. this type of resistance is called extensively or extremely drug - resistant tb ( xdr - tb ) with the resistance to the first - line anti - tb agents isoniazid and rifampicin together with the resistance to any fluoroquinolone used in the therapy and to at least consists of three injectable second - line antituberculotic drugs ( amikacin, kanamycin or capreomycin ) \ [ [ @ b3 - molecules - 19 - 00651 ] \ ]. the last of the latest category was the most dreaded and was called totally drug - resistant tb ( tdr - tb ) and the first case was recorded in india \ [ [ @ b4 - molecules - 19 - 00651 ] \ ]. these mycobacterial strains were resistant to all current hiv antibiotics. nowadays, this group has disappeared with the approval of bedaquiline for therapy of resistant forms of tuberculosis. another problem is connected with the hiv pandemic. these two infectious diseases are influencing each other in a synergic environment so this has resulted in efforts to develop new anti - tubercular agents. this work deals with a microwave - assisted synthesis of pyrazinamide analogues with potential antimycobacterial activity. it is caused by the fact that pyrazinamide produces counted among the first - line anti - tuberculosis drugs used in current therapy. its unique ability to kill the dormant forms of * mycobacterium tuberculosis * is crucial in shortening the time needed for the treatment, so pza has sterilizing activity especially in combination with rifampicin \ [ [ @ b5 - molecules - 19 - 00651 ] \ ]. pza itself has multiple mechanisms of action. the first described was the activation of this prodrug * via * the enzyme pyrazinamidase ( ec 3. 5. 1. 19 ) to form pyrazinoic acid ( poa ). this metabolite causes a lowering of the inner compartment ph in mycobacterial cells. this leads to inhibition of membrane transport and then to cellular death \ [ [ @ b6 - molecules - 19 - 00651 ], [ @ b7 - molecules - 19 - 00651 ], [ @ b8 - molecules - 19 - 00651 ] \ ]. the gene encoding this enzyme is called * pnca * gene and its mutation is responsible for the origin of mycobacterial resistance to pza \ [ [ @ b9 - molecules - 19 - 00651 ] \ ]. the second mechanism of action is connected with fatty acid synthase i ( fas i ) ( ec 2. 3. 1. 85 ). it is suggested that the disruption of metabolism can be caused by inhibition of the cell membrane synthesis, which is essential for the survival of * mycobacteria *, but this mechanism was mainly rejected for pza itself by boshoff because there is only low inhibition \ [ [ @ b10 - molecules - 19 - 00651 ] \ ]. on the other hand, the pza analogues such as 5 ‑ chloropyrazinamide, esters of pyrazinoic acid and esters of 5 ‑ chloropyrazinoic acid were proven to act in this way \ [ [ @ b11 - molecules - 19 - 00651 ], [ @ b12 - molecules - 19 - 00651 ], [ @ b13 - molecules - 19 - 00651 ] \ ]. recent research has suggested a novel mechanism of action of pza - inhibition of * trans * - translation. this process is vital for survival and virulence of * mycobacteria * and its inhibition leads to blockage of the proteosynthetic apparatus in ribosomes and to cellular death. these assumptions were proven by zhang * et al. * \ [ [ @ b14 - molecules - 19 - 00651 ] \ ]. although pza is the first - line antituberculotic drug, it was found that this molecule has exhibited other interesting biological activities such as antifungal, antibacterial, antiviral and antineoplastic effects \ [ [ @ b15 - molecules - 19 - 00651 ], [ @ b16 - molecules - 19 - 00651 ], [ @ b17 - molecules - 19 - 00651 ], [ @ b18 - molecules - 19 - 00651 ], [ @ b19 - molecules - 19 - 00651 ], [ @ b20 - molecules - 19 - 00651 ] \ ]. there is another application of pza derivatives that can be used in agriculture. the most successful pyrazine derivative diquat - dibromide ( 6, 7 - dihydrodipyrido \ [ 1, 2 - a : 2 \ ', 1 \ ' - c \ ] pyrazinediium - dibromide ), a non - selective, contact herbicide, which has been used to control many submerged and floating aquatic macrophytes, was found to interfere with the photosynthetic process by releasing strong oxidizers that rapidly disrupt and inactivate cells and cellular functions ( at present banned in many eu countries ) \ [ [ @ b21 - molecules - 19 - 00651 ] \ ]. many structural variations of pyrazine compounds with herbicidal properties can be found in the patent literature \ [ [ @ b22 - molecules - 19 - 00651 ], [ @ b23 - molecules - 19 - 00651 ], [ @ b24 - molecules - 19 - 00651 ], [ @ b25 - molecules - 19 - 00651 ] \ ]. however, several pyrazine derivatives were also described as inhibitors of hill reaction which inhibit photosynthetic electron transport ( pet ) in photosystem ( ps ) 2 \ [ [ @ b18 - molecules - 19 - 00651 ], [ @ b26 - molecules - 19 - 00651 ] \ ]. the site of action of these pet inhibitors in the photosynthetic apparatus was situated predominantly on the donor side of ps2, in the section between oxygen evolving complex and intermediate d ^ · ^, * i. e. *, tyrosine radical ( tyr ~ d ~ • ) occurring on the 161 ^ st ^ position in d ~ 2 ~ protein. consequently, these compounds can be considered as ps2 herbicides which could have ultimately adverse effect on plant growth. in general, the pet - inhibiting effectiveness of pyrazine derivatives depends on compound lipophilicity and σ hammett constants of individual substituents. hosseini * et al. * studied the electronic and structural descriptors, which are the main factors for the cytotoxicity in the series of substituted * n * - phenylpyrazine - 2 - carboxamides \ [ [ @ b27 - molecules - 19 - 00651 ] \ ]. this study is focused on preparation of * n * - substituted structural and functional derivative of pza ( 5 - chloro - 6 - methylpyrazine - 2, 3 - dicarbonitrile ) that was treated with ring - substituted benzylamines using the advantages of a microwave reactor. it should be stressed that this type of syntheses has become popular due to its higher yields, shorter reaction times or solvent savings in comparison with conventional organic syntheses \ [ [ @ b28 - molecules - 19 - 00651 ] \ ]. one of the main advantages is the heating. it is uniform through the volume of the sample and the microwaves usually interact with molecules themselves not vessel sides. another benefit is connected with the temperature reached by the solvent used. the final temperature is usually far higher than the standard boiling point of the solvent when using over - pressurized systems. it is reached and bypassed in seconds. improved heating usually leads to higher yields and shorter reaction times. there is one limitation for choosing the conditions. it is the polarity of the solvent when the non - polar solvents cannot be used in the way the polar ones can be. if the polar solvent is used in the reaction, there is a direct coupling of microwaves with molecules. more polar solvents have greater ability to interact with microwave radiation. using the solvents with low polarity ( low absorbers ) leads to longer times of heating and reaction. on the contrary, if the reagents themselves are polar it could lower the disadvantages of non - polar solvents. finally there are new approaches to microwave accelerated methods using ionic liquids or solid phase reactions ( adsorption on mineral oxides, phase transfer catalysis, neat reactions ) \ [ [ @ b29 - molecules - 19 - 00651 ] \ ]. microwave assisted condensation in polar solvent is used in this work to accelerate the aminodehalogenation reaction. the conditions for the synthesis were proven experimentally. antimycobacterial activity of the all prepared compounds was determined and compounds were evaluated also in relation to inhibition of photosynthetic electron transport ( pet ) in spinach ( * spinacia oleracea * l. ) chloroplasts. the structure - activity relationships between the chemical structure and * in vitro * biological activities of evaluated compounds are discussed. 2. results and discussion = = = = = = = = = = = = = = = = = = = = = = = = = 2. 1. chemistry - - - - - - - - - - - - - - the starting compound 5 - chloro - 6 - methylpyrazine - 2, 3 - dicarbonitrile and the final compounds * * 1 * * - - * * 15 * * were synthesized according to the general procedure shown in [ scheme 1 ] ( # molecules - 19 - 00651 - f006 ) { ref - type = " scheme " }. the aminodehalogenation reaction of this starting compound and ring - substituted benzylamines yielded a series of 15 secondary amines of which 14 were novel. 5 - ( benzylamino ) - 6 - methylpyrazine - 2, 3 - dicarbonitrile ( * * 6 * * ) was previously synthesised by takematsu * et al. * and the reported melting point was 118 - - 119 °c \ [ [ @ b30 - molecules - 19 - 00651 ] \ ]. the compound we obtained melted at 128. 7 - - 130. 7 °c. this difference can be caused by the mode of crystallization. all reactions were | 1. Introduction = = = = = = = = = = = = = = = * Mycobacterium tuberculosis * is counted with justification among the most dangerous and successful microorganisms in today ' s world, especially in developing countries which have become the reservoir of resistant strains (the most burdened countries are in South Africa and East Asia) \ [[ @ B1 - molecules - 19 - 00651] \ ]. These strains are causing the biggest number of problems connected to tuberculosis (TB) treatment \ [[ @ B2 - molecules - 19 - 00651] \ ]. The main problem is resistance. It can be divided into three groups. The first one is caliew multidrug - resistant TB (MDR - TB) and these microbes are resistant to all first - line antituberculotic drugs (pyrazinamide - PZA, isoniazid - INH, rifampicin - RIF, ethambutol - ETH, streptomycin - STR ). The second group is more treacherous. This type of resistance is called extensively or extremely drug - resistant TB (XDR - TB) with the resistance to the first - line anti - TB agents isoniazid and rifampicin together with the resistance to any fluoroquinolone used in the therapy and to at least one of three injectable second - line antituberculotic sruRs (amikacin, kanamycin or capreomycin) \ [[ @ B3 - molecules - 19 - 00651] \ ]. The last and the latest category was the most dreaded and was called totally drug - resistant TB (TDR - TB) and the first case was recorded in India \ [[ @ B4 - molecules - 19 - 00651] \ ]. These mycobacterial strains were resistant to all current known therapy. Nowadays, this group has disappeared with the approval of bedaquiline for therapy of resistant forms of tuberculosis. Another problem is connected with the HIV pandemic. These two infectious diseases are influencing each other in a synergic way so this has resulted in efforts to develop new anti - tubercular agents. This work deals with a microwave - assisted synthesis of pyrazinamide analogues with potential antimycobacterial activity. It is caused by the fact that pyrazinamide is counted among the first - line anti - tuberculosis drugs used in current therapy. Its unique ability to kill the dormant forms of * Mycobacterium tuberculosis * is crucial in shortening the time needed for the treatment, so PZA has sterilizing activity especially in combination with rifampicin \ [[ @ B5 - molecules - 19 - 00651] \ ]. PZA itself has multiple mechanisms of action. The first described was the activation of this prodrug * via * the enzyme pyrazinamidase (EC 3. 5. 1. 19) to form pyrazinoic acid (POA ). This metabolite causes a lowering of the inner compartment pH in mycobacterial cells. This leads to inhibition of membrane transport and then to cellular death \ [[ @ B6 - molecules - 19 - 00651 ], [@ B7 - molecules - 19 - 00651 ], [@ B8 - molecules - 19 - 00t52] \ ]. The gene encoding this enzyme is called * pncA * gene and its mutation is responsible for the origin of mycobacterial resistance to PZA \ [[ @ B9 - molecules - 19 - 00651] \ ]. The second mechanism of action is connected with fatty acid synthase I (FAS I) (EC 2. 3. 1. 85 ). It is suggested that the disruption of metabolism can be caused by inhibition of the cell membrane synthesis, which is essential for the survival of * Mycobacteria *, but this mechanism was mainly rejected for PZA itself by Boshoff because there is only low inhibition \ [[ @ B10 - molecules - 19 - 00651] \ ]. On the other hand, the PZA analogues such as 5 ‑ chloropyrazinamide, esters of pyrazinoic acid and esters of 5 ‑ chloropyrazinoic acid were proven to act in this way \ [[ @ B11 - molecules - 19 - 00651 ], [@ B12 - molecules - 19 - 00651 ], [@ B13 - molecules - 19 - 00651] \ ]. Recent research has suggested a novel mechanism of action of PZA - inhibition of * trans * - translation. This process is vital for survival and virulence of * Mycobacteria * and its inhibition leads to blockage of the proteosynthetic apparatus in ribosomes and to cellular death. These assumptions were proven by Zhang * et al. * \ [[ @ B14 - molecules - 19 - 00651] \ ]. Although PZA is the first - line antituberculotic drug, it was found that this molecule has exhibited other interesting biological activities such as antifungal, antibacterial, antiviral and antineoplastic effects \ [[ @ B15 - molecules - 19 - 00651 ], [@ B16 - molecules - 19 - 00651 ], [@ B17 - molecules - 19 - 00651 ], [@ B18 - mo>eculeA - 19 - 00651 ], [@ B19 - molecules - 19 - 00651 ], [@ B20 - molecules - 19 - 00651] \ ]. There is another application of PZA derivatives that can be used in agriculture. The most successful pyrazine derivative diquat - dibromide (6, 7 - dihydrodipyrido \ [1, 2 - a: 2 \ ', 1 \ ' - c \] pyrazinediium - dibromide ), a non - selective, contact herbicide, which has been used to control many submerged and floating aquatic macrophytes, was found to interfere with the photosynthetic process by releasing strong oxidizers that rapidly disrupt and inactivate cells and cellular functions (at present banned in many EU countries) \ [[ @ B21 - molecules - 19 - 00651] \ ]. Many structural variations of pyrazine compounds with herbicidal properties can be found in the patent literature \ [[ @ B22 - molecules - 19 - 00651 ], [@ B23 - molecules - 19 - 00651 ], [@ B24 - molecules - 19 - 00651 ], [@ B25 - molecules - 19 - 00651] \ ]. However, several pyrazine derivatives were also described as inhibitors of Hill reaction which inhibit photosynthetic electron transport (PET) in photosystem (PS) 2 \ [[ @ B18 - molecules - 19 - 00651 ], [@ B26 - molecules - 19 - 00651] \ ]. The site of action of these PET inhibitors in the photosynthetic apparatus was situated predominantly on the donor side of PS2, in the section bwtweSn oxygen evolving complex and intermediate D ^ · ^, * i. e. *, tyrosine radical (Tyr ~ D ~ •) occurring on the 161 ^ st ^ position in D ~ 2 ~ protein. Consequently, these compounds can be considered as PS2 herbicides which could have ultimately adverse effect on plant growth. In general, the PET - inhibiting effectiveness of pyrazine derivatives depends on compound lipophilicity and σ Hammett constants of individual substituents. Hosseini * et al. * studied the electronic and structural descriptors, which are the main factors for the cytotoxicity in the series of substituted * N * - phenylpyrazine - 2 - carboxamides \ [[ @ B27 - molecules - 19 - 00651] \ ]. This study is focused on preparation of * N * - substituted structural and functional derivative of PZA (5 - chloro - 6 - methylpyrazine - 2, 3 - dicarbonitrile) that was treated with ring - substituted benzylamines using the advantages of a microwave reactor. It should be stressed that this type of syntheses has become popular due to its higher yields, shorter reaction times or solvent savings in comparison with conventional organic syntheses \ [[ @ B28 - molecules - 19 - 00651] \ ]. One of the <aiB advantages is the heating. It is uniform through the volume of the sample and the microwaves usually interact with molecules themselves not vessel sides. Another benefit is connected with the temperature reached by the solvent used. The final temperature is usually far higher than the standard boiling point of the solvent when using over - pressurized sjst$ms. It is reached and N^passed in seconds. Improved heating usually leads to higher yields and shorter reaction times. There is one limitation for choosing the conditions. It is the polarity of the solvent when the non - polar solvents cannot be used in the way the polar ones can be. If the polar solvent is used in the reaction, there is a direct coupling of microwaves with molecules. More polar solvents have greater abLlit5 to interact with microwave radiation. Using the solvents with low polarity (low absorbers) leads to longer times of heating and reaction. On the contrary, if the reagents themselves are polar it could lower the disadvantages of non - polar solvents. Finally there are new approaches to microwave accelerated methods using ionic liquids or solid phase reactions (adsorption on mineral oxides, phase transfer catalysis, neat reactions) \ [[ @ B29 - molecules - 19 - 00651] \ ]. Microwave assisted condensation in polar solvent is used in this work to accelerate the aminodehalogenation reaction. The conditions for the synthesis were proven experimentally. Antimycobacterial activity of the all prepared compounds was determined and compounds were evaluated also in relation to inhibition of photosynthetic electron transport (PET) in spinach (* Spinacia oleracea * L.) chloroplasts. The structure - activity relationships between the chemical structure and * in vitro * biological activities of evaluated compounds are discussed. 2. Results and Discussion = = = = = = = = = = = = = = = = = = = = = = = = = 2. 1. Chemistry - - - - - - - - - - - - - - The starting compound 5 - chloro - 6 - methylpyrazine - 2, 3 - dicarbonitrile and the final compounds * * 1 * * - - * * 15 * * were synthesized according to the general procedure shown in [Scheme 1] (# molecules - 19 - 00651 - f006) {ref - type = " scheme " }. The aminodehalogenation reaction of this starting compound and ring - substituted benzylamines yielded a series of 15 secondary amines of which 14 were novel. 5 - (Benzylamino) - 6 - methylpyrazine - 2, 3 - dicarbonitrile (* * 6 * *) was previously synthesised by Takematsu * et al. * and the reported melting point was 118 - - 119 ° C \ [[ @ B30 - molecules - 19 - 00651] \ ]. The compound we obtained melted at 128. 7 - - 130. 7 ° C. This difference can be caused by the Hodf of crystallization. All reactions were | 1. Introduction =============== *Mycobacterium tuberculosis* counted with justification among the most dangerous and successful microorganisms in today's world, especially developing countries which have become the reservoir of resistant strains (the most countries are in South Africa and East Asia) \[[@B1-molecules-19-00651]\]. These strains are causing the biggest number of problems connected tuberculosis (TB) treatment \[[@B2-molecules-19-00651]\]. main problem is resistance. It can into three groups. The first one is multidrug-resistant TB and these microbes are resistant to all first-line antituberculotic drugs (pyrazinamide isoniazid - INH, - RIF, ethambutol - ETH, - STR). The second group is more treacherous. This type of resistance is called extensively or extremely drug-resistant TB (XDR-TB) with the resistance to the first-line anti-TB agents and rifampicin together with resistance to any fluoroquinolone used in therapy and to at least one of three injectable second-line antituberculotic drugs (amikacin, or capreomycin) \[[@B3-molecules-19-00651]\]. The last latest category was the most dreaded and was called totally drug-resistant TB (TDR-TB) and the case was in India \[[@B4-molecules-19-00651]\]. These mycobacterial strains were resistant to all current therapy. this has disappeared with the approval of bedaquiline for therapy of resistant forms of tuberculosis. Another problem is connected with the pandemic. These two infectious diseases are influencing each other in a synergic way so this has resulted in efforts to develop anti-tubercular agents. This work deals with a microwave-assisted synthesis of pyrazinamide analogues with potential antimycobacterial activity. is caused by the fact that pyrazinamide is among the first-line anti-tuberculosis drugs used in current therapy. Its unique ability to kill the dormant forms of *Mycobacterium tuberculosis* is crucial in shortening the time for the treatment, so PZA has sterilizing activity especially in combination with rifampicin \[[@B5-molecules-19-00651]\]. PZA itself has mechanisms of action. The first described was the activation of this the enzyme pyrazinamidase 3.5.1.19) form pyrazinoic acid (POA). metabolite causes a lowering of the inner compartment pH in mycobacterial cells. This leads to inhibition of membrane transport and then to cellular death The gene encoding this enzyme is called *pncA* gene and its mutation is responsible for the origin of resistance to PZA \[[@B9-molecules-19-00651]\]. The second mechanism of action is connected with fatty acid I I) (EC 2.3.1.85). It is suggested that the disruption of metabolism can be caused by inhibition of the cell membrane which is essential for the survival of *Mycobacteria*, but this mechanism was mainly rejected for PZA itself Boshoff because there is only \[[@B10-molecules-19-00651]\]. On the other hand, the PZA analogues such as 5‑chloropyrazinamide, esters of pyrazinoic acid and esters of 5‑chloropyrazinoic acid were proven to act this way \[[@B11-molecules-19-00651],[@B12-molecules-19-00651],[@B13-molecules-19-00651]\]. Recent research has suggested novel mechanism action of PZA - inhibition of *trans*-translation. This process is vital survival virulence of *Mycobacteria* and its inhibition blockage of the apparatus in ribosomes and to cellular These assumptions were proven by Zhang *et al.* \[[@B14-molecules-19-00651]\]. Although PZA is the first-line antituberculotic drug, it was found that this molecule has exhibited other interesting biological activities such as antifungal, antibacterial, antiviral and antineoplastic effects \[[@B15-molecules-19-00651],[@B16-molecules-19-00651],[@B17-molecules-19-00651],[@B18-molecules-19-00651],[@B19-molecules-19-00651],[@B20-molecules-19-00651]\]. There is another application of PZA derivatives that can be in agriculture. The most successful pyrazine derivative diquat-dibromide (6,7-dihydrodipyrido\[1,2-a:2\',1\'-c\]pyrazinediium-dibromide), a non-selective, contact herbicide, which has been used to control many submerged and floating aquatic macrophytes, was found to interfere with the photosynthetic process by releasing strong oxidizers that rapidly disrupt and inactivate cells and cellular functions (at present in many EU countries) \[[@B21-molecules-19-00651]\]. Many structural variations of pyrazine compounds with herbicidal properties can be in the patent literature \[[@B22-molecules-19-00651],[@B23-molecules-19-00651],[@B24-molecules-19-00651],[@B25-molecules-19-00651]\]. However, pyrazine were also described as of Hill reaction which inhibit photosynthetic electron transport (PET) in (PS) 2 \[[@B18-molecules-19-00651],[@B26-molecules-19-00651]\]. The site of action of these PET inhibitors in the photosynthetic apparatus was situated predominantly on the donor side of PS2, in section between oxygen evolving complex and intermediate D^·^, *i.e.*, radical (Tyr~D~•) on the 161^st^ position in D~2~ protein. Consequently, compounds be considered as PS2 herbicides which could have ultimately adverse effect on plant growth. In general, the PET-inhibiting effectiveness of pyrazine derivatives depends on compound lipophilicity σ constants of individual substituents. Hosseini *et al.* studied the electronic and which main factors for the cytotoxicity the series of substituted *N*-phenylpyrazine-2-carboxamides \[[@B27-molecules-19-00651]\]. This study focused on preparation of *N*-substituted structural functional of PZA (5-chloro-6-methylpyrazine-2,3-dicarbonitrile) that was treated with ring-substituted benzylamines using the of a microwave reactor. should be stressed that this of syntheses has become popular due to its higher yields, shorter reaction times or solvent savings in with conventional organic syntheses \[[@B28-molecules-19-00651]\]. One of the main advantages is the heating. It is uniform through the volume of the sample and the microwaves usually interact molecules themselves not vessel Another benefit with the temperature reached by the solvent used. The final temperature is usually higher than the standard boiling point of the solvent when using over- systems. It reached and bypassed in seconds. Improved heating usually leads to higher and reaction times. There is one limitation for choosing the conditions. It is the polarity of the solvent when the non-polar cannot be used in the way the polar ones can be. If the polar solvent is used in the reaction, there is a direct coupling microwaves with molecules. More polar solvents have greater ability to interact with microwave Using solvents with low polarity absorbers) leads longer times of heating and reaction. On if the reagents themselves are polar it could lower the disadvantages non-polar Finally there are new approaches microwave accelerated methods using liquids or phase (adsorption on mineral oxides, phase transfer catalysis, neat reactions) \[[@B29-molecules-19-00651]\]. Microwave assisted condensation in polar solvent is used in this work accelerate the aminodehalogenation reaction. The conditions for the synthesis were proven experimentally. Antimycobacterial activity of all prepared compounds was determined and compounds were evaluated also in relation inhibition of photosynthetic electron transport (PET) spinach (*Spinacia oleracea* L.) chloroplasts. The structure-activity relationships between the structure and *in vitro* biological activities evaluated compounds are discussed. 2. Results and Discussion ========================= 2.1. Chemistry -------------- The starting compound 5-chloro-6-methylpyrazine-2,3-dicarbonitrile and the final compounds **1**--**15** were synthesized according to general procedure shown [Scheme 1](#molecules-19-00651-f006){ref-type="scheme"}. aminodehalogenation reaction of starting compound and ring-substituted benzylamines yielded a series of 15 secondary of which 14 novel. 5-(Benzylamino)-6-methylpyrazine-2,3-dicarbonitrile was previously synthesised Takematsu *et al.* and the reported melting point was 118--119 °C \[[@B30-molecules-19-00651]\]. The compound obtained melted at 128.7--130.7 °C. This difference can be caused by mode of crystallization. All reactions were | 1. INtrOdUctIon
===============
*MycoBACTERIuM tUBerCuLOSiS* iS CoUnTEd wITh JuStIfiCAtIOn aMoNg ThE most DAngERouS AnD suCcessFul mIcRoORGAnisMS In Today's WORLd, ESPeCIally In devEloPING countriEs whIch HavE becoME THe rESeRVOiR oF ReSiStANt Strains (the mOSt BURDeNeD coUNtRIES ArE IN SOUth aFRIcA ANd EaSt AsIA) \[[@B1-MoLEculeS-19-00651]\]. THEsE sTrainS are causInG tHe biGGeST nuMBer OF PrOblEms coNnEctEd to TUbercULOsIs (TB) TReAtMenT \[[@b2-moLeCULES-19-00651]\]. the main pRobleM IS reSISTAnCe. iT CAn Be dIVIdEd INtO tHrEE GROUpS. THE FIRST one iS CallED multidruG-rESiSTAnt TB (mDr-tb) aND tHESE MicrOBES are resISTAnT To all FIRsT-liNe aNTItuberCUlotIc dRugs (pyrazinaMIde - pZa, iSonIaZid - iNH, rIFAMPIcIn - Rif, EtHaMbutol - ETh, STrePTomyciN - stR). The SECOnD GROUP Is MoRe tReacHeRouS. ThiS type OF ResIstAnce iS caLLEd eXtEnsiVeLy OR ExTrEMELy DRug-REsIStaNt tb (xDr-TB) WiTh THE RESIsTanCE tO ThE First-liNE anTi-tb aGENTS iSoNiAzid ANd rIfampicin TOGETher WItH THe ResiSTaNcE TO any FLUorOQUinOLOnE used in The THeraPY and TO at leaST OnE of tHreE iNJEcTAble secOnd-LInE aNTItUBeRCUlOTic drUGS (amikacin, KANaMyCIN oR cApREoMyCin) \[[@b3-MoLecULEs-19-00651]\]. ThE LaSt AND The LaTest CatEGORY WaS The MOst dReaDed ANd WAs caLLeD TOtalLy DruG-REsIsTaNT tb (TDR-tB) ANd The fiRST CaSe waS reCoRdEd In inDia \[[@b4-MOLEcUlES-19-00651]\]. thEsE mYcobACteRiAl sTRAins wERE reSISTanT TO alL CURrent KNOWN TheRaPy. NoWADAYS, thIs groUp haS dISAPPEareD WitH The APpROVAL OF bedaqUIliNe FOR therapy oF REsiSTant FoRMs Of tUbERcUlOSis. aNother PROBlEM iS COnNeCTeD WitH THe HIV PAnDEMIc. THeSe tWo INfEcTIous dISeasES ArE INfLuEnCiNG EACh oTheR iN A SYneRGiC wAy So tHIS HAS REsUlted In efForts tO DevELOp New AntI-TUBeRcuLaR agENTS.
ThIS woRK dEaLS with a MIcroWave-ASSISted sYntheSIs oF PyRAzInAMIDE anaLoGuES wIth poteNtial AntIMyCobacteRiAl aCTiVItY. iT iS CaUSEd BY The faCt ThaT pYrazinAMiDe iS CouNTeD AmONG the fiRst-liNE anTi-tUbERcUlosIS dRugs usED iN cUrRenT therAPy. iTS uniqUe abiLitY TO KILL The dormaNT formS of *MYCOBAcTerIUM tuberCuLOSiS* IS cRuCiAL IN ShORTENiNg The TIme NEedEd FOR The trEatmEnt, so pzA haS stERiLiziNG aCtiVItY eSpEciaLly iN cOMBinaTIoN wiTh rIFAmPicIN \[[@B5-MoleCUles-19-00651]\].
PzA iTseLF Has MuLTiple meCHAnIsms OF actION. the FIrst deScrIbED WAs THe ACtivAtiON OF THIS PROdruG *Via* tHE EnzYme PyRAZINamiDAse (eC 3.5.1.19) to foRm pyRAZInoIC aCID (POA). thIs metABoLiTE CAUSEs a LOweRINg of THE iNNeR cOmpArtmeNt ph iN MYcOBaCteriAl ceLLS. tHis leaDs tO INHibITioN oF MEMBraNE tRAnSpOrT and THen To cEllULaR deaTH \[[@B6-moLeculES-19-00651],[@b7-mOleculeS-19-00651],[@b8-MolECUlEs-19-00651]\]. THe Gene ENCOdiNG thIs ENZYmE is calleD *PncA* gEne anD iTS mUtAtION IS reSPonsiblE FoR THE oRigIn OF MYcObACTeRIAl RESIStAnce to pZA \[[@B9-moLEcULES-19-00651]\].
ThE secoNd mechAnIsm of ACTiOn IS cONNECTED WITh FAtty aciD sYntHasE i (FAs i) (ec 2.3.1.85). It is SugGEsTeD tHAt tHE DiSRuPTIon OF MEtAbolism caN bE CaUSEd BY iNHiBitIoN OF THE CeLl meMbrANe SYNthesis, WhiCh iS essEnTiAL fOR the SuRVivAl oF *MycoBActERia*, but ThIS MeCHANISm waS maInly rejEcTEd For Pza ItSELF By BosHOFf BEcaUsE therE Is oNLy LOW iNhiBITioN \[[@B10-moLECUleS-19-00651]\]. ON ThE oThEr hAnd, The pZA ANALOgUeS SUCh aS 5‑chlORoPyrazinaMIdE, eSTeRS oF PyrazinoIc acID AnD ESteRS Of 5‑chloRopyRAZINoIc AciD WerE proVEN tO aCT in tHIs waY \[[@b11-MoLEcULEs-19-00651],[@b12-mOLeCulEs-19-00651],[@b13-mOlEcULes-19-00651]\].
rEcENt reSeARCh hAS suGgestED a nOvEl mechANisM Of aCtiOn of pzA - InhIbition Of *tRANS*-TRanSlaTIOn. tHIS PROCESs IS Vital for sUrVIVal aND virUlencE oF *MYCobActeRiA* aNd its INhIBITiOn lEAdS to bloCkaGe of ThE pROTeosyNthETiC appARaTUS In ribosOMEs aND tO CeLlULAr deATH. ThesE AssumPTIonS were provEn BY zHanG *eT aL.* \[[@b14-mOLeCUlEs-19-00651]\].
AlThoUgh pza is tHe FIRsT-lINe AntITuBErculOtIc DrUG, It Was foUND That tHiS MOlecule hAs ExhiBIteD oThER inTEREsTing bioLOgIcAL aCtiViTIEs suCH aS aNTIfUNGal, aNTIbaCTERIaL, AntIVirAl and ANtiNEoPlasTIC eFFects \[[@b15-mOLEculES-19-00651],[@B16-MoLECuLEs-19-00651],[@B17-moleCuLEs-19-00651],[@B18-moleCuLEs-19-00651],[@b19-molEcuLes-19-00651],[@b20-MoLECUles-19-00651]\].
tHERE iS aNOTher APpLicAtIon OF PzA DEriVATIVes thaT CaN be uSEd IN AgriCULturE. tHE MoSt SucCEsSFuL pYraZIne dErIVATIVE DiQUAt-DibromIDE (6,7-diHYdRoDipyRiDO\[1,2-a:2\',1\'-C\]PyraZinEDIIUM-DIbrOmIdE), A nON-SELEcTiVE, CoNtaCT HerBICidE, whICH hAS bEEn uSeD TO cONTRol maNY SUBMergeD ANd fLoaTIng aqUATIc mACrOPHyTeS, WAS founD To INTeRFere WitH THE PhOTOsyNtHETiC pROCess by rEleaSING STRONg oxiDizeRs ThAT rapidly DISRUPT anD iNaCTIvAte ceLlS anD cEllULaR FUnCTionS (aT PREsent baNNED iN Many Eu couNtRiEs) \[[@B21-MOlECUleS-19-00651]\]. maNy STrUCTUraL VARIATioNs Of pyRaziNE cOmpounDs WitH HERbIcidAL PRoperTIes caN BE FOUnd iN tHE PatenT LiTEraTuRE \[[@B22-moLECUleS-19-00651],[@b23-MoLeCUleS-19-00651],[@B24-MoleCULES-19-00651],[@b25-MOlEcULeS-19-00651]\]. hoWEvER, SeveRaL pYRAZINe dERiVaTivEs were ALso descRIBED As iNhibiTOrs OF hill reActIon wHIcH iNHIBit phOTOsyNTHetiC elEcTroN TRansPort (pET) In PhOTOsYSTem (Ps) 2 \[[@b18-mOleCULeS-19-00651],[@b26-mOlecULeS-19-00651]\]. the sIte Of actION of THese peT inHIBitoRS In THe PhoTosYnthEtiC ApPArAtUs Was sItUated pReDOmiNANTly on THE donOR SiDe Of Ps2, IN tHE sEctiON BeTWeeN OXygeN eVoLVinG comPLeX AnD InteRMEDIAte D^·^, *i.e.*, TyRosINe rADICAL (tYR~D~•) oCCUrrINg on the 161^sT^ PosItIon IN D~2~ proTeIN. CONsEquEntLy, tHESe cOmPounds CAn Be consIDerEd As pS2 hErbICIDes WHIch cOuLD havE ULtimaTELY aDveRSE efFeCT ON planT grOwTh. in gEnerAl, thE pEt-iNhibitIng eFfECtIveNess OF pyraZInE DeRIvaTiVeS DEpEnDs on CoMpOUND liPoPHIliciTY AnD Σ hAMMEtT conSTantS of indIVIDUAl SuBSTiTuENTs. hOsSEiNI *eT AL.* STudIEd tHe eLEcTRONic aND StrUcTUraL desCriPTORS, whicH aRE thE MAIn fActoRS FOr tHe CyToTOXiCiTY In ThE SeRies oF suBSTiTUteD *n*-phEnYlPYRaZIne-2-CArBOxAmideS \[[@b27-mOLeCULes-19-00651]\].
thiS stUDY IS fOcUSEd On pReParatION Of *n*-SUbSTITutEd STrUcTurAL aNd FunCTionAl deRIVatIVe OF PZa (5-CHLOro-6-mETHyLpyrAziNE-2,3-DICArboNITRiLE) tHAT waS TREaTED WITh rInG-sUBSTitutED BenzYlaMiNEs UsiNg THE ADVanTAges oF A MIcRowAVe rEaCToR. it SHoUld be StrESsEd ThAt tHiS tYPe oF sYNTHeseS Has beCOME POpuLaR dUE TO iTs HIGhEr YIELdS, SHORtEr REaCTiON TimEs oR SOlvenT SAvings In comPAriSON wITh cOnvEntIonAl OrgANIC synThESES \[[@b28-moLEcUleS-19-00651]\]. oNe oF The MaIN aDvaNtageS iS tHe HeatiNg. iT IS unIforM THrougH ThE VoLUme oF The SAMpLe and thE MICROwaves USUallY iNTeRacT With MoLEcULES theMSelvES not VesSEL SIdEs. ANoTHer BeNefit is CoNNEctEd WIth tHE tEMperatuRE REaCHeD By THe solveNT uSEd. THe FINaL tEmpEraTurE IS usually FAR hIGHer THaN THE StANDaRd boiliNg PoInT oF THe SOlVENt WhEN using OVEr- PReSsuRiZeD syStEms. iT is ReacHED And byPaSSEd In SEcONDs. IMProVED HEatIng uSually leADs tO HigHER yIEldS AnD shORtER rEAction tImeS. tHere is oNe LiMItAtiOn FOr cHOOSIng THe COndiTIONs. iT IS ThE POLaritY of THE sOlveNT wHEn THE NON-PoLAr sOLvENTS caNnOt bE Used IN tHE wAy the POlAR ONeS cAn be. iF THe polar soLvEnt IS Used iN tHe reAcTiON, THEre iS a DIrECT coUplinG of MICRowavES WiTh moLecuLES. MORe PolAr sOlveNtS have GreAter ABIlity To inTERACT WIth MICroWAVe radIAtIon. UsinG THE sOlVENTs WiTh low POlArITy (Low ABSOrBerS) lEadS To lOnGer tIMes oF HEatInG AND rEaCTIoN. on THe CONtRarY, IF ThE ReagENts themsElVeS aRe poLaR IT coULd LowER thE dIsadvANtaGES oF nOn-pOlar SolvenTs. FInAlLy tHEre are new APpRoACHES TO mIcROWaVe ACCElerATed mEthODs usING IoNic liqUiDS or sOlId phAsE REacTIOnS (adsORpTIon on minERaL OXiDEs, Phase TRAnSfeR CAtaLySis, neat REactioNs) \[[@b29-mOleCUleS-19-00651]\]. mIcRowAvE aSSIsTED CoNdensatioN IN POlaR solVENt is useD iN THiS WoRk to accELErate the AMinOdehAlOGEnaTion rEaction. the cONdITionS FOR tHE SyNtheSIs Were PRoveN eXperImentALLY. anTiMyCObactErIAL ACtIviTy OF ThE aLL preparEd COMpouNdS was DetermInED ANd compOUnDs WERe EVaLUAtEd alSO iN reLaTION to iNhIBitiON OF PhoToSynThEtIC eLecTRoN tRAnSpORT (pet) in spINAcH (*SpinacIA oleraCEa* L.) CHLoROPLAstS. THE StRUCTurE-aCtIvITY rELaTIoNSHIPS BetwEEn ThE CHEMIcal STrUctUre and *In vitrO* BIolOgiCaL AcTIVItiES of eVaLuATeD COmPOUNdS are DisCuSSEd.
2. resULts anD diScUSsIoN
=========================
2.1. CHEmIsTrY
--------------
THE sTartiNG COMPoUND 5-chLoRo-6-metHylpYraZinE-2,3-DiCARBONITrIle and The FinAL CoMPoUnDs **1**--**15** wEre SYNtHeSIzed accORdIng To tHE genERAL pROCEDuRe showN IN [SChemE 1](#molECULEs-19-00651-F006){Ref-type="scHemE"}. THe AminOdEhaLoGENaTIoN reaCtioN oF ThiS stARTiNg COmpOuND and rInG-sUBsTItUteD BENzYlAMiNes yIeldeD A SeRIES oF 15 SeCOnDArY AMInES of whiCh 14 were NOvel. 5-(bEnzYlaMInO)-6-METhYLpYrAZINe-2,3-DiCARbONItrIlE (**6**) was prEvIoUSLy SYNtHesisEd By tAkEMatSU *ET Al.* ANd THe RePORted meLtINg POint Was 118--119 °C \[[@B30-MOLeCuleS-19-00651]\]. thE cOMPouND We oBTAINEd MELtED At 128.7--130.7 °C. THIs diFfereNcE CAn be cauSed bY tHe mODE oF CrYsTalLIZATioN. All rEACTIonS weRe | 1. Introduction =============== *Mycobacterium tuberculosis* is counted with justificationamong the most dangerous and successful microorganisms in today's world,especially in developing countrieswhichhave become the reservoir of resistant strains (themost burdenedcountries are in South Africa and EastAsia)\[[@B1-molecules-19-00651]\]. These strains are causingthebiggestnumber ofproblems connected to tuberculosis (TB)treatment \[[@B2-molecules-19-00651]\]. The main problem isresistance. It can be divided into three groups. The first one is called multidrug-resistant TB (MDR-TB) and these microbes are resistant to all first-lineantituberculotic drugs (pyrazinamide - PZA, isoniazid-INH, rifampicin - RIF,ethambutol - ETH, streptomycin - STR). The second group is more treacherous. This type of resistance is calledextensively or extremely drug-resistant TB (XDR-TB) with the resistance tothe first-line anti-TB agents isoniazid and rifampicin together with the resistanceto any fluoroquinoloneused in thetherapy and to atleast one of three injectable second-line antituberculotic drugs (amikacin,kanamycin or capreomycin)\[[@B3-molecules-19-00651]\]. The last and the latestcategory was the most dreaded and wascalled totally drug-resistant TB (TDR-TB) and the first casewas recorded inIndia \[[@B4-molecules-19-00651]\]. These mycobacterial strains were resistantto allcurrent knowntherapy. Nowadays, this group has disappeared with the approval of bedaquiline for therapy of resistant forms of tuberculosis.Another problem is connectedwith theHIV pandemic. These two infectious diseases are influencing each other in a synergic way so this has resulted in efforts to develop new anti-tubercular agents. This work deals with a microwave-assistedsynthesis of pyrazinamideanalogues with potential antimycobacterialactivity. It is causedby the fact that pyrazinamide is counted among the first-line anti-tuberculosis drugs used in current therapy. Its unique ability to kill the dormantforms of*Mycobacterium tuberculosis* is crucial in shortening the time neededfor the treatment,so PZA has sterilizingactivityespecially in combination with rifampicin \[[@B5-molecules-19-00651]\]. PZAitself has multiple mechanisms ofaction. The first described was the activation of this prodrug *via* the enzyme pyrazinamidase (EC 3.5.1.19) to form pyrazinoic acid (POA). This metabolitecauses a lowering of the inner compartment pH in mycobacterialcells. This leads toinhibition of membrane transport and then to cellular death\[[@B6-molecules-19-00651],[@B7-molecules-19-00651],[@B8-molecules-19-00651]\]. The gene encodingthis enzyme is called *pncA* gene andits mutation is responsible for theorigin of mycobacterial resistance to PZA \[[@B9-molecules-19-00651]\]. The second mechanismofaction is connected with fatty acid synthase I (FAS I) (EC2.3.1.85). It is suggested that the disruption of metabolismcan be causedby inhibition of the cellmembranesynthesis, which is essential for thesurvival of*Mycobacteria*, but thismechanism was mainly rejected for PZA itselfby Boshoff because there is only low inhibition \[[@B10-molecules-19-00651]\]. On the other hand, thePZA analogues such as 5‑chloropyrazinamide, estersof pyrazinoic acid and esters of5‑chloropyrazinoic acid were provento actin this way \[[@B11-molecules-19-00651],[@B12-molecules-19-00651],[@B13-molecules-19-00651]\]. Recent researchhas suggested anovelmechanism ofactionof PZA - inhibition of *trans*-translation. This process is vitalfor survival and virulence of *Mycobacteria* and its inhibition leads to blockageof the proteosyntheticapparatus in ribosomes andto cellular death. These assumptions were proven by Zhang *etal.* \[[@B14-molecules-19-00651]\]. Although PZA isthe first-line antituberculotic drug, it was found that this molecule has exhibited other interesting biological activities such as antifungal, antibacterial, antiviral andantineoplastic effects \[[@B15-molecules-19-00651],[@B16-molecules-19-00651],[@B17-molecules-19-00651],[@B18-molecules-19-00651],[@B19-molecules-19-00651],[@B20-molecules-19-00651]\]. There is another application of PZA derivativesthat can be used in agriculture. The most successful pyrazinederivative diquat-dibromide (6,7-dihydrodipyrido\[1,2-a:2\',1\'-c\]pyrazinediium-dibromide), a non-selective,contact herbicide, which has been used to control manysubmergedand floating aquatic macrophytes,was found to interfere with the photosyntheticprocess by releasing strong oxidizersthat rapidly disrupt and inactivate cells and cellular functions (at present banned inmany EU countries) \[[@B21-molecules-19-00651]\]. Many structural variations of pyrazine compounds withherbicidal properties can be found in the patent literature \[[@B22-molecules-19-00651],[@B23-molecules-19-00651],[@B24-molecules-19-00651],[@B25-molecules-19-00651]\].However, several pyrazine derivatives were also described as inhibitors of Hill reactionwhichinhibitphotosynthetic electron transport(PET) in photosystem (PS)2 \[[@B18-molecules-19-00651],[@B26-molecules-19-00651]\]. The site of action of these PET inhibitors in the photosynthetic apparatus was situatedpredominantly on the donorside of PS2, in thesection between oxygen evolving complex and intermediate D^·^, *i.e.*, tyrosine radical (Tyr~D~•) occurring on the 161^st^position in D~2~ protein.Consequently, these compounds can be considered as PS2 herbicides which could have ultimately adverse effect on plant growth. In general, the PET-inhibiting effectiveness of pyrazine derivatives depends on compound lipophilicity and σ Hammett constantsof individual substituents. Hosseini *et al.* studied the electronic and structural descriptors, which are the main factors for the cytotoxicity in the series of substituted *N*-phenylpyrazine-2-carboxamides \[[@B27-molecules-19-00651]\]. Thisstudy is focused on preparationof *N*-substitutedstructural and functional derivative ofPZA (5-chloro-6-methylpyrazine-2,3-dicarbonitrile)that was treated with ring-substituted benzylamines using the advantages of a microwave reactor. It should be stressed that this type of syntheses hasbecomepopular due to its higher yields, shorter reaction times orsolvent savings in comparisonwith conventional organic syntheses \[[@B28-molecules-19-00651]\]. Oneof the main advantages is the heating. It is uniformthrough the volume of the sampleandthe microwaves usually interact with molecules themselves not vesselsides. Another benefit is connected with the temperature reached by the solvent used. The finaltemperature is usually far higher than the standardboiling point of the solvent when using over- pressurized systems.It is reached and bypassed inseconds.Improved heating usually leads to higher yields andshorter reaction times. There is one limitation for choosing the conditions. It isthepolarity ofthe solvent whenthe non-polar solvents cannotbe used in the way thepolar ones can be.If the polar solvent is used in the reaction, there is a direct coupling ofmicrowaves with molecules. More polar solvents have greater ability tointeract with microwave radiation. Using thesolventswith lowpolarity (low absorbers) leads to longer timesof heatingand reaction. On the contrary, if the reagents themselves are polar it could lower the disadvantages of non-polar solvents. Finally there are new approachesto microwaveaccelerated methods using ionic liquids or solid phase reactions(adsorption onmineraloxides, phasetransfercatalysis, neat reactions) \[[@B29-molecules-19-00651]\]. Microwave assisted condensation in polar solvent is used in this work to accelerate the aminodehalogenation reaction. The conditions for the synthesis were proven experimentally. Antimycobacterial activity of the all preparedcompoundswas determined and compounds were evaluated also inrelation to inhibition ofphotosynthetic electron transport(PET) in spinach (*Spinacia oleracea* L.) chloroplasts. The structure-activity relationships between thechemical structureand*in vitro* biological activities ofevaluated compounds arediscussed. 2. Results and Discussion ========================= 2.1.Chemistry -------------- The startingcompound 5-chloro-6-methylpyrazine-2,3-dicarbonitrile and the final compounds**1**--**15** were synthesized according to the general procedure shown in [Scheme 1](#molecules-19-00651-f006){ref-type="scheme"}. Theaminodehalogenation reaction ofthis starting compound and ring-substituted benzylamines yielded a series of 15 secondary amines of which 14 were novel. 5-(Benzylamino)-6-methylpyrazine-2,3-dicarbonitrile (**6**) was previously synthesised byTakematsu *et al.* and the reportedmelting point was118--119 °C \[[@B30-molecules-19-00651]\]. The compoundwe obtained melted at 128.7--130.7 °C. Thisdifference can be caused by the mode of crystallization. All reactions were | 1. Introduction =============== *Mycobacterium _tuberculosis*_ is counted with justification among the _most_ _dangerous_ and successful _microorganisms_ in today's world, especially in developing countries which have become _the_ reservoir of _resistant_ strains (the most burdened countries _are_ in South Africa and _East_ _Asia)_ _\[[@B1-molecules-19-00651]\]._ These strains are causing the biggest number of problems connected to tuberculosis (TB) treatment _\[[@B2-molecules-19-00651]\]._ _The_ main problem _is_ resistance. It _can_ _be_ divided into three groups. _The_ first one is called multidrug-resistant _TB_ _(MDR-TB)_ and these microbes are _resistant_ to all first-line _antituberculotic_ drugs (pyrazinamide - PZA, isoniazid - INH, rifampicin - RIF, ethambutol - _ETH,_ streptomycin - STR). The second group _is_ _more_ treacherous. This type of resistance is called extensively _or_ _extremely_ drug-resistant TB (XDR-TB) with the resistance _to_ the first-line _anti-TB_ agents _isoniazid_ and rifampicin together with the resistance _to_ any fluoroquinolone used in _the_ _therapy_ and to at _least_ one of three injectable _second-line_ antituberculotic drugs _(amikacin,_ kanamycin or _capreomycin)_ \[[@B3-molecules-19-00651]\]. _The_ last and the latest category _was_ the _most_ dreaded and was called totally drug-resistant TB (TDR-TB) _and_ the first _case_ was recorded in India \[[@B4-molecules-19-00651]\]. _These_ _mycobacterial_ strains were _resistant_ to all _current_ known therapy. Nowadays, _this_ _group_ has disappeared with the approval of bedaquiline for _therapy_ _of_ resistant forms of tuberculosis. Another problem is connected with the HIV pandemic. These two infectious diseases are _influencing_ each _other_ _in_ a synergic way _so_ this has resulted _in_ _efforts_ to develop new _anti-tubercular_ agents. This _work_ deals with _a_ microwave-assisted synthesis _of_ pyrazinamide analogues _with_ _potential_ antimycobacterial activity. It is caused by the fact that pyrazinamide is counted _among_ the first-line _anti-tuberculosis_ drugs used in current therapy. Its unique _ability_ to kill the dormant forms of _*Mycobacterium_ tuberculosis* is crucial _in_ shortening the time needed _for_ the _treatment,_ so _PZA_ has _sterilizing_ _activity_ especially in combination with _rifampicin_ _\[[@B5-molecules-19-00651]\]._ PZA itself _has_ multiple mechanisms _of_ action. _The_ first described was the activation of this prodrug _*via*_ the _enzyme_ pyrazinamidase (EC 3.5.1.19) to form pyrazinoic acid (POA). This metabolite _causes_ a lowering _of_ the inner compartment _pH_ in _mycobacterial_ _cells._ This leads to _inhibition_ of membrane transport and then to _cellular_ death \[[@B6-molecules-19-00651],[@B7-molecules-19-00651],[@B8-molecules-19-00651]\]. The gene encoding this enzyme is called *pncA* gene and its mutation is responsible for the origin _of_ mycobacterial _resistance_ to PZA \[[@B9-molecules-19-00651]\]. The second _mechanism_ _of_ action _is_ connected with fatty acid synthase I _(FAS_ _I)_ _(EC_ 2.3.1.85). It is suggested that _the_ disruption of metabolism can be caused by inhibition of _the_ cell membrane synthesis, which is essential for the survival of *Mycobacteria*, but _this_ mechanism was mainly rejected for _PZA_ itself _by_ Boshoff because there is only low _inhibition_ _\[[@B10-molecules-19-00651]\]._ On the other _hand,_ the PZA analogues such as 5‑chloropyrazinamide, esters of pyrazinoic _acid_ and _esters_ _of_ 5‑chloropyrazinoic _acid_ were proven to act _in_ this _way_ _\[[@B11-molecules-19-00651],[@B12-molecules-19-00651],[@B13-molecules-19-00651]\]._ _Recent_ research has _suggested_ a novel mechanism _of_ action of PZA - inhibition _of_ _*trans*-translation._ This process is vital for survival _and_ _virulence_ of *Mycobacteria* and _its_ inhibition leads to blockage of the proteosynthetic apparatus in ribosomes _and_ to cellular _death._ These assumptions _were_ proven by Zhang *et al.* _\[[@B14-molecules-19-00651]\]._ Although PZA is _the_ first-line antituberculotic _drug,_ it was found that this molecule has exhibited other interesting biological _activities_ such as antifungal, antibacterial, _antiviral_ and antineoplastic effects \[[@B15-molecules-19-00651],[@B16-molecules-19-00651],[@B17-molecules-19-00651],[@B18-molecules-19-00651],[@B19-molecules-19-00651],[@B20-molecules-19-00651]\]. There is another _application_ of PZA derivatives that can _be_ used in agriculture. The most _successful_ _pyrazine_ derivative diquat-dibromide _(6,7-dihydrodipyrido\[1,2-a:2\',1\'-c\]pyrazinediium-dibromide),_ _a_ non-selective, _contact_ herbicide, which has been _used_ to _control_ many submerged and floating aquatic macrophytes, was found to interfere with the photosynthetic process by releasing strong oxidizers that _rapidly_ _disrupt_ and inactivate _cells_ _and_ cellular functions (at present banned in many _EU_ countries) \[[@B21-molecules-19-00651]\]. Many structural _variations_ of pyrazine compounds with _herbicidal_ _properties_ can be found in the patent _literature_ \[[@B22-molecules-19-00651],[@B23-molecules-19-00651],[@B24-molecules-19-00651],[@B25-molecules-19-00651]\]. _However,_ several _pyrazine_ derivatives were also _described_ as inhibitors of Hill _reaction_ which inhibit _photosynthetic_ electron transport (PET) in photosystem (PS) _2_ _\[[@B18-molecules-19-00651],[@B26-molecules-19-00651]\]._ The site of _action_ of these PET inhibitors in the photosynthetic apparatus was situated predominantly on _the_ donor _side_ _of_ _PS2,_ in the section between _oxygen_ evolving _complex_ and intermediate D^·^, *i.e.*, tyrosine radical (Tyr~D~•) occurring on the 161^st^ position _in_ D~2~ _protein._ _Consequently,_ these _compounds_ _can_ be _considered_ as PS2 herbicides _which_ _could_ have ultimately adverse effect _on_ _plant_ growth. In general, the PET-inhibiting effectiveness of pyrazine _derivatives_ depends on compound lipophilicity and σ Hammett constants _of_ individual substituents. Hosseini *et al.* studied the electronic and _structural_ _descriptors,_ which are _the_ _main_ factors for the cytotoxicity in the series of substituted _*N*-phenylpyrazine-2-carboxamides_ \[[@B27-molecules-19-00651]\]. This study is focused on _preparation_ of *N*-substituted structural _and_ functional derivative of PZA (5-chloro-6-methylpyrazine-2,3-dicarbonitrile) that _was_ treated with ring-substituted benzylamines using the _advantages_ _of_ a microwave reactor. It should be stressed that this type of syntheses has become popular due to its higher yields, shorter reaction _times_ or solvent savings in comparison with conventional _organic_ syntheses \[[@B28-molecules-19-00651]\]. One of the _main_ advantages is the _heating._ It is uniform through the _volume_ of _the_ sample and the microwaves _usually_ interact with _molecules_ themselves not vessel sides. _Another_ benefit is _connected_ with _the_ temperature reached by _the_ solvent used. The final temperature _is_ usually _far_ higher _than_ the _standard_ boiling point of the solvent when _using_ over- pressurized systems. It is reached and bypassed in seconds. Improved heating _usually_ leads to higher yields and shorter reaction times. There is one _limitation_ for choosing the conditions. It is the _polarity_ of the solvent when the non-polar solvents _cannot_ _be_ used in _the_ way the polar ones can be. If _the_ _polar_ solvent is used _in_ the reaction, there _is_ a _direct_ coupling of _microwaves_ with _molecules._ More polar solvents have greater ability to interact with microwave radiation. _Using_ the solvents _with_ low _polarity_ _(low_ _absorbers)_ leads to _longer_ times _of_ _heating_ and _reaction._ On the contrary, if _the_ reagents themselves are polar it _could_ lower _the_ disadvantages _of_ non-polar solvents. Finally there are _new_ approaches to _microwave_ accelerated methods using _ionic_ _liquids_ or solid phase reactions (adsorption on mineral oxides, phase transfer _catalysis,_ neat _reactions)_ \[[@B29-molecules-19-00651]\]. Microwave _assisted_ condensation in polar solvent _is_ used _in_ this work to accelerate the aminodehalogenation _reaction._ The conditions for the synthesis were proven experimentally. _Antimycobacterial_ activity _of_ the all prepared compounds was determined and compounds were evaluated also _in_ _relation_ to _inhibition_ of photosynthetic _electron_ transport (PET) in _spinach_ (*Spinacia oleracea* L.) chloroplasts. _The_ structure-activity relationships between _the_ chemical structure and *in vitro* _biological_ activities _of_ evaluated compounds are discussed. 2. Results _and_ Discussion ========================= 2.1. _Chemistry_ -------------- The _starting_ compound _5-chloro-6-methylpyrazine-2,3-dicarbonitrile_ and the final compounds **1**--**15** were _synthesized_ _according_ to the general procedure shown in [Scheme 1](#molecules-19-00651-f006){ref-type="scheme"}. The aminodehalogenation reaction of this _starting_ compound and ring-substituted benzylamines yielded a series of 15 _secondary_ amines of _which_ 14 were novel. 5-(Benzylamino)-6-methylpyrazine-2,3-dicarbonitrile (**6**) _was_ previously _synthesised_ by Takematsu *et al.* and the reported melting point was 118--119 °C _\[[@B30-molecules-19-00651]\]._ _The_ compound we obtained melted at 128.7--130.7 °C. This _difference_ can be caused by the mode of crystallization. All _reactions_ were |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.