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+    "id": "PMC2527529",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527529\nAUTHORS: Anbarasu Kumarasamy, Edathara C. Abraham\n\nABSTRACT:\nBackgroundSignificant portion of αA-crystallin in human lenses exists as C-terminal residues cleaved at residues 172, 168, and 162. Chaperone activity, determined with alcohol dehydrogenase (ADH) and βL-crystallin as target proteins, was increased in αA1–172 and decreased in αA1–168 and αA1–162. The purpose of this study was to show whether the absence of the C-terminal residues influences protein-protein interactions with target proteins.Methodology/Principal FindingsOur hypothesis is that the chaperone-target protein binding kinetics, otherwise termed subunit exchange rates, are expected to reflect the changes in chaperone activity. To study this, we have relied on fluorescence resonance energy transfer (FRET) utilizing amine specific and cysteine specific fluorescent probes. The subunit exchange rate (k) for ADH and αA1–172 was nearly the same as that of ADH and αA-wt, αA1–168 had lower and αA1–162 had the lowest k values. When βL-crystallin was used as the target protein, αA1–172 had slightly higher k value than αA-wt and αA1–168 and αA1–162 had lower k values. As expected from earlier studies, the chaperone activity of αA1–172 was slightly better than that of αA-wt, the chaperone activity of αA1–168 was similar to that of αA-wt and αA1–162 had substantially decreased chaperone activity.Conclusions/SignificanceCleavage of eleven C-terminal residues including Arg-163 and the C-terminal flexible arm significantly affects the interaction with target proteins. The predominantly hydrophilic flexible arm appears to be needed to keep the chaperone-target protein complex soluble.\n\nBODY:\nIntroductionThe major proteins in the vertebrate eye lens are α-, β-, and γ-crystallins the predominant one being the α-crystallin. α-Crystallin consists of two nearly homologous subunits, namely, αA- and αB-crystallins and both having a molecular mass of 20 kDa in the monomer form and contain 173 and 175 amino acid residues respectively [1]–[3]. Both αA- and αB-crystallins belong to the class of small heat shock proteins [4] and function as molecular chaperones having the ability to prevent aggregation of partially unfolded proteins [5]–[7]. The model structure of α-crystallin consists of a globular N-terminal domain and a C-terminal domain containing an exposed C-terminal arm rich in hydrophilic amino acids, whereas the C-terminal stretch of 80–100 residues known as the ‘α-crystallin/sHsp domain’ are highly conserved [8].C-terminal cleavage of αA-crystallin at residues 162, 168, and 172 has been reported earlier [9]–[15]. The major post-translational modification which occurs in human αA-crystallin is the loss of the C-terminal serine residue [9], [14], [15]. Enhanced cleavage of the C-terminal residue of αA-crystallin in diabetic human lenses has been reported in our earlier study, the average level of the truncated αA-crystallin increased from 30% to 50% [15]. Aziz et al\n[16] have recently reported modification in the oligomeric structure and chaperone function of the various truncated human αA-crystallins. Interestingly, the truncated αA1–172 exhibited significant increase in its oligomeric size as well as chaperone activity. The oligomeric size of αA1–168 was similar to that of αA-wild type (αA-wt) whereas the chaperone activity was moderately decreased. αA1–162, on the other hand, showed substantial decrease in the oligomeric size as well as the chaperone activity. If indeed chaperone to target protein binding is an essential step for αA-crystallin to operate as a molecular chaperone, characterization of the interaction of the truncated αA-crystallins with target proteins should show why their chaperone function is altered as a result of the truncation. In this study, we have used fluorescence resonance energy transfer (FRET) to study chaperone - target protein interaction using ADH and βL-crystallin, two widely used target proteins [16], [17], and recombinant αA-wt and C-terminal truncated αA-crystallins.ResultsLevels of fluorescence labeling of human αA-wt, C-terminal truncated αA-crystallins and the target proteinsThe level of subunit exchange between two different proteins was determined by FRET. In order to determine the in vitro FRET level two fluorescent dyes with overlapping fluorescence spectra are necessary. SITS and LYI are widely used fluorescent dyes with spectral overlaps and safe from structural alterations due to fluorescent tags [18]–[20]. So, in the present study the amine specific fluorescent probe SITS was attached to human αA-wt and the C-terminal truncated αA-crystallins and the cysteine specific LYI fluorescent probe was attached to the target proteins ADH and βL-crystallin. The level of labeling was determined spectrophotometrically using molar extinction coefficients of 47,000 mol−1 cm−1 at 336 nm for SITS and 11,000 mol−1 cm−1 at 426 nm for LYI. The level of labeling was about 0.8, 1.12, 1.17, 1.04 and 7.1 and 1.56 moles for αA-wt, αA1–172, αA1–168, αA1–162, ADH and βL-crystallin respectively. Differences in the level of labeling of the two different fluorophores were taken into account while computing the data.Subunit exchange between labeled αA-crystallins and the target protein ADHThe interaction between the SITS -labeled human αA-wt and the C-terminal truncated αA-crystallins and the LYI- labeled target protein ADH was initiated by mixing equimolar concentration of the proteins in 20 mM MOPS buffer with 100 mM NaCl and 10 mM EDTA at 37°C. EDTA was used for unfolding ADH so that αA-crystallin will bind to ADH under the same condition as used for the chaperone assay. The rate of subunit exchange with ADH was determined by FRET analysis. Figure 1 shows the fluorescence spectra showing the time dependent decrease in SITS emission intensity at 426 nm and a concomitant increase in LYI fluorescence at 515 nm. After 30 min at 37°C, there was no remarkable change in the emission intensity at 515 nm due to the achievement of stable equilibrium (data not shown). The fluorescence emission spectra illustrate that the highest increase in the acceptor spectrum at 515 nm was observed in αA-wt when compared to the C-terminal truncated αA-crystallins (Fig. 1). We have calculated the rate of subunit exchange from the increase in acceptor fluorescence intensity after taking into account differences in the levels of tagging of the various proteins by the probes. Figure 2A shows the plot of Ft/ F0 of LYI at 515 nm as a function of time, where Ft and F0 are the emission intensities at time t and zero respectively. The rate constant was obtained by fitting the data to the exponential function Ft/F0 = A1+A2 e-kt, where A1 and A2 are constants and k is the rate constant for subunit exchange. The increase in the relative fluorescence intensity at 515 nm is due to fluorescence resonance energy transfer from donor SITS-labeled human αA-wt and C-terminal truncated αA-crystallins' fluorophore to the acceptor LYI-labeled ADH during the interaction. The maximum relative fluorescence intensity was seen with αA-wt whereas αA 1–172 had lower fluorescence intensity, αA1–168 showed further decrease in the fluorescence intensity, and αA1–162 showed the lowest. The subunit exchange rates or the k values are summarized in Table 1 which is in agreement with the above observation. For instance, αA-wt had the highest k value (2.073) whereas the k values of αA1–172, αA1–168, and αA1–162, respectively, were 6, 24, and 43% lower. Figure 2B illustrates the decrease in the relative fluorescence at 426 nm when the fluorescence resonance energy is transferred to the acceptor target protein. The rate constant (k) values (Table 1) were higher when collected and calculated at donor energy (at 426 nm) than when calculated at acceptor energy (at 515 nm). This is believed to be was due to fluorescence quenching because no direct transfer of fluorescence energy occurs where there is no sufficient contact between the residues carrying the two probes. The amount of loss due to quenching is comparatively lower in both αA-wt and αA1–172 than in αA1–168 and αA1–162.10.1371/journal.pone.0003175.g001Figure 1Time –dependent spectral changes in the emission spectra of SITS-labeled αA- crystallins interacting with LYI- labeled ADH.The emission spectra of fluorescence- labeled αA crystallin excited at 336 nm were recorded at every 2 min intervals at 0, 2,4,6,8,10,12,14,16,18,20,22,24,26,28 and 30 minutes after mixing of SITS – labeled αA - wt and its C-terminal truncated αA1–172, αA1–168, and αA1–162 with LYI – labeled ADH in 1∶1 ratio, with a final protein concentration of 1 mg/ml at 37°C. The decrease in fluorescence intensity at 426 nm of SITS- labeled αA- crystallins and concomitant increase in fluorescence intensity at 515 nm of LYI- labeled ADH is indicative of energy transfer.10.1371/journal.pone.0003175.g002Figure 2A: Relative fluorescence intensity at 515 nm due to subunit exchange of SITS – labeled αA-crystallins with LYI – labeled ADH.Time – dependent increase in acceptor fluorescence emission intensity is due to subunit exchange as evident from energy transfer from the SITS – labeled protein to the LYI – labeled protein. SITS – labeled αA wt (♦), αA1–172(▪), αA1–168 (▴) and αA1–162 (•) was incubated with LYI – labeled ADH. Each curve was analyzed with the best curve fit of the data to the exponential function Ft/F0 = A1+A2 e−kt. B: Relative fluorescence intensity at 426 nm due to subunit exchange of SITS – labeled αA-crystallins with LYI – labeled ADH. Time – dependent decrease in donor fluorescence emission intensity is due to subunit exchange as evident from energy transfer from the SITS – labeled protein to the LYI – labeled protein. SITS – labeled αA wt (♦), αA1–172(▪), αA1–168 (▴) and αA1–162 (•) was incubated with LYI – labeled ADH. Each curve was analyzed with the best curve fit of the data to the exponential decay function Ft/F0 = A1+A2 e−kt.10.1371/journal.pone.0003175.t001Table 1Subunit exchange rate constant (k) of αA-wt and its C-terminal truncated forms interacting with ADH when increase and decrease respectively (Mean±SE).CrystallinSubunit exchange rate constant at 515 nm (×10−4 S−1)Subunit exchange rate constant at 426 nm (×10−4 S−1)αA wt+ADH2.073±0.1698.035±0.373αA1–172+ADH1.947±0.1497.549±0.327αA1–168+ADH1.585±0.1127.170±0.462αA1–162+ADH1.190±0.1115.737±0.403Subunit exchange between αA-crystallins and the target protein βL-crystallinThe interaction between the SITS- labeled human αA-wt and the C-terminal truncated αA-crystallins and the LYI- labeled target protein βL-crystallin was initiated by mixing equimolar concentration of the proteins in 20 mM MOPS buffer with 100 mM NaCl at 62°C. Higher temperature was necessary to unfold βL-crystallin. Figure 3 shows the fluorescence spectra and Figure 4A and 4B show the increase and decrease in relative fluorescence intensity or Ft/F0 at 515 nm and 426 nm, respectively as a function of time. The k values which reflect the data in Figure 4A and 4B were summarized in Table 2. Interestingly, the acceptor gain rate constant (k) value for αA1–172 was maximal at 2.422 and it was about 8% higher than the value for αA-wt and about 41% higher than the values for αA1–168 and αA1–162. The same trend was noticed in donor k values determined at 426 nm also. The C-terminal truncated αA1–172 was maximal at 6.391 which was about 24% higher than that of αA-wt and 34% and 54% higher than those of αA1–168 and αA1–162 respectively. As mentioned above, here also at the time of energy transfer from donor fluorophore to the acceptor fluorophore some energy loss was noticed. The amount of energy lost was about 54–66%. The subunit exchange rate constant (k) values clearly showed that in αA-crystallin and the counterparts with ADH at both emission intensities (at 426 & 515 nm) the αA-wt showed the highest interaction compared to the C-terminally truncated αA-crystallins and among the C-terminally truncated αA-crystallins, αA1–172 showed higher k value followed by αA1–168 and αA1–162 in that order. However, the subunit exchange rate constant (k) values in αA-crystallin and counterparts with βL crystallin were higher in αA1–172 followed by αA-wt, αA1–168 and αA1–162. Nevertheless, the loss of energy (due to quenching) was least in αA-wt with both target proteins (ADH & βL crystallin) followed by αA1–172, αA1–168 and αA1–162.10.1371/journal.pone.0003175.g003Figure 3Time –dependent spectral changes in the emission spectra of the SITS-labeled αA- crystallins interacting with LYI- labeled βL- crystallin.The emission spectra of fluorescence- labeled αA crystallin excited at 336 nm were recorded at every 2 min intervals at 0, 2,4,6,8,10,12,14,16,18,20,22,24,26,28 and 30 minutes after mixing of SITS – labeled αA - wt and C-terminal truncated αA-crystallins with LYI – labeled βL- crystallin in a 1∶1 ratio, with a final protein concentration of 1 mg/ml at 62°C. The decrease in fluorescence intensity at 426 nm of SITS-labeled αA- crystallins and concomitant increase in fluorescence intensity at 515 nm of LYI- labeled ADH is indicative of energy transfer due to exchange of subunits between the two labeled proteins.10.1371/journal.pone.0003175.g004Figure 4A: Relative fluorescence intensity at 515 nm of SITS – labeled αA-crystallins interacting with LYI – labeled βL- crystallin.Time – dependent increase in acceptor fluorescence in emission intensity is due to subunit exchange. Increase in the relative fluorescence intensity at 515 nm is due to energy transfer from the SITS – labeled protein to the LYI – labeled protein. SITS – labeled αA- wt (♦), αA1–172(▪), αA1–168 (▴) and αA1–162 (•) were incubated with LYI – labeled βL- crystallin. Each curve was analyzed with the best curve fit of the data to the exponential function Ft/F0 = A1+A2 e−kt. B: Relative fluorescence intensity at 426 nm of SITS – labeled αA-crystallins interacting with LYI – labeled βL- crystallin. Time – dependent decrease in donor fluorescence in emission intensity is due to subunit exchange. Decrease in the relative fluorescence intensity at 426 nm is due to energy transfer from the SITS – labeled protein to the LYI – labeled protein. SITS – labeled αA- wt (♦), αA1–172(▪), αA1–168 (▴) and αA1–162 (•) were incubated with LYI – labeled βL- crystallin. Each curve was analyzed with the best curve fit of the data to the exponential function Ft/F0 = A1+A2 e−kt.10.1371/journal.pone.0003175.t002Table 2Subunit exchange rate constant (k) of αA-wt and its C-terminal truncated forms interacting with βL-crystallin when increase and decrease respectively (Mean±SE).CrystallinSubunit exchange rate constant at 515 nm (×10−4 S−1)Subunit exchange rate constant at 426 nm (×10−4 S−1)αA wt+��L-crystallin2.233±0.0104.826±0.876αA1–172+βL-crystallin2.422±0.0396.391±0.501αA1–168+βL-crystallin1.419±0.0084.183±0.287αA1–162+βL-crystallin1.435±0.0043.462±0.267Chaperone activity of αA-wt and the C-terminal truncated αA-crystallinsAlthough the chaperone activity of the truncated human αA-crystallins were reported earlier [16] the chaperone activity assay was repeated in the present study using the ratio of 1∶1 which is the same ratio as used in the FRET analysis. With ADH as the target protein, αA1–172 showed about 18% better chaperone activity than αA-wt, αA1–168 had nearly the same chaperone activity as αA-wt and αA1–162 showed nearly 80% loss (Fig. 5). With βL-crystallin as the protein, both αA1–172 and αA1–168 showed normal chaperone activity whereas αA1–162 showed nearly 60% loss in chaperone activity (Fig. 6).10.1371/journal.pone.0003175.g005Figure 5Effect of C-terminal truncation of αA-crystallin on its chaperone activity as measured with ADH as the target protein.The chaperone activity of αA-wt (▪) and C-terminal truncated αA1–172,(▴) αA1–168 (•) and αA1–162 (−) ADH alone (♦) determined with ADH as the target protein at 1∶1 ratio (0.14 mg/ml) denatured with EDTA at 37°C.10.1371/journal.pone.0003175.g006Figure 6Effect of C-terminal truncation of αA-wt on its chaperone activity as measured with βL-crystallin as the target protein.The chaperone activity of αA-wt (▪) and C-terminal truncated αA1–172,(▴) αA1–168 (•) and αA1–162 (−) βL-crystallin alone (♦) determined with ADH as the target protein at 1∶1 ratio (0.14 mg/ml) at 62°C.Materials and MethodsFluorescence probesLucifer yellow iodoacetamide (LYI) and 4-acetamido-4′-isothiocyanatostilbene-2,-2′-disulfonic acid (SITS) were obtained from Molecular Probes (Eugene, OR, USA). Alcohol dehydrogenase (ADH) and βL-crystallin were purchased form Sigma-Aldrich (St. Louis, USA).Cloning, site-directed mutagenesis, expression and purification of human αA-crystallin wild type (αA-wt), and C-terminal truncated αA-crystallinsCloning of human αA-crystallin and subsequent subcloning into the pET-23d(+) expression vector has been described previously [21]. To generate the different C-terminally truncated human αA-crystallins lacking 1 (αA1–172), 5 (αA1–168), and 11(αA1–162) residues, stop codons were introduced at desired sites as described previously [16]. The pET-23d(+) expression vector harboring DNA constructs of human αA-wt and C-terminally truncated αA1–172, αA1–168, and αA1–162 were expressed in BL21 (DE3) PLysS E. coli cells. The expressed proteins were purified by Sephacryl S-300 HR size exclusion chromatography, the peak fractions collected were concentrated and repurified by molecular sieve HPLC using a 600 mm×7.8 mm BIOSEP-SEC 4000 column (Phenomenex). The purity of the protein preparations was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli [22].Labeling of human αA-wt, C-terminal truncated αA-crystallins and target proteins ADH and βL-crystallin with fluorescence probesLabeling of αA-crystallin and C-terminal truncated αA-crystallins with SITS and the target proteins ADH and βL-crystallin with LYI was done as described previously [20], [21]. Briefly, 10 fold excess of solid SITS was added to 1 ml of a protein solution (3 mg/ml) in 20 mM MOPS buffer containing 100 mM NaCl (pH 7.9) and the reaction was allowed to proceed for about 16 h at room temperature (25°C) in the dark. The unlabeled fluorescent dye was removed from the fluorescently labeled protein on a Sephadex G-25 column equilibrated with 20 mM MOPS buffer containing 100 mM NaCl (pH 7.9). Elution was performed with the same buffer, pooled and concentrated. LYI-labeled protein was prepared as described above with the same buffer in 20 fold excess of the reagent. The extent of labeling was determined spectrophotometrically using molar extinction coefficients of 47,000 mol−1 cm−1 at 336 nm for SITS and 11,000 mol−1 cm−1 at 426 nm for LYI and corrected for the contribution of the dye at 280 nm.Measurement of the rate of subunit exchange among αA-crystallins and the target proteinsThe rate of subunit exchange was measured by the method of fluorescence resonance energy transfer (FRET). The subunit exchange reaction was initiated by mixing equal amounts (0.5 mg/ml) of SITS -labeled αA-wt and C-terminal truncated αA-crystallins (αA1–172, αA1–168 & αA1–162) with the same amount of LYI-labeled target proteins: 1)ADH in 20 mM MOPS buffer containing 100 mM NaCl and 10 mM EDTA (pH 7.9) at 37°C and 2) βL-crystallin in 20 mM MOPS buffer containing 100 mM NaCl (pH 7.9) at 62°C. The fluorescence emission spectra were obtained with Shimadzu RF-5301PC spectrofluorophotometer (Columbia, MD) at an excitation wavelength of 336 nm. Decrease in SITS emission intensity at 426 nm and increase in LYI emission intensity at 515 nm were recorded at every 2 min intervals and the subunit exchange rate constant (k) was calculated by curve fitting an exponential function Ft/ F0 = A1+A2 e−kt where A1 and A2 are constants and k is the rate constant for subunit exchange.Determination of Chaperone activityThe ability of αA-wt and the C-terminal truncated αA-crystallins to prevent the extent of EDTA induced aggregation of ADH and thermal aggregation of βL-crystallin was determined as described before [16], [17]. αA-wt and the C-terminal truncated αA-crystallins were mixed with equal amounts of target proteins (1∶1 ratio) at a total concentration of 0.14 mg/ml. ADH was induced to unfold with 10 mM EDTA in 50 mM PBS buffer (pH 7.9) at 37°C and the βL-crystallin unfolding was thermally induced at 62°C. The extent of aggregation was measured by monitoring the light scattering at 360 nm using Shimadzu UV 160 spectrophotometer equipped with a temperature regulated cell holder.DiscussionWe have shown earlier that cleavage of 1, 5, and 11 C-terminal residues affects or improves chaperone activity depending on the number of residues cleaved and the chaperone assay system [16]. Chaperone activity of the truncated αA-crystallins can vary by two major factors: accessibility to the chaperone sites which are responsible for the chaperoning process or due to enhanced or decreased affinity to the substrate which is being unfolded. The present study focused on the latter aspect by studying the binding of the wild-type and the truncated αA-crystallins with the putative target proteins. The data show that the binding kinetics (k value) reflect the chaperone activity differences with the exception that αA1–172 showed higher chaperone activity, but, lower k value. Thus, we can conclude that the binding affinity to target proteins does not always concur with the chaperone activity data. However, it is noteworthy that all the three truncated αA-crystallins have shown decreased target protein binding.In an earlier study, we have determined the oligomeric size, secondary and tertiary structures and chaperone activity of recombinant human αA-wt and the various C-terminal truncated αA-crystallins [16]. αA1–172, which is the major form of the truncated human αA-crystallin, had a molecular mass of 866 kDa as compared to 702 kDa for αA-wt, when the molecular mass was determined by dynamic light scattering. The chaperone activity of αA1–172 was higher than that of αA-wt, when ADH, insulin and βL-crystallin were used as target proteins and the αA-crystallin : target protein ratio varied between 1∶1 and 1∶20. In this study, we have used equal amounts of α-crystallin and target protein, ie, 1∶1 ratio, in all the assays to avoid artificially exaggerated differences between αA-wt and the truncated αA-crystallins. As shown in Figures 5 and 6, αA1–172 had slightly better chaperone activity than αA-wt, which confirms our previous observation [16]. However, αA-crystallin-target protein binding studies gave conflicting results. For instance, the relative fluorescence intensity due to FRET and the k values were lower in αA1–172 (Figs. 2 & 4; Tables 1 & 2). Both the present study and the earlier study [16] have shown αA1–168 having nearly the same chaperone activity as αA-wt. however, the FRET studies gave contradicting results as the relative fluorescence intensity as well as the k values were significantly lower (Figs. 2 & 4; Tables 1 & 2). With both the ADH and the βL-crystallin target protein methods, αA1–162 showed the lowest chaperone activity (Figs. 5 & 6) as shown earlier. Interestingly, αA1–162 also showed the lowest level of relative fluorescence intensity and k value during FRET studies. Thus, the cleavage of 11 C-terminal residues of αA-crystallin, which is known to affect its secondary and tertiary structures [16], severely affects its binding to target proteins which in turn affect its ability to function as a molecular chaperone.By using FRET analysis, we have recently investigated the effect of cleavage of the C-terminal residues of human αA-crystallin on subunit exchange with αB-crystallin forming heteroaggregates [21]. The subunit exchange rate or the k value for αA1–172 interacting with αB-wt was decreased by 50% compared to αA-wt interacting with αB-wt. Likewise, the k value was decreased 40% when αA1–168 interacted with αB-wt, whereas interaction of αA1–162 with αB-wt showed 84% decrease in the k value. Thus, cleaving 11 residues including lysine-163 had shown the most effect. The importance of lysine-163 in maintaining the oligomeric structure of αA-crystallin has been demonstrated in a recent study [23]. In fact, in αA1–162 the secondary and tertiary structures were significantly altered and the molecular mass was substantially decreased [16]. Such changes affect binding to αB-crystallin and heterooligomerization [21] and also seem to affect binding to target proteins decreasing the chaperone activity.\n\nREFERENCES:\n1. Van Der OuderraFJDe JongWWBloemendalH\n1973\nThe amino- acid sequence of the alphaA2 chain of bovine alpha-crystallin.\nEur J Biochem\n39\n207\n222\n4770792\n2. Van Der OuderraFJDe JongWWHilderinkABloemendalH\n1974\nThe amino-acid sequence of the alphaB2 chain of bovine alpha-crystallin.\nEur J Biochem\n49\n157\n168\n4477528\n3. De JongWWTerwindtECBloemendalH\n1975\nThe amino-acid sequence of the A chain of human alpha-crystallin.\nFEBS Lett\n58\n310\n313\n817940\n4. IngoliaTDCraigEA\n1982\nFour small Drosophila heat shock proteins are related to each other and to mammalian α-crystallin.\nProc Natl Acad Sci USA\n79\n2360\n2364\n6285380\n5. HorwitzJ\n1992\nα-Crystallin can function as a molecular chaperone.\nProc Natl Acad Sci USA\n89\n10449\n10\n1438232\n6. WangKSpectorA\n1994\nThe chaperone activity of bovine alpha-crystallin. Interaction with other lens crystallins in native and denatured states.\nJ Biol Chem\n269\n13601\n13608\n7909809\n7. RaoPVHuangQLHorwitzJZiglerJS\n1995\nEvidence that alpha-crystallin prevents non-specific protein aggregation in the intact eye lens.\nBiochim Biophys Acta\n1245\n439\n447\n8541324\n8. WistowG\n1985\nDomain structure and evolution in α-crystallins and small heat shock proteins.\nFEBS Lett\n18\n1\n6\n9. TakemotoLJ\n1995\nIdentification of the in vivo truncation sites at the C-terminal region of alpha-A-crystallin from aged bovine and human lens.\nCurr Eye Res\n14\n837\n841\n8529423\n10. LundALSmithJBSmithDL\n1996\nModifications of the water-insoluble human lens alpha-crystallins.\nExp Eye Res\n63\n661\n672\n9068373\n11. ColvisCGarlandD\n2002\nPosttranslational modification of human αA-crystallin: correlation with electrophoretic migration.\nArch Biochem Biophys\n397\n319\n323\n11795889\n12. MaZHansonSRLampiKJDavidLLSmithDLSmithJB\n1998\nAge-related changes in human lens crystallins identified by HPLC and mass spectrometry.\nExp Eye Res\n67\n21\n30\n9702175\n13. MiesbauerLRZhouXYangZSunYSmithDL\n1994\nPosttranslational modifications of water-soluble human lens crystallins from young adults.\nJ Biol Chem\n269\n12494\n12502\n8175657\n14. TakemotoLJ\n1998\nQuantitation of specific cleavage sites at the C-terminal region of alpha-A-crystallin from human lenses of different age.\nExp Eye Res\n66\n263\n266\n9533852\n15. ThampiPHassanASmithJBAbrahamEC\n2002\nEnhanced C-terminal truncation of alphaA- and alphaB-crystallins in diabetic lenses.\nInvest Ophthalmol Vis Sci\n43\n3265\n3272\n12356833\n16. AzizASanthoshkumarPSharmaKKAbrahamEC\n2007\nCleavage of the C-terminal serine of human αA-crystallin produces αA1–172 with increased chaperone activity and oligomeric size.\nBiochemistry\n46\n2510\n2519\n17279772\n17. ShroffNPBeraSCherian-ShawMAbrahamEC\n2001\nSubstituted hydrophobic and hydrophilic residues at methionine-68 influence the chaperone-like function of αB-crystallin.\nMol Cell Biochem\n220\n127\n133\n11451372\n18. BeraSAbrahamEC\n2002\nThe αA-crystallin R116C mutant has a higher affinity for forming heteroaggregates with αB-crystallin.\nBiochemistry\n41\n297\n305\n11772029\n19. KallurLSAzizAAbrahamEC\n2008\nC-Terminal truncation affects subunit exchange of human αA-crystallin with αB-crystallin.\nMol Cell Biochem\n308\n85\n91\n17909943\n20. GhahghghaeiARekasAPriceWECarverJA\n2007\nThe effect of dextran on subunit exchange of the molecular chaperone αA-crystallin.\nBiochim Biophys Acta\n1774\n102\n111\n17118727\n21. ShroffNPCherian-ShawMBeraSAbrahamEC\n2000\nMutation of R116C results in highly oligomerized αA-crystallin with modified structure and defective chaperone-like function.\nBiochemistry\n39\n1420\n1426\n10684623\n22. LaemmliUK\n1970\nCleavage of structural proteins during the assembly of the head of bacteriophage T4.\nNature\n227\n680\n685\n5432063\n23. RajanSChandrashekarAzizAAbrahamEC\n2006\nRole of arginine-163 and the 163 REEK 166 motiff in the oligomerization truncated αA-crystallins.\nBiochemistry\n45\n15684\n15691\n17176090"
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batch_9/PMC2527561.json

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+{
+    "id": "PMC2527561",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527561\nAUTHORS: Timothy H Sulahian, Amy Imrich, Glen DeLoid, Aaron R Winkler, Lester Kobzik\n\nABSTRACT:\nBackgroundScavenger receptors are important components of the innate immune system in the lung, allowing alveolar macrophages to bind and phagocytose numerous unopsonized targets. Mice with genetic deletions of scavenger receptors, such as SR-A and MARCO, are susceptible to infection or inflammation from inhaled pathogens or dusts. However, the signaling pathways required for scavenger receptor-mediated phagocytosis of unopsonized particles have not been characterized.MethodsWe developed a scanning cytometry-based high-throughput assay of macrophage phagocytosis that quantitates bound and internalized unopsonized latex beads. This assay allowed the testing of a panel of signaling inhibitors which have previously been shown to target opsonin-dependent phagocytosis for their effect on unopsonized bead uptake by human in vitro-derived alveolar macrophage-like cells. The non-selective scavenger receptor inhibitor poly(I) and the actin destabilizer cytochalasin D were used to validate the assay and caused near complete abrogation of bead binding and internalization, respectively.ResultsMicrotubule destabilization using nocodazole dramatically inhibited bead internalization. Internalization was also significantly reduced by inhibitors of tyrosine kinases (genistein and herbimycin A), protein kinase C (staurosporine, chelerythrine chloride and Gö 6976), phosphoinositide-3 kinase (LY294002 and wortmannin), and the JNK and ERK pathways. In contrast, inhibition of phospholipase C by U-73122 had no effect.ConclusionThese data indicate the utility of scanning cytometry for the analysis of phagocytosis and that phagocytosis of unopsonized particles has both shared and distinct features when compared to opsonin-mediated phagocytosis.\n\nBODY:\nBackgroundLung infection is responsible for more disability-adjusted life years lost than any other disease [1] and high levels of inhaled dusts have been linked in several epidemiological studies to increases in ear and airway infections, cardiovascular disease, lung cancer and mortality [2-5]. Alveolar macrophages (AMs) are a first line of defense against inhaled bacteria and environmental dusts. Therefore, understanding the mechanism by which AMs defend against inhaled insults is crucial. Since contact with inhaled particles often takes place before an antibody response has occurred or with particles for which specific antibodies are not readily made, the AM relies on innate receptors to recognize inhaled particles.Scavenger receptors (SRs) are a key component of the innate immune system. In addition to their well-known role in low-density lipoprotein metabolism, SRs play a critical role in AM clearance of inhaled particles by binding and allowing the cells to internalize unopsonized microorganisms, apoptotic bodies and environmental dusts [6,7]. General blockade of SRs using polyanionic inhibitors results in a dramatic reduction of AM uptake of residual oil fly ash, ambient air particles, diesel dust, iron oxide, titanium dioxide, silica, Escherichia coli and Staphylococcus aureus [8-11]. Specific blockade and transfection of members of the SR family have shown these receptors to be capable of binding several Gram-positive and Gram-negative bacteria as well as isolated lipopolysaccharide and lipotechoic acid [12-21]. In addition, mice deficient in SR-A or MARCO demonstrate reduced bacterial clearance, increased pulmonary inflammation and increased mortality following an intranasal challenge with Streptococcus Pneumoniae [10,22]. Furthermore, MARCO can bind CpG DNA [23], whereas blockade of MARCO with a monoclonal antibody dramatically reduces AM uptake of titanium dioxide, iron oxide, silica and latex beads [24,22,25]. SR-A and MARCO, therefore, are clearly critical components of pulmonary host defense. However, it is important to point out that AMs also express several other less well-characterized SRs including LOX-1, SR-PSOX and SRCL [10]. These SRs are capable of binding bacteria [26-28] and might also contribute to the AM response to inhaled insults.While it is clear that SR-initiated uptake of inhaled particles is critically important for lung defense, it is currently not known which signaling pathways are necessary for SR-mediated phagocytosis. In contrast, phagocytosis of opsonized particles (via Fc or complement receptors) has been well characterized [29]. Many characteristics of opsonin-mediated phagocytosis are shared by both Fc and complement receptors (such as signaling by tyrosine kinase, protein kinase C (PKC), phosphoinositide-3 kinase (PI-3K), mitogen activated protein kinases (MAPK) and phospholipase Cγ (PLCγ)). In contrast, some characteristics are unique to one receptor pathway (such as sensitivity of complement-mediated uptake to microtubule inhibitors) [30]. Many of these opsonin-mediated phagocytic signaling pathways have also been implicated in non-phagocytic SR-mediated responses such as cytokine production and lipoprotein endocytosis [31-38]. We hypothesized that these pathways would also be necessary for SR-mediated phagocytosis. To test this, we employed a battery of well-established signaling inhibitors and a novel high-throughput fluorescence phagocytosis assay.AMs are known to express a wide array of SRs with overlapping ligand specificities. Therefore, it is likely that inhaled particles are simultaneously bound by multiple SR family members. Since the underlying biology of the particle-AM interaction is more complicated than a simple one ligand/one receptor interaction, we chose a target particle (latex spheres) that likewise binds multiple SRs to more closely model the true physiology of particle-AM interactions. It should be noted that the latex sphere has long been used as a model for inhaled particulates and is similar to 'real world' particles in terms of its SR-mediated uptake by AM [10,39,9,25,42].MethodsCell isolation, differentiation and characterizationDiscarded platelet apheresis collars were obtained from the Kraft Family Blood Donor Center at the Dana-Farber Cancer Institute (Boston, MA, USA). Buffy coats were harvested from these collars and enriched for monocytes using the RosetteSep Monocyte Enrichment kit (Stem Cell Technologies, Vancouver, BC, Canada). Monocytes were then cultured in Vuelife bags (American Fluoroseal, Gaithersburg, MD, USA) for 11 days at 5% CO2 and 37°C in RPMI/10% FBS/20 μg/ml gentamicin supplemented with 20 ng/ml human granulocyte/macrophage-colony stimulating factor (GM-CSF, Peprotech, Rocky Hill, NJ, USA). GM-CSF matured MØ (GM-MØ) were then harvested and resuspended at 1 × 106/ml in RPMI/10% FBS. 1 × 105 cells were dispensed into black-walled 96 well Micro-Clear plates (Greiner Bio-One, Monroe, NC, USA). For some experiments, the number of cells per well was altered but the volume remained constant. After plating and adherence, GM-MØ were incubated for 40–44 hours, and then used to measure particle binding and internalization.Some GM-MØ were characterized by flow cytometry before being plated for experiments. Cells were stained with anti-PSOX (10 μg/ml, provided by Dr. Kimihisa Ichikawa, Sankyo, Tokyo, Japan), anti-LOX-1 (10 μg/ml, Cell Sciences, Inc., Canton, MA, USA), anti-SR-A (10 μg/ml, provided by Dr. Motohiro Takeya, Kumamoto University, Kumamoto, Japan), anti-CD68 (10 μg/ml, Dako, Carpinteria, CA, USA), anti-CD14 (3.3 μg/ml), anti-HLA-DR (10 μg/ml), anti-HLA-DQ (10 μg/ml) or equal concentrations of isotype matched control antibodies (all from BD Biosciences, Rockville, MD, USA) in PBS with 2 mg/ml bovine serum albumin (BSA) and 4 mg/ml human IgG (both from Sigma, St. Louis, MO, USA). This step was followed by staining with 20 μg/ml Alexafluor 488 labeled F(ab')2 goat anti-mouse antibodies (Invitrogen, Carlsbad, CA, USA) and fixation in PBS with 1% paraformaldehyde. Other cells were stained with 10 μg/ml PLK-1 (anti-MARCO [10]) or control IgG that had been biotinylated using biotin-X-NHS (Calbiochem, San Diego, CA, USA). This was followed by secondary staining with 7.5 μg/ml streptavidin-phycoerythrin (Invitrogen) and fixation as described above. Cellular fluorescence was measured using a Coulter Epics Elite flow cytometer (Beckman Coulter, Miami, FL, USA).Cells were also evaluated for their ability to bind unopsonized latex beads in the presence or absence of SR inhibitors. One hundred microliters of GM-MØ (suspended at 2 × 106/ml in HBSS/0.3% BSA) were plated in each well of a low adherence 96-well plate (Corning, Corning NY, USA). One hundred microliters of 20 μg/ml polyinosinic acid (poly (I)), 20 μg/ml chondroitin sulfate (both from Sigma), 20 μg/ml PLK-1 mAb or 20 μg/ml mIgG3 isotype control (eBioscience, San Diego, CA, USA) were added and cells were allowed to incubate for 10 minutes at 37°C. One hundred microliters of green fluorescent latex beads (1 μm, Invitrogen) were added at a concentration of 1 × 108/ml in HBSS/0.3%BSA with or without 10 μg/ml poly(I), 10 μg/ml chondroitin sulfate, 10 μg/ml PLK-1 or 10 μg/ml mIgG3. This corresponds to a 50:1 bead to cell ratio. Cells were incubated for 30 minutes at 37°C, with gentle pipetting every 10 minutes to resuspend the cells and beads. After incubation, the assay was stopped by chilling cells on ice and analyzing fluorescence by flow cytometry.For mouse studies, primary AMs were isolated from C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME, USA). Immediately before bronchialveolar lavage, mice were euthanized by an overdose of Phenobarbital. The lungs were lavaged six times with 0.8 ml of ice-cold PBS. Cell purity and yield was determined using a hemocytometer. Murine AMs were cultured in black-walled 96 well Micro-Clear plates in RPMI/10% FBS for 40–44 hours before phagocytosis assays were performed as described for GM-MØ.Preparation of biotinylated latex beadsBiotin-BSA was generated by incubating 50 mg of tissue culture grade BSA (Sigma) with 30 mg biotin-X-NHS in 10 ml PBS for one hour at room temperature. Unconjugated biotin was removed by extensive dialysis. Green fluorescent carboxylated latex beads (1 μm, Invitrogen) were centrifuged at high speed and washed twice in 2-(N-morpholino)ethanesulfonic acid (MES, Calbiochem) buffer (19.2 mg/ml, pH 6.0). Beads were suspended at 5 × 109 per ml in MES buffer. Water-soluble carbodimide (WSC, Calbiochem) was freshly dissolved in MES buffer and beads were incubated at room temperature for one hour with 10 mg/ml WSC. Beads were washed twice in 0.5 × PBS and resuspended in water. An equal volume of biotin-BSA was added for a final concentration of 2 mg/ml BSA in 0.5 × PBS. Beads were incubated overnight at room temperature and then centrifuged at high speed. Beads were then resuspended in 0.5 × PBS with 40 mM glycine and incubated for one hour. Finally, beads were washed twice in PBS containing 0.2% BSA and 0.01% sodium azide and stored at 4°C.Internalization assayAll reagents and buffers were at room temperature when added to cells and all incubations were performed in warm (37°C) humid air unless otherwise noted. All fluorescent dyes were purchased from Invitrogen. Cells were incubated with CellTracker Blue at 100 μM in HBSS with Ca++ and Mg++ (Cambrex, East Rutherford, NJ, USA) for 40 minutes followed by a 30 minute recovery period in assay buffer (HBSS/0.3% BSA). Inhibitors (Table 1) or DMSO were then added for 20 minutes. Poly(I), cytochalasin D, nocodazole, staurosporine, wortmannin and herbimycin A were purchased from Sigma. All other inhibitors were purchased from Calbiochem. GM-MØ were then incubated for 20 minutes with bead suspension (2 × 108 beads/ml) +/- inhibitors for bead binding and internalization. Cells were then washed 2 × 250 μl with assay buffer, covered with fresh buffer +/- inhibitors and incubated for an additional 20 minutes to allow for further bead internalization (the cells were, therefore, incubated with inhibitors for a grand total of 60 minutes). After this the cells were washed and extracellular beads were labeled on ice for 30 minutes using streptavidin-Texas Red (20 μg/ml in assay buffer). After a final wash with 250 μl assay buffer, cells were fixed with 4% paraformaldehyde in PBS. The fixative was removed after 30 minutes and cell nuclei were stained for 30 minutes with 3 μg/ml of Hoechst 33342. The Hoechst dye was then removed and wells were filled with 100 μl of 4% paraformaldehyde in PBS for storage.Table 1Summary of Inhibitors Tested.InhibitorTargetFinal ConcentrationVehiclePoly(I)SR binding10 μg/mlPBSCytochalasin Dfilamentous actin15 μMDMSONocodazolemicrotubules25 μMDMSOStaurosporineprotein kinases1 μMDMSOChelerythrine ClPKC25 μMDMSOGö 6976PKC10 μMDMSOWortmanninPI-3K0.04 μMDMSOLY294002PI-3K200 μMDMSOGenisteintyrosine kinases100 μMDMSOHerbimycin Atyrosine kinases80 μMDMSOMEK inhibitor IMEK200 μMDMSOJNK inhibitor IJNK4 μMPBSJNK controlinactive analog of JNK inhibitor I4 μMPBSU-73122phospholipase C10 μMDMSOU-73343inactive analog of U-7312210 μMDMSOImage Acquisition and Data AnalysisImages of adherent cells were collected using the Pathway HT bioimager (BD Biosciences). Cells were both illuminated through and fluorescence emission was collected from the bottom of the plate using a 20 × NA075 lens (Olympus, Center Valley, PA, USA) and a field size of approximately 300 μm square. All images were collected using flat field correction and 2 × 2 binning of pixels. Auto focus was carried out using the fluorescence emission of Hoechst and CellTracker Blue, which share the same excitation and emission spectra. Confocal images of bead fluorescence (488 BP excitation, 515 LP dichroic, 515 LP emission filters), Texas Red (560 BP excitation, 595 LP dichroic, 645 LP emission filters) and Hoechst/CellTracker Blue (380 BP excitation, 400 LP dichroic, 435 LP emission filters) were collected every 1.7 μm for a total of 10 sections. The dyes were illuminated sequentially and the confocal images collected were collapsed, creating new images with clear definition of all beads within each cell.Cell segmentation for each image was achieved using a combination of the Hoechst signal (to identify single cells) and the CellTracker Blue signal (to define the cell borders). Using the collapsed stacks of confocal images, software was developed to define the cells (blue emission image), count the number of beads per cell (green emission image) and determine if the beads are outside the cell (red emission image) using custom software developed in MATLAB (The Mathworks, Inc., Natick, MA, USA). Hoechst/CellTracker Blue images were processed to reduce noise, enhance contrast and correct for non-uniform field brightness. A gradient-facilitated watershed segmentation algorithm was used to identify and label individual cells. Cell sizes (profile areas) were calculated as the number of pixels in segmented cell objects (collapsed stack images). Cell volumes were calculated as the sum of the cell profile areas of the individual confocal images comprising collapsed stacks. Green fluorescent (all beads) and red fluorescent (external beads) images were sharpened and contrast enhanced. Watershed segmentation was used to identify and label individual bead objects. Labeled bead objects within the \"all beads\" image were classified as \"internal\" if they had less than 20% overlap with an external bead object. Bead objects sharing one or more pixel with any cell object were considered to be associated with that cell. All partial cell images along the edges of the field were omitted from analysis.Bead binding was calculated as the average number of cell-associated beads per cell. Typically between 1200 and 1800 total cells were counted per donor per condition. Percent internalization was calculated as the number of internalized beads divided by the total number of cell-associated beads for each cell, then multiplied by 100. Significant differences were calculated for the poly(I) data using Students paired t-test. For the cell density data, the Spearman correlation test was performed. For all other data, significant differences were calculated using one-way ANOVA followed by Bonferroni's multiple comparison of all means. An unpaired ANOVA was used in the analysis of the protein tyrosine kinase data in Figure 8. For all other data, a paired ANOVA was used. Prism 4 for the Macintosh (Graphpad Software, San Diego, CA, USA) was used for all graphing and statistical calculations.ResultsCharacterization of GM-MØMonocytes are typically matured into MØ in vitro using M-CSF. However, AM are unusual in that they require GM-CSF, but not M-CSF, for their development in vivo [43-47]. Therefore, we followed the GM-CSF-based differentiation protocol of Akagawa, et al., designed to produce monocyte-derived MØ with a distinctly AM-like phenotype (GM-MØ) [48]. Both AM and GM-MØ have been shown to produce lower levels of H2O2, express higher levels of catalase and are more resistant to H2O2 toxicity when compared to M-CSF derived MØ. Furthermore, AM and GM-MØ (but not M-CSF derived MØ) express HLA-DQ and are resistant to HIV infection, but susceptible to Mycobacterium tuberculosis infection [48,49]. Finally, we are confident that GM-MØ are an appropriate model for primary AMs in that several of the inhibitors described in this communication (genistein, herbimycin A, wortmannin, nocodazole and staurosporine) were also tested for their ability to inhibit phagocytosis of beads by primary murine AM. In all cases, the results were comparable to those obtained using GM-MØ (data not shown).It should be noted that, unlike murine bone marrow, incubation of human monocytes with GM-CSF alone does not produce dendritic cells, as evidenced by the morphology and surface marker expression of GM-MØ. GM-MØ were harvested after 11 days of culture in GM-CSF-supplemented media and immunolabeled to measure surface expression of general macrophage markers as well as markers which can differentiate between alveolar/GM-MØ and the more traditional M-CSF matured MØ. As shown in Figure 1, greater than 90% of GM-MØ stain positive for the MØ surface proteins CD14 and HLA-DR and demonstrate a MØ-like morphology when analyzed by light microscopy, confirming their identity as MØ. These cells are also positive for both HLA-DQ and MARCO (Figure 2), a phenotype consistent with both GM-MØ and primary AMs [10,48,25,52]. In addition, GM-MØ were labeled for SRs known to be present on primary AMs [53,54,10]. As shown in Figure 2, GM-MØ are weakly positive for CD68 and strongly positive for MARCO, PSOX and SR-A.Figure 1Characterization of GM-MØ. CD14 (A), HLA-DR (B) and HLA-DQ (C) expression were evaluated by flow cytometry. Solid lines represent the fluorescence of stained cells, while dashed lines represent the results from control antibodies. Data are representative of experiments performed on cells from three donors. Cytocentrifuge preparations illustrating monocyte and macrophage morphology before (D) and after (E) maturation with GM-CSF were captured at equal magnification (200×).Figure 2Characterization of SRs expressed by GM-MØ. MARCO (A), PSOX (B), LOX-1 (C), SR-A (D) and CD68 (E) expression as well as fluorescent bead binding in the presence of poly(I) (F) or an anti-MARCO mAb (G) were evaluated by flow cytometry. Data are representative of experiments performed on cells from three (A-E) or five (F and G) donors.Our findings also confirm that SRs are involved in the binding of unopsonized latex beads. As shown in Figures 2F and 2G, bead uptake is dramatically inhibited by either the broad SR blocker poly(I) or the MARCO-specific SR blocker mAb PLK-1. These agents reduced the fluorescent bead signal by 80% and 62% respectively, whereas their control reagents (chondroitin sulfate and mIgG3) had no effect. Taken together, these data suggest that GM-MØ accurately model primary AMs in their expression of a wide range of SRs and that their interaction with unopsonized beads involves MARCO (and likely other SRs as well).High throughput direct measurement of phagocytosisA high throughput phagocytosis assay was developed to provide rapid and direct measurement of both particle binding and internalization. For this assay, GM-MØ are first incubated with CellTracker Blue, which provides a uniform label of the whole cell to facilitate cytometric identification. The GM-MØ are then allowed to bind and ingest biotinylated green fluorescent latex beads, followed by incubation with streptavidin-Texas Red to label external beads. Analysis with a scanning cytometer produces images in which beads that are bound, but not internalized, are clearly distinguishable from those which are internalized. Figures 3A–D are typical examples of images produced by this technique. In Figures 3A and 3B, phagocytosis has been inhibited by cytochalasin D treatment. As a result, all of the beads are extracellular and appear as yellow, due to the colocalization of red and green fluorescence. In contrast, the cells in Figures 3C and 3D have been allowed to internalize beads. In these images, some beads are extracellular (appearing as yellow) while others have been internalized (appearing as green). The cells in these images can be automatically identified ('segmented') and the number of beads per cell counted using a combination of commercial and custom software (Figure 3B and 3D).Figure 3Binding and phagocytosis assay. Adherent GM-MØ were treated with cytochalasin D (A and B) to block internalization or vehicle control (DMSO, C and D) before incubation with biotinylated green fluorescent latex beads and labeling of extracellular beads with streptavidin-Texas Red. Images obtained by scanning cytometry show intracellular beads in green, while extracellular beads are in yellow (due to the colocalization of red and green fluorescence). Panels A and C depict the fluorescence images obtained by the scanning cytometer, while panels B and D depict the same images after automated segmenting and bead identification. Scale bars represent 20 μm.In order to validate this technique, GM-MØ were cultured with known inhibitors of SR binding and phagocytosis before being incubated with fluorescent beads. We observed a nearly complete (96%) reduction in the number of beads bound by cells in the presence of the SR blocker poly(I) (Figure 4A). In contrast, the actin destabilizer cytochalasin D has no effect on total bead binding, but decreases the number of beads internalized by 90% when compared to the DMSO control (Figures 4B and 4C). To compare the results of software image analysis to human quantitation of the same images, beads per cell were manually counted for 50 cells in both the control and cytochalasin D treated conditions. These results were quite similar to those obtained by software analysis and are shown in Table 2. Hence, our software quantification technique is capable of accurately counting and distinguishing between beads that have been internalized and beads that have been bound, but not internalized.Table 2Manual vs. Computer Enumeration of Cell-Associated Beads.Internalized BeadsExtracellular BeadsControlCytochalasin DControlCytochalasin DManual1261057221Computer1301169234Figure 4Quantification of bead binding and internalization. Adherent GM-MØ were pretreated with either poly(I) (A) or cytochalasin D (B and C) before incubation with fluorescent latex beads. Cells were analyzed for total bead binding (A and B) and percent internalization (C). Bars represent the means of four (A) or three (B and C) donors +/- the standard deviation. For each donor, three fields from each of three replicate wells were analyzed. *p < 0.01 **p < 0.001 when compared to either the control or DMSO conditions.Binding and internalization are differentially affected by cell densityTo determine the optimal cell concentration for this assay, we compared results using a range of cell densities. The data collected indicate that cell density affects cell size, and has considerable and opposing effects on bead binding and internalization (Figure 5). Higher plating densities are associated with reduced cell size, as measured by pixels per cell profile in collapsed stack images, (r = -0.972, p < 0.001). Comparison of cell sizes and cell volumes calculated from confocal slices confirmed that cell size accurately reflects cell volume (n = 95 cells, r = 0.971, p < 0.0001, data not shown). As cell density increases, the number of beads bound per cell decreases significantly (r = -0.853, p < 0.001). This could be due to reduced cell size and/or increased cell-to-cell contact, thereby reducing the cellular surface area available for bead binding. In contrast, increasing the cell density dramatically augments the percentage of bound beads that are internalized (e.g., increasing the density of the cells from 54 to 180 cells per field resulted in a 60% increase in the percentage of internalized beads (r = 0.622, p < 0.05)), an observation that cannot easily be explained by a reduction in cell size. Taken together, these data indicate that the density used for in vitro analysis of GM-MØ has a significant influence on phagocytic parameters. For all subsequent experiments, cells were plated at 1 × 105 cells per well.Figure 5Bead binding and internalization are cell density dependent. GM-MØ were plated at 5 × 104 to 1.25 × 105 cells/well and cultured overnight before incubation with fluorescent latex beads. At the end of the experiment, cell density for each well was calculated as the average number of cells per field from three fields. Cells were analyzed for total bead binding (A), cell size (B) and bead internalization (C). Points represent the means of three wells each. Lines represent the best-fit linear regression. Data are pooled from three donors.Microtubule destabilization inhibits SR mediated internalizationAlthough filamentous actin is required for phagocytosis in general, the requirement for microtubules depends upon which phagocytic receptor is involved. For example, inhibiting microtubule function blocks complement receptor-mediated, but not Fc receptor-mediated, particle internalization [30,55]. In order to determine if SR-mediated phagocytosis requires microtubules, GM-MØ were analyzed for their ability to bind and internalize latex beads in the presence of the microtubule destabilizer nocodazole. Nocodazole treatment has no effect on the total number of beads bound per cell (data not shown), suggesting that SRs do not require microtubules for particle binding. In contrast, nocodazole treatment reduces the proportion of internalized beads by 50% when compared to the DMSO control (Figure 6). We conclude that SR-mediated internalization is similar to complement receptor-mediated phagocytosis in that they both require functional microtubules.Figure 6Microtubule destabilization inhibits SR mediated phagocytosis. Adherent GM-MØ were pretreated with nocodazole before incubation with fluorescent latex beads. Cells were analyzed for the percent internalization of bound beads. Bars represent the means of five donors +/- the standard deviation. For each donor, three fields from each of three replicate wells were analyzed. **p < 0.001 when compared to either the control or DMSO conditions.Effect of signaling pathway inhibitors on SR-mediated phagocytosisA large number of signaling molecules have been implicated in MØ phagocytosis [56,57]. However, most of this work has been performed using IgG or (to a lesser extent) complement opsonized particles. Very little is known about which signaling pathways are required for SR-mediated phagocytosis. Our strategy was to analyze these pathways using a panel of relevant pharmacologic inhibitors, an approach facilitated by the high throughput assay described above.Tyrosine kinases and PKC are both known to be involved in Fc-receptor mediated phagocytosis [57]. Therefore, we tested the effect of protein tyrosine kinase and PKC inhibitors on SR-mediated phagocytosis (Figures 7 and 8). Inhibition of PKC with staurosporine results in a significant reduction in the number of beads internalized. However, staurosporine is known to inhibit a number of other protein kinases in addition to PKC. In order to definitively show that PKC is required, the PKC specific inhibitors chelerythrine chloride and Gö 6976 were used. These inhibitors cause dramatic (77% and 86%, respectively) reductions in bead internalization. Similarly, treatment with the protein tyrosine kinase inhibitors genistein and herbimycin A result in a 51% and 64% reduction in internalization, respectively. These data show that PKC and tyrosine kinase activities are important for non-opsonic phagocytosis.Figure 7Protein kinase C blockers inhibit SR mediated phagocytosis. Adherent GM-MØ were pretreated with either staurosporine (A), chelerythryine chloride or Gö 6976 (B) before incubation with fluorescent latex beads. Cells were analyzed for the percent internalization of bound beads. Bars represent the means of five (A) or three (B) donors +/- the standard deviation. For each donor, three fields from each of three replicate wells were analyzed. *p < 0.01 and **p < 0.001 when compared to either the control or DMSO conditions.Figure 8Protein tyrosine kinase blockers inhibit SR mediated phagocytosis. Adherent GM-MØ were pretreated with either genistein or herbimycin A before incubation with fluorescent latex beads. Cells were analyzed for the percent internalization of bound beads. Bars represent the means of at least four donors +/- the standard deviation. For each donor, three fields from each of three replicate wells were analyzed. **p < 0.001 when compared to either the control or DMSO conditions.The MAPK family of protein kinases is critical for Fc receptor mediated phagocytosis as well as cell cycle progression and a number of other cytoskeletal processes. Since PKC and tyrosine kinases are known to stimulate MAPK [58], inhibitors of the JNK and ERK MAPK pathways were tested for their ability to inhibit SR-mediated phagocytosis. Inhibition of either of these MAPK pathways blocks internalization. The JNK inhibitor reduces bead internalization by 28% while the inactive analog used as a control does not cause a statistically significant reduction (Figure 9A). Inhibition of the ERK pathway was achieved using an inhibitor of the upstream kinase, MEK. Treatment with this inhibitor reduces phagocytosis by 42% when compared to DMSO control (Figure 9B).Figure 9MAPK blockers inhibit SR mediated phagocytosis. Adherent GM-MØ were pretreated with inhibitors of either JNK (A) or MEK (B) before incubation with fluorescent latex beads. Cells were analyzed for the percent internalization of bound beads. Bars represent the means of five (A) or six (B) donors +/- the standard deviation. For each donor, three fields from each of three replicate wells were analyzed. **p < 0.001 when compared to either the control, JNK control or DMSO conditions.In addition to the protein kinases mentioned above, the lipid modifying enzymes PI-3K and PLCγ have also been shown to play a role in MØ phagocytosis [57]. Therefore, the PI-3K inhibitors wortmannin and LY294002 and the PLCγ inhibitor U-73122 were used to block these enzymes before challenging GM-MØ with latex beads As shown in Figure 10A, wortmannin inhibits bead internalization by 59%, while LY294002 causes an even greater inhibition (78%) (Figure 10B). These data demonstrate that PI-3K is required for optimal SR-mediated phagocytosis. However, unlike PI-3K, PLCγ does not appear to be necessary, as U-73122 is unable to block internalization at the concentration tested (Figure 11).Figure 10Effect of PI-3K inhibitors on SR mediated phagocytosis. Adherent GM-MØ were pretreated with wortmannin (A) or LY294002 (B) before being fed fluorescent latex beads. Cells were analyzed for the percent internalization of bound beads. Bars represent the means of five (A) or three (B) donors +/- the standard deviation. For each donor, three fields from each of three replicate wells were analyzed. **p < 0.001 when compared to either the control or DMSO conditions. *p < 0.05 when compared to the control condition.Figure 11Effect of a PLCγ inhibitor on SR mediated phagocytosis. Adherent GM-MØ were pretreated with U-73122 or the inactive analog U-73343 before being fed fluorescent latex beads. Cells were analyzed for the percent internalization of bound beads. Bars represent the means of four donors +/- the standard deviation. For each donor, three fields from each of three replicate wells were analyzed.Interestingly, while most of the inhibitors shown in Figures 7, 8, 9, 10, 11 block internalization, none of them have a significant effect on particle binding, cell size or the number of cells per field (data not shown). This indicates that SRs do not require PKC, tyrosine kinase, MAPK, PI-3K or PLCγ signaling to effectively bind unopsonized particles. In addition, the fact that cell size and number are unaffected by the inhibitors used demonstrates that these inhibitors did not affect cell viability. This is confirmed by the observation that the inhibitors used do not alter cellular morphology or increase staining with propidium iodide (data not shown).DiscussionWhile the ligand binding characteristics of SRs have been characterized [6], very little is known about the signaling pathways utilized during SR-mediated phagocytosis. In order to address this, we developed a high-throughput phagocytosis assay capable of distinguishing between internalized and non-internalized cell-associated particles. Using this assay, we tested a battery of signaling inhibitors that are known to block opsonin-mediated phagocytosis for their effect on opsonin-independent phagocytosis. We found that microtubules, PKC, tyrosine kinases, MAPKs and PI-3K are required for optimal SR-mediated phagocytosis. Furthermore, cell density has a significant impact on both particle binding and internalization.As primary human AM are difficult to obtain in large quantities, we took advantage of a previously published in vitro human monocyte differentiation scheme that produces MØ that are phenotypically and physiologically similar to human AM. In order to confirm our findings, we tested a subset of inhibitors (genistein, herbimycin A, wortmannin, nocodazole and staurosporine) for their effect on bead phagocytosis by primary murine AMs. Every inhibitor tested significantly decreased bead internalization. This demonstrates that, at the very least, protein tyrosine kinases, PKC, PI-3K and microtubules are necessary for bead phagocytosis by primary murine AM. These findings are identical to those obtained using GM-MØ and further establish these cells as a useful model of primary AM.Most currently available phagocytosis assays rely on subtracting the number of particles associated with cells in which internalization has been blocked from the number of particles associated with cells in which internalization has not been blocked. The agents used to block phagocytosis are typically cytoskeletal or mitochondrial poisons such as cytochalasin D or sodium azide (although incubation at low temperature has also been used) [59-61]. Built into these indirect techniques is the assumption that the agent used to block internalization is effective in the particular cells being studied, yet does not alter the number of bound extracellular beads.In some cases (particularly for receptors of unopsonized targets), this assumption is erroneous, resulting in either an under- or overestimation of particle internalization. For example, our two-color direct approach definitively demonstrates that cytochalasin D is an extremely effective blocker of phagocytosis in GM-MØ (Figure 3D). However, it does not alter the total number of cell-associated beads (Figure 3C). Since the total number of cell-associated beads is the sum of the internalized beads and the beads that have been bound but not internalized, these data indicate that cytochalasin D treatment does indeed alter the number of bound extracellular beads under our experimental conditions. In this case, using the indirect single-color technique would have led to a dramatic underestimation of bead internalization by the untreated cells. The opposite problem would have been encountered if a low temperature incubation had been used to block internalization. This is because, unlike opsonized particles, the binding of unopsonized beads is temperature dependent ([42] and our unpublished results).Given the limitations of the indirect assays mentioned above, we chose to utilize a direct phagocytosis assay based on previously developed two-color fluorescence assays [62,42]. These assays use one intrinsic fluorescent dye to identify all particles and a second non-cell permeable stain applied after internalization to identify particles that have not been internalized. These techniques allow the investigator to distinguish between internalized and extracellular particles without relying on interventions that alter the biology of the cell. While these assays overcome the pitfalls of the indirect assays, they introduce new difficulties for data collection. For example, analysis by flow cytometry can provide exact bead per cell counts for up to three (or perhaps four) cell-associated beads per cell. This is due to the high intensity and low bead-to-bead variability of the intrinsic fluorescent dye. However, at higher bead loads, the absolute number of beads per cell cannot be determined, as the fluorescent peaks begin to overlap [63]. Furthermore, the higher variability and lower intensity of staining with the extracellular dye precludes precise bead per cell counts at even very low bead loads (our unpublished data). As a result of these issues, results are typically reported as a ratio of fluorescence intensities (not absolute bead number) when flow cytometry is used as a read out. The alternative to flow cytometry (counting beads by eye using a fluorescent microscope) is tedious and incompatible with high throughput.In order to overcome these limitations, we developed a system using scanning cytometer technology that can automatically count the number of beads associated with any given cell and distinguish between internalized and extracellular beads. This system allows the investigator to express his or her data as the number of beads per cell and not simply as fluorescence intensity, even for cells with high bead loads. While this assay is similar in many respects to one recently developed by Steinberg and colleagues for analysis of opsonized phagocytosis [64], it differs in that our method involves collecting a set of confocal images spanning the entire thickness of the cell that are then collapsed into a single image for analysis. This technique allows all of the cell-associated beads to be in focus for the final analysis. In contrast, we have found that using conventional fluorescence microscopy does not allow all of the cell-associated beads to remain in focus simultaneously and therefore excludes some beads from analysis (data not shown).The confocal-based phagocytosis assay described in this report was used to test the hypothesis that SR-mediated phagocytosis is similar to complement-mediated phagocytosis in respect to its sensitivity to a microtubule inhibitor. Phagocytosis of opsonized particles by Fc or complement receptors share a number of characteristics, including dependence on actin filaments and the accumulation of signaling and actin binding proteins at the site of the forming phagosome [56]. However, fundamental differences exist between these two modes of phagocytosis [65,55,68]. These differences have led some to characterize them as type I (Fc receptor-mediated) and type II (complement receptor-mediated). Microtubule poisons such as nocodazole paralyze complement-mediated, but not Fc receptor-mediated, particle internalization [55,30]. In this report we present the first evidence that SR-mediated phagocytosis exhibits a characteristic of type II phagocytosis in that nocodazole significantly inhibits internalization.This report is also the first to show that tyrosine kinases, PKC, PI-3K and MAPKs are necessary for SR-mediated phagocytosis by MØ. The requirement for PI-3K and tyrosine kinases is consistent with a recent report showing that PI-3K and the Src kinase Lyn are both required for SR-A-mediated MØ spreading [69]. Furthermore, treatment of MØ cell lines with soluble SR ligands results in the tyrosine phosphorylation of Src kinases, PLCγ and PI-3K as well as a tyrosine kinase dependant activation of PKC [34,33,31,32], suggesting that tyrosine kinase activation may occur relatively early in the SR signaling cascade. Consistent with the inhibition of phagocytosis reported here, inhibition of tyrosine kinases blocks the induction of urokinase-type plasminogen activator (uPA) and IL-1 expression by THP-1 cells in response to SR ligands [31,32]. Similarly, pharmacological blockade of PKC inhibits SR-mediated increases in uPA expression, myelin endocytosis, prostaglandin E2 release and ERK activation [32,36,35].It is surprising to note that the PLCγ inhibitor U-73122 does not affect bead internalization, as U-71322 has previously been shown to inhibit myelin endocytosis by CR3-/- microglia [35] and PKC activation in response to oxidized LDL (oxLDL) [33]. However, the experimental conditions in these reports differ greatly from those described here as the authors use either primary murine microglia or LPS primed P388D1 cells. The signaling pathways and receptors utilized by these murine cells could be quite different from those utilized by our primary unprimed human GM-MØ. Furthermore, while PLCγ is an important activator of conventional PKC, atypical PLCγ-independent PKC isoenzymes have been shown to be important in a number of immune cell functions [70]. Our finding that PKC blockers inhibit internalization, but a PLCγ blocker does not, raises the possibility that GM-MØ utilize atypical PKC isoenzymes as second messenger signals for SR-mediated phagocytosis. While this has yet to be formally demonstrated, it is supported by our finding that an inhibitor of the atypical PKC isoenzyme activator PI-3K [70] blocks internalization.Finally, the MAPK family of proteins are known to play an important role in MØ phagocytosis and have been implicated as downstream signaling molecules for SRs. Stimulation of SRs with fucoidan, oxLDL or poly(I) results in the activation of JNK and ERK MAPK pathways [37,31,38]. Furthermore, Lamprou and colleagues reported that inhibition of these pathways results in a reduction of latex bead internalization by medfly hemocytes [71,72]. The results of our experiments are consistent with these reports in that the inhibition of JNK and ERK pathways results in a reduction of bead internalization. This suggests that some of the pathways utilized during SR-mediated phagocytosis are conserved across a broad spectrum of species.It is important to note that none of the signaling inhibitors tested in this report had any measurable effect on cell viability, size, density or bead binding. It is known that SR-A-mediated acetylated low density lipoprotein binding and cell adhesion require G proteins [73,74]. This, combined with the previous observation that particle binding by SRs is highly temperature dependent, suggests that it contains an active component. However, our data suggests that this active binding mechanism does not require actin filaments, microtubules, PKC, PI-3K, tyrosine kinases, MAPKs or PLCγ even though many of these pathways are necessary for internalization. Our finding that cytochalasin D has no effect on bead binding stands in contrast to the report of Post, et al. in which cytochalasin D was shown to inhibit SR-A-mediated cell attachment by 35% [73]. This discrepancy may reflect the differences between the cytoskeletal requirements for particle binding vs. firm anchorage to a substrate.ConclusionWe have developed a novel high-throughput assay for particle phagocytosis that we used to test the signaling pathways and cytoskeletal components required for unopsonized phagocytosis by human monocyte-derived MØ. We found that filamentous actin, microtubules, PKC, tyrosine kinases, PI-3K, MEK and JNK are required for optimal particle internalization while an inhibitor of PLCγ has no effect.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsTHS conceived and designed the experiments, generated the biotin-conjugated beads and authored the manuscript. AI participated in experimental design and manuscript authoring and carried out the binding and phagocytosis assays. GD developed the custom data analysis software and participated in manuscript authoring. ARW generated the GM-MØ protocol and participated in manuscript revision. LK contributed to the conception and design of the experiments and played a significant role in the revision of the manuscript. All authors read and approved the final manuscript.\n\nREFERENCES:\nNo References"
+}

batch_9/PMC2527570.json

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+{
+    "id": "PMC2527570",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527570\nAUTHORS: Ali Montazeri, Azita Goshtasebi, Mariam Vahdaninia\n\nABSTRACT:\nBackgroundThe aim of this study was to investigate the relationship between educational level and self-reported health in an Iranian population, in order to provide evidence on social inequalities in health from a country in which such data need to be collected.MethodsThis population-based study was carried out in Tehran, Iran. Individuals aged 15 years and over were interviewed. Self-reported health was measured by asking each individual to respond to the question: \"In general how would you describe your health at present?\" We used years of formal education as a measure of socioeconomic status and categorized the answers in five levels. Logistic regression analysis was used to estimate odds ratios and 95% confidence intervals indicating the contribution of educational level to self-reported health, adjusting for age, gender, marital status, and chronic diseases.ResultsIn all, 4163 individuals were interviewed. The mean age of the respondents was 35.1 years (SD = 16.0); 52% were female; the mean duration of formal education was 10.0 years (SD = 4.5); and 31% rated their health 'less than good'. Overall, women rated their health more poorly than men (P < 0.0001), and the findings showed that those with higher education rated their health significantly better than those with lower educational levels after adjusting for the age, gender, marital status and chronic diseases. The odds ratio for having 'less than good' self-rated health in those at the lowest educational level compared with those at the highest was 2.65 (95% CI = 1.88–3.73).ConclusionThe findings indicated an inverse relationship between educational level and self-rated health, and that age, gender, and chronic conditions had independent effects on self-reported health status. The findings of this first study from Iran suggest that health inequalities in developing countries such as Iran need to be addressed and policies for tackling the problem should be considered. In this respect, less well-educated people and women should be seen as the first target populations. It seems that although expanding the educational system might help the state to provide people with more educational options, it is also necessary to ensure that equal opportunities and access to quality education are provided for those from lower socioeconomic backgrounds; otherwise the current situation might cost the government more in the long term because of poor health among disadvantaged groups.\n\nBODY:\nBackgroundIn recent years, compelling evidence has been obtained for an inverse relationship between health and socioeconomic status over time and in different countries [1,2]. This association has been found for all indicators of socioeconomic level whether they are based on occupation, education or income [3,4]. Studies have shown that socioeconomic levels have both direct and indirect effects on health [5]. However, the magnitude of health disparities across socioeconomic levels varies within and between countries [6]. It has been suggested that reducing health inequalities in disadvantaged groups may offer great potential for improving the health status of the population as a whole [7]. Thus, the World Health Organization now considers the reduction of health inequalities to be one of the top priorities [8].The aim of this study was to describe self-reported health by educational level in an Iranian population. It is believed that self-rated health is a valuable measure in health-related inequality research because it is based on individuals' own assessments of the trajectories of their social and familial histories and on how they perceive their health status, and it reflects the availability of resources and environmental factor that may ultimately affect health [3,9,10]. We believe the same argument may apply to the general public in Iran, so the findings from this study might be a good starting point for future research on the topic here and in other developing countries in Asia. In addition, Iran has a complex educational system and educational attainment could reflect individuals' socioeconomic positions. Therefore our hypothesis was that educational achievements contribute to self-rated health. To our knowledge, this is the first paper from Iran that reports on the topic. It may therefore add to the existing evidence on international variations in socioeconomic inequalities in self-reported health. It may also facilitate the reduction of health inequalities in the population by raising awareness among research communities and providing evidence for policymakers, affecting national level policies, though at present there are no such policies in force in Iran. In Europe there are national level policies to promote health equity within and between the countries [11]. It has been suggested that information and knowledge-sharing has a key role in linking evidence about the social and environmental causes of health inequalities to local actions and challenges [12].MethodsDesignThis study was based on information taken from a cross-sectional population-based survey on quality of life carried out in Tehran, Iran. To select a representative sample of the general population aged 15 years and over a stratified multi-stage area sampling was applied. Every household within 22 different districts in Tehran had the same probability of being sampled. For the first stage, units (blocks) were randomly selected after stratifying by district and size of residence. Then the homes to be sampled within each block were selected by random routes. Finally, the last-stage sampling units (the individuals) were selected randomly from all persons living in the same home.Self-reported healthSelf-reported health was measured by asking each individual to respond to the question: \"In general how would you describe your health at present?\" There were five response categories; 'excellent', 'very good', 'good', 'fair', and 'poor'. For analysis we combined the categories 'excellent', 'very good' and 'good' to yield a measure of self-reported health of 'good or better than good' and the categories 'fair' and 'poor' to yield a measure of 'less than good'. There is evidence that using a general question on self-reported health measured on an ordinal scale is a valid instrument for determining individuals' perceptions in studies of health inequalities [12].EducationWe used years of formal education as a measure of socioeconomic status. It is argued that educational level is a variable that can be applied to the entire population [13]. It has been shown that stratification by education is probably the best measure of socioeconomic status when results from different populations are compared [4,14]. In addition, since income information in Iran is not reliable and people usually have more than one job at the same time, we did not collect or use data on income or occupation as a measure of socioeconomic position. However, education was categorized into five levels: no education, first level (1 to 5 years), second level (6–9 years), third level (10–12 years) and fourth level (more than 12 years).In principle, everybody in Iran should have free access to education even at higher levels. However, since Iran has a young population (50% under 30 years old) and the state could not respond to the potential need, methods of payments for education currently vary: completely free of charge, partially paid and completely paid. All these are governed by the state and overall a unique formal system is followed: primary education (1–5 years), high school (6–9 and 10–12 years), and college (13–14 years)/or university (13–16 years and more). The success rate for entering higher education is usually high for those who can afford paid education (either partially or completely paid).Chronic diseasesData on chronic diseases were collected by asking each respondent to indicate whether he or she suffered from any medically diagnosed chronic condition including cardiovascular diseases, diabetes, musculoskeletal disorders, cancers, neurological or psychological diseases and chronic respiratory diseases.Statistical analysisThe potential variation in self-reported health by educational level was estimated by odds ratios, using logistic regression and adjusting for age, gender, and chronic conditions. We also entered martial status into the logistic regression model since there were significant differences between men and women with regard to marital status. Self-reported excellent/very good/good health (i.e. good or better than good) versus fair/poor health (i.e. less than good) was the comparison [3].EthicsThe study received ethical approval from the Iranian Institute for Health Sciences Research and all participants gave oral consent.ResultsIn all, 4804 individuals were approached and 4163 (87%) agreed to be interviewed. Of those who did not participate in the study, 230 were female and the remaining 411 were male. The main reason for non-participation was that after two approaches most of these individuals were not available in their homes. Only a few refused to take part in the study because of dislike (n = 64). The characteristics of the study sample are shown in Table 1. The mean age of the respondents was 35.1 (SD = 16.0) years. Fifty-two percent were female, mostly married (58%), and the mean duration of formal education for the whole study sample was 10.0 years (SD = 4.5). Sixty-nine percent of the respondents rated their health 'good or better than good' (excellent, very good, or good), while 31% rated their health 'less than good' (fair or poor). Seven percent of the respondents (n = 296) indicated that they suffered from a medically diagnosed chronic condition.Table 1The characteristics of the study sampleAll (n = 4163)Male (n = 1997)Female (n = 2166)PNo. (%)No. (%)%No. (%)Age group< 0.000115–241420 (34)681 (34)739 (34)25–441614 (39)721 (36)893 (41)45–64882 (21)446 (22)436 (20)≥ 65247 (6)149 (8)98 (5)Mean (SD)35.1 (16.0)36.1 (16.9)34.1 (15.1)< 0.0001Marital status< 0.0001Single1601 (38)827 (47)774 (36)Married2406 (58)1149 (52)1257 (58)Widowed/divorced156 (4)21 (1)135 (6)Educational level (years)< 0.0001No education280 (7)100 (5)180 (8)First level (1–5)475 (11)211 (11)264 (12)Second level (6–9)901 (22)460 (23)441 (21)Third level (10–12)1695 (41)783 (39)912 (42)Fourth level (>12)812 (19)443 (22)369 (17)Mean (SD)10.0 (4.5)10.4 (4.3)9.6 (4.5)< 0.0001Self-reported health< 0.0001Excellent/very good1450 (35)803 (40)647 (30)Good1405 (34)655 (33)750 (34)Fair/poor1308 (31)539 (27)769 (36)Chronic diseases0.004Yes296 (7)117 (6)179 (8)No3867 (93)1880 (94)1987 (92)Diseases (n = 296)0.01Hypertension31 (11)7 (6)24 (13)Other cardiovascular diseases78 (26)43 (37)35 (20)Diabetes22 (7)8 (7)14 (8)Musculoskeletal disorders95 (32)34 (29)61 (34)Cancer5 (2)0 (0)5 (3)Neurological and psycho-logical diseases45 (15)19 (16)26 (15)Chronic respiratory diseases20 (7)6 (5)14 (8)As indicated in Table 1, women rated their health more poorly than men (χ2 = 56.9, df = 2, P < 0.0001), were younger (mean age 36.1 vs. 34.2 years, P < 0.0001) and were less educated than men (χ2 = 38.9, df = 4, P < 0.0001). There were also significant differences between men and women in having chronic conditions (χ2 = 16.4, df = 6, P = 0.01).The most important findings of the study are shown in Table 2. There was a significant association between educational level and self-reported health: those with higher education rated their health significantly better than those with lower educational levels. The odds ratio for having 'less than good' self-reported health for those with the lowest educational level compared with those with the highest was 2.65 (95% CI = 1.88–3.73).Table 2The odds ratio for 'less than good' self-rated health obtained from logistic regression analysis on all the study sample, on men and on womenOR95% CIPAll (n = 4163)Age1.051.04–1.06< 0.0001GenderMale1.0 (ref.)Female1.651.41–1.93< 0.0001Marital statusSingle1.0 (ref.)Married1.571.25–1.96< 0.0001Widowed/divorced2.331.45–3.73< 0.0001Chronic diseasesNo1.0 (ref.)Yes2.722.03–3.65< 0.0001Educational level (years)Fourth level (> 12)1.0 (ref.)Third level (10–12)1.561.25–1.94< 0.0001Second level (6–9)1.851.45–2.35< 0.0001First level (1–5)1.861.42–2.43< 0.0001No education2.651.88–3.73< 0.0001Male (n = 1997)Age1.051.04–1.06< 0.0001Marital statusSingle1.0 (ref.)Married1.270.90–1.790.17Widowed/divorced8.282.52–27.20.001Chronic diseasesNo1.0 (ref.)Yes2.441.58–3.78< 0.0001Educational level (years)Fourth level (> 12)1.0 (ref.)Third level (10–12)1.260.92–1.720.14Second level (6–9)1.431.02–1.980.03First level (1–5)1.621.10–2.390.01No education3.071.80–5.24< 0.0001Female (n = 2166)Age1.051.04–1.06< 0.0001Marital statusSingle1.0 (ref.)Married1.721.28–2.32< 0.0001Widowed/divorced1.911.09–3.350.02Chronic diseasesNo1.0 (ref.)Yes2.992.00–4.48< 0.0001Educational level (years)Fourth level (> 12)1.0 (ref.)Third level (10–12)1.911.38–2.63< 0.0001Second level (6–9)2.351.65–3.34< 0.0001First level (1–5)2.131.44–3.14< 0.0001No education2.601.63–4.12< 0.0001Separate analysis of the data for males and females showed different pictures for the variables studied. Poorer self-reported health in men was not significantly associated with marriage or third level education (10–12 years); for women poorer self-reported health was strongly associated with all levels of education and marital status. However, the contribution of education to 'less than good' self-rated health was simultaneously more gradual and steeper in males than in females (Table 2).DiscussionThis was a population-based study of the relationship between educational level and self-reported health in Tehran, Iran. There was a distinct pattern of self-reported health among those with different educational levels, showing a dose-response relationship between education and the risk of 'less than good' self-rated health status. However, one might argue that a sample from the urban capital (Tehran) is not necessarily representative of the entire country. In general this is true, but since Tehran has became a multicultural metropolitan area with a mixture of different socioeconomic and ethnic backgrounds, a sample from the general population in Tehran could at least be regarded as representative of the urban population of Iran [15]. It has been suggested that studies of self-reported health including quality of life assessments provide information on the health of a population that are usually invisible in traditional analyses of population health [16]. Responses to questions of this type (self-reported health or self-reported morbidity) may vary according to mode of administration, but studies have shown that such responses are nevertheless valid [17].The findings from the present study indicate that in general people with a higher educational level rated their health status more highly than people with a lower educational level. There are several explanations for education-related health inequalities within and between countries. The most straightforward is that the effect of education varies from one place to another for unknown reasons, which would make it difficult to account for differences in the magnitude of the association between education and various health indicators in different countries [18]. In addition, international studies have shown that educational health inequalities vary in magnitude between countries [19]. A recent publication from the Eurothine Project [20], which compares inequalities in health in more than 20 European countries, suggests that although income and education are 'upstream' determinants of health inequalities, this is the feature of European welfare regimes that could provide evidence for the magnitudes of educational health inequalities between countries. They found that South European welfare regimes had the largest health inequalities while countries with Bismarckian welfare regimes tended to have the smallest. Although the other welfare regimes ranked relatively close to each other, the Scandinavian regimes were placed less favorably than the Anglo-Saxon and East European [21].Another possible explanation is that in societies where everyone has the same access to education, educational level is not a good indicator of socioeconomic position. Thus, even if there is an inverse relationship between education and self-reported health, this does not demonstrate inequality in health in such societies [4]. On the other hand, it can be argued educational attainment could reflect dissimilarities among individuals in terms of work conditions, economic status, lifestyle and the use of health care services, so education has a significant impact on the observed inequalities in health among different socioeconomic subgroups of populations [22]. Others have suggested that educational inequalities in health might be attributable to the fact that education reflects the different life course accumulations of material and psychosocial hazards to which people have been exposed [23]. Since not everyone has the same access to education in Iran, we suspect that the latter explanation applies to the variation in self-reported health by educational level. Interestingly, a study using data from the 2003 US Current Population Health Survey indicated that people in the very high income bracket tend to report slightly worse health, which may be explained by their lower education [24].We found that women rated their health more poorly than men. This was definitely not due to age since women were younger than men on average. Thus one might argue that education contributes to the observed differences between men and women in self-reported health. Studies have shown that education is one of the most important contributors to gender inequalities in health [22]. There are also several other explanations for such observed differences: economic dependence, employment, marital status, family position and family demands are among the factors found to contribute to gender differences in self-rated health [25,26]. However, it is argued that diversity in life style is not the most important reason for gender differences in social inequalities in health [27].We found that women reported having significantly more chronic medical conditions. Studies from some other developing and transitional countries have yielded similar results, with some exceptions [28,29]. For instance, a Syrian study identified gender-specific determinants of poor self-rated health including being married, low socioeconomic status, and not having social support for women; and smoking, and low physical activity for men [28]. However, the present study showed that married men and particularly married women were worse off (in terms of health) than their single counterparts. One might argue that the married were merely older than the single, and being older implies worse health.In addition to gender, the results from the present study clearly indicate that age has an independent effect on self-reported health. It is therefore argued that age, sex and social class make distinct contributions to specific morbidities and should be recognized as a transparent and robust approach to the assessment of morbidity-based inequality [30].Finally, the limitations of this study should be considered in interpreting the results. The design was cross sectional and therefore could not indicate whether education really causes inequality in self-rated health or whether existing inequality due to other factors causes poorer health, and this in turn leads to lack of success in appropriate education. Furthermore, we used binary logistic regression analysis and dichotomized a 5-level ordinal scale to yield 'good or better than good' and 'less than good' self-rated health. Thus, excellent, very good and good self-ratings of health (for example) are assumed to be the same, but in fact they are not. There are other ways of analyzing such data and tackling this problem, for example by multinomial regression analysis, where ordinal data can be used without collapsing categories. However, each of these approaches to analysis has its own limitations. The study did not collect data on other measures such as health behaviors (diet, exercise, smoking, etc.) or measures such as blood pressure or body mass index. The contributions of these variables to self-rated health remain unknown. Collecting such data is recommended for future studies. We used years of formal education as a measure of socioeconomic position; it would be useful if reliable data on income could be collected for future investigations on the topic.ConclusionThe findings from this investigation provide further evidence for the education-related health inequalities. They suggest that health inequalities in developing countries such as Iran need to be addressed and policies for tackling the problem should be considered. In this respect, less well-educated people and women should be seen as the first target populations. It seems that although expanding the educational system might help the state to provide more educational options, it is also necessary to be sure that equal opportunities and access to quality education are provided for those from lower socioeconomic backgrounds; otherwise the current situation might cost the government more in the long term because of poor health in disadvantaged groups.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsAll authors contributed to the study design and data collection. MV contributed to the data entry and analysis, AG contributed to writing of the first draft and AM supervised the study, analyzed the data and wrote the final manuscript. All authors read and approved the paper.\n\nREFERENCES:\nNo References"
+}

batch_9/PMC2527610.json

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+{
+    "id": "PMC2527610",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527610\nAUTHORS: Michael A Mastrangelo, William J Bowers\n\nABSTRACT:\nBackgroundSeveral transgenic animal models genetically predisposed to develop Alzheimer's disease (AD)-like pathology have been engineered to facilitate the study of disease pathophysiology and the vetting of potential disease-modifying therapeutics. The triple transgenic mouse model of AD (3xTg-AD) harbors three AD-related genetic loci: human PS1M146V, human APPswe, and human tauP301L. These mice develop both amyloid plaques and neurofibrillary tangle-like pathology in a progressive and age-dependent manner, while these pathological hallmarks are predominantly restricted to the hippocampus, amygdala, and the cerebral cortex the main foci of AD neuropathology in humans. This model represents, at present, one of the most advanced preclinical tools available and is being employed ever increasingly in the study of mechanisms underlying AD, yet a detailed regional and temporal assessment of the subtleties of disease-related pathologies has not been reported.Methods and resultsIn this study, we immunohistochemically documented the evolution of AD-related transgene expression, amyloid deposition, tau phosphorylation, astrogliosis, and microglial activation throughout the hippocampus, entorhinal cortex, primary motor cortex, and amygdala over a 26-month period in male 3xTg-AD mice. Intracellular amyloid-beta accumulation is detectable the earliest of AD-related pathologies, followed temporally by phospho-tau, extracellular amyloid-beta, and finally paired helical filament pathology. Pathology appears to be most severe in medial and caudal hippocampus. While astrocytic staining remains relatively constant at all ages and regions assessed, microglial activation appears to progressively increase temporally, especially within the hippocampal formation.ConclusionThese data fulfill an unmet need in the ever-widening community of investigators studying 3xTg-AD mice and provide a foundation upon which to design future experiments that seek to examine stage-specific disease mechanisms and/or novel therapeutic interventions for AD.\n\nBODY:\nBackgroundAlzheimer's disease (AD) represents the most common age-related neurodegenerative disorder and cause of dementia worldwide. The prevalence of AD is predicted to increase significantly to affect over 100 million people worldwide by the year 2050 [1]. With this dire prediction, it has become imperative to dissect the pathophysiologic mechanisms intrinsic to AD in an effort to eventually devise disease course-modifying therapies. Individuals afflicted with AD harbor two pathological signatures within their brains: extracellular amyloid plaques and neurofibrillary tangles (NFTs), which are identifiable only upon post-mortem examination. Extracellular plaques are comprised of proteinaceous aggregates of amyloid beta (Aβ) peptides, ubiquitin, various proteoglycans, proteases, serum-related molecules, as well as numerous other proteins [2]. The major amyloidogenic components of plaque, Aβ 1–40 and 1–42 peptides, are the proteolytically liberated products that arise from the enzymatic processing of amyloid precursor protein (APP), a type 1 transmembrane protein. NFTs are the result of intraneuronal hyperphosphorylated paired helical filaments of the microtubule-associated protein tau. The seminal work by Drs. Heiko and Eva Braak demonstrated that these pathologies proceed in a definable temporal and spatial pattern within the human brain [3]. Stage A of amyloid accumulation represents the presence of amyloid patches in the basal neocortex and in poorly myelinated temporal areas such as perirhinal and entorhinal areas; the spreading of amyloid deposition to neocortical areas and the hippocampus is indicative of Stage B, while Stage C includes appearance of amyloid deposits in highly myelinated areas of the cortex and neocortex. The evolution of NFTs in the AD brain proceeds through six distinct stages that to some extent overlap with those of amyloid deposition. Stage I is defined by NFT appearance in cell projections comprising the trans-entorhinal region of the temporal lobe, whereas evidence of NFT pathology in the entorhinal region, hippocampus/temporal pro-neocortex is indicative of Stages II and III, respectively. Stages IV-VI of NFT formation includes progression to the neocortex and areas adjoining the neocortex.To elucidate the varying pathophysiologic mechanisms underlying AD progression and to assess potential disease-modifying therapeutics in a preclinical in vivo setting, investigators have turned to transgenic mouse models harboring mutated human genes associated with the familial forms of AD. Although no single transgenic model recapitulates the human disease in all aspects of neuropathology and behavior, some assumptions can be made as to which model best fits specific criteria of AD. Amyloid-based transgenic mouse models exist that overexpress wild-type or mutant forms of APP (i.e., Tg2576; [4]), leading to extracellur Aβ peptide accumulation into plaque-like deposits, synaptic loss, microgliosis, astrocytosis, and cerebrovascular angiopathy [5,4,10]. Most of these models exhibit differential behavioral phenotypes related to significant learning and memory impairment, spatial deficits, and at times, increased aggression. At least nine transgenic mouse models have been created to study consequences of pathogenic tau expression [11-16]. All models show pathology of varying severity, including models overexpressing normal human tau.The triple-transgenic Alzheimer's disease (3xTg-AD) mouse, created in the laboratory of Dr. Frank LaFerla, represents one of the most state-of-the-art and biologically relevant mouse model for AD described to date. The 3xTg-AD mouse model was generated by co-microinjection of the human APPswe and tauP301L genes, both under the transcriptional control of a modified Thy1.2 promoter, into single-cell homozygous mutant PS1M146V knock-in mouse embryos [17]. These mice develop intracellular Aβ, amyloid plaques and NFTs in a progressive and age-related pattern, where the pathologies are predominantly restricted to the hippocampus, amygdala, and the cerebral cortex [18]. These mice also exhibit deficits in synaptic functioning, including long-term potentiation (LTP) [17], and learning/memory behaviors that are similarly manifested in an age-dependent manner [19]. These early papers describing the derivation and initial characterization of pathological progression in 3xTg-AD mice was limited in terms of temporal and regional evolution of particular AD-related hallmarks. Moreover, it has been difficult to glean from those reports as to whether one gender was exclusively studied or whether experimental groups consisting of mixed genders were employed [17].A detailed regional and temporal assessment of the subtleties of disease-related brain pathologies that arise in 3xTg-AD mice over much of their lifespan has yet to be reported. Absence of such information may lead one to improperly initiate long-term experiments designed to address specific hallmarks of AD-related pathology. To that end, we have systematically examined the temporal and spatial progression of human APPswe transgene expression, appearance of intracellular and extracellular Aβ1–42, human tauP301L transgene expression, appearance of pathogenic phospho-tau, and evidence of microglial activation and astrogliosis in male 3xTg-AD mice from 2 to 26 months of age.ResultsHuman APPswe Transgene ExpressionIn the present study, we sought to immunohistochemically document the temporal and regional evolution of AD-related transgene expression, amyloid deposition, tau phosphorylation, astrogliosis, and microglial activation throughout the hippocampus, amygdala, primary motor cortex, and entorhinal cortex over a 26-month period in male 3xTg-AD mice (Antibodies employed in this study are shown in Table 1 and Nissl-stained brain regions of interest depicted in Figure 1). To sufficiently garner detailed insight into how AD-related pathologies arise in 3xTg-AD mice, animals were sacrificed at 2, 3, 6, 9, 12, 15, 18, and 26 months of age for subsequent immunohistochemical processing (N = 4 per time point). Amyloid pathology that develops in these mice derives from the proteolytic processing of the human APP transgene product that harbors the Swedish double mutation (K595N/M596L; [20]) and M146V knock-in mutation in presenilin 1 [21,22] that, in combination, lead to the marked overproduction and progressive accumulation of the fibrillogenic peptide, Aβ1–42 [23]. The hAPPswe transgene, as well as the tauP301L transgene, is under the transcriptional control of Thy1.2 gene promoter, which results in transgene expression specifically within neuronal populations beginning early in post-natal development and continuing into adulthood [24]. Human APPswe transgene expression, as assessed using the APP Y188 antibody (AbCam), which recognizes the NPXY amino acid motif of the hAPP protein localized amino terminal to the cleavage fragment of Aβ, was detectable in 3xTg-AD mouse brain beginning at the 2-month time point throughout the pyramidal neurons of the hippocampus (rostral, intermediate, and caudal; Figure 2), layer II and III neurons of the entorhinal cortex (Figure 3A–H), and primary motor cortex (Figure 4A–H). Staining intensities for hAPPswe transgene product qualitatively appear to stabilize from 6 months and older in all of the regions of the hippocampus and entorhinal cortex examined (Figures 2 and 3A–H). Interestingly, in the amygdala, hAPPswe expression is not detected by the Y188 antibody consistently until 6 months of age (Figure 5A–H).Figure 1Representative Nissl-stained brain sections from 3xTg-AD mice depicting regions examined by immunohistochemistry in this study. Coronal mouse brain sections (30 μm) were prepared from 3xTg-AD mice sacrificed at 6 months of age and were processed for Nissl staining. Primary motor cortex (PMC) at Bregma 0.5 mm (A), amygdala (Amy) at Bregma -0.8 mm (B), CA1 hippocampal sections at Bregma -1.8 mm (C), at Bregma -2.5 mm (D), and at Bregma -2.8 mm (E), as well as entorhinal cortex (Ent) at Bregma -2.8 mm (E) are outlined by dotted boxes to illustrate the sub-regions of the brains examined in this study. Photomicrographs were obtained at 1.25×.Figure 2Human amyloid precursor protein transgene expression is readily detectable within the pyramidal cell layer of 3xTg-AD mouse hippocampus from 2 to 26 months of age. Coronal mouse brain sections (30 μm) were prepared from 3xTg-AD mice sacrificed at 2 (A, I, Q), 3 (B, J, R), 6 (C, K, S), 9 (D, L, T), 12 (E, M, U), 15 (F, N, V), 18 (G, O, W), and 26 months of age (H, P, X) and were processed for immunohistochemistry to detect human amyloid precursor protein (hAPP) A4 using the Y188 monoclonal antibody. CA1 hippocampal sections at Bregma -1.8 mm (A–H), at Bregma -2.5 mm (I-P), and at Bregma -2.8 mm (Q-X), were examined for regional and temporal patterns of hAPPswe transgene expression and photomicrographs were obtained at 10×. The inset in panel X represents a 40× digitally magnified image of the photomicrograph for better visualization of stained cell morphology. Scale bar in D represents 200 μm.Figure 3Entorhinal cortex-resident human APPswe transgene expression and Aβ peptide deposition evolve on similar timescales as observed in the 3xTg-AD hippocampus. Coronal mouse brain sections (30 μm) were prepared from 3xTg-AD mice sacrificed at 2 (A, I, Q), 3 (B, J, R), 6 (C, K, S), 9 (D, L, T), 12 (E, M, U), 15 (F, N, V), 18 (G, O, W), and 26 months of age (H, P, X) and were processed for immunohistochemistry to detect the human Swedish mutant amyloid precursor protein (hAPPswe) transgene product using the Y188 monoclonal antibody (A–H), human amyloid precursor protein (hAPP) and Aβ peptides using the 6E10 monoclonal antibody (I-P), and extracellular Aβ1–42 deposition using the 12F4 monoclonal antibody (Q-X). Entorhinal cortex was examined for patterns of immunopositivity and photomicrographs were obtained at 10×. The insets in panels H, P, and X represent 40× digitally magnified images of designated photomicrographs for better visualization of immunopositive cell/pathology. Scale bar in D represents 200 μm.Figure 4Primary motor cortex-resident human APPswe transgene expression and Aβ peptide deposition evolve on similar timescales as compared to the 3xTg-AD hippocampus. Coronal mouse brain sections (30 μm) were prepared from 3xTg-AD mice sacrificed at 2 (A, I, Q), 3 (B, J, R), 6 (C, K, S), 9 (D, L, T), 12 (E, M, U), 15 (F, N, V), 18 (G, O, W), and 26 months of age (H, P, X) and were processed for immunohistochemistry to detect the human Swedish mutant amyloid precursor protein (hAPPswe) transgene product using the Y188 monoclonal antibody (A–H), human amyloid precursor protein (hAPP) and Aβ peptides using the 6E10 monoclonal antibody (I-P), and extracellular Aβ1–42 deposition using the 12F4 monoclonal antibody (Q-X). Primary motor cortex was examined for patterns of immunopositivity and photomicrographs were obtained at 10×. The insets in panels H, P, and X represent 40× digitally magnified images of designated photomicrographs for better visualization of immunopositive cell/pathology. Scale bar in D represents 200 μm.Figure 5Amygdala-resident human APPswe transgene expression is slightly delayed, while Aβ peptide deposition evolves on a similar timescale as compared to the 3xTg-AD hippocampus. Coronal mouse brain sections (30 μm) were prepared from 3xTg-AD mice sacrificed at 2 (A, I, Q), 3 (B, J, R), 6 (C, K, S), 9 (D, L, T), 12 (E, M, U), 15 (F, N, V), 18 (G, O, W), and 26 months of age (H, P, X) and were processed for immunohistochemistry to detect the human Swedish mutant amyloid precursor protein (hAPPswe) transgene product using the Y188 monoclonal antibody (A–H), human amyloid precursor protein (hAPP) and Aβ peptides using the 6E10 monoclonal antibody (I-P), and extracellular Aβ1–42 deposition using the 12F4 monoclonal antibody (Q-X). Amygdala was examined for patterns of immunopositivity and photomicrographs were obtained at 10×. The insets in panels H, P, and X represent 40× digitally magnified images of designated photomicrographs for better visualization of immunopositive cell/pathology. Scale bar in D represents 200 μm.Table 1Antibodies Employed in Present StudyTarget EpitopeAntibody (Supplier)ReferencehAPPRabbit monoclonal clone Y188-Amyloid precursor protein A4, corresponding to the NPXY motif of hAPP(Abcam)hAPP/AβMouse monoclonal clone 6E10Götz J, et al. [70]Aβ amino acid residues 1–16; also cross-reacts with hAPP(Signet)Hock C, et al. [71]Oddo S, et al. [72]hAβ 1–42 (extracellular) C-terminus of Aβ 1–42Mouse monoclonal clone 12F4Parvathy, S., et al. [73](Covance/Signet)hAβ 1–42 (intracellular) C-terminus of Aβ 1–42Rabbit polyclonal anti Aβ 1–42D'Andrea, et al [27](Biosource/Invitrogen)hTauMouse monoclonal HT7Oddo et al [18]Human Tau amino acid residues 159–163(Pierce)Mercken M et al [74]Phosphorylated TauMouse monoclonal AT180Greenberg and Davies [39]Human Tau phosphorylated residue Thr231(Pierce)Oddo S et al. [18]Paired Helical FilamentsMonoclonal mouse anti-PHF-1Ksiezak-Reding, et al. [37]Human Tau phosphorylated on amino acid residues Ser396 and Ser404 associated with paired helical filaments(Dr. Peter Davies, Albert Einstein School of Medicine)Clinton, et al. [68]F4/80Rat anti mouse F4/80Janelsins et al. [52](AbD Serotec)Cell surface glycoprotein on mature macrophages that is a member of the EGF-TM7 familyGFAPRabbit polyclonal anti-GFAPShaftel S, et al. [75]Cell surface marker (glial fibrillary acidic protein) for mature astrocytes(Dako Cytomation)Patterns of Intracellular and Extracellular Aβ Peptide AccumulationAssessment of Aβ peptide deposition was performed using two antibodies with differing specificity: 6E10, which recognizes amino acid residue 1–16 of beta-amyloid, but also reacts with that identical epitope within non-proteolytically processed hAPP; and monoclonal antibody 12F4, which is specifically reactive to the C-terminus of Aβ1–42. The expression of APP/Aβ as detected by 6E10 reveals similar cell-associated patterns of staining to those observed in sections stained with the hAPP-specific antibody (Y188) through 12 months of age (Figures 3I–P, 4I–P, 5I–P, and 6). The co-staining of APP/Aβ begins to appear in CA1 neurons at approximately 3 months of age in the caudal most region of the hippocampus (Figure 6R), neurons in layer II and III of the entorhinal cortex (Figure 3J), and neurons of the primary motor cortex (Figure 4J). Evidence of 6E10 staining in the amygdala does not become appreciable until 6 months of age (Figure 5K). The intracellular expression of APP/Aβ is confined to the pyramidal layer of the hippocampus and all other brain regions until development of extracellular deposition begins to be apparent at ages greater than 15 months (Figures 3O, 4O, 5O, and 6V, G, O). With all cell-associated immunopositive signal, APP/Aβ staining is limited to the cell bodies, with little to no staining of fibers residing in the radiatum of the hippocampus. Plaques that appear at 18 and 26 months of age are found in the stratum oriens, stratum lucidium and radiatum of the hippocampus, and layers V and VI of the entorhinal cortex, with a majority of the extracellular plaques developing within the subiculum.Figure 66E10 immunohistochemistry reveals differential cell-associated hAPP/Aβ accumulation patterns and markedly late-stage extracellular plaque deposition in 3xTg-AD mouse hippocampus. Coronal mouse brain sections (30 μm) were prepared from 3xTg-AD mice sacrificed at 2 (A, I, Q), 3 (B, J, R), 6 (C, K, S), 9 (D, L, T), 12 (E, M, U), 15 (F, N, V), 18 (G, O, W), and 26 months of age (H, P, X) and were processed for immunohistochemistry using the 6E10 monoclonal antibody to detect both human amyloid precursor protein (hAPP) and Aβ peptides. CA1 hippocampal sections at Bregma -1.8 mm (A–H), at Bregma -2.5 mm (I–P), and at Bregma -2.8 mm (Q-X), were examined for regional and temporal patterns of hAPPswe transgene/Aβ peptide expression and photomicrographs were obtained at 10×. The inset in panel X represents a 40× digitally magnified image of the photomicrograph for better visualization of stained cell morphology. Scale bar in D represents 200 μm.To confirm that the extracellular plaque-like deposition pattern was the result of accumulated Aβ1–42 peptide, adjacent sections were stained with the anti-Aβ1–42 antibody 12F4 (Figures 3Q–X, 4Q–X, 5Q–X, and 7). Using a peptide competition experiment, this antibody was shown to specifically recognize extracellular Aβ1–42 (Figure 7A-D). It is readily apparent that Aβ1–42-reactive deposits accumulate in the hippocampus starting at the subiculum/CA1 interchange at 15 months of age (Figure 7Z). Prior to this age, Aβ1–42 accumulation in the hippocampus is below the threshold of detection for this antibody in male 3xTg-AD mice. Interestingly the more rostral areas of the hippocampus even at the 15-month time point do not show any Aβ1–42 reactivity (Figure 7J, R). By 18 months of age, the Aβ1–42 burden is rather significant with large dense plaques apparent in the caudal hippocampus at the area of the subiculum/CA1 interchange (Figure 7AA) with smaller deposits appearing in the stratum oriens and radiatum flanking the pyramidal layer of the hippocampus (Figure 7K, S). By 26 months the dense cored extracellular Aβ1–42 deposits have spread throughout the hippocampus. Intriguingly, Aβ1–42 reactivity is lacking in the cells that comprise the pyramidal layer of the CA1, which robustly stained with 6E10 at the same age (Figure 6H, P, X). Patterns of Aβ1–42 deposition in the entorhinal cortex begins in the deeper layers at 18 months of age (Figure 3W) and spreads to the more superficial layers, encompassing the entire entorhinal cortex by 26 months (Figure 3X). Similar observations of extracellular Aβ 1–42 deposition can be seen at 18 and 26 months in amygdala and primary motor cortex (Figures 4W, X, and 5W, X).Figure 7Extracellular Aβ 1–42 deposition is not immunohistochemically detectable in male 3xTg-AD mouse hippocampus until 15 months of age. A monoclonal antibody specific for human Aβ1–42 (12F4; Signet) was incubated with 20 month-old 3xTg-AD mouse brain sections alone (A), or with a 200-fold molar excess of the cognate reverse peptide (B), forward peptide (C), or no-primary control (D) according to a protocol designed to detect extracellular Aβ1–42. Coronal mouse brain sections (30 μm) were prepared from 3xTg-AD mice sacrificed at 2 (E, M, U), 3 (F, N, V), 6 (G, O, W), 9 (H, P, X), 12 (I, Q, Y), 15 (J, R, Z), 18 (K, S, AA), and 26 months of age (L, T, AB) and were processed for immunohistochemistry using the 12F4 antibody to detect extracellular Aβ1–42 peptide accumulation. CA1 hippocampal sections at Bregma -1.8 mm (E-L), at Bregma -2.5 mm (M-T), and at Bregma -2.8 mm (U-AB), were examined for regional and temporal patterns of extracellular Aβ1–42 deposition and photomicrographs were obtained at 10×. The inset in panel AB represents a 40× digitally magnified image of the photomicrograph for better visualization of stained cell morphology. Scale bar in D represents 200 μm.An early role of intracellular Aβ in neuronal dysfunction has been proposed (reviewed by [25]). Oddo et al. previously reported 3xTg-AD mice first show evidence of intraneuronal Aβ1–42 accumulation at ages when cognitive deficits begin to surface [19], and that as these mice age, they exhibit decreases in intraneuronal Aβ immunoreactivity with a concomitant increase in extracellular plaque load [26]. Historically, it has been technically difficult to detect intracellular Aβ with high confidence of specificity. To further detail the evolution of intracellular Aβ pathology in 3xTg-AD mice, we employed an immunohistochemical staining method optimized for visualization of intracellular Aβ1–42 peptide [27,28], which more readily unmasks intracellular Aβ peptide epitopes than standard formic acid epitope retrieval methods used for extracellular plaque immunohistochemistry. Using this methodology the employs a different anti-Aβ 42 antibody (Biosource/Invitrogen Aβ1–42), we have been able to reproducibly detect intracellular Aβ1–42 and demonstrated antibody specificity with cognate peptide competition tests (Figure 8A–D). The Covance/Signet 12F4 anti-Aβ1–42 antibody was not used with microwave pretreatment to detect intraneuronal Aβ. With formic acid pretreatment, the 12F4 antibody only labeled extracellular plaques and not Aβ localized intraneuronally. Evidence of intracellular Aβ1–42 immunopositivity using the Biosource/Invitrogen Aβ1–42 antibody was found in 3xTg-AD mouse brains beginning as early as 2 months of age (Figure 8E, M, U), and stably present throughout the time points assessed. The majority of intracellular Aβ1–42 expressing cells were detected outside of the pyramidal layer of the hippocampus, and found within the stratum oriens of the hippocampus (Figure 8E–T), the subiculum (Figure 8U-AB), and the corpus callosum (Figure 8M–T).Figure 8Intracellular Aβ 1–42 accumulation is immunohistochemically detectable by 3 months of age in 3xTg-AD mouse hippocampus. A polyclonal antibody specific for human Aβ1–42 (Biosource/Invitrogen) was incubated with 20 month-old 3xTg-AD mouse brain sections alone (A), or with a 200-fold molar excess of the cognate reverse peptide (B), forward peptide (C), or no-primary control (D) according to a protocol designed to detect intracellular Aβ1–42 [27]. Arrows indicate immunopositive cells, while \"*\" depicts non-specific signal due to precipitant. Coronal mouse brain sections (30 μm) were prepared from 3xTg-AD mice sacrificed at 2 (E, M, U), 3 (F, N, V), 6 (G, O, W), 9 (H, P, X), 12 (I, Q, Y), 15 (J, R, Z), 18 (K, S, AA), and 26 months of age (L, T, AB) and were processed for immunohistochemistry to detect intracellular Aβ1–42 peptide accumulation using the Biosource/Invitrogen anti-Aβ1–42 polyclonal antibody. CA1 hippocampal sections at Bregma -1.8 mm (E-L), at Bregma -2.5 mm (M-T), and at Bregma -2.8 mm (U-AB), were examined for regional and temporal patterns of intracellular Aβ1–42 and photomicrographs were obtained. The insets in panels U-AB represent digitally magnified images of designated photomicrographs for more optimal visualization of stained cell morphology. Scale bar in D represents 50 μm.Progression of Tau PathologyTau, which is expressed as 6 soluble isoforms from a genetic locus found on chromosome 17, is a microtubule-associated protein with numerous functions within the neuron [29-31]. One such cellular role is its ability to stabilize and promote the polymerization of microtubules [32-35]. This function has led to the hypothesis that the inability of tau to adequately bind and promote polymerization of microtubules would result in diminished transport within a neuron. Since it has been shown that the abnormal morphologic entity in AD brains known as the neurofibrillary tangle is comprised primarily of tau [36], it has been proposed that abnormalities of tau, directly or indirectly, play a central role in the pathogenesis of AD by progressively leading to a loss of fast axonal transport. A number of abnormalities of tau have been identified or suggested in AD neurons. These abnormalities include formation of tau into abnormal straight filaments or paired helical filaments [37-39], aggregations of paired helical filaments into the larger entities known as the neurofibrillary tangles (NFTs) [40], hyperphosphorylated tau [41-43], truncated tau [44], and the inability of tau to bind microtubules due to phosphorylation of key epitopes within the binding domain [45,46]. The human tauP301L mutation, which is included as one of the transgenes harbored in 3xTg-AD mice, is commonly used in mouse models for studying human tauopathies, such as progressive supranuclear palsy, corticobasal degeneration, and frontal temporal dementia (reviewed in [47]). In these mouse models, intraneuronal inclusions of tau arise as a result of a number of progressive phosphorylation events on serine, threonine, and tyrosine residues [31,48], and eventually evolve into NFTs.Human tauP301L transgene product can be detected using the HT7 antibody in a very limited number of 3xTg-AD pyramidal neurons in the CA1 of the hippocampus starting at 2 months of age (Figure 9A, I, Q). It was not until 6 months of age that a majority of pyramidal neurons harbored immunohistochemically detectable human tau. At both the 6 and 9-month time points (Figure 9C, K, S and 9D, L, T, respectively), the staining of axonal projections extending into the stratum radiatum began to intensify. Qualitatively, the staining for human tauP301L transgene product in cell bodies and processes appeared to diminish starting at 12 months of age (Figure 9E, M, U), remaining relatively constant at subsequent ages. Such changes in apparent levels of human tau protein could correspond to age-related increases in the phosphorylation state of tau, leading to steric hindrance and/or structural alteration of the HT7 epitope. When we examined the entorhinal cortex, HT7-positive cells were not detectable until 12 months of age (Figure 10E), and no qualitative signs of human tau transgene product accumulation, as determined by intensification of immunopositive signal, were evident as the ages of the mice increased (Figure 10F, G, H). As in the case for hAPPswe transgene expression, the human tauP301L transgene product was detectable in the amydala beginning at 6 months of age and its levels did not appear to significantly fluctuate as the 3xTg-AD mice aged (Figure 11A–H). Primary motor cortex appeared to be the earliest of those examined to exhibit human tauP301L transgene expression, where HT7-stained neurons were consistently detectable starting at 3 months of age (Figure 12A–H).Figure 9Human tauP301L transgene expression exhibits regionally and temporally disparate staining patterns in the 3xTg-AD mouse hippocampus. Coronal mouse brain sections (30 μm) were prepared from 3xTg-AD mice sacrificed at 2 (A, I, Q), 3 (B, J, R), 6 (C, K, S), 9 (D, L, T), 12 (E, M, U), 15 (F, N, V), 18 (G, O, W), and 26 months of age (H, P, X) and were processed for immunohistochemistry using the HT7 monoclonal antibody to detect human tauP301L transgene expression. CA1 hippocampal sections at Bregma -1.8 mm (A–H), at Bregma -2.5 mm (I–P), and at Bregma -2.8 mm (Q–X), were examined for regional and temporal patterns of human tauP301L and photomicrographs were obtained at 10×. The inset in panel X represents a 40× digitally magnified image of the photomicrograph for better visualization of stained cell morphology. Scale bar in D represents 200 μm.Figure 10Tau pathological hallmarks do not arise in the entorhinal cortex until 26 months of age. Coronal mouse brain sections (30 μm) were prepared from 3xTg-AD mice sacrificed at 2 (A, I, Q), 3 (B, J, R), 6 (C, K, S), 9 (D, L, T), 12 (E, M, U), 15 (F, N, V), 18 (G, O, W), and 26 months of age (H, P, X) and were processed for immunohistochemistry to detect the human tau P301L mutant transgene product using the HT7 monoclonal antibody (A–H), human phospho-tau (Thr231) using the AT180 monoclonal antibody (I–P), and paired helical filament pathology using the PHF-1 monoclonal antibody (Q–X). Entorhinal cortex was examined for patterns of immunopositivity and photomicrographs were obtained at 10×. The insets in panels H, P, and X represent a 40× digitally magnified images of designated photomicrographs for better visualization of immunopositive cell/pathology. Scale bar in D represents 200 μm.Figure 11The phospho-tau (Thr231) epitope is readily detectable, but paired helical filament pathology is virtually absent in the amygdala of 3xTg-AD mice. Coronal mouse brain sections (30 μm) were prepared from 3xTg-AD mice sacrificed at 2 (A, I, Q), 3 (B, J, R), 6 (C, K, S), 9 (D, L, T), 12 (E, M, U), 15 (F, N, V), 18 (G, O, W), and 26 months of age (H, P, X) and were processed for immunohistochemistry to detect the human tau P301L mutant transgene product using the HT7 monoclonal antibody (A–H), human phospho-tau (Thr231) using the AT180 monoclonal antibody (I–P), and paired helical filament pathology using the PHF-1 monoclonal antibody (Q–X). Amygdala was examined for patterns of immunopositivity and photomicrographs were obtained at 10×. The insets in panels H, P, and X represent a 40× digitally magnified images of designated photomicrographs for better visualization of immunopositive cell/pathology. Scale bar in D represents 200 μm.Figure 12Tau pathological hallmarks exhibit disparate staining patterns in the primary motor cortex of 2–26 month-old 3xTg-AD mice. Coronal mouse brain sections (30 μm) were prepared from 3xTg-AD mice sacrificed at 2 (A, I, Q), 3 (B, J, R), 6 (C, K, S), 9 (D, L, T), 12 (E, M, U), 15 (F, N, V), 18 (G, O, W), and 26 months of age (H, P, X) and were processed for immunohistochemistry to detect the human tau P301L mutant transgene product using the HT7 monoclonal antibody (A–H), human phospho-tau (Thr231) using the AT180 monoclonal antibody (I–P), and paired helical filament pathology using the PHF-1 monoclonal antibody (Q–X). Primary motor cortex was examined for patterns of immunopositivity and photomicrographs were obtained at 10×. The insets in panels H, P, and X represent 40× digitally magnified images of designated photomicrographs for better visualization of immunopositive cell/pathology. Scale bar in D represents 200 μm.Oddo and colleagues recently showed evidence that numerous phospho-tau epitopes are immunohistochemically detectable in 3xTg-AD mice by 15 months of age [49]. To assess the status of one important phospho-tau epitope not examined in that prior report and its evolution in the 3xTg-AD mouse brain as a function of age, we examined the phosphorylation of tau at residue Thr231 using the AT180 antibody. We could detect a limited number of immunopositive neurons in the pyramidal layer of the hippocampus as early as 6 months of age (Figure 13C, K), with a majority of AT180-positive cells residing in more caudal regions (Figure 13S). Robust AT180 positivity was apparent at 9 months of age in cells of the pyramidal layer, and fibers extending into the stratum radiatum of the hippocampus (Figure 13D, L, T). As with the HT7 detection there appears to be a waning of AT180 staining at 12 months. However, at more advanced ages (26 months), AT180-positive signals re-intensified, suggesting that these mice could exhibit a cycling phenomenon of tau phosphorylation profiles. AT180-positive cells were not detectable until 26 months in the entorhinal cortex of 3xTg-AD mice (Figure 10P), strongly suggesting that these mice are not experimentally suitable for studying the effects of pathogenic tau in neuronal networks comprising this brain region. The amygdala stains with AT180 beginning at 6 months and this immunopositivity remains high at later ages (Figure 11K–P), whereas the primary motor cortex less consistently stained for this phospho-tau epitope (Figure 12I–P), in that immunopositive signal could be detected only at 9, 12, and 26 months of age.Figure 13The phospho-tau epitope Thr231 is immunohistochemically detectable in 3xTg-AD mouse hippocampus by 6 months of age. Coronal mouse brain sections (30 μm) were prepared from 3xTg-AD mice sacrificed at 2 (A, I, Q), 3 (B, J, R), 6 (C, K, S), 9 (D, L, T), 12 (E, M, U), 15 (F, N, V), 18 (G, O, W), and 26 months of age (H, P, X) and were processed for immunohistochemistry using the AT180 monoclonal antibody to detect phospho-tau (Thr231) expression. CA1 hippocampal sections at Bregma -1.8 mm (A–H), at Bregma -2.5 mm (I–P), and at Bregma -2.8 mm (Q–X), were examined for regional and temporal patterns of human phospho-tau and photomicrographs were obtained at 10×. The inset in panel X represents a 40× digitally magnified image of the photomicrograph for better visualization of stained cell morphology. Scale bar in D represents 200 μm.As tau transitions to a more hyperphosphorylated state, it undergoes a self-assembly process into intertwining 4-nm paired helical filament (PHF) structures, further diminishing the ability of tau to preserve microtubule network integrity [50]. The monoclonal antibody PHF-1 (kindly provided by Dr. Peter Davies) recognizes PHF structural epitopes with robust affinity with trace reactivity towards unmodified normal human tau [51]. We were able to detect limited tau PHFs beginning in 15 month-old mice within the caudal CA1 region and subiculum (Figure 14W), but it was not until mice reached 26 months of age that we were able to consistently detect PHF-1 positive structures throughout the hippocampus (Figure 14H, P, X). Limited numbers of PHF-1 immunopositive cells were detectable in the entorhinal cortex and amygdala only at 26 months of age (Figures 10X and 11X), while PHF-1 immunopositive structures were barely detectable in primary motor cortex only at 26 months of age (Figure 12X).Figure 14Paired helical filament pathology does not arise until 18–26 months of age in 3xTg-AD mouse hippocampus. Coronal mouse brain sections (30 μm) were prepared from 3xTg-AD mice sacrificed at 2 (A, I, Q), 3 (B, J, R), 6 (C, K, S), 9 (D, L, T), 12 (E, M, U), 15 (F, N, V), 18 (G, O, W), and 26 months of age (H, P, X) and were processed for immunohistochemistry using the PHF-1 monoclonal antibody to detect phospho-tau (Ser396 and Ser404) associated with paired helical filament pathology. CA1 hippocampal sections at Bregma -1.8 mm (A–H), at Bregma -2.5 mm (I–P), and at Bregma -2.8 mm (Q–X), were examined for regional and temporal patterns of PHF-1 immunopositivity and photomicrographs were obtained at 10×. The inset in panel X represents a 40× digitally magnified image of the photomicrograph for better visualization of stained cell morphology. Scale bar in D represents 200 μm.Age-related Patterns of Microglia and Astrocyte StainingInflammatory processes have long been posited as serving integral roles in initiating and/or propagating AD-associated pathology within the human brain, as the elaboration of inflammatory cytokine expression and other markers of inflammation is more pronounced in individuals with known AD pathology. We previously reported significant enhancement of pro-inflammatory cytokine and chemokine expression and concomitant increases in region-specific microglial cell numbers, prior to the onset of overt amyloid pathology in young 3xTg-AD mice [52]. Herein, we assessed the status of two abundant non-neuronal cells traditionally activated in the setting of AD: microglia and astrocytes. The role of microglia and their accumulation at the sites of dense neuritic plaques has been described [53-55]. Immunohistochemical analysis of 3xTg-AD hippocampal brain tissue using an antibody specific for the microglia/macrophage surface marker, F4/80, revealed a qualitative enhancement of microglia staining from 2 (Figure 15A, I, Q) to 3 months of age (Figure 15B, J, R). The pattern of microglial distribution appeared rather uniform throughout the hippocampus from 3 to 15 months of age (Figure 15). Beginning at 18 months in the most caudal sections of hippocampus (Figure 15W) and continuing at 26 months (Figure 15X), there was a marked change in the distribution of microglia, with these cells appearing to assemble into dense aggregates, reminiscent of amyloid plaque-like structures. Microglial staining patterns in the entorhinal cortex were similar to those in hippocampus, except that aggregation of F4/80-positive cells was not overtly evident in 18 and 26 month-old 3xTg-AD mice (Figure 16A–H). F4/80-positive microglia in both the amygdala and primary motor cortex exhibited age-related distributions similar to those of the hippocampal formation (Figures 17A–H and 18A–H, respectively).Figure 15Microglial staining patterns modulate as a function of age in the 3xTg-AD mouse hippocampus. Coronal mouse brain sections (30 μm) were prepared from 3xTg-AD mice sacrificed at 2 (A, I, Q), 3 (B, J, R), 6 (C, K, S), 9 (D, L, T), 12 (E, M, U), 15 (F, N, V), 18 (G, O, W), and 26 months of age (H, P, X) and were processed for immunohistochemistry using the F4/80 monoclonal antibody to detect brain-resident microglia/macrophages. CA1 hippocampal sections at Bregma -1.8 mm (A–H), at Bregma -2.5 mm (I–P), and at Bregma -2.8 mm (Q–X), were examined for regional and temporal patterns of F4/80 immunopositivity and photomicrographs were obtained at 10×. The inset in panel X represents a 40× digitally magnified image of the photomicrograph for better visualization of stained cell morphology. Scale bar in D represents 200 μm.Figure 16Entorhinal cortex microglial and astrocytic staining patterns evolve on a similar timescale as observed in the 3xTg-AD hippocampus. Coronal mouse brain sections (30 μm) were prepared from 3xTg-AD mice sacrificed at 2 (A, I), 3 (B, J), 6 (C, K), 9 (D, L), 12 (E, M), 15 (F, N), 18 (G, O), and 26 months of age (H, P) and were processed for immunohistochemistry to detect activated microglia using an anti-F4/80 specific monoclonal antibody (A–H) and astrocytes using an anti-GFAP specific monoclonal antibody (I–P). Entorhinal cortex was examined for patterns of immunopositivity and photomicrographs were obtained at 10×. The insets in panels H and P represent 40× digitally magnified images of designated photomicrographs for better visualization of immunopositive cells. Scale bar in D represents 200 μm.Figure 17Amygdala-resident microglial and astrocytic staining patterns evolve on a similar timescale as observed in the 3xTg-AD hippocampus. Coronal mouse brain sections (30 μm) were prepared from 3xTg-AD mice sacrificed at 2 (A, I), 3 (B, J), 6 (C, K), 9 (D, L), 12 (E, M), 15 (F, N), 18 (G, O), and 26 months of age (H, P) and were processed for immunohistochemistry to detect activated microglia using an anti-F4/80 specific monoclonal antibody (A–H) and astrocytes using an anti-GFAP specific monoclonal antibody (I–P). Amygdala was examined for patterns of immunopositivity and photomicrographs were obtained at 10×. The insets in panels H and P represent 40× digitally magnified images of designated photomicrographs for better visualization of immunopositive cells. Scale bar in D represents 200 μm.Figure 18Primary motor cortex microglial and astrocytic staining patterns evolve on a similar timescale as observed in the 3xTg-AD hippocampus. Coronal mouse brain sections (30 μm) were prepared from 3xTg-AD mice sacrificed at 2 (A, I), 3 (B, J), 6 (C, K), 9 (D, L), 12 (E, M), 15 (F, N), 18 (G, O), and 26 months of age (H, P) and were processed for immunohistochemistry to detect activated microglia using an anti-F4/80 specific monoclonal antibody (A–H) and astrocytes using an anti-GFAP specific monoclonal antibody (I–P). Primary motor cortex was examined for patterns of immunopositivity and photomicrographs were obtained at 10×. The insets in panels H and P represent 40× digitally magnified images of designated photomicrographs for better visualization of immunopositive cells. Scale bar in D represents 200 μm.Astrocytes are involved in many different functions in the brain, including structural integrity of the blood brain barrier, support of neuronal synapses by ion regulation and removal of glutamate [56]. Although it is believed that they are not directly responsive to primary insults, astrocytes react to inflammatory events in the brain, relying upon pro-inflammatory molecules elaborated from activated microglia [55]. Once signaled to do so, astrocytes can perpetuate inflammatory events in the brain via expression of iNOS and the enzyme argininosuccinate synthetase [57,58]. Glial fibrillary acidic protein (GFAP) is often employed as a marker of astrocytic activation. GFAP-expressing astrocytes were readily visible in 3xTg-AD mice at 2 months of age throughout the hippocampus (Figure 19A, I, Q), with limited signs of activation in the entorhinal cortex, amygdala, and primary motor cortex at this age (Figures 16I, 17I, and 18I). There appeared to be a qualitative decline in staining intensity in rostral hippocampal regions beginning at 15 months of age (Figure 19F) and continuing through 26 months of age (Figure 19H). However, the overall pattern of activated astrocyte staining within the hippocampus remained relatively constant as a function of age. More robust GFAP-positive astrocyte staining in the entorhinal cortex was more apparent at the 18- and 26-month time points (Figure 16O, P), while GFAP staining was less detectable, but constant at ages greater than 18 months in amygdala and primary motor cortex (Figures 17O, P and 18O, P, respectively).Figure 19GFAP-positive astrocyte staining remains relatively constant as a function of age in 3xTg-AD mouse hippocampus. Coronal mouse brain sections (30 μm) were prepared from 3xTg-AD mice sacrificed at 2 (A, I, Q), 3 (B, J, R), 6 (C, K, S), 9 (D, L, T), 12 (E, M, U), 15 (F, N, V), 18 (G, O, W), and 26 months of age (H, P, X) and were processed for immunohistochemistry using a GFAP-specific monoclonal antibody to detect brain-resident astrocytes. CA1 hippocampal sections at Bregma -1.8 mm (A–H), at Bregma -2.5 mm (I–P), and at Bregma -2.8 mm (Q–X), were examined for regional and temporal patterns of GFAP immunopositivity and photomicrographs were obtained at 10×. The inset in panel X represents a 40× digitally magnified image of the photomicrograph for better visualization of stained cell morphology. Scale bar in D represents 200 μm.Discussion and conclusionThe 3xTg-AD mouse serves as an informative preclinical model employed ever increasingly in the examination of mechanisms underlying AD, as well as for the vetting of experimental AD-modifying therapeutics. The present study was designed to immunohistochemically document the evolution of transgene expression, amyloid deposition, pathogenic tau phosphorylation, astrogliosis, and microglial activation throughout the hippocampus and entorhinal cortex over a 26-month period in 3xTg-AD mice. A summary of pathological progression and qualitative severity scoring in these mice is illustrated in Table 2. Previously published reports employing the 3xTg-AD model have primarily focused upon mechanisms/pathologies pertaining to either early or late-stage disease, and have provided limited insight into the overall progression of evolving AD-related pathologies using a systematic immunohistochemical approach. Gaining a detailed understanding as to how hAPPswe and tauP301L transgene expression relates temporally and spatially to the appearance of pathogenic Aβ peptide- and hyperphosphorylated tau-related pathologies enables the informed design and implementation of future studies. Moreover, given that these AD-related pathologies exhibit subtle regional differences within the 3xTg-AD mouse brain, the information gleaned from systematic immunohistochemical assessment hones the focus of endpoint analyses on brain regions more or less severely impacted depending upon the hypotheses being tested.Table 2Qualitative assessment of pathological progression by region in male 2–26 month-old 3xTg-AD mice.AntigenAge (months)hAPP236912151826Primary motor cortex++++++++Amygdala--++++++++++Rostral Hippocampus CA1++++++++++++Medial Hippocampus CA1++++++++++++Caudal Hippocampus CA1/Subiculum++++++++++++Entorhinal Cortex++++++++++++hAPP/AβPrimary motor cortex++++++++++++++++Amygdala++++++++++++++++Rostral Hippocampus CA1--++++++++++++Medial Hippocampus CA1--++++++++++++Caudal Hippocampus CA1/Subiculum-++++++++++++++Entorhinal Cortex--++++++++++hAβ 1–42 (Extracellular)Primary motor cortex------+++Amygdala------+++++Rostral Hippocampus CA1------+++Medial Hippocampus CA1------+++Caudal Hippocampus CA1/Subiculum-----++++++Entorhinal Cortex------++++hAβ 1–42 (Intracellular)Primary motor cortex+/-+/-++++++++++Amygdala+/-+/-++++++++++Rostral Hippocampus CA1+/-+++++++++++++Medial Hippocampus CA1+++++++++++++++Caudal Hippocampus CA1/Subiculum+++++++++++++++Entorhinal Cortex+/-+++++++Human TauPrimary motor cortex+/-+++++++Amygdala--++++++Rostral Hippocampus CA1+/-+/-++++++Medial Hippocampus CA1+/-+/-++++++++Caudal Hippocampus CA1/Subiculum+/-+/-++++++++Entorhinal Cortex-----+/-+/-+/-Phospho-hTau (Thr231)Primary motor cortex---+++/-+/-+Amygdala--+++++++++Rostral Hippocampus CA1--+/-+++/-+/-+Medial Hippocampus CA1--+/-++++/-++Caudal Hippocampus CA1/Subiculum--++++++Entorhinal Cortex-------+/-Paired Helical FilamentsPrimary motor cortex--------Amygdala--------Rostral Hippocampus CA1------+/-+Medial Hippocampus CA1------+/-+Caudal Hippocampus CA1/Subiculum------++Entorhinal Cortex-------+/-MicrogliaPrimary motor cortex++++++++++Amygdala+++++++++Rostral Hippocampus CA1++++++++++++++Medial Hippocampus CA1++++++++++++++Caudal Hippocampus CA1/Subiculum++++++++++++++++Entorhinal Cortex++++++++++++++AstrocytesPrimary motor cortex++++++++Amygdala+/-+/-+/-+/-+/-+/-+/-+/-Rostral Hippocampus CA1++++++++Medial Hippocampus CA1++++++++Caudal Hippocampus CA1/Subiculum++++++++Entorhinal Cortex++++++++We found that patterns of intracellular Aβ peptide immunoreactivity do not correlate with the patterns of human APPswe transgene expression in 3xTg-AD mice. Both Aβ1–42-specific antibodies that were employed, which have been pre-absorbed to eliminate binding to non-Aβ1–42 species, exhibit distinct patterns of staining that lie outside of the pyramidal cell layer of the hippocampus and layer II/III of the entorhinal cortex. These cell layers, however, show heavy immunoreactivity for human APPswe transgene product. A number of groups have demonstrated that APP is synthesized at the cell body and undergoes anterograde axonal transport to distal compartments where it is ultimately proteolytically processed (reviewed in [59]). Neurons emanating from layer III of the entorhinal cortex extend bilaterally into the CA1 and CA3 of the hippocampus and subiculum [60], whereas pyramidal neurons from the CA1 of the hippocampus project into layer V of the entorhinal cortex and to areas of the subiculum [61]. This evidence taken together with the observed patterns of hAPPswe transgene expression within cells that comprise the perforant pathway in 3xTg-AD mice, it is reasonable to propose that the Aβ1–42 cleavage product is generated and accumulates at termini.We should note that the cells harboring intracellular Aβ are purported to be neurons, but whether they represent the sole cell type physiologically influenced by intracellular Aβ peptide accumulation is unlikely. It is known that other cell types [62], including microglia [53] and astrocytes [63], are able to pinocytize and phagocytize extracellular Aβ1–42. These cells may represent viable targets for the deleterious effects attributed to intracellular Aβ peptides. Cummings and colleagues demonstrated previously that Aβ peptides, produced initially by neurons and deposited at neuronal terminals, are readily able to form higher order aggregates [64]. This may also explain the spatial alterations found with in the hippocampus where plaque burden is observed at earlier ages in the more caudal regions of the 3xTg-AD brain. Hence, the anatomical interconnectivity of these projections and biogenesis and subsequent proteolytic processing of hAPPswe likely underlie the dissonant patterns of Aβ and hAPPswe immunoreactivity observed in the CA1, subiculum, and the entorhinal cortex of 3xTg-AD mice.It was somewhat surprising that extracellular Aβ1–42 plaques were not readily detectable in male 3xTg-AD mice in our study until 15 months in the caudal hippocampus and 18 months in cortical structures, findings that are in conflict with Oddo and colleagues that stated in their original 2003 report that 3xTg-AD mice exhibit amyloid plaques beginning at 6 months of age in the frontal cortex [17]. These disparate results may be the result of the antibodies employed or the genders of mice studied in each study, but such details are absent from the original report making it difficult to resolve this issue. Other explanations may exist, including loss of phenotype due to a progressive loss of transgene copies with successive breeding, different founder/line of mice provided to outside investigators compared to the mice described in the original report, as well as differences in housing conditions. The mice used in the present study were pathogen-free and were maintained in a pathogen-free facility throughout the duration of the experiments. Moreover, we have not observed any overt reduction in transgene copy numbers over the 12+ generations we have housed 3xTg-AD mice within our animal colony. However, determining the differences in pathological progression and elucidating the underlying cause(s) is important for generation of consistent data sets across different laboratories that are meaningful and provide generally applicable mechanistic insight into AD-related processes.Similar to the hAPPswe transgene product, human tauP301L, as detected with the HT7 antibody, appears immunohistochemically at 2–3 months of age in 3xTg-AD mice. This is not surprising given that both hAPPswe and tauP301L transgenes are transcriptionally controlled by the Thy 1.2 promoter, and that the transgenes are genetically linked due to the method by which the 3xTg-AD mice were derived [17]. Using phospho-epitope specific antibodies, Sahara and colleagues showed that tau proceeds through a series of post-translational phosphorylation events that progressively lead to increased insolubility and decreased functionality [31]. We were able to detect phosphorylation at the Thr231 amino acid residue of tau, an indication of pathogenic progression, as early as 6 months of age in cells residing within the pyramidal layer of the hippocampus. This evidence suggests that tau dysfunction contributes to AD-related pathophysiology in 3xTg-AD mice at ages earlier than purported in previously published studies [18]. Oddo and colleagues reported that the Thr231 phospho-epitope of tau is not detectable until 12 months of age. Given our findings, designs of future studies seeking to dissect the differential influences of APPswe and tauP301L on brain physiology need to take into account the overlapping temporal expression patterns of the 3xTg-AD transgenes.We have previously demonstrated that various inflammatory events correspond to the presentation of early (< 6 months of age) intracellular Aβ pathology in 3xTgAD mice [52]. Specifically, the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) and chemokine monocyte chemoattractant protein-1 (MCP-1) are expressed at heightened levels in a region-specific pattern, and these enhanced molecule expression profiles correlate with increased numbers of microglia specifically within 3xTg-AD mice as compared to age-matched, non-transgenic control mice. As such, it was imperative in the present study to continue to monitor alterations in glial marker expression as these mice age as staining intensity and/or pattern changes could provide insight into pathophysiology. We observed a clustering of F4/80-positive microglia/monocyte cells within areas exhibiting heavy amyloid burden in 3xTg-AD mice at 15 to 26 months of age. It is likely that this pattern of microglial accumulation is indicative of association with Aβ-containing plaques similar to that observed in human AD brain. Interestingly, 3xTg-AD mice do not show noticeable age-related enhancement GFAP-positive astrocyte staining intensities, which markedly differentiates this model from what has been documented in other AD mouse models, including the PDAPP mouse [65]. These differences in activation of astrocytes may inherently relate to the relative strength and/or cell type expression specificities of the promoters employed to drive each of the respective AD-related transgenes (reviewed by [66]).Other investigators have shown that amyloidogenic mouse models of AD exhibit gender-related differences in severity of pathology [67]. In this study we present a time course of AD-related pathological progression solely for male 3xTg-AD mice in order to more finely assess intermediate ages for subtle region-specific differences. While Clinton et al. have shown that sexual dimorphisms in cognition and stress responses are apparent between male and female 3xTgAD mice [68], that study did not include an extensive histological comparison of brains from the two genders. Carroll and colleagues more recently reported that female 3xTg-AD mice exhibit an earlier onset of AD pathology and this may be a consequence of progesterone and estrogen-mediated signaling mechanisms [69]. Given these gender-specific differences in time of onset and severity of behavioral phenotype it is imperative that future experimental therapeutics be vetted in both male and female 3xTg-AD mice. Moreover, the underlying neuroinflammatory state of each gender is likely disparate and may markedly impact the efficacy and/or safety profile of a particular therapeutic, especially if that modality is immune-based in nature. The 3xTg-AD model provides an informative platform on which to test new therapeutic modalities, but the regional, temporal, and gender-specific subtleties and limitations of this model must be fully appreciated before this model is elevated to \"golden standard\" status in the field of AD research.MethodsTransgenic MiceThe 3xTg-AD mice (B1 line) were kindly provided by Frank LaFerla (University of California, Irvine; [17]). All mice were housed and bred in accordance with University of Rochester requirements for animal welfare and care. Homozygous 3xTg-AD mice were monogamously mated to produce offspring, which were housed until sacrificed at the designated age. Mice were sacrificed via pentabarbitol overdose and subsequently transcardiac perfused with heperanized saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB). Brains were removed and post-fixed overnight in 4% paraformaldehyde in 0.1 M PB, followed by equilibration in 20% sucrose in 0.1 M phosphate-buffered saline (PBS) and then 30% sucrose in 0.1 M PBS. Brains were coronally sectioned on a freezing stage sliding microtome (Microm, Walldorf, Germany) at 30 μm and stored in cryoprotectant at -20°C until immunohistochemical processing.Nissl StainingBrain sections were washed with 0.15 M PB for 2 h to remove the cryoprotectant, and mounted on Superfrost® Plus slides (VWR International, West Chester, PA) and allowed to dry completely. The slides were subsequently hydrated in dH20 for 5 min. before being stained with 0.02% Cresyl violet Acetate in 0.25% Acetic acid for 30 min. Sections were rinsed in 3 changes of dH20, and placed in 50% ethanol for 1 min. followed by 70% for 1 min. to destain. Sections were allowed to dry and then cleared by dipping in xylene before being coverslipped.AntibodiesThe following antibodies were used at the designated working dilutions: anti-amyloid precursor protein A4, corresponding to the NPXY motif of hAPP, (Clone Y188; AbCam, Cambridge, MA, 1:750); anti-hAPP/amyloid-beta reactive to amino acid residue 1–16 of beta-amyloid (6E10; Covance, Berkeley, CA; 1:1000); anti-amyloid beta 1–42 clone 12F4 reactive to the C-terminus of beta-amyloid and specific for the isoform ending at amino acid 42 (Covance/Signet, Berkeley, CA, 1:1000); anti-amyloid beta 1–42 polyclonal antibody for intracellular amyloid-beta staining (Invitrogen, Carlsbad, CA, formerly Biosource, Hopkinton, MA 1:1000); anti-human tau HT7, reactive to residues 159 to 163 (Pierce, Rockford, IL; 1:200); anti-human phosphorylated tau AT180, specific for htau phosphorylated at the Thr231 residue (Pierce, Rockford, IL; 1:200); anti-human phosphorylated tau PHF-1 (gift from Dr. Peter Davies, Albert Einstein College of Medicine; 1:30); anti-glial fibrillary acidic protein GFAP (Dako Cytomation, Glostrup, Denmark; 1:1000); and an antibody specific for the microglial/monocytic cell surface marker F4/80 (AbD Serotec, Raleigh, NC; 1:500). Specificity of the anti-Aβ1–42 antibodies (12F4 from Covance/Signet for extracellular Aβ staining and anti-Aβ1–42 polyclonal from Biosource/Invitrogen for intracellular Aβ staining) was confirmed using peptide competition experiments. The results of these assessments are illustrated in Figures 7 and 8, respectively.ImmunohistochemistryBrain sections were washed with 0.15 M PB for 2 h to remove the cryoprotectant, then incubated with 3% H2O2 in 0.15 M PB for 20 min. to quench endogenous peroxidase activity. For Aβ peptide-specific stains, the sections were treated with 70% formic acid for 15 min. for epitope retrieval. For the intracellular Aβ1–42stain, we employed a microwave/Target buffer (Dako Cytomation, Glostrup, Denmark) epitope retrieval method as described previously [27]. Briefly, the brain sections were washed and peroxidase activity quenched. The sections were mounted on to slides and allowed to dry. The Target buffer was heated to 98°C in a microwave (GE, Louisville, KY), the slides submerged into the buffer and placed in the microwave, twice for 3 min. at 450 W, and allowed to rest for 5 min. between microwave steps. Brain sections were similarly processed for immunohistochemistry as detailed below. The sections were washed and permeabilized in 0.15 M PB and 0.4% Triton X-100, followed by blocking in 0.15 M PB with 10% normal goat serum, and 0.4% Triton X-100. After blocking, the sections were incubated in 0.15 M PB with 1% normal goat serum, and 0.4% Triton X-100, with the designated primary antibody. The sections were washed with 0.15 M PB, followed by an incubation with the appropriate secondary biotin-conjugated secondary antibodies (Vector Labs, Burlingame, CA; 1:1000) in 0.15 M PB with 1% normal goat serum, and 0.4% Triton X-100. The sections were washed with 0.15 M PB with 1% normal goat serum and 0.4%Triton X-100, and incubated in the avidin-biotin complex (Vector Labs Vectastain ABC System as per manufacturer's protocol, Vector Labs, Burlingame, CA). Sections were washed in 0.15 M PB followed by rinses in dH2O. The sections were developed with nickel-enhanced DAB (Vector Labs, Burlingame, CA). Sections were mounted on Superfrost®Plus slides (VWR International, West Chester, PA) cover-slipped and viewed using an Olympus AX-70 microscope and motorized stage (Olympus, Center Valley, PA) and the MCID 6.0 Imaging software (Interfocus Imaging subsidiary of GE Healthcare, Cambridge, England).Qualitative scoring of immunohistochemical staining intensitiesTime points were compared to one another within a particular antibody staining group. Images were analyzed using the relative optical density (ROD) score in the MCID 6.0 Imaging software (Interfocus Imaging subsidiary of GE Healthcare, Cambridge, England). Sections of all the mice, corresponding to the areas of study, were scored according to the following schema: (-) indicates no staining present, (+/-) indicates limited number of cells/structures showing evidence of staining, (+) denotes consistent expression of the marker, (++) represents an elevated expression measured in ROD of Δ 0.075 to 0.250, and (+++) represents a further increase in staining as indicated by a change in ROD of Δ 0.251 greater than (+) staining intensities.AbbreviationsAD: Alzheimer's disease; APP: amyloid precursor protein; APPswe: amyloid precursor protein Swedish mutation; PHF: paired helical filament; PS1: Presenilin 1; Aβ: Beta-amyloid; TNF-α: Tumor necrosis factor-alpha; MCP-1: monocyte chemoattractant protein-1; Tg: Transgenic; PB: Phosphate buffer; PBS: Phosphate-buffered saline.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsMAM performed brain sectioning and immunohistochemistry, and aided in the preparation of the manuscript. WJB conceived the design of the study, aided in the preparation of the manuscript, and provided critical analysis of the manuscript. Both authors read and approved the final manuscript.\n\nREFERENCES:\nNo References"
+}

batch_9/PMC2527612.json

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+{
+    "id": "PMC2527612",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527612\nAUTHORS: Matthew N Ward, Allison M Churcher, Kevin J Dick, Chris RJ Laver, Greg L Owens, Megan D Polack, Pam R Ward, Felix Breden, John S Taylor\n\nABSTRACT:\nBackgroundComparisons of functionally important changes at the molecular level in model systems have identified key adaptations driving isolation and speciation. In cichlids, for example, long wavelength-sensitive (LWS) opsins appear to play a role in mate choice and male color variation within and among species. To test the hypothesis that the evolution of elaborate coloration in male guppies (Poecilia reticulata) is also associated with opsin gene diversity, we sequenced long wavelength-sensitive (LWS) opsin genes in six species of the family Poeciliidae.ResultsSequences of four LWS opsin genes were amplified from the guppy genome and from mRNA isolated from adult guppy eyes. Variation in expression was quantified using qPCR. Three of the four genes encode opsins predicted to be most sensitive to different wavelengths of light because they vary at key amino acid positions. This family of LWS opsin genes was produced by a diversity of duplication events. One, an intronless gene, was produced prior to the divergence of families Fundulidae and Poeciliidae. Between-gene PCR and DNA sequencing show that two of the guppy LWS opsins are linked in an inverted orientation. This inverted tandem duplication event occurred near the base of the poeciliid tree in the common ancestor of Poecilia and Xiphophorus. The fourth sequence has been uncovered only in the genus Poecilia. In the guppies surveyed here, this sequence is a hybrid, with the 5' end most similar to one of the tandem duplicates and the 3' end identical to the other.ConclusionEnhanced wavelength discrimination, a possible consequence of opsin gene duplication and divergence, might have been an evolutionary prerequisite for color-based sexual selection and have led to the extraordinary coloration now observed in male guppies and in many other poeciliids.\n\nBODY:\nBackgroundUnderstanding the molecular basis of characters shaped by selection is a major goal of evolutionary genetics. Of particular interest are genes that encode conspicuous secondary sexual traits in males and the genes that influence female preference for such traits [1]. Among fish; sticklebacks (genus Gasterosteus), cichlids, and poeciliids, including the guppy (Poecilia reticulata) and swordtails (genus Xiphophorus), are the most important models for the study of sexual selection driven by female choice. In each of these taxa, female mate choice is influenced by male coloration and in each group, male coloration and female preference have a genetic basis [2-6].Mapping studies designed to uncover genes responsible for species- and population-level color variation in cichlids and sticklebacks are underway [7] but to date none have been identified. While it is also the case that no female preference loci have been uncovered in fish, many cichlid species and some populations possess unique opsin genes that provide strong candidates. Indeed, the only DNA sequences that have been found to differ among the 200 to 500 endemic Lake Victoria haplochromine species are long wave-sensitive (LWS) opsins [8,9]. In the cichlid genus Pundamilia, LWS opsin sequence and expression appears to be tuned to specific male color morphs [10]. Thus, it appears that variation in LWS opsin genes influences female mate choice and speciation in this family [11].Opsin genes encode membrane-bound receptors that are expressed primarily in rod and cone cells of the retina. Each opsin protein is associated with a chromophore and when exposed to light, this complex changes shape leading to rod or cone cell hyperpolarization [12]. The detection of light at the receptor level requires input from just one type of opsin-chromophore receptor. However, discriminating among colors (wavelengths) involves the interpretation of signals from multiple adjacent retinal cone cells expressing different opsins. These different opsins often have names that reflect the wavelength of light to which they are most sensitive. For instance, short wave-sensitive (SWS), middle wave-sensitive (MWS), and long wave-sensitive opsins (LWS) are most sensitive to blue, green and red light, respectively. Gene duplication and divergence has generated this opsin diversity. For example, the human MWS opsin is a duplicate (or paralog) of the LWS opsin locus and now differs at three of the five amino acid positions known to influence wavelength sensitivity [13-16]. Zebrafish also have a pair of LWS opsin genes with different five key-site haplotypes [17].The purpose of this study was to characterize LWS opsin gene sequence variation in guppies and in closely related species. We focused on this gene because microspectrophotometry (MSP) data indicated that guppies express more than one type of LWS opsin [18,19] and because orange is an important component of female mate choice for these fish [4,20-22]. While two recent studies have reported LWS opsin gene variation in guppies [23,24], one focused only on short amplicons from a single fish and both presented incomplete data on the key-site amino acids known to influence spectral sensitivity. Genomic sequences, transcript expression levels, and data from other poeciliids have also not been reported to date.We show that guppies (Poecilia reticulata) and three species in the guppy sister group (Micropoecilia) have four LWS genes. Sequence variation at the five key sites indicates that three of these LWS opsins are most sensitive to different wavelengths of light providing Poecilia with a larger repertoire of LWS pigments than any other fish taxon. One of the guppy LWS opsins appears to be a single-exon gene, likely arising from a retrotransposition event. This gene was sequenced in all poeciliids surveyed except Tomeurus gracilis and has also been reported in the killifish, Lucania goodei (family Fundulidae). Two LWS opsins are linked, oriented in a tail-to-tail fashion, and separated by approximately 3.3 Kbp. The fourth is found only in the genus Poecilia. This is a hybrid or mosaic sequence in the guppies surveyed here from Cumaná Venezuela. All four LWS opsins in the guppy were amplified from RNA isolated from adult eyes, but qPCR experiments show much variation among these duplicates in the level of expression.MethodsGenomic PCR and sequencingAll species surveyed are in the family Poeciliidae. Long wave-sensitive (LWS) opsin genes were amplified from DNA isolated from Tomeurus gracilis (one individual), Xiphophorus pygmaeus (one individual), and from four species in the genus Poecilia: P. reticulata (14 individuals), P. picta (four individuals) P. parae (three individuals) and P. bifurca (three individuals). The genus Tomeurus is the sister group to a clade that includes Poecilia and Xiphophorus, and most other poeciliids [25,26]. Poecilia picta, P. parae, and P. bifurca, occur in the sister taxon to the guppy [27]. They were in a separate genus previous to Rosen and Bailey's [28] revision of the poeciliids and we refer to them collectively as Micropoecilia. Poecilia reticulata (the guppy) was sampled from a population collected in Cumaná, Venezuela and bred in our laboratory aquarium. The Cumaná guppy has also been referred to as Endler's guppy, but is closely related to other guppy populations [29]. PCR reactions were run using genomic DNA isolated from fish euthanized with buffered MS222 (Sigma® A5040) or from specimens preserved in 95% ethanol.Initially, PCR and sequence data were obtained using primers ForBeg, Fw1a and Rev5, which are complementary to conserved regions of fish LWS opsin genes in exon I (ForBeg), exon II (Fw1a), and exon V (Rev5) (see Additional files 1 and 2). After uncovering multiple LWS opsin sequences in Poecilia, we attempted to PCR-amplify DNA between guppy opsin genes. The between-gene PCR experiment was initiated because LWS opsins occur in tandem in human, zebrafish, and medaka. It employed the reverse complement of a forward primer close to the 5' end of the gene (Fw1a Comp) and the reverse complement of a reverse primer close to the 3' end of the gene (Rev8 Comp). Sequence data from amplicons derived from primers ForBeg, Fw1a and Rev5, and the success of between-gene PCR allowed us to develop gene-specific primers, including reverse primers complementary to 3' UTR sequences. Primers complementary only to guppy LWS 'variant 6' were designed from sequence data recently published by Weadick and Chang [24]. PCR amplicons were cut and purified from agarose gels using a QIAquick® Gel Extraction Kit and were cloned using the pGEM® – T Easy Vector System II kit (Promega™). Sequencing of insert-positive clones utilized labeled M13 forward and reverse primers and a LI-COR sequencer at the Centre for Biomedical Research at the University of Victoria. A list of PCR primer sequences, PCR reaction conditions and a primer map can be found in Additional file 1, Additional file 2, and Additional file 3, respectively.Southern Blot HybridizationDNA from one lab-reared Cumaná guppy was extracted using a Qiagen® DNeasy Tissue Kit and digested with the four-cutter restriction enzyme BfaI (New England Biolabs®). 423 bp DIG labeled probes were prepared from guppy genomic DNA using the PCR DIG Probe Synthesis Kit (Roche®) and primers Fw100 and Rev4 (see Additional file 1). The amplicons from this PCR reactions were purified using the QIAquick® Gel Extraction Kit (Qiagen®). BfaI does not cut any of the LWS opsins in the region complementary to the probes. Southern blot hybridization was carried out using a modified protocol from the Roche® DIG application manual for filter hybridization. Digested DNA was blotted onto a Bio-Rad™ Zeta-Probe® Blotting membrane using the Bio-Rad™ Model 785 Vacuum Blotter. This was followed by UV exposure (120 mJ) and probe hybridization at 40°C overnight. The blot was washed in 2× SSC at room temperature and then in 1× SSC at 65°C and visualized using the DIG Luminescent Detection Kit for Nucleic Acids (Roche®).LWS opsin gene expression using RT-PCRPrior to quantitative PCR (qPCR) experiments (see below) we tested the hypothesis that all four LWS opsin loci were expressed using reverse transcriptase (RT)-PCR. Three guppies were euthanized in buffered MS222. A single eye from each individual was placed in 1.0 mL PureZOL™ (Bio-Rad®) with 3 mm tungsten carbide beads and homogenized for five minutes in a Retsch MM301 Mixer Mill. Total RNA was extracted using the Aurum™ Total RNA Fatty and Fibrous Tissue kit from BioRad®. The iScript™ kit (Bio-Rad®) was used to generate single-stranded cDNA. LWS opsin transcripts were PCR amplified and cloned using the pGEM® – T Easy Vector System II kit (Promega™) and then sequenced with labeled M13 primers. Primers reported by Meyer and Lydeard [25] that amplify XSrc were used as a positive control. Primer sequences and PCR conditions can be found in Additional files 1 and 2.Quantitative PCRData from cichlids and zebrafish indicate that cone opsin expression is highest at the end of the day [30,31]. Guppies were maintained in a 14:10 hr light and dark cycle and qPCR experiments were performed on cDNA samples obtained from three fish in the last hour of the subjective day. Total RNA was extracted from the eyes of these fish (one adult male and two adult females) using the Retsch MM301 Mixer Mill and the Aurum™ Total RNA Fatty and Fibrous Tissue kit from BioRad®. Synthesis of cDNA for qPCR experiments utilized the SuperScript™ III First-Strand Synthesis SuperMix kit for qRT-PCR (Invitrogen™) and 1 μg of total RNA from the three samples. To determine the concentration of each transcript in the three cDNA samples, we used the Invitrogen™ SYBR® GreenER™ qPCR SuperMix Universal kit to prepare triplet qPCR reactions. qPCR was carried out in a Stratagene® Mx4000® Multiplex Quantitative PCR machine with the following locus-specific primer pairs: a/sExon2 and LWS1IntRev; A180SpecFwd and rev8; pExon2 and LWS2IntRev; and fw100 plus revA (see Additional file 1). A 1:10 ROX Reference Dye normalized the fluorescent reporter signal. qPCR conditions consisted of 1 cycle at 95°C (9 minutes); 50 cycles of 95°C (15 seconds), 60°C (30 seconds), 72°C (45 seconds); 1 cycle of 95°C (1 minute); and a 40-step melting curve analysis (initial temperature 55°C, increasing 1°C every 30 seconds). Each gene was also PCR amplified, cloned using the pGEM® – T Easy Vector System II kit (Promega™), sequenced to confirm identity, and then utilized for qPCR at concentrations of 1 ng, 1 × 10-3ng and 1 × 10-5ng per 16 μL reaction. Ct values from these plasmid templates were then used to generate a standard curve and to estimate qPCR efficiency (qPCReff = [10(-1/slope) – 1] × 100; Table 1). The plasmid template reactions were also run in triplicate. Dissociation curves (Fluorescence [-R'(T)] over T°C) and gel electrophoresis confirmed the presence of single amplicons in all qPCR reactions.Table 1Key-site haplotypes for LWS opsin duplicates in fish and humans.Key Site PositionGuppy LWS S180Guppy LWS A180Guppy LWS P180Guppy LWS S180rKillifish LWS AKillifish LWS BRice fish LWS ARice fish LWS BZebrafish LWS 1Zebrafish LWS 2Cave fish LWS g101Cave fish LWS g103Human LWSHuman MWS180SAPSSSSSAAAASA197HHHHHHHHHHHHHH277YYFYYYYYYFFFYF285TTATTTTTTTAATA308AAAAAAAAAAAAAAExpected λmax (nm)~560~553~531 +P~560~560~560~560~560~553~546~531~531~560~531The λmax of each LWS opsin is estimated from the spectral shift predicted by each key-site substitution. The influence of the proline residue at position 180 is not known.Phylogenetic analysesThe Tetraodon nigroviridis LWS opsin amino acid sequence AAT38457.1 was employed as a query sequence in a BLASTp search [32] to identify homologs in the NCBI nr database. All hits with bitscores > 300 were aligned with one another and with the new data using the MPI version of ClustalW [33,34]. Subsequent sequence manipulations including multiple sequence alignments, toggle translations, hand editing, and delimitation of intron/exon boundaries utilized BioEdit v.7.0.5.3. [35]. A short nucleotide multiple sequence alignment (390 bp) that included coding sequences from exons IV and V was used to determine relationships among our new guppy LWS opsin genes and those from guppies of the Oropuche and Quare Rivers in Trinidad reported by Hoffmann et al. [23]: OR6-4 D09/DQ168660.1 and OR6-3 EO8/DQ168659.1 and QUEm5 LO6/DQ168661.1, and from the Paria River (Trinidad) guppy reported by Weadick and Chang [24]: DQ865167.1, DQ865168.1, DQ865169.1, DQ865170.1, DQ865171.1, and DQ865172.1. Maximum parsimony (MP) and Neighbor-joining (NJ) trees [36], which were based upon Tamura-Nei [37] distance estimates were reconstructed using MEGA v.4 [38]. Both analyses utilized all codon positions. Support for nodes was assessed using 1,000 bootstrap reiterations.The MP and NJ analyses were repeated using an alignment of nucleotide sequences that varied in length from 619 to 1095 bp. LWS opsin sequences in this analysis included the following species and acquisition numbers: Zebrafish (Danio rerio), AB087803.1 and AB087804.1; Medaka (Oryzias latipes) AB223051.1 and AB223052.1; Bluefin killifish (Lucania goodei) AY296740.1 and AY296741.1; Blind cave fish (Astyanax mexicanus) M90075.1, U12024.1, and U12025.1; Sea chub (Girella punctata) AB158261.1; Nile tilapia (Oreochromis niloticus) AF247128.1; Fugu (Takifugu rubripes) AY598942.1; Spotted green pufferfish (Tetraodon nigroviridis) AY598943.1; Turbot (Scophthalmus maximus) AF385826.1; Winter flounder (Pseudopleuronectes americanus) AY631039.1; Goldfish (Carassius auratus) L11867.1; Coho salmon (Oncorhynchus kisutch) AY214145.1; Ayu smelt (Plecoglossus altivelis), AB098702.1 and AB107771.1; Atlantic halibut (Hippoglossus hippoglossus) AF316498.1; Carp (Cyprinus carpio) AB055656.1; human (Homo sapiens) NM_020061.3 and NM_000513.1; Arctic lamprey (Lethenteron japonicum) AB116381.1; and our new sequences from the Cumaná guppy (Poecilia reticulata), Picta or 'swamp guppy' (Poecilia picta), Parae (Poecilia parae), Bifurca (Poecilia bifurca), the Pygmy swordtail (Xiphophorus pygmaeus) and Tomeurus (Tomeurus gracilis).ResultsHybrid or mosaic sequencesA large number of LWS opsin-like sequences (up to 17 per guppy) were uncovered after cloning and sequencing the products of PCR reactions utilizing primers Fw1a and Rev5. These sequences included suspected recombinants, that is, sequences that could have been generated by the ordered concatenation of fragments of other sequences produced in the same PCR reaction. Template switching during PCR and/or mismatch repair of cloned heteroduplex molecules has been shown to generate such artefacts [39-42]. To test the hypothesis that PCR and cloning could generate LWS opsin sequences not found in the guppy genome, we used primers Fw1a and Rev5 to re-amplify DNA from a two-sequence template (i.e., two insert-bearing plasmids). Five different sequences were uncovered from the two-template PCR reaction; one copy of each of the two templates and three recombinant sequences. These two-template experiments confirmed speculation by Hoffmann et al. [23] and Weadick and Chang [24] that LWS opsin genes in poeciliids are susceptible to PCR and/or cloning artefacts that generate artificial hybrid sequences.To determine the minimum number of genuine LWS opsin sequences in our dataset we first considered variation at polymorphic positions. Two sequences (e.g., from two different loci or from two alleles at one locus) could serve as a PCR template for the generation of an enormous diversity of hybrid sequences via template switching or mismatch repair. However, among such a set of hybrid sequences there would be only two variants (substitutions or indels) at a given polymorphic site. LWS opsin sequences derived from individual fish using primers Fw1a and Rev5 included three different intron II haplotypes and a position in exon III that was polymorphic for three different nucleotides. Remarkably, this exon III variation translated into amino acid variation at position 180; the first of the five sites known to influence spectral sensitivity (see below). Gene duplication is the only explanation for the occurrence of three haplotypes in a single individual. We set out to strengthen this evidence for LWS opsin gene duplication by amplifying DNA between the genes (see next section).Between-gene PCR and sequencingPCR using primers designed to amplify between-gene DNA (Fw1a Comp and Rev8 Comp) produced a ~4 Kbp product in the guppy and in the three species of the guppy sister group Micropoecilia (P. picta, P. parae, and P. bifurca). These amplicons were cloned in three of these four species (cloning of this amplicon was unsuccessful in P. bifurca) and approximately 1500 bp were sequenced from each end of the clone insert. Each end of the insert contained the last intron and exon of an LWS opsin gene and approximately 790 bp beyond the stop codon. The explanation for this sequence pattern, confirmed by subsequent PCR experiments using only Rev8 Comp, was that this fragment was amplified with Rev8 Comp acting as a forward and a reverse primer and that it contained the ends of two LWS loci oriented in an inverted (tail-to-tail) fashion. The between-gene fragment did not amplify from X. pygmaeus or T. gracilis. In the guppy, additional primers were designed and the entire intergenic sequence was characterized. It was 3329 bp long, 66% A/T, and contained a short compound microsatellite; (TGGA)10(TA)9.LWS opsins in the family PoeciliidaeGiven the evidence for the artificial generation of opsin sequence variation during PCR or cloning, and the observation that artefacts produced by template switching and/or mismatch repair do not appear to be reproducible [41,43], only haplotypes recovered from multiple independent PCR and cloning experiments were assumed to represent genuine opsin sequences. Additional primers were designed from these reliable sequences and from the sequences obtained by the between-gene PCR experiments described above (see Additional file 3).Initially, three different LWS opsin sequences were identified in guppy (P. reticulata). These three genes were delimited by variation at codon 180: TCT (serine), GCT (alanine), and CCT (proline), and by unique intron II and intron V mutations. Codon 180 is one of the five key positions that influence wavelength sensitivity [16] and the variation uncovered here is reflected in the names we have given to each of the loci; LWS S180 (S for serine), LWS A180 (A for alanine), and LWS P180 (P for proline).Seven LWS S180 sequences were obtained from six different Cumaná guppies with five of these including the start codon, all exons and introns, and part of the 3' UTR (see Additional file 3). Thirteen LWS A180 sequences were obtained from seven guppies. Only one was full-length but six included sequence from exon II to the 3' UTR. The LWS A180 sequence appears to be a naturally occurring hybrid in the Cumaná guppy; the first five exons and four introns are most similar to LWS S180, whereas the last intron and exon are identical to the LWS P180 locus. As the two regions of this hybrid LWS A180 sequence will give conflicting phylogenetic signals, the LWS A180 sequences were truncated in the phylogenetic analyses reported below (i.e. only the first five exons were utilized). Seven LWS P180 sequences were obtained from seven guppies (see Additional file 2). The ForBeg primer, which includes the start codon, combined with any of the reverse primers, did not amplify the LWS P180 locus. Therefore, LWS P180 sequences spanned exon II to the 3' UTR. In addition to the proline residue at site 180, the LWS P180 locus has amino acids substitutions at two other key sites. In guppies, LWS P180 also possessed a variable-length tetranucleotide microsatellite in intron III. PCR experiments using Fw100 and a primer complementary only to Weadick and Chang's [24] variant 6 (RevA) uncovered a fourth LWS opsin gene. While we did not amplify or sequence the first exon or intron, we show that the rest of this gene is intronless, suggesting that variant 6 is a single-exon gene, arising from a retrotransposition. It has a serine at position 180 (codon: TCG) and is renamed LWS S180r (S for the serine at position 180 and r for retrotransposition). Finally, our southern blot shows four bands (Fig. 1), consistent with the PCR-based hypothesis that the Cumaná guppy has four LWS loci. These four LWS opsins encode the following five key-site haplotypes: SHYTA (LWS S180 and LWS S180r), AHYTA (LWS A180), and PHFAA (LWS P180) and are thus expected to be most sensitive to three different wavelengths of light [16] (Table 1).Figure 1Southern blot hybridization for determination of LWS opsin copy-number in Poecilia reticulata. Four bands (labeled 1–4) correlate with four LWS loci from a single Cumaná guppy. BfaI (New England Biolabs®) was the restriction enzyme used. A generic DIG-labeled probe was designed to target all LWS loci (see methods). Two size markers (given in base-pairs) are shown to the right of the blot.Long portions of the four LWS genes found in the guppy were also amplified and sequenced from P. picta and P. bifurca. Three of these opsins, LWS S180, LWS S180r, and LWS P180, were sequenced from P. parae. We did not obtain the 3' end of LWS A180 from any of these three species. Therefore the mutation producing the hybrid sequence consistently recovered from Cumaná guppy cannot yet be mapped onto the poeciliid phylogeny. Xiphophorus pygmaeus had three LWS opsin genes: LWS S180, LWS S180r, and LWS P180. Only one LWS opsin sequence (LWS S180) was recovered from Tomeurus gracilis. All sequences have been deposited in GenBank under accession numbers EU329428 – EU329486 (see Additional file 2).Phylogenetic analysis of LWS opsin gene duplication in PoeciliidaeThe guppy LWS opsin sequences obtained here and those reported by Hoffman et al. [23] were added to the 390 bp alignment reported by Weadick and Chang [24]. Phylogenetic analyses sorted these guppy LWS opsins into three well-supported clades; LWS S180r, LWS P180 and LWS S180 plus LWS A180. Weadick and Chang's [24] variant 6 clustered with the single exon gene LWS S180r, and variant 5 clustered with the LWS P180 gene. This last result was anticipated before phylogenetic reconstruction because both sequences encode a phenylalanine at position 277 and an alanine at position 285. The remaining guppy LWS opsin genes; variants 1–4 from Weadick and Chang [24], the three LWS opsins from Hoffman et al. [23] (LWS_OR6-4_D09, LWS_OR6-3_E08, LWS_QUEm5_L06) and our LWS S180 and LWS A180 genes, formed the third clade. Over this 390 bp alignment, these genes are almost identical (mean percent identity = 98.6%). We suspect that the Weadick and Chang [24] variants 1–4 and the three Hoffman et al. [23] LWS genes include alleles at the LWS A180 and LWS S180 loci. This guppy-only LWS opsin sequence comparison also revealed that Weadick and Chang's [24] variant 5 is a recombinant or mosaic sequence; the first 221 bp are identical to variant 4, and the last 170 bp are identical to our LWS P180 sequence. In our phylogenic analysis, this gene occurred in the LWS P180 clade because the region where it is identical to variant 4 has few phylogenetically informative characters.Maximum parsimony analysis of the longer multiple sequence alignment (see Additional file 4) with the new poeciliid sequences and LWS opsins from a diversity of ray-finned fish produced a single tree (Fig. 2). The NJ tree included all of the nodes from the MP tree that had bootstrap support >65% and many of the nodes with lower support. Unlike the MP tree in Fig. 2, the NJ analysis placed the Tomeurus LWS opsin as the sister sequence to a clade with the Xiphophorus and Poecilia LWS P180, LWS S180 and LWS A180 genes. This reconstruction makes more sense than the MP tree with respect to poeciliid taxonomy; morphological and molecular data indicate that Poecilia and Xiphophorus are more closely related to one another than either is to Tomeurus. However, we present the MP tree because the neighbor-joining tree also placed the bluefin killifish LWSA gene at the base of the LWS S180r clade and the Xiphophorus LWS S180 gene at the base of the LWS P180 clade. The number of gene duplication events and gene losses required to reconcile such a topology with the well-supported taxonomic relationships among these species makes these components of the NJ topology very unlikely.Figure 2Phylogenetic analysis of LWS opsin genes. A Maximum Parsimony (MP) bootstrap consensus tree of long wavelength-sensitive (LWS) opsins from representative ray-finned fish lineages. The percentage of trees in which the associated taxa clustered in the bootstrap re-analyses (1000 replicates) is shown. Nodes in less than 50% of the bootstrap trees were collapsed. All codon positions were included and gaps treated as missing data. There were 1101 positions in the alignment and 520 were parsimony informative. The tree was rooted with the arctic lamprey LWS gene. Duplication events marked with an asterix. Phylogenetic analyses utilized MEGA4 [77] and the alignment from Additional file 4.The MP tree indicates that the guppy LWS opsin repertoire is a consequence of gene duplication events that occurred i) prior to the divergence of families Fundulidae and Poeciliidae, ii) in the common ancestor of Xiphophorus and Poecilia, and iii) within the genus Poecilia. Mechanisms of LWS opsin gene duplication include retrotransposition (producing LWS S180r) and inverted tandem duplication (producing the gene pair, LWS S180 and LWS P180). Formation of the hybrid LWS A180 locus may have involved quasipalindrome correction [44]. These three duplication events have provided species in this genus with a larger repertoire of LWS opsin pigments than any other fish taxon.LWS gene evolution in teleostsRelationships among higher taxonomic groups were well resolved in the tree reconstructed from LWS opsin sequences. There is high (>75%) bootstrap support for monophyly of Cyprinidontiformes (the bluefin killifish and all poeciliids), Pleuronectiformes (sea chub, turbot and flounder), Percomorpha, and the family Cyprinidae (goldfish, carp and zebrafish). One of the blind cavefish (Astyanax mexicanus) LWS genes (R007) is the sister sequence to those from goldfish, carp and zebrafish, which is not surprising as all species are in the taxon Ostariophysi. However, there were also two cavefish LWS genes at the base of the actinopterygian clade (G101 and G103). These genes might be derived from a gene produced during the fish-specific whole genome duplication event [45]. Long Branch Attraction (LBA) occurs when rapidly evolving sequences are attracted to the base of a tree [46] and is an alternative explanation for the position of the cavefish duplicates in our analysis. However, LBA is an artefact that is usually correlated with poor taxonomic sampling, which is not the case here.Among LWS sequences available for all ray-finned fishes, there is much variation at the five sites that influence spectral sensitivity most (see Additional file 4). Three different amino acids were observed at position 180 (A, S, or P), two at position 277 (Y or F), and two at position 285 (T or A). SHYTA is believed to be the ancestral five-site haplotype for vertebrates [16]. Serine to alanine substitutions at position 180 are common, but only guppies, turbot, and the spotted green pufferfish have a proline in this position. Lamprey, though not a ray-finned fish, also has a proline at position 180. Amino acid variation at positions 197 and 308 are also known to influence spectral sensitivity, however, all ray-finned fish surveyed to date possess only H197 and A308. LWS opsins have been duplicated at least six times in ray-finned fishes; twice within Poeciliidae, once prior to the divergence of Fundulidae and Poeciliidae, once in medaka (Oryzias latipes), once in zebrafish (Danio rerio), and again in the blind cave fish (Astyanax mexicanus). Only in poeciliids and zebrafish (and humans), has the duplication been followed by a substitution at one or more of the key sites (Table 1).LWS opsin gene expressionRT-PCR experiments (see Additional file 2) show that all four transcripts were expressed at the same time in the eyes of adult guppies. We then used qPCR to compare transcript copy numbers. In three adult guppies, LWS A180 was expressed at a much higher level than LWS S180 and LWS S180r (approximately 26 and 127 times greater, respectively). LWS P180 was expressed at very low levels, with approximately 5338 times less transcript abundance than LWS A180 (Fig. 3). The amount of cDNA in the three samples was estimated by comparing critical threshold (Ct (dR)) values between samples and standard curves prepared from plasmids containing each transcript (Tables 2 and 3). Standard curve log fit values are shown in Table 3.Figure 3Quantitative PCR amplification plot and logarithm histogram of original copy number. Four LWS transcripts from the cDNA of three adult guppies were quantified using qPCR with the amplification plot shown. Ct (dR) values are given in Table 2. A histogram showing the averaged logarithm value of original transcript copy number is shown (with standard error bars included).Table 2Quantitative PCR data.IndividualTranscriptCt (dR)Original Copy NumberMaleA18024.356758MaleS18029.54121MaleS180r33.1239MaleP18039.011+Female #1A18025.463126Female #1S18030.1078Female #1S180r33.9024Female #1P18039.581+Female #2A18024.496131Female #2S18027.93416Female #2S180r32.3663Female #2P18037.511+Ct (dR) values are shown for each LWS opsin transcript from three adult guppies. Original copy number was calculated using Ct (dR) values and the standard curve data shown in Table 3.Table 3Standard curve log fit values for quantitative PCR.(Plasmid) Standard CurveLinear EquationEfficiency (%)RSqA180Y = -3.315(logX) + 37.05100.30.999S180Y = -2.992(logX) + 35.77115.90.996S180rY = -3.670(logX) + 38.9687.30.985P180Y = -3.319(logX) + 36.68100.10.999The linear equation, efficiency and r-squared (RSq) values for the qPCR standard curve are shown. Plasmid copy numbers used as standards were determined by calculating the number of moles in each sample multiplied by Avogadro's number. A standard curve of Ct (dR) was used to calculate original copy number of mRNA transcripts in the diluted cDNA samples.DiscussionVariation at key sites among LWS opsin gene duplicates in PoeciliidaeThe guppy (Poecilia reticulata) and species in its sister group Micropoecilia possess four LWS opsin genes that we have named LWS S180, LWS S180r, LWS A180, and LWS P180. The first two genes encode proteins with the five key-site haplotype, SHYTA, and the second two genes encode proteins with key-site haplotypes, AHYTA and PHFAA, respectively. Three of these genes; LWS S180, LWS S180r, and LWS P180, were also amplified and sequenced from pygmy swordtail (Xiphophorus pygmaeus) genomic DNA. We found only the LWS S180 gene in Tomerus gracilis. Serine (S) and alanine (A) are common residues at position 180, but proline (P) is rare. This proline residue encoded by LWS P180 might disrupt the transmembrane domain [47,48] and compromise opsin protein function [48,49]. However, several observations suggest that it is functional. First, LWS P180 is at least 44 million years old, as it evolved before the divergence of Poecilia and Xiphophorus [26], and it has no other amino acid substitutions that are expected to disrupt function. Second, the LWS P180 locus has diverged from paralogous LWS opsins in ways that are expected to enhance color vision; positions 277 and 285 have experienced a tyrosine to phenylalanine and threonine to alanine substitution, respectively. These are the same key-site substitutions involved in the evolution of an MWS opsin from an LWS opsin in humans. Third, this gene is expressed, albeit at very low levels. Finally, LWS opsins from arctic lamprey, turbot, and the spotted green pufferfish also have a proline at position 180 and no other substitutions likely to disrupt protein function.All four LWS opsins uncovered in this study are predicted to have unique roles in color vision. With three different five key-site haplotypes, they are predicted to be most sensitive to three different wavelengths of light. Also, despite encoding a gene with the same key-site haplotype as LWS S180, the LWS S180r opsin differs from all other LWS opsins at amino acid positions known to play a role in binding and activating transducin [24].Southern blot experiments in our study revealed four bands (Fig. 1) consistent with the hypothesis that Cumaná guppies have four LWS opsin loci. Hoffman et al. [23] produced a southern blot with only three bands and suggested that guppies have a minimum of two LWS opsin genes. Variation in LWS opsin gene number among populations may be another trait guppies share with humans [50-52].Two of the four LWS opsin genes described here (LWS S180 and LWS A180) were reported in Hoffman et al.'s [23] study of guppies from the Quare and Oropuche Rivers in Trinidad. Although sequence data reported by Weadick and Chang [24] did not include all five key sites, our phylogenetic analysis indicates that Weadick and Chang [24] sequenced portions of all four loci from their Paria River guppy. The phylogenetic relationships among guppy LWS opsin paralogs reported by Weadick and Chang [24] differ from those shown here. Both topologies were produced using maximum parsimony, but by surveying more individuals, more species and by obtaining longer sequences we have produced a larger set of parsimony-informative characters. This is most evident when considering the relationship between LWS P180 and Weadick and Chang's variant 5, and variant 4. The large number of differences between variant 5 and variant 4 are apparent in Weadick and Chang's [24] tree where maximum likelihood branch lengths have been superimposed on the MP topology. Nonetheless, in their analysis, these two sequences form a monophyletic group. However, the sister sequence relationship between variant 5 and variant 4 disappears with the addition of LWS P180 genes from other Poecilia species and Xiphophorus pygmaeus because many of the unique nucleotides (autopomorphies) in variant 5 become synapomorphies (shared derived traits) in this larger dataset. Also, two of the three characters that had united variant 5 and variant 4 in Weadick and Chang's [24] MP analysis (adenines at positions 126 and 213 in their alignment), appear to be homoplasious when compared to a much larger set of LWS P180 and LWS S180 sequences. The origin of these apparent homoplasies is intriguing and is discussed below.Mechanisms of LWS duplication in poeciliidsThe first duplication event that expanded the guppy LWS opsin repertoire produced two genes that have retained SHYTA five key-site haplotype: LWS S180 and LWS S180r. The later duplicate is missing introns II-V and is likely a product of retrotransposition. Partial cDNA sequences for each gene have been reported [24] but their intron-exon structure was unknown until now. It is not clear how LWS S180r has retained retinal expression, although it may occur in the vicinity of other LWS opsins (and their regulatory modules) in the guppy genome. Of interest, gene duplication by retrotransposition has also produced a pair of fish rhodopsin 1 (RH1) genes called errlo and single-exon rho [53]. errlo and rho occur on different chromosomes. Therefore, the observation that rho is expressed in the retina (rod cells) [53] demonstrates that upstream regulatory elements can be retained during retrotransposition.In medaka (Oryzias latipes) and zebrafish (Danio rerio), duplicated LWS opsins are linked and oriented in a head-to-tail manner [54,55]. Phylogenetic analysis shows that independent mutations produced these gene pairs [54]. Our study also characterized an LWS opsin tandem duplication event. Duplication of the LWS S180 gene early during the evolution of poeciliids, produced an LWS opsin that retained the SHYTA haplotype (LWS S180) and an LWS opsin that evolved a PHFAA haplotype (LWS P180). These two genes are linked, but in an inverted (i.e., tail-to-tail) orientation. Several models have been proposed to explain the formation of inverted duplicates. Secondary rearrangement after duplication by unequal sister chromatid exchange is one. Another is intra-chromosomal replication slippage in trans [56]. This occurs when the DNA polymerase reverses direction using either the nascent strand (intra-molecular strand switch) or opposite strand (inter-molecular strand switch) as a template. By running backwards, a duplicate of the just-completed sequence is produced in an inverted orientation before the polymerase switches back to the correct template. The DNA downstream of LWS S180 has strings of adenines and thymines (data not shown) that might have facilitated strand switching by the polymerase during DNA replication [57]. The inverted arrangement of LWS opsins in the Poecilia genome might make them even more prone to additional duplication events [58]. Therefore, variation in LWS gene number (among species or populations) would not be surprising. Xiphophorus pygmaeus also has the LWS P180 gene. Post-duplication gene transposition or the expansion of intergenic DNA are possible explanations for our failure to amplify DNA between LWS P180 and LWS S180 in this species.The third and most recent LWS gene duplication uncovered in our study lead to the production of LWS S180 and LWS A180. In the Cumaná guppy, the first five exons and four introns of LWS A180 are most similar to LWS S180, and the last intron, exon, and 3' UTR are identical to LWS P180. These data suggest that in this population LWS A180 is a hybrid gene; its formation might have been facilitated by the inverted tandem orientation of LWS S180 and LWS P180 [44]. As mentioned above, variant 5, reported by Weadick and Chang [24] is also a hybrid sequence, with approximately one half of the sequence identical to the LWS S180 gene and the other half identical to LWS P180. As is the case for LWS A180 reported here in the Cumaná guppy, the position of variant 5 in the tree based upon the 390 bp alignment depends upon which fragments are used in the phylogenetic analysis (i.e., it would occur in the LWS S180/A180 clade if only the first 220 bp were utilized). As there are many more phylogenetically informative characters in the second half of this sequence, variant 5 was placed in the LWS P180 clade when the entire sequence was used, despite being identical to LWS A180 sequences over the first 220 bp. The observation that variant 5 has not diverged from either of its progenitor sequences and that it was recovered only once by Weadick and Chang [24] leads us to the conclusion that while both halves of the sequence can be found in the guppy genome, their concatenation is an artefact produced by template switching or mismatch repair during cloning.In the larger alignment (Fig. 2), LWS S180 and LWS A180 are not partitioned into monophyletic clades. For instance, the P. bifurca sequence that clusters with the LWS S180 genes has an alanine at position 180. One explanation for this observation is that the P. bifurca LWS A180 sequence is an allele of the LWS S180 locus. A similar situation occurs in non-African humans where a common allele of the LWS opsin locus (which typically has the SHYTA haplotype) has an alanine in position 180 and thus, an AHYTA five key-site haplotype [59,60].The evolutionary consequences of LWS opsin duplication in guppiesThe evolutionary implications of opsin gene duplication and divergence depend largely upon the expression patterns of these genes. In several species, the possession of a large opsin family allows the retina to be spectrally tuned for different environments and/or life stages. For example, eels (Anguilla anguilla) have two rhodopsins, each tuned to slightly different wavelengths. They express a green-shifted locus as juveniles in fresh water and a paralogous blue-shifted locus when they return to the ocean and mature [61]. The lamprey (G. australis) also adjusts its spectral sensitivity by changing opsin gene expression as it moves between marine and riverine environments [62]. In cichlids, opsin gene expression varies during development [63]. Of particular note is the observation that variation in LWS opsin sequence and expression is associated with variation in water turbidity [9,64]. This has lead to the hypothesis that species- and population-level differences in opsin gene sequence and expression represent adaptations for foraging in either turbid or clear water and that these differences in spectral sensitivity may drive and/or maintain divergence in male coloration via sexual selection [11]. Guppies, however, do not move very far during their lifetime [65] and thus differential use of opsin gene duplicates in different habitats is an unlikely explanation for the evolution of LWS opsin gene diversity in this taxon.The simultaneous expression of opsin paralogs with different sensitivities might expand the region of the spectrum where the guppy possesses high sensitivity and expand the range of detectable wavelengths. This enhancement and broadening of wavelength sensitivity can occur when individual cone cells express more than one opsin gene [66] or when adjacent cone cells express different opsins (e.g., humans and transgenic mice – see below). MSP data in guppies showing cells with a broad range of sensitivities in the long wave region of the spectrum [18,19] are consistent with the hypothesis that the sensitivity of some cones is a consequence of the co-expression of different LWS opsins. By providing guppies with a broad region of maximum wavelength sensitivity, LWS opsin gene duplication and divergence might make multi-colored male guppies appear brighter (more conspicuous) [67] to other guppies, but not to predators with wavelength sensitivity limited (by LWS opsin gene copy number) to a narrower region of the spectrum.Expression of different LWS opsins in adjacent cones not only improves overall spectral sensitivity, but is the basis of wavelength discrimination. Observations from humans and mice are consistent with the hypothesis that opsin gene duplication and divergence can lead to better color discrimination even without any associated revisions to neuroanatomy. Among human women who are heterozygous at either the LWS or MWS locus, some appear to have a pattern of X-inactivation that leads to tetrachromacy. These women see an average of 10 colors in a spectrum, whereas trichromatic women typically see seven [[68], but see reference [69]]. In mice, the hypothesis that extra opsin genes can improve wavelength discrimination was supported by data from females expressing an SWS opsin and two LWS opsins (an endogenous LWS gene on one X chromosome and a human LWS opsin on the other). These knock-in mice performed better in wavelength discrimination tests than wild-type mice with a single LWS gene [70].Sexual selection in guppies favors males with more red, orange, and yellow color patches [71-73] suggesting that females use color diversity (chroma) to evaluate males. This may be because males with more chroma are more conspicuous [67]. However, the 'extra' chroma is a consequence of the guppy visual system and this conspicuousness may not, therefore, apply to predators. Finally, if LWS opsin gene duplication improves motion detection, as proposed by White et al. [74] then female guppies might also be 'pre-adapted' to evaluate the well-characterized sigmoid display, a behavior that consists of the male arching its body into a S-shape and oscillating the long axis of the body both horizontally and vertically [75].LWS expression in the guppyGene expression data will help us to test alternative hypotheses about the adaptive value of LWS opsin diversity. To expand the range of maximum sensitivity and enhance wavelength discrimination, it is necessary that different opsins be expressed at the same time. All four LWS opsin gene transcripts were amplified from cDNA derived from adult eyes in our lab and by Weadick and Chang [24]. However, our qPCR experiments on three adults (1 male, 2 females) showed that most of the LWS opsin mRNA in the Cumaná guppy retinas was derived from the LWS A180 gene. Human SWS (blue) cone cells make up only 15% of the retina cone cell repertoire, yet play an important role in wavelength discrimination. Therefore, qPCR data showing unequal expression among LWS opsin paralogs in three adults do not rule out a role for LWS opsin gene duplication and divergence in wavelength discrimination in guppies, but does indicate the need for further investigation. We are currently using qPCR to examine LWS expression in a larger sample of adults and in fish at different stages of development. Finally, duplicated opsins are sometimes expressed in different regions of the retina [55,76]. In-situ hybridization experiments are underway to test the hypothesis that different LWS opsin paralogs have unique expression domains within the guppy retina, as is the case in zebrafish [76].ConclusionGene duplication and divergence has provided Poecilia and its close relatives with four distinct LWS opsins; a larger repertoire than any other fish. Phylogenetic analyses suggest that three of these LWS opsins (LWS S180r, LWS P180 and LWS S180) were present very early in poeciliid evolution and are predicted to occur in all of the approximately 239 species of the subfamily Poeciliinae. Adult guppies express all four LWS paralogs simultaneously, albeit at varying levels. As a consequence of these gene duplications, the potential for a broad region of high sensitivity and/or enhanced wavelength discrimination in the long-wave portion of the visible spectrum, may have facilitated a red-orange color bias for sexual selection within the guppy.Authors' contributionsJST supervised the study. MNW and JST designed and implemented wet-lab experimentation and data-analysis. AMC discovered the LWS P180 locus and carried out the template switching/mismatch repair experiment. KJD carried out the between-gene PCR and sequencing experiments. CRJL and GLO assisted with RT-PCR, qPCR and sequencing of the S180r locus. FB and JST obtained all samples. MDP and PRW assisted in RT-PCR. MNW and JST wrote the manuscript with editing assistance from FB.Supplementary MaterialAdditional file 1List of LWS primer names and sequences used for PCR, RT-PCR and qPCR. Primer names and numbers correspond to reaction conditions shown in Additional file 2. Primer numbers corresponds to amplicons shown in Additional file 3. Sequences are given in the 5'to 3' orientation. Primers were synthesized by Operon® Biotechnologies, suspended in sterile buffered TE (pH 7.0) and stored for no longer than one year at -20°C.Click here for fileAdditional file 2Sequence data and PCR conditions for specific primer combinations used for all six species of Poeciliidae. Primer names are given with corresponding primer number from Additional file 1. Amplicons are shown in Additional file 3 along with corresponding n value. 0.5 U iProof™ DNA polymerase (BioRad®) was used for each reaction with 5× iProof™ HF Buffer, 10 mM dNTP mix, ~100 ng template DNA, 0.5 μM of both forward and reverse primers and dH2O. An Eppendorf® silver block thermal cycler was used for all PCR reactions. Each reaction included an initial denaturation of 94°C for 30 seconds and a final extension of 72°C for 600 seconds. If >1 bands were found after gel electrophoresis, expected sized amplicons were cut and purified using a QIAquick® Gel Extraction Kit.Click here for fileAdditional file 3Primer and sequence maps for LWS opsin loci in Poeciliidae. Large colored boxes and horizontal bars represent exons and introns, respectively. Black and solid horizontal lines represent genomic sequences amplified using various PCR primer combinations (see legend; see Additional files 1 and 2). Dotted horizontal lines represent mRNA transcripts acquired through RT-PCR from cDNA samples. \"n\" represents the number of times a given sequence of near perfect identity had been acquired. Key site amino acids are shown within their respective exons. Inter-species and individual sequence differences due to SNP's, small strings of nucleotides, or codon selection (see discussion) are omitted and a consensus map has been created. Yellow exons and introns are highly similar to the guppy LWS S180 locus. Blue coloration represents sequences of high identity to the guppy LWS P180 locus. This use of color is exploited to highlight the hybrid nature of the LWS A180 gene in the guppy. Finally, green coloration represents the diverged, single exon LWS S180r locus (originally detected by Weadick and Chang [24]).Click here for fileAdditional file 4Exon alignment of LWS genes used in phylogenetic analysis. 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+{
+    "id": "PMC2527617",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527617\nAUTHORS: Sébastien Tempel, Matthew Jurka, Jerzy Jurka\n\nABSTRACT:\nBackgroundRepbase is a reference database of eukaryotic repetitive DNA, which includes prototypic sequences of repeats and basic information described in annotations. Repbase already has software for entering new sequence families and for comparing the user's sequence with the database of consensus sequences.ResultsWe describe the software named VisualRepbase and the associated database, which allow for displaying and analyzing all occurrences of transposable element families present in an annotated genome. VisualRepbase is a Java-based interface which can download selected occurrences of transposable elements, show the distribution of given families on the chromosome, and present the localization of these occurrences with regard to gene annotations and other families of transposable elements in Repbase. In addition, it has several features for saving the graphical representation of occurrences, saving all sequences in FASTA format, and searching and saving all annotated genes that are surrounded by these occurrences.ConclusionVisualRepbase is available as a downloadable version. It can be found at .\n\nBODY:\nBackgroundText Transposable elements (TEs) are short DNA sequences (less than 25000 bp) that use different strategies to replicate and insert at different genomic locations. They can be grouped based on their mechanism of transposition. Class I mobile genetic elements, or retrotransposons, proliferate in the genome by being transcribed to RNA and then back to DNA by reverse transcriptase, while class II mobile genetic elements move directly from one position to another within the genome using a transposase to \"cut and paste\" them within the genome [1]. TEs can represent a large fraction of the genomic DNA in eukaryotic species. For example, more than 45% of the human genome is composed of remains of TEs [2].Several databases have been created to collect and organize TEs. Some of them are focused on a particular family of TEs, sometimes on a single genome. For example, the Gypsy Database contains elements from the Gypsy LTR retroelement family [3], while PBmice is specialized in the piggyBac transposon in the mouse genome [4]. As far as we know, there are only four databases of TEs which are not specialized in a single family or genome: Repbase [5], MIPS Repeat Element Database (mips-REdat) [6], the Plant Repeat Database Project of TIGR [7] and TREP database [8]. Only Repbase covers TEs from all known eukaryotic species. The Plant Repeat Database Project  represents 11 plant genomes and the mips-REdat  is limited to plant genomes. The TREP database contains the transposable elements of cereal genomes. The sequences can be downloaded from all these databases and used for screening by BLAST [9], Censor [10] or RepeatMasker [11].Repbase is a relational database that can be searched or browsed for individual TEs, which can then be downloaded in EMBL or FASTA formats [5]. New data can be contributed through the Java interface known as RepbaseSubmitter [10]. A popular version of Repbase known as RepeatMasker libraries is widely used to annotate TEs in genomic sequences [11].Sequencing and annotation of complete eukaryotic genomes revealed the massive impact of TEs on genomic structure and evolution [2,12]. This stimulated broad interest in detailed biological studies of TEs in the genomic context. We created a specialized browser which facilitates such studies. Currently, there are browsers which allow browsing the existing annotations of TEs. Specifically, the UCSC Genome Browser [13] permits viewing of annotations of TEs and can display them at different resolutions. The annotated TEs are classified in four categories (SINEs, LINEs, LTRs and DNA transposons). However, the UCSC browser does not permit analysis of individual families and subfamilies of TEs. Another popular browser, ENSEMBL [14], has similar limitations. Moreover, these browsers do not display similarities of TEs to their consensus sequences, which is essential for dating of different layers of TE-derived repetitive elements.ResultsDatabaseWe have created six tables named: Exons, Introns, Genes, NonCodingZones, Transposons and Updates. The names reflect the type of data stored in the tables. The last table stores updates of the interface and data. The first four tables were created based on genome annotations and, in addition to coordinates of the annotated sequences, they include names of the species and chromosomes. The Genes and NonCodingZones tables also specify the orientation (strand + or -), the name, and the type (gene, pseudogene, tRNA or miscRNA) of annotations. The annotations of the genomes were downloaded in the GBS/GBK format file from the NCBI website . Currently, our browser contains data for sixteen genomes, but new genome annotations will be added in the future pending demand.The \"Transposon\" table was created using Censor [15], and the latest version of Repbase. It contains eight fields which include: the name of the genome, chromosome, family, superfamily, beginning and end coordinates of each TE, sequence orientation and percent of identity to the reference sequences from Repbase.Interface descriptionVisualRepbase can be accessed using a Repbase login and password. When the user enters a valid login and password, the main interface opens. All updates will be listed in a pop-up message. The VisualRepbase interface is composed of three sections (see Figure 1).Figure 1Screenshot of VisualRepbase graphical display. The interface contains three sections. The top section permits selection of families of TEs and eukaryotic genomes, or individual chromosomes. The middle section displays a variety of options for graphical presentation, the help menu, and output format options. The third section displays chromosomes with a graphical representation of TEs and other annotated features, tables of occurrences, and sequence information. This figure illustrates an example of two TE families from Repbase (AtREP1; AtREP3) on chromosome 2 of Arabidopsis thaliana. On the first line the selected families are displayed, including sequence orientation. On the second line other repeats from Repbase are displayed in orange, and their orientation is indicated. On the third line, other genomic annotations are displayed in addition to the selected families. The annotations include exons (in red), introns and UTRs (in framed white), and other annotated non-coding regions (in pink). Their orientations are also indicated. The table of occurrences lists the names of selected families, colors they are displayed in, chromosome name, species, number of elements on this particular chromosome, and the total number in all chromosomes displayed in the window (in this case all chromosomes of the Arabidopsis thaliana genome). The sequence is displayed in the bottom text window in FASTA format, and appears after clicking on the red rectangle which represents an exon.Searching transposable elementsThe first section contains the text field and the genome tree field. In the text field, the user enters the chosen name of the family or superfamily of TEs. Currently the maximum number of families one can search for at the same time is twenty. If the complete name is not known, one can type a part of the name and include an asterisk at any position. For example, typing ATREP*3, retrieves all Repbase family names beginning with \"ATREP\" and ending with \"3\" (in this case ATREP3 and ATREP13). The genome tree field permits selection of a particular genome (or genomes), and the chromosomes within that genome(s). The \"all\" option allows selection of all chromosomes for the given genome. The user can also enter his own sequences into the interface if he clicks on the \"Your Sequence(s)\" icon. A new window appears which contains a text field where the user enters the name of his sequence and three buttons where the user enters the other information necessary for the interface (Figure 2). The \"SEARCH\" button executes the query and the interface downloads the result of the query.Figure 2Integration of the own sequences. When the user clicks on the \"Your Sequence(s)\" icon, a new window appears. This window contains a text field where the user enters the name of his sequence and three buttons where the user enters the other information necessary for the interface. The first button selects FASTA or STADEN as the sequence format. The second button selects the transposable element annotation file. The last button selects the optional annotation file.Visual results of the searchThe second section of VisualRepbase is designed for a different type of search. It is composed of a graphical window, a table of the occurrences, and a text field displaying the sequence of the selected item. The graphical window can be split into two parts: the \"occurrences graph\" and the \"distribution graph\" of the selected biological item(s). The user can switch between these two graphs with the \"Occurrences/Distribution\" button (see Figure 1).In the \"occurrences graph\" the orientation and position of TEs on the chromosome are displayed on different lines and each family/subfamily is displayed in a different color (see Figure 1). For each selected family/subfamily of the TEs genomic annotation can be displayed in different colors, and includes: exons in red, introns in white, all non protein coding DNA such as tRNA genes, pseudogenes and snoRNAs in pink, and the non-translated parts of the genes are in white, framed in red. The annotations of TEs are displayed in two colors: superfamilies of selected TEs in light orange and others superfamilies in orange. The orientations of the TEs and genes are indicated by pointed triangles and arrows, respectively. The interface contains three buttons for adjusting the resolution (see Figure 1). The \"global view\" button changes the width of the graph until the largest of the selected chromosomes is visible in the graphical window. Each click on the \"Zoom in\" button multiplies the chromosome width by a factor of two until the chromosome unit size reaches 100 bp. The \"Zoom out\" button divides the chromosome width by two, until the width of largest among the selected chromosomes is contained in the graphical window.The \"distribution graph\" displays the density distribution of the selected TEs on the same chromosome(s) as the \"occurrences graph\", and it also permits adjustment of the view of the graphical window with the three buttons described above. Each density point corresponds to the number of occurrences within two actual units of the chromosome displayed on the screen (see Figure 3).Figure 3Distribution of AtREP3 family on chromosome 3. The graph shows the density distribution of the AtREP3 family (blue line), all RepBase TEs (orange line) and other annotations (red line), plotted against the chromosome length (x axis). The density distribution values for the selected family (ATREP3) are displayed on the left, and the corresponding numbers for other annotations are displayed on right in matching colors. The y axis is split in five intervals, and the density distribution values for the three items listed in the figure are normalized.The table of occurrences under the graph contains six columns: \"Family of TE\", \"Color\", \"Chromosome\", \"Genome\", \"Occurrences/chromosome\", and \"All chromosomes\". Each line of this table gives the number of occurrences of a given selected TE for one chromosome and for all selected chromosomes, and gives the correspondence between the TE family and the color in the graph (see Figure 1).The last element of the \"distribution graph\" is a text window which presents detailed information such as the name and position of the TE in FASTA format. Clicking on a particular biological item displays its sequence in the text window.Options and saving of resultsThe last section of VisualRepbase presents different options for saving and drawing the interface, and the user help menu. There are two menus which display the different annotations and the TE superfamilies present for the selected chromosome. From the annotation menu, the user can choose to draw any subset of the types of annotations: protein coding genes, exons, pseudogenes, tRNA genes, miscRNAs and cis-regulatory modules. These types of annotations can be independently selected. From the Repbase menu, the user can choose to draw any Repbase family, all Repbase families, or only the superfamilies of selected TEs (see Figure 1).The drawing option is a slide bar which displays occurrences of selected families of TEs as a function of their sequence identity to the reference sequence from Repbase.The interface has two help buttons: \"Manual\" and \"List of TE\". The \"Manual\" button opens the user manual. The other button displays the list of families and subfamilies of TEs present in each genome stored in the database (see Figure 4). Clicking on the family name adds it to the list of analyzed Tes.Figure 4A tree menu of transposable elements. This menu appears after clicking on the \"List of TE\" button in the main menu. It corresponds to the visual tree of all families of TE present in the database. The tree permits selection by genomes, superfamilies and families.The \"Results to Save\" menu contains five different options for saving the results and one option for accelerating the future queries. The first option allows the user to save a complete picture of all chromosomes selected. The second option saves a picture of the contents visible in the graphical window at the moment that the option is selected. For both options the picture is saved in PNG format. The third option (see Figure 1) allows writing of the table of occurrences present below the graphical window to a text file. Using the fourth option one can store all sequences of TEs present in all selected chromosomes in FASTA format. The last option also permits generation of a text file containing the closest gene at the left and right of each TE and the distance between them. In some cases, the TEs are inserted in introns or they overlap with the 5' UTRs or the 3'UTRs (UnTranslated Regions) of a gene. If the TE is inserted in an intron, the software indicates that the occurrence is within the gene and it does not record the second closest gene. In the case of the 5' or 3' overlap between the TE and a gene, the software lists the overlapping gene as the closest gene on the side where the overlap occurs. The last option permits saving of all different annotations of all selected chromosomes. The interface saves them in the file called \"Annot G C.vrb\" where G is the name of genome and C the name of chromosome. This file is saved in the same directory in which the interface was launched. During the next run of VisualRepbase for the same chromosome, the program will use the stored information instead of the original database, which speeds up the analysis.Significance of the interface for biological studiesVisualRepbase can be used not only for displaying or saving the occurrences of a TE family, but it also permits studying biological problems such as the relationships between TEs and host genes. Here we give two examples illustrating its value for biological studies.Figure 3 presents the distribution of the AtREP3 family [16] (blue), compared to all Arabidopsis TE families from Repbase (orange), and all genes on chromosome 3 from Arabidopsis thaliana. In this example, the red curve shows that the distribution of genes along chromosome 3 is uniform, except the heterochromatin centromere region, which contains less genes. In contrast, the TEs are more abundant in the heterochromatin than in the euchromatin regions (Figure 3). The distribution of the AtREP3 family is similar in the arm of the chromosome to other repeats from Repbase, but the distribution close to the centromere is very different. The distribution in the centromere is low like the distribution of the gene, but the distribution around the centromere is high. On closer inspection we found that the centromeric region is composed mostly of Gypsy-derived repeats.The visual presentation of sequence divergence also allows one to quickly estimate the age of TE families and the age difference between different families [17]. We compared occurrences of CHARLIE1 (green) and L4 (blue) (Figure 5) on human chromosomes 11 and 16 for two different sequence identity values. In Figure 5 we show the repeat distribution with 50% identity to the consensus in the first graph and higher than 75% identity in the second graph. The number of CHARLIE1 occurrences are similar in these two graphs, but the number of L4 occurrences drop dramatically from the higher range of similarities (see Figure 5). This shows that the L4 family is more divergent than the CHARLIE1 family, and it also indicates that it is older [17].Figure 5Number of transposable elements as a function of their similarity to consensus sequences. This figure shows the two graphs of occurrences of CHARLIE and L4 families on human chromosomes 11 and 16, for two different similarity values (50 and 75%). Each bar corresponds to one occurrence of the TE. The second graph (with 75% identity to consensus), shows that the number of L4 occurrences has decreased while the number of CHARLIE1 occurrences is similar to those in the first graph.ConclusionVisualRepbase has been developed to facilitate studies of transposable elements in the genomic context. This is the first tool that permits a dynamic display of individual families and subfamilies in terms of their age and location in the genome. In particular, the interface permits direct visualization and comparison of older and younger layers of TE families. It facilitates inter-chromosomal and inter-genomic comparisons of TEs. VisualRepbase is also likely to stimulate studies of transposable elements associated with gene regulation.Availability and system requirementsText Project name: VisualRepbaseProject home page: Operating system(s): Any, with Java Virtual Machine 1.4 or aboveProgramming language: JavaLicense: GPLAny restrictions to use by non-academics: None.Authors' contributionsST was responsible for programming and designing of the interface and execution of the study. MJ was responsible for the creation and update of the database and helped in the preparation of the manuscript. JJ contributed to design, supervised the study, and edited the manuscript. All authors read and approved the final manuscript.\n\nREFERENCES:\nNo References"
+}

batch_9/PMC2527683.json

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+{
+    "id": "PMC2527683",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527683\nAUTHORS: Paule Bénit, Sergio Goncalves, Emmanuel Philippe Dassa, Jean-Jacques Brière, Pierre Rustin\n\nABSTRACT:\nBackgroundDespite the considerable progress made in understanding the molecular bases of mitochondrial diseases, no effective treatments have been developed to date. Faithful animal models would be extremely helpful for designing such treatments. We showed previously that the Harlequin mouse phenotype was due to a specific mitochondrial complex I deficiency resulting from the loss of the Apoptosis Inducing Factor (Aif) protein.Methodology/Principal FindingsHere, we conducted a detailed evaluation of the Harlequin mouse phenotype, including the biochemical abnormalities in various tissues. We observed highly variable disease expression considering both severity and time course progression. In each tissue, abnormalities correlated with the residual amount of the respiratory chain complex I 20 kDa subunit, rather than with residual Aif protein. Antioxidant enzyme activities were normal except in skeletal muscle, where they were moderately elevated.Conclusions/SignificanceThus, the Harlequin mouse phenotype appears to result from mitochondrial respiratory chain complex I deficiency. Its features resemble those of human complex I deficiency syndromes. The Harlequin mouse holds promise as a model for developing treatments for complex I deficiency syndromes.\n\nBODY:\nIntroductionMost of the energy produced in the cell comes from oxidative phosphorylation (OXPHOS), which is catalyzed by the respiratory chain (RC) embedded in the mitochondrial inner membrane [1]. The respiratory chain comprises the proton-pumping respirasome [2] associating complexes I, III, and IV and a number of dehydrogenases including complex II. Electrons from respiratory substrates competitively converge from the various dehydrogenases to the CIII-associated quinone pool and are then conveyed to cytochrome oxidase and molecular oxygen by cytochrome c loosely bound to the mitochondrial inner membrane. The pumped protons are used to produce ATP from ADP and inorganic phosphate via the enzyme ATPase, which associates with adenylate and phosphate carriers to form the ATP synthasome [3]. OXPHOS dysfunction underlies a wide spectrum of human diseases, known as mitochondrial diseases. Respiratory chain complex I (RCCI) deficiency contributes 30% to 40% of all cases of mitochondrial disease [4]. Features of RCCI deficiency include optic atrophy, cerebellar ataxia, retinitis pigmentosa, growth retardation, and hypertrophic cardiomyopathy [5]. The mechanism, tissue-specificity, and course of mitochondrial diseases are poorly understood [6].The number of reported human mitochondrial diseases has increased considerably in recent years. The underlying mutations affect either the mitochondrial DNA or the nuclear genes, of which more than 100 have been incriminated [7]. Treatments are extremely limited [8]. A major obstacle to the development of effective treatments is the absence of reliable animal models. Obtaining mice with mitochondrial DNA (mtDNA) mutations has proved challenging, and the animals rarely transmit the mutant mtDNA to their progeny, which limits their usefulness as tools for designing treatments [9]. Targeting nuclear genes encoding OXPHOS components has proved more efficient. However, extinction of a majority of the nuclear OXPHOS genes caused early embryonic death or produced no major phenotypic abnormalities. Cre-Lox recombination, in contrast, has been successful in producing mitochondrial abnormalities in specific tissue types. Thus, studies of Tfam-KO and frataxin-KO mice [10], [11] have shed light on a number of pathological mechanisms. However, the severe biochemical abnormalities in the target tissues fail to replicate human mitochondrial disease, limiting the value of these animal models for designing treatments. Recently, targeting the NDUFS4 subunit of complex I produced mice that exhibit several of the features seen in patients with RCCI [12]. However, the severe RCCI deficiency in these mice causes death at 7 weeks of age, and the animals do not exhibit the extraordinary variability in disease symptoms and course that characterizes human RCCI deficiency.We recently established the occurrence of partial complex I deficiency in the Harlequin (Hq) mouse [13], which is characterized by progressive cerebellar ataxia [14]. Other features in these animals include early fur abnormalities, optic tract dysfunction with retinitis pigmentosa, and a risk of hypertrophic cardiomyopathy [14]–[16]. The Hq phenotype is due to a proviral insertion in the X-linked gene encoding the mitochondrial protein Apoptosis Inducing Factor (Aif) [14]. The insertion causes a partial decrease in the Aif protein to about 20% of the amount seen in wild-type mice. Gross development is not significantly affected, but the animals exhibit the typical features of mitochondrial disease. Total loss of Aif function caused abnormal cell death, presumably related to RCCI deficiency, during embryonic development but did not affect the temporal progression of patterning [17].Although the involvement of Aif in RCCI assembly and stability is still being investigated [18], [19], the characteristics of the Hq mouse make it a potential model to investigate complex I deficiencies. With the aim to establish the value of this model, our main objective was to study in details the course of the abnormalities in Hq mice and the tissue-specific biochemical features at various time points. We found that the temporal progression of complex I deficiency differed across tissues and that the involvement of each tissue correlated with the residual amount of complex I 20 kDa subunit. Although oxidative injury has been suggested as a key mechanism in the Hq phenotype [16], antioxidant enzymes were normal except in skeletal muscle, where they were moderately increased.ResultsI. The Harlequin phenotype courseThe onset of most of the gross phenotypic abnormalities varied widely across individuals. Although all the mutant animals eventually developed the disease phenotype, the time-course ranged from early ataxia and death to the slow development of functional impairment over several months. These differences were detected by monitoring body weight, fur abnormalities, and ataxia.Weight loss and growth retardation are common among patients with RC disorders [20], [21], we therefore first studied this parameter in wild type and Hq mice. B6CBACa Aw-J/A-Pdcd8 mice are relatively small. Thus, in our study, the wild type mice achieved their adult weight of 27±2 g for males (n = 19) and 22±2 g for females (n = 22) within 4 months. Body weight varied widely in the hemizygous Hq/Y males (Fig. 1A) and homozygous Hq/Hq females (Fig. 1C), with some Hq individuals having similar weights to those of wild type animals. No growth retardation occurred in the heterozygous females (Fig. 1B). At 1 month of age, about half the Hq males had marked growth retardation with a greater than 30% decrease in body weight compared to wild type animals (Fig. 1D). Most of these animals had normal body weights (within 30% of the control mean) at 3 months of age and significantly decreased body weights at 6 months of age compared to controls. However, 30% of Hq animals had no significant growth retardation at 6 mo of age. Growth retardation probably started before birth, as birth weight was significantly lower in the Hq population than in the wild type population (1.2±0.2 g (n = 15) and 1.5±0.2 g (n = 13) respectively; p<0.001). The sex ratio was normal in all the litters and the number of Hq individuals was consistent with Mendelian inheritance. About 30% of Hq animals died during the 6-month study, with most deaths occurring between 15 and 30 d of age. The only developmental abnormality found in the Hq population was ocular hypoplasia with absence of the optic nerve, which was noted in 40% of Hq individuals at 1 mo of age. Ocular hypoplasia occurred at the severe end of the disease spectrum and was present in many of the animals that died early on. At 3 and 6 mo of age, ocular hypoplasia was present in 20% and 10% of Hq animals, respectively. Ocular hypoplasia with absence of the optic nerve has been previously reported in human mitochondrial disease [22].10.1371/journal.pone.0003208.g001Figure 1Time-course of weight loss in Harlequin mice.A: Weight changes with age in Harlequin hemizygous males (Hq/Y; n = 19) and wild-type males (B6CBACa Aw-J/A-Pdcd8 strain; n = 14). B, C: Weight changes with age in wild-type (n = 6) and heterozygous females (Hq/Aif+; n = 6) (B) and in homozygous females (Hq/Hq; n = 6) (C). D: Weight deficiency in Hq mice (n = 25) compared to wild type mice (n = 20) at various ages. Dark squares and open circles indicate control and Hq mice respectively.Paucity of fur was first described as the hallmark of Hq mice [23]. We noted complete baldness in most of the Hq animals initially (Fig. 2A) followed by some hair growth resulting in a patchy or near-normal coat. At 3 mo of age, about half the Hq animals were bald over more than 30% of their body surface area and about 30% had near-normal fur. At 6 mo of age, 10% of Hq animals had near-normal fur.10.1371/journal.pone.0003208.g002Figure 2Variability of baldness and ataxia severity in Harlequin mice.A: Baldness in Hq mice. Open bars indicate severe baldness (more than 70% of the body surface area) at ages when controls had their full adult coat. Gray bars indicate patchy baldness (less than 30% of the body surface area). Patch distribution varied across animals. Dark bars represent animals with a full coat, many of which had sparser hair than the controls. The arrow shows that a substantial proportion of Hq mice had nearly normal hair at 3 and 6 mo of age. B: Severe ataxia in Hq animals (open bars; three or more falls in 5 minutes) was detected only at 6 mo of age. The arrow shows that two Hq animals had only mild ataxia at 6 mo of age.Ataxia was detected in most of the animals at 3 mo of age. Severe ataxia defined as more than three falls within 5 minutes was noted only late in the disease, at 6 mo of age (Fig. 2B). At 3 mo of age a few animals had an unsteady gait but none had severe ataxia. At 6 mo of age, 90% of Hq males and females had marked gait ataxia but 8% had no clinical evidence of ataxia (Fig. 2B; arrow).We looked for correlations linking growth retardation, baldness, and ataxia in the Hq population (Fig. 3, A–C). Plotting body weight against percentage of surface area without fur at 6 mo of age (Fig. 3A) showed that weight was highest in the animals that had nearly complete coats and vice versa. The animals with higher body weights were also those with less severe ataxia (Fig. 3B). Ataxia was more severe in the animals with earlier disease onset (Fig. 3C). Thus, body weight, baldness, ataxia, and age at onset seemed to vary in lockstep.10.1371/journal.pone.0003208.g003Figure 3Correlations among the severities of the major abnormalities in Harlequin mice.A: Baldness (% body surface area) plotted against body weight in Hq males (n = 12). B, C: Ataxia (number of falls per min) plotted against body weight (B) or against the age at the first recorded sign of ataxia (C).Motor coordination used as a rough estimate of neurological involvement was tested using a Rotarod device (Fig. 4). Performance varied widely in the Hq and wild type animals at 3, 4, 5, and 6 mo of age. A significant difference between Hq and wild type animals was noticeable from 4 mo of age onward, with some of the Hq animals performing very poorly at 4 mo of age.10.1371/journal.pone.0003208.g004Figure 4Variable Rotarod performance in control and Harlequin mice.Plots of individual results of three tests per animal (3 mo: 16 wild-type and 13 Hq mice; 4 mo: 11 wild-type and 13 Hq animals; and 5 and 6 mo: 15 wild-type and 11 Hq animals) showing the wide variability of values in both the Hq and control populations at each time point. The mean and standard deviation are shown for each distribution. Despite the major variability, t-tests showed significantly poorer performance in the Hq mice at 4 mo of age.II. Complex I defect and its consequences in Harlequin mouse tissuesWe previously reported partial loss of complex I activity by about 40% of the value in controls in the whole brain of Hq mice, with variations in the severity of the deficiency across tissues and cells [13]. Here, we show that complex I activity was about 50% of the control value in the cerebellum of Hq mice (Fig. 5A). At 6 mo of age, complex I deficiency was noted in all the brain territories investigated (cerebellum, thalamus, and cortical-enriched fraction), as well as the optic nerves (50% residual activity) and retinas (25% residual activity) (Fig. 5A–D). Complex I deficiency was detected at 1 mo of age, before the occurrence of cerebellar atrophy or major ataxia (Fig. 5), in any of the 20 Hq animals at this time point. In other organs, complex I activity was normal (heart, liver, and testis) or only slightly reduced (by 20% in the spinal cord and 10% in the kidney). Complex I activity was decreased by about 30% in the skeletal muscle (Fig. 5E). In most of the affected tissues, complex I deficiency worsened over time, albeit with a variable course (Fig. 5). The deficiency reached its greatest level by 1 mo of age in the cerebellum and skeletal muscle. In the thalamus, cortex, and optic nerve, in contrast, the deficiency was modest or absent at 1 mo of age and reached 50% by 6 mo of age. Complex I activity in the retinas was decreased by about 30% by 1 mo of age but improved subsequently, probably as a result of bias due to gradual selection of animals with normal eyes.10.1371/journal.pone.0003208.g005Figure 5Differences in the time-course and severity of complex I deficiency across tissues from Harlequin mice.A–F: Complex I activity in homogenates prepared from cerebellum (A), thalamus (B), cortex (C), optic nerves (D), skeletal muscle (E), and retinas (F). Complex I activity was measured by spectrophotometry [40] and normalized for the activity of complex V, which was not affected by Aif depletion. The open and dark symbols refer to the control and Hq mice, respectively. Continuous lines indicate changes in complex I activity over time in various tissues of Hq mice. The dotted line indicates changes in complex I activity over time in Hq retinas, presumably ascribed to biased sample collection. Numbers of animals were as follows: 15 d of age, 3 controls and 3 Hq mice; 1 mo of age, 5 controls and 5 Hq mice; 3 mo of age, 5 controls and 5 Hq mice; 6 mo of age, 10 controls and 10 Hq mice. *p<0.005; **p<0.001.Oxidative insult has been suggested as a key factor in the pathogenesis of the Hq phenotype [16]. Superoxide dismutase (SOD) and catalase activities are induced by oxidative insult in vivo. We compared SOD and catalase activities at 6 mo of age in control (n = 10) and Hq (n = 10) mice (Fig. 6A). Moderate delayed increases were found in skeletal muscle (to about 150% and 200% of control values for SOD and catalase, respectively, at 6 mo of age). However, the activities of these two enzymes were not increased in any of the other tissues investigated in this study.10.1371/journal.pone.0003208.g006Figure 6Antioxidant enzymes and Complex I 20 kDa subunit in various tissues of Harlequin mice.A: Superoxide dismutase (manganese- and copper, zinc-dependent SODs) activity and catalase activity measured as described in Materials and Methods in skeletal muscle tissue from Hq mice at various ages. B: Western blot analysis of Aif, complex I 20 and 39 kDa subunits (ND6 and NDUFA9, respectively) and porin in skeletal muscle of Hq mice (lines 1, 2) and control mice (3, 4). C: Plot of residual complex I 20 kDa subunit in mouse tissues against residual complex I activity, showing a significant correlation. D: Plot of residual Aif in mouse tissues against residual complex I activity, indicating no significant correlation.Since the extent of complex I deficiency varied across tissues, we measured residual complex I using the 20 kDa subunit of complex I as a marker. We reported previously that this subunit, now recognized as NDUFB8 (see Material and Methods), was decreased in case of Aif deficiency [24]. Western blot analysis of skeletal muscle samples from Hq mice showed a 60% decrease (Fig. 6B) in the 20 kDa subunit compared to controls, indicating a 30% decrease in complex I activity (Fig. 5E). Western blot analysis was also performed with samples of heart, liver, kidney, cerebellum, cortex, and spinal cord. We plotted the residual complex I activities in these tissues, as measured previously, against residual 20 kDa subunit (Fig. 6C). A significant correlation was found between these two parameters. In particular, tissues without complex I deficiency (e.g. heart, liver) contained normal amounts of 20 kDa subunit.We measured Aif content in various tissues of Hq mice and controls (Fig. 7). In all studied tissues, one major (>98%) band at 57 kDa reacted with antibody to the Aif internal region. Heart samples from control animals showed two bands, at 57 and about 30 kDa, in nearly identical proportions (Fig. 7A). In heart samples from Hq animals, in contrast, the 57 kDa was extremely faint (Fig. 7A–B). When we compared the residual amount of Aif (57 kDa) in various tissues of Hq mice (n = 6) and controls (n = 3), we found that Aif was barely detectable in the retinas and was reduced to 20%–40% in most tissues except for the liver, where the level was about 60% of the control value. We found no correlation between the amount of residual Aif protein and tissue involvement (Fig. 6D).10.1371/journal.pone.0003208.g007Figure 7Aif in Harlequin mice.A: Western blot analysis of Aif in the heart of Hq mice (lines 1–6) and control mice (lines 7–9). In the heart, two bands reacted with the Aif antibody. The band with the highest molecular weight was produced by the Aif1 isoform (molecular weights of the unprocessed and processed forms, 67 and 57 kDa, respectively). The molecular weight of the other band is consistent with the Aif sh2 isoform. This second form was not detected in any of the other tissues studied (cerebellum, spinal cord, cortex, retinas, skeletal muscle, kidney, or liver). Porin was used as a loading marker. B: Proportion of the two Aif isoforms identified in the heart of control mice (open symbol) and Hq mice (dark symbol) mice. C: Aif (57 kDa) content in various tissues of control and Hq mice normalized for porin content. **p<0.005; ***p<0.001.DiscussionThe Hq phenotype is caused by severe Aif protein deficiency in hemizygous males and homozygous females. Aif gene impairment is due to an ecotropic proviral insertion in the first intron of the gene, as shown by Klein et al. [14]. Aif deficiency leads to RCCI deficiency in affected tissues of Hq mice [13], in ES cells [13] and embryos [17] of Aif knock-out mice, in Aif knockdown human HeLa cells [13], in mouse tissues (skeletal muscle, heart, liver) with tissue-specific ablation of the Aif gene [24], [25], and in Aif knockout Drosophila\n[26]. These conditions are characterized by RCCI deficiency without evidence of oxidative stress except in the skeletal muscle, where SOD and catalase activities are increased [24]. Evidence of antioxidant enzyme induction has led to the suggestion that Aif may protect the mitochondria against oxidation [16]. Primary complex I deficiency has been reported to result in oxidative stress [27], but there is no evidence that oxidative stress can specifically cause complex I deficiency. Impairment of mitochondrial antioxidant enzymes (e.g., manganese-dependent SOD) results instead in a generalized deficiency of the RC, including the iron-sulfur cluster-containing enzymes (complexes I, II, and III) and the Krebs cycle enzyme aconitase [28]. Accordingly, treatment of Aif-depleted human cells (Hep3B and HeLa cell lines) with antioxidants (N-acetyl-cysteine or MitoQ) failed to restore complex I integrity [29], suggesting primary loss of complex I, which might increase superoxide leakage. Conversely, the (pro-oxidant) superoxide-generating NADH oxidase activity of Aif may be required for normal muscle regeneration [30]. Taken in concert, the data suggest that any antioxidant effects of Aif may be confined to the close vicinity of complex I, so that Aif acts as a complex I maintenance protein. Antioxidant enzyme induction in muscle-specific Aif knockout mutants [24] and in skeletal muscle from our Hq mice appears to be tissue-specific and may reflect either an additional role for Aif in muscle or a tissue-specific consequence of RCCI. Although the role for Aif remains debated [26], [29], [30], the effects of Aif deficiency are similar to those of deficiency in any complex I assembly/maintenance factor. NDUFS4 knockout mice exhibit severe RCCI with features similar to those of Hq mice (growth retardation, blindness, ataxia, and baldness) but greater disease severity leading to death at 7 weeks of age after only 2 weeks with symptoms. Thus, complex I deficiency seems to be the key determinant of the Hq and NDUFS4 knockout models.Multivisceral disease develops in Hq mice over time. As with human complex I-associated diseases, the brain and optic tract are affected [5], whereas the liver is spared. We failed so far to obtain a clear analogy between the standard Leigh syndrome images observed in a number of patients with complex I deficiency and the Hq mice. Similarly, T2-weighted MRI images showing signal hyper intensity have not been reported for the NDUFS4 mutant mice. Disease progression follows the same pattern in all NDUFS4 knockout mice but varies considerably across Hq mice. As with humans, both genetic and non-genetic factors may explain this variability, since the animals do not have a pure genetic background (B6CBACa Aw-J/A-Pdcd8) and the phenotypic consequences of mitochondrial dysfunction depend on a number of unidentified factors. Marked inter-individual variability also occurs in human mitochondrial diseases. Thus, the Hq model, although difficult to study, constitutes a good model of human mitochondrial diseases. Obviously, as long as the intimate mechanism linking Aif depletion and impaired complex I deficiency is not fully depicted, this model will be best used to test for efficiency of compounds/strategies aiming at counterbalancing the consequences of complex I deficiency.Reduced birth weight was the earliest abnormality in Hq animals compared to their wild type littermates and suggested intra-uterine growth retardation (IUGR). Similarly, Aif-KO mouse embryos had severe growth retardation contrasting with a normal temporal progression of patterning [31]. Among humans with RCCI deficiency, 23% had low birth weights [20]. In mice, complete Aif gene deletion not only caused early deaths by embryonic day E9 related to RCCI deficiency [32], but also resulted in marked embryonic growth retardation [17]. Body weight in our Hq animals was low at birth and subsequently remained lower than in the control animals. The mechanism of IUGR associated with RC deficiencies remains unclear. In humans, early antenatal expression of respiratory chain deficiencies (including RCCI deficiency) reflects the time-course of disease-causing-gene expression in utero\n[20]. In utero, respiratory chain deficiency may result in decreased ATP formation and/or in alteration of apoptotic events controlled by the mitochondria.Hair abnormalities occur in a substantial proportion of human patients with mitochondrial disorders. In 8 of 25 children with mitochondrial diseases, slow-growing, sparse, fragile hair was an early manifestation [33]. This fact, together with the hair abnormalities seen in NDUFS4 knockout mice, supports a role for RCCI deficiency in the hair abnormalities of Hq mice. The correlation in our Hq mice between the severity of baldness and the severity of growth retardation also suggests a role for RCCI deficiency in the hair abnormalities, as delayed growth is probably due to RCCI deficiency.The severity of cerebellar ataxia varied widely in our Hq mice, probably reflecting a variable degree of neuron loss. Similar variability occurs in humans with mitochondrial disorders [6]. The greater severity of ataxia at 6 mo of age in a subset of our Hq animals compared to an earlier study [14] suggests a role for unidentified environmental factors and a possible influence of the mixed genetic background. Interestingly, complex I deficiency in the cerebellum was readily evidenced at 15 d of age, whereas cerebellar ataxia was detected only after several weeks in the most severely affected animals. Thus, cerebellar function persisted for several weeks despite the presence of complex I-deficient mitochondria.Our study does not shed new light on the mechanism of the tissue specificity associated with complex I deficiency. However, our data establish that residual complex I measured using the 20-kDa subunit as a marker correlates with the residual complex I activity in affected tissues. In contrast, residual complex I activity was not correlated with residual Aif protein. Five Aif isoforms have been identified to date [34], suggesting a possible explanation for the variability of tissue involvement. In control hearts, a shorter isoform contributes as much as 50% of total Aif. The normal complex I activity in the heart may be ascribable to the presence of this short Aif isoform, whose electrophoretic properties resemble those of the Aif-sh 2 [34].Taken together, our results indicate that Hq mice replicate the main features of RCCI deficiency in humans, including tissue-specificity, course, and interindividual variability. The Hq model is likely to prove valuable for investigating treatments aimed at RCCI deficiency.Materials and MethodsAnimalsHomozygous (Hq/Hq) females and hemizygous (Hq/Y) males were obtained by mating Hq/X females with either Hq/Y or control males obtained from The Jackson Laboratory (Bar Harbor, ME). The Hq strain was B6CBACaAw-J/A-Pdc8Hq/J. All mice used in this study were F1 mice bred from founders having a mixed genetic background. The mice were housed with a 12-h light/dark cycle and free access to food (3% lipids, 16% protein; SAFE A-04 chow; UAR Epinay sur Orge, France) and water.Genotype determinationMice were genotyped using multiplex PCR with five primers: two for sex determination (SRY: 5′-TGGGACTGGTGACAATTGTC-3′ and 5′-GAGTACAGGTGTGCAGCTCT-3′), two for the wild-type Aif allele (Aif 1F: 5′AGTGTCCAGTCAAAGTACCGG-3′; Aif 1R: 5′-CTATGCCCTTCTCCATGTAGTT-3′), and one for the Aif allele harboring the proviral insertion (Hq allele) (Aif RV: 5′-CCCGTGTATCCAATAAAGCCTT-3′).Phenotype determinationThree hallmarks of the Hq phenotype [14] were studied: baldness, growth retardation, and cerebellar ataxia. Baldness was assessed as the percentage of body surface area without hair. Weight was determined daily from birth through day 7 of postnatal life and weekly thereafter. Cerebellar ataxia was assessed as the number of falls per minute of walking, with severe ataxia being defined as three or more falls within 5 minutes; and by testing on a computer-driven Rotarod device (Imetronic; Pessac, France). The mice were trained at 3, 4, 5, and 6 mo of age, for 3 consecutive days after a previous day of training, at speeds that increased from 4 to 40 rpm/min.Tissue homogenate preparation and enzyme assaysFrozen tissue (cortex, cerebellum, thalamus, spinal cord, optic nerve, retinas, heart, liver, kidney, skeletal muscle, testis) homogenates were prepared using a 1 ml glass-glass potter in 500 µl of extraction buffer composed of 0.25 mM sucrose, 40 mM KCl, 2 mM EGTA, 1 mg/ml BSA, and 20 mM Tris-HCl (pH 7.2). Large cellular debris was separated by centrifugation (1500 g×5 min). RCCI activity was measured as recently described using a Cary 50 spectrophotometer (Varian Australia, Victoria, Australia) [35]. Catalase activity and total SOD level were quantified as already described [36], [37]. Protein was estimated using the Bradford assay [38].Western blot analysisWestern blot analysis was performed on mitochondria-enriched supernatant (900 g×5 min) prepared from tissue homogenate separated by SDS-PAGE, and blotted onto a PVDF membrane. The membranes were blocked for 1 h in TBST tween 0.01% and then incubated for 1–3 h with different antibodies (AIF1∶1000, chemicon; complex I subunits 20 kDa and 39 kDa∶1∶1000, Molecular Probes and porin 1∶1000, MitoSciences. It must be mentioned that future controversy about the identity of the 20 kDa antibody may occur, because different lines of evidence suggest that, in fact, the protein recognized by the antibody provided by Molecular Probes is not ND6, but a different complex I membrane arm subunit, NDUFB8, with a similar molecular weight as ND6 (Dr Jose Antonio Enriquez, University of Zaragoza, Spain, personal communication to Dr Fernandez-Moreira) [39]. The horseradish peroxidase-conjugated secondary antibody (1∶5000) was incubated for 1 h at room temperature in 5% milk powder in TBST buffer, 0.01% Tween 20. Peroxidase activity was visualized with the ECL plus (GE healcare) according to the manufacturer's instructions. Chemiluminescent signal were captured on autoradiography and used to assess protein content. The relative protein levels of AIF and Complex I 20 kDa subunit were assessed by their chemiluminescent signals compared with that of the porin protein.StatisticsWe evaluated mean differences in study variables between Hq and wild-type populations by using appropriate t-tests (SigmaStat software, Richmond, CA) to compute the means, standard deviations, and p values as indicated. Values of p less than 0.001 were considered statistically significant.\n\nREFERENCES:\n1. TzagoloffA\n1982\nMitochondria\nNew York\nPlenum Press\n2. SchaggerHPfeifferK\n2000\nSupercomplexes in the respiratory chains of yeast and mammalian mitochondria.\nEmbo J\n19\n1777\n83\n10775262\n3. ChenCKoYDelannoyMLudtkeSJChiuW\n2004\nMitochondrial ATP synthasome: three-dimensional structure by electron microscopy of the ATP synthase in complex formation with carriers for Pi and ADP/ATP.\nJ Biol Chem\n279\n31761\n8\n15166242\n4. BénitPLebonSCholMGiurgeaIRötigA\n2004\nMitochondrial NADH Oxidation Deficiency in Humans Current Genomics.\n5\n137\n146\n5. BénitPDELebonSGiurgeaIRustinP\n2007\nSisoMIG\nin Complex I and alternative dehydrogenases,\nKerala, India\nTransworld Research Network, Research Signpost\n157\n178\n6. BriereJJChretienDBenitPRustinP\n2004\nRespiratory chain defects: what do we know for sure about their consequences in vivo?\nBiochim Biophys Acta\n1659\n172\n7\n15576049\n7. ZevianiMCarelliV\n2007\nMitochondrial disorders.\nCurr Opin Neurol\n20\n564\n71\n17885446\n8. DiMauroSHiranoMSchonEA\n2006\nApproaches to the treatment of mitochondrial diseases.\nMuscle Nerve\n34\n265\n83\n16810684\n9. LarssonNGRustinP\n2001\nAnimal models for respiratory chain disease.\nTrends Mol Med\n7\n578\n81\n11733222\n10. WangJWilhelmssonHGraffCLiHOldforsA\n1999\nDilated cardiomyopathy and atrioventricular conduction blocks induced by heart-specific inactivation of mitochondrial DNA gene expression.\nNat Genet\n21\n133\n7\n9916807\n11. PuccioHSimonDCosseeMCriqui-FilipePTizianoF\n2001\nMouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits.\nNat Genet\n27\n181\n6\n11175786\n12. KruseSEWattWCMarcinekDJKapurRPSchenkmanKA\n2008\nMice with mitochondrial complex I deficiency develop a fatal encephalomyopathy.\nCell Metab\n7\n312\n20\n18396137\n13. VahsenNCandeCBriereJJBenitPJozaN\n2004\nAIF deficiency compromises oxidative phosphorylation.\nEmbo J\n23\n4679\n89\n15526035\n14. KleinJALongo-GuessCMRossmannMPSeburnKLHurdRE\n2002\nThe harlequin mouse mutation downregulates apoptosis-inducing factor.\nNature\n419\n367\n74\n12353028\n15. van EmpelVPBertrandATvan der NagelRKostinSDoevendansPA\n2005\nDownregulation of apoptosis-inducing factor in harlequin mutant mice sensitizes the myocardium to oxidative stress-related cell death and pressure overload-induced decompensation.\nCirc Res\n96\ne92\ne101\n15933268\n16. van EmpelVPBertrandATvan OortRJvan der NagelREngelenM\n2006\nEUK-8, a Superoxide Dismutase and Catalase Mimetic, Reduces Cardiac Oxidative Stress and Ameliorates Pressure Overload-Induced Heart Failure in the Harlequin Mouse Mutant.\nJ Am Coll Cardiol\n48\n824\n32\n16904556\n17. BrownDYuBDJozaNBénitPMenesesJ\n2006\nLoss of Aif function causes cell death in the mouse embryo but the temporal progression of patterning is normal.\nProc Ntal Acad Sci \n(in press)\n18. PorterAGUrbanoAG\n2006\nDoes apoptosis-inducing factor (AIF) have both life and death functions in cells?\nBioessays\n28\n834\n843\n16927311\n19. CheungECJozaNSteenaartNAMcClellanKANeuspielM\n2006\nDissociating the dual roles of apoptosis-inducing factor in maintaining mitochondrial structure and apoptosis.\nEmbo J\n25\n4061\n73\n16917506\n20. von Kleist-RetzowJCCormier-DaireVViotGGoldenbergAMardachB\n2003\nAntenatal manifestations of mitochondrial respiratory chain deficiency.\nJ Pediatr\n143\n208\n12\n12970634\n21. MunnichARustinP\n2001\nClinical spectrum and diagnosis of mitochondrial disorders.\nAm J Med Genet\n106\n4\n17\n11579420\n22. KatzBJZhaoYWarnerJETongZYangZ\n2006\nA family with X-linked optic atrophy linked to the OPA2 locus Xp11.4-Xp11.2.\nAm J Med Genet A\n23. BarberB\n1971\nTwo new mutations.\nMouse News Lett\n45\n34\n35\n24. JozaNOuditGYBrownDBenitPKassiriZ\n2005\nMuscle-specific loss of apoptosis-inducing factor leads to mitochondrial dysfunction, skeletal muscle atrophy, and dilated cardiomyopathy.\nMol Cell Biol\n25\n10261\n72\n16287843\n25. PospisilikJAKnaufCJozaNBenitPOrthoferM\n2007\nTargeted deletion of AIF decreases mitochondrial oxidative phosphorylation and protects from obesity and diabetes.\nCell\n131\n476\n91\n17981116\n26. JozaNGalindoKPospisilikJABenitPRangachariM\n2008\nThe molecular archaeology of a mitochondrial death effector: AIF in Drosophila.\nCell Death Differ\n27. KoopmanWJVerkaartSVischHJvan der WesthuizenFHMurphyMP\n2005\nInhibition of complex I of the electron transport chain causes O2−. -mediated mitochondrial outgrowth.\nAm J Physiol Cell Physiol\n288\nC1440\n50\n15647387\n28. MelovSCoskunPPatelMTuinstraRCottrellB\n1999\nMitochondrial disease in superoxide dismutase 2 mutant mice.\nProc Natl Acad Sci U S A\n96\n846\n51\n9927656\n29. ApostolovaNCerveraAMVictorVMCadenasSSanjuan-PlaA\n2006\nLoss of apoptosis-inducing factor leads to an increase in reactive oxygen species, and an impairment of respiration that can be reversed by antioxidants.\nCell Death Differ\n13\n354\n7\n16195738\n30. QiuXZYuLLaiGHWangLYChenB\n2008\nMitochondrial AIF protein involved in skeletal muscle regeneration.\nCell Biochem Funct\n31. BrownDYuBDJozaNBenitPMenesesJ\n2006\nLoss of Aif function causes cell death in the mouse embryo, but the temporal progression of patterning is normal.\nProc Natl Acad Sci U S A\n103\n9918\n23\n16788063\n32. JozaNSusinSADaugasEStanfordWLChoSK\n2001\nEssential role of the mitochondrial apoptosis-inducing factor in programmed cell death.\nNature\n410\n549\n54\n11279485\n33. BodemerCRotigARustinPCormierVNiaudetP\n1999\nHair and skin disorders as signs of mitochondrial disease.\nPediatrics\n103\n428\n33\n9925836\n34. DelettreCYusteVJMoubarakRSBrasMRobertN\n2006\nIdentification and characterization of AIFsh2, a mitochondrial apoptosis-inducing factor (AIF) isoform with NADH oxidase activity.\nJ Biol Chem\n281\n18507\n18\n16644725\n35. BenitPGoncalvesSDassaEPBriereJJMartinG\n2006\nThree spectrophotometric assays for the measurement of the five respiratory chain complexes in minuscule biological samples.\nClin Chim Acta\n36. ClaiborneA\n1985\nCatalase activity\nBoca Raton, , Florida, , USA\nCRC Press\n37. MarklundSMarklundG\n1974\nInvolvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase.\nEur J Biochem\n47\n469\n74\n4215654\n38. BradfordMM\n1976\nA rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.\nAnal Biochem\n72\n248\n54\n942051\n39. Fernandez-MoreiraDUgaldeCSmeetsRRodenburgRJLopez-LasoE\n2007\nX-linked NDUFA1 gene mutations associated with mitochondrial encephalomyopathy.\nAnn Neurol\n61\n73\n83\n17262856\n40. BenitPSlamaARustinP\n2008\nDecylubiquinol impedes mitochondrial respiratory chain complex I activity.\nMol Cell Biochem\n314\n45\n50\n18414996"
+}

batch_9/PMC2527785.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2527785",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527785\nAUTHORS: N G Hurst\n\nABSTRACT:\nNo Abstract\n\nBODY:\nSir,We thank Professor Jung for his interest in our work and congratulate him on both his previous published work and his more recent small demonstrative study.As he correctly states, serum analysis was employed, without clot activators, for our own pilot study, initiated before his and other authors' publications on choice of substrate. Technical details in minutiae form were not included in the final submitted paper for reasons of brevity and space constraints. However, it is almost universally agreed that, without similar space constraints, further details, particularly pertaining to pre-study planning, serum acquisition and post-venesection processing would be useful. Furthermore, description of quality control measures and internal control methods would be useful adjuncts for those wishing to replicate work done.However, Professor Jung may have overlooked the underlying premise and methodology of our study in that this represented a comparative analysis of total detectable levels of serum MMP9, collected under tightly controlled protocols, for the prediction of the presence of significant colorectal pathology.The absolute levels obtained were less critical in this study than the relative levels seen within the population measured, harbouring colorectal neoplasia or otherwise. No assertion is made that the absolute serum MMP9 levels are applicable to the general population as a whole, nor cohorts in different regions/countries.In addition, choice of assay employed in measurement has significant effects on the absolute levels reported, whether total/active/proenzyme or complexed protein is measured. Once again, in a study design as employed in our pilot study, relative levels within a population providing its own controls are more useful than imported levels obtained under potentially varied conditions elsewhere.Professor Jung's final comments regarding sample type of choice are noted with appreciation, and further discussion will no doubt ensue within the research group.\n\nREFERENCES:\nNo References"
+}

batch_9/PMC2527794.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2527794",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527794\nAUTHORS: P Martín-Martorell, S Roselló, E Rodríguez-Braun, I Chirivella, A Bosch, A Cervantes\n\nABSTRACT:\nThis is a phase II institutional exploratory trial of biweekly irinotecan and cetuximab administration regimen in metastatic colorectal cancer patients progressing to at least one previous chemotherapy line. A total of 40 patients were treated between November 2005 and November 2007 with irinotecan 180 mg m−2 and cetuximab 500 mg m−2 q2w (every 2 weeks), in every 21-day cycles, until unacceptable toxicity or progressive disease. An overall response rate of 22.5% was obtained (two complete and seven partial responses). The disease control rate was 60%. The time to progression was 3.4 months and the overall survival was 8 months. The toxicity compared very favourably to weekly cetuximab combination schedules. Grade 3/4 adverse effects were observed in 12 patients. Overall, our results turn up very similar both in terms of toxicity and efficacy to those obtained by weekly and biweekly administration regimens.\n\nBODY:\nCetuximab is an epidermal growth factor receptor (EGFR)-directed IgG1 chimeric monoclonal antibody showing antitumour activity in the treatment of advanced colorectal cancer. Cetuximab binds to the extracellular domain of EGFR when it is in the inactive configuration, competes for receptor binding by occluding the ligand-binding region, and thereby blocks ligand-induced EGFR tyrosine kinase activation (Ciardello and Tortora, 2008)In a randomised phase II trial comparing a combination of weekly cetuximab and biweekly irinotecan with weekly cetuximab monotherapy, this monoclonal antibody proved to have consistent antitumour activity in patients with advanced colorectal cancer refractory to irinotecan (Cunningham et al, 2004). This study led to the approval of cetuximab by the regulatory authorities for the treatment of irinotecan refractory metastatic colorectal cancer. Other trials have shown that cetuximab improves survival over best supportive care alone (Jonker et al, 2007) and might offer some advantage in patients receiving first or second line therapy (Van Cutsem et al, 2007, Pessino et al, 2008, Sobrero et al, 2008). Moreover, it has also proved to be active in combination with oxaliplatin-based regimens, both in the first (Tabernero et al, 2007) and successive lines of treatment (Souglakos et al, 2007)In all those studies, cetuximab was administered weekly with an initial intravenous infusion of 400 mg m−2 on day 1 with subsequent weekly doses of 250 mg m−2. Although this regimen is undoubtedly active, the weekly administration of cetuximab is out of step with administration of the chemotherapy regimens with which cetuximab is commonly combined. Irinotecan is often administered at a dose of 180 mg m−2 every 2 weeks. Similarly, in the first-line setting, combinations of infusional 5-fluorouracil (5-FU)/folinic acid plus irinotecan (FOLFIRI) or oxaliplatin are frequently administered on an every 2 weeks basis. The option to synchronise the administration of cetuximab and concomitant chemotherapy would reduce the impact of treatment administration on patients' lives and simplify treatment administration for health-care workers. It is also reasonable to assume that a simplified schedule may reduce the costs associated with cetuximab administration.In a preliminary reported phase I trial, it was shown that cetuximab can be safely administered at 500 mg m−2 every 2 weeks, with similar pharmacokinetic and pharmacodynamic behaviour compared with the weekly schedule (Tabernero et al, 2006). The biweekly dosing may facilitate the administration of this drug, by making this therapy more convenient for patients (Tabernero et al, 2008). This publication reports on a phase II trial designed to explore the antitumour activity of combined irinotecan and cetuximab, both administered in a biweekly fashion.Materials and methodsStudy designThis was an institutional prospective, single-arm phase II trial exploring the antitumour activity of biweekly administration of cetuximab and irinotecan in patients with metastatic colorectal cancer who had progressed to at least one previous line of chemotherapy for advanced disease. The primary end point was response rate. Secondary end points were toxicity, time to progression, duration of response and overall survival. Patients were recruited between November 2005 and November 2007. Analysis of data took place on January 2008. All patients gave their informed consent before treatment and the trial was performed according to the Institutional Review Board.Selection of patientsEligibility criteria were histologically confirmed colorectal adenocarcinoma, with progressive metastatic disease to at least one previous line of chemotherapy. Patients had to be at least 18 years or older, have a performance status of 0–2, adequate bone marrow reserve (Hb ⩾8.0 g dl−1, neutrophil count ⩾1.5 × 109/l, platelet count ⩾100 × 109/l), hepatic and renal function (total bilirubin <1.5 UNL, ASAT and ALAT <2.0 UNL and serum creatinine <2 mg dl−1). Epidermal growth factor receptor immunohistochemistry and k-ras status were not required in the eligibility criteria.TherapyEligible patients were treated with cetuximab 500 mg m−2 intravenous infusion on day 1 (during 2 h on the first infusion and during 1 h on subsequent cycles if no adverse reaction had occurred on the previous administration), followed by irinotecan 180 mg m−2 intravenous infusion on day 1 (during 30 min in all cycles). Before cetuximab, all patients received dexchlorphenamine maleate at a dose of 5 mg intravenously. Antiemetic prophylaxis with dexamethasone and ondansetron was given before irinotecan. Patients were evaluated with blood count, complete serum biochemistry and CEA before day 1 initially. If no relevant toxicity occurred, this was then done every other cycle. Treatment was continued until documented disease progression or unacceptable toxicity, whichever occurred first.Evaluation of safety and responseEvaluation of disease was carried out according to RECIST criteria (Therasse et al, 2000) every 3–5 cycles. Toxicity was evaluated according to NCI-CTCAE (version 3.0), before every treatment of the first three cycles, and every other cycle thereafter if no relevant toxicity had appeared. Irinotecan dosage was reduced by 25% if ⩾grade 3 diarrhoea was observed. Cetuximab administration was delayed if cutaneous toxicity ⩾grade 3 was observed and restarted when it had reduced to grade 2.Statistical analysisTimes to event variables were calculated according to Kaplan–Meier methods using StatSoft (version 6). Descriptive variables of patient characteristics and toxicity were calculated directly from the database. Time to tumour progression (TTP) was defined as the time to documented progression from the start of the treatment, duration of response (DR) as the time from first objective response to documented progression and overall survival (OS) was considered from the start of treatment to date of data analysis or date of loss from follow-up for patients alive. Patients without disease progression who discontinued the study for any reason were censored at the last on study tumour assessment date. All efficacy and safety analyses were evaluated at an exploratory level.ResultsBetween November 2005 and November 2007, a total of 40 patients were recruited. Patient characteristics are shown in Table 1. Median age at diagnosis was 61 years. The median performance status at the start of treatment was 1. Patients had received a median of one previous chemotherapy line for advanced disease, but 27.5% had two previous lines and 20% got this therapy as fourth or further line. The median number of metastatic sites was 2. Almost all patients were pretreated with oxaliplatin- and fluoropyrimidine-based combinations. Half of them received previous treatment with bevacizumab and 25% received irinotecan-based therapies. The median follow-up time for the patients alive was 5 months (range: 4–24.5). A total of 322 treatment cycles were administered, which amounts to a median of 7 cycles per patient (range: 2–29).EfficacyA total of 39 patients were assessed for response. There was an overall response rate of 22.5% (two complete and seven partial responses, CI 95%: 9.6–35.4%). Stable disease was observed in 15 cases (37.5%). The disease control rate was 60% (CI 95%: 44.9–75.1%). Progressive disease was observed in 15 patients. One patient was not assessable because she died before evaluation. The median TTP was 3.4 months (range: 0.7–23.9). Figure 1 shows the progression-free survival curve. The median OS from the start of treatment was 8 months (range: 0.7–26.1). Figure 2 shows the Kaplan–Meier OS curve. Eight out of the nine responding patients had progressed at the time of analysis. The median duration of response was 5.0 months (range: 2–20).SafetyThe biweekly administration of cetuximab and irinotecan proved to be tolerable. Considering maximum reported toxicity by patient, the only grade 4 observed event was diarrhoea in two patients (5%). Two more patients suffered from grade 3 diarrhoea (5%). Grade 3 skin rash was seen in three patients (7.5%). Grade 3 anaemia and neutropoenia were reported in one (2.5%) and three (7.5%) cases, respectively. Only one case of grade 3 nausea was seen. Neither grade 4 myelosuppression nor severe infusional anaphylactic reactions were observed. Concerning grade 2 events, paronychia was reported in 30% of patients, alopecia in 7.5%, skin rash in 17.5% and asthenia in 15%. No treatment related deaths occurred.DiscussionThe aim of this exploratory trial was to investigate the antitumour activity of the combination of irinotecan and cetuximab given in a biweekly fashion, as well as its safety profile with respect to the standard weekly regimen used in most trials with cetuximab. The efficacy data obtained in the different studies of irinotecan and cetuximab given weekly or biweekly are listed in Table 2. The response rate seen in this trial is very similar to that observed with weekly cetuximab administration in the BOND trial (Cunningham et al, 2004). Also time to progression (3.4 vs 4.1 months) and overall survival (8 vs 8.6 months) illustrate the similarities of both schedules. Our results are also comparable to those obtained in a large confirmatory trial accruing more than 1000 patients (Wilke et al, 2006). Safety between the different schedules is compared in Table 3. In terms of toxicity, the biweekly regimen also proved to be tolerable. Grade 3 or 4 events were mainly observed only in eight (20) and two (5%) of our patients, respectively. Pfeiffer et al reported very similar results using a biweekly cetuximab and irinotecan regimen. Their results using the biweekly schedule were comparable to those of a 65 patients historical cohort receiving the weekly schedule at the same participating institutions.Different phase I and II trials have studied the pharmacokinetic and pharmacodynamic behaviour of cetuximab in different dosing schedules. Fairly predictable linear pharmacokinetic has been demonstrated for cetuximab. Multiple dose studies have demonstrated that the pharmacokinetic parameters – CL, AUC, t1/2 and volume of distribution at steady state (Vss) – are similar after single and multiple doses of cetuximab at the approved dosing regimen. The AUC also shows a linear relationship to the dose and frequency of administration (Humblet et al, 2005). The data regarding pharmcokinetic and pharmacodynamic behaviour of cetuximab at different dosages and frequency schedules support the feasibility of biweekly cetuximab administration in combination with irinotecan (Tabernero et al, 2006).Furthermore, the different trials using the simplified biweekly administration have all yielded very consistent results, both with regard to the TTP and OS, as well as to the response rate. Both our results and those recently published by Pfeiffer et al, show very similar efficacy data compared to the weekly schedule. Administering irinotecan and cetuximab together every 2 weeks would render conveniency both for patients and for the health resources, without compromising efficacy or having a deleterious effect on toxicity. It therefore appears as a very reasonable strategy that would be worth testing in future trials.\n\nREFERENCES:\n1. Ciardello F, Tortora G. EGFR antagonists in cancer treatment. N Eng J Med\n2008; 358: 1160–1174\n2. Cunningham D, Humblet Y, Siena S, Khayat D, Bleiberg H, Santoro A, Bets D, Mueser M, Harstrick A, Verslype C, Chau I, van Cutsem E (2004) Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med\n351: 337–34515269313\n3. Humblet Y, Peers M, Bleiberg H, Stupp R, Sessa C, Roth A, Nippgen J, Nolting A, Stuart P, Giaccone G (2005) An open label, phase I study of cetuximab to assess the safety, efficacy and pharmacokinetics (PK) of different cetuximab regimens in patients with epidermal growth factor receptor (EGFR)-expressing metastatic colorectal cancer (mCRC). J Clin Oncol\n23(16 Suppl): 279s\n4. Jonker DJ, O'Callaghan CJ, Karapetis CS, Zalcberg JR, Tu D, Au HJ, Berry SR, Krahn M, Price T, Simes RJ, Tebbutt NC, van Hazel G, Wierzbicki R, Langer C, Moore MJ (2007) Cetuximab for the treatment of colorectal cancer. N Engl J Med\n357: 2040–204818003960\n5. Pessino A, Artale S, Sciallero S, Guglielmi A, Fornarini G, Andreotti IC, Mammoliti S, Comandini D, Caprioni F, Benicelli E, Andretta V, Siena S, Sobrero A (2008) First-line single-agent cetuximab in patients with advanced colorectal cancer. Ann Oncol\n19: 711–71618073221\n6. Pfeiffer P, Nielsen D, Bjerregaard J, Qvortrup C, Tilmaz M, Jensen B (2008) Biweekly cetuximab and irinotecan in patients with advanced colorectal cancer after failure to irinotecan, oxaliplatin and 5-fluorouracil. Ann Oncol; e-pub ahead of print; doi:10.1093/annonc/mdn020\n7. Sobrero AF, Maurel J, Fehrenbacher L, Scheithauer W, Abubakr YA, Lutz MP, Vega-Villegas E, Eng C, Steinhauer EU, Prausova J, Lenz HJ, Borg C, Middleton G, Kröning H, Luppi G, Kisker O, Zubel A, Langer C, Kopit J, Burris III HA (2008) EPIC: phase III trial of cetuximab plus irinotecan after fluoropyrimidine and oxaliplatin failure in patients with metastatic colorectal cancer. J Clin Oncol\n26: 2311–231918390971\n8. Souglakos J, Kalykaki A, Vamvakas L, Androulakis N, Kalbakis K, Agelaki S, Vardakis N, Tzardi M, Kotsakis AP, Gioulbasanis J, Tsetis D, Sfakiotaki G, Chatzidaki D, Mavroudis D, Georgoulias V (2007) Phase II trial of capecitabine and oxaliplatin (CAPOX) plus cetuximab in patients with metastatic colorectal cancer who progressed after oxaliplatin-based chemotherapy. Ann Oncol\n18: 305–31017079693\n9. Tabernero J, Pfeiffer P, Cervantes A (2008) Administration of cetuximab every 2 weeks in the treatment of metastatic colorectal cancer: an effective more convenient alternative to weekly administration? The Oncologist\n13: 113–11918305055\n10. Tabernero J, van Cutsem E, Díaz-Rubio E, Cervantes A, Humblet Y, André T, van Laethem JL, Soulié P, Casado E, Verslype C, Sastre Valera J, Tortora G, Ciardiello F, Kisker O, de Gramont A (2007) Phase II trial of cetuximab in combination with fluorouracil, leucovorin, and oxaliplatin in the first-line treatment of metastatic colorectal cancer. J Clin Oncol\n25: 5225–523218024868\n11. Tabernero J, Cervantes C, Martinelli E, Vega-Villegas E, Rojo F, Pérez-Fidalgo A, Casado E, Ciardiello F, Zubel A, Baselga J (2006) Optimal dose of cetuximab (C) given every 2 weeks (q2w): A phase I pharmacokinetic (PK) and pharmacodynamic (PD) study of weekly (q1w) and q2w schedules in patients (pts) with metastatic colorectal cancer (mCRC). J Clin Oncol\n24(18 Suppl): 142s\n12. Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, Rubinstein L, Verweij J, Van Glabbeke M, van Oosterom AT, Christian MC, Gwyther SG (2000) New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst\n92: 205–21610655437\n13. Van Cutsem E, Nowacki M, Lang I, Cascinu S, Shchepotin I, Maurel J, Rougier P, Cunningham D, Nippgen J, Köhne C (2007) Randomized phase III study of irinotecan and 5-FU/FA with or without cetuximab in the first-line treatment of patients with metastatic colorectal cancer (mCRC): The CRYSTAL trial. J Clin Oncol\n25: 4000\n14. Wilke H, Glynne-Jones R, Thaler J et al. (2006) MABEL – a large multinational study of cetuximab plus irinotecan in irinotecan resistant metastatic colorectal cancer. J Clin Oncol\n24(18 Suppl): 158s"
+}

batch_9/PMC2527796.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2527796",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527796\nAUTHORS: L Pusztai, B Leyland-Jones\n\nABSTRACT:\nNo Abstract\n\nBODY:\nSeveral biomarkers have been reported to be associated with survival in breast cancer over the past decades. Unfortunately, it is often difficult to distinguish between the prognostic and predictive values of these proposed markers and independent validations are frequently lacking. Patients with early-stage breast cancer receive various combinations of treatments including surgery, post-operative radiation therapy, adjuvant endocrine treatment and chemotherapy. Each of these can have an impact on survival, and most biomarker studies included heterogeneously treated patient cohorts. The classical methodology of assessing the true prognostic value of a marker was restricted to patients who underwent local regional therapy alone and received no systemic adjuvant treatment.However, the clinical utility of such prognostic markers in the absence of systemic therapy is limited in this era. Endocrine sensitivity in newly diagnosed stage I–III breast cancer may be best evaluated in patients who received adjuvant endocrine treatment but no chemotherapy after surgery and the chemotherapy response predictive value of a marker may be best examined in the preoperative treatment setting where tumour response can be measured directly. Undoubtedly, the best samples to assess interaction between markers and treatment outcome are from randomized clinical trials that compare different treatment modalities. Unfortunately, many such trials from the past with good outcome data lack tumour banks: it is difficult to obtain specimens from large cohorts of homogeneously treated patients and specimens from randomized clinical trials are an even rarer research commodity. This motivates systematic efforts to collect biological specimens in cancer clinical trials today.Traditional markers also require separate assays to be performed for each marker on the limited tissue resources. Therefore, it is not surprising that few prognostic or predictive markers have completed the necessary validation steps on the right type of clinical specimens to convince physicians about their clinical value (Simon, 2005). In this issue of the journal, Epping et al (2008) report on that PRAME mRNA expression is prognostic in early-stage breast cancer. They observed that this gene had higher expression in tumours that relapsed within 5 years in the absence of systemic adjuvant therapy. The bimodal distribution of PRAME expression offered an opportunity to define a natural cutoff in the data to assign high or low expression status for cases (for most biomarkers that show a near normal distribution, defining optimal cutoff values to assign low or high expression status is more difficult). In a second independent cohort of patients, who also received no systemic adjuvant therapy (n=185), they confirmed that cancers with higher PRAME expression had poorer survival. When PRAME expression was correlated with survival in a third cohort of patients who received adjuvant chemotherapy (n=110), no association with outcome was seen. This suggests that high PRAME expression is predictive of poor prognosis in the absence of adjuvant chemotherapy, but is also predictive of greater sensitivity to chemotherapy (ie poor prognosis is no longer observed if patients receive adjuvant chemotherapy). Markers with similar characteristics have been reported previously including proliferative activity, histologic grade or the Oncotype DX recurrence score.Perhaps the most important feature of this article is that these provocative observations were made entirely through in silico analysis of publicly available gene expression data (that was initially generated by these investigators), without any additional experiments. In the past few years, a remarkable transformation has begun to evolve in the biomarker field. Comprehensive high-throughput genomic analytical tools including mRNA gene expression profiling are increasingly applied to human cancers in an attempt to discover predictors of clinical outcome. When the main results from these studies are published, it is required by most journals to make the genomic data public. An important central repository for these data sets is the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo). Among many others, there are now publicly available comprehensive gene expression data sets from cohorts of breast cancers patients who did not receive any systemic adjuvant therapy, as well as from oestrogen receptor-positive patients who were treated with adjuvant endocrine therapy only, and from patients who received preoperative chemotherapy.These databases provide an unprecedented opportunity to rapidly evaluate almost any mRNA expression-based marker separately for prognostic, endocrine and chemotherapy response predictive values in silico (Andre et al, 2007). Studies that used to require many years to complete for immunohistochemistry-based markers can now be completed in a few weeks by a well trained investigator in basic bioinformatics methods. Not only can single genetic markers be assessed, but also any number of combinations of genes that might represent cellular regulatory pathways or other molecular functions can be tested. The multiple data sets also provide an opportunity for marker optimisation in one data set and independent validation in others.One important caveat to this approach is that results generated by in silico analysis of publicly available genomic data are only as reliable as the source data itself. It is often difficult, or impossible, to validate the accuracy of clinical annotations. Technical noise due to variable quality of measurements can be substantial and systematic differences in the analytical methods including array platforms and data normalisation can make comparisons across data sets challenging. For these reasons, it is unlikely that any biomarker discovered solely through in silico analysis would gain wide clinical acceptance in the foreseeable future. This is a preliminary approach to the value of the markers and future studies should follow the reporting recommendations for tumour marker prognostic studies (REMARK, McShane et al, 2005) and standardised end points and events in clinical trials (STEEP, Hudis et al, 2007). It is also important to consider that if the intended clinical assay is different from the high throughput platform, validation of the new assay (eg, PCR or immunochemistry) vs the original microarray findings is necessary and inter- and intra-assay variation should also be provided. Nevertheless, genomic data sets and the rapidly expending bioinformatic tools to analyse them provide a unique opportunity to quickly evaluate biomarker concepts with minimal cost and identify promising markers for prospective validation on precious, nonrenewable clinical specimens.\n\nREFERENCES:\n1. Andre F, Hatzis C, Anderson K, Sotiriou C, Mazouni C, Mejia J, Wang B, Hortobagyi GN, Symmans WF, Pusztai L (2007) Microtubule-associated protein-tau is a bifunctional predictor of endocrine sensitivity and chemotherapy resistance in estrogen receptor-positive breast cancer. Clin Cancer Res\n13: 2061–206717404087\n2. Epping MT, Hart AAM, Glas AM, Krijgsman O, Bernards R (2008) PRAME expression and clinical outcome of breast cancer. Br J Cancer (in press)\n3. Hudis CA, Barlow WE, Costantino JP, Gray RJ, Pritchard KI, Chapman JA, Sparano JA, Hunsberger S, Enos RA, Gelber RD, Zujewski JA (2007) Proposal for standardized definitions for efficacy end points in adjuvant breast cancer trials: The STEEP system. J Clin Oncol\n25: 2127–213217513820\n4. McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM (2005) Reporting recommendations for tumor marker prognostic studies. J Clin Oncol\n23: 9067–907216172462\n5. Simon R (2005) Roadmap for developing and validating therapeutically relevant genomic classifiers. J Clin Oncol\n23: 7332–734116145063"
+}

batch_9/PMC2527800.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2527800",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527800\nAUTHORS: S Raguz, E Yagüe\n\nABSTRACT:\nResistance to cancer chemotherapeutic treatment is a common phenomenon, especially in progressive disease. The generation of cellular models of drug resistance has been pivotal in unravelling the main effectors of resistance to traditional chemotherapy at the molecular level (i.e. intracellular drug inactivation, detoxifying systems, defects in DNA repair, apoptosis evasion, membrane transporters and cell adhesion). The development of targeted therapies has also been followed by resistance, reminiscent of an evolutionary arms race, as exemplified by imatinib and other BCR-ABL inhibitors for the treatment of chronic myelogenous leukaemia. Although traditionally associated with the last stages of the disease, recent findings with minimally transformed pretumorigenic primary human cells indicate that the ability to generate drug resistance arises early during the tumorigenic process, before the full transformation. Novel technologies, such as genome profiling, have in certain cases predicted the outcome of chemotherapy and undoubtedly have tremendous potential for the future. In addition, the novel cancer stem cell paradigm raises the prospect of cell-targeted therapies instead of treatment directed against the whole tumour.\n\nBODY:\nGilman and co-workers were the first to introduce chemotherapy into clinical practice at the end of the Second World War when they used nitrogen mustard to treat a patient with advanced malignant lymphoma. After an initial regression of the disease, a second course of therapy was given at a reduced dose due to the toxicity of the treatment, with an associated lesser therapeutic effect. By the time the third treatment was given, the tumour no longer responded to the chemotherapeutic agent (Goodman et al, 1946). Since then, chemotherapy has been one of the main therapeutic strategies in cancer treatment, but, to paraphrase Paul Ehrlich, resistance has followed as a faithful shadow.Chemotherapeutic strategies have used a variety of drugs and hormonal agents that interfere with the basic machinery of the cell. Subsequent improved understanding of the molecular alterations present in the cancer cell has enabled the development of targeted therapies for some forms of cancer. Interestingly, resistance appears not only to traditional chemotherapy but also to targeted therapies such as tamoxifen, which targets the oestrogen receptor (ER) in breast cancer (Ali and Coombes, 2002); imatinib, which targets the kinase activity of the translocated BCR-ABL in chronic myelogenous leukaemia (CML) (Weisberg et al, 2007); or gefitinib, which inhibits epidermal growth factor receptor (EGFR) kinase (Engelman et al, 2007).Mechanisms of resistance to traditional chemotherapyTwo main groups of factors contribute to the development of drug resistance. The first group includes pharmacological and physiological factors such as drug metabolism and excretion, inadequate access of the drug to the tumour, inadequate infusion rate and inadequate route of delivery. These are extremely important issues not only in clinical practice but also fundamental in drug development (Garattini, 2007). The second group includes cell- or tissue-specific factors. The cytochrome P450 enzymes, a multigene family of constitutive and inducible haem-containing oxidative enzymes from the liver, play an important role in the metabolism of a diverse range of xenobiotics and are often overexpressed in a variety of solid tumours in which they can contribute to drug resistance. Drug analogues of DNA precursors such as 5-fluorouracil and cytosine arabinoside require metabolic activation, and resistance can arise from modification of these activation pathways. Altered topoisomerase I and II activity prevents drugs such as doxorubicin, etoposide and camptothecin from binding the topoisomerase–DNA complex, allowing the broken strands to be repaired. Many anticancer drugs, such as platinum compounds, alkylating agents and nitrosoureas, cause direct damage to the structural integrity of the DNA, and resistance to these compounds results from activation of DNA repair systems. Regulation of cell death by evasion of apoptosis, necrosis, mitotic catastrophe (Mansilla et al, 2006) or evasion of senescence (Dimri, 2005) contributes towards drug resistance. In addition, the differential expression of membrane proteins such as solute carriers, channels and ATP-binding cassette (ABC) transporters (Gottesman et al, 2002; Huang et al, 2004) have all been demonstrated to play an important role in drug resistance. Although these mechanisms have been clearly demonstrated in cell culture, their relevance with the clinical outcome of chemotherapy is less clear (Cimoli et al, 2004; Uggla et al, 2007) and has only been demonstrated for some of these mechanisms (Clarke et al, 2005).Molecular mechanisms of resistance to targeted chemotherapyCML and imatinibChronic myelogenous leukaemia was the first human cancer to be associated with a consistent chromosomal abnormality, the Philadelphia chromosome, a translocation that juxtaposes the 3′ sequence from the ABL1 proto-oncogene on chromosome 9 with the 5′ sequence from the BCR gene on chromosome 22. The resultant chimaeric BCR-ABL protein is a constitutively active protein tyrosine kinase with an important role in the regulation of cell growth (Melo and Barnes, 2007). Traditional therapy for CML includes initial allogenic stem-cell transplantation and interferon-α, followed by second-line treatment with hydroxyurea or busulfan in non-responsive patients. Imatinib mesylate (formerly STI571; Gleevec, Novartis, Basel, Switzerland) is a potent and highly specific competitive inhibitor of the BCR-ABL tyrosine kinase. Initially, it had a high rate of cytogenetic and haematologic responses in patients with chronic-phase CML in whom previous therapy had failed, and its use has revolutionised the management and clinical expectations of CML patients. Unfortunately, not long after its initial use, resistance to imatinib was demonstrated in CML patients (Gorre et al, 2001). Approximately 50% of imatinib-resistant CML patients carry a resistance-associated point mutation in BCR-ABL, which interferes with imatinib binding. More than 50 different resistance-associated point mutations have been described to date. These findings have spurred the development of second-generation BCR-ABL inhibitors such as BMS-354825, which has a two-log increased potency relative to imatinib and retains its inhibitory activity against 14 of 15 imatinib-resistant BCR-ABL mutants tested (Shah et al, 2004). However, resistance to some of these second-generation inhibitors, such as nilotinib and dasatinib, has already been described. This indicates that the potential for new drug-resistant point mutations in BCR-ABL persists and justifies the continued development of more potent BCR-ABL inhibitors (Weisberg et al, 2007). In the remaining 50% of imatinib-resistant patients with no BCR-ABL mutations, BCR-ABL gene amplification or overexpression at the mRNA and protein levels has been detected in clinical samples (Hochhaus et al, 2002). In addition, chromosomal aberrations, reduced intracellular uptake of imatinib and the disease phase have all been implicated in imatinib resistance (Nimmanapalli and Bhalla, 2002).Breast cancer, tamoxifen, aromatase inhibitors and trastuzumabBreast cancer accounts for one in four of all female cancers, making it by far the most common cancer in women in the western world, where one in nine women will develop the disease at some stage in their lives. Breast cancer treatment involves surgical removal of the tumour, although this is ineffective if malignant cells have escaped from the site of the primary tumour. Discovery of the involvement of the ovarian hormone oestrogen and its mechanism of action (Dickson and Lippman, 1995) paved the way for the development of therapies for ER-positive patients that inhibit oestrogen action. These therapies include tamoxifen, which blocks the ER, and the oestrogen synthetase (aromatase) inhibitors formestane and exemestane, which inhibit oestrogen synthesis (Ali and Coombes, 2002). Despite the huge improvement in cancer survival due to tamoxifen treatment, some patients relapse and the use of sequential therapy with exemestane after 2–3 years of tamoxifen treatment has improved disease-free survival, as compared with the standard 5 years of tamoxifen treatment alone (Coombes et al, 2004). The use of endocrine agents has markedly reduced the number of deaths from breast cancer over the past decades. However, in many cases, these therapies fail due to recurrent endocrine-resistant tumours, and much effort is being made to elucidate the mechanisms that underlie resistance to endocrine therapies (Weinberg et al, 2005). Altered growth factor signalling, notably EGFR (Schiff et al, 2005) and insulin-like growth factor I receptor (IGF-IR) make a significant contribution to the development of antioestrogen resistance, and these have been reviewed recently (Baselga, 2006).Up to 25% of patients diagnosed with breast cancer have tumours that overexpress the EGFR-2 (HER2 or Erb B-2). HER2-positive breast cancer is highly proliferative, difficult to treat and confers a poor prognosis. Trastuzumab is a monoclonal antibody targeted against the HER2 tyrosine kinase receptor. The majority of patients with metastatic breast cancer, who initially respond to trastuzumab, develop resistance within 1 year of treatment initiation, and in the adjuvant setting, 15% of patients still relapse despite trastuzumab-based therapy. Preclinical studies have indicated several molecular mechanisms that could contribute to the development of trastuzumab resistance. One major determinant to resistance is increased signalling via the phosphatidylinositol 3-kinase/Akt pathway. This results in the activation of multiple receptor pathways, including HER2-related receptors and non-HER receptors such as the IGF-IR, which appear to be involved in a cross talk with HER2 in resistant cells (Berns et al, 2007). Alternatively, the loss of function of the tumour suppressor PTEN, the negative regulator of Akt, results in an increase in Akt signalling that leads to decreased trastuzumab sensitivity. Decreased interaction between trastuzumab and its target receptor HER2, which is due to steric hindrance of HER2 by cell-surface proteins such as mucin-4 (MUC4), can block the inhibitory actions of trastuzumab. Novel therapies targeted against these aberrant molecular pathways offer hope that the effectiveness and duration of response to trastuzumab can be greatly improved (Baselga, 2006; Nahta et al, 2006). As only about one-third of breast cancer patients overexpressing HER2 respond to trastuzumab monotherapy, the identification of predictive biomarkers that can more accurately select responders or non-responders is vital, not only to improve its therapeutic index, but also to gain insight into the molecular pathways involved in trastuzumab resistance and to rationally design successful combination therapies.The ability to acquire drug resistance arises early during the tumorigenesis processThe use of drug-resistant derivatives from human and other mammalian cell lines has been of paramount importance for the unravelling of many of the mechanisms of cancer drug resistance highlighted above. However, they have proved less successful in identifying the ultimate upstream regulators controlling these events, and as such, how drug resistance arises is still unresolved. Most cellular models of drug resistance have been developed from transformed cell lines isolated from patients at a late stage in cancer progression and whose tumours already exhibit a plethora of karyotypic and physiologic abnormalities. Recently, several groups have shown that it is possible to transform primary human cells into fully tumorigenic cells by altering a small number of defined pathways ex vivo. This cellular model of tumorigenesis (Figure 1A) was first described by Hahn et al (1999) in human BJ fibroblasts and embryonic kidney epithelial cells by expressing the catalytic subunit of telomerase (to avoid replicative senescence), SV40 large T-antigen (which binds and inactivates the tumour suppressors p53 and pRb controlling the DNA repair and G1 cell cycle checkpoints, respectively) and small t-antigen (which binds and inactivates PP2A, a serine/threonine phosphatase involved in several signalling pathways) and oncogenic ras. Primary human epithelial cells from the mammary gland, prostate, ovary, trachea and bronchia have now been transformed by introducing these or similar sets of genes (Boehm and Hahn, 2005). Although somatic p53 missense mutations are found in approximately 50% of human cancers, the p53 pathway can also be inactivated in wild-type p53-carrying tumours by p53 destabilisation via indirect mechanisms such as MDM2/MDMX amplification. In addition, most wild-type p53 types of cancer harbour alternative genetic alterations such as mutations in APC in colon cancer, BRCA1 and BRCA2 in breast cancer, and B-RAF in melanoma. As the p53 network is closely linked to many other cellular pathways, it is likely that defects in any of these pathways could alter p53 function (Soussi and Wiman, 2007).The development of these minimally transformed cells has, for the first time, allowed us to ask whether the capacity to develop drug resistance arises before or after tumorigenic transformation, and what is the minimum number of altered pathways required to permit this event. In a series of progressively transformed embryonic skin fibroblasts, it has been found that the minimum number of genetic transformations necessary for a primary cell to become drug resistant, in addition to hTERT expression, is inactivation of the pathways controlled by p53 and pRb (Yagüe et al, 2007), confirming the pivotal roles of p53 and pRb in deciding cell fate after drug treatment: senescence, apoptosis or drug resistance (Dimri, 2005). Thus, in this cell model, the ability to acquire drug resistance is not, as previously supposed, a late event in tumorigenesis resulting from gross genetic instability, but is intrinsic to the early steps in the tumorigenic pathway necessary for transformation and can arise earlier than the full malignant transformation (Figure 1B).These findings with minimally transformed fibroblasts need to be confirmed and extended to other epithelial cancer cell models and to drugs with different modes of action to generalise their relevance. However, they have opened the possibility to analyse the ultimate controllers in the development of drug resistance.Cancer stem cells and drug resistanceThe cancer stem cell (CSC) hypothesis states that many, if not all, cancers contain a minority population of transformed self-renewing stem cells. These CSCs are responsible for sustaining the tumour as well as giving rise to proliferating but progressively differentiating cells constituting the tumour mass (Burkert et al, 2006; Li and Neaves, 2006). Cancer stem cells retain the essential property of self-protection through the activity of multiple drug resistance transporters such as ABCB1 (P-glycoprotein) and/or ABCG2 (Breast Cancer Resistance Protein-1, BCRP1). The latter is responsible for the side-population (SP) phenotype detected in both normal and acute myeologenous leukaemia (AML) haematopoietic stem cells (Wulf et al, 2001). As Mdr1a/1b−/− (P-glycoprotein-deficient) mice are able to display a normal SP phenotype that disappears when Abcg2 is knocked down (Zhou et al, 2002), expression of ABCG2 and Hoechst 33342 efflux are two of the best markers of these cells. To date, the existence of CSCs has been demonstrated in AML and CML, in brain and gastrointestinal tumours, and in lung and breast cancer (de Jonge-Peeters et al, 2007).A connection between CSCs and drug resistance is thought to exist due to the expression of many of the membrane transporters. In fact, the whole drug resistance concept has been revised incorporating the CSC paradigm (Dean et al, 2005; Donnenberg and Donnenberg, 2005). According to the acquired resistance stem-cell model, CSCs, which express drug transporters, are present in the original tumour mass and survive chemotherapy, whereas the committed but variably differentiated cells are killed. These cells reform a heterogeneous drug resistant tumour composed of CSCs and a committed but variably differentiated offspring. In addition, mutation in the surviving CSCs can arise expanding the drug-resistant phenotype. However, the CSC hypothesis does not account for resistance that develops in certain cancers following chemotherapy in which all cancer cells (not just stem cells) become resistant. Such intrinsic drug resistance is in many cases, such as colon and liver cancer, due to the function of ABC transporters, which are already highly expressed in the healthy tissues.Drug resistance in the clinic and its reversalCell culture systems and animal models have been pivotal in defining the main molecular and cellular mechanisms responsible for the drug-resistance phenotype. With them it has been relatively straight forward to demonstrate that a particular molecule (i.e. P-glycoprotein or p53) is the effector of drug resistance, due to the ease of performing knockout or ectopic expression experiments. However, the situation in the clinic is far more complicated, not only because of the lack of sensitivity and the absence of appropriate detection techniques from clinical samples, but also because the association of a particular drug-resistance effector does not necessarily correlate with an alteration in the chemotherapy response (Cimoli et al, 2004). Despite this, a negative correlation has been unequivocally demonstrated between P-glycoprotein expression and chemotherapy response in AML (Gottesman et al, 2002) and breast cancer (Clarke et al, 2005).Since the discovery of P-glycoprotein in the early 1980s, most agents tested for the reversal of multidrug resistance in the clinic have aimed at inhibiting P-glycoprotein function. The first generation of P-glycoprotein inhibitors included verapamil, quinine and cyclosporine, which were already approved for other medical purposes. Although these compounds proved to be ineffective or toxic at the doses required to attenuate P-glycoprotein function, some clinical trials indicated that modulation of P-glycoprotein function could be achieved (Gottesman et al, 2002). This encouraged the development of a second-generation of modulators, such as the cyclosporine analogue PSC-833 (Valspodar), aimed at avoiding the toxic side effects seen in the first generation. However, the development of second-generation inhibitors has now been discontinued, mainly due to their limited success in clinical trials (PSC-833 induced pharmacological interactions that limited drug clearance and metabolism of the chemotherapeutic agent, thereby elevating plasma concentrations beyond acceptable toxicity). Third-generation inhibitors have been designed for low pharmacokinetic interaction, and inhibition of cytochrome P450 3A has been avoided with compounds such as laniquidar (R101933), oc144-093 (ONT-093), zosuquidar (LY335979), elacridar (GF-120918) and tariquidar (XR9576). A further generation of inhibitors acts on a broader range of ABC transporters. These include biricodar (VX-710) and GF-120918, which modulate not only P-glycoprotein but also MRP1 and ABCG2, respectively. Most clinical trial end points have not been analysed yet; for an extended discussion of P-glycoprotein inhibitors in the clinic see Szakacs et al (2006).There are many different possible reasons for the failure of phase III clinical trial targeting P-glycoprotein. These include multifactorial mechanisms of resistance, toxicity of the inhibitors and unfavourable pharmacological interactions, as well as a poor clinical trial design. The latter is exemplified by the phase III clinical trial using tariquidar as an adjunctive treatment in combination with first-line chemotherapy for patients with non-small cell lung carcinoma, in which there is no strong evidence to suggest that in this type of cancer, P-glycoprotein is expressed to a significant extent (Szakacs et al, 2006). Even AML patients, in which P-glycoprotein expression affects the outcome of chemotherapy, are not routinely phenotyped, and current efforts to develop simple, intercentre reproducible protocols to detect P-glycoprotein in AML blasts have been developed (Pallis et al, 2005). In such a way, trial organisers can have the choice of whether to give P-glycoprotein modulators to an unsorted cohort or to P-glycoprotein-positive patients only.Other alternative approaches to target P-glycoprotein-mediated drug resistance could involve the development of agents to interfere with any one of the regulatory steps in P-glycoprotein expression: transcription, mRNA turnover, translation, protein processing and turnover. Of these, the only one under trial in soft tissue sarcoma (currently phase II) is ecteinascidin 743, a natural product isolated from the marine organism Ecteinascidia turbinate. Ecteinascidin 743 interferes with the activation of ABCB1 via the stress-responsive enhanceosome complex (Le Cesne et al, 2005).Genome-wide expression profiling has been used to study drug resistance, and the foreseeable complexity of their mechanisms has been revealed. In addition to the corroboration of traditionally associated genes, these studies have given new insight into the regulatory networks controlling drug resistance (Turton et al, 2001). The most interesting application of the novel genomic technologies has been in the clinical arena, where molecular signatures have been used not only to characterise neoplastic transformation (Sotiriou et al, 2003) and resistant tumours (Jansen et al, 2005) but also, and most importantly, to predict the outcome of chemotherapy (Alaoui-Jamali et al, 2004).Whether these novel technologies will gain acceptance in the routine diagnosis of cancer will depend greatly on whether their current costs can be reduced (Sotiriou and Piccart, 2007).Conclusions and perspectivesMany years have passed since the first description of cancer as an evolutionary process (Nowell, 1976). With the establishment of the CSC paradigm, new insights into the relative high frequency of cancer in humans and the pitfalls of many cancer treatments have been put forward based on Darwinian selection (Greaves, 2007). Although there are many differences between neoplastic and organismal evolution, the lack of cellular controls to maintain genomic stability, telomere length, repair of DNA damage or cell cycle regulation discussed above set up the conditions for genetic diversity as a source of clonal evolution. Treatment with chemotherapeutic agents promotes an evolutionary arms race exemplified by the resistance to imatinib and the generation of novel derivatives in CML. Studies using cell model systems have shown that chemotherapy resistance is intrinsic to the tumorigenesis process and can even arise before malignant transformation. 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Melo JV, Barnes DJ (2007) Chronic myeloid leukaemia as a model of disease evolution in human cancer. Nat Rev Cancer\n7: 441–45317522713\n29. Nahta R, Yu D, Hung MC, Hortobagyi GN, Esteva FJ (2006) Mechanisms of disease: understanding resistance to HER2-targeted therapy in human breast cancer. Nat Clin Pract Oncol\n3: 269–28016683005\n30. Nimmanapalli R, Bhalla K (2002) Mechanisms of resistance to imatinib mesylate in Bcr-Abl-positive leukemias. Curr Opin Oncol\n14: 616–62012409651\n31. Nowell PC (1976) The clonal evolution of tumor cell populations. Science\n194: 23–28959840\n32. Pallis M, Fisher J, Truran L, Grundy M, Russell N, Burnett A (2005) Reproducible measurements of AML blast p-glycoprotein function in 2 center analyses. Blood\n105: 1367–136815659619\n33. Schiff R, Massarweh SA, Shou J, Bharwani L, Arpino G, Rimawi M, Osborne CK (2005) Advanced concepts in estrogen receptor biology and breast cancer endocrine resistance: implicated role of growth factor signaling and estrogen receptor coregulators. Cancer Chemother Pharmacol\n56(Suppl 1): 10–20\n34. Shah NP, Tran C, Lee FY, Chen P, Norris D, Sawyers CL (2004) Overriding imatinib resistance with a novel ABL kinase inhibitor. Science\n305: 399–40115256671\n35. Sotiriou C, Neo SY, McShane LM, Korn EL, Long PM, Jazaeri A, Martiat P, Fox SB, Harris AL, Liu ET (2003) Breast cancer classification and prognosis based on gene expression profiles from a population-based study. Proc Natl Acad Sci USA\n100: 10393���1039812917485\n36. Sotiriou C, Piccart MJ (2007) Taking gene-expression profiling to the clinic: when will molecular signatures become relevant to patient care? Nat Rev Cancer\n7: 545–55317585334\n37. Soussi T, Wiman KG (2007) Shaping genetic alterations in human cancer: the p53 mutation paradigm. Cancer Cell\n12: 303–31217936556\n38. Szakacs G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM (2006) Targeting multidrug resistance in cancer. Nat Rev Drug Discov\n5: 219–23416518375\n39. Turton NJ, Judah DJ, Riley J, Davies R, Lipson D, Styles JA, Smith AG, Gant TW (2001) Gene expression and amplification in breast carcinoma cells with intrinsic and acquired doxorubicin resistance. Oncogene\n20: 1300–130611313874\n40. Uggla B, Tina E, Nahi H, Paul C, Hoglund M, Sirsjo A, Tidefelt U (2007) Topoisomerase IIalpha mRNA and protein expression vs in vitro drug resistance and clinical outcome in acute leukaemia. Int J Oncol\n31: 153–16017549416\n41. Weinberg OK, Marquez-Garban DC, Pietras RJ (2005) New approaches to reverse resistance to hormonal therapy in human breast cancer. Drug Resist Updat\n8: 219–23316054421\n42. Weisberg E, Manley PW, Cowan-Jacob SW, Hochhaus A, Griffin JD (2007) Second generation inhibitors of BCR-ABL for the treatment of imatinib-resistant chronic myeloid leukaemia. Nat Rev Cancer\n7: 345–35617457302\n43. Wulf GG, Wang RY, Kuehnle I, Weidner D, Marini F, Brenner MK, Andreeff M, Goodell MA (2001) A leukemic stem cell with intrinsic drug efflux capacity in acute myeloid leukemia. Blood\n98: 1166–117311493466\n44. Yagüe E, Arance A, Kubitza L, O'Hare M, Jat P, Ogilvie CM, Hart IR, Higgins CF, Raguz S (2007) Ability to acquire drug resistance arises early during the tumorigenesis process. Cancer Res\n67: 1130–113717283147\n45. Zhou S, Morris JJ, Barnes Y, Lan L, Schuetz JD, Sorrentino BP (2002) Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc Natl Acad Sci USA\n99: 12339–1234412218177"
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+    "text": "This is an academic paper. This paper has corpus identifier PMC2527832\nAUTHORS: J K Field, S W Duffy\n\nABSTRACT:\nTo take lung cancer screening into national programmes, we first have to answer the question whether low-dose computed tomography (LDCT) screening and treatment of early lesions will decrease lung cancer mortality compared with a control group, to accurately estimate the balance of benefits and harms, and to determine the cost-effectiveness of the intervention.\n\nBODY:\nLung cancer kills more people worldwide than other malignancy. The number of deaths in the western world has fallen in the past years and this is likely to be due to a greater public awareness as well as successes in smoking cessation programmes. Unfortunately, the tobacco epidemic is still growing in Southeast Asia and China as the tobacco industry has now concentrated its sales in these regions. However, there is now a large ex-smoking population in the United States and Europe, who remain at a very high risk of developing lung cancer, which is dependent on their smoking duration before tobacco cessation. This group of individuals now exceeds current smokers in both the United States and Europe and will continue to do so over the next two to three decades. National health-care programmes would have a responsibility, if there were a proven screening tool, to provide a mechanism by which these high-risk individuals are identified and targeted for lung cancer screening. Screening must be instigated before patients develop any symptoms, as surgical resection at an early stage of the disease remains the only realistic option for a cure.Chest X-ray and sputum cytology lung cancer screeningThe earliest lung screening trial was undertaken in London with over 55 000 individuals randomised to chest X-ray every 6 months for 3 years or chest X-ray at the beginning and end of the 3-year period (Brett, 1969). No mortality difference was found between the two groups. Three major trials in the United States and one in Czechoslovakia were developed in the 1970s, as outlined in Table 1. The results of these large trials were disappointing as none of these studies showed any reduction in lung cancer mortality utilising chest X-ray, with or without, sputum cytology. However, some design features of these trials have been criticised on the basis of active early detection measures in the control arm in many of the studies, possible suboptimal selection of study populations, and of arguably inadequate sample sizes (Prorok et al, 2000). Many of these criticisms have now been taken on board by the current lung cancer screening trials.One current trial, which has ‘usual care’ only in the control arm, is the lung component of the NCI PLCO (Prostate, Lung Colorectal and Ovarian) screening trial. In this trial, smokers are offered annual chest X-ray for 3 years, and non-smokers two annual repeat screens; the results of this study are not expected until 2010.Low-dose computed tomography lung cancer screening: observational studiesLow-dose computed tomography (LDCT) offers a major advance in imaging technology, which was first introduced in the 1990s (Naidich et al, 1990) and later by Reeves and Kostis (2000). This is more sensitive than chest X-ray and has enabled the detection of lung tumours less than 1 cm; thus, allowing a complete scan on the thorax in less than 30 s. Randomised trials of this technology as a screening tool have not as yet been completed, but there have been a number of demonstration projects (Table 2). Early studies of note include the Early Lung Cancer Action Project (ELCAP) (Henschke et al, 1999) in 1000 high-risk smokers; the Mayo Clinic Project with 1520 individuals aged 50 years having annual sputum cytology and spiral CT screening (Swensen et al, 2000); and a 3-year mass screening programme using a mobile CT unit in Japan (Sone et al, 1998).The ELCAP study enroled 1000 symptom-free individuals aged 60 years or more with >10 pack-years history of smoking, who were fit to undergo surgery into a study. All individuals underwent an annual spiral CT and chest X-ray. The lung cancer detection rate was 2.7% in the first year and 0.7% in the second year (incidence), and this study also demonstrated that the sensitivity of low-dose spiral CT for early lung cancer was far greater than for chest X-rays. The majority of ‘screen-detected’ tumours were at an early stage and suitable for surgery. This seminal paper by Henschke and co-workers (Henschke et al, 1999) re-ignited interest and debate in developing new lung cancer screening trials in the United States and Europe. Other demonstration projects found similar results (Table 2). The Early Lung Cancer Action Project has since been expanded to a major international collaboration, I-ELCAP, with more than 30 000 screenees (see below), with similar findings to the original New York project (Henschke et al, 2006). The authors also estimated a very high case survival rate for stage I tumours undergoing surgery. There is, however, considerable debate around the interpretation of increased survival in LDCT-diagnosed cancers, as longer survival does not necessarily equate to reduced mortality (Twombly, 2007). In addition to concerns about self-selection for surgery (or for no surgery) among stage 1 patients, the major reservation relates to overdiagnosis of tumours, which would not have been life threatening and would never have come to clinical attention in the absence of screening. The previous generation of chest X-ray trials suggested a measure of overdiagnosis (Kubik et al, 2000; Marcus et al, 2000). The much greater sensitivity of LDCT has, in turn, led to fears of an increased risk of overdiagnosis. The most balanced arguments to date concerning the IELCAP findings have been in a recent BMJ editorial (McMahon and Christiani, 2007). The authors' view is that the objective of lung cancer screening is to reduce lung cancer mortality, and it is not possible to confidently conclude this from the IELCAP study.The one other large observational analysis is by Bach et al (2007) whose conclusions were diametrically opposed to those from by the IELCAP Consortium. Bach and colleagues used data from 3246 current or former smokers who entered into screening studies in the United States and in Italy, with follow-up for a median of 3.9 years. They used a model of predicted risk of lung cancer mortality to estimate the expected numbers of lung cancer deaths and compared these with the corresponding observed deaths; they found no decrease in the number of diagnoses of advanced lung cancers or deaths from lung cancer (38 deaths due to lung cancer observed and 38.8 expected; RR 1.0; 95% CI: 0.7–1.3; P=0.90). The authors concluded that there was no evidence of a mortality advantage with LDCT screening from this study. However, their exclusion of deaths from tumours diagnosed early in the period of observation has been criticised, as have been various other assumptions and procedures in their approach.LDCT lung cancer screening: randomised trialsThe EU-US spiral CT Collaboration was initiated in 2001 in Liverpool. Subsequent meetings throughout Europe resulted in the development of collaborative protocols on radiology, pathology, minimum datasets, treatment, as well as core LDCT protocol. This provided a mechanism by which the different trial groups could work together with the ultimate aim to pool their data, thereby enhancing the overall power of these studies and potentially reporting earlier; the concept of which was formulated in the ‘Liverpool Statement 2005’ (Field et al, 2006).The randomised trials of LDCT are summarised in Table 3. The first major RCT lung cancer screening trial utilising LDCT was the National Lung cancer Screening Trial (NLST), which is a combination of two trials, one set up by the US National Cancer Institute (NCI) and the other by the American College of Radiology Imaging Network (ACRIN). The NLST started in 2002 and completed enroling in 2004. This study has over 50 000 former and current smokers randomised to annual LDCT or annual chest X-ray for 3 years. The major objective of this was to determine whether LDCT reduces lung cancer mortality compared with a chest X-ray arm. (http://www.cancer.gov/NLST). This trial will be completed in 2009 and aims to report in 2012; it is designed to have a 90% power to detect a mortality reduction of 20%.The NELSON trial was launched in 2003 in the Netherlands and Belgium (van Iersel et al, 2007), and now incorporates centres in Denmark. This trial is designed to compare lung cancer mortality in a group randomised to LDCT screening with a control group, without screening. This trial aims to report in 2014 and with 20 000 recruits and is designed to have a power of 80%, significance level of 0.05 to detect a mortality reduction of 20%; a 95% compliance in the screen group, a 5% contamination rate in the control group and 10 years follow-up after randomisation. A great deal of attention was focused on the selection of patients for NELSON in order to focus on the highest risk groups and thus reduce the cost of the RCT but retain the power of the study. Potential study participants were approached by letter with a questionnaire on their smoking exposure and whether they wished to be included in the trial. The questionnaire was initially sent to 335 441 men and women aged 50–75 years old. On the basis of this data set the selection criteria were developed, depending on the duration of smoking, duration of smoking cessation in ex-smokers, number of cigarettes smoked per day, and the mean estimated expected lung cancer mortality rate. In this trial, LDCT screening takes place in years 1, 2, and 4, with 10 years of follow-up. The trial has 20 000 individuals randomised in equal numbers to LDCT or ‘usual care’.A number of small trials have been initiated in anticipation of combination with partner studies or a future meta-analysis. These include the ItaLung and Dante Trials in Italy (Picozzi et al, 2005; Infante et al, 2007).The French randomised pilot study, Depiscan, comparing LDCT and chest X-ray recently reported its baseline findings (Blanchon et al, 2007); in this the selection of participants was undertaken by General Practioners (GPs) and occupational physicians. Eligible subjects were males and females aged 50–75 years with either a current or former smoking history of at least 15 cigarettes per day for 20 years. The screening was undertaken annually for 2 years. The objective was to enrol 1000 subjects; 765 have been recruited with 621of these having complete imaging baseline data. Non-compliance was an important issue in this study and the recruitment took twice as long as envisaged. Eight lung cancers were detected in the LDCT arm (2.4%) and one (<1%) in the chest X-ray arm.National lung cancer screening programmeTo date, we do not have the results of any randomised trials, which can provide adequate evidence to justify the instigation of a National Lung Cancer Screening Programme. The results of the NLST and NELSON studies are eagerly awaited. The unanswered question that remains in the United Kingdom is whether either of these studies will provide adequate information on their own to justify the implementation of a UK National Screening Programme? Although the combined US study is large and should have precise results, the use of an active screening regime in the control group may raise problems of interpretation. The NELSON study has adequate power for a substantial benefit in a high-risk group, but a lower baseline lung cancer mortality or smaller benefit than anticipated may jeopardise a conclusive result.The UK National Screening Committee has determined 22 criteria for the viability, effectiveness, and appropriateness of a screening programme (http://www.nsc.nhs.uk/uk_nsc/uk_nsc_ind.htm); 20 of which are relevant to LDCT lung cancer screening. Black et al (2007) have undertaken a systematic review of the literature to ascertain whether there was evidence for any clinical effectiveness utilising LDCT for lung cancer screening. This extremely detailed review was undertaken at the time when there was a paucity of real data, and thus their conclusions were drawn from two small trials with very variable results. Not surprisingly, their conclusion stated that there was insufficient evidence at the time to support LDCT screening.The current lack of evidence and the possibility of inconclusive results from relatively small group of current trials would suggest that a UK trial would make a valuable contribution to the research effort worldwide and answer questions particularly pertinent to the UK health environment. It is a salutary fact that four decades after the development of this ‘technology’, we still do not have experimental evidence for or against the implementation of this screening modality. Lung cancer kills more individuals in the United Kingdom than any other malignancy. Our responsibility is not only to determine whether LDCT screening and treatment of early lesions will decrease lung cancer mortality compared with a control group without screening but also to test this against the criteria outlined by the UK Screening Committee, especially those concerning cost-effectiveness. A useful aid to cost-effectiveness is the ability to select a population at sufficiently high risk to give a substantial harvest of tumours in return for the screening activity. The Liverpool Lung Project Risk Model provides an opportunity for this (Field et al, 2007; Cassidy et al, 2008). The risk groups selected are those for whom the benefits of the screening will outweigh the likely harms.The cost-effectiveness of lung cancer LDCT screening has been estimated by a number of groups, which were reviewed by Black et al (2006), who found the current estimates difficult to interpret and certainly not definitive. In response to a request from the UK National Cancer Research Institute, Whynes (2008) developed a simple and transparent economic model based on UK costings and the empirical clinical data are currently available. The UK cost-effectiveness model used a simple, deterministic approach to the modelling of a screening regimen. The model required only a limited number of parameters. The expected mortality gain as a result of screening was estimated by combining published survival data from screened and unscreened cohorts with routinely published national mortality figures. Conservative costs were estimated where there was uncertainty over any specific parameter, thus probably resulting in less cost-effective screening. The incremental cost-effectiveness ratio of a single CT screen among a high-risk male population was calculated to be around £14 000 per quality-adjusted life year gained, if the anticipated mortality benefit was indeed observed. Sensitivity analysis was carried out with a range of differing assumptions, providing a range of cost-effectiveness ratios as high as £21 000 or as low as around £6000. In the United Kingdom, the National Institute and Clinical Excellence (NICE) evaluated both clinical and cost-effectiveness when deciding on recommendations to implement new interventions. Currently, NICE considered ICERs below £20 000 per QUALY as definitely acceptable and costs up to £30 000 as suitable for consideration.The approval of any future lung cancer screening trial will evidently be dependent on costings in line with current political health economics; however, this defining factor was not applicable for either breast cancer screening, which was set up after the Forest Report in 1985 (Gerard et al, 1997), or cervical cancer screening, which was set up in 1992 (Quinn et al, 1999). The most efficient way of controlling cost, however, will be to screen those individuals who are at high risk of developing the disease. There has been increasing interest in developing methods for individual risk prediction for lung cancer. Models have been developed for use within high-risk groups (Bach et al, 2003), and for the general population (van Klaveren et al, 2002), which rely only on age and smoking. Epidemiological risk factors usually show poor discrimination between those ‘who do’ and ‘do not’ develop disease (Wald et al, 1999), but lung cancer is an exception, in that a high proportion of cases are attributable to one risk factor, smoking. The predictive accuracy of lung cancer risk models may be further improved by the addition of other epidemiological risk factors, including smoking history variables, environmental tobacco smoke, family history of cancer, prior respiratory disease, and occupational exposures (dust and asbestos) (Cassidy et al, 2007, 2008; Spitz et al, 2007). The Liverpool Lung Project (LLP) (Field et al, 2005) has recently developed a method to calculate absolute risk of lung cancer over a defined period, based on data from a case–control study of lung cancer in Liverpool. Significant risk factors in the final model were smoking duration, family history of lung cancer, history of non-pulmonary malignant tumour, history of pneumonia, and occupational exposure to asbestos. These factors were combined with published age- and sex-specific incidence rates to give absolute probability of lung cancer development within 5 years. In comparison with previous lung cancer prediction models, the LLP risk model has distinctive strengths. First, the predictor variables are all explicitly defined and can be readily assessed at the time of patient presentation, and secondly, patients can be assigned to their appropriate risk class on the basis of information from the initial history alone. The LLP Risk Model requires rigorous validation in a separate population.ConclusionCurrently, the treatment of advanced lung cancer is inadequate and, thus, there is an urgent pressure to implement screening programmes in many countries. In the United Kingdom, no decision will be made until we have the results of the current international RCT trials and, hopefully, those from a future UK lung cancer screening RCT. However, time is not on our side with over 32 000 individuals a year dying from lung cancer in the United Kingdom, and this statistic alone should accelerate progress in reaching a conclusion concerning the feasibility of lung cancer screening.\n\nREFERENCES:\n1. Bach PB, Jett JR, Pastorino U, Tockman MS, Swensen SJ, Begg CB (2007) Computed tomography screening and lung cancer outcomes. JAMA\n297: 953–96117341709\n2. Bach PB, Kattan MW, Thornquist MD, Kris MG, Tate RC, Barnett MJ, Hsieh LJ, Begg CB (2003) Variations in lung cancer risk among smokers. J Natl Cancer Inst\n95: 470–47812644540\n3. Black C, Bagust A, Boland A, Walker S, McLeod C, De Verteuil R, Ayres J, Bain L, Thomas S, Godden D, Waugh N (2006) The clinical effectiveness and cost-effectiveness of computed tomography screening for lung cancer: systematic reviews. Health Technol Assess\n10(3): iii–iv, ix–x, 1–90\n4. Black C, de Verteuil R, Walker S, Ayres J, Boland A, Bagust A, Waugh N (2007) Population screening for lung cancer using computed tomography, is there evidence of clinical effectiveness? A systematic review of the literature. Thorax\n62: 131–13817287305\n5. 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Sobue T, Moriyama N, Kaneko M, Kusumoto M, Kobayashi T, Tsuchiya R, Kakinuma R, Ohmatsu H, Nagai K, Nishiyama H, Matsui E, Eguchi K (2002) Screening for lung cancer with low-dose helical computed tomography: anti-lung cancer association project. J Clin Oncol\n20: 911–92011844811\n37. Sone S, Takashima S, Li F, Yang Z, Honda T, Maruyama Y, Hasegawa M, Yamanda T, Kubo K, Hanamura K, Asakura K (1998) Mass screening for lung cancer with mobile spiral computed tomography scanner. Lancet\n351: 1242–12459643744\n38. Spitz MR, Hong WK, Amos CI, Wu X, Schabath MB, Dong Q, Shete S, Etzel CJ (2007) A risk model for prediction of lung cancer. J Natl Cancer Inst\n99: 715–72617470739\n39. Stephenson SM, Mech KF, Sardi A (2005) Lung cancer screening with low-dose spiral computed tomography. Am Surg\n71: 1015–101716447470\n40. 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J Natl Cancer Inst\n99: 190–19517284710\n44. van Iersel CA, de Koning HJ, Draisma G, Mali WP, Scholten ET, Nackaerts K, Prokop M, Habbema JD, Oudkerk M, van Klaveren RJ (2007) Risk-based selection from the general population in a screening trial: selection criteria, recruitment and power for the Dutch-Belgian randomised lung cancer multi-slice CT screening trial (NELSON). Int J Cancer\n120: 868–87417131307\n45. van Klaveren RJ, de Koning HJ, Mulshine J, Hirsch FR (2002) Lung cancer screening by spiral CT. What is the optimal target population for screening trials? Lung Cancer\n38: 243–25212445745\n46. Wald NJ, Hackshaw AK, Frost CD (1999) When can a risk factor be used as a worthwhile screening test? BMJ\n319: 1562–156510591726\n47. Whynes DK (2008) Could CT screening for lung cancer ever be cost effective in the United Kingdom? Cost Eff Resour Alloc\n6: 518302756\n48. Yau G, Lock M, Rodrigues G (2007) Systematic review of baseline low-dose CT lung cancer screening. Lung Cancer\n58: 161–17017723250"
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+    "id": "PMC2528012",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528012\nAUTHORS: John A Cunningham, Clayton Neighbors, Cameron Wild, Keith Humphreys\n\nABSTRACT:\nBackgroundHelping the large number of problem drinkers who will never seek treatment is a challenging issue. Public health initiatives employing educational materials or mass media campaigns have met with mixed success. However, clinical research has developed effective brief interventions to help problem drinkers. This project will employ an intervention that has been validated in clinical settings and then modified into an ultra-brief format suitable for use as a public health intervention. The major objective of this study is to conduct a randomized controlled trial to establish the effectiveness of an ultra-brief, personalized feedback intervention for problem drinkers.Methods/designProblem drinkers recruited on a baseline population telephone survey conducted in a major metropolitan city in Canada will be randomized to one of three conditions – a personalized feedback pamphlet condition, a control pamphlet condition, or a no intervention control condition. In the week after the baseline survey, households in the two pamphlet conditions will be sent their respective pamphlets. Changes in drinking will be assessed post intervention at three-month and six-month follow-ups. Drinking outcomes will be compared between experimental conditions using Structural Equation Modeling. The primary hypothesis is that problem drinkers from households who receive the personalized feedback pamphlet intervention will display significantly improved drinking outcomes at three and six-month follow-ups as compared to problem drinkers from households in the no intervention control condition. Secondary hypotheses will test the impact of the intervention on help seeking, and explore the mediating or moderating role of perceived drinking norms, perceived alcohol risks and the problem drinker's social reasons for drinking.DiscussionThis trial will provide information on the effectiveness of a pamphlet-based personalized feedback intervention for problem drinkers in a community setting.Trial registrationClinicalTrials.gov registration #NCT00688584.\n\nBODY:\nBackgroundThe ratio of problem drinkers to those seriously dependent on alcohol is about 4:1 [1]. As Cahalan [2] has noted, \"clinically defined alcoholics constitute only a relatively small proportion of those whose drinking creates significant problems for themselves and society\" (p. 363). The majority of these problem drinkers, while being at risk for health problems and other psychosocial consequences [3], will never access any treatment services. (estimated ratio of treated to untreated problem drinkers ranges from 1:3 to 1:14 in Canada and the United States) [4-9]. Many individuals with drinking problems do not approach alcohol treatment facilities, often because of stigma, embarrassment or because they don't think of their drinking as a problem [5,10-12]. Further, when asked why they have not sought formal help or treatment, problem drinkers overwhelmingly indicate that they \"want to change on their own\" [10,13]. The present study proposes to test the effects of a self-change intervention that circumvents some of the barriers of traditional treatment by allowing problem drinkers to work on their alcohol problems on their own in private without approaching alcohol treatment facilities.In 1990, the Institute of Medicine [1] recommended a broadening of the range of services for people with alcohol problems. Providing self-help interventions to individuals who have not accessed treatment services is one way to accomplish this important public health goal and ensure that a broader range of services is available. Although such interventions might only have a limited impact on an individual level, they can still be beneficial as part of a continuum of care for individuals unlikely to enter formal alcohol treatment programs [1,14,15]. Further, when viewed from a public health perspective [16], minimal interventions have the potential for a significant population-level impact as such interventions are low cost and can be provided to a large number of drinkers. Public health impact is typically conceptualized as the reach of the intervention X efficacy per unit cost [17]. The low cost and potential for broad reach of self-help interventions, coupled with the high base rate of the behavior suggests that such interventions can have a significant public health impact.Public Health 'Educational' Initiatives to Reduce Problem DrinkingAs problem drinking is one of the five leading contributors to the global burden of disease [18], there is considerable need to address the impact of drinking from a public health perspective. In their authoritative review of public health initiatives for problem drinking, Babor and colleagues [19] concluded that policy initiatives such as taxation, limiting access, and drinking and driving laws have the best research base for demonstrating an impact on reducing alcohol consumption. Educational initiatives were judged to be ineffective. Public education initiatives are distinct from school education programs and cover the domain of public marketing campaigns such as responsible drinking advertisements, banner advertisements and other media initiatives. Babor and colleagues concluded that there was no evidence for such public educational initiatives having a measurable impact on drinking. Other reviews on this topic have been less negative [20], although none make the claim that such campaigns can lead to reductions in alcohol consumption. Rather, the role of these educational campaigns is to cause changes in attitudes towards drinking and to provide public support for control initiatives, such as taxation and drinking-driving laws, so that these control initiatives can cause reductions in alcohol consumption.Is the conclusion that public health educational initiatives cannot directly cause reductions in alcohol consumption a definitive one? We do not believe so. Our argument is that educational initiatives would benefit from looking at the brief intervention literature. As will be outlined below, there is substantial evidence that brief alcohol interventions can have a significant impact on problem drinking [21,22]. The difficulty, from a public health perspective, is how to deliver these efficacious interventions to a large enough group of problem drinkers in order to have a measurable impact on the population level of alcohol consumption. This high level of impact has been demonstrated within 'special' populations, such as college drinkers [23]. However, the challenge becomes greater when we consider ways to impact on the general population of problem drinkers in entire countries such as Canada and the United States because most problem drinkers will never access any type of treatment for their drinking [8,9]. One approach has been to promote the use of brief interventions by medical professionals in general practice settings [24,25]. Unfortunately, many drinkers in the general population may never receive a preventive alcohol intervention in the context of primary health care [26,27]. What other options for intervention exist? There is substantial effort underway to establish and evaluate interventions situated on the Internet [28]. The Internet has the potential for wide spread impact because a growing number of people, including problem drinkers, will access health related information [29]. However, although the potential is there, not all problem drinkers will actively seek out interventions on the Internet [30,31]. We argue that there is advantage to creating a range of different, research validated interventions that have the potential for population level impact. Brief interventions by health professionals are one possible avenue. The Internet is another. The current study seeks to evaluate a third, an ultra-brief intervention in the form a self-test personalized feedback pamphlet. By diversifying our options for helping problem drinkers in the general population, we have the potential of being able to impact the prevalence of alcohol problems. This is a worthy public health goal.Efficacy Trials of Personalized Alcohol Feedback Paper and Pencil Self-Change InterventionsPersonalized alcohol feedback interventions are designed to increase motivation for behavior change [32,33]. Such materials provide normative feedback to individuals – providing a personalized summary of an individual's drinking and comparing it to the consumption of the average male or female in the general population. Normative feedback is theorized to promote change in alcohol use because many heavy drinkers overestimate the consumption of others. Consequently, normative feedback acts as a powerful source of social comparison, motivating heavy drinkers to re-evaluate their consumption patterns [34].Personalized alcohol feedback has been found to promote behavior change in drinkers [32,35-45]. In the college student literature brief interventions utilizing relevant personalized feedback, as opposed to more general social norms campaigns, have consistently been found efficacious whether delivered in-person, by computer, or mail [23,46]. In particular, mailed feedback, similar to the intervention in this study has been found effective in reducing drinking and preventing onset and escalation of drinking in college students [32,35-37,45,47]. Analogous mailed feedback has also been effective in reducing symptoms of depression [48]. A recent meta-analysis by Carey and colleagues [49] also identified normative feedback as an effective intervention to reduce problem drinking in college students.Personalized alcohol feedback interventions are particularly well suited for pamphlet-based delivery. This is because such interventions can be translated into a simple, self-test format. The other advantage of personalized feedback interventions is their brevity – both as far as the assessment required and in the time required to complete them. Such features are important as pamphlets sent directly to households have to attract the readers' attention quickly and be easy to complete. Neighbors and colleagues [44] have also demonstrated that even the most minimal of normative interventions can have an impact on drinking at a six-month follow-up among college students, lending confidence to the possibility of creating an effective, ultra-brief personalized feedback pamphlet. Further, a recent study conducted by this research group has demonstrated an impact on drinking by our ultra-brief, personalized feedback pamphlet at a six-month follow-up, providing evidence for a sustained effect of this approach (study described in more detail in the Preliminary Studies section of this application) [38].Do personalized alcohol feedback interventions work because they modify perceived drinking norms?Several researchers have applied self-regulation theory [50] as a model to help explain the impact of personalized feedback interventions [32,51,52]. By overestimating the prevalence of heavy consumption among peers, heavy drinkers are thought to view their own behavior as normative rather than abnormal or inappropriate. Personalized normative feedback is theorized to develop discrepancy in the recipient by providing information showing that their own drinking is not normative [41,45,53]. Alerting heavy drinkers to the fact that their own drinking is abnormal (i.e., developing discrepancy) is theorized to result in problem recognition and may instigate behavior change [44,51,54]. If this process does mediate the impact of normative feedback interventions then, for the intervention to work, it must modify recipients' perceptions of how much others actually do drink. The modification of these perceptions would then lead to an increase in perceptions of discrepancy between their own and others' drinking. Because of the importance of understanding why normative feedback interventions work, the current study will include perceived drinking norms as a hypothesized mediator of the impact of the personalized feedback pamphlet intervention.Does Perceived Risk Mediate the Effects of Safe Drinking Interventions?The motivational impact of perceived risk is clearly shown by considering four cognitive theories of health protective behavior. In particular, the health belief model [55], protection motivation theory [56], the theory of reasoned action [57], and subjective expected utility theory [58] are all identical insofar as they each assume \"that anticipation of a negative health outcome and the desire to avoid this outcome or reduce its impact creates motivation for self-protection\" [p. 234, [59]]. We have also conducted correlational research exploring why some drinkers perceive risk associated with their drinking while others do not. These studies concluded that there is a need to consider perceived risk as well as objective problem status when designing and evaluating interventions to help heavy drinkers [60]. One of the studies described in the Preliminary Studies section below also indicates the mediating role that perceived risk may play in drinkers' reactions to safe drinking interventions [39].Why might perceived risk mediate the impact of personalized feedback interventions? As was discussed earlier, such interventions provide normative feedback to respondents, comparing their drinking to others in the general population. As many heavy drinkers overestimate how much others drink, this normative information often comes as a surprise to them, allowing them to make the social comparison that they drink more than others [34]. According to cognitive theories of health protective behavior, this information leads to changes in health behavior because it results in an increase in respondents' perceived vulnerability. The recipient recognizes that he or she is engaged in a risky health behavior (i.e., drinking more than others). This increase in perceived risk then motivates respondents to reduce their drinking. The current study will test whether changes in perceived risk mediate reductions in drinking. Although not necessarily contradictory, the mediator hypotheses of perceived drinking norms and of perceived risk come from different theories of change. This study will measure both these constructs and evaluate their validity as potential mediators of the impact of personalized feedback interventions. Secondary analyses will also explore the possible interrelationship between perceived risk and drinking norms as hypothesized mediators of the impact of the ultra-brief intervention.One alternative explanation of the role of perceived risk is that it may also act as a modifier of respondents' reactions to safe drinking interventions. That is, problem drinkers may vary in their perceived risk about drinking at baseline, before they receive the intervention. Those problem drinkers who perceive some risk with regard to their drinking may utilize the intervention and reduce their drinking. Those problem drinkers who perceive no risk associated with their drinking may disregard the intervention materials and thus display no reduction in drinking.Is normative feedback more important for problem drinkers who drink for social reasons?Using the theoretical underpinnings of expectancy research [61,62] and the concept of social drinking motives [63], Neighbors and colleagues [44] suggested that the fact that people vary in the extent to which they drink for social reasons might have matching implications for normative feedback interventions. By social reasons, Neighbors and colleagues (2004) were referring to drinking because of anticipated social positive reinforcement. Normative feedback may be more important for people who drink primarily for social reasons as compared to those who don't because people who drink for social reasons may anticipate more social benefits from their drinking. Neighbors et al. [44] found evidence for this moderating effect on the impact of normative feedback information in a sample of college students. The present study will explore the moderating role of social reasons for drinking in order to identify those who might benefit most from pamphlet-based personalized feedback interventions.Preliminary StudiesThere are two previous studies that have employed this same personalized feedback pamphlet.Preliminary Study # 1 – Pamphlet Personalized Alcohol Feedback TrialThis pilot study demonstrated the potential of the ultra-brief intervention for problem drinkers to be used in the present study [39]. The \"Evaluate Your Drinking\" pamphlet contains a self-test that allows the reader to compare his or her personal drinking to that of other Canadians (see Methods section for a more detailed description of this pamphlet).The pamphlet was sent by unaddressed ad mail to households, randomized by block to receive or not receive the brief intervention. A random digit dialing telephone survey was conducted in the following month to assess differences in drinking between experimental conditions. The primary result was a 3-way interaction between pamphlet condition, perceived risk, and problem drinking status. The pattern of means indicated that the manner in which perceived risk mediated the impact of pamphlet conditions differed, depending on whether respondents met objective criteria for problem drinking. For non-problem drinkers there was no significant difference between intervention conditions, irrespective of whether the person perceived his or her drinking to be of some or no risk. However, among problem drinkers who perceived no risk, there was a trend for those who received the intervention to drink more as compared to those who did not receive the intervention (p < .06). In contrast, among problem drinkers who perceived some risk, respondents who received the pamphlet were drinking less than those who did not (p < .05).The results of this trial indicated that perceived risk might act as either a mediator or a moderator of respondents' reactions to safe drinking interventions. This finding is important to confirm because it points to one of the factors that may differentiate those who continue problem drinking from those who reduce their drinking. However, the post-test only design employed in this research did not allow causal statements to be made regarding the effects of respondents' perceived risk because this variable, along with all others, was assessed after the intervention was administered. Thus, there was no way of knowing whether those with greater perceived risk about alcohol consumption at the one-month follow-up experienced an increase in their perceived risk after receiving the intervention or had higher perceived risk scores already at baseline. To address this issue, the current study will conduct an appropriate evaluation of the role of perceived risk, assessing it at baseline as well as at the three-month follow-up, and then evaluating the potential mediating or moderating effect of perceived risk on problem drinkers' drinking outcomes at six-month follow-up.It is also important to conduct an appropriate evaluation of the role of perceived risk because of the possibility indicated in this pilot study that respondents with no perceived risk might react negatively to receiving the pamphlet. This post-test only pilot study can only be taken as the most preliminary of evidence that those respondents without any perceived vulnerability with respect to their drinking might react negatively to the pamphlet by drinking more. However, we feel that the possible 'boomerang' effect of this ultra-brief intervention among problem drinkers with no perceived risk is an important one to investigate. This is because confirmation of this pattern of results would indicate the need to target interventions, such as this normative feedback pamphlet, to problem drinkers who are concerned about their drinking. Thus, we feel that the potential benefits of conducting this test outweigh the ethical risks associated with conducting a trial where there is the potential of a small negative impact among some participants.Preliminary Study # 2 – Replicating the impact of the ultra-brief interventionWild and colleagues conducted a trial that further supported the ultra-brief pamphlet intervention [38]. Drinkers included in a general population telephone survey, who indicated that they were hypothetically interested in receiving self-help materials, were recruited through a general population telephone survey (n = 1720). Respondents who agreed to a six-month follow-up were randomly assigned to receive or not receive the same pamphlet intervention that was used in Study # 1. Residualized change score analysis found that, among respondents who met criteria for problem drinking at baseline, the intervention group showed a 10% reduction in per-occasion binge drinking (i.e., consuming 5 or more standard drinks per occasion), compared to controls (p < .01). Notably, we observed no iatrogenic effects of providing the pamphlet to drinkers who did not meet criteria for hazardous drinking at baseline, i.e., no escalation of drinking among no-problem drinkers after receiving the ultra-brief intervention.Although the results of this study are encouraging, only respondents who were interested in receiving self-help materials were recruited for the intervention. For the present study, we intend to evaluate the impact of the ultra-brief pamphlet intervention, whether the respondent is specifically interested in receiving self-help materials or not (note: while still maintaining fully informed consent). This is a challenging undertaking but we feel that such a goal is essential if we are to evaluate the effectiveness of the intervention within the same setting that interventions, such as the one to be used in the present study, would be employed in real life. It should also be noted that only recruiting respondents who say they are interested in self-help materials has the additional limitation of excluding many respondents who perceive no risk associated with their drinking. In Preliminary Study # 1, 76% of the respondents who perceived no risk associated with their drinking also said they were not interested in self-help materials. Thus, we must employ a design that recruits all problem drinking respondents, whether interested in self-help materials or not, in order to fully explore the public health impact of this intervention approach, and to conduct an adequate test of the potential role of perceived vulnerability as a mediator and a moderator of the intervention effects. One final limitation of the Wild et al. study was that there is no way to tell whether the observed impact on drinking was due to the content of the personalized feedback pamphlet or simply because the respondent received any alcohol-related pamphlet at all. Thus, the current study incorporates a second control condition in which the households of a randomized third of the participants will receive a popular educational pamphlet on alcohol that contains no personalized feedback content.What is the principal research objective?The principal research objective is to evaluate the efficacy of a pamphlet-based self-help intervention among problem drinkers in the general population. The development of an effective, research-based pamphlet of this type is important because of its potential for use in public health initiatives where low-cost and wide distribution are key considerations. In order to mimic the use of a personalized feedback pamphlet in a public health initiative, the pamphlet will be sent unaddressed to households rather than to specific individuals. In addition, because it is important to determine whether it is the content of the personalized feedback pamphlet or just the receipt of any pamphlet that leads to reductions in drinking, a control pamphlet condition will be included in the study.Methods/designAimThe proposed research will evaluate the efficacy of a pamphlet-based personalized feedback intervention for problem drinkers in the general population.The hypotheses regarding the efficacy of the pamphlet-based intervention are:Hypothesis 1: Respondents from households who receive the personalized feedback pamphlet-based intervention will display significantly improved drinking outcomes at three and six-month follow-ups as compared to respondents from households in the no intervention control condition.Hypothesis 2: Respondents from households who receive the personalized feedback pamphlet-based intervention will display significantly improved drinking outcomes as compared to respondents from households who receive the control pamphlet.In addition, two mediator hypotheses will be tested:Hypothesis 3: Respondents who receive the intervention and reduce their estimates about how much others drink between baseline and three-month follow-up will display significantly improved drinking outcomes at six-month follow-up as compared to respondents who receive the intervention but experience no decrease in their perceived drinking norms.Hypothesis 4: Respondents who receive the intervention and experience an increase in their perceived vulnerability to experience harm because of their alcohol consumption between baseline and three-month follow-up will display significantly improved drinking outcomes at six-month follow-up as compared to respondents who receive the intervention but experience no increase in their perceived risk.Finally, two moderator hypotheses will be tested:Hypothesis 5: The impact of the intervention will be greater among drinkers who believe that they are personally vulnerable to negative outcomes at baseline assessment, compared to drinkers who do not believe that they are personally 'at risk' before receiving the intervention.Hypothesis 6: Respondents who drink for social reasons will be more likely to reduce their drinking as a result of the intervention as compared to respondents who do not drink for social reasons.DesignIn an initial telephone interview, sociodemographic information will be collected. Current drinkers will complete a standardized epidemiological assessment of problem drinking and alcohol consumption, and will answer items to assess their perceived drinking norms, their perceived risk regarding drinking and their social reasons for drinking. Respondents identified as problem drinkers will be asked if they are willing to participate in a three-month and a six-month follow-up. Respondents will be offered a $20 honorarium for completion of each of the three-month and six-month follow-ups. Verbal informed consent will be gathered and respondents will be told that some households will be receiving a pamphlet. Problem drinking will be defined as a score of eight or more on the Alcohol Use Disorders Identification Test (AUDIT) [64,65]. The initial telephone survey will be conducted with a random sample of respondents from households within a major metropolitan city in Canada. One current drinker 19 years or older (legal drinking age in Ontario, Canada) will be selected to participate from each household by soliciting participation from the adult in the household who had the most recent birthday who also drinks alcohol at least once per month. All households with respondents agreeing to participate in the follow-up interviews will be randomized into three groups – personalized feedback pamphlet condition, control pamphlet condition and no intervention control condition. In the week after the baseline survey, all households that contain respondents in the two pamphlet conditions will be sent their respective pamphlets. Three months after the intervention mailing, respondents who agree to the follow-ups will be administered a second telephone interview assessing the same drinking and mediator terms as the baseline survey (modified to refer to the past three months). Similar drinking outcome measures will be made six months after the intervention mailing with the items framed to refer to the past three-months. In order to ensure that all study participants receive some form of intervention, households in the no intervention control condition will be sent the personalized feedback intervention pamphlet after the six-month follow-up.Rationale for the choice of study designWhy do we believe that the proposed study design is the best for testing the impact of this personalized feedback pamphlet? The intent of this project is to evaluate an ultra-brief intervention for problem drinkers that can be used in public health initiatives. As such, it is important that the intervention pamphlet be evaluated in a setting that mimics how it will be used. Thus, the pamphlets will be sent addressed to the household rather than addressed to the respondents because public health initiatives are often non-specific in their target recipients. The proposed research design has the advantage of allowing the pamphlet to be sent to all households in the intervention condition, irrespective of whether any of the respondents are specifically interested in receiving self-help materials. In order to conduct a study where intervention materials are sent directly to the person by name, for ethical reasons the respondent must at least state that he or she is hypothetically interested in receiving such materials. A study in which materials are sent unsolicited to households has been judged ethical because receiving such materials unsolicited (and unaddressed to a specific individual) is not an unusual occurrence given that public health initiatives are ongoing in the Toronto metropolitan area. However, informed consent is given because all respondents are told about the possibility of their household receiving the pamphlet. Further, it is important to be able to send materials to all households because interest in self-help materials covaries with respondents' perceived risk. In the pilot study described above, 76% of problem drinkers who perceived no risk associated with their drinking were not interested in self-help materials. This group comprised 32% of all problem drinkers in the sample. Thus, although the proposed method reduces the power of the intervention because not all respondents will see the pamphlet, it is still the best design for our purposes because it allows recruiting some problem drinkers with no perceived risk regarding their alcohol consumption. Studying the reactions of problem drinkers who do not perceive any risk associated with their drinking to intervention materials is of great importance because results from the pilot study for this proposal (Study 1 in the preliminary studies section) indicated that this group could react to the intervention materials by drinking more. Although the results of Study 1 were not conclusive because of its post-test only design and the fact that this 'boomerang' effect in reaction to the materials was a trend rather than a statistically significant result, we feel that the study of these types of unintentional effects is essential. The personalized feedback pamphlet is intended for wide distribution in the general public. If we confirm that this ultra-brief intervention can have a negative impact on some sub-groups of problem drinkers then this finding would speak to the need to target the intervention to only those drinkers who already voice some concern regarding their drinking. As there is insufficient evidence to-date that could confirm or refute this hypothesis, we feel that the need to test this hypothesis appropriately outweighs the potential ethical dilemma of causing a small unintended increase in alcohol consumption among a population of drinkers who are not seeking treatment and who meet criteria for a rather liberal definition of problem drinking (AUDIT score of 8 or more).Informed Consent ProcedureThe study was approved by the standing ethics review committee of the Centre for Addiction and Mental Health. Telephone calls will be made by trained interviewers from the Institute of Social Research at York University (Toronto). The interviewers will introduce themselves, indicate they are calling from York University and that they are calling on behalf of the Centre for Addiction and Mental Health, which is conducting research on drinking. They will determine the number of monthly drinking adults in the household, randomly select one of them (according to most recent birthday) and before they start the interview they will read the following script:\"I would like to assure you that all information you provide, including your answers, identity, and any other information will remain completely confidential. You do not have to answer any questions you do not want to and if you decide to stop the interview, and wish us to do so, we will destroy all the information you have given us. On average, the interview will take about 15 minutes. Just to let you know, from time to time my supervisor may listen in to make sure we are doing the research correctly. The survey is voluntary, but your participation is very important if the results are to be accurate. Is now a good time to start the interview?\"At the end of the baseline survey, all problem drinkers (AUDIT ≥ 8) will be asked to participate in two, 15 minute surveys, the first in about three months time and the second in six months time. Participants will be offered $20 for the completion of each of the three-month and six-month follow-ups. Potential respondents will be told that these surveys will ask about their current drinking. Further, they will be informed that \"the Centre for Addiction and Mental Health is in the process of mailing a safe-drinking pamphlet to some households in Toronto. I do not know if this pamphlet is being sent to your household, but if you do see it, the six-month follow-up survey will ask about your impressions of the materials.\" Respondents who agree to the follow-up will provide their name, address and telephone number. Because the baseline interview contact is made by telephone, verbal agreement to participate in the study will act as providing informed consent. Respondents will also be informed of the request to provide a collateral on the six-month follow-up at this time.The Personalized Feedback Pamphlet (\"Evaluate Your Drinking\")The \"Evaluate Your Drinking\" self-test pamphlet was modeled after the Drinker's Check-up [32,40,66] and the Fostering Self-Change intervention [33]. The pamphlet starts with an encouragement for the reader to evaluate his or her own drinking. The reader is then asked to record his or her drinking for each day of a typical week and to sum this information to calculate the number of drinks usually consumed per week (a 'standard drinks' chart is provided to help the reader). Next, information on the drinking patterns of males and females in the general Canadian population is provided. The reader is encouraged to compare his or her personal drinking to that of other Canadians and a graph is presented of the likelihood of adverse effects associated with different levels of consumption. The pamphlet concludes with a menu of options and encourages those readers who are concerned about their drinking to take the next step towards changing their alcohol consumption. Incorporated in this menu are low-risk drinking guidelines and a toll-free telephone number for individuals who would like to call to receive a free referral to a local treatment agency. To enhance impact and readability, the pamphlet was professionally produced in a multi-color, glossy format. The pamphlet was modeled after research-validated personalized feedback interventions and provides an easily completed normative feedback self-test for the reader in an attractive and eye-catching format [see Additional file 1].Control Condition Pamphlet (\"Do You Know ... Alcohol\")The control pamphlet called 'Do you know .... Alcohol' is disseminated by the Centre for Addiction and Mental Health (CAMH). This pamphlet provides good quality information about alcohol, consequences of its misuse and safe drinking guidelines (same guidelines as are used in the personalized feedback pamphlet). The \"Do you know\" pamphlet is one of the most popular distributed by CAMH and is a high quality pamphlet that is a good example of educational materials disseminated about alcohol [see Additional file 2].Content of the Baseline Survey(1) The Alcohol Use Disorders Identification Test will be used to measure level of alcohol consumption and severity of alcohol problems (a score of 8 or more on the AUDIT indicates a past 12 month alcohol problem) [64,65]. The AUDIT has been validated for use in telephone surveys [67]. Usual quantity of drinking will be assessed as a continuous variable. An additional item will ask the highest number of drinks the respondent recalled having on any one occasion in the past three months. Finally, respondents' drinking will also be assessed by asking the number of drinks consumed on each day of the last week.(2) Six items assessing whether in the past 12 months alcohol had a harmful effect on respondents' (1) friendships/social life, (2) physical health, (3) outlook on life (happiness), (4) home life or marriage, (5) work, studies, or employment opportunities, or (6) financial position [68].(3) Perceived drinking norms will be measured using a modified version of the Drinking Norms Rating Form [69]. Respondents will be asked how often they think a typical person their age and gender drinks and how often they consume five or more drinks on one occasion (same category response options as AUDIT). Further, respondents will be asked to estimate how much the typical person their age and gender usually drinks on one occasion.(4) Perceived risk for drinking-related problems will be assessed using a six-item scale with questions about perceived vulnerability to harm from the drinkers' own perspectives [70].(5) Following the procedure used by Neighbors [44], social reasons for drinking will be measured using the Social Rewards subscale of the Drinking Motives Questionnaire [63] a five item scale that asks respondents how often they are motivated to drink for positive social outcomes. Also, social outcomes expectancies and subjective evaluations of social effects of alcohol will be assessed using the sociability subscales of the Comprehensive Effects of Alcohol Scale [71].(6) Formal addictions treatment utilization will be measured using a single item from the National Longitudinal Alcohol Epidemiological Survey [72]; \"Have you ever gone anywhere or seen anyone for a reason that was related in any way to your drinking – a physician, counselor, Alcoholics Anonymous, or any other community agency or professional? Include help for combined alcohol and other drug use if alcohol was the major problem for which you sought help.\"(7) A series of demographic characteristics will be assessed: age, gender, ethnicity, marital status, education, and employment status.Problem drinkers (AUDIT score of 8 or more) will be recruited to participate in a three-month and a six-month follow-up telephone survey.Three-month Follow-up SurveyThe three-month follow-up will occur roughly three-months after the intervention pamphlet is sent (or equivalent time for those in the control condition).(1) Drinking in the last three months will be assessed using the same five items employed on the baseline survey, the first four framed for the past three months (frequency of consumption, drinks per occasion, frequency of 5+ consumption, highest number of drinks on one occasion) and the last asking how much the person drank on each day of the last week.(2) Six items assessing whether in the past 3 months alcohol had a harmful effect on respondents' (1) friendships/social life, (2) physical health, (3) outlook on life (happiness), (4) home life or marriage, (5) work, studies, or employment opportunities, or (6) financial position [68].(3) Mediator variables, perceived drinking norms and perceived risk will be measured using the same items as on the baseline survey.(4) Formal addictions treatment utilization will be measured using the same single item as the baseline survey, modified to ask about the last three months.Six-month Follow-up Survey(1) Using the same measures of alcohol consumption as the baseline survey, respondents' drinking over the time since the three-month follow-up will be assessed.(2) The same six items assessing any harmful effects of alcohol in the past 3 months alcohol.(3) Formal addictions treatment utilization will be measured using the same single item as the baseline survey, modified to ask about the last three months. In addition, respondents in the personalized feedback pamphlet condition will be asked if they called the telephone number provided on the pamphlet in order to assess whether the pamphlet stimulates additional help seeking.(4) Knowledge of whether the household received any drinking-related materials will be assessed. Respondents will be asked if their household received either of the two pamphlets that were mailed. If they received one they will be asked which one and whether they read it. Those who received the \"Evaluate Your Drinking\" pamphlet and read it will be asked if the did the self-test contained in the pamphlet.Collateral ConfirmationAt the six-month follow-up respondents will be asked to provide the name of a collateral to confirm their drinking self-reports. Collaterals identified for participation will be mailed a letter explaining that they have been nominated to act as a collateral. Collaterals will then be contacted by telephone to confirm their willingness to talk about the respondent's drinking. The 10-minute telephone interview will cover the respondent's drinking in the past three months and any use of treatment services for alcohol problems during the past three months (mirror items of the respondents' six-month follow-up survey). Collaterals will be offered a $20 honorarium for completing this interview.Data AnalysisPower AnalysesThe power analysis conducted to estimate the sample size required to test the hypotheses of this study used the procedures suggested by Cohen [73] for estimating statistical significance. That is, what is the sample size required to detect an increase in the variance explained due to the inclusion of the personalized feedback pamphlet intervention into the model (Hypothesis 1)? Based on pilot study results, a 1% increase in explained variance can be expected (a small effect size of f = 0.10; note – corresponded to a reduction of two drinks per week in the pilot study). Following the convention that studies should be designed to have a statistical power of at least 80%, and that hypotheses be tested at the .05 level of significance, SamplePower 1.0 [74] was used to estimate the required sample size. These specifications resulted in a final sample (required after attrition) of N = 390 in each condition (N = 1170 total). Given that the inclusion of perceived risk as a moderator resulted in an increase in explained variance of 9% (also in the pilot test), this same sample size would result in a power of better than 99% to test for the mediator and moderator hypotheses (assuming a similar effect size). Results presented by Neighbors and colleagues [44] found similar effect sizes for the mediator hypothesis regarding perceived drinking norms and the moderator hypothesis regarding drinking for social reasons, indicating that the proposed sample size will be adequate to test these hypotheses as well.Although Structural Equation Modeling [75], the analysis method to be used, allows for the sophisticated treatment of missing data, it is still important to assure an adequate number of respondents are followed-up. Based on previous experience [76], it is estimated that 80% of respondents will be followed-up on the six-month follow-up. This means that about 1830 respondents who agree to the follow-up will need to be recruited on the baseline survey in order to account for respondent attrition. We are assuming a worst-case scenario of an 80% follow-up rate on each of the three-month and six-month follow-up interviews but that the 20% lost on each follow-up survey will be different respondents. Thus, to obtain 1170 respondents with complete follow-up data, a follow-up rate of 80% × 80% = 64% is assumed. Given that previous research [77] has indicated that 75% of problem drinkers will agree to be followed-up, the baseline survey will need to include 2440 respondents who are problem drinkers (AUDIT score of 8 or more). As 20% of the baseline sample will be problem drinkers, the full baseline survey will need to screen 12200 respondents who consume alcohol at least once per month (based on previous experience, 20% of monthly alcohol drinkers have an AUDIT score of 8 or more). This will allow a final sample size of 1170 problem drinkers participating in the six-month follow-up. In summary, from 12,200 respondents interviewed at baseline 20% (n = 2440) will be problem drinkers, 75% of whom (n = 1830) will agree to participate in the follow-up interviews, and an 80% follow-up rate for each follow-up survey will give total data for 1170 respondents.Analysis PlanThe analysis plan will follow the description of the use of Structural Equation Modeling (SEM) [75] for the analyses of experimental studies provided by Neighbors et al. [44] and by Russell et al. [78]. SEM with full information maximum likelihood will be used. Effect sizes will be reported [79,80].DiscussionThe primary goal of this project is to evaluate the efficacy of an ultra-brief self-help intervention for non-treatment seeking problem drinkers in the general population. One strength of the proposed study is that it merges population-based methods with a randomized controlled trial. Thus, a general population survey will be employed using a random digit dialing method in order to recruit a good cross-section of problem drinkers from the Toronto population. Respondents will be randomly assigned to condition, allowing for causal inference about any differences observed.The information from the proposed trial may help illuminate effective ways of promoting change among problem drinkers who do not seek formal treatment. If the project finds support for the efficacy of a pamphlet-based feedback intervention, it would provide justification to substantially increase the accessibility of this self-help method for problem drinking. This would be accomplished by sending the pamphlet directly to the public in bulk mailing, to health care settings (treatment centers, hospitals, doctors' offices), and to other social services such as unemployment agencies and welfare departments. As problem drinking is common in all these settings, a research evaluated ultra-brief intervention made freely available to all those in need would help broaden the base of treatment for alcohol problems.AbbreviationsAUDIT: Alcohol Use Disorders Identification Test; CAMH: Centre for Addiction and Mental Health; SEM: Structural Equation Modeling.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsAll authors have made an intellectual contribution to this research protocol. JAC is the Principal Investigator of the project and wrote-up the protocol. All authors have contributed to the drafting process, and all authors have read and approved the final manuscript.Pre-publication historyThe pre-publication history for this paper can be accessed here:Supplementary MaterialAdditional file 1An electronic copy of the intervention pamphlet, Evaluate Your Drinking, to be used in the research trial.Click here for fileAdditional file 2An electronic copy of the control condition pamphlet, Do you know .... Alcohol, to be used in the research trial.Click here for file\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2528015",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528015\nAUTHORS: Satish Kumar, PBSV Padmanabham, Rajasekhara R Ravuri, Kiran Uttaravalli, Padmaja Koneru, P Aditi Mukherjee, B Das, M Kotal, D Xaviour, SY Saheb, VR Rao\n\nABSTRACT:\nBackgroundThe \"out of Africa\" model postulating single \"southern route\" dispersal posits arrival of \"Anatomically Modern Human\" to Indian subcontinent around 66–70 thousand years before present (kyBP). However the contributions and legacy of these earliest settlers in contemporary Indian populations, owing to the complex past population dynamics and later migrations has been an issue of controversy. The high frequency of mitochondrial lineage \"M2\" consistent with its greater age and distribution suggests that it may represent the phylogenetic signature of earliest settlers. Accordingly, we attempted to re-evaluate the impact and contribution of earliest settlers in shaping the genetic diversity and structure of contemporary Indian populations; using our newly sequenced 72 and 4 published complete mitochondrial genomes of this lineage.ResultsThe M2 lineage, harbouring two deep rooting subclades M2a and M2b encompasses approximately one tenth of the mtDNA pool of studied tribes. The phylogeographic spread and diversity indices of M2 and its subclades among the tribes of different geographic regions and linguistic phyla were investigated in detail. Further the reconstructed demographic history of M2 lineage as a surrogate of earliest settlers' component revealed that the demographic events with pronounced regional variations had played pivotal role in shaping the complex net of populations phylogenetic relationship in Indian subcontinent.ConclusionOur results suggest that tribes of southern and eastern region along with Dravidian and Austro-Asiatic speakers of central India are the modern representatives of earliest settlers of subcontinent. The Last Glacial Maximum aridity and post LGM population growth mechanised some sort of homogeneity and redistribution of earliest settlers' component in India. The demic diffusion of agriculture and associated technologies around 3 kyBP, which might have marginalized hunter-gatherer, is coincidental with the decline of earliest settlers' population during this period.\n\nBODY:\nBackgroundThe \"out of Africa\" model postulating a single \"southern route\" dispersal of \"Modern human\" from Horn of Africa to the Persian/Arabian Gulf and further along the tropical coast of the Indian Ocean to southeast Asia and Australasia has largely taken ground in the recent years [1-3]. This most likely involved the exodus of a founding group of several hundred individuals, who might have made the crossing from northeastern Africa, probably over the mouth of the Red Sea some time after the appearance of lineage L3 ~85,000 years ago, followed by a period of mutation and drift during which macrohaplogroups M, N, and R evolved and the ancestral L3 was lost [4]. Subsequently the same three founder macrohaplogroups, with the population expansion most likely occurring on Indian coast [5,6] shows a rapid coastal dispersal from ~66,000 years ago around the Indian Ocean littoral and on to Australasia by ~63,000 years ago [4] resulting in the non overlapping distribution of the derived haplogroups within M and N and its subclade R in south Asia, eastern Asia and Australasia.However, the presence of the diversity of basal clades with in mtDNA macrohaplogroup M in India exceeds that in eastern Eurasia; and numerous so-called M* lineages occur in India but not in east Asia. Whereas estimated age of the M macrohaplogroup in India 54.1 thousand years (ky); [7] on the other hand is considerably low as compared to its east Eurasian counterparts (east Asia 69.3 ± 5.4 ky; Oceania 73.0 ± 7.9 ky; southeast Asia 55.7 ± 7.4 ky) [7,8]. The reason could be the molecular diversity and so as the coalescence age of the Indian M subhaplogroups themselves, which vary substantially as indicated in the studies of Sun et al. [7] and Thangaraj et al. [9].Nested within this model, there could be two plausible scenarios:1. The number of drift events in middle/early upper-Paleolithic populations (earliest settlers) has shaped the present day mtDNA phylogenetic structure of Indian populations. 2. Either the ancestral M existed for a minimum interval of ~30,000 to ~20,000 years, during which the younger lineages branched off sequentially or second emigrational event most likely occurring ~30,000 to ~20,000 years ago from the west of the subcontinent has given rise or brought the younger lineages, thereby accounting for the different numbers of mutations accumulated to the present.The latter has been complicated by the fact that if not all, most of these lineages are autochthonous to India and arose essentially simultaneously from ancestral M as argued by Macaulay et al. [4]. Furthermore, since the only haplogroup of M lineages found in the substantial number to the west of the subcontinent are members of the M1 fragment, it also seems unlikely that the so appeared younger lineage of macrohaplogroup M has originated much farther west.Owing to the aforesaid ambiguity in population structure coupled with the west Eurasian contribution into the Indian maternal gene pool as a consequence of migrations during the last 10,000 years before present (ybp) [10,11] the origin and settlement of Indian people still remains intriguing.Of the known M lineages in India, M2 with an estimated age of ~50,000 years is the oldest [7,12] and largest sub-haplogroup, which almost accounting for one tenth of the Indian macrohaplogroup M [11,13]. The distribution of M2 is significantly more pronounced in southern part of India as compared to north, a cline similar to that of M in general [5,11]. Moreover Metspalu et al. [11] also noted that frequency of M2 among the Brahmin and Kshatriyas of Andhra Pradesh is not significantly different from that of other caste and tribal populations of the region. However it is absent among the Brahmins and Kshatriyas of the northern states of India, while the frequency reaches nearly 3 % among other caste and tribal populations of the region. The high frequency of M2 consistent with its greater age and distribution suggests that it may represent the phylogenetic signature of earliest settlers who colonized India through southern route.To explore the past population dynamics, impact and contribution of Middle/EarlyUpper-Paleolithic settlers in shaping the genetic diversity and structure of contemporary Indian populations, we have sequenced 72 complete mitochondrial genomes of M2 lineage from 16 relic tribal populations of India.ResultsOf the screened 2768 mtDNAs from 24 tribes of India the macrohaplogroup M accounted for 69.39 %, which is consistent with the earlier reports [5,11,14]. The frequency distribution of macrohaplogroup M varies significantly (P <0.0001) among studied tribes with a cline towards southern and eastern regions of India as shown in Table 1 and Figure 1. In tribes (MaThakur, KaThakur, Kathodi, Katkari) of western region, macrohaplogroup M frequency is significantly low (~50% or less; P <0.011) as compared to the other studied regions of India. Unexpectedly Dungri Bhil representing the north-westernmost region shows a high frequency of M (76.1%) as compared to its other western counterparts.Table 1Sampling details and mtDNA lineage distribution in India.LocationPopulationPopulation CodeLinguistic AffiliationanbFrequency (%)McNon MdM2M2bIndia276865.3934.619.572.96Western62947.2252.7813.350.00DungriBhillDBIE11866.1033.903.390.00KathakurKATIE22032.7367.279.090.00KathodiKTHIE12051.6748.3321.670.00KatkariKKIE5046.0054.0016.000.00MathakurMTKIE12151.2448.7621.490.00Central71169.9030.1015.754.36AndhANDIE11559.1340.8711.300.00Hill KolamHKDR/IE12377.2422.7618.709.75KamarKMRIE11168.4731.539.910.00KorkuKRKAA11070.0030.0021.8215.45MadiaMADDR/IE14075.7124.2920.711.43NihalNHLIE11266.9633.0410.710.00East33673.2126.794.461.48MalpahariaMLPAA11475.4424.566.142.63MundaMUNAA/MU10274.5125.493.920.98PaudiBhuiyaPBDR/IE12070.0030.003.330.83North East86367.3232.680.120.00DirangMonpaDMTB10078.0022.000.000.00GallongGALTB10850.0050.000.000.00LachungpaLCTB10479.8120.190.000.00LepchaLPTB10958.7241.280.000.00ShertukpenSKPTB10382.5217.480.000.00Sonowal KachariSKIE11256.2543.750.890.00TotoTTTB10280.3919.610.000.00WanchooWANTB12557.6042.400.000.00South22982.5317.4723.1420.08Betta KurubaBKDR11574.7825.2239.1335.65Jenu KurubaJKDR11490.359.657.024.39Togataveera*TGDR#####Reddy*REDR#####a As per Gordon 2005[47]; b Number of samples screened; c Includes all haplogroups within macrohaplogroup M; d Includes all other than macrohaplogroup M; * Caste populations data from Sun et al. [7]; # Data not available; \"AA\" Austro-Asiatic; \"DR\" Dravidian \"IE\" Indo-European; \"MU\" Mundari; \"TB\" Tibeto-Burman.Figure 1Map of the Indian subcontinent indicating approximate locations of studied populations and mtDNA haplogroup distribution. '*' Approximate location of the populations studied by Sun et al. [7], the mtDNA sequences of which were used in this study.For the earliest settlers' component among the studied tribes, 1810 samples of macrohaplogroup M were screened for the motif that confirms haplogroup M2 within M as described in methods. Our results indicate that M2 is completely absent among the eight tribes of northeast India, expect one M2 in Sonowal Kachari. Avoiding northeast tribes, the M2 haplogroup frequency is about 13.86 % among the studied tribes. Its frequency is ~10 to ~20 % in tribes of western and central India. The frequency declines gradually to farther north and east. In southern region tribes, Betta Kuruba shows highest frequency (i.e. 39.13%) whereas the adjacent Jenu Kuruba tribe shows frequency of only 7.02 %. The distribution of subclade M2b varies greatly from complete absence among Indo-European speakers of western and central India to as high as 35.65 % among Betta Kuruba. Irrespective of region, its frequency is high (>50% of total M2) in all Dravidian speakers, except Madia tribe of central region whose linguistic affiliation is not very clear. Similarly it's frequency is high in Korku, an Austro-Asiatic tribe of central India. In eastern region M2b frequency remains low (<50% of M2).Defining the M2 substructureThe reconstructed phylogenetic tree based on our newly sequenced 72 mtDNAs of M2 haplogroup and 4 additional M2 complete sequences from the literature [7] is given in Figure 2. Out of the four defining mutations of macrohaplogroup M, one transition at nucleotide position (np) 14783 shows reversion in one of our samples. Besides the commonly occurring 16319 transition, M2 in our samples is defined by the motif 447G-1780-8502-11083-15670-16274 as also described in Kivisild et al., Rajkumar et al., Sun et al. and Thangaraj et al. [6,7,12,9]. Though one major branch in our tree lack mutation at np 16274 but due to its presence in most M2 samples of this study, as well reported elsewhere [6,7,11] we considered this mutation as a basal polymorphism of M2 as suggested in Sun et al. [7] and lack of the 16274 variant in some samples [[6,12], this study] indicates a back-mutation event. Similarly lack of mutation at np 11083 in one of our samples is also treated as reversion event.Figure 2Phylogenetic reconstruction of 76 complete mtDNAs of M2 Lineage. Mutations were scored relative to the rCRS [58]. Sample details and population codes has been given in Table 1. Four additional complete mtDNA sequence of M2 lineage (labeled as R102, T3, T11 and T27) are acquired from published sources [7] has been used for tree reconstruction. Suffixes A, C, G, and T indicate transversions, \"d\" signifies a deletion and a plus sign (+) an insertion; recurrent mutations are underlined. The prefix \"@\" indicates back mutation. The coalescent estimates were calculated as per [16] and [17] presented as bold and Italic respectively.The M2 tree shows an initial deep split into two sister clades M2a and M2b. No third clade, as indicated in Rajkumar et al. [12] has been found. The clade M2a is defined by transition at np 7961, 12810 and contains three independent basal branches M2a1, M2a2 and M2a3, in contrast to the earlier reports [6,7,11] where M2a defining motif largely constitute mutations of its sub-braches. M2a1 is defined by the motif 204-5252-8396-9758-16270-16352, in which transition at np 8396 show parallelism in two samples of M2a2 branch and transition at np 16352 shows a reversion event. M2a2 is defined by the motif of four diagnostic 7702-11041A-12657-13708 and two recurrent 16240C-16311 mutations. The branch M2a3 is defined by the motif of one recurrent np 146 and three specific 5426-5774-7762 mutations. The further divergence within these branches of M2a exhibit probable pattern of more shared haplotypes within populations of geographic proximity followed by population specific haplotypes and a few shared haplotypes among geographically apart populations.Unlike M2a, M2b instead of early branching represented by a single deep root defined by the motif 152-182-195-522,523d-1453-2831T-3630-5744-6647-9899-13254-14766-16183C-16189-16193+C-16320 which, of late shows branching pattern similar to the sub-branches of M2a. The M2b1 defined by the transition at np 6260-5420 harbour population of eastern region. Whereas, M2b2 defined by transition at np 16295, harbours Dravidians. Other braches within M2b are more or less population specific. In this study, spread of M2b by enlarge restricted to Dravidians and tribes of eastern region. The root of M2b in our tree differs in two positions to the earlier definition of Sun et al. [7] i.e. transition at np 182 is present in all of our M2b samples so we treated this as basal mutation and lack of this in one sample of Sun et al. [7] could be better explained by reversion event, second our all M2b samples has poly 'A' at np 16180–16182 and twelve 'C's thereafter. Hence in our tree an additional 'C' at np 16184–16193 has been treated as insertion at np 16193, than transversion (A16182C) reported by Sun et al. [7].Age estimates and Phylogenetic implicationsCoalescent age estimates were calculated by Rho (ρ) statistics [15] using two different mutation rates [16] and [17] shows a marginal time difference when standard deviation is taken into account, the later has been considered because of robustness in view of natural selection [17]. The average sequence divergence of the 76 M2 coding-region sequences from the root of M2 calculated as per [17] corresponds to a coalescence time estimate of 36.5 ± 1.6 thousand years (ky). The founder age estimate for Indian mtDNA lineages using M2 data, 50.0 ± 1.5 ky is well within the lower bound range of earlier estimates (i.e. sometime before 50 kyBP) of modern human dispersal into Arabia and southern Asia [1,2,4,17-21], and perhaps more close to the estimates of [17].The two clades of M2 show differential branching patterns. M2a with coalescent age 21.6 ± 2.3 ky splits into its three deep rooting branches M2a1, M2a2 and M2a3. M2a2 is specific to Kathodi/Katkari tribe, whereas M2a1 and M2a3 encompass almost all the studied tribes. M2a1, M2a2 and M2a3 show coalescent estimates of 7 to 9 ky. The clade M2b doesn't show branching event earlier than estimated coalescence time of 12.6 ± 2.8 ky. In our samples we could not find M2b among Indo-European speakers of west and central India. The Dravidian speaking tribes of south extending up to central India and tribes of eastern region irrespective of linguistic affiliation, harbour both clades (i.e. M2a and M2b) of M2, presenting a time depth of ~37 ky.Diversity indicesDiversity indices and demographic parameters estimated for studied tribes are given in Table 2. The M2 lineage, haplotype diversity among Indian tribes ranged from 0.40 to 1.00 and nucleotide diversity from 0.0001 to 0.002. Though four geographical regions of India did not differ significantly (Mann-Whitney U-test) in haplotype diversity it was comparatively higher in west (0.90–1.00) followed by central (0.83–1.00), eastern (0.83–1.00) and southern tribes (0.40–1.00). Nucleotide diversity in east (0.0010–0.0019) was significantly higher than west (0.0001–0.0009; Z = 2.24, P = 0.025) and central tribes (0.00016–0.0011; Z = 2.65, P = 0.039), intermediate nucleotide diversity values were observed in south India (0.0006–0.002), they were not significantly different from west and central India (Z = 1.71; P = 0.087) or east India (Z = 0.44; P = 0.662). These patterns of genetic diversity were further strengthened by the analysis of mean pairwise differences (MPD). MPD of west (1.67–16.00) and central tribes (2.67–18.00) were significantly lower (Z = 2.41, P = 0.016) than the MPD from east (17.17–32.67), whereas MPD from south (11.20–30.00) were not significantly different from east, west and central tribes (Z = 1.39, P = 0.166). Thus observed mtDNA diversity indicate to the fact that haplotype/haplogroup frequency is a poor parameter of deep rooting ancestry rather it is the product of recent population growth. Similarly, the diversity parameters are also influenced by the past demographic events and any phylogenetic inference drawn on such parameter should keep in view the past demographic events, particularly for India where such event has been predicted previously [22].Table 2Diversity and demographic parameters deduced from complete mtDNA sequences of M2 lineage in India.LocationPopulation codenaHaplotype diversitybNucleotide diversitybMPDbcFu's FsIndia760.99 ± 0.0030.0015 ± 0.000724.64 ± 10.94-24.03Western220.99 ± 0.0160.0008 ± 0.000412.96 ± 6.07-7.07DB41.00 ± 0.1770.0001 ± 0.00011.67 ± 1.21-1.74KAT50.90 ± 0.1610.0002 ± 0.000163.80 ± 2.29-0.13KTH51.00 ± 0.1260.0007 ± 0.000513.00 ± 7.080.41KK41.00 ± 0.1770.0009 ± 0.000616.00 ± 9.090.87MTK41.00 ± 0.1770.0002 ± 0.000184.16 ± 2.61-0.77Central290.99 ± 0.0120.0014 ± 0.0007323.97 ± 10.85-4.46AND60.93 ± 0.1220.0009 ± 0.000515.07 ± 7.871.49HK40.83 ± 0.2220.0011 ± 0.000718.00 ± 10.183.64KMR51.00 ± 0.1260.0002 ± 0.000164.00 ± 2.39-1.72KRK60.93 ± 0.1210.0006 ± 0.000411.13 ± 5.901.00MAD41.00 ± 0.1770.0003 ± 0.00025.00 ± 3.06-0.52NHL40.83 ± 0.2220.00016 ± 0.00012.67 ± 1.780.56East110.98 ± 0.0460.0016 ± 0.000827.38 ± 13.010.01MLP41.00 ± 0.1770.0013 ± 0.000822.00 ± 12.371.22MUN31.00 ± 0.2720.0019 ± 0.001532.67 ± 19.872.37PB40.83 ± 0.2220.001 ± 0.000717.17 ± 9.733.56South140.91 ± 0.0590.0012 ± 0.000620.71 ± 9.742.77BK50.40 ± 0.2370.0006 ± 0.000411.20 ± 6.147.28JK50.80 ± 0.1640.001 ± 0.000719.00 ± 10.185.38TG*31.00 ± 0.2720.002 ± 0.001430.00 ± 18.282.28RE*1####a Number of complete mtDNA sequences; b ± Standard Deviation; c Mean Pairwise Differences; * Sequence data from Sun et al. [7]; # Unable to calculate due to single sequence.Past population dynamicsAs indicated in our results and previously [22] the demographic history of populations in different geographic regions might have played pivotal role in shaping the complex net of population phylogenetic relationships in Indian subcontinent. The demographic history of M2 lineage as a surrogate of the middle/early upper Palaeolithic component of Indian populations was reconstructed using Bayesian skyline plot (BSP) [23]. Figure 3 (panel 'A') shows the BSP of M2 lineage produced using 76 complete mtDNA sequences along with plot (panel 'B') using only coding region. Although the two analyses are very similar, the second is confined to slow evolving region of mtDNA [24] which is likely to define lineages that have existed in the population prior to a putative bottleneck, thus increasing the sensitivity of BSP to detect more complex demographic trends. As the analysis is based on only single lineage it provides insight into the demographic event limiting to the age of the lineage (i.e. 37–45 kyBP). Most striking is the population decline, observed during Last Glacial Maximum i.e. 23 to 19 kyBP [25] and Late Glacial Aridity i.e. 18 to 14 kyBP [26], followed by many fold population growth in a comparatively short period of time. If such demographic event had affected the earliest settlers of India it would have resulted in several implications of phylogenetic interest. Firstly, reduction of genetic diversity across all the lineages in which, lineages with a smaller population spread would have been affected the most. Second, it might have mechanized some sort of unifying effect where smaller lineages are eliminated or at least reduced to margins of extinction and lineages of larger spread remained among all the post bottleneck populations. The Post Glacial rapid population growth achieved some sort of plateau by 7 to 3 kyBP followed by another decline which was to its maximum around ~1000 to 1500 BP. Now the question is whether the observed demographic trend was uniform throughout India or it was as complex as reported by the earlier studies [22]. A similar analysis for each studied geographical region of India is presented in panel C to F of Figure 3. Due to the small sample size in each geographic region BSP produces low resolution; however rapid post glacial population growth is evident in east, south and central India, followed by a population decline from 3 to 1 kyBP. The rapid regain after this period has been observed in central region; however such regains are marginal in other two regions. The demographic past of ancient lineage among western tribes was quite different- a population growth from ~7–8 kyBP continued to present. The negative values of Fs that differ significantly from zero indicative of population's demographic expansion [27] also support the recent population expansion in western region (Fu's Fs = -7.07; P = 0.004).Figure 3Bayesian skyline plots showing demographic histories of earliest settlers' component. The thick solid line is the median estimate, and the grey area overlay show the 95% highest posterior density (HPD) limits. Panel 'A'- The Bayesian skyline plot (m = 10) for India total, derived from complete mtDNA sequences (n = 76). Panel 'B'- The Bayesian skyline plot (m = 10) for India total, derived from coding region (577–16023) mtDNA sequences (n = 76). The time estimates (yBP) were calculated as per [16]. For comparison, the cold and arid period around the Last Glacial Maximum are also indicated on panel A & B. Panel 'C to F' shows Bayesian skyline plots (m = 10) derived from complete mtDNA sequences of eastern (n = 11), central (n = 29), southern (n = 14) and western (n = 22) regions of India respectively.Genetic StructureThe above results are indicative of some genetic structure in Indian populations, to investigate that, AMOVA was used (Table 3). In the total samples (model 1) 49.13% of the variance was found within populations and 50.87% among populations. Studied tribes were then grouped according to geographic proximity (model 2), linguistic affinities (model 3) and to the results suggested, namely two groups separating Indo-European speakers of west and central India from all others (model 4). Under the models 2 and 3, 45–48 % of the variance was found within populations, 36–39% among populations within groups and 13–18 % among groups. The model 4 more appropriately reflects the genetic structure with variance among groups 29.93% exceeds the variance among populations within groups 27.79%.Table 3Analysis of Molecular Variance (AMOVA).Model tested for population structureAmong groupsAmong populations with in groupWith in populationsVaraP-valueVaraP-valueVaraP-value1.Total--50.87<0.00149.13-2.Geographical criteria13.660.04338.81<0.00147.53<0.0013.Linguistic criteria17.650.02036.80<0.00145.54<0.0014.Indo-European speakers of west & central India vs. all others29.93<0.00127.79<0.00142.28<0.001a VarianceDiscussionA rapid coastal migration along the \"southern route\" from Africa into southern Asia, some time before 50 kyBP has been strongly suggested by studies on present day world populations (especially those based on mitochondrial DNA) [1,11,18-21,28]. The founder analysis of the mtDNAs in this study suggests 50.0 ± 1.5 kyBP for such arrival which is well within the lower bound range of earlier estimates and perhaps more consistent with the earliest and most pronounced population expansion in southern Asia around 52 kyBP suggested in [28]. Magnitude of this southern Asian growth phase suggests that over half of the global human population lived in Indian subcontinent between ~45 to 20 kyBP and population size peaked at over 60% around 38kyBP [28]. These population expansion estimates are largely in agreement with high mtDNA diversity and star like non-overlapping pattern of numerous lineages of macrohaplogroup M reported previously [4,6,9,7].Though the stage upto here is clear, the contribution and role of this sizably large earliest settlers' component in the contemporary Indian populations, coupled with later migrations during the last 10 thousand years (ky) from west and east of the subcontinent has been an issue of controversy. Cordaux et al. [22] based on the non-overlapping pattern of mtDNA phylogeny between India and east Eurasia has supported the argument of Cavalli-Sforza et al. [29] that in India the genetic traces of early migrations along the southern route were erased by the subsequent migrations, which shaped the present-day mtDNA gene pool of India. However presence of numerous autochthonous lineages in India emerging directly from the root of the founder macrohaplogroups M, N and R [4,6,7,9] during the estimated population growth period in southern Asia (~45 to 20 kyBP)[28] indicates the presence of large component of earliest settlers in the contemporary Indian populations.In the quest of finding the carriers of the genetic legacy of the earliest settlers among the contemporary Indian populations, some previous studies on mtDNA variation by calculating nucleotide diversity and expansion time (as per methods of Slatkin et al. [30]) for different linguistic groups of India, distinguished Austro-Asiatic speaking tribes as the oldest and the carriers of the said legacy [31,32]. Basu et al. [14] also supported the view by reporting that the frequency of the ancient haplogroup M2 among the Austro-Asiatic tribal populations is as high as 19% and they lack the younger haplogroup M4. However Metspalu et al. [11], so as this study, rejects such claims as linguistic groups of India do not cluster into distinct branches of the Indian mtDNA tree, [[6,10,13], this study] calculating the beginning of expansion for those groupings is problematic, whereas lack of coding region information in Basu et al. [14] have lead to an over estimation of M2 frequency. Moreover our results indicate that M2 frequency variation among the studied tribes can be better explained by recent population expansion/demographic events than as a function of deep rooting ancestry. The nucleotide diversity though appears better parameter, is also predisposed to influence of past demographic events. The phylogenetic inferences based on such parameters should be strictly viewed in reference to the demographic events, particularly for India.Our analysis of mtDNA variation in populations of India indicate that the Dravidian tribes extending from southern to central India and tribes of eastern India irrespective of the linguistic affiliation shows equally deep rooted M2 ancestry ~37 ky (Figure 2), comparable nucleotide diversity (Table 2) and similar past demographic history (Figure 3). However Indo-European tribes of western and central India except Kathodi/Katkari and Andh tribes harbour only M2a1 branch representing a time depth of ~8 ky. Kathodi/Katkari and Andh tribe encompasses other braches of M2a, but lacks M2b. All these Indo-European tribes shows appreciable frequency of M2 (Table 1), but they are low on nucleotide diversity (Table 2). Thus it would be highly speculative to tag any one or a group of populations based on linguistics or geography as the representatives of earliest settlers, rather it indicate to the fact that earliest settlers' component is more pronounced in the areas extending from southern to eastern India, and shows decline towards north and northwest India, a cline similar to that of M in general [[5,11], this study]. However a decline of earliest settlers' component across tribe to higher caste gradient may also be accepted in the respective regions as indicated in [13].The time depth of M2 lineage and diversity indices in Indo-European speakers of western region extending up to central India posits the expansion of earliest settlers' component into these areas during the post Last Glacial Maximum(LGM) growth (~12 to 7 kyBP) of population (Figure 3) or perhaps little later (Figure 3-pannel 'F'). However this requires further investigation. It is only during this rapid growth; regional and population specific branching patterns appear on the more or less homogenous M2 phylogeny. The possible explanation would be the earliest settlers of India prior to this rapid population growth had lived in an extended enclave and there had been continuous gene flow across population boundaries. The second but more plausible reason of such homogeneity could be that earliest settlers by virtue of large population size during ~45 to 20 kyBP [28] and Indian ecological setting which, favoured tendency to isolate and subjugate [33] might have been differentiated into populations distributed far apart as suggested in recent studies [4,9]. But during the LGM and late glacial aridity, climate across India and south Asia generally seems to have been much more arid than present. Geomorphological indicators from the landmass of India suggest dune mobility in the northwest [34], and greatly reduced river flow in north central India during the span of time that covered the full glacial [35]. Offshore indicators of salinity (due to runoff from the land) suggest that LGM aridity was substantially greater than at present. Indicators of upwelling intensity in the Indian Ocean suggest that the summer monsoon was much weaker than present at the LGM, but reaching its weakest at around 15,800 – 12,500 C14 years ago, that is 17,800-13,800 calibrated or 'real' years ago [26]. During this period of cold and more arid conditions rainforest retreated and was replaced by dry grasslands. However, some monsoon forests and woodlands in southern India and scrub, open woodland in eastern India probably existed in presently moist forest climates. This appears to be harsh conditions for an hunting gathering based subsistence, thus to fight the adverse, probably shrinking populations might have come close to each other in a more habitable area allowing a free gene flow between populations, whereas ancestral population of the Kathodi/Katkari M2a2 lineage appears to have remained isolated during this period. In the post LGM growth period, though population spread over wide geographical regions. Maternal gene flow is evident in the geographical neighbors suggesting fluidic population specific boundaries until recently at least among the tribes.The next important event on the Indian scene is the beginnings of agriculture and use of pottery [36-41]. Cultivation of plants/agriculture diffused from the Fertile Crescent within the past 10,000 years. The steady advance beyond this stage seems however to have been primarily driven by the crop-animal complex derived from the mid-east, reaching the tip of southern India around 3 to 2 kyBP [42,43]. The diffusion of pottery traditions, which arise in response to the need to store and cook grains, shows evidence of the influences from northwest and northeast, with the western influence predominating over much of the country. Thus the Black and Red ware reflects western, while the Corded ware Chinese influence [44-46]. Two other technological innovations, known to have originated outside of India, the domestication of horse, around 6 kyBP on the shores of Black Sea in present Ukraine, and the use of iron around 5 kyBP in Anatolia in present day Turkey, appears in the Indian archeological records (around 2 kyBP) soon after the agriculture [42]. The recent study investigating the cultural or demic diffusion model of agriculture in India supported the demic diffusion model which predicts a substantial genetic input from migrating agriculturalists [47]. The advent of agriculture and perhaps migrating agriculturists brought about dramatic changes in the economy, technology and demography of human societies. Human habitat in the hunting-gathering stage was essentially on hilly, rocky and forested regions, which had ample wild plant and animal food resources. Agriculture led to the emergence of villages and towns and perhaps brought with it the division of society into occupational groups. [43]. Crop cultivation resulted in the loss of the traditional habitat of hunter-gatherers by deforestation, fragmenting and marginalizing numerous such populations, many of whom were assimilated into agriculturally based subsistence economies [48], thereby catalyzing some sort of regional similarities across tribe caste continuum. Our results on reconstructed past population demography indicating decline of earliest settlers' population (female population here) during this period in almost all the geographical regions except western (Figure 3) is consistent with the above proposition and suggests that demic diffusion of these technologies were rapid, perhaps involving large migrating populations with these technologies.The highest frequency of east Eurasian- specific mtDNA haplogroups [11,22] and absence of M2 an earliest settlers component (Table 1) among Tibeto-Burman speaking tribes of northeastern states of India suggests that, despite the more recent migrations to India, these populations remained relatively isolated, explaining the close correlation between genetic and linguistic results [49,50]. This contrasts with the situation observed in other regions of India, where linguistic structure shows very little concordance with the genetic structures.ConclusionThe time depth and diversity of M2 lineage among the studied tribes suggests that the tribes of southern and eastern region along with Dravidian and Austro-Asiatic speakers of central India are the modern representatives of earliest settlers of India via proposed southern route. The LGM and late glacial (~23 to 14 kyBP), climatic conditions across India and south Asia seems to be much more arid and harsh for an hunting gathering based subsistence, thus mechanized reduction and bringing earliest settlers' population closer in a more habitable area allowing a free gene flow, followed by a rapid three fold population growth around 12-7 kyBP when climatic conditions improved, thereby inducing some sort of homogeneity and redistribution of earliest settlers' component in wide geographical regions. The next important event on the Indian scene appears to be demic diffusion of agriculture and associated technologies around 3 kyBP, resulted in the loss of the traditional habitat of hunter-gatherers by deforestation, fragmenting and marginalizing such populations, many of whom were assimilated into agricultural based subsistence economy, as evident in the decline of earliest settlers' component in all the geographical regions except western.MethodsPopulation SamplesThe approximate location of the 24 tribal populations from which 2768 mitochondrial DNAs (mtDNAs) were sampled is shown in Figure 1. Each sample comprises unrelated healthy donors from whom appropriate informed consent was obtained. The ethical clearance for the study was obtained from the organizational ethical clearance committee of Anthropological Survey of India. Further details of the whole sample collection are reported in Table 1.About the PopulationsThe population of India is culturally stratified broadly into tribal and non-tribal. It is generally accepted that the tribal people, who constitute 8.2% of the total population [51] are the original inhabitants of India [52,53]. There are an estimated 461 tribal communities in India [54], who speaks about 750 dialects [55] which can be classified into one of the following four language families: Indo-European (IE) Austro-Asiatic (AA), Dravidian (DR) and Tibeto-Burman (TB).Considering two assumptions, (i) The M2 is one of the major matrilineal lineages contributed by the southern route migrants in the Indian populations and (ii) The tribal people being the original/earliest inhabitants of the subcontinent could have larger representation of such contribution. We have screened 24 relic tribal populations (see details in Figure 1 and Table 1) who by virtue of their habitat, socio-economic and cultural boundaries probably less influenced by the so called modern populations.MtDNA molecular analysesThe collected 2768 samples from 24 tribes were first screened for M macrohaplogroup. Those belongs to M (1810 in total) were typed for mtDNA motif C447G, T1780C, A8502G, G16319A which defines M2 haplogroup [6,7,9,11,12]. In our sample C447G and A8502G polymorphisms are specific to M2, whereas T1780C and G16319A are also found in the background of haplogroups other than M2 (our unpublished data).Out of total samples screened, 265 mtDNAs belong to M2 haplogroup distributed among 17 tribes with varying frequency. Avoiding Sonowal Kachari where only one M2 sample was found, 3–6 M2 samples were randomly selected from each of the 16 tribes for complete mtDNA sequencing (72 in total).DNA was extracted from all the collected 4–5 ml blood samples using standard phenol-chloroform methods [56] with minor modifications. For screening and complete mtDNA sequencing, DNA was PCR amplified following standard protocols and using the PCR primers and conditions of Rieder et al. [57]. Successful amplification was verified by electrophoresis on 1% ethidium bromide-stained agarose gels. Samples were prepared for sequencing by an ExoI/SAP cleanup to remove single-stranded DNA and unincorporated nucleotides. PCR product was sequenced with both forward and reverse primers using BigDye Terminator v3.1 sequencing kits from Applied Biosystems on an Applied Biosystems 3730 automated DNA analyzer. Contig assembly and sequence alignment was accomplished with SeqScap v2.5 software from Applied Biosystems. Mutations were scored relative to the revised Cambridge Reference Sequence (rCRS) [58] with each deviation confirmed by manual checking of electropherograms. All (n = 72) mtDNA complete genome sequences have been submitted to GenBank (accession numbers EU443443–EU443514).Statistical analysisPhylogeny Reconstruction and Age EstimationBesides our newly sequenced 72 mtDNAs of M2 haplogroup, 4 additional M2 complete genome sequences from the literatures [7] were employed for tree reconstruction. The phylogenetic tree was reconstructed from median-joining networks rooted to L3 using NETWORK 4.2.0.1 software [59]. The tree was checked manually to resolve homoplasies. The coalescent age estimates were calculated by Rho (ρ) statistics [15] and two different mutation rates i.e. one base substitution (one mutation other than indel) in the coding region (577 – 16023) per 5,140 years [16] and one synonymous transition per 6,764 year [17] calibrated on the basis of an assumed human-chimp split of 6.5 million years ago. Standard errors for coalescence estimates were calculated following Saillard et al. [15].Estimates of Population Structure and evolutionary relatednessThe 76 aligned complete mtDNA sequences were analyzed for haplotype, nucleotide diversity (± SD), and mean pair-wise differences (± SD). Analyses of Molecular Variance (AMOVA) [60] were also performed to evaluate the genetic structure of the populations. The aforesaid analysis has been performed using software package ARLEQUIN version 3.0 [61].Estimates of past Population DynamicsWith the prior assumption of M2 as the signature of the earliest migrants of modern humans in Indian subcontinent, we have tried to reconstruct the demographic history of earliest settlers from Most Recent Common Ancestor (MRCA), using Bayesian skyline model [23] of effective population size. Effective population size is a compound population genetic parameter generally considered linearly proportional to census population size – in this analysis, the population of breeding females. It is influenced by many factors, including local extinction, recolonization and various forms of nonrandom mating (62). The model assumes that regional populations are isolated. Estimates of effective populations were derived from the 76 complete mtDNA sequence data belonging to M2 haplogroup using Markov Chain Monte Carlo (MCMC) (63) sampling with 10 groups (m = 10) in software packages BEAST v1.4 [64] and Tracer v1.3 [65], available from http://beast.bio.ed.ac.uk/. The plots were obtained using stepwise (constant) model. The substitution model was selected by comparison of Akaike Information Criterion scores (AIC). The analysis was run for 30 million iterations with the first 10% discarded as burn-in, genealogies and model parameters were sampled at every 1,000 iterations thereafter.AbbreviationsAA: Austro-Asiatic; IE: Indo-European; DR: Dravidian; TB: Tibeto-Burman; ky: Thousand Years; kyBP: Thousand Years Before Present; yBP: Years Before Present; mtDNA: Mitochondrial DNA; rCRS: Revised Cambridge Reference Sequence; np: Nucleotide Position; PCR: Polymerase Chain Reaction; MPD: Mean Pairwise Differences; AMOVA: Analysis of Molecular Variance; MRCA: Most Recent Common Ancestor; MCMC: Markov Chain Monte Carlo; SD: Standard Deviation.Authors' contributionsSK, KU, PK and PBSVP carried out initial screening and complete mtDNA sequencing of the data. SK and RRR did sequence alignment and all the phylogenetic analysis. PAM, BD, MK, DX and SYS contributed samples. SK drafted the manuscript. VRR conceived the study, participated in its design and coordination also helped to improve the manuscript. 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+    "id": "PMC2528016",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528016\nAUTHORS: Dong-Dong Wu, David M Irwin, Ya-Ping Zhang\n\nABSTRACT:\nBackgroundHair is unique to mammals. Keratin associated proteins (KRTAPs), which contain two major groups: high/ultrahigh cysteine and high glycine-tyrosine, are one of the major components of hair and play essential roles in the formation of rigid and resistant hair shafts.ResultsThe KRTAP family was identified as being unique to mammals, and near-complete KRTAP gene repertoires for eight mammalian genomes were characterized in this study. An expanded KRTAP gene repertoire was found in rodents. Surprisingly, humans have a similar number of genes as other primates despite the relative hairlessness of humans. We identified several new subfamilies not previously reported in the high/ultrahigh cysteine KRTAP genes. Genes in many subfamilies of the high/ultrahigh cysteine KRTAP genes have evolved by concerted evolution with frequent gene conversion events, yielding a higher GC base content for these gene sequences. In contrast, the high glycine-tyrosine KRTAP genes have evolved more dynamically, with fewer gene conversion events and thus have a lower GC base content, possibly due to positive selection.ConclusionMost of the subfamilies emerged early in the evolution of mammals, thus we propose that the mammalian ancestor should have a diverse KRTAP gene repertoire. We propose that hair content characteristics have evolved and diverged rapidly among mammals because of rapid divergent evolution of KRTAPs between species. In contrast, subfamilies of KRTAP genes have been homogenized within each species due to concerted evolution.\n\nBODY:\nBackgroundThe availability of the rapidly increasing number of genome sequences provides opportunities for investigators to study evolutionary patterns that potentially account for morphological characteristics, and suggest the genetic basis for variation in phenotypes. In particular, gene families in which duplications, rate variation and pseudogenization occur frequently are likely involved in functional innovation and adaptation [1]. Examples of such gene families are those involved in the perception systems, for example, the odorant receptors [2-4], the vomeronasal receptors [5-8], and the sweet/umami and bitter receptors [9-12]. Furthermore, these studies should facilitate our understanding of the general evolutionary trends in genomic complexity and lineage-specific adaptation [1]. Here, we studied the evolutionary patterns of the keratin associated protein (KRTAP) gene family, whose encoded proteins are major components of hair, with the goal of revealing the underlying basis for unique mammalian hair and its phenotypic diversity.Hair is a unique character found on all mammals, but not on other animals, where it plays a crucial role in the retention of heat within these homoiotherms and presumably contributed significantly to the rapid radiation of mammals and their rise to become the dominant terrestrial vertebrate [13]. Other functions of hair include sexual dimorphism, attraction of mates, and protection of skin [14]. An interesting event in hair evolution has been its loss in humans [15,16], however; humans actually have a similar density of hair follicles to that seen in apes [15]. Comparative studies have concluded that hair presents similar structure and modality of growth throughout mammals [17-19]. For example, the overall ultrastructure of hair and the distribution of keratins in monotremes are similar to that of marsupial and placental mammals [18], and the localization of acidic and basic keratins in marsupial hairs is similar to that in placentals [19]. However, most studies have focused on keratins rather than the keratin associated proteins.The major components of hair are alpha-keratins and keratin associated proteins, each of which are encoded by multigene families. The alpha-keratins include two multigene subfamilies, type I (acidic) and type II (basic) [20,21], and form the intermediate filament cytoskeleton of all epithelia providing stability against stress [22]. In humans, the alpha-keratin gene family has been extensively studied demonstrating that there are 54 functional genes that are clustered on chromosomes 12q13.13 and 17q21.2 that show differing expression patterns during hair development [23]. Hair keratins form an intermediate filament (IF) network by co-polymerization of type I and type II members, in trichocytes, which are cells that populate the central hair-forming compartment of the anagen hair follicle [24,25]. In the hair cortex, hair keratins IFs are embedded in an in interfilamentous matrix, which consists of hair keratin-associated proteins (KATAP, usually abbreviated as KAP) [26]. KRTAP contains two major groups: high/ultrahigh cysteine (HS) and high glycine-tyrosine (HGT) that are considered to have originated independently, and are essential for the formation of rigid and resistant hair shafts through their extensive disulfide bond cross-linking with the abundant cysteine residues of hair keratins or hydrophobic interactions with keratins [23,26-28]. The genes have been grouped into 27 subfamilies, termed KRTAP1 to KRTAP27, based upon phylogeny [23,29]. In humans, about 100 KRTAP genes are identified, that are in five tandemly arranged clusters (chromosomal regions 11p15.5, 11q13.4, 17q21.2, 21q22.1, and 21q22.3) [30-34]. Previous research on KRTAP genes has focused on their function and expression, with little emphasis on the origin and evolution of this gene family. Here, we investigate the evolution of the KRTAP gene family, including phylogeny and classification, and the mechanisms involved, such as gene duplication, gene conversion, in our endeavor to resolve hair's evolutionary history and to explain the diversity observed in extant mammals. We find different repertoires among mammals which potentially explain the differing hair features of different lineages. An expanded KRTAP gene repertoire was found in rodents. Surprisingly, despite the lack of hair, human had a similar number of genes with other primates. Large-scale gene conversion events were detected in high cysteine KRTAP but fewer in high glycine/tyrosine, and the latter genes evolve more dynamically. Compared to the conserved structure and modality of keratins within mammals, the significant divergence of characteristics of hair among placental, marsupial and monotreme species is likely due to interspecific divergence of KRTAP sequences.ResultsInventory of KRTAP genes in mammalsThe recent rapid increase in the availability of comparative genomic data is facilitating the illumination of evolutionary features of organisms. Particularly, mammals are well represented with data from: placental mammals including primates – human [35], chimpanzee [36] and rhesus macaque [37]; rodents – mouse [38] and rat [39]; carnivore – dog [40]; a marsupial, the opossum [41]; and a monotreme, the duck-billed platypus [42].Some gene sequences in GenBank http://www.ncbi.nlm.nih.gov have been submitted independently by several researchers. KRTAP genes have been shown to have size polymorphism within populations [43-45], and sequences from different individuals at these loci are diverse and may not align well. For instance, we identified that a KRTAP gene cluster in Contig: NT_113931 actually corresponds to the KRTAP region on Chr17 of the human genome, except that it differs in sequence length for several KRTAP genes. Therefore, we removed the genes from Contig: NT_113931 from our analyses. Furthermore, since many of the draft genome sequences were generated by whole genome shotgun assembly, we took care to identify redundant KRTAP sequences and excluded these from our analysis.Since KRTAP genes are clustered as tandem arrays at just a few chromosomal locations and the genome sequences have high coverage (except platypus), we should be able to obtain near-complete and non-redundant KRTAP gene inventories from these mammalian genomes after in-depth screening and examination (summarized in additional file 1). In contrast to the mammalian genomes, we were unable to identify any KRTAP-like sequences from the chicken, lizard, Xenopus tropicalis or zebrafish genomes. We also note that the keratin associated protein 10-4 annotated in the chicken genome in GenBank (gene id: 425968) is actually an oncogenic transcription factor (JAC), and is not KRTAP-like nor has homology with any mammalian KRTAP sequences. As an alternative strategy to identify KRTAP genes in the chicken, we used Mapviewer from NCBI http://www.ncbi.nlm.nih.gov/mapview/ to identify genes flanking the KRTAP gene clusters in mammals and searched the chicken genome using BLAST to identify orthologous genomic regions. While conserved synteny of the flanking genes was observed within the chicken genome, KRTAP-like genes could not be found. These results indicate that the KRTAP gene clusters are unique to mammals and have been inserted into in the ancestral mammalian genome.We classified our identified KRTAP gene sequences into two categories, intact and pseudo-genes. Pseudogenes are defined as coding sequences that are disrupted by frameshifts and/or stop codons. In placental mammals, rodents appear to contain an expanded KRTAP gene repertoire with a lower level of pseudogenization relative to other mammals both in high (including ultrahigh) cysteine and high glycine KRTAPs (fig. 1). Surprisingly, although humans have the lowest number of intact genes (101) and the highest number of pseudogenes (21, 17.2% of total number), the complete repertoire (122) is still similar in number to that of other mammals. Potentially, changes in the levels of expression of KRTAP genes may account for the relative hairlessness of humans. Opossum has a slightly larger repertoire of genes, and the repertoire in platypus is probably much larger than that we reported, since a more complete genome sequence is required to accurately define gene number.Figure 1Summary of the evolution of the KRTAP gene repertoires in eight mammals. Major events in the evolution of the KRTAP gene family are summarized. The number of KRTAP genes in each species is indicated on the right with \"all\", \"HS\", and \"HGT\" indicating patterns for all, high/ultrahigh cysteine, and high glycine-tyrosine KRTAPs, respectively. The columns represent the numbers of intact, interrupted and all genes and the percentage of pseudogenization calculated as the ratio of number of disrupted gene to all genes, for each of the categories of genes. Stars in the phylogeny indicate the origin of subfamilies, while circles indicate losses.Classification of KRTAP gene family, and tandem cluster in the genomesWithout the influence of evolutionary constraint, pseudogenes evolve faster then functional genes. Accordingly, substitution bias could generate errors in constructing phylogenies; therefore all pseudogenes were excluded from our preliminary analysis on subfamily classification. The high levels of divergence between species and/or subgroups and the homogenization within them caused by frequent gene conversion (see following text), may also introduce a bias into the construction of a phylogeny of all of the high cysteine KRTAPs. To circumvent this problem, we first constructed a neighbor-joining tree of just the human high cysteine KRTAP protein sequences, and used this phylogeny to identify subfamilies (Additional file 2: figure 1). The majority of genes for each subfamily were found as subfamily-specific tandem cluster in the genome (Additional file 2: figure 1 and Additional file 1). Genes with close relationships in our phylogeny tended to be most closely linked in the genome. This positive correlation between phylogeny and chromosomal location has previously been observed in several other gene families [46,47]. Thus we used chromosomal location in combination with phylogeny to refine our subfamily classification. In addition we also considered amino acid sequence composition to generate our final classification. Next we constructed phylogenies of all of the high cysteine KRTAPs for each species alone (Additional file 2: figures 2–8), or combined with the human sequences (Additional file 2: figures 9–15), as well as a phylogeny of the combined mouse and rat sequences (Additional file 2: figure 16). From these phylogenies, together with chromosomal location and amino acid composition, we could identify several new subfamilies not reported previously, which we name subfamilies 28–35. Additionally, we grouped the previously characterized subfamilies 14 and 15 into subfamily 13 based on our phylogeny. In a similar manner, we grouped the glycine-tyrosine rich KRTAPs into 6 subfamilies (subfamilies 6, 7, 8, 19, 20 and 21) based on the phylogenetic tree (fig. 2, and Additional file 2: figure 17). The previously defined human subfamily 22 was combined with subfamily 19. Our recommendations have been endorsed by the HUGO Gene Nomenclature Committee [48].Figure 2Phylogenetic tree of high glycine-tyrosine KRTAP genes of all mammals. Simplified phylogeny of all of the high glycine-tyrosine KRTAP genes generated by the neighbor-joining algorithm using p-distances. Genes of each subfamily are represented by different colors. Numbers on branches are the reliabilities of the branches which are calculated by interior branch tests with 1,000 replications. The bars indicate six subfamilies (6, 7, 8, 19, 20 and 21) of HGT-KRTAP genes.Our phylogeny of intact genes indicated that genes within subfamilies are clustered in the genomic location (fig. 3, and Additional file 1). When pseudogenes were included in the phylogenetic analysis they were also found to be most closely related to their genomic neighbors. To classify interrupted genes that were annotated in unmapped (i.e., unassembled) genomic locations, BLAST was performed against the non-redundant database in GenBank to identify the best hit KRTAP genes, and phylogenetic classification of these were used to help classify the pseudogenes.Figure 3Summary of the chromosomal distribution of KRTAP genes in the human genomes. The relative genomic location of each KRTAP gene is shown for chromosomes 2, 11, 17 and 21. Each gene, and distances between genes are not to scale. Arrowheads indicate the direction of transcription. The clusters are also labeled.It should be noted that there is typically only about one gene per species for subfamilies 16, 24–27, and 29 (table 1), thus, these subfamilies were not used for our subsequent analysis for gene conversion.Table 1Numbers of KRTAP gene in each subfamily in eight mammalian species.subfamilyhumanchimpanzeerhesus macaquemouseratdogopossumplatypusHS-KRTAP14(0)4(0)4(0)4(0)4(0)4(0)4(0)4(1)24(0)4(0)4(1)4(0)4(0)4(0)4(0)4(0)34(1)4(0)4(1)4(0)4(0)4(0)4(0)5(0)414(3)14(3)14(5)18(2)16(3)8(0)30(6)19(3)514(2)15(2)18(1)18(0)20(1)14(2)9(0)7(1)911(0)11(1)9(2)7(2)7(2)7(1)001013(1)13(1)13(0)14(0)14(0)14(0)13(0)12(0)111(0)1(0)1(0)1(0)1(0)1(0)1(0)1(0)125(1)5(1)5(1)5(0)6(0)7(0)6(2)0138(2)6(2)9(3)14(4)13(3)9(3)05(0)161(0)1(0)1(0)1(0)1(0)1(0)1(0)1(0)171(0)1(0)1(0)1(0)1(0)01(0)1(0)241(0)1(0)1(0)1(0)1(0)1(0)00251(0)1(0)1(1)001(0)00261(0)1(0)2(0)1(0)1(0)1(0)03(0)271(0)1(0)1(0)1(0)1(0)1(0)002810(2)11(1)10(0)13(0)12(0)10(0)10(0)9(0)291(0)1(0)1(0)1(0)1(0)1(0)1(0)1(0)3000019(2)19(0)000310003(1)3(0)0003200000007(3)33000000017(2)340001(0)1(0)000350001(0)0000HGT-KRTAP63(0)3(0)5(1)6(0)6(0)6(0)0071(1)1(0)1(0)1(0)1(0)1(0)1(0)083(2)3(2)1(0)1(0)1(0)1(0)1(0)2(0)1914(5)10(3)15(4)12(1)12(1)14(6)00202(0)2(0)3(0)32(0)21(1)8(4)42(6)9(1)214(1)6(0)5(0)4(1)3(0)10(0)011(1)The number in parentheses indicates the number of disrupted genes. HS-KRTAP and HGT-KRTAP indicate high cysteine and high glycine/tyrosine KRTAP respectively.Chromosome distribution of KRTAPKRTAP genes are distributed mainly at five genomic regions in placental and marsupial genomes: Cluster 1 contains genes from subfamilies KRTAP 1, 2, 3, 4, 9, 17, 16, and 29. Cluster 2 contains genes from subfamilies 13, 24–27 and all glycine-tyrosine rich KRTAPs. Cluster 3 possesses genes from subfamilies 10 and 12. Cluster 4 encodes genes of subfamily 28. Cluster 5 corresponds to genes of subfamily 5 (fig. 3). Some variation in gene distribution is observed in some species. In rodents, the new subfamilies 30 and 31 have been inserted into the genomic locations of subfamilies 4 and 9 respectively. Paralogous KRTAP gene clusters have been mapped to human chromosomes 11q13 and chr11p15, with genes residing in these two clusters intermingled in the phylogeny (fig. 3.). Thus suggests that the KRTAP gene cluster at 11q13 is derived from 11p15 potentially representing a segmental duplication. The new chromosome 11q13 gene cluster is unique to primates as an orthologous region is also found in the chimpanzee and rhesus macaque genomes but not in others mammals (additional file 1). In a similar manner the dog genome has generated a new cluster on chromosome 31 that includes three genes, one from subfamily 10 and two from subfamily 12. Similar mechanisms for the origin of new genes at new genomic locations have been observed for other gene families [49,50].Amino acid composition comparison of KRTAP subfamiliesPrevious research has classified the keratin associated proteins by their amino acid composition into three major groups: high-sulfur (~16–30% cysteine), ultra-high sulfur (> 30% cysteine), and high-glycine/tyrosine [26]. Subfamilies 1, 2, 3, 10, 12, 16, 29 and 31, belong to the high-sulfur group; subfamilies 4, 5, 9, 17, 28, 30, 32 and 33 are ultra-high sulfur (table 2). Many high cysteine genes also have a high content of serine. Interestingly, subfamilies 11, 13, 24–27, 29, 34 and 35 have high serine content but relative low cysteine (table 2). The newly identified subfamilies 28 and 30, for which there is no functional or expression data, have the highest cysteine content (39.5%, 50.1% respectively).Table 2Amino acid composition of KRTAPs subfamily genes in mammals.HS-KRTAPsubfamilyCGLPQSTY126.5710.041.679.226.8415.558.041.88229.14.421.4414.46.0310.339.250.56319.934.728.0115.62.98.5310.841.53437.372.98110.575.4716.217.910.51535.8623.670.15.013.3919.250.650.27935.262.651.0211.047.1712.7413.951.321027.612.553.34136.118.764.620.521113.18.084.088.247.4514.7512.162.431222.862.572.1713.816.1821.124.021.331311.4710.615.737.414.1721.315.887.491736.0631.440.264.494.239.913.170249.775.057.459.383.9917.557.317.182575.075.568.216.2819.083.866.282611.39.048.4711.454.0118.085.143.24278.714.076.597.817.8118.067.731.382839.5433.660.011.63.935.82.691.022916.276.013.3411.428.3316.77.842.393019.352.072.4314.565.0717.126.192.153126.531.023.0611.94.7615.0810.660.113238.723.41.9116.174.26108.940.213332.185.422.6515.291.529.173.190.113450.734.650.179.5411.114.428.550.23359.198.384.3211.084.0521.628.115.683610.344.68.0511.496.920.696.91.15HGT-KRTAP613.6140.264.870.190.057.590.2922.8778.8119.164.797.280.1911.496.1312.2685.7223.973.947.1608.592.1520.04196.0736.524.221.380.3310.80.3319.962013.937.614.611.730.265.720.2324.22117.236.820.931.030.2412.60.8720.98The average amino acid content (%) of high cysteine (HS) and high glycine/tyrosine (HGT) KRTAP subfamily genes is shown. C (cysteine), G (glycine), L (leucine), P (proine), Q (glutamine), S (serine), T (threonine) and Y (tyrosine) are single letter codes for amino acids that are abundant in some KRTAP proteins.Concerted evolution yields a high GC (Guanine and Cytosine) content in high cysteine KRTAP gene familyThe evolutionary patterns observed in a multigene family can be attributed to two traditional models: concerted evolution and the birth-and-death process [47]. Members of a gene family under concerted evolution evolve in a concerted manner rather than independently, as a mutation occurring in one member will spread through the entire gene family by the repeated occurrence of unequal crossover and/or gene conversion [47]. Concerted evolution therefore results in the distance between pairs of genes remaining low. Considering the abundant species-specific clusters in the KRTAP gene phylogeny, many should be candidates for concerted evolution. Gene conversion plays a parallel role to unequal crossing over, with their major difference being that the latter can change the copy number of a gene; however, it is difficult to distinguish between these two mechanisms.We identified potential gene conversion events within each subfamily using the methods implemented in the GeneConv program [51] which identifies identical fragments shared between pairs of nucleotide sequences. We found a large number of KRTAP gene pairs for which gene conversion events are suggested in the high/ultrahigh cysteine subfamilies, but significantly fewer, only 4 pairs, in high glycine-tyrosine subfamilies (χ2 = 73.85, p << 10-10) (summarized in additional file 3). The distribution of gene conversion events differs between species, suggesting that different levels of gene conversion occur in each species. For example, 32 gene pairs in rat subfamily 10 suggested evidence for gene conversion, but only 7 pairs were identified for this subfamily in the mouse. The RDP2 program also detected a large number of recombination events within the high cysteine KRTAP genes but fewer in the high glycine/tyrosine genes (additional file 4), and typically suggested that different gene pairs were involved. The identification of different gene pairs by the two methods may reflect differences in how the two programs identify recombination events.Evidence for concerted evolution was also found in changes in base composition of the genes. Gene conversion is a nonreciprocal recombination process in which a DNA segment of a recipient gene is copied from a donor gene and occurs during the repair of double strand breaks by recombination [52]. Recently, it has been discovered that gene conversion introduces a GC nucleotide bias into sequences, the biased gene conversion (BCG) concept, resulting in the enrichment of GC content in DNA sequences undergoing concerted evolution [52,53] resulting in a positive correlation between rate of gene conversion and GC content. Therefore we examined the GC content of KRTAP coding sequences, which we propose scales with the rate of gene conversion. To evaluate the contribution of concerted evolution, we also scaled the level of gene conversion by distance between paralogs in each subfamily. It has been reported that the frequency of gene conversion correlates negatively with the divergence between gene pairs [54]. Synonymous nucleotide sites are expected to evolve neutrally and can be used to evaluate the relative evolutionary divergence between pairs of genes. Intriguingly, high cysteine KRTAP genes contain extremely high GC content, and the GC content is negatively correlated with the divergence within the high cysteine KRTAP gene subfamilies (fig. 4). Comparatively, high glycine/tyrosine KRTAP contain relatively lower GC content (~50%), and a higher synonymous substitution rate (fig. 4), consistent with the detection of fewer gene conversion events.Figure 4Correlation between GC (Guanine and Cytosine) content and divergence between KRTAP genes. The synonymous substitution rates (dS) among paralogs within each subfamily and codon GC content (GC%) (A) and third codon GC content (GC3%) (B) of each subfamily is plotted. Dots and circles represent high cysteine KRTAP (HS) and high glycine-tyrosine KRTAP (HGT), respectively. The linear regression formulae for GC and dS are shown.Phylogenetic relationships can also suggest sequence homogenization, and potentially uncover the level of gene conversion. In the tree of human and chimpanzee KRTAP protein sequences, large quantities of one-to-one orthologies were identified (additional file 2: figure 9), suggesting that intraspecific gene conversion in the past 5 MYRs ago has not obscured orthologous relationships of the human and chimpanzee genes. The number of these one-to-one orthologies is reduced as phylogenetic distances between species pairs increases (additional file 2: figures 9–15).More dynamical evolution of high glycine/tyrosine KRTAPsHigher GC content in high cysteine KRTAP compared to high glycine/tyrosine genes reveals that strong concerted evolution is occurring in former, and that they evolve in a more stably manner. Accordingly, we compared the evolutionary dynamics of the two kinds of KRTAPs by calculating the Pearson correlation coefficient of the number of genes within each subfamily between species. As expected, the coefficient value is significantly higher for high cysteine KRTAP than for high glycine/tyrosine genes (p < 10-4 by Wilcoxon signed ranks test) (fig. 5A), and the two groups of coefficient values are positively correlated (fig. 5B). In particular, subfamily 20 evolved with a very dramatic variation in gene numbers, e.g. mouse and opossum have 30 and 42 genes respectively, but humans only have 2. As the number of gene conversion events detected was close to zero, the false positive rate for detecting positive selection in subfamily 20 should be very low. Positive selection, which was detected on the rat and opossum lineages by likelihood ratio tests (table 3), thus is proposed to be the major force for the dynamic evolution of subfamily 20 and possibly for other high glycine/tyrosine KRTAP genes.Figure 5Evolutionary dynamics of KRTAP genes. A comparison of the evolutionary dynamics as evaluated by Pearson correlation coefficient (r) of gene numbers of each subfamily between species is shown. A) The coefficient value for the high cysteine KRTAP and high glycine/tyrosine genes is shown with standard errors of the estimates. The correlation coefficient of the high cysteine genes is significantly higher than for the high glycine/tyrosine genes. B) A linear regression of the coefficients between high cysteine KRTAP and high glycine/tyrosine KRTAP genes. The two groups of coefficient values are positively correlated at the 1% level.Table 3Likelihood ratio tests for positive selection in subfamily 20 by the site-specific models M1a vs M2a within mouse, rat, and opossum.2ΔLd.f.Parameters estimated under positive selection modelp-valuemouse2.8192p0 = 0.686, p1 = 0.000, p2 = 0.314ω0 = 0.107, ω1 = 1.000, ω2 = 1.4800.244rat13.9102p0 = 0.657, p1 = 0.280, p2 = 0.064,ω0 = 0.108, ω1 = 1.000, ω2 = 13.3709.54E-04opossum18.5752p0 = 0.535, p1 = 0.411, p2 = 0.054ω0 = 0.096, ω1 = 1.000, ω2 = 6.8949.26E-05DiscussionWe have described the near-complete inventories of KRTAP genes from the genomes of human, chimpanzee, and rhesus macaque representing primates, mouse and rat representing rodents, dog representing carnivore, opossum representing marsupial, and platypus as a monotreme, and conducted comprehensive analyses of the evolutionary patterns for this gene family, including phylogenetic classification, and the detection of gene conversion.Among the mammals that we studied, the genomes of each species have members of most of the subfamilies suggesting that the majority of KRTAP gene subfamilies originated and diverged before the mammalian radiation (fig. 1). Accordingly, the mammalian ancestor should also have had a high diversity of KRTAP genes and enjoyed a similar range and spectrum of hair characteristics that is seen in modern mammalian species. The genes for subfamilies that emerged in the early mammals account for about 74% total number of genes in present mammalian genomes. We advocate that the rapid emergence of the KRTAP gene family correlates with the evolution of mammalian hair and that the rapid emergence of plentiful hair contributed to the successful radiation of homothermal mammals by helping them to retain body heat since hair acts as an insulator [13]. In contrast, Homo sapiens has recently lost body hair function, presumably because humans can obtain heat and can keep cold out by using clothing. Despite a lack of hair, humans actually have a similar density of hair follicles to apes [15], which possibly explains why human do not have a significant fewer number of KRTAP genes. Perhaps, the changes in human hair are due to the reduction in expression of the KRTAP genes. In contrast to humans, rodents have an expanded KRTAP gene family. Perhaps, mouse and rat need more hair as they are adapted to the nocturnal environment. Despite the proposed similar range and diversity of hair characteristics in the ancestor of mammals, the current traits, and content, of hair likely diverged significantly from their ancestral characteristics as KRTAP sequences have diverged dramatically among species due to gene conversion.Both gene conversion and unequal crossing over have occurred during the evolution of the KRTAP. The evolution of several KRTAP gene subfamilies, such as subfamilies 1, 2 and 3 fit well with a model of punctuated equilibrium (table 1), where morphological divergence as well as speciation occurs in a burst-like manner with rapid evolutionary change followed by long periods of constancy [55]. These KRTAP genes show evidence for fewer gene duplication or loss events, suggesting that a low level of unequal crossing over has occurred within these subfamilies, and that gene conversion may predominate. In addition, the syntenic arrangement of genes of these subfamilies has not been disturbed by post-duplication rearrangement events, suggesting a strong constraint has been exerted upon their distribution. In contrast, other KRTAP genes, such as subfamilies 4 and 5 (table 1) have evolved relatively dynamically likely including unequal crossing over as changes in the copy number of this subfamily is observed between species.Changes in gene numbers within subfamilies between mammals may explain differences in the observed hair morphology between species. No genes of subfamily 9 were identified in opossum or platypus, suggesting that this subfamily originated after the divergence of placental and marsupial mammals. In a similar manner, genes of subfamily 12 emerged before the divergence of marsupials but after the divergence from monotremes (fig. 1). Intriguingly, subfamily 13 has been lost on the marsupial lineage, and the subfamilies 24, 26 and 27 which are adjacent to subfamily 13 in the genome, are also not found in the opossum genome. We used Mapviewer from NCBI to identify genes flanking this cluster in the human genome and searched the opossum genome using BLAST to identify an orthologous genomic region. An ortholog of the human gene that flanks the human KRTAP genes is only about 13 kbp away from the remainder of the KRTAP cluster in the opossum genome, which indicates the loss of this region in the opossum was due to a deletion event. Subfamily 30, which is a member of the ultra-high sulfur (~50%) group, along with subfamily 34 are unique to rodents, thus may be partially responsible for the unique characteristics of hair in rodents. Subfamily 35 is mouse-specific, while subfamilies 32 and 33 are platypus-specific, which could account for species-specific hair characteristics.The high-glycine/tyrosine (HGT) gene repertoire evolve more dynamically within mammals with increased levels of pseudogenization (fig. 1), for example, mice possesses 56 genes, while primates have considerably fewer genes with only 27 and 25 genes in human and chimpanzee respectively. The gene number in platypus is likely to be underestimated because HGT cluster in platypus is shorter than that of other mammals (only about 100 kbp in platypus, but ~500 kbp in human, ~800 kbp in mouse, and ~350 kbp in opossum), and the incomplete nature of this genome sequence. However, the dynamics of HGT subfamilies does not appear to be due to unequal crossing over, as unequal crossing over should also generate a GC content bias that is not observed in these genes (personal communication, Gabriel Marais).Tandemly arrayed paralogous genes with similar function can provide combinatorial complexity to biological diversity [56]. This extraordinary evolutionary feature has been observed in many multigene families responsible for processes that face enormous external signals. For instance, the sensory system such as the olfactory receptor, vomeronasal receptor and sweet/umami receptor, bitter receptor genes, require a huge combination of diverse receptors for the diverse ligands they encounter in a tremendous range of molecular environments [57]. Another classic example is the immunoglobulin and T-cell receptor protein superfamily which use recombination to generate large quantities of antigen recognition complexes to allow an immune responses to rapidly evolving pathogens [58]. We therefore speculate that an analogous process occurs within the KRTAP gene family, where the KRTAP and keratin proteins have combined in unique combinations to generate the high diversity of hair phenotypes that are observed both between and within species, and even within individuals.ConclusionWe have described the near-complete inventories of KRTAP genes in eight mammalian genomes. We found that the KRTAP family was unique to mammals, KRTAP gene repertoire was expanded in rodents, and surprisingly, humans had a similar number of genes as other primates, inconsistent with the hairlessness of humans. The high glycine-tyrosine KRTAP genes have evolved more dynamically, with fewer gene conversion events and thus have a lower GC content compared with high cysteine KRTAPs. We propose that the mammalian ancestor should have a diverse KRTAP gene repertoire, and that hair content characteristics have evolved and diverged rapidly among mammals because of rapid divergent evolution of KRTAPs between species caused by concerted evolution.MethodsRetrieval of sequencesSome KRTAP genes have previously been annotated to possess introns, which generally complicates gene prediction and identification. All of the intron-containing KRTAP genes have short introns, and the sequences of these introns are similar to the repeated regions found within the exons, and many are predicted to be alternatively spiced. All of the introns could be included in a primary transcript that can be translated in-frame with the coding exons, they just have longer repetitive regions. Therefore, we hypothesize that all of the KRTAP genes can generate an mRNA sequence that is intron-less.We identified KRTAP gene repertoires in the genome assemblies from human (Homo sapiens) (build36.2), chimpanzee (Pan troglodytes) (build2.1), rhesus macaque (Macaca mulatta) (build1.1), mouse (Mus musculus) (build36.1), rat (Rattus norvegicus) (RGSC v3.4), dog (Canis familiaris) (build2.1), opossum (Monodelphis domesticus) (MonDom5) and platypus (Ornithorhynchus anatinus) (build1.1). We used the BLASTn algorithm [59] to search these genomes using all known human KRTAP genes as queries. Each newly identified putative KRTAP gene was used as a query using BLAST http://blast.ncbi.nlm.nih.gov/Blast.cgi against the non-redundant GenBank database to check whether their best hit was a KRTAP gene. The chicken (Gallus gallus) (Build 2.1), zebrafish (Danio rerio) genomes (Zv6) in NCBI [60], lizard (Anolis carolinensis) genome at UCSC http://genome.ucsc.edu/ and the Xenopus tropicalis genome at JGI [61] genomes were also searched using BLAST for KRTAP-like sequences. Sequences that possessed an interrupting stop codons and/or frame-shifts caused by insertions or deletions were denoted as pseudogenes.Phylogenetic reconstructionIn order to classify the members of the KRTAP gene family, protein sequences were used to construct phylogenetic trees using the neighbor-joining method with p-distances with MEGA3.0 http://www.megasoftware.net[62] after alignment with ClustalW http://www.ebi.ac.uk/Tools/clustalw/index.html[63]. The reliability of the trees was evaluated by the interior branch tests with 1,000 replications.Detection of recombinationWe employed the GeneConv program http://www.math.wustl.edu/~sawyer/geneconv/[51] to identify potential gene conversion events in the KRTAP coding sequences. Gene conversion is a process where a segment of DNA from one allele of a gene is copied and replaces the sequence in another allele or gene. Accordingly, GeneConv extends a method previously described by [51] and detects this process by identifying shared fragments between pairs of sequences. Global Bonferroni corrected P values were calculated to evaluate the statistical significance of the observed fragment lengths and are compared to a simulated distribution (10,000 iterations) of the same number of sequences with similar variation. Lower P values suggest a greater probability that a gene conversion event has occurred. GeneConv has a significant limitation in that it is unable to distinguish between gene conversion and unequal crossing over events, but to date no other effective bioinformatic method have been described that can distinguish between these two types of events. We therefore, can not distinguish gene conversion and unequal crossing over.The divergence among paralogs could also reflect the level of conversion which can homogenize paralogs. Nucleotide sequences were back-translated from protein sequences after alignment by ClustalW [63]. Alignments were modified manually if necessary. Approximate synonymous substitution rate (Ks) values within subfamilies were calculated by the modified Nei-Gojobori (p-distance) method with a transition/transversion ratio of 2 [64].Recombination events were also detected using the RDP2 software package http://darwin.uvigo.es/rdp/rdp.html[65]. Evidence for recombination was detected by running RDP, BootScan, MaxChi and Chimaera with 1,000 permutations. Sequences were considered linear. The highest acceptable P value cut-off was set to 0.01. Bonferroni correction was employed.Adaptive evolution analysisThe site-specific models M1a and M2a implemented in PAML http://abacus.gene.ucl.ac.uk/software/paml.html were used to detect potentially positively selected sites in the subfamily [66-68]. Considering the high false positive rate of likelihood ratio tests, particularly when there is frequent recombination [69,70], we only detected positive selection in subfamily 20, a family in which gene conversion does not appear to occur and has expanded dramatically on the mouse, rat and opossum lineages.Statistical analysisWe calculated the potential number of gene pairs where gene conversion could occur by ∑Nij(Nij-1)/2, where Nij is the intact gene numbers in subfamily i within species j, and N must be higher than 2 for gene conversion to be detected by the GeneConv program. The chi test was used to detect statistical significance in the difference in number of gene conversion events occurring between high cysteine and high glycine/tyrosine KRTAP. The Pearson correlation coefficient of the number of genes in each subfamily was determined for pairs of species (table 1). The evolutionary dynamics of the high cysteine and high glycine/tyrosine KRTAP genes was evaluated by comparing the values of the Pearson correlation coefficients.Authors' contributionsDDW, DMI and YPZ designed the research and outlined the manuscript together, and DDW drafted the manuscript. ALL authors have read and approved the final manuscript.Supplementary MaterialAdditional file 1table 1. KRTAP genes in the human, chimpanzee, rhesus macaque, dog, mouse, rat, opossum, and platypus genomes.Click here for fileAdditional file 2figure 1–figure 17. Figure 1–Figure 16 are the phylogenetic trees of high/ultrahigh cysteine KAPs of human (Figure 1), chimpanzee (Figure 2), rhesus macaque(figure 3), dog (figure 4), mouse (figure 5), rat (figure 6), opossum (figure 7), platypus(figure 8), human and chimpanzee (figure 9), human and rhesus macaque (figure 10), human and dog (figure 11), human and mouse (figure 12), human and rat (figure 13), human and opossum (figure 14), human and platypus (figure 15), mouse and rat (figure 16). Figure 17 is the phylogenetic tree of high glycine/tyrosine KRTAPs. h represents human, c is chimpanzee, rh is rhesus macaque, d-dog, m-mouse, r is rat, o is opossum, and p is platypus. The values on the branches are reliabilities, which are evaluated by the interior branch tests with 1,000 replications. Only values higher than 50% are noted.Click here for fileAdditional file 3table 2. Pairs of genes with significant statistical support for gene conversion. 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batch_9/PMC2528138.json

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+{
+    "id": "PMC2528138",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528138\nAUTHORS: S Franceschi, L Dal Maso, M Rickenbach, J Polesel, B Hirschel, M Cavassini, A Bordoni, L Elzi, S Ess, G Jundt, N Mueller, G M Clifford\n\nABSTRACT:\nBetween 1984 and 2006, 12 959 people with HIV/AIDS (PWHA) in the Swiss HIV Cohort Study contributed a total of 73 412 person-years (py) of follow-up, 35 551 of which derived from PWHA treated with highly active antiretroviral therapy (HAART). Five hundred and ninety-seven incident Kaposi sarcoma (KS) cases were identified of whom 52 were among HAART users. Cox regression was used to estimate hazard ratios (HR) and corresponding 95% confidence intervals (CI). Kaposi sarcoma incidence fell abruptly in 1996–1998 to reach a plateau at 1.4 per 1000 py afterwards. Men having sex with men and birth in Africa or the Middle East were associated with KS in both non-users and users of HAART but the risk pattern by CD4 cell count differed. Only very low CD4 cell count (<50 cells μl−1) at enrolment or at HAART initiation were significantly associated with KS among HAART users. The HR for KS declined steeply in the first months after HAART initiation and continued to be low 7–10 years afterwards (HR, 0.06; 95% CI, 0.02–0.17). Thirty-three out of 52 (63.5%) KS cases among HAART users arose among PWHA who had stopped treatment or used HAART for less than 6 months.\n\nBODY:\nAt the beginning of the HIV epidemic, Kaposi sarcoma (KS) was one of the most common manifestations of AIDS (Dal Maso et al, 1995; Biggar et al, 1996), present during the mid-1980s in 25% of individuals at the time of AIDS diagnosis in the United States, but decreased steadily through the late 1980s and mid-1990s, down to 2% after the advent and widespread use of highly active antiretroviral therapy (HAART) in 1996 (Engels et al, 2006).A similar temporal pattern was observed for KS in Australia (Grulich et al, 2001) and in European countries (Dal Maso et al, 1995; Rezza et al, 2000; Franceschi et al, 2003; Mocroft et al, 2004; Clifford et al, 2005), but detailed data on the long-term trends of KS incidence in Europe are limited. We therefore took advantage of the more than 20 years of follow-up data available from the Swiss HIV Cohort Study (SHCS) to assess changes in the incidence of and risk factors for KS before and after HAART use.Materials and methodsThe SHCS is an ongoing study that has been enrolling people with HIV/AIDS (PWHA) over 16 years of age since 1988, with some retrospective enrolment going back to 1984, from seven large hospitals in Swiss cities (Basel, Bern, Geneva, Lausanne, Lugano, St Gallen, and Zurich) (www.shcs.ch). Follow-up visits take place every 6 months and all AIDS-defining events, including KS diagnosis and death, are recorded. The present study included PWHA enrolled up to 30 September 2005, and information recorded in the SHCS database up to 31 March 2006. People with HIV/AIDS were excluded from the present study if they (1) did not have information on date of birth, gender, or HIV transmission category (number (n)=54), (2) were diagnosed with KS at enrolment or earlier (n=368), or (3) had no follow-up visits (n=131).A total of 597 KS cases were included in our present study: 545 were identified from the SHCS database, and 52 through record linkage with eight Swiss Cantonal Cancer Registries (Clifford et al, 2005). Six of these cancer registries (Basel, Geneva, Ticino, St Gallen and Appenzell, Vaud, and Zurich) overlap directly with six of the seven cantons covered by SHCS hospitals (all except Bern). The Neuchâtel and Valais Cancer Registries do not directly overlap with SHCS hospitals, but some residents of these cantons are followed in a neighbouring SHCS hospital. Places of birth were classified as Europe (Switzerland and the rest of Europe, 87.1% of PWHA) and Africa or the Middle East (8.1%). The few SHCS participants born outside Europe, but in countries where KS is not endemic (e.g., the Americas and Asia; Hengge et al, 2002), were included in the Europe category. Conversely, the few PWHA born in the Caribbean were included in the Africa/Middle East category. Histological confirmation was mentioned in the majority of KS cases, but presentation site (i.e., skin only vs other) was available only for 382 (64%) KS cases.Highly active antiretroviral therapy was defined as a combination of at least three drugs, including a protease inhibitor or a non-nucleoside transcriptase inhibitor, or three nucleosides including abacavir. Individuals who had used HAART for more than 1 month were classified as users. Treatment interruption was defined as in a previous report from the SHCS (Taffé et al, 2002), as absence of any antiretroviral drug in PWHA who were previously receiving HAART. Taffé et al (2002) evaluated the impact of interruptions of less than 3 months on the progression of HIV infection, whereas we focused on the impact of interruptions of 3 months or more on KS incidence. CD4 cell counts at enrolment in the SHCS and, among HAART users, at, or within 6 months before HAART initiation were retrieved.For each participant, person-years (py) at risk were calculated between enrolment and KS diagnosis, death, or last follow-up visit, whichever occurred first. Incidence rates per 1000 py were standardised for gender and age based on the enrolled population in the overall study period, using the direct method (Breslow and Day, 1987). Ninety-five percent confidence intervals (CI) of incidence were computed according to the Poisson distribution (Breslow and Day, 1987). The effect of various risk factors on KS onset was assessed using hazard ratios (HR) and corresponding 95% CI, estimated by means of the Cox proportional hazard model (Cox, 1972), and adjusted for SHCS centre, gender, age (in 5-year groups), HIV transmission category (MSM and non-MSM) and, when mentioned, CD4 cell count at enrolment or at HAART initiation (<50, 50–99, 100–199, 200–349, ⩾350 cells μl−1, and unknown). Calendar period, HAART use, and months after HAART initiation and after treatment interruption were introduced as time-dependent variables.This study was approved by the local ethics committees of the collaborating SHCS clinics and of the International Agency for Research on Cancer.ResultsThe present study included 12 638 PWHA who were KS-free at enrolment and among whom 597 incident KS cases were identified (8.2 per 1000 py; 95% CI, 7.6–8.9). Fifteen per cent of PWHA had AIDS at enrolment and an additional 3119 (24.7%) developed it during follow-up. Among the latter, KS was the AIDS-defining illness in 268, whereas 329 KS cases developed in PWHA who had already manifested another AIDS-defining illness.Figure 1 shows KS temporal trends: overall KS incidence was 33.3 per 1000 py in 1984–1986 and did not change significantly in the subsequent periods until 1996–1998, when it fell to 5.1 (95% CI, 3.9–6.5) per 1000 py. Kaposi sarcoma incidence further decreased to 1.4 per 1000 py in 1999–2001 and remained constant thereafter. Temporal trends in KS incidence were chiefly driven by men having sex with men (MSM), but they were consistent among other HIV transmission categories (Figure 1).A large proportion (48.4%) of available py derived from HAART users and Table 1 shows the incidence and HR of KS by various characteristics separately among non-users and users of HAART. Incidence of KS decreased from 15.0 per 1000 py in non-users to 1.3 per 1000 py in users (HR, 0.11; 95% CI, 0.08–0.14). Among non-users of HAART, intravenous drug users (HR, 0.09; 95% CI, 0.06–0.13), and heterosexuals and other HIV transmission categories (HR, 0.27; 95% CI, 0.20–0.36) showed a lower KS incidence than MSM. The HR for KS was increased among PWHA older than 35 years (HR, 1.53; 95% CI, 1.29–1.82) and those born in Africa/Middle East (HR, 1.84; 95% CI, 1.10–3.06). Kaposi sarcoma risks among non-users of HAART steeply increased with decreasing CD4 cell count (HR for <50 vs ⩾350 cells μl−1, 12.85; 95% CI, 9.59–17.23). These associations were also present, but were weaker, among HAART users with the exception of the association with place of birth that became stronger (HR for Africa/Middle East vs Europe among HAART users, 6.49; 95% CI, 2.79–15.11). In contrast with non-users, no change in the HR for KS was seen among HAART users with CD4 cell counts in the range of 50–⩾350 cells μl−1 and the only significant risk increase was found for CD4 cell count less than 50 cells μl−1 at enrolment (HR, 3.26; 95% CI, 1.53–6.91). On account of the rarity of KS among HAART users, HRs showed, however, broad CIs (Table 1).Among HAART users, CD4 cell count at treatment initiation below 50 cells μl−1 (HR vs ⩾350 cells μl−1, 5.36; 95% CI, 2.08–13.80) (data not shown) was even more strongly associated with KS risk than CD4 cell count at enrolment. Furthermore, KS risk was greatly increased among PWHA who had stopped using any antiretroviral drugs for at least 3 months (HR, 8.14; 95% CI, 4.01–16.54) (Table 1). Additional adjustment for CD4 cell count at HAART initiation did not modify the HR for treatment interruption (HR, 9.45; 95% CI, 4.64–19.25, data not shown).Skin was reported as the presentation site in the majority of KS cases (74.3%). Temporal trends and associations with HAART use and CD4 cell count (overall and in separate strata by HAART use) did not differ by KS presentation site (data not shown).Figure 2 shows the HR for KS in different periods after HAART initiation compared with non-users. The HR of KS was already reduced by 76% in the first 5 months of use and declined to 0.06 (95% CI, 0.02–0.17) in the subsequent 6 months of use. The reduction in KS risk persisted unchanged up to 84–119 months after HAART initiation (HR, 0.06; 95% CI, 0.02–0.16) (Figure 2).Finally, the 52 HAART users who developed KS were individually reviewed and classified into the following groups: (1) no antiretroviral drug in the 3 months before KS diagnosis; (2) recent initiation of HAART (<6 months before KS diagnosis); (3) severe immunodeficiency (CD4 cell count <100 cells μl−1 at KS onset while on HAART for ⩾6 months); and (4) none of the above (data not shown). Eighteen (34.6%) KS cases had not been taking any antiretroviral drug for 3 months or more, and in 10 instances for 12 months or longer. Recent initiation of HAART was identified in 15 KS cases, among whom five of the nine KS were from Africa/Middle East. Severe immunodeficiency was identified among 10 KS cases. Nine KS cases, all from the MSM transmission category, could not be assigned to any of the three categories above. Five of them (aged 35, 49, 52, 56, and 63 years) had a CD4 cell count ⩾400 cells μl−1 (i.e., 405, 440, 557, 596, 782) at KS diagnosis.DiscussionOur study shows the dramatic decline of KS incidence in the SHCS following the advent of HAART. This expands an earlier report (Ledergerber et al, 1999) from this cohort showing that by 1998 the KS decline was at least as large as that seen for opportunistic infections. Similar reductions in the incidence of KS among PWHA were seen in many other studies (International Collaboration on HIV and Cancer, 2000) although the decrease started earlier (i.e., even before the introduction of HAART) in the United States (Biggar et al, 1996; Engels et al, 2006) and Australia (Grulich et al, 2001) than in Europe (Dal Maso et al, 1995). The prevalence of KS herpesvirus, the cause of KS (IARC, 1997), may have been especially high in the first wave of HIV infection in MSM in the United States and Australia (Osmond et al, 2002).Highly active antiretroviral therapy became rapidly available to SHCS participants and, by 1997, 80% of them were using three antiretroviral drugs or more (www.shcs.ch). The proportion of SHCS participants with CD4 cell counts <350 cells μl−1 who had never been treated was small (<3%) in 2006 in all HIV transmission categories. Despite the widespread use of HAART and the introduction after 1996 of successively more potent antiretroviral drugs, KS incidence in the SHCS seems to have reached a plateau after 2001, as reported among AIDS patients in the United States (Engels et al, 2006).In addition to making KS a relatively rare event, HAART use has also diminished the variation in KS risk by host characteristics, including gender, age group, and to some extent, HIV transmission category and CD4 cell count at enrolment as compared with that found among non-users. Only a count <50 cells μl−1 at enrolment or HAART initiation was associated with an increased HR for KS. Reduced importance of CD4 cell count at enrolment in HAART users vs non-users was also seen in the SHCS for non-Hodgkin's lymphoma, but the impact of HAART on non-Hodgkin's lymphoma was weaker (HR, 0.26; 95% CI, 0.20–0.33) than on KS, and, hence, non-Hodgkin's lymphoma incidence (1.9; 95% CI, 1.6–2.6 per 1000 py) became higher than KS incidence among HAART users (Polesel et al, 2008).Kaposi sarcoma risk was already reduced by over 90% after 1 year of HAART and it did not show any sign of increasing again for at least 10 years. The decline of non-Hodgkin's lymphoma risk after HAART initiation was more gradual than for KS, but equally prolonged (Polesel et al, 2008).Approximately one-third of HAART users in the SHCS had one or more interruptions of antiretroviral treatment (Taffé et al, 2002) due in most cases either to intolerance to drugs, or social factors (i.e., being an intravenous drug user, poor education, etc.), and not to treatment failure. In our study, the absence of any antiretroviral treatment for 3 months or more was associated with an eight-fold increased KS risk, thus confirming the danger of treatment interruption already reported with respect to progression to AIDS or death (Holkmann et al, 2007). Significantly higher KS incidence among PWHA who were assigned to the CD4 cell-guided intermittent antiretroviral treatment arm than those assigned to the continuous treatment arm was also shown in a randomised clinical trial (Silverberg et al, 2007).Of the 52 KS among HAART users, 23 arose among people who had either stopped using HAART at or had initiated treatment less than 6 months before KS diagnosis. Recent initiation of HAART in the SHCS seemed especially important among KS cases born in Africa/Middle East, suggesting possible delays in the diagnosis or treatment of HIV infection. Ten KS cases arose in PWHA whose CD4 cell count was very low despite concurrent HAART use whereas 5 MSM developed KS despite being on HAART and having CD4 cell counts at which AIDS-related KS is seldom seen (Biggar et al, 2007). The occurrence of KS cases in PWHA with high CD4 cell counts and undetectable viral loads has already been reported in the United States after 1996 (Maurer et al, 2007; Krown et al, 2008). It is possible that, with the ageing of PWHA, those who are co-infected with KS herpesvirus may develop KS despite good control of HIV infection.Weaknesses of our study are the lack of information on year of HIV seroconversion and on the presence of KS herpesvirus co-infection. Furthermore, we evaluated HAART use by intention-to-treat, that is, without subtracting all periods where treatment had been stopped, so its efficacy may be underestimated. 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+}

batch_9/PMC2528141.json

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+{
+    "id": "PMC2528141",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528141\nAUTHORS: A W Hsing, L C Sakoda, A Rashid, J Chen, M C Shen, T Q Han, B S Wang, Y T Gao\n\nABSTRACT:\nThough obesity is an established risk factor for gall bladder cancer, its role in cancers of the extrahepatic bile ducts and ampulla of Vater is less clear, as also is the role of abdominal obesity. In a population-based case–control study of biliary tract cancer in Shanghai, China, odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for biliary tract cancer in relation to anthropometric measures, including body mass index (BMI) at various ages and waist-to-hip ratio (WHR), adjusting for age, sex, and education. The study included 627 patients with biliary tract cancer (368 gall bladder, 191 bile duct, 68 ampulla of Vater) and 959 healthy subjects randomly selected from the population. A higher BMI at all ages, including early adulthood (ages 20–29 years), and a greater WHR were associated with an increased risk of gall bladder cancer. A high usual adult BMI (⩾25) was associated with a 1.6-fold risk of gall bladder cancer (95% CI 1.2–2.1, P for trend <0.001). Among subjects without gallstones, BMI was also positively associated with gall bladder cancer risk. Regardless of BMI levels, increasing WHR was associated with an excess risk of gall bladder cancer risk, with those having a high BMI (⩾25) and a high WHR (>0.90) having the highest risk of gall bladder cancer (OR=12.6, 95% CI 4.8–33.2), relative to those with a low BMI and WHR. We found no clear risk patterns for cancers of the bile duct and ampulla of Vater. These results suggest that both overall and abdominal obesity, including obesity in early adulthood, are associated with an increased risk of gall bladder cancer. The increasing prevalence of obesity and cholesterol stones in Shanghai seems at least partly responsible for the rising incidence of gall bladder cancer in Shanghai.\n\nBODY:\nBiliary tract cancers, which include cancers of the gall bladder, extrahepatic bile duct, and ampulla of Vater, are rare but highly fatal (Hsing et al, 2006). Apart from gallstones, the aetiology of biliary cancers is obscure (Wistuba and Gazdar, 2004; Hsing et al, 2007c). Obesity is closely linked to both gallstones and gall bladder cancer (Jorgensen, 1989; Kato et al, 1992; Stampfer et al, 1992; Hsing et al, 2006), although its relationship with other biliary cancers is less clear. It is also unclear at what age excess body fat most affects disease risk, as measurements of body size have been mainly related to a single point in time, and whether obesity independently influences biliary cancer risk, given the close connection between obesity and gallstone risk and between gallstones and biliary tract cancer. In addition, the role of abdominal obesity or body fat distribution in biliary tract cancer are unclear, although abdominal obesity is more closely associated with lipid and hormone metabolism (Giorgino et al, 2005).Thus, to clarify further the role of overall and abdominal obesity in biliary tract cancer, in a large population-based case–control study in Shanghai, China, we have examined the roles of body mass index (BMI), both usual (adult) and at various time points in life, and waist-to-hip ratio (WHR).Materials and methodsThe study protocol was approved by the Institutional Review Boards of the US National Cancer Institute (NCI) and the Shanghai Cancer Institute (SCI). Details of the study have been reported elsewhere (Hsing et al, 1998, 2007a, 2007b, 2007c, 2007d). Patients with primary biliary cancer (ICD-9, 156) newly diagnosed between June 1997 and May 2001 were identified through a rapid-reporting system established between the Shanghai Cancer Institute and 42 collaborating hospitals in urban Shanghai. Judging from incidence data reported to the Shanghai Cancer Registry, this system captured over 95% of all biliary cancer cases diagnosed within the study period. Both independent and consensus review of histologic data by expert pathologists positively confirmed the reported diagnoses for more than 70% of the cancer cases. The remaining cancer cases were confirmed through clinical review of medical imaging data from computed tomography, magnetic resonance imaging, ultrasonography, or endoscopic retrograde cholangiopancreatography. In addition, healthy individuals were randomly chosen as population controls, frequency-matched on the age and sex of the cancer case, using the demographic information recorded on the personal registration cards of the Shanghai Resident Registry.In-person interviews were conducted using a structured questionnaire to elicit information on potential risk factors, including demographic characteristics, cigarette and alcohol use, medical history, diet, and family history of cancer. All interviews were tape-recorded and reviewed to verify that these data were recorded accurately and to ensure that each interview was conducted properly. In addition, 5% of the study subjects were randomly selected and re-interviewed, with a concordance rate of over 90% on responses to key questions across the two interviews. Of those eligible, 627 cancer patients and 959 population control subjects agreed to participate and provided written informed consent at enrollment, a response rate of over 90% for cases and 82% for controls. Among participating controls, 85% also consented to abdominal ultrasound screening for gallstone detection. Individuals were defined as having biliary stones if they had previously undergone cholecystectomy or had gallstones.At the interview, subjects were asked for their adult height, current weight; usual weight 5 years before interview, usual weight during specific age decades (i.e., at ages 20–29, 30–39, 40–49, 50–59 years), maximum adult weight (excluding pregnancy weight, for women), age at and duration of maximum weight, main area of weight gain on the body, and history of adult weight cycling (⩾7 kg gain or loss). Physical measurements of standing height, weight, and waist and hip circumference were also taken at the interview. Each measure was taken two times. When the difference between the two measurements exceeded predetermined tolerance limits (height, 2 cm; weight, 1 kg; waist and hip circumference, 2 cm), an additional measurement was taken. Final measurements for height, weight, and waist and hip circumference were determined by averaging the two closest values.Statistical analysisBMI, weight in kilograms divided by the square of height in meters (kg/m2), was used to assess overall obesity. Self-reported data were used to calculate BMI at specific age periods and maximum adult BMI, respectively. The change in BMI over time, defined as the difference in BMI between age intervals (e.g., from the ages 20–29 to 30–39 years), was also computed. Subjects were classified into the BMI categories established by the World Health Organization (WHO) for Asian populations, with the BMI category of 18.5–22.9 kg/m2, defined as ‘normal’ and used as the reference group (WHO, 2000). Body mass index categories of ‘overweight’ (23.0–24.9) and ‘obesity’ (⩾25) were combined when the number of cases in either category was small. For other anthropometric measures, such as height and BMI change, subjects were categorized according to either the tertile or quartile distributions of the controls. WHR, waist circumference divided by hip circumference, was used as a measure of abdominal adiposity. As bile duct and ampulla of Vater cases are diagnosed at a late stage, data on waist and hip circumference are less reliable, and WHR was not computed for these two subsites.Unconditional logistic regression was used to calculate odds ratios and 95% confidence intervals for each cancer subsite associated with BMI and other anthropometric measures, adjusting for age, sex, and education. Gall bladder cancer patients were compared with controls without a history of cholecystectomy (n=49), whereas patients with bile duct or ampullary cancer were compared with all control subjects. Additional covariates, including cigarette smoking, alcohol use, and history of diabetes and hypertension, were evaluated as potential confounding variables but were not included in the final models, as they did not appreciably change the risk estimates. Sex-specific analyses were performed to evaluate differences between men and women in risk associated with body size. Tests for linear trend in risks of cancer were conducted when appropriate, with anthropometric variables evaluated on a continuous scale. All tests were two-sided, with P<0.05 defined as statistically significant.ResultsTable 1 shows selected characteristics of study subjects. As shown, more women were diagnosed with gall bladder cancer than men, whereas slightly more men were diagnosed with cancers of the extrahepatic bile duct and ampulla of Vater. Cases and controls were similar in age. Relative to controls, patients with gall bladder cancer were less educated. Gall bladder cancer case patients were less likely to smoke cigarettes and drink alcohol regularly than control subjects, whereas smoking was more common among those with bile duct and ampullary cancers. Cancer patients were more likely to have gallstones and diabetes.Table 2 shows the risk of biliary cancer by subsite in relation to body size. Quartile cutoffs were used for gall bladder cancer, whereas tertiles were used for extrahepatic bile duct and ampulla of Vater cancers because of their smaller numbers of cases. All BMI-related variables were positively associated with gall bladder cancer risk, after adjustment for age, gender, and education level. BMI in early adulthood (ages 20–29 years) remained significant after adjustment for gallstones (OR=2.04, 95% CI: 0.9–4.8, P for trend=0.01) or among subjects without gallstones (OR=3.7, 95% CI 1.1–12.1) (data not shown). Although larger waist circumference was only slightly associated with an increased risk of gall bladder cancer, a high WHR (>0.897) was associated with a 4.7-fold risk (95% CI 3.1–7.2, P for trend <0.0001). There was no clear association with cancers of the extrahepatic bile duct or ampulla of Vater. Timing and duration of maximum adult weight, primary location of weight gain, and weight cycling did not appear to be related to risk for stones or cancer (data not shown), although too few subjects reported a history of weight cycling for reliable assessment.Table 3 presents gall bladder cancer risk cross-classified by four levels (quartiles) of BMI and three levels (tertiles) of WHR. Regardless of BMI similarly increasing levels of WHR were strongly associated with excess risk of gall bladder cancer, with WHR having a greater impact than BMI. The highest risk is seen for subjects with both a high BMI and a high WHR, with those in the highest categories of BMI (⩾25) and WHR (>0.87) having an 8.3-fold risk of gallstones (95% CI 4.6–14.9) and a 12.6-fold risk of gall bladder cancer (95% CI 4.8–33.2), relative to subjects with a low BMI and WHR.DiscussionIn this population-based study, WHR and BMI at all ages, including early adulthood, were associated with an increased risk of gall bladder cancer. These results suggest that both overall and abdominal obesity play an important role in the aetiology of gall bladder cancer. It seems likely that the increasing prevalence of obesity and cholesterol stones in Shanghai is at least partly responsible for the rising incidence of gall bladder cancer in Shanghai. These findings for gall bladder cancer are consistent with those of previous studies (Zatonski et al, 1997; Wolk et al, 2001; Calle et al, 2003; Samanic et al, 2004; Engeland et al, 2005). We found that the effect of obesity on gall bladder cancer is not mediated entirely by gallstones, as the BMI effect persisted after adjustment for gallstones. Among those without gallstones, BMI is also associated with gall bladder cancer risk. However, it should be noted that the statistical power for analysis stratified by gallstones was limited, since less than 20% of the gall bladder cancer patients had no history of stones and less than 25% of the controls had a history of stones. Although gallstones are closely linked to both BMI and gall bladder cancer, in assessing the effect of BMI, it may not be necessary to adjust for gallstones, which are thought to be an important factor in the causal pathway. Thus, adjustment for gallstones would attenuate the true association between obesity and biliary cancer risk. However, the gallstone-adjusted results suggest that obesity may also increase the risk of gall bladder cancer through pathways not related to gallstone pathogenesis. In addition to affecting lipid metabolism, obesity can affect the risk of gallstones and gall bladder cancer through adverse changes in the metabolism of endogenous hormones, including sex steroids, sex hormone-binding globulin, insulin growth factor-I, and inflammatory mediators, such as insulin and cytokines; all of these stimulate cell proliferation and inhibit apoptosis, thereby enhancing the potential for tumour growth (Coussens and Werb, 2002).Despite the fact that our study is the largest population-based study to date, we found no clear association between obesity and cancer of the bile duct or ampulla of Vater, possibly because of their small numbers. Of the five available studies assessing bile duct cancer risk specifically (Chow et al, 1994, 1999; Samanic et al, 2004; Oh et al, 2005), one reported an increased risk in white men but not white women, two small studies reported no association between obesity and ampullary cancer (Chow et al, 1999), whereas one large study showed elevated risk of ampullary cancer in US black men (Samanic et al, 2004). In two studies, tumours of the extrahepatic bile duct and/or the ampulla of Vater were combined with gall bladder cancer and were not evaluated separately (Lew and Garfinkel, 1979; Engeland et al, 2005). As molecular and epidemiologic studies suggest that these three subsites have different aetiologies, future studies should evaluate the role of obesity in extrahepatic bile duct and ampulla of Vater separately from gall bladder cancer.In this report, we used the WHO-recommended BMI cutoffs for Asians, as they have been shown to have different body composition and bone density (Pan et al, 2004). It has also been shown that at a much lower level of BMI, relative to Caucasians, Asians have a high prevalence of diabetes, metabolic syndrome, and other adverse outcomes, suggesting that lower BMI cutoffs should be used for these relatively lean populations. Based on the WHO Asian-specific cutoffs, the prevalence of obesity (BMI⩾25 kg/m2) in the Shanghai population is 25% but it is only 4.1% if the conventional cutoffs (BMI >30 kg/m2) are used. It should be noted that although median BMI in our population is low (22.9 kg/m2), the prevalence of abdominal obesity (WHR>0.9) is quite high (50%).The validity of our results hinges largely on the validity of self-reported BMI, which appeared to be quite good, given the fact that, among our population controls, self-reported current weight correlated well with measured weight (r=0.89, P<0.0001) and there is a good correlation between reported BMI and measured WHR, suggesting that misclassification by BMI is minimal. The validity of recalling weight for each decade in adulthood is likely to be lower than that of current weight. However, in our study correlations between reported weights in various decades of life were high (>0.8) and misclassification, if any, is likely to be non-differential in cases and controls.Several strengths of the study should be noted. Selection bias was minimal because of population-based design with high case ascertainment and high participation rates. Misclassification of cases was minimal, given the nearly complete confirmation of case status, achieved by comprehensive pathology and clinical review. As the largest study to date, we were able to investigate the role of obesity by anatomic subsite.One major limitation of our study is the potential disease effect on the reporting and measurement of body size. As cancer can cause wasting and weight loss, cases might be expected to have lower body weight than controls. However, we found that cases actually had a higher BMI than controls, suggesting that the weight, especially usual adult weight, in our cases had not been greatly affected by cancer. We cannot generalize our results to western populations, because of the large differences in prevalence of obesity and the fact that we were unable to evaluate the effect of BMIs greater than 30, as only 4% of the controls had a BMI that large.In summary, this population-based study in Shanghai confirmed that overall obesity is an important risk factor for gall bladder cancer and found that obesity in early adulthood and abdominal obesity are also relevant. Given the epidemic of obesity worldwide and the substantial burden of gallstones in most populations, a strategy to slow down the rising obesity trend and to minimize the burden of gallstones and biliary cancer would be important to develop.\n\nREFERENCES:\n1. Calle EE, Rodriguez C, Walker-Thurmond K, Thun MJ (2003) Overweight, obesity, and mortality from cancer in a prospectively studied cohort of US adults. N Engl J Med\n348: 1625–163812711737\n2. Chow WH, Johansen C, Gridley G, Mellemkjaer L, Olsen JH, Fraumeni Jr JF (1999) Gallstones, cholecystectomy and risk of cancers of the liver, biliary tract and pancreas. Br J Cancer\n79: 640–64410027343\n3. Chow WH, McLaughlin JK, Menck HR, Mack TM (1994) Risk factors for extrahepatic bile duct cancers: Los Angeles County, California (USA). Cancer Causes Control\n5: 267–2728061176\n4. Coussens LM, Werb Z (2002) Inflammation and cancer. Nature\n420: 860–86712490959\n5. Engeland A, Tretli S, Austad G, Bjorge T (2005) Height and body mass index in relation to colorectal and gallbladder cancer in two million Norwegian men and women. Cancer Causes Control\n16: 987–99616132807\n6. Giorgino F, Laviola L, Eriksson JW (2005) Regional differences of insulin action in adipose tissue: insights from in vivo and in vitro studies. Acta Physiol Scand\n183: 13–3015654917\n7. Hsing AW, Bai Y, Andreotti G, Rashid A, Deng J, Chen J, Han TQ, Wang BS, Zhang BH, Shen MC, Fraumeni Jr JF, Gao YT (2007a) Family history of gallstones and the risk of biliary tract cancer and gallstones: a population-based study. Int J Cancer\n121: 832–83817450525\n8. Hsing AW, Gao YT, Devesa SS, Jin F, Fraumeni Jr JF (1998) Rising incidence of biliary tract cancers in Shanghai, China. Int J Cancer\n75: 368–3709455795\n9. Hsing AW, Gao YT, McGlynn KA, Niwa S, Zhang M, Han TQ, Wang BS, Chen J, Sakoda LC, Shen MC, Zhang BH, Deng J, Rashid A (2007b) Biliary tract cancer and stones in relation to chronic liver conditions: a population-based study in Shanghai, China. Int J Cancer\n120: 1981–198517278101\n10. Hsing AW, Rashid A, Devesa SS, Fraumeni Jr JF (2006) Biliary tract cancer. In Cancer Epidemiology and Prevention, Schottenfeld D and Fraumeni Jr JF (eds) 3rd edn Oxford University Press: New York, pp 787–800\n11. Hsing AW, Rashid A, Sakoda LC, Deng J, Han TQ, Wang BS, Shen MC, Fraumeni Jr JF, Gao YT (2007c) Gallstones and the risk of biliary tract cancer: a population-based study. Br J Cancer\n97: 1577–158218000509\n12. Hsing AW, Sakoda LC, Chen J, Rashid A, Chu L, Deng J, Wang BS, Shen MC, Chen E, Rosenberg P, Zhang M, Andreotti G, Welch R, Yeager M, Fraumeni Jr JF, Gao YT, Chanock S (2008) Variants of inflammation-related genes and the risk of gallstones and biliary tract cancer: a population-based study in China. Cancer Res\n68: 6442–645218676870\n13. Hsing AW, Zhang M, Rashid A, Gao YT, McGlynn KA, Wang B, Shen M, Zhang B, Deng J, Fraumeni Jr JF, O'Brien T (2007d) Hepatitis B and C infection in relation to biliary stones and cancer: a population-based study. Int J Cancer\n122: 1849–1853\n14. Jorgensen T (1989) Gall stones in a Danish population. Relation to weight, physical activity, smoking, coffee consumption, and diabetes mellitus. Gut\n30: 528–5342785475\n15. Kato I, Nomura A, Stemmermann GN, Chyou PH (1992) Prospective study of clinical gallbladder disease and its association with obesity, physical activity, and other factors. Dig Dis Sci\n37: 784–7901563324\n16. Lew EA, Garfinkel L (1979) Variations in mortality by weight among 750,000 men and women. J Chronic Dis\n32: 563–576468958\n17. Oh SW, Yoon YS, Shin SA (2005) Effects of excess weight on cancer incidences depending on cancer sites and histologic findings among men: Korea National Health Insurance Corporation Study. J Clin Oncol\n23: 4742–475416034050\n18. Pan WH, Flegal KM, Chang HY, Yeh WT, Yeh CJ, Lee WC (2004) Body mass index and obesity-related metabolic disorders in Taiwanese and US whites and blacks: implications for definitions of overweight and obesity for Asians. Am J Clin Nutr\n79: 31–3914684394\n19. Samanic C, Gridley G, Chow WH, Lubin J, Hoover RN, Fraumeni Jr JF (2004) Obesity and cancer risk among white and black United States veterans. Cancer Causes Control\n15: 35–4314970733\n20. Stampfer MJ, Maclure KM, Colditz GA, Manson JE, Willett WC (1992) Risk of symptomatic gallstones in women with severe obesity. Am J Clin Nutr\n55: 652–6581550039\n21. Wistuba II, Gazdar AF (2004) Gallbladder cancer: lessons from a rare tumour. Nat Rev Cancer\n4: 695–70615343276\n22. Wolk A, Gridley G, Svensson M, Nyren O, McLaughlin JK, Fraumeni JF, Adam HO (2001) A prospective study of obesity and cancer risk (Sweden). Cancer Causes Control\n12: 13–2111227921\n23. World Health Organization/International Association for the Study of Obesity/International Obesity Task Force (2000) The Asia-Pacific Perspective: Redefining Obesity and its Treatment. Health Communications Australia Pty Ltd.: Brisbane\n24. Zatonski WA, Lowenfels AB, Boyle P, Maisonneuve P, Bueno de Mesquita HB, Ghadirian P, Jain M, Przewozniak K, Baghurst P, Moerman CJ, Simard A, Howe GR, McMichael AJ, Hsieh CC, Walker AM (1997) Epidemiologic aspects of gallbladder cancer: a case-control study of the SEARCH Program of the International Agency for Research on Cancer. J Natl Cancer Inst\n89: 1132–11389262251"
+}

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+{
+    "id": "PMC2528158",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528158\nAUTHORS: F Graziano, A Ruzzo, F Loupakis, D Santini, V Catalano, E Canestrari, P Maltese, R Bisonni, L Fornaro, G Baldi, G Masi, A Falcone, G Tonini, P Giordani, P Alessandroni, L Giustini, B Vincenzi, M Magnani\n\nABSTRACT:\nWe investigated the association between thymidylate synthase (TS) germline polymorphisms and response to 5-fluorouracil-based chemotherapy in 80 patients with liver-only metastatic colorectal cancer (MCRC). The tandem repeat polymorphism (VNTR) in TS 5′-untranslated region (5′-UTR), which consists of two (2R) or three (3R) 28-bp repeated sequences, with or without a G/C nucleotide change in 3R carriers (3G or 3C) and a 6-bp insertion/deletion (6+/6−) in the TS 3′-UTR, was studied. The distinction between high (2R/3G, 3C/3G and 3G/3G) and low (2R/2R, 2R/3C and 3C/3C) TS expression genotypes according to the 5′-UTR VNTR+G/C nucleotide change showed significant association with tumour response (P=0.01). In particular, high TS expression genotypes were found in 8 out of 34 patients (23.5%) with complete or partial response and in 24 out of 46 patients (52%) with stable disease and disease progression. Liver-only MCRC patients are a homogeneous and clinical relevant subgroup that may represent an ideal setting for studying the actual influence of TS polymorphisms.\n\nBODY:\nFunctional polymorphisms in the 5′-untranslated region (5′-UTR) and the 3′-UTR of the thymidylate synthase (TS) gene have been identified in the last decade (Marsh, 2005). A variable number of 28-bp tandem repeated sequence (VNTR) in TS 5′-UTR determines two (2R) or three (3R) alleles (Horie et al, 1995) and three common genotypes (2R/2R, 2R/3R and 3R/3R). Upregulated TS protein levels were found to be associated with the 3R allele (Kawakami et al, 1999; Yu et al, 2008). A G/C single-nucleotide polymorphism (SNP) in the 3R allele was found to determine two additional alleles (3G or 3C) at this locus (Kawakami and Watanabe, 2003; Mandola et al, 2003), and according to their functional role, it allows a distinction between high (2R/3G, 3C/3G and 3G/3G) and low (2R/2R, 2R/3C and 3C/3C) TS expression genotypes in vivo (Morganti et al, 2005; Yawata et al, 2005). A more recently discovered TS genetic variant is a 6-bp insertion/deletion (6+/6) in 3′-UTR (Ulrich et al, 2000). TS 3′-UTR genotypes (6+/6+−, 6+/6− and 6−/6−) seem to be associated with variable TS mRNA levels (Mandola et al, 2004); however, the functional effect of the 3′-UTR polymorphism is not well defined yet (Calascibetta et al, 2004).5-Fluorouracil is a fundamental drug in the treatment of patients with colorectal cancer, and TS levels are considered an important factor for explaining the differences in 5-fluorouracil antitumour activity (Popat et al, 2004). Therefore, the TS functional polymorphisms are under investigation for the possibility of optimising chemotherapy (Yong and Innocenti, 2007). Studies in patients with metastatic colorectal cancer showed that carriers of the TS 5′-UTR 3R (3G) and/or the TS 3′-UTR 6+ alleles had adverse clinical outcomes (Pullarkat et al, 2001; Etienne et al, 2002; Park et al, 2002; Marcuello et al, 2004; Stoehlmacher et al, 2004; Martinez-Balibrea et al, 2007); however, such an association was not always detected (Lecomte et al, 2004; Jakobsen et al, 2005; Ruzzo et al, 2007a, 2007b). Heterogeneity in clinical experimental conditions (Sorbye et al, 2007), in tumour burden (Köhne et al, 2002) and in genetic/molecular features in the presence of a multisite metastatic disease (Yokota, 2000) may explain variable results in these pharmacogenetic studies.We hypothesised that the 20–30% of patients with liver-only metastatic colorectal cancer (MCRC) (Mandalà et al, 2007) may represent a favourably homogeneous and clinically relevant setting for evaluating the role of TS polymorphisms for predicting response to 5-fluorouracil-based chemotherapy. For this purpose, we performed an analysis of TS polymorphisms in patients with liver-only MCRC who were previously enrolled in two prospective pharmacogenetic studies including 312 patients treated with first-line FOLFOX (bolus/infusional 5-fluorouracil coupled with oxaliplatin) or FOLFIRI (bolus/infusional 5-fluorouracil coupled with irinotecan) regimens (Ruzzo et al, 2007a, 2007b). FOLFOX and FOLFIRI regimens are equally active and they produce comparable response rates in first-line chemotherapy. In both the regimens, 5-fluorouracil is used at the same dose and schedule (Colucci et al, 2005). The primary end point of the study was the association between TS polymorphism and tumour response.Materials and methodsStudy populationThree hundred and twelve patients with metastatic colorectal cancer were prospectively enrolled in two previous pharmacogenetic studies (Ruzzo et al, 2007a, 2007b) and they underwent first-line chemotherapy including leucovorin 100 mg m−2 administered as a 2-h infusion before 5-fluorouracil 400 mg m−2 administered as an intravenous bolus injection and 5-fluorouracil 600 mg m−2 as a 22-h infusion immediately after FU bolus injection on days 1 and 2, every 2 weeks. Eighty patients (25.6%) had liver-only metastatic disease and they were included in this analysis. Ten of the 80 patients had history of liver surgery for metastasectomy and they were with liver-only relapse.The 80 studied patients had cytologically or histologically confirmed metastatic colorectal cancer and the presence of at least one measurable lesion. Pretreatment evaluation included a complete medical and clinical–physical examination, KPS evaluation, baseline measurement of tumour size based on CT scan, serum chemistries and CEA. Objective response was evaluated after four cycles of treatment and then every 2 months according to the RECIST criteria (Therasse et al, 2000). For the purpose of this study, radiology studies of the 80 patients were reviewed for confirming the treatment outcomes. Patients’ characteristics and their outcomes were unknown to investigators performing genetic analyses. The study was approved by local ethical committees and patients provided signed informed consent.Analysis of polymorphismsA blood sample from each enrolled patient was used for genotyping and it was collected before starting chemotherapy. Genomic DNA was extracted from 200 μl whole blood using the QiaAmp kit (Qiagen, Valencia, CA, USA). All polymorphisms were investigated using a PCR-restriction fragment length polymorphism technique. The assays for studying polymorphisms were performed as described previously (Ruzzo et al, 2007a, 2007b).Statistical analysesThe primary end point of the study was the association between TS polymorphisms in patients with liver-only MCRC and response to 5-fluorouracil-based chemotherapy. Genotype frequencies were checked for agreement with those expected under the Hardy–Weinberg equilibrium. Genotypes for each polymorphism were analysed as three-group categorical variable in a codominant model and they were also grouped according to the recessive and additive model. Patients were categorised as responders (patients with complete or partial response) and non-responders (patients with stable disease or disease progression). The χ2-test was used for comparing proportions. Statistical significance was defined as P<0.05. A Bonferroni correction of the P-value for multiple comparisons was used where applicable.The SHEsis software platform (http://202.120.7.14/analysis/myAnalysis.php) was used to estimate haplotype frequencies and the presence of linkage disequilibrium (LD). Linkage disequilibrium exists between two SNPs, if their variants appear together more often than expected (non-random inheritance). Linkage disequilibrium was estimated using r2, with r2=1 indicating complete LD and r2=0 indicating absent LD. Haplotype frequencies were reconstructed in the study population of responders and non-responders. Association of haplotypes with clinical outcome was estimated by comparing haplotype distributions among dichotomised patients using the χ2-test.ResultsThe characteristics of the 80 studied patients and the overall frequencies of genotypes are shown in Table 1. All patients were assessable for response and they received a minimum of four cycles of chemotherapy. In the 80 patients, there was one complete response (1.2%), 33 partial responses (41.2%), 30 stable diseases (37.6%) and 16 progressions (20%). Median age was 63 years (minimum 38 years and maximum 75 years). Liver metastases were synchronous in 22 patients (27.5%) and metachronous in 68 patients (72.5%). The frequencies of genotypes are in Hardy–Weinberg equilibrium and they are consistent with those observed in Caucasian ethnicity (Archive of Genetic Association Studies accessible at: http://geneticassociationdb.nih.gov/).No significant association between clinicopathological features and tumour response was found (Table 2). The analysis of the three polymorphisms and response is shown in Table 3. The Bonferroni-adjusted P-value for the three comparisons is 0.05/3, P=0.016. The TS 5′-UTR VNTR with G/C polymorphism in 3R alleles showed association with treatment outcome (P=0.011). In particular, high TS expression genotypes (2R/3G, 3C/3G and 3G/3G) were found in 8 out of 34 patients with complete or partial response (23.5%) and 24 out of 46 patients with stable disease and disease progression (52%). The 5′-UTR VNTR and the 3′-UTR 6-bp insertion/deletion (6+/6−) did not show association with tumour response. To further evaluate these two variants, their distribution was explored in recessive and additive models also, but without finding significant associations (Table 4).The TS 5′-UTR and TS 3′-UTR loci showed mild LD (r2=0.17). As shown in Table 5, non-3G haplotypes were prevalent in responders and 3G haplotypes in non-responders, with significantly different distribution of the 3G/6− haplotype.Eleven responsive patients underwent liver surgery for resection of the residual metastatic disease (13.7%). Clear resection margins with removal of all known metastatic lesions were attained in these patients, with 10 of them carrying one of the low TS expression genotypes (2R/2R, 2R/3C and 3C/3C). At the time of data analysis (March 2008), 78 patients suffered from disease progression (97.5%). For addressing an exploratory analysis of time to progression in patients with high and low TS expression genotypes, time to event distributions were studied using the Kaplan–Meier method. As shown in Figure 1, the results support the influence of the TS 5′-UTR VNTR with G/C SNP on the outcome of these patients.DiscussionTo the best of our knowledge, this is the first analysis of TS polymorphisms in patients with liver-only MCRC. In comparison with previous studies (Pullarkat et al, 2001; Etienne et al, 2002; Park et al, 2002; Lecomte et al, 2004; Marcuello et al, 2004; Stoehlmacher et al, 2004; Jakobsen et al, 2005; Martinez-Balibrea et al, 2007; Ruzzo et al, 2007a, 2007b), we evaluated a homogeneous and low-burden disease population that was exposed to the same regimen of 5-fluorouracil including both bolus and infusional administration of the drug (Hoshino et al, 2005).Heterogeneity in clinical features in patients with metastatic disease (Sorbye et al, 2007) may represent a major limitation for observing actual pharmacogenetic effects of functional germline polymorphisms. Notably, the number of metastatic sites plays a relevant prognostic role in patients with metastatic colorectal cancer (Köhne et al, 2002), and differences in clinical outcomes can be observed between patients with different metastatic sites (Köhne et al, 2002). The planning of this study was also motivated by the fact that colorectal cancer commonly metastasises to the liver, and such a single-organ involvement, instead of a multisite metastatic disease, can accurately be monitored during chemotherapy for the assessment of response (Trillet-Lenoir et al, 2002). Low-burden metastatic disease may also limit the impact of heterogeneity in molecular/genetic alterations as they accumulate during tumour progression and metastatisation (Yokota, 2000). Loss of heterozygosity (LOH) is to be included among these possible genetic changes. Owing to LOH at the TS locus on chromosome 18 in cancer cells, carriers of the germline heterozygous 2R/3R genotype can acquire the 2R/loss genotype in their tumours (Kawakami et al, 2002; Uchida et al, 2004a, 2004b). Therefore, the lower responsiveness of germline 2R/3R carriers could not be displayed because heterozygous patients with tumour 2R/loss genotype behave as 2R/2R patients (Uchida et al, 2004a, 2004b). This phenomenon implies that responsiveness to 5-fluorouracil-based therapy may depend on the tumour rather than on the germline status of the genotypes (Uchida et al, 2004a, 2004b). However, our findings suggest that the double assessment of the VNTR plus G/C nucleotide change with dichotomisation of patients into carriers of high (2R/3G, 3C/3G and 3G/3G) and low (2R/2R, 2R/3C and 3C/3C) TS expression genotypes may not suffer from the possible presence of LOH. The finding of the association between TS 5′-UTR VNTR+G/C and tumour response may not only reflect a better functional characterisation of 3C and 3G alleles, but also a less extensive influence of tumour LOH on the germline assessment for high-TS expression genotypes (2R/3G, 3C/3G and 3G/3G).In our opinion, TS polymorphisms deserved the present investigation more than other genetic variants with putative influence on 5-fluorouracil activity (i.e. methylenetetrahydrofolate reductase gene polymorphisms). In addition to the fact that TS is the target enzyme of 5-fluorouracil, it has been observed that TS levels may be dynamic, with upregulation after fluoropyrimidine exposure (Uchida et al, 2004a, 2004b; Mauritz et al, 2007). In particular, this effect was described in liver metastases from colorectal cancer in patients who received bolus 5-fluorouracil (Mauritz et al, 2007). Therefore, TS polymorphisms may influence the outcome to 5-fluorouracil chemotherapy, not only for their role in determining different baseline levels of TS activity (Marsh, 2005), but also for modulating the enhancement of TS levels in response to 5-fluorouracil. In fact, the 5-fluorouracil-induced upregulation of TS mRNA may be greater in carriers of high-expression TS genotypes than in carriers of low-expression TS genotypes. We cannot rule out, however, that a double assessment of TS and methylenetetrahydrofolate reductase polymorphisms may improve the predictive role of the single analysis of TS polymorphisms.Another reason for studying pharmacogenetics in liver-only MCRC patients is related to the lack of predictive factor for response to neoadjuvant chemotherapy. Liver surgery can provide long-term survival for liver-only, metastatic colorectal cancer patients, but liver metastasectomy is feasible in only 15–25% of the patients. Neoadjuvant chemotherapy can provide response rates as high as 50%, allowing liver metastasectomy in about 10–15% of patients initially deemed unresectable. Tumour response to preoperative chemotherapy seems to be associated with outcome following liver resection for colorectal metastases (Folprecht et al, 2005) and, if genetically predictable, it could be improved by the selective choice of available drugs. In this study, 10 of the 11 responsive patients who underwent liver surgery were carriers of low TS expression genotypes (2R/2R, 2R/3C and 3C/3C). Actually, we performed an analysis of TS polymorphisms for response to 5-fluorouracil and we did not address this study to pharmacogenetics for liver metastases resectability and survival after preoperative chemotherapy. These end points require a prospective study, including a baseline multidisciplinary evaluation of the unresectable liver disease and long-term follow-up.In conclusion, the homogeneous subgroup of patients with liver-only metastatic disease allowed the predictive role of TS polymorphisms to stand out. In fact, the association between polymorphisms and tumour response was included in the secondary end points of our two previous pharmacogenetic studies (Ruzzo et al, 2007a, 2007b), but these analyses failed to demonstrate a predictive role for the genetic variants. The clinical setting assumes a relevant role for exploring the pharmacogenetic associations in patients with metastatic cancer and additional studies are warranted for confirming our findings.\n\nREFERENCES:\n1. Calascibetta A, Cabibi D, Martorana A, Sanguedolce G, Rausa L, Feo S, Dardanoni G, Sanguedolce R (2004) Thymidylate synthase gene promoter polymorphisms are associated with TSmRNA expressions but not with microsatellite instability in colorectal cancer. Anticancer Res\n24: 3875–388015736425\n2. Colucci G, Gebbia V, Paoletti G, Giuliani F, Caruso M, Gebbia N, Cartenì G, Agostara B, Pezzella G, Manzione L, Borsellino N, Misino A, Romito S, Durini E, Cordio S, Di Seri M, Lopez M, Maiello E, Montemurro S, Cramarossa A, Lorusso V, Di Bisceglie M, Chiarenza M, Valerio MR, Guida T, Leonardi V, Pisconti S, Rosati G, Carrozza F, Nettis G, Valdesi M, Filippelli G, Fortunato S, Mancarella S, Brunetti C, Gruppo Oncologico Dell’Italia Meridionale (2005) Phase III randomized trial of FOLFIRI versus FOLFOX4 in the treatment of advanced colorectal cancer: a multicenter study of the Gruppo Oncologico Dell’Italia Meridionale. J Clin Oncol\n23: 4866–487515939922\n3. Etienne MC, Chazal M, Laurent-Puig P, Magné N, Rosty C, Formento JL, Francoual M, Formento P, Renée N, Chamorey E, Bourgeon A, Seitz JF, Delpero JR, Letoublon C, Pezet D, Milano G (2002) Prognostic value of tumoral thymidylate synthase and p53 in metastatic colorectal cancer patients receiving fluorouracil-based chemotherapy: phenotypic and genotypic analyses. J Clin Oncol\n20: 2832–284312065560\n4. Folprecht G, Grothey A, Alberts S, Raab HR, Köhne CH (2005) Neoadjuvant treatment of unresectable colorectal liver metastases: correlation between tumour response and resection rates. Ann Oncol\n16: 1311–131915870084\n5. Horie N, Aiba H, Oguro K, Hojo H, Takeishi K (1995) Functional analysis and DNA polymorphism of the tandemly repeated sequences in the 5′-terminal regulatory region of the human gene for thymidylate synthase. Cell Struct Funct\n20: 191–1977586009\n6. Hoshino S, Yamashita Y, Maekawa T, Shirakusa T (2005) Effects on DNA and RNA after the administration of two different schedules of 5-fluorouracil in colorectal cancer patients. Cancer Chemother Pharmacol\n56: 648–65215959779\n7. Jakobsen A, Nielsen JN, Gyldenkerne N, Lindeberg J (2005) Thymidylate synthase and methylenetetrahydrofolate reductase gene polymorphism in normal tissue as predictors of fluorouracil sensitivity. J Clin Oncol\n2: 1365–1369\n8. Kawakami K, Ishida Y, Danenberg KD, Omura K, Watanabe G, Danenberg PV (2002) Functional polymorphism of the thymidylate synthase gene in colorectal cancer accompanied by frequent loss of heterozygosity. Jpn J Cancer Res\n93: 1221–122912460463\n9. Kawakami K, Omura K, Kanehira E, Watanabe Y (1999) Polymorphic tandem repeats in the thymidylate synthase gene is associated with its protein expression in human gastrointestinal cancers. Anticancer Res\n19: 3249–325210652619\n10. Kawakami K, Watanabe G (2003) Identification and functional analysis of single nucleotide polymorphism in the tandem repeat sequence of the thymidylate synthase gene. Cancer Res\n63: 6004–600714522928\n11. Köhne CH, Cunningham D, Di CF, Glimelius B, Blijham G, Aranda E, Scheithauer W, Rougier P, Palmer M, Wils J, Baron B, Pignatti F, Schöffski P, Micheel S, Hecker H (2002) Clinical determinants of survival in patients with 5-fluorouracil-based treatment for metastatic colorectal cancer: results of a multivariate analysis of 3825 patients. Ann Oncol\n13: 308–31711886010\n12. Lecomte T, Ferraz JM, Zinzindohoué F, Loriot MA, Tregouet DA, Landi B, Berger A, Cugnenc PH, Jian R, Beaune P, Laurent-Puig P (2004) Thymidylate synthase gene polymorphism predicts toxicity in colorectal cancer patients receiving 5-fluorouracil-based chemotherapy. Clin Cancer Res\n10: 5880–588815355920\n13. Mandalà M, Mosconi S, Quadri A, Milesi L, Labianca R (2007) Neoadjuvant chemotherapy for patients with liver metastases from colorectal cancer. Exp Rev Anticancer Ther\n7: 887–897\n14. Mandola MV, Stoehlmacher J, Muller-Weeks S, Cesarone G, Yu MC, Lenz HJ, Ladner RD (2003) A novel single nucleotide polymorphism within the 5′ tandem repeat polymorphism of the thymidylate synthase gene abolishes USF-1 binding and alters transcriptional activity. Cancer Res\n63: 2898–290412782596\n15. Mandola MV, Stoehlmacher J, Zhang W, Groshen S, Yu MC, Iqbal S, Lenz HJ, Ladner RD (2004) A 6 bp polymorphism in the thymidylate synthase gene causes message instability and is associated with decreased intratumoral TS mRNA levels. Pharmacogenetics\n14: 319–32715115918\n16. Marcuello E, Altés A, del Rio E, César A, Menoyo A, Baiget M (2004) Single nucleotide polymorphism in the 5′ tandem repeat sequences of thymidylate synthase gene predicts for response to fluorouracil-based chemotherapy in advanced colorectal cancer patients. Int J Cancer\n112: 733–73715386371\n17. Marsh S (2005) Thymidylate synthase pharmacogenetics. Invest New Drugs\n23: 533–53716267625\n18. Martinez-Balibrea E, Manzano JL, Martinez-Cardus A, Moran T, Cirauqui B, Catot S, Taron M, Abad A (2007) Combined analysis of genetic polymorphisms in thymidylate synthase, uridine diphosphate glucoronosyltransferase and X-ray cross complementing factor 1 genes as a prognostic factor in advanced colorectal cancer patients treated with 5-fluorouracil plus oxaliplatin or irinotecan. Oncol Rep\n17: 637–64517273745\n19. Mauritz R, van Groeningen CJ, Smid K, Jansen G, Pinedo HM, Peters GJ (2007) Thymidylate synthase and dihydropyrimidine dehydrogenase mRNA expression after administration of 5-fluorouracil to patients with colorectal cancer. Int J Cancer\n120: 2609–261217330233\n20. Morganti M, Ciantelli M, Giglioni B, Putignano AL, Nobili S, Papi L, Landini I, Napoli C, Valanzano R, Cianchi F, Boddi V, Tonelli F, Cortesini C, Mazzei T, Genuardi M, Mini E (2005) Relationships between promoter polymorphisms in the thymidylate synthase gene and mRNA levels in colorectal cancers. Eur J Cancer\n41: 2176–218316182121\n21. Park DJ, Stoehlmacher J, Zhang W, Tsao-Wei D, Groshen S, Lenz HJ (2002) Thymidylate synthase gene polymorphism predicts response to capecitabine in advanced colorectal cancer. Int J Colorectal Dis\n17: 46–4912018454\n22. Popat S, Matakidou A, Houlston RS (2004) Thymidylate synthase expression and prognosis in colorectal cancer: a systematic review and meta-analysis. J Clin Oncol\n22: 529–53614752076\n23. Pullarkat ST, Stoehlmacher J, Ghaderi V, Xiong YP, Ingles SA, Sherrod A, Warren R, Tsao-Wei D, Groshen S, Lenz HJ (2001) Thymidylate synthase gene polymorphism determines response and toxicity of 5-FU chemotherapy. Pharmacogenomics J\n1: 65–7011913730\n24. Ruzzo A, Graziano F, Loupakis F, Rulli E, Canestrari E, Santini D, Catalano V, Ficarelli R, Maltese P, Bisonni R, Masi G, Schiavon G, Giordani P, Giustini L, Falcone A, Tonini G, Silva R, Mattioli R, Floriani I, Magnani M (2007a) Pharmacogenetic profiling in patients with advanced colorectal cancer treated with first-line FOLFOX-4 chemotherapy. J Clin Oncol\n25: 1247–125417401013\n25. Ruzzo A, Graziano F, Loupakis F, Santini D, Catalano V, Bisonni R, Ficarelli R, Fontana A, Andreoni F, Falcone A, Canestrari E, Tonini G, Mari D, Lippe P, Pizzagalli F, Schiavon G, Alessandroni P, Giustini L, Maltese P, Testa E, Menichetti ET, Magnani M (2007b) Pharmacogenetic profiling in patients with advanced colorectal cancer treated with first-line FOLFIRI chemotherapy. Pharmacogenomics J\n8(4): 278–28817549067\n26. Sorbye H, Köhne CH, Sargent DJ, Glimelius B (2007) Patient characteristics and stratification in medical treatment studies for metastatic colorectal cancer: a proposal for standardization of patient characteristic reporting and stratification. Ann Oncol\n18: 1666–167217660495\n27. Stoehlmacher J, Park DJ, Zhang W, Yang D, Groshen S, Zahedy S, Lenz HJ (2004) A multivariate analysis of genomic polymorphisms: prediction of clinical outcome to 5-FU/oxaliplatin combination chemotherapy in refractory colorectal cancer. Br J Cancer\n91: 344–35415213713\n28. Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, Rubinstein L, Verweij J, Van Glabbeke M, van Oosterom AT, Christian MC, Gwyther SG (2000) New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst\n92: 205–21610655437\n29. Trillet-Lenoir V, Freyer G, Kaemmerlen P, Fond A, Pellet O, Lombard-Bohas C, Gaudin JL, Lledo G, Mackiewicz R, Gouttebel MC, Moindrot H, Boyer JD, Chassignol L, Stremsdoerfer N, Desseigne F, Moreau JM, Hedelius F, Moraillon A, Chapuis F, Bleuse JP, Barbier Y, Heilmann MO, Valette PJ (2002) Assessment of tumour response to chemotherapy for metastatic colorectal cancer: accuracy of the RECIST criteria. Br J Radiol\n75: 903–90812466256\n30. Uchida K, Hayashi K, Kawakami K, Schneider S, Yochim JM, Kuramochi H, Takasaki K, Danenberg KD, Danenberg PV (2004a) Loss of heterozygosity at the thymidylate synthase (TS) locus on chromosome 18 affects tumor response and survival in individuals heterozygous for a 28-bp polymorphism in the TS gene. Clin Cancer Res\n10: 433–43914760062\n31. Uchida K, Hayashi K, Kuramochi H, Takasaki K (2004b) Changes in intratumoral thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) mRNA expression in colorectal and gastric cancer during continuous tegafur infusion. Int J Oncol\n19: 341–346\n32. Ulrich CM, Bigler J, Velicer CM, Greene EA, Farin FM, Potter JD (2000) Searching expressed sequence Tag databases: discovery and confirmation of a common polymorphism in the thymidylate synthase gene. Cancer Epidemiol Biomarkers Prev\n9: 1381–138511142426\n33. Yawata A, Kim SR, Miyajima A, Kubo T, Ishida S, Saito Y, Nakajima Y, Katori N, Matsumoto Y, Fukuoka M, Ohno Y, Ozawa S, Sawada J (2005) Polymorphic tandem repeat sequences of the tymidylate gene correlates with cellular-based sensitivity to fluoropyrimidine antitumor agents. Cancer Chemother Pharmacol\n56: 465–47215918040\n34. Yokota J (2000) Tumor progression and metastasis. Carcinogenesis\n21: 497–50310688870\n35. Yong WP, Innocenti F (2007) Translation of pharmacogenetic knowledge into cancer therapeutics. Clin Adv Hematol Oncol\n5: 698–70617982411\n36. Yu KH, Wang WX, Ding YM, Li H, Wang ZS (2008) Polymorphism of thymidylate synthase gene associated with its protein expression in human colon cancer. World J Gastroenterol\n14: 617–62118203297"
+}

batch_9/PMC2528306.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2528306",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528306\nAUTHORS: L. Lee, A. Keller, M. Clemons\n\nABSTRACT:\nThe occurrence of a solitary sternal metastasis from breast cancer is relatively uncommon, and its treatment is controversial. Most case reports on the role of sternal resection in what is termed a “solitary sternal metastasis” tend to present a rather optimistic outcome.Here, we report the case of a premenopausal woman with axillary lymph node–positive, triple-negative breast cancer treated with mastectomy followed by adjuvant chemotherapy and radiation therapy. She developed a radiologically isolated sternal recurrence 3 years later, which was treated with partial sternectomy. The present case report reviews the use of sternectomy for breast cancer recurrence and highlights the need for thorough clinical and radiologic evaluation to ensure the absence of other systemic disease before extensive surgery is undertaken.\n\nBODY:\n1. CASE REPORTA 35-year-old woman with a history of breast cancer diagnosed 3 years earlier was referred to us for discussion of bisphosphonate use after surgical excision of an isolated sternal metastasis. Her initial breast cancer was treated with left mastectomy and axillary node dissection. Pathology revealed multifocal, poorly-differentiated invasive ductal carcinoma with three tumour masses (2.3 cm, 2.0 cm, and 1.1 cm). Of 14 lymph nodes, 5 were involved. Margins were negative. Immunohistochemistry was negative for estrogen, progesterone, and human epidermal growth factor (her2/neu) receptors. This patient received adjuvant epirubicin/5-fluorouracil/cyclophosphamide (fec-100) chemotherapy for 6 cycles and locoregional radiation treatment. Subsequently, she also had a prophylactic contralateral mastectomy and bilateral transrectus abdominal muscle flap reconstructions. Genetic testing was negative for BRCA1 and BRCA2 mutations.Three years after completion of adjuvant systemic therapy, this woman presented to her family physician with new-onset chest and neck tenderness during pregnancy. Investigations were deferred for 6 months until she was postpartum. A computed tomography scan of the neck and chest revealed a 5-cm soft-tissue mass with manubrial destruction consistent with recurrent disease (Figure 1). Restaging revealed no other foci of metastatic disease, and a bone scan showed uptake only in the manubrium. Given the clinical and radiologic impression of an isolated focus of recurrence, the patient elected to have a partial sternectomy.Pathology revealed a 5.4-cm, poorly differentiated invasive ductal carcinoma infiltrating the manubrium with involvement of the left sternocleidomastoid muscle and left clavicular head. Three lymph nodes were positive (one sternocleidomastoid and two thymic). Margins were clear. Once again, immunohistochemistry was triple-negative.There were no postoperative complications, and after a 6-week recovery, the woman was referred to our team for a second opinion regarding bisphosphonate use following surgical excision of the sternal metastasis. At presentation, she was asymptomatic. On physical examination, the scars from her breast reconstructions and sternal surgery were evident. She also had two palpable 0.5-cm right supraclavicular nodes.Restaging radiologic imaging performed 6 weeks after her surgery confirmed sub-centimetre right supraclavicular lymph nodes and also an enlarged 1.6-cm left supraclavicular lymph node (Figure 2). Fine-needle aspiration of the latter node was positive for triple-negative, poorly differentiated invasive ductal carcinoma.2. DISCUSSIONIn patients with breast cancer, the presence of either sternal involvement or an isolated sternal metastasis is relatively uncommon, with reported incidences of 5.2% and 1.9%–2.4% respectively 1,2. Sternal involvement may occur either from direct invasion by enlarged internal mammary lymph nodes or from hematogenous spread. However, in contrast with vertebral lesions, which tend to result in multicentric bony disease from spread through the paravertebral plexus 3, some sternal lesions have been observed to remain solitary with time and may be amenable to surgical resection with curative intent 4,5.Sternectomy for isolated breast cancer recurrence remains a controversial issue, and the literature consists predominantly of retrospective case series. Noguchi et al. 6 performed sternal resections with parasternal and mediastinal lymph node dissection on 9 patients before chemo-endocrine therapy. Eventual relapse in 8 patients revealed that lymph node dissection had no effect on locoregional control. Nevertheless, the dissection provided prognostic information, because all patients with involved parasternal and mediastinal lymph nodes relapsed and died within 30 months, but 3 patients without lymph node involvement survived for more than 6 years.Lequaglie et al. 7 performed radical, curative-intent sternectomies in a subgroup of 28 patients with isolated breast cancer recurrence and found that the 10-year overall survival in the group was 41.8%. These authors suggested that sternectomy could be curative in carefully selected patients.An isolated sternal metastasis should, however, be regarded with caution, because it is more likely to herald systemic disease than to be truly solitary. Kwai et al. 1 demonstrated that 54% of patients with breast cancer and solitary sternal metastasis developed other foci of distant disease within 20 months. The predominance of pulmonary metastasis and distant skeletal disease found is their study was attributed to drainage of the internal mammary nodes into the subclavian vein.Consequently, patients with breast cancer presenting with a solitary sternal metastasis require thorough restaging evaluation to rule out other foci of metastatic disease. Treatment should be based on a multimodality approach. With an isolated recurrence, local therapy with radiation would be appropriate. In the presence of distant disease, systemic options should be offered. Surgical resection should be reserved for palliation or for instances in which the other treatment modalities are not possible.Furthermore, the natural history of the initial breast cancer and the prognosis following recurrence should be considered. An important aspect of the present case is that our patient had a triple-negative breast cancer with axillary node involvement. Compared with other breast cancers, this disease subtype is highly aggressive, with an increased rate of early development of distant recurrence that peaks at 3 years after diagnosis 8. Progression is rapid, with a median survival of only 9 months following recurrence. In addition, the extensive literature on relapsed breast cancer demonstrates that a shorter disease-free interval from time of initial treatment is associated with worse prognosis 9.3. CONCLUSIONSAlthough resection of a so-called isolated sternal metastasis is surgically feasible, it should be assessed and discussed with patients on a case-by-case basis. In the case of our patient, her history of a treated lymph node–positive tumour with triple-negative phenotype and recurrence within 3 years following mastectomy, chemotherapy, and radiotherapy should realistically have predicted this tragic outcome of widespread relapse within weeks following her extensive palliative surgery.FIGURE 1Computed tomography of the thorax, performed preoperatively, shows a soft-tissue mass with manubrial destruction (arrows). (A) Soft-tissue window. (B) Bone window.FIGURE 2Computed tomography of the thorax, performed postoperatively, shows a 1.6-cm left supraclavicular lymph node deep to the sternocleidomastoid muscle.\n\nREFERENCES:\nNo References"
+}

batch_9/PMC2528939.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2528939",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528939\nAUTHORS: Yuji Yoshiike, Ryoichi Minai, Yo Matsuo, Yun-Ru Chen, Tetsuya Kimura, Akihiko Takashima\n\nABSTRACT:\nRecent in vitro and in vivo studies suggest that destabilized proteins with defective folding induce aggregation and toxicity in protein-misfolding diseases. One such unstable protein state is called amyloid oligomer, a precursor of fully aggregated forms of amyloid. Detection of various amyloid oligomers with A11, an anti-amyloid oligomer conformation-specific antibody, revealed that the amyloid oligomer represents a generic conformation and suggested that toxic β-aggregation processes possess a common mechanism. By using A11 antibody as a probe in combination with mass spectrometric analysis, we identified GroEL in bacterial lysates as a protein that may potentially have an amyloid oligomer conformation. Surprisingly, A11 reacted not only with purified GroEL but also with several purified heat shock proteins, including human Hsp27, 40, 70, 90; yeast Hsp104; and bovine Hsc70. The native folds of A11-reactive proteins in purified samples were characterized by their anti-β-aggregation activity in terms of both functionality and in contrast to the β-aggregation promoting activity of misfolded pathogenic amyloid oligomers. The conformation-dependent binding of A11 with natively folded Hsp27 was supported by the concurrent loss of A11 reactivity and anti-β-aggregation activity of heat-treated Hsp27 samples. Moreover, we observed consistent anti-β-aggregation activity not only by chaperones containing an amyloid oligomer conformation but also by several A11-immunoreactive non-chaperone proteins. From these results, we suggest that the amyloid oligomer conformation is present in a group of natively folded proteins. The inhibitory effects of A11 antibody on both GroEL/ES-assisted luciferase refolding and Hsp70-mediated decelerated nucleation of Aβ aggregation suggested that the A11-binding sites on these chaperones might be functionally important. Finally, we employed a computational approach to uncover possible A11-binding sites on these targets. Since the β-sheet edge was a common structural motif having the most similar physicochemical properties in the A11-reactive proteins we analyzed, we propose that the β-sheet edge in some natively folded amyloid oligomers is designed positively to prevent β aggregation.\n\nBODY:\nIntroductionProtein misfolding diseases are characterized by the formation of amyloid, which occurs through misfolding promoted by the conversion of a protein from its native to non-native state. Under the appropriate conditions, any proteins can form generic amyloid [1]. The ability of an oligomeric entity in amyloid to seed the polymerization of other proteins indicates that amyloid may disrupt cellular functions by interfering with the folding of other proteins [2]–[5, Kayed et al., Society for Neuroscience Abstracts 2005;35:893.6]. Therefore, it is important to understand the molecular mechanism(s) underlying the seeding function of amyloid oligomers, whose pathogenic significance in protein misfolding diseases have been well supported. Because amyloid oligomers innately tend to aggregate, high-resolution elucidation of their structures through conventional physical techniques has been challenging. Novel insight on the structure of amyloid oligomers was made possible by the development of an anti-amyloid oligomer conformation-dependent antibody, A11 [2]. The fact that various amyloid oligomers are A11 immunopositive suggests that amyloid oligomers share a common structure and implies that various protein-misfolding diseases may have a common pathogenic mechanism [2]. In the present study, we identified proteins that contain the amyloid oligomer conformation by using the A11 antibody as a probe. One group of A11-reactive proteins commonly displayed anti-β-aggregation activity as opposed to the expected β-aggregation promoting activity of misfolded amyloid oligomers.ResultsAmyloid oligomer conformation in chaperonesA11 is an antibody that specifically detects the conformation of amyloid oligomers regardless of their amino acid sequence [2]. To determine whether proteins that contain the amyloid oligomer conformation exist in bacteria, we examined the A11 immunoreactivity of Escherichia coli cell lysates. We chose to examine bacterial proteins because extensive structural information about these proteins already exists in the protein data bank (PDB). We subjected E. coli DH5α cell lysates to PAGE analysis. To reduce false-positive signals, we used sample buffer that contained neither SDS nor reducing agents to solubilize the lysates although the disruption of conformation was predicted to some extent during SDS-PAGE. One gel was Western blotted with A11 to identify the molecular weights of A11-immunoreactive proteins (Figure 1A), and another gel was stained with coomassie brilliant blue (CBB). The bands in the CBB-stained gel corresponding to the A11-immunoreactive bands on the immunoblot were analyzed by tandem mass spectrometry (MS-MS). We identified one of these A11-immunoreactive bands to contain GroEL, a bacterial chaperonin [6]. Unlike other proteomics studies that have used sequence-dependent antibodies, in our study, the proteins detected in the cell lysates through this simple approach remain unconfirmed candidates of A11-reactive proteins. Indeed, the conformational specificity of A11 for each protein (purified and native forms) needs to be analyzed, since the cell lysis procedure might have affected protein conformation to unknown degrees.10.1371/journal.pone.0003235.g001Figure 1Amyloid oligomer conformation in chaperones.(A) E. coli competent cells (strain DH5α; TOYOBO, Japan) were freeze-thawed three times by freezing the cells on ice at −180°C then thawing them at 37°C. The lysates were then mixed with sample buffer lacking SDS and reducing agents and then subjected to PAGE analysis. One gel was immunoblotted with A11 antibody. A11-immunoreactive bands (A–G) were excised from the CBB-stained gel, treated with trypsin, and analyzed by tandem-mass spectrometry. A candidate A11-reactive protein from band B was GroEL. The difference in staining patterns between the A11 immunoblot and CBB-stained gel indicates A11 antibody-binding selectivity. Note also that the proteins in cell lysates reacted with the A11 antibody are not necessarily all natively folded. Purified GroEL (23 µM) was mixed with sample buffer lacking both reducing agents and SDS, and then run on (B) a gel in a regular SDS-containing buffer or (C) a gel in a buffer lacking SDS (native PAGE). Gels were blotted onto a membrane that was subsequently probed with A11 under the same conditions. Note that the mobility of the molecular weight standard is different in (B) and (C). (D) The following solutions (3 µL each) were dot blotted onto a nitrocellulose membrane: 20 µM Aβ40 monomer, 20 µM Aβ40 oligomer, H2O, purified 23 µM GroEL, 37 µM Hsp27, 20 µM Hsp40, 14 µM Hsp70, 12 µM Hsp90, 10 µM Hsp104, and 10 µM Hsc70. GroEL, Hsp27, Hsp40, Hsp90, and Hsc70 were purchased from Stressgen (Canada). Hsp70 was from Sigma (USA). Hsp104 was from ATGen (South Korea). Each protein solution was blotted onto a single membrane that was probed with A11 under the same conditions. The individual dot blots were arranged into a single column.To confirm the A11 immunoreactivity of GroEL, we performed Western analysis on purified GroEL and observed that A11 strongly immunostained monomeric GroEL (Figure 1B). Although the sample buffer used in this PAGE analysis lacked SDS and reducing agents, the running buffer did contain SDS. In true native PAGE, which is performed without SDS, A11 mostly immunostained a high molecular weight band representing a GroEL oligomer complex, which is the expected functionally active form of GroEL in bacterial cytosol (Figure 1C). A11 antibody also strongly immunostained purified GroEL on dot blots (Figure 1D). This enabled us to exclude the possibility that the PAGE processing misfolded GroEL molecules that potentially could react with A11 and lead to false A11 positives. A11 antibody also immunostained purified recombinant human chaperones, including heat shock protein (Hsp) 27, 40, 70, 90; yeast Hsp104; and bovine heat shock cognate (Hsc) 70, albeit with varying intensity (Figure 1D). As expected, A11 antibody immunostained β-amyloid (Aβ) oligomer prepared from Aβ(1–40) but not monomeric Aβ. These results suggest that certain chaperone structures share a common amyloid oligomer conformation. Among the A11-reactive candidates, we were interested in identifying chaperones that normally display anti-aggregation activity as opposed to proteins, such as misfolded pathogenic amyloid oligomers, that induce β-aggregation of other proteins [2]–[5, Kayed et al., Society for Neuroscience Abstracts 2005;35:893.6].Proteins containing the amyloid oligomer conformation affect Aβ aggregation in opposite waysTo confirm the seeding property of pathogenic Aβ oligomer, we assessed the β-aggregation kinetics of Aβ40 in the presence of preformed Aβ oligomer using a thioflavin T (ThT) fluorescence assay [7] (Figure 2A), and found that Aβ oligomer induced Aβ aggregation in a dose-dependent manner. To determine whether the chaperones we assessed with A11 antibody were functionally active and thus natively folded or misfolded like pathogenic amyloid oligomers, we examined the effects of Hsp27, Hsp70, and Hsp90 on Aβ-aggregation kinetics using the ThT assay [7] (Figure 2B–D). Aβ, a type of amyloid protein observed in Alzheimer's disease (AD), has been pathologically linked to these three chaperones [8]. As with other published studies [9], [10], we found that all three chaperones—Hsp27, Hsp70, and Hsp90—suppressed the β aggregation of Aβ in a dose-dependent manner to varying degrees. We did not observe fibrillar amyloid structures in Aβ samples incubated with each of these chaperones under an electron microscope (data not shown). To verify this chaperone activity [6], we examined whether the anti-β-aggregation properties of Hsp was energy dependent (Figure 2E, F). Adding ATP to samples containing Hsp70 or Hsp70/Hsp40 at concentrations that alone do not show anti-β-aggregation activity caused Hsp70 and Hsp70/Hsp40 to significantly suppress Aβ aggregation. Most of the purified recombinant chaperones used here, including Hsp70 and Hsp40, have tested positive for their ATPase activity. The energy-dependent anti-β-aggregation activity of these chaperone samples indicates that A11-immunoreactive chaperones are indeed functional; thus, a significant number of chaperone molecules in these purified samples are natively folded [4], [11]. In other words, the native structures of these chaperones possess the amyloid oligomer conformation. Therefore, there are at least two classes of A11-reactive proteins that contain the amyloid oligomer conformation: pathogenic amyloid oligomers that promote β aggregation and chaperones that suppress β aggregation.10.1371/journal.pone.0003235.g002Figure 2Contrasting effects of Aβ oligomer and chaperones on Aβ aggregation.(A) Aβ40 (final concentration: 5 µM) dissolved in aCSF was mixed with 0, 0.5, 5, or 50 nM Aβ40 oligomer (i.e., “seed”) in H2O. Results from a sample containing only 50 nM Aβ40 oligomer is also shown. ThT fluorescence intensity indicates the level of Aβ aggregation. (B) Aβ40 (final concentration: 10 µM) dissolved in H2O was mixed with 0, 10, 100, or 1000 nM Hsp27 in buffer (1.08 mM Tris-HCl [pH 7.5], 541 µM NaCl, 54.1 µM EDTA, and 54.1 µM DTT). (C) Aβ40 (final concentration: 10 µM) dissolved in H2O was mixed with 0, 10, 100, or 1000 nM Hsp70 in buffer (2.7 mM KCl, 1.5 mM KH2PO4, 137 mM NaCl, and 8.1 mM Na2HPO4). (D) Aβ40 (final concentration: 5 µM) dissolved in H2O was mixed with 0, 5, 50, or 500 nM Hsp90 in buffer (3.9 mM Tris-HCl [pH 7.5], 7.8 mM NaCl, and 78.2 µM DTT). (E) Anti-β-aggregation activity of Hsp40 and Hsp70. Aβ40 (5 µM) dissolved in aCSF was mixed with either 10 nM Hsp70 in buffer A (15.1 µM KCl, 8.4 µM KH2PO4, 767 µM NaCl, and 45.3 µM Na2HPO4) or a mixture of 10 nM Hsp70 in Buffer A and 10 nM Hsp40 in Buffer B (10.8 µM KCl, 6.0 µM KH2PO4, 548 µM NaCl, and 32.4 µM Na2HPO4) in the presence or absence of 500 µM Mg-ATP. (F) Bar graph showing ThT fluorescence intensity of Aβ samples with or without chaperones after 17 h of incubation. Statistical significance was calculated by using a Student's t-test. *p = 0.035; **p = 0.00046. Data are presented as means+/−SEM.Heating affects the amyloid oligomer conformation and anti-β-aggregation activity of Hsp27Next, we examined how heat treatment alters the amyloid oligomer conformation of Hsp27 and the Hsp27-mediated suppression of Aβ aggregation (Figure 3). We assessed Hsp27 because of its strong A11 immunoreactivity on both dot blot and native-PAGE/Western blot analyses, and because of its pathological association with Aβ [8]. We heated Hsp27 dissolved in buffer to 80°C for 15 and 30 min and then subjected the sample to native-PAGE followed by Western blotting with A11 antibody. Heat treatment abolished A11 immunoreactivity of Hsp27 (Figure 3A), even though the peptide backbone of Hsp27 was intact (SDS-PAGE of heat-treated samples, Figure 3B). ThT assay of samples containing Aβ and heat-treated Hsp27, however, showed that Hsp27-mediated suppression of Aβ aggregation disappeared concurrently with the reduction of amyloid oligomer conformation resulting from the heat treatment (Figure 3C). The heat-denaturing experiments simply suggested that the conformation-specific binding of A11, and thus the amyloid oligomer conformation—not non-specific interactions—was consistently observed as natively folded Hsp27 prevented β aggregation. These results certainly did not demonstrate that the A11-reactive domain within Hsp27 is responsible for suppressing Aβ aggregation. Both the structure and functional mechanisms of Hsp27 are unknown. Since the spherical and oligomeric forms of Hsp27 are active states [12], Hsp27 could co-oligomerize with Aβ while suppressing amyloidogenesis. Heat-treated Hsp27 may have also formed amyloid that complexed with Aβ. Taken together, these data identify the amyloid conformation as the generic structural feature of natively folded chaperones directly correlating with the anti-β-aggregation activity of chaperones.10.1371/journal.pone.0003235.g003Figure 3Effects of heat treatment on amyloid oligomer conformation in Hsp27 and the anti-β-aggregation activity of Hsp27.(A) Western blot of Hsp27 probed with A11 anti-amyloid oligomer conformation-dependent antibody. Hsp27 samples were heat treated at 80°C for 15 or 30 min. Heat-treated and untreated Hsp27 samples (10 µM) were mixed with sample buffer lacking SDS and reducing reagents, subjected to native PAGE using 4–20% gels, and separated proteins were immunoblotted with A11 antibody. Note that A11-immunoreactive Hsp27 bands migrated to a high molecular-weight level in native PAGE, because Hsp27 exists as large oligomers having chaperone-like activity [8], [12]. (B) Heat-treated and untreated Hsp27 samples (10 µM) were mixed with sample buffer containing SDS and β-mercaptoethanol, and then subjected to SDS-PAGE using 4–20% gels. Gels were stained with coomassie brilliant blue (CBB). (C) Aβ-aggregation kinetics of samples containing heat-treated Hsp27. One buffer sample and three Hsp27 samples were diluted 100 times with H2O. Aβ40 (final concentration: 5 µM) dissolved in aCSF was mixed with buffer (54 µM Tris-HCl [pH 7.5], 27 µM NaCl, 2.7 µM EDTA, and 2.7 µM DTT); untreated 50 nM Hsp27; 15-min heat-treated Hsp27; or 30-min heat-treated Hsp27. All samples were incubated in the presence of 10 µM ThT; triplicate measurements were taken every 30 min at excitation and emission wavelengths of 440 nm and 486 nm [7], respectively. Data are presented as means+/−SEM.A11 immunoreactivity and anti-β-aggregation activity in non-chaperone proteinsAnti-β-aggregation activity is not unique to chaperones. Thus, if chaperones with anti-β-aggregation activity contain the amyloid oligomer conformation, we assumed that other proteins known to suppress Aβ aggregation might also contain the amyloid oligomer conformation. One such protein is transthyretin (TTR), which has been shown to sequester Aβ and prevent amyloid formation [13], [14]. We confirmed the suppressive effect of TTR on Aβ aggregation in our model system (Figure 4A). Another protein with anti-Aβ-aggregation properties is α2-macroglobulin (α2MG). Although the genetic association between α2MG and AD remains controversial [15], the anti-β-aggregation activity of α2MG against Aβ has been reported to occur in vitro\n[16], [17], as we also observed (Figure 4B). As expected, A11 antibody immunostained dot blots of TTR and α2MG (Figure 4C), indicating that both proteins possess the amyloid oligomer conformation. Although TTR is a protein that forms amyloid in pathogenic conditions, its A11 immunoreactivity has never been reported [18]. If a significant number of TTR molecules somehow misfold and bind to A11 in solution, they are more likely to induce or cross-seed Aβ aggregation, as other misfolded pathogenic amyloid oligomers [2]–[5, Kayed et al., Society for Neuroscience Abstracts 2005;35:893.6]. In our experiment, this did not occur. Instead TTR suppressed Aβ aggregation (Figure 4A) [13], [14], indicating that the amyloid oligomer conformation within TTR is not misfolded but is natively folded.10.1371/journal.pone.0003235.g004Figure 4Dose-dependent suppression of Aβ aggregation by A11-immunoreactive non-chaperone proteins.(A) Aβ40 (final concentration: 5 µM) dissolved in aCSF was mixed with 0, 50, 500, or 5000 nM TTR in 10 mM Tris-HCl (pH 7.4) with 0.9% NaCl. ThT fluorescence indicates the level of Aβ aggregation. (B) Aβ40 (final concentration: 5 µM) dissolved in H2O was mixed with 0, 5, 50, or 500 nM α2MG in buffer (1.45 mM Tris-HCl [pH 8.0], 9.4 mM glycine, and 5.8 mM trehalose). (C) Dot blot of 100 µM TTR, 13.8 µM α2MG, 17.2 µM DT, and 15.2 µM ETA (3 µl each) probed with A11 antibody [2]. (D) and (E) Aβ40 (final concentration: 5 µM) dissolved in aCSF was mixed with buffer alone; 5, 50, or 500 nM DT; or ETA. For (D), the buffer contained 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA. For (E), the buffer contained 10 mM sodium phosphate (pH 7.5) and 150 mM NaCl. (F) Dot blot of 100 µM pdE (approx. MW: 50 kDa); 10 mM plE (approx. MW: 1000 Da); and 50 µM Aβ40 oligomer (3 µl each) probed with A11, secondary antibody alone without primary antibody, and 4G8 antibody (which reacts to amino acid residues 17–24 of human Aβ). (G) and (H) Aβ40 (final concentration: 5 µM) dissolved in PBS was mixed with buffer alone; 0.05, 0.5, 5 µM pdE; or plE. Data are presented as means+/−SEM.Analysis of the crystal structures of the TTR and α2MG receptor-binding domains reveal that both contain a β sandwich with a jelly-roll topology [19], [20]. The crystal structures of diphtheria toxin (DT) and exotoxin A (ETA) also have a homologous jelly-roll-like topology [21], [22]. As expected, A11 antibody also immunostained both DT and ETA (Figure 4C). Surprisingly, these amyloid oligomer conformation-containing toxins also suppressed Aβ aggregation in vitro in a dose-dependent manner, even though they have no known physiological relationship to Aβ (Figure 4D, E). These results suggest that A11 immunoreactivity is not only found in chaperones but is also found among certain non-chaperone proteins possessing anti-β-aggregation activity.Comparative analyses require control experiments. Thus in the present study, we tested whether A11-immunonegative proteins fail to inhibit β aggregation. Different chain lengths of D-poly glutamic acids (pdE) and L-poly glutamic acids (plE) did not exhibit A11 immunoreactivity or inhibit β aggregation, instead they promoted β aggregation (Figure 4F–H). These results support the conformational specificity of A11 antibody binding. Indeed, plE mostly exists as a random coil at neutral pH [23], the same pH we used in our control experiment. These results also suggest that A11 reactivity, and thus amyloid oligomer conformation, is not the only functional determinant of β-aggregation-promoting activity. Because we did not test the anti-β-aggregation activity of all purified and folded A11-non-reactive proteins, we could not exclude the possibility that an A11-non-reactive protein possessing anti-β-aggregation activity exists. For the same reason, our current results cannot exclude the possible existence of A11-reactive folded proteins that do not possess anti-β-aggregation activity. Therefore, we do not advance the idea that A11 immunoreactivity is a prerequisite for anti-β-aggregation activity.A11 affects GroE-assisted luciferase refolding and Hsp70-decelerated Aβ nucleationTo determine whether the A11-binding site plays a functional role in protein folding, we first investigated the effects of A11 antibody on GroEL/ES (GroE)-assisted firefly luciferase refolding. Luciferase samples were heated at 45°C for 5 min. After cooling the samples at room temperature for 5 min and adding luciferin substrate, GroEL/ES and ATP facilitated luciferase refolding, which was evident by the increased luminescence in the samples (Figure 5A). Luminescence was not observed in heat-treated samples co-incubated with GroEL/ES and ATP in the presence of A11. This suggests that A11 prevented GroEL/ES-assisted refolding of heat-denatured luciferase. Surprisingly, A11 itself assisted luciferase refolding to a similar extent as GroEL/ES.10.1371/journal.pone.0003235.g005Figure 5A11-mediated inhibition of GroE-assisted luciferase refolding and Hsp70-mediated decelerated Aβ nucleation.(A) In a sample of GroE (final concentrations: 1.25 µM GroEL and 2.1 µM GroES) and luciferase (final concentration: 100 nM), about 30% of luciferase activity was recorded relative to the activity of luciferase samples without heat-treatment, indicating that GroE-assisted partial refolding of heat-denatured luciferase had occurred. Luciferase activity was not observed when heat-treated luciferase was co-incubated with both GroE and A11 (final concentration: 0.1 mg/mL). A11 antibody assisted the refolding of luciferase (n = 15 for GroE and n = 6 for GroE/A11 and A11 alone, respectively). Data are presented as means+/−SEM. (B) Three microliters of 250 nM GroEL, 2.5 µM luciferase, and H2O were blotted onto a nitrocellulose membrane. The membrane was incubated either with the A11 antibody in 5% skim milk or with just skim milk. (C) Aβ40 (final concentration: 5 µM) dissolved in aCSF was mixed with or without Hsp70 (final concentration: 75 nM) and A11 (final concentration: 300 nM). All samples were incubated in the presence of 5 µM ThT. Triplicate measurements were taken every 30 min. Average fluorescence values are plotted. Error bars were omitted from the graph for clarity.Antibody-assisted refolding of proteins, including luciferase, has been reported previously in cases in which an antibody specific to a given protein facilitates antigen folding [24], [25]. Therefore, we surmised that the A11-assisted refolding of luciferase we observed might involve A11 binding to luciferase, which also possesses a β motif [26]. Thus, we tested the A11 immunoreactivity of purified luciferase by dot blot (Figure 5B). The luciferase sample exhibited A11 immunoreactivity that was weaker than that of the positive GroEL control blotted on the same membrane. These results provide a possible explanation for the assisted refolding of luciferase by A11: Like other antibodies having chaperone-like activity, A11 may induce luciferase refolding through specific interactions. If this is the case, then the diminished refolding of luciferase in samples containing both chaperonin and A11 is more likely due to the interaction of A11 with chaperonin rather than the interaction of A11 with luciferase.Next, we examined how A11 affects Hsp70-mediated suppression of Aβ aggregation by assessing Aβ aggregation kinetics (Figure 5C). The presence of Hsp70 decelerated the nucleation of Aβ aggregation. This effect was suppressed by A11, even though elongation was not substantially affected. This result indicates that the A11-binding site on Hsp70 may play a role in inhibiting nucleation but not elongation during Aβ aggregation. Interestingly, A11 alone failed to substantially affect Aβ aggregation at the concentration used. It will be interesting to examine in further detail whether A11 assists or prevents the oligomerization of Aβ and other amyloidogenic proteins. The present results indicate that A11-binding sites on chaperones may be functionally important.Deduction of A11-epitope and amyloid oligomer conformation through structural similarity searchOur analysis of A11 immunoreactivity and anti-β-aggregation activity of purified proteins led us to identify at least ten A11-reactive folded proteins with reported crystal structures. These included associated protein fragments and corresponding homologues from different species. Information about A11-immunoreactive proteins and their atomic structures may help us deduce the conformation shared by amyloid oligomers. To determine the A11 epitope and thus amyloid oligomer conformation, we searched each of the ten crystal structures for areas sharing the most surface physicochemical properties (see Methods). For the similarity search, we placed two main restrictions according to the known properties of amyloid oligomer conformations. First, the search was limited to only structures having a β strand, which is a generally accepted basic component of amyloid. Second, the search was limited to only areas that are less influenced by amino acid side chains, because A11 immunoreactivity is conformation dependent not amino-acid-sequence dependent [2]. Areas showing the most similarity are shown in red for each of the ten crystal structures (Figure 6 and Figure S1). All areas were located at the side edge of the β-sheet conformation, which comprises most of larger β-structural motifs such as the β-sandwich motif. Some of the potential A11-binding areas were located at or near the suggested substrate-binding domain of chaperones (e.g., GroEL, Hsp70, Hsc70, Hsp40) [27]–[30], while other potential A11-binding areas were not (e.g., ClpB [Hsp104] and Hsp90) [31]–[33]. The Aβ-binding site of TTR, previously identified through site-directed mutagenesis as well as through molecular modeling [13], [34], partially matched the area proposed here as the A11 epitope. Interestingly, the displacement of this particular terminal β strand by a conformational change promotes fibril formation of TTR itself [35]. These observations support the hypothesis that the β-sheet edge, a common feature of the ten crystal structures we assessed, plays a key role in A11-reactive proteins. Indeed, the negative design of the β-sheet edge supposedly helps natural β-sheet proteins to avoid β aggregation [36]. Here, we propose that β-sheet edge in natively folded amyloid oligomers not only protects proteins from undergoing β aggregation but also prevents interacting molecules from undergoing β aggregation. Thus, in some cases this can be considered to be a positive design. How the amyloid oligomer conformation influences protein conformation still needs to be determined in the future. This can be achieved by investigating the detailed molecular interactions between chaperone/chaperone-like proteins and the A11 antibody and between these proteins and amyloidogenic proteins.10.1371/journal.pone.0003235.g006Figure 6Sites with physicochemical properties of greatest similarity among ten A11-reactive proteins.The crystal structures of ten A11-reactive proteins were aligned to identify the areas where the physicochemical properties of solvent-exposed atoms show the highest similarity (red). (A) GroEL [1aon]. (B) α2MG [1ayo]. (C) Hsp40 [1c3g]. (D) DnaK (Hsp70) [1dkz]. (E) TTR (prealbumin) [1dvq]. (F) ETA [1ikq]. (G) DT [1mdt]. (H) ClpB (Hsp104) [1qvr]. (I) Hsc70 [1yuw]. (J) Hsp90 [2cg9]. The PDB codes of the structures used in the structural similarity search are shown in square brackets.DiscussionBy using the A11 antibody, an antibody known to detect the conformation of amyloid oligomers [2], we unexpectedly observed the amyloid oligomer conformation in a group of proteins whose folds were functionally characterized as native on the basis of their anti-β-aggregation activity. This contrasts with the misfolded pathogenic amyloid oligomers that promote β aggregation [2]–[5, Kayed et al., Society for Neuroscience Abstracts 2005;35:893.6]. Because we propose a novel corollary to the original claim that A11 recognizes a common structure shared by oligomeric intermediates formed by a diverse range of amyloidogenic proteins [2], our results were carefully examined.Could the recognition by A11 of the identified proteins be a non-specific effect caused, for example, by a small fraction of partially misfolded A11 antibody? We considered that this was unlikely because heat-denatured Hsp27 did not bind A11 (Figure 3). A11 reacted with some non-chaperone proteins (Figure 4). Moreover, none of the identified A11-immunoreactive proteins bound secondary antibody in samples lacking A11 (data not shown).Could the anti-β-aggregation activity of the identified proteins be induced by colloidal or crowding effects? We considered this to be unlikely because Hsp70/40 exhibited energy-dependent anti-β-aggregation activity (Figure 2E). Heat-treated Hsp27 (Figure 3C) and polyglutamate (Figure 4G,H) did not show the anti-β-aggregation activity. In addition, crowded GroE/A11 mixtures did not facilitate luciferase refolding more than less crowded GroE or A11 solutions (Figure 5A). Along similar lines, crowded Hsp70/A11 mixtures did not enhance anti-β-aggregation activity more than less crowded Hsp70 solutions (Figure 5C).Could A11 be recognizing traces of misfolded amyloidogenic intermediates of these proteins? We considered that this was unlikely because if the contaminating molecules of such misfolded amyloid intermediates were substantially present in the sample, then we would expect the A11-reactive proteins, especially non-chaperone proteins like toxins, not to suppress β aggregation (Figure 4D, E). Rather, we would expect them to promote β aggregation as Aβ oligomers did (Figure 2A), because misfolded amyloid oligomers characteristically promote oligomerization and fibrillization of other proteins, a suggested pathogenic mechanism underlying protein misfolding diseases [2]–[5, Kayed et al., Society for Neuroscience Abstracts 2005;35:893.6]. A functional property that was commonly observed in the group of A11-reactive proteins was anti-β-aggregation activity, which differentiated these proteins from misfolded pathogenic amyloid oligomers, which promote β-aggregation. Together with the conformation-specific nature of A11 reactivity, this phenomenon led us to conclude that a significant number of molecules in the purified samples were neither misfolded nor unfolded, but were most likely natively folded.Does the notion of a “common structure” mean anything if it so diverse it encompasses the folded and misfolded proteins? Under the right conditions, any protein can form amyloid [1]. The formation of amyloid structures by poly amino acids supports the premise that amyloid formation occurs independent of specific side-chain contacts or amino acid sequences [37]. Although plE forms amyloid at pH 4.1, it exists mostly as a random coil at physiological pH [23]. Indeed, plE dissolved in a neutral pH buffer showed neither A11 immunoreactivity nor anti-β-aggregation activity, which is consistent with the conformational specificity of A11 binding (Figure 4F–H). The difference in the staining patterns in immunoblots probed with A11 and in CBB-stained gels of bacterial cell lysate samples (Figure 1A) indicates that A11 does not bind just any protein. Not all A11-reactive proteins in the bands were necessarily natively folded or retained their in vivo conformation, since lysing the cells can significantly affect the conformation of some proteins. We believe that A11 specificity does exist, as demonstrated in the original study [2]. It is most important to note, however, that the specificity of A11 is completely different from that of other antibodies that recognize specific amino acid sequences in that A11 reactivity depends on the three-dimensional conformation of a protein since A11 specifically binds oligomeric intermediates but not unfolded monomers or fibers of amyloidogenic proteins [2]. Thus, A11 reactivity is independent of an antigen's amino acid sequence, because A11 specifically binds oligomeric intermediates formed by a diverse range of amyloidogenic proteins. The conformational-dependent binding specificity of A11 also implies that the amyloid oligomer represents a generic conformation [2]. On the basis of this particular generic property of the A11 antibody, we believe that A11 antibody is also capable of binding a group of natively folded proteins, as we observed in the present study.What is the functional significance of having an amyloid oligomer conformation within natively folded proteins? Function-blocking experiments clarified the functional importance of A11-binding sites on chaperones (Figure 5). To further tackle this issue structurally, we took a computational approach. A functional property shared by pathogenic amyloid oligomers and chaperones is that both affect the conformation of unfolded proteins. This property might depend on the structural plasticity that is shared by amyloid and the substrate-binding domain of chaperonin, called the apical domain [38]–[41]. The structural plasticity of amyloid is partially based on its polymorphism, the ability of one polypeptide to form aggregates of different structures [39]. Indeed, the polymorphic nature of amyloid sets it apart from other globular folded proteins with unique structures. Thus, it is unlikely that the amyloid oligomer conformation is represented by a unique structure.On the other hand, amyloid also exhibits isomorphism, the ability of different polypeptides to display similar morphologies [2], [39]. Taking advantage of this property, we searched for areas containing similar physicochemical properties on the surfaces of the crystal structures of A11-reactive folded proteins (Figure 6 and Figure S1). Our analysis results were consistent with the proposed structures of amyloid protofilaments or precursors [42]–[46]. Although the non-native amyloid precursor of β2-microglobulin is highly native-like, its edge strands contain perturbations that normally protect β-sandwich proteins from self-association [43]–[46]. Structurally, the edges of a completely regular β-sandwich motif are inherently β-aggregation prone [36]. Coincidentally, our structural similarity search revealed that the β-sheet edge of GroEL was located in the apical domain. A pioneering study on GroEL suggested that chaperonins act by stabilizing non-native intermediates against off-pathway misfolding and aggregation [27]. Because β-sheet structures are primarily formed through main-chain hydrogen bonds [4], [6], the induction of β-strand formation in a substrate through the β-sheet edge in the apical domain of GroEL may normally function to prevent non-specific side-chain contacts and thus non-specific aggregation, but in certain cases promote β aggregation as reported previously [47], [48]. We consider this idea to be speculative but consistent with the formation of β-strands by non-native substrates in a complex structure with Hsp40, and consistent with Hsp40-dependent amyloid assembly in cells [30], [49].Finally, we would like to mention evolutionary aspects of ‘native’ amyloid oligomers. The presence of the amyloid oligomer conformation within a natively folded protein indicates that the folding observed in this type of conformation is based on amino acid sequence, which is encoded in that particular protein's gene according to Anfinsen's hypothesis [11]. On the other hand, the unprecedented diversity of proteins containing amyloid oligomer conformations also indicates that the folding in these conformations may be caused by something else, especially in pathogenic conditions [1], [2], [4]. In evolution, aggregative properties of some proteins may have pressured the selection of anti-aggregation properties in some genes as a preventive measure. On the basis of the generic anti-aggregation characteristics of certain A11-reactive folded proteins, such as the chaperone family, we hypothesize that (1) the amyloid oligomer conformation has evolved over time as a means to prevent non-specific protein aggregations, and that (2) based on the structural commonality, amyloidogenic proteins may sporadically deviate from this system of preventing protein aggregation in some conditions such as aging in the extended life span of humanity today that has not been caught up by evolution.Materials and MethodsImmunoblot (Western blot and dot blot)Separated proteins by SDS-PAGE were transferred onto nitrocellulose membranes by using a wet system at 4°C. Alternatively, 3 µL of sample solution was dot-blotted onto membranes. After being blocked with milk, the blotted membrane was probed with anti-amyloid oligomer antibody A11 (1∶1000; Biosource, USA) [2] or 4G8 (1∶1000; Signet, USA). A11 and 4G8 antibody immunoreactivity were detected with HRP-conjugated anti-rabbit IgG (1∶2500) or anti-mouse IgG (1∶5000), respectively, followed by ECL. A replicated membrane probed with the secondary antibody alone did not display signals. Each panel displaying an immunoblot was from a single membrane and thus a single experiment. For presentation purposes, the squares containing each dot blot were arranged in a single column or row for figures.Aβ oligomer preparationAβ oligomer was prepared as described previously [2]. Briefly, lyophilized Aβ40 peptide (Peptide Institute, Japan) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (Wako, Japan) on ice and aliquoted to be frozen until use. Aβ was spin-vacuumed just prior to the experiment; dissolved in HFIP solution (final concentration: 10% (v/v) HFIP); and kept at room temperature under constant stirring. After 2 days, the tube was transferred to and maintained at 4°C.Thioflavin T (ThT) assayLyophilized Aβ40 peptide was first dissolved in HFIP on ice. Most of the HFIP was vacuum evaporated before Aβ was dissolved in H2O; PBS; or artificial cerebrospinal fluid (aCSF: 124 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgSO4, 1.25 mM NaH2PO4, 26 mM NaHCO3, and 10 mM glucose). Solubilized Aβ40 (5 µM) was mixed with a protein dissolved in a respective buffer. Note that different buffer conditions resulted in different aggregation kinetics of Aβ. ThT (10 µM unless otherwise noted) was added to each solution to assess the formation of β-sheet structures. Samples were incubated at 37°C in 96-well plates; triplicate measurements were taken every 30 min with an ARVO multilabel spectrofluorometer (PerkinElmer, USA). Plates were shaken for 10 sec prior to every measurement. Excitation and emission wavelengths were 440 and 486 nm, respectively [7]. In each experiment, triplicate wells containing protein sample at each concentration without Aβ were made. Results from the measurement of these wells were not included in the figures because the values were significantly smaller than those of wells with Aβ.Luciferase refolding assayTwenty microliters of 250 nM luciferase (Sigma) in 100 mM Tris-HCl (pH 7.4) and 5 µL of 20 mM DTT in H2O were mixed in each well of a 96-well plate. For heat treatment, plates were sealed and placed in an incubator maintained at 45°C. After 5 min, the plates were taken out and kept at room temperature for 5 min. Next, 2.5 µL of 40 mM DTT (Nacalai Tesque), samples representing four different conditions, and 2.5 µL of 80 mM Mg-ATP (Sigma) in H2O were added to each well. The four samples were as follows: (1) 15 µL of H2O; (2) 5 µL of H2O, 5 µL of 12.5 µM GroEL, and 5 µL of 21 µM GroES; (3) 5 µL of 1 mg/mL A11, 5 µL of 12.5 µM GroEL, and 5 µL of 21 µM GroES; and (4) 5 µL of 1 mg/mL A11, and 10 µL of H2O. The plate was incubated at 30°C for 1 h. As soon as 5 µL of 2 mM luciferin (Nacalai Tesque) dissolved in 100 mM Tris-HCl (pH 7.4) was added to each well, the luminescence was measured with an ARVO multilabel spectrofluorometer (PerkinElmer). The average luminescence value from H2O-incubated wells was subtracted from the luminescence value measured from other wells. The percentage of luminescence recorded in each well was calculated, taking as 100% the average value measured from wells that were not heat treated. The percentages determined for each condition were averaged and defined as percent (%) luciferase activity. GroE represents the combination of both GroEL and GroES.Structural similarity searchTen PDB files of A11-immunoreactive proteins were retrieved. In the preliminary search, 214 potential sites on the surfaces of ten structures were detected among the areas where the main β-sheet chain was exposed to the surface (Table 1). Atoms in these sites were first classified into six physicochemical types using the PATTY algorithm [50]. Structural alignment was carried out by aligning the spatial coordinates of atoms at every two sites and identifying pairs of atoms of the same physicochemical type. We then calculated similarity scores for these aligned structures. Twenty-three sites with relatively high similarity scores were determined using a clustering algorithm. For each structure, we selected sites having the highest similarity score. We then constructed a figure by performing multiple structure alignments on the ten sites having the highest similarity scores. Surface atoms were determined by calculating accessible surface areas (>0 Å2) at 1.4 Å of probe atoms. The atoms were clustered using the single-linkage clustering algorithm with a cutoff value of 4 Å. Clusters formed by less than four atoms were removed. Surface atoms that existed within 4 Å from atoms of the remaining clusters were included. Each cluster was defined as a single site.10.1371/journal.pone.0003235.t001Table 1Potential surface sites on ten A11-reactive proteins in which the main β-sheet chain is exposed to the surface.PDB codeNumber of sites1aon1141ayo81c3g61dkz51dvd71ikq71mdt151qvr131yuw132cg926Total214Similarity scoreEach atom located on a structure's surface was given a feature vector. The feature vector describes the local physicochemical environment of a surface atom. Atoms were first classified into six physicochemical types using the PATTY algorithm [50]: cation (AT1), anion (AT2), hydrogen-bond donor (AT3), hydrogen-bond acceptor (AT4), hydrophobic (AT5), and none of these (AT6). An atom that was both a hydrogen-bond donor and an acceptor (‘polar’ atom, according to the PATTY algorithm) was treated as 0.5 donor and 0.5 acceptor. The feature vectors were defined for each atom according to this atom-type classification. The feature vector C\ni of atom i was defined as:in which the summation was over all the solvent-accessible atoms (including atom i) of the protein,  if dij/dc≤1, and atom j is of type ATx; otherwise, . If atom j is both a donor and an acceptor and x = 3 or 4, a = 0.5; otherwise, a = 1. dij is the distance (in Å) between atom i and atom j. The parameter values dc = 3.2 were used. The similarity between each pair of atoms from the two regions was assessed by sij = TC(i, j), where atoms i and j were from regions a and b, respectively; and TC(i,j) is the Tanimoto coefficient:\nFinally, a similarity score S was defined for the two regions a and b: S = Σsij/min{Na,Nb}, where sij is defined as above; Na and Nb are the numbers of atoms of regions a and b, respectively; and the summation was over all atom pairs in the alignment except for those pairs of different atom types. Possible S values ranged from 0 to 1, where 1 represents the maximum similarity.Supporting InformationFigure S1Sites having the most similar physicochemical properties in the molecular structures of ten A11-reactive proteins. Because the aligned site in 1aon was hidden from view at the viewing angle we used for the other structures, only 1aon is shown from a different angle from other molecules. (A) GroEL [1aon]. (B) α2MG [1ayo]. (C) Hsp40 [1c3g]. (D) DnaK (Hsp70) [1dkz]. (E) TTR (prealbumin) [1dvq]. (F) ETA [1ikq]. (G) DT [1mdt]. (H) ClpB (Hsp104) [1qvr]. (I) Hsc70 [1yuw]. (J) Hsp90 [2cg9].(8.92 MB TIF)Click here for additional data file.\n\nREFERENCES:\n1. FandrichMFletcherMADobsonCM\n2001\nAmyloid fibrils from muscle myoglobin.\nNature\n410\n165\n166\n11242064\n2. KayedRHeadEThompsonJLMcIntireTMMiltonSC\n2003\nCommon structure of soluble amyloid oligomers implies common mechanism of pathogenesis.\nScience\n300\n486\n489\n12702875\n3. 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+{
+    "id": "PMC2528940",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528940\nAUTHORS: Susham S. Ingavale, Yun C. Chang, Hyeseung Lee, Carol M. McClelland, Madeline L. Leong, Kyung J. Kwon-Chung\n\nABSTRACT:\nCryptococcus neoformans is an environmental fungal pathogen that requires atmospheric levels of oxygen for optimal growth. For the fungus to be able to establish an infection, it must adapt to the low oxygen concentrations in the host environment compared to its natural habitat. In order to investigate the oxygen sensing mechanism in C. neoformans, we screened T-DNA insertional mutants for hypoxia-mimetic cobalt chloride (CoCl2)-sensitive mutants. All the CoCl2-sensitive mutants had a growth defect under low oxygen conditions at 37°C. The majority of mutants are compromised in their mitochondrial function, which is reflected by their reduced rate of respiration. Some of the mutants are also defective in mitochondrial membrane permeability, suggesting the importance of an intact respiratory system for survival under both high concentrations of CoCl2 as well as low oxygen conditions. In addition, the mutants tend to accumulate intracellular reactive oxygen species (ROS), and all mutants show sensitivity to various ROS generating chemicals. Gene expression analysis revealed the involvement of several pathways in response to cobalt chloride. Our findings indicate cobalt chloride sensitivity and/or sensitivity to low oxygen conditions are linked to mitochondrial function, sterol and iron homeostasis, ubiquitination, and the ability of cells to respond to ROS. These findings imply that multiple pathways are involved in oxygen sensing in C. neoformans.\n\nBODY:\nIntroduction\nCryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening meningoencephalitis primarily in immunocompromised patients [1]. C. neoformans is an obligate aerobe and its natural environment includes pigeon droppings, soil contaminated with avian guano [1] and decaying tree barks [2],[3]. In laboratory conditions, atmospheric levels of oxygen (∼21%) are required for optimal growth of C. neoformans and lower oxygen concentrations lead to a significant reduction in cell growth [4]. Upon inhalation, C. neoformans disseminates to central nervous system and causes life-threatening meningoencephalitis mostly in patients with immune deficiency. It is well known that oxygen concentrations in the human brain and other anatomical sites are significantly lower compared to atmospheric levels [5]. Thus, in order to establish infection in the brain, C. neoformans needs to sense and adapt to low oxygen conditions. Even though the mechanisms involved in oxygen sensing and adaptation to low oxygen conditions have been studied in humans and other organisms, this important aspect towards understanding the pathobiology of C. neoformans remains elusive.In most eukaryotic organisms, molecular oxygen is essential for oxidative phosphorylation and biosynthetic processes. To survive in low oxygen conditions or hypoxia, organisms have evolved oxygen-sensing mechanisms that activate a complex set of responses. In mammals, a major effector of the hypoxic transcriptional response is the hypoxia inducible factor (HIF1α). Under high oxygen conditions, HIF1α is continuously degraded through hydroxylation while in low oxygen conditions, it is not hydroxylated thus avoiding degradation and activating the target genes [6]–[9].In mammalian systems, cobalt chloride has been widely used as the hypoxia-mimicking agent. Studies done so far have shown that the CoCl2 mediated hypoxia-mimicking response is induced through the stabilization of HIF1α in the presence of oxygen [10]–[13]. The absence of HIF1 homolog in Saccharomyces cerevisiae indicates a different mode of oxygen sensing [14]. While CoCl2 has been shown to have pleiotropic effects on cellular mechanisms in fungi, only a few of those have been linked to oxygen sensing [15]–[17].Recent work from our laboratory has established a link between sterol synthesis, oxygen sensing and CoCl2 sensitivity in C. neoformans\n[18],[19]. Under low sterol or low oxygen conditions, C. neoformans homologs of the mammalian SREBP (sterol regulatory element-binding protein) transcription factor and its binding partner SCAP (SREBP cleavage-activating protein), named Sre1 and Scp1 respectively, regulate the expression of several genes involved in ergosterol biosynthesis and iron homeostasis. Mutations in SRE1 and SCP1 genes resulted in reduced growth under low oxygen condition and these mutants were not able to establish infection in the mouse brain [19]. Interestingly, both sre1 and scp1 mutants show reduced growth on media containing CoCl2. Further characterization of these mutants demonstrated that the response to CoCl2 in C. neoformans mimics certain aspects of the low oxygen condition by targeting enzymes in the sterol biosynthetic pathway [18].In C. neoformans, apart from the genes involved in ergosterol biosynthesis, not many other genes have been identified that are required for adaptation to low oxygen as well as to high concentration of CoCl2. To identify genes involved in low oxygen response, our laboratory has taken two different approaches; 1) screening for the mutants directly under low oxygen conditions (manuscript in preparation) and 2) screening for the mutants that are sensitive to hypoxia-mimicking agent. In this study, we have explored the possibility of using CoCl2 as an effective chemical to screen for other factors involved in oxygen sensing. Using a genetic screening approach, we have isolated cobalt chloride hypersensitive mutants. Through characterization of these mutants, we have shown a link between the ability of cells to respond to CoCl2 and/or low oxygen conditions and mitochondrial function, sterol homeostasis, sensitivity to reactive oxygen species (ROS) and ubiquitination.Materials and MethodsStrains, media, and growth conditions\nC. neoformans serotype D genomic sequencing strain; B-3501A (http://www-sequence.stanford.edu/group/C.neoformans/index.html) was used as the wild type strain. All the strains in this study were derived from this strain. The strains were maintained on YES and YES+geneticin (100 µg/ml) where necessary. YES medium consists of 0.5% (w/v) yeast extract, 2% glucose and supplements containing uracil, adenine, leucine, histidine and lysine (225 µg/ml). For CoCl2 screening and sensitivity assays YES+0.7 mM CoCl2 was used. For all FACS experiments and O2 consumption assays yeast cells were grown to log phase (OD600 = 0.5) at 30°C in YES medium then 1 mM CoCl2 was added and grown further for 4 hrs at 30°C. Low-oxygen conditions (1% O2) were maintained using an Invivo2 400 workstation (Ruskinn) at 37°C.Construction and screening of library for CoCl2-sensitive mutantsSince the frequency of random integration is very high by Agrobacterium tumefaciens mediated transformation (ATMT), a T-DNA insertion library of C. neoformans serotype D genomic strain (B-3501A) was made using ATMT [20]. The plasmid pYCC716 containing T-DNA fragment was used to create A. tumefaciens strain C603. This plasmid has a NEO gene conferring geneticin resistance. ATMT of C. neoformans was carried out as previously described [20]. To make a library, 30,000 individual transformants were picked and inoculated in 96 well plates containing YPD+50 µg/ml geneticin+200 µM cefotaxime. After 48 hr of growth at 30°C, glycerol stocks were made and stored at −80°C.To identify the genes involved in the sensitivity to CoCl2, we screened T-DNA insertion library of C. neoformans. The primary screening for mutants sensitive to CoCl2 was carried out by replica spotting of the library consisting of 30,000 clones on YES+0.7 mM CoCl2 and YES+geneticin plates. Mutants that failed to grow on CoCl2 medium were selected for further analysis. A total 37 mutants (including 5 redundant isolates) were selected based on their sensitivity to CoCl2. Transformants selected for further analyses were streaked on selective YES+geneticin+cefotaxime medium.Mapping of T-DNA insertion siteIsolation and analysis of genomic DNA was carried out as described previously [21]. To identify the number of insertion events in these transformants, Southern hybridization was done using the ORF of the NEO gene as a probe. Radioactive probes were prepared using StripEZ kit (Ambion, Austin, TX) according to manufacturer's manual. By using vectorette system (Sigma, Woodlands, TX), T-DNA insertion site was mapped in C. neoformans mutants and genomic sequence flanking the insertion site was obtained. BLAST analysis of these sequences was done to reveal which loci have been affected.Construction of complementation strainsThe wild-type gene for each affected gene was PCR amplified from B-3501A, cloned and sequenced. For complementation, using biolistic transformation method [22] T-DNA insertion mutants were transformed with the respective wild-type gene using NAT selection marker. Stable transformants were selected after repeated transfer on YES agar. PCR was used to identify integrative transformants containing an intact wild type gene and Southern blot analysis was used to confirm the transformation.Growth assaysPlate assays were carried out to determine if the mutants are sensitive to an external source of ROS-generating chemicals. YES plates containing 25 mM of sodium dihydrogen citrate, pH 4.0 (buffered YES) were made with 0.5 mM of H2O2 and 1mM of NaNO2. For other chemicals such as paraquat (0.25 mM) and diethyl maleate (2 mM), plates were made using regular YES. Cultures were either streaked or spotted and incubated at 30 and 37°C. For growth phenotype analysis serial dilutions of cultures were spotted on YES plates and incubated at 30°C, 37°C in ambient oxygen level and at 37°C under 1% O2 for 3–4 d.Confocal microscopyCells were grown overnight in YES medium, diluted in fresh YES to obtain initial OD600 of 0.2–0.3 and incubated for an additional 4 h. After 4 h, cells were harvested and resuspended in YES medium containing the MitoTracker Red CMXRos stain (Molecular probes, Eugene, OR) in the final concentration of 100 nM. Cells were incubated at 30°C for 1 h. After staining, cells were washed 3 times with 1× PBS and resuspended in 1× PBS. For mitochondrial DNA staining, cells were suspended in 10 mM HEPES buffer, pH 7.4, containing 5% glucose. SYTO 18 mitochondrial stain (Molecular probes, Eugene, OR) was added to a final concentration of 10 µM and cells were incubated at room temperature for 15–20 min. Before proceeding for microscopy, cells were washed and suspended in HEPES buffer. Images were collected on a Leica SP5 confocal microscope (Leica Microsystems, Exton, PA, USA) using a 100× oil immersion objective NA 1.4 zoom 4. Fluorochromes were excited using an Argon laser for SYTO18 and a 561 nm diode laser for MitoTracker Red CMXRos. Differential interference contrast (DIC) images were collected simultaneously with the fluorescence images using the transmitted light detector. Images were processed using Leica LAS-AF software (version 1.8.2 build 1465).ROS measurementCells were grown overnight in YES medium at 30°C, diluted in fresh YES and allowed to grow till OD600 reaches 0.4–0.5. Dichlorodihydrofluorescein diacetate (H2DCFDA) (Molecular Probes, Eugene, OR) at a final concentration of 10 µM was added for an additional 2 h to load the dye into cells. After 2 h, one set was supplemented with 1 mM CoCl2 and both sets were further incubated at 30°C for 4 h. Cells were harvested, washed 3 times with 1× PBS and an equal number of cells (2×106) were resuspended in 1 ml of PBS. Fluorescence was then analyzed using a Becton- Dickinson FACScan Flow Cytometer. The parameters for Fluorescence-activated cell sorting (FACS) were set at excitation of 488 nm and for emission FITC channel using standard FL-1 filter (530/30 nm).Oxygen consumption assaysCells were grown overnight in YES medium at 30°C, diluted in fresh YES to get starting OD600 of 0.2–0.3 and incubated for an additional 4 h. After 4 h, one set was supplemented with 1 mM CoCl2 and both sets were further incubated at 30°C for 4 h. Cells were harvested and washed with PBS and an equal number of cells (5×107) were resuspended in 2 ml of YES and YES+CoCl2. Cells were then loaded into a sealed 2.0 ml glass chamber. Changes in oxygen tension were measured at room temperature with a Clark-type oxygen electrode, and the respiratory rate was calculated as a change in oxygen tension over time. In order to quantify mitochondrial respiration oxygen consumption, respiratory inhibitor Antimycin A (1 µg/ml) was used to block mitochondrial respiration.Gene expression profilingMicroarray slides were purchased from an academic consortium co-ordinated by T. Doering, C. Hull and J. Lodge at the University of Washington-St Louis. Arrays contain 7,738 70 bp DNA oligomers designed to uniquely recognize each gene in the C. neoformans serotype D genome plus control oligomers. Each oligomer is printed in duplicate. Overnight cultures of wild-type (B-3501A) strain was refreshed and grown in YES for 5 h and incubated for an additional 2 h in the presence or absence of 0.6 mM CoCl2. Wild-type strain does not show any growth inhibition at this concentration when treated only for 2 h. RNA was extracted from yeast cells using Trizol (Invitrogen, Carlsbad, CA), treated with RNAse-free DNase (Ambion, Austin, TX) for the removal of genomic DNA, and purified with RNeasy MinElute cleanup kit (Qiagen, Valencia, CA). RNA was labeled and hybridized as described before [18]. Data were collected using a GenePix 4000B scanner. For data analysis, features were examined and flagged using Genepix Pro 6.0 software (Axon Instruments, Foster City, CA) and confirmed manually. Data were further analyzed in mAdb database at http://mAdb.niaid.nih.gov (NIAID). Three biological repeats were performed using three independent RNA sets isolated from cells cultured on different days and the dye-reverse hybridizations were performed for all 3 sets. One set of RNA was also subjected to technical repeats. All statistically significant genes were identified by significance analysis of microarrays (SAM) using a mean false discovery rate (FDR) of 5% [23]. Only statistically significant genes were used for data analysis. To simplify presentation of the results, Figure 8 shows categorization of statistically significant genes identified by SAM, that have average changes greater than 2-fold. Table S1 lists the statistically significant genes identified by SAM that have average changes of greater than 2-fold in the wild-type strain upon CoCl2 treatment. All statistically significant genes identified by SAM are given in Table S2 in the supplementary material. The array data has been submitted at Gene Expression Omnibus (GEO) site (accession no. GSE11390).ResultsConstruction of T-DNA insertional library and screening for cobalt chloride sensitive mutants\nAgrobacterium tumefaciens–mediated transformation is a very useful technique to study Cryptococcus. The high frequency of random integrations by ATMT [20],[24] enabled the construction of a T-DNA insertional library using the C. neoformans serotype D genomic strain (B-3501A). The majority of transformants harbored a randomly integrated single copy of T-DNA and were mitotically stable (unpublished data). The library consists of 30,000 individual transformants.To identify the genetic loci involved in the sensitivity to CoCl2, the T-DNA insertion library was screened for mutants sensitive to CoCl2. A total of 37 mutants that failed to grow on CoCl2 medium were selected for further analysis. Sequence analysis of the genomic sequence flanking the insertion site revealed that the T-DNA was inserted in a wide array of genes (Table 1). Based on the sensitivity towards CoCl2, mutants could generally be categorized into 2 types: mutants that either showed complete growth inhibition or those that exhibited partial growth on media containing 0.7 mM CoCl2 (Table 1). From the mutants listed in Table 1, mutants 146G2, 92D9, 135G10, 132H6, 17B1, 238D8, and 72B10 showed partial growth on medium containing cobalt chloride. All other mutants were hypersensitive to cobalt chloride and did not show any growth on CoCl2 containing plates (Table 1). Figure 1 shows the sensitivity pattern of some of these mutants from each group. To establish a link between a mutation and the CoCl2 sensitive growth phenotype, some of the mutants were complemented with wild-type gene. These complemented strains, when grown on CoCl2 containing plates, were no longer hypersensitive to CoCl2 and the growth was comparable to the wild-type strain (Figure 2). This indicates the defects in these genes result in the sensitivity towards cobalt chloride. In this screening, both sre1 and scp1 mutants were isolated several times and it has previously been demonstrated that the deletion of each of these two genes leads to CoCl2 sensitivity and these genes are involved in oxygen sensing in C. neoformans\n[18]. The repeated isolation of sre1 and scp1 mutants in the screen strongly attests to the suitability of this screening approach in the identification of genes responsible for growth under low oxygen conditions.10.1371/journal.ppat.1000155.g001Figure 1Cobalt chloride sensitive phenotype of T-DNA insertional mutants.Ten fold serial dilutions of wild type and T-DNA insertional mutant strains were spotted on YES and YES+CoCl2 plates and incubated at 30°C for 3 d.10.1371/journal.ppat.1000155.g002Figure 2Complementation of CoCl2 sensitivity phenotype.Individual mutant was transformed with the corresponding wild type gene. Original and complemented strains were patched on YES and YES+CoCl2 plates and incubated at 30°C for 3 d.10.1371/journal.ppat.1000155.t001Table 1A list of CoCl2 sensitive mutants isolated from T-DNA insertion library and their growth phenotypes.Clone no.Locus IDDescriptiona\nGrowthb onCoCl2\n37°C/1%O2\nH2O2\nNaNO2\nParaquatDEM\nSterol Biosynthesis Pathway\n10E11CNC00950SCAP1\n\n−\n\n−/+\n\n−\n\n+/−\n\n−/+\n\n+\n87A1CNJ02310SREBP1\n\n−\n\n−/+\n\n−\n\n−\n\n+/−\n\n+\n146G2CNM00870Squalene synthase\n−/+\n\n−\n\n−\n\n−\n\n−\n\n−\n\nGenes Involved in Mitochondrial Function/Energy Metabolism\n10D12CNC05260H+ transporting ATP synthase\n−\n\n−\nnd*nd*\n−\n\n−\n46E10CNA00760tRNA (guanine-N2-)-methyltransferase\n−\n\nTs\n\n−/+\n\n−\n\n−\n\n−\n66G6CNC04010HIG_1_N domain family\n−\n\nTs\n\n−/+\n\n−\n\n−/+\n\n−\n69G9CNE00180t-RNA Lysine\n−\n\n−\n\n−\n\n−/+\n\n−\n\n−/+\n92D9CNJ01940Dihydrofolate reductase\n−/+\n\n−\n\n−\n\n+/−\n\n−\n\n−/+\n135G10CND04070NADH∶ubiquinone oxidoreductase\n−/+\n\n−\n\n−\n\n−\n\n−\n\n−\n161B3CNM01080ATP∶ADP antiporter\n−\n\n−\n\n−/+\n\n+/−\n\n+\n\n+/−\n\nVesicular Transport\n132H6CNF00890Importin beta-4 subunit\n−/+\n\nTs\n\n−/+\n\n−/+\n\n−\n\n−\n155B3CNA06920V ATPase subunit H\n−\n\n−\n\n−\n\n−\n\n−\n\n−\n252C2CNC07180Vacuolar protein sorting 54\n−\n\n−\n\n−\n\n−/+\n\n−/+\n\n+/−\n239E3CNJ03270Clathrin heavychain 1\n−\n\nTs\n\n−\n\n−\n\n−\n\n−\n\nRegulatory Function Related\n27C2CNK03380Fungal Zn(2)-Cys(6) domain family\n−\n\n−\n\n+/−\n\n−/+\n\n+\n\n−\n215F4CNF01510Nonsense-mediated mRNA decay factor\n−\n\n−\n\n+\n\n+\n\n−/+\n\n+/−\n238D8CNN00160Two-component protein-histidine kinase\n−/+\n\n−/+\n\n+/−\n\n+\n\n−/+\n\n+\n293A4CNM01040C6 transcription factor\n−\n\n−\n\n−\n\n+\n\n+/−\n\n−/+\n\nEnzymes/Transporters\n72B10CNG01540Hexose transport related protein\n−/+\n\nTs\n\n−\n\n−\n\n−\n\n−/+\n103H12CNN01350ATP dependent clp protease\n−\n\nTs\n\n−\n\n−\n\n−\n\n−\n148C4CNF00750Seroheme synthase\n−\n\n−\n\n−\n\n−\n\n−\n\n+\n262F2CNM00800Amino acid transporter\n−\n\nTs\n\n−\n\n−\n\n−\n\n−\n297F11CNF03950Myo-inositol oxygenase\n−\n\n−\n\n−\n\n+/−\n\n+/−\n\n+/−\n\nMutants with Multiple Insertion Sites\n2\n29A6CNA07630Hypothetical\n−\n\n−\nnd*nd*\n−\n\n−\n94F1CNH00220Ubiquitin protein ligase\n−\n\nTs\n\n−\n\n−\n\n−\n\n−/+\n209A11CNH03300Microtubule binding protein\n−\n\n−\n\n−/+\n\n−\n\n+/−\n\n−/+\n234E1CNF03720C-22 sterol desaturase\n−\n\n−\n\n��\n\n−/+\n\n−/+\n\n−\nCNG02960-Hypothetical--CNG02950-Dihydrodipicolinate synthetase\nHypothetical and Expressed Proteins\n17B1CNB03300Hypothetical protein\n−/+\n\nTs\nnd*nd*\n−\n\n−\n48B7CNL04460Hypothetical\n−\n\n−\n\n−\n\n−/+\n\n−/+\n\n+/−\n67A3CNA00940Expressed protein\n−\n\nTs\n\n−/+\n\n+\n\n+/−\n\n−\n95C11CNF01170Expressed protein\n−\n\n−\n\n−\n\n−\n\n+\n\n−/+\n262A5CNG02200Hypothetical\n−\n\n−\n\n+/−\n\n−\n\n−/+\n\n+/−\naAnnotations were obtained from NCBI database (http://www.ncbi.nlm.nih.gov) with additional hand editing based on BLAST searches.b\n+, growth similar to WT; +/−, slight reduction in growth compared to WT; −/+, significant reduction in growth compared to WT; −, no growth. Ts, temperature sensitive, mutants did not grow at 37°C. *, phenotype could not be determined because these mutants did not grow at pH 4.0.1Multiple mutants were obtained with same insertion sites.2Based on Southern analysis, these mutants have 2 or more T-DNA insertions. Only the loci that were able to produce PCR product and sequenced were shown.CoCl2-hypersensitive mutants are unable to grow in low oxygen conditionsTo determine if the CoCl2 sensitive mutants also showed reduced growth in low oxygen conditions, cells were spotted on YES plates and incubated under different conditions: 30°C+ambient air (21% O2), 37°C+21% O2, and 37°C+1% O2. 37°C was chosen in this set of experiments in order to mimic the human body temperature. In the selected mutant pool, 10 of 32 mutants either failed to grow or grew poorly at 37°C, exhibiting a temperature-sensitive (Ts) phenotype (Figure 3A and Table 1). Most interestingly, the remaining non-Ts mutants showed reduced or no growth in low oxygen conditions (Figure 3B and Table 1). These results showed that screening for CoCl2 sensitivity is a useful approach to find mutants sensitive to low oxygen and suggested a strong correlation between CoCl2 sensitivity and sensitivity to low-oxygen conditions in C. neoformans. To identify the cellular processes involved in CoCl2 and/or low oxygen sensitivity, we categorized these mutants into different functional groups as shown in Table 1. Clearly, the processes involved in CoCl2 and/or low oxygen sensitivity are complex.10.1371/journal.ppat.1000155.g003Figure 3CoCl2 sensitive mutants exhibit hypoxia sensitive phenotype.(A). Mutants showing temperature sensitive phenotype. Yeast cells were serially diluted and spotted on YES and incubated at 30°C and 37°C for 3 d. (B). Mutants showing hypoxia sensitive phenotype. To check growth in low oxygen conditions, serial dilutions of wild type and mutant cells were spotted on YES and plates were incubated at 30°C, 37°C, and 37°C+1%O2 for 3 d.Mitochondrial membrane permeability defectWe noted that seven mutants listed in Table 1 are directly related to mitochondrial function. In the mammalian system, cobalt chloride is known to affect mitochondrial function [11], [25]–[27]. Cobalt has also been shown to target mitochondria and induce respiratory deficiency in yeast [15],[17],[28],[29]. Various aspects of mitochondrial dysfunction can be detected by analyzing the mutants for perturbed mitochondrial membrane potential and efficiency of respiration. First, we examined all the cobalt chloride sensitive mutants for any perturbation in mitochondrial membrane potential by using mito-tracker dyes such as CMXRos. Confocal microscopy results showed that six different mutants were unable to retain the mito-tracker dye indicating the presence of dysfunctional mitochondria (Figure 4A, red stain). We observed heterogeneity in the cell size of these strains that maybe resulted from mutation caused by insertion of T-DNA. Four mutants belonged to the group involved in mitochondrial function in Table 1 (10D12, 161B3, 69G9, and 66G6). Both genes affected in 10D12 and 161B3 mutants have been studied in detail in S. cerevisiae\n[30]–[32]. Locus CNC05260 (mutant 10D12) encodes subunit f of the F(0) sector of mitochondrial F1F0 ATP synthase. The other locus, CNM01080 (mutant 161B3), encodes ATP∶ADP antiporter that catalyzes the exchange of ADP and ATP across the mitochondrial inner membrane. In the mutant 69G9, a tRNA lysine gene involved in mitochondrial protein synthesis [33],[34] is disrupted. The locus CNC04010 (mutant 66G6) encodes a hypothetical protein that has hypoxia induced protein conserved region (HIG_1_N domain). Two mutants (146G2 and 67A3) that also failed to accumulate the CMXRos dye did not belong to the group involved in mitochondrial function (Table 1). Mutant 146G2, containing a T-DNA insertion in ERG9 gene encoding squalene synthase (CNM00870), also showed a defect in mitochondrial membrane potential. As the name suggests, this enzyme is involved in ergosterol biosynthesis and joins the two farnesyl pyrophosphate moieties to form squalene [35]. The hypothetical protein encoded by CNA00940 (mutant 67A3) has two trans-membrane domains and a weak homology to the t-RNA synthetase subunit. To determine whether these mutants lacked mitochondria, yeast mitochondrial DNA specific dye Syto18 was used for staining. Accumulation of Syto18 in the cells indicated that all 6 mutants contained mitochondrial DNA (Figure 4B, green stain). All the other mutants listed in Table 1 were also analyzed for reduced membrane potential by staining with CMXRos. The staining profile of these mutants was similar to that of wild type cells indicating that mitochondrial membrane potential was not disturbed in the rest of mutants (unpublished data).10.1371/journal.ppat.1000155.g004Figure 4Confocal microscopy for mitochondrial staining.(A) Cells were grown to log phase and stained with mito-tracker dye CMXRos. Red color in the wild type shows the normal membrane permeability and diminished red color in the mutants suggested defect in mitochondrial membrane permeability. (B) Cells were grown to log phase and stained with SYTO18 dye for mitochondrial DNA. Green color indicates the presence of mitochondrial DNA. Experiments were done at least two times using mito-tracker red and SYTO18 dyes for these strains. Scale bar represents 3 µm.CoCl2-sensitive mutants have respiratory defectsAs mentioned above, another way to analyze mitochondrial dysfunction is to assess the efficiency of respiration, which can be accomplished by measuring oxygen consumption. From the mutants listed in Table 1, all of the mutants in “mitochondrial function/energy metabolism” category and selected mutants from other categories were chosen to assay the rate of oxygen consumption. As presented in Figure 5, 9 mutants, 155B3, 146G2, 10D12, 135G10, 161B3, 69G9, 94F1, 46E10, and 92D9 showed a greater than 40% reduction in the rate of oxygen consumption compared to that of wild type in the absence of any chemical treatment. Four of these mutants, 146G2, 10D12, 161B3, and 69G9 also had a mitochondrial membrane potential defect as demonstrated in Figure 4. Even though mitochondrial membrane potential seemed unperturbed in the other 5 mutants, the reduction in oxygen consumption indicated those mutations seemed to have caused severe respiratory defects. Interestingly, mutant 66G6 had a mitochondrial membrane potential defect but showed only close to a 20% reduction in oxygen consumption rate compared to wild type in the absence of any chemical treatment. Among the rest of the mutants, for instance, 95C11 and 215F4 showed only a ∼15%–20% reduction while mutants 297F11, 48B7, and 72B10 had similar rates of oxygen consumption compared to that of the wild type.10.1371/journal.ppat.1000155.g005Figure 5Oxygen consumption is reduced in yeast grown in CoCl2-containing medium.Yeast cells were grown as described in Materials and Methods. A total of 5×107 washed cells were suspended in 2 ml media, and changes in oxygen tension were measured at room temperature using a Clark type electrode. Samples were measured with no addition of chemical (control), with antimycin A (A+), with CoCl2 (Co+), or with CoCl2 and antimycin A (Co+, A+).To understand if the reduced oxygen consumption is due to a defect in the core respiration process, antimycin A was used. Antimycin A blocks mitochondrial respiration by inhibiting complex III of the electron transport chain and the use of antimycin A revealed the rate of oxygen consumption in the core respiration process. In wild-type cells, addition of antimycin A resulted in a 30% reduction in oxygen consumption. In mutants 155B3, 146G2, 10D12, 135G10, 161B3, and 69G9, the addition of antimycin A did not induce further reduction compared to the control samples (Figure 5). Similarly, mutants 94F1, 66G6, and 95C11 showed no significant difference in respiration rates between antimycin A treated and non-treated samples (p<0.02). These results suggest the reduction in the oxygen consumption in these mutants is due to the block in the antimycin A sensitive pathway and the genes targeted in these mutants are responsible for proper functioning of core respiration. It is not clear why in mutants 215F4 and 297F11 the oxygen consumption rate was close to the wild type and yet not sensitive to antimycin A treatment. In a second group of mutants, respiration was sensitive to antimycin A treatment. In the mutant 46E10 antimycin A caused 80% reduction in oxygen consumption while in mutants 72B10, 92D9, and 48B7 it was 30%, 40%, and 65%, respectively. This significant reduction in oxygen consumption in the presence of antimycin A in these 4 mutants indicates unlike above-mentioned mutants, cells are able to respire through the core electron transport chain.Next, the cells were treated with cobalt chloride and assayed for oxygen consumption in the presence and absence of antimycin A. Compared to antimycin A, treatment with cobalt chloride showed an increased reduction in oxygen consumption in wild type. Addition of antimycin A to the cells grown in the presence of CoCl2 did not show any further reduction in the rate of oxygen consumption in the wild type as well as in all the mutants. This suggested that cobalt chloride strongly inhibits both antimycin A sensitive and antimycin A insensitive respiration pathways in all strains. Unlike in mutants where CoCl2 showed a significant effect on respiration compared to antimycin A alone, the reduction in the rate of respiration due to antimycin A or CoCl2 or in combination of both was similar in mutants 46E10 and 48B7. This indicates that in these mutants, antimycin A insensitive respiration pathways are more severely affected than the respiration through antimycin A sensitive core electron transport chain (ETC). These results indicate that 12 of the 15 genes affected in cobalt chloride–sensitive mutants influence oxygen consumption either through antimycin A sensitive mitochondrial respiration or antimycin A insensitive respiration.Mutants show elevated ROS levels and are sensitive to oxidantsCobalt is one of the transition metals known to generate a spectrum of reactive oxygen species (ROS) in the Fenton reactions and subsequent lipid peroxidation [36],[37]. Generation and accumulation of ROS in the cellular environment due to cobalt chloride creates an imbalance in the oxidative state of the cells. This ROS-induced oxidative stress could be another factor that is leading to cobalt chloride sensitivity in the mutants. To investigate the possibility that the mutants have abnormal ROS production, the fluorescent dye H2DCFDA was employed. This probe increases its fluorescence when oxidized by ROS. Cells preloaded with H2DCFDA were treated with cobalt chloride and analyzed by FACS along with non-CoCl2–treated cells. Upon CoCl2 treatment, we observed only a slight increase in ROS levels in the wild type cells. One interesting observation to note here is that in four mutants, their intrinsic ROS levels were higher (1.5- to 2.3-fold increase in the fluorescence intensity) than that of wild type even without CoCl2 exposure (10D12, 66G6, 155B3, and 161B3; Figure 6). Compared to cobalt chloride treated wild-type cells, some mutants such as 46E10, 92D9, 95C11, and 135G10 showed an increase in ROS levels after incubation with CoCl2 (1.2- to 2.2-fold increase in fluorescence intensity; Figure 6). Therefore, most of the mutants have increased ROS levels either with or without CoCl2 treatment.10.1371/journal.ppat.1000155.g006Figure 6CoCl2-sensitive mutants accumulate reactive oxygen species (ROS).Wild-type and mutant cells were grown to log phase at 30°C, loaded with H2DCFDA and exposed to CoCl2 for 4 h. ROS accumulation was assessed by flow cytometry. Each panel represents the FACS analysis result of each strain; filled peaks (black) represent fluorescence intensity of untreated cells while line peaks (grey line) represent the fluorescence intensity of CoCl2-treated cells on the FL1-H scale. All the FACS experiments were done multiple times, and data presented here are a representative of the experiments.Cellular antioxidant system influences the ability of cells to tolerate various oxidants. Unlike in wild-type cells, if the antioxidant system is not functioning properly in the mutants, they can exhibit hypersensitivity towards ROS and RNS (reactive nitrogen species) generating chemicals. To determine how CoCl2-sensitive mutants respond to the external source of ROS/RNS, growth was monitored on media containing H2O2, sodium nitrite, paraquat, and diethyl maleate. These four reagents act on different cellular targets and generate reactive oxygen/nitrogen species. H2O2 has the ability to directly damage nucleic acids, proteins, and lipids [38],[39]. The effect of H2O2 was analyzed at pH 4.0 and both 30°C and 37°C. 28 of the 29 mutants showed increased sensitivity towards H2O2 at 37°C compared to 30°C (Figure 7A and Table 1). Few mutants such as 95C11, 69G9, and 94F1 showed increased sensitivity even at 30°C. Sodium nitrite is known to generate nitric oxide (NO) at pH 4.0 [40],[41]. Nitric oxide reacts with different chemicals inside the cells forming reactive nitrogen species (RNS). The targets for RNS include protein-bound metal centers, thiols, DNA, lipids etc. [42]. NO has also been shown to inhibit complex III and complex IV of the respiratory chain [43],[44]. Based on plate assays, NO affected the growth in 25 out of 29 CoCl2-sensitive mutants (Figure 7B and Table 1). Paraquat (1,1′-dimethyl-4,4′-bipyridinium dichloride) is widely used to induce superoxide generation in cells. After its entry in mitochondria, paraquat is reduced by complex I leading to superoxide generation and subsequently extensive mitochondrial damage [45]. Figure 7C and Table 1 show that 28 of the 32 tested mutants are sensitive to paraquat. Diethyl maleate (DEM) is a glutathione-depleting agent and glutathione is one of the important non-enzymatic antioxidants that binds and inactivates ROS directly to protect cells against the toxic effects of ROS [46]. Exposure of cells to DEM leads to protein denaturation and increased intracellular ROS levels [47]. The majority of mutants tested (28 of the 32) showed sensitivity towards DEM (Figure 7D and Table 1). Furthermore, not all the mutants showed sensitivity to all four chemicals (Table 1). This was expected because based on the gene affected, different antioxidant systems would produce different outcomes in growth inhibition. Importantly, all the mutants showed sensitivity to at least one of the four ROS/RNS generating chemicals suggesting that all the CoCl2 sensitive mutants had a defect in handling the stress generated by ROS/RNS-producing reagents.10.1371/journal.ppat.1000155.g007Figure 7CoCl2-sensitive mutants are sensitive to ROS-generating reagents.Serial dilutions of yeast cells were spotted onto buffered, pH 4.0 YES with and without 0.5 mM H2O2 and incubated at 30°C and 37°C for 3 d (A), and with and without 1.0 mM NaNO2 and incubated at 30°C for 3 d (B). Wild-type and mutant strains were streaked on YES containing 0.25 mM paraquat (C) and YES+2 mM diethyl maleate plates (D). Plates were incubated at 30°C for 3 d.Transcriptional profile of cobalt chloride treated wild-type cellsTo gain an insight into the molecular basis for the effect of cobalt chloride treatment on C. neoformans cells, a whole genome microarray experiment was performed. Cells were harvested after 2 h of CoCl2 treatment. All genes with statistically significant differences in expression between wild type cells with CoCl2 and without CoCl2 were identified by significance analysis of microarray (SAM) using a mean false discovery rate (FDR) of less than 5% as described in Experimental Procedures. Of the 6,660 detectable genes analyzed, 979 genes were identified as significant genes by a mean FDR of 5% with SAM analysis. Of the 979 genes, 314 genes were up- or down-regulated by at least 2-fold. 42% of these genes have been annotated as hypothetical or expressed genes. The remaining 182 genes were categorized into different groups based on their annotated functions and presented in the form of a pie chart (Figure 8). The complete list of 314 genes is provided in the supplementary material (Table S1).10.1371/journal.ppat.1000155.g008Figure 8Pie chart.Pie chart shows the functional categories and distribution of the 182 genes whose expression has changed more than 2-fold in response to CoCl2. Annotations were obtained from the NCBI database (http://www.ncbi.nlm.nih.gov), with additional hand editing based on BLAST searches. Numbers in parentheses refer to the percentage of genes in each class and do not include the genes that are annotated hypothetical or expressed.As the figure suggests, genes involved in mitochondrial functions form a major category (30%). Upon examination of the transcriptional profile of genes involved in mitochondrial function, it is apparent that CoCl2 repressed the expression of genes involved in the electron transport chain. Table 2 lists the genes encoding ETC components (complex I through IV) that were down-regulated at least 2-fold upon cobalt chloride treatment. Along with ETC component genes, CoCl2 treatment also led to a significant reduction in the expression of the gene encoding alternative oxidase (AOX1). Alternative oxidase bypasses complex III and IV by carrying out ubiquinol oxidation and reduction of dioxygen to water [48],[49]. As shown above, the respiration rate was reduced by ∼65% when the wild-type cells were treated with CoCl2. The possible reason for this significant reduction may be in part due to the down-regulation of ETC genes as well as alternative oxidase in response to CoCl2.10.1371/journal.ppat.1000155.t002Table 2Effect of CoCl2 on expression of respiration-related genes.Locus IDFold Change*\nDescriptiona\n\nComplex I\nCNH02730−1.54NADH-ubiquinone oxidoreductase (subunit D)CNB01310−2.10NADH-ubiquinone oxidoreductase (subunit G)CND04070−1.93NADH-ubiquinone oxidoreductase 51 kDa subunit (NuoF)CNF03360−1.45NADH-ubiquinone oxidoreductase 30.4 kDa subunit, putativeCND01070−1.61NADH dehydrogenase (ubiquinone)CNC07090−1.91NADH dehydrogenase (subunit E) putativeCNE03960−1.62NADH ubiquinone oxidoreductase (subunit NDUFA12) putativeCNM01810−1.69ETC complex I subunit conserved region, putativeCNM02270−1.77ETC complex I subunit conserved region, putativeCNE02800−1.70NADH dehydrogenase (subunit B), putativeCNH01030−1.18NADH dehydrogenase 10.5 K chain\nComplex II\nCNA03530−1.80Succinate dehydrogenase (ubiquinone) (Sdh3 subunit)CNG03480−3.40Succinate dehydrogenase iron-sulfur (Sdh2 subunit)CNI03270−1.54Succinate dehydrogenase flavoprotein subunit precursor (Sdh1)CNB00800−2.01Mitochondrial inner membrane protein (Sdh4 subunit)CNJ00140−3.12Succinate dehydrogenase/fumarate reductase, flavoprotein subunitCND02060−1.57Succinate-semialdehyde dehydrogenase I, GabDCNF03900−1.32Succinate-semialdehyde dehydrogenase I, AlddhCNA05580−1.27Complex I protein (LYR family)\nComplex III\nCNF00630−1.40Electron transporter, cytochrome c\n1\nCNH02740−1.17ubiquinol-cytochrome C reductase complex (Qcr9 subunit)CNG00860−1.19Mitochondrial processing peptidase beta (Cor1 subunit)CNB01620−1.88L-lactate dehydrogenase, cytochrome b\n2 (Cyb2)CND04430−1.12ubiquinol-cytochrome C reductase complex (Qcr7 subunit)CNF03560−1.27ubiquinol-cytochrome C reductase complex (Qcr6 subunit)\nCytochrome\nCNA06950−3.46Electron carrier, Iso-1-cytochrome c, (Cyc1)\nOther Enzymes\nCNA01500−2.35Alternative oxidase 1 (Aox1)CND02080−2.27FMN-dependent dehydrogenase, putativeCNA04420−2.69oxidoreductase (Dehydrogenases (flavoproteins), FixC)CND02030−1.09Methylmalonate-semialdehyde dehydrogenase [acylating], putativeCNM01770−1.56Aconitate hydratase (AcnA_Mitochondrial)CNK02510−1.34uroporphyrinogen-III synthase, (HemD)CNI03590−1.69phosphoenolpyruvate carboxykinaseCNB04260−2.275-aminolevulinate synthase, (Hem1)\nAlternative Respiration Related\nCNI003602.06NADH dehydrogenase, (FAD-containing subunit, classII)CND022801.43oxidoreductase (aldehyde/oxo group of donors, NAD or NADP as acceptor)CNC062202.39glycerate-and formate-dehydrogenase (NAD or NADP as acceptor)CNI023603.46NADPH dehydrogenase 2 (Oye2)CNB036402.58oxidoreductase (NADH dehydrogenase, FAD-containing subunit)CNM003701.13Aryl-alcohol dehydrogenaseCNA050001.63NADH dehydrogenase (FAD-containing subunit, class II), putaiveCND007201.76NADH-flavin reductase, putativeCND023801.63NADPH dehydrogenase, putativeCNC002001.12Methylase in ubiquinone/menaquinone biosynthesis, putative (UbiE)CNA043401.81Mitochondrial hypoxia responsive domain protein, putativeCNE028202.62Mitochondrial ATPase, putative (AFG1_ATPase)CNE032601.02Transcriptional activator (Hap3)aAnnotations were obtained from NCBI database (http://www.ncbi.nlm.nih.gov) with additional hand editing based on BLAST searches.*: Fold change values have been presented as log2 values.Along with the genes involved in mitochondrial function are other groups of genes required for ergosterol biosynthesis, iron/copper homeostasis, oxidative stress, proteasome/ubiquitination function, carbohydrate metabolism, various transporters, specific transcription/translation, and some cellular metabolic processes. These data suggest that CoCl2 can induce a wide range of response in C. neoformans.DiscussionThis study investigated the role of CoCl2 as an effective chemical to screen for probable pathways involved in oxygen sensing in C. neoformans. We show that most of the CoCl2 sensitive mutants are also sensitive to low oxygen conditions. These mutants fall into various functional categories including; mitochondrial function, sterol biosynthesis, vesicular transport, carbohydrate metabolism, and other cellular functions. Based on the characterization, we report cobalt chloride sensitivity and/or sensitivity to low oxygen conditions are strongly influenced by mitochondrial function, ability of cells to deal with ROS production, ubiquitination, and sterol homeostasis. These data highlight the complex nature of CoCl2 hypersensitivity and/or oxygen sensing and adaptation process in C. neoformans.Many mutants obtained in our CoCl2 sensitivity screen were unable to retain Mitotracker dye indicating defective membrane potential. Genes affected in 3 of these mutants (10D12, 161B3, and 146G2) are related to structural components of the mitochondrial membrane. These results suggested proper maintenance of mitochondrial membrane potential is essential for C. neoformans to tolerate the high concentration of CoCl2 and to adapt to low oxygen conditions.Another major defect in many of our CoCl2 sensitive mutants is the impaired oxygen consumption. It is clear that CoCl2 not only affected antimycin A sensitive core respiration but also inhibited antimycin A insensitive alternative respiration. Importantly, many of the cobalt chloride sensitive mutants have a defect in oxygen consumption through either mode of respiration, core or alternative. Cobalt chloride has the ability to induce a respiratory deficiency in cells [15],[17]. In response to specific inhibitors and oxidative stress, fungi are known to utilize alternative components of the respiratory chain consisting of alternative NADH dehydrogenases to bypass complex I and an alternative oxidase to bypass complex III and IV [44]. Although we do not have mutants that belong to the alternative respiration pathway, we isolated a complex I NADH∶ubiquinone oxidoreductase mutant (135G10). In addition, four more mutants defective in mitochondrial membrane potential (146G2, 10D12, 69G9, and 161B3) also showed only antimycin insensitive respiration. These data indicate that the presence of an intact respiration system is required for C. neoformans to handle the high concentrations of CoCl2. If respiration is affected so severely in the presence of CoCl2, it is possible that CoCl2 treatment affects the expression of genes encoding respiration related proteins. As described in Table 2, expression of a wide array of genes for subunits in complex I through III along with other genes involved in aerobic metabolism (FIXC, HEMD, HEM1) were down-regulated upon CoCl2 treatment. In addition, our array data showed that the AOX1 gene was significantly repressed, while 10 different alternative dehydrogenases that belong to class II NADH∶ubiquinone oxidoreductases were up-regulated in CoCl2 treated cells. Two of the oxidoreductases listed in Table 2, (CNA05000 and CNI02360) have been shown to be up-regulated in a SRE1-dependent manner under 1% oxygen [19]. Interestingly, in S. cerevisiae under normoxic conditions aerobic genes are needed to encode proteins involved in aerobic metabolism such as oxidative phosphorylation while in low oxygen conditions hypoxic response genes are induced so as to allow the cells to utilize oxygen efficiently. These gene products involved in oxygen utilizing pathways include alternate cytochrome subunits and oxidases, and enzymes such as reductases and desaturases for heme, sterol, and fatty acid biosynthesis [50]. Our data here indicate in C. neoformans CoCl2 affects the expression of genes that are partially overlapping with genes expressed in oxygen limiting conditions. Perhaps, by regulating the expression of these genes in response to CoCl2, the cells can utilize oxygen more efficiently.Many of the mutants in this study showed sensitivity to various ROS producing agents and a change in the intracellular ROS levels either with or without CoCl2 treatment. This increase in ROS levels in the mutants as well as hypersensitivity towards oxidants could be the cumulative effect of mutations they are harboring and the presence of the ROS inducing chemicals in the environment. To eliminate ROS from the cellular environment, a number of antioxidant systems such as superoxide dismutase, catalase, thioredoxin, glutathione, mannitol are in play [51],[52]. The sensitivity towards ROS generating chemicals in the mutants indicates that either the antioxidant systems are not functioning properly in these strains or irrespective of functional antioxidant system, cells are unable to handle the elevated levels of ROS. There may be some correlation between the ability of various chemicals to increase ROS levels and their increased toxicity to a CoCl2 sensitive mutant. In Saccharomyces cerevisiae, mitochondrial function has been shown to be required for resistance to oxidative stress [53]. Hence, there is a possibility that some of the mutants are sensitive to oxidants due to defects in mitochondrial function and not due to the antioxidant status of the cell. The mitochondrial respiratory chain is the main site of cellular ROS production. It has been reported that disruption of the electron transport chain leads to an increase in ROS levels in mammals as well as in S. cerevisiae\n[54]–[56]. Among our category of mitochondrial function mutants, although 135G10, the complex I NADH∶ubiquinone oxidoreductase mutant did not show elevated levels of ROS in log phase cells, this strain displayed increased ROS levels upon CoCl2 treatment compared to the wild type. Furthermore, two strains with a mitochondrial membrane potential defect (10D12 and 161B3) show increased levels of ROS in log phase cells even in the absence of CoCl2. These data support the notion that a perturbance in mitochondrial function can lead to intracellular accumulation of reactive oxygen species. Mitochondrial ROS generated during hypoxia and in the presence of CoCl2 have been implicated in cellular oxygen sensing in metazoans as well as in S. cerevisiae suggesting a role of mitochondria in oxygen sensing [56]–[58]. Therefore, like in hypoxic conditions in other systems, it is probable that CoCl2 also induces mitochondrial ROS levels in C. neoformans leading to the cross talk between mitochondria and the nucleus, which ultimately results in changes of gene expression profile. Since the mutants showing abnormal ROS levels are also sensitive to low oxygen conditions, it is possible that in oxygen depriving conditions, due to a mutation in a respective gene in the mutant, signals transduced through ROS are not able to activate a cascade of genes that sustain growth under low oxygen conditions.Ubiquitination has been shown to be involved in targeting of nuclear-encoded pre-proteins into mitochondria, intracellular localization of macromolecules and degradation of superfluous/denatured proteins [59],[60]. In S. cerevisiae, in addition to these general functions, functional proteasomes and ubiquitin-dependent pathways are necessary for degradation of aerobic proteins (iso-1-cytochrome c) and modification of hypoxic proteins (Spt23, Mga2) [61],[62]. When C. neoformans cells were treated with CoCl2, we observed up-regulation of genes involved in proteasome function and ubiquitination suggesting similar mechanisms may be taking place (Table S1). In addition, complementation of clone 94F1 with CNH00220 locus encoding ubiquitin protein ligase rescued the CoCl2 sensitive phenotype (Figure 2), supporting the involvement of ubiquitination in handling the stress induced by cobalt chloride and/or low oxygen conditions in C. neoformans.One of the early studies that suggested antagonism between iron and cobalt in fungal systems was carried out in the 1950s using Neurospora crassa as a model system [15]. Further studies in the same system showed growth inhibition due to cobalt could be reversed to some extent by iron [63] and cobalt induced iron deficiency resulted in the formation of siderochrome [16],[64]. In S. cerevisiae, even at sub-lethal cobalt concentrations, iron regulon is induced resulting in immediate expression of iron transporter genes to increase intracellular iron content. This response is similar to iron starvation conditions [65]. In mammalian systems, it is known that Co2+ can be substituted for the iron in porphyrin ring of oxygen-requiring enzymes leading to the lower affinity for oxygen and mimicking hypoxia environment [6],[66],[67]. In light of these studies in yeast, molds and mammals, gene expression profile in relation to iron/copper homeostasis made sense. Our data showed when wild-type cells are exposed to CoCl2, genes encoding high affinity iron permeases (FRE3, CFT1, CFO1, CFT2) as well as siderophore transporters (SIT1) were significantly up-regulated (Table S1), mimicking the iron deprivation response, although none of our CoCl2 mutants showed significant growth reduction in iron limiting conditions (unpublished data). As suggested in other systems, cobalt may also exert its antagonistic effects on iron metabolism in C. neoformans by competing with Fe2+ thereby affecting heme dependent and heme independent proteins. Transcription profile of cells treated with CoCl2 also revealed that genes involved in sterol regulatory pathways such as ERG1, ERG3, ERG25, OLE1, NCP1, SCS7 are significantly induced (Table S1). These are oxygen-responsive genes and have been shown to be induced in low oxygen conditions in C. neoformans as well as in S. cerevisiae\n[19],[68]. Recent studies in C. neoformans showed the deletion of high affinity iron permeases (Cft1 and Cft2) leads to sterol synthesis defects [69]. Previous work from our laboratory identified an oxygen-sensing mechanism in C. neoformans, in which Sre1p and Scp1p are required for normal growth under low oxygen [19]. In our CoCl2 sensitive mutant screen, we identified two additional mutants related to sterol biosynthesis; squalene synthase (146G2) and C22-sterol desaturase (234E1), which are also sensitive to low oxygen conditions. Thus, in C. neoformans also iron-cobalt antagonism may disrupt iron and sterol homeostasis affecting the ability of cells to survive under high CoCl2 concentrations and/or low oxygen conditions.Here, we have explored the possibility of using CoCl2 to understand the mechanism of adaptation to low oxygen conditions in C. neoformans. We propose CoCl2 may be mimicking hypoxia like conditions by inhibiting many of the mitochondria-related functions. These data have highlighted the complexity of oxygen sensing in C. neoformans. The unpublished data from our lab suggest involvement of a respiratory chain is only one aspect of oxygen sensing and there are multiple pathways involved in oxygen sensing which are not related to CoCl2 sensitivity (unpublished data). Further studies on the complex interactions between sterol and iron homeostasis, mitochondrial function and oxygen sensing, using this diverse set of CoCl2 sensitive mutants will shed light on the pathobiology of C. neoformans, especially in the brain environment.Supporting InformationTable S1Gene expression upon 0.6 mM CoCl2 treatment.(444 KB XLS)Click here for additional data file.Table S2All statistically significant genes obtained by SAM analysis that were affected by 0.6 mM CoCl2 in wild type.(78 KB XLS)Click here for additional data file.\n\nREFERENCES:\n1. Kwon-ChungKJBennettJE\n1992\nMedical Mycology\nPhiladelphia, PA\nLea & Febiger\n2. 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batch_9/PMC2528945.json

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+{
+    "id": "PMC2528945",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528945\nAUTHORS: Ying-Jie Lu, Jane Gross, Debby Bogaert, Adam Finn, Linda Bagrade, Qibo Zhang, Jay K. Kolls, Amit Srivastava, Anna Lundgren, Sophie Forte, Claudette M. Thompson, Kathleen F. Harney, Porter W. Anderson, Marc Lipsitch, Richard Malley\n\nABSTRACT:\nAlthough anticapsular antibodies confer serotype-specific immunity to pneumococci, children increase their ability to clear colonization before these antibodies appear, suggesting involvement of other mechanisms. We previously reported that intranasal immunization of mice with pneumococci confers CD4+ T cell–dependent, antibody- and serotype-independent protection against colonization. Here we show that this immunity, rather than preventing initiation of carriage, accelerates clearance over several days, accompanied by neutrophilic infiltration of the nasopharyngeal mucosa. Adoptive transfer of immune CD4+ T cells was sufficient to confer immunity to naïve RAG1−/− mice. A critical role of interleukin (IL)-17A was demonstrated: mice lacking interferon-γ or IL-4 were protected, but not mice lacking IL-17A receptor or mice with neutrophil depletion. In vitro expression of IL-17A in response to pneumococci was assayed: lymphoid tissue from vaccinated mice expressed significantly more IL-17A than controls, and IL-17A expression from peripheral blood samples from immunized mice predicted protection in vivo. IL-17A was elicited by pneumococcal stimulation of tonsillar cells of children or adult blood but not cord blood. IL-17A increased pneumococcal killing by human neutrophils both in the absence and in the presence of antibodies and complement. We conclude that IL-17A mediates pneumococcal immunity in mice and probably in humans; its elicitation in vitro could help in the development of candidate pneumococcal vaccines.\n\nBODY:\nIntroduction\nStreptococcus pneumoniae (pneumococcus) is an “extracellular” pathogen, generally considered to be killed by phagocytic ingestion, which is facilitated by opsonic antibodies. The success of anti-pneumococcal serum therapy using passive transfer of serotype-specific antibodies [1] and of vaccinations based on purified or conjugated capsular antigens [2],[3] clearly shows that anticapsular antibodies protect humans against pneumococcal colonization and disease. There is good epidemiologic evidence for the importance of such immunity in certain common serotypes [4],[5]. However, we and others have found that factors other than anticapsular antibodies may play a role in the natural development of protection against pneumococcal colonization and disease. First, the reduction in pneumococcal disease incidence after the first year of life occurs simultaneously for both rare and common serotypes, suggesting the acquisition of one rather than many individual immune responses [6]. Similarly, the duration of carriage of many serotypes declines steeply between the first and second birthdays for many serotypes [7]. Since experience with conjugate vaccines has suggested that anticapsular antibodies reduce incidence of carriage but leave duration unaffected [8], this observation also suggests a mechanism of acquired immunity other than anticapsular antibodies. Moreover, the declines in carriage duration and invasive disease incidence precede by several years the detection of naturally-acquired anticapsular antibody in most children [6],[7]. Experimental [9],[10] and observational [4],[11] studies in adults have found little or no evidence that higher anticapsular antibody concentrations are associated with greater protection from colonization. Pneumococci also express non-capsular antigens common among serotypes, and certain of these have been found to elicit antibodies with protective potential in animal models. The role of such antibodies in human immunity has been evaluated [12],[13],[14],[15],[16],[17].Surprisingly however, recent studies have shown that immunity in mice to pneumococcal colonization acquired from prior exposure to live bacteria [18] or a killed, whole-cell vaccine [WCV, consisting of killed pneumococcal whole cell antigen (WCA) with cholera toxin (CT) as an adjuvant] [19] is independent of antibodies of any specificity, and clearance of longstanding carriage in previously unexposed animals can likewise be antibody-independent [20]. Immunity had been shown to be dependent on the presence of CD4+ T cells at the time of challenge [18],[19], but the co-participation of specific immune factors other than antibody was not ruled out.Here we show that intranasal immunization with the WCV confers protection against experimental pneumococcal colonization via the chemoattractant and neutrophil activating cytokine IL-17A, in a neutrophil-dependent fashion. Methods were devised to assay expression of IL-17A in vitro using peripheral blood samples. IL-17A expression by peripheral blood of WCV-immunized mice is highly correlated with subsequent protection against colonization, and expression by human cells, including those from adults and children, can be shown as well. Finally, we developed a surface phagocytosis assay with which we show that IL-17A enhances pneumococcal killing by human polymorphonuclear cells in the absence as well as presence of opsonins.The data indicate the possibility that IL-17A responses play a role in naturally-acquired immunity to pneumococcus in humans and that assay of this cytokine in vitro may assist in the evaluation of certain candidate pneumococcal vaccines that target mucosal colonization.ResultsPrior exposure of mice to killed or live pneumococci reduces the duration of experimental pneumococcal carriageThe duration of carriage was followed after intranasal challenge with serotype 6B pneumococci 4 weeks post-exposure to WCV. Both WCV-vaccinated and control mice immunized with CT alone were colonized one day after challenge. In mice immunized with WCV however, carriage became significantly reduced after 4 days compared to controls given cholera toxin (CT) adjuvant alone (median density of colonization on day 4 in WCV- vs. CT-immunized mice 251 vs. 3720 cfu/nasal wash, P = 0.029 by Mann-Whitney U test) and was undetectable by day 6 (0/4 WCV-immunized mice had detectable colonies on day 6 vs. 4/4 mice that received CT, P = 0.029 by Fisher's Exact test, Figure 1A). A similar differential was observed in mice that had been repeatedly exposed to live pneumococci vs. saline controls: the density of colonization became significantly different by day 4 after inoculation (Figure 1A). By day 6, similar to what we observed in WCV-immunized mice, 0/4 mice exposed to live pneumococci had detectable colonies compared to 4/4 saline controls (P = 0.029 by Fisher's exact test). When data from all time points were compared, mice immunized with WCV or exposed to live pneumococci had a significantly shorter time to clearance compared to their respective CT or saline controls (P = 0.0001 for comparison of WCV vs. CT and P = 0.004 for comparison of live exposure vs. saline). Thus the protection by prior pneumococcal exposure involves not immediate blockage of colonization but rather an accelerated clearance over days. Subsequent studies compared WCV-vaccinated with control animals 7 days after the intranasal challenge.10.1371/journal.ppat.1000159.g001Figure 1Duration of carriage and effect of adoptive transfer following immunization with killed or live pneumococci.A. Effect of intranasal immunization with WCV or live exposure upon density of pneumococcal colonization in C57BL/6 mice. Density of colonization in mice immunized with WCV vs. CT alone or repeatedly exposed to live pneumococcal strain 0603 vs. saline alone at various time points (n = 4 per time point) following challenge. By day 4, both the incidence and density of carriage were significantly lower in mice immunized with WCV or exposed to live pneumococcus compared to mice immunized with CT or saline, respectively. * P<0.05. The dashed line represents the lower limit of detection of bacterial colonization. B. Effect of adoptive transfer of CD4+ T cells from WCV-immunized mice on pneumococcal colonization of recipient, unimmunized RAG1−/− mice. Each data point represents the density of nasopharyngeal colonization in cfu/ nasal wash for each mouse one week post-challenge. The horizontal bar shows the geometric mean cfu/nasal wash for each group and the dashed line represents the lower limit of detection of bacterial colonization. C57BL/6 mice were immunized with WCV or CT as indicated. Four weeks after the last immunization, CD4+ T cells were harvested from splenocytes of mice and infused into naïve, unimmunized RAG1−/− mice; challenge of these mice and quantification of colonization was then performed. RAG1−/− mice that received CD4+ T cells from WCV-immunized mice had significantly lower density of colonization than mice that received cells from CT-immunized mice (*** P<0.0001 by Mann-Whitney U).CD4+ T cells transfer acquired immunity to pneumococcal colonization to RAG1−/− miceSeveral previous studies showed that acquired immunity to pneumococcal colonization in mice can be antibody-independent and dependent on CD4+ T cells [19],[20],[21],[22]. Here, adoptive transfer showed that CD4+ T cells are not only necessary but also sufficient for the accelerated clearance induced by WCV: unimmunized Rag1−/− mice (lacking both B and T cells) were infused with CD4+ T cells from mice immunized with WCV or CT alone. The RAG1−/− mice that received CD4+ T cells from WCV-immunized wild-type mice had significantly reduced density of colonization by day 7 compared to mice infused with CD4+ T cells from mice immunized with CT alone (P = 0.0001 by Mann-Whitney U, Figure 1B).Acquired immunity to pneumococcal colonization is associated with the TH17 subset of CD4+ T cellsTo evaluate which CD4+ T cell subset is responsible for protection, IFN-γ, IL-4 or IL-17A receptor (IL-17AR) knockout mice were immunized with WCV vs. CT alone. IFN- γ- and IL-4-deficient mice immunized with WCV were significantly protected against colonization both with respect to proportion of colonized mice (P<0.001 by Fisher's Exact test for comparison of % of colonization in WCV- and CT-immunized IFN-γ- or IL-4-deficient mice) and density of colonization (P≤0.001 compared to their respective CT controls, Figure 2A). In contrast, mice with a targeted deletion of the IL-17A receptor were not protected (P>0.5 vs. CT controls for % colonized mice or density of colonization, Figure 2A). It is noteworthy that IL-17AR-knockout mice in the CT control group had, on average, a ten-fold greater density of colonization than the corresponding IFN-γ or IL-4 deficient mice, suggesting that IL-17A may also be involved in resistance to colonization in naïve mice.10.1371/journal.ppat.1000159.g002Figure 2Role of T-helper-subset-associated cytokines in protection from nasopharyngeal colonization.A. Mice defective in IFN-γ, IL-4 or IL-17A receptor were immunized as described, then challenged with pneumococcal strain 0603. Mice with IFN-γ or IL-4 deficiency were significantly protected by WCV (P<0.001 vs. respective CT controls) whereas IL-17A receptor deficient mice were not protected (P>0.5 vs. CT). Dashed line represents the lower limit of detection of bacterial colonization. B. Expression of IL-17A from splenocytes of WCV-immunized mice. Cultured splenocytes from mice immunized with WCV (black columns) or CT alone (white columns) were stimulated for 72 hours with medium alone, Concanavalin A (5 µg/ml), WCA (10 µg dry weight) after which IL-17A production was measured by ELISA. Significantly more IL-17A was expressed following WCA stimulation of WCV-immunized vs. CT-immunized mice, although response to concanavalin A was similar. C. Effect of CD4+ T cell depletion upon IL-17A expression from splenocytes. Splenocytes (without or with CD4+ T cell depletion) from mice immunized with WCV were stimulated for 72 hours with medium alone or WCA after which IL-17A was measured by ELISA. IL-17A expression in splenocytes following WCA stimulation was significantly higher in the presence of CD4+ T cells compared to stimulation with medium alone or when CD4+ T cells were depleted. Repletion of CD4+ T cells restored the response. ** P<0.01 compared to cells stimulated with medium alone. D. IL-17A intracellular staining of splenocytes from WCV immunized mice. Splenocytes from WCV immunized mice were stimulated with WCA, blocked with monensin, harvested and stained for CD4+ and intracellular IL-17A as described. There is a statistically significant increase in CD4+ IL-17A positive cells following stimulation with WCA, which is not observed in the CD4- population. No increase in IL-17A positive cells could be detected in cells from unimmunized mice (data not shown). **P = 0.008 for comparison of frequency of IL-17A-positive cells in absence and presence of WCA stimulation among CD4+ cells. Data shown here are representative of three experiments, including at least 5 mice per experiment. E. Expression of IL-17A from NALT of WCV- vs. CT-immunized mice. Cultured splenocytes from mice immunized with WCV (black columns) or CT alone (white columns) were stimulated for 72 hours with medium alone or with WCA (10 µg dry weight) after which IL-17A production was measured by ELISA. Significantly more IL-17A was expressed following WCA stimulation of WCV-immunized vs. CT-immunized mice. **P<0.01 for comparison of IL-17A in WCV vs. CT-immunized mice following stimulation with WCA.Splenocytes from mice immunized with WCV expressed significantly more IL-17A in response to WCA in vitro than cells from CT control animals (Figure 2B). We have previously shown that immunization with WCV confers protection against NP colonization in a CD4+ T cell dependent fashion [19]; additional experiments were performed here to confirm that IL-17A is produced by CD4+ T cells. Depletion of CD4+ T cells effectively abrogated IL-17A expression from WCA-stimulated splenocytes, such that there was no detectable increase in IL-17A production compared to splenocytes stimulated with medium alone; repletion of CD4+ T cells restored the IL-17A response seen with nondepleted splenocytes stimulated with WCA (Figure 2C). Moreover, intracellular cytokine staining experiments confirmed that IL-17A production originates primarily from CD4+ cells (Figure 2D). Splenocytes from WCV-immunized animals were stimulated with WCA and analyzed by flow cytometry. The frequency of IL-17A positive cells increased 2.3 fold among CD4+ T cells (3.37% in WCA stimulated cells vs. 1.49% in cells stimulated with medium alone, P = 0.008 by Mann-Whitney U) whereas the non-CD4+ population was unaffected (1.5% vs. 1.7% for stimulation with WCA vs. medium alone in CD4- cells, P = 0.5). Similar analyses were performed using splenocytes from mice immunized with CT alone; no increase in IL-17A positive cells was noted, either in the CD4+ or CD4- population (data not shown). Collectively, these results indicate that IL-17A is produced by CD4+ T cells.Next, we harvested nasal associated lymphoid tissue (NALT) from WCV- and CT-immunized mice. Cells were incubated for 3 days in the presence of medium alone or with WCA, after which IL-17A expression was measured by ELISA. NALT cells from WCV-immunized mice showed significantly greater IL-17A production than CT controls in response to stimulation with WCA (Figure 2E).To test whether the capacity to produce IL-17A predicted an individual mouse's protection, a total of 90 mice were intranasally immunized with CT (1 µg) plus a range of doses of WCA ranging from 1 to 100 µg and blood samples were taken 7 days before challenge and stimulated with WCA in vitro for IL-17A production. The IL-17A concentrations following 6 days of culture in vitro varied from undetectable (<0.02 ng/ml) to about 6 ng/ml, and the cfu of pneumococci/nasal wash 7 days post-challenge varied from undetectable (<1.6 cfu/nasal wash) to about 3000; there was a strong inverse correlation (Spearman ρ = −0.62, P <0.0001, Figure 3); 95% of mice with pre-challenge IL-17A concentrations above 0.3 ng/ml were free of pneumococcal colonization.10.1371/journal.ppat.1000159.g003Figure 3Correlation of IL-17A expression and density of nasopharyngeal colonization in mice.Three weeks after immunization of mice (n = 90) with CT with doses of WCA ranging from 1 to 100 µg, and one week before pneumococcal challenge, blood samples were obtained and stimulated with WCA (10 µg) for 6 days, after which supernatants were collected and assayed for IL-17A concentration. The correlation between density of colonization (cfu/nasal wash) 7 days after challenge and pre-challenge IL-17A expression was evaluated. IL-17A expression was significantly correlated with density of colonization.Neutrophil-like cells are required for acquired immunity to pneumococcal carriageBecause IL-17 A induces neutrophil recruitment and activation [23], we evaluated whether neutrophils were required for protection against colonization. Mice intranasally immunized with WCV (or CT alone) were challenged, with or without administration of monoclonal antibody RB6-8C5 (which targets neutrophil-like cells) at the time of challenge. Several experiments were performed to ensure that treatment with this antibody did not affect CD4+ T cell number or function. Evaluation of splenocytes of antibody-treated animals showed no reduction in the CD4+ T cell population (data not shown). Furthermore, we confirmed that IL-17A production from the peripheral blood or from NALT of WCV-immunized mice was not affected by treatment with RB6-8C5 antibody. The peripheral blood IL-17A expression from immunized, neutrophil-depleted mice was similar to that of immunized, nondepleted mice (median IL-17A whole blood expression in neutrophil depleted vs. non-depleted mice: 1059 vs. 1290 pg/ml, P = 0.7 by Mann-Whitney U test); similarly, there was no reduction in NALT IL-17A expression from immunized mice following neutrophil depletion (median IL-17A expression from NALT in depleted vs. nondepleted mice 23.6 vs. 19.8 pg/ml, P = 0.69 by Mann-Whitney U test).Neutrophil depletion significantly diminished protection by immunization (Figure 4A). WCV-immunized and neutrophil depleted mice had both higher proportion of colonized mice (9/14 vs. 3/15 colonized mice for neutrophil-depleted vs. non-depleted WCV-immunized mice respectively, P = 0.025 by Fisher's Exact test) and density of colonization (median 12.8 cfu/nasal wash vs. 0.8 cfu/nasal wash respectively, P = 0.05 by Mann-Whitney U). While WCV-immunized, neutrophil-depleted mice had reduced colonization density compared to mice that received CT alone (median colonization density 453 cfu/nasal wash, P = 0.006 by Mann-Whitney U), the percentage of remaining neutrophils was strongly negatively correlated with recovered cfu from challenged mice (Spearman ρ = −0.77, P = 0.001, Figure 4B), suggesting that residual protection was accounted for in large part by incomplete neutrophil depletion.10.1371/journal.ppat.1000159.g004Figure 4Effect of neutrophils on WCV-induced protection against pneumococcal colonization.A. Effect of neutrophil depletion on WCV-induced protection from nasopharyngeal colonization. Each data point represents the density of nasopharyngeal colonization in cfu/nasal wash for each mouse. The horizontal bar shows the geometric mean cfu/nasal wash for each group and the dashed line shows the lower limit of detection of bacterial colonization. Mice were immunized with CT or WCV as indicated; just prior to the time of challenge, mice were randomized to receive antineutrophil antibody vs. saline. Proportion of colonized mice and density of colonization was determined 7 days post challenge. WCV-immunized mice that received saline treatment were significantly better protected than WCV-immunized mice that received antineutrophil antibody, with a lower proportion of colonized mice (P = 0.025 by Fisher's Exact) and density of colonization (P = 0.05 by Mann-Whitney U). B. Correlation between neutrophil count and density of pneumococcal colonization. Neutrophil counts following neutrophil depletion were assayed at the time of sacrifice and plotted against density of colonization. There was a strong negative association between neutrophil counts and colonization density (Spearman ρ = −0.75). C. Histopathology of nasopharyngeal tissue following nasopharyngeal challenge of CT- (left panel) and WCV-immunized (right panel) mice. Seven days post pneumococcal challenge, mice were euthanized, heads stored in formalin, and H&E sections of nasopharyngeal tissue prepared and examined under light microscopy at 60× magnification. The presence of a dense neutrophilic infiltrate in the submucosa at the junction of the olfactory and respiratory epithelium was noted in WCV-immunized mice following pneumococcal nasopharyngeal challenge but not in CT-immunized mice. The two slides shown are representative of a total of 15 examined specimens (8 WCV-immunized and 7 CT controls, all at day 7 post pneumococcal challenge). Lesions like those represented here were observed in 6/8 immunized mice and 0/7 controls.Consistent with these results, blinded review of histopathology of nasopharyngeal tissue of 6/8 WCV-immunized mice seven days after challenge with pneumococcus showed a distinct neutrophilic infiltrate in the submucosa at the junction of the olfactory and respiratory epithelium (Figure 4C, right panel), which is not seen in CT-immunized subsequently challenged with pneumococci (left panel) (presence of infiltrate in 6/8 WCV immunized mice vs. 0/7 CT controls, P = 0.007). Thus the data support a role for IL-17A acting upon neutrophils in protection against pneumococcal colonization in mice.IL-17A expression in human samples following pneumococcal stimulationNext we determined whether IL-17A responses to pneumococcus could be measured in humans. Tonsillar mononuclear cells (from 8 donors) were stimulated with medium alone, WCA obtained from a pneumolysin-negative strain (WCA(ply-)) or WCA from the wild-type strain. IL-17A expression measured at 72 hours was significantly higher following stimulation with WCA than with medium alone (Fig 5A); this increase was abrogated when a pneumolysin-negative WCA was used as stimulus, consistent with prior findings in humans and in mice regarding the association between T cell-mediated responses to this toxin and prevention of pneumococcal colonization ([24] and unpublished data). Furthermore, whole blood from unimmunized adult human volunteers, presumed to have been naturally exposed to pneumococcus, produced IL-17A in response to WCA in vitro (Fig 5B). Eighteen subjects produced a range of IL-17A concentrations from about 4 to 200 pg/ml, with a geometric mean of 20 pg/ml. Of these volunteers, 11 were parturient women, whose geometric mean IL-17A expression was 18 pg/ml. Umbilical cord blood was available in each of these cases; IL-17A in these samples was at the lower limit of detection of the assay (4 pg/ml) in all but one case, significantly lower than that of all adult subjects or parturient women (P<0.001 and P<0.01, respectively by Mann-Whitney U).10.1371/journal.ppat.1000159.g005Figure 5Effect of exposure to pneumococcus on IL-17A expression from human tissues and cells.A. Expression of IL-17A from tonsillar mononuclear cells from children. Tonsillar cells (n = 8) were cultured as described and stimulated with WCA or WCA derived from an isogenic, pneumolysin-negative strain (WCAply-). Stimulation with WCA was associated with significantly increased IL-17A expression compared to exposure to medium alone (P = 0.008 by Wilcoxon signed rank test), whereas stimulation with WCAply- did not increase IL-17A production. B. Expression of IL-17A from peripheral blood of adults and umbilical cord blood. Peripheral blood samples from adults (healthy adult volunteers (n = 7), parturient women (n = 11) and umbilical cord blood (n = 11) were stimulated with WCA for 6 days after which IL-17A concentration was assayed by ELISA. IL-17A production was significantly greater in adults than cord blood (P<0.001 by Mann-Whitney U test).IL-17A enhances in vitro phagocytic killing of pneumococciWe evaluated whether IL-17A enhances in vitro killing of pneumococci by human neutrophils in different assays. Having reported previously that WCV induced protection in antibody-deficient mice, we developed a surface phagocytic killing assay to evaluate whether -17A could potentiate killing of non-opsonized pneumococci. Neutrophils isolated from healthy volunteers were pre-incubated with recombinant human IL-17A, then overlaid on pneumococci that had been plated onto blood agar. The overlay of IL-17A in the absence of neutrophils did not result in any killing, consistent with studies in which the addition of IL-17A to culture medium did not affect growth of pneumococci and arguing against any direct killing effect of the cytokine or contaminant present in the preparation (as shown in Figure 6B). In the presence of neutrophils, IL-17A induced dose-dependent killing of pneumococci (Figure 6A). Thus IL-17A potentiated in vitro neutrophil killing of pneumococcus, in the absence of antibodies or complement.10.1371/journal.ppat.1000159.g006Figure 6IL-17A-mediated enhanced killing of S. pneumoniae.A and B. Effect of human IL-17A on surface phagocytic killing of S. pneumoniae. A. Isolated neutrophils from healthy adult volunteers were incubated with recombinant human IL-17A at the indicated concentrations and evaluated in a surface phagocytic killing assay with pneumococcal strain 0603; colonies were counted after overnight incubation at 37°C with 5% CO2. IL-17A induces a dose-dependent enhancement of neutrophil killing of S. pneumoniae (P = 0.01 for 1 or 10 µg of IL-17A vs. no added IL-17A). B. Supernatant obtained from neutrophils after incubation with IL-17A did not have any demonstrable antipneumococcal effect, whereas washed neutrophils after incubation with IL-17A demonstrated enhanced killing. C. Effect of human IL-17A on opsonophagocytic killing of S. pneumoniae. Neutrophils purified from the peripheral blood of healthy adult volunteers were incubated with pneumococci anticapsular antibodies, complement, and a range of concentrations of IL-17A as indicated for 90 minutes, following which viable counts were obtained by plating on blood agar plates. Each line represents a different volunteer. IL-17A enhanced killing of pneumococci in a dose-dependent fashion in 6/6 subjects. *P = 0.016 by Wilcoxon matched pairs test.To evaluate whether the potentiating effect of IL-17A is the result of lysis or degranulation of neutrophils, we performed trypan blue exclusion studies of neutrophils with and without preincubation with IL-17A at the highest dose studied (10 µg/ml). There was no difference in viability when IL-17A was added; over 95% of cells remained viable after 30 minutes of incubation. Furthermore, neutrophils preincubated with IL-17A then washed showed the same enhancement of pneumococcal killing as incubated neutrophils that were not washed. In contrast, the supernatant obtained after incubation of neutrophils and IL-17A had no detectable antipneumococcal activity (Figure 6B). Taken together, these data argue against a toxic or degranulating effect of IL-17A on neutrophils and are more consistent with enhancement of phagocytic activity of neutrophils by this cytokine.Because capsular and noncapsular antipneumococcal antibodies gradually increase with age in children [6],[25],[26], we also evaluated whether IL-17A would potentiate opsonophagocytic killing of S. pneumoniae in the presence of limiting amounts of antibody. Bacterial polysaccharide immune globulin (BPIG) is a plasma concentrate from volunteers immunized with bacterial polysaccharides including pneumococcal serotype 6B [27]. To evaluate whether IL-17A would synergize with antipneumococcal antibodies, we added IL-17A to an opsonophagocytic assay using a suboptimal dose of BPIG. When IL-17A was added to the reaction, killing of pneumococcus was enhanced in a dose-dependent fashion in all individuals tested (Figure 6C). Killing was significantly increased when either 1 µg or 10 µg/ml IL-17A were added (P = 0.016 by Wilcoxon matched pairs test). There was no significant pneumococcal killing in the assay in the absence of any of the principal components, including PMNs, antibody (BPIG) or complement (data not shown). Additionally, as in the surface phagocytosis assay, IL-17A alone had no effect on growth of bacteria in vitro or survival in our assay, implying that direct killing of pneumococci by IL-17A is very unlikely.DiscussionThe importance of CD4+ T cells in protection against pulmonary infections became clearly evident with the advent of the HIV epidemic. Infected individuals have a dramatically increased risk of infections with opportunistic pulmonary pathogens such as Mycobacterium tuberculosis or Pneumocystis jiroveci and this risk is inversely related to the number of circulating CD4+ T cells [28],[29]. For S. pneumoniae, HIV infection confers a 50-fold increased risk of infection, which is also inversely related to CD4+ T cell count [30],[31]. Most recently, a study in Zambian mothers has demonstrated that HIV infection is associated with a significantly increased risk of colonization and reduced time to new colonization [32]. Various hypotheses have been advanced to explain why CD4+ T cell-deficiency is associated with such a high risk of infection with pneumococcus, a primarily extracellular encapsulated bacterium; these include reduced opsonic activity of anticapsular antibodies [33], loss of memory B cells [34], and alteration of innate pulmonary immunity [35]. To date, however, it is fair to state that the paradox remains unexplained. The data presented here suggest that a loss of TH17 cells may also contribute to this increased susceptibility.While the ability of pneumococcal conjugate vaccine-induced anticapsular antibodies to protect against pneumococcal colonization is clear, less is known about the natural development of immunity to pneumococcal colonization. There appear to be both antibody-dependent and antibody-independent mechanisms that reduce the likelihood or duration of carriage. Several studies have documented a homotypic anticapsular serum antibody response to colonizing pneumococcal serotypes [4],[36],[37],[38]. In a longitudinal study examining the relationship between antibodies and carriage in adults, Goldblatt et al. showed that, among six serotypes tested, anticapsular antibody concentration to serotype 14 was significantly associated with reduced odds of carriage [4]. More recently, by analyzing longitudinal carriage data from Israeli toddlers in daycare, we found a lower risk of colonization with type 6A, 14, and 23F after previous exposure to the homologous type [5]. For types 14 and 23F, this specific protection correlated with increased serotype-specific antibody concentration. On the other hand, as in the previous study [4], there was no evidence for such a correlation with several other serotypes. Several studies have argued against the role of anticapsular antibody. For example, the risk of acquisition of new pneumococcal strains in adults with chronic obstructive pulmonary disease was associated with higher preacquisition concentrations of anticapsular and noncapsular pneumococcal antibodies [11]. These results suggest that in this population, antipneumococcal antibodies are markers of prior exposure and perhaps greater susceptibility rather than predictors of protection. Finally, in the sole published example of experimental pneumococcal colonization of humans, antibodies to the capsular polysaccharide did not predict protection against colonization [39].We, and others, have presented data supporting a role of CD4+ T cells, independently of antibody, in reducing pneumococcal colonization in mouse models [18],[19],[20],[22],[40]. In studies involving immunization with the pneumococcal zwitterionic conserved cell wall polysaccharide, we showed that neutralization of IL-17A significantly reduced protection, albeit not completely [21]. Zhang et al. then showed that reduced CD4+ T cell proliferative responses to the pneumococcal toxin pneumolysin were associated with nasopharyngeal pneumococcal carriage in children [24]. We subsequently showed that immunization with a mixture of a pneumolysin nontoxic mutant and two other pneumococcal proteins elicits TH17 cells and confers protection against colonization in a CD4+ T cell-dependent, antibody-independent fashion [22]. Others have argued against this possibility, proposing instead a TH1, IFN-γ mediated mechanism, based on the finding that IL-12p40-deficient mice cleared pneumococcal colonization as well as wild-type mice [20]. Since that report, it has become clear that IL-23, which is lacking in IL-12p40 deficient mice, is not absolutely required for the generation of TH17 cells as once was thought, but instead participates in their maintenance or expansion [41],[42].Here we present evidence that acquired CD4+ TH17 cells reduce the duration of experimental colonization with S. pneumoniae in a manner reminiscent of the age-dependent decline in duration of carriage [7] and that this mechanism occurs independently of key TH1 or TH2 cytokines, IFN- γ and IL-4 respectively. We show that CD4+ T cells are sufficient to provide the protection against colonization, which is abrogated in the absence of the IL-17A receptor and highly dependent on neutrophils, one of the main targets of this cytokine. Recombinant human IL-17A enhances both antibody-independent and –dependent killing of S. pneumoniae in vitro. Importantly, IL-17A expression can be induced by exposure to pneumococcal antigens of tonsillar cells from children and peripheral blood from healthy adult volunteers, but not in umbilical cord blood, consistent with the view that this responsiveness may be the result of prior exposure to the pathogen.IL-17A signaling been shown to participate in host defense against extracellular pathogens, such as Klebsiella and Candida in naïve mice [43],[44]. Prior to this report, there have been two demonstrations of a role of TH17 cells in vaccine-induced immunity and in both cases, whole organisms, killed or live, were used. Higgins et al. showed that protection against Bordetella pertusssis with a whole cell vaccine induced TH17-dependent protection and Khader et al. presented similar findings with the mycobacterial protein ESAT6-induced protection against M. tuberculosis\n[45],[46],[47]. The data derived from both mouse and human studies in the present report thus add to the growing evidence that TH17 cells contribute to immunity to respiratory pathogens.Numerous attempts have been made to define correlates of protection against pneumococcal carriage and have focused on the humoral response to pneumococcal capsular or noncapsular antigens [4],[15],[48]; although associations between levels of antibodies in saliva and reduced risk of otitis media have been reported [48], no reliable correlate has been identified. Here we show that the IL-17A response in immunized mice is highly correlated with reduced carriage; in particular above a certain concentration, colonization beyond 7 days is very unlikely. Naturally-exposed humans have low, but measurable IL-17A responses, which could be evaluated in response to immunization with candidate pneumococcal vaccines, such as the whole cell vaccine currently under development. The demonstrated association between carriage and reduced T cell proliferative responses to pneumolysin in childhood [24] provides further support for a functional T cell assay such as the one proposed here.Materials and MethodsBacterial strains and immunogens\nS. pneumoniae strain 0603 is a serotype 6B clinical strain [49]. Frozen mid-log phase aliquots were diluted to ∼106 cfu/10 µl of intranasal inoculum for challenge. The whole cell antigen (WCA) was derived from strain Rx1AL-, a capsule- and autolysin-negative mutant and prepared as described [49]. A pneumolysin-negative WCA (WCA(ply-)) was derived from an isogenic, pneumolysin-negative strain of Rx1AL- using methods previously described [18]. The final vaccine mixture (whole cell antigen WCA + adjuvant CT) for routine immunization contained 100 µg (dry weight) of WCA plus 1 µg of CT (List Biological Laboratories, Campbell, CA) per 10 µl dose. For potency comparisons, lower amounts of WCA were used (ranging from 0.1 µg to 10 µg). For all experiments, control mice were immunized nasally with 1 µg of CT in 10 µl saline.Animal modelsThe animal model used in these experiments has been previously described [49]. C57BL/6J mice or mutants in the same background (female, age 6 weeks, Jackson Laboratories, Bar Harbor, ME) were randomized to receive 10 µl of whole cell vaccine or adjuvant alone intranasally twice at one week interval. Three weeks following the last inoculation, mice were anesthetized for retro-orbital blood sampling. One week later, mice were challenged intranasally with ∼106 cfu of strain 0603. At 1 week after challenge, the mice were euthanized by CO2 inhalation; an upper respiratory wash was done by instilling sterile, nonbacteriostatic saline retrograde through the transected trachea and collecting the first 6 drops (about 0.1 ml) from the nostrils. Following collection of nasopharyngeal samples, the heads of WCV- and CT-immunized mice were removed and placed in formalin prior to histopathological preparation with hematoxylin and eosin (H&E) staining.To evaluate the time to eradication of carriage, in a separate experiment, 4 mice from groups of 16 each were sacrificed at 1, 2, 4 and 6 days post inoculation and sequential dilutions of nasal washes were plated. To test whether CD4+ T cells are sufficient for protection, adoptive transfer experiments were performed. Splenocytes from wild type C57Bl/6 mice immunized with WCV or CT alone were harvested 2 months after the last immunization and CD4+ T cells were purified by magnetic bead positive selection (Miltenyi Biotec, Auburn, CA). A total of 3×106 CD4+ T cells were injected retro-orbitally in naïve RAG1−/− mice (B6.129S7-Rag1tm1Mom/J) that lack both B and T cells. The following day, these mice were challenged intranasally with strain 0603; one week later, density of colonization was determined as described above.To determine which T cell subset is responsible for protection, mice in the C57BL/6 background and deficient in IFN-γ (B6.129S7-Ifngtm1Ts/J, Jackson Laboratories, Bar Harbor, ME) IL-4 (B6.129P2-Il4tm1Cgn/J, Jackson Laboratories) or the IL-17 receptor (B6.129 IL17Ra−/−\n[50]) were immunized and challenged as described above. For neutrophil depletion experiments, mice were immunized as described above; on days −1, +1 and +4 relative to challenge, mice were injected intraperitoneally with 100 µg of antineutrophil monoclonal antibody RB6-8C5 (purified from myeloma cell line by Bio Express, Lebanon, NH), a rat anti-mouse IgG2b directed against Ly-6G on the surface of murine myeloid (and limited subpopulations of lymphoid) lineage cells; in pilot experiments, this regimen resulted in >90% depletion of neutrophils in most mice, although variability was observed. Because of this variability, peripheral neutrophil counts were determined at the time of euthanasia and correlated with the number of recovered pneumococci from that animal.Measurement of IL-17A secretion by splenocytesCellular suspensions of splenocytes were obtained by passing spleens from immunized or control mice through a 70-µm cell strainer (BD Biosciences, Bedford, MA). After washing and removal of red blood cells by hemolysis, cells were plated into 24-well tissue culture plates at a concentration of 5×106 cells/well in 500 µl of DMEM/F12 with L-glutamine supplemented with 10% fetal calf-serum, 50 µM 2-mercaptoethanol (Sigma), and 10 µg/ml ciprofloxacin. Following 72-hour stimulation with concanavalin A (5 µg/ml, Sigma) or WCA (equivalent to 106 cfu/ml), supernatants were collected following centrifugation and stored at −80°C until analyzed by ELISA for IL-17A concentration (R&D Systems, Minneapolis, MN). Supernatants were analyzed in duplicate and read against a standard, following directions provided by the manufacturer.For CD4+ T cell depletion, splenocytes were harvested as described above. CD4+ T cells were depleted from half of each spleen by magnetic bead selection (Miltenyi Biotec, Auburn, CA) following instructions by the manufacturer. Flow cytometry confirmed removal of >95% CD4+ T cells (data not shown). Cells were seeded at the same concentration as described above (5×106 cells/well). In some cases, we repleted CD4+ T cells from depleted splenocytes, by adding 106 CD4+ T cells in the relevant wells.Intracellular staining for IL-17ASplenocytes were harvested, seeded, and stimulated with medium or WCA (10 µg/ml) as described above. Twenty-four later, monensin (BD GolgiStop, BD Biosciences) was added as per the manufacturer's instructions and cells were harvested 12 hours later. Cells were washed, stained with anti-CD4+ antibody (antiCD4+-PE, BD Biosciences) in the presence of Fc block, permeabilized with Perm/Wash buffer (BD Biosciences), and incubated with antimouse IL17A Alexa Fluor-647 (eBioscience) for 30 minutes. Intracellular cytokine staining for IL-17A was compared in CD4- or CD4+ cells in medium alone or following stimulation with WCA. Samples were analyzed on a Cytomation MoFlo (Beckman Coulter, Fullerton, CA), and results analyzed with Summit Version 4.3 (Dako, Fort Collins, CO).Measurement of IL-17A secretion by NALTNALT was harvested from immunized and control mice as described [51]. Mice were euthanized humanely, bled via intracardiac puncture to avoid blood contamination, and placed on a dissection board. The mouth was opened wide to expose the palate, which was cut carefully, so that the strips of NALT could be easily peeled off. These strips of cells were collected in medium (DMEM/F12 with L-glutamine supplemented with 10% fetal calf-serum, 50 µM 2-mercaptoethanol (Sigma), and 10 µg/ml ciprofloxacin) on ice. Cells were passed through a 70 µm strainer as described above and plated at 3×105 cells/well in a 96-well tissue culture plate in a total volume of 100 µl. Cells were stimulated with medium with or without added WCA (10 µg/ml) for a total of 3 days, after which supernatants were collected and assayed for IL-17A concentration by ELISA as above.Measurement of IL-17A secretion by whole bloodFor whole blood assays, blood of mice or humans at a final concentration of 10% was incubated in DMEM/F12 with L-glutamine supplemented with 10% fetal calf-serum, 50 µM 2-mercaptoethanol (Sigma), and 10 µg/ml ciprofloxacin in the absence or presence of killed pneumococcal antigen (corresponding to 107 cfu/ml for mice and 106 cfu/ml for human samples). Supernatants were collected after 6 days and the concentration of IL-17A measured as above for mice and, for human samples, by IL-17A ELISA (eBioscience Inc, San Diego, CA).Human subjects and samplesFor peripheral blood, samples were obtained at Children's Hospital Boston (for healthy adult volunteers) or from Cambridge Health Alliance, Cambridge, MA (for parturient women or umbilical cord) after written informed consent had been obtained. The studies were approved by the Children's Hospital Boston and Cambridge Health Alliance research ethics committees. For tonsillar specimens, tonsils were obtained from children who were 2 to 12 years old (median age, 5 years), were undergoing tonsillectomy for hypertrophy, and were otherwise healthy at Bristol Royal Hospital for Children, Bristol, United Kingdom. Patients who were immunized against pneumococcus previously, who had received antibiotics within 2 weeks of the operation or steroids, or who had an immunodeficiency or serious infection were excluded. The study was approved by the South Bristol local research ethics committee and written informed consent was obtained in all cases.Agar surface phagocytic killing without opsoninsThis assay approximates the “surface phagocytosis” described by Smith and Wood [52]. Neutrophils were isolated from heparinized blood by density gradient centrifugation (Histopaque, Sigma) following manufacturer's instructions. Neutrophils were washed extensively then resuspended in Hanks' Balanced Solution (+ Ca2+ and Mg2+) with 0.2% bovine serum albumin (Sigma), then co-incubated for 30 minutes at 37°C with recombinant human IL-17A (R&D Biosystems) at different concentrations. In some experiments, the cells were harvested by centrifugation and the supernatant collected, to examine whether the potentiating effect of IL-17A could be detected with the supernatant alone. Between 8–10 replicates of 10 µl of a bacterial suspension containing on average 100 cfu of strain 0603 were plated onto blood agar and the fluid allowed to adsorb into the agar for 15 min; 15 µl of the neutrophil suspension was overlaid and allowed to adsorb; the plates were incubated at 37°C with 5% C02 overnight after which colonies were counted.Phagocytic killing in suspension with suboptimal opsonizationNeutrophils were isolated from whole blood as described above, washed twice with cold Hanks Balanced Salt Solution (HBSS-) (Mediatech, Herndon, VA), and resuspended to a final concentration of 6×106 cells/ml in cold HBSS containing calcium and magnesium (HBSS+) (Cellgro Mediatech, Herndon, VA) then held on ice until used. Cell counts were determined on a standard hemocytometer by counting viable cells (as determined by an absence of blue staining in the presence of Trypan Blue (Cellgro Mediatech, Herndon, VA)). S. pneumoniae (strain 0603 [49]) was diluted in HBSS+ to a final concentration of 5×104 bacteria/ml and incubated with antibodies to pneumococcal polysaccharide (Bacterial Polysaccharide Immune Globulin, BPIG-8, a kind gift of Dr. George Siber, consisting of concentrated IgG obtained from serum of adult volunteers immunized with pneumococcal, Haemophilus and meningococcal polysaccharide vaccines [27]) diluted in HBSS+. The reaction was incubated at 37°C for 15 minutes rotating at 200 RPM to promote bacterial opsonization. After bacterial opsonization, the opsonophagocytic killing reaction was initiated with the addition of baby rabbit complement (Pelfreez Biologicals, Rogers, AR) and neutrophils (ratio of 1∶200 bacteria∶cells) with or without recombinant human IL-17A (R&D Systems, Minneapolis, MN) at 0.01, 0.1 or 1 µg/ml. A 1∶1600 dilution of BPIG was chosen to give sub-optimal bacterial killing (<50% killing when compared to the same conditions without BPIG) in the presence of complement and neutrophils. The opsonophagocytic killing assay was performed in a 96-well round-bottom plate (Thermo Fisher Scientific, Waltham, MA) at 37°C for 90 minutes rotating at 200 RPM. After incubation, the opsonophagocytic reaction was diluted two fold and aliquots of each reaction were plated on blood agar plates then incubated at 37°C with 5% CO2 overnight.Isolation and culture of tonsillar mononuclear cellsMononuclear cells were isolated by using methods described previously [53],[54]. Tonsillar MNC were washed in sterile phosphate-buffered saline (PBS) and resuspended at a concentration of 4×106 cells/ml in RPMI medium containing HEPES, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (Sigma, Dorset, United Kingdom). Cells were cultured in 96-well culture plates (Corning Inc, Corning, NY), and cell culture supernatants were collected at predetermined times and stored at −70°C until assays for human IL-17A were performed by sandwich ELISA (R&D Biosystems).Statistical analysisIncidence of carriage was compared by Fisher's exact test and colonization density in challenged mice was compared by the Mann-Whitney U test. Statistical significance of the difference between time-to-clearance curves was assessed as follows. For each group i (i = WCV, CT, live, or naïve), the proportion of mice cleared at each time point t, pi(t), was calculated. Using the max-min formula for isotonic regression [55], these proportions were smoothed to assure they were nondecreasing in t, yielding smoothed proportions qi(t). Then, a test statistic was calculated to quantify the distance between the smoothed curves for two groups (e.g., WCV vs. CT): . The significance level of this test statistic was estimated by permuting the group identifiers of the cleared mice at each time point, fixing the total number of mice in each group and the total number cleared at each time point. 100,000 replicates of the permuted data were obtained, and T was calculated for each. The p value was calculated as the fraction of these 100,000 permutations having a test statistic strictly less than that calculated for the data. The correlation between neutrophil count or IL-17A concentration and colonization density was determined by Spearman rank correlation. 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+}

batch_9/PMC2528948.json

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+{
+    "id": "PMC2528948",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528948\nAUTHORS: Dael Geft, Shmuel Schwartzenberg, Ori Rogowsky, Ariel Finkelstein, Jacob Ablin, Sofia Maysel-Auslender, Dov Wexler, Gad Keren, Jacob George\n\nABSTRACT:\nBackgroundCirculating CD34+ endothelial progenitor cells (EPCs) are capable of differentiating into mature endothelial cells to assist in angiogenesis and vasculogenesis. We sought to quantify the numbers of apoptotic progenitors in patients with congestive heart failure.Methods and ResultsPeripheral blood mononuclear cells were isolated by Ficoll density-gradient from 58 patients with various degrees of heart failure and 23 matched controls. Apoptosis in progenitor CD34+ cells was assessed using the Annexin V-PE/PI detection kit, and FACS analysis was performed with triple staining for CD34, annexin-V and propidium iodide. The percentage of early and late apoptotic progenitor cells was determined in the subject groups and was correlated with clinical characteristics. While there was no significant difference in total CD34 positive cells or early apoptotic progenitors between control subjects and CHF patients (p = 0.42) or between severe and mild/moderate CHF groups (p = 0.544), there was an elevated number of late apoptotic progenitors in the severe CHF group compared with the mild/moderate CHF group (p =  0.03). Late apoptotic progenitors were significantly increased in CHF patients as compared to matched controls. There was also an inverse correlation between late apoptotic progenitors and ejection fraction (r = −0.252, p = 0.028) as well as a positive association with NYHA class (r = 0.223, p = 0.046).ConclusionSevere heart failure patients exhibited higher numbers of late apoptotic progenitors, and this was positively associated with NYHA class and negatively correlated with ejection fraction. This finding may shed light on the numerous factors governing the pathophysiology of CHF.\n\nBODY:\nIntroductionOver the past few decades, researchers as well as clinicians have made great strides in understanding the pathophysiological mechanisms of heart failure. Whereas heart failure was once thought of as a series of symptoms simply due to a poorly functioning heart, it is now understood to be a syndrome whose causes are both multifactorial and complex [1]. Several diverse mechanisms contribute to this syndrome including structural and functional abnormalities of the heart, vascular disease, biological and neurohormonal factors, oxidative stress, genetics, environment and coexisting conditions [1]. Yet, while these advancements in understanding have indeed led to better treatment of heart failure, it remains a major cause of morbidity and mortality worldwide.More recently, considerable evidence has shown that heart failure is associated with tissue ischemia and endothelial dysfunction, as assessed by impaired flow-mediated dilatation, as well as increases in specific plasma markers such as von Willebrand factor and soluble thrombomodulin [2]–[5]. A newer method to identify endothelial damage and dysfunction is the quantification of circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) in the peripheral circulation. CECs are mature endothelial cells that have detached from the intimal monolayer of blood vessels in response to endothelial injury [6], whereas EPCs are immature, bone-marrow derived cells with the capacity to transform into mature endothelial cells and promote postnatal angiogenesis and vasculogenesis [7]–[9]. EPCs can be characterized by the expression of surface markers, such as CD34, CD133 and VEGFR-2 (KDR or Flk-1) in various combinations [10]. It has, in fact, recently been shown by us that patients with heart failure have elevated circulating EPCs, which may be an independent predictor of mortality in CHF [11].There are small membrane particles, known as endothelial microparticles, which are associated with endothelial cell damage and apoptosis. These endothelial microparticles have been shown to be elevated in conditions such as acute coronary syndrome (ACS) and myocardial infarction [12]–[14]. Recently, we identified, for the first time, a new population of apoptotic progenitor cells (APCs) which were elevated in patients with ACS [15]. The apoptotic progenitors can be divided into early, reversible apoptotic cells and late, irreversible apoptotic cells. In this study, we sought to quantify the number of apoptotic progenitor cells in patients with heart failure. In so doing, we learned that while CHF patients did not exhibit higher levels of total or early apoptotic progenitors than controls, the more severe CHF patients exhibited elevated numbers of late apoptotic progenitors compared to those with less severe CHF.Materials and MethodsStudy SubjectsWe studied a total of 58 patients (median age 76.5) arbitrarily with various classes of heart failure according to the New York Heart Association (NYHA) classification. The control group comprised of 23 subjects with a similar profile of age (a median of 74 years, range 42–81), gender, a normal ejection fraction by echocardiography and no evidence of heart failure. The incidence of risk factors for atherosclerosis including diabetes, hypertension, smoking and treated hyperlipidemia did not differ between the study and control groups. With regard to medication use, study group patients had significantly increased use of warfarin and renal failure was more common as compared with controls. In addition, as seen in Table 1, we subdivided the 58 CHF patients into two subgroups: 33 patients with mild/moderate CHF (NYHA class I–II) and 25 patients with advanced CHF (NYHA class III–IV). There were no significant differences between the demographic characteristics of these two subgroups.10.1371/journal.pone.0003238.t001Table 1Baseline characteristics CHF and control patients.GroupControlsNYHA I–IINYHA III–IV\nP\nCharacteristicsn = 23n = 33n = 25Demographic dataMale/Female15/825/818/7Median age (range)74 (42–81)73 (48–89)74 (47–79)nsCurrent Smoker2 (9%)6 (18%)4 (16%)ComorbiditiesHypertension12 (52%)21 (63%)14 (56%)nsDiabetes Mellitus12 (52%)20 (61%)14 (56%)nsHyperlipidemia16 (65%)26 (79%)15 (60%)nsDrug TreatmentStatin16 (65%)27 (82%)13 (52%)nsBeta Blocker6 (26%)25 (76%)19 (76%)<.01ACEI/ARB16 (65%)27 (82%)19 (76%)nsSpironolactone023 (70%)17 (68%)<.01Furosemide033 (100%)25 (100%)<.001 <.01Coumadin014 (42%)10 (40%)<.01MeasurementsEjection Fraction*\n >40%13 (39%)10 (40%) ≤40%20 (61%)15 (60%)Creatinine >1.520 (61%)16 (64%) ≤1.513 (39%)9 (36%)Ischemic vs. Non-IschemicΔ\n Ischemic16 (64%) Non-ischemic20 (36%)*LV ejection fraction was estimated by 2D echocardiography.ΔIschemic cardiomyopathy refers to patients with ischemic heart disease (prior MI, PTCA or CABG) and ejection fraction <40%.Of the CHF patients, 43 were male and 15 were female. Of the controls, 15 were male and 8 were female. Table 1 summarizes demographic and clinical characteristics of the patient population. Institutional ethics committee approved the study and informed consent was obtained from all patients.Preparation of Blood SamplesPeripheral blood mononuclear cells (PBMNCs) were isolated from 20 ml of freshly drawn heparinized blood using Isopaque-Ficoll (Amersham Biosciences, Buckinghamshire, United Kingdom) gradient centrifugation.Flow Cytometry evaluation of early and late apoptotic progenitor CD34+ cellsAfter Ficoll gradient separation, PBMNCs were washed with phosphate-buffered saline (PBS), and 106 cells were stained with (FITC)-anti-CD34 MAb for 30 minutes at 4°C in 100 microliters of FACS staining buffer (PBS and 2% fetal calf serum (FCS). Apoptosis in progenitor CD34+ progenitor cells was assessed using Southern Biotech ApoScreen Annexin V Apoptosis detection kit (Annexin V-PE, Propidium Iodide (PI) solution and Annexin V binding buffer). This assay involves staining peripheral blood mononuclear cells with Annexin V-PE (a phospholipid-binding protein binding to disrupted cell membranes) in combination with propidium iodide (PI, a vital dye binding to DNA penetrating into apoptotic cells). FACS analysis of CD34+ progenitor cells that are in early (annexin V+/PI−) or late (annexin V+/PI+) apoptotic phase was performed.The percentage of apoptotic CD34+ progenitor cells (out of total circulating CD34+ progenitor cells) was assessed by staining peripheral blood mononuclear cells for 3 color FACS analysis employing (FITC)-anti-CD34 MAb (IQ products), Annexin V-PE and Propidium Iodide (SouthernBiotech). The cells were then washed again with PBS and resuspended in 100 microliters of Annexin V-PE binding buffer and incubated with 5 microliters of Annexin V-PE for 15 minutes at room temperature. Without washing, another 200 microliters of binding buffer and 5 microliters of PI solution were added, and 800,000 cells were acquired by flow cytometry (FACSCalibur, Becton Dickinson) and analyzed by CellQuest software (BD Bioscience). All analyses and readings were made by technicians who were blinded to the study questions.Determination of Erythropoietin, Thrombomodulin/CD141 and Antibodies to Oxidized LDL levelsELISA kits were used to detect levels of Erythropoietin (Stem Cell Technologies) and Thrombomodulin/CD141 (Diaclone). Antibody to oxidative LDL levels were detected by ELISA method with anti-human antibody and solutions created in the laboratory [19].Statistical AnalysisAll data was summarized and displayed as mean (SD) for the continuous variables and as number of patients plus the percentage in each group for categorical variables. The one-way Kolmogorov-Smirnov test was used to assess the distributions. Levels of late apoptotic cells could not be converted to normal distribution, thus we categorized the variables into tertiles.For all categorical variables the Chi-Square statistics was used for assessing the statistical significance between the groups of CHF severity, while for all continuous variables, the independent samples one way ANOVA test was used. Regression analysis and analysis of variance for APCs by independent variables was performed in all cases.All above analyses were considered significant at p<0.05 (two tailed). The SPSS 15 statistical package was used to perform all statistical evaluations (SSPS Inc., Chicago, IL, USA).ResultsWe employed a novel assay for determining apoptotic CD34 cells. Progenitor CD34+ cells were initially gated from the side scatter/CD34 dot plot according to the Milan protocol [20]–[21]. As can be seen in Figure 1a, the progenitor CD34+ cells can be outlined and gated using this FACS dot plot. The percentage of apoptotic CD34+ progenitor cells was then determined by FACS analysis of Annexin V/PI staining. In apoptotic cells, the membrane phospholipids phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the extracellular environment. Annexin V is a 36 kDa Ca2+ dependent phospholipid-binding protein that has a high affinity for PS and binds to cells when PS is exposed to the external cellular environment, which occurs when membrane integrity is affected in early phase of apoptosis [16]–[17]. Propidium Iodide (PI) is a vital dye binding to DNA, a process implying disrupted cellular membrane and exposed DNA, compatible with late, irreversible cell necrosis. Cells that are viable are Annexin V-PE and PI negative; cells that are in early apoptosis are Annexin V-PE positive and PI negative (lower right quadrant–figure 1b); and cells that are in late apoptosis or necrosis are both Annexin V-PE and PI positive (upper right quadrant–figure 1b) [17]–[18].10.1371/journal.pone.0003238.g001Figure 1Representative flow cytometric dot plots: SSC/CD34 scatter (1a) and flow cytometric evaluation of progenitor CD34+ apoptotic cell percentage (1b).Dots in the lower right quadrant represent early apoptotic cells (Annexin-V positive and PI negative) while dots in the upper right quadrant represent late apoptotic cells (Annexin-V and PI positive).As can be appreciated in Table 1, there were no significant statistical differences between the demographic characteristics of the two study groups (mild-moderate CHF and severe CHF) and the control group which comprised ACS patients not having CHF. With regard to the number of early apoptotic cells, no statistically significant difference was found between the age matched controls vs. CHF groups (once adjusted for age, gender and risk factors) or between the two CHF groups (mild/moderate vs. severe) (figure 2). Within the CHF groups, however, there was a positive correlation between the number of early apoptotic progenitor cells and levels of hemoglobin (r = 0.279, p = 0.016) and total CD34+ cells (r = 0.261, p = 0.023). With regard to the late apoptotic cells, there was an elevated number of cells in the severe CHF group compared to the mild/moderate CHF group (p =  0.013), as shown in figure 3. Interestingly, there was a positive association between late apoptotic cells and NYHA class (r = 0.223, p = 0.046) as well as a negative correlation between late apoptotic cells and ejection fraction (r = −0.252, p = 0.028). There was also a negative association between hyperlipidemia and total CD34+ progenitors in the CHF groups. Tables 2 and 3 demonstrate the parameters tested for correlations and associations. As shown in table 2, neither erythropoietin (r = 0.072; p = 0.3), thrombomodulin/CD141 (r = 0.09; p = 0.25), nor antibodies to oxidized LDL levels (r = −0.13; p = 0.19) correlated with early apoptotic progenitor cells.10.1371/journal.pone.0003238.g002Figure 2Early apoptotic progenitor CD34+ cell percentage (out of total circulating progenitor CD34+ cells) in mild/moderate and severe CHF groups.P-value is >0.05 after adjustment for variables.10.1371/journal.pone.0003238.g003Figure 3Late apoptotic progenitor CD34+ cell percentage (out of total circulating progenitor CD34+ cells) in mild/moderate (NYHA class 1–3) and severe (NYHA class 4) CHF groups.Chi-square P value of the distribution of tertiles between NYHA 1–3 & NYHA = 4 is 0.038.10.1371/journal.pone.0003238.t002Table 2Correlations between early apoptotic/late apoptotic/total CD34+ EPCs and various parameters.ParameterEarly Apoptotic CellsLate Apoptotic CellsTotal CD34+ cellsrp-valuerp-valuerp-valueAge0.0130.4600.0520.350−0.1230.180Heart failure duration−0.0630.318−0.0180.447−0.0580.332LVEF−0.1070.211−0.2520.028*\n−0.1520.127Creatinine0.0330.403−0.0270.420−0.0490.357Erythropoietin0.0720.2980.1570.1230.0310.409Thrombomodulin0.0930.2470.0910.2510.0310.411OxLDL antibodies−0.1170.194−0.0370.3910.0410.380Hemoglobin0.2790.016*\n−0.0270.4200.1760.092CD34% of total0.2610.023*\n0.2130.053*Statistically Significant.10.1371/journal.pone.0003238.t003Table 3Associations between early apoptotic/late apoptotic/total CD34+ EPCs and various parameters.ParametersEarly apoptotic cellsLate apoptotic cellsTotal CD34+ cellsrp-valuerp-valuerp-valueGender−0.0270.420−0.1240.176−0.2140.053Hyperlipidemia0.1300.165−0.1860.082−0.2360.038*\nSmoking0.0940.241−0.0520.3510.1700.101Hypertension0.0170.450−0.0740.291−0.1570.120Diabetes Mellitus0.1080.210−0.0230.433−0.1130.200Ischemic heart disease0.0400.384−0.0810.274−0.1060.215TIA/CVA0.0650.3130.1740.096−0.1030.221PTCA0.1090.209−0.2430.033*\n0.1240.178CABG0.0710.3000.0280.417−0.1130.199NYHA Class−0.1740.0960.2230.046*\n0.0980.232*Statistically Significant.DiscussionOver the past decade, since the isolation of a circulating angioblast (later referred to as EPC) from adults with the capacity to differentiate into mature endothelial cells in response to ischemia [9], [22], much work has been done to further classify and characterize the role of endothelial progenitor cells. EPCs are released from the bone marrow in response to endothelial damage in order to facilitate in angiogenesis and vasculogenesis. Previous studies have shown elevated numbers of EPCs in ACS [23]–[25] as well as in heart failure [11], presumably due to the release of angiogenic factors and activation of multiple neurohormonal axes triggered by tissue ischemia [7]. It is also known that all major cardiovascular risk factors negatively influence these factors [26].In CHF there is increased oxidative stress due to an imbalance between reactive oxygen species (including the superoxide anion, hydrogen peroxide, and the hydroxyl radical) and endogenous antioxidant defense mechanisms [32]. Oxidative stress may damage cellular proteins and cause myocyte apoptosis and necrosis. Markers of oxidative stress that are increased in CHF include plasma-oxidized low-density lipoproteins, malondialdehyde and myeloperoxidase (an index of leukocyte activation), urinary levels of biopyrrins (oxidative metabolites of bilirubin), and isoprostane levels in plasma and urine [32].Endothelial cell (EC) apoptosis is an additional biomarker of endothelial damage and hemostasis that has more recently been explored [27]. The number of circulating endothelial microparticles positively correlates with the severity of coronary endothelial dysfunction, suggesting a close relationship between coronary endothelial-dependent vasodilation and EC apoptosis [12]. In addition, their functional properties such as their procoagulant activity, involvement in inflammation and direct effect on endothelial dysfunction play a large role [28]. We have recently described a novel assay to detect and quantify circulating apoptotic CD34+ progenitor cells showing that they were elevated in ACS patients compared with healthy controls [15].Similar to ACS, CHF is associated with myocardial and peripheral tissue ischemia. Thus, CHF patients were found to exhibit endothelial dysfunction that is correlated with disease severity [29]. Previous studies have demonstrated elevated levels of EPCs in heart failure patients [11], yet levels of apoptotic EPCs in these patients have not been determined so far. In our study, we sought to quantify these apoptotic progenitor cells in patients with heart failure. In so doing, we divided these apoptotic cells into two groups: early, reversible apoptotic CD34+ cells and late, irreversible apoptotic progenitors. The late apoptotic progenitor cells represent cells whose plasma membrane is no longer intact. Our results show that, while there was no significant difference in numbers of early or late apoptotic cells between CHF patients and healthy controls, there was an elevated number of late apoptotic progenitors in the more severe CHF patients compared to the less severe heart failure patients. Furthermore, there was a negative correlation between late apoptotic progenitors and ejection fraction as well as a positive association between late apoptotic CD34 cells and NYHA class.Several possible mechanisms could be postulated to explain these findings. Oxidative stress is known to cause endothelial dysfunction, and, as mentioned previously, CHF is associated with increased oxidative stress [32]. It has been demonstrated by Dernbach et al that EPCs are equipped with antioxidative enzyme systems, allowing for improved survival of cells undergoing severe oxidative stress [30]. Oxidized LDL has been shown to increase the rate of EPC senescence/apoptosis [31]. Antibodies to oxidized LDL are thought to mirror oxidative stress and have been shown to be increased in patients with CHF [19]. Although it would be plausible to explain the increase in late apoptotic cells in the more severe CHF (low EF and high NYHA class) patients as being due to increased oxidative stress, we did not find a statistically significant correlation between antibodies to oxidized LDL and apoptotic progenitor cells. Therefore, the relationship of additional indirect markers of oxidative stress which are known to be increased in CHF [32] should be further explored in future studies.Erythropoietin and thrombomodulin have also been shown to play a role in endothelial function and heart failure. Both these markers, erythropoietin being a mobilizer of progenitor cells and thrombomodulin being associated with endothelial dysfunction, were not found to correlate with the number of apoptotic CD34 cells.In view of the interplay between cytokines enhancing progenitor cell mobilization and those precipitating their apoptosis, our results support the hypothesis that increasing severity of heart failure shifts the balance towards enhanced progenitor cell apoptosis. The lower the ejection fraction, the poorer the forward flow, which may increase tissue ischemia and therefore endothelial damage.It should be mentioned, however, that the relatively small number of circulating CD34+ cells poses a question as to the true functional importance of these cells and imposes a difficulty in determining their accurate number. In addition, our study is limited by the relatively small sample size.In conclusion, we found that patients with advanced CHF have higher levels of late apoptotic progenitors than those with mild/moderate CHF and that levels of late apoptotic progenitors were positively associated with NYHA class and had a negative correlation with ejection fraction. These findings support the hypothesis that increasing severity of heart failure shifts the balance towards enhanced progenitor cell apoptosis. Therefore, apoptotic progenitor cells could be evaluated in future studies as a potential predictive biomarker in CHF.\n\nREFERENCES:\n1. JessupMBrozenaS\n2003\nHeart Failure.\nN Engl J Med\n348\n2007\n2018\n12748317\n2. GiannattasioCAchilliFGrappioloAFaillaMMelesE\n2001\nRadial artery flow-mediated dilatation in heart failure patients: effects of pharmacological and nonpharmacological treatment.\nHypertension\n38\n1451\n1455\n11751734\n3. VarinRMulderPTamionFRichardVHenryJP\n2000\nImprovement of endothelial function by chronic angiotensin-converting enzyme inhibition in heart failure: role of nitric oxide, prostanoids, oxidant stress, and bradykinin.\nCirculation\n102\n351\n356\n10899101\n4. ChinBSConwayDSChungNABlannADGibbsCR\n2003\nInterleukin-6, tissue factor and von Willebrand factor in acute decompensated heart failure: relationship to treatment and prognosis.\nBlood Coagul Fibrinolysis\n14\n515\n521\n12960603\n5. Nonaka-SarukawaMYamamotoKAokiHTakanoHKatsukiT\n2003\nIncreased urinary 15-F2t-isoprostane concentrations in patients with non-ischaemic congestive heart failure: a marker of oxidative stress.\nHeart\n89\n871\n874\n12860861\n6. BoosCLipGBlannA\n2006\nCirculating endothelial cells in cardiovascular disease.\nJournal of the American College of Cardiology\n48\n1538\n1547\n17045885\n7. RafiiSLydenD\n2003\nTherapeutic stem and progenitor cell transplantation for organ vascularization and regeneration.\nNat Med\n9\n702\n712\n12778169\n8. UrbichCDimmelerS\n2004\nEndothelial progenitor cells: characterization and role in vascular biology.\nCirc Res\n95\n343\n353\n15321944\n9. AsaharaTMuroharaTSullivanASilverMvan derZR\n1997\nIsolation of putative progenitor endothelial cells for angiogenesis.\nScience\n275\n964\n967\n9020076\n10. HristovMWeberC\n2004\nEndothelial progenitor cells: characterization, pathophysiology, and possible clinical relevance.\nJ Cell Mol Med\n8\n498\n508\n15601578\n11. MichowitzYGoldsteinEWexlerDShepsDKerenGGeorgeJ\n2007\nCirculating endothelial progenitor cells and clinical outcome in patients with congestive heart failure.\nHeart\n93(9)\n1046\n50\n17277352\n12. WernerNWassmannSAhlersPKosiolSNickenigG\n2006\nCirculating CD31+/Annexin V+ apoptotic microparticles correlate with coronary endothelial function in patients with coronary artery disease.\nArterioscler Thromb Vasc Biol\n26\n112\n116\n16239600\n13. Bernal-MizrachiLJyWJimenezJJPastorJMauroLM\n2003\nHigh levels of circulating endothelial microparticles in patients with acute coronary syndromes.\nAm Heart J\n145\n962\n970\n12796750\n14. BoulangerCMScoazecAEbrahimianTHenryPMathieuE\n2001\nCirculating microparticles from patients with myocardial infarction cause endothelial dysfunction.\nCirculation\n104\n2649\n2652\n11723013\n15. SchwartzenbergSDeutschVMaysel-AuslanderSKissilSKerenGGeorgeJ\n2007\nCirculating apoptotic progenitor cells: a novel biomarker in patients with acute coronary syndromes.\nArterioscler Thromb Vasc Biol\n27(5)\ne27\n31\n17332488\n16. RaynalPPollardHB\n1994\nAnnexins: the problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipids-binding proteins.\nBiochim Biophys Acta\n1197\n63\n93\n8155692\n17. KoopmanGReutelingspergerCPKuijtenGAKeehnenRMPalsST\n1994\nAnnexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis.\nBlood\n84\n1415\n1420\n8068938\n18. MartinSJReutelingspergerCPMcGahonAJRaderJAvan SchieRC\n1995\nEarly redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl.\nJ Exp Med\n182\n1545\n1556\n7595224\n19. GeorgeJWexlerDBarakTShepsDKerenGGeorgeJ\n2006\nUsefulness of anti-oxidized LDL antibody determination for assessment of clinical control in patients with heart failure.\nEur J Heart Fail\n8\n58\n62\n16061420\n20. 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FischerDRossaSLandmesserUSpiekermannSEngberdingN\n2005\nEndothelial dysfunction in patients with chronic heart failure is independently associated with increased incidence of hospitalization, cardiac transplantation, or death.\nEur Heart J\n25\n65\n69\n30. DernbachEUrbichCBrandesRPHofmannWKZeiherAMDimmelerS\n2004\nAntioxidative stress-associated genes in circulating progenitor cells: evidence for enhanced resistance against oxidative stress.\nBlood\n104\n3591\n3597\n15161665\n31. ImanishiTHanoTSawamuraTNishioI\n2004\nOxidized low-density lipoprotein induces endothelial progenitor cell senescence, leading to cellular dysfunction.\nClin Exp Pharmacol Physiol\n31\n407\n413\n15236625\n32. BraunwaldE\n2008\nBiomarkers in Heart Failure.\nNew England J Med\n358\n2148\n2159\n18480207"
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+{
+    "id": "PMC2528949",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528949\nAUTHORS: Jean-Claude Dreher, Etienne Koechlin, Michael Tierney, Jordan Grafman\n\nABSTRACT:\nBackgroundA major question in understanding the functional organization of the brain is to delineate the functional divisions of the prefrontal cortex. Of particular importance to the cognitive capacities that are uniquely human is the fronto-polar cortex (Brodmann's area 10), which is disproportionally larger in humans relative to the rest of the brain than it is in the ape's brain. The specific function of this brain region remains poorly understood, but recent neuroimaging studies have proposed that it may hold goals in mind while exploring and processing secondary goals.Principal FindingsHere we show that the extent of damage to the fronto-polar cortex predicts impairment in the management of multiple goals. This result reveals that the integrity of the fronto-polar cortex is necessary to perform tasks that require subjects to maintain a primary goal in mind while processing secondary goals, an ability which is crucial for complex human cognitive abilities.Conclusion/SignificanceThese results provide important new insights concerning the cerebral basis of complex human cognition such as planning and multitasking.\n\nBODY:\nIntroductionSome of the most complex cognitive abilities of humans, such as planning, are commonly attributed to a disproportionate enlargement of the human frontal lobe during evolution. However, recent comparative studies of the relative size of the frontal cortex taken as a whole indicate that the human frontal cortex is not larger in comparison to those of the great apes [1], [2]. Rather, the specific cognitive capacities of humans may be due to differences in specific individual cortical areas (such as the frontopolar cortex), as well as to richer interconnectivity between the frontal lobe and other higher-order association areas, none of which require an increase in the overall relative size of the frontal lobe during hominid evolution.Of particular importance in the cognitive capacities that are uniquely human seems to be the most anterior part of the prefrontal cortex, namely the fronto-polar cortex (Brodmann's area 10), which is larger in humans relative to the rest of the brain than it is in the ape's brain [1], [2]. The specific function of this brain region remains poorly understood, but one recent hypothesis states that its role is to hold goals in mind while exploring and processing secondary goals [3], [4], [5], [6] – a process that we refer to as multitasking in the remainder of this paper-. Neither keeping in mind a goal over time (working memory) nor successively allocating attentional resources between alternative goals (dual-task performance) could by themselves selectively activate the fronto-polar cortex while a highly specific super-additive effect was demonstrated in the frontopolar cortex when subjects held in mind goals while processing secondary goals at the same time [3].This functional hypothesis about a key role of Brodmann's area (BA) 10 in multitasking is based on the results of functional neuroimaging studies that can only support inferences about the association of brain regions with a specific cognitive process. In contrast, neuropsychological studies are crucial for inferring whether a brain region is necessary to mediate a cognitive process.Here we examined patients with focal prefrontal cortex lesions to test whether the fronto-polar cortex is necessary for multitasking. Our hypothesis was that the extent of damage to the fronto-polar cortex should correlate with impairment in this process. The results confirmed this hypothesis by demonstrating that the extent of damage to Brodmann's area 10 correlated with impaired multitasking.Materials and MethodsParticipantsWe tested 13 patients with focal frontal lobe lesions (see \nTable 1\n and \nFigs 1\n\n–\n\n\n3\n\nfor patient's demographic and lesion's sites). Patients were divided into two groups on the basis of the location of their lesion: one group had lesions that included the fronto-polar cortex (n = 7, 4 males, mean age = 49±6) and one group had lesions that excluded the fronto-polar cortex (n = 5, 3 males, mean age = 49±3.5). The two groups of patients did not differ in age (t = 0.03, P = 0.98), level of education (t = 0.29, P = 0.8) and Full Scale WAISIII IQ scores (t = 0.74, P = 0.48). In addition, 7 control subjects (5 males, mean age = 50.8±6) were matched in age (t = 0.2, P = 0.82, n.s) and level of education (t = 0.72, P = 0.48) with the patients with fronto-polar lesions. All subjects were screened for a prior history of neurological disease, substance abuse, and psychiatric disorder.10.1371/journal.pone.0003227.g001Figure 1(A). Location and degree of lesion overlap in patients with fronto-polar cortex lesions.(B). Location and degree of lesion overlap in control patients without fronto-polar cortex lesions. Slices are oriented in radiological convention (i.e. the left side of the image is the right hemisphere). Lighter colors denote the degree to which lesions involve the same structure in multiple subjects. The darker color at the bottom of the color scale indicates no overlap between brain region.10.1371/journal.pone.0003227.g002Figure 2Reconstruction of lesions for each patient with frontopolar cortex damage (P1–P7) based on computerized tomography and MRI scans.The shaded area represents the lesion. Axial slices from ventral (left) to dorsal (right). According to radiological convention right is left.10.1371/journal.pone.0003227.g003Figure 3Reconstruction of lesions for each patient without frontopolar cortex damage (NFP1–NFP5) based on computerized tomography and MRI scans.The shaded area represents the lesion. Axial slices are displayed in radiological convention from ventral (left) to dorsal (right).10.1371/journal.pone.0003227.t001Table 1Demographic of patients and healthy control subjects.GroupAgeLevel of Educa-tionSexrHandTime since lesion (years)WAIS-III Full Scale IQWMS - III Working Memory Index ScoreWMS - III Auditory Immediate Index ScoreWMS - III Auditory Recognition Delayed Index ScoreFluency Total Raw ScoreBNT Total Raw ScoreBDI-2 Total Raw ScoreNART Full IQ\nFP Lesions\nFP14711MR3180108898931341FP26220MR71218812010574599FP35612MR33102968010027573FP43914FR1310411111495305617FP52818FR13132124117100775533FP66216FR3113710811411057543FP74912MR399796656514525\nNo FP lesion\nNFP15214ML32999110290295719NFP25516FR1101778095371614NFP35410MR32981089210522513NFP44515MR8118115748531547NFP53816FR11009310811038583\nControls\nC15013MR0110C24512MRNANAC36312MR2112C46216ML4102C55512MR091C65012MR0121C72818FR2112The etiology of fronto-polar patients was as followed: 3 were Vietnam veterans who suffered a penetrating brain injury, one patient had a resection of a malignant tumor, one had an aneurysm of the right anterior communicating artery and two patients had closed head injuries. Reconstruction of lesions for each patient with or without frontopolar cortex damage is provided in figures 2 and 3. All subjects were screened for a prior history of neurological disease, substance abuse, and psychiatric disorder. The control subjects were paid for their participation. Subjects provided written informed consent approved by the NINDS Institutional Review Board.Lesion analysisThe penetrating brain injury patients were scanned using a standard CT sequence since they had retained metal in their brain. Other patients were scanned by MRI using a three-dimensional set acquisition in the axial plane with a SPGR T1-weighted sequence and a T2-weighted axial sequence. All lesions were traced using the Analysis of Brain Lesions software package [7] and normalized in Talairach space to the Damasio's template [8]. Then, the percentage of the approximate Brodmann areas contained within the boundaries of the lesions was computed for each subject using this standardized, semi-automated software that can determine the extent of brain lesions in terms of cytoarchitectonic regions in Talairach space [7] (see \nFig. 1\n).Behavioral paradigmThe tasks were identical to those used in a previous fMRI study [3] and were designed to systematically vary keeping in mind a main goal over time (working memory) and allocation of attentional resources between alternative subgoals (dual-task) (\nFig. 4\n). The experiment consisted of 6 runs in which 3 tasks (delay, dual and multi-tasking, described hereafter) were administered in pseudo-random order (28 trials by task, inter-stimuli interval = 3 s). This pseudo-random order was built in such a way that each condition appears at all serial positions within a run and two conditions appeared once or twice in immediate succession to prevent confounding order effects. The first run was used for training and is not included in the present analysis.10.1371/journal.pone.0003227.g004Figure 4Behavioural tasks.Single-letters (upper or lower-case) from the word “tablet” were successively presented and subject's decisions were recorded using two single response-buttons, one for each hand. Delay condition: subjects decided whether two successively presented upper-case letters were also in immediate succession in the word “tablet” by pressing the right button for yes and the left button if they were not in succession, and they had to ignore lower case letters that were presented in order to delay the response required for upper-case letters. Dual-task condition: subjects decided whether two successively presented letters were also in immediate succession in the word “tablet” by pressing the right button for yes and the left button if they were not in succession, this time both for upper and lower case letters, except that they had to decide whether every first letter indicating a case change was the letter T (or t). Multi-tasking condition: subjects responded to upper case letters exactly as in the delay condition and to lower case letters exactly as in the dual task condition. Thus, the multitasking condition requires maintenance of the primary task information in memory (primary goal) so that it can be returned to after completing a secondary task (subgoal).Subjects responded to visually presented letters (500 ms duration, 3000 ms stimulus-onset-asynchrony) by pressing response buttons with their right (match) or left (no match) hand, respectively. Subjects were given standard instructions to respond quickly and accurately. Single-letters (upper or lower-case) from the word “tablet” (i.,e A, B, E, L, T, a, b, e, l, t) were successively presented and subject's decisions were recorded using the two single response-buttons. Matching proportions were maintained between 40 and 43% of trials in each condition. In all conditions lower-case letters were pseudorandomly presented in 64% of trials and the mean SOA between two successive upper-case letters was strictly maintained at 6.3 s. The tasks were administered using the Expe6 software package [9].\nIn the delay condition, subjects decided whether two successively presented upper-case letters were also in immediate succession in the word “TABLET” by pressing the right button for yes and the left button if they were not in succession, and they had to ignore lower case letters that were presented in order to delay the response required for upper-case letters.\nIn the dual-task condition, subjects decided whether two successively presented letters were also in immediate succession in the word “tablet” or “TABLET” (this time both for upper and lower case letters), by pressing the right button for yes and the left button if they were not in succession, except that they had to decide whether every first letter indicating a case change was the letter T (or t).\nIn the multitasking condition, subjects responded to upper case letters exactly as in the delay condition and to lower case letters exactly as in the dual task condition. Thus, the multitasking condition requires maintenance of the primary task information in memory (primary goal) so that it can be returned to after completing a secondary task (subgoal). In other words, for successive upper case letters, or for successive lower case letters, subjects decided whether the current letter followed immediately the previously presented letter in the word “TABLET” or ‘tablet” by pressing the right button for yes and the left button if they were not in succession, and they had to decide whether every first letter indicating a case change was the letter T (or t).ResultsBehavioral performanceFirst, a 2*3 repeated measures ANOVA was conducted on correct reaction time (RT) (<3000 ms) and on error rates with group (patients with fronto-polar cortex lesions and age-matched controls) as the between-subject factor and with conditions (delay, dual task, multi-tasking) as the within-subject factor (\nFig. 5\n). In this group analysis of variance, for response times, there was a main effect of task [F(2, 36) = 10.6, P<0.0005], indicating that additional processes are engaged successively in the delay, dual-task and multi-tasking conditions. No main effect of group [F(1,36) = 2.0, P = 0.16] and no group*task interaction [F(2,36) = .25, P = .8] were observed.10.1371/journal.pone.0003227.g005Figure 5Behavioral performance.(A). Graph representing responses times (for correct responses) in patients with fronto-polar cortex lesions (blue), in patients without fronto-polar cortex lesions (green) and in controls (red). (B). Percentage of errors in the three conditions (delay, dual and multi-tasking) in patients with fronto-polar lesions (blue), in patients without fronto-polar cortex lesions (green) and in normal controls (red). Error bars represent the 95% confidence intervals.For error rates, patients with lesions affecting the fronto-polar cortex made more errors than controls (main effect of group [F(1, 36) = 5.01, P<0.05]). There was also a main effect of task [F(2, 36) = 9.8, P<0.0005]. Overall, the multi-tasking condition led to more errors than the delay condition (F(1,26) = 21.5, P<0.0001) but did not differ from the dual-task condition [F(1,26) = 1.2, P = 0.3]. We did not observe any group*task interaction [F(2,36) = 0.5, P = 0.6] on performance accuracy. However, when performing a new 2*2 analysis of variance grouping the dual task and multi-tasking conditions together (both involving putting information into sequences) compared to the delay condition, we found a significant difference between patients with frontopolar lesions and controls [F(1,26) = 5.04, P<0.05]. No between group difference was observed when performing another 2*2 analysis of variance grouping the delay and multi-tasking conditions together (both involving working memory) compared to the dual task condition [F(1,26) = 1.24, P = 0.27].We also directly compared the performance of the two groups of patients. For response times, there was a main effect of task [F(2,30) = 11.6, P<0.0005], confirming additional engagement of processes from the delay to the dual-task and to the multi-tasking conditions. No main effect of group [F(1,30) = .74, P = 0.39] and no group*task interaction [F(2,30) = .63, P = .53] were observed for response times. For error rates, there was a trend towards significance in patients with lesions affecting the fronto-polar cortex compared to patients without lesion of the frontopolar cortex (main effect of group [F(1,30) = 3.1, P = 0.08]). There was also a main effect of task [F(2,30) = 16.9, P<0.00005] due to the lower error rate in the delay condition. No group*task interaction [F(2,30) = 0.59, P = 0.55] was observed on performance accuracy. When we performed another 2*2 analysis of variance grouping the dual task and multi-tasking conditions together (both involving switching between tasks) compared to the delay condition, we found a trend towards significance between patients with versus without frontopolar lesions [F(1,32) = 3.68, P = 0.06]. These data suggest that frontopolar lesions impair switching processes, both compared to controls and compared to patients without frontopolar lesions. No difference between patient group was observed when performing another 2*2 analysis of variance grouping the delay and multi-tasking conditions together (both involving working memory) compared to the dual task [F(1,32) = 0.96, P = 0.33].Correlations between performance and damage to the fronto-polar cortexIn order to test our specific hypothesis that the fronto-polar cortex (Brodmann's area 10) is necessary for performing a subgoal while maintaining primary goal related information in memory, we correlated the proportion of damage to each approximate Brodmann's area contained within the boundaries of the lesion with the error rates in the multi-tasking condition. Only the left BA 10 (Spearman rank correlation coefficient R1 = 0.94, P<0.005) showed a significant positive correlation with performance (6 patients had left frontopolar damage) (\nFig. 6.A\n). No other Brodmann's area was significantly correlated with performance in the multi-tasking condition. Moreover, lesion size of the left BA 10 did not show a significant correlation with error rates in the dual-task condition (Spearman rank correlation R2 = 0.65, P = 0.16, n.s) (\nFig. 6.B\n). This demonstrates that variability of frontopolar cortex patients in multi-tasking performance is primarily explained by the size of the lesion in BA 10.10.1371/journal.pone.0003227.g006Figure 6Relationships between lesion size and behavior.(A) A positive significant correlation was observed between the percentage of errors in the multi-tasking condition and the percentage of damage to the left Brodmann's area 10 in patients with fronto-polar cortex lesions (Spearman rank correlation coefficient R = 0.94, P<0.005). (B) No significant correlation was observed between the lesion sizes of the left BA 10 with error rate in the dual-task condition. (C) No significant correlation was observed between total volume of brain lesions and error rate in the multi-tasking condition.Note that this analysis did not include the only patient with lesion restricted to the right fronto-polar cortex because there may be a functional lateralization of this brain region. However, when including the data of this subject, the correlation between error rates in the dual-task condition and the size of fronto-polar lesions was still non-significant (R = 0.1, P = 0.5) and the correlation between error rates in the branching condition and size of fronto-polar lesions remained significant (Spearman rank correlation coefficient R = 0.82; P<0.05).Further analyses of the total volume of brain lesions revealed no relationship between the total volume of damaged tissue (which included extra fronto-polar damage) and error rates in the multi-tasking condition (R = 0.36, P = 0.43), ruling out the possibility of a confound between total lesion size and lesions of the fronto-polar cortex (\n\nFig. 6.C\n\n). Moreover, task difficulty or mental effort alone can not explain our findings since error rates in the multi-tasking and the dual-task condition did not differ significantly in patients with fronto-polar cortex lesions ((F1,19) = 0.4, P = 0.5).DiscussionBased on recent neuroimaging findings [3], [4], we could have expected that patients with frontopolar lesions would exhibit a specific increase of errors only in the multi-tasking condition. The fact that we did not observe such a group by task interaction may be due to the extent of the lesions of the frontopolar group, which included, but were not restricted to, the frontopolar region. This may also explain why performance is altered in the dual task condition, thereby masking the behavioral effect of more restricted frontopolar lesions. Note that it is also possible that the frontopolar cortex contains multiple subregions, each contributing to different processes, and/or that brain regions outside the frontopolar cortex are part of a functionally distributed network that is necessary to perform multitasking computations.Although we did not find a group*task interaction on performance accuracy, likely due to group size and low statistical power, our results suggest that what is crucially impaired in patients with frontopolar lesions is the ability to put information into sequences as required by the dual-task and the multi-tasking conditions rather than simply holding a goal in working memory (such as in the delay condition). Indeed, when performing an analysis of variance grouping the dual task and multi-tasking conditions together (both involving putting information into sequences) compared to the delay condition, we did find a significant difference between patients with frontopolar lesions and controls and a trend towards significance between patients with frontopolar lesions versus without frontopolar lesions.Our main finding is that only the extent of the lesion of the left fronto-polar cortex showed a positive correlation with performance in the multitasking condition. This demonstrates that damage to the frontopolar cortex is necessary to impair multitasking, i.e. a process dependent upon the ability to put tasks in pending sequences. These results provide new evidence that specific executive functions are subserved by distinct prefrontal regions [10], [11], [12], [13], [14], [15], contradicting the view that the functions of distinct prefrontal regions cannot be distinguished [16]. Thus, variability of frontopolar cortex patients in multi-tasking performance is primarily explained by the size of the lesion in BA 10. This correlation between left BA 10 lesion volume and performance is consistent with the linguistic nature of the task. Nevertheless, since there was only one subject with strictly unilateral right frontopolar damage among our subjects, we cannot be certain about whether the effect is truly lateralized.Although the lesion size of the left BA 10 did not show a significant correlation with error rates in the dual-task condition (Spearman rank correlation R = 0.65, P = 0.16, non significant), it could be argued that the significant correlation observed between lesion size in the fronto-polar cortex and multi-tasking (R = 0.94, p<0.005) does not prove that the effect is specific. However, the inference we tested was that the correlation coefficient was significantly different from zero in the multitasking condition and/or in the dual task condition, not that the correlation in the multitasking condition was significantly higher than the correlation in the dual task. Thus, it would only be statistically justified to compare correlation coefficients (between overlapping pair of variables) if both of these correlations coefficients were significantly different from zero (which is not the case). To further ensure the specificity of our findings, we tested the robustness of our results by recomputing the correlation coefficient in the multitasking and dual-task conditions for each possible n-1 subset of data sample (see supplementary tables S1 and S2). These tables show that the significance of the correlation coefficient remains P<0.05 in the multitasking condition and is non-significant in the dual-task condition. This demonstrates that variability of frontopolar cortex patients in multitasking is primarily explained by the size of the lesion in BA 10 and that the size of the left BA 10 lesion is a good anatomical predictor of multitasking but not of dual-task related errors.Although there may be many functional subregions within the frontopolar cortex, we believe that BA 10 is particularly important for multitasking. This process may be involved in a number of functions previously associated with the frontopolar cortex besides multitasking [17], including integrating the outcomes of two or more separate cognitive operations in the pursuit of a higher behavioural goal [18], processing of internally generated information [19], [20], memory retrieval [21], [22], carrying out delayed intentions (prospective memory) [5], [6], relational integration [23], [24], [25], integration of diverse information content [26] and exploratory decisions [27].It should be noted that there is a fundamental qualitative difference between multitasking and the dual task. Multitasking combines not only a dual-task component but also a working memory component. It successively allocates processing resources between concurrent tasks, as in dual-task performance and it keeps relevant information in working memory to allow a return to the main task after completing a secondary task. In contrast to multitasking, which specifically involves the fronto-polar cortex, dual task performance induces higher inferior and middle frontal sulcus activity as compared to single task performance [12], [28], [29], [30]. However, our results are not conclusive about other regions than the frontopolar cortex because we did not test patients with specific dorsolateral prefrontal cortex lesions.A general model, integrating the recent cascade model by Koechlin et al. [31] and the multi-tasking view of the fronto-polar cortex has recently been proposed in a review paper [32]. This general model explains at an information processing level, using information theory, what is called branching (renamed here as multitasking). The overview of this general model is that cognitive control operates according to three nested levels of control processes (contextual, episodic and multi-tasking) implemented from posterior to polar prefrontal regions. In this model, H(a) measures the total amount of control information required for selecting action “a” and is processed in the premotor cortex. H(a) is the sum of two control terms: bottom-up information conveyed by a stimulus S (I(s,a), sensorimotor control) and the remaining top-down information Q(a|s) processed in the posterior lateral PFC and measuring cognitive control. Cognitive control, in turn, is the sum of two control terms: bottom-up information conveyed by the context c in which stimulus s occurs (I(c,a|s), contextual control); and the top-down remaining information Q(a|s,c) processed in the anterior lateral PFC. Finally, this latter control term is the sum of bottom-up information conveyed by a past event u (I(u,a)|s,c), episodic control) and the remaining top-down information processed in the polar lateral PFC (multi-tasking control). Multi-tasking control is related to the information conveyed by events preceding u and maintained in a pending state until completion of the ongoing episode. Thus, according to this model, during execution of the current episode u, the most anterior portions of the PFC maintain (in a distractor-resistant fashion) pending information from a yet more temporally distant episode, enabling this information to be flexibly retrieved when this episode is re-instantiated. This model explains the pattern of prefrontal activations observed in several experimental paradigms, including learning [33], episodic memory [34], working memory [35] and task switching paradigms [4], [10], [13], [14]. In these experiments, caudal and rostral LPFC activations were observed, depending on whether the executive control of behavior was based on contextual or episodic signals.A recent review, consistent with our interpretation of frontopolar function, is that a common process across these studies may be that the frontopolar cortex is recruited to integrate the results of two or more cognitive operations, fulfilling a higher behavioural goal [18]. This view predicts that the process of integration should be reflected in frontopolar activity beyond the activity observed for processing the component elements to be integrated. Confirming this prediction, a highly specific super-additive effect was previously demonstrated in the frontopolar cortex using fMRI when subjects held in mind goals while processing secondary goals at the same time [3].To conclude, we have demonstrated that managing subgoals while maintaining information about primary goals is a process that is critically and selectively disrupted with increasing size of fronto-polar cortex damage. From an evolutionary point of view, it is interesting to note that during hominoid evolution, the frontopolar cortex (area 10) may have undergone not only a shift in its extent but also of its topographic location and a specific increase in connectivity with other higher-order association areas (the supragranular layers having more space available for connections with other higher-order association area) [1], [2]. It has recently been proposed that the frontopolar cortex may be the only prefrontal region that is predominantly (and possibly exclusively) interconnected with supramodal areas in the prefrontal cortex and anterior temporal cortex [18], allowing the frontopolar cortex to dynamically monitor and assign positional priority to information received from more caudal areas of supramodal cortex. Since the frontopolar cortex in the human brain appears to have evolved in size and organization, this suggests that complex functions requiring the temporary interruption of a current plan to achieve subgoals (such as planning of future actions and reasoning) associated with this part of the cortex have become particularly important during hominid evolution.Supporting InformationTable S1Correlation coefficient in the branching condition for each possible n-1 subset of data sample.(0.03 MB DOC)Click here for additional data file.Table S2Correlation coefficient in the dual-task condition for each possible n-1 subset of data sample.(0.02 MB DOC)Click here for additional data file.\n\nREFERENCES:\n1. SemendeferiKLuASchenkerNDamasioH\n2002\nHumans and great apes share a large frontal cortex.\nNat Neurosci\n5\n272\n276\n11850633\n2. SemendeferiKArmstrongESchleicherAZillesKVan HoesenGW\n2001\nPrefrontal cortex in humans and apes: a comparative study of area 10.\nAm J Phys Anthropol\n114\n224\n241\n11241188\n3. KoechlinEBassoGPietriniPPanzerSGrafmanJ\n1999\nThe role of the anterior prefrontal cortex in human cognition.\nNature\n399\n148\n151\n10335843\n4. BraverTSBongiolattiSR\n2002\nThe role of frontopolar cortex in subgoal processing during working memory.\nNeuroimage\n15\n523\n536\n11848695\n5. BurgessPWQuayleAFrithCD\n2001\nBrain regions involved in prospective memory as determined by positron emission tomography.\nNeuropsychologia\n39\n545\n555\n11257280\n6. BurgessPWScottSKFrithCD\n2003\nThe role of the rostral frontal cortex (area 10) in prospective memory: a lateral versus medial dissociation.\nNeuropsychologia\n41\n906\n918\n12667527\n7. MakaleMSolomonJPatronasNJDanekAButmanJA\n2002\nQuantification of brain lesions using interactive automated software.\nBehav Res Methods Instrum Comput\n34\n6\n18\n12060992\n8. DamasioAR\n1995\nBrain anatomy in computerized images.\nPressOU\nNew York\n9. PallierCDupouxELiddlePFFrackowiakR\n1997\nEXPE: an expandable programming language for on-line psychological experiments.\nBehav Res Methods Instrum Comput\n29\n322\n327\n10. DreherJCKoechlinEAliSOGrafmanJ\n2002\nThe roles of timing and task order during task switching.\nNeuroimage\n17\n95\n109\n12482070\n11. DreherJCBermanKF\n2002\nFractionating the neural substrate of cognitive control processes.\nProc Natl Acad Sci U S A\n99\n14595\n14600\n12391312\n12. DreherJCGrafmanJ\n2003\nDissociating the roles of the rostral anterior cingulate and the lateral prefrontal cortices in performing two tasks simultaneously or successively.\nCereb Cortex\n13\n329\n339\n12631562\n13. BrassMvon CramonDY\n2002\nThe Role of the Frontal Cortex in Task Preparation.\nCereb Cortex\n12\n908\n914\n12183390\n14. WylieGRJavittDCFoxeJJ\n2006\nJumping the gun: is effective preparation contingent upon anticipatory activation in task-relevant neural circuitry?\nCereb Cortex\n16\n394\n404\n15917482\n15. WoodJNGrafmanJ\n2003\nHuman prefrontal cortex: processing and representational perspectives.\nNat Rev Neurosci\n4\n139\n147\n12563285\n16. DuncanJOwenAM\n2000\nCommon regions of the human frontal lobe recruited by diverse cognitive demands.\nTrends Neurosci\n23\n475\n483\n11006464\n17. BurgessPWVeitchEde Lacy CostelloAShalliceT\n2000\nThe cognitive and neuroanatomical correlates of multitasking.\nNeuropsychologia\n38\n848\n863\n10689059\n18. RamnaniNOwenAM\n2004\nAnterior prefrontal cortex: insights into function from anatomy and neuroimaging.\nNat Rev Neurosci\n5\n184\n194\n14976518\n19. ChristoffKGabrieliJDE\n2000\nThe frontopolar cortex and human cognition: evidence for a rostrocaudal hierarchical organization within the human prefrontal cortex.\nPsychobiology\n28\n168\n186\n20. ChristoffKReamJMGeddesLPGabrieliJD\n2003\nEvaluating self-generated information: anterior prefrontal contributions to human cognition.\nBehav Neurosci\n117\n1161\n1168\n14674837\n21. TulvingEKapurSMarkowitschHJCraikFIHabibR\n1994\nNeuroanatomical correlates of retrieval in episodic memory: auditory sentence recognition.\nProc Natl Acad Sci U S A\n91\n2012\n2015\n8134341\n22. DuzelECabezaRPictonTWYonelinasAPScheichH\n1999\nTask-related and item-related brain processes of memory retrieval.\nProc Natl Acad Sci U S A\n96\n1794\n1799\n9990104\n23. ChristoffKPrabhakaranVDorfmanJZhaoZKrogerJK\n2001\nRostrolateral prefrontal cortex involvement in relational integration during reasoning.\nNeuroimage\n14\n1136\n1149\n11697945\n24. PrabhakaranVSmithJADesmondJEGloverGHGabrieliJD\n1997\nNeural substrates of fluid reasoning: an fMRI study of neocortical activation during performance of the Raven's Progressive Matrices Test.\nCognit Psychol\n33\n43\n63\n9212721\n25. GreenAEFugelsangJAKraemerDJShamoshNADunbarKN\n2006\nFrontopolar cortex mediates abstract integration in analogy.\nBrain Res\n1096\n125\n137\n16750818\n26. PrabhakaranVNarayananKZhaoZGabrieliJD\n2000\nIntegration of diverse information in working memory within the frontal lobe.\nNat Neurosci\n3\n85\n90\n10607400\n27. DawNDO'DohertyJPDayanPSeymourBDolanRJ\n2006\nCortical substrates for exploratory decisions in humans.\nNature\n441\n876\n879\n16778890\n28. HerathPKlingbergTYoungJAmuntsKRolandP\n2001\nNeural correlates of dual task interference can be dissociated from those of divided attention: an fMRI study.\nCereb Cortex\n11\n796\n805\n11532885\n29. StelzelCSchumacherEHSchubertTD'EspositoM\n2006\nThe neural effect of stimulus-response modality compatibility on dual-task performance: an fMRI study.\nPsychol Res\n70\n514\n525\n16175414\n30. SzameitatAJSchubertTMullerKVon CramonDY\n2002\nLocalization of executive functions in dual-task performance with fMRI.\nJ Cogn Neurosci\n14\n1184\n1199\n12495525\n31. KoechlinEOdyCKouneiherF\n2003\nThe architecture of cognitive control in the human prefrontal cortex.\nScience\n302\n1181\n1185\n14615530\n32. KoechlinESummerfieldC\n2007\nAn information theoretical approach to prefrontal executive function.\nTrends Cogn Sci\n11\n229\n235\n17475536\n33. KoechlinEDanekABurnodYGrafmanJ\n2002\nMedial Prefrontal and Subcortical Mechanisms Underlying the Acquisition of Motor and Cognitive Action Sequences in Humans.\nNeuron\n35\n371\n381\n12160754\n34. BucknerRLKoutstaalW\n1998\nFunctional neuroimaging studies of encoding, priming, and explicit memory retrieval.\nProc Natl Acad Sci U S A\n95\n891\n898\n9448256\n35. RoweJBToniIJosephsOFrackowiakRSPassinghamRE\n2000\nThe prefrontal cortex: response selection or maintenance within working memory?\nScience\n288\n1656\n1660\n10834847"
+}

batch_9/PMC2529271.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2529271",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529271\nAUTHORS: Muhammad Najm Khan, Avi Agrawal, Paul Strauss\n\nABSTRACT:\nColonic Intussusception although common in children, is a rare cause of acute intestinal obstruction in adults. The etiology, clinical presentation and management of this condition is different in adults as compared to children. Pre-operative diagnosis is usually difficult due to the non specific and intermittent nature of the symptoms. CT scan can be a helpful adjunct in establishing the diagnosis. We present a case report of adult ileocolic intussusception with classical radiological signs and operative findings. A brief literature review is also presented with emphasis on the controversy of reduction of the intussusception before resection.\n\nBODY:\nCase reportA 74 year old male presented with a four month history of colicky right upper quadrant pain, altered bowel habits and weight loss. He did not have any rectal bleeding. On examination there were signs of anaemia and a palpable mass in the right upper quadrant. Base line blood tests including FBC were normal. An abdominal USS showed a 5 × 7 cm ill defined bowel related mass in the right upper quadrant. The liver was normal.A subsequent barium enema showed an intra-luminal filling defect measuring 8 × 10 cm in the proximal transverse colon with no flow of barium proximally (Figure 1). CT scan of the abdomen showed significantly thickened bowel loops with fat density within a proximal segment and a target lesion, consistent with a neoplasm or a lipoma causing Intussusception (Figure 2).Figure 1Barium enema showing a classical \"claw sign\".Figure 2CT scan of abdomen. Red arrow demonstrates a \"target lesion\" diagnostic of intussusception.A provisional diagnosis of colonic carcinoma was made although the tumour markers including CEA and CA 19.9 were normal. The patient underwent a laparotomy where an ileocolic intussusception was found at the level of hepatic flexure (Figure 3). A right hemicolectomy was carried out with a hand sewn end to end ileocolic anastomosis. The specimen was opened to reveal a protruding polypoidal mass in the caecum (Figure 4). The patient made an uneventful post operative recovery and was discharged seven days later. The histology of the specimen showed this to be a benign submucosal lipoma of the caecum protruding into the caecal lumen.Figure 3Operative picture demonstrating ileocolic intussusception.Figure 4Resected specimen opened up to show the presence of intussusception.DiscussionIntussusception is an uncommon cause of intestinal obstruction and more than 95% of cases occur in the paediatric age group [1]. Intussusception in adults is a rare pathology its incidence is around 2–3 per 1000,000 per year [2]. Due to this rare nature of the disease there are no large scale/multi-centre studies or meta-analyses published to investigate the management of adult intussusception. The aetiology, presentation and management of intussusception in adults is different from children. In children intussusception is usually idiopathic or secondary to a viral illness. However in adults in more than 90% of cases a lead point can be identified causing the intussusception [3,4]. This is usually a polyp or a tumour and in majority of these cases the colonic tumours are malignant [5,6].The clinical presentation is very non-specific which makes this a difficult condition to diagnose. Abdominal pain, nausea, diarrhoea and bleeding per rectum are the common symptoms. Rarely this can present with acute intestinal obstruction. The classical triad of abdominal pain, sausage shaped palpable mass and passage of red current jelly stools seen in children is rarely observed in adults [6,7]. The use of investigations including a barium enema, ultrasound scan, and computed tomography can be helpful to establish the diagnosis [2,3,7-9]. CT scan has been reported to have a diagnostic accuracy of around 80% [10]. The classical finding on a CT scan is a target lesion or target sign which represents the outer intussuscepiens and the inner intussusceptum (Figure 2, 3). The dense intussuscepted mass comprising of swollen bowel and mesentery within the lumen of the bowel is responsible for the characteristic target lesion seen on the CT scan [7,11,12]. Ultrasound scan is a less invasive and reproducible investigation. The classical features include a donut sign in transverse view and pseudo-kidney sign on longitudinal view [13]. The examination is of limited value in the presence of significant amount of air in the intestine. A few studies have reported the use of colonoscopy in preoperative diagnosis particularly in the cases presenting with symptoms of large bowel obstruction [14]. However the examination is technically challenging and the diagnosis is difficult to make.Benign lesions account for almost 25% cases of intussusception in adults. The commonest benign lesion is a lipoma in the colon. These are solitary submucosal lesions with 75% occurring in the right colon. Small lipomas are asymptomatic. Other benign lesions include adenomatous polyps and Peutz-Jeghers polyps. However in more than two thirds of cases there is a malignant tumour in the colon or small bowel resulting in intussusception [15-17].Operative intervention is required in all cases of adult intussusception and unlike children conservative treatment does not work [2,6]. This usually involves segmental colonic resection. The optimal treatment for adult intussusception is slightly controversial. The type of procedure depends upon the location of intussusception, pre-operative diagnosis and condition of the intestine at the time of laparotomy. A few authors have described intra-operative reduction of intussusception before resection [5]. However most authors do not recommend this due to a higher incidence of malignancy in these cases [2,6-8,18,19] and hence the risk of tumour embolisation and seedling.In most cases of adult colonic intussusception, primary resection without reduction should be performed particularly in those more than 60 years of age due to a higher risk of malignancy. In cases of small bowel intussusception reduction before resection should be carried out only if there is a pre-operative diagnosis of benign etiology, the bowel is viable or it entails resecting massive lengths of small bowel with the risk of short gut syndrome [12,20].ConclusionIntussusception is a rare cause of acute abdomen in adults. A high index of suspicion and appropriate investigations (USS, Barium enema and CT scan) can result in prompt diagnosis. Unlike children 75% of cases are due to a malignant tumour in the small bowel or colon. The extent of resection and operative technique depend upon the age of the patient, results of investigations (benign or malignant) and the length of the bowel involved.Authors' contributionsMNK carried out the literature review and drafted the paper. AA designed the paper, literature review and reviewed the manuscript. PS literature review, provided the figures and reviewed the manuscript. All authors have read and approved the final manuscript.ConsentWritten informed consent was obtained from the patient for publication of this  case report and accompanying images. A copy of the written consent is available  for review by the Editor-in-Chief of this journal.\n\nREFERENCES:\nNo References"
+}

batch_9/PMC2529278.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2529278",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529278\nAUTHORS: Alicja Sieminska, Krzysztof Buczkowski, Ewa Jassem, Ewa Tkacz\n\nABSTRACT:\nBackgroundA better understanding of the genetic determinants of tobacco smoking might help in developing more effective cessation therapies, tailored to smokers' genotype. Insertion/deletion polymorphism in the promoter region of the serotonin transporter gene (5-HTTLPR) has been linked to vulnerability to smoking and ability to quit. We aimed to determine whether 5-HTTLPR genotype is associated with smoking behavior in Caucasians from Northern Poland and to investigate other risk factors for tobacco smoking.Methods5-HTTLPR genotypes were determined in 149 ever smokers (66 females; mean age 53.0 years) and 158 gender and ethnicity matched never smoking controls (79 females; mean age 45.0 years) to evaluate the association of this polymorphism with ever smoking status. Analysis of smokers was performed to evaluate the role of 5-HTTLPR in the age of starting regular smoking, the number of cigarettes smoked daily, pack-years, FTND score, duration of smoking, and the mean length of the longest abstinence on quitting. Genotype was classified according to the presence or absence of the short (S) allele vs. the long (L) allele of 5-HTTLPR (i.e., S/S + S/L vs. L/L). Logistic regression analysis was also used to evaluate correlation between ever smoking and several selected variables.ResultsWe found no significant differences in the rates of S allele carriers in ever smokers and never smokers, and no relationship was observed between any quantitative measures of smoking and the polymorphism. Multivariate analysis demonstrated significant association between the older age (OR = 4.03; 95% CI: 2.33–6.99) and alcohol dependence (OR = 10.23; 95% CI: 2.09–50.18) and smoking.Conclusion5-HTTLPR seems to be not a major factor determining cigarette smoking in Poles. Probably, the risk of smoking results from a large number of genes, each contributing a small part of the overall risk, while numerous non-genetic factors might strongly influence these genetic undergrounds of susceptibility to smoking.\n\nBODY:\nBackgroundTobacco smoking is the commonest addiction to psychoactive substances and, being an extremely harmful behavior, grossly compounds to general morbidity and mortality. Growing evidence from classic twin studies supports the significant influence of genetic factors in smoking initiation and persistence, as well as the ability to quit smoking [1,2]. Currently, studies focus on investigating candidate genes to gain a better understanding of a molecular basis for smoking behavior [3]. However, the precise contribution of individual genes in such a complex behavior remains uncertain. It is most likely that many genes influence it, including those that are involved in neurotransmitter pathways and nicotine metabolism [4].Studies indicate that nicotine increases serotonin release in the brain, while nicotine withdrawal has an opposite effect [5,6]. It was hypothesized that smoking habits may be associated with diminished serotonin (5-HT) neurotransmission determined by genetic polymorphism [7]. The human serotonin transporter gene (5-HTT), being involved in serotonin reuptake, has appeared as a plausible candidate gene for susceptibility to smoking, the more so since its link with psychological traits relevant to smoking behavior was demonstrated [8,9], as well as with alcohol dependence, which increases the risk of tobacco smoking [10,11]. A 44-bp insertion/deletion polymorphism within the promoter region (5-HTTLPR) has been identified with two allelic variants, the long (L) and the short (S) form, which affect the transcriptional efficiency of the 5-HTT gene [12]. Lerman et al. [7] first hypothesized that S allele might exert a protective effect against smoking and evaluated the association of smoking practices and smoking cessation with 5-HTTLPR. However, they did not find any significant differences in the distribution of 5-HTT genotypes between smokers and non-smokers in either Caucasian or African Americans. One of the possible explanations for the negative results of this initial case-control study could be the relatively small sample size (268 current smokers vs. 230 never smokers), which could decrease the statistical power to detect small gene effect. However, in contrast to these results, Ishikawa et al. [13] found in a Japanese sample of a similar size that individuals with S/S genotype were less inclined to smoke and/or could more easily stop smoking than others. Results of subsequent studies, including larger sample studies of over one thousand subjects [14], which attempted to replicate these findings [14-17], have not been consistent, which might result from different ethnic backgrounds being associated with different degrees of linkage disequilibrium with other genes loci [18]. Thus, the hypothesized association between the low-activity S allele and smoking remains controversial, although a recent positron emission tomography study provided new evidence of this possible link [19]. Therefore, there is the need for further replication of association studies to define the role of the 5-HTTLPR in vulnerability to smoking.Since there have been no investigations into the association of 5-HTTLPR and smoking carried out so far in a Polish population, we conducted the present study. The purpose was to determine whether 5HTTLPR genotype is associated with smoking behavior in Caucasians from Northern Poland, as well as to investigate other risk factors for tobacco smoking.Materials and methodsThe study sample and measuresThe study sample was completed among patients and staff of the Academic Clinical Center in Gdansk and outpatients of the Department of Family Medicine, the Nicolaus Copernicus University of Torun and Collegium Medicum in Bydgoszcz (NCUT-CMB). They were asked to complete a questionnaire referring to socio-demographic data (age, gender, educational level) and categorical definitions of smoking status. Never smoker was someone, who either had never smoked at all or had never been daily smoker and had smoked less than 100 cigarettes (or the equivalent amount of tobacco) in his lifetime [20]. Ever smokers were defined as individuals who had smoked at least 100 cigarettes in their lifetime [20]. Current smokers were defined as individuals who, at the time of the survey, smoked cigarettes either daily or occasionally [20]. Former smokers were defined as those who had quit smoking at least 1 year before the study. To verify recent non-smoking status, the measurement of carbon oxide in exhaled air was performed in former smokers with the use of Micro CO smokelyser (Bedfont Instruments, Kent, UK). The level of education was recorded as primary, vocational, high and university. Three hundred and ten adult subjects, including 150 ever smokers (cases) and 160 gender matched never smoking controls, were recruited. All of them were Caucasians from the North of Poland.Several quantitative measures of smoking behavior were also completed with the use of the questionnaire. They included: age of starting regular smoking, number of cigarettes smoked, and number of years of smoking. Pack-years were calculated using the average number of cigarettes smoked daily and the number of years smoked. Current daily smokers were also asked to complete the Fagerstrom Test for Nicotine Dependence [21] and give the longest abstinence period on quitting attempt. All reported periods of maximal abstinence were further calculated into days.In addition, information on recent or prior treatment due to any psychiatric disorders (diagnosis and medications) and on alcohol dependence was obtained from respondents by self-report.From all participants of the study, 8-mm of venous blood sample was collected into heparinized tubes. Samples were frozen and stored at -80°C until required for molecular genotypic analyses.The institutional research ethics committees at the Medical University of Gdansk and the Nicolaus Copernicus University of Torun approved all study procedures, and all subjects provided written, informed consent prior to participation in the study.GenotypingGenomic DNA was extracted from lymphocytes by an enzymatic method using a commercial kit Blood DNA Prep Plus (A&A Biotechnology, Gdynia) and used as a template for the PCR. 5-HTTLPR genotyping was performed with the use of oligonucleotide primers flanking the 5-HTTLPR as described by Heils et al. [12], and with a few modifications [22]. The set of primers used was as follows:sense: 5'-GGCGTTGCCGCTCTGAATGC-3';antisense: 5'-GAGGGACTGAGCTGGACAACCAC-3'.Statistical analysesThe chi-squared (χ2) test was used to asses the deviations of genotype distribution from the Hardy-Weinberg equilibrium and for group comparisons of frequencies of allele and genotype. Logistic regression analysis, using STATISTICA 7.1 software (StatSoft Inc., USA), was used to estimate correlations. Variables which appeared to be associated with any increased risk for smoking status in the univariate analysis were analyzed by multivariate analysis. The association between these variables and smoking was expressed as crude and adjusted odds ratios (Ors) with 95% confidence intervals (95% CIs). Student's test t was used to compare means for continuous variables. Because of the non-normal distribution of most parameters, nonparametric Mann-Whitney U test was applied for two-group comparisons. Data have been expressed as means ± standard deviation (SD). A significance level of 0.05 was set for a type 1 error in all analyses.ResultsThree participants did not undergo genotyping successfully; they were, therefore, excluded from further investigation. As a result, the final sample for analysis consisted of 307 subjects, including 149 ever smokers (66 females; mean age 53.0 ± 11.2 years) and 158 never smokers (79 females; mean age 45.0 ± 16.2 years).Distribution of 5-HTTLPR genotypes did not deviate significantly from the Hardy-Weinberg expectation, as determined by the chi-square test (χ2 = 0.80, df = 1; p = 0.37)With carriers defined as subjects who tested positive for the presence of the allelic variants, whether homozygous or heterozygous (i.e., S/S, S/L), we found that 58% of the sample (n = 178) carried S allele (n = 42 homozygous), while 42% (n = 129) did not (i.e., L/L). Frequencies of L and S allele, as well as short allele carriers and non-carriers, did not differ significantly in ever and never smokers (χ2 = 1.71, df = 1; p = 0.19, and χ2 = 1.03; df = 1; p = 0.31, respectively). Similarly, there were no significant difference in the distribution of 5-HTTLPR L and S alleles and genotypes in current and never smokers, as well as in current and former smokers. Allele frequencies and genotypes for the 5-HTT gene by smoking status can be found in Table 1.Table 1Distribution of 5-HTTLPR L and Seles and genotypes according to smoking status in the surveyed population from the North of PolandSmoking statusNo. (%) of alleleNo. (%) of genotypeLSL/LL/SS/SEver smokers199 (66.8)99 (33.2)67 (45)65 (43.6)17 (11.4)Current smokers131 (66.2)67 (33.8)43 (43.4)45 (45.5)11 (11.1)Former smokers68 (68)32 (32)24 (48)20 (40)6 (12)Never smokers195 (61.7)121 (38.3)62 (39.2)71 (44.9)25 (15.8)In the smokers group, no association was observed between any quantitative measures of smoking and the polymorphism (Table 2).Table 2Values for smoking characteristics by 5-HTTLPR genotype in ever smokersCharacteristicMean (SD)P valueS/S+S/LL/LDuration of smoking27.4 ± 11.926.9 ± 12.70.76No. of cigarettes smoked daily19.6 ± 10.518.2 ± 8.90.58No of pack/years27.3 ± 20.022.4 ± 12.80.32Age of proceeding to regular smoking19.9 ± 4.520.5 ± 5.30.76FTND score*5.5 ± 2.55.3 ± 2.00.66Duration of the longest abstinence in quitting attempts*368.6 ± 1102.3211.6 ± 388.20.90*only current daily smokers were analysedOut of the total of 307 participants, 23 subjects (7.5%; 15 females) reported current or prior treatment because of psychiatric disorders, including depression or anxiety-related disorders (20 subjects) and schizophrenia (3 subjects). Cited medications were in agreement with self-reported psychiatric diagnoses and included inhibitors of selective serotonine transporter (15 subjects), tricyclic antidepressants (4 subjects), neuroleptics (3 subjects), and anxiolytics (1 subject). There were 6 (26%) never smokers and 17 (74%) current smokers, while there were no former smokers among them. The rate of ever smokers among subjects reporting mental health problems was significantly higher than in other subjects (74% vs. 46.5%; p = 0.011). Seventeen subjects (5.5%; 3 females) in the study group mentioned alcohol dependence in the self-report. There were 15 ever smokers (88%) and two never smokers among them (12%). The frequency of smoking in alcoholics was significantly higher in comparison to non-alcoholic subjects (88% vs. 46%; p = 0.0018). We found no differences in frequencies of S and L alleles between subjects with psychiatric disorders or alcohol dependence and remaining subjects (p = 0.64 and p = 0.91, respectively).In univariate logistic regression analyses, the following variables appeared to be associated with an increased risk for ever smoking status (i.e., smoking initiation): older age, a history of psychiatric disorders and alcohol dependence. Gender was not considered in these analyses because of the gender-matching of the study sample.Multivariate regression analysis demonstrated a significant association between the older age and alcohol dependence and smoking, while adjusted odds ratio for smoking for subjects with psychiatric disorders nearly reached statistical significance (Table 3).Table 3Multivariate analysis of the association between ever tobacco smoking and selected variablesVariablesCrude ORs (95% CI)Adjusted ORs (95% CI)*S/S + S/L genotype0.79 (0.50–1.25)0.78 (0.46–1.33)Age ≥ 50 years3.13 (1.96–5.00)4.03 (2.33–6.99)Lower education (primary/vocational)1.36 (0.82–2.24)0.98 (0.54–1.79)Psychiatric disorder +3.26 (1.25–8.55)2.90 (0.98–8.55)Alcohol dependence +8.73 (1.95–39.13)10.23 (2.09–50.18)*Adjusted ORs are adjusted for all other items in the multivariate modelThe results of the separate analyses on the association between 5-HTTLPR and smoking, performed in more homogenous groups of ever and never smokers obtained by excluding subjects with psychiatric disorders and/or alcohol dependence, did not differ significantly from results of analyses performed in the whole study sample. Frequencies of L and S allele, as well as short allele carriers and non-carriers did not still differ significantly in ever and never smokers (χ2 = 1.87, df = 1, p = 0.17, and χ2 = 1.43, df = 1, p = 0.23, respectively). Similarly, we consistently found no association between any quantitative measures of smoking and the polymorphism in the smokers group (Table 4). Further, the results of multivariate analysis of the association between ever tobacco smoking and 5-HTTLPR, age, and the level of education (adjusted ORs and 95% CI: 0.76, 0.43–1.34; 4.72, 2.63–8.46; 1.07, 0.57–2.01, respectively) were consistent with those when it was performed in the whole study sample.Table 4Values for smoking characteristics by 5-HTTLPR genotype in ever smokers after excluding subjects with self-reported psychiatric disorders and/or alcohol dependenceCharacteristicMean (SD)P valueS/S+S/LL/LDuration of smoking24.8 ± 11.028.0 ± 13.10.18No. of cigarettes smoked daily18.0 ± 9.217.1 ± 8.50.73No of pack/years22.9 ± 15.016.1 ± 17.40.73Age of proceeding to regular smoking20.6 ± 4.320.5 ± 4.70.76FTND score*5.1 ± 2.45.2 ± 2.00.79Duration of the longest abstinence in quitting attempts*413.9 ± 1482.3265.9 ± 446.40.29*only current daily smokers were analysedDiscussionIn the surveyed population from the North of Poland, we found 36% of carriers of the 5-HTTLPR short variant allele. This rate was approximate to 34% found by another Polish research group [23], somewhat lower than those reported in two studies conducted in European Americans, where S allele was found in 40% and 43% of subjects, respectively, and somewhat higher than the rates found in African American participants of these studies, i.e., 30% and 31%, respectively [7,24]. In turn, the considerably lower rate of S allele was found in two studies conducted in Japanese populations – 16% and 19% [13,24]. These differences between 5-HTTLPR allele frequencies in various populations reflect their racial and ethnic genetic differentiation [24].We found no association between 5-HTTLPR and smoking status, as well as any quantitative measure of smoking, such as the number of cigarettes smoked daily, the number of pack-years, FTND score, or the duration of the longest abstinence period on quitting. To date, only a few studies, in contrast to our findings, supported a link between 5-HTTLPR and smoking, and demonstrated that individuals with the L allele were more inclined to smoke and/or had more difficulties with quitting smoking than others [13,15]. Other studies failed to replicate these positive results, including the more recent report of Trummer et al., who found additionally that neither smoking status nor Fagerstrom Tolerance Questionnaire score, pack-years, number of cigarettes smoked daily or previous attempts to quit smoking were related to 5-HTT genotypes [7,14]. It should be noted, however, that several factors may have a substantial impact on the outcome of association studies and contribute to their inconsistency [3,18]. The most important is population heterogeneity, as regards ethnicity, gender or age, and possible stratification. Further, interacting effects, such as environment and personality, are considered to play an important role [25]. Between-study heterogeneity results also from the various categorical definitions of never smokers, current smokers and former smokers adopted in particular studies, as well as from differences in the mean number of cigarettes smoked per day or mean values of other quantitative measures of smoking behavior in smokers used in studies. If we compare, for instance, our survey to the Israeli study [15] which reported a highly significant association between 5-HTTLPR genotype and smoking behavior, irrespective of dependence level, several essential differences regarding both populations might be found. Apart from population ancestry, the younger age of the participants of the Israeli survey seems to be one of them, with the average age of ever smokers being 29 years. In addition, smokers in this study group were mainly light smokers, not biologically dependent on nicotine (i.e., they had a FTND score lower than 6), while, in our study population, half of current smokers were highly addicted subjects. It is likely that these differences could influence the results of these two studies. Moreover, probably the effect of polymorphism is related to socio-cultural settings and ethnicity and may be less marked in populations of such countries like Poland, where smoking was a \"national habit\" for decades [26]. Ever smokers in our material were recruited mostly from individuals who had started smoking at a particular socio-political period of Polish history, which influenced the style of life of Poles with an extremely high consumption of cigarettes [26]. We found that subjects aged 50 years or older had an over three times higher risk of smoking than younger subjects. It is possible that possession of S allele, hypothesized to increase dopamine availability in midbrain, was not enough to protect from smoking in such a disadvantageous environment. Thus, non-replication of association studies may result from a small effect of a single gene and the relatively greater influence of a number of environmental factors on smoking. The link of the 5-HTTLPR or other candidate gene polymorphism with smoking might emerge in a given population when smoking rates decline due to the predominance of non-genetic factors, including positive changes in the attitudes to smoking of the population as a whole.It appears essential in case-control genetic association studies to precise defining of smoking phenotypes. The method of classification of ever smokers may have an impact on the results of the study [27]. It is suggested, that a significant amount of information on the resistance to regular smoking might be emerged with defining individuals who have smoked 1–99 cigarettes in their lifetime as ever smokers, not never smokers according to WHO [18,27]. It is also suggested, that genuine never smokers, who had never smoked even one cigarette (or even one puff) may be highly genetically informative [3]. However, in our study conducted in the population of extremely high prevalence of smoking by decades, subjects having smoked up to 100 cigarettes in their lifetime were considered never smokers.Because of the divergent results of association studies, evidence for a substantial role of the 5-HTT gene in smoking behavior is not strong to date. Additionally, meta-analysis of studies on association between the 5-HTTLPR and smoking behavior [3] did not suggest an effect of this polymorphism on initiating, adopting and persisting with smoking. However, when only the studies which reported data on the 5-HTTLPR polymorphism that pertained to smoking cessation were analyzed, a significant effect was revealed. It is likely that the presence of a variant allele may be associated with an increased likelihood of successful smoking cessation. In our study, the comparison of the mean length of the self-reported longest abstinence on quitting did not reveal significant differences between S allele carriers and non-carriers, which indirectly indicated that ability to quit might not be related to the 5-HTTLPR genotype. Other genes, non-genetic factors, personality and motivation to quit might contribute a greater effect in smoking cessation.Growing evidence indicates that smoking behavior and ability to quit are influenced by personality traits, including neuroticism, and psychiatric disorders or alcohol dependence [28,29]. On the other hand, several studies demonstrated the association between the 5-HTTLPR and alcohol dependence or neuroticism, as well as depression and anxiety, which correlate well with this personality trait [10,11,30,31]. Data suggested also that smoking behavior is influenced by an interaction between neuroticism and 5-HTTLPR genotype [32]. More recently, however, a number of studies demonstrated that although neuroticism and depression vulnerability were associated with smoking behavior, genotype did not affect this relationship [33,34].Undergoing treatment for alcohol addiction and/or the presence of psychiatric disorders was an exclusion criterion imposed on subjects' eligibility for some of the earlier association studies on 5-HTTLPR to avoid a possible confounding effect on the distribution of genotypes in smokers and non-smokers [7,13]. In the present study, this exclusion criterion was not adopted, although for example the results of other Polish researchers indicated an importance of careful inclusion of probands in studies on association between 5-HTTLPR and personality traits [22,35], which in turn may affect smoking status. The fact that we found no differences in the frequencies of S and L alleles between subjects with psychiatric disorders or alcohol dependence and remaining subjects might suggest the lack of associations between psychiatric disorders and alcohol dependence and 5-HTT genotype. However, the group size of subjects reporting these problems seems too small to generalize the results of this study, especially as regards the link of 5-HTTLPR with separate psychiatric diagnoses. In several studies which focused on the investigation of a possible link of 5-HTTLPR with affective disorders, a positive association was found [30,31], while other studies have not supported this [36-38]. Thus, the relationship between this polymorphism and affective disorders remains uncertain. Our study let us only support earlier observations that individuals with psychiatric disorders and alcohol dependence are more likely to smoke cigarettes [29].Several limitations of the study should be pointed out. Firstly, the study sample was relatively small, while sample size in thousands of subjects might be more sufficient to detect small genetic effects, which are likely for single loci and complex smoking behavior. However, several studies on the link of 5-HTTLPR with smoking have been recently conducted in smaller samples, which included several hundreds of subjects, and positive association was reported [15-17]. This incited us to investigate the potential association between 5-HTTLPR and smoking in the sample of a similar size in Polish population. Secondly, we have not genotyped a SNP within the 5-HTTLPR [39], which could modify the effect of the L allele. Further, the smoking status has been verified with the measurement of exhaled CO concentration only in former smokers (i.e., those smokers who had quit smoking at least 1 year before the study) to avoid the classification error attributed to self-report [40]. In turn, we did not expect this bias toward a socially desirable response in never smokers and current smokers, therefore biochemical verification of smoking status was not performed in these groups. Finally, participants' self-reports of their psychiatric disorders were not confirmed by a formal clinical interview or checking medical documentation, but only confronted with cited pharmacological treatment. However, the main purpose of the present study was to assess the relationship between 5-HTTLPR and smoking, and psychiatric disorders in general, as well as alcohol dependence, served only as covariates in our analyses.ConclusionIn spite of potential limitations, the results of our study allow us to conclude that 5-HTTLPR is not a major factor determining cigarette smoking in Poles. Probably, the risk of smoking results from a large number of genes, each contributing a small part of the overall risk, while numerous non-genetic factors might strongly influence these genetic undergrounds of susceptibility to smoking. A better understanding of the genetic determinants of smoking needs further investigation into the interactions of genes involved in synthesis, release, uptake and receptor function for a variety of neurotransmitters, as well as into establishing the interactions between the 5-HTTLPR polymorphism and psychological traits.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsAS conceived the study, participated in its design and coordination, performed the statistical analysis and drafted the manuscript. KB participated in the data collection phase, helped to interpret findings and contributed to the text. EJ participated in the design, coordination and supervision of the study and helped to draft the manuscript. ET participated in the data collection phase. All authors reviewed drafts of the manuscript and approved the final version before submitting it for publication.Pre-publication historyThe pre-publication history for this paper can be accessed here:\n\nREFERENCES:\nNo References"
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batch_9/PMC2529296.json

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+{
+    "id": "PMC2529296",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529296\nAUTHORS: Jing Shi, Meina Liu, Qiuju Zhang, Mingshan Lu, Hude Quan\n\nABSTRACT:\nBackgroundWith rapid economic growth and globalization, lifestyle in China has been changing dramatically. This study aimed to describe the male and female adult Chinese population health status.MethodsThe Chinese Third National Health Services Survey was conducted in 2003 to collect information about health status and quality of life from randomly selected residents. Of the 193,689 respondents to the survey (response rate 77.8%), 139,831 (69,748 male and 70,083 female) respondents who were 18 years of age or older were analyzed.ResultsAmong the respondents, fewer males than females rated their overall wellbeing as being poor or very poor (4.8% versus 6.2%), reported illness in the last 2 weeks (14.1% versus 17.4%), presence of physician diagnosed chronic disease (15.0% versus 17.7%) and at least one functional problem in seven items of the quality of life (26.9% versus 32.8%). More males than females were currently smoking (52.4% versus 3.4%) and drank alcohol more than three times per week (16.5% versus 1.1%). Physically inactive rate was similar between males and females (85.8% versus 87.0%). Fewer rural respondents reported chronic disease than urban respondents (13.0% versus 19.9% for males and 15.5% versus 22.8% for females). In all seven items of the quality of life measured, rural respondents reported less problems than urban respondents (26.2% versus 28.7% for males and 32.0% versus 34.7% for females).ConclusionMales had better health status than females in terms of self-perceived wellbeing, presence of illness, chronic disease, and quality of life. However, smoking and frequent alcohol drinking was more prevalent among males than that among females. In contrast with the social-economic gradient in health commonly found in the literature, the wealthier urban population in China was not found to be healthier than the rural population in terms of physician diagnosed chronic disease.\n\nBODY:\nBackgroundChina's population of 1.3 billion accounts for 20% of the world population, making it the most populated country in the world. With rapid economic growth and globalization, Chinese people's lifestyle has been changing dramatically towards being more physically inactive, eating more fast food, and being overall more stressed compared their old lifestyle. Because of the rapid change in lifestyle, population health is quickly shifting from a high mortality rate due to infectious and domestic diseases, related to 'poverty' before implementation of 'open door policy' in 1980, to a currently greater life expectancy and higher prevalence of chronic and non-communicable diseases brought on by 'affluence' [1].In the last five decades, the Chinese population has become healthier as measured by decreased mortality rate and increased life expectancy. For example, infant mortality has decreased from 80.8/1000 live births in 1958 to 21.5/1000 live births in 2004 [2], maternal mortality has declined from 88.9/100000 live births in 1990 to 48.3/100000 live births in 2004, and life expectancy has increased from 57.0 years in 1957 to 71.8 years in 2004 [3]. The most recent data showed that heart disease, cancer and stroke are the major causes of death, accounting for 65% of all deaths and infectious disease has become the least important cause of death, only accounting for 3% of all deaths in Chinese population aged 40 or above [4].Life expectancy is an internationally used health measure because many countries, including China, periodically calculated it using vital data. However, such measure mirrors mortality, and particularly infant mortality, but is insensitive to nonfatal and psychosocial conditions that contribute indirectly to death. Hence, it is imperative to assess population health using indicators that reflect contemporary health issues. This study aimed to describe the male and female adult Chinese population health status in multiple dimensions, including well-being, morbidity, quality of life, and health behaviors of smoking, alcohol consumption and physical activity, using data from the most recent National Health Services Survey from the Chinese government. Our descriptive study on Chinese population health status has application for international organizations because China's large population will influence global health status. Our study provided important information for generating research questions for future studies.MethodsStudy PopulationWe derived data from the China Third National Health Services Survey, which collected data through face-to-face interviews from September 18 to October 20, 2003. Of the 193,689 respondents surveyed, we included individuals who were 18 years of age or older and excluded 3,677 respondents with missing values, resulting in a total of 139,831 (69,748 males and 70,083 females) respondents in our analysis.The national survey employed a multiple stage cluster sampling method to select the sample randomly. The mainland of China was clustered according to the government administrative geographic system (i.e., county, town and village in rural areas, and city, community, and neighbourhood in urban areas). Firstly, 95 counties and cities were randomly selected from rural and urban areas. Secondly, 5 towns and 5 communities were randomly selected in each county and city, respectively. Thirdly, 2 villages in each town and 2 neighbourhoods in each community were randomly selected. Fourthly, 60 households were randomly selected in each village and neighbourhood, respectively, resulting in about 57,000 households. All family members aged 15 years or older were invited to participate in the face-to-face interview.Data collectionMedical doctors and nurses conducted the survey. Before the survey, interviewers were trained and practiced interviewing; their understanding and knowledge about the survey method and content were examined through testing. During the survey, interviewers visited each household up to three times on different days and times. Interviewers explained the purposes and confidentiality of the survey, and then invited family members to participate. Respondents could choose not to participate and their participation in the survey was accepted as oral consent. The completeness of questionnaires was checked by a district survey manager at the end of every day. If there was missing information on the survey, individuals would be re-surveyed if possible. After the survey, 5% of households were randomly selected and re-surveyed on 14 questions to examine survey quality; the agreement was 95%. The survey response rate for adults was 77.8% [5].Demographic CharacteristicsDemographic variables included age, sex, marital status, education; rural/urban residence, and geographic region. Educational level was categorized into five categories, illiterate (it was defined as people who could not read newspaper or magazines, or write a short note), elementary school (i.e. those who attended up to 6 years of schooling or were not illiterate for those without schooling), junior high school (i.e. schooling 7 – 9 years), senior high school (i.e. schooling 10–12 years), and college or university or higher (i.e. complete or incomplete of post-secondary school). Residence was divided based on rural and urban area and then economic development. Rural area included towns and villages. Based on economic development, Eastern China, the most developed region, included 11 provinces and metropolitans such as Beijing, Shanghai, and Liaoning. Middle China included 8 provinces, such as provinces of Heilongjiang, Shanxi, and Hunan. Western China, the least developed region, included 12 provinces such as Yunnan, Tibet, In-Mongolia, and Ningxia.Health Status IndicatorsSelf-perceived overall wellbeing was assessed using a five -point Likert-type scale of being excellent, good, fair, poor or very poor. Presence of illness in the last two weeks and physician-diagnosed chronic disease in the last six months was recorded. The two-week illness was surveyed by asking: \"Have you had any physical and mental discomforts during the last two weeks?\" Chronic disease referred to disease diagnosed by medical doctors and occurring in the last 6 months prior to the survey, or chronic disease that was diagnosed more than 6 months prior to the survey but reoccurred within the last 6 months and received treatment. Non-physician diagnosed chronic disease was not included because the validity of self-diagnosed medical conditions depends on the level of the respondent's knowledge and their perceptions on the definition of 'disease' and 'health'. Physician diagnosed chronic disease was further confirmed by asking diagnosis location including community clinics, county hospital, city hospital, provincial hospital, military hospital, and others. Respondent reported up to three specific chronic diseases. The reported diseases were coded and classified using the disease classification scheme designed by China Ministry of Health for the survey.Quality of life was measured using a seven-item instrument. Respondents were asked about presence and level of severity of their dysfunction and disability in the last 30 days in 1) ability about washing or dressing themselves, 2) ability to do job work or housework, 3) feeling of pain or physical discomfort, 4) ability of concentration on work or study and memory, 5) ability of recognizing familiar people from 20 meters away (with glasses for those wearing glasses), 6) emotional discomfort due to restlessness, and 7) anxiety or depression. Under each item, five itemized answers about presence and severity were provided, including: none, mild, moderate, severe, and extremely severe.Health determinantInformation about smoking, alcohol consumption and physical exercise was collected. For smoking, the survey asked: \"Are you currently smoking?\" (with answer: Yes, No) Under the survey question of \"Do you drink alcohol?\", the three answers were provided: \"No or rarely\", \"Sometimes\" (defined drinking < 3 times per week), and \"Frequently\" (defined drinking ≥ 3 times per week). For exercise, a question of \"What is the sport or exercise that you have been regularly doing in the last 6 months?\" was asked with providing a list of recreational physical activities, such as running, Tai Chi, Wushu, dancing, and playing balls. Regularity of exercise was not defined in the survey and determined by respondent's perception.Statistical AnalysisProportion was employed to describe respondents in demographic characteristics, health status and health determinants. Because of the large sample size and multiple categories in some variables, the P-value for sex difference was not reported. Frequencies of variables in the survey were not weighted because sampling weight was not available. The same sampling method had been used in the previous two National Health Services Surveys in China. Analyses of previous surveys suggest that this sampling method is adequate to generate a nationally representative sample [5]. The survey respondent age and sex composition was comparable with the 2000 census. Finally, multiple logistic regressions were used to generate risk adjusted P-value for gender difference in health indicators after adjustment for demographic characteristics and correction of clustering of individuals within family using the repeated measure [6,7].The data were analyzed at the health information centre of the Ministry of Health in Beijing. Confidentiality of the survey was protected through storing the data on password protected computers at the Ministry, and removing personal identifiable information (such as name and address) from the database available for researchers and examining analysis outputs for release of aggregated data by the centre staff.ResultsDemographic CharacteristicsDemographic characteristics for respondents are presented in Table 1. A majority of the respondents were married (80.3%) and resided in rural areas (71.2%). There was a similar proportion of male and female (49.9%. versus 50.1%). More males than females were unmarried (14.8% versus 9.2%) but had higher education (illiterate rate: 12.2% for males and 27.7% for females). The composition by age, rural/urban and region was similar between males and females.Table 1Characteristics of the survey respondents aged 18 years or older in ChinaVariablesTotalN (% of 139831)MaleN (% of 69748)FemaleN (% of 70083)Age 18–3443055 (30.8)21475 (30.8)21580 (30.8) 35–4431770 (22.7)15646 (22.5)16124 (23.0) 45–5430023 (21.5)15088 (21.6)14935 (21.3) 55–6416942 (12.1)8746 (12.5)8196 (11.7) ≥ 6518041 (12.9)8793 (12.6)9248 (13.2)Marital status Married112274 (80.3)55622 (79.8)56652 (80.8) Unmarried16736 (12.0)10324 (14.8)6412 (9.2) Divorce1584 (1.1)938 (1.3)646 (0.9) Widow9237 (6.6)2864 (4.1)6373 (9.1)Education Illiterate27905 (20.0)8494 (12.2)19411 (27.7) Elementary school38332 (27.4)19374 (27.7)18958 (27.1) Junior high school45654 (32.7)25788 (37.0)19866 (28.3) Senior high school15024 (10.7)8787 (12.6)6237 (8.9) College or university12916 (9.2)7305 (10.5)5611 (8.0)Residence area Urban40244 (28.8)19516 (28.0)20728 (29.6) Rural99587 (71.2)50232 (72.0)49355 (70.4)Region of China East of China48554 (34.7)23957 (34.4)24597 (35.1) Middle of China39056 (27.9)19541 (28.0)19515 (27.8) West of China52221 (37.4)26250 (37.6)25971 (37.1)Health Status and DeterminantOf the respondents, 5.5% rated their overall wellbeing as being poor or very poor, 15.8% reported illness in the last 2 weeks and 16.3% reported presence of chronic disease (see Table 2). Compared to males, more females rated their overall wellbeing as being poor or poorer (4.8% versus 6.2%, risk adjusted P < 0.001), and reported presence of illness in the last 2 weeks (14.1% versus 17.4%, risk adjusted P < 0.001) and chronic disease (15.0% versus 17.7%, risk adjusted P < 0.001). Males had lower prevalence of heart disease (1.4% versus 2.4%), hypertension (3.2% versus 4.0%) and rheumatologic arthritis (0.8% versus 1.5%) than females. However, prevalence of chronic pulmonary disease was slightly higher for males than that for females (1.7% versus 1.1%).Table 2Self-perceived overall physical and emotional wellbeing, illness, and morbidity in the respondents aged 18 years or older in ChinaTotalN (% of 139831)MaleN (% of 69748)FemaleN (% of 70083)Physical and emotional wellbeing Excellent49088 (35.1)25816 (37.0)23272 (33.2) Good50996 (36.5)26156 (37.5)24840 (35.5) Fair32055 (22.9)14447 (20.7)17608 (25.1) Poor6724 (4.8)2873 (4.1)3851 (5.5) Very poor968 (0.7)456 (0.7)512 (0.7)Combination of poor and very poor*7692 (5.5)3329 (4.8)4363 (6.2)MorbidityPresence of illness in the last 2 weeks before the survey*22050 (15.8)9865 (14.1)12185 (17.4)Presence of physician diagnosed chronic disease in the last 6 months before the survey*22808 (16.3)10432 (15.0)12376 (17.7) Infectious and parasitic disease485 (0.4)303 (0.4)182 (0.3) Cancer231 (0.2)122 (0.2)109 (0.2) Diabetes1062 (0.8)472 (0.7)590 (0.8) Heart disease*2644 (1.9)997 (1.4)1647 (2.4) Stroke1257 (0.9)693 (1.0)564 (0.8) Chronic pulmonary disease*1911 (1.4)1153 (1.7)758 (1.1) Hypertension*4989 (3.6)2203 (3.2)2786 (4.0) Peptic ulcer707 (0.5)446 (0.6)261 (0.4) Chronic liver disease192 (0.1)123 (0.2)69 (0.1) Chronic renal disease258 (0.2)83 (0.1)175 (0.3) Rheumatologic arthritis*1598 (1.1)534 (0.8)1064 (1.5)* Note: P value < 0.001 for males versus females after adjustment for age, marital status, education, urban/rural residence and geographic region.In all seven items of the quality of life, 29.9% reported at least one problem (see Table 3). The rate was significantly lower for males than that for females (26.9% versus 32.8%, risk adjusted P < 0.001). Males were more likely than females to report no problems on all seven items (such as 86.6% versus 81.8% for pain, 88.1% versus 84.3% for concentration or memory, 90.6% versus 87.5% for vision, 88.2% versus 84.3% for emotional discomfort and 89.3% versus 86.1% for anxiety/depression).Table 3Quality of life in the respondents aged 18 years or older in ChinaQuality of lifeTotalN (% of 139831)MaleN (% of 69748)FemaleN (% of 70083)Problem about washing or dressing yourself in the last 30 days No problem131927 (94.4)66277 (95.0)65650 (93.7) Mild problem5193 (3.6)2216 (3.1)2977 (4.2) Moderate problem1666 (1.2)734 (1.1)932 (1.3) Severe problem777 (0.6)389 (0.6)388 (0.6) Extremely severe problem268 (0.2)132 (0.2)136 (0.2)Problem about usual activities such as work, or housework in the last 30 days No problem126403 (90.4)64061 (91.9)62342 (89.0) Mild problem8876 (6.3)3621 (5.2)5255 (7.5) Moderate problem2723 (2.0)1186 (1.7)1537 (2.2) Severe problem1362 (1.0)644 (0.9)718 (1.0) Extremely severe problem467 (0. 3)236 (0.3)231 (0.3)Level of pain and physical discomfort in the last 30 days No pain or physical discomfort117701 (84.2)60364 (86.6)57337 (81.8) Mild pain or physical discomfort16228 (11.6)6835 (9.7)9393 (13.4) Moderate pain or physical discomfort4373 (3.1)1847 (2.7)2526 (3.6) Severe pain or physical discomfort1274 (0.9)580 (0.8)694 (1.0) Extremely severe pain or physical discomfort255 (0.2)122 (0.2)133 (0.2)Problem about concentration or memory in the last 30 days No problem120534 (86.2)61433 (88.1)59101 (84.3) Mild problem14232 (10.2)6173 (8.8)8059 (11.5) Moderate problem3765 (2.7)1553 (2.2)2212 (3.2) Severe problem1027 (0.7)467 (0.7)560 (0.8) Extremely severe problem273 (0.2)122 (0.2)151 (0.2)Problem of recognizing a familiar person in 20 meter or more away (with glasses for people wearing glasses) No problem124449 (89.0)63157 (90.6)61292 (87.5) Mild problem9895 (7.1)4354 (6.1)5541 (7.9) Moderate problem3772 (2.7)1503 (2.2)2269 (3.2) Severe problem1316 (0.9)558 (0.8)758 (1.1) Extreme problem399 (0.3)176 (0.3)223 (0.3)Problem about emotional discomfort due to restlessness in the last 30 days No problem120632 (86.3)61542 (88.2)59090 (84.3) Mild problem15504 (11.1)6680 (9.6)8824 (12.6) Moderate problem2938 (2.1)1185 (1.7)1753 (2.5) Severe problem586 (0.4)258 (0.4)328 (0.5) Extremely severe problem171 (0.1)83 (0.1)88 (0.1)Anxiety or depression in the last 30 days No anxiety or depression122612 (87.7)62263 (89.3)60349 (86.1) Mild anxiety or depression13230 (9.4)5786 (8.3)7444 (10.6) Moderate anxiety or depression3040 (2.2)1271 (1.8)1769 (2.5) Severe anxiety or depression764 (0.6)349 (0.5)415 (0.6) Extremely severe anxiety or depression185 (0.1)79 (0.1)106 (0.2)Presence of mild, moderate, severe or extremely severe problem on any one of seven items above*41756 (29.9)18753 (26.9)23003 (32.8)* Note: P value < 0.001 for males versus females after adjustment for age, marital status, education, urban/rural residence and geographic region.Respondents aged 65 years or older had much poorer health status than those aged less than 65 years old among males and females (see Table 4). A similar proportion of rural and urban respondents rated their health status as being poor or very poor (4.8% versus 4.7% for males and 6.3% versus 6.1% for females), and reported the presence of illness in the last 2 weeks (14.2% versus 14.0% for males and 17.3% versus 17.6% for females). However, fewer rural respondents reported chronic disease than urban respondents (13.0% versus 19.9% for males and 15.5% versus 22.8% for females). In all seven items of the quality of life measured, rural respondents reported fewer problems than urban respondents (26.2% versus 28.7% for males and 32.0% versus 34.7% for females).Table 4Self-perceived overall physical and emotional wellbeing, illness, morbidity and quality of life by age and region in the male and female respondents aged 18 years or older in ChinaMale AgeFemale AgeMale ResidenceFemale ResidenceQuality of life< 65N (%)≥ 65N (%)< 65N (%)≥ 65N (%)RuralN (%)UrbanN (%)RuralN (%)UrbanN (%)Total N (denominator)60955879360835924850232195164935520728Perceived poor or very poor physical and emotional wellbeing1905 (3.1)1424 (16.2)2589 (4.3)1774 (19.2)2419 (4.8)910 (4.7)3106 (6.3)1257 (6.1)Presence of illness in the last 2 weeks before the survey7198 (11.8)2667 (30.3)9194 (15.1)2991 (32.3)7142 (14.2)2723 (14.0)8533 (17.3)3652 (17.6)Presence of physician diagnosed chronic disease in the last 6 months before the survey6899 (11.3)3533 (40.2)8575 (14.1)3801 (41.1)6541 (13.0)3891 (19.9)7659 (15.5)4717 (22.8)Presence of mild to extremely severe problem in the last 30 days on any one of seven items below13173 (21.6)5580 (63.5)16376 (26.9)6627 (71.7)13144 (26.2)5609 (28.7)15814 (32.0)7189 (34.7) Washing or dressing yourself1815 (3.0)1656 (18.8)2322 (3.8)2111 (22.8)2559 (5.1)912 (4.7)3291 (6.7)1142 (5.5) Usual activities such as work, or housework3113 (5.1)2574 (29.3)4320 (7.1)3421 (37.0)4179 (8.3)1508 (7.7)5584 (11.3)2157 (10.4) Pain and physical discomfort6219 (10.2)3165 (36.0)8688 (14.3)4058 (43.9)6689 (13.3)2695 (13.8)8880 (18.0)3866 (18.7) Concentration or memory4720 (7.7)3595 (40.9)6525 (10.7)4457 (48.2)5723 (11.4)2592 (13.3)7360 (14.9)3622 (17.5) Recognizing a familiar person in 20 meter or more away (with glasses for people wearing glasses)3248 (5.3)3343 (38.0)4352 (7.2)4439 (48.0)4634 (9.2)1957 (10.0)5973 (12.1)2818 (13.6) Emotional discomfort due to restlessness5531 (9.1)2675 (30.4)7525 (12.4)3468 (37.5)5668 (11.3)2538 (13.0)7479 (15.2)3514 (17.0) Anxiety or depression5476 (9.0)2009 (22.9)7171 (11.8)2563 (27.7)5260 (10.5)2225 (11.4)6845 (13.9)2889 (13.9)Of the respondents, 27.9% were smoking, 8.8% drank alcohol frequently and 13.6% exercised regularly (see Table 5). Compared to males, females were significantly less likely to smoke (52.4% versus 3.4%, risk adjusted P < 0.001), drink alcohol (frequent alcohol consumption 16.5% versus 1.1%, risk adjusted P < 0.001) but less likely to do regular exercise (14.2% versus 13.0%, risk adjusted P < 0.001). Smoking and frequent alcohol consumption rate were particularly high among males aged 35 to 64 years and regular exercise rate was especially high among male and female seniors (age ≥ 65 years) and among urban respondents (see Table 6).Table 5Prevalence of smoking, alcohol consumption and physical activity in the respondents aged 18 years or older in ChinaFactorsTotalN (% of 139831)MaleN (% of 69748)FemaleN (% of 70083)Currently smoking*38943 (27.9)36544 (52.4)2399 (3.4)Frequency of alcohol consumption* # No or rarely109283 (78.1)42749 (61.3)66534 (95.0) Sometimes (< 3 times per week)18258 (13.1)15507 (22.2)2751 (3.9) Frequently (≥ 3 times per week)12290 (8.8)11492 (16.5)798 (1.1)Regular exercise in the last 6 months*&19057 (13.6)9932 (14.2)9125 (13.0)* Note: P value < 0.001 for males versus females after adjustment for age, marital status, education, urban/rural residence and geographic region.#P-value < 0.001 for frequent alcohol drinkers versus none, rare or sometimes drinkers.&Regularity of exercise was determined based on respondent's perception.Table 6Prevalence of smoking, frequent alcohol consumption and physical activity by age and gender in the respondents aged 18 years or older in ChinaMaleFemaleSmokingN (%)Frequently drinking alcohol*N (%)Regular exercise#N (%)SmokingN (%)Frequently drinking alcohol*N (%)Regular exercise#N (%)Age 18–348966 (41.8)1656 (7.7)2478 (11.5)218 (1.0)62 (0.3)1961 (9.1) 35–449504 (60.7)2902 (18.6)1525 (9.8)409 (2.5)183 (1.1)1405 (8.7) 45–549425 (62.4)3517 (23.3)1807 (12.0)580 (3.9)230 (1.5)2015 (13.5) 55–644842 (55.4)1914 (21.9)1723 (19.7)464 (5.7)147 (1.8)1763 (21.5) ≥ 653807 (43.3)1503 (17.1)2399 (27.3)728 (7.9)176 (1.9)1981 (21.4)Residence Rural27245 (54.2)8736 (17.4)2789 (5.6)1615 (3.3)572 (1.2)1759 (3.6) Urban9299 (47.7)2756 (14.1)7143 (36.6)784 (3.8)226 (1.1)7366 (35.5)* Note: Frequent drinker was defined as drinking ≥ 3 times per week.# Regularity of exercise was determined based on respondent's perception.DiscussionThis study highlighted the Chinese adult population health status as the following: 1) only a small proportion of Chinese adults perceived their health as being poor; 2) chronic diseases were high, particularly hypertension, heart disease, chronic pulmonary disease and diabetes; 3) one third of Chinese had a functional problem; 4) prevalence of emotional and/or mental health problems surpassed prevalence of physical functional problems; 5) smoking and alcohol abuse was very common in men; 6) most of the Chinese surveyed were physical inactive and 7) male health status was better than female health status.Non-communicable, rather than communicable, diseases are the major burden in China and the burden has dramatically increased in the last decade as that in some developing countries [8-10]. Compared to the self-reported health conditions in 1993 [2], our analysis of 2003 national survey data showed that prevalence of hypertension and stroke had doubled and prevalence of diabetes had tripled while the prevalence of pulmonary disease and infectious disease had declined by half. In reality, the prevalence of chronic diseases should be higher than our reports because of unawareness of their presence. For example, we reported prevalence of 3.6% for hypertension and 0.8% for diabetes. Based on previous report of unawareness rate of 55.3% for hypertension [11] and 66.6% for diabetes in China [12], the prevalence should be 8.1% for hypertension and 2.4% for diabetes.In contrast with the social-economic gradient in health commonly found in the literature [13-16], the wealthier urban population in China is not found to be healthier than the rural population in terms of physician diagnosed chronic disease. Our findings are consistent with a previous report in China [17]. That study measured glucose tolerance among 42,751 residents who were randomly selected from 11 provinces in China, and reported diabetes prevalence rates of 5.8% in municipal areas, 2.9% in high income rural areas and 1.8% in low income rural areas [17].There are several possible explanations for the above findings. First, the rural population had lower incidence of chronic disease compared with the urban population. The urban population, who has benefited most from China's economic development, has experienced a dramatic lifestyle change in the past two decades. Compared with before, they are becoming more physically inactive (commuting by cars rather than bicycles), and eating more fast food and high protein/fat food. Prior to the implementation of the \"open door policy\" in the 1980s, China's population health was characterized with a high prevalence of infectious diseases as a result of poverty. It has now shifted to a high prevalence of chronic and non-communicable diseases, brought on by 'affluence.' Such change is much more dominant in urban than rural populations. Of all daily sources of energy, cereals accounted for 61.4% and meat 10.8% for rural residents, compared with 48.5% and 17.6% respectively for urban residents [18].The second possible explanation is that compared with the urban population, the rural population had a higher mortality rate (6.1/1000 versus 5.6/1000) [19], with a shorter duration from disease occurrence to death, and thus a lower life expectancy (69.5 versus 75.2 years) [20,21]. It was reported that the rural population had higher rates of heart disease and stroke specific mortality than the urban population (330.7 versus 279.5/100,000 person-years for heart disease and 304.1 versus 256.1/100,000 person-years for stroke) [4]. The higher mortality rate is related to lower insurance coverage and lower ability to afford treatment among rural population compared with their counterparts in urban areas [22,23]. China's current healthcare system relies heavily on a non-regulated market to reduce government health expenditure and allows public hospitals to determine the price of services within a certain range [24]. The rural population utilized physicians more than the urban population (52.0% versus 43.0%), but utilized hospitals far less (7.6% versus 11.1%, p < 0.001) when they were ill [25]. More people in rural than in urban areas opted for no treatment when suffering from an illness and were more likely to die earlier.The third possible explanation is that rural populations were more likely than the urban population to be unaware of the presence of chronic disease. China's national physical measurement study [12] reported that the unawareness of diabetes was 71% and 62% for rural and urban populations, respectively.Quality of life has been less studied in China although it is an important parameter of population health status. The reason may be due to unawareness of its importance and unavailability of well-developed and validated instruments in the Chinese language. A few previous reports on quality of life either in English or Chinese [26-28] focused on general populations in small geographic areas or patients receiving certain healthcare services. Our study findings of about 30% population with functional problems could not be compared to previous Chinese studies. Compared to a Canadian report [29], our study indicated that Chinese in mainland China had 5% more physical problems but 5% fewer emotional functional problems than Chinese in Canada. However, we have noticed that differences between these two studies in social demographic characteristics and cultural influence were not adjusted.Health promotion for better nutrition, tobacco and alcohol reduction, increase in exercise, and hypertension control is critical for avoiding population health declining and promote quality of life. Smoking and alcohol abuse was very common in male population, particularly those in the middle age group, but was rare among females. This is related to the Chinese culture, which accepts male smoking and drinking but not female [30]. China's recent health promotion activities have achieved a decline in the male smoking rate from 70% in 1996 to 52% in 2003 [31]. However, many people are still unaware of the dangers in smoking; the proportion who were unaware of smoking's dangers was over 60% in some provinces and higher in rural than urban area [31]. Physical inactivity was common for both men and women. Interestingly, Chinese seniors were more active than the younger populations.Hypertension is an important risk factor for many chronic diseases, particularly for stroke, heart disease and chronic renal disease. He and colleagues [4] found that hypertension contributed 11.7% to total mortality, smoking 7.9% and physical inactivity 6.8%, resulting in a total of 28.4% (when combined) to mortality in the Chinese adult population. However, these factors were very poorly controlled. About 30% of hypertensive patients took antihypertensive medication with 8.1% achieved blood pressure control [11], and 27.2% of diabetics took medication with 9.7% controlled diabetes [12]. The huge gap between presence, awareness, treatment and control of hypertension strongly indicates imperative needs for a national education program that targets the public, clinicians and decision makers to eliminate the gap. Reforming the healthcare system towards the universal insurance coverage is also essential to remove financial barrier to access the system.There are four major limitations in the study. First limitation was that validity of self-report health condition was suboptimal. Our prevalence of chronic disease was likely underestimated as stated above. Second limitation was that we did not conduct risk factorial analysis for health status due to the nature of the cross sectional survey. Third limitation is that we did not assess child health status. The reason for that is the survey did not include children under age 15. Fourth limitation is that we only analyzed three major risk factors but were unable to assess other important risk factors of diet and obesity.ConclusionOur analysis demonstrated that males had better health status than females in terms of presence of self-perceived poor wellbeing, illness, chronic disease, and poor quality of life. However, smoking and frequent drinking of alcohol was more prevalent among males than that among females. Our results also indicated that prevalence of chronic illnesses was higher among the urban residents, as compared with rural residents among males and females. Along with reduction of the risk factors to chronic disease, promotion of emotional and mental health should be considered to increase quality of life. Further research on measuring mental health is imperative. Without intervening preventable risk factors for chronic diseases (i.e. reducing hypertension, smoking, alcohol abuse, and physical inactivity), the Chinese population health status will deteriorate even faster as the population ages rapidly due to one-child per family policy in the last thirty years.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsJS designed the study and drafted the manuscript. ML performed the statistical analysis, interpreted the results and participated in coordination. QZ performed the statistical analysis and interpreted the results. MLu participated in the study design and interpretation of the results. HQ conceived the study, participated in its design and drafted the manuscript. All authors read and approved the final manuscript.Pre-publication historyThe pre-publication history for this paper can be accessed here:\n\nREFERENCES:\nNo References"
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batch_9/PMC2529314.json

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+{
+    "id": "PMC2529314",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529314\nAUTHORS: Shijun Fu, Haiguang Zhao, Jiantao Shi, Arhat Abzhanov, Keith Crawford, Lucila Ohno-Machado, Jianqin Zhou, Yanzhi Du, Winston Patrick Kuo, Ji Zhang, Mier Jiang, Jason Gang Jin\n\nABSTRACT:\nBackgroundPeripheral arterial disease (PAD), a major manifestation of atherosclerosis, is associated with significant cardiovascular morbidity, limb loss and death. However, mechanisms underlying the genesis and progression of the disease are far from clear. Genome-wide gene expression profiling of clinical samples may represent an effective approach to gain relevant information.ResultsAfter histological classification, a total of 30 femoral artery samples, including 11 intermediate lesions, 14 advanced lesions and 5 normal femoral arteries, were profiled using Affymetrix microarray platform. Following real-time RT-PCR validation, different algorithms of gene selection and clustering were applied to identify differentially expressed genes. Under a stringent cutoff, i.e., a false discovery rate (FDR) <0.5%, we found 366 genes were differentially regulated in intermediate lesions and 447 in advanced lesions. Of these, 116 genes were overlapped between intermediate and advanced lesions, including 68 up-regulated genes and 48 down-regulated ones. In these differentially regulated genes, immune/inflammatory genes were significantly up-regulated in different stages of PAD, (85/230 in intermediate lesions, 37/172 in advanced lesions). Through literature mining and pathway analysis using different databases such as Gene Ontology (GO), and the Kyoto Encyclopedia of Gene and Genomics (KEGG), genes involved in immune/inflammatory responses were significantly enriched in up-regulated genes at different stages of PAD(p < 0.05), revealing a significant correlation between immune/inflammatory responses and disease progression. Moreover, immune-related pathways such as Toll-like receptor signaling and natural killer cell mediated cytotoxicity were particularly enriched in intermediate and advanced lesions (P < 0.05), highlighting their pathogenic significance during disease progression.ConclusionLines of evidence revealed in this study not only support previous hypotheses, primarily based on studies of animal models and other types of arterial disease, that inflammatory responses may influence the development of PAD, but also permit the recognition of a wide spectrum of immune/inflammatory genes that can serve as signatures for disease progression in PAD. Further studies of these signature molecules may eventually allow us to develop more sophisticated protocols for pharmaceutical interventions.\n\nBODY:\nBackgroundPeripheral arterial occlusive disease (PAD) is a major manifestation of atherosclerosis and is commonly found in elderly patients. Epidemiological studies have shown that PAD affects 8 to 10 million adults in the United States [1]. Most patients with PAD are asymptomatic. The disease is primarily diagnosed by an ankle brachial index (ABI) < 0.9. The most common symptom of mild-to-moderate PAD is intermittent claudication, which is present in about one third of symptomatic patients [1]. In addition to leg symptoms, patients with PAD are at an increased risk for developing new coronary events and eventually death from cardiovascular disease. Although conventional procedures such as stents, arterectomies, angioplasty, and bypass surgery have been successful in improving clinical symptoms of PAD to a large extent [2], ultimately elimination of the disease may require sophisticated protocols of pharmaceutical interventions, which may depend on better understanding of molecular mechanisms involved in the disease.Previous studies have implicated the involvement of the immune system in atherosclerosis formation and progression. Animal models have been used to test the contributions of components of the immune system [3,4]. Cellular involvement of macrophages was found to be important in the formation and progression of atherosclerosis in animal models [4]. In addition, various immune-related genes have been examined in an atherosclerosis animal model, and genes such as CXCR6, CXCL10, CXCR3 and CXCL16/scavenger receptor have been shown to be involved in the progression of atherosclerosis in animal models [5-8]. In humans, many immune cells such as macrophages, lymphocytes, mast cells, and T cells are found in atherosclerosis [9]. These findings suggest that the immune system plays important roles in atherogenesis. However, data available to date are primarily derived from studies of atherosclerosis in the coronary or/and the carotid arteries, whereas data derived from clinical samples of PAD appear to be particularly limited.In the past decade, microarray analysis using high-throughput screening technology has emerged as an important tool to study gene expression patterns and to study molecular events in complex diseases [10-12]. In this study, Affymetrix GeneChips were used to perform gene expression profiling of femoral atherosclerotic lesions to fully characterize the peripheral arterial wall gene expression patterns associated with atherosclerosis. By statistical analysis, hundreds of known and novel genes were identified that differentially express in PAD. Genes involved in immune/inflammatory responses appeared to be significantly enriched in the set of genes up-regulated in different stages of PAD. To further examine the expression patterns of individual genes in the context of particular biological or molecular pathways, gene functional enrichment was performed using Gene Ontology and KEGG database. The results revealed that immune system-related categories and pathways were significantly overrepresented in the progression of the disease, suggesting that up-regulation of immune/inflammatory genes may be critical components of the disease progression expression signature associated with atherosclerosis. These findings may provide new insights and foster a better understanding of the mechanism of PAD.ResultsPatient classification and outcomeHistological characterization of 30 collected peripheral artery samples was conducted based on the criteria of the American Heart Association. Of these samples, 15 were classified as grade III (intermediate lesions), one as grade IV and fourteen as grade V (advanced lesions). Among them, 11 intermediate lesions samples (grade III) and 14 advanced lesions samples (grade V) had RNA of sufficient quality and quantity for hybridization. Representative images of the different stages are shown in Figure 1. Further details of these 25 samples are listed in Table 1. As shown, there was no significant difference between the intermediate lesions and the advanced lesions group except for indications of hypertension. In the intermediate lesions group, 4 patients (36.4%) presented with hypertension, while 9 hypertensive patients (64.3%) were found in the advanced lesions group.Table 1Patient characteristics for the 25 samples in the microarray analysisIntermediate lesions group (n = 11)Advanced lesions group (n = 14)Age*76.1 ± 4.6876.6 ± 3.79Male, n (%)8(72.7)12(85.7)Hypertension†, n (%)Yes4(36.4)9(64.3)no7(63.6)5(35.7)Hypercholesterolemia, n (%)Yes9(81.8)11(78.6)no2(18.2)3(21.4)Diabetes, n (%)Yes7(63.6)10(71.4)no4(36.4)4(28.6)*as mean ± SD.† P < 0.05 (Student t test)Figure 1Histological characteristics of various severities of femoral atherosclerotic lesions in PAD patients. HE stain analysis of histological characteristics of collected femoral non-atherosclerotic arteries and atherosclerotic arteries. 8 um cryostat sections were stained with hematoxylin and eosin, dehydrated in graded alcohol, and cover-slipped with permanent mounting solution after xylene clearing. Three representative samples are listed: normal artery (A, B, C), intermediate lesions (D, E, F), and advanced lesions (G, H, I).Differentially regulated genes in intermediate lesionsIdentifying differential expression genes was achieved for different stages by using Significance Analysis of Microarrays (SAM) with a false discovery rate (FDR) of 0.5%. Comparative analysis revealed that 366 genes were differentially expressed in intermediate lesions when compared to normal femoral arteries, of which 230 genes were up-regulated and 136 were down-regulated [see Additional file 1]. The 100 most differentially expressed genes between intermediate lesions and normal femoral arteries are shown in Figure 2A. Notably, in the up-regulated genes, up to 85 genes have been reported to be involved in immune response, such as HLA-DQB1, HLA-DRB1, CCR1, CXCR4, C1QB and TLR7 [see Additional file 2]. In addition, a large number of genes known to encode proteins crucial for proteolysis (CTSB, CTSC, CTSD and CTSS) and cell proliferation (BTG1, BTG2, CDKN1A, and MCM5) appeared to be significantly changed. Since BTG1 and BTG2 are known to be involved in anti-proliferation activities, it can be of interest to further investigate their potential roles in PAD in detail. MCM5 is heavily involved in chromosomal stability. Among the down-regulated genes, those involved in calcium signaling (CAMK2G), transport (SLC22A3, CYP1A1 and ATP5H), metabolism (GCSH and PLA2G4A), and protein amino acid dephosphorylation (PTPN20) were found to be significantly down-regulated. The Gene Ontology functional categories in which intermediate lesions are overrepresented are illustrated in Table 2 and additional data [see Additional file 3]. As shown in the table, the most significant biological process categories in the up-regulated genes are immune response, humoral immune response, inflammatory response, and T cell proliferation (Z-score>5). For down-regulated genes, the significant ones mainly represent metabolism and catabolism-related categories.Table 2Biological process categories overrepresented in intermediate lesionsGO NameZ-scoreP-valueFor up-regulated genes immune response14.4910.000antigen processing, endogenous antigen via MHC class I3.8210.014antigen processing, exogenous antigen via MHC class II5.9680.001cellular defense response5.6690.000cell-mediated immune response3.0930.038T-helper 1 type immune response3.2140.035humoral immune response9.4510.000complement activation3.6080.016inflammatory response8.5150.000neutrophil chemotaxis6.6290.004leukocyte adhesion4.7670.013immune cell activation3.2680.011immune cell migration6.250.000cell surface receptor linked signal transduction2.0820.042integrin-mediated signaling pathway2.6330.035intracellular signaling cascade4.3670.000MAPKKK cascade2.4130.037myeloid cell differentiation2.8760.042lipid transport3.2550.011Phagocytosis4.7570.005Apoptosis2.0910.049cell proliferation4.2570.000T cell proliferation5.4890.003oxygen transport3.2550.016icosanoid metabolism3.5240.010lipoprotein metabolism3.2910.017For down-regulated genescellular catabolism2.7080.014amine catabolism3.5990.011glycine catabolism8.4570.003glycine metabolism5.8170.008ubiquitin cycle2.0920.041protein folding3.0880.008Endocytosis2.5210.032GO analysis was applied to differentially expressed genes in intermediate lesions compared with normal controls. Similar significant categories were not included to reduce redundancy. The first half of the table indicates categories highly significant for up-regulated genes; the second half of the table shows categories highly significant for down-regulated genes. Calculated p-values and Z-scores for each category are shown. The corresponding cell component categories and molecular function categories are available in additional data [see Additional file 3]Figure 2Heatmap of the 100 most differentially expressed genes in intermediate lesions and advanced lesions, respectively. SAM analysis reveals genes with differential expression in PAD. This analysis compared plaques from within arteries of either intermediate (n = 11) or advanced lesions (n = 14) to normal control group, respectively. Heatmap representation of the 100 most differentially expressed in intermediate lesions (A) and advanced lesions (B). Samples are displayed in columns and genes in rows. Gene expression is represented as a color, normalized across each row, with brighter red for higher values and brighter green for lower values. Gene symbols are listed to the right. N (Normal control group), Int (intermediate lesions group), Ad(advanced lesions group). The list of differentially expressed genes in intermediate lesions and advanced lesions is provided [see Additional file 1 and Additional file 4].Differentially regulated genes in advanced lesionsWhen advanced lesions were compared to normal femoral arteries, 447 genes were identified, of which 172 genes were up-regulated and 275 were down-regulated [see Additional file 4]. The list of the 100 most differentially expressed genes is shown in Figure 2B. Interestingly, up to 37 genes involved in the immune system response, such as CCR1, CX3CR1, TLR1 and TLR7, were found to be up-regulated in advanced lesion [see Additional file 5], which might suggest that these immune/inflammatory related genes could serve as expression signatures characterizing different stages of PAD. In addition, genes constituting a major portion of the vascular extracellular matrix were significantly up-regulated in advanced lesions, including COL1A1, COL3A1, COL1A2, COL5A1, COL6A1, COL6A3 and LAMB1, suggesting that these genes could be involved in the femoral artery occlusion in PAD. GO analysis further confirmed the above findings, by highlighting categories of immune response, humoral immune response, inflammatory response and I-kappaB kinase/NF-kappaB cascades (Z-score>5) (Table 3 and Additional file 6). For down-regulated genes, those involved in ion transport (GRIA2 and SLC22A3) and protein folding (DNAJB5) appeared to be the most significantly down-regulated in advanced lesions. GO analysis showed that the most significant categories for down-regulated genes were response to protein stimulus, RNA metabolism, and protein folding (Table 3 and Additional file 6).Table 3Biological process categories overrepresented in advanced lesionsGO NameZ-scoreP-valueFor up-regulated genes immune response6.3050.000cellular defense response2.940.026humoral immune response5.3920.000inflammatory response6.1790.000immune cell migration4.7780.014intracellular signaling cascade4.2970.001small GTPase mediated signal transduction2.3720.024I-kappaB kinase/NF-kappaB cascade6.4080.000T cell proliferation4.1860.016induction of apoptosis3.7090.007oxygen transport4.7780.003amine transport3.0510.024anion transport3.3730.006phosphate transport6.1390.000organic acid transport2.6340.039nucleoside monophosphate metabolism5.8530.001cell adhesion3.9580.000For down-regulated genesresponse to protein stimulus6.790.000response to unfolded protein6.790.000integrin-mediated signaling pathway4.360.003Apoptosis2.8380.003muscle cell differentiation3.7710.032vasculature development3.7950.005neuron differentiation3.9930.003vitamin transport3.7710.023RNA metabolism3.4760.000RNA splicing3.4970.005glutamine metabolism4.9010.004macromolecule metabolism2.2140.023protein folding5.6430.000cell-matrix adhesion2.6050.036peptide hormone secretion5.4010.004GO analysis was applied to differentially expressed genes in advanced lesions compared with normal controls. Some other selected categories are shown. The first half of the table indicates categories highly significant for up-regulated genes; the second half of the table shows categories highly significant for down-regulated genes. Calculated p-values and Z-scores for each category are shown. The corresponding cell component categories and molecular function categories are available in additional data [see Additional file 6]In parallel, further data analysis revealed that many genes were over-represented in both intermediate and advanced lesions vs. normal controls. Of these genes, 68 were found to be commonly up-regulated and 48 were found to be commonly down-regulated (Figure 3). The list of commonly up-regulated genes is available [see Additional file 7]. Some of these overlapping genes, such as CTSB, CCR1, ALOX5, and SPP1, have been previously reported to play important roles in atherogenesis [13-16]. Accordingly, these commonly regulated genes can therefore be important for the progression of PAD. In contrast, a much larger number of genes appear to be characteristically expressed in either intermediate lesions or advanced lesions, which may therefore serve as stage-specific signatures of PAD.Figure 3Over-represented genes in both intermediate lesions and advanced lesions. A. The genes whose expressions were significantly changed in intermediate lesions and advanced lesions, respectively, are shown in a Venn diagram. B, C. Heatmap representation of commonly up-regulated genes (B) and commonly down-regulated genes (C) in overlapping genes, respectively. Samples are displayed in columns and genes in rows. Gene expression is represented as a color, normalized across each row, with brighter red for higher values and brighter green for lower values. Gene symbols are listed to the right. N (Normal control group), Int (intermediate lesions group), Ad (advanced lesions group).Differential gene expression in disease progressionIntermediate lesions and advanced lesions represent different stages in disease progression of PAD. Identification of genes that exhibit characteristic expression patterns in different stages may provide information relevant to the progression of PAD. For this reason, expression profiles of normal arteries, intermediate lesions and advanced lesions were analyzed by the SAM multiclass method. Out of this analysis, 614 genes appeared to be differentially expressed in the progression of PAD with a FDR<0.5% [see Additional file 8]. Hierarchical clustering analysis suggested that the expression patterns of the genes could be assigned to three major groups (Figure 4). The first group represents those genes commonly expressed in both intermediate and advanced lesions (Cluster II). GO terms indicate that these genes are mainly involved in the immune response, inflammatory response, cellular defense and various signaling pathways (Table 4 and Additional file 9). These results further support the notion that the immune system may play an important role in the progression of PAD. The second group represents specifically down-regulated genes in advanced lesions (Cluster II). GO terms indicate that these genes are primarily involved in cell cycle, apoptosis, multicellular organism development and protein folding (Table 4 and Additional file 9). Genes in the third group are represented by those down-regulated in both intermediate lesions and advanced lesions (Cluster III). GO terms indicate that these genes are mainly involved in neurogenesis, protein modification, RNA splicing, and blood pressure regulation (Table 4 and see Additional file 9). In addition, we have performed data analysis restricted to male subjects. Up to 85% genes identified in male subjects are the same as those identified in the total samples (data not shown), which suggests that the potential gender-biases is minimal. Taken together, genes commonly up-regulated in intermediate and advanced stages are typically represented by those involved in immune and inflammatory responses, implicating enhanced immune response activities during the progression of the disease, whereas down-regulated genes in the both disease stages are primarily represented by those involved in various aspects of cell proliferation and differentiation.Table 4GO discovered categories for disease progression manner analysisGO NameZ-ScoreP-valueFor Cluster Iimmune response8.9080.000inflammatory response7.6430.000cellular defense response5.9380.001chemotaxis7.6110.000cell surface receptor linked signal transduction2.9370.009cytokine and chemokine mediated signaling pathway4.3780.004JAK-STAT cascade3.2540.020MAPKKK cascade3.0620.006endocytosis1.9560.049angiogenesis4.4520.000cell adhesion4.5800.000ion homeostasis3.0900.010For Cluster IIapoptosis3.8710.002cell cycle4.1080.000cell growth2.2850.037epidermal cell differentiation3.0750.044protein folding3.6770.001macromolecule metabolic process3.0220.005RNA metabolic process5.3780.000transcription4.5930.000peripheral nervous system development2.9500.046multicellular organismal development3.9270.000vitamin transport4.4260.008response to stimulus4.5790.000circadian rhythm5.0900.001For Cluster IIIneurogenesis2.6120.014neuron recognition3.3150.046cell motility2.0930.042glycogen metabolic process2.9990.025protein modification process3.1660.001RNA processing2.1830.042RNA splicing2.5910.021phosphate metabolic process2.3460.024blood pressure regulation4.0280.008heart contraction2.9010.022GO analysis was applied to differentially expressed genes in each cluster group. Some selected categories are shown. The Calculated p-values and Z-scores for each category are shown. The detailed GO results are provided in the additional data [see Additional file 9].Figure 4Hierarchical clustering analysis of the differentially expressed genes in disease progression. The differentially expressed genes analyzed by hierarchical clustering method in disease progression. The genes were classified into three major clusters by visual inspection. Clustering method: Average linking; Similarity measure: Euclidean distance. Samples are displayed in columns and genes in rows. Gene expression is represented as a color, normalized across each row, with brighter red for higher values and brighter green for lower values. N (Normal control group), Int (intermediate lesions group), Ad (advanced lesions group). The list of differentially expressed genes in disease progression is provided [see Additional file 8].Validation of gene transcription by real-time PCRReal-time PCR is still the gold standard for quantitative analysis of mRNA. In order to validate the microarray results, RT- PCR was carried out on the same set of samples that were analyzed by the microarray approach. The results were highly correlated with those from the array data. (The correlation coefficient for microarray and RT-PCR was 0.835 ± 0.076). Representative RT-PCR results of 6 genes are shown in Figure 5.Figure 5Real-time PCR and the relative expression level of six genes. Six genes mRNA in normal femoral artery (N, black round), intermediate lesions (Int, ascending triangle) and advanced lesions (Ad, descending triangle) were determined by real-time PCR and presented as a ratio to GAPDH mRNA. mRNA abundance in intermediate lesions or advanced lesions was differentially expressed (*P < 0.05, and **P < 0.01, respectively) when normal samples were used for comparison.Transcription factors enrichment analysisTranscription factors appear to play important roles in the development or progression of atherosclerosis [17,18]. To address whether specific transcription factors are involved in the regulation of genes associated with the progression of PAD, we conducted a transcription factor binding site enrichment study by analyzing cross-species conserved binding sites in promoter regions of genes differentially regulated during progression of PAD. Through the Fisher Exact test, binding sites of transcription factor AP-1 and CREB appeared to be significantly enriched (q-value < 0.05). AP-1 is a transcription factor known to be involved in various cellular processes. In atherosclerosis, it has been reported in gene regulation of microphages, vascular smooth muscle cells and epithelial cells [19,20]. In disease progression, AP-1 was enriched to regulate expression of 72 genes (Figure 6A and Additional file 10). The enrichment of AP1 binding sites in regulated genes associated with PAD progression may therefore suggest an important role played by this transcription factor in the development of PAD. Through literature mining, indeed, some of the potential targets of AP1 appear to be previously reported as target genes of AP-1 [21-24]. CREB is a member of the leucine zipper family of DNA binding proteins. This transcription factor binds as a homodimer to the cAMP-responsive element and induces transcription of genes in response to hormonal stimulation [25]. A total of 55 genes were recognized as potential targets of CREB (Figure 6B and Additional file 11). Although further studies are need to elucidate detailed roles played by AP1 and CREB in PAD progression, significantly enriched binding sites and highly correlated with signature genes of PAD progression suggest that these two transcription factors may play critical roles in the development of PAD.Figure 6Enriched transcription factors and their putative target genes in disease progression. Putative targets of transcription factors were curated based on results by Xiaohui Xie. Fisher Exact test showed that two transcription factors (AP-1 and CREB) were significantly enriched in disease progression (q-value <0.05). AP-1 and CREB were enriched to regulate 72 and 55 genes expression, respectively. A, B. The top 20 putative target genes of AP-1 and CREB were listed, respectively. A list of the enriched transcription factor and their putative targets is provided [see Additional file 10 and Additional file 11].Pathways identification by overabundant genesA pathway analysis database, KEGG, was then applied to genes differentially regulated in intermediate and advanced lesions. Several overrepresented pathways were identified, and the enriched pathways appeared not to be independent of one another, many genes involved in one pathway could be also involved in another pathway. This interaction is illustrated in Figure 7, and pathway abbreviations can be found in Table 5. As demonstrated, many immune-related pathways were significantly over-represented in intermediate and/or advanced lesions including TLR, NK, BCR, FER, APP, CCC and LTEM pathways. These findings, on the one hand, provide evidence supporting previous hypotheses that immune/inflammatory responses play important roles in the development of PAD, and on the other hand, demonstrate that particular components of immune/inflammatory systems can be crucial for the genesis and progression of PAD. For instance, TLR and NK pathways are shown to be particularly overrepresented in both intermediate lesions and advanced lesions, highlighting their functional importance in the disease. The TLR pathway is shown in Figure 8 with the differentially regulated genes indicated.Table 5KEGG biological pathways for differentially expressed genes in different stagesKEGG Pathway NamePathway IDGenes involved P-ValueFor intermediate lesionsType I Diabetes Mellitus(TIDM)Hsa0494090.002Antigen Processing and Presentation(APP)Hsa04612130.003Complement and Coagulation Cascades(CCC)Hsa04610120.003Cell Adhesion Molecules(CAM)Hsa04514170.005Toll-Like Receptor Signaling Pathway(TLR)Hsa04620130.013Natural Killer Cell Mediated Cytotoxicity(NK)Hsa04650150.029For advanced lesionsFocal Adhesion(FA)Hsa04510250.000ECM-Receptor Interaction(ECM)Hsa04512150.000Toll-Like Receptor Signaling Pathway(TLR)Hsa04620130.002Regulation of Actin Cytoskeleton(RAC)Hsa04810200.005Fc Epsilon RI Signaling Pathway(FER)Hsa04664100.012MAPK Signaling Pathway(MAPK)Hsa04010220.019Natural Killer Cell Mediated Cytotoxicity(NK)Hsa04650130.021Long-Term Potentiation(LTP)Hsa0472080.030Cell Communication(CC)Hsa01430120.032B Cell Receptor Signaling Pathway(BCR)Hsa0466280.042Genes identified from SAM under FDR<1% were tested for overrepresentation within the pathways for the KEGG under the modified Fisher Exact test assumption; p-Value and genes involved in the pathway are provided for each pathwayFigure 7Interactions of KEGG pathways for differentially expressed genes in different stages of PAD. Pathways are enriched in intermediate lesions (A) and advanced lesions (B), respectively. Many genes involved in one pathway could also be involved in another pathway. A, B. Networking displayed the interaction of pathways in intermediate lesions and advanced lesions, respectively.Figure 8Over-representation of Toll-like receptor signaling pathway genes. Analysis of over-representation of differentially expressed genes in pathway from KEGG. The Toll-like receptor signaling pathway is illustrated with significantly regulated genes highlighted.Protein validation of TLR7 expressionMembers of the Toll receptor family are key mediators of innate immunity. They respond to various pathogen-associated stimuli and transduce complex signaling responses that are required for inflammation and for the subsequent development of adaptive immunity [26]. In atherosclerosis, TLR-mediated signaling cascades are observed in macrophages, mast cells and endothelial cells [27,28]. Data shown in this setting demonstrate that genes involved in TLR-mediated pathway are significantly up-regulated in intermediate or advanced lesions, including TLR1, TLR2, TLR7, and MyD88. TLR1 and TLR2 have been previously reported to be significantly regulated in atherosclerosis and their functional roles have been widely investigated in atherosclerosis [29,30]. However, the expression of TLR7 in atherosclerosis has not been reported before. TLR7 mediates innate responses by recognizing oligonucleotide based (RNA-) molecular patterns in endocytic compartments. Our data show that it is significantly up-regulated in both intermediate and advanced lesions. Western-blot analysis was performed to further validate its expression on the protein level (Figure 9). The function of TLR7 in atherogenesis is currently under further investigation.Figure 9Significant expression of TLR7 in femoral atherosclerotic lesions. Western blot analysis of TLR7 in atherosclerotic femoral arteries (As, n = 3) and normal femoral arteries (N, n = 3). The protein level of Cathepsin S, whose expression was previously validated in atherosclerosis, was also examined in femoral atherosclerotic lesions. Beta – actin served as a loading control.DiscussionIn the present study, we first examined the gene expression profiles of PAD. Data analysis identified a number of genes that might be significantly correlated with different levels of PAD severity. The list of differentially expressed genes in intermediate and advanced lesions contains many genes which can be important for atherosclerosis. Most of these genes have not been reported to be related to atherosclerosis before. For example, MAP4K4 is a member of the serine/threonine protein kinase family. It has been shown to specifically activate MAPK8/JNK and mediate the TNF-alpha signaling pathway [31,32]. In this study, it was significantly and consistently up-regulated in both intermediate and advanced lesions.A large multidisciplinary study is currently underway to comprehensively assess PAD at multiple levels [33], The goal of that study is to investigate 300 symptomatic patients with PAD undergoing medical management with or without vascular intervention by lower extremity angioplasty/stenting or vein graft bypass, and to test the hypothesis that the systemic inflammatory response after vascular intervention influences the local milieu responsible for vascular repair and adaptation [33]. Identification of genes through the work may be significant in the selection of candidate genes that can be investigated through these cases-control genetic epidemiology studies. Our research supports the idea that immune responses play a key role in the development of PAD.In this report, immune related genes were shown to be significantly expressed during the development of PAD. Gene functional analysis further revealed that immune related categories and pathways were significant enriched in the different stages of PAD. In these immune related genes, several genes have been shown to modulate the development of atherosclerosis in mice models. For example, IgG Fc receptors (FcgammaRs) play a role in activating the immune system and in maintaining peripheral tolerance. Previous research suggested that Fcγ receptor deficiency protects against atherosclerosis in Apolipoprotein-E knockout mice [34]. The results suggest that broad-range inhibitors of immune and inflammatory responses can be considered as potential targets for the treatment of PAD. However, gene expression patterns of immune related genes can be different in different stages of PAD. For example, in intermediate lesions, MHC class II molecules were significantly up-regulated including HLA-DMA, HLA-DMB, HLA-DPB1, HLA-DQB1, HLA-DRA, HLA-DRB1 and HLA-DRB5. MHC class II molecules are normally restricted to a subset of antigen presenting dendritic cells, B cells, macrophages, and thymic epithelium cells [35]. These cells can be detected close to CD4+ T cells and present peptides to the T cells. The results suggest that there can be an ongoing immune activation in the intermediate lesions. However, MHC class II molecules were not differentially expressed in advanced lesions, even with a higher false discovery rate, which may suggest that the HLA-mediated immune activation may occur mainly in the progression stages of PAD. In addition, complement molecules were also significantly up-regulated in intermediate lesions, not in advanced lesions. Previous studies have implicated that activation of the complement system is probably associated with the initiation and progression of atherosclerosis [36,37]. Our data thus provide direct evidence from clinical samples demonstrating that complement system mainly play a role in the development stages of PAD. It is therefore conceivable that different and complex immune/inflammatory responses may take place at different stages of PAD.Atherosclerosis is a systemic, multifocal disease leading to various symptoms and clinical events including cardiovascular disease, cerebrovascular disease, and peripheral arterial disease. Our results reveal that many genes identified in the report are also expressed in coronary or carotid atherosclerotic lesions. For example, C3AR1 and C5R1 are receptors of C3 (C3a) and C5a respectively. A recent study shows that C3AR1 and C5R1 are expressed in human atherosclerotic coronary plaques [38]. Double immunofluorescence staining has shown that the plaque of cells that express both C3aR and C5aR are macrophages, T cells, endothelial cells, and sub-endothelial smooth muscle cells. In addition, gene expression changes between atherosclerosis from coronary and carotid artery samples have been measured by microarray technology in recent years. One study using microarray found that 82 genes were differentially expressed in both animal model and human coronary artery atherosclerosis disease [39]. Our data confirmed 29 genes and 18 genes had significantly different expression in intermediate lesions and advanced lesions, respectively. Moreover, these genes had expression trends similar to the ones found in our data, but our data showed higher fold-changes. In these overlapping genes, 14 were reported to be involved in immune response. Another microarray study found that 206 genes were differentially expressed in aortic atherosclerosis samples [40]. Our data confirms 43 genes and 32 genes had significantly different expression in intermediate and advanced lesions (FDR<1%), respectively. Importantly, in these overlapping genes, 15 were reported to be involved in immune response. Taken together, the results suggested that immune response is a common feature in atherosclerosis-related diseases. Our microarray study differs from prior microarray studies in the array type, sample type, sample classification, and analytical techniques. Nevertheless, the high level of overlapping genes suggests that there are similar molecular mechanisms in the development of peripheral arterial disease and other atherosclerosis-related diseases.Several limitations of our approach should be noted. First, hybridization-based microarrays, despite their immense potential, have inherent shortcomings related to deficient standardization of methods employed in normalization, statistical analysis, and so on [41,42]. In this study, we have attempted to limit these shortcomings by selecting subjects who were phenotypically similar to each other except for hypertension. In addition, the initial phases of data analysis, we used different normalization and statistical methods to identify differentially expressed genes. After choosing SAM, we used a rigorous false discovery rate to minimize false positive results. Expression patterns were validated by confirming mRNA expression patterns with conventional molecular techniques. We attempted, based on current literature, to suggest a potential functional role for genes whose expression was markedly altered. Second, atherosclerosis is a slow, progressive disease that may start in childhood; entirely normal arteries can only be obtained from young donors, a factor that can affect gene expression measurements. Although previous research and our data analysis suggest that age had very little effect on genes, further work is needed to identify age-related genes. Third, the relatively small number of patients did not allow us to assess serial changes in the disease development in more detail as would have been possible in animal models [36]. Furthermore, we do not know to what extent the observed changes in gene expression translate into protein synthesis and function, and which genes cause atherosclerosis. Future studies are needed to address these issues.ConclusionWe first examined the gene expression profiles of PAD; the results from this analysis provide an initial step towards a better understanding of molecular mechanisms underlying PAD development. Differences in immune-related responses were observable at the gene expression level. These findings may be significant for understanding the molecular basis of PAD and investigating pharmacological approaches for the prevention and amelioration of atherosclerosis in PAD.MethodsTissue HarvestAfter obtaining informed consent, primary femoral artery specimens containing atherosclerotic lesions were taken from 30 patients undergoing surgical bypass or limb amputation at Shanghai Ninth People's Hospital. The specimens were immediately rinsed once with PBS and cut longitudinally by the surgeon. Three quarters of the samples were stored at once in – 80°C for subsequent total RNA extraction. The remaining samples were embedded in OCT medium and snap frozen for further morphological analysis. Clinical patient parameters were also registered. For controls, five normal femoral arteries were obtained from healthy donors during organ transplantation (male, mean 31.6 years; range 22–45 years). These five samples were without clinical or gross macroscopic signs of atherosclerotic disease. The Local Ethical Committee approved all procedures in this investigation, and proper protocol was followed throughout the entire course of the experiment.HistologyFor each sample, cryostat sections of 8 um were stained with hematoxylin for 10 min and eosin for 2 min, dehydrated in graded alcohol, and cover-slipped with permanent mounting solution after xylene clearing.RNA Isolation and QuantificationTotal RNA was isolated from the samples using a Trizol reagent (Invitrogen, Carlsbad, CA) and cleaned up using RNeasy Micro Kit (Qiagen, Valencia, CA) techniques. In brief, for each tissue, at least 100 mg sample was pulverized under liquid nitrogen. After complete disruption of the tissues, the Trizol reagent was added in the amount of 1 ml/100 mg. Total RNA was extracted using the protocol supplied with the Trizol reagent. After isolation, the RNA was cleaned up using the RNeasy Micro Kit. To remove any contaminating genomic DNA, a DNase step was included, following the manufacturer's protocol. The RNA quantity and quality were determined by an Agilent Bioanalyzer 2100 and an Eppendorf Biophotometer. Any RNA samples that showed degradation was excluded from the study.Microarray ExperimentOne microgram of total RNA was used for generating biotin labeled cRNA. The labeling reaction was performed according to the standard Affymetrix® protocol to generate a biotin-labeled cRNA probe. The samples were hybridized to the Affymetrix® Human Genome -U133A Genechip, stained, washed and scanned according to the standard Affymetrix® protocol. The computer data files to be used in data analysis (*.dat, *.cel, *.chp) were generated with the Affymetrix GeneChip Operating Software (GCOS) Version 1.4 (Affymetrix®), using the statistical algorithm provided. All chip samples were scanned using the same instrument and followed the same protocol. Data quality assessment was then performed following the guidance in Affymetrix data analysis fundamentals manual. All quality control results met Affymetrix recommended criteria.Data process and analysisThe probe level intensity data were transferred to ArrayAssist® Software (StrataGene; La Jolla, CA) for further analysis. For comparison of differential gene expression between different stage groups, the background was removed and data were normalized in accordance to the GC-RMA method [43]. GC-RMA takes into account the GC content of the probe sequences when comparing the expression intensities of the different probesets. Then, the processed gene expression data were transformed into log base 2 and filtered to delete the genes whose detection calls were \"absent\" in all samples.Microarray data analysis was carried out to identify individual genes that were significantly expressed between classes by the software package SAM (please see Availability & requirements for more information), using Δ = 0.5. Results from the difference analysis were clustered and displayed using the Cluster3.0 and Treeview1.1.0 software (please see Availability & requirements for more information). Each list of differentially expressed genes was analyzed in the context of Gene Ontology (GO) in order to identify groups of genes with similar functions, or processed using MAPPFinder (Gene MicroArray Pathway Profiler; please see Availability & requirements for more information). For each gene ontology term, the probability values were computed based on a hypergeometric distribution test by comparing (a) the number of genes annotated by the gene ontology term in a given list of differentially expressed genes with (b) the expected number of such genes. Z-score>0 and p-values < 0.05 were considered significant categories.Similar methods were used to identify curated pathways that were significantly over-represented in the data using KEGG database by using DAVID (please see Availability & requirements for more information). For each pathway, the probability values were computed based on a modified Fisher exact test. EASE p-values < 0.05 were considered significant categories. The enriched pathways are not entirely separate from one another. For example, many genes involved in MAPK signaling pathway can also be involved in other pathways, such as NK pathway. The interconnectedness information was manually extracted from the pathway. Because the nature and complexity of these interactions varied from pathway to pathway, a simple line connecting two pathways was used to represent their interaction. The interaction map was generated for the interaction of enriched pathways using CytoScape software.Transcription factor enrichment analysis was also performed. The putative targets of transcription factors from TRANSFAC (v7.4) were discovered by Xie et al [44] and downloaded from the supplementary web site (please see Availability & requirements for more information). All the RefSeq IDs were converted to Entrez Gene ID according to the mapping table downloaded from NCBI web site (please see Availability & requirements for more information). Enrichment of transcription factor targets was performed as described previously [45]. The interaction map was generated for the interaction of enriched transcription factors and their putative target genes using CytoScape softwareReal-time QPCR AnalysisOne microgram of total RNA was reverse transcripted using random hexamers and superscript -II reverse transcriptase (Invitrogen, Carlsbad, CA). QPCR was performed by using ABI prism 7900 (ABI, Foster City, CA) and SYBR Green Detection (Toyobo, Japan). Primers were designed by using the Primer Express 2.0 software and verified by using a BLAST search. Sequences of the primers are listed [see Additional file 12]. The experimental conditions followed the manufacturer's protocol and the data were analyzed with sequence Detection Software 2.0 (ABI, Foster City, CA). Relative expression of mRNA was calculated with the comparative CT method. To standardize the amount of input RNA, the GAPDH gene was included. For each sample, the experiment was performed in triplicate.Western BlottingProteins were extracted after RNA isolation according to the Introvigen protocol (Invitrogen, Carlsbad, CA) and measured using a Bio-Rad DC protein assay (Bio-Rad, Richmond, CA, USA). Aliquots of protein (100 μg of protein each) were resolved on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (Millipore, Medford, MA, USA). The membrane was incubated with a primary antibody overnight at 4°C and then with a secondary antibody conjugated with alkaline phosphatase (1 h at room temperature), which was detected by a chemiluminescence method. The following polyclonal primary antibodies were used: anti-human TLR7 (1:300, IMGENEX, San Diego, CA), anti-human CTSS (1:400, Abcam Inc), anti-human beta-action (1:10000, Abcam Inc).StatisticsThe statistical significance of real-time results was examined with the nonparametric Mann-Whitney test, using GraphPad Prism 4. In the experiment, p values < 0.05 were considered significantly different between the lesions group and the normal artery group.Availability & requirementsSAM software package: Cluster3.0 and Treeview1.1.0 software: MAPPFinder (Gene MicroArray Pathway Profiler): DAVID: Xie et al Supplementary Information: NCBI web site: Authors' contributionsSJF, experiment design and conduction, and manuscript drafting; HGZ, clinical sample processing; JTS, transcription factor enrichment analysis; AA, WPK, KC and LO–M, data analysis and manuscript revising; JQZ, data analysis; YZD, data analysis and literature support; JZ and MEJ, experimental design and manuscript revising; JGJ, experiment design, data analysis and manuscript revising. All the authors read and approved this version of the manuscript.Supplementary MaterialAdditional File 1Table 1. 366 differentially expressed genes in intermediate lesions relative to normal femoral arteries. Affymetrix Probe Set ID, Gene Title, Gene Symbol, GO Biological Process, GO Molecular Function, GO Cellular Component, Unigene, Entrez Gene, Ensembl, Chromosome Number, Socre(d), Fold Change and q-value(%) which is the lowest FDR are listed in the table.Click here for fileAdditional File 2Table 2. Immune-related genes in intermediate lesions relative to normal femoral arteries, Affymetrix Probe Set ID, Gene Title, Gene Symbol, GO Biological Process, GO Molecular Function, GO Cellular Component, Unigene, Entrez Gene, Ensembl, Chromosome Number, Socre(d), Fold Change and q-value(%) which is the lowest FDR are listed in the table.Click here for fileAdditional File 3Table 3. Cell component and molecular function categories overrepresented in intermediate lesions, the first half of the table indicates categories highly significant for up-regulated genes; the second half of the table shows categories highly significant for down-regulated genes. The calculated p-values and Z-scores for each category are shown.Click here for fileAdditional File 4Table 4. 447 differentially expressed genes in advanced lesions relative to normal femoral arteries. Affymetrix Probe Set ID, Gene Title, Gene Symbol, GO Biological Process, GO Molecular Function, GO Cellular Component, Unigene, Entrez Gene, Ensembl, Chromosome Number, Socre(d), Fold Change and q-value(%) which is the lowest FDR are listed in the table.Click here for fileAdditional File 5Table 5. Immune-related genes in advanced lesions relative to normal femoral arteries, Affymetrix Probe Set ID, Gene Title, Gene Symbol, GO Biological Process, GO Molecular Function, GO Cellular Component, Unigene, Entrez Gene, Ensembl, Chromosome Number, Socre(d), Fold Change and q-value(%) which is the lowest FDR are listed in the table.Click here for fileAdditional File 6Table 6. Cell component and molecular function categories overrepresented in advanced lesions, the first half of the table indicates categories highly significant for up-regulated genes; the second half of the table shows categories highly significant for down-regulated genes. The calculated p-values and Z-scores for each category are shown.Click here for fileAdditional File 7Table 7. 68 commonly up-regulated genes in intermediate lesions and advanced lesions, Affymetrix Probe Set ID, Gene Title, Gene Symbol, GO Biological Process, GO Molecular Function, GO Cellular Component, Unigene, Entrez Gene, Ensembl, Chromosome Number, Socre(d), Fold Change and q-value(%) which is the lowest FDR are listed in the table.Click here for fileAdditional File 8Table 8. 614 differentially expressed genes in disease progression. Affymetrix Probe Set ID, Cluster ID, Gene Title, Gene Symbol, GO Biological Process, GO Molecular Function, GO Cellular Component, Unigene, Entrez Gene, Ensembl, Chromosome Number, Socre(d), Contrast which is the standardized mean difference between the gene's expression in that class versus its overall mean expression, and q-value(%)which is the lowest FDR are listed. In the table, contrast1, 2 and 3 represent the standardized mean difference in normal femoral arteries, intermediate lesions, and advanced lesions, respectively.Click here for fileAdditional File 9Table 9. The detail GO overrepresented categories for disease progression in each cluster, the calculated p-values and Z-scores for each category are shown in the table.Click here for fileAdditional File 10Table 10. 72 putative AP-1 target genes in disease progression. Affymetrix Probe Set ID, Gene Title, Gene Symbol, GO Biological Process, GO Molecular Function, GO Cellular Component, Unigene, Entrez Gene, Ensembl, Chromosome Number, Socre(d), Contrast, and q-value(%) are listed.Click here for fileAdditional File 11Table 11. 55 putative CREB target genes in disease progression. Affymetrix Probe Set ID, Gene Title, Gene Symbol, GO Biological Process, GO Molecular Function, GO Cellular Component, Unigene, Entrez Gene, Ensembl, Chromosome Number, Socre(d), Contrast, and q-value(%) were listed.Click here for fileAdditional File 12Table 12. The primer sequences of selected genes for real-time PCR.Click here for file\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2529325",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529325\nAUTHORS: Zhike Zi, Yanan Zheng, Ann E Rundell, Edda Klipp\n\nABSTRACT:\nBackgroundIt has long been recognized that sensitivity analysis plays a key role in modeling and analyzing cellular and biochemical processes. Systems biology markup language (SBML) has become a well-known platform for coding and sharing mathematical models of such processes. However, current SBML compatible software tools are limited in their ability to perform global sensitivity analyses of these models.ResultsThis work introduces a freely downloadable, software package, SBML-SAT, which implements algorithms for simulation, steady state analysis, robustness analysis and local and global sensitivity analysis for SBML models. This software tool extends current capabilities through its execution of global sensitivity analyses using multi-parametric sensitivity analysis, partial rank correlation coefficient, SOBOL's method, and weighted average of local sensitivity analyses in addition to its ability to handle systems with discontinuous events and intuitive graphical user interface.ConclusionSBML-SAT provides the community of systems biologists a new tool for the analysis of their SBML models of biochemical and cellular processes.\n\nBODY:\nBackgroundWith growing interest in systems biology, mathematical models have been widely used to study metabolic networks, gene regulatory networks and cell signaling pathways [1-6]. These mathematical models are used to reproduce experimental data and predict unobserved behaviors of the system. However, many sources of uncertainty including errors, inconsistency and noise of experimental data, absence of parameter information, incomplete representation of underlying process details, and poor understanding of the biological mechanisms impose a limit on model confidence. Furthermore, intrinsic variability or noise of the system such as the occurrence of stochastic events also affects the output of the model. Therefore, it is important not only to understand the dynamical properties of the model with particular parameter values, but also to further investigate the effect of their perturbations on the system [7]. Sensitivity analysis is a powerful approach for investigating which parameters in a model have the strongest effect on overall behavior. In addition to identifying key parameters in a model, sensitivity analysis is valuable in pinpointing parameters, which should be in the focus of experimental perturbation [8].Sensitivity analysis has been widely utilized for the systems biology research [2,7,9-16]. However, it is time consuming for researchers to apply different algorithms to their specific models. In order to automate sensitivity analysis for different types of systems biology models, we developed a free software tool named SBML-SAT: a systems biology markup language (SBML) based sensitivity analysis tool. SBML is a language developed by the systems biology community to represent and exchange models of biochemical reaction networks [17]. SBML is being used by a large group of software developers and researchers. More than 120 software systems have so far been developed for supporting SBML . Although a few existing software systems such as COPASI [18] and SBToolbox [19] incorporate local sensitivity analysis, a powerful, flexible and broadly applicable sensitivity analysis platform is still lacking. In particular, some important features missing from the existing software systems are described below.Firstly, some mathematical models of biological system include discontinuous events, such as the division of cells, removal of biological signal at a specific time or blocking protein synthesis during an experiment. Most existing SBML supported software systems (except for SBML-PET [20], MathSBML [21], SBTOOLBOX2 [19], etc.) do not support models involving such discontinuous events. The broad applicability of these software systems is thus limited.Secondly, none of the existing SBML software packages allows for global sensitivity analysis. A few of the existing software systems can run local sensitivity analysis which introduces a small perturbation of one parameter for each simulation. Therefore, local sensitivity analysis investigates sensitivity of the model outputs with respect to a particular point in the parameter space. However, a single \"true\" point of parameter set may not occur in nature. It is likely that biological parameters such as rate constants and initial concentrations are variable in a large range depending on the specific cell types and cellular environments. For this reason, a global sensitivity analysis is valuable to explore sensitivities of model outputs to simultaneous variations of all the parameters over a large range and examine possible non-linear effects of the parameters as well as their interactions [7].Thirdly, the results of sensitivity analysis correspond to specific model outputs. The specific model outputs of interest usually vary from case to case. In some cases, users may want to study the integrated or maximum response of certain species, while in other cases interest may be placed on particular time dependent or steady state responses of the system. Thus, a good sensitivity analysis software platform should provide various options for specifying model outputs.Here, we present the software system SBML-SAT that encompasses all of the above capabilities. It is worth pointing out that the purpose of this paper is not to explain the technical details of the software (described in the manual file) or the published algorithms, nor to present any particular biological findings. Rather, we provide an overview of the software, its validation with a variety of mathematical models for biological systems and demonstrate its broad applicability.ImplementationOverview of the software systemSBML-SAT is designed to run simulation, steady state analysis, robustness analysis, as well as local and global sensitivity analysis for ordinary differential equations (ODE) based biological models. SBML-SAT meets the needs mentioned in the rational section with the following features:Inspired by our previous work in SBML-PET project [20], we enabled SBML-SAT to support a variety of models including assignment rules and events, even for complicated event scenarios such as bisecting mass in case of cell division. Therefore, SBML-SAT will have a wide applicability for different types of models.In addition to the implementation of traditional local sensitivity analysis, SBML-SAT provides four different global sensitivity analysis methods, including multi-parametric sensitivity analysis [7,12], partial rank correlation coefficient analysis [9,22], SOBOL's method [15,23] and weighted average of local sensitivities [2]. Furthermore, steady state analysis and robustness analysis are also available in this tool. The algorithms for these different types of analyses are briefly described in the following section.The sensitivity analysis can be performed with respect to any ODE model variable (species amount or concentration) and reaction rate; these quantities are referred to as the object of the sensitivity analysis in SBML-SAT. The model output, which the sensitivity analysis is performed on, is defined through a functional operation on the object. The predefined model outputs in SBML-SAT are: steady state response, maximum response, integrated response, and time dependent response. The steady state response is only applicable for model objects that are ODE variables as the sensitivity analysis is computed with respect to the equilibrium solution of the system (when all derivatives of the ODE variables are algebraically set to zero). The maximum response is the maximum value of the object Xi (state variable or reaction rate) over the time course simulated:(1)maxt∈{t0, t1, ..., tend}[Xi](t)The integrated response corresponds to the area under the curve when plotting Xi versus time. SBML-SAT approximates the integrated response for object Xi by the discrete summation [24]:(2)Ii=∫t0tend[Xi]dt≌∆t2([Xi](t0)+2[Xi](t1)+....+2[Xi](tn−1)+[Xi](tn))The time dependent response performs multiple sensitivity analyses based on the values of the object, Xi, at selected time points during the simulated time course.SBML-SAT for Windows, Mac, and Linux can be freely downloaded from its website . The manual documentation file including a detailed tutorial for the usage of SBML-SAT is also available in the website. The future updates of SBML-SAT will be released on the website as well. Like most other SBML supported software systems, SBML-SAT requires a link to libSBML and utilizes SBMLToolbox [25], which allows us to import SBML into MATLAB [26]. Once the SBML model is imported into SBML-SAT, a MATLAB file will be automatically generated, which includes the ODEs of the model. This is very helpful for the user, who wants to code in MATLAB for other purposes. To speed up the process of solving the ODEs, we employed the CVODE module of SUNDIALS (Suite of Nonlinear and Differential/Algebraic Equation Solvers) as the ODE Solver [27]. An interface to setting the options of CVODE solver is also available in SBML-SAT. Both SBMLToolbox and SUNDIALS [28] can be freely downloaded.In order to run the analysis in SBML-SAT, the users need to represent their models in SBML format which can be easily done using the existing software systems such as CellDesigner [29], COPASI [18] and SBMLeditor [30]. Then, the users can load the SBML models to SBML-SAT and perform a variety of analyses.Simulation, robustness analysis and sensitivity analysis can be easily implemented using SBML-SAT's graphical user interface (Figure 1). SBML-SAT allows the user to browse the model information, to save the model as well as to simulate and analyze the model. Simulation and sensitivity analysis results can be exported as text files, making it convenient for post-processing. In addition to the export function, SBML-SAT provides automatic visualization of the results. Furthermore, SBML-SAT is smart in remembering the user's settings for the corresponding tasks. The user can save his/her project settings as a project file and load it again to SBML-SAT for further analysis later.Figure 1GUI of SBML-SAT. The graphic user interface (GUI) of SBML-SAT provides an easy way for the user to run the simulation, steady state analysis, sensitivity analysis and robustness analysis for SBML models.Local sensitivity analysisLocal sensitivity analysis is a study of the changes in the model outputs with respect to parameter (factor) variations around a local point in the parameter space, which are quantified by the sensitivity coefficients. Mathematically, the sensitivity coefficients are the first order derivatives of model outputs with respect to the model parameters:(3)Sij=∂Oi∂pjwhere Oi is the i-th model output and pj is the j-th parameter. This is called \"Unnormalized Sensitivity\" in SBML-SAT. SBML-SAT employed centered difference approximation to compute the sensitivity coefficients in the following way [31]:(4)Sij=∂Oi∂pj≈Oi(pj+∆pj)−Oi(pj−∆pj)2∆pj.When the model output and parameters are non-zero, the normalized sensitivity coefficients are defined as:(5)Sijnormalized=     ∂OiOi    ∂pjpj≈    Oi(pj+∆pj)−Oi(pj−∆pj)Oi    2∆pjpj.The new model outputs are calculated by a small perturbation (Δpj) of parameter pj while keeping all the other parameter values the same: SBML-SAT computes one-at-a-time (OAT) local sensitivity coefficients.The proper choice of perturbation size is a delicate issue as it depends on the nature of the model and the numerical solution method. The perturbation should be small enough to achieve a negligible error in the centered difference approximation, and large enough to be unaffected by the numerical inaccuracies of the ODE solver. Too large parameter perturbation violates the implied linearity of the approximations in (4) and (5) and will provide inaccurate results. The user can modify the perturbation coefficient in the \"Sensitivity Analysis\" panel of SBML-SAT. The default perturbation is 0.1% of the corresponding parameter value, ie. Δpj = 0.001 × pj.Global sensitivity analysisAs mentioned in the rationale section, there are many sources of uncertainty in the model parameter values. Global sensitivity analysis is a useful way to investigate the global effects of parameters on the model output by simultaneously perturbing all the parameters within a parameter space. In the SBML-SAT tool, four different global sensitivity analysis methods are available. Each method has a distinct mathematical rationale and can be used for different purposes.(1) Multi-Parametric Sensitivity Analysis (MPSA): This method was first proposed by Hornberger et al [32] in the field of hydrology and further applied to modeling of biological systems by Cho et al. [12] and Zi et al. [7]. MPSA can be used to study the relative importance of the parameters with respect to the model output. The basic idea of MPSA is to map the uncertainty of the parameters into the model output by randomly generating parameter values from predefined distributions (without prior knowledge, uniform distributions are assumed). SBML-SAT uses Latin Hypercube Sampling (LHS) method to sample the parameter values under the given ranges of the parameters [7]. The LHS method is an efficient method to sample random parameter vectors while guaranteeing that individual parameter ranges are evenly covered [33]. The ranges of the parameter distributions are usually determined from the available literature or guided by experience of the researchers.For each randomly generated parameter set, the objective function is computed by the sum of square errors between the model outputs from the random parameter set and the reference parameter set. The next step is to classify each parameter set as acceptable or unacceptable by comparing its objective function value to the average of all the objective function values. If the objective function value is smaller than the average, the parameter set is classified as \"acceptable\"; otherwise it is \"unacceptable\". Then, the cumulative frequency is calculated for both acceptable and unacceptable cases for each selected parameter with increasing parameter values. Finally, the sensitivity of the parameter is measured by the maximum vertical distance of the two cumulative frequency curves according to the Kolmogorov-Smirnov statistics [7]. The calculated MPSA sensitivities are between 0 and 1, where a value closer to 1 indicates a relatively higher importance of the parameter variation to the overall corresponding model output.(2) Partial Rank Correlation Coefficient Analysis (PRCC): The PRCC method is a rank transformed linear regression analysis that is routinely used for analysis of systems with a nonlinear and monotonic relationship between the system inputs and outputs [22]. Linear regression analysis methods best fit a straight line to input and output values. When nonlinear, monotonic relationships exist between system input and outputs, poor linear regression fits can be alleviated by performing the linear regression analysis on a rank ordered list of the model output and input values. PRCC calculates the sensitivity indices from the Pearson correlation coefficients between the model output and input parameters as well as each pair of parameters after rank transformation [9]. Interactions among different parameters are eliminated by evaluating multiple regression models on a subset of parameters that excludes a single parameter. The calculated PRCC sensitivity indices are a standardized sensitivity measurement between -1 and 1 with 0 indicating an input to which the model output is completely insensitive. SBML-SAT computes PRCC as implemented in [15] with LHS sampling of the parameter space.(3) SOBOL's Method: SOBOL's method is a variance based method that makes no assumptions on the relationship between the system inputs and outputs. It is computationally expensive since it utilizes a large number of model simulations with parameter values sampled from the parameter space by the winding stair algorithm. The variance of the numerous model outputs is estimated by Monte Carlo integrations. The model output variance is apportioned into summands of partial variances from combinations of input parameters with increasing dimensionality [23]. The total effects sensitivity indices quantify all of the effects that a parameter, in combination with any other parameter(s), has on the model output. They are defined as the ratio of the sum of the related partial variances to the overall variance of the model output. The larger the fraction, the higher is the corresponding sensitivity. SBML-SAT calculates the total effect sensitivity indices.(4) Weighted Average of Local Sensitivities: In this approach, local sensitivity indices are calculated at multiple random points within the parameter space; a weighted average of the local sensitivity indices serves to provide some approximation of the global parameter sensitivities. Bentele et al. [2] proposed a Boltzmann-Distribution weighting function, exp(-E/kbT), where E is the error between the model simulation and experimental data and kbT is a customizable scaling factor. Herein we define E as the least squares error (LSE) between the perturbed model simulation and reference model simulation and kbT as the minimum LSE. Based on this weighting function, the random points in the parameter space with low LSE contribute the most to the calculated global sensitivity indices.Steady state analysisSBML-SAT uses two different methods to check the existence of a steady state for the SBML model. The first strategy is to set the ordinary differential equations to zero and solve the algebraic system by KINSOL, which is part of the software family called SUNDIALS and is an algebraic system solver based on Newton-Krylov method [27]. Another method is called quasi steady state method, which runs the simulation for a very long time and check the rate of change of the ODE variables (such as species and other state variables) at different time points. When the rates of change for all the variables are smaller than a certain threshold (1 × 10-10), a quasi steady state is reached. The latter method is useful for steady state analysis of models that include events and implicit mass conservation rules. These two methods will only find a single steady state to which the initial condition converges. Other existing steady states as well as the steady state of oscillatory and unstable system will not be detected. SBML-SAT automatically selects the method for steady state analysis. If the model doesn't have events, SBML-SAT will use the algebraic method to detect the steady state of the model. Otherwise the second quasi steady state method will be used.Robustness analysisRobustness is one of the fundamental properties of biological systems, which allows the system to maintain its behavior against random perturbations [34,35]. SBML-SAT employs a method proposed in previous studies to investigate the robustness of model output against the total parameter variation, TPV, which is defined as [36-38]:(6)TPV=∑n=1L|log10(knkn0)|where kn are the perturbed model parameters randomly generated by the LHS method; kn0 are the corresponding reference parameter values in the model; L is the total number of parameters that are randomly varied.To measure robustness, we use the robustness metric Routput,TPVM, which quantifies the change in a function of the system (model output) induced by TPV:(7)Routput,TPVM=−∑p=1N|log10(fpf0)|N.where f0 and fp are the model output which describes the biological function under non-perturbed condition (reference model) and perturbed condition (parameters varied model), respectively. N is the total number of perturbations or model simulations. M denotes the model for the corresponding system. When the reference model output is zero, the following alternative definition is used:(8)Routput,TPVM=−∑p=1N(fp−f0)2N.According to the definition of (7) and (8), the robustness score of a biological system (model) usually assumes a negative value. The closer it is to zero, the more robust the system (model) is against the perturbations (parameter variations). When the robustness score of a system is zero, it means this system is absolutely robust against the imposed perturbations.The difference between the robustness scores of two systems (models) with respect to a certain model output against the perturbations can be evaluated as:(9)∆Routput,TPVM1,M2=Routput,TPVM1−Routput,TPVM2.The comparison of the robustness scores of two systems/models is meaningful only when the evaluated model output of the two systems/models are the same and perturbations are operated in the same way.ResultsIn this section, we will demonstrate the functions and broad applicability of SBML-SAT using a variety of mathematical models for the biological systems. All of the models presented here are pre-encoded in SBML format and most of them are taken from the BioModels Database [39]. At the start of each subsection, a brief description of the instructions to operate SBML-SAT for each function are provided to enable the reader to further envision the interaction with the software tool and facilitate its use.Simulation of SBML modelsTo simulate a pre-constructed SBML model, the user loads the SBML model, sets the time course for simulation, and selects \"Run Simulation\".SBML-SAT provides an easy way to run a simulation and visualize the simulation results for SBML models. The output screen for SBML-SAT model simulation is shown in Figure 2. In order to test the wide applicability of SBML-SAT, we ran simulations for a variety of models from the BioModels Database, which include biophysical models, signaling pathways, gene expression and metabolic networks. The results shown in Figure 3 demonstrates that SBML-SAT appropriately simulates both continuous SBML models (signaling pathway, gene expression and metabolic models), as well as those with discontinuous events (cell cycle model) with different degrees of complexity and nonlinearity.Figure 2Plot of simulation result in SBML-SAT. The plot function enables the user to visualize the time course profiles of species and reaction rates. This graph shows the simulation result of the MAPK cascade model [43] (BioModels ID: BIOMD0000000010).Figure 3Simulation of different types of models in SBML-SAT. (A) Simulation result of the fission yeast cell cycle model (events included, BioModels ID: BIOMD0000000111), identical to Fig. 4 of [44]. (B) Simulation result of a NF-κB signalling pathway model (BioModels ID: BIOMD0000000140), identifical to Fig. 2F of [1]. (C) Simulation result of a T cell gene expression model (BioModels ID: BIOMD0000000122), identical to Fig. 4a of [45]. (D) Simulation result of a metabolic model (BioModels ID: BIOMD0000000106), identical to Fig. 2A of [46].Sample local sensitivity analysisTo conduct the local sensitivity analysis, the user• loads the SBML model,• sets the time course,• chooses the parameter(s),• defines the perturbation coefficient, and• selects the objects (ODE variables or reaction rates) and the model output operation for the analysis,• select the appropriate analysis approach to run.The result of a SBML-SAT normalized local sensitivity analysis on the MAPK cascade model (BioModels ID: BIOMD0000000010) is shown in Figure 4. For this analysis, the objects of the sensitivity analysis were the state variables associated with the various phosphorylated forms of the MAPK cascade elements and the model output analyzed were the integrated responses. The parameters perturbed were the initial concentrations of each form with the default perturbation coefficient. These results indicate that the integrated response of the MAPK concentration was the most sensitive to the initial concentration of MAPK.Figure 4Local sensitivity analysis in SBML-SAT. Local sensitivity analysis of the integrated response of MAPK cascade model [43] (BioModels ID: BIOMD0000000010) with respect to variation of initial conditions.Sample global sensitivity analysisThe user interface and operation for performing global sensitivity analyses is similar to that for the local sensitivity analysis: the user specifies the time course, object(s) and parameter(s) as well as the model output(s) for global sensitivity analysis. In addition, the user chooses the global sensitivity analysis method, and sets the variation range of the parameter values. The user must also define the number of Monte-Carlo simulations to be performed to base the analysis upon: this is highly dependent upon the nature of the model, the number of parameters (factors) to be analyzed, and the size of the parameter space (factor levels). The user needs to try different \"Number of Simulations\". If the analysis results are not significantly changed by the increasing of \"Number of Simulations\", then the results are assumed to be reliable and accurate enough. Once all these settings are done, SBML-SAT is ready to perform the specified global sensitivity analysis. The time required to complete the analysis varies from several minutes to several hours. It depends on the complexity of the model and the number of Monte-Carlo simulations.We use a model of the receptor trafficking network to demonstrate how to implement global sensitivity, steady state and robustness analyses in SBML-SAT. The general model of receptor trafficking networks is composed of the de novo production of surface receptor, ligand-receptor interaction, internalization, recycling and degradation of both empty and occupied receptors (Figure 5). The symbols of the parameters in the model and their corresponding biological processes are listed in Table 1. Detailed information about this model is available in our previous work [24,40].Figure 5Scheme of receptor trafficking network model. Schematic description of the receptor trafficking network. The symbols L, Rs, LRs, Ri, LRi represent the ligand, unbound cell surface receptor, cell surface ligand-receptor complex, internalized unbound receptor and internalized ligand-receptor complex, respectively. The parameter information is listed in Table 1.Table 1Parameters for the model of the receptor trafficking networkSymbols of ParametersCorresponding Biological Processesk1de novo synthesis of surface receptork2formation of ligand-receptor complexk3dissociation of ligand-receptor complexk4recycling of internalized unbound receptork5internalization of unbound surface receptork6recycling of internalized ligand-receptor complexk7internalization of surface ligand-receptor complexk8dephosphorylation of ligand-receptor complexk9degradation of unbound receptork10degradation of ligand-receptor complexThe results of global sensitivity analysis of the integrated response of the state variables in the receptor trafficking model using all four different methods are shown in Figure 6A–D. The exact values of the sensitivity indices obtained by different methods are not comparable because of their distinct definitions. However, the ranks or relative importance of the parameters to the model output are similar among different global sensitivity analysis methods. The results suggest that the rates of ligand-receptor complexes formation (parameters k2 and k3) are very important to the integrated response of ligand concentration (L). In contrast, the integrated response of ligand-receptor complexes (LRs and LRi) are shown to be mainly affected by the rates of the internalization, recycling and dephosphorylation of the occupied receptors (parameters k6, k7 and k8). The MPSA global sensitivity analysis result of the time dependent response (Figure 6E) indicates that k2 is the key regulator for Rs behavior at the early stage (before 20 minutes), but its effect is reduced significantly at a later stage. Upon further analysis, the MPSA global sensitivity analysis of the steady state response (Figure 6F) shows that the steady state of Rs is not very sensitive to k2.Figure 6Global sensitivity analysis in SBML-SAT. Results of different types of global sensitivity analysis for the receptor trafficking model. (A) MPSA analysis, (B) PRCC analysis, (C) SOBOL's total effect sensitivity analysis, and (D) WALS analysis of integrated response. (E) MPSA analysis of the time dependent response. (F) MPSA analysis of the steady state response.Sample steady state analysisA steady state analysis of a user loaded SBML model simply requires to select such analysis from the icons or pull down menu. SBML-SAT initially tries to algebraically solve the system of ODEs for equilibrium solutions. If that fails, the model is simulated over an extended time period to approach the stable steady state related to the initial conditions provided.The results of the steady state analysis of the model of receptor trafficking network are provided in Figure 7. At steady state, all the ligand molecules are taken up by the receptors and eventually degraded, while the internalized and surface receptors that remain unbounded by ligand reach non-zero equilibriums. This information helps to interpret the steady-state global sensitivity analysis results shown in Figure 6F.Figure 7Steady state analysis in SBML-SAT. Steady state analysis of the receptor trafficking network model in SBML-SAT.Sample robustness analysisTo conduct a robustness analysis, the user• loads the SBML model,• sets the time course,• chooses the parameter(s),• defines the variation range of the parameter(s), and• selects the objects (ODE variables or reaction rates) and the model output operation for the analysis and eventually• runs the analysisFigure 8 shows the result of robustness analysis of the receptor trafficking model. The steady state concentrations of different forms of receptors are plotted as a function of the total parameter variation (TPV) and the quantitative robustness metric is provided in the subplot title. The results indicate that the steady state concentrations of unbound receptors are less robust to parameter perturbations than the internalized unbound receptor concentration. Not surprisingly, the ligand-bound receptors' concentrations are very robust to the parameter perturbations since their steady state solutions are zero.Figure 8Robustness analysis in SBML-SAT. Robustness analysis of the steady state response of the receptor trafficking model against simultaneous variations of the parameter values. The red circles correspond to the reference model output. The blue points correspond to the model outputs under perturbed parameter values. (Specifications for SBML-SAT, \"Model Output\": \"Steady State Response\"). (A-B) Robustness of unbound receptor steady state concentration. (C-D) Robustness of ligand bound receptor steady state concentration.Discussion and conclusionCurrently, a SBML model editor module is not available in SBML-SAT. Fortunately, many existing free software packages such as CellDesigner, SBMLeditor and COPASI, share a common functionality for constructing and editing SBML models. The users can easily generate their models with these free software packages and then run a variety of analyses in SBML-SAT by importing the model in SBML format. Although SBML-SAT doesn't provide a SBML editor for model construction, it provides a convenient track for modifying the initial conditions of the state variables and parameter values in the model. Moreover, delay differential equation models are not supported in SBML-SAT, as in most existing software systems. In practice, delay differential equations can be solved in approximation by converting to ordinary differential equations using the linear chain transformation [41]. Therefore, users can still apply SBML-SAT to their delay differential equation models.There are more than 120 SBML-supporting software packages for kinetic analysis of biological models and this number continues to grow. However, a powerful, flexible and broadly applicable software package for global sensitivity analysis and robustness analysis has been lacking. In reality, it is difficult and time consuming to implement different sensitivity analysis algorithms especially the global sensitivity analysis methods. Here we introduced, a free Matlab-based software tool, SBML-SAT, for both local and global sensitivity analysis of SBML models. With a user-friendly graphic interface, this tool allows the user to run sensitivity analysis, steady state analysis and robustness analysis for a variety of model outputs. Models involving events are also supported in SBML-SAT. Furthermore, created in Matlab, the most popular software used in the community of systems biology [42], SBML-SAT has a good cross-compatibility with different platforms. Taken all together, we can expect that SBML-SAT will have a broad applicability among systems biologists.Availability and requirementsProject name: SBML-SAT: A Systems Biology Markup Language (SBML) based Sensitivity Analysis ToolProject homepage: Operating system(s): Windows, Linux, MacProgramming language: MatlabOther requirements: SBMLToolbox, SUNDIALS TBLicense: noneAny restrictions to use by non-academics: noneAbbreviationsSBML: Systems Biology Markup Language; SBML-SAT: Systems Biology Markup Language based Sensitivity Analysis Tool; MPSA: Multi-Parametric Sensitivity Analysis; PRCC: Partial Rank Correlation Coefficient; WALS: Weighted Average of Local Sensitivities; GUI: Graphic User Interface; LSE: Least Squares Error; TPV: Total Parameter VariationAuthors' contributionsZZ proposed the project, designed the GUI interface and wrote all the source code of the software. AR and YZ contributed some algorithms for global sensitivity analysis methods. ZZ, YZ, AR and EK wrote the manuscript and tested the software. All authors have read and approved the final manuscript.\n\nREFERENCES:\nNo References"
+}

batch_9/PMC2529340.json

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+{
+    "id": "PMC2529340",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529340\nAUTHORS: Guimin Gao, Wen Wan, Sijian Zhang, David T Redden, David B Allison\n\nABSTRACT:\nBackgroundInvestigators are actively testing interventions intended to increase lifespan and wish to test whether the interventions increase maximum lifespan. Based on the fact that one cannot be assured of observing population maximum lifespans in finite samples, in previous work, we constructed and validated several tests of difference in the upper parts of lifespan distributions between a treatment group and a control group by testing whether the probabilities that observations are above some threshold defining 'old' or being in the tail of the survival distribution are equal in the two groups. However, a limitation of these tests is that they do not consider how much above the threshold any particular observation is.MethodsIn this article we propose new methods which improve upon our previous tests by considering not only whether an observation is above some threshold, but also the magnitudes by which observations exceed the threshold.ResultsSimulations show that the new methods control type I error rates quite well and that the power of the new methods is usually higher than that of the tests we previously proposed. In illustrative analyses of two real datasets involving rodents, when setting the threshold equal to 110 (100) weeks for the first (second) datasets, the new methods detected differences in 'maximum lifespan' between groups at nominal alpha levels of 0.01 (0.05) for the first (second) datasets and provided more significant results than competitor tests.ConclusionThe new methods not only have good performance in controlling the type I error rates but also improve the power compared with the tests we previously proposed.\n\nBODY:\nBackgroundInvestigators are actively testing interventions intended to increase lifespan [1]. Caloric restriction (CR) is the intervention most well established as able to increase lifespan in experimental models [2], and investigators are now seeking other interventions that may mimic the life-prolonging effects of CR without requiring a reduction in caloric intake [3]. It is frequently said that CR not only increases average lifespan, but also 'maximum' lifespan [4]. Many researchers in the field of aging therefore wish to test whether other interventions increase maximum lifespan.Recognizing this and the fact that one cannot be assured of observing population maximum lifespans in finite samples, Wang et al. [5] constructed and validated several tests (hereafter, the 'Wang-Allison tests') of differences in the upper parts of lifespan distributions by building on the work of Redden et al. [6] in the area of quantile regression. Wang et al. also showed that a commonly used test for differences in maximum lifespan that involved comparing the means of the top p% (e.g., top 10%) of each of two samples (e.g., a treatment and a control sample) was not valid in that it had an excessive type-1 error rate. Nevertheless, there is appeal to using the full continuity of information in the upper tails of the sample distribution, and colleagues have recently suggested to us that a limitation of the Wang-Allison tests is that they only treat individual lifespans as being above or below some threshold defining 'old' or being in the tail of the survival distribution. That is, the Wang-Allison tests do not consider how much above the threshold any particular observation is, only whether the observation is above the threshold. We acknowledge this limitation and in response, we herein develop new tests that utilize the continuity of information among observations that exceed the threshold of interest, are more powerful than competing tests, including the Wang-Allison tests, in most cases, and remain valid under the null hypothesis of no effect on 'maximum' lifespan.MethodsDevelopment of the testsConsider an experiment with two groups, treatment and control. The extension to more than two groups is straightforward (see discussion section). Let X be an indicator variable taking the value 1 for observations in the treatment group and 0 for observations in the control group. Let Y denote survival time. Let τ denote some threshold chosen by the investigator to denote an extreme portion of the distribution. In survival studies, τ can be chosen in advance to correspond to an age considered 'old' (e.g., 30 months in mice) or set to some high sample percentile (e.g., the 90th). Critically important, τ must be set to the same value for the two groups. That is, if τ is to be defined by an upper sample quantile, it should be the upper sample quantile of both of the two groups combined, not of each group separately.Although not described in exactly these terms in the paper by Wang et al. [5], the Wang-Allison tests essentially create a new variable, W, where for the ith subject, Wi ≡ 0 if Yi ≤ τ, and Wi ≡ 1 if Yi > τ, and subsequently tests whether W is associated with X using an appropriate test statistic.Thus, the Wang-Allison tests test the following null hypothesis:H0,A : P (Y > τ|X = 1) = P(Y > τ|X = 0).A problem with the Wang-Allison tests is that, hypothetically, P (Y > τ|X = 1) may equal P (Y > τ|X = 0) and yet the average magnitude by which lifespans exceed τ when X = 1 may be radically different than when X = 0. This is exemplified in the hypothetical frequency distributions depicted in Figure 1. Note that these hypothetical distributions are not intended to be realistic, but only to clarify the point.Figure 1The left graph is the density for control group (X = 0), 0.9*Weibull(5.73, 106.6)*I(X ≤ 130) + 0.1*Weibull(5.40, 100.06)*I(X > 130), and the right graph is the density for treatment group (X = 1), 0.9*Weibull(5.73, 106.6)*I(X ≤ 130) + 0.1*Weibull(5.45, 130.06)*I(X > 130), where P(Y > τ|X = 1) = P(Y > τ|X = 0) and yet the average magnitude by which lifespans exceed τ when X = 1 is different than when X = 0. τ is 90th percentile of the all observations in treatment and control groups.Let X1 and X0 denote the numbers of observations with Yi > τ in the treatment group and control group, respectively. The Wang-Allison tests use the test procedures for two independent binomial proportions [7] and these procedures require that X1 and X0 are independent. In the Wang-Allison tests, if the threshold is set in advance according to prior knowledge, X1 and X0 can satisfy the requirement of independence. But if τ is set to be the 90-th percentile, X1 and X0 may not be independent, this creates a theoretical problem. However, on an empirical level, our simulations show that in the sample sizes we considered, this is not an apparent problem because the Wang-Allison tests have very high power and can control type I error quit well in the simulation studies and are practical for the lifespan studies). When X1 and X0 are not independent, simulation studies (including estimation of power and type I error) are an effective way to evaluate the methods (such as Wang-Allison tests) using the test procedures for two independent binomial proportions.An alternative to testing H0,A is to test the following conceptually related but mathematically distinct null hypothesis:H0,B : μ (Y|Y > τ ∩ X = 1) = μ (Y|Y > τ ∩ X = 0),where μ (•) denotes the population mean (or expectation) of (•). Though appealing, a problem with testing H0,B is that when P (Y > τ|X = 1) >> P (Y > τ|X = 0) or P (Y > τ|X = 1) <<P (Y > τ|X = 0), for any finite sample with equal initial assignment to the two groups, E [n0] <<E [n1] or E [n0] >> E [n1], where E [n0] denotes the expected number of observations in the control group for which Y > τ, and E [n1] denotes the expected number of observations in the treatment group for which Y > τ. This imbalance between E [n0] and E [n1] will greatly reduce the power to reject H0,B. In fact, in the extreme, when either P (Y > τ|X = 1) or P (Y > τ|X = 0), there will be zero power to reject H0,B (actually, it is appropriate to say that H0,B is undefined in such cases). Such a situation is exemplified in the hypothetical frequency distributions depicted in Figure 2. Again, these hypothetical distributions are not intended to be realistic, but only to clarify the point.Figure 2The left graph is the density for control group (X = 0), 0.9*Weibull(5.07, 93.52)*I(X ≤ 130) + 0.1*Weibull(5.40, 100.06)*I(X > 130), and the right graph for treatment group (X = 1), 0.6*Weibull(5.07, 93.52)*I(X ≤ 130) + 0.4*Weibull(5.40, 100.06)*I(X > 130), where P(Y > τ|X = 1) ≠ P(Y > τ|X = 0), μ (Y |Y > τ ∩ X = 1) = μ (Y |Y > τ ∩ X = 0), and μ (•) denotes the population mean of (•). τ is 90th percentile of the all observations in treatment and control groups.Thus, one can conceive situations in which the power to reject H0,A will be zero and yet the upper tails of the distribution are clearly different. Similarly, one can conceive situations in which the power to reject H0,B will be zero and yet again the upper tails of the distribution are clearly different. Hence, we propose a single-step union-intersection test [8] of the following compound null hypothesis:H0,C : [P(Y > τ|X = 1) = P(Y > τ|X = 0)] ∩ [μ(Y|Y > τ ∩ X = 1) = μ (Y|Y > τ ∩ X = 0)].We construct the test of H0,C with the following simple procedure. Define a new variable Z such that Zi ≡ I(Yi > τ)Yi, where I(•) denotes the indicator function taking on values of one if (•) is true and zero otherwise. One can then simply conduct an appropriate test (several candidates will be considered below) of whether the population mean of Z is significantly different between the treatment and control groups. This approach (hereafter new tests), has several desirable properties. First and foremost, when an appropriate test statistic is used, the approach will be valid. That is, unlike the conditional t-tests (CTTs) commonly used and shown to be invalid by Wang et al. [5], when H0,C is true, it will only be rejected 100*α% of the time at the nominal α level even if f(Y|Y ≤ τ ∩ X = 1) ≠ f(Y|Y ≤ τ ∩ X = 0), where f(•) denotes the probability density function of (•).Note that expectation (or population mean) of Z, μ(Z) = P(Y > τ) μ(Y | Y > τ). Therefore the new test for H0,C is really testing whetherP(Y > τ | X = 1) μ (Y | Y > τ ∩ X = 1) = P(Y > τ | X = 0) μ (Y | Y > τ ∩ X = 0),while the method for H0,B is testing whether μ (Y | Y > τ ∩ X = 1) = μ(Y | Y > τ ∩ X = 0) and the method for H0,A is testing whether P(Y > τ | X = 1) = P(Y > τ | X = 0). The mean difference of μ(Z) between two groups consists of two components: the difference between probabilities P(Y > τ | X = 1) and P(Y > τ | X = 0) and the difference between expectations μ (Y | Y > τ ∩ X = 1) and μ (Y | Y > τ ∩ X = 0). The test for H0,A focuses on the first component and the test for H0,A focuses on the second one, while the test for H0,C is related to both components.We also note that Dominici and Zeger [9] studied similar mean difference components for two groups (cases and controls) by estimating the mean difference Δ(v) for the two groups conditional on a vector of covariates v for zero-inflated data through smooth quantile ratio estimation with regression,Δ(v) = P(Y > 0 | X = 1, v) μ (Y | Y > 0, X = 1, v) - P(Y > 0| X = 0, v) μ (Y | Y > 0, X = 0, v),where, Y is nonnegative random variable denoting the health expenditures. While Dominici and Zeger [9] estimate the mean difference of nonnegative random variables (Y) for two groups, our methods test the mean difference of random variables (Y) which are greater than threshold τ.Evaluation of the testsWe evaluate the tests via computer simulation. For each scenario simulated, we evaluate the tests at the 2-tailed .05 α level and at the 2-tailed .01 α level using 5,000 simulated datasets per scenario (except for permutation tests where we use 1,000 datasets per scenario and 1,000 random permutations by Monte Carlo sampling for each dataset). In simulation 1, we first evaluate performance in simulation under the null hypothesis H0,C (i.e., both H0,A and H0,B are true) and yet f (Y|Y ≤ τ ∩ X = 1) is radically different from f (Y|Y ≤ τ ∩ X = 0). After showing that the tests remain valid even in these extreme circumstances, we compare their power in several scenarios (simulations 2–4) described below. For each scenario, we assumed that there were two groups with an equal number of subjects per group. We ran scenarios with 50, 80, or 100 subjects in each of the two groups, realistic sample sizes for animal model longevity research.We simulated data using a concatenation of Weibull distributions to flexibly emulate the data observed in a real study [10] of obese animals (control; X = 0) versus animals that were obese and then lost weight via CR (treatment; X = 1). Specifically, For example, in simulations 1–4, we simulated Y from the following distribution:f(Y|X=j)=rj[bj,Laj,L(Yaj,L)bj,L−1e−(Yaj,L)bj,L]I(Y≤130)+(1−rj)[bj,Uaj,U(Yaj,U)bj,U−1e−(Yaj,U)bj,U]I(Y>130),where j = 0 to 1, lifespan (Y) is measured in weeks, aj,L and bi,L are the parameters of a Weibull distribution for the lower 90% of the distribution, and aj,U and bi,U are the parameters of a Weibull distribution for the upper 10% of the distribution. rj is a proportion parameter, for example rj = 0.9. The specific values of the parameters used are provided in Figure 3.Figure 3Parameter values and distributions for component Weibull distributions used in each simulation.Delineation of tests to be evaluatedEach of the tests listed below was implemented in two manners, first with τ set in advance to a fixed lifespan value (130 weeks), and second with τ set at the sample 90th percentile of the two groups combined. In real-life situations, one usually does know the threshold of interest a priori. We do recognize that we will not have such knowledge in all cases. It is for this reason that when analyzing the simulated data, we also consider a threshold of the 90th percentile of the data allowing for an ad hoc data-based determination of a threshold.Tests of H0,A (Wang-Allison tests)For comparative purposes, the first category of tests we evaluated were the tests denoted QT3 and QT4 in Wang et al [5] which are, respectively, Boschloo's test and an exact unconditional test based on the observed difference divided by its estimated standard error under the null hypothesis (score statistic) and are described in more detail by Mehrotra et al. [7]. These were the two tests that Wang et al. [5] had found performed best as tests of H0,A.Tests of H0,BIn testing H0,B, subjects were only included in the analysis when their lifespans exceeded τ. Distributions of survival times (lifespans) are rarely Gaussian and, even if they were nearly Gaussian after, for example, log transformation, the distribution of just the tail portion (i.e., f (Y|Y > τ) would not be. Hence, in constructing tests we relied on nonparametric statistical methods. Specifically, we used the Wilcoxon-Mann-Whitney (exact) test [11,12] and a permutation test (with t-statistic) as described by Good [13] to test for differences in lifespan among those subjects whose lifespans exceeded τ.Tests of H0,C (new tests)In testing H0,C, all subjects were analyzed, but the variable analyzed was Z as defined above. Because the distribution of Z cannot be normally distributed, we again used the Wilcoxon-Mann-Whitney test and a permutation test to test for differences in Z.For a dataset with n1 (n2) subjects in treatment (control) group, the permutation test can be performed in the following way: First put all the (n1 +n2) subjects together, and then generate 1000 replicated datasets. For each replicated dataset, we randomly sample n1 subjects from the (n1 +n2) subjects and assign them to treatment group, and assign the left n2 subjects to control group. We run T-test on the observed dataset and the 1000 replicated datasets. Let T0 be the T value for the observed dataset, then p-value for the permutation test is calculated as the proportion of replicated datasets with absolute T values greater than or equal to the absolute valued of T0.ResultsResults are displayed in Tables 1 to 5. As can be seen, the new methods for tests of H0,C controls type I error rates quite well. The power of the new methods are always higher than or very close to that of the methods for tests of H0,A (Wang-Allison tests) and are higher than that of the methods for tests of H0,B (Wilcoxon-Mann-Whitney tests and permutation tests for observations above the threshold τ) in some of the simulations.Table 1Performance (type 1 error rates) of the tests in simulation 1 under H0,C (i.e., both H0,A and H0,B are true) and yet f (Y|Y ≤ τ ∩ X = 1) is radically different from f (Y|Y ≤ τ ∩ X = 0) (see Figure 3 for details of simulation).TestSample Size (N) Per Group5080100α = .05α = .01α = .05α = .01α = .05α = .01Tests of H 0,A (Wang-Allison tests)QT3 with τ set to 130.0.032 (.027, .036)#0.008 (.005, .011)0.041 (.036, .046)0.006 (.003, .009)0.040 (.035, .045)0.006 (.003, .009)QT3 with τ set to sample 90th percentile0.026 (.022, .030)0.026* (.020, .032)0.080 (.072, .088)0.007 (.004, .010)0.040 (.035, .045)0.010 (.006, .014)QT4 with τ set to 130.0.038 (.033, .043)0.008 (.005, .011)0.051 (.045, .057)0.009 (.006, .012)0.047 (.041, .053)0.007 (.004, .010)QT4 with τ set to sample 90th percentile.0.026 (.022, .030)0.026 (.020, .032)0.083 (.075, .091)0.026 (.020, .032)0.040 (.035, .045)0.010 (.006, .014)Tests of H0,BWilcoxon-Mann-Whitney** with τ set to 130.0.041 (.036, .046)0.017 (.012, .022)0.044 (.038, .050)0.008 (.005, .011)0.046 (.040, .052)0.008 (.005, .011)Wilcoxon-Mann-Whitney with τ set to sample 90th percentile.0.049 (.043, .055)0.014 (.010, .018)0.065 (.058, .072)0.015 (.011, .019)0.080 (.072, .088)0.018 (.013, .023)Permutation test with τ set to 130.0.050 (.036, .064)0.009 (.001, .017)0.050 (.036, .064)0.011 (.002, .020)0.064 (.049, .079)0.015 (.005, .025)Permutation test with τ set to sample 90th percentile.0.077 (.060, .094)0.016 (.006, .026)0.078 (.061, .095)0.022 (.010, .034)0.083 (.066, .100)0.019 (.008, .030)Tests of H0,C (new tests)Wilcoxon-Mann-Whitney with τ set to 130.0.042 (.036, .048)0.007 (.004, .010)0.049 (.043, .055)0.010 (.006, .014)0.051 (.045, .057)0.008 (.005, .011)Wilcoxon-Mann-Whitney with τ set to sample 90th percentile.0.055 (.049, .061)0.015 (.011, .019)0.060 (.053, .067)0.015 (.011, .019)0.061 (.054, .068)0.015 (.011, .019)Permutation test with τ set to 130.0.052 (.038, .066)0.015 (.005, .025)0.047 (.034, .060)0.009 (.001, .017)0.057 (.043, .071)0.007 (.000, .014)Permutation test with τ set to sample 90th percentile.0.045 (.032, .058)0.017 (.006, .028)0.062 (.047, .077)0.011 (.002, .020)0.068 (.053, .084)0.018 (.007, .029)#2-tailed 95% confidence interval.*The bolded values are those simulated type I error rates which are significantly higher than the nominal α at the 2-tailed 95% confidence level (i.e., the lower bound of the interval is higher than α). Note that for the permutation tests we used 1000 replicated datasets and for other tests we used 5000 replicated datasets.**In all the simulation studies (Tables 1-5), we used Wilcoxon-Mann-Whitney exact test.Table 2Performance of the tests in simulation 2, H0,A is true, H0,B is false and f (Y|Y ≤ τ ∩ X = 1) is radically different from f (Y|Y ≤ τ ∩ X = 0) (see Figure 3 for details of simulation).TestSample Size (N) Per Group5080100α = .05α = .01α = .05α = .01α = .05α = .01Tests of H0,A (Wang-Allison tests)QT3 with τ set to 130.0.0320.0080.0410.0060.0400.006QT3 with τ set to sample 90th percentile.0.0340.0340.1040.0090.0620.018QT4 with τ set to 130.0.0380.0080.0510.0090.0470.007QT4 with τ set to sample 90th percentile.0.0340.0340.1040.0330.0620.018Tests of H0,BWilcoxon-Mann-Whitney with τ set to 130.0.2640.0900.5040.2610.6310.368Wilcoxon-Mann-Whitney with τ set to sample 90th percentile.0.160.0510.3140.1430.4060.220Permutation test with τ set to 1300.3370.1110.6080.3320.7370.456Permutation test with τ set to sample 90th percentile.0.1970.0470.4230.2040.5250.284Tests of H0,C (new tests)Wilcoxon-Mann-Whitney with τ set to 130.0.0510.0080.0620.0120.0560.010Wilcoxon-Mann-Whitney with τ set to sample 90th percentile.0.1070.0290.0900.0280.1240.035Permutation test with τ set to 130.0.0610.0130.0550.0120.0650.014Permutation test with τ set to sample 90th percentile.0.1090.0320.0970.030.1290.046Table 3Performance of the tests in simulation 3, H0,B is true, H0,A is false and f (Y|Y ≤ τ ∩ X = 1) is radically different from f (Y|Y ≤ τ ∩ X = 0) (see Figure 3 for details of simulation).TestSample Size (N) Per Group5080100α = .05α = .01α = .05α = .01α = .05α = .01Tests of H0,A (Wang-Allison tests)QT3 with τ set to 130.0.2440.1010.4120.1810.4900.258QT3 with τ set to sample 90th percentile.0.1020.1020.3320.0510.2970.143QT4 with τ set to 130.0.2660.1020.4180.1870.5140.274QT4 with τ set to sample 90th percentile.0.1020.1020.3320.1510.2970.143Tests of H0,BWilcoxon-Mann-Whitney with τ set to 130.0.0460.0130.0490.0110.0450.008Wilcoxon-Mann-Whitney with τ set to sample 90th percentile.0.0480.0190.0440.010.0410.009Permutation test with τ set to 130.0.0420.0070.0460.0120.0640.013Permutation test with τ set to sample 90th percentile.0.0460.0090.0460.0120.0440.01Tests of H0,C (new tests)Wilcoxon-Mann-Whitney with τ set to 130.0.2760.1110.4200.2010.5170.271Wilcoxon-Mann-Whitney with τ set to sample 90th percentile.0.1820.070.2780.1040.350.154Permutation test with τ set to 130.0.2910.1010.4270.2030.5150.28Permutation test with τ set to sample 90th percentile.0.1690.0670.2640.1070.3630.173Table 4Performance of the tests in simulation 4, H0,B is false, H0,A is false and f (Y|Y ≤ τ ∩ X = 1) and f (Y|Y ≤ τ ∩ X = 0) are identical (see Figure 3 for details of simulation).TestSample Size (N) Per Group5080100α = .05α = .01α = .05α = .01α = .05α = .01Tests of H0,A (Wang-Allison tests)QT3 with τ set to 130.0.2440.1010.4120.1810.4900.258QT3 with τ set to sample 90th percentile.0.3630.3630.7350.3370.7530.600QT4 with τ set to 130.0.2660.1020.4180.1870.5140.274QT4 with τ set to sample 90th percentile.0.3630.3630.7350.5550.7530.600Tests of H0,BWilcoxon-Mann-Whitney with τ set to 130.0.4090.1720.6840.4110.8040.56Wilcoxon-Mann-Whitney with τ set to sample 90th percentile.0.2450.1420.330.1440.4340.176Permutation test with τ set to 130.0.5170.2440.810.5680.9130.728Permutation test with τ set to sample 90th percentile.0.1690.0390.4280.1900.5690.249Tests of H0,C (new tests)Wilcoxon-Mann-Whitney with τ set to 130.0.3740.1710.5280.2800.6290.373Wilcoxon-Mann-Whitney with τ set to sample 90th percentile.0.6020.3530.7340.5520.8650.724Permutation test with τ set to 130.0.3930.1770.5240.2880.6260.377Permutation test with τ set to sample 90th percentile.0.6190.3650.7260.5530.8520.704Table 5Performance of the tests in simulation 5, H0,B is false, H0,A is false and f (Y|X = 1) = 1.2f (Y|X = 0) (see Figure 3 for details of simulation).TestSample Size (N) Per Group5080100α = .05α = .01α = .05α = .01α = .05α = .01Tests of H0,A (Wang-Allison tests)QT3 with τ set to 130.0.6630.3490.9250.7540.9650.883QT3 with τ set to sample 90th percentile.0.8150.8150.9960.8850.9970.986QT4 with τ set to 130.0.7650.3490.9410.7970.9810.906QT4 with τ set to sample 90th percentile.0.8150.8150.9960.9690.9970.986Tests of H0,BWilcoxon-Mann-Whitney with τ set to 130.0.0010.0000.0060.0000.0100.000Wilcoxon-Mann-Whitney with τ set to sample 90th percentile.0.0160.0000.0350.0020.0580.009Permutation test with τ set to 1300.0010.0000.0360.0030.0610.005Permutation test with τ set to sample 90th percentile.0.0320.0020.0820.0170.1240.041Tests of H0,C (new tests)Wilcoxon-Mann-Whitney with τ set to 130.0.5560.2390.9200.7420.9790.897Wilcoxon-Mann-Whitney with τ set to sample 90th percentile.0.9320.7670.9950.9640.9990.992Permutation test with τ set to 130.0.8520.6460.9600.8500.9930.940Permutation test with τ set to sample 90th percentile.0.9420.7860.9950.9580.9970.986Table 1 shows the type I error rate of the tests (in simulation 1) when the null hypothesis H0,C is true (i.e., both H0,A and H0,B are true) and yet f (Y|Y ≤ τ ∩ X = 1) is radically differentfrom f (Y|Y ≤ τ ∩ X = 0). The type I error rates of the new methods are comparable to those of the methods for tests of H0,A and those of the methods for tests of H0,B . It is note worthy that there is a slight but fairly consistent excess of type I errors when the sample 90th percentile is used rather than a fixed cutoff point. This is because the sample 90th percentile is a random variable and when it falls below its population level, the null hypothesis is no longer strictly true in our simulations. That is, the tests remain valid tests of differences in distributions above the actual value used but should not be strictly interpreted as tests of differences in distributions above the 90th (or any other percentile). In practice, this distinction is probably trivial.In simulation 2 (see Table 2), where H0,A is true, H0,B is false and f (Y|Y ≤ τ ∩ X = 1) is radically different from f (Y|Y ≤ τ ∩ X = 0), the new methods for tests of H0,C and the methods for tests of H0,A have lower power than that of the corresponding methods for tests of H0,B, however, the new methods for tests of H0,C can slightly improve the power compared to the methods for tests of H0,A.Table 3 shows the power of the tests in Simulation 3, where H0,B is true, H0,A is false and f (Y|Y ≤ τ ∩ X = 1) is radically different from f (Y|Y ≤ τ ∩ X = 0). The new methods for tests of H0,C and the methods for tests of H0,A have very similar power which is much higher than that of the corresponding methods for tests of H0,B.From simulation 4 (see Table 4), where H0,B is false, H0,A is false and f (Y|Y ≤ τ ∩ X = 1) and f (Y|Y ≤ τ ∩ X = 0) are identical, we can find that the new methods for tests of H0,C always have higher power than the corresponding methods for tests of H0,A. When τ being set to the 90th percentile of the sample, the new methods also have higher power than the corresponding methods for tests of H0,B.Finally, we conducted a set of simulations under what we perceived to be the most realistic situations. Here both H0,A and H0,B are false, f (Y|Y ≤ τ ∩ X = 1) is quite different from f (Y|Y ≤ τ ∩ X = 0), and the distributions have no discontinuities. In other words, there is just a simple reduction in the hazard rate when X = 1. Table 5 presents the power of the tests in Simulation 5, where f (Y|X = 1) = 1.2f (Y|X = 0). In this simulation, the methods for tests of H0,B almost have no power because the control group always has no or few observations above the threshold τ . The new methods for tests of H0,C, when using a permutation test, have power higher than or equal to that of the methods for tests of H0,A.Illustration with real dataTo illustrate the methods, we applied them to two real datasets. In both of these datasets, prior research had shown differences in overall survival rate and we tested for differences in 'maximum lifespan' herein. The first was a subset of data reported by Vasselli et al [10]. The subset of the data consists of two groups of Sprague-Dawley rats, those kept on a high-fat diet ad libitum throughout life and becoming obese (EO-HF) and those kept on a high-fat diet ad libitum until early-middle adulthood, becoming obese, and subsequently reduced to normal weight via caloric restriction, but on the same high-fat diet (WL-HF). Each group had 49 rats (see Figure 4 for the histograms for the data). The second dataset was from a study comparing the lifespan of Agouti-related protein-deficient (AgRP(-/-)) mice to wildtype mice (+/+) as reported by Redmann & Argyropoulos [14]. This dataset consists of 16 mice with genotype '+/+' and 21 mice with genotype '-/-' (see Figure 5 for the histograms for this dataset). From Figure 4, we can see the upper tails of the histograms of the two groups are different. Similar results can be found in Figure 5.Figure 4The left (right) graph is the histogram of lifespan for WL-HF (EO-HF) group in the data from Vasselli et al. [10].Figure 5The left (right) graph is the histogram of lifespan for group with genotype '+/+' ('-/-') in the data from Redmann & Argyropoulos [14].Results (p values of tests) are shown in Table 6. As can be seen, when setting τ equal to 110 (100) for the first (second) datasets, both the methods for tests of H0,A and the new methods for tests of H0,C can detect the differences in 'maximum lifespan' between groups at nominal alpha levels of 0.01 (0.05) for the first (second) datasets. But the methods for tests of H0,B cannot detect the difference for all different values of τ . The following description may provide some explanation to these results. For the first dataset, when set τ = 110, the proportions of the observations greater than τ in the EO-HF group and WL-HF group (i.e., estimations of P(Y > τ | X = 0) and P(Y > τ | X = 1)) are 0.061 and 0.306, respectively. These two proportions are significantly different and not surprisingly, the methods for tests of H0,A can detect the difference in 'maximum lifespan' between the two groups. Second, the sample means of the observations greater than τ in the two groups (i.e., estimations of μ (Y | Y > τ ∩ X = 1) and μ (Y | Y > τ ∩ X = 0)) are 117.8 and 122.9, respectively, and there is no much difference between these sample means. However the sample means of the Z-values in the two group (i.e., the estimations of P(Z | X = 0) and P(Z | X = 1)) are 7.210 and 37.633, respectively, and are greatly different, where, Zi ≡ I(Yi > τ)Yi. These may explain that the methods for tests of H0,B cannot reject the null but the new methods for tests of H0,C can detect the difference in 'maximum lifespan' between the two groups. Similarly, for the second dataset, when set τ = 100, the proportions of the observations greater than τ in the group with genotype '+/+' and group with genotype '-/-' are 0.188 and 0.571, respectively. The sample means of the observations greater than τ in the two groups are 109.3 and 110.9, respectively. The sample means of the Z-values in the two groups are 20.5 and 63.4 respectively.Table 6Results (p values of tests) of application to two real datasets.TestData from Vasselli et al. [10]1Data from Redmann & Argyropoulos [14]2Tests of H0,A (Wang-Allison tests)QT3 with τ set to 110/100#.0.0020.027QT3 with τ set to sample 90th percentile.0.0380.186QT4 with τ set to 110/100.0.0020.022QT4 with τ set to sample 90th percentile.0.0330.146Tests of H0,BWilcoxon-Mann-Whitney with τ set to 110/100.0.2890.868Wilcoxon-Mann-Whitney with τ set to sample 90th percentile.0.750N/A*Permutation test with τ set to 110/100.0.2810.738Permutation test with τ set to sample 90th percentile.0.634N/A*Tests of H0,C (new tests)Wilcoxon-Mann-Whitney with τ set to 110/100.0.0010.022Wilcoxon-Mann-Whitney with τ set to sample 90th percentile.0.0260.243Permutation test with τ set to 110/100.0.0010.014Permutation test with τ set to sample 90th percentile.0.0240.072Notes: In each dataset, males and females have been combined. 1For the data from Vasselli et al. [10] two groups of rats (EO-HF and WL-HF) are compared; each group has 49 observations.2The data from Redmann & Argyropoulos [14] consists of 16 mice with genotype '+/+' and 21 mice with genotype '-/-'.# For Data from Vasselli et al. [10]τ is set to 110; for data from Redmann & Argyropoulos [14]τ is set to 100.*Only one group has observations above the threshold τ.From Table 6 we can also see that in almost all situations the p-values of the new methods for tests of H0,C are somewhat smaller than those of the methods for tests of H0,A. This is consistent with the simulations showing greater power of the new methods.DiscussionHerein, we proposed new methods for testing the difference of 'maximum' lifespan between groups (e.g., treatment and control). By defining a new variable Z such that Zi ≡ I (Yi > τ)Yi for each observation and then applying Wilcoxon-Mann-Whitney test or better still a permutation test to Z, the new methods achieve far better performance when considered across a broad range of circumstances in terms of both Type-1 error rates and power. In the new methods, we use the Wilcoxon-Mann-Whitney test or permutation test. One could also choose to use a bootstrap test in place of these two tests. However, additional simulations would likely be warranted to evaluate its performance relative to the permutation test we have evaluated herein.It is straightforward to extend the new methods to more than two groups. For example, one could use the Kruskal-Wallis Test to replace the Wilcoxon-Mann-Whitney test, or use permutation testing for multiple groups to replace that for two groups.We have shown that the new methods are effective by simulation studies when the sample size (N) of each group is 50, 100, or 200. We expect that these methods will be also be relatively more powerful than existing competitors for much larger sample sizes, such as N = 500 or even N = 5000. There are some mouse data sets (like those of the National Institute of Aging's Intervention Testing Program) where N > 500, and worm and fly data sets in which N may sometimes even exceed 5000. We expect that the new methods are equally applicable to the analysis of such data.Finally, we note that the tests proposed here are described for the context of testing for differences in lifespan. However, there is nothing intrinsic to them that limits their use to survival data. They could be applied to any situation in which one wanted to test for group differences in the tails of distributions.Competing interestsThe authors declare they have no competing interests.Authors' contributionsDBA participated in all parts of the work of the study (including the study design, methodology development, simulations, data acquisition, and manuscript drafting). He wrote major sections of the original manuscript. He revised final version of the manuscript. DTR provided consulting on the statistical issues in the study and manuscript editing. SZ provided assistance in programming for simulation studies. WW provided consulting on simulation and prepared the figures. GG did all simulation studies and real data analyses and drafted the sections of Results, Illustration with real data, and Discussion of the manuscript and participated in revision of the manuscript.Pre-publication historyThe pre-publication history for this paper can be accessed here:\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2529400",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529400\nAUTHORS: Hao Feng, Xiaonan Dong, Ashley Negaard, Pinghui Feng\n\nABSTRACT:\nThe Kaposi's sarcoma-associated herpesvirus (KSHV) genome encodes a G protein-coupled receptor (vGPCR). vGPCR is a ligand-independent, constitutively active signaling molecule that promotes cell growth and proliferation; however, it is not clear how vGPCR is negatively regulated. We report here that the KSHV K7 small membrane protein interacts with vGPCR and induces its degradation, thereby dampening vGPCR signaling. K7 interaction with vGPCR is readily detected in transiently transfected human cells. Mutational analyses reveal that the K7 transmembrane domain is necessary and sufficient for this interaction. Biochemical and confocal microscopy studies indicate that K7 retains vGPCR in the endoplasmic reticulum (ER) and induces vGPCR proteasomeal degradation. Indeed, the knockdown of K7 by shRNA-mediated silencing increases vGPCR protein expression in BCBL-1 cells that are induced for KSHV lytic replication. Interestingly, K7 expression significantly reduces vGPCR tumorigenicity in nude mice. These findings define a viral factor that negatively regulates vGPCR protein expression and reveal a post-translational event that modulates GPCR-dependent transformation and tumorigenicity.\n\nBODY:\nIntroductionKaposi's sarcoma-associated herpesvirus (KSHV, also known as human herpesvirus 8) is believed to be the etiologic agent for Kaposi's sarcoma (KS) [1]. KSHV infection is also linked to primary effusion lymphoma [2] and multicentric Castleman's disease, rare lymphoproliferative malignancies of B-cell origin 3,4. The KSHV genome encodes over 80 viral polypeptides, many of which are capable of promoting cell proliferation and/or modulating host responses, when expressed in gene transfer experiments (for review see reference [5]). One such gene product consistently detected in KS lesions is the viral G protein-coupled receptor (vGPCR, or open reading frame 74) [6],[7].vGPCR is a homolog of the human interleukin-8 receptor and possesses promiscuous chemokine-binding activity [8]. In tissue culture, vGPCR expression activates various signaling pathways and up-regulates the transcription of numerous cellular and viral genes that encode cytokines, signaling molecules, and transcription factors that culminate in promoting cell proliferation and endothelial tube formation [9]–[15]. Additionally, vGPCR transgenic mice developed tumors that resemble human KS lesions [9],[16],[17]. Although ligand binding is not required for vGPCR-mediated signaling, cognate chemokines appear to modulate vGPCR activity in tissue culture and in mice as well [8],[18]. Despite the fact that proliferative and prosurvival activities of vGPCR have attracted extensive attention in the past, accumulating evidence suggests that tightly regulated expression and signaling are important for vGPCR function in KSHV infection. Indeed, over-expression of vGPCR induced cell death in COS-1 cells and constitutive expression of vGPCR was toxic to PEL cells [6],[19]. Furthermore, vGPCR is predominantly translated from a bicistronic mRNA transcript downstream of K14 (vOX2), presumably reducing vGPCR protein expression [6],[20]. These observations suggest that KSHV likely has evolved mechanisms to achieve a temporary expression of the constitutively active vGPCR during lytic infection. A post-translational degradation is one of these mechanisms.Regulated protein degradation is important for a variety of cellular events including cell cycle, apoptosis, signal transduction, immune response, and development. Cellular GPCR can be degraded either by the ubiquitin-proteasome system (UPS) or by the lysosome. Within the lysomsome, proteins are cleaved by diverse acidic proteases upon fusion with endosomes or autophagosomes. For UPS substrates, proteins destined for destruction are tagged with ubiquitin through sequential actions of the E1 activating enzyme, E2 conjugating enzyme, and E3 ligase [21]. Relying on the UPS, the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway is a major route to remove mis-folded proteins post-translationally, and plays an essential role for ER quality control. Indeed, alteration of ERAD pathways has been implicated in diverse clinical presentations such as neurodegeneration and cystic fibrosis. Furthermore, viruses usurp components of this pathway to evade host recognition and possibly modulate other host responses [22]–[24].We previously identified a small membrane protein, K7, which induces protein degradation of IκB and p53. K7 specifically interacts with the ubiquitin-associated domain of cellular protein linking integrin-associated protein and cytoskeleton (PLIC1) and antagonizes PLIC1, thereby promoting protein degradation [25]. Additionally, K7 was shown to deregulate cellular apoptosis by targeting Bcl-2 and an ER resident calcium modulating cyclophilin ligand [26],[27]. Although these data imply that K7 inhibits apoptosis to facilitate viral replication, its biological roles in KSHV infection remain obscure. We report here that K7 interacts with vGPCR and induces its proteasomeal degradation. The knockdown of K7 by shRNA-mediated silencing increased vGPCR protein expression in BCBL-1 cells that are induced for KSHV lytic replication. Biochemical and confocal microscopy analyses support that K7 retains vGPCR in the ER, thereby facilitating the proteasome to degrade vGPCR. Consequently, K7 significantly reduces vGPCR transformation in vitro and tumorigenicity in nude mice. These data establish a negative regulation of vGPCR protein expression and tumorigenicity by KSHV K7.ResultsK7 Interacts with KSHV vGPCRTo understand K7's functions, we searched for cellular interacting proteins with K7 as bait using the yeast two-hybrid screen. One clone contained a partial sequence of a putative G protein-coupled receptor that encodes its last four transmembrane (TM) domains. Since the KSHV genome encodes a vGPCR, we speculated that K7 interacts with vGPCR. To test this possibility, whole cell lysates of 293T cells transiently transfected with plasmids expressing vGPCR-Flag and/or K7-V5 were precipitated with the M2 anti-Flag antibody and precipitates were analyzed by immunoblot with anti-V5 (K7) antibody. Indeed, K7 was readily detected in immune complexes containing vGPCR (Figure 1A, left panels). Notably, vGPCR expression greatly increases K7 protein expression and the glycosylated form (the slower migration band) is only detected in the presence of vGPCR. Reciprocally, vGPCR was also precipitated by anti-V5 (K7) antibody (Figure 1A, right panels). Of note, the interaction between K7 and vGPCR was also identified by the yeast two-hybrid screen with a high throughput approach [28]. To further characterize the vGPCR-K7 interaction, K7 mutants that contain various deletions as described in our previous publications [25],[26] were used for a co-immunoprecipitation (co-IP) assay. The internal hydrophobic region (amino acid 22–74) containing the putative TM domain was sufficient to interact with vGPCR (Figure 1B). Unfortunately, K7 mutants lacking the TM domain were expressed at an undetectable level compared to the wild type (wt) K7. Thus, we failed to obtain any deletion mutant that no long interacts with vGPCR. Nevertheless, these data indicate that K7 interacts with vGPCR and suggest that its predicted TM domain is important for this interaction.10.1371/journal.ppat.1000157.g001Figure 1KSHV K7 interacts with vGPCR.(A) K7 interaction with vGPCR by co-IP. (Left) 293T cells were transfected with plasmids containing K7-V5 and vGPCR-Flag. Proteins precipitated with anti-Flag antibody were resolved by SDS-PAGE and analyzed by immunoblot with anti-V5 (top panel) and anti-Flag (middle panel) antibodies. WCLs were analyzed by immunoblot with anti-V5 (K7) antibody. Note: the K7 doublet indicates its glycosylated and unglycosylated forms. (Right) Proteins precipitated with anti-V5 antibody were analyzed by immunoblot with anti-Flag (top panel, peroxidase-conjugated) and anti-V5 (middle panel) antibodies. WCLs were analyzed by immunoblot with anti-Flag (vGPCR) antibody. H+L, the heavy and light chains of IgG; IB, immunoblot. (B) The K7 hydrophobic region is sufficient for its interaction with vGPCR. (Top) Diagram shows the structure of K7 protein and its residue numbers designed for the deletion analysis. Transfection of 293T cells with plasmids as indicated and IP with anti-Flag antibody were performed. Precipitated proteins were analyzed by immunoblot with anti-V5 (top panel) and anti-Flag (middle panel) antibodies. WCLs were analyzed with anti-V5 antibody. Δ3-21, deletion of amino acid 3 to 21; 5K/R, all lysine residues changed to arginine (for more details, please see [25]). (C) The putative K7 TM domain is necessary for its interaction with vGPCR. The putative K7 TM domain was replaced by the Stp C TM. Transfection of 293T cells with plasmids as indicated and IP were performed as in (A). Precipitated proteins were analyzed by immunoblot with anti-Flag (left panel) and anti-V5 (right top panel) antibodies. WCLs were analyzed by immunoblot with anti-V5 (right middle panel) anti-Flag (right bottom panel) antibodies. TMStpC denotes the K7 mutant that contains a StpC TM domain. To achieve equivalent protein expression, 3-fold more plasmid containing K7TMStpC than that containing the wt K7 was used for transfection. (D) The putative K7 TM domain is sufficient to interact with vGPCR. Transfection of 293T cells with plasmids as indicated and IP were performed as in (A). Precipitated proteins were analyzed by immunoblot with anti-GFP (top panel) and anti-Flag (middle panel) antibodies. WCLs were analyzed by immunoblot with anti-GFP antibody (bottom panel). L, the light chain of IgG.K7 contains a putative TM domain and vGPCR is a seven-membrane-spanning protein, therefore we examine whether the predicted K7 TM domain is necessary for this interaction. The K7 mutant whose putative TM region was replaced by a heterologous TM from the Saimiri transforming protein C (Stp C), designated K7TMStp C, was constructed and expressed in 293T cells. We found that K7TMStp C failed to interact with vGPCR under the same co-IP conditions (Figure 1C). Of note, K7TMStp C was expressed and localized to intracellular organelles similarly to the wt K7 (unpublished data). Furthermore, appending the putative TM region (amino acids 23–45) of K7 to GFP renders it capable of binding vGPCR (Figure 1D). Thus, these data collectively support that K7 interacts with vGPCR and that the putative K7 TM region is necessary and sufficient for this interaction.K7 and vGPCR proteins are confined to distinct intracellular organelles. Particularly, vGPCR was reported to reside primarily in the trans-Golgi network (TGN) [7], whereas K7 localizes to both the ER and mitochondrial compartments [26],[27]. To examine the intracellular distribution of vGPCR and K7, indirect immuno-fluorescence microscopy was performed. To this end, human lymphoid BJAB and HeLa cells were transfected with plasmids expressing vGPCR-Flag and K7-V5, and analyzed by confocal microscopy. In both HeLa and BJAB cells, vGPCR predominantly localizes to a subcellular structure reminiscent of the TGN, while K7 distributes throughout the cytoplasm mainly as punctate vesicles (Figure 2A and 2B). In support of the interaction between K7 and vGPCR, K7 had an intracellular staining pattern similar to that of vGPCR in both HeLa and BJAB cells (Figure 2C). Despite the overall colocalization between K7 and vGPCR, there are some regions that either K7 or vGPCR is predominant, likely reflecting their distinct intracellular compartments that vGPCR and K7 reside in when they are separately expressed (Figure 2C, insets of BJAB cells).10.1371/journal.ppat.1000157.g002Figure 2Intracellular localization of vGPCR and K7.Human HeLa and lymphoid BJAB cells were transfected as described in Materials and Methods. At 16 h after transfection, cells were fixed, permeabilized, and stained with rabbit anti-Flag and mouse anti-V5 antibodies. (A) vGPCR intracellular localization in human HeLa and BJAB cells. An inset in (A) and (B) represents an image of the other channel. (B) K7 intracellular localization in human HeLa and BJAB cells. (C) Intracellular co-localization of vGPCR (green) and K7 (red) in human HeLa and BJAB cells. Insets represent enlarged (3-fold) view of the boxed regions. Representative sections and their overlays (for panels in [C] only) are shown. Scale bar represents 12.5 µm.Overlapped Expression of vGPCR and K7The fact that K7 interacts with vGPCR prompted us to investigate the temporal expression kinetics of K7 and vGPCR in KSHV lytic replication. Both K7 and vGPCR were reported to be expressed early during KSHV lytic reactivation and/or de novo infection [6],[7],[27],[29]; however, the relative temporal expression of vGPCR and K7 remains unclear. Our interaction study suggests that vGPCR and K7 are possibly expressed at the same time. Thus, we examined mRNA levels of vGPCR and K7 by reverse-transcriptase (RT)-polymerase chain reaction (PCR). The KSHV latently infected PEL cell lines BCBL-1 (KSHV only) and JSC-1 (KSHV and EBV co-infected) were treated with TPA to induce KSHV lytic replication. Alternatively, lytic replication was reactivated by Rta expression that was induced by doxycycline using the BCBL-1/T-Rex_Rta cell line [30]. RT-PCR analyses were performed using primers specific for vGPCR, K7, the polyadenylated nuclear RNA (PAN), and cellular β-actin. When treated with TPA (20 ng/ml), lytic replication was initiated in both BCBL-1 and JSC-1 cells which was indicated by potent induction of PAN transcripts (Figure 3). The residual PAN RNA in untreated BCBL-1 cells and BCBL-1/T-Rex_Rta cells (lane 3 of left two sets in Figure 3) are likely due to spontaneous lytic replication of KSHV or leaky Rta expression in these PEL cells, respectively. Upon TPA induction, vGPCR transcripts peaked at 72 h, which coincided with the highest mRNA level of K7 in BCBL-1 cells. Upon Rta expression induced by doxycycline addition, vGPCR was highly expressed as early as 12 h post induction and was sustained for more than 24 h (Figure 3, middle panels), while K7 transcripts gradually increased and peaked at 36 h after TPA induction when vGPCR mRNA started to decline. This indicates that K7 expression predominantly overlaps with that of vGPCR in response to the KSHV lytic switch protein, Rta. Similar results were obtained for TPA-induced JSC-1 cells in which vGPCR was highly expressed at 12 and 24 h after treatment. Meanwhile, K7 was highly expressed at 24 h after induction (Figure 3, right panels). The most abundant lytic transcript of KSHV, PAN, was significantly induced by TPA and sustained high transcript levels in BCBL-1 and JSC-1 cells throughout the entire induction period. This was more pronounced by Rta induction (Figure 3 third panel from top), while cellular β-actin transcript remained the same. Overall, these data indicated that K7 and vGPCR are expressed at the same time and suggest that the interaction between these two molecules is biologically relevant.10.1371/journal.ppat.1000157.g003Figure 3Overlapped expression of K7 and vGPCR during KSHV lytic replication.CBL-1, JSC-1, and BCBL-1/T-Rex_Rta cells were induced for KSHV lytic replication as described in Materials and Methods. RT-PCR analyses were performed using primers for vGPCR, K7, PAN, and cellular β-actin. No reverse transcriptase reaction was performed with total RNA of 36 h after treatment of each panel; h, hour after treatment.K7 Reduces vGPCR Protein ExpressionWe have consistently observed that K7 co-expression significantly reduces the protein level of vGPCR (Figure 1A and 1B), suggesting that K7 modulates vGPCR biosynthesis. Because our previous data implicate K7 in regulating protein degradation [25], we speculated that K7 induces the degradation of vGPCR. To examine K7's effect on vGPCR protein expression, human endothelial ECV cells were transiently transfected with a plasmid expressing vGPCR-Flag and increasing amounts of a plasmid expressing K7-V5. Whole cell lysates were analyzed by immunoblot for vGPCR protein expression. The result shows that K7 reduces vGPCR protein in a dose-dependent manner (Figure 4A). The specificity of K7 is further supported by the observation that the K7TMStp C chimera, a mutation that abolished its interaction with vGPCR, failed to suppress vGPCR protein expression (Figure 4B).10.1371/journal.ppat.1000157.g004Figure 4K7 reduces vGPCR protein expression.(A) K7 reduces vGPCR protein expression in a dose-dependent manner. Human endothelial ECV cells were transfected with plasmids containing vGPCR-Flag and K7-V5 as indicated. WCLs were analyzed by immunoblot with anti-Flag (vGPCR, top panel), anti-V5 (K7, middle panel), and anti-tubulin (bottom panel) antibodies. (B) The putative K7 TM domain is necessary for vGPCR downregulation. Transfection of ECV cells and immunoblot analyses were performed as in (A). Data represents three independent experiments. (C) K7 reduces vGPCR-mediated activation of NF-κB, NF-AT, and AP-1 transcription factors. 293T cells were transfected with reporter plasmid cocktail, and plasmids containing vGPCR and K7. Luciferase activity normalized against β-galactosidase activity is shown. Error bars denote standard deviation and data represent three independent experiments. (D) K7 knockdown by shRNA-mediated silencing. (Left Top) The relative genomic locations of K7 coding sequence and the transcribed region of PAN were shown. The numbers indicate the nucleotide position according to a published KSHV genome sequence (accession number: U75698). Bars represent relative location of sequences base paired with four shRNAs within the 5′ untranslated region of K7. The order of shRNAs on the diagram is: shRNA#2, #3, #1, and #4. (Left Bottom) A diagram shows the experimental design of lentivirus infection and KSHV lytic replication induced by TPA. (Right) RT-PCR analyses were performed using gene specific primers for K7, β-actin, vGPCR, and PAN as described in Materials and Methods. Numbers indicate intensity of K7 band measured by densitometry. Data represent two independent experiments.Previous publications have convincingly shown that vGPCR activates a number of signaling pathways, leading to the activation of NF-AT, NF-κB, and AP-1 transcription factors [19],[31],[32]. To further correlate K7's effect on vGPCR protein expression, the transcription activation of NF-AT, NF-κB, and AP-1 response elements by vGPCR were measured by luciferase assays in transiently transfected 293T cells. Consistent with published data, vGPCR activated NF-κB, NF-AT, and AP-1 transcription factors by approximately 4, 25, and 4.5 fold, respectively. In contrast, K7 exhibited no effect on the transcription of NF-κB, NF-AT, and AP-1 (Figure 4C). In agreement with our observation that K7 reduces vGPCR protein, K7 suppressed the transcription activation by vGPCR to approximately two-fold for NF-κB and AP-1, and eight-fold for NF-AT, respectively (Figure 4C). These data indicate that K7 reduces vGPCR protein expression and mitigates vGPCR-activated downstream signaling.Although our studies clearly indicate that K7 reduces vGPCR protein expression, these experiments relied on exogenous protein expression. To corroborate K7-reduced vGPCR protein expression during KSHV infection, the shRNA-mediated silencing experiments were designed to knock down K7 expression and vGPCR protein level was examined by confocal microscopy. Both K7 and vGPCR are expressed in the lytic phase during KSHV infection. Given the fact that K7 open reading frame overlaps with the transcribed region of PAN (or T1.1), four pairs of short hairpin RNA (shRNA) molecules targeting the 5′ untranslated region of K7 transcripts were cloned (Figure 4D) and lentiviral particles were produced in 293T cells. Lentivirus was then used to infect KSHV-positive BCBL-1 cells that were subsequently treated with TPA to induce KSHV lytic replication. A scrambled shRNA was used as a control for all silencing experiments. Among the shRNAs, K7 shRNA#1 and #3 significantly reduced the level of K7 transcripts, while these two shRNA molecules had no discernable alteration on mRNA levels of PAN and vGPCR, when compared to BCBL-1 cells expressing the scrambled shRNA (Figure 4D, right panels). Densitometry of RT-PCR products showed that K7 shRNA#3 and shRNA#1 had a silencing efficiency of 60% and 50% (Figure 4D). Semi-quantitative PCR analyses using serial dilution of cDNA templates further support that K7 transcripts were reduced by 60%–70% (Figure S1). Notably, the knockdown of K7 did not significantly affect cell viability after lytic induction, suggesting that additional viral proteins such as vBcl-2 and vFLIP play a redundant antiapoptotic role. BCBL-1 cells infected with lentiviruses expressing K7 shRNA#1, shRNA#3, or the scrambled shRNA were induced with TPA for KSHV lytic replication. At 48 h after induction, cells were fixed and subjected to confocal microscopy analysis to examine vGPCR protein level. As shown in Figure 5, the knockdown of K7 significantly increased vGPCR protein expression (second and third rows from the top), while the ER resident protein calreticulin was not affected. The vGPCR-positive cells increased from 20% in BCBL-1 cells expressing the scrambled shRNA to 65% in BCBL-1 cells expressing K7 shRNA#3 and 45% in BCBL-1 cells expressing K7 shRNA#1 (Figure 5, middle panels). Furthermore, merged images clearly indicate the increased vGPCR protein expression upon K7 knockdown, because image color shifted from red (calreticulin) in BCBL-1 cells expressing the scrambled shRNA to green (vGPCR) in BCBL-1 cells expressing K7 shRNA (Figure 5, right panels). Taken together, these findings support the conclusion that K7 suppresses vGPCR protein expression in tissue culture and in KSHV lytic infection.10.1371/journal.ppat.1000157.g005Figure 5Increased vGPCR protein expression by shRNA-mediated K7 knockdown.Lentivirus infection and KSHV lytic replication induced with TPA were performed as in Figure 4D. Cells were fixed and stained with anti-vGPCR (green) and anti-calreticulin (red) antibodies. vGPCR-positive and vGPCR-negative cells in 5 randomly selected fields were counted to obtain the percentage shown in the middle panels. For BCBL-1 cells induced with TPA, images to their right represent enlarged (2.5-fold) view of the boxed regions. Representative sections and their overlays are shown. Scale bar represents 12.5 µm.K7 Induces Proteasome-Dependent Degradation of vGPCROur previous publication indicated that K7 induces protein degradation dependent on the UPS [25]. To investigate the mechanism by which K7 downregulates vGPCR protein expression, the half-life of vGPCR was measured by a pulse chase experiment. Transient transfection of ECV cells expressing vGPCR or vGPCR and K7 were pulse labeled with [35S]-methionine/cysteine (Met/Cys). After extensive washing, ECV cells were chased with cold medium. Precipitated vGPCR was quantified by autoradiography and its half-life was calculated. As shown in Figure 6A, vGPCR has a half-life of about 6.5 h and K7 expression reduced its half-life to approximately 3.4 h, indicating that K7 promotes vGPCR degradation. Cellular GPCRs are 7-membrane-spanning proteins that can be degraded through the lysosome or the UPS [33]. To examine whether K7-induced vGPCR degradation is dependent on the proteasome or the lysosome, vGPCR protein stability was examined by a pulse chase experiment with either a lysosome inhibitor (chloroquine) or proteosome inhibitors (lactacystin and MG132). It was found that lactacystin and MG132, but not chloroquine, completely blocked K7-induced vGPCR degradation, indicating that this process relies on the proteolytic activity of the proteasome (Figure 6B).10.1371/journal.ppat.1000157.g006Figure 6K7 induces proteasome-dependent degradation of vGPCR.(A) K7 reduces the half-life of vGPCR. ECV cells were transfected with plasmids expressing vGPCR-Flag and K7-V5. Pulse chase, IP, and autoradiography analyses were performed as described in Materials and Methods. The fully glycosylated vGPCR band was quantified and its half-life was calculated. Data (left panel) represent three independent experiments and error bars denote standard deviation. (B) K7-induced vGPCR degradation is dependent on the proteasome. Transfection and pulse chase experiments with ECV cells were performed as described in (A) except cells were harvested at time points as indicated. The numbers at the bottom indicate the relative intensity (top row) of vGPCR band compared to the initial chase time point and standard deviation (bottom row). Data represent three independent experiments. Lac: lactacystin (10 µM); MG: MG132 (20 µM); Ch: chloroquine (50 µM). (C) K7 increases vGPCR ubiquitination. NIH3T3/lenti-puro (Vec) or NIH3T3/lent-vGPCR-Flag (vGPCR) were transfected with plasmids expressing K7-V5 and HA-tagged Ubiquitin (wt), K48R (R48), or K63R (R63). At 36 h after transfection, cells were treated with lactacystin (20 µM) for 6 h. vGPCR was precipitated with anti-Flag sepharose and eluted with Flag peptide for immunoblot with anti-HA (ubiquitin, first panel from left), or eluted with loading buffer for immunoblot with anti-Flag (vGPCR, second panel). WCLs were analyzed by immunoblot with anti-HA (ubiquitin; third panel) and anti-V5 (K7; fourth panel) antibodies. Arrowheads indicated ubiquitinated vGPCR species (second panel).Proteasome substrates are often marked with polyubiquitin chains that facilitate delivery to and subsequent degradation by the proteasome. To further corroborate the proteasome-dependence of K7-induced vGPCR degradation, vGPCR ubiquitination was examined by immunoprecipitation and immunoblot. vGPCR was precipitated with anti-Flag sepharose and analyzed by immunoblot with anti-HA (ubiquitin) antibody. Consistent with the increased degradation of vGPCR, K7 promoted vGPCR polyubiquitination in the presence of a proteasome inhibitor, lactacystin (Figure 6C, first panel from left). Recent findings have shown that K48-linkage ubiquitin chains mediate protein degradation and K63-linkage ubiquitin chains are involved in signal transduction. Thus, these ubiquitin mutants were included in the vGPCR ubiquitination assay. Indeed, the K48R mutant, but not the K63R mutant, completely abolished vGPCR ubiquitination induced by K7 (Figure 6C). Of note, the protein level of precipitated vGPCR and vGPCR in whole cell lysate in the presence of K7 is significantly lower than vGPCR alone (Figure 6C, second panel, lanes 2–5, and Figure S2). These data collectively support the conclusion that K7 increases vGPCR ubiquitination and promotes its proteasomeal degradation.K7 Retains vGPCR in the ER to Induce its DegradationTo further define the molecular action of K7 in inducing vGPCR degradation, vGPCR intracellular localization was analyzed by confocal microscopy using human HeLa cells. Consistent with a previous report [7], vGPCR primarily localized to the TGN stained by anti-TGN46 antibody (Figure 7A). Upon K7 expression, vGPCR localized to intracellular structures that resemble the ER and nuclear membrane (Figure 7B), suggesting that K7 retains vGPCR in the ER compartment. Indeed, HeLa cells expressing both K7 and vGPCR revealed that these two proteins colocalized significantly with protein disulfide isomerase (PDI), an ER resident protein (Figure 7C), supporting the notion that K7 retains vGPCR in the ER. Furthermore, K7 expression reduced vGPCR localization in the TGN when intracellular distribution of vGPCR and K7 was examined in relation to TGN46 (Figure 7D). These results clearly indicate that K7 retains vGPCR in the ER and suggest that K7 induces vGPCR degradation via the ER-associated degradation pathway.10.1371/journal.ppat.1000157.g007Figure 7K7 retains vGPCR in the ER.(A) vGPCR localizes to the TGN. HeLa cells were transfected with plasmids containing vGPCR-Flag. At 16 h after transfection, cells were fixed and stained with mouse anti-Flag (vGPCR, green) and sheep anti-TGN46 (red) antibodies. For both (A) and (B), images at the bottom represent enlarged (3-fold) view of the boxed regions. Representative sections and their overlays are shown. Scale bar represents 12.5 µm. (B) K7 alters vGPCR intracellular localization. HeLa cells were transfected with plasmids containing vGPCR-Flag and K7-V5, and fixed as in (A). Cells were stained with rabbit anti-Flag (vGPCR, green) and mouse anti-V5 (K7, red) antibodies. (C) K7 retains vGPCR in the ER. HeLa cells were transfected with plasmids expressing HA-vGPCR and K7-V5, and fixed as described in (A). Cells were stained with rabbit antibody to protein disulfide isomerase (PDI, blue) and mouse anti-V5 (K7, red) antibody. After staining with corresponding secondary antibody and extensive washing, cells were further stained with Alexa 488-conjugated anti-HA antibody (vGPCR, green). Images on the right represent enlarged (3-fold) view of the boxed regions. Representative sections and their overlays are shown. Scale bar represents 12.5 µm. (D) The intracellular localization of K7 and vGPCR in relation to the TGN. HeLa cells were transfected with plasmids containing vGPCR-Flag and K7-V5. Cells were fixed and stained with mouse monoclonal anti-V5 (K7, green) and sheep anti-TGN46 (red) (left panels), or rabbit polyclonal anti-Flag (vGPCR, green) and sheep anti-TGN (red) (right panels). Representative images and their overlays are shown. Scale bar represents 12.5 µm.vGPCR and K7 Inhibit Cell Growth in vitroTo examine K7's effect on vGPCR biological functions, NIH3T3 cell lines stably expressing K7, vGPCR, and vGPCR+K7 were established with lentivirus infection. As shown in Figure 8A, K7 detectably reduced vGPCR protein expression without affecting its mRNA levels (Figure 8B). Of note, vGPCR did not further increase K7 protein after treatment by the proteasome inhibitor MG132 (Figure 8A). During the course to establish these stable cell lines, we noticed that NIH3T3 cells expressing vGPCR grow more slowly than the control NIH3T3 cells. In contrast to what was reported [34], NIH3T3/vGPCR cells had a doubling time of approximately 31 h that is significantly longer than 22.6 h of NIH3T3/vector cells. RT-PCR analysis indicated that vGPCR is expressed at similar levels in NIH3T3, and reactivated BCBL-1 and JSC-1 cells (Figure S3). This observation rules out the possibility that the inhibitory effect on cell growth is due to over-expression. Interestingly, K7 expression also increased NIH3T3 doubling time to roughly 28.2 h. Consistent with K7-reduced vGPCR protein expression, K7 co-expression slightly decreases the doubling time of NIH3T3 cells to 30 h (Figure 8C). Due to K7's inhibitory effect on cell growth and vGPCR-increased K7 expression (unpublished data), the subtle difference in cell growth may be significant. Given the inhibitory effect of vGPCR on cell growth, we suspect that NIH3T3 cells expressing higher vGPCR will gradually decrease when continuously cultured without selection. To test this, NIH3T3/vGPCR and NIH3T3/vGPCR+K7 cells were passaged for a week and RT-PCR analyses were performed to assess the mRNA levels of vGPCR. Indeed, the vGPCR mRNA level significantly decreased after 1 wk of passage and K7 reduced the vGPCR loss (Figure 8D). Semi-quantitative RT-PCR and real-time PCR analyses revealed that the vGPCR mRNA in NIH3T3/vGPCR+K7 was approximately 5-fold of that in NIH3T3/vGPCR cells at day 7 (Figure 8E and S4). The rapid loss of vGPCR transcripts suggests that NIH3T3 cells that lost vGPCR have a growth advantage.10.1371/journal.ppat.1000157.g008Figure 8The effect of vGPCR and K7 on cell growth in vitro.(A) K7 reduces vGPCR protein expression in stable NIH3T3 cells. Whole cell lysates of NIH3T3/vector, NIH3T3/vGPCR, NIH3T3/K7, and NIH3T3/vGPCR+K7 were precipitated with anti-HA and immunoblotted with anti-HA antibody (top panel). For K7 expression, above stable cells were treated with MG132 for 6 h before harvest, K7 was precipitated with anti-Flag and analyzed by immunoblot with anti-Flag antibody (bottom panel). (B) K7 does not reduce vGPCR mRNA level. The mRNA level of vGPCR in stable cell lines as described in (A) was analyzed by RT-PCR and β-actin PCR product serves as a loading control. (C) vGPCR and K7 inhibit cell growth. NIH3T3 stable cell lines described in (A) were cultured in complete DMEM containing puromycin (1 µg/ml) and counted at 24 h and 48 h. The doubling time was measured as derscribed in Materials and Methods. Data represent three independent measurements and error bars denote standard deviation. (D) K7 reduces the loss of vGPCR transcripts in NIH3T3 cells. NIH3T3 stable cell lines as described in (A) were passaged up to 7 d and RT-PCR analyses were performed with primers specific for vGPCR, β-actin, and K7. (E) PCR analyses with serial dilution of cDNA templates from NIH3T3/vGPCR and NIH3T3/vGPCR+K7 were performed using vGPCR-specific primers. The ratio denotes fold of serial dilutions. (F) The effect of vGPCR and K7 on apoptosis. NIH3T3 stable cell lines were treated with TNF-α (5 ng/ml) and CHX (1 µg/ml) for 24 h; cell viability measured by trypan blue staining is shown. Data represent 3 independent experiments, and error bars denote standard deviation; *p<0.03 relative to NIH3T3/vector cells as calculated by Student's t-test.We and others have shown that K7 inhibits apoptosis induced by various stress stimulations [25]–[27]. To examine whether vGPCR affects K7's antiapoptotic function, NIH3T3 stable cells were stimulated with TNF-α and cyclohexamide and cell viability was measured by trypan blue staining as described previously [25]. It was found that vGPCR expression had no significant effect on cell survival upon TNF-α stimulation, while K7 expression increased cell survival rate by 20% compared to NIH3T3/vector cells (Figure 8F). Interestingly, vGPCR co-expression with K7 further promotes cell survival rate by approximately 30%, indicating that vGPCR potentiates K7's antiapoptotic effect. This is consistent with our observation that vGPCR increases K7 protein expression (unpublished data). These results indicate that K7 reduces vGPCR-induced stress and suggest that K7 likely co-operates with vGPCR to promote cell survival during KSHV lytic replication.K7 Negatively Regulates vGPCR TumorigenicityIn a mouse pathogenesis model, vGPCR is sufficient to induce tumor formation in nude mice and vGPCR transgenic mice developed lesions that resemble human KS, suggesting its potential contribution to KSHV-associated malignancies [17],[18],[34]. To assess K7's effect on vGPCR tumorigenicity, NIH3T3 stable cells expressing K7, vGPCR, or vGPCR+K7 were mixed with NIH3T3 cells and colony formation on soft agar was examined. Similar to the human cytomegalovirus US28 [35], vGPCR-expressing cells stimulated anchorage-independent growth of NIH3T3 cells, whereas neither NIH3T3/vector, nor NIH3T3/K7 cells supported colony formation (Figure 9A). In support of the observation that K7 suppressed vGPCR protein expression, NIH3T3/vGPCR+K7 cells formed smaller colonies than NIH3T3/vGPCR cells (Figure 9A, left panels). Furthermore, K7 expression also reduced the number of colonies from 258 of NIH3T3/vGPCR to 131 of NIH3T3/vGPCR+K7 (Figure 9A, right diagram). To further investigate K7's effect on vGPCR tumorigenicity in vivo, these stably transfected cells were injected into nude mice and tumor growth was assessed. Mice injected with NIH3T3/vGPCR developed visible tumors within two weeks and all mice harbored tumors after 6 wk. Neither NIH3T3/vector cells nor NIH3T3/K7 cells induced apparent tumor in nude mice. In agreement with results from the soft agar assay, K7 significantly reduced vGPCR capacity to promote tumor growth in nude mice as shown by the number of mice harboring tumor and tumor weight (Figure 9B). All four nude mice injected with NIH3T3/vGPCR developed tumors after 6 wk, whereas only two mice injected with NIH3T3/vGPCR+K7 developed tumors, which were substantially smaller (Figure 9B). The mean weight of tumors derived from NIH3T3/vGPCR cells is approximately 8-fold higher than that of tumors derived from NIH3T3/vGPCR+K7 cells (Figure 9B and unpublished data). Interestingly, we found that K7 transcripts were expressed at a higher level in the smaller tumor than the bigger tumor, suggesting that K7 inhibits the vGPCR-dependent tumor growth in vivo (Figure 9C). This result is consistent with the observation that K7 expression reduces vGPCR tumorigenicity (Figure 9B). In contrast, the vGPCR transcript was expressed more abundantly in tumors derived from NIH3T3/vGPCR+K7 cells than those derived from NIH3T3/vGPCR cells (Figure 9C). This likely represents the relative expression of vGPCR in stable NIH3T3 cells before mice injection. Overall, K7 negatively regulates vGPCR tumorigenicity in vitro by a soft agar assay and in vivo in nude mice.10.1371/journal.ppat.1000157.g009Figure 9K7 negatively regulates vGPCR tumorigenicity.(A) K7 reduces vGPCR activity to stimulate anchorage-independent growth of NIH3T3 cells. Colonies under microscope were photographed (left panels, 4×) or counted (right graph) after a 2-wk incubation. Data represent 3 independent experiments. Error bars denote standard deviation; *p<0.02 relative to NIH3T3/vGPCR cells as calculated by Student's t-test. (B) K7 reduces vGPCR tumorigenicity in nude mice. Cells were injected into nude mice subcutaneously, and mice were killed and photographed (left panel) 6 wk later. Tumor weight was measured (right graph). The numbers in parenthesis indicate the number of mice developed tumor among 4 tested animals. Arrows indicate location of tumor and data represent 4 independent measurements for each group. (C) vGPCR and K7 expression in tumors. RT-PCR analyses were performed as described in Materials and Methods using primers specific for vGPCR, K7, and cellular β-actin. PCR products resolved on agarose gel were photographed. B, the bigger tumor; S, the smaller tumor; Pos, a positive control of K7.DiscussionWe report here that KSHV K7 interacts specifically with vGPCR and induces the rapid degradation of vGPCR, thereby reducing vGPCR protein expression. The putative K7 TM domain is necessary and sufficient for its interaction with vGPCR, indicating a specific interaction between vGPCR and K7. However, the K7/vGPCR interaction may involve multiple residues within the putative TM domain of K7 because further mutational analyses within this domain failed to identify critical residues that are essential for this interaction (unpublished data). Alternatively, additional cellular components such as membrane proteins or lipids could be involved, as our co-IP procedure does not exclude this possibility. Nevertheless, these data support the conclusion that K7 interacts specifically with vGPCR.We have previously shown that K7 antagonizes cellular PLIC1, a factor that inhibits proteasome-mediated protein degradation, and induces rapid degradation of p53 and IκB [25]. Our current study enlists vGPCR as an additional proteasome substrate whose degradation is accelerated by K7. The specificity of K7-induced degradation appears to be derived from an interaction with either a proteasome substrate such as vGPCR or a key component of the UPS pathway such as PLIC1. It is possible that binding of K7 to cellular PLIC1 also contributes to K7-dependent reduced expression of vGPCR, given that PLIC1 has been shown to promote protein expression of multiple transmembrane proteins [36],[37]. Indeed, we have observed that PLIC1 overexpression increases vGPCR protein, while the knockdown of PLIC1 by shRNA-mediated silencing greatly reduces vGPCR protein expression. These data indicate that PLIC1 is a positive regulator for vGPCR expression (unpublished data). Future experiments will determine whether K7 binding to PLIC1 is sufficient for suppressing vGPCR protein expression.Confocal microscopy analyses and biochemical assays examining vGPCR protein degradation support the conclusion that K7 retains vGPCR in the ER and allows vGPCR to be removed by the proteasome. The rapid degradation of vGPCR induced by K7 also correlates with increased ubiquitination upon treatment with a proteasome inhibitor. vGPCR appears to carry polyubiquitin chains and K7-induced polyubiquitination of vGPCR is specifically inhibited by the K48R ubiquitin mutant, but not by the K63R ubiquitin mutant (Figure 6C). Interestingly, the K63R mutant significantly increased unmodified- as well as ubiquitinated-vGPCR protein. This is likely due to the inhibitory effect of K63R ubiquitin on vGPCR signaling that is presumably coupled to vGPCR degradation. For example, the K63R mutant may inhibit signaling downstream vGPCR such as NF-κB activation, therefore stabilizing vGPCR. Alternatively, vGPCR polyubiquitin chains may contain a mixture of K63- and K48-linkages. The fact that the K48R mutant abolished, while the K63R mutant increased vGPCR ubiquitination suggests that K48-linkage is necessary to initiate ubiquitination, whereas K63-linkage is important for degradation. These intriguing possibilities are not mutually exclusive and require further experimental investigation. Our data, however, do not exclude the possibility that vGPCR undergoes ubquitination-independent proteasomeal degradation. In transfected cells, K7 consistently altered vGPCR intracellular distribution, showing a more diffused ER/nuclear membrane pattern that was confirmed by staining with anti-PDI antibody. This observation suggests that K7 retains vGPCR in the ER in order to induce vGPCR degradation. This also implies that K7 likely engages the ERAD pathway to facilitate vGPCR degradation in similar ways employed by human cytomegalovirus US11 and murine γ-herpesvirus 68 mK3 [23],[24],[38],[39]. Future experiments will be directed to test whether K7-induced protein degradation is dependent on any critical components of the ERAD pathway.Interaction with K7 was found to reduce vGPCR protein, thereby dampening vGPCR-mediated signaling. Both vGPCR and K7 are expressed during KSHV lytic replication and it appears that K7 and vGPCR share an identical or overlapped expression profile. The observation that the K7 transcript peaks at a later time point than the vGPCR transcript raises the possibility that K7 serves as a negative regulatory factor to shut off vGPCR protein during KSHV lytic infection. Indeed, the knockdown of K7 by shRNA-mediated silencing increased vGPCR protein without altering vGPCR transcription level in BCBL-1 cells that are induced for KSHV lytic replication (Figure 5). Interestingly, K7 protein expression was substantially increased when co-expressed with vGPCR (unpublished data), revealing a negative feedback loop that culminates in dampening vGPCR protein expression. These observations are consistent with the notion that diverse regulatory mechanisms operate to achieve a temporary expression of vGPCR in KSHV infection. In addition to the K7-reduced vGPCR expression, known mechanisms also include the bicistronic translation and the vMIP-mediated regulation [18],[40]. Interestingly, modulation by its cognate chemokines is important for vGPCR tumorigenicity in transgenic mice [18]. Our findings that K7 interacts with vGPCR and directs it for proteasome-mediated degradation further support the notion that KSHV has evolved intricate mechanisms to regulate vGPCR activity. Additionally, K7 expression provides antiapoptotic activity under various conditions [25]–[27] and vGPCR co-expression potentiates K7's antiapoptotic activity (Figure 8F). This implies that K7 can cooperate with vGPCR in the tumorigenesis of KSHV infection, analogous to the paradigm in which Bcl-2 cooperates with c-myc [41]. However, our transformation assay in vitro and tumor growth in nude mice ruled out this possibility. Together with the biscitronic translation and modulation by vMIP chemokines, vGPCR downregulation by K7 raises an intriguing speculation that KSHV has evolved these mechanisms to monitor vGPCR pathogenicity, permitting a persistent infection within its host.K7 expression suppressed vGPCR transformation on soft agar assay and more pronouncedly reduced vGPCR tumorigenicity in nude mice. Although K7 reduced vGPCR protein expression by approximately two-fold (Figures 6A and 8A), it was found that K7 inhibited vGPCR tumorigenicity by more than 8-fold (Figure 9B). This suggests that additional mechanisms, other than reduced protein expression, may contribute to K7's effect on vGPCR tumorigenicity. One likely mechanism is a K7-dependent retention of vGPCR in the ER, given that vGPCR predominantly localizes to the TGN and cell surface under normal circumstances. Conceivably, vGPCR functions in the TGN and on the cell surface are abolished by K7 expression. Interestingly, we have found that vGPCR is tyrosine sulfated in the TGN and tyrosine sulfation is important for vGPCR tumorigenicity (unpublished data). In addition to tyrosine sulfation, post-translational modifications in the ER (such as ubiquitination and glycosylation) altered by K7 may cause impaired vGPCR signaling and tumorigenicity. These mechanisms are not mutually exclusive and warrant further investigations of post-translational events underlying vGPCR tumorigenicity.Mounting evidence points to vGPCR expression inducing a stress in mammalian cells including KSHV infected PEL cells [6],[13]. Indeed, our vGPCR-expressing NIH3T3 cells have a longer doubling time than control NIH3T3/vector cells (Figure 8C). Furthermore, NIH3T3/vGPCR cells gradually lost vGPCR expression when continuously passaged in vitro, suggesting that NIH3T3 cells gain a growth advantage by reducing vGPCR expression. Indeed, K7 alleviated vGPCR-mediated inhibition of NIH3T3 growth and the rate of vGPCR transcript loss (Figure 8C–8E). In contrast, vGPCR expression was necessary for tumorigenicity in nude mice, and K7-reduced vGPCR expression correlated with less transformation in vitro and tumorigenicity in vivo (Figure 9A and 9B). Interestingly, the endothelial progenitor cell line containing Bac36 (a KSHV Bacmid) behaves similarly to NIH3T3/vGPCR cells, demonstrating reduced cell growth in vitro and increased tumor formation in vivo [42]. The seemingly paradox between in vitro stress and in vivo tumorigenicity may be explained by a paracrine mechanism supported by accumulating studies [9],[43],[44]. In fact, vGPCR-induced tumor formation is highly dependent on growth factors and chemokines that stimulate the angio-proliferation of neighboring cells [43],[44]. In KS lesions, vGPCR expressing cells presumably stimulate the proliferation of spindle cells that are latently infected by KSHV. The fact that slower growth of NIH3T3 stable cell lines in vitro correlates with higher tumorigenicity in vivo suggests that the nude mice model primarily assesses the paracrine function of vGPCR. This is also supported by our in vitro transformation assay where the proliferation of regular NIH3T3 cells was examined in the presence of NIH3T3/vGPCR cells (Figure 9A). Additionally, it is not unprecedented that oncogenic proteins exploit cellular stress responses to induce tumor formation. Perhaps, these stress responses represent various barriers that oncogenesis has to overcome. For example, H-RAS triggers the ER-associated unfolded protein response, cellular senescence and sensitizes cells to apoptosis [45],[46]. Similarly, the myc-mediated stress is overcome by Bcl-2 expression [41]. Taken together, the fact that the stress in tissue culture accompanies the tumorigenicity in vivo for many oncogenic proteins suggests that the stress response may serve as an indicator for tumorigenicity in vivo. Similar to vGPCR, K7 also reduces NIH3T3 growth and it will be interesting to examine K7's tumorigenicity in nude mice.All members of the beta- and gamma-herpesvirus family encode up to four GPCRs in their genomes. Some of them have been shown to constitutively activate signaling events downstream of various G proteins (for review see [47]). Although it was demonstrated that KSHV vGPCR can be uncoupled from downstream signal activation by overexpressed G protein-coupled receptor kinase 5 and arrestins [48], it is largely unknown how these unconventional viral GPCRs are differentially regulated as opposed to cellular GPCRs under normal physiological conditions. This study established an example of post-translational regulation of vGPCR pathogenicity by which a viral factor-induced degradation greatly influences its tumorigenicity. Similar regulatory mechanisms may exist for other viral GPCRs of herpesviruses. Therefore, viral factors that modulate these viral GPCRs likely have a profound effect on various biological activities during herpesvirus infection.Materials and MethodsPlasmidsUnless specified, all constructs were derived from pcDNA5/FRT/TO (Invitrogen). A DNA fragment corresponding to the KSHV vGPCR was amplified from BCBL-1 genomic DNA by polymerase chain reaction (PCR) and cloned into pcDNA5/FRT/TO between BamHI and XhoI. For protein expression, either the HA epitope or the Flag epitope was inserted upstream or downstream of vGPCR coding sequence, respectively. Plasmids expressing wild-type and mutant K7 polypeptides were described in previous publications [25],[26]. For lentiviral expression, K7-Flag was cloned into pCDH-EF-puro (System Bioscience) between EcoRI and BamHI. HA-vGPCR was digested with EcoRI and BglII, and ligated to pCDH-EF-puro or pCDH-EF-CopGFP that was digested with EcoRI and BamHI. To generate the K7TMStpC, the K7 transmembrane (TM) domain was replaced with a heterologous TM segment of Stp C by PCR-based mutagenesis using overlapping PCR primers. All constructs were sequenced for verification.For the shRNA-mediated knockdown of K7, four pairs of synthetic DNA oligos were annealed and cloned into pLKO.1 (Sigma) that was digested with AgeI and EcoRI. The pLKO.1 expressing the scrambled shRNA was purchased from Sigma. Plasmids expressing HA-tagged wt and mutant ubiquitin were a kindly gift from Dr. James Z.J. Chen (UT Southwestern).Cell Culture and TransfectionHEK293T (293T), HeLa, and NIH3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 5 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. BJAB, JSC-1, BCBL-1, and BCBL-1/T-Rex_Rta cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, 5 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. BCBL-1 cells were treated with phorbol-12-teradecanoate-13-acetate (TPA, 20 ng/ml) to induce lytic replication. HeLa cells were transfected with Fugene 6 (Roche), 293T cells were transfected with calcium phosphate (Clontech), ECV cells were transfected with lipofectamine (Invitrogen), and BJAB cells were transfected with electroporation at 220 V/975 µF. The stable BCBL-1/T-Rex_Rta inducible cell line was maintained and induced as previously described [30].Immunoprecipitation and ImmunoblotImmunoprecipitation and immuno-blot analyses were performed as previously described [26]. Immunoblot detection was performed with anti-V5 antibody (1∶5000, Invitrogen), anti-Flag M2 antibody (1∶5000, Sigma), anti-HA (1∶2000, Covance), anti-tubulin (1∶250, Santa Cruz), or anti-actin (1∶30,000, Abcam). Proteins were visualized with chemical luminescent detection reagent (Pierce) and a Fuji LAS-3000 camera.Reverse Transcriptase (RT)-PCROne million KSHV latently infected BCBL-1 or JSC-1 cells were treated with either TPA (20 ng/ml) to induce viral lytic replication and harvested at various time points. Alternatively, KSHV lytic replication was induced in BCBL-1/T-Rex_Rta stable cells with doxycline (1 µg/ml). Total RNA was extracted with RNAeasy column (Qiagen, CA) and digested with DNase I at 37°C for 1 h. After phenol/chloroform extraction, 1 µg of total RNA was used for first-strand cDNA synthesis using an oligo(dT) primer. Then, 1 µl of cDNA was added to 19 µl of PCR mixture and gene of interest was amplified using specific primers. PCR products were resolved on agarose gel and photographed. For each gene of interest, dilution of original cDNA and cycle number were determined to warrant that PCR products were generated within the linear range of PCR reaction. Total RNA from tumor tissues was extracted with triazol (Invitrogen, CA) and ethanol precipitation as previously described.Protein StabilityTransiently transfected ECV cells were pulse labeled with 35S-methionine/cysteine (Met/Cys) for 30 min. After extensive washing with phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4), cells were chased with cold medium up to 16 h. At various time points, cells were harvested, washed with cold PBS, resuspended in RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP40, 5 mM EDTA/EGTA), and lysed by passing through 26-G syringe for 15 times. Centrifuged supernatant was pre-cleared with protein A/G agarose and mixed with 2 µg of anti-Flag M2 antibody. Incubation was carried out at 4°C for 4–6 h. Protein A/G agarose was added and incubation was further extended for 90 min. After extensive washing with RIPA buffer, precipitated proteins were resolved by SDS-PAGE and analyzed by autoradiography. The relative intensity of a selected protein band was quantified and its half-life was calculated. When vGPCR degradation route was investigated, 20 µM of lactacystin and MG132 (proteasome inhibitors) or 50 µM of chloriquine (a lysosome inhibitor) was added during the chase period. IP and autoradiography were performed similarly.Luciferase Reporter AssayThe reporter cocktail consists of plasmids expressing fire fly luciferase (50 ng/µl) and β-galactosidase (100 ng/µl). While β-galactosidase expression is driven by a housekeeping glucophosphokinase promoter, the expression of fire fly luciferase is under control of response elements of NF-κB, NF-AT, and AP-1 transcription factor. 293T cells were transiently transfected with 2.5 µl of reporter cocktail, and 200 ng of plasmids expressing vGPCR and K7. For each transfection, the total amount of plasmid was balanced with an empty vector (pcDNA5/FRT/TO). At 36 h after transfection, cells were harvested and lysed on ice. Centrifuged supernatant was used to measure luciferase and β-galactosidase activity according to manufacturer's protocol (Promega).Apoptosis AssayNIH3T3 stable cells were treated with vehicle (DMSO), cyclohexamide (CHX, 1 µg/ml), or TNF-α (5 ng/ml) plus CHX (1 µg/ml) for 24 hours. Cells were harvested and live cells were scored by trypan blue staining as previously described [25]. Viable cells treated with drugs divided by viable cells treated by DMSO was used to obtain cell viability in percentage.Immunofluorescence MicroscopyBJAB, HeLa, or BCBL-1 cells were fixed with paraformaldehyde and permeabilized with Triton X-100 (0.2% in PBS). After stained with primary and secondary antibodies, cells were analyzed by immunofluorescence microscopy as previously described [26],[49]. vGPCR in BCBL-1 cells was detected with a gift rabbit polyclonal antibody provided by Dr. Gary Hayward [7]. For commercial antibodies, mouse monoclonal anti-Flag antibody (1∶1500), rabbit polyclonal anti-Flag antibody (1∶400, Sigma), mouse monoclonal anti-V5 antibody (1∶500, Invitrogen), sheep anti-TGN46 (1∶200, Serotec), rabbit anti-PDI (1∶200, Calbiochem) were used. All conjugated secondary antibodies were obtained from Molecular Probes and diluted at 1∶1000 (Alexa 488-conjugated) or 1∶500 (Alexa 568 or Alexa 647-conjugated).Knockdown of K7 by shRNA-mediated silencingFour shRNA seuquences were designed using Dharmacon software and cloned into pLKO.1. These sequences are: 5′ TCATCCGTATTGTGTATAT 3′; 5′ CATCGTGAGTTGGTTAATA 3′; 5′ TGGCTACTCTGCTCGATTA 3′; 5�� TGAAGGATGATGTTAATGA 3′. Together with packaging plasmids DR8.9 and VSV-G, pLKO.1 plasmids expressing various K7 shRNA molecules were transfected into 293T cells with Fugene 6 (Roche). Lentivirus expressing the scrambled shRNA was produced similarly. Filtered lentivirus was used to infect BCBL-1 cells at 20 MOI in medium containing 10 µg/ml polybrene. To increase infection efficiency, cells were centrifuged at 1,800 rpm, 30°C for 1 h and incubation was further extended for up to 12 h. The infection was repeated once and cells were selected with puromycin at 1 µg/ml. At 48 h later, BCBL-1 cells were treated with TPA (20 ng/ml) to induce KSHV lytic replication.Cell Growth and Soft Agar AssayNIH3T3 cells were infected with lentiviruses to establish stable cell lines expressing K7 with puromycin selection. Then, NIH3T3/puro and NIH3T3/K7 cells were further infected with lentivirus expressing GFP or vGPCR. This lentiviral infection was repeated once to obtain stable cells expressing K7, vGPCR, or vGPCR and K7. Cells were cultured in complete DMEM medium containing puromycin (1 µg/ml). To measure the doubling time, 2×105 cells were plated and cells were counted at 24 h and 48 h later. The soft agar assay was performed as described by Liang et al [50]. Stable NIH3T3 cells (5×104) were mixed with 1×105 normal NIH3T3 cells and cultured for two weeks in regular culture medium without puromycin.Tumor Formation In VivoAll animal experiments were performed according to the National Institutes of Health principles of laboratory animal care and approved by the University of Texas Southwestern Medical Center. Stable NIH3T3 cells (3×106/site) expressing GFP, K7, vGPCR, or vGPCR and K7 were injected subcutaneously into the flanks of 6- to 8-wk-old mice (athymic, nu/nu, Jackson Laboratory).Supporting InformationFigure S1Knockdown of K7 During KSHV Lytic Reactivation. (A) The knockdown efficiency of K7 by shRNA-mediated silencing. BCBL-1 cells were infected with lentivirus and induced for lytic reactivation as diagrammed in Figure 4D. RT-PCR analyses were performed with serial dilution of cDNA template (shown in Figure 4D) as indicated by the ratio. (B) K7 knockdown on cell viability in KSHV lytic reactivated cells. Lentivirus infection and lytic reactivation were performed as in (A). Cells were harvested and cell viability was assessed by trypan blue staining.(0.12 MB TIF)Click here for additional data file.Figure S2Reduced Expression of vGPCR by K7 Mutants and Lactacystin Treatment. (A) Glycosylation and ubiquitination are dispensable for K7’s ability to reduce vGPCR protein expression. Whole cell lysates of ECV cells transfected with plasmids containing vGPCR and K7 as indicated were analyzed by immunoblot with anti-Flag (vGPCR, top panel), anti-actin (middle panel), and anti-V5 (K7, bottom panel). Of note, the K7(5K>R) and K7(N108Q) carry 6xHIS downstream of the V5 epitope that reduces their detection by immunoblot. Ub, ubiquitinated K7; gly, glycosylated K7. (B) The effect of lactacystin treatment on K7-reduced vGPCR expression. Human ECV cells were transfected with plasmids containing vGPCR or K7 and treated for 6 h with DMSO or lactacystin (20 μM). Whole cell lysates were analyzed by immunoblot with anti-Flag (vGPCR, top panel), anti-actin (middle panel), and anti-V5 (bottom panel).(0.17 MB TIF)Click here for additional data file.Figure S3Relative Expression Levels of vGPCR in Reactivated BCBL-1 Cells and NIH3T3 Stable Cells. Total RNA was extracted and RT-PCR analyses were performed as described in Materials and Methods using gene specific primers to vGPCR and β-actin. BCBL-1 cells were induced for lytic reactivation by TPA (20 ng/ml, 48 h) or BCBL-1/T-Rex_Rta cells were treated with doxycycline (1 μg/ml, 72 h) before harvest. No PCR product was etected for controls without RT (data not shown). The primers for β-actin locate within a highly conserved region of the human and mouse β-actin gene.(0.11 MB TIF)Click here for additional data file.Figure S4vGPCR mRNA Levels in NIH3T3 Stable Cells by Real-Time PCR. The primers were designed using Primer Express v1.5 (Applied Biosystems). 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+}

batch_9/PMC2529405.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2529405",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529405\nAUTHORS: Kenji Hashimoto, Shingo Nishiyama, Hiroyuki Ohba, Masaaki Matsuo, Tatsuhiko Kobashi, Makoto Takahagi, Masaomi Iyo, Takeru Kitashoji, Hideo Tsukada\n\nABSTRACT:\nBackgroundThe α7 nicotinic acetylcholine receptors (nAChRs) play an important role in the pathophysiology of neuropsychiatric diseases such as schizophrenia and Alzheimer's disease. However, there are currently no suitable positron emission tomography (PET) radioligands for imaging α7 nAChRs in the intact human brain. Here we report the novel PET radioligand [11C]CHIBA-1001 for in vivo imaging of α7 nAChRs in the non-human primate brain.Methodology/Principal FindingsA receptor binding assay showed that CHIBA-1001 was a highly selective ligand at α7 nAChRs. Using conscious monkeys, we found that the distribution of radioactivity in the monkey brain after intravenous administration of [11C]CHIBA-1001 was consistent with the regional distribution of α7 nAChRs in the monkey brain. The distribution of radioactivity in the brain regions after intravenous administration of [11C]CHIBA-1001 was blocked by pretreatment with the selective α7 nAChR agonist SSR180711 (5.0 mg/kg). However, the distribution of [11C]CHIBA-1001 was not altered by pretreatment with the selective α4β2 nAChR agonist A85380 (1.0 mg/kg). Interestingly, the binding of [11C]CHIBA-1001 in the frontal cortex of the monkey brain was significantly decreased by subchronic administration of the N-methyl-D-aspartate (NMDA) receptor antagonist phencyclidine (0.3 mg/kg, twice a day for 13 days); which is a non-human primate model of schizophrenia.Conclusions/SignificanceThe present findings suggest that [11C]CHIBA-1001 could be a novel useful PET ligand for in vivo study of the receptor occupancy and pathophysiology of α7 nAChRs in the intact brain of patients with neuropsychiatric diseases such as schizophrenia and Alzheimer's disease.\n\nBODY:\nIntroductionThe most of neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels composed of α and β subunits that assemble to form pentamers with a variety of physiological and pharmacological properties. Two major subtypes exist in the brain, those comprised of α4β2 and those comprised of α7 subunits [1], [2]. The former contribute >90% of the high affinity binding sites for nicotine in the rat brain, and the low affinity binding sites (α7 subunits) for nicotine are recognized by their nanomolar affinity for α-bungarotoxin. Several lines of evidence suggest that α7 nAChRs play a role in the pathophysiology of neuropsychiatric diseases such as schizophrenia, Alzheimer's disease, anxiety, depression, and drug addiction, and that α7 nAChRs are the most attractive therapeutic targets for these diseases [3]–[11]. Studies using postmortem human brain samples have demonstrated alterations in the levels of α7 nAChRs in the brains of patients with schizophrenia [12], [13] and Alzheimer's disease [14]–[16]. It is thus of great interest to examine whether α7 nAChRs are altered in the living brain of patients with neuropsychiatric diseases such as schizophrenia and Alzheimer's disease. It is also of interest to measure the receptor occupancy of potential therapeutic α7 nAChR drugs in the intact human brain.The distribution, density, and activity of receptors in the living brain can be visualized noninvasively by radioligands labeled for positron emission tomography (PET), and the receptor binding can be quantified by appropriate tracer kinetic models, which can be modified and simplified for particular applications [17]–[19]. The PET ligands ([11C]nicotine and 2-[18F]fluoro-A85380) for α4β2 nAChRs have been used in clinical studies [20]–[22]. However, there have been no clinical studies using PET ligands for α7 nAChRs in the human brain. Therefore, it is very important to develop a safe PET ligand for quantification of α7 nAChRs in the human brain. Very recently, researchers at Sanofi-Aventis developed the novel selective α7 nAChR agonist SSR180711 (4-bromophenyl 1,4-diazabicyclo(3.2.2) nonane-4-carboxylate)(Figure 1) [23], [24], which is under clinical study.10.1371/journal.pone.0003231.g001Figure 1Synthesis of [76Br[SSR180711 and [11C]CHIBA-1001.Here, we developed two novel PET ligands, [76Br]SSR180711 and [11C]CHIBA-1001, for in vivo imaging of α7 nAChRs in the human brain. Using conscious monkeys, we evaluated the two PET ligands for in vivo imaging of α7 nAChRs in the non-human primate brain. Furthermore, we evaluated the usefulness of [11C]CHIBA-1001 in a non-human primate model of schizophrenia.ResultsReceptor affinity and specificitySSR180711 displaced specific binding of [3H]α-bungarotoxin to the rat and human α7 nAChRs with Ki values of 22 and 14 nM, respectively [23], and SSR180711 (10 µM) was found to be devoid of activity (inhibition lower than 50%) for a 100 standard receptor binding profile [23]. In our assay, the IC50 values of SSR180711 and CHIBA-1001 for [125I]α-bungarotoxin (0.5 nM) binding to the rat brain homogenates were 24.9 and 45.8 nM, respectively. Furthermore, CHIBA-1001 (1 µM) was found to be devoid of activity (inhibition lower than 50%) for a 28 standard receptor binding profile (See Supplemental Table S1 and S2).Synthesis of [76Br]SSR180711 and [11C]CHIBA1001[76Br]SSR180711 and [11C]CHIBA-1001 were synthesized by bromination and methylation of the precursor, respectively (Figure 1). The radiochemical purity and specific activity of [76Br]SSR180711 were approximately 100% and 8.11±1.65 GBq/µmol (mean±SD of 9 experiments), respectively. The radiochemical yields and yields of [76Br]SSR180711 were 16.7±6.14% and 0.21±0.09 GBq (mean±SD of 9 experiments), respectively. The radiochemical purity and specific activity of [11C]CHIBA-1001 were 98.6±1.68% (mean±SD of 12 experiments) and 343.7±36.1 GBq/µmol (mean±SD of 12 experiments), respectively. The radiochemical yields and yields of [11C]CHIBA-1001 were 9.49±1.45% and 1.88±0.33 GBq (mean±SD of 12 experiments), respectively.Conscious monkey PET studiesBaseline PET scans showed rapid brain penetration and accumulation of [76Br]SSR180711 in the monkey brain (Figures 2–\n4). The peak time of radioactivity in the hippocampus was about 60 min after administration of the radioligand. Furthermore, the peak time of radioactivity in the other brain regions (occipital cortex, temporal cortex, frontal cortex, striatum, thalamus, and cerebellum) was about 30–40 min after administration of the radioligand. The distribution of radioactivity in the brain regions after administration of the radioligand was consistent with the distribution of α7 nAChRs in the monkey brain [25]–[27]. Uptake of radioactivity in the brain regions after intravenous administration of [76Br]SSR180711 was significantly decreased by pretreatment with the α7 nAChR agonist SSR180711 (5.0 mg/kg, i.v., 30 min)(Figures 2–\n4). Uptake of radioactivity (during 70–91 min) in the brain regions except the cerebellum (low receptor density) after intravenous administration of [76Br]SSR180711 was significantly decreased by pretreatment with the α7 nAChR agonist SSR180711 (5.0 mg/kg, i.v., 30 min)(Figures 4A). However, the distribution of radioactivity in the brain regions after intravenous administration of [76Br]SSR180711 was not altered by pretreatment with the selective α4β2 nAChR agonist A85380 (1.0 mg/kg, i.v., 30 min)[28], [29](Figures 2, 3 and 4B).10.1371/journal.pone.0003231.g002Figure 2Representative PET images in the brains of a rhesus monkey after intravenous administration of [76Br]SSR180711.Upper: Control monkey (saline pre-treated). Middle: Pretreatment with SSR180711 (5.0 mg/kg, 30 min before). Lower: Pretreatment with A85380 (1.0 mg/kg, 30 min before)10.1371/journal.pone.0003231.g003Figure 3Representative time-activity curves of radioactivity (expressed as % Dose/mL) in several brain regions of a rhesus monkey after intravenous administration of [76Br[SSR180711 in control (saline pre-treated) monkey, SSR180711 (5.0 mg/kg, 30 min before)-pretreated monkey, and A85380 (1.0 mg/kg, 30 min before)-pretreated monkey.10.1371/journal.pone.0003231.g004Figure 4Effects of SSR180711 and A85380 on the uptake of the radioactivity in the monkey brain after intravenous administration of [76Br[SSR180711.(A): Uptake values (expressed as % Dose/mL) of [76Br[SSR180711 in several brain regions under control (saline pre-treated) group (during 70–91 min post-injection) and SSR180711 (5.0 mg/kg, 30 min before) treated groups. Data were the mean±S.D. of three monkeys. *p<0.05, **p<0.01 as compared to control group (Paired t-test). (B): Uptake values (expressed as % Dose/mL) of [76Br[SSR180711 in several brain regions under control (saline pre-treated) group (during 70–91 min post-injection) and A85380 (1.0 mg/kg, 30 min before) treated groups. Data were the mean±S.D. of three monkeys. CERE: cerebellum, HIPP: hippocampus, OCC: occipital cortex, STR: striatum, THA: thalamus, TEM: temporal cortex, FC: frontal cortexBaseline PET scans showed rapid brain penetration and accumulation of [11C]CHIBA-1001 in the monkey brain (Figures 5–\n7). The peak time of radioactivity in the other brain regions (occipital cortex, temporal cortex, frontal cortex, striatum, thalamus, and cerebellum) was about 10 min after administration of [11C]CHIBA-1001, whereas the peak time of radioactivity in the hippocampus was about 30 min after administration. The distribution of radioactivity in the striatum, thalamus, hippocampus, occipital cortex, temporal cortex, and frontal cortex 40–60 min after administration of the radioligand was higher than that in the cerebellum, consistent with the distribution of α7 nAChRs in the monkey brain [25]–[27]. Uptake of radioactivity (during 70–91 min) in the brain regions except the cerebellum (low receptor density) after intravenous administration of [11C]CHIBA-1001 was decreased by pretreatment with SSR180711 (5.0 mg/kg) although these differences failed to reach statistical significance because of small number (n = 3) of monkey (Figures 7A). Furthermore, a preliminary study indicated that the uptake of radioactivity in the brain regions after intravenous administration of [11C]CHIBA-1001 was also decreased by pretreatment with another α7 nAChR agonist A844606 (5.0 mg/kg, i.v., 30 min before) [30] (Supplemental Figure S1). However, the uptake of radioactivity in the brain regions after intravenous administration of [11C]CHIBA-1001 was not altered by pretreatment with the selective α4β2 nAChR agonist A85380 (1.0 mg/kg, i.v., 30 min)[28], [29](Figures 5, 6, and 7B).10.1371/journal.pone.0003231.g005Figure 5Representative PET images in the brains of a rhesus monkey after intravenous administration of [11C]CHIBA-1001.Upper: Control monkey (saline pre-treated). Middle: Pretreatment with SSR180711 (5.0 mg/kg, 30 min before). Lower: Pretreatment with A85380 (1.0 mg/kg, 30 min before)10.1371/journal.pone.0003231.g006Figure 6Representative time-activity curves of radioactivity (expressed as % Dose/mL) in several brain regions of a rhesus monkey after intravenous administration of [11C]CHIBA-1001 in control (saline pre-treated) monkey, SSR180711 (5.0 mg/kg, 30 min before)-pretreated monkey, and A85380 (1.0 mg/kg, 30 min before)-pretreated monkey.10.1371/journal.pone.0003231.g007Figure 7Effects of SSR180711 and A85380 on the uptake of the radioactivity in the monkey brain after intravenous administration of [11C]CHIBA-1001.(A): Uptake values (expressed as % Dose/mL) of [11C]CHIBA-1001 in several brain regions under control (saline pre-treated) group (during 70–91 min post-injection) and SSR180711 (5.0 mg/kg, 30 min before) treated groups. Data were the mean±S.D. of three monkeys. (B): Uptake values (expressed as % Dose/mL) of [11C]CHIBA-1001 in several brain regions under control (saline pre-treated) group (during 70–91 min post-injection) and A85380 (1.0 mg/kg, 30 min before) treated groups. Data were the mean±S.D. of three monkeys. CERE: cerebellum, HIPP: hippocampus, OCC: occipital cortex, STR: striatum, THA: thalamus, TEM: temporal cortex, FC: frontal cortexIn the described in discussion section, it is likely that [11C]CHIBA-1001 is superior to [76Br]SSR180711 because of high brain uptake and lower half-life of [11C]. Therefore, [11C]CHIBA-1001 was used in the subsequent studies.Phencyclidine (PCP)-treated monkeysThe N-methyl-D-aspartate (NMDA) receptor antagonist phencyclidine (PCP) has been used as an animal model of schizophrenia, since it has been shown to cause schizophrenia-like symptoms in humans [31]–[35]. We performed two PET scans, one before (baseline) and one 1-day after subchronic administration of PCP (0.3 mg/kg, twice a day for 13 days). Subchronic administration of PCP did not alter the time-curve of the radioactivity or the percentage of unmetabolized fraction in the plasma of monkeys (Figure 8). Interestingly, subchronic administration of PCP decreased the binding of [11C]CHIBA-1001 in several regions (frontal cortex, temporal cortex, occipital cortex, striatum, thalamus, and hippocampus) of the monkey brain; the difference of binding in the frontal cortex was statistically significant (t = 5.73, df = 3, p = 0.011) between the two groups (Figure 8C), consistent with a previous report using mice [35].10.1371/journal.pone.0003231.g008Figure 8Effects of subchronic administration of PCP on the binding in monkey brain after intravenous administration of [11C]CHIBA-1001.(A): Radioactivity in the plasma of control (baseline; n = 4) and PCP-treated (n = 4) groups after intravenous administration of [11C]CHIBA-1001. Data were the mean±S.D. of four monkeys. (B): Percentage of unmetabolized fraction in the plasma of control and PCP-treated groups after intravenous administration of [11C]CHIBA-1001. Data were the mean±S.D. of four monkeys. (C): Receptor binding in the several brain regions of control and PCP-treated groups. Data were the mean±S.D. of four monkeys. *p<0.05 as compared to control (baseline) group (Paired t-test).DiscussionIn the present study, we have developed two PET ligands, [76Br]SSR180711 and [11C]CHIBA-1001. It is likely that [11C]CHIBA-1001 is superior to [76Br]SSR180711 for the following reasons. First, [11C]CHIBA-1001 can be synthesized using an in house cyclotron, whereas [76Br]SSR180711 cannot. Second, the radiation exposure dose in humans by [11C]CHIBA-1001 PET study is lower than that of [76Br]SSR180711 because of the short half-life (the half-lives of [11C] and [76Br] are 20.4 min and 16.2 hours, respectively). Third, the short half life allows several repetitions of [11C]CHIBA-1001 PET in a single day. Fourth, brain uptake of [11C]CHIBA-1001 is higher than that of [76Br]SSR180711.We have demonstrated that [11C]CHIBA-1001 is a novel PET ligands for in vivo imaging of α7 nAChRs in the non-human primate brain. First, an in vitro receptor binding study showed that CHIBA-1001 is a highly selective ligand at α7 nAChRs, since this ligand was found to be devoid of activity for the standard receptor binding profile. Second, an in vivo PET study using conscious monkeys demonstrated a high accumulation into the brain after intravenous administration of [11C]CHIBA-1001. The regional distribution of radioactivity in the monkey brain after intravenous administration of [11C]CHIBA-1001 is consistent with the distribution of α7 nAChRs in the monkey brain [25]–[27]. Furthermore, the uptake of radioactivity in the monkey brain regions was blocked by pretreatment with the selective α7 nAChR agonist SSR180711 and A844606, but not the selective α4β2 nAChR agonist A85380. Third, we found a reduction of [11C]CHIBA-1001 binding in the frontal cortex of the monkey brain after subchronic administration of PCP.Recently, we reported that the repeated administration of PCP (10 mg/kg/day for 10 days) significantly decreased the density of α7 nAChRs in the frontal cortex of the mouse brain [35], consistent with our monkey data. The precise mechanism(s) underlying how repeated PCP administration could modulate α7 nAChRs in the brain are currently unknown. It has been reported that the immunoreactivity of α7 nAChRs in the prefrontal cortex of schizophrenics was significantly decreased compared to that in normal controls [36]. Interestingly, α7 nAChR agonists can increase the release of glutamate from the presynaptic terminals, resulting in stimulation of the NMDA receptors on the postsynaptic neurons, suggesting that stimulation at α7 nAChRs may potentiate the NMDA receptors [7], [37], [38]. Taken together, these findings suggest that α7 nAChRs may interact with the NMDA receptors in the brain, although further study on the cross-talk between α7 nAChRs and NMDA receptors in the brain is necessary [7], [37], [38].A postmortem human brain study demonstrated decreased expression of hippocampal α7 nAChRs in schizophrenic patients [12], suggesting that schizophrenic patients have fewer α7 nAChRs in the hippocampus, a condition which may lead to the failure of cholinergic activation of the inhibitory interneurons, manifesting clinically as decreased gating of responses to sensory stimulation [12]. Deficient inhibitory processing of the P50 auditory evoked potential is a pathophysiological feature of schizophrenia [3], [39]–[41] and Alzheimer's disease [42], and it has been suggested that α7 nAChRs play a critical role in this phenomenon [40], [41], [43], [44]. In the present study, using [11C]CHIBA-1001 and PET, we could detect the reduction of α7 nAChRs in the frontal cortex in a non-human primate PCP model of schizophrenia although semi-quantitative analysis using Logan plot analysis was performed in this study. Taken together, these results suggest that it would be of great interest to examine whether α7 nAChRs are altered in the intact brain of patients with schizophrenia and Alzheimer's disease by using [11C]CHIBA-1001 and PET.Based on the above findings, α7 nAChRs are the most attractive target for potential therapeutic drugs in several neuropsychiatric diseases [3]–[11], [43], [44]. A number of pharmaceutical industries have developed selective α7 nAChR agonists for the treatment of neuropsychiatric diseases, including schizophrenia and Alzheimer's disease, and clinical trials of some drugs have been started. Using [11C]CHIBA-1001 and PET, it will be possible to measure the relationship between the receptor occupancy and the dose of α7 nAChR agonists in the human brain, since this radioligand can be used for quantitative occupancy assessment of α7 nAChRs.In conclusion, the present study presents the successful in vivo characterization of α7 nAChRs in the conscious monkey brain using [11C]CHIBA-1001 and PET. Therefore, in vivo PET imaging of α7 nAChRs in the intact human brain provides a method for quantitative study of α7 nAChR-related pathophysiology in neuropsychiatric diseases. In addition, the in vivo determination of receptor occupancy allows for the demonstration of target engagement and assessment of titration for potential dose regimens. A clinical PET study in healthy human subjects using [11C]CHIBA-1001 is currently underway.Materials and MethodsSynthesis of the precursor and CHIBA-1001SSR180711, CHIBA-1001 and the precursor, 4-(tributylstannyl)phenyl 2,5- diazabicyclo[3.2.2]nonane -2-carboxylate (Figure 1), were synthesized as described in the Supplemental Method S1.[125I]α-Bungarotoxin bindingThe binding assay using [125I]α-bungarotoxin was performed as described in a previous report [45] with a slight modification (See Supplemental Method S2).Synthesis of [75Br]SSR180711 and [11C]CHIBA-1001[76Br]SSR180711 and [11C]CHIBA-1001 were synthesized by bromination and methylation of the precursor, respectively (See Supplemental Method S3).SubjectsEleven young-adult male rhesus monkeys (Macaca mulatta) weighing from 4 to 6 kg were used for PET measurements. The monkeys were maintained and handled in accordance with the recommendations of the US National Institutes of Health and also the guidelines of the Central Research Laboratory, Hamamatsu Photonics (Hamamatsu, Shizuoka, Japan). The animal experimental procedure was approved by the Animal Care and Use Committee of Hamamatsu Photonics and Chiba University. Over the course of 3 months, the monkeys were trained to sit on a chair twice a week. The magnetic resonance images (MRI) of all monkeys were obtained with a Toshiba MRT-50A/II (0.5T) under anesthesia with pentobarbital. The stereotactic coordinates of PET and MRI were adjusted based on the orbitomeatal (OM) line with monkeys secured in a specially designed head holder [46]. At least 1 month before the PET study, an acrylic plate, with which the monkey was fixed to the monkey chair, was attached to the head under pentobarbital anesthesia as described previously [47].PET scansPET data were collected on a high-resolution PET scanner (SHR-7700; Hamamatsu Photonics K.K., Hamamatsu, Japan) with a transaxial resolution of 2.6-mm full-width at half-maximum (FWHM) and a center-to-center distance of 3.6 mm [48]. The PET camera allowed 31 slices for imaging to be recorded simultaneously. After an overnight fast, animals were fixed to the monkey chair with stereotactic coordinates aligned parallel to the OM line. A cannula was implanted in the posterior tibial vein of the monkey for administration of [76Br]SSR180711 or [11C]CHIBA-1001. [76Br]SSR180711 or [11C]CHIBA-1001 was injected through the posterior tibial vein cannula 30 min after administration of saline (control), SSR180711 (5.0 mg/kg, i.v.), or A85380 (1.0 mg/kg, i.v.; Sigma-Aldrich Co., Ltd., St Louis, MO). PET images were acquired over 91 min (10 s×6 frames, 30 s×6 frames, 1 min×12 frames, and 3 min×25 frames). Summation images from 70 to 91 min postinjection were constructed. PET scans were reconstructed using filtered backprojection in a 100×100 matrix, with a voxel size of 1.2 mm×1.2 mm×3.6 mm. Each MRI was coregistered to a summation image. Due to the very short half-life of 11C (20.4 min), a time lag of at least 3 hr between scans provided sufficient decay time of radioactivity in monkeys (approximately 1/400 of the injected dose). Therefore, the level of radioactivity associated with the previous injection of labeled compound would not interfere with the next scan as previously reported [49], [50].Next, we examined the effects of subchronic administration of the NMDA receptor antagonist phencyclidine (PCP: 0.3 mg/kg, i.m., twice a day for 13 days) on the distribution of [11C]CHIBA-1001 binding in the monkey brain. In the control (n = 4), PET scans were performed before PCP administration. One day after subchronic administration of PCP, PET scans were performed as described above.To assess the semi-quantitative analysis of PET data, arterial samples were obtained every 8 s from injection to 64 s, and then again at 1.5, 2.5, 4, 6, 10, 20, 30, 45, 60, and 90 min after [11C]CHIBA-1001 injection. Blood samples of [11C]CHIBA-1001 were centrifuged to separate the plasma, weighed, and subjected to radioactivity measurement. For metabolite analysis, methanol was added to some plasma samples, the resulting solutions were centrifuged, and the supernatants were developed with a thin-layer chromatography (TLC) plate (AL SIL G/UV; Whatman, Kent, UK) using a mobile phase of dichloromethane:diethyl ether:ethanol:triethylamine (20:20:2:2). At each sampling time point for analysis, the ratio of radioactivity in the unmetabolized fraction to that in the total plasma (metabolite plus unmetabolite) was determined using a phosphoimaging plate (BAS-1500 MAC; Fuji Film Co., Tokyo, Japan). The metabolite-corrected plasma curve was obtained.Kinetic analysisTime-activity curves of radioactivity in each region of interest (ROI) in the brain and metabolite-corrected arterial plasma were determined. Analysis of the Logan plot provides the linear function of the free receptor concentration, which is known as the distribution volume [51]. In reversibly labeled compounds, the Logan plot becomes linear after a certain period of time with a slope (K) that is equal to the steady-state distribution volume. In the preliminary semi-quantitative analysis, the ratios of K in each ROI (K (ROI)) to K in the cerebellum (K (CE)) were calculated to determine the binding of α7 nAChRs in the monkey brain.Statistical analysisStatistical analysis of the control (baseline) and drug (SSR180711 or A85380) -treated groups was performed by paired t-test. Statistical analysis of the control (baseline) and PCP-treated groups was also performed by paired t-test. Significance was set at p<0.05.Supporting InformationFigure S1Effects of the another alpha7 nAChR agonist A844606 on the uptake of the radioactivity in the monkey brain after intravenous administration of [11C]CHIBA-1001. Representative time-activity curves of radioactivity (expressed as % Dose/mL) in several brain regions of a rhesus monkey after intravenous administration of [11C]CHIBA-1001 in control (saline pre-treated) monkey, and A844606 (1.0 mg/kg, 30 min before)-pretreated monkey.(0.14 MB TIF)Click here for additional data file.Method S1Preparation of SSR180711, CHIBA-1001 and precursor.(0.08 MB DOC)Click here for additional data file.Method S2[125I]alpha-Bungarotoxin binding(0.03 MB DOC)Click here for additional data file.Method S3Synthesis of [75Br]SSR180711 and [11C]CHIBA-1001(0.03 MB DOC)Click here for additional data file.Table S1Inhibition effect of CHIBA-1001 (10 uM) on radioligand binding to various receptors(0.07 MB DOC)Click here for additional data file.Table S2Inhibition effect of CHIBA-1001 (1 µM) on radioligand binding to various receptors(0.04 MB DOC)Click here for additional data file.\n\nREFERENCES:\n1. 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+{
+    "id": "PMC2530873",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2530873\nAUTHORS: Jean-Michel Ubeda, Danielle Légaré, Frédéric Raymond, Amin Ahmed Ouameur, Sébastien Boisvert, Philippe Rigault, Jacques Corbeil, Michel J Tremblay, Martin Olivier, Barbara Papadopoulou, Marc Ouellette\n\nABSTRACT:\nGene expression and DNA copy number analyses using full genome oligonucleotide microarrays of Leishmania reveal molecular mechanisms of methotrexate resistance.\n\nBODY:\nBackgroundThe protozoan parasite Leishmania is distributed worldwide and is responsible for a wide spectrum of diseases, including cutaneous, mucocutaneous and visceral leishmaniasis. No vaccines are presently available against Leishmania infections [1] and treatments rely primarily on chemotherapy. The chemotherapeutic arsenal is limited and resistance to the mainstay of pentavalent antimonials has reached epidemic proportions in parts of India [2]. Several studies dealing with drug resistance in Leishmania have highlighted the plasticity of the Leishmania genome [3,4]. The antifolate methotrexate (MTX) has been one of the first and most widely used drugs for understanding drug-induced plasticity and resistance mechanisms [5-8]. While Leishmania is sensitive to MTX, the drug is not used clinically to treat leishmaniasis. However, Leishmania is a folic acid auxotroph and studies of MTX resistance mechanisms have highlighted several novel aspects of folate metabolism in this parasite that could be exploited for drug interventions [9,10]. Indeed, the development of novel antifolate molecules for Leishmania and related parasites has been ongoing in several laboratories [11-13].Leishmania resists MTX by a number of mechanisms. Leishmania has the capacity to transport folic acid, but this activity is often impaired in MTX resistant cells [8,14-17]. The main Leishmania folate transporter FT1 has been isolated [18,19] and is part of a large family of folate biopterin transporter (FBT) proteins with 14 members in Leishmania (AA Ouameur et al., unpublished data). Rearrangements of FBT genes are correlated with MTX resistance [19-21]. A frequent mechanism of drug resistance in Leishmania is gene amplification [3]. Small chromosomal regions of 20-70 kb that are part of one of the 36 Leishmania chromosomes are amplified as part of extrachromosomal elements [3]. These elements are usually formed by recombination between repeated homologous sequences [22-24]. Amplification of the gene coding for the target dihydrofolate reductase-thymidylate synthase (DHFR-TS) has been described in MTX resistant parasites [5,6,25-29]. Work on MTX resistance also led to the characterization of the pteridine reductase PTR1, whose main function is to reduce pterins. However, when overexpressed it can also reduce folic acid and lead to MTX resistance by by-passing DHFR-TS activity [30-33]. The PTR1 gene is frequently amplified as part of extrachromosomal circular or linear amplicons [6,16,22,34-38]. In addition to these three main mechanisms of resistance, perturbation in folate metabolism [39,40], in one carbon metabolism [41] or in DNA metabolism [42] have also been associated with MTX resistance. Several of these mutations can co-exist in the same cell, demonstrating that resistance can be a complex multi-gene phenomenon. Genome wide expression profiling scans represent a useful tool for understanding complex resistance mechanisms and may lead either to the discovery of novel resistance mechanisms and/or could provide clues about mechanisms of gene rearrangements.Indeed, DNA microarrays have been useful for investigating the mode of action of drugs [43] and mechanisms of resistance (reviewed in [44-46]). DNA microarrays for Leishmania have evolved from random genomic DNA clones [47-50], cDNA clones [51,52], targeted PCR fragments [29], selected 70-mer oligonucleotides [53,54] to full genome microarrays [55,56]. Targeted microarrays have been used previously for the study of drug resistance in Leishmania [29,52,54,57]. We present here the generation of full genome DNA microarrays for both L. major and L. infantum and their use in the study of one L. major and one L. infantum MTX resistant mutant. These genome wide expression profiling experiments illustrate the complexity of resistance mechanisms present in the same cell. They allowed the definition of the precise mechanisms leading to the formation of extrachromosomal circular and linear amplicons, the definition of gene deletion events and revealed the involvement of aneuploidy in the complex genotype of MTX resistance.ResultsRNA expression profiling in methotrexate resistant Leishmania cellsCompletion of the L. major genome has allowed the generation of arrays containing 60-mer oligonucleotide probes designed by NimbleGen Systems [55,56] and in this work, we present the generation of a full genome DNA microarray composed of 70-mer oligonucleotide probes suitable for both L. major and L. infantum analysis (see Materials and methods for a full description of the arrays). These full genome arrays were used for deciphering how Leishmania resists the antifolate model drug MTX. Two MTX resistant mutants, L. major MTX60.4, which has previously been studied with small targeted arrays [29], and L. infantum MTX20.5, were studied using the full-genome microarrays. Mutants of both species are highly resistant to MTX (Figure 1a), and since they were selected in a stepwise fashion, it is likely that multiple resistance mechanisms may exist in these mutants and could thus be uncovered by these arrays. The resistant cells had a similar generation time as the wild-type parent cells.Figure 1Methotrexate susceptibility in Leishmania cells. (a) Leishmania cells were grown in M199 medium and their growth was monitored at 72 hours by measuring their OD600 nm with varying concentrations of MTX. White circles, L. major wild-type cell; black circles, L. major MTX60.4; white squares, L. infantum wild-type cells; black squares, L. infantum MTX20.5. (b) The mutant L. major MTX60.4 was grown in the absence of drug for 5, 12, 25, 30 and 42 passages. The average of triplicate measurements is shown.The DNA microarrays were first validated by hybridizing fluorescently labeled digested DNA of wild-type L. major and L. infantum cells. The arrays were found to yield uniform and reproducible results (not shown) and were deemed appropriate for RNA expression profiling experiments. Total RNAs were thus purified for both wild-type and mutant strains, used to synthesize fluorescent probes, and hybridized to the microarrays as described in Materials and methods. Scanning and normalization led to expression data that were first represented as scatter plots. As evident from these plots (inserts in Figure 2a,b), most genes in both species are equally expressed between the sensitive and resistant strains. Indeed, the bulk of expression (RNA level) ratios between sensitive and resistant strains were close to 1. Nonetheless, there were notable differences. First, the RNA levels of a total of 61 genes were found to be modulated (cut-off of 2, p < 0.05) in the L. infantum MTX20.5 mutant compared to the wild-type strain (Figure 2a; Table S1 in Additional data file 1) and the expression levels of 75 genes were changed significantly (cut-off of 2, p < 0.05) in the L. major MTX60.4 mutant compared to the wild-type strain (Figure 2b; Table S1 in Additional data file 1). Secondly, a majority of genes whose expression was modulated by more than two-fold had increased expression levels in L. infantum MTX20.5 but the majority of another set of genes had decreased expression levels in L. major MTX60.4 (inserts of Figure 2; Table S1 in Additional data file 1). If the expression modulation cut-off was changed from 2 to 1.5 (p < 0.05), we found 251 and 372 genes that were differentially expressed in L. infantum MTX20.5 and L. major MTX60.4, respectively (Figure 2). Surprisingly, few differentially expressed genes were found to be modulated similarly in both mutants (Figure 3; Table S1 in Additional data file 1). One notable exception is a region of chromosome 6 that corresponds to a six gene locus including the DHFR-TS gene. DHFR-TS is the main target for MTX and its gene was frequently found amplified in L. major MTX resistant mutants as part of extrachromosomal circles (reviewed in [3,4]).Figure 2Modulation of gene expression in Leishmania cells resistant to methotrexate. DNA microarrays were analyzed as described in Materials and methods and the software GeneSpring version GX3.1 was used to represent fold modulation either on a chromosome by chromosome basis (1 to 36) or as a scatter plot (inserts) for both (a) L. infantum MTX20.5 and (b) L. major MTX60.4. Vertical bars refer to individual genes on each chromosome and their location above or below the strand represents the transcribed strand. Transcription in Leishmania leads to polycistronic RNAs. Red (increased expression) and blue (decreased expression) dashed lines in the scatter plots indicate 1.5-fold differences in gene expression, with the y-axis representing the expression ratios between the mutant and wild-type cells and the x-axis the signal intensity in the mutant. The color scale indicates the modulation of hybridization signals in the resistant mutants compared to wild-type cells. The spots corresponding to genes that are part of the DHFR-TS amplicons are circled in the scatter plots. The entire data set was deposited in GEO under the accession number series GSE9949.Figure 3Validation of DNA microarray expression data by qRT-PCR. The mean log10 ratios of selected genes from microarray expression data (grey bars) are compared to qRT-PCR data (black bars) for (a) L. infantum MTX20.5 and (b) L. major MTX60.4. The microarray data are the average of four biological replicates (with two dye swaps), while the qRT-PCR data are the average of three biological replicates repeated two times each. The asterisk indicates that the related gene transcript was not detected by qRT-PCR. The upper panel shows the expression of orthologous genes where the expression changes in the two species; the middle panel shows the modulation in the expression of FBT genes; the lower panel shows the expression of individual genes specific for each mutant.The DNA microarray data were supported by selected quantitative real-time reverse transcription PCR (qRT-PCR) assays in both the L. major and L. infantum mutants (Figure 3). In only two cases we found a discrepancy between the two techniques. LmjF04.0160 and its orthologue LinJ04_V3.0160 were found down-regulated in both mutants using DNA microarrays, but this was confirmed only in the L. major mutant by qRT-PCR (Figure 3). The other discrepancy between microarray and qRT-PCR data was for FT1, but this is explained by a gene deletion event (see below). The only other gene that was modulated similarly in the two mutants was the ABC protein gene ABCA2 and this was confirmed by qRT-PCR (Figure 3). Other genes were modulated in both mutants but in different ways. While LmjF31.0720 was down-regulated in L. major MTX60.4, its orthologue LinJ31_V3.0750 in L. infantum MTX20.5 was overexpressed (Figure 3). Otherwise, genes differentially expressed were specific to individual mutants.The differential gene expression of the MTX resistant mutants was also represented in a chromosome by chromosome fashion (Figure 2). This has permitted us to visualize regions that are differently expressed (red/orange, corresponding to overexpressed genes in the mutants). Two regions were clearly overexpressed in the L. infantum MTX20.5 mutant. One region was on chromosome 6 (DHFR-TS loci) and the second was in the left portion of chromosome 23 (Figure 2a). For the L. major MTX60.4 mutant, we also saw an increase in expression of selected genes present on chromosome 6 (DHFR-TS loci), but we also observed a number of whole chromosomes (for example, chromosome 22; colored predominantly red in Figure 2b).Extrachromosomal circular amplification of DHFR-TSDHFR-TS is present on chromosome 6 and by close examination of the expression data derived from the arrays we were able to precisely define the genes with increased expression in both the L. major and L. infantum mutants. In L. infantum, the genomic region overexpressed is delimited by genes LinJ06_V3.0860 and LinJ06_V3.0910 (Figure 4a). Most interestingly, the same region is overexpressed in L. major MTX60.4 (Figure 4a). As Leishmania is devoid of control for the initiation of transcription (no pol II promoter has yet been isolated in this parasite [58]), it is possible that the amplification of a small genomic region containing the DHFR-TS gene is responsible for the increased gene expression as determined by DNA microarrays. This was tested by hybridization of a blotted pulsed-field gel electrophoresis (PFGE) gel with a DHFR probe. Wild-type cells gave rise to two hybridizing bands, suggesting that the two homologous chromosomes 6 have different sizes (Figure 4b, lanes 1 and 3), a well established phenomenon in Leishmania [59]. The two mutants had an extra band hybridizing to the DHFR probe, which with its hybridizing smear is characteristic of extrachromosomal circles (Figure 4b, lanes 2 and 4). The genesis of circular DNA in Leishmania has been studied and is often due to homologous recombination between direct repeats bordering the regions amplified [22-24]. Close examination of the sequences flanking the regions amplified indeed pointed to the presence of repeated sequences (Figure 4a). The repeated sequences were highly similar between L. major (575 bp) and L. infantum (837 bp) (Figure S1 in Additional data file 2). To provide evidence that the DHFR-TS containing circles were generated through homologous recombination between these direct repeated sequences, we used two primers (6a and 6b in Figure 4a,c) that should give rise to a PCR amplification product only when an extrachromosomal circle is formed (Figure 4c). Indeed, when using this primer pair, PCR fragments of the expected size were observed in L. infantum MTX20.5 and L major MTX60.4 (Figure 4d, lanes 2 and 4) while no amplification was observed in the wild-type cells (Figure 4d, lanes 1 and 3). The difference in size of the PCR fragments between L. major and L. infantum is due to the difference in size of the repeats in the two species (Figure S1 in Additional data file 2). Sequencing of the PCR generated amplicon derived from L. major MTX60.4 [GenBank:EU346088] confirmed the scenario of homologous recombination between the repeated sequences (Figure S1d in Additional data file 2).Figure 4Extrachromosomal circular amplification of a genomic region of Leishmania chromosome 6 that includes the DHFR-TS locus. (a) Genomic organization of the DHFR-TS locus in both L. infantum MTX20.5 and L. major MTX60.4. Relative gene expression data (RNA) were determined using DNA microarrays and relative hybridization data were obtained by comparative genomic hybridization (DNA). Asterisks indicate that the microarray data of these genes were not found to be reliable. Direct repeats are shown with thick arrows and the approximate position of primers 6a and 6b are indicated with half arrows. (b) Chromosome size blot of Leishmania cells hybridized to a DHFR-TS probe. Sizes were determined using a yeast molecular weight marker (Biorad. Hercules, CA, USA). (c) Model for the formation of the extrachromosomal DHFR-TS circular DNA generated through homologous recombination between direct repeats (Figure S1 in Additional data file 2). (d) PCR with primers 6a and 6b to support the model shown in (c). Lane 1, L. infantum wild-type cells; lane 2, L. infantum MTX 20.5; lane 3, L. major wild-type cells; lane 4, L. major MTX60.4.Linear amplification of PTR1In mutant L. infantum MTX20.5 we observed a region of chromosome 23 that was overexpressed (increased RNA levels; Figure 2a). This region contains the gene for pteridine reductase 1 (PTR1), a well established MTX resistance gene whose product can reduce folic acid, hence by-passing the need for DHFR-TS [30,31]. Similarly to the DHFR-TS loci, the microarray expression data have allowed the precise determination of the region that was overexpressed, which started at the telomeric end and extended 120 kb up to gene LinJ23_V3.0380 (Figure 5a). The putative presence of telomeric sequences would suggest a linear amplification instead of a circular amplification. Hybridization of a chromosome PFGE blot has shown that PTR1 hybridized to the approximately 800 kb chromosome in both wild-type and resistant cells but also to a smaller linear amplicon of approximately 230 kb in L. infantum MTX20.5 (Figure 5b). This amplicon also hybridized to a telomere probe (Figure 5b). The size of the amplicon suggests that the amplified region was duplicated. The LinJ23_V3.0390 gene is clearly not overexpressed and thus not part of the amplicon (Figure 5a). Three genes, LinJ23_V3.0360, LinJ23_V3-0370 and Lin23_V3.0380, were less overexpressed than the other genes that are part of the amplicon (Figure 5a). Examination of the sequences where expression changed enabled the detection of inverted homologous repeats of 578 bp (Figure S2 in Additional data file 2) between LinJ23_V3.0350 and Lin23_V3.0360, and between LinJ23_V3.0380 and Lin23_V3.0390 (Figure 5a). Interestingly, similar repeats of 574 bp with 91% identity were found at the same position in the L. major genome [60]. The presence of these inverted repeats and the microarray expression data would suggest the formation of a linear amplicon with large inverted duplications that was formed by annealing of the identical 578 bp inverted repeats (Figure 5c). To obtain support for this scenario, we used PCR primer pairs (23a and 23b, or 23c and 23d) that would lead to a PCR product only if the rearrangement had occurred at the level of the inverted repeats (as, for example, during a block in DNA replication). Indeed, we obtained a product of the expected size with these pairs of primers in L. infantum MTX20.5 but no product was obtained from DNA derived from wild-type cells (Figure 5d). The nucleotide sequence of the PCR amplicon obtained with primer pair 23a/23b [GenBank:EU346089] is entirely consistent with the model shown in Figure 5c (Figure S2 in Additional data file 2).Figure 5Linear amplification of PTR1 as a large inverted duplication. (a) Genomic organization of the PTR1 locus in L. infantum and relative gene expression data as determined by DNA microarrays in L. infantum MTX20.5. Note that all genes from the telomere up to LinJ23_V3.0380 showed increased levels of expression in the MTX20.5 mutant compared to wild-type cells. (b) Chromosome size PFGE of Leishmania cells. Ethidium bromide (Et-Br) stained gel, or blotted gels hybridized to a PTR1 probe or to a probe containing the telomeric repeats are shown. Sizes were determined using a yeast molecular weight marker (Biorad). (c) Model for the formation of the extrachromosomal PTR1 linear amplicon generated through annealing of homologous inverted repeats (Figure S2 in Additional data file 2). This annealing could be facilitated by a block in replication. (d) PCR with primer pairs 23a and 23b or 23c and 23d to support the model shown in (c). Lane 1, L. infantum wild-type cells; lane 2, L. infantum MTX20.5.Decrease in gene expression due to deletion of folate transporter genesLeishmania spp. have a large gene family of conserved folate transporters with 14 FBT members (AA Ouameur et al., unpublished data). Part of this family located on chromosome 10 is shown in Figure 6a. Microarray expression data indicated that FT1, coding for the main Leishmania folate transporter [18,19], is down-regulated in L. major MTX60.4 but not in L. infantum MTX20.5 (Figure 3). The level of conservation of the various FBTs precluded that the 70-mer oligonucleotides spotted on the arrays would discriminate several of these closely related genes. The use of qRT-PCR to confirm the microarray data indicated that FT1 may be absent (Figure 3). This was suggestive of a gene deletion event and indeed a Southern blot of L. major MTX60.4 DNA hybridized with a probe recognizing the majority of FBT genes confirmed this extensive gene rearrangement (Figure 6b) and bands corresponding to LmjF10.0380, LmjF10.0385 (FT1) and LmjF10.0390 were either lacking or rearranged. Using PCR primers (labeled F and R in Figure 6a,c), we were able to demonstrate that FT1 (LmjF10.0385) was deleted following an event of homologous recombination between conserved sequences between LmjF10.0380 and LmjF10.0390 (Figure 6c). Indeed, primers F and R gave rise to a PCR fragment of 2.2 kb in L. major MTX60.4 (Figure 6d, lane 2) while under the conditions tested no fragments were found with L. major wild-type cells. Sequencing of the amplicon [GenBank:EU346090] validated the scenario of homologous recombination between two FBT genes leading to the diploid deletion of FT1 (Figure 6c; Figure S3 in Additional data file 2).Figure 6Mechanism of deletion of the main folate transporter gene FT1 in L. major selected for MTX resistance. (a) A portion of the L. major chromosome 10 showing some of the FT genes. Approximate location of PvuI sites (crosses) and their size are shown. Primers F and R are indicated by half arrows. The relative hybridization data obtained from RNA expression profiling (RNA) and comparative genomic hybridization (DNA) are shown. Due to conservation between the FT genes, the 70-mer probes for LmjF10.0380, FT1 and LmjF10.0390 are not discriminatory. (b) Southern blot of Leishmania total DNA digested with PvuI and hybridized to a probe recognizing conserved sequences of most FBT genes (indicated by bars underneath the genes in (a,c)). The genes corresponding to some hybridizing bands are indicated. (c) Model for the deletion of FT1 mediated by the homologous recombination of the conserved sequences between the folate transporter genes LmjF10.0380 and LmjF10.0390 (Figure S3 in Additional data file 2). (d) PCR with primers F and R to support the model shown in (c). Lane 1, L. major wild-type cells; lane 2, L. major MTX60.4.Selection for MTX resistance and chromosome aneuploidyAnalysis of gene expression on a chromosome by chromosome basis (Figure 2) suggested that the expression of whole chromosomes is modulated in L. major MTX60.4. Indeed, the majority of genes present on chromosomes 11 and 12 appeared down-regulated while the expression of genes located on chromosomes 7, 22, 28 and 32 seemed up-regulated (Figure 2). Chromosome 6 of L. infantum MTX20.5 also appears to be in more than two copies. This chromosome-wide uniform modulation of expression was represented more thoroughly for selected chromosomes by plotting the fold modulation in gene expression along the chromosome (Figure 7). The normalized microarray data indicated that genes of chromosomes 22 and 28 were overexpressed 1.7- and 1.5-fold, respectively, in the resistant strain L. major MTX60.4 compared to the wild-type strain. The expression of genes on chromosomes 11 and 12 seemed, in general, to be 50% underexpressed in the mutant strain compared to wild-type cells (Figure 7).Figure 7Chromosome aneuploidy in L. major selected for MTX resistance. The relative expression ratio of each individual gene of chromosomes (a) 22, (b) 28, (c) 11 and (d) 12 of L. major MTX60.4 was contrasted with the expression levels of the same genes in L. major wild-type cells, which were arbitrarily set at 1. Quantitative Southern blots were performed; two distant probes per chromosome were hybridized to HpaII digested DNA from L. major wild-type (lane 1), and L. major MTX60.4 (lane 2) (only one hybridization is shown for chromosomes 11 and 12). The hybridization signals of an α-tubulin (α-tub) probe, whose related gene is unchanged in the resistant strain, were used to standardize all the hybridization signals. HpaII digested total DNA from revertant L. major MTX60.4 parasites after 5, 12, 25, and 30 passages without MTX (lanes 3, 4, 5, and 6, respectively) were added, showing the progressive loss of aneuploid chromosomes in revertants.A number of hypotheses can explain this whole chromosome-specific gene regulation and we tested whether the copy number of specific chromosomes changed upon MTX selection in L. major MTX60.4. Quantitative Southern blot analyses with two distinct probes derived from chromosome 22 revealed that if the wild-type cells contain two homologous copies of chromosome 22 (Leishmania is a diploid organism), L. major MTX60.4 had four copies (Figure 7a, lanes 1 and 2). Similarly, L. major MTX60.4 had three copies of chromosome 28 compared to wild-type cells (Figure 7b, lanes 1 and 2). The probes used are physically far apart, indicating a change in ploidy of the whole chromosome. However, this change in chromosome copy number was not observed for chromosomes 11 and 12 (Figure 7c,d). Aneuploidy of specific chromosomes and drug resistance has been described in cancer cells (reviewed in [61]) and fungi [62,63]. To test this possibility, we generated a revertant line of L. major MTX60.4 by successive passages in the absence of MTX; under these conditions, resistance to the drug decreased (Figure 1b). Revertant cells were not as sensitive as wild-type cells to MTX but this is expected as a deletion of FT1 (Figure 6) will lead to resistant parasites [19]. The aneuploidy of chromosomes 22 and 28 regressed to diploidy (similar to wild-type diploidy) after 30 passages, thus circumstantially linking resistance levels (Figure 1b) and copy number of these chromosomes (Figure 7a,b, lanes 2-6). With the cells now diploid, additional passages (for example, passage 42) did not decrease resistance further.Comparative genomic hybridizationSince several of the changes in RNA levels were correlated with gene amplification or gene deletion, we undertook a comparative genomic hybridization (CGH) study using the full genome array. The DNA of mutant L. major MTX60.4 was labeled and changes in copy number in comparison to sensitive wild-type cells were measured using CGH. The CGH data are represented in a chromosome by chromosome fashion in Figure S4 in Additional data file 3. A qualitative correlation was observed between CGH and RNA-based hybridization (Figure 8). Indeed, amplification of the DHFR-TS locus, derived from chromosome 6, was easily detected by both techniques and quantification of the DNA amplification was compared to RNA levels (Figure 4). The deletion of FT1 was also detected by CGH and the latter technique was found to be quantitative. Indeed, the 70-mers recognizing FT1 recognized three conserved FT genes. In the MTX60.4 mutant two of these genes are deleted, hence explaining the ratio of 0.33 obtained by CGH (Figure 6). Polyploidy was also easily detected by CGH (Figure 8). Indeed, a similar qualitative pattern of hybridization intensities was obtained for both RNA expression profiling and CGH (Figure 8). Interestingly, while RNA expression profiling showed that chromosome 11 was down-regulated, quantitative Southern blots indicated that the copy number of the chromosome remained unchanged (Figure 7). This was also confirmed by CGH (Figure 8). There are some differences, however, between RNA expression profiling and CGH. For example, the latter technique showed that chromosome 2 is polyploid (Figure S4 in Additional data file 3) but this is likely due to the dynamic process of cell culture and parasite evolution, as DNA and RNA were prepared 1.5 years apart, rather than a difference in the techniques.Figure 8Comparison of relative hybridization data between RNA expression profiling and comparative genomic hybridization. RNA or genomic DNA derived probes were prepared from L. major MTX60.4 and the sensitive parent strain and hybridized to DNA microarrays. A subset of whole chromosome comparisons showing the correlation between RNA and DNA hybridization data are depicted. Examples shown are: chromosome 1 used as a no change control; chromosome 6 and the overexpression/amplification of the DHFR-TS locus (for quantification see Figure 4); and chromosome 22, where DNA and RNA are increased. For chromosome 11, RNA is decreased while DNA appears the same but the latter was also confirmed by Southern blots (Figure 7).DiscussionThe use of DNA microarrays is now useful to understand both the mode of action of drugs and the mechanisms of drug resistance (reviewed in [44-46]). Since Leishmania has no control at the level of transcription initiation [58], it is unlikely that drug response profiling using microarrays will be helpful to understand the mode of action of drugs in Leishmania. Results using MTX as a lead drug and qRT-PCR to monitor key genes, such as DHFR-TS, PTR1, and FT1, appeared to confirm this lack of RNA modulation of target genes upon drug exposure (unpublished observations). This is unfortunate, as the mode of action of most anti-Leishmania drugs is unknown. Nonetheless, microarrays are likely to be useful for studying resistance in Leishmania since it is often mediated by gene amplification [3,4] and we show here that DNA arrays hybridized to cDNAs were most valuable for detecting gene amplification events (Figures 2, 4, and 5). Since resistance is mostly correlated with gene amplification, we also used CGH and found a good qualitative correlation between RNA expression profiling and CGH (Figure 8). The technique of CGH was found to be technically simpler, but since there are clear examples of modulation in RNA level (for example, increased RNA stability) without changes in copy number of DNA in drug resistant Leishmania [64-66] (Figure 3, and Figure 7 for chromosomes 11 and 12), hybridization with cDNAs is likely to be more comprehensive. Nonetheless, modulation in RNA levels without changes in copy number of a gene is an infrequent event in drug resistant Leishmania. The use of both L. infantum and L. major MTX resistant mutants validated the design of our multi-species array but has also illustrated that the cellular resistance genotype can be complex and differ considerably between different mutants selected for resistance to the same drug. The modulation in expression of a few genes was common to both mutants, and only ABCA2 and DHFR-TS could be confirmed by qRT-PCR (Figure 3). Down-regulation of the ABC protein gene ABCA2 has never been described in MTX resistant Leishmania cells and additional investigations would be required to test whether it has any role in MTX resistance.DHFR-TS was the first amplified gene studied in a protozoan parasite [5] but its exact mechanism of amplification has never been reported. In addition to detecting gene amplification events, microarray data, whether derived from RNA expression profiling or CGH, were also useful in mapping the exact regions that were amplified. We show that DHFR-TS is amplified in L. major MTX60.4 as an extrachromosomal circle through homologous recombination between non-coding repeated sequences (Figure 4). This is consistent with other loci that were also found to be amplified by homologous recombination between relatively long repeated sequences [22-24]. Blast searches have shown that these exact repeated sequences are found only on chromosome 6. Remarkably, the same similar repeated sequences (albeit with different sizes) have also been conserved in L. infantum (Figure S1 in Additional data file 2). The same observation was made for the inverted repeats close to PTR1 that were conserved between L. major and L. infantum. L. major and L. infantum are thought to have diverged 0.5 million years ago [67] and it thus seems that there is considerable selective pressure to keep these repeated sequences intact. Since folates and pterins are important for Leishmania growth, it is possible that the presence of these repeats may allow a strategy to rapidly increase DHFR-TS or PTR1 levels in conditions of limited substrates. With its lack of transcription initiation control, Leishmania may utilize this alternative strategy of flanking key metabolic genes by repeated sequences to amplify these genes when required. Consistent with this proposal, DNA amplification has been observed in Leishmania cells subjected to nutrient shocks [68].PTR1 is a well established MTX resistance gene product [30,31] and the amplification of its gene was first reported as part of extrachromosomal circles [6,34-36]. Linear amplification of PTR1 with inverted duplications was described later [16,24,37] and linear amplicons could be precursors of circular amplicons [38]. Linear amplicons derived from other loci than the PTR1 region with inverted duplications have also been described in Leishmania [69-73]. The microarray hybridization data have enabled the elaboration of a plausible model for the generation of a linear amplicon that contained large inverted duplications formed at the site of inverted repeats (Figure 5). This is consistent with other models of gene amplification in Leishmania [16,37] where inverted repeats seem to be a major pathway to generate amplified large DNA palindromes (inverted duplications), as described in Tetrahymena [74], yeast [75] and mammalian cancer cells [76,77]. One of the large inverted duplications extends from the inverted repeats, where rearrangement has occurred, to the telomeric sequences (Figure 5). These data exclude the necessity of chromosomal breaks/rearrangements at two independent positions, but it remains to be determined whether a double-stranded break, a single-stranded break or blocks in replication are facilitating inverted repeat annealing.Gene deletions were thought to be associated with MTX resistance in Leishmania [19,20] but had not yet been characterized at the molecular level. The microarray data, either derived from RNA expression profiling or CGH, has led to the observation that a diploid non-conservative deletion occurred by homologous recombination between two members of the large FBT gene family (Figure 6). The mechanism of gene deletion thus resembles the mechanism of amplification. Usually, amplification in Leishmania is conservative, and only a few instances of non-conservative amplification (loss of one allele) have been described in it [3,22,23]. In the L. major MTX60.4 mutant, we observed a diploid deletion of the FT1 gene (Figure 6). It is not known whether the second allele is deleted by homologous recombination or by a gene conversion event such as a loss of heterozygosity, but there is a strong selection pressure to delete FT1, the main folate (and MTX) transporter in Leishmania. Without FT1, cells can become resistant to MTX but folates or related molecules will still need to be transported. It will be of interest to determine whether the fusion FBT protein produced by the recombination event (Figure 6) is active or not.The microarray approach has shown that modulation of gene expression could (rarely) be due to differential RNA expression without changes in copy number (Figure 3) [29]; it could be more frequently due to gene amplification (Figures 4 and 5) and, as determined now, to gene deletion (Figure 6). Two novel strategies were highlighted through the use of microarrays. In the L. major MTX60.4 mutant, the entire set of genes of chromosomes 11 and 12 is down-regulated while all the genes present on chromosomes 22 and 28 and possibly a few other chromosomes are overexpressed. The mechanism underlying an upregulation in gene expression results from a change in chromosome ploidy (Figure 7). Changes in ploidy have been observed when attempting to inactivate essential genes in Leishmania [78], but not in resistant parasites. We recently observed a similar phenomenon with other resistant Leishmania cells (P Leprohon et al., unpublished data), suggesting that chromosome aneuploidy is part of the Leishmania arsenal for responding to drug pressure. There was a good correlation between resistance levels and the copy number of these supernumerary chromosomes (Figures 1 and 7), linking this genetic event to the resistance phenotype. Obviously, additional studies will be required to determine which gene(s) is (are) responsible for resistance. A putative mechanism for increasing the levels of a gene product in Leishmania would thus be to generate supernumerary chromosomes. This may occur when direct or inverted repeats are absent in the vicinity of a gene conferring a selective advantage. While this is plausible, especially for an organism lacking control at the level of transcription initiation, this drug induced aneuploidy has been well documented in cells with transcriptional control, such as cancer cells (reviewed in [61]) or fungi [62,63]. The mechanism of down-regulation of whole chromosome expression does not seem to involve a change in chromosome number (Figures 7 and 8) and may involve epigenetic factors that will need to be investigated.ConclusionThe microarray approach was useful in highlighting several mechanisms used by resistant cells to modulate the copy number of genes by: gene deletion or extrachromosomal circular or linear amplicons; through supernumerary chromosomes; and by decreasing the expression of whole chromosomes by a mechanism that remains to be identified. In the case of the first two events, the rearrangements have occurred at the site of repeated (direct or inverted) sequences. It is possible that these repeats are not randomly distributed to allow the amplification of specific chromosomal regions. Using DNA microarrays it was shown that inverted duplications are frequent in cancer cells; these are not randomly distributed, and a subset are associated with gene amplification [79]. The availability of DNA microarrays for Leishmania has highlighted the role of repeated sequences and of chromosome ploidy in responding to environmental changes. Aneuploidy has been suggested as an important cause of cancer specific drug resistance [61] and further work should reveal the potential importance of this phenomenon in drug resistance in Leishmania.Materials and methodsCell cultureThe wild-type strain L. major LV39 and the mutants L. major MTX60.4 have been described previously [65]. The L. infantum strain (MHOM/MA/67/ITMAP-263) was selected in vitro in a stepwise fashion starting with its EC50 (0.5 μM) with doubling concentrations of MTX when cells were adapted to yield L. infantum MTX20.5 growing at 20 μM of MTX. All cells were grown in M199 medium supplemented with 10% heat-inactivated fetal bovine serum and 5 μg/ml hemin at 25°C.DNA manipulationChromosomes in agarose blocks were prepared and separated by PFGE as described previously [38]. For Southern blot and PCR, genomic DNA was isolated using the DNAzol technique (Invitrogen, Carlsbad, CA, USA) as recommended by the manufacturer. Southern blots, hybridization, and washing conditions were done following standard protocols [80]. For chromosome copy number investigation, Southern spots were quantified using ImageQuant 5.2 (GE Healthcare, Upsala, Sweden) and the reference gene α-tubulin was used for normalization.L. infantum and L. major DNA oligonucleotides full genome microarray designThe recent completion of the sequence of the L. major [81] and L. infantum [82] genomes, allowed the generation of multispecies high-density oligonucleotide microarrays. Our analysis of open reading frame sequence conservation between L. major and L. infantum revealed that these two species share 91-96% nucleotide identity, suggesting that interspecies microarray probes can be designed. Therefore, 70-mer oligonucleotides were designed for each open reading frame of L. infantum and L. major using automated bioinformatic procedures. The genomes of both species were first compared using BLAST and homologous genes were grouped together. Probes were designed with consistent thermodynamic properties. Probes were initially designed for L. infantum with the added requirement that the region targeted by the probes had perfect homology between both species. For common probes, up to 2 mismatches (out of 70 nucleotides) were tolerated. In the case that more than two mismatches were present in a given gene between L. infantum and L. major, a new probe was designed specifically for L. major (956 probes). The microarray included a total of 8,978 70-mer probes that recognized with no mismatches all L. infantum genes (8,184, GeneDB version 3) and also all L. major genes (8,370, GeneDB version 5.1) with a small percentage of the probes having at most 2 mismatches. Also, 372 control probes were included in the microarray for assessing synthesis variability, and location of the probe within a given open reading frame and of mismatches on hybridization. The probes were synthesized in 384-well plates by Invitrogen. The microarrays were printed on SuperChip (Erie Scientific, Portsmouth, NH, USA) using a BioRobotics MicroGrid (Genomic solutions Inc, Ann Arbor, MI, USA). Each probe was printed in duplicate. Our microarray platform is described in the Gene Expression Omnibus (GEO) with accession number GPL6855.Total RNA preparation and labelingTotal RNA was isolated from 108 Leishmania cells during the mid-log phase using RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany). The RNA preparation was treated with TURBO DNase (Ambion, Austin, TX, USA) to avoid any genomic contamination. The purity, integrity and quantity of the RNA were assessed on the Agilent 2100 bioanalyzer with the RNA 6000 Nano LabChip reagent set (Agilent Technologies, Santa Clara, CA, USA). For each probe, 10 μg of RNA were converted to aminoallyl-dUTP incorporated cDNA using random hexamers (Roche, Basel, Switzerland) and the SuperScript III RNase H Reverse Transcriptase (Invitrogen). Probes were thereafter coupled to the fluorescent dye Alexa Fluor555 or Alexa Fluor647 (Invitrogen) following the manufacturer's recommendations. Fluorescent probes were then purified with MinElute Spin Columns (QIAGEN) and quantified spectrophotometrically.Genomic DNA preparation and labelingGenomic DNA from 108 cells was isolated using the DNAzol technique (Invitrogen) as recommended by the manufacturer. Total DNA was then fragmented by successive passages through 22G1\" and 27G 1/2\" needles (Becton Dickinson Franklin Lakes, NJ, USA). Fragmented DNA was then double digested with PvuII and MseI restriction enzymes. Digested DNA was purified by phenol-chloroform, followed by an ethanol precipitation. For each probe, 4 μg of purified fragmented and digested genomic DNA were converted to fluorescently labeled DNA using Cy5- or Cy3-dCTP (Amersham, Piscataway, NJ, USA), random hexamers (Roche) and the exo- Klenow DNA polymerase (NEB, Ipswich, MA, USA). Fluorescent probes were then purified with ArrayIt columns (TeleChem International, Sunnyvale, CA, USA) and quantified spectrophotometrically.Microarray hybridizationPrehybridization and hybridization were performed at 42°C under immersion (Corning chambers, Corning, NY, USA). Slides were prehybridized for 90 minutes in PreHYB Solution (5× Denhardt, 30% formamide, 6× SSPE, 0.5% SDS, 100 μg/ml salmon sperm DNA). Then, slides were first washed 2 times at 42°C for 5 minutes in 2× SSC, 0.1% SDS with gentle agitation. Subsequent washes were at room temperature, 3 minutes each, in 1× SSC, 0.2× SSC and 0.05× SSC. Slides were then dipped in 100% isopropanol and dried by centrifugation. For hybridization, Alexa Fluor555 and 647 cDNA probes were dried and resuspended in the HYB solution (2.5× Denhardt, 30% formamide, 6× SSPE, 0.5% SDS, 100 μg/ml salmon sperm DNA, 750 μg/ml yeast tRNA), then mixed, denatured 5 minutes at 95°C and cooled slowly to 42°C. Mixed probes were applied on the array under a lifterslip. Hybridization was performed for 16 h. Washes after hybridization were the same as those described for the prehybridization.Fluorescence detection, data processing and statistical analysisThe Perkin Elmer ScanArray 4000XL Scanner was used for image acquisition (Perkin Elmer, Waltham, MA, USA). GenePix Pro 6.0 image analysis software (Axon Instruments, Union City, CA, USA) was used to quantify the fluorescence signal intensities of the array features. Four different RNA preparations of each mutant and their respective wild-type strain were analyzed, including dye-swaps. Raw data from GenePix were imported in R 2.2.1 for normalization and statistical analyses were performed using the LIMMA (version 2.7.3) package [83-85]. Before processing, probes were flagged according to the hybridization signal quality [86]. Weights were assigned to each array in order to give less weight to arrays of lesser quality [87]. Data were corrected using background subtraction based on convolution of normal and exponential distributions [88]. Intra-array normalization was carried out using the 'print-tip loess' statistical method and inter-array normalization was done by using the 'quantiles of A' method for each array [89]. Statistical analysis was done using linear model fitting and standard errors were moderated using a simple empirical Bayes [83]. Multiple testing corrections were done using the FDR method with a threshold p-value of 0.05. Only genes statistically significant with an absolute log ratio greater than 0.58 (log2 1.5) were considered as differentially expressed. Species comparison was performed only on probes that had less than two mismatches when hybridized to either Leishmania species. GeneSpring GX 3.1 was used for the generation of scatter plots and for chromosome by chromosome analysis. The entire data set has been deposited in GEO under the accession number series GSE9949. The comparative genomic hybridization data are deposited under reference number GSE11623.qRT-PCRThree independent RNA preparations were conducted for each condition. First-strand cDNA was synthesized from 2 μg of total RNA using the Superscript III RNase H Reverse Transcriptase enzyme and random hexamers (Roche) according to the manufacturer's instructions. The resulting cDNA samples were stored at -20°C until use. Control PCR amplification was carried out using primers from different internal controls (GAPDH and actin) to evaluate the uniformity of cDNA synthesis in different samples. Primers, TaqMan probes, experimental procedures and quantification for qRT-PCR of the folate transporter genes was as described (AA Ouameur et al., unpublished data) using the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) for normalization. For all other genes, equal amounts of cDNA were run in triplicate and amplified in a 15 μl reaction containing 7.5 μl of 2× Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 10 nM of Z-tailed forward primer, 100 nM of reverse primer, 250 nM of Amplifluor Uniprimer probe (Chemicon Int., Temecula, CA, USA), and 1 μl of cDNA target. Reactions were performed at the Gene Quantification core laboratory of the Centre de Génomique de Québec using the Applied Biosystems Prism 7900 Sequence Detector [90]. Amplification was normalized to two genes showing a highly stable expression in wild-type and resistant strains: LinJ18_V3.0630/LmjF18.0620 encoding a putative 60S ribosomal protein L10a, and LinJ36_V3.0850/LmjF36.2500 encoding a chromatin assembly factor 1 subunit b-like protein.AbbreviationsCGH, comparative genomic hybridization; DHFR, dihydrofolate reductase; DHFR-TS, DHFR-thymidylate synthase; FBT, folate biopterin transporter; FT, folate transporter; GEO, Gene Expression Omnibus; MTX, methotrexate; PFGE, pulsed-field gel electrophoresis; PTR, pteridine reductase; qRT-PCR, quantitative real-time reverse transcription PCR.Authors' contributionsJM carried out the molecular genetic studies and all the microarray hybridizations performed in this study, participated in the bioinformatic analyses of microarray data and drafted the manuscript. AHO helped in the design of qRT-PCR assays. DL developed and optimized the comparative genomic hybridization protocol. PR designed the 70-mer Leishmania oligonucleotide microarrays. FR performed the microarray normalization and statistical analysis. SB developed the LIMS that was used to integrate microarray results storage and analysis. JC, MOl, MOu, BP and MJT are part of a CIHR group grant and have supervised all the experiments presented in this paper. All authors read and approved the final manuscript.Additional data filesThe following additional data are available with the online version of this paper. Additional data file 1 contains Table S1, which lists the differential expression measured by the full-genome microarray analysis. Additional data file 2 contains supplementary Figures S1-S3. Additional data file 3 contains supplementary Figure S4, which shows the results of the comparative genomic hybridization analyses of L. major MTX60.4 versus the respective wild-type cells.Supplementary MaterialAdditional data file 1Differential expression measured by the full-genome microarray analysis.Click here for fileAdditional data file 2Figure S1 shows the direct repeats flanking the DHFR-TS locus of L. major and L. infantum chromosome 6, and also provides the circular junction sequence formed by homologous recombination. Figure S2 shows the inverted repeats present on chromosome 23 of L. infantum, and provides the sequence of the new junction formed through the inverted duplication. Figure S3 shows the sequence of the L. major chimera gene LmjF10.0380/0390.Click here for fileAdditional data file 3Results of the comparative genomic hybridization analyses of L. major MTX60.4 versus the respective wild-type cells.Click here for file\n\nREFERENCES:\nNo References"
+}

batch_9/PMC2531076.json

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+{
+    "id": "PMC2531076",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531076\nAUTHORS: Marco Racchi, Daniela Uberti, Stefano Govoni, Maurizio Memo, Cristina Lanni, Sonya Vasto, Giuseppina Candore, Calogero Caruso, Loriana Romeo, Giovanni Scapagnini\n\nABSTRACT:\nOn March 19, 2008 a Symposium on Pathophysiology of Ageing and Age-Related diseases was held in Palermo, Italy. Here, the lectures of M. Racchi on History and future perspectives of Alzheimer Biomarkers and of G. Scapagnini on Cellular Stress Response and Brain Ageing are summarized. Alzheimer's disease (AD) is a heterogeneous and progressive neurodegenerative disease, which in Western society mainly accounts for clinica dementia. AD prevention is an important goal of ongoing research. Two objectives must be accomplished to make prevention feasible: i) individuals at high risk of AD need to be identified before the earliest symptoms become evident, by which time extensive neurodegeneration has already occurred and intervention to prevent the disease is likely to be less successful and ii) safe and effective interventions need to be developed that lead to a decrease in expression of this pathology. On the whole, data here reviewed strongly suggest that the measurement of conformationally altered p53 in blood cells has a high ability to discriminate AD cases from normal ageing, Parkinson's disease and other dementias. On the other hand, available data on the involvement of curcumin in restoring cellular homeostasis and rebalancing redox equilibrium, suggest that curcumin might be a useful adjunct in the treatment of neurodegenerative illnesses characterized by inflammation, such as AD.\n\nBODY:\nBackgroundOn March 19, 2008 a Symposium on Pathophysiology of Ageing and Age-Related diseases was held in Palermo, Italy. Here, the lectures of M. Racchi on History and future perspectives of Alzheimer Biomarkers and of G. Scapagnini on Cellular Stress Response and Brain Ageing are summarized.Alzheimer's disease (AD) is a progressive neurodegenerative disorder affecting aged people; AD prevalence is approximately 1% between 65 and 69 years and is higher than 50% in individuals above 95 years. It is characterized by irreversible cognitive and physical deterioration. With increasing life expectancy across the world, dementia is a rapidly growing socioeconomic and medical problem. The confirmatory diagnosis of AD is based on the recognition and quantification of senile plaques and neurofibrillary tangles, which are the hallmarks of the disease [1].AD prevention is an important goal of ongoing research. Two objectives must be accomplished to make prevention feasible: i) individuals at high risk of AD need to be identified before the earliest symptoms become evident, by which time extensive neurodegeneration has already occurred and intervention to prevent the disease is likely to be less successful and ii) safe and effective interventions need to be developed that either reduce or slow the accumulation of AD neuropathology or lead to a decrease in clinical expression of this pathology [2].p53 as a putative peripheral marker for ADThe treatment of AD remains a major challenge because of an incomplete understanding of the events that lead to the selective neurodegeneration typical of Alzheimer's brains. Nowadays the attention is focused on one side on the study of the β-amyloid (Aβ) precursor protein (APP) metabolism's pharmacological modulation, and on the other one to develop disease-modifying or -arresting compounds. The first purpose is that to reduce the development of Aβ in the hope of reducing the formation of a potentially neurotoxic peptide whereas examples of the second concern the use either of monoclonal antibodies direct to inflammatory mediators or of β-sheet breakers [2-6].In view of existing and emerging therapeutic compounds, the focus has increasingly shifted to accurate detection of the earliest phase of illness and, to date, there is an increasing interest to develop techniques allowing an accurate detection of the earliest stages of the disease. Candidate biochemical markers for AD should be molecules representing some of the cerebral pathogenetic processes typical of AD or representing altered metabolic or cellular processes as shown by several studies performed either on brain or on peripheral tissues from affected patients. A wide variety of different proteins such as inflammatory markers, markers of oxidative stress, apolipoproteins, and markers of neuronal degeneration in blood and cerebrospinal fluid (CSF) have been examined [7,8]. Most of these studies have, however, yielded conflicting results. The cerebrospinal fluid has been the focus of research for diagnostic markers in AD pathology due to its direct contact with the extracellular space of the brain [7]. The more encouraging results come from the studies on the measurement of different isoforms of Aβ in CSF, particularly Aβ 1–42 [9], due to its role in the early pathogenesis of AD. Most studies showed that Aβ 1–42 concentrations are lower in the CSF of AD [7,8]. Unfortunately, plasma Aβ 1–40 and Aβ 1–42 did not correlate with the disease. In fact the results from these studies are often contradictory [10].The biological markers can be classified as primary (specific), such as Aβ, or secondary to the disease, or they can simply be epiphenomenal in nature. In search of secondary markers, Uberti et al. demonstrated an intriguing correlation between p53 and AD by using cell lines derived from these patients [11]. Fibroblasts of sporadic AD patients represent an important starting point in the research for novel biomarkers because of their various abnormalities in metabolic and biochemical processes, which reflect some of the events in the AD brain. They described and demonstrated an abnormal response of AD fibroblasts to an acute oxidative injury; in particular, fibroblasts from AD patients were found to be less vulnerable to the oxidative injury induced by H2O2 in comparison with fibroblasts from non-AD subjects. On the basis of immunoprecipitation studies with conformation-specific p53 antibodies, which discriminated folded vs. unfolded p53 tertiary structure, they found that in fibroblasts from AD patients a significant amount of total p53 assumes an unfolded tertiary structure in comparison with fibroblasts from control elderly subjects. Sequence analysis of the p53 gene allowed to exclude the possibility that the mutant p53 found in AD fibroblasts was the result of gene mutation. Thus, these data suggest that one of the peripheral events associated to the disease is responsible for generating such p53 isoform [11,12].In the attempt of investigating on the mechanism of such alteration, they assessed the contribution of APP metabolic products to the change in p53 conformational state. They found that the exposure to nanomolar concentrations of beta-amyloid (Aβ) 1–40 peptide induced the expression of an unfolded p53 protein isoform in fibroblasts derived from non-AD subjects. These data suggest that the tertiary structure of p53 and the sensitivity to p53-dependent apoptosis are influenced by low concentrations of soluble Aβ. On this basis, they hypothesised that low amounts of soluble Aβ induce early pathological changes at cellular level that may precede the amyloidogenic cascade. One of these changes is the induction of a novel conformational state of p53 [11,13].In addition and most importantly, Lanni et al. [14] were able to develop a rapid, easy and quantitative flow cytometric approach for the discrimination of conformational mutant p53-bearing cells from AD patients compared to non-AD controls, using small volumes of blood. Using this technique, they processed 75 AD, 66 controls, 15 subjects affected by another neuroinflammatory disease, Parkinson's disease and 3 subjects affected with other types of dementia (2 vascular dementia; 1 progressive supranuclear palsy) and confirmed the previous findings: AD subjects expressed higher levels of unfolded p53 in comparison with controls and subjects with other neurological diseases. The levels of conformationally altered p53, both in controls and AD patients, correlated with age but not with the lenght of illness or with the Mini Mental State Examination value. Interestingly, the sensitivity and specificity within different age intervals were more significant in subjects up to 70 years of age compared with the corresponding values for individuals older than 70 years. Within this specific age interval (≤ 70 years), the Authors worked out a sensitivity of 90% to discriminate AD patients from nondemented aged individuals at a specificity value of 77%. A comparison of these sensitivity and specificity values with those published in several studies, which evaluated the diagnostic power of CSF markers for AD (Total-tau, Phospho-tau and Abeta 1–42), reveal that p53 measurement is more sensitive (90% compared to respectively 81.4%, 81.3% and 85.9%), but less specific (77% compared to respectively 91.5%, 91.2% and 88.5%) [7].On the whole, these data strongly suggest that the measurement of conformationally altered p53 in blood cells has a high ability to discriminate AD cases from normal ageing, Parkinson's disease and other dementias. In spite of the fact that the method described in this study has a lower specificity value compared to CSF biomarkers, its high sensitivity in subjects up to 70 years and the non invasive nature of the test, permit its proposal as an adjunctive marker. Accordingly, p53 analysis may be used in the clinical evaluation of mild cognitive impairment cases or to improve a clinical diagnosis of AD, which should be based on cumulative information derived from clinical examination, brain neuroimageing techniques and biochemical markers either from CSF or blood. In a disease where therapeutic treatments are at most symptomatic, early treatment and therefore early prediction of future pathology is particularly important. Whether this different expression of conformationally altered p53 will be suitable as an adjunctive diagnostic tool in early stage AD in larger and independent populations of patients is matter of further investigations.On the other hand, p53 is a hot topic in AD research. Interestingly, it has been hypothesised that oxidative modification of p53 could be involved in the neuronal loss observed in neurodegenerative conditions [15,16].Cellular stress responseOxidative stress has been implicated in a variety of pathophysiological conditions, including neurodegenerative disorders. Irrespective of the source and mechanisms that lead to the generation of reactive oxygen species, mammalian cells have developed highly regulated inducible defensive systems, whose cytoprotective functions are essential in terms of cell survival. When appropriately activated, each one of these systems has the possibility to restore cellular homeostasis and rebalance redox equilibrium. Activation of antioxidant pathways is particularly important for tissue with relatively weak endogenous antioxidant defenses, such as the brain. Increasing evidences, in fact, support the notion that reduction of cellular expression and activity of antioxidant proteins and consequent augment of oxidative stress are fundamental causes for aging processes and neurodegenerative diseases [17]. Among the molecules belonging to stress protein family, Heme oxygenase-1 (HO-1) has been the object of intensive studies in the brain for its potential role in protecting neurons against cell death. HO enzymes provide the first and rate-limiting step in heme degradation, to give biliverdin, gaseous carbon monoxide and free iron. All the byproducts of HO activity play a significant role in physiological cell functions [18]. In the CNS, the HO system has been reported to be very active [19,20] and its modulation seems to play a crucial role in the pathogenesis of neurodegenerative disorders. Deregulation of the HO system has been associated with the pathogenesis of Alzheimer's disease, multiple sclerosis and brain aging [21,22]. Many studies clearly demonstrate that activation of HO-1 in neurons is strongly protective against oxidative damage and cell death [23]. Thus, modulation of HO-1 should represent a potential pharmaceutical strategy for the treatment of neurodegenerative disorders. A number of experimental and epidemiological studies have recently supported the beneficial effects of some commonly used natural products in preventing various pathologic conditions ranging from cardiovascular diseases to cancer. Spices and herbs often contain phenolic substances with potent antioxidative and chemopreventive properties [24]. Scapagnini et al. have previously shown that curcumin (1,7-bis [4-Hydroxy-3-methoxyphenyl]-1,6-heptadiene-3,5-dione), a natural phenolic agent, extracted from the rhizome of Curcuma Longa, strongly induced HO-1 expression and activity in rat astrocytes [25]. The Authors have then extended their findings demonstrating curcumin ability to induce HO-1 in cultured hippocampal neurons [26]. The results indicate that curcumin activates HO-1 and phase II enzymes expression in astrocytes and neurons, probably by activation of transcription factor Nrf2, and this activation is able to effort a significant cytoprotection in cultured neurons exposed to oxidative stress. The involvement of curcumin in restoring cellular homeostasis and rebalancing redox equilibrium, suggests that it might be a useful adjunct also in the treatment of neurodegenerative illnesses characterized by inflammation, such as AD. This idea has been reinforced by epidemiological studies showing that, in India where this spice is widely used in daily diet, there is a reduced age-adjusted prevalence of AD (in patients between 70 and 79 years of age is 4.4-fold less than that of the United States) [27]. Consistent with its possible use in neurodegenerative diseases, curcumin has been reported to decrease oxidative damage and amyloid deposition in a transgenic mouse model of Alzheimer's disease, and to reverse Aβ-induced cognitive deficits and neuropathology in rats [28,29]. Other plant-derived phenolic agents with analogous chemical structures to curcumin have been demonstrated to strongly activate HO-1 expression and to defend cells against oxidative stress. In particular, Scapagnini et al. have shown that ethyl ferulate, resveratrol (a phitoalexin derived from grape) and caffeic acid phenethyl ester (CAPE), are able to protect neurons via HO-1 induction [30]. These and other studies identify a novel class of natural substances that could be used for therapeutic purposes as potent inducers of HO-1 in the protection of tissues against inflammatory and neurodegenerative conditions. It needs to be emphasized that curcumin, and other plant constituents eventually become part of the human diet and can be consumed daily as herbal supplements. Further in vitro and in vivo studies using curcumin-like molecules will give important information on the feasibility of developing new pharmacological strategies for maximizing heme oxygenase activity in targeted tissues as an alternative to or in combination with HO-1 gene therapy.However, curcumin studies are a growing area in AD research [31] as well as in other pathological conditions. Various preclinical cell culture and animal studies suggest that curcumin has potential as an antiproliferative, anti-invasive, and antiangiogenic agent; as a mediator of chemoresistance and radioresistance; as a chemopreventive agent; and as a therapeutic agent in wound healing, diabetes, AD, Parkinson disease, cardiovascular disease, pulmonary disease, and arthritis [32].ConclusionA major goal of ongoing research in AD is to improve early detection by developing tools to move diagnosis backward in disease temporal course, i.e. before the clinical manifestation of the disease, where a treatment might play a decisive role in preventing or significantly retarding the manifestation of the disease [2,33]. On the whole, data here reviewed strongly suggest that the measurement of conformationally altered p53 in blood cells has a high ability to discriminate AD cases from normal ageing, Parkinson's disease and other dementias. On the other hand, available data on the involvement of curcumin in restoring cellular homeostasis and rebalancing redox equilibrium, suggest that curcumin might be an useful adjunct in the treatment of neurodegenerative illnesses characterized by inflammation, such as AD.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsMR, DU, SG, MM, CL carried out all the studied on conformational p53, SV, GC, CC took care of pharmacogenomic approach and drafted the manuscript, LR, GS carried out the curcumin experiments. All authors read and approved the final manuscript.\n\nREFERENCES:\nNo References"
+}

batch_9/PMC2531078.json

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+{
+    "id": "PMC2531078",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531078\nAUTHORS: Robert L Findling, Jean A Frazier, Vivian Kafantaris, Robert Kowatch, Jon McClellan, Mani Pavuluri, Linmarie Sikich, Stefanie Hlastala, Stephen R Hooper, Christine A Demeter, Denise Bedoya, Bernard Brownstein, Perdita Taylor-Zapata\n\nABSTRACT:\nBackgroundLithium is a benchmark treatment for bipolar illness in adults. However, there has been relatively little methodologically stringent research regarding the use of lithium in youth suffering from bipolarity.MethodsUnder the auspices of the Best Pharmaceuticals for Children Act (BPCA), a Written Request (WR) pertaining to the study of lithium in pediatric mania was issued by the United States Food and Drug Administration (FDA) to the National Institute of Child Health and Human Development (NICHD) in 2004. Accordingly, the NICHD issued a Request for Proposals (RFP) soliciting submissions to pursue this research. Subsequently, the NICHD awarded a contract to a group of investigators in order to conduct these studies.ResultsThe Collaborative Lithium Trials (CoLT) investigators, the BPCA-Coordinating Center, and the NICHD developed protocols to provide data that will: (1) establish evidence-based dosing strategies for lithium; (2) characterize the pharmacokinetics and biodisposition of lithium; (3) examine the acute efficacy of lithium in pediatric bipolarity; (4) investigate the long-term effectiveness of lithium treatment; and (5) characterize the short- and long-term safety of lithium. By undertaking two multi-phase trials rather than multiple single-phase studies (as was described in the WR), the feasibility of the research to be undertaken was enhanced while ensuring all the data outlined in the WR would be obtained. The first study consists of: (1) an 8-week open-label, randomized, escalating dose Pharmacokinetic Phase; (2) a 16-week Long-Term Effectiveness Phase; (3) a 28-week double-blind Discontinuation Phase; and (4) an 8-week open-label Restabilization Phase. The second study consists of: (1) an 8-week, double-blind, parallel-group, placebo-controlled Efficacy Phase; (2) an open-label Long-Term Effectiveness lasting either 16 or 24 weeks (depending upon blinded treatment assignment during the Efficacy Phase); (3) a 28-week double-blind Discontinuation Phase; and (4) an 8-week open-label Restabilization Phase. In December of 2006, enrollment into the first of these studies began across seven sites.ConclusionThese innovative studies will not only provide data to inform the labeling of lithium in children and adolescents with bipolar disorder, but will also enhance clinical decision-making regarding the use of lithium treatment in pediatric bipolar illness.Trial RegistrationNCT00442039\n\nBODY:\nBackgroundIn January of 2002, the United States Congress passed the Best Pharmaceuticals for Children Act (BPCA) [1] into law with the intent of improving the safety and efficacy of medications in pediatric populations. Ultimately, the initial goal of the BPCA was to establish a process for studying on-patent and off-patent drugs for use in pediatric populations. The legislation also calls for the scientific investigation of pediatric therapeutics through the conducting of pediatric studies and research to learn more about the efficacy and safety of medications in children. This occurs through a partnership of the National Institutes of Health (NIH) and the Food and Drug Administration (FDA) [2]. The Director of the NIH has delegated the authority to implement the drug development program to the Director of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), and the NICHD administers the research program through the Obstetric and Pediatric Pharmacology Branch of the Center for Research for Mothers and Children, working in cooperation with the other NIH Institutes and Centers with other significant pediatric research portfolios.To identify off-patent medications in need of further study, the BPCA asks the NICHD, in consultation with the FDA and experts in pediatric drug development, to develop a process for prioritizing needs in pediatric therapeutics by publishing a priority list. As a result, the U.S. FDA and NICHD began collaborations to identify and prioritize medications that were to be studied in pediatric populations [2]. In 2003, the first list of drugs for which pediatric studies were needed was generated. It consisted of 20 medications, including lithium [2].Lithium is a benchmark treatment for adult patients with bipolar disorder (BD). Lithium has been found to be efficacious in alleviating acute mania and preventing manic and mixed mood relapses in adults [3-6]. As a result, lithium is indicated in the United States for the acute and maintenance treatment of mania in BD in adults. Unfortunately, definitive randomized controlled trials of lithium have not been performed in pediatric populations that would lead to labeling of lithium for children and/or adolescents suffering from mania or mixed states in BD.Despite the paucity of data, it should be noted that preliminary studies have found that open label treatment with lithium may be effective in the treatment of children and adolescents with bipolar disorders [7,8]. In addition, lithium is a recommended treatment for manic or mixed states for youth with BD according to published treatment guidelines for pediatric BD [9]. A major consideration regarding the study of lithium as a treatment for youths suffering from BD is the untested assumption that lithium dosing procedures and therapeutic drug level monitoring that are used in adults are applicable to children and adolescents.MethodsIn an effort to better characterize lithium's use and efficacy in children as well as develop pediatric labeling under the auspices of the BPCA, a Written Request (WR) pertaining to lithium was issued by the FDA to the NICHD in 2004. A Written Request is a letter issued by FDA to the holder of the New Drug Application (NDA) that outlines how a pediatric study should be conducted and includes the study population, numbers of patients, study design, outcome measures, format, and time line of submission.The study design outlined in the WR was informed by recommendations included in a published consensus paper regarding the study of mania in pediatric patients [10]. The WR for lithium noted that three studies should be executed in children and adolescents ages 7–17 years with acute mania in order to inform the labeling of lithium for this population. These three studies included a Pediatric Pharmacokinetic and Tolerability study, a Pediatric Efficacy and Safety study, and a Pediatric Long-term Safety study.In the Pediatric Pharmacokinetic and Tolerability study, the pharmacokinetics of lithium would be examined. The WR mandated that at least 18 pediatric patients (9 males and 9 females) be enrolled in this study. In addition, an evidence-based dosing paradigm would be developed that would achieve target serum levels but also minimize toxicity. Moreover, the dosing schedule results from this study would then be utilized in the subsequent Efficacy and Long-term Safety treatment trials.According to the WR, following the completion of the Pediatric Pharmacokinetic and Tolerability study, the Pediatric Efficacy and Safety study was to be initiated. This trial would last for a minimum of 6 to 8 weeks, and would consist of a randomized, double-blind, parallel-group, placebo-controlled acute study. As directed by the WR, this study would have a sufficient number of male and female patients to detect a difference between lithium and placebo, equivalent to the median effect size seen in adult trials. After this second trial was completed, the Pediatric Long-term Safety Study would commence so that long-term safety data could be collected. It was required that at least 100 patients be exposed to lithium for no less than 6 months for this study. Specific areas of attention for both the Efficacy and Long-term studies included safety assessments with special emphasis being placed on the examination of putative short- and long-term effects of lithium on cognition, growth, thyroid, and renal function.Accordingly, the NICHD issued a Request for Proposals (RFP) on February 10, 2005 soliciting submissions for the study of lithium as described in the WR. The RFP indicated that the key purposes of the lithium studies were to: (1) establish evidence-based dosing strategies for lithium in children and adolescents; (2) characterize the pharmacokinetics and biodisposition of lithium in youth; (3) examine the acute efficacy of lithium in pediatric bipolarity; (4) investigate the long-term effectiveness of lithium treatment; and (5) comprehensively and meticulously characterize the short- and long-term safety of lithium in children and adolescents. RFPs are peer-reviewed and all proposals submitted are scored based upon technical merit in response to the criteria set forth in the RFP. The offerors who submit the proposal with the highest technical score and business proposals are then awarded a contract to perform the clinical studies. The NICHD then submits an Investigational New Drug Application (IND) to the FDA for the proposed studies. The data generated from these trials will be submitted to the FDA and it is anticipated that the label of lithium will be changed to reflect the outcome of these important clinical trials.ResultsSubmission and Development of StudiesIn order to respond to this RFP, the Collaborative Lithium Trials (CoLT) group was formed. The current CoLT team, which is lead by investigators from Case Western Reserve University (P.I. Findling), also includes investigators from Cincinnati Children's Hospital Medical Center/University of Cincinnati (P.I. Kowatch), Cambridge Health Alliance (P.I. Frazier), Children's Hospital & Regional Medical Center Seattle Washington (P.I. McClellan), University of North Carolina (P.I. Sikich), University of Illinois at Chicago (P.I. Pavuluri), and The Feinstein Institute for Medical Research of the North Shore–Long Island Health System (P.I. Kafantaris). These sites were selected specifically based upon the sites' investigators' established scientific expertise in pediatric bipolar disorder as well as clear evidence of being able to consistently, successfully, and safely recruit youths into prospective pediatric bipolar treatment studies. Proposals were submitted for competitive review in April of 2005.As a result of the CoLT group's submission, these investigators were subsequently awarded this government contract to study lithium in juvenile mania. As part of the work that was to be conducted under the auspices of this contract from the NICHD, collaboration with the Best Pharmaceuticals for Children Act-Coordinating Center (BPCA-CC; Premier Research; Medical Director, B. Brownstein, M.D.) and the CoLT team was established. In addition to the BPCA-CC, the CoLT team also began to collaborate with the NICHD Project Officer (P. Taylor-Zapata, M.D.) in order to propose final study designs to the FDA prior to initiating the requisite clinical trials.During this protocol refinement process, the CoLT team integrated feedback and input from the NICHD and the BPCA-CC into the study protocols. Although it was originally indicated that three distinct studies were to be performed to meet the goals of the WR, the CoLT team, BPCA-CC, and NICHD collaboratively created two multi-phase trials that would both: (1) ensure that the data that were outlined in the WR were obtained, and (2) allow feasibility of implementation to be enhanced. Each of these two multi-phase studies consists of four phases. The designs of both of these clinical trials were subsequently reviewed by the FDA in February, 2006. Enrollment into the first of these studies began in December of 2006.Lithium Formulations and Daily DosingIt should be noted that throughout these studies, immediate release lithium carbonate will be used due to its availability as a generic formulation. In addition, patients will receive treatment in 300 mg dose increments and for doses of 900 mg or greater, lithium will be given in thrice daily divided doses.Ethical Approval and Informed ConsentThese studies will be conducted in full accordance with the principles of the Declaration of Helsinki (52nd WMA General Assembly, Edinburgh, Scotland, October 2000). Additionally, prior to enrollment, these studies will be approved by all sites' Institutional Review Board for Human Investigation, and an independent Data Safety Monitoring Board (DSMB) will monitor the studies.Written informed consent will be acquired from all participants' legal guardians. Additionally, all participating youths will provide written assent prior to the initiation of any study related procedures.Inclusion and Exclusion CriteriaSimilar entry criteria for each of these outpatient clinical trials will be employed. In short, medically healthy children and adolescents (ages 7–17 years) with bipolar I disorder experiencing a manic or mixed episode may be eligible to enroll. These inclusion and exclusion criteria were developed in order to permit many youths suffering from mania to enroll. However, it was felt that the participation of some youths with selected comorbidities might confound the results of this work. For that reason, a limitation of the CoLT trials is that the data collected may not be applicable to all patients with bipolar I disorder. The inclusion and exclusion criteria for both studies are shown in Tables 1 and 2.Table 1Inclusion CriteriaInclusion Criteria1. Subjects aged 7 years to 17 years, 11 months old at time of first dose2. Patients must meet DSM-IV diagnostic criteria, as assessed by a semi -structured assessment (KSADS-PL) and a separate clinical interview with a child/adolescent psychiatrist for manic or mixed episodes in bipolar I disorder3. Score of > 20 on the YMRS at screening and baseline4. The patient and legal guardian must understand the nature of the study and be able to comply with protocol requirements. The legal guardian must give written informed consent and the youth, written assent.5. Patients with comorbid conditions [attention deficit hyperactivity disorder (ADHD), conduct disorder] may participate.6. If female: is premenarchal, or is incapable of pregnancy because of a hysterectomy, tubal ligation, or spousal/partner sterility. If sexually active and capable of pregnancy, has been using an acceptable method of contraception (hormonal contraceptives, intrauterine device, spermicide and barrier) for at least one month prior to study entry and agrees to continue to use one of these for the duration of the study. If sexually abstinent and capable of pregnancy, agrees to continued abstinence or to use of an acceptable method of birth control (either intrauterine device or spermicide and barrier) should sexual activity commence7. Has a negative quantitative serum ß-human chorionic gonadotrophin hormone pregnancy test at screening and a negative qualitative urine pregnancy test at baseline, if female8. Patients with a history of substance abuse may participate if they agree to abstain from drugs during the trial and have a negative drug screen at screening or prior to baseline.9. The subject is willing and clinically able to wash out of exclusionary medication during the screening period. Prior to the administration of lithium, patients will not have used any of the following mediations: antipsychotics, monoamine oxidase inhibitors, antidepressants within the preceding two weeks; stimulants within the preceding week; or fluoxetine or depot antipsychotics in the past month (no stable patients will be asked to discontinue medications)10. ECG and blood work including CBC, prothrombin/partial thromboplastin time, fibrinogen, and thyroid function showing no clinically significant abnormalitiesTable 2Exclusion CriteriaExclusion Criteria1. Patient who is clinically stable on current medication regimen for bipolar disorder.2. A current or lifetime diagnosis of Schizophrenia or Schizoaffective Disorder, a Pervasive Developmental Disorder, Anorexia Nervosa, Bulimia Nervosa, or Obsessive-Compulsive Disorder3. Current DSM-IV diagnosis of Substance Dependence4. Positive drug screen at screening and on retest 1–3 weeks later5. Patients with symptoms of mania that may be attributable to a general medical condition, or secondary to use of medications (e.g., corticosteroids)6. Evidence of any serious and/or unstable neurological illness for which treatment under the auspices of this study would be contra-indicated7. Any serious, unstable medical illness or clinically significant abnormal laboratory assessments that would adversely impact the scientific interpretability or unduly increase the risks of the protocol8. Current general medical condition including neurological disease, diabetes mellitus, thyroid dysfunction, or renal dysfunction that might be affected adversely by lithium, could influence the efficacy or safety of lithium, or would complicate interpretation of study results9. Evidence of current serious homicidal/suicidal ideation such that in the treating physician's opinion it would not be appropriately safe for the subject to participate in this study10. Evidence of current active hallucinations and delusions such that in the treating physician's opinion it would not be appropriately safe for the subject to participate in this study11. Concomitant prescription of over-the-counter medication or nutritional supplements that would interact with lithium or the subject's physical or mental status12. Concurrent psychotherapy treatments provided outside the study initiated within 4 weeks prior to screening13. Previous adequate trial with lithium (at least 4 weeks with lithium serum levels between 0.8–1.2 mEq/L)14. History of allergy to lithium15. Psychiatric hospitalization within 1 month of screening16. Clinician's judgment that subject is not likely to be able to complete the study as an outpatient due to psychiatric reasons17. History of lithium intolerance18. Females who are currently pregnant or lactating19. Sexually active females who, in the investigators' opinion, are not using an adequate form of birth control.20. Subjects who are unable to swallow the study medication21. Subjects for whom a baseline YMRS score of < 20 is anticipated22. Subjects with an IQ less than 70 (determined using the Wechsler Abbreviated Scales of Intelligence {WASI}Vocabulary and Matrix Reasoning Subscales) [33]Overview of Studies 1 and 2Due to their anticipated sample size of approximately 260 patients and their methodological rigor, when completed, the CoLT studies should provide definitive data about the acute efficacy and long-term treatment with lithium in children and teenagers with bipolar mania/mixed states. A brief description of each of these two studies is presented below.Study 1 includes an initial open-label phase lasting 8 weeks during which a variety of dosing paradigms will be explored and data for pharmacokinetic analyses will be obtained. This phase is then followed by a 16 week open-label, long-term stabilization phase and a subsequent double-blind discontinuation phase. During this discontinuation phase, eligible patients are randomly assigned to either continue with lithium treatment or receive placebo for up to 28 weeks. Subsequently, the final phase of this first study includes a restabilization phase that allows subjects who suffer a mood symptom relapse during the discontinuation phase to re-initiate open-label lithium therapy. It is planned that this first study will enroll 60 subjects who will receive up to a maximum of 52 weeks of treatment.Study 2, which will begin subsequent to completion of the first study, begins with an 8-week, double-blind, placebo-controlled phase where 200 subjects will be randomized to receive either lithium or placebo. Similar to the first study, this initial phase will be followed by an open-label long-term phase. However, unlike the analogous phase in the first study, participation in this open-label long term phase in this trial will be of either 16 or 24 weeks in duration depending on the treatment that the patient received during the double-blind phase of this protocol. The long-term treatment phase will be followed by two subsequent phases: a discontinuation phase and a restabilization phase, identical to those described in Study 1. As in Study 1, this study will allow patients to receive treatment with lithium for up to 52 weeks in duration. These studies are described in more detail below. Our hypothesis in Study 1 is that rational dosing strategies for lithium in children and adolescents will be able to be developed. Our hypotheses in Study 2 are (1) that lithium will reduce manic symptoms to a greater extent than placebo acutely and (2) that few participants treated with lithium will withdraw because of adverse effects. Both studies will address the hypotheses (1) that lithium will have long-term efficacy for reducing bipolar symptoms and (2) that lithium will be safe and generally well-tolerated for up to one year.Study 1 (see Figure 1)Figure 1Study 1 of CoLT.As noted above, Study 1 is comprised of four phases that will address the following key components of the WR and the RFP: (1) establishing evidence-based dosing strategies for lithium in youth; (2) characterizing the pharmacokinetics and biodisposition of lithium in children and adolescents; (3) investigating the long-term effectiveness of lithium treatment; and (4) comprehensively characterizing the short- and long-term safety of lithium in children and adolescents. Patients' continuation into subsequent phases of this study is dependent upon their response to their current treatment in the phase of study in which they are participating. Throughout both studies, the a priori response criteria found in Table 3 will be used in order to determine eligibility to participate in subsequent study phases.Table 3A priori response criteria used throughout both studiesResponse CriteriaNon-ResponsePartial ResponseResponse< 25% reduction in baseline YMRS25–49% reduction in baseline YMRS≥ 50% reduction in baseline YMRSORANDANDCGI-I ≥ 4CGI-I ≤ 3CGI-I = 1 or 2Pharmacokinetic PhaseThe Pharmacokinetic Phase of Study 1 is an 8-week, open-label, randomized, escalating dose clinical trial that has two key objectives. The first is to characterize the pharmacokinetic profile of lithium. The second is to develop evidence-based dosing strategies for lithium in children and adolescents with bipolar I disorder. Three different starting doses of lithium carbonate will be explored: 300 mg, 600 mg, and 900 mg. Additionally, two different dose escalation strategies will be examined. In one, the dose of lithium will be increased weekly by 300 mg. In the other, the dose of lithium will be increased by 300 mg twice weekly.In this initial phase of Study 1, subjects will be assigned to one of three treatment arms. A total of approximately 60 subjects, in which approximately 20 subjects will be assigned to each of the three study arms, will be enrolled and dosed. Initially, subjects will be assigned to one of two different treatment groups: \"Arm I\" or \"Arm II\".In Arm I of this initial phase, subjects will be given a starting dose of 300 mg or 600 mg, depending on their weight. All subjects that weigh less than 30 kg will be assigned to Arm I and will receive a starting dose of 300 mg/day. All initial subjects weighing greater than or equal to (≥) 30 kg, after being stratified by age and sex, will be randomly assigned to Arms I and II in approximately equal numbers. All subjects whose weight is ≥ 30 kg and are assigned to Arm I will have a starting dose of 600 mg/day (divided twice daily). In Arm II, subjects who are randomized to this treatment arm will receive a starting dose of 900 mg (divided thrice daily). An evaluation of data collected from subjects who are treated in Arm I and Arm II will provide information about the appropriate starting dose of lithium in children/adolescents. Subjects randomized to Arms I and II may have their dose increased by 300 mg weekly, based upon response and tolerability. After 10 subjects are enrolled in Arm II and have completed the 8 weeks of treatment, and if at least 8 of the first 10 subjects dosed have tolerated the study drug for at least 8 weeks, enrollment into the third arm (Arm III) may begin. Only subjects weighing ≥ 30 kg will be permitted to enter into Arm III. Subjects enrolled into Arm III will have a starting dose of 900 mg, divided thrice daily.In order to examine a second dose escalation strategy, subjects in Arm III will have their lithium dose increased twice weekly depending upon the effectiveness and tolerability of lithium in this cohort. As a result of this study arm, the speed that the lithium dose may be increased (weekly vs. twice weekly) in children and adolescents will be determined.Final dosing for subjects will be determined based upon both response and side effect profile for Arms I-III. For the purposes of this study, subjects will continue to have their dose of lithium increased until any of the following criteria are met: (1) a subject meets response criteria (Clinical Global Improvement Scale (CGI-I) [11] of ≤ 2 and ≥ 50% decrease in the Young Mania Rating Scale (YMRS) [12]; (2) the patient experiences side effects that significantly impact functioning; (3) the serum lithium level is > 1.4 mEq/L [13]; or (4) if the dose exceeds 40 mg/kg/day (with the exception of subjects weighing less than 23 kg who may receive up to 900 mg/day). Based upon the Pharmacokinetic Phase, information will be obtained about the most appropriate starting dose and the speed by which the lithium dose can be increased. This dosing strategy will then be employed in the acute randomized controlled trial that is to be performed under the auspices of Study 2 (below).Pharmacokinetic SamplingIn addition to these dosing procedures, subjects in Arms I and II will undergo blood sampling procedures in order to characterize first-dose pharmacokinetic (PK) parameters for lithium. Blood samples will be obtained prior to dosing and at 0.5, 1.5, 1, 1.5, 2, 4, 8, 12, and 24 hours post-dose. Additionally, one half of the subjects will provide a single blood sample for PK analyses at 48 hours post-dose, and one half will provide a single blood sample at 72 hours post-dose.Furthermore, subjects in Arms I and II will have additional PK samples collected at 2 more time points over the next 16 weeks. The time points for these samples to be collected will be determined by random assignment. These additional samples are collected over a 12 hour period, including a 0 and 12 hour sample and 3 randomly assigned additional samples at the following possible time points: 0.5, 1, 1.5, 2, 4, or 8 hours post-dose.Long-Term Effectiveness PhaseOnce the Pharmacokinetic Phase has ended, subjects who continue to be eligible and who demonstrate at least a partial response (reduction in YMRS score of ≥ 25% and a CGI-I score ≤ 3) and are able to tolerate at least 600 mg lithium/day will be eligible to continue with their current dose for 16 weeks in a Long-Term Effectiveness Phase (LTE). Following a standardized algorithm, adjunctive medications may be added during this phase. Of note, a maximum of only 2 adjunctive medications are allowed to be prescribed at the same time to study subjects once participation in this study phase begins. Patients who are prescribed other agents besides lithium with therapeutic serum concentration levels will have their medication levels monitored throughout their participation in this study.The standardized algorithm includes a sequence of medications to treat residual symptoms of psychosis, mania and hypomania, depression, anxiety, and ADHD (prioritized in that order). These treatment algorithms were developed in order to limit variability across subjects and to provide reasonably interpretable preliminary information regarding adjunctive pharmacotherapy in patients treated with lithium. The rationale concerning the choice of adjunctive medications for residual symptom treatment was informed by various adult data in bipolar I and II disorder, as well as limited data that exist in children and adolescents with bipolar disorder (for a review, see Smarty and Findling 2007) [14]. When no adult or juvenile data existed, algorithms were derived by investigators' consensus, based upon their clinical experiences and consideration of which widely used treatments lacked study to support or refute their use.Psychotic symptoms are to be treated with risperidone, and if needed, followed by a trial of quetiapine, and then aripiprazole. Furthermore, unresponsive manic and hypomanic symptoms will be initially treated with valproate. If the manic or hypomanic symptoms do not respond to valproate, then quetiapine will be started, followed by a trial of aripiprazole.To address residual depressive symptoms, lamotrigine will be the first line treatment followed by a trial of quetiapine. If the patient is non-responsive to treatment with quetiapine, concomitant treatment with citalopram will be initiated to address the depressive symptoms. Additionally, patients who experience anxiety symptoms will initially be treated with valproate. Subsequently, unresponsive anxiety symptoms will be addressed with concomitant treatment with quetiapine followed by lamotrigine.Finally, adjunctive ADHD treatment will be begin with a long acting methylphenidate compound. If it is necessary to initiate another ADHD treatment due to unresponsiveness or intolerance to this initial treatment, a long acting mixed amphetamine salt preparation may be started. The final treatment option for comorbid ADHD symptoms is atomoxetine. These adjunctive interventions will provide additional treatment options for youth with BD for which lithium monotherapy does not address residual mood and other psychiatric symptoms.At the end of this phase, subjects will be categorized as \"responders,\" \"partial responders,\" and \"non-responders\" based upon a priori criteria (Table 3). Subjects who have 6 out of the last 8 consecutive weeks starting at week 8 (the last two weeks must be final 2 weeks of participation in the LTE Phase) without symptom relapse and who have therapeutic lithium levels are eligible for continuation into the Discontinuation Phase.Discontinuation and Restabilization PhasesThe third phase, Discontinuation Phase, is a 28-week, double-blind phase where subjects are randomized to receive either continued treatment with lithium or placebo. Subjects who are randomized to receive placebo will have their dose of lithium gradually discontinued. It should be noted that the ethical issues associated with a medication discontinuation paradigm were carefully reviewed. However, in the absence of maintenance treatment data for pediatric bipolar disorder, the investigators believed that the discontinuation phase of the study provided clinical equipoise between the potential risks of side effects related to long-term lithium exposure, and the potential risks for relapse in patients randomized to placebo. While patients are enrolled in Discontinuation Phase, patients may continue to receive the adjunctive medication at the same dose that was prescribed during the Long-Term Effectiveness Phase. During the Discontinuation Phase, if subjects experience a significant deterioration in clinical status, they are offered 8 weeks of open-label lithium treatment re-initiated in a Restabilization Phase.Study 2 (see Figure 2)Figure 2Study 2 of CoLT.Like Study 1, Study 2 is comprised of four phases that will investigate the acute efficacy of lithium in pediatric bipolarity, examine the long-term effectiveness of lithium treatment, and allow for both the short- and long-term safety of lithium in youth as outlined in the WR. As in Study 1, patients will continue into subsequent phases of the study based upon their response to their response to their current phase of treatment.Efficacy PhaseThe first phase of Study 2 is the 8-week, double-blind, parallel-group, placebo-controlled Efficacy Phase. During the Efficacy Phase, approximately 200 subjects will be randomized to receive 8 weeks of treatment with either lithium carbonate or placebo. The starting lithium dose and the rate at which lithium will be titrated upwards will be based upon the results of the Pharmacokinetic Phase in Study 1. At the end of 8 weeks of treatment, response status will be evaluated in all participating subjects.Long-Term Effectiveness PhaseSimilar to Study 1, at the end of the first 8 weeks of the study and depending on their response to blinded treatment, the patient may proceed to the Long-Term Effectiveness Phase. However, subjects who are responders to placebo or non-responders to lithium treatment will not continue into the Long-Term Effectiveness Phase. In Study 2, the length of participation in this phase will be dependent upon the treatment and response status of subjects in the Efficacy Phase. This is done in order to ensure all subjects receive open label lithium for 24 weeks prior to possible participation in the Discontinuation Phase. As a result, eligible subjects will continue into two parallel treatment arms of different lengths of time in the Long-Term Effectiveness Phase. The two arms include a 16-week arm and a 24-week arm. Those subjects who received lithium in the Efficacy Phase and showed partial or full response will be eligible to continue in the 16-week arm. During the 16-week arm, adjunctive medications may be added following the standardized algorithm noted above as needed.Those subjects who received placebo during the Efficacy Phase and are either partial responders or non-responders (YMRS reduction of < 25% or a CGI-I score ≥ 4) will receive open-label lithium treatment in the 24-week treatment arm of the Long-Term Effectiveness Phase. After an initial 8 weeks of treatment with open-label lithium, these subjects will be assessed; if subjects are at least partial responders, they will continue treatment with lithium for the remaining 16 weeks. During the final 16 weeks of the 24-week arm, adjunctive psychotropic agents will be permitted per the aforesaid treatment algorithms. If subjects are non-responders at the end of 8 weeks of treatment in the 24-week arm, they will be discontinued from the study.Furthermore, subjects who received placebo during the Efficacy Phase will be randomized to possibly receive psychosocial treatment at the onset of the 24-week Long-Term Effectiveness Phase in order to explore whether additional benefit to open-label lithium initiation occurs when combined with psychotherapy. In addition, in order to explore the neurological effects of lithium in juveniles, electro-encephalograms (EEG) will be obtained in the 24-week treatment arm for a randomly chosen subset at baseline, end of week 8, and the end of the Long-Term Effectiveness Phase participation.As in Study 1, patients will be eligible for participation into the Discontinuation Phase if they have had at least 6 of the last 8 weeks (including the last 2 weeks) of the LTE Phase without symptom relapse and have therapeutic lithium levels.Discontinuation and Restabilization PhasesThe Discontinuation and Restabilization phases in Study 2 are identical to these phases in Study 1. Half of the responders will remain on lithium maintenance treatment, and the other half will undergo gradual tapering of their lithium dose by the substitution of placebo capsules for active lithium capsules for 28 weeks. Subjects who experience significant deterioration in clinical status during the Discontinuation Phase will be offered 8 weeks of treatment with open-label lithium in the Restabilization Phase. Data from these final study phases (Discontinuation and Restabilization) will be combined from both Study 1 and Study 2 for statistical analyses.Study Teams and Maintaining the BlindIn order to maintain the blind throughout the two trials, two different study teams will be assembled at each of the sites analogous to the study design implemented by the RUPP research group [15]. At each CoLT site, there will be two groups of clinicians and coordinators that compose the \"blinded\" team and an \"unblinded\" team. At a minimum, each team will include a child and adolescent psychiatrist and a study coordinator. The blinded study team will manage all aspects of study enrollment with the exception of reviewing lithium levels and making lithium concentration-based dose adjustment decisions during placebo-controlled phases. The unblinded teams will be responsible for reviewing the lithium levels and making dosing adjustments in the blinded phases of the two studies.Patient AssessmentsAs requested by the WR, the YMRS will be the primary outcome measure owing to its ability to detect the effects of medication treatment of mania [16]. Patient diagnoses will be based upon results of the Kiddie Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime Episode (K-SADS-PL) [17]. In addition, the WR indicated that the secondary measures should assess attention-deficit/hyperactivity disorder (ADHD) symptomatology, aggressive behavior, irritability, substance abuse, clinical global improvement, and family, school, peer relationships and quality of life. The measures that will be used to assess these domains during the trials are shown in Table 4.Table 4Mood Symptomatology and Life Satisfaction Measures obtained in the CoLT trialsMeasureDomainReferenceInterview with parents & child/adolescentYoung Mania Rating Scale (YMRS)Manic SymptomsYoung et al. [12]Children Depression Rating Scale (CDRS-R)Depression SymptomsOverholser et al. [34]Brief Psychiatric Rating Scale – for Children (BPRS-C)PsychosisHughes et al. [35]Children's Global Assessment Scale (CGAS)Global OutcomeShaffer et al. [36]Clinical Global Impressions Scale–Severity (CGI-S)Severity of illnessNIMH [11]Clinical Global Impressions Scale–Improvement (CGI-I)Improvement of illnessNIMH [11]Drug Use Severity Inventory (DUSI)Substance UseTarter et al. [37]Irritability, Depression, and Anxiety (IDA) (selected items)IrritabilitySnaith et al. [38]The Pediatric Anxiety Rating Scale (PARS)AnxietyThe Research Units on Pediatric Psychopharmacology Anxiety Study Group [39]Social Adjustment Inventory for Children & Adolescents (SAICA)Social Development, Academic AchievementJohn et al. [40]Suicide Severity Rating Scale (SSRS)-LifetimeLifetime suicidal ideation and behaviorPosner et al. [41]Suicide Severity Rating Scale (SSRS)Suicidal ideation and behaviorPosner et al. [41]Parent ReportGeneral Behavior Inventory – Parent Report Mania and Depression Short FormManic and Depression SymptomsYoungstrom et al. [42]AD/HD Rating Scale-IV (ARS-IV)ADHD symptomsDuPaul et al. [43]Child Mania Rating Scale-Parent (CMRS-P)Manic symptomsPavuluri et al. [44]Nisonger Child Behavior Rating Form (NCBRF) Parent VersionAggressionAman et al. [45]Caregiver Strain Questionnaire (CSQ)Parental StressBrannan et al. [46]Family Environment Scale (FES)Family RelationshipsMoos & Moos [47]Neurocognitve TestingThe WR also required that possible cognitive and neurological effects of lithium be evaluated. Lithium can potentially improve certain cognitive functions, but can be deleterious in other domains. The purpose of this adjunctive testing is to provide an evidence-based understanding of the neurocognitive effects of lithium pre- and post-acute trial and post-maintenance trial. The data collected will provide a comprehensive characterization of lithium-specific effects on neurocognitive function that has not been available to date. Therefore, in Study 2, all subjects will undergo a neurocognitve battery at baseline prior to receiving lithium or placebo, at week 8, and after 24 weeks of lithium treatment.The goal of this testing is to determine the integrity of fine-motor, attention, verbal memory, and selected executive function domains pre- and post-acute and maintenance lithium trials. It is hypothesized that positive improvement will be noted in domains of attention, verbal memory, visual memory, and selected executive functions (e.g., set-shirting, inhibition) post-treatment. In contrast, based upon data from adult studies, it is hypothesized that lithium may negatively affect fine-motor speed and control and cognitive processing speed, but results may vary based upon response to lithium and serum levels. In addition, testing will help to determine the integrity of affective regulation, including affective inhibition, pre- and post-acute and maintenance trials. It is hypothesized that the affective regulation of this sample will improve from baseline to the proposed post-acute and maintenance trial time points.Given the available literature on the pathophysiology of bipolar disorder, these assessment domains were selected to coincide with brain regions where the effects of this disorder would be most expected to occur (i.e., hippocampal and pre/frontal brain regions). In addition, tasks were selected to: (1) be age-appropriate and child friendly; (2) have adequate statistical applicability to the various ages and ability levels of this population; (3) be psychometrically sound (i.e., reliable, valid); and (4) theoretically driven by the available literature that has examined lithium usage in children and adults, as well as the extant literature on pediatric bipolar disorder. Further, given the 8-week differential between some of the tasks, measures that evidenced minimal practice effects over this time frame were selected. The neurocognitive tests that will be used in this trial are shown in Table 5.Table 5Neurocognitive measures to be collected in the CoLT studyDomainIntellectualFine MotorAttentionMemoryExecutiveAffective ProcessingWASI 2 Subtest (WASI) [33]Grooved Pegboard [48-50]Vigil Auditory CPT [53]WRAML-2 Verbal Memory (2 Subtests) [54]D-KEF Verbal Fluency (Conditions 1–3) [51,52]Affective Stroop Task [55]Delis-Kaplan Executive Function SystemVigil Visual CPT [53]WRAML-2 Delayed Verbal Memory [54]D-KEF Figural Fluency (Condition 1) [51,52]Affective N Back Memory Task [56](D-KEFS) Trail Making (Condition 4) [51,52]D-KEF Color-Word [51,52]Safety AssessmentsAdverse Event MonitoringSubjects and their guardians will be directly queried about the presence of adverse events throughout the CoLT trials. In addition, to facilitate the careful monitoring of adverse events, multiple assessments will be utilized throughout both studies. These include the Neurological Examination for Lithium (NELi), a modified Side Effects Form for Children and Adolescents (SEFCA) [18], and the Neurological Rating Scale (NRS) [19].The NeLi, which was developed specifically for this trial, includes an examination of neurological events that have been associated with Li treatment. These neurological symptoms include: (1) Tremor; (2) a Finger-nose Test; (3) Tandem Walk; (4) Gait; (5) Grip Strength; and (6) the Romberg Test.The SEFCA is a 54-item scale that rates both the frequency and severity of adverse events. In addition, the SEFCA used in the CoLT studies will be supplemented by selected UKU Side Effect Rating Scale [20] items including queries regarding concentration difficulties, increased fatigability, sleepiness/sedation, reduced salivation, and memory difficulties. Furthermore, items that pertain to acne, motor in-coordination, muscle weakness, and confusion will be added to the SEFCA from the Safety Monitoring and Uniform Report Form (SMURF) [21].Laboratory and Electrocardiogram (ECG) MonitoringOver the course of the CoLT trials, laboratory and ECG testing will be performed periodically. The chemistry profile that will be used throughout the CoLT trials will measure blood concentrations of sodium, potassium, chlorine, carbon dioxide, blood urea nitrogen (BUN), creatinine, calcium and glucose. Furthermore, blood concentrations of total protein, albumin, alkaline phosphate, alkaline transferase, alkaline aspartate, and total bilirubin will be obtained.To study prospectively the effects of lithium on metabolism and lipid profile, fasting total cholesterol, triglycerides, high density lipo-proteins (HDL), low density lipo-proteins (LDL), and cholesterol/HDL ratio will be acquired on all subjects at specified times during the CoLT trials. Additionally, a Complete Blood Count (CBC) with differential will be performed periodically. A urinalysis and urine drug toxicology screen will be assessed at various time points during both CoLT trials.Lithium has been found to interfere with the production of thyroid hormones including the inhibition of the thyroid stimulating hormone (TSH)-responsive adenylate cyclase and PKC in thyroid cells [22-24]. Therefore, thyroid function tests, including TSH (thyroid stimulating hormone), triiodothyronine, and thyroxine will be regularly obtained in theses studies. It should be noted that the CoLT trials also incorporate an algorithm for the assessment and management of TSH elevation/hypothyroidism should either event occur during the auspices of these trials.In addition, research indicates that about 5% of subjects treated with lithium may develop kidney dysfunction as indicated by impaired renal function tests [25]. For this reason, creatinine clearance will be measured at various time points throughout the CoLT trials in order to further assess renal function.Lithium Serum LevelsMonitoring lithium serum concentrations is critical for the safe use of this agent. It has been suggested that the therapeutic serum concentration range for treatment with lithium lies between 0.3 and 1.3 mEq/L, with 1.5 mEq/L representing the lower limit for intoxication [13]. Additionally, it has been recognized that lithium has a narrow therapeutic index and near-toxic doses are required to achieve the optimal therapeutic effect [26,27]. Therefore, the chosen maximum lithium level after which dose increases would not be permitted was set at 1.4 mEq/L. Lithium serum concentrations will be obtained weekly during the first 8 weeks of treatment and will be monitored regularly thereafter.Electrocardiogram (ECG)The cardiovascular effects of orally administered lithium have been reported as being generally benign. However, lithium has been shown to prolong sinus node recovery time [28,29]. Therefore, an ECG will be utilized prior to patients receiving lithium and throughout the CoLT trials in order to monitor cardiac function.Electroencephalogram (EEG)As stated above, an EEG will be obtained from a randomly assigned subset of subjects who participate in the 24-week long Long-Term Effectiveness Phase at baseline, at the end of week 8, and at the 24-week time point. In addition, if a subject experiences significant deterioration in neurological or cognitive status, an EEG will also be obtained. Furthermore, if a patient experiences moderate or severe headaches that are temporally related to medication/placebo, dysarthria, ataxia, cognitive dulling, or confusion that are possibly or probably related to the study medication an EEG will be obtained.RBC/Plasma Lithium (Li+) RatioRBC/plasma Li+ ratio is considered to have clinical implications in: (1) predicting clinical response; (2) risk for toxicity; (3) possibly, time course for response; and (4) optimal dosage for long-term prophylaxis. Researchers have found that high in vivo RBC/plasma Li+ ratios are a result of a RBC membrane defect that causes a deficiency of Li+ - Na+ counter flow [30]. In addition, there is preliminary evidence that this membrane defect is autosomal dominant in transmission, and leads to high intracellular Li+ level compared to serum level [30,31]. A high Li+ ratio is considered to be predictive of a positive lithium response [32].Therefore, during the 24-week Long-Term Effectiveness arm, a subset of 24 randomly subjects will have weekly serum and whole blood samples to allow for a Li+ ratio to be computed. In addition, subjects who experience ataxia, dysarthria, reduced motor coordination, listlessness/sedation, slurred speech, tremors, confusion, or delirium that is both possibly or probably related to the study medication, and is noted to be of moderate or severe intensity, will have a serum and a whole blood sample in order to assess the RBC/plasma Li+ ratios.Throughout the trials, a Data Safety Monitoring Board (DSMB) will be involved in monitoring the trials' progress.ConclusionIn summary, the innovative and multidisciplinary CoLT studies will provide the data to allow for evidence based dosing of lithium in the children and adolescents with bipolar disorder. In addition, if lithium is shown to have an acceptable acute- and long-term efficacy and safety profile in children and adolescents, this knowledge could substantively influence the treatment choices of clinicians who provide care to those vulnerable children and teenagers suffering from bipolar disorder.Competing interestsDr. Findling receives or has received research support, acted as a consultant and/or served on a speaker's bureau for Abbott, Addrenex, AstraZeneca, Bristol-Myers Squibb, Forest, GlaxoSmithKline, Johnson & Johnson, Lilly, Neuropharm, Novartis, Organon, Otsuka, Pfizer, Sanofi-Aventis, Sepracore, Shire, Solvay, Supernus Pharmaceuticals, and Wyeth. Dr. Frazier receives or has received research support from Bristol Myers Squibb, GlaxoSmithKline, Eli Lilly and Company, Johnson and Johnson, Neuropharm, Otsuka, and Pfizer. Dr. Kowatch receives or has received research support, acted as a consultant, served on an advisory board, and/or served on a speaker's bureau for Abbott, Astra-Zeneca, Bristol-Myers Squibb, CABF, Creative Educational Concepts, GlaxoSmithKline, Medscape, NICHD, NIMH, Sanofi-Aventis, and the Stanley Research Foundation. Dr. Pavuluri's work unrelated to this manuscript is supported by NIH/NCRR K23 RR018638-01, NIMH MH077852, NIMH P50 HD055751, DANA Foundation, NARSAD, American Foundation for Suicide Prevention, Colbeth Foundation, GlaxoSmithKline- NeuroHealth, Abbott Pharmaceuticals and Janssen Research Foundation. Dr. Sikich receives or has received research support from Eli Lilly, Janssen, Pfizer, Bristol Myers Squibb, Otsuka and Neuropharm. Dr. Hooper has acted as a consultant to Lilly. Dr. Taylor-Zapata is the project officer for the funding institute, the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), for this study. The other authors have no financial ties to disclose.Authors' contributionsAll authors have made substantial contribution to the conception and design of the study, have been involved in the drafting and/or critical revising of this manuscript, and all authors have given final approval of this manuscript.\n\nREFERENCES:\nNo References"
+}

batch_9/PMC2531092.json

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+{
+    "id": "PMC2531092",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531092\nAUTHORS: Danny Landau, Judith Jackson, Gisselle Gonzalez\n\nABSTRACT:\nIntroductionPhendimetrazine is a medication currently being used to help patients with weight loss. It shares a chemical structure with amphetamines. As such, it shares some of the same toxicities, which can include cardiac toxicity. This case highlights this principle.Case presentationa 54 year old Caucasian female presented to our urgent care facility with complaints of chest pains and other symptoms suggestive of acute coronary syndrome. Ultimately, she was transferred to the emergency room. After evaluation there, it appeared she was having demand ischemia from prescription diet pillsConclusionThis case report demonstrates the potential dangers of amphetamine based diet pills. There have been other cases of cardiomyopathies related to phendimetrazine, but it is something that is rarely recognized in an outpatient setting. A case such as this demonstrates the importance of obtaining a careful medication history in all patients and in recognizing diet pills with an amphetamine base can cause cardiac toxicity.\n\nBODY:\nCase presentationA 54 year-old Caucasian female presented to our urgent care facility complaining of nausea and vomiting, sense of impending doom and vague chest pain radiating toward her left side for about five hours. She never had similar symptoms in the past. She also denied anything that could have precipitated these symptoms. Her only past medical history was significant for spina bifida. Her medications included occasional Fiorinal (unknown dose), Xanax 0.5 mg as needed, and Phendimetrazine (unclear dose). Her social history was significant for smoking 1/2 pack per day cigarette use. She denied alcohol use. Family history was non contributory. She worked from home. Her physical exam showed a tachycardia of around 100 beats per minute, respiratory rate of 16, temperature of 98.1, and O2 saturation of 100% on room air. She was approximately 5'7\" and 145 pounds. In general, she was an anxious appearing, diaphoretic woman in moderate distress, she had no elevated JVD at 30 degrees, her heart was tachycardic, but otherwise without murmur, gallops, or rubs, her lungs were clear, abdomen soft, and she had no peripheral edema. An EKG was checked which appears below (figure 1). After examination, there was concern for acute coronary syndrome (ACS). She was given nitroglycerin with relief of her chest discomfort. She was also given aspirin to chew. EMS was called and she was transferred to a local emergency room. She was hospitalized there for three days and after her discharge, we got permission from her to request records. While hospitalized, she was ruled out for ACS with negative troponins. She was also given beta blockade which resolved her tachycardia and her T wave changes on EKG. The next morning, she had an adenosine stress test which revealed normal uptake with no areas of ischemia and an ejection fraction of 55%. She was monitored for one more day and then discharged with instructions to discontinue her diet pills.Figure 1An EKG taken from the patient while they were having chest pain. It demonstrates T wave depression in lateral leads.DiscussionPhendimetrazine is a medication currently being used for weight loss, with potential for illicit use. It has a similar chemical composition of amphetamines, which is thought to account for its clinical actions [1]. Amphetamines are well recognized as an etiology of cardiac ischemia, however phendimetrazine is more rarely described in the literature as causing cardiac events. [2,3]. Acute effects include hyperpyrexia, mydriasis, chest pain, arrhytmias, delirium, and, rhabdomylosis, among others [2]. Long term use has been associated with dilated cardiomyopathies, some of which have resolved with discontinuation of the medication [3]. In this particular case, it appears she may have developed a demand ischemia from the medication. It is not known how much of the drug she was taking. Initially, she was resistant to accepting that phendimetrazine could induce side effects, and there was suspicion that she could have been taking more of the drug that recommended. In addition, she was not prescribed the medication and would not admit to where she obtained it. As the public seems to have more focus on using medications to induce weight loss, this may be a more recognized complication and heart conditions should likely be monitored prior to starting amphetamine based weight loss pills.ConclusionDue to potentially detrimental effects of this medication, phendimetrazine should be used cautiously in many situations. As it shares its chemical structure with amphetamines, it also shares many of the side effects and the potential for abuse/addiction. There have been other reports in literature describing adverse outcomes from phendimetrazine as well as other weight loss medications. Therefore, cautious use is warranted.AbbreviationsACS: Acute Coronary Syndrome.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsDL, JJ, GG have all been involved in and approve of the writing of this case presentation.ConsentWritten informed consent was obtained from the patient for publication purposes. A copy can be obtained if requested by the Editor in Chief of this journal.\n\nREFERENCES:\nNo References"
+}

batch_9/PMC2531095.json

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+{
+    "id": "PMC2531095",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531095\nAUTHORS: Paolo Zanatta, Enrico Bosco, Piero Di Pasquale, Agarwal Nivedita, Carlo Valfrè, Carlo Sorbara\n\nABSTRACT:\nBackgroundEarly postoperative stroke is an adverse syndrome after coronary bypass surgery. This report focuses on overcoming of cerebral ischemia as a result of haemodynamic instability during heart enucleation in off-pump procedure.Case presentationA 67 year old male patient, Caucasian race, with a body mass index of 28, had a recent non-Q posterolateral myocardial infarction one month before and recurrent instable angina. His past history includes an uncontrolled hypertension, dyslipidemia, insulin dependent diabetes mellitus, epiaortic vessel stenosis. The patient was scheduled for an off-pump procedure and monitored with bilateral somatosensory evoked potentials, whose alteration signalled the decrement of the cardiac index during operation.The somatosensory evoked potentials appeared when the blood pressure was increased with a pharmacological treatment.ConclusionDuring the off-pump coronary bypass surgery, a lower cardiac index, predisposes patients, with multiple stroke risk factors, to a reduction of the cerebral blood flow. Intraoperative somatosensory evoked potentials monitoring provides informations about the functional status of somatosensory cortex to reverse effects of brain ischemia.\n\nBODY:\nBackgroundPostoperative stroke is a serious adverse event after coronary artery bypass surgery (CABG) and may be increased in patients with multiple risk factors for cerebral ischemia [1]. The off-pump procedure can reduce neurological complications avoiding the use of cardiopulmonary bypass and aortic manipulation [2,3]. However, this can cause hemodynamic instability related to a low cardiac output, low vascular resistance, preload variation and a physical obstruction of the venous return, with subsequent hypotension [4].Reduced cerebral perfusion can be further aggravated in patients with significant carotid stenosis [5].Intraoperative neurophysiological assistance provides information on the brain's functional reserve allowing the anesthesiologist and the surgeon to perform a neuroprotective strategy [6,7].Case presentationA 67 year old male patient, Caucasian race, body mass index of 28, with two-vessel disease not amenable to angioplasty, was scheduled for an off-pump procedure, consisting in a left internal mammary artery graft on anterior descending coronary artery and venous grafts on the obtuse marginal. His medical history included one month before, a non-Q posterolateral myocardial infarction and recurrent instable angina; complete occlusion of the right internal carotid and left vertebral artery and 50% stenosis of the left internal carotid artery; uncontrolled hypertension; dyslipidemia, and insulin dependent diabetes mellitus complicated with lower limb sensory neuropathy. Seven years ago, the patient had suffered a stroke because of closing right carotid artery, without clinical effects.Brain Magnetic Resonance Imaging Scan diagnosed a suffering circle of Willis. The preoperative echocardiography revealed a mild posterolateral hypokinetic wall movement with normal ejection fraction. The chest X-ray showed moderate aortosclerosis of the ascending aorta. The neurologic examination was negative with the exception of altered tactile sensibility of the legs bilaterally.During operation we used the Pressure Invasive Continuos Cardiac Output technology  to monitor in continuous, the cardiac output. Epicardial echocardiography was obtained to exclude any atheromatous plaques in the ascending aorta.SEPs (somatosensory evoked potentials) from median nerve by electrical stimulation were recorded in continuous, after general anesthesia induction. The median nerve were bilaterally stimulated with subdermal needle electrods at the wrist. The recording electrods were placed at the homolateral Erb's point and at the C3'/C4' at opposite side the stimulation site. The stimulation rate was 3.7 Hz. After a baseline obtained with a 300 stimulus average, the ongoing average was obtained with 30 stimulus.ResultsThe SEPs and the hemodynamic parameters did not change until the end of the first graft.During the heart displacement to perform the second coronary anastomosis, the cardiac index (CI) markedly decreased (from 2.9 to 1.8 l/min/m2), without arterial pressure variation and the right SEP disappeared (Table 1), (Figure 1). The left SEP amplitude was reduced by 30%. No variation were noted on the Erb's recording.Table 1SEP, hemodynamic and respiratory variable recordings and the neuroprotection strategy during the operative steps.Post anaesthesia inductionHeart enucleation\"Anestesiologist reaction\"End surgeryRightLeftRightLeftRightLeftRightLeftN20/P25 latency (msec)22.6723.83-25.54 ± 0.0823.86 ± 0.1325.52 ± 0.0825.1523.35N20/P25 amplitude (uV)1.922 ± 0.092.51 ± 0.130.021.83 ± 0.0071.03 ± 0.32 ± 0.041.67 ± 0.031.68 ± 0.01CF (pulse/min)80606565SAP (mmHg)135130173146MAP (mmHg)849111495DAP (mmHg)58707667CVP (mmHg)18242718CI (l/min/m2)2.91.81.82.6SVRI (dyn.sec.m2/cm5)1820297738662369PaCO2 (mmHg)38394040.5SaO2 (mmHg)100100100100T (°C)36.23534.835neuro-protectionNorephi (mcg/Kg/h)--0.08-MAC (Et isoflurane)0.50.511FiO2 (%)505010050Hb (g/dl)10101212Volume load (liter)0.5 HES.+ 0.5 bloodN20/P25 = cortical SEP, CF = cardiac frequency, SAP = systolic arterial pressure, MAP = mean arterial pressure, DAP = diastolic arterial pressure, CVP = central venous pressure, CI = cardiac index, SVRI = systemic vascular resistence index, PaCO2 = arterial pressure of CO2, SaO2 = oxygen saturation, Norephi = norephinefrine, MAC = minimum alveolar concentration, FiO2 = inspiratory fraction of oxygen, Hb = haemoglobin, T = temperature, HES = HydroxyethylstarchFigure 1SEP (N20/P25) amplitude variation during the intraoperative steps. The right SEP disappeared when the heart is enucleated. Both SEPs have a non significant wave amplitude attenuation at the end of surgery. N20/P25 complex is the most important scalp-recorded cortical component that has a negative peak about 20 msec followed by a positive peak at about 25 msec.After 10 minutes, with establishment of a neuroprotection strategy, the right SEP reappeared (Table 1) (Figure 1). The systolic blood pressure was increased to 173 mmHg using norepinephrine intravenous boluses of 15 mcg. The inspiratory fraction of oxygen was set at 100% until the end of last anastomosis. The volemia was also increased by administering 500 ml of Hydroxyethylstarch and two packed red cells. Furthermore, the brain metabolism was reduced by increased the end tidal minimal alveolar concentration (MAC) of isoflurane until 1. The CI didn't change during this time.A norepinephrine infusion of 0.08 mcg/Kg/h was then started and maintained until the end of the surgery (Table 1). When the heart was replaced in the pericardium the CI went up to 2.4 l/min/m2.At end surgery the cortical SEP amplitude in the right and left hemispheres was respectively 13% and 33% lower from baseline values which are still in the normal range [7]. No significative variation on the SEP latency were noted during the case.The patient was estubated after 5 hours without any neurologic impairment.DiscussionCerebral ischemia in off-pump cardiac surgery occurs due to brain hypoperfusion induced by heart dislocation and possible macroembolic events during partial clamping of the aorta. In our patient, the SEP amplitude disappeared after heart enucleation because of reduced brain oxygen delivery. This variation allowed us to increase Cerebral Blood Flow and arterial concentration of oxygen by enhancing the haemoglobin concentration and the inspiratory fraction of oxygen. A bolus of norepinephrine was administered to increase cerebral perfusion pressure. Brain vascular resistances were reduced by increasing the MAC of isoflurane that reduced also the brain oxygen consumption. The increasing dose of volatile agent did not influence the ability to use SEP to monitor the effect of treatment but produced an attenuation of wave amplitude recorded until the end of surgery like few authors have reported [8].In literature there is no papers about the use of SEPs for monitoring the brain function in high risk patients for cerebral ischemia submitted to off pump cardiac surgery while there is one paper about the use of electroencephalogram in this setting [6].The SEPs are a reliable method to monitor brain function during surgery and they present some advantages in respect to the Electroencephalogram, because they are particularly resistant to the anaesthesia, moderate hypothermia and enviromental electrical interference because of averaging [7].We chose SEP because the brain generators of the cortical SEP (N20/P25) are situated within the middle cerebral artery territory which covers 60% of the brain. Thus, continuous monitoring of the cortical SEP not only provides information on the integrity of the Central Nervous System, but also indirectly on the level of cerebral flow necessary to maintain minimal cortical function.A 50% reduction of the N20 amplitude and a 20% increase in its latency is considered a clear sign of brain ischemia, in absence of ischemic arm, global hypoxia and bolus of anesthetic drugs [7].ConclusionThe low cardiac index produced by the heart enucleation during the CABG off-pump, increases the risk of cerebral hypoperfusion. Intraoperative SEP monitoring seems to be a reliable method to perform a neuroprotection strategy and prevent cortical damage also in off pump coronary artery bypass grafting. Further studies are necessary to confirm this hypothesis.AbbreviationsCABG: Coronary artery bypass surgery; SEPs: Somatosensory evoked potentials; CI: Cardiac index; MAC: Minimum alveolar concentration; N20/P25: Cortical SEP.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsPZ conceived the work, collected and analyzed the data and write the article. EB analyzed the data. PDP analyzed the data. AN helped to write the article. VC conceived the work and analized the data. SC analized the data. All authors read and approved the final manuscript.ConsentWritten informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.\n\nREFERENCES:\nNo References"
+}

batch_9/PMC2531099.json

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+{
+    "id": "PMC2531099",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531099\nAUTHORS: Armin RW Elbers, Johan Popma, Sandra Oosterwolde, Piet A van Rijn, Piet Vellema, Eugène MA van Rooij\n\nABSTRACT:\nBackgroundIn August 2006 a major epidemic of bluetongue virus serotype 8 (BTV8) started off in North-West Europe. In the course of 2007 it became evident that BTV8 had survived the winter in North-West Europe, re-emerged and spread exponentially. Recently, the European Union decided to start vaccination against BTV8. In order to improve the understanding of the epidemiological situation, it was necessary to execute a cross-sectional serological study at the end of the BT vector season. Cattle were the target species for cross-sectional serological studies in Europe at the end of 2006 and 2007. However, there was no information on the BTV8-seroprevalence in sheep and goats.ResultsOn the basis of our cross-sectional study, the estimated seroprevalence of BTV8-exposed locations in the Netherlands in 2006 was 0% for goats (95% confidence interval: 0 – 5.6%) and 7.0% for sheep (95% confidence interval: 3.5 – 12.9%). The estimated seroprevalence of BTV-8 exposed locations in 2007 was 47% for goats (95% confidence interval: 36 – 58%) and 70% for sheep (95% confidence interval: 63 – 76%). There was a wide range in within-location seroprevalence in locations with goats and sheep (1 – 100%). A gradient in seroprevalence was seen, with the highest level of seroprevalence in the southern Netherlands, the area where the epidemic started in 2006, and a decreasing seroprevalence when going in a northern direction.ConclusionThere is a much higher estimated seroprevalence of locations with goats exposed to BTV8 than can be inferred from the rather low number of reported clinical outbreaks in goats. This is probably due to the fact that clinical signs in infected goats are far less obvious than in sheep. The wide range in within-location seroprevalence observed means that the proportion of animals protected in 2008 by a natural infection in 2006 and/or 2007 can differ highly between flocks. This should be taken into account when vaccinating animals.\n\nBODY:\nBackgroundIn August 2006 a major epidemic of bluetongue virus serotype 8 (BTV8) started off in North-West Europe, including the Netherlands, Belgium, Germany, Luxembourg and the North of France [1,2]. In order to improve the understanding of the epidemiological situation of this disease, it was necessary to execute a cross-sectional serological study at the end of the vector season of 2006. The Community legal framework on bluetongue monitoring and surveillance was laid down in Council Directive 2000/75/EC and Commission Decision 2005/393/EC and these are in line with the Terrestrial Animal Health Code of the OIE. Cattle were the target species for the cross-sectional serological study at the end of 2006 [3,4].Many hoped that the winter season of 2006/2007 would halt the BTV epidemic, assuming that the chain of transmission would be broken by the dying off of infected adult vectors and a halt in the life cycle of the vector because of low temperatures. However, in the course of 2007 it became evident that BTV8 somehow had survived the winter in North-West Europe and a re-emerging epidemic spread exponentially within the original affected countries. Moreover, BTV8 was introduced into the United Kingdom, Denmark, Czech Republic and Switzerland [5].The scale of the epidemic in 2007 was so huge that the European Union decided to start vaccination against BTV8 in 2008. The vaccination campaign aims to achieve a 80% or more coverage of animals protected (either by vaccination or by immunity acquired through natural infection) [6].The sentinel monitoring system, set up at the beginning of 2007 to detect re-emergence of BTV8, already provided some insights into the extend of the BTV8 spread in the cattle population in 2007. However, with respect to goats and sheep there was no information on the BTV8-seroprevalence in North-West Europe. This paper presents the seroprevalence and geographical spread of BTV8 on animal and herd levels in goats and sheep in the Netherlands in 2006 and 2007.MethodsBlood samples from Dutch sheep and goats were serologically tested at the Central Veterinary Institute (CVI) in Lelystad for antibodies against BTV8 using a competitive ELISA (Institute Pourquier, Montpellier, France). This ELISA has a high sensitivity (~100%) and specificity (>99.8%) [7]. The blood samples were collected in the framework of obligatory and voluntary health programmes (e.g. certified disease-free programmes within the European Union) executed by the Dutch Animal Health Service. For the 2006 seroprevalence estimates, we used blood samples collected in the first months of 2007. For the 2007 seroprevalence estimates, we used blood samples collected in the last months of 2007. Locations with animal sampled were selected proportional to the population distribution of locations with goats and sheep in the Netherlands in the different provinces. Sample size within locations for the health programmes was set at detecting at least a prevalence of disease of 5%. In smaller flocks this often meant that almost all animals within the flock were sampled.There are approximately 41,000 locations with sheep and 23,000 locations with goats in the Netherlands registered at the Animal Health Service. Based on these data and an a priori estimated seroprevalence of BTV8-infected locations with goats of 5% and a maximum acceptable error in the estimated prevalence of 5% and a 95% confidence level required for the estimated prevalence, a sample size of approximately 70 locations with goats was calculated using WinEpiscope version 2.0 [7] for our cross-sectional study in 2006. For 2007, we adjusted the a priori estimated seroprevalence of BTV-8 infected locations with goats to 30% with a maximum acceptable error in the estimated prevalence of 10% and a 95% confidence level required for the estimated prevalence, resulting in a sample size of approximately 81 locations with goats for 2007. For the locations with sheep, we used an a priori estimated prevalence of BTV8-infected locations with sheep of 10% and a maximum acceptable error in the estimated prevalence of 5%, resulting in a calculated sample size of approximately 140 locations with sheep for 2006. For 2007, we adjusted the a priori estimated seroprevalence to 50% with a maximum acceptable error in the estimated prevalence of 7.5% and a 95% confidence level required for the estimated prevalence, resulting in a sample size of approximately 170 locations. Since there is also an unknown number (not registered) of small-holder locations in the Netherlands with goats and sheep with a small numbers of animals (1 to 5 animals per location), we arbitrarily increased the actual sample size with approximately 15 to 20%. Exact confidence intervals for the estimated seroprevalence were calculated according to Fleiss [9].ResultsSeroprevalence of BTV8-infected locations with goats and sheep in 2006A total of 1,975 goat sera from 83 locations with goats and 2,555 sheep sera from 143 locations with sheep were collected. All goat samples were seronegative, a total of 17 sheep samples (0.7%) from 10 locations with sheep (7.0%) were seropositive. The BTV8-seropositve locations with sheep were located in the provinces of Limburg, North Brabant, Zeeland, and Gelderland. Within-location seroprevalence ranged from 3 to 50% (almost all sheep present in the flocks were sampled). The location of the provinces in the Netherlands is shown in Figure 1. Based on our sampled population we estimated the proportion of BTV8-seropositive locations in the Netherlands with sheep and goats. On a national level the estimated seroprevalence of BTV8-exposed locations in 2006 was 0% for goats (95% confidence interval: 0 – 5.6%) and 7.0% for sheep (95% confidence interval: 3.5 – 12.9%).Figure 1Geographical location of the twelve provinces in the Netherlands (DR: Drente; FL: Flevoland; FR: Friesland; GL: Gelderland; GR: Groningen; L: Limburg; NB: North Brabant; NH: North Holland; OV: Overijssel; UT: Utrecht; ZH: South Holland; ZL: Zeeland).Seroprevalence of BTV8-infected locations with goats and sheep in 2007A total of 1,995 goat sera from 81 locations with goats and 4,252 sheep sera from 214 locations with sheep were collected. A total of 204 goat samples (10.2%) from 38 locations with goats (46.9%) were seropositive. The BTV8-seropositve locations with goats in 2007 were located in the provinces of North Holland, South Holland, Utrecht, Gelderland, North Brabant, and Limburg.A total of 1,627 sheep samples (38.3%) from 149 locations with sheep (69.6%) were seropositive. The BTV8-seropositve locations with sheep were located in all provinces of the Netherlands in 2007. On a national level the estimated seroprevalence of BTV8-exposed locations in 2007 was 47% for goats (95% confidence interval: 36 – 58%) and 70% for sheep (95% confidence interval: 63 – 76%). The median within-location seroprevalence on BTV8-infected locations with goats was 21% (min – max: 1 – 100%) and with sheep 67% (min – max: 1 – 100%) (Figure 2).Figure 2Distribution of within-location seroprevalence on locations with antibodies against bluetongue virus serotype 8 in goats (N = 38) and sheep (N = 149) in the Netherlands in 2007.DiscussionThere is very sparse information on seroprevalence of BTV-infected locations with goats and sheep during outbreaks. A cross-sectional study in Kazakhstan showed a within-herd seroprevalence in cattle, sheep and goats varying between 0 and 100% [10]. A cross-sectional study in sheep flocks in Northern Pakistan [11] indicated 90% of the flocks seropositive. In these seropositive sheep flocks, within-flock seroprevalence ranged from 12 to 100% (median: 47%). A cross-sectional serological study in Queensland, Australia, found within-flock seroprevalences in infected sheep flocks ranging from 1 – 42% and in infected goat flocks ranging from 5 – 16% [12].During the 2006-epidemic in North-West Europe, no clinically affected goats were reported [3]. The results of our seroprevalence study is in line with those findings. However, in week 35 of 2007, the first clinical disease in goats caused by BTV8 in North-West Europe was reported from the Netherlands [13]: in a holding containing 600 milking goats, 10 goats demonstrated clinical signs of BT, starting with acute drop in milk yield and pyrexia, followed by edema of lips and face, crusts on lips and muzzle, nasal discharge, conjunctivitis and erythema of the udder. Up to the end of 2007, a total of 25 holdings reported clinical disease (BT indicative) in goats in the Netherlands. The results of our seroprevalence study indicate a seroprevalence of locations with goats of approximately 50% in the Netherlands in 2007. This is much higher than can be inferred from the 25 locations that reported clinical outbreaks in goats in 2007. This is probably due to the fact that clinical signs in infected goats are far less obvious than in sheep [14].In the first year (2006) of the BTV8-epidemic in the Netherlands, a total of 270 sheep flocks and 200 cattle herds reported clinical disease. In 2007, the epidemic really took off: about 3,200 clinical outbreaks were reported by locations with sheep on a total of 6,500 outbreaks reported. The results of the seroprevalence study in sheep in 2006 and 2007 are in line with the number of clinical outbreaks reported.In our study we see a wide range in within-location seroprevalence in locations with goats and sheep. This means that the proportion of animals protected in 2008 by a natural infection in 2006 and/or 2007 can differ highly between locations. Two options for a vaccination strategy are open: either one vaccinates all ruminants on the locations with animals irrespective of the proportion of animals protected by a natural infection in 2006 or 2007 or one determines the proportion of animals protected by a natural infection within the flock: if a low to moderate proportion of animals is naturally protected one has a good reason to vaccinate all the animals; if a high proportion of animals is naturally protected, this might be a reason not to vaccinate the flock.ConclusionSeroprevalence of BTV8-exposed locations with goats and locations with sheep was much higher in 2007 than in 2006. There was a much higher estimated seroprevalence of locations with goats exposed to BTV8 than can be inferred from the rather low number of reported clinical outbreaks in goats. This is probably due to the fact that clinical signs in infected goats are far less obvious than in sheep. There was a wide range in within-location seroprevalence in locations with goats and sheep. This means that the proportion of animals protected in 2008 by a natural infection in 2006 and/or 2007 can differ highly between flocks. This should be taken into account when vaccinating animals.Authors' contributionsARWE, PAvR and EMAvR designed the study. PV facilitated the use of sera collected by the Animal Health Service. JP and SO performed the laboratory analyses. ARWE performed the data analyses and drafted the manuscript. JP, PAvR, PV and EMAvR commented on the draft. All authors read the manuscript and approved the manuscript.\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2531118",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531118\nAUTHORS: Claudia Patrignani, Maria Chiara Magnone, Patrizia Tavano, Michele Ardizzone, Valeria Muzio, Béatrice Gréco, Paola F Zaratin\n\nABSTRACT:\nBackgroundThe present study has investigated the protein tyrosine phosphatase H1 (PTPH1) expression pattern in mouse brain and its impact on CNS functions.MethodsWe have previously described a PTPH1-KO mouse, generated by replacing the PTP catalytic and the PDZ domain with a LacZ neomycin cassette. PTPH1 expression pattern was evaluated by LacZ staining in the brain and PTPH1-KO and WT mice (n = 10 per gender per genotype) were also behaviorally tested for CNS functions.ResultsIn CNS, PTPH1 is expressed during development and in adulthood and mainly localized in hippocampus, thalamus, cortex and cerebellum neurons. The behavioral tests performed on the PTPH1-KO mice showed an impact on working memory in male mice and an impaired learning performance at rotarod in females.ConclusionThese results demonstrate for the first time a neuronal expression of PTPH1 and its functionality at the level of cognition.\n\nBODY:\nBackgroundTyrosine phosphorylation plays an important role in several signaling pathways regulating cell growth, differentiation, cell cycle, apoptosis and neuronal functions [1,2]. The phosphorylation/dephosphorylation balance is controlled by protein tyrosine kinases and phosphatases. PTPs can be distinguished into four classes: 1) classical PTPs that can be subdivided into transmembrane, receptor-like enzymes, and the intracellular, nonreceptor PTPs, 2) dual-specificity PTPs (Ser and Tyr phosphatases), 3) low molecular weight PTP and 4) the Asp-based PTPs (Tyr/Ser phosphatase activity) [3].Classical PTPs have been reported to play a key role in neural functions, from development to cognitive function. For example, RPTPs such as PTPδ, PTPσ, LAR, and especially PTPRO, are important players in axonal growth and guidance during development [4]. Studies on PTPσ-KO (RPTP) mice have shown involvement of this PTP in the regulation of the developing hypothalamo-pituitary axis [5,6] and in the development of the CNS architecture [7]. PTPBL-KO (non receptor like PTP-NRPTP) mice display impaired motor nerve repair in a model of sciatic nerve crush lesion [8] and PTPMEG (NRPTP) interacts with key intracellular players leading to the stimulation of the channel activity of NMDA receptors [9].In the present study we focused our attention on a NRPTP, PTPH1, and on its possible role on neural functions. Indeed PTPH1 has been shown to be expressed in the CNS [10] but little is currently known on its potential impact on CNS functions. PTPH1 (also called PTPN3) belongs to a sub-family of non receptor cytosolic PTPs characterized by the presence of a FERM domain (band 4.1, ezrin, radixin, moesin) at its N-terminus, responsible for the interaction with transmembrane proteins and/or phospholipids in the cell membrane [11-13]. In addition PTPH1 has a PDZ domain in the central part responsible for the interaction with other proteins, whereas the single catalytic domain is located at the C-terminus.PTPH1 activity has been involved in a variety of cellular functions including TCR-signaling [14-16], cell cycle regulation [11,16,17], endoplasmic reticulum assembly [18], cardiac sodium channel modulation [19] and TNFα converting enzyme inhibition [20].Recently, our group has demonstrated that PTPH1 dephosphorylates GHR in vitro and in cellular assays [21] and results in an increase of body weight in the functional PTPH1-knockout (KO) mice via modulation of IGF1 secretion [22] thus demonstrating its in vivo relevance.PTPH1 has been shown in the rat to be highly expressed in thalamic nuclei as well as various cortical areas [10]. However, no information is currently available on its impact on CNS functions. To address this question we have further characterized our PTPH1-KO mice line through behavioral and anatomical approaches. PTPH1 expression and localization was evaluated by LacZ staining in the brain and a behavioral test battery evaluated PTPH1 loss on CNS functions such as locomotor activity (open field), anxiety-like behavior (open field and elevated plus maze), motor ability, coordination and learning (accelerating rotarod), spatial working memory (Y maze) and nociceptive sensitivity (hot plate).MethodsAnimalsPTPH1-KO and wild type littermates (F2 generation, 87.5% C57Bl/6 – 12.5% 129S6SvEv) aged 3–4 months were used for behavioral phenotyping. Mice were individually caged and maintained in a 12:12 hours light: dark cycle (lights on at 7 am) at 21 ± 1°C with food and water available ad libitum. Protection of animals used in the experiment was in accordance with Directive 86/609/EEC, enforced by the Italian D.L. No. 116 of January 27, 1992. Physical facilities and equipment for accommodation and care of animals were in accordance with the provisions of EEC Council Directive 86/609. Tail snips from mice were collected and genotyped as previously reported [22].PTPH1 KO designPTPH1-KO mice were generated using the Velocigene technology [23], as described in details elsewhere [22]. Briefly a mouse BAC containing the PTPH1 gene was modified: an in-frame LacZ reporter sequence and a neomycin-selectable marker replaced exons 15 to 22 encoding for the PDZ and the catalytic domain of PTPH1. BAC electroporation into embryonic stem cells was performed. F1 heterozygous mice were bred to generate F2 PTPH1-KO mice. Line breeding and animal care were performed in Charles River Italy and France.LacZ staining procedure and immunohistochemistryPTPH1-KO and WT mice, 12 months old, n = 2, male and females, were sacrificed by ip overdose of thiopental (5%), perfused with paraformaldehyde 4%, then washed in PBS and incubated overnight at 37°C in the solution containing the substrate for beta-galactosidase (beta-gal, encoded by the LacZ cassette) coupled to a NBT salt. The organs and the tissues in the sections display a green/blue staining where PTPH1 gene is normally expressed. After rinsing into PBS, organs were postfixed in PFA 4% for 1 hour, then incubated in 50% glycerol overnight at 4°C and finally maintained in 70% glycerol at room temperature. LacZ staining was observed through a low magnification microscope and described by an operator blind to the genotypes.LacZ staining was also performed on CNS sections. Mice (n = 3, 12 months old) were sacrificed by ip injection of an overdose of thiopental (5%), perfused with PBS and PFA 4%. Brains were removed and postfixed overnight at 4°C in PFA 4%, then placed overnight at 4°C in 15% and finally in 30% sucrose buffer. The brains were then included in O.C.T. (Tissue-Tek) and sections were cut on slides with a cryostat at 20 μm thickness. The slides were incubated in LacZ staining solution (see above) overnight at 37°C, washed thrice in PBS (5 min each) and either counterstained with H&E (Merck KGaA) [23] or co-expressed with NeuN immunostaining. Briefly, sections were incubated for 3 hours at room temperature in blocking solution (Vectastain Kit), washed in TBS (Tris-buffered saline), incubated overnight at 4°C with a solution containing the primary antibody mouse anti-mouse Neuronal Nuclei (Chemicon MAB377, 1/1000). The staining was revealed by ABC kit secondary antibody (mouse Vectastain Kit), and DAB (Sigma). After dehydration, sections were transferred onto coverslips. LacZ staining and co-expression with NeuN-immunoreactivity (NeuN-ir) was observed by microscopy and described by an operator blind to the genotypes.Semiquantitative RT-PCR for beta galactosidase geneSemiquantitative RT-PCR for PTPH1 and beta-gal gene expression was performed in different brain areas of PTPH1-KO and WT mice in order to confirm the presence of beta-gal expression in the KO tissues, replacing PTPH1 PDZ and catalytic domain. Brains from KO and WT mice (n = 5, 6 months old) were freshly removed and rinsed in HBSS. Hippocampus, cerebellum, cortex, striatum, midbrain and olfactory bulbs were dissected. Total RNA was extracted using Trizol Reagent (Invitrogen) and cleaned-up by RNAeasy columns from Qiagen. 5 μg of total RNA were used to perform the RT-PCR reaction (SuperScript II RT kit, Invitrogen). The primer sequences for LacZ amplification were the following: LacZ – forward 5'-GAT GTA CGT GCC CTG GAA CT/reverse 5'-GGT CCC ACA CTT CAG CAT TT. In order to load equally the reaction mixes, a 300 bp fragment of Histone 2A was amplified as a house keeping gene with the following primers: H2Az forward – 5' CGT ATT CAT CGA CAC CTG AAA; H2Az reverse – 5' CTG TTG TCC TTT CTT CCC GAT.Behavioral phenotyping test batteryNeurological functions of PTPH1-WT and KO mice (males and females, 11 weeks-old, n = 10 per gender per genotype) were assessed through a behavioral test battery.The sequence of the test battery was chosen from the least invasive to the most ones. The schedule of the testing sessions included one week of recovery from one test to the next, as reported in Table 1.Table 1Schedule of the behavioral test battery.Age (wks)8910111213141510M+10FarrivalquarantineadaptationOpen FieldEPMRotarodY-mazeHot plateOpen fieldAfter one hour of adaptation in the testing room, each mouse was placed in an open field chamber (50 cm2 wide with white floor and walls) (ViewPoint Life Sci. Inc.) to test locomotor activity and anxiety-like behaviors. Locomotion was recorded for one hour by a video camera and analyzed automatically by VideoTRACK® software (ViewPoint Life Sci. Inc.). Locomotor activity was evaluated by calculating the total path length traveled, whereas the relative time spent in the center was taken as indicative of anxiety-like behavior [24]. The tests were performed in two sessions with equivalent group representation.Elevated plus mazeAfter one hour of adaptation in the testing room, anxiety-like behavior was tested for each mouse by EPM within one session. The apparatus consists of four arms (29.5 cm long and 5 cm wide each). Two arms are open whereas the 2 others are limited by 2 black walls (20 cm high). The number of entries of each mouse in the open and closed arms was recorded by a video camera during a period of 5 minutes and analyzed by the SMART Video-Tracking Software (ViewPoint Life Sci. Inc.). The total number of entries into the arms is an index of locomotion, whereas the percentage of time spent and percentage of entries in the closed arms is an index of anxiety-like behaviors [25].Accelerated rotarodMotor ability, coordination and learning were evaluated by using an Accelerated Rotarod apparatus for mice (Cat. # 7650 by Jones and Roberts, distr. by Basile Instr., Italy). The apparatus was placed within the animal colony room and was cleaned after each trial. Mice were tested for their abilities to maintain a balance on a rotating bar, which accelerated from 4 to 40 rpm/min in a 5 min trial. Latency to fall off was measured within one session and all mice underwent four trials (one every 30 min) [26-28]. The differences at the rotarod performances in WT and KO were assessed by a single set of trials [27,28]. This set-up allows a major focus on the early phases of motor learning, involving a strong activation of prefrontal cortex and of the associative areas of basal ganglia and cerebellum [29,30].Y-maze alternationAfter one hour of adaptation in the testing room, mice were tested on a Y maze apparatus (40 cm long/8 cm wide arms with transparent walls) to investigate spatial working memory [31]. The number and the sequence of the arm entries for each mouse were recorded during 5 minutes. The locomotion index was calculated as the overall number of arm entries, whereas the working memory index was calculated as following: number of exact alternations (entries into three different arms consecutively)/possible alternations (i.e. the number of arms entered minus 2) × 100.Hot plateThermal sensitivity was assessed by a hot plate apparatus for mice (Cat. # 7280 by Biol. Research Apparatus, distr. by Basile Instr., Italy) and lasted a maximum of 45 seconds, time at which damages could occur [32]. The apparatus was placed in the animal colony room and all the mice were tested within one session. Animals were placed on a surface heated at 52.5°C and the latency (seconds) to shake or lick the paw was recorded by the operator.StatisticsStatistical comparisons were performed by unpaired two-tailed T-test (p < 0.05) and two-way ANOVA (p < 0.05) followed by post-hoc test as necessary. In the accelerated rotarod, two-way ANOVA with repeated measures followed by T-test was used. Results are expressed as mean ± SEM.ResultsLacZ staining in whole mountIn PTPH1-KO adult animals, LacZ staining in the brain was observed in the cerebellum, hippocampus and in the thalamic nuclei. In addition a strong staining was observed in the cerebral cortex, tenia tecta and septum (Figures 1a, 1b).Figure 1PTPH1-KO adult mouse brain. a: whole brain, dorsal view, staining in cerebellum (Cb) and cortex (Co); b: LacZ staining on brain, sagittal view: detection in the tenia tecta (Tt), cortex (Co), thalamus (Th), hippocampus (H), retrosplenial cortex (Rc), septum (S) and in the granule cell layer of cerebellum.LacZ staining in sections and RT-PCR resultsNo gross cytoarchitectural brain differences were observed by simple visual observation at the microscope in the cortex, hippocampus and thalamus in PTPH1-KO mice compared to WT littermates.LacZ staining was performed on frozen brain sections to confirm and to describe the expression of PTPH1 at the brain structural level (Table 2).Table 2Qualitative estimation of LacZ staining intensity in the different brain areas.Brain AreaIntensity of LacZ stainingBrain AreaIntensity of LacZ stainingCerebral cortex+Dorsal Tenia Tecta++Retrosplenial cortex++Septohippocampal nu+CA1 oriens layer+++VPL+CA1 radiatum layer+++MDL++CA1 pyramidal cell layer+AV++CA2 oriens layer-VPM++CA2 radiatum layer-VL+CA2 pyramidal layer+VM++CA3 oriens layer+Po++CA3 radiatum layer+/-LD+CA3 pyramidal layer+Rt+DG granular cell layer-DLG++DG molecular layer+++VPPC++DG hilus-PF-Fascicola cinereum++cerebellum+Indisium griseum+-: absent; +/-: faint; +: present; ++: intense; +++: very intense staining. Cornu Ammonis (CA), Dentate Gyrus (DG), mediodorsal lateral (MDL), anteroventral (AV), ventromedial (VM) ventral posteromedial (VPM), ventrolateral (VL), ventral posterolateral (VPL), laterodorsal (LD), posterior (Po) and reticular (Rt), ventral posteromedial parvicel (VPPC) thalamic nuclei, lateral geniculate nucleus (DLG), parafascicular thalamic nuclei (PF), nuclei (nu).In cortical regions, LacZ was expressed in the external pyramidal (III) and internal granular layer (IVA) of the cerebral cortex (Figures 2a, 2b), in the retrosplenial cortex (Figures. 3a, 3b 3c, 4a and 4b) and indusium griseum (Figures 3a, 3b). In the cerebellum, in spite of a strong staining in the whole mount (Figure 2), only a faint LacZ signal was observed in sections (Figures 5a, 5b) in particular in the granule cells, close to the nuclei. The RT-PCR on cortical and cerebellar extracts confirmed the presence of LacZ expression in these brain areas (Figure 6).Figure 2a: PTPH1-KO cerebral cortex (10×, scale bar: 220 μm). b: positive cytoplasmatic and perinuclear LacZ staining (blue dots) in the external pyramidal (III) and internal granular layer (IVA) (63×, scale bar: 10 μm).Figure 3PTPH1-KO cerebral cortex. a: LacZ detection in retrosplenial cortex (Rc) and indusium griseum (ig) staining (4×, scale bar: 80 μm). b: detail of the Rc and ig (40×, scale bar: 20 μm); c: positive cytoplasmatic staining of the neurons of Rc (63× scale bar: 10 μm); the interneural LacZ signals are due to the presence of trans-sectioned axons and dendrites.Figure 4PTPH1-KO cerebral cortex. a: colocalization of NeuN-ir and LacZ staining signal in the Rc (100×, scale bar: 4.5 μm).; b: detail of the cytoplasmatic signal of LacZ in neurons.Figure 5PTPH1-KO cerebellar cortex. a: faint LacZ staining in the granule cell layer (20×, scale bar: 16.5 μm); b: perinuclear staining in the granule cell layer of the cerebellum (63×; scale bar: 5 μm).Figure 6RT-PCR for beta-galactosidase expression in brain extracts. Beta-gal mRNA is expressed in PTPH1-KO cerebellum, cortex, hippocampus and substriatal regions (midbrain, thalamic nuclei, pontine region); no beta-gal expression detected in WT brain extracts (first lane of each block); histone H2A gene was used as positive control (second lane).In subcortical regions, LacZ was detected in the anterior ventral, mediodorsal, ventrolateral, anteromedial and central lateral thalamic nuclear groups (Figure 7a). In more caudal thalamic areas, LacZ was again detectable in the posterior thalamic nuclear group (Po), and to a lesser extent in posteromedial, in posterolateral and in reticular thalamic nuclei and also in the dorsal lateral geniculate nuclei (Figures 7b, 7c, 7d and 7e). In the tenia tecta, LacZ staining visible in the whole mount preparation was confirmed (Figures 2, 8a, 8b). The RT-PCR on substriatal regions including the thalamus, the midbrain and the pontine areas confirmed the presence of LacZ expression in some of these brain areas (Figure 6). To exclude any potential impact of LacZ blood signal contamination in brain areas, RT-PCR on 5 to 20 μl of whole blood was carried out and did not reveal any significant signal [Additional files 2, 3].Figure 7PTPH1-KO thalamus [76]. a: LacZ expression detected in several thalamic nuclei (4×; scale bar: 165 μm): mediodorsal (MD), central lateral (CL), anteroventral (AV), anteromedial (AM), ventromedial (VM) ventral posteromedial (VPM) and ventrolateral (VL) thalamic nuclear groups. MHb: medial habendular nuclei. b: LacZ expression detected in the ventral posteromedial thalamic nuclei (VPM) and it is present also in ventrolateral (VL), ventromedial (VM), ventral posterolateral (VPL), laterodorsal (LD), posterior (Po) and reticular (Rt) thalamic nuclei (2.5×, scale bar: 130 μm). c: LacZ is expressed in the dorsal lateral geniculate nucleus (DLG) and in the lateroposteral thalamic nuclear group. In this caudal section LacZ staining is more intense in the posterior nucleus, but present also in VPM, VPL and VPPC (ventral posteromedial parvicel) thalamic nuclei (2.5×, scale bar: 13 μm). d: Detail of beta-gal expression in neural cell body of VPL and VPM at 40× (scale bar: 20 μm) and e: at 63× (scale bar: 10 μm).Figure 8PTPH1-KO adult mouse brain. a: beta-gal expression detected in the dorsal tenia tecta (Dtt) and in the septohippocampal nuclei (SHi) (4×; scale bar: 165 μm). b: Detail of cytoplasmatic LacZ staining in the Dtt and SHi (10×; scale bar: 70 μm).In the hippocampus, LacZ expression was observed in the cytoplasm of a few pyramidal cells and through the fibers of the oriens and radiatum layer in a rostral caudal spread (Figure 9a). In rostral sections, LacZ was expressed in the septohippocampal nuclei (Figure 8a). In more caudal sections LacZ was present in the CA1 and CA3, and in a lesser extent in the CA2 (Figures 9b, 9c and 9d). In the CA3 LacZ was strongly expressed in the oriens and pyramidal cell layer (Figure 9d), but its intensity was reduced in the radiatum and oriens compared to CA1 (Figure 9b). No staining was detected in the lacunosum-molecular layer in CA1, CA2 and CA3 (Figures 9b, 9c and 9d). The dentate gyrus showed a strong positive LacZ signal in the molecular layer, but not in the hilus (Figure 9e).Figure 9PTPH1-KO hippocampus [76]. a: Hippocampus at 4×; b: CA1 area of hippocampus shows very intense LacZ staining in both oriens and radiatum layers and to a less extent in the pyramidal cell layer (20×; scale bar: 90 μm). c: CA2 area of hippocampus displays LacZ-positive staining in the pyramidal cell layer. d: CA3 area shows an intense beta-gal expression in the oriens and pyramidal cell layer, and in a less extent in the radiatum (20×). e: The dentate gyrus (DG) displays a strong LacZ staining in the molecular layer and not in the hilus (20×) (scale bar: 20 μm). pyr: pyramidal cell layer; oriens: oriens layer; rad: radiatum layer; mol: molecular layer: gr: granule cell layer; lac/mol: lacunosum-molecular layer.Behavioral phenotypingAs previously demonstrated, PTPH1-KO mice were healthy, reproduced normally and did not show any phenotypic traits distinguishing them from their WT littermates by simple visual observations [22,33]. An increased body weight has been detected in PTPH1-KO mice compared to WT littermates, more pronounced in male mice and probably due to an enhanced GHR sensitivity, that leads to increased IGF-1 mRNA and protein expression in liver and plasma, respectively [22].In EPM, open field test and hot plate tests (anxiety-related behavior and thermal pain sensitivity), PTPH1-KO male and female mice did not show any significant differences in comparison with their WT littermates (data not shown).In the accelerated rotarod and Y-maze test, significant differences were observed between PTPH1-KOs and WTs based on gender and genotype factors. In the accelerating rotarod test PTPH1-WT mice did not show any gender differences (P2WAY = 0.5824 (WT gender vs WT activity); PAUC = 0.3218 (PTPH1-WT male vs female)) (Figure 10a). PTPH1-KO male mice displayed an overall significant better performance compared to their matched female littermates (P2WAY = 0.007 (KO gender vs KO activity) (Figure 10b). Post-hoc T-test analyses showed that the difference was significant at the second trial of the test (P0 = 0.109; P30 = 0.015; P60 = 0.067; P90 = 0.835), and the area under the curve for PTPH1-KO male mice was significantly higher (by 50%) compared to the matched values of the female littermates (P = 0.0194) (Figure 10c).Figure 10Rotarod test on PTPH1-WT and KO mice (n = 10) males and females. a: WT males and WT females do not display any significant different performance at the rod (P2WAY = 0.5824) b: KO males and KO females display a significant different performance (P2WAY = 0.007) (post-hoc T-test: P0 = 0.109; P30 = 0.015; P60 = 0.067; P90 = 0.835). c: 50% difference in the area under the curve represented in figure 10a (unpaired T-test, P=0.0194). d: Female KO mice display a worse performance at the rod compared to WT females (P0 = 0.171; P30 = 0.002; P60 = 0.028; P90 = 0.025) e: No significant difference in the performance on the rod between male KO and WT mice. a, b, d, e: All the data were analyzed by Two-way Anova followed by T-test; *: p < 0.05; **: p < 0.01.Considering this gender effect, the follow up analysis was carried out in males or females assessing genotype effects on activity. PTPH1-KO female mice performed significantly worse compared to their matched WT littermates, starting from the second trial and onwards (P0 = 0.171; P30 = 0.002; P60 = 0.028; P90 = 0.025) (Figure 10d). No significant differences were observed in PTPH1-KO male mice compared to their matched WT littermates (P0 = 0.92; P30 = 0.363; P60 = 0.222; P90 = 0.135) (Figure 10e).In the Y-maze test, no differences were detected between PTPH1-KO and WT female mice either in working memory (Pfemale = 0.972) or in locomotion indices (Pfemale = 0.73; Figures 11a, 11b). On the other hand, PTPH1 KO male mice displayed a significantly higher working memory index (percentage of exact alteration; Pmale = 0.041) but similar locomotion activity (total arm entries) (Pmale = 0.348) compared to their matched WT littermates (Figures 11a, 11b).Figure 11Y-maze behavioral test on PTPH1-WT and KO mice (n = 10) males and females. a: Male KO mice display higher working memory index compared to WT male littermates (Pmale = 0.041); no differences recorded in the female mice. b: No significant differences recorded in the locomotion index, represented by the total arm entries between PTPH1-WT and KO males and females. T-test, *: p < 0.05.DiscussionPTPs are key factors in multiple signaling pathways, leading to modulated functional activities in various cell types [34,35]. Among all PTP forms, PTPH1 has been shown in vitro to modulate cardiac sodium channel Nav1.5 [19], that it is also known to be expressed in the axons of cerebral cortex, cerebellum, thalamus and brain stem [36]. Moreover, PTPH1 contains a domain with high sequence homology with the members of the band 4.1 superfamily protein, FERM. This domain mediates the linkage of actin filaments to the plasma membrane [37], and therefore may be involved in cytoskeleton-membrane interactions, crucial for axon functionality. To further understand the potential role of PTPH1 in neural functions in vivo, we first investigated its expression pattern in embryonic and adult PTPH1-KO mice CNS by LacZ staining, and second its role in CNS functions by behavioral phenotype characterization.In rat embryonic stage Es19, PTPH1 expression through FISH analyses has already been shown in the dorsal thalamic nuclei, which give rise to the thalamo-cortical connections in adulthood [10]. Thus, it has been suggested to play a role in the maintenance of these connections in adults. We replicated these data in PTPH1-KO mice at Es14 and Es16 embryonic stages. PTPH1 is expressed in the hypothalamic area and but also in the dorsal root ganglia of the spinal cord, excluding the spinal cord itself [Additional file 1] [38]. Moreover, at postnatal P1, PTPH1 expression is also present in peripheral organs such as muscles and intestines as in the adults [22]. On the other hand, the CNS expression at P1 appears weaker than in the adults suggesting a pattern of PTPH1 expression corresponding to specific developmental stages of the CNS as well as peripheral organs (data not shown). These changes in expression may play a role in various developmental functions that need to be further understood.In PTPH1-KO adults, LacZ is expressed in different CNS areas such as cerebral and retrosplenial cortices (Figures 1, 2, 3 and 4), hippocampus (Figure 9), thalamus (preferentially ventral thalamus) (Figure 7), cerebellum (Figure 5) and in the region of the tenia tecta (Figures 1, 8). This data confirms previously observed expression patterns in the rat brain by Sahin et al. [10] and extends the observation to other brain regions. We, furthermore, demonstrate that PTPH1 is expressed within the cytoplasm and close to the cell membrane of neurons in most of the brain area investigated (Figures 4a, 4b). It is known that the FERM domain is indeed necessary for PTPH1 localization close to the plasma membrane in Jurkat T cells [14] and it could be responsible for the punctate expression pattern of PTPH1 in the cytosol of the neurons (Figure 4b) [39]. This supports the concept that PTPH1 may be involved in cytoskeleton-membrane interaction within extended neuronal population in the CNS, potentially playing a role in various neuronal functions.Indeed the neural expression of PTPH1 in CA1, CA3 and DG of the hippocampus (Figures 9a, 9b, 9c, 9d and 9e), in the retrosplenial cortex (Figures 3, 4) and in a series of thalamic nuclei (Figures 7a, 7b, 7c, 7d and 7e) suggests an involvement of PTPH1 in the modulation of the memory circuit. Both hippocampus and retrosplenial cortex are key regions in the spatial working memory functions [40-46]. Moreover, several thalamic nuclei have also been shown to be important in the memory process [47,48]. For example, a strong loss of dorsomedial and ventral posterior thalamic neurons is associated with severe cognitive and memory disabilities in patients affected by traumatic brain injury [49]. Lesions in the lateral thalamus may lead to important working memory defects in rodents [50]. The anterior thalamic nuclei project via the retrosplenial cortices to the hippocampus [51,52], thus underlying the importance of both these circuits and of PTPH1 in the mnemonic process.Another interesting PTPH1-positive area is the indusium griseum (Figures 3a, 3b) whose role in the adult brain is not clear. It is thought to be part of the limbic system, receiving afferents from the entorhinal and pyriform cortex and projecting to the septohippocampal nuclei, olfactory tubercle (presumably the tenia tecta) and the medial frontal cortex [53,54]. The expression of PTPH1 in these specific regions suggests a potential role in the processing/integration of memory and sensory information to the SHi and likely the cortex.Indeed PTPH1 expression is also detectable in the pyramidal neurons in layer III and IVA of the cerebral cortex of the mouse (Figures 2a, 2b), in agreement with Sahin's findings in the rat brain. The middle layers (III and IV) of the cerebral cortex are key sites for thalamic inputs [55,56] especially for VPM and VPL, primary thalamic nuclei for somato-sensory information integration [57]. Furthermore a strong cortico-cortical communication has been assessed between these two layers [58], thus suggesting a role for PTPH1 as key regulator in the transmission of the thalamo-cortical and cortico-cortical information.The cerebellar cortex is also positive for PTPH1 expression, in particular in the cytoplasm of granule cells (Figure 5b). The cerebellum is known to be the main structure for motor learning functions. In particular, the cerebellar cortex seems to be involved in the early learning phases of motor activities [59,60] that include also a strong activation of other areas such as prefrontal cortex and basal ganglia [29,30]. PTPH1 expression in the granule cells seems to indicate a potential involvement in the processing of afferent information to the purkinje cells, since it is known that afferents fibers to the cerebellar cortex will project in part through the granule cell layer.PTPH1 expression pattern observed in our analysis points out a potential involvement of this phosphatase in numerous CNS processing functions such as locomotion, sensorial integration, learning and memory. In this study, the behavioral phenotyping of the PTPH1-KO mice allowed us to test these hypotheses in vivo. Indeed, as already demonstrated by our group [22] and also by others [33], PTPH1-KO mice are healthy and do not display any phenotype, distinguishing them from their matched WT littermates, detectable by simple visual observation. Therefore PTPH1-WT and KO mice underwent a battery composed by five behavioral tests, from the least to the most invasive (Table 1), with the tolerable limitation of the handling bias.Behavioral testing revealing locomotor dysfunctions, such as open-field, EPM and Y-maze did not highlight differences between the two genotypes (Figure 11b), suggesting that PTPH1 does not play a critical role in the integration of locomotor information.Anxiety-like behaviors measured by open-field (as path in the center) and EPM (as time spent in the open arms), exploiting rodents natural aversion to open space, did not show any differences between the two genotypes (data not shown), leading to the conclusion that PTPH1 may not be involved in the integration of thalamo-limbic information, key paths for anxiety behavior processing. Similar conclusions can be drawn from the lack of difference between the genotypes regarding integration of nociceptive information, based on hot plate test.In the behavioral test, that partly depends on working memory performances (Y maze), PTPH1-KO male mice showed a slightly better short-term memory than their WT littermates (Figure 11a). Thus, PTPH1 may be involved in the integration of memory information. This was further strengthened by results obtained with a test assessing learning and coordination, the rotarod. Contrary to other behaviors where little differences have been observed, learning and coordination capacities in PTPH1-KO female mice are significantly impaired (Figures 10b, 10c). The low rotarod performance on the early trials, compensated by the last trial, is suggestive of a delay in learning acquisition (Figures 10b, 10d).As reported in Pilecka et al., our PTPH1-KO mice express the non-catalytic part of PTPH1 in frame with the enzymatically active part of LacZ gene. LacZ is widely used as a reporter for promoter activity in KO mice and all those mice express a modified protein, whose full function is not known. So far it was never reported a function of LacZ alone in cognition and we consider quite unlikely that this is the case in our mice. Thus, it is very likely that the behavioral phenotype we detect in our mice is linked to the deletion of the catalytic domain of PTPH1.The impairment in learning and coordination of PTPH1-KO female mice may be resulting from the involvement of PTPH1 in the GH signaling pathway [21]. Indeed our group has already shown that PTPH1-KO mice display higher GHR response in vivo and consequently a higher expression of its down-stream effector hormone, the IGF1 in liver and plasma [22]. GHR is highly expressed in most areas of the CNS, in particular in the choroid plexus, hippocampus, putamen, thalamus and hypothalamus. Similarly IGF1 and IGF1-receptors are localized predominantly in hippocampus, but also in amygdala, cerebellum and cortex [61]. Although IGF1 is considered a neuroprotective hormone, it can be produced in the CNS, it is primarily synthesized in the liver and can cross the blood-brain barrier [62-65]. The GH-IGF1 axis is also known to influence cognitive functions due to several neuroprotective effects on the hippocampus [66]. Furthermore it has been recently pointed out that old conditional liver-IGF1-KO mice display impaired spatial learning and memory [67]. The presence of PTPH1 in key CNS regions, as well as the consequent deregulation of the GH-IGF1 axis in KO mice, strengthens the concept that the PTPH1 network (CNS and downstream peripheral effectors) may be involved in cognitive functions.The behavioral tests assessing working memory and specifically learning revealed not only a genotype effect but also a gender effect, as mentioned above. Sex hormones are known to modulate the somatotropic system [68,69]. In humans, testosterone has an important effect on GH axis, in part by its aromatization to estradiol. Administration of estrogens, or aromatized androgen, modulates GH axis neuroregulation [69,70]. In particular, chronic E2 administration has been shown to reduce GH-induced IGF1 increased expression in liver and plasma via a negative feedback mechanism, while acute E2 administration leads to the expected GH-induced IGF1 release [71]. Furthermore, it has been reported that estrogens play not only regulatory functions on neuroendocrine systems but can also have stimulatory or inhibitory impacts on the inter-connectivity of the hippocampal structure depending on the gender [72-75], meaning that the same stimulus can have opposite effects in male vs female mice. Thus, the cognitive behavioral differences observed in our KO mice are underlying the potential impact of the PTPH1 network on neuroendocrine regulation as well as on cellular architecture within specific brain regions.ConclusionIn conclusion, we have demonstrated that PTPH1 is expressed in neural populations present in adult brain areas mainly involved in locomotor and cognitive functions. The behavioral assessments have allowed us to reveal PTPH1 functionality especially within cognitive domains. Better understanding the interplay between various phosphatases regulating CNS functions, which now includes PTPH1, will be key in the future to unravel some of the complexity of CNS signaling pathways necessary for information processing.List of abbreviationsPTPH1: protein tyrosine phosphatase H1; KO: knock-out; WT: wild type; CNS: central nervous system; PTKs: protein tyrosine kinases; PTPs: protein tyrosine phosphatases; RPTPs: receptor-like protein tyrosine phosphatases; NRTPTs: nonreceptor PTPs; FERM: 4.1, Ezrin, Radixin, Moesin; TACE: TNFα converting enzyme;GH: growth hormone; GHR: growth hormone receptor; IGF1: insulin-like growth factor 1; BAC: bacterial artificial chromosome; ip: intraperitoneal; PBS: phosphate buffered saline; PFA: paraformaldehyde; NBT: nitrobluetetrazolium; beta-gal/LacZ: beta-galactosidase; NeuN: Neuronal Nuclei; HBSS: Hank's balanced salts solution; H2A: Histone 2A; EPM: Elevated plus maze; AUC: area under the curve; Es: embryonic stage; E2: estradiol; CA: Cornu Ammonis; DG: Dentate Gyrus; MDL: mediodorsal lateral thalamic nuclei; AV: anteroventral thalamic nuclei; VM: ventromedial thalamic nuclei; VPM: ventral posteromedial thalamic nuclei; VL: ventrolateral thalamic nuclei; VPL: ventral posterolateral thalamic nuclei; LD: laterodorsal thalamic nuclei; Po: posterior thalamic nuclei; Rt: reticular thalamic nuclei; VPPC: ventral posteromedial parvicel thalamic nuclei; DLG: lateral geniculate nucleus; PF: parafascicular thalamic nuclei; nu: nuclei; SHi: septohippocampal muclei; Tt: tenia tecta; Ig: indusium griseum.Competing interestsThe present work is part of CP’s PhD program at the University of Eastern Piedmont, in close collaboration with MerckSerono International S.A.. MCM, PT, VM, BG, PFZ are employed by MerckSerono International S.A., which is  involved in the discovery and the commercialization of therapeutics for the prevention and treatment of human diseases.Authors' contributionsThe study was devised by CP and MCM and carried out by CP. PT was responsible for the genotyping of all the adult animals that have been used in this study. MA performed the LacZ staining experiment on adult mice. VM and BG have been deeply involved in the first editing of the manuscript and all the authors contributed to modifications in subsequent drafts. PFZ has been involved in critically revising the manuscript and has given the final approval of the version to be published. All the authors read and approved the final version of the manuscript.Supplementary MaterialAdditional file 2Semiquantitative RT-PCR for beta galactosidase gene in blood samples. a: white and red blood cells count in PTPH1-WT and KO mice; no major differences were found in the hematological composition in WT and KO mice. b: Beta-gal mRNA signal was present in PTPH1-KO hippocampus and cortex and not in the WTs; histone H2A gene was used as positive control. c: RT-PCR for beta-gal/H2A on 4 increasing amounts of whole blood (WB): 5, 10, 15 and 20 μl. No signal for beta-gal or H2A was detectable using 5 and 10 μl of WB, due to the low amount of total RNA; a faint signal for H2A was detectable on 15 and 20 μl of WB and a faint band for beta-gal was present only in KO mice, representing the maximum blood contamination in the whole mouse brain. Thus, blood contamination is minimum and it cannot interfere with the main source of signal.Click here for fileAdditional file 3Additional methods. This document provides the methods and the references that have been used to perform the experiments represented in Additional file 2Click here for fileAdditional file 1LacZ staining on PTPH1-WT and KO embryos. PTPH1-WT embryos do not show any staining either at embryological stage 14 (Es14) or at Es16. PTPH1-KO embryos display a positive LacZ staining in the hypothalamic area and but also in the dorsal root ganglia of the spinal cord, excluding the spinal cord itself.Click here for file\n\nREFERENCES:\nNo References"
+}

batch_9/PMC2531127.json

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+{
+    "id": "PMC2531127",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531127\nAUTHORS: Nilüfer Karadeniz, Kristin Mrasek, Anja Weise\n\nABSTRACT:\nBackgroundComplex chromosomal rearrangements (CCRs) are defined as structural chromosomal rearrangements with at least three breakpoints and exchange of genetic material between two or more chromosomes. Complex chromosomal translocations are rarely seen in the general population but the frequency of occurrence is anticipated to be much higher due balanced states with no phenotypic presentation. Here, we report a severely mentally retarded fertile male patient in whom further delineation of CCR involving chromosomes 1, 4 and 2 was carried out by using high resolution multicolor banding (MCB) technique. As a FISH based novel chromosome banding approach, high resolution MCB allows for the differentiation of chromosome region specific areas at band and subband levels.ResultsCytogenetic studies using high resolution banding of the proband necessitated further delineation of the breakpoints because of their uncertainty: 46,XY,t(1;4;2)(p21~31;q31.3;q31). After using high resolution MCB based on microdissection derived region-specific libraries, the exact nature of chromosomal rearrangements for chromosomes 1, 2 and 4 were revealed and these breakpoints were located on 1p31.1, 1q24.3 and 4q31.3 giving rise to a balanced situation.ConclusionFurther delineations are certainly required to provide detailed information about the relationship between balanced CCRs and their phenotypes in order to offer proper counseling to the families concerned. Carriers must be investigated with high resolution banding and molecular cytogenetic techniques to determine the exact locations of the breakpoints. High resolution MCB is an alternative and an efficient method to other FISH based chromosome banding techniques and can serve in clarifying the nature of CCR.\n\nBODY:\nBackgroundStructural chromosomal abnormalities are estimated to occur in around 0.5% of newborn infants, using moderate level of resolution in conventional cytogenetic analysis [1]. Complex chromosomal rearrangements (CCRs) are defined as structural chromosomal rearrangements with at least three breakpoints and exchange of genetic material between two or more chromosomes. It is therefore not surprising to see CCR rarely in constitutional karyotypes. Moreover, some CCRs cannot be interpreted with standard cytogenetic methods at all [2]. Complex chromosomal rearrangements are extremely rare but are often associated with mental retardation, congenital abnormalities, recurrent abortions and infertility [3]. More than 130 constitutional CCRs have been documented so far [4]. 12 of these were related with fertile men including the case we present [5]. Providing genetic counseling for CCRs is very important and this can be offered before or after pregnancy as well as at the time of prenatal diagnosis [6].Since the introduction of fluorescence in situ hybridization (FISH) techniques using whole chromosome painting probes [7] in human cytogenetics, progress has been achieved concerning the ability to characterize chromosomal subregions by molecular cytogenetic methods. Recently, high resolution MCB technique was developed [8] making it possible to identify different chromosome region specific areas at band and subband levels.Here we report a fertile male with mental retardation carrying balanced complex chromosomal rearrangements, involving chromosomes 1, 4 and 2. We also provide advice for genetic counseling of the fertile CCR carrier by discussing the possible mechanisms underlying the origin of CCR.ResultsBanding cytogenetic revealed a normal karyotype for the wife and a complex rearranged one for the spouse. A CCR involving chromosomes 1, 2 and 4 was detected and his karyotype was characterized as 46, XY, t (1; 4; 2) (p21~31; q31.3; q31) (see Fig 1). After performing FISH by using MCB (see Fig 2), the breakpoints were localized to 1p31.1, 2q24.3 and 4q31.3.Figure 1shows image of the GTG banded metaphase. Image belongs to GTG banded metaphases from the father. Arrow indicates the breakpoints on each chromosome.Figure 2shows MCB pattern of the cytogenetic result. It shows multicolor banding (MCB) applying probe-sets for chromosomes 1, 2 and 4 characterized the breakpoints as 1p31.1, 2q24.3 and 4q31.3. The corresponding results are shown here. For each chromosome depicted the MCB-pseudo-colors as well as the underlying fluorescence profiles are shown (for details of MCB evaluation see [22]. The first of the 5 columns shows which probe set was used (MCB 1, 2 or 4). The second column shows the normal chromosomes #1, #2 and #4. The arrowhead shows the breakpoint as present in the derivative sister-chromosomes. Third to fifth columns show the derivative chromosomes (der) 1, 2 and 4. MCB 1 stains parts of der(1) and der(4), MCB 2 parts of der(1) and der(2) and MCB 4 parts of der(2) and der(4). Parts not stained by the corresponding MCB probe-sets are pseudo-colored in gray. Overall, the complex chromosomal rearrangement was balanced, according to molecular cytogenetics.DiscussionAs existing difficulty of precise definition of CCR using standard cytogenetic methods [2], detection of CCR in the chromosomes of a patient causes anxiety for patient and clinician, especially when it is balanced that can lead to genetic imbalance [9]. Because precise identification of all the chromosomes involved in a CCR is the prerequisite to every appropriate genetic counseling. By combining high resolution techniques of chromosome banding with FISH we have an essential tool to determine whether a complex abnormal karyotype is apparent or not, this is especially important for prenatal diagnosis [10].These rearrangements are usually ascertained by routine chromosome analysis of a child with mental retardation and congenital abnormalities [11], recurrent abortions in female [12], and infertility in man [13]. Although most carriers of balanced translocations are phenotypically normal, in a small proportion (~6%) of these phenotypic abnormalities are reported [9,14]. Madan et al [1997] analyzed 60 cases with balanced CCRs [6]. They found that for female CCR carriers the risk of abortions and abnormal livebirths is 52.6% and for male carriers the risk of abortion and abnormal livebirths is 60%, with a combined frequency of 53.7%. While liveborn infants possessing normal chromosomes have incidences of 31.6%, liveborn infants carrying balanced chromosomes have incidences of 50% [6]. If chromosomal rearrangement is detected in a phenotypically normal individual, then this rearrangement is generally assumed to be truly balanced. These often represent familial cases. If, however, a chromosomal rearrangement is detected in a phenotypically abnormal individual; then usually a submicroscopic imbalance or other genetic defects exist. This situation often represents de novo cases. The incidence of live born infants with unbalanced chromosomes and variable degrees of phenotypic abnormalities is 18.4% [6,15]. An abnormal phenotype with apparently balanced rearrangements may be the result of chromosomal breakage disrupting a gene leading to abnormal gene expression or the presence of a submicroscopic deletion or duplication [9]. The change of location and/or orientation of translocated genes can also influence the activity of regulatory sequences co-operating with the breakpoint flanking translocated genes [16]. Recently Goumy et al [2006] described a boy with mild developmental delay and psychotic disorder. He had balanced complex rearrangements but no molecular abnormalities were detected by using FISH with whole chromosome painting (WCP), comparative genomic hybridization (CGH) and array-CGH [4]. The results of array CGH belong to De Gregori et al. [2007] showed that 16 of 18 patients had imbalances while all cases had been interpreted as balanced by conventional cytogenetics. 11 of 16 CCRs associated with deletion. The phenotypic abnormalities of apparently balanced de novo CCRs are mainly due to cryptic deletions. There was no association between the severity of the pathology and the number of deletions or their sizes [17].Moreover, the exact cytogenetic mechanisms underlying the origin of CCRs are unclear. A major catastrophe within the gamete (spermatogenesis) appears a vague yet plausible patognomonic mechanism for CCRs [15]. Simple three-way translocations are predicted to form hexavalents at meiosis. By focusing solely on symmetric segregation (3:3), up to 20 possible gametic combinations could be devised among which only two were balanced. The number of unbalanced gametes increased significantly together with the possibility of asymmetric segregation and recombination during meiosis [5]. Lespinasse et al. [2003] analyzed the localization of 90 chromosome breakpoints in 24 CCRs delineating random involvements of specific chromosomes in CCRs. However, they observed a non-random distribution of specific breakpoints at 1q25, 4q13, 6q27, 7p14, 9q12, 11p11, 12q21, 13q31 and 18q21 [13]. Recently De Gregori et al. [2007] screened 59 balanced translocations including CCRs by using array comparative genome hybridization and 18 of these were found to be de novo balanced complex translocations. At the 22 breakpoints identified using a specific customized array, they could not find any specific DNA sequences. Thus, they were unable to determine the mechanisms underlying the concurrent breakage of several chromosomes with losses of parts of the broken portions and their random assortment. Considering that all the men fathering children with unbalanced translocation or CCRs were fertile, they came up with the following hypothesis: during spermatogenesis some cells escape the mechanism responsible for correct crossing-over; these undergo chaotic breaks resulting in the reunion of several chromosomes and thereby exposing the broken portions to exonuclease degradation [17].The couple we present in here has only one living child out of four pregnancies. Their male child does not have any clinical abnormalities or any developmental delay. But we do not have any objective findings to confirm whether this child bears any chromosomal abnormalities. Even if the detected CCR looks balanced with MCB, the carrier of this CCR has severe mental deficiency. Several studies reported apparently balanced chromosomal rearrangements to be associated with significant risks of mental retardation and malformation [11,16]. Based on a review of apparently balanced translocations, Warburton [1982] concluded that the presence of a de novo apparently balanced translocation is associated with an increased risk of mental retardation with an odds ratio of 6.0–7.0 [18]. Moreover, the vast majority of male carriers show reduced fertility [19,20]. Disturbances in spermatogenesis as well as pre- and post implantation losses are discussed as reasons for this phenomenon [6]. Zahed et al. [1998] suggested that the scarcity of the number of transmitting males with CCRs is usually attributed to either a lower risk of producing abnormal progeny therefore, a lower probability of ascertainment, or to infertility attributed to problems in chromosome pairing at spermatogenesis [21]. There are only few reports on fertile male CCR carriers referred for cytogenetic evaluation due to spontaneous abortions of spouses or due to abnormal offsprings, which were well reviewed by Grasshoff et al. [2003] as presented here [5].Identification of submicroscopic aberrations (below 3 Mb) and more detailed molecular profiling of the rearrangements require precise mapping of the breakpoints with other methods such as florescence in situ hybridization (FISH) with locus-specific probes or array CGH [9]. Since the establishment of FISH technique in human cytogenetic, much progress has been achieved concerning the ability to characterize chromosomal subregions by molecular cytogenetic methods. Recently, high resolution multicolor banding (MCB) technique was developed. By producing changing florescence intensity ratios along the chromosomes, MCB approach allows the differentiation of chromosome region specific areas at the band and subband levels and is based on region specific microdissection libraries [8]. MCB technique is a high resolution alternative suited to clarify the changes appearing in complex chromosomal rearrangements [22]. We used MCB techniques for certain determination of the breakpoints on each chromosome and the detection of possible deletions. According to MCB results the proband has balanced complex chromosomal translocations. Liehr et al [2002] suggested that the MCB-technique is a high resolution alternative to other FISH based chromosome banding approaches and it suits to clarify the changes appearing in CCRs [22].ConclusionFurther delineations are certainly required to provide more information about the relationships between balanced CCRs and their phenotypes. Determination of certain breakpoints is also important for counseling the patients. With these, correct prenatal diagnosis and efficient genetic counseling can be possible for the carriers of CCR. The couples with CCR should be also informed about the possible outcomes of the progeny and the fact that exact risk of malformation is still unknown and that phenotypically normal child can still have a high risk of reproductive problems. The carriers must be investigated with high resolution banding and molecular cytogenetic techniques in order to see whether the CCR is truly balanced or not and if balanced where these breakpoints are located. Finally, high resolution MCB techniques by themselves can be used as alternative methods to determine exact locations of the breakpoints.Materials and methodsClinical case reportA couple was referred to us following three pregnancy losses out of four pregnancies. The mother was 26 years old and had one living child from her third pregnancy. The first one was lost at the first trimester, the second one was aborted at the third trimester due to fetal abnormality, the third one was finally born 4 years ago as a healthy male child, and the fourth one was lost at the first trimester again. The father was 33 years old, he had mental deficit since birth while his spermiogram was normal. There were no functional motor deficits apart from the severe mental retardation he suffered necessitating continuous support. The examination of their male child did not reveal any clinical evidence about any abnormality. The parents did not give consent to further evaluate their living child with chromosomal analysis.Banding cytogeneticsCytogenetic investigations from the couple were performed on peripheral blood samples using a high resolution technique after cell culture synchronization and BrdU incorporation [23].Molecular cytogeneticsHigh resolution multicolor banding (MCB) based on microdissection derived region-specific libraries for chromosomes 1, 2 and 4 was carried out to further delineate the nature of chromosomal rearrangements as described before [22]. Each of the 20 metaphase spreads were analyzed by using a fluorescence microscope (Axioplan 2 mot, Zeiss) equipped with appropriate filter sets to discriminate between a maximum of five fluorochromes and the counterstain DAPI (Diaminophenylindol). Image capturing and processing were carried out using an ISIS mFISH imaging system (MetaSystems, Altlussheim, Germany) for the evaluation of MCB.List of abberationsCCRs: comples chromosomal rearrengements; CGH: comparative genomic hybridization; FISH: fluoresence in situ hybridization; MCB: multi colour banding; WCP: whole chromosome painting.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsNK evaluated the family with examination, counseling and cytogenetically, and prepared the revised MS. KM and AW did the molecular cytogenetic analysis and interpretation of the MCB results. All authors' read and approved the final manuscript.\n\nREFERENCES:\nNo References"
+}

batch_9/PMC2531200.json

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+{
+    "id": "PMC2531200",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531200\nAUTHORS: Peter Hinh, Run Wang\n\nABSTRACT:\nIntroduction. Post-prostatectomy erectile dysfunction affects a considerable number of men and is a significant quality of life issue. There has been a substantial amount of research on the treatment of post-prostatectomy ED, and now there is a rising interest in the concept of penile rehabilitation. The goal of penile rehabilitation is to moderate the destructive processes that occur after prostatectomy in order to preserve erectile function, either through spontaneous or assisted means. Methods. We reviewed published data and experiences of post-prostatectomy penile rehabilitation using regimented interventions of phosphodiesterase inhibitors, vacuum erectile device, and intracavernosal agents, and we present and analyze the research conducted. Results. These studies show improved objective and subjective clinical outcomes in regards to physical parameters, sexual satisfaction, and rates of spontaneous erections. Conclusion. These studies are often limited by small size, study period, and study design. There continues to be a need for large, randomized, placebo controlled trials with adequate followup to fully evaluate the efficacy and cost-effectiveness of the various proposed penile rehabilitation regiments before a clear standard can be established.\n\nBODY:\n1. INTRODUCTIONErectile dysfunction following prostatectomy remains a\nsignificant quality of life issue for men undergoing prostatectomy. It is\nestimated to affect 26–100% of patients\nafter surgery [1]. Even with advancements in understanding the anatomy of\nthe prostate and the neurovascular bundle [2], a considerable number of men\nundergoing prostatectomy will have resulting erectile dysfunction.Progress has been made in identifying the events that contribute to erectile\ndysfunction after prostatectomy. The changes of neuropraxia, ischemic and\nhypoxic insults, fibrotic remodeling, and apoptosis are all believed to\ncontribute to erectile dysfunction [3, 4]. These events can occur even in\nattempts at meticulous dissection to preserve the neurovascular bundle. The etiologies of cavernous nerve neuropraxia\ninclude mechanical stretch injury during retraction, ischemia from accessory\nvessel disruption in dissection, thermal injury from electrocautery use, and\ninflammation from surgical trauma. This\nneuropraxia can prevent erections, and the perpetual lack of erection can\nitself set up a cascade of deleterious processes. Chronic impotence reduces\nblood flow to the corporeal bodies, which leads to fibrosis and transformation\nof the trabecular smooth muscle through collagen [5]. Further hypoxic insults\nalso may trigger apoptosis [6]. Therefore,\nthe goal of penile rehabilitation is to set up an environment that moderates\nthese processes in attempt of preserving penile function and earlier return of\npotency. Regimented usage of erectile\naids aims to improve the circulation of oxygen and maintain the structure of\nthe corporeal bodies.However, the research has yet to be translated into a\ncoherent clinical strategy for penile rehabilitation. As such, there are\nno currently accepted guidelines for penile rehabilitation regiments.\nCertainly, there exist several popular options that are currently in use.\nIn practice, three principle modalities of treating post-prostatectomy erectile\ndysfunction are employed. This paper will review the efficacy of these\noptions in this patient population for the purposes of penile rehabilitation.2. PHOSPHODIESTERASE 5 INHIBITORSThe introduction of phosphodiesterase inhibitors (PDEi) revolutionized\nthe treatment of erectile dysfunction. Since entering the market in 1998,\nthese medications have become nearly synonymous with erectile\ndysfunction. Their ease of use and relatively safe profile have made them\npervasive in the treatment of erectile dysfunction. They have also been\nextensively investigated. A Cochrane meta-analysis looking at many large,\nrandomized clinical trials concluded that PDEi are efficacious in the treatment\nof erectile dysfunction and are generally safe [7]. However, their role\nand their administration in penile rehabilitation after prostatectomy remain\nundefined. A number of clinical studies\nhave investigated PDEi use in this population for this intention.One of the first studies on PDEi in rehabilitation looked\nat objective data to support this use. \nSchwartz et al. conducted a study on 40 men who had undergone nerve\nsparing prostatectomy [8]. Prior to prostatectomy, all men had\npercutaneous biopsy of cavernous tissue to serve as baseline reference. They were divided into receiving either 50 or\n100 mg of Sildenafil every other night. Participant then underwent percutaneous\nbiopsy of cavernous tissue at 6 months to compare with baseline tissue. Investigators found that the 50 mg group did\nnot experience any loss of smooth muscle compared with baseline, and the 100 mg\ngroup actually showed an increase of smooth muscle content when compared to the\nbaseline. There was no control group, and no clinical correlation between\nsmooth muscle preservation and erectile function is made in this study. A prior animal study had shown that cavernosal\nsmooth muscle does atrophy after prostatatectomy [9], though this effect is\nsomewhat mediated with unilateral nerve sparing and it is unclear to what\nextent this atrophy would occur with nerve sparing prostatectomy.Bannowski et al. conducted a randomized trial following 43\nmen who underwent nerve-sparing radical prostatectomy [10]. All men provided baseline International Index of Erectile Function (IIEF) scores prior\nto surgery. After catheter removal following\nsurgery, the men underwent testing for nocturnal tumescence the following\nevening measured by the rigiscan. 41 of\n43 were found to have spontaneous erections on rigiscan, and these men were\nthen randomized to receive sildenafil daily or no treatment. They were then followed and evaluated with\nIIEF at 6, 12, 24, 36, and 52 weeks. The\nresults show that the daily treatment group had significantly higher IIEF score\nby 36 and 52 weeks. Additionally, 47% of\nthe daily treatment groups were able to achieve spontaneous, unassisted\nerection sufficient for penetration. \nThis compares to 28% of the control group who were able to have such\nerections. Both groups were also allowed\nSildenafil on demand, and accounting assisted erections, 86% of the daily group\nhad erections sufficient for penetration, compared to 66% of the control\ngroup. The study made no mention of any\nparticipant drop out, and there was no placebo control. However, it does appear that daily Sildenafil\ndoes improve return of spontaneous erections and can augment response to on-demand use of Sildenafil.A stringent, randomized,\ndouble-blinded, placebo controlled study was performed by McCullough et al.\nevaluating the efficacy of daily Sildenafil in men after bilateral nerve-sparing\nradical prostatectomy [11]. This study\nincluded 54 men with baseline normal EF and NPTR (nocturnal penile tumescence\nand rigidity with duration of rigidity >55% of maximal rigidity using\npenile plethysmography). After a\npretreatment period of 4 weeks, they were then randomized to either receive\nnightly 100 mg Sildenafil (N = 18), 50 mg Sildenafil (N = 17), or placebo (N = 19). The groups were then analyzed at\n16, 28, and 40 weeks. Then after 40\nweeks, all medications were discontinued and the groups were again analyzed at\n48 weeks. At each point, the\nparticipants were evaluated by NPTR and IIEF . \nThe study found that the groups receiving daily Sildenafil were able to\nhave return of rigidity (R > 55%) at seven times the nadir value compared to minimal\nimprovement in the control group. This\nimprovement of erection was also seen by the investigators for RAU (rigidity-activated unit—a time-intensity measurement that\nrepresents the area under the rigidity curve during a qualified\nevent), with the additional finding that the 100 mg group experienced continued\nimprovement after the discontinuation phase, while the 50 mg group began to\nexperience decline in RAU. Importantly,\nthe men on daily Sildenafil were five times more likely to have return of spontaneous,\nunassisted erection sufficient for intercourse compared to placebo. The authors were able to link objective\nmeasurements of erections with subjective, clinical response. They noted that tip rigidity >55% clearly\nseparated responders versus nonresponders (responders defined as recovery of\nspontaneous, unassisted sufficient erections). \nThis study may mark objective validation as an important component of\nfuture clinical trials. These initial\nresults are from a subset analysis performed on men who showed normal EF and\nNPTR on baseline, and we await further data and analysis on the entire study\npatients.Taken together,\nthese studies seem to indicate that phosphodiesterase inhibitors have a role in\npenile rehabilitation for men after prostatectomy. There may also be a dose-dependent relationship between the medication and outcomes. The studies also confirm the tolerability and\nsafety of such a regiment, as discontinuation rates were very minimal and no\nadverse events were reported.3. VACUUM ERECTILE DEVICEThe Vacuum erectile device assists erections by drawing blood flow into the\ncavernous sinuses through negative pressure, physically causing an\nerection. A constrictive band can also be placed at the base of the\npenis, preventing backflow and maintaining corporal pressures. This\ndirect mechanism of action can circumvent the limitation of oral agents, which\nrequires an intact and functioning neuronal connection to produce\nerections. This can be a significant factor even in men undergoing nerve\nsparing prostatatectomy, as neuropraxia still occurs and can diminish the\neffectiveness of PDEi.This\ntreatment modality can also be extended to men who have undergone nonnerve\nsparing prostatectomy, though not in the context, in penile rehabilitation with\nthe expectation of return of potency.If not for potency itself, VED usage has also been advocated due to its\npossible efficacy in preventing penile shrinkage and maintaining length.\nStudies have shown significant shrinkage of penile length, with one\nstudy finding that nearly 20% of men experience a loss of length greater than\n15% [12]. In another study examining penile shortening after\nprostatectomy, Gontero et al. followed 126 men who had undergone prostatectomies\nand measured penile length prior to surgery, at the time of catheter removal,\nand then at 3, 6, and 12 months [13]. They found that the greatest amount of\nshrinkage occurs in the immediate postoperative period, though shortening\ncontinues at a lesser rate throughout the entire study period. These\nauthors hypothesize that early hypoxia leads to increased expression of TGF-B\nand Collagen I and III fibers. This study also finds that the return of erectile\nfunction, defined as an IIEF of 15, was associated with mitigation of the\nshrinkage, as well as having a nerve sparing surgery. Several studies looking at the efficacy of\nvacuum erectile device in preserving erectile function have also examined\npreserved penile length as a secondary endpoint.Raina et al. randomized 109 post-prostatectomy men to\neither early VED use daily (N = 74) versus no erectogenic aid (N = 35) [14]. The men were to use the constriction band\nonly during intercourse to maintain rigidity. Participants were followed\nwith SHIM and IIEF scores for comparison. For the group using VED, 80%\nwere able to achieve penetration with use of VED, and this group, not\nsurprisingly, had a significantly higher SHIM and IIEF group compared to no\ntreatment. The discontinuation rate was 18%, and the majority of the drop\nout was for discomfort. In the context\nof penile rehabilitation, at 9 months this study found that 17% of those\nadhering to daily VED were able to have spontaneous erections sufficient for\nerections at 9 months, compared to 11% (n = 4) of the control group that had such\nerections. In regard of the effect of\nVED on penile length, the men who adhered to VED regiment experienced less\nsubjective penile shrinkage, with 23% reporting less length compared to 85% of\nthe men who quit treatment and 65% of the control. No objective data was collected concerning\nlength. The authors conclude that early\nuse of VED with the purpose of penile rehabilitation improves sexual and\npartner satisfaction and allow for earlier return of spontaneous erections. Although,\nthe rate of return of spontaneous erection is low for both groups, those\nnumbers include men who had undergone nonnerve sparing prostatectomies. This distinction is necessary, as penile\nrehabilitation is more directed for NS men and the inclusion of nonnerve sparing\nprostatectomy patients diluted the response to rehabilitation.The timing of when to initiate VED has been questioned,\nwith some advocating an earlier intervention. Köhler et al. randomized 28\nmen to either receive early VED regiment (1 month after RP) or delayed VED\nregiment (6 months) [15]. The regiment consisted of 10 minutes of VED\nusage without the constriction band. IIEF was measured at baseline, 1, 3,\n6, 9, and 12 months. The mean followup was 9.5 months and the results\nanalyzed at 3 and 6 months showed that the early intervention group had a statistically higher\nIIEF score. At this point, the comparison shows that VED does improve\nIIEF scores among those who use VED versus those controls that do not. At beyond 6 months, the delayed group began using of VED, and at\nshort followed up the\ntwo groups converged with no statistical difference in IIEF \ncategorization. No patients in this\nstudy had return of spontaneous erections sufficient for penetration at that\nfollowup. This paper was presented as a\npilot study, and more outcomes are expected to follow, especially data\nconcerning return of spontaneous erections. There are still some important\npoints that can be gleamed from this study. For one, the authors reported\ncomplete compliance with this regiment, suggesting this short regiment (two five-minute cycles) could\nbe feasibly implemented. Importantly,\nthese researchers also looked at penile length and found that the group\nperforming VED regiment did not experience penile shrinkage, while the group on\ndelayed VED showed significant penile shrinkage at 3 (mean loss 1.87 cm) and 6\nmonths (mean 1.82 cm). However, after\nbeginning VED in the delayed group, the loss decreased to a mean of 1 cm and no longer remained\nstatistically significant. Again, this is short-term followup data in the delayed group, and\nfurther improvement in shrinkage may still yet be seen. We still await data concerning return of\nspontaneous erection from this study.The value of VED in penile\nrehabilitation remains uncertain. Daily\nregimented use of VED requires a motivated patient and does improve sexual\nsatisfaction in those who responds. If\nthe stated goal of penile rehabilitation is the return of preexisting potency,\nthen further studies are needed to show that VED improves the rate of return of\nerection. However, VED use may also be advocated for its effects on preventing\npenile shrinkage after prostatectomy.4. INTRACORPOREAL INJECTIONIntracorporeal injection of vasoactive agents increase blood flow into the\ncavernous sinuses locally, either through increasing cAMP, by antagonizing alpha-adrenergic\nreceptors, or by direct smooth muscle relaxation. Like VED, they also do\nnot require an intact, functional nervous system to produce erections.\nThus, they can also be offered in men who have undergone nonnerve sparing surgery\nand men who do not respond to oral agents.Montorsi et al. conducted a randomized trial\ninvestigating whether a regiment of intracavernosal injections improves\nerectile function in post-prostatectomy men [16]. 30 men with established\npreoperative potency were randomized to either receive a regiment of 3 times\nper week injections of alprostadil for 12 weeks versus a control group that did\nnot receive erectogenic treatment. The groups were then assessed after 3 months\nfor sexual history, for Doppler response after alprostadil administration, and\nfor nocturnal tumescence. Of the ICI\nregiment group, 80% completed the 12 weeks of treatment with a 20% drop-out\nrate and a 17% complication rate. Of\nthese men, 67% at 3 months were able to have spontaneous erections sufficient\nfor penetration. This favorably compares to 20% of the control group men that\nwere able to have such erections. The authors do note that in this group some\nstill continued to use ICI to achieve erections. However, the authors\nconsidered it a complete response since the majority of sexual encounters\noccurred without ICI use (average of one in 4.2 attempts). The study also found\nthat having normal penile hemodynamics was strongly associated with ICI\ncomplete response. The study suffers from small size and short followup.\nHowever, it was still able to show an improvement in spontaneous erections for\nregimented ICI and that regimented ICI is generally well tolerated.Mulhall et al. conducted a trial\nthat may be more clinically applicable, even though it was not randomized [17]. In\nthis trial, post-prostatectomy men were committed to penile rehabilitation with\nSildenafil or ICI if there was no response to Sildenafil, versus no penile rehabilitation\nprogram, but they were\nnot restricted from using erectile aids. \nThe rehabilitation group used either Sildenafil or ICI three times per week. Analysis at 18\nmonths revealed that 52% of rehabilitation men were able to have functional\nerections, compared to 19% of control group men. Additionally, the rehabilitation group had\nsignificantly higher IIEF scores. These\nresults are impressive, especially considering that the control group was also\nable to use erectile aids, including both Sildenafil and ICI, though not in a\nregimented manner. The study included men with nonnerve sparing\nprostatectomies, which generally are not considered candidates for erectile rehabilitation,\nthough the authors do note that there was not a differential distribution\nbetween the groups. Additionally, the\naverage length of time before sexual consultation and therefore the start of\nrehabilitation were 4.2 months. Some would suggest that this\nlength of time is too long removed from the surgery, and the insults of\nhypoxia, fibrosis, and apoptosis may have already occurred and be irreversible\nat this point. It is also important to note that selection bias may be very\nconsiderable in this study, in that only men who prospectively committed\nthemselves were included in the rehabilitation group and the study relied on\nmainly self-reported, subjective data.5. COMBINATION THERAPIESStudies have examined the feasibility and efficacy of\nemploying two treatment modalities during penile\nrehabilitation. Nandipati et al. incorporated both intracavernosal\ntherapy and PDEi in a group of 22 men who underwent nerve sparing prostatectomy\n[18]. All participants received Sildenafil 50 mg daily (25 mg if subjects\ncomplained of headaches). For ICI, 18\npatients received PGE 1–4 micrograms and\n4 received trimix injection, with ICI being done two to three times per\nweek. These patients were then analyzed at 3, 6, 9, and 12 months with IIEF . Doppler studies were performed for dose\noptimizations of ICI and at intervals to increase dosage for response. The study reported a mean followup of six\nmonths. At this point, investigators found that 21 of 22 patients were sexually\nactive, while 12 of the 21 were using ICI alone and 9 of 21 were using combination\ntherapy. Of the 18 patients using PGE, 12 were able to lower their dosage;\nwhile 1 of the 4 patients on trimix was able to do so. 11 of the 22 men\nhad return of spontaneous erection, though none graded the erections sufficient\nfor penetration. The authors concluded\nthat the addition of Sildenafil could reduce the amount of ICI necessary to\nachieve erections. This study had a lower rate of return of functional\nerections compared to other studies looking at nightly PDEi 1 [11] or regimented\nICI alone [16], and this difference may be explained by the\nshorter followup and the small number of participants. Without proper study design, in such\ncombination therapy studies, it is difficult to assign particular findings to\nspecific intervention.6. NOVEL THERAPIESOther therapies outside of these three mainstream\nmodalities have been investigated for penile rehabilitation after\nprostatectomy. Recently, investigators in Korea looked at statins for treating\nerectile dysfunction after prostatectomy [19]. The basis for this\nhypothesis stems from the known protective effect on vascular endothelium and\nincreased NO activity. The researchers randomized 50 men post-prostatectomy\nto receive 10 mg of atorvastatin for 90 days. All men were then to use\nSildenafil 50 mg per day on demand. The men all had superior function prior to\nthe surgery with IIEF 25. At 6 months of followup, the study found that\nthe statin group had more patients categorized at potent ( IIEF greater than 16)\nwith 11 in the statin group and 6 in the control group. Additionally, more men in the statin group were able\nto achieve vaginal penetration without PDEi than in the control group (8 versus\n4), though this significance did not reach statistical significance. The\nstudy was neither blinded nor placebo-controlled. It is important to note that the\ninclusion criteria were extremely stringent, and patients with significant cormidities were excluded. This may affect the\napplicability or generalizability of the results.7. CONCLUSIONSexual potency after prostatectomy remains a significant quality of life issue\nafter prostatectomy. There exist many studies on the efficacy of the\nvarious treatment options on this patient population. There are much less data looking on the\neffects of regimented usage of PDEi, VED, and/or ICI in improving erectile\nfunction in this group, and no guidelines exist to help steer the\nclinician. We are still in need of large, randomized, controlled, clinical\ntrial with adequate, long-term followup to evaluate this question. Moreover, even after each treatment can be\nestablished to be efficacious in penile rehabilitation, the exact regiment amount and\nduration will still be open to further investigations for optimization. This is\na especially important question in dealing with phosphodiesterase inhibitors,\nwhere the cost of treatment is substantial. \nHowever, even though currently sparse, the consistent, growing body of\nevidence does support penile rehabilitation in improving return of sexual\nfunctioning. As awareness of penile\nrehabilitation increases and becomes more accepted, it is becoming more\ndifficult to conduct placebo or nonintervention controlled trial. There are\nstill several ongoing trials evaluating penile rehabilitation, including a\nlarge multicenter study examining penile rehabilitation with medicated urethral system for erection (MUSE), and several of the studies presented here will further analyze with additional data from which the urology community may further define when and how to\nimplement penile rehabilitation in post-prostatectomy men.Currently, the body of evidence does\nseem to suggest a beneficial role for penile rehabilitation after prostatectomy\nin improving return of potency. Such a program should begin with a detailed\nevaluation on the preoperative sexual performance characteristic of the patient\nand then a thorough\ndiscussion of the available rehabilitation regiments. The practitioner should consider factors that\nare important to the patient including ease of use and compliance, patient\nmotivation, conditioning, cost and patient expectations about sexual function,\nand penile length. Penile rehabilitation\nmay continue to remain investigative until more standardized clinical data\nbecomes available.\n\nREFERENCES:\n1. BurnettALAusGCanby-HaginoEDErectile function outcome reporting after clinically localized prostate cancer treatmentThe Journal of Urology2007178259760117570435\n2. WalshPCPartinAWWeinAJKavoussiLRNovickACPartinAWPetersCAAnatomic radical retropubic prostatectomyCampbell-Walsh Urology20079th editionPhiladelphia, Pa, USASaunders2956 pages\n3. McculloughARRehabilitation of erectile function following radical prostatectomyAsian Journal of Andrology2008101617418087645\n4. WangRPenile rehabilitation after radical prostatectomy: where do we stand and where are we going?The Journal of Sexual Medicine200744, part 21085109717466058\n5. MorelandRBIs there a role of hypoxemia in penile fibrosis: a viewpoint presented to the Society for the Study of ImpotenceInternational Journal of Impotence Research19981021131209647948\n6. McVaryKTPodlasekCAWoodDMcKennaKEApoptotic pathways are employed in neuropathic and diabetic models of erectile dysfunctionThe Journal of Urology2006175387 pagesAbstract 1203\n7. FinkHAMac DonaldRRutksIRNelsonDBWiltTJSildenafil for male erectile dysfunction: a systematic review and meta-analysisArchives of Internal Medicine2002162121349136012076233\n8. SchwartzEJWongPGraydonRJSildenafil preserves intracorporeal smooth muscle after radical retropubic prostatectomyThe Journal of Urology20041712, part 177177414713808\n9. UserHMHairstonJHZelnerDJMcKennaKEMcVaryKTPenile weight and cell subtype specific changes in a postradical prostatectomy model of erectile dysfunctionThe Journal of Urology200316931175117912576876\n10. BannowskyASchulzeHvan der HorstCHautmannSJünemannK-PRecovery of erectile function after nerve-sparing radical prostatectomy: improvement with nightly low-dose sildenafilBJU International2008101101279128318284406\n11. McculloughARLevineLAPadma-NathanHReturn of nocturnal erections and erectile function after bilateral nerve-sparing radical prostatectomy in men treated nightly with sildenafil citrate: subanalysis of a longitudinal randomized double-blind placebo-controlled trialThe Journal of Sexual Medicine20085247648418086170\n12. SavoieMKimSSSolowayMSA prospective study measuring penile length in men treated with radical prostatectomy for \nprostate cancerThe Journal of Urology200316941462146412629384\n13. GonteroPGalzeranoMBartolettiRNew insights into the pathogenesis of penile shortening after radical prostatectomy and the role of postoperative sexual functionThe Journal of Urology2007178260260717570431\n14. RainaRAgarwalAAusmundsonSEarly use of vacuum constriction device following radical prostatectomy facilitates early sexual activity and potentially earlier return of erectile functionInternational Journal of Impotence Research2006181778116107868\n15. KöhlerTSPedroRHendlinKA pilot study on the early use of the vacuum erection device after radical retropubic prostatectomyBJU International2007100485886217822466\n16. MontorsiFGuazzoniGStrambiLFRecovery of spontaneous erectile function after nerve-sparing radical retropubic prostatectomy with and without early intracavernous injections of alprostadil: results of a prospective, randomized trialThe Journal of Urology1999161619141915\n17. MulhallJLandSParkerMWatersWBFlaniganRCThe use of an erectogenic pharmacotheraphy regimen following radical prostatectomy improves recovery of spontaneous erectile functionThe Journal of Sexual Medicine20052453254016422848\n18. NandipatiKRainaRAgarwalAZippeCDEarly combination therapy: intracavernosal injections and sildenafil following radical prostatectomy increases sexual activity and the return of natural erectionsInternational Journal of Impotence Research200618544645116482200\n19. HongSKHanBKJeongSJByunS-SLeeSEEffect of statin therapy on early return of potency after nerve sparing radical retropubic prostatectomyThe Journal of Urology2007178261361617570410"
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+{
+    "id": "PMC2532957",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2532957\nAUTHORS: M Turpeinen, K Nikander, A S Pelkonen, P Syvänen, R Sorva, H Raitio, P Malmberg, K Juntunen-Backman, T Haahtela\n\nABSTRACT:\nObjective:To compare the effect of inhaled budesonide given daily or as-needed on mild persistent childhood asthma.Patients, design and interventions:176 children aged 5–10 years with newly detected asthma were randomly assigned to three treatment groups: (1) continuous budesonide (400 μg twice daily for 1 month, 200 μg twice daily for months 2–6, 100 μg twice daily for months 7–18); (2) budesonide, identical treatment to group 1 during months 1–6, then budesonide for exacerbations as needed for months 7–18; and (3) disodium cromoglycate (DSCG) 10 mg three times daily for months 1–18. Exacerbations were treated with budesonide 400 μg twice daily for 2 weeks.Main outcome measures:Lung function, the number of exacerbations and growth.Results:Compared with DSCG the initial regular budesonide treatment resulted in a significantly improved lung function, fewer exacerbations and a small but significant decline in growth velocity. After 18 months, however, the lung function improvements did not differ between the groups. During months 7–18, patients receiving continuous budesonide treatment had significantly fewer exacerbations (mean 0.97), compared with 1.69 in group 2 and 1.58 in group 3. The number of asthma-free days did not differ between regular and intermittent budesonide treatment. Growth velocity was normalised during continuous low-dose budesonide and budesonide therapy given as needed. The latter was associated with catch-up growth.Conclusions:Regular use of budesonide afforded better asthma control but had a more systemic effect than did use of budesonide as needed. The dose of ICS could be reduced as soon as asthma is controlled. Some children do not seem to need continuous ICS treatment.\n\nBODY:\nMost children with asthma experience their first symptoms before 7 years of age.1 Studies of adults and children with asthma have shown that some functional reversibility may be lost if anti-inflammatory treatment is postponed.2–4 The anti-asthmatic effect of inhaled corticosteroids (ICS) has been demonstrated in long-term intervention studies,5–10 and these findings have led to ICS becoming the mainstay of treatment for persistent asthma.11 12 However, high-dose ICS may have systemic effects such as reduction in height velocity6–8 13 and adrenal insufficiency.14In an 18-month intervention, we compared two budesonide therapeutic regimens with a control group treated with a fixed dose of disodium cromoglycate (DSCG). The study was designed to evaluate the anti-asthmatic efficacy and systemic effect of daily versus as-needed budesonide in the treatment of early, mild persistent asthma in children.METHODSChildren between 5 and 10 years, all Caucasians, were included, if they presented symptoms such as wheezing, prolonged cough or shortness of breath, suggesting asthma for at least 1 month before entry into the study, and significant bronchial reversibility. The latter was defined as at least a 20% diurnal variation in repeatable peak expiratory flow (PEF) measurements, or at least a 15% increase in PEF at least three times within 2 weeks of home recording, or at least a 15% increase in forced expiratory volume in 1 second (FEV1) 15 min after inhalation of a β2-agonist, or at least a 15% decline in FEV1 in an outdoor exercise test in the clinic.15 According to the symptoms and lung-function tests, the majority of children could be categorised as having mild persistent asthma.16 Children with acute asthma, an FEV1<50%17 and with treatment during the preceding 2 months with ICS, cromones, leukotriene modifiers or long-acting β2-agonists were excluded. The total cumulative doses of previously used ICS must not have exceeded 36 mg, 12 mg of nasal corticosteroids or oral doses equivalent to 200 mg prednisolone.The 18-month study was of a controlled, randomised, double-blind, parallel-group, single-centre design including a 2-week run-in period. Two blinded treatment regimens were initiated with inhaled budesonide via a dry-powder inhaler (Pulmicort Turbuhaler, AstraZeneca, Lund, Sweden), and one open-label treatment regimen was initiated with DSCG via a pressurized metered dose inhaler (pMDI; Lomudal with Fisonair spacer, Aventis Pharma, Holmes Chapel, UK). Patients were randomised as to treatment in balanced blocks as generated by a computer program. During the 2-week run-in period, all enrolled patients received a short-acting β2-agonist, terbutaline (Bricanyl Turbuhaler, 0.25 mg/dose, AstraZeneca, Lund, Sweden) as needed. After the run-in period, children were assigned to one of three treatment groups: (1) continuous budesonide group, receiving budesonide (400 μg twice daily for the first month, then 200 μg twice daily for 5 months) followed by low-dose budesonide (100 μg twice daily for 12 months); (2) budesonide/placebo group, where patients received identical budesonide treatment as Group 1 for the first 6 months followed by placebo for 12 months; and (3) DSCG control group, where patients received DSCG 10 mg three-times daily for 18 months (fig 1).Figure 1Study design. The daily dose of budesonide was divided into two doses, DSCG into three doses.All patients were given rescue medication of terbutaline 0.25 mg per dose as needed. For all groups, during exacerbations of asthma, study medication was replaced by budesonide 400 μg twice daily for 2 weeks. Children were withdrawn from the study and given individually tailored therapy if treatment of their exacerbations remained insufficient.The study was performed in accordance with the Declaration of Helsinki, and was approved by the local ethics committee. Written, informed consent was obtained from each patient’s parent(s) or legal guardian and from the patient.For the budesonide treatment groups, treatment compliance was recorded using a home spirometer (Vitalograph Data Storage spirometer, Vitalograph, Buckingham, UK), which recorded the peak inspiratory flow via Turbuhaler (PIFTBH) each time a dose of the drug was taken.18 In the DSCG group, the returned pMDI drug canisters were counted and weighed every 3 months.The primary efficacy variable was morning PEF. Secondary efficacy variables were FEV1, the number of asthma exacerbations, asthma-free days and rescue medication use. Morning PEF was measured daily at home. FEV1 was measured at the clinic visit every third month. An asthma exacerbation was defined as an increase in symptoms that were not controlled with six doses of rescue terbutaline per 24 h that caused the parent to contact the clinic. All parents were provided with a 24-h emergency telephone number. At the clinic, patients were examined by a paediatrician, who decided whether an exacerbation had occurred and, if so, replaced the regular medication with a 2-week course of budesonide 400 μg twice daily. The treatment of an exacerbation was considered insufficient if the symptoms did not subside during the 2-weeks’ budesonide inhalations that caused the parent to contact the clinic. If an oral or parenteral corticosteroid was needed, the child received individual treatment and was withdrawn from the study.All patients recorded their PEF rate daily, as measured by a home spirometer, before taking study medication. They also recorded their asthma symptoms using a visual analogue scale (0–10) and use of rescue medication. An asthma-free day was defined as a 24-h period without use of rescue medication and with a symptom score <2. Standard laboratory spirometry (Spirotrac III, Vitalograph) was performed at all clinic visits.19The primary indicator of systemic effect was the standing-height velocity, which was measured at each clinic visit using a stadiometer (Holtain, Crymych, UK) following a standardised procedure. Children with Tanner stage I-II at baseline were included. Tanner stage of sexual development is scored from I (pre-adolescence) to V (adult characteristics).20 Standing height was compared with Finnish reference values.21 Other indicator was body mass index (kg/m2).The sample size was determined by power calculations for morning PEF. A clinically significant change was assumed to be 40 l/min over an 18-month period. With 60 patients per treatment group, there was a 90% chance of detecting a difference of 24 l/min between treatments. The analysis of growth was performed on the complete study population (excluding pubertal children). All other variables were analysed using intention-to-treat principles, that is, all patients who had taken at least one dose of study medication and had data for the required period(s). Withdrawn patients were handled using last value extended, within period. Comparisons between the combined budesonide groups and the DSCG group were made for months 1–6; from 7–18 months comparisons were made between all three groups.For most variables, treatment groups were compared using analysis of variance (ANOVA) with fixed factor treatment and baseline values as covariates. For growth variables, sex was included as an additional factor in the analysis. Time to first asthma exacerbation and time to withdrawal were compared using the log-rank test. The number of exacerbations was compared using a Poisson regression model with fixed factor treatment, time in study as an offset and adjustments made for overdispersion.RESULTSA total of 176 children were enrolled in the study. There were no significant differences between treatment groups in any baseline measures (table 1). During the run-in period, the mean use of terbutaline was about one dose every 2 days in all treatment groups. Three patients were withdrawn because of asthma deterioration during continuous budesonide treatment after 6 months of the study. In the budesonide /placebo group, nine children were withdrawn because of asthma deterioration, all after 6 months of treatment. In the DSCG group, eight children were withdrawn during the first 6 months of the study, and four children thereafter (continuous budesonide versus DSCG; p = 0.026). One child on placebo and one child on DSCG were hospitalised, because of deterioration in their asthma. The numbers of patients withdrawn from the treatment groups for reasons not related to asthma were three in the continuous budesonide group, three in the budesonide/placebo group, and four in the DSCG group. The flow of the participants through the trial is presented in the fig 2. The mean treatment compliance for the three treatment groups decreased linearly from an initial level of ∼90% to a mean level of ∼60% towards the end of the study. This was matched by a subsequent reduction in the amount of drug used during the study. Children in the continuous budesonide and budesonide/placebo treatment groups achieved a mean PIFTBH of 60 l/min during the study period.Figure 2The flow of the participants through the trial.Table 1Baseline characteristics of treatment groups*Treatment groupContinuous budesonide (n = 58)Budesonide/placebo (n = 58)Disodium cromoglycate (n = 60)Age (years)7.0 (5 to 10)6.7 (5 to 10)6.9 (5 to 10)Male (%)596654Tanner pubertal stage I/II58/158/161/2Standing height (cm)128.4 (108 to157)125.1 (106 to 148)125.6 (105 to 148)Standing height, standard deviation scores (SDS)0.04 (−0.32 to 0.54)0.03 (−0.30 to 0.39)0.04 (−0.43 to 0.32)Body mass index (kg/m2)17.516.916.9Skin prick test positive (n)354136Duration of symptoms (months†)12.8 (1.1 to 70.5)11.3 (2.0 to 76.4)11.7 (3.0 to 70.8)Wheeze ever (n)354233Asthma symptom score (0–10)‡1.5 (0.0 to 5.5)1.7 (0.0 to 4.5)1.9 (0.0 to 5.7)Rescue medication, dose/24 h‡0.47 (0 to 4.0)0.55 (0 to 3.7)0.68 (0 to 2.8)Morning PEF rate (l/min)‡182 (78 to 301)176 (68 to 313)184 (94 to 363)Morning PEF (% predicted value)‡76 (43 to 105)77 (42 to 112)79 (54 to 107)FEV1 (L†)1.43(0.89 to 2.15)1.32 (0.72 to 2.36)1.37 (0.63 to 2.45)FEV1 (%† predicted value)87 (57 to 111)82 (52 to 107)83 (57 to 107)FVC (% predicted value)90 (64 to 112)87 (57 to124)89 (56 to 120)*Values are means with range in parentheses, unless otherwise stated; †no correlation between duration of the symptoms and FEV1. ‡Data from the run-in period. FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; PEF, peak expiratory flow rate.After 6 months, the morning PEF values (l/min) of the budesonide groups improved by 6.6% and by 6.1% in the DSCG group. After 18 months, the increase was 10.3% in the continuous, 10.0% in the budesonide/placebo and 12.5% in the DSCG group. No significant differences were observed between the groups at any time point. After 6 months of treatment, improvement in FEV1 in litres in the clinic was significantly greater in the budesonide groups than in the DSCG group (9.6 versus 5.9%; p = 0.012). From baseline to 18 months, FEV1 improved by 18.2%, in the continuous, by 16.9% in the budesonide/placebo and by 17.3% in the DSCG group without any significant differences.Over the 18-month study period, 364 exacerbations of asthma were recorded in 133 patients. During the first 6 months of treatment, children receiving budesonide had significantly fewer exacerbations compared with children in the DSCG group (table 2). During months 7–18, the continuous budesonide group (ie, children on low-dose budesonide) had significantly fewer exacerbations than either the budesonide/placebo group (ie, children given the placebo) or the DSCG group (table 2).Table 2Number of exacerbation episodesTreatmentNo of patients analysed*Exacerbations/patient** (95% CI)p ValueMonths 1–6Budesonide1150.32 (0.22 to 0.46)DSCG601.24 (0.95 to 1.63)<0.001Months 7–18Bud/Bud570.97 (0.70 to 1.34)Bud/placebo (Budesonide as needed)581.69 (1.31 to 2.18)DSCG511.58 (1.20 to 2.08)Bud/Bud vs Bud/placebo0.008Bud/Bud vs DSCG0.023Bud/placebo vs DSCG0.728*The total effective number of patients analysed. **The mean number of exacerbations divided by the number of patients in the group. Bud, budesonide.The median time to the first exacerbation was significantly longer for both the continuous budesonide (344 days) and the budesonide/placebo (268 days) groups compared with the DSCG group (78 days) (p<0.001 for each) (fig 3). After 180 days, the median time to the next exacerbation was 233 days for the continuous budesonide group, 138 days for the budesonide/placebo group (ie, during placebo) and 131 days for the DSCG group (continuous budesonide and DSCG; p = 0.03).Figure 3Kaplan–Meier plot of the time to first exacerbation for the continuous budesonide (O, n = 57), budesonide/placebo (□, n = 58) and disodium cromoglycate (Δ, n = 60) treatment groups during the 18-month study. The median time to the first exacerbation was significantly longer for both the continuous budesonide (344 days) and the budesonide/placebo (268 days) groups compared with the DSCG group (78 days) (p<0.001 for each). The vertical line indicates the time point (180 days) when budesonide treatment was changed to the low-dose regimen or to placebo. After 180 days, the median time to the next exacerbation was 233 days for the continuous budesonide group, 138 days for the budesonide/placebo group and 131 days for the DSCG group (continuous budesonide and DSCG; p = 0.03).At 6 months, the mean number of asthma-free days increased more in budesonide group than in the DSCG group (table 3). During months 7–18, the mean number of symptom-free days increased significantly in the continuous budesonide group compared with the DSCG group.Table 3Asthma-free days after the run-in periodTreatmentNo of patients analysed*Mean change in asthma-free days, % ** (95% CI)p ValueMonths 1–6Budesonide114+20.1 (+14.9 to +25.4)DSCG60+4.1 (−3.2 to +11.3)0.001Months 7–18Bud/Bud55+29.2 (+21.2 to +37.2)Bud/placebo (Budesonide as needed)58+19.6 (+11.8 to +27.4)DSCG51+11.6 (+3.3 to +19.9)Bud/Bud vs Bud/placebo0.092Bud/Bud vs DSCG0.003Bud/Placebo vs DSCG0.166*The total effective number of patients analysed; **Mean change in asthma-free days compared with the baseline.During the first 6 months, compared with the run-in period, both budesonide groups used significantly less rescue terbutaline (−0.29 doses/day) than the DSCG group (−0.07 doses/day) (p = 0.012). During months 7–18, the decline was –0.29 doses/day in the continuous budesonide group, −0.22 doses/day in the budesonide/placebo group and −0.18 doses/day in the DSCG group, with no significant differences between the groups.From baseline to 6 months, the mean standing–height velocity in the budesonide groups was 2 cm/year slower than in the DSCG group (p<0.001). From 7 to 18 months, height velocity increased in both budesonide groups, with the mean height velocity being greater for the budesonide/placebo group (ie, during placebo) than the continuous budesonide group (6.2 versus 5.6 cm; p = 0.016). After 18 months of treatment, children receiving DSCG had grown, on average, 1.0 cm more than children in the continuous budesonide group (8.8 versus 7.8 cm; p = 0.008) and 0.6 cm more than children in the budesonide/placebo group (ie, during placebo) (8.8 versus 8.2 cm; p = 0.048). Development of standing height is presented as standard deviation scores (SDS) in fig 4. No significant differences in body mass index were observed between treatment groups at any time point.Figure 4Mean change in standing height (SDS) over the 18-month study period for the continuous budesonide (◊, n = 50), budesonide/placebo (□, n = 45) and disodium cromoglycate (DSCG) (Δ, n = 43) treatment groups. 1–6 months, both budesonide groups versus DSCG, p<0.001; 7–18 months, continuous budesonide group versus budesonide/placebo group, p = 0.016. Note the fast height velocity during months 7–18 in the budesonide/placebo group.DISCUSSIONThe artificial nature of the research protocol in our study, as in many other asthma studies with drug interventions, does not pay attention to the evolution of individual disease. Exclusion criteria used in this study affect the selection of children. During the study, the selection was further affected by the withdrawal criteria. Furthermore, a “true” placebo group is impossible to arrange because asthma exacerbations cannot be left untreated, and glucocorticoids used for the treatment of exacerbations might influence the individual evolution of asthma.We consider that, clinically, the dominant phenotype of the children examined is mild persistent asthma according to the present guidelines. Some children with moderate persistent asthma were included, as consecutive patients fulfilling the inclusion criteria were allocated to the treatment groups. Within the treatment groups, every patient received fixed doses for a predetermined time despite the individual phenotype of asthma. However, in our study, the treatment regimen could be modified individually by 2-week courses of budesonide given as needed.Cessation of inhaled budesonide maintenance treatment has previously been shown to result in a worsening of the disease and a decline in lung function in children with persistent moderate to severe asthma.22 In the present study of newly detected mild persistent asthma, a proportion of children had a low number of exacerbations during this intermittent treatment with budesonide. The exacerbation rate during months 7 to 18 in this budesonide/placebo group was similar to that of the DSCG group. In the present study, two weeks’ budesonide given when needed, after the initial regular treatment with budesonide, seems to produce an anti-exacerbation effect comparable with the continuous use of DSCG. However, most withdrawals in the DSCG were early, in contrast to late withdrawals in the regular budesonide and budesonide/placebo groups. This might select more mild phenotypes of asthma to the DSCG group for the last 12 months of treatment and artificially improve the results of DSCG compared with placebo or low-dose budesonide treatments.While treatment of patients in the DSCG group was open, exacerbations were diagnosed and treated in the same way as in the other two treatment groups. Treatment in the DSCG group was not associated with measurable systemic effects. However, it was associated with the highest number of asthma exacerbations and withdrawals from the study. The initially high number of exacerbations suggests that DSCG is not suitable to start treatment of newly detected childhood asthma.No significant differences between treatment groups were observed in the morning PEF values at any time point during the study. This suggests that morning PEF is not a very sensitive efficacy parameter in long-term studies in children with mild asthma as suggested previously.7 During the first 6 months of the study, FEV1 in litres improved significantly more in the budesonide groups than in the DSCG group. However, at the end of the study the differences in FEV1 disappeared despite significant differences in the number of exacerbations. This is in agreement with previous observations of changes in FEV1 in litres between the treatments with budesonide, nedocromil and placebo.7 The use of FEV1 values measured in litres has been recommended because predicted values depend on height, which may be affected by ICS.7Our results confirm previous observations of a small initial decline in height velocity during treatment with ICS used at comparable doses, followed by normal height velocity.7 Decline in height velocity without catch-up growth has been recently observed even during regular low ICS dosage.8 However, another study suggests that children treated regularly with budesonide attain their predicted final adult height.23 In the present study, height velocity was dose-related; during the low-dose budesonide and placebo treatments, the systemic effect of the initial high-dose budesonide were reduced. In the present 18 months follow-up study, standing height velocity was normalised during low-dose budesonide treatment within 1 year of commencement of treatment. The height velocity increased, however, more rapidly during the placebo treatment than during the low-dose budesonide treatment, suggesting catch-up of the initial loss in standing height.While long-term maintenance therapy with low-dose ICS is recommended for mild persistent asthma,7 8 24 25 some children do not seem to need continuous inhaled corticosteroid treatment. Advantages of this treatment strategy include a reduced risk of ICS-related growth suppression. Intermittent courses of inhaled or oral corticosteroids have been suggested recently for adults with mild persistent asthma.26Regular use of budesonide afforded better exacerbation control but more systemic effect than intermittent use of budesonide given as needed or regular DSCG treatment. No significant differences in the morning PEF and FEV1 in litres or in asthma-free days were observed between the regular or intermittent budesonide treatments during months 7–18. These findings suggest that the overall anti-asthmatic effect of the intermittent budesonide treatment might be intermediate between the regular low-dose ICS and DSCG treatments. The dose of ICS could be reduced as soon as asthma is controlled. Some children do not seem to need continuous ICS treatment.What is already known on this topicIt is still debated whether mild asthma in adults needs regular treatment with inhaled corticosteroids.What this study addsSome children who achieve good initial control of their mild asthma does not seem to need continuous treatment with inhaled corticosteroids.\n\nREFERENCES:\n1. ÅbergNEngströmI Natural history of allergic diseases in childen. Acta Paediatr Scand 1990;79:206–112321483\n2. HaahtelaTJärvinenMKavaT  Effects of reducing or discontinuing inhaled budesonide in patients with mild asthma. N Engl J Med 1994;331:700–7058058076\n3. AgertoftLPedersenS Effects of long-term treatment with an inhaled corticosteroid on growth and pulmonary function in asthmatic children. Respir Med 1994;88:373–818036306\n4. ZeigerRSDawsonCWeissS Relationship between duration of asthma and asthma severity among children in the Childhood Asthma Management Program. J Allergy Clin Immunol 1999;103:376–8710069869\n5. van Essen-ZandvlietEEHughesMDWaalkensHJ Effects of 22 months of treatment with inhaled corticosteroids and/or beta-2-agonists on lung function, airway responsiveness, and symptoms in children with asthma. Am Rev Respir Dis 1992;146:547–541355640\n6. SimonsFE A comparison of beclomethasone, salmeterol and placebo in children with asthma. Canadian Beclomethasone Dipropionate–Salmeterol Xinafoate Study Group. N Engl J Med 1997;337:1659–659385125\n7. Childhood Asthma Management Program (CAMP) Research Group Long-term effects of budesonide or nedocromil in children with asthma. N Engl J Med 2000;343:1054–6311027739\n8. PauwelsRAPedersenSBusseWW  Early intervention with budesonide in mild persistent astma: a randomized, double-blind trial. Lancet 2003;361:1071–612672309\n9. SelroosOPietinahoALofroosAB  Effect of early vs late intervention with inhaled corticosteroids in asthma. Chest 1995;108:1228–347587421\n10. O'ByrnePMPedersenSBusseWW  Effects of early intervention with inhaled budesonide on lung function in newly diagnosed asthma. Chest 2006;129:1478–8516778264\n11. National Institutes of Health: National Heart, Lung, and Blood Institute, National Asthma Education and Prevention Programme Expert panel report II: Guidelines for the diagnosis and management of asthma. Bethesda, Md: National Institutes of Health, 1997\n12. BarnesPJ Efficacy of inhaled corticosteroids in asthma. J Allergy Clin Immunol 1998;102:531–389802359\n13. DoullIJFreezerNJHolgateST Growth of pubertal children with mild asthma treated with inhaled beclomethasone dipropionate. Am J Respir Crit Care Med 1995;151:1715–97767512\n14. ToddGRGAceriniCLRoss-RusselR  Survey of adrenal crisis associated with inhaled corticosteroids in the United Kingdom. Arch Dis Child 2002;87:457–6112456538\n15. ERS Task Force Clinical exercise testing with reference to lung disease: indications, standardization and interpretation strategies. Eur Respir J 1997;10:2662–899426113\n16. Global initiative for asthma Pocket guide for asthma management and prevention in children. National Institutes of Health, National Heart, Lung and Blood Institute, 2002\n17. KoillinenHWanneONiemiV  Spirometric and peak expiratory flow reference values of healthy Finnish children. Finnish Med J 1998;53:395–402\n18. PelkonenASHakulinenALTurpeinenM Bronchial lability and responsiveness in schoolchildren born very preterm. Am J Respir Crit Care Med 1997;156:1178–849351619\n19. American Thoracic Society Standardization of spirometry (1994 update). Am J Respir Crit Care Med 1994;152:1107–36\n20. TannerJM Growth at adolescence. Edn 2. Oxford: Blackwell, 1962\n21. SorvaRPerheentupaJTolppanenEM A novel format for a growth chart. Acta Paediatr Scand 1984;73:527–96464740\n22. WaalkensHJvan Essen-ZandvlietEEHughes MD etal Cessation of long-term treatment with inhaled corticosteroid (budesonide) in children results in deterioration. Am Rev Respir Dis 1993;148:1252–78239161\n23. AgertoftLPedersenS Effect of long-term treatment with inhaled budesonide on adult height in children with asthma. N Engl J Med 2000;343:1064–911027740\n24. VisserMJPostmaDSArendsLR  One-year treatment with different dosing schedules of fluticasone propionate in childhood asthma. Am J Respir Crit Care Med 2001;164:2073–711739137\n25. PowellHGibsonPG Cochrane Database Syst Rev 2004;(2):CD00410915106238\n26. BousheyHASorknessCAKingTS  Daily versus as-needed corticosteroids for mild persistent asthma. N Engl J Med 2005;352:1519–2815829533"
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batch_9/PMC2533285.json

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+{
+    "id": "PMC2533285",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533285\nAUTHORS: Teck-Hock Toh, Sing-Jill Chow, Tzer-Hwu Ting, Jill Sewell\n\nABSTRACT:\nStrengths and Difficulties Questionnaire (SDQ) is a brief behavioural screening questionnaire for children and teenagers aged 3 to 16 years. It is available in 66 languages, and gaining more popularity world wide. Chinese translation of SDQ is available and has been used in clinical practice and research. We undertook the exercise to back-translate the current Chinese translation and it showed a number of differences compared to the original English SDQ. The differences and concerns include: (1) the flow and grammar of Chinese translation as well as wrongly written Chinese characters; (2) translated words that have deviated from the original meaning; (3) significant numbers of wording that are somewhat different from the original English version; (4) addition of auxiliary verbs that do not exist in original English version; and (5) the current Chinese SDQ is a general questionnaire for all age groups that does not observe the differences of wording that exist in the English versions.An accurate translated Chinese version is important for researchers, clinicians and educators who work in the Chinese communities. There is an urgent need to review the translation of the Chinese SDQ version before more studies use it in the field.\n\nBODY:\nFull TextThe results of a study in China on the validity, reliability and normative scores of a Chinese translation of the Strengths and Difficulties Questionnaire (SDQ) were recently published in your online journal on 29 April 2008 by Du et al [1]. Findings on psychometric properties were mixed, especially in the areas of peer problems and self-rating by adolescents. Concern was also raised about the validity of the Chinese translation. This does not surprise us because we believe the answers lie in the Chinese translation of the SDQ.The SDQ is a brief behavioural screening questionnaire for children and teenagers aged 3 to16 years [2]. It was first tested in the United Kingdom and copy-righted by Goodman in 1997 [3]. Several versions are available and each version may include one to three of the following: a) 25-item psychological attributes, b) 5-question impact supplement, and c) seven follow-up questions. It is available in 66 languages, which include three English versions for the USA, United Kingdom and Australia that differ slightly in the wording used and age specification [2].The Centre for Clinical Trials and Epidemiological Research at the Chinese University of Hong Kong and Iris Tan Mink had contributed greatly in the translation, back-translation and validation of the Chinese version [2]. Currently the Chinese translation has three versions available for parent, teacher and student respectively and each version consists of the 25-item psychological attributes and impact supplement only. They were presumably translated from the United Kingdom's English version because the wording matches more closely than the other English versions [2]. It is available in traditional form of Chinese writing, commonly used in Hong Kong and Taiwan. Chinese communities in China and South East Asian countries use the simplified form of writing.Kou J, Du Y and Xia L published an article in Chinese in 2005 which concluded that the Chinese SDQ can be used to assess children and adolescents in Shanghai. This was derived from a validity and reliability study involving parents of 2128 students, using the three versions of Chinese SDQ and a retest 6 weeks later involving 47 of these parents [4].Despite reported findings by Du Y and Kou J [1,4], we feel strongly that the current Chinese translation of SDQ has a number of differences compared to the original English SDQ. It is challenging and unscientific to compare any finding as a result of using two questionnaires with different languages and meaning. We therefore question the conclusion by Du Y et al about the use of Chinese version of SDQ in China.We recognize that translation of scientific and clinical materials is not an easy task. We believe much effort has been put forward in the first translation by people in the Chinese University of Hong Kong and Iris Tan Mink. Their contribution should be recognized and appreciated. However, the current Chinese translation of SDQ should be critically appraised and reviewed to provide a more accurate translated Chinese version of SDQ that is reliable for its users in the field.An exercise was undertaken by two authors of this letter (Toh TH and Ting TH) to back-translate the current Chinese SDQ independently. Ting TH had no prior knowledge of the SDQ before the translation. Both back-translations were similar, and they are presented in Additional files 1, 2 and 3. The differences and concerns we found are as follow:1. The flow and grammar of the current Chinese SDQ are not smooth, with wrongly written Chinese characters.2. Some translated word has deviated from the original meaning.3. Significant numbers of wording, which include the word \"True\", used as the answer of all the 25 items, are somewhat different from the original UK English version.4. Auxiliary verb \"will\", \"can\" and \"very\" were added in many of the 25-item psychological attributes and the significance of adding these verbs is unclear.5. The current Chinese SDQ is a general questionnaire for all age groups, and does not observe some differences of wording that exists in the English versions.Examples and explanation of these major differences and concerns are included in Table 1.Table 1Summary of differences found between original english (UK) SDQ and Chinese translationMajor Differences and ConcernsAffected Items/Questions & ExamplesImplications/Suggestions1Chinese grammar/flow and wrongly written Chinese characters○ Items 2, 7, 12 & 23 in Parent/Teacher version○ Items 2, 12, 14, 17 & 23 in Student version○ Question 1 to 4 of the impact supplement in all three versions○ Two wrongly written Chinese characters (Item 15 in Student version and Question 4 in impact supplement, Parent/Teacher versions)Can be improved and rephrased to a more comprehensible language and more easily understood by a lay audience2Deviation of translated word○ Items 4, 7, 9, 12 & 17 in Parent/Teacher version*○ Items 4, 8, 9, 10, 11, 12, 20 & 25 in Student version*○ Question 1, 3 & 4 of the impact supplement in all three versions*○ Introductory paragraph of the Student version*These words need to be reviewed and matching the original English version3Translated word that is \"somewhat different\"○ The answers to the 25 items, \"true\"†○ Items 3, 5, 6, 8, 13, 24 & 25 in Parent/Teacher version‡○ Items 6, 18 & 23 in Student version‡○ Question 1 of the impact supplement in all three versions‡These words require further consideration and the significance of the differences is unclear.4Addition of auxiliary verbs (\"will\", \"can\" and \"very\")○ Items 16, 21 & 22 in Parent version○ Items 16 & 21 in Teacher version○ Items 16, 17, 21, 22 & 24 in Student versionThe significance of these verbs is unclear, ideally they should be removed5Age-unspecific versionsThe English versions are divided into different age groups, with some differences in wording. E.g., \"often argumentative with adults\" in the 3–4 years old group is represented by \"often lies or cheats\" in the 4–16 years old group. Current Chinese SDQ does not observe these differences because one version is used for all age groups. In this example, the item concerned was translated as \"often lies or cheats\" only.* For examples, instead of \"fights\", it was translated as \"quarrel\" and \"argue\"; instead of \"upset\", \"unwell\" and \"sad\" were used. \"I have one good friend or more\", was translated as \"I have one or a few good friends\"; and \"do the difficulties upset or distress your child?\" became \"are these difficulties perplexing/puzzling/disturbing you?\"† \"Not True\", \"Somewhat True\" and \"Certainly True\" – the answers to the 25 items, \"true\" was translated as \"tallying/accord or keeping with\"‡ For examples \"often seems worried\" was translated as \"often exhibit/display sign of anxiety\"; \"tearful\" was translated as \"crying\".An online search on 12 June 2008, involving PsycINFO 1806, Ovid MEDLINE(R) 1996, CINAHL 1982 and EMBASE 1996 using \"SDQ or Strengths and Difficulties Questionnaire\" and \"Chinese or Mandarin or China or Taiwan or Hong Kong\" as key words have shown numerous publications quoting the use of SDQ Chinese translations in China and Hong Kong. It was used as a measurement tool for interventional trials [5,6] and descriptive epidemiological studies [7,8]. Clinicians have also used it as a screening tool to prioritise psychiatry services [9] and to compare findings on psychometric properties of parent ratings on the Chinese version of the Swanson, Nolan, and Pelham IV scale [10].ConclusionIt is obvious that SDQ will gain increasing popularity world wide, and an accurate translated Chinese version is important for researchers, clinicians and educationists who work in the Chinese population. There is an urgent need to review the translation of the Chinese SDQ version before more studies use it in the field. A more complete set of Chinese SDQ versions in both traditional and simplified Chinese forms of writing should be made available on the SDQ website.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsBack-translation of the Chinese SDQ was performed by Toh TH and Ting TH. In addition all authors have contributed towards the writing and approval of this letter.Availability & requirementsSupplementary MaterialAdditional file 1Appendix A.Click here for fileAdditional file 2Appendix B.Click here for fileAdditional file 3Appendix C.Click here for file\n\nREFERENCES:\nNo References"
+}

batch_9/PMC2533292.json

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+{
+    "id": "PMC2533292",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533292\nAUTHORS: Jacques Beco\n\nABSTRACT:\nBackgroundLevator plate sagging (LPS), usually called descending perineum syndrome, is one of the main defects encountered in perineology. This defect is classically associated with colo-proctologic functional troubles (dyschesia and anal incontinence) but can also induce perineodynia, gynaecological and lower urinary tract symptoms.MethodsA retrospective case series of nine female patients (mean age: 44.3) underwent an isolated retro-anal levator plate myorrhaphy (RLPM) to treat symptomatic LPS confirmed by rectal examination and/or Perineocaliper®. An anti-sagging test (support of the posterior perineum) must significantly improve the symptoms that were resistant to conservative treatment. The effect of the procedure on the symptoms of the 3 axes of the perineum (urological, colo-proctologic and gynecological) and on perineodynia was evaluated during a follow up consultation more than 9 months after surgery. The effect of RLPM on the position of the anal margin and on the levator plate angle was studied using rectal examination, Perineocaliper® and retro-anal ultrasound.ResultsBefore surgery, anti-sagging tests were positive for dyschesia, urinary urgency and pain. After a mean follow-up of 16.1 months, RLPM resolved or improved 2/2 cases of stress urinary incontinence, 3/5 of urinary urgency, 3/4 of dysuria, 3/3 of anal incontinence, 7/8 of dyschesia, 3/4 of cystocele, 4/5 of rectocele, 5/8 of dyspareunia and 6/7 of perineodynia. Rectal examination showed a complete suppression of sagging in 4 patients and an improvement in the 5 others. The mean reduction of perineal descent was 1.08 cm (extremes: 0–1.5). Using retro-anal ultrasound of the levator plate, the mean reduction of sagging was 12.67 degrees (extremes: 1 – 21).ConclusionAnti-sagging tests can be used before surgery to simulate the effect of RLPM. This surgical procedure seems to improve stress urinary incontinence, frequency, nocturia, urgency, dysuria, anal incontinence, dyschesia, dyspareunia, perineodynia, cystocele and rectocele. These results must be confirmed by a larger case series.\n\nBODY:\nBackgroundPerineology is a new speciality that deals with the functional troubles of the three axes of the perineum [1-4]. This interdisciplinary and holistic field is approached from the angles of anatomy, biomechanics and physiology, avoiding at all costs any side effects (primum non nocere). There are seven basic, defect specific, useful surgical procedures that apply in perineology [2]. Retro-anal levator plate myorrhaphy (RLPM) is dedicated to treating the \"levator plate sagging\" defect.The usual name for this levator plate sagging is descending perineum syndrome (DPS). This syndrome is well described by Parks in 1966 [5]. For this author this title is mainly descriptive, as perineal descent on straining is both the cause of symptoms and the most obvious physical sign.For Parks, the main symptoms of DPS are dyschesia (partial and intermittent obstruction by the anterior rectal wall), pain (dull aching pain in the perineum or sacrum after defecation), bleeding or passage of mucus (prolapse of the anterior rectal wall) and anal leakage. The physical signs of this syndrome on external examination are a low position of the anus at rest or a perineal descent on straining (more than 3 cm). During this straining, the anal mucosa may pout. On rectal examination, during straining, the pubo-rectalis descends sharply and the anterior rectal wall pushes down on the examining finger. Muscle tone is easily overcome by posterior traction.For Parks, the first step in the treatment of this syndrome consists of preventing further damages by avoiding straining during defecation and emphasizing pelvic floor reeducation.For the most significant cases with rectal prolapse, Parks has developed a new surgical procedure called \"post-anal perineorrhaphy\" [6]. This procedure, also known as \"post-anal repair\", has been used by many other authors to treat faecal incontinence [7-13].In 1982, Nichols used a \"retro-rectal levatorplasty\" to treat an uncommon type of genital prolapse characterized by descent of the anus and sagging of the levator plate associated with severe constipation [14].In 1987, Shafik presented his experience with \"levatorplasty\" in the treatment of complete rectal prolapse [15].To improve the process of defecation by reducing levator plate sagging, Nichols [14,16] proposed using a special toilet seat with a small opening and Lesaffer [17,18] created a \"perineum device\" to support the perineum. These proposals were the first \"anti-sagging tests\".More recently, Beco [19] demonstrated that besides dyschesia and anal incontinence, a perineal descent of more than 1.5 cm, measured with a Perineocaliper® during straining in the gynaecological position (with thighs flexed to 90 degrees), significantly increased the frequency of urinary incontinence, dyspareunia, dysuria, cystocele and rectocele. The frequency of the 3 clinical signs of pudendal neuropathy [20] was also significantly increased.The first aim of this study is to show the diagnostic importance of \"anti-sagging tests\" on the symptoms of dyschesia, dysuria, dyspareunia, urinary incontinence, urgency and perineodynia experienced in levator plate sagging while standing. These tests can be performed by the patient herself or by the examining practitioner.The second aim is to evaluate the effect of a simplified retro-anal levator plate myorrhaphy (RLPM) on different symptoms and on perineal position and descent during straining.Methods1. Studied populationBetween March 2000 and January 2007, 104 retro-anal levator plate myorrhaphies (RLPM) were performed to treat levator plate sagging. To study the effect of RLPM, only 9 cases of isolated procedures were taken into account. The mean follow up was 16.1 months (extremes: 10–39 months). The mean age of patients was 44.3 years (extremes: 29–63) and mean parity 2.11 (extremes: 1–3). Five patients had a history of abdominal hysterectomy and 2 of surgery for prolapse. Five patients had difficult deliveries (2 vacuum extractors and 3 forceps). The approval for this study was granted by the CHC – Clinique Sainte Elisabeth Ethic Committee and each patient signed a written informed consent before surgery.2. Pre-operative evaluationIn addition to the classical history and clinical examination of the three axes of the perineum (urological, gynecological and colo-proctologic), special attention was given to diagnose pudendal neuropathy and levator plate sagging.History-Urinary symptomsStress and urge urinary incontinences are evaluated according to a 4 level ordinal scale depending on the amount of the leakage: 0 = no incontinence, 1 = mild incontinence (few drops of urine), 2 = moderate incontinence (moderate amount) and 3 = severe incontinence (large amount). The number of pads used per day is included.Daily frequency is based on the mean time (in minutes) between 2 micturitions. It is considered abnormal if this time is less than 90 minutes.Nocturnal frequency is evaluated by the number of micturitions during the night. The patient suffers from nocturia if there is more than 1 micturition per night.Urgency was evaluated according to a 3 level ordinal scale: 0 = no urgency, 1 = occasional urgency, 2 = constant urgency.Dysuria is evaluated with the same 3 level ordinal scale.- Colo-proctologic symptomsFor anal incontinence a four level ordinal scale was used: 0 = no incontinence, 1 = gas incontinence, 2 = liquid incontinence, 3 = solid incontinence.The patient presents dyschesia if the defecation process is abnormal, including formation of plugs, the need for enemas, glycerin suppositories or digital manipulation to evacuate stool. The importance of dyschesia has been evaluated according to a three level scale: 0 = no dyschesia, 1 = occasional dyschesia and/or mild difficulties to defecate, 2 = continuous or severe.- Dyspareunia and other perineodyniaThe importance of dyspareunia has been evaluated according to a 3 level scale: 0 = no dyspareunia, 1 = mild dyspareunia, 2 = severe dyspareunia.For perineodynia (perineal pain), the intensity of pain is evaluated using a classical visual analog scale going from 0 to 10. The different characteristics of pain are also studied.Clinical examinationThe entire examination is done in gynecological position (with the thighs flexed to 90 degrees).- Basic examinationCystocele, rectocele, enterocele and uterus descent have been graded from 0 to 3 according to the French classification [21,22] during Valsalva's maneuver and with a speculum moving away the vaginal wall in front of the prolapse (0 = no descent, 1 = in the vagina, 2 = at the level of vulvae skin, 3 = outside the vagina).In case of dyspareunia or perineodynia, the most prominent painful areas are explored by vaginal and rectal examination.- Pudendal neuropathyThe three clinical signs of pudendal neuropathy (abnormal pinprick sensibility, pain over the pudendal nerve during rectal examination and positive skin rolling test) were searched in the 9 patients [20,23].- Levator plate saggingTo evaluate levator plate sagging, we have used 3 methods: rectal examination, the Perineocaliper® and retro-anal ultrasound.During rectal examination, the position of the levator plate is evaluated with the index finger at rest and during Valsalva's maneuver. The two first phalanges of this finger are in the rectum in close contact with the levator plate. During straining, a small amount of traction on the finger is used. A three levels ordinal scale is used: 0 = no sagging of the levator plate (90° angle between anal canal and levator plate plane), 1 = moderate sagging of the levator plate (between 0 and 2) and 2 = complete levator plate sagging (180° angle between anal canal and levator plate plane).The Perineocaliper® (Duchateau SA, Liège, Belgium) has been developed to evaluate the position of anal margin with respect to the ischial tuberosities at rest and during a Valsalva's maneuver in the gynecological position (with thighs flexed to 90 degrees) (Figure 1).Figure 1Use of Perineocaliper® (Duchateau SA, Liège, Belgium). A: position of anal margin 2 cm higher than the ischial tuberosities (value = + 2 cm) at rest and in gynecological position (with thighs flexed to 90 degrees). B: during Valsalva's maneuver (red arrow) the anal margin is 1 cm below the ischial tuberosities (value = - 1 cm). View from the top as during clinical measurement. In this case, perineal descent = 3 cm (difference between A and B). 1 = anal margin. 2 = ischial tuberosity. 3 = level of the ischial tuberosities = reference or zero level.If the anal margin is located higher than the ischial tuberosities the value is positive. If it is located below, the value is negative. Perineal descent corresponds to the difference between the position at rest and during straining.Retro-anal ultrasound has been done with an end-fire transvaginal probe (Hitachi®) emitting at 6.5 MHz [24] The patient is lying in gynecological position (with the thighs flexed to 90 degrees). The probe must be perfectly horizontal with its tip located in the midline 1 or 2 cm in front of the coccyx to obtain a sagittal section (Figure 2).Figure 2Retro-anal ultrasound of the levator plate. A: position of the probe between anus and coccyx (levator plate or ano-coccygeal raphe in red). B: ultrasonographic image: 1 = posterior rectal wall, 2 = levator plate, 3 = coccyx. Dotted lines represent structures which are not in the section plane: thin lines = limits of the levator hiatus, thick line = right pudendal nerve. Small red ring = anal sphincter.The practitioner has to be very cautious to avoid any lifting or supportive effect of the levator plate with the probe at rest and during straining. The angle between the levator plate (\"ano-coccygeal raphe\" in the midline) and the vertical plane has been measured at rest and during Valsalva's maneuver. The angle of sagging corresponds to the difference between these 2 values.- Anti-sagging testsThe aim of the \"anti-sagging tests\" is to reduce the sagging of the levator plate, which simulates the effect of retro-anal levator plate myorrhaphy (Figure 3).Figure 3Anti-sagging test on a sagittal section. A: sagging of the levator plate – descending perineum syndrome (levator plate or ano-coccygeal raphe in red) during Valsalva's maneuver. B: anti-sagging test: support of the posterior perineum restores normal position. Dotted lines represent structures which are not in the section plane: thin lines = limits of the levator hiatus (increase of its size if perineal descent during Valsalva's maneuver), thick lines = right pudendal nerve (stretching induced by perineal descent). Small red ring = anal sphincter.For dyschesia and dysuria, the patient has to move back as much as possible on the toilet seat during defecation and micturition to support the levator plate (between coccyx and anus).For dyspareunia, if vaginal examination reproduces the classical pain induced by intercourse (usually at the level of the pubo-rectalis muscle, utero-sacral ligaments, transverse muscle or vaginal scar), the test consists of lifting the posterior perineum (between coccyx and anus) with two or three fingers and to evaluate the effect on pain. The patient can also try this test during intercourse (or use sexual positions where the buttocks are higher than the head).The anti-sagging test can be tried by the practitioner and taught to the patient while standing for urinary urgency or perineodynia. During urodynamic exploration, it is possible to try the effect of this test on the urgent need to urinate, on bladder capacity or even on urinary stress incontinence.The \"anti-sagging test\" is positive if there is a very clear improvement of the symptom studied during this maneuver.3. Indication for surgeryWhen diet, drugs and physiotherapy fail, surgery is indicated when the anti-sagging test dramatically improves the resistant symptoms associated with complete levator plate sagging on rectal examination (180° angle between anal canal and levator plate plane) and/or a perineal descent of more than 1 cm (measured with the Perineocaliper®).4. Surgical procedureDuring the 48 hours before surgery, the patient has to eat a residue free diet and must take paromomycine 2 g per day. To complete the intestinal preparation, an enema is done the evening before surgery.The patient is installed in a gynecological position with hyperflexion of the thighs. The surgical procedure begins with a sagittal incision 4 cm long between anus and coccyx (Figure 4a). The two ischio-rectal fossae are opened with the tip of the scissors laterally to the ano-coccygeal ligament (also called \"intermediate loop of the external anal sphincter\" [25]). The space between the ano-coccygeal ligament and the levator plate is opened with the finger. The scissors are passed from left to right over the ano-coccygeal ligament to isolate this ligament (Figure 4b). It is cut after having marked its two extremities with a suture (Figure 4c).Figure 4Retro-anal levator plate myorrhaphy: surgical steps. A: Skin incision. B: Scissors introduced between levator plate and ano-coccygeal ligament (also called intermediate loop of the external anal sphincter). C: The ano-coccygeal ligament ready to be cut (two extremities marked with a thread). D: Levator plate myorrhaphy between coccyx and ano-rectal junction until suppression of the sagging (checked by rectal examination). E: Ano-coccygeal ligament repaired. F: Skin closure with a Y-shaped multi-tubular drain at the posterior edge of the incision.The levator plate myorrhaphy began in front of the coccyx. Before putting stitches, it is necessary to \"clean\" the muscle with the finger by removing the adherent grease as much as possible. This step is necessary to be sure to put the stitches into the muscle. The first stitch is very important. It is located just in front of the coccyx. It must take a good bit (1 cm; with a dexon® 2, needle GS-21) of the levator plate on each side to tighten the muscle but without excessive tension not to tear it. Traction on this stitch to the rear checks the solidity of this first point of myorrhaphy and presents the two edges of the levator plate to be sutured. Usually two to four stitches of dexon® 2 (with some \"figure of eight\" if possible) are necessary to suppress completely the sagging (Figure 4d). To avoid rectal injury, the stitches can be put with a finger in the rectum to check the position of the needle. The last stitches must remain behind the level of the anal canal to avoid damage to the rectal branches of the pudendal nerves.At the end of the procedure, rectal examination confirms the absence of sagging of the levator plate (90° angle between anal canal and levator plate plane). The ano-coccygeal ligament is repaired with two to three stitches of dexon® 2 (Figure 4e). A Y-shaped multi-tubular drain is put into the two ischio-rectal fossae and exits through the posterior edge of the initial incision. The subcutaneous tissue is then closed followed by closure of the skin with simple stitches of vicryl rapide® 2.0 (Figure 4f). The drains are usually removed the 5th day after surgery. Antibiotic coverage is continued for 5 days.After surgery, the patient is told to avoid heavy lifting, constipation (use of soft laxatives), and chronic cough and to move back on the toilet seat to support her posterior perineum during defecation and voiding, for a minimum of two months and ideally for the rest of her life.5. Evaluation of the procedureThe effects of RLPM have been evaluated more than 9 months after surgery by using the same history, clinical examination and retro-anal ultrasound as before the operation.Results1. Anti-sagging tests before surgeryBesides a clear levator plate sagging, a positive anti-sagging test on a severe perineal symptom is crucial in the indication of a RLPM. The results of the pre-operative anti-sagging tests of this case series are presented in Table 1.Table 1Results of the anti-sagging tests before surgeryDuring clinical examinationSubjectivePatientsFollow-up (months)PainUrgencyDyschesiaCk12Improved--Dz18- (**)-ImprovedRx12Improved--Re19ImprovedImproved(*)-Hy39Improved--Ro11Improved--Ns12Improved-ImprovedSn12Improved-ImprovedHs10Improved-ImprovedThe test is positive if the symptoms are significantly improved.(*) during bladder filling.(**) – = not done.No test for dysuria and stress urinary incontinence done in this population.2. Effect on symptoms of the three axes of the perineum and on perineodyniaUrological axis (Table 2)Table 2Effect of RLPM on the urological axisPatientsFollow-up (months)SUI (0–3)Frequency (min)Nocturia (nr/night)Urgency (0–2)Dysuria (0–2)BeforeAfterBeforeAfterBeforeAfterBeforeAfterBeforeAfterCk120060180200020Dz1810120120000000Rx1200180180000000Re190060180002000Hy3900120120111100Ro1120240240000000Ns1200120120102021Sn1200120180111010Hs1000120180011111SUI = stress urinary incontinencemin = minutesIn this case series no patient had urge incontinenceIn this case series, no patients had urge incontinence. The 2 cases of stress urinary incontinence were cured (one used 1 pad per day and the other 3 pads per day). The 2 cases with frequency and the one with nocturia were cured. Dysuria was cured in 2 patients, improved in one and was unchanged in the other one. Urgency was cured in 3 patients and unchanged in two.Colo-proctologic axis (Table 3)Table 3Effect of RLPM on the colo-proctologic axisAnal incontinence (0–3)Dyschesia (0–2)PatientsFollow-up (months)BeforeAfterBeforeAfterCk120020Dz182120Rx120020Re191020Hy390022Ro110000Ns120021Sn121020Hs100020Two cases of gas incontinence were cured and one of liquid incontinence improved. Dyschesia was completely cured in 6 patients, improved in one and was unchanged in one case.Gynecological axis (Table 4)Table 4Effect of RLPM on the gynecological axisDyspareunia (0–2)Vaginal examination (location of pain)Cystocele (0–3)Rectocele (0–3)PatientsFollow-up (months)BeforeAfterBeforeAfterBeforeAfterBeforeAfterCk1220Left PR(right absent)Reduced0000Dz1800--0010Rx1220Right PRLeft scarReduced0000Re192N.I.PR bilateral01110Hy3922Right PR00000Ro1120Vaginal cuff01010Ns122N.I.PR bilateralVaginal cuff1011Sn1221Left PRReduced0000Hs1020US ligaments02010PR = pubo-rectalisUS = utero-sacralN.I. = no intercourse (other reason than pain)Dyspareunia was cured in 4 patients, improved in one and was unchanged in one. Two patients did not have intercourse after surgery for other reasons than pain. Three cystoceles were cured (two grade 1 and one grade 2). One was unchanged. Four grade 1 rectocele disappeared and one was unchanged. Pain during vaginal examination disappeared in 4 patients and was reduced in another four.Perineodynia (Table 5)Table 5Perineodynia before and after surgeryVisual analog scaleLocationWorse whilePatientsFollow-up (months)BeforeAfterBeforeAfterBeforeAfterCk1253LeftLeftSitting Walking StairsSitting StairsDz1800----Rx1272RightRightSitting StandingSittingRe1980Bilateral0Sitting Standing Defecation0Hy3933BilateralBilateralStandingStandingRo1182Bilateral0Defecation Post-coitalPost-coitalNs1200----Sn1242LeftLeftSittingSittingHs1040Proctalgia fugax0--The intensity of perineodynia (perineal pain), its location and its aggravating circumstances before and after surgery are presented in Table 5. Pain was cured in 2 patients, improved in 4 and was unchanged in one.The summary of the effects of RLPM on perineodynia and on the urological, colo-proctologic and gynaecological axes is presented in Table 6.Table 6Summary of the effects of RLPM on the functional troubles of the perineumSymptoms, SignsCuredImprovedFailureUnknownSUI200Frequency200Nocturia100Urgency302Dysuria211Anal incontinence210Dyschesia611Cystocele301Rectocele401Dyspareunia4112 (no sex)Perineodynia241SUI = stress urinary incontinence.3. Effect on the three clinical signs of pudendal neuropathyThe effects of RLPM on the 3 clinical signs of pudendal neuropathy are showed in Table 7. On the 12 positives with abnormal clinical signs, 5 were normalized by surgery and 7 did not change. On the 9 with negative (normal) clinical signs, 4 became abnormal after RLPM.Table 7Clinical signs of pudendal neuropathy before and after surgeryAbnormal sensibilityPainful pudendal nervePositive skin rolling testPatientsFollow-up (months)BeforeAfterBeforeAfterBeforeAfterCk12111101Dz18010000Rx12------Re19001010Hy39000101Ro11------Ns12101000Sn12111110Hs101111110 = normal (negative) test1 = abnormal (positive) test4. Effect on levator plate position and saggingThe objective evaluation of the position of the levator plate and anal margin at rest and during straining before and after RLPM is showed in Table 8.Table 8Objective evaluation of the levator plate and anal margin positions at rest and during straining before and after surgeryPatientsPerineocaliper (cm)Retro-anal ultrasound (degrees)Rectal examination (0–2)Rest BeforeRest AfterStrain BeforeStrain AfterDescent BeforeDescent AfterRest BeforeRest AfterStrain BeforeStrain AfterSag BeforeSag AfterSag beforeSag afterCk-10-3-0,520,53516602425820Dz02-1,51,51,50,5--5620--10Rx--------4725--21Re-1,5-1-2-1,50,50,5------21Hy0,51-1,50,520,53122582927721Ro------29255335241021Ns------24175628321120Sn1,50,5-1-0,52,5128314343151221Hs0,50,5-101,50,5251833258720Mean00.5-1.66-0.081,660,5828,6621,550.7528.6221.839,161.880.55Evaluation of LPS by rectal examination after surgery showed a complete suppression of sagging in 4 patients and a reduction in the 5 others.With the Perineocaliper®, the position of the anal margin at rest was 0.5 cm (extremes: -1–2) higher after surgery. During Valsalva's maneuver (strain), it was 1.58 cm (extremes: 0.5–3) higher. The reduction of perineal descent was therefore 1.08 cm (extremes: 0–1.5).The mean reduction of angulation of the levator plate using retro-anal ultrasound at rest was 7.16 degrees (extremes: -3 – 19). During straining the mean reduction of angulation was 22.13 degrees (extremes: 0 – 36). The mean reduction of sagging was 12.67 degrees (extremes: 1 – 21).5. ComplicationIn this case series, one patient (case \"Ro\") had a complete rupture of the RLPM during heavy lifting 12 months after surgery. Stress urinary incontinence, cystocele, rectocele, perineodynia after defecation and dyspareunia returned rapidly after the incident.DiscussionIn perineology, only one specialist must treat all the symptoms of the three compartments of the perineum by using low risk and defect specific procedures.By treating surgically only seven basic defects with their dedicated procedure it is possible to improve most of the functional troubles encountered in this area.Abnormal levator plate sagging is one of these basic defects that must be treated in perineology [2]. This defect is quite frequent but rarely isolated.The other ones are:- relaxation of the sub-urethral vaginal hammock with hypermobility of the bladder neck (explaining mainly genuine stress urinary incontinence).- rupture or weakness of the anterior part of the pelvic fascia – Halban's fascia (inducing cystocele).- rupture or weakness of the posterior part of the pelvic fascia – Denonvilliers fascia, including its fixation to the utero-sacral ligaments(favouring high rectocele, enterocele, uterine descent and cuff prolapse)- weakness of the perineal body (increasing the risk of all prolapses but especially of low rectocele)- rupture of the anal sphincter (with anal incontinence)- pudendal neuropathy [20].In the 104 patients treated by RLPM, only 9 (8,6%) had an isolated procedure and represent the studied population. In the 95 other cases, one or many of the six other defect specific procedures were done together to achieve a complete restoration of the perineum. These cases were excluded from the study because each of the associated procedures can have an important effect by itself on perineal function and anatomy.Because this study focus on the defect called levator plate sagging, its first issue is to define when this sagging is abnormal. Three methods were used to evaluate this defect: the Perineocaliper®, rectal examination and retro-anal ultrasound.In 1982, Henry et al studied the relationship of the anal verge to the ischial tuberosities in patients with descending perineum syndrome and compared them with normal subjects. They do not specify the name of the instrument used [26]. Some authors utilize the name \"perineometer\" for this instrument and perform perineometry with it [27]. Kegel had used this name many years earlier for an instrument that is introduced in the vagina to measure the increase of pressure induced by a perineal contraction [28]. The name \"Perineocaliper\" has been introduced because the measurement done in perineology corresponds to a depth or step measurement obtained with a Vernier caliper and the instrument has practically the same shape as a brake or skinfold caliper. With this instrument it is possible to define precisely the position of anal margin at rest, during Valsalva's maneuver or during pelvic floor contraction.By using the Perineocaliper® in a control group of 143 female patients, mean aged 55 years (extremes 26–81) without any symptoms, the normal position of the anal margin at rest was 0.03 cm above the ischial tuberosities (SD = 0.99) and during Valsalva 0.56 cm below these bones (SD = 0.98). The mean descent of the anal margin was 0.59 (SD = 0.54) (unpublished data). According to these data, a descent of more than 1.67 cm is unusual in a control group.In a recent study, Beco [19,24] demonstrated that besides dyschesia and anal incontinence, a perineal descent of more than 1.5 cm measured with a Perineocaliper® during Valsalva's maneuver in a gynaecological position significantly increases the frequency of urinary incontinence, dyspareunia, dysuria, cystocele and rectocele. The frequency of the 3 clinical signs of pudendal neuropathy [20] was also significantly increased (Table 9).Table 9Frequency of the 6 main perineological symptoms and of the 3 signs of pudendal neuropathy according to the perineal descent measured with a Perineocaliper® [19,24].Perineal descentNumber of casesUrinary incontinenceFaecal incontinenceProlapse 2–3DysuriaDyschesiaDyspareunia3 signs(n = 991)(n = 566)(n = 41)(n = 256)(n = 93)(n = 261)(n = 234)(n = 152/820)-158000400200-0,5742,85042,85014,2814,280022751,541,3221,5810,1324,2214,5313,870,525750,19(NS)3,50(NS)20,62(NS)6,22(NS)15,95(NS)17,89(NS)17,28(NS)130860,06(p<0.05)4,54(p<0.05)25(NS)8,76(NS)25,97(NS)27,59(p<0.001)18,41(NS)1,57660,52(NS)3,94(NS)34,21(p<0.05)9,21(NS)35,52(NS)32,89(p<0.001)23,33(NS)28275,60(p<0.001)12,19(p<0.001)43,90(p<0.001)13,41(NS)48,78(p<0.001)36,58(p<0.001)27,94(p<0.01)2,51566,666,6633,332053,3346,6655,5531181,819,0954,5427,2772,7254,5444,443,5250050505000The p values were obtained using chi-squared tests; comparison with descent = 0. A perineal descent of 2 cm (compared to a 0 cm descent) leads to a significant increase in the frequency of urinary incontinence, faecal incontinence (solid stools), genital prolapse (grade 2 and more) and dyschesia. The same threshold exists for the 3 clinical signs of pudendal neuropathy (820 cases). For dyspareunia the threshold seems to be at 1 cm and in case of dysuria, the difference is significant between 0.5 cm and 2 cm of descent (p < 0.05).Therefore, it seems that a perineal descent of more than 1.5 cm during a Valsalva's maneuver is abnormal.Henry [26], using an old version of the same instrument, found different values in his control group of 55 women (mean age 48 years): at rest + 2.5 cm (SD = 0.6), during \"bears down\" like in defecation + 0.9 cm (SD = 1). There are three main differences between the two studies: the mean age of the control group (48 versus 55 years), the position of the patient during the measurement (left lateral versus gynecological position) and the type of effort (\"bears down\" like in defecation versus Valsalva's maneuver). They could explain the different results obtained.Measuring the descent of the anal margin is an indirect method of evaluating levator plate sagging. In fact, theoretically the descent of the anal margin is greater in cases of a longer levator plate for the same angle. Therefore it seems logical to evaluate the angle itself.This evaluation is possible by rectal examination as proposed by Shafik [15]. The three levels ordinal scale used in this study was very useful in the indication of RLPM (grade 2 = complete sagging) and at the end of the myorrhaphy (grade 0 = no sagging). Other methods like colpocystodefecography, magnetic resonance imaging or ultrasound must be used to obtain more precise data.For Costalat et al [29] using colpocystodefecography in a sitting position, the normal angulation of the posterior rectal wall at rest is less than 20 degrees with the horizontal. During defecation, the angle of sagging must be less than 20 degrees.For Hsu et al [30] using dynamic nuclear resonance magnetic imaging in a supine position, the mean angulation of the levator plate with the vertical plane at rest was 36.2 degrees (SD = 12.3) and during Valsalva's maneuver 44.3 degrees (SD = 15.2). With respect to the control group, there was a significant increase of 9.1 degrees in the levator plate angle during Valsalva in case of prolapse.Retro-anal ultrasound (done in a gynecological position) is a new easily available method that enables the study of the levator plate angle. In a control group of 40 female patients (unpublished data), with a mean age of 51 years (extremes: 23–81) the mean value of the angles at rest was 19.7 degrees (SD = 8.8 degrees) and during Valsalva 30.5 degrees (SD = 10.7 degrees). The mean sagging angle (difference between Valsalva and rest angles) was 10.8 degrees (SD = 8.2 degrees). This method has been used to evaluate the changes in angles induced by surgery, but not as an inclusion criteria for surgery because it is still being validated.Anti-sagging tests must be used to prove that perineal descent is the cause of one symptom. By supporting the posterior perineum, these tests suppress levator plate sagging and simulate the effect of RLPM. It must be used to confirm the indication to perform a RLPM, but it may also be used as a part of the non-surgical treatment of a descending perineum syndrome. Especially in case of dyschesia or dysuria, the improvement of micturition or defecation by moving back as much as possible on the toilet seat can be so important that it can stop the vicious circle: straining => perineal descent (stretching of the pudendal nerve) => straining. Ideally, the back part of the toilet seat must be large enough and only slightly tilted to support perfectly the posterior perineum. The same approach was proposed by Lesaffer [17,18] and Nichols [14,16] who suggest the use of special toilet seats or supports to improve defecation.Overload of the \"suspensor structures\" (pubo-rectalis, utero-sacral ligaments, vaginal scars, transverse muscles) induced by levator plate sagging is a newly discovered cause of perineodynia and dyspareunia which must be differentiated from pudendal neuropathy [31] or muscular pain [32]. Contrary to the two other causes, pain induced by levator plate sagging is usually increased while standing and reduced while sitting. It is suppressed by the anti-sagging test. This test is also very helpful to confirm that perineal descent is the cause of dyspareunia.The same differential diagnosis must be done in cases of urinary frequency, urgency and urge incontinence where pudendal neuropathy [20,33] and muscular trigger points [34] may also be involved. Again, in cases of levator plate sagging, the symptoms are worse in a standing position and disappear when the posterior perineum is lifted with the hand.Some surgical procedures able to reduce levator plate sagging have been described in the literature. A comparison between these procedures and RLPM is presented in Table 10.Table 10Surgical procedures used to treat levator plate saggingPost-anal repair (Parks [6])Retro-rectal levatorplasty (Nichols [14,16])Levatorplasty (Shafik [15])Retro-anal levator plate myorrhaphyRetro-anal incisionU-shapedMidlineU-shapedMidlineDissection planeIntersphinctericRetro-analRetro-analRetro-analOpening of the pelvisIncision of Waldeyer's fasciaOpening of the retro-rectal or pre-sacral spaceNoNoMyorrhaphyLevator plate, pubo-rectalis and external sphincterLevator plate and pubo-rectalisLevator plateLevator plateRectal neck attached to the levator plateNoNoYesNoPosterior wall of the rectum sewn to the presacral fasciaNoYesNoNoThe intersphincteric plane has been avoided because this approach can damage the thin anal sphincter and the operating field is reduced. After having used retro-anal U-shaped skin incision (Shafik's levatorplasty [15]) in our first cases (not included in this case series), we decide to make midline skin incisions which are less painful. Only myorrhaphy of the levator plate has been done because the pubo-rectalis is included in the upper part of the anal sphincter and is not available for suture by the retro-anal approach. Furthermore, stitches in this muscle would damage it and therefore reduce its contractile force. To reduce the risk of injury to the rectum, the rectal neck was not attached to the edge of the levator plate (this step is probably necessary in cases of rectal prolapse [15]) and the pelvis was not opened.In the literature, the main indication of post-anal repair was idiopathic anal incontinence. The long term results vary from 90% cured in the old studies to 35% cured in the more recent ones [7-10]. Nichols [14] used retro-rectal levatorplasty to treat isolated dyschesia with anal descent. Shafik [15] proposed levatorplasty to treat dyschesia associated with complete rectal prolapse.In this short case series, besides anal incontinence and dyschesia, RLPM has been effective on most of the functional troubles of the perineum. Of course the number of cases is too small to draw definitive conclusions. This new solution may though be very useful to treat difficult problems such as urinary urgency, dysuria, dyspareunia or perineodynia. For stress urinary incontinence, RLPM is not a first line treatment because less invasive and more specific procedures exist but it could be helpful in some difficult cases.Athanasiadis [9] found no significant difference in pelvic descent and ano-rectal angle after post-anal repair. After the same surgical procedure, Orrom [11] and Van Tets [12] did not find any difference either in the ano-rectal angle. In this study, levator plate sagging was reduced independently from the evaluation method used. This difference may be due to the smaller number of cases, to a shorter follow-up or to the different surgical approach.Jameson [8] and Athanasiadis [9] didn't find any significant change in the motor latencies of the pudendal nerve after post-anal repair. In this study, the effect on pudendal neuropathy is unclear. By reducing the stretching on the pudendal nerve, RLPM should normally improve pudendal nerve function. This result has been observed clinically in 50% of the cases. The appearance of some clinical signs of pudendal neuropathy after surgery is quite surprising. Maybe it is linked to the formation of adhesions in the ischio-rectal fossae or the appearance of trigger points in the pelvic floor muscles. Further studies are necessary to better understand this side effect.RLPM is one defect specific treatment of levator plate sagging, but of course other surgical procedures could reduce perineal descent as well. The patient with rupture of the RLPM has been re-operated successfully. The procedure used was part of a full defect restoration project [2] and included the treatment for rectocele, cystocele and urinary incontinence using the vaginal route. Without any statistical proof, this multi-layer operation seems to be more logical to reduce the load on the RLPM. In fact each layer (anterior vaginal wall, sub-urethral support, posterior vaginal wall, perineal body) absorbs a part of the pressure, therefore reducing the tension on the levator plate myorrhaphy. Conversely, RLPM should reduce the load on genital prolapse repairs and the risk of recurrence.In our full experience with RLPM, the main risk is ischio-rectal fossae infection (not encountered in this case series). This risk seems to be reduced by the use of a multi-tubular drain for 5 days, antibiotic coverage and intestinal preparation before surgery.ConclusionDyschesia and anal incontinence are only a small part of the problems induced by pathological sagging of the levator plate. In fact, perineal descent of more than 1.5 cm significantly increases the frequency of all the functional troubles related to the perineum.RLPM can treat levator plate sagging, perineal descent and all the symptoms associated with this defect (stress urinary incontinence, frequency, urgency, dysuria, anal incontinence, dyschesia, dyspareunia, perineodynia and prolapse). Anti-sagging tests must be used to confirm the role of this sagging in the origin of these symptoms. A clear positive test is mandatory before surgery is indicated. Sometimes though, simply moving back on the toilet seat during defecation and micturition is enough to treat the patient's problem, avoiding the need for physiotherapy or surgery.Many symptoms induced by levator plate sagging may be seen in cases of pudendal neuropathy or pelvic floor muscle trigger points. The perineology specialist must be aware of all these etiologies to obtain good results for his patients.AbbreviationsRLPM: retro-anal levator plate myorrhaphy; LPS: levator plate sagging; DPS: descending perineum syndrome; SD: standard deviation; SUI: stress urinary incontinence.Competing interestsThe author has designed the instrument used to measure perineal descent and is the owner of the registered mark Perineocaliper®. The society which sell this instrument did not participate financially to this study.Authors' contributionsJB carried out all of the work for this study.Pre-publication historyThe pre-publication history for this paper can be accessed here:\n\nREFERENCES:\nNo References"
+}

batch_9/PMC2533303.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2533303",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533303\nAUTHORS: Raj Kumar Sharma, Vir Kumar Jain\n\nABSTRACT:\nThe current work attempts to highlight the various life threatening complications of Meckel's diverticulum and to present the surgical strategies used in the emergency conditions so far in the form of a review of the works presented in the literature. Our aim behind this presentation is to cover the possible indications, methods, their complications and the outcome of these surgical techniques. For this, we made an extensive literature search using Google and Pubmed with the words-\"Meckel's diverticulum\", \"Complications\", \"Management\" and \"Emergency surgery\". All the relevant articles containing the surgical aspects of symptomatic Meckel's diverticulum till May 2008 were collected and analyzed. Meckel's diverticulum is the remains of the prenatal yolkstalk (Vitellointestinal duct). Although it generally remains silent but life threatening complications may arise making it an important structure for having a detailed knowledge of its anatomical and pathophysiological properties to deal with such complications.\n\nBODY:\nIntroductionMeckel's diverticulum was first described by Fabricius Hildanus in 1598. The name derives from the German anatomist Johann Friedrich Meckel who described the embryological and pathological features in 1809 [1]. Although it generally remains silent but life threatening complications may arise making it an important structure for having a detailed knowledge of its anatomical and pathophysiological properties to deal with such complications.The literature is replete with the description of Meckel's diverticulum for its clinical presentations and complications. A search of Pubmed with the word \"Meckel's diverticulum\" shows 2835 articles till May 2008. However, the emergency surgery part of the symptomatic Meckel's diverticulum is still deficient in the literature. This seems to be so because most of the articles have been in the form of case reports or case series, so that the management strategies have been highly individualized based on both the patients condition and on the surgeons perspective. This seems to be the appropriate approach in an emergency condition. We intend to underscore some general principles used by these surgeons in their emergency surgeries. The current work attempts to highlight the various life threatening complications of Meckel's diverticulum and to present the surgical strategies used in the emergency conditions so far in the form of a review of the works presented in the literature. Our aim behind this presentation is to cover the possible indications, methods, their complications and the outcome of these surgical techniques. For this, we made an extensive literature search using Google and Pubmed with the words-\"Meckel's diverticulum\", \"Complications\", \"Management\" and \"Emergency surgery\". All the relevant articles containing the surgical aspects of symptomatic Meckel's diverticulum till May 2008 were collected and analyzed.Meckel's diverticulum is the remain of the prenatal yolkstalk (Vitellointestinal duct). The yolk sac of the developing embryo is connected to the primitive gut by the yolk stalk or vitelline (i.e. omphalomesenteric) duct. This structure normally regresses between the fifth and seventh weeks of fetal life. If this process of regression fails, various anomalies can occur. The spectrum of defects includes a Meckel diverticulum, a fibrous cord attaching the distal ileum to the abdominal wall, an umbilical-intestinal fistula, a mucosa-lined cyst, or an umbilical sinus. Of these, Meckel's diverticulum is the most common congenital anomaly of the gastrointestinal tract in humans occurring in approximately 2% of the population with equal incidence in males and females [2]. It is located on the antimesentric border of the ileum 45 to 60 cm proximal to the ileocecal valve and is usually 3–5 cm long [3] [fig 1]. It possesses all the three layers of the intestinal wall and has its own blood supply from the superior mesenteric artery, which makes it vulnerable to infection and obstruction like appendix[3]. Since cell lining of vitelline duct are pluripotent, we may get heterotopic gastric mucosa (50%), pancreatic mucosa(5%) and less commonly colonic mucosa, endometriosis, hepatobiliary tissue, which are responsible for other complications like hemorrhage, chronic peptic ulceration and perforation [2,4,5].Figure 1Incidentally found Meckel's diverticulum.Majority of the meckel's diverticulum remain silent and are diagnosed incidentally during small bowel contrast study, laparoscopy or laparotomy done for unrelated conditions, or until complications arise from the diverticulum [6-8]. A person with Meckel's diverticulum has a 4 – 6% lifetime risk of developing a complication [1,9]. The most common clinical presentation is gastrointestinal bleeding, which occurs in 25% – 50% of the patients having complication [3]. Other complications include obstruction, intussusception, diverticulitis and perforation. Age wise statistics reveals that hemorrhage is the most common presentation in children aged 2 years or younger[3,10] and intestinal obstruction being the commonest among adults[11], although some studies have found reverse [12]. Overall, the complications have been found more common in males, with the ratios varying in different studies from 1.8:1 to 3:1 [13-15]. The pathogenesis of ulceration in a Meckel's diverticulum is secondary to peptic ulceration from heterotopic gastric mucosa. Although colonization of Helicobacter pylori in this ectopic gastric mucosa has been reported but its role in pathogenesis of complication is yet to established [16].Complications and their managementsBleedingLower gastrointestinal hemorrhage is the most common presentation in children with a symptomatic Meckel diverticulum, with incidence rates recorded as high as 50% [17]. The average age of presentation is 2 years but it may occur in older children and adults [3,10]. The presence of heterotopic gastric and pancreatic mucosa within the Meckel's diverticulum, which secretes acid and highly alkaline pancreatic secretion respectively, causing ulceration of adjacent ileal mucosa is the main pathophysiology behind this. However, in adults, other rare causes of bleeding from Meckel's diverticulum have been noted which include the stromal tumors of the same [14]. Children often present with dark red or maroon stools or stools with blood or mucus, whereas adults usually present with melena and crampy abdominal pain. This is perhaps attributable to slower colonic transit time in adults [18]. The bleeding is typically painless and it may be brisk or massive giving the appearance of stool a bright red, brick red or black. Other causes of lower gastrointestinal bleeding in children which include polyps, clotting disorders, arteriovenous malformations, and Crohn's disease, need to be excluded by proper investigations. Technetium -99m pertechnetate radioisotope scintigraphy has been utilized universally for the diagnosis of bleeding Meckel's diverticulum and is at present the investigation of choice in a suspected Meckel diverticulum bleed [19]. In this method the injected radioisotope is readily taken up by the ectopic gastric mucosa within the Meckel diverticulum. The diagnostic sensitivity has been reported as high as 85% with a specificity of 95% and accuracy of 90% in the pediatric age group[2]. The accuracy of such scanning may be increased with the use of pentagastrin (to stimulate the uptake of the radioisotope), histamine-blockers (to inhibit the secretion of the pertechnetate once it is taken up), and glucagon (to inhibit peristalsis and thereby decrease \"wash-out\" of the pertechnetate). Capsule endoscopy has proved to be of diagnostic value in some cases of bleeding Meckel's diverticulum, however, the reports are very few and a concluding statement regarding its diagnostic value cannot be made at this time[20-22]. Colonoscopy cannot diagnose the bleeding from a Meckel's diverticulum because the colonoscope usually cannot reach the part of the small intestine in which the Meckel's diverticulum is located. But we can think of this pathology when we get blood filled colon without another source of bleeding particularly if accompanied by an abnormal radioisotope scan. Angiography may be useful in the evaluation of an adult patient with occult or intermittent gastrointestinal bleeding for the localization of the site of bleeding, specific diagnosis, and therapeutic preoperative embolization. A vascular blush may also be identified at the site of the Meckel diverticulum. When active hemorrhage is occurring at the time of angiography, luminal extravasation of contrast material will be present [4,23].When bleeding is massive and can not be controlled by conservative methods, then this is an emergency situation which needs to be dealt promptly with surgical resection of Meckel's diverticulum after initial resuscitation of the patient with blood transfusion. The approach can be through laparotomy, laparoscopy or laparoscopic assisted[24,25]. The aim of the surgery is to resect the Meckel's diverticulum, all ectopic gastric mucosa, and any ulcerated adjacent ileum to prevent recurrent bleeding. During surgery if we find a narrow base without any mass in the lumen, then a wedge resection of the diverticulum with transverse closure of the ileum is the ideal method. We can use linear stapler in this situation to close the ileum. But when the base is wide or mass of ectopic tissue is palpable or when there is inflammation, it is preferable to resect the involved bowel followed by end-to-end ileoileostomy [4]. The rationale of this procedure is derived from the observation that in short Meckel's diverticulum, ectopic mucosa can be seen even in the proximal portion, as compared to long diverticulum where the mucosa has been found mostly in the apical region [26].Intestinal obstructionIntestinal obstruction due to Meckel's diverticulum is the most common presentation in adult and is the second most common in children[11,10]. There are various mechanism by which it can cause intestinal obstruction like (a) Volvulus of small intestine around a fibrous band extending from Meckel's diverticulum to umbilicus. (b) Intussusception – in which Meckel diverticulum sags into the bowel lumen and then serves as a lead point to allow telescoping of the small intestine into first the distal ileum and then in to the large intestine causing ileo-ileal and ileocolic type of intussusception. (c) Littre's hernia – Incarceration of the diverticulum in hernia, (inguinal and femoral) causing intestinal obstruction. (d) Entrapment of small bowel beneath the blood supply of the diverticulum, also known as a mesodiverticular band (e) Stricture secondary to chronic diverticulitis [4] (f) Meckel's diverticulum lithiasis – The formation of stone in Meckel's diverticulum can cause small bowel obstruction by two mechanisms; firstly, it can cause impaction in the terminal ileum after its extrusion from the diverticulum and secondly by promoting local inflammation of the diverticulum and intussusception [27,28] (g) Band extending between the diverticulum and the base of the mesentery, forming a loop in which a part of ileum may get stuck causing obstruction [29]. In our experiences we encountered one such case in which this band forming mechanism was associated with another rare complication of gangrenous change[30]. (h) Other mechanisms involve rare causes of obstruction like tumors (Lipomas, Carcinoid tumors and others), impacted meconium in neonates causing inflammatory adhesions of Meckel's diverticulum to surrounding structures leading to volvulus [31], cecal volvulus around the band extending from Meckel's diverticulum to umbilicus [32], gall stone ileus [33], obstruction secondary to phytobezoar formation in Meckel's diverticulum [22].Whatever be the cause of obstruction, the presentation is strikingly similar. The patient typically presents with the features of small bowel obstruction like absolute constipation, spasmodic abdominal pain, vomiting which may be bilious and abdominal distention. In case of intussusception we may get the features of acute obstruction associated with an urge to defecate, early vomiting and occasionally, the passage of the classical currant jelly stool[2]. Plain x-ray abdomen may reveal dilated bowel loops and multiple air fluid levels. If this condition is left untreated, it leads to strangulation and ischemic necrosis of the wall of the bowel loop. Gas under diaphragm on plain erect x-ray may be found in that situation. Hence intestinal obstruction should be treated as an emergency situation warranting immediate exploratory laparotomy after initial resuscitation. During exploration if we get volvulus around a fibrous band, untwisting of bowel along with division of band should be done. In case of intussusception, attempts to reduce such mass may be difficult, warranting resection of intussuscepted mass followed by primary anastomosis. However in Litter's hernia, Meckel's diverticulum should be resected after reducing it followed by hernial repair[34]. For a mesodiverticular band, the small bowel is reduced, and the diverticulum along with its blood supply should be resected. Enterolith in Meckel diverticulum should be resected en bloc with primary anastomosis[28]. Thus in cases of intestinal obstruction, the main aim of surgery is still to remove the culprit i.e. Meckel diverticulum along with correction of associated pathology, independent of the chosen surgical approach being either open or laparoscopic.DiverticulitisDiverticulitis represents 20% of the symptomatic Meckel's diverticulum[4] and is common in adult patients [2,3,11]. Clinical manifestation mimics acute appendicitis and should be considered in the differential diagnosis of a patient with right lower quadrant pain. This feature necessitates the exploration of distal ileum when normal looking appendix is found during operation of suspected acute appendicitis. Pathophysiology is analogous to that of acute appendicitis, with inflammation being secondary to stasis and bacterial infection, which occurs due to the obstruction of the lumen by Enterolith or foreign body or by parasites (Ascaris lumbricoides or Taenia saginata) [35]. Alternatively, peptic ulceration of ileal mucosa due to ectopic gastric mucosa can cause diverticulitis. It may also result from diverticular torsion that causes secondary ischemia and inflammatory change [36]. If this condition is left untreated, it usually leads to perforation and peritonitis.This condition should be dealt with a surgical approach that can be open or laparoscopic, with resection of diverticulum at its base and closure perpendicular to the axis of intestine to minimize the risk of subsequent stenosis. And if perforation has occurred, thorough peritoneal toileting is done after resection.PerforationIt is difficult to diagnose the site of perforation prior to exploration although duodenal and ileal perforation can be distinguish to a lesser extent by observing the nature of the aspirates from abdomen i.e. whether it is bilious or feculent. Various etiologies that can lead to perforation of Meckel's diverticulum are (a) Progression of diverticulitis, (b) Ulceration of adjacent ileal mucosa secondary to acid produced by ectopic gastric mucosa, (c) Secondary to ingested foreign body like fish bone [37,38], chicken bone and bay leaf [39], (d) Traumatic [40]. (e) Perforation associated with tumors like Leiomyoma in Meckel's diverticulum has also been reported [41]. It typically presents with the features similar to that of the perforation of other hollow viscera, with features of either localized or generalized peritonitis. Perforation of Meckel's diverticulum is usually managed by initial resuscitation and antibiotics followed by diverticulectomy or segmental resection along with peritoneal irrigation.TumorTumors in Meckel's diverticulum are very rare occurrences, with incidence of only 0.5% to 1.9% [42]. These tumors can be benign or malignant. Lipoma, Neuromuscular and vascular hamartoma are among the benign group [43-45]. In the malignant group, carcinoids are the most common tumor occurring with 44% of incidence [46,42]. Others are mesenchymal tumors (including gastro intestinal stromal tumors, leiomyosarcomas and peripheral nerve sheath tumors 35%), adenocarcinomas (16%) [42] and Desmoplastic small round cell tumor [47,46,50].These tumors can have various manifestations like acute abdominal pain, perforation, bleeding, intussusception and intestinal obstruction making it an emergency situation [45,18]. Lipoma can be dealt with simple diverticulectomy. Since Carcinoid is associated with metastasis early in course (in 25% of cases), solitary, localized, asymptomatic nodules less than 1 cm are generally managed with diverticulectomy or segmental resection. Larger or multiple lesions require wide excision of bowel and mesentery, and hepatic resection may be required for metastatic disease [51,21].Method of performing open diverticulectomy or segmental resection.As stated above the treatment of symptomatic Meckel's diverticulum should be prompt surgical resection of the diverticulum or resection of segment of adjacent ileum bearing the diverticulum. Segmental ileal resection is required for the treatment of patients with bleeding because bleeding site is usually in the adjacent ileum.After opening the abdomen either through midline or right lower incision, cecum and terminal ileum is identified. Ileum is followed proximally where we find Meckel's diverticulum approximately 2 feet from the ileocecal valve. After delivering the diverticulum with ileum into the wound, if mesodiverticulum found, it should be divided and ligated between the clamps. The lumen of diverticulum is emptied of its contents and the base is clamped with two noncrushing clamps and then excised between the clamps. Then the inner layer is stitched in full thickness with 000 vicryl in continuous manner. When this layer is complete the clamp is removed and second seromuscular layer is stitched with inverting interrupted Lembert sutures of 000 mersilk. However, this is an old procedure and trend has changed towards single extra mucosal seromuscular closure. This can be further simplified if stapling device is available [1]. With the advent of endostapling, these procedures are now readily done using the laparoscope.If the indication of diverticulectomy is bleeding then segmental ileal resection should be done. It is also indicated if tumor is detected or if the base is inflamed or perforated. The diverticulum is excised along with 2 to 3 cm of adjacent ileum. Then single layer end- to- end anastomosis is performed using 000 mersilk and lumen is tested for its patency between thumb and index finger.Role of laparoscopic surgery in the management of complicated Meckel's diverticulum.Laparoscopy as a minimally invasive approach has emerged as both diagnostic as well as therapeutic means to deal with various surgical conditions including Meckel's diverticulum. Its ability to visualize whole of the abdomen makes it a diagnostic choice for various undiagnosed intraabdominal pathologies. There are several studies stating the safe and effective use of laparoscopy in case of complicated Meckel's diverticulum [52,25,55]. It can be used in undiagnosed acute abdominal pain, in obstruction [56-58] and perforation [53,58]. But its role in removing bleeding Meckel's diverticulum has been argued by some due to the fact that, the base of the diverticulum and the ileum cannot be palpated, hence ectopic mucosa could be left behind thus leaving the risk of recurrence, but others have successfully dealt with this situation [53,59,60]. The criteria of external appearance to deal with such condition has been studied, with conclusion that long diverticula can be removed by simple transverse resection with a stapling device because long diverticula usually have ectopic tissue at its distal end, but in short diverticula it can occur in almost any area so ileal resection with end-to-end anastomosis or wedge resection after exteriorization is recommended in short type of diverticula [26]. Another study presented the external appearances in terms of height to diameter ratio (HDR) of 2 as a cut off value to decide whether to do simple transverse resection or ileal resection with end-to-end anastomosis [61]. The development of endostapling devices has made the resection safer, faster and more efficient. The most important advantage is its simultaneous cutting and closing properties making the chance of contamination very less [62].Generally the laparoscopic approach is same as for the appendicectomy. It involves the creation of pneumoperitoneum under direct vision followed by placement of the 10 mm umbilical trocar, 5 mm suprapubic trocar and 5 mm trocar in right lower quadrant of the abdomen based on the principle of \"triangulation\". Suprapubic port is used for the 5 mm (30°) laparoscope, umbilical port for the right hand instrument and right lower quadrant port for the left hand instrument. If the decision of resection is made then, the 10 mm umbilical port can be enlarged to 12 mm port for the use of endostapler, which is fired at the base of the diverticulum perpendicular to the long axis of the ileum[62]. Alternatively it can also be done by bringing the small bowel out through the enlarged umbilical port site for the palpation and resection.Some complications of Meckel's diverticulum often need additional laparoscopic interventions. For example, the management of volvulus and intussusception involve techniques like laparoscopic derevolving and desussception respectively. [63,64,42]. Similarly, laparoscopic approaches have greatly simplified the management of Littre's hernia. Both excision of the Meckel's diverticulum using an endoscopic stapling device and repair of this hernia with Permacol, or other meshes have been done, which has been recorded as case reports[34,65,17].Thus in the present scenario, as compared to the conventional laparotomy, the laparoscopic management of the complicated Meckel's diverticulum has been claimed to be safe, cost effective and efficient, with added advantages of precise operative diagnosis, fewer complications and shorter recovery period [53,58].Limitation of laparoscopic surgeryAlthough it has been substantially proved that laparoscopic surgery is safe and efficient, having all other advantages of minimally invasive surgery, still it has some limitations. The most important one is its unavailability, this being specially true in developing countries where it also gives burden of high cost.Another technical limitation during surgery is confrontation with either too short or very broad-based Meckel's diverticulum. If its base is too short there is danger of including too much of the ileum during stapling or leaving behind its part when it is of very broad base [62,55]. Such situations can be dealt with resection of Meckel's diverticulum using a Harmonic scalpel and thereafter closing the enterotomy with intracorporeal vicryl sutures. Another safe method to accomplish excision is to exteriorize the diverticulum via a minilaparotomy incision, resection and closing the enterotomy with sutures.Laparoscopic assisted surgeryWith the advent of laparoscope, both extracorporeal and intracorporeal resection of Meckel's diverticulum may be performed [52]. The indications are almost similar to that of laparoscopic surgery but it scores over it in terms of some additional benefits. In laparoscopic-assisted transumbilical Meckel's diverticulectomy (LATUM), a transumbilical 10 mm trocar is inserted in an open fashion, then using 10 mm operative laparoscope, the terminal ileum is exteriorized through umbilicus with an atraumatic instrument and then diverticulectomy or segmental resection can be performed [60]. Thus this technique also allow palpation of Meckel's diverticulum which aids in ruling out any mass or thickening of base thus providing the more complete assessment for presence of any ectopic gastric mucosa [66]. This technique also prevents the use of costly staplers [25,59] making it more cost effective.Complication of surgeryComplications are generally the same as that of other operations like bleeding, infection, intra abdominal abscess formation, wound dehiscence, incisional hernia and post operative adhesive intestinal obstruction. Surprisingly, morbidity (20%) and mortality (3%) of diverticulectomy in the asymptomatic group by any of these procedures have been found to be higher than morbidity (13%) and mortality (0%) in the symptomatic group. This highlights the earlier impression that these complications are the sequels of open abdominal surgery rather than of diverticulectomy [67,12]. These complications have to be dealt in the similar ways as for other surgeries. But special emphasis should be given for the recurrence of bleeding which occurs when some of ectopic gastric mucosa are left behind. To prevent this, ileal resection with end-to-end anastomosis should be done to exclude all of the ectopic gastric mucosa.ConclusionMeckel's diverticulum is the most common congenital anomaly of the gastrointestinal tract but the life time risk of developing complications in this vestigial organ is 4 – 6%. Complications which require emergency treatment includes bleeding, obstruction, diverticulitis and perforation and the appropriate knowledge of various pathophysiologies by which a Meckel's diverticulum can cause complication should be kept in mind for the better management and to prevent recurrences. The diagnosis of symptomatic Meckel's diverticulum needs a high degree of suspicion as the preoperative clinical and investigational diagnosis is difficult to be made with accuracy. Open surgery has long been used as an effective method to deal with complicated Meckel's diverticulum. However, in this age of minimally invasive surgery, laparoscopic management of complicated Meckel's diverticulum is safe, cost effective and efficient as proved by various reports and studies. Although both the methods have their own limitations, still the choice of management depends on patients condition, surgeon's experiences, and availability of the laparoscopic instruments.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsRKS conceptualized the review, drafted the review, analyzed the literature where ever was needed. VKJ conducted extensive literature review, manipulated the draft, contributed to the analysis. All Authors' read and approved the final manuscript.Supplementary MaterialAdditional File 1Statement of consentClick here for file\n\nREFERENCES:\nNo References"
+}

batch_9/PMC2533310.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2533310",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533310\nAUTHORS: Wei Xiong, Nathaniel Hupert, Eric B Hollingsworth, Megan E O'Brien, Jessica Fast, William R Rodriguez\n\nABSTRACT:\nBackgroundMathematical modeling has been applied to a range of policy-level decisions on resource allocation for HIV care and treatment. We describe the application of classic operations research (OR) techniques to address logistical and resource management challenges in HIV treatment scale-up activities in resource-limited countries.MethodsWe review and categorize several of the major logistical and operational problems encountered over the last decade in the global scale-up of HIV care and antiretroviral treatment for people with AIDS. While there are unique features of HIV care and treatment that pose significant challenges to effective modeling and service improvement, we identify several analogous OR-based solutions that have been developed in the service, industrial, and health sectors.ResultsHIV treatment scale-up includes many processes that are amenable to mathematical and simulation modeling, including forecasting future demand for services; locating and sizing facilities for maximal efficiency; and determining optimal staffing levels at clinical centers. Optimization of clinical and logistical processes through modeling may improve outcomes, but successful OR-based interventions will require contextualization of response strategies, including appreciation of both existing health care systems and limitations in local health workforces.ConclusionThe modeling techniques developed in the engineering field of operations research have wide potential application to the variety of logistical problems encountered in HIV treatment scale-up in resource-limited settings. Increasing the number of cross-disciplinary collaborations between engineering and public health will help speed the appropriate development and application of these tools.\n\nBODY:\n1. BackgroundThree decades after its start, the global HIV epidemic is now the target of perhaps the most ambitious global health program ever conceived, dwarfing in its complexity and requirement for sustainability the campaign to eradicate smallpox, which is widely considered one of the crowning public health achievements of the late 20th century. In the last ten years, the campaign to scale up HIV care and antiretroviral therapy (ART) for millions of people with AIDS living in resource-limited countries has gained substantial commitments in financing [e.g., the Global Fund to Fight AIDS, Tuberculosis and Malaria (GFATM), the World Bank's Multi-Country AIDS Program (MAP), the United States' President's Emergency Plan for AIDS Relief (PEPFAR) program, private donors such as the Bill & Melinda Gates Foundation], operational support [e.g., Joint United Nations Programme on HIV (UNAIDS), World Health Organization (WHO)], and procurement support [e.g., the Clinton Foundation HIV/AIDS Initiative (CHAI)].While over 30 million people are living with HIV and over 2 million new infections are estimated to occur each year, HIV care and treatment has expanded significantly in this decade: it is estimated that by December 2006 more than 2 million people, or 28% of people living with HIV in low- and middle-income countries, had access to antiretroviral treatment, a substantial improvement from 2% coverage just three years earlier [1-3]. These figures confirm the success of early efforts to scale up global HIV treatment, but serious obstacles to continued expansion remain to be addressed. For example, approximately 95% of HIV-infected people live in developing countries [4], which typically face challenges of underfunding, limited materiel resources, and severe human resource shortages in the health sector [5]. Increasing ART coverage in these settings will require sophisticated logistics and managerial skill at every point of health service delivery from the local to the transnational levels. The operational challenges include developing adequate and efficient physical infrastructures, providing long-term financial sustainability, and eliminating constraints to treatment capacity, most notably in human resources and pharmaceuticals and diagnostics supply chains.These challenges were noted in a recent review of the PEPFAR program: \"The continuing challenge for the U.S. Global AIDS Initiative is to simultaneously maintain the urgency and intensity that have allowed it to support a substantial expansion of HIV/AIDS services in a relatively short time while also placing greater emphasis on long-term strategic planning and increasing the attention and resources directed to capacity building for sustainability [5].\" Below we describe one scientific field that holds promise to help maintain the acceleration of HIV treatment scale-up: the discipline of Operations Research (OR), the applied science of maximizing the effective use of limited resources [6].OR offers a plethora of modeling techniques that may be used to assess, operationalize, and evaluate HIV treatment scale-up activities with the goal of transforming existing health systems in target countries to achieve efficiency and sustainability in the provision of life-long, effective treatment for people with HIV. While OR techniques and models have been successfully utilized in many areas to maximize the effective use of scarce resources, the application of mathematical modeling to HIV scale-up to date typically has focused on policy-level issues (e.g., program costs) as opposed to operational-level issues [7-10]. We believe that application of these tools to practical decisions about resource allocation and health care organization may increase the success of existing programs and help to expand access to HIV care and treatment in resource-limited countries. This paper reviews current challenges to expanded HIV care and treatment that may be amenable to OR-based interventions; discusses potential benefits and pitfalls of the use of applied models in longitudinal HIV care and treatment; and concludes with a summary to guide the future application of OR to scale-up activities.2. The practical challenges of HIV treatment scale-upGlobal scale-up of HIV care and treatment involves complex issues in logistics, the science that deals with the procurement, distribution, maintenance, and replacement of materiel and personnel [11]. During the WHO's \"3 by 5\" campaign, many stakeholders provided logistical support, including procurement and distribution of antiretroviral drugs and other supplies, creation of infrastructure, and the training and recruitment of the healthcare work force. Not surprisingly, significant logistical challenges remain. Here we focus on three categories of logistical challenges that are key barriers to achieving universal access to HIV prevention and treatment: pharmaceutical supply chain management, laboratory service infrastructure and planning, and healthcare workforce development.2.1 Pharmaceutical supply chain managementThe increasing demand of global AIDS treatment poses unprecedented challenges for supply chain management, since each point of dispensing antiretroviral (ARV) drugs (i.e., clinic, hospital, community outreach worker) must have access to a customized and predictable supply of ARVs and other drugs at all times. The robustness of this supply chain is essential to the success of any AIDS treatment program, and it must take into account a number of unique features and constraints of antiretroviral treatment.At the clinical level, HIV treatment scale-up in most low- and middle-income countries embraces the WHO's \"public health approach\" to ART use, where treatment regimens are standardized according to widely accepted consensus guidelines [12]. Unavoidably, customization of care takes place at multiple points, from country-level protocols that adapted WHO guideline to regional needs, to patient-level adjustments in treatment due to co-morbidities, such as tuberculosis, or adverse events, such as stavudine-related neuropathy. The degree of customization varies considerably from program to program; in Brazil, for example, clinicians have access to most antiretroviral medications, while in Malawi individual health facilities receive shipments of a prescribed, narrow formulary [13,14]. In all settings, key patient-level variables (e.g., the presence of anemia or neuropathy) should trigger a range of possible drug substitutions that will lead to measurable differences in demand at an individual clinic or regional level of care. The ideal pharmaceutical supply chain that is responsive to these needs will have, at a minimum, the following features [15]:(1) an inventory control system(2) a logistics management information system(3) a storage system, and(4) a distribution systemMajor efforts are underway to assist countries and care providers with all four of these aspects of supply chain development and management. To be successful, these efforts need to overcome a number of constraints, some of which are unique to ART and some of which apply generally to medical material management. ARVs are notable for their short shelf-lives and, for some – particularly the non-heat stable form of protease inhibitors – the need for a \"cold chain\" to provide refrigeration. Standard ART formularies for children and adults require multi-drug regimens that often involve products manufactured by more than one manufacturer, potentially complicating efforts to insure not only continuity but parity of supplies. For many other medical conditions, including many infectious diseases, drug substitution due to stockouts (e.g., prescribing one antibiotic for another that is not available) is common and has little clinical impact. Stockouts of ARVs, in contrast, can have important health consequences because of the limited availability and medical compatibility of substitute drugs, and the serious risks of intermittent adherence in terms of viral resistance. Finally, although the cost of first-line ARVs has declined dramatically in recent years, ART is one of the most expensive global public health interventions ever undertaken, amplifying the impact of supply chain inefficiencies [16].The ideal supply chain will synchronize aggregate patient-level drug demand with the flow of pharmaceuticals from suppliers, reducing inventory investment along the chain, and improving patient care by minimizing supply shortages. Sustainable and flexible supply chains with capacity for forecasting, procurement, distribution, and information sharing can improve drug utilization and decrease waste, yet even the best-designed supply chains may experience interruptions caused by shortage, damage, drug expiration, and miscommunication. Optimizing supply chain management (SCM) has been a central focus of the OR community since the concept of SCM first appeared in the engineering literature in the mid-1980's, bridging previously independent lines of research into inventory control, product management, and industrial processes control [17,18].2.2 Laboratory service infrastructure and planningLaboratory-based diagnostic services are necessary for the proper identification of HIV disease and are used for longitudinal monitoring of all HIV-infected patients. Laboratory data – notably the CD4 cell count – can guide the timing of therapeutic interventions and ensure the maximum level of safety and monitoring for treatment failure when antiretroviral drugs are being delivered [1]. However, access to hospital-based laboratory facilities for ART monitoring is a challenge in most resource-limited countries; to compound matters, outpatient laboratory services especially are scarce in rural areas [19]. Despite the importance of laboratory capacity for the effective delivery of ART, there have been only a handful of systematic approaches developed and implemented in resource-limited countries to ensure sustainable, practical laboratory service for HIV treatment scale-up [20].As with the creation of effective ARV supply chains, the development and support of clinical laboratory infrastructure to support ART scale-up in resource-limited countries will need to address a number of logistical constraints. Foremost among these is the expense of currently available HIV laboratory testing equipment and reagents, their need for regular maintenance, and the skilled labor force required both to operate and maintain them. Currently, many countries operate a decentralized model of laboratory capacity with little interaction between laboratories at national, regional, and district levels [20]. In some of these countries, many facilities dispensing ART already experience shortages of functioning equipment, reagents, and technicians to conduct regular maintenance and technical support. When faced with insufficient laboratory capacity, health planners have two options: add additional laboratory capacity by purchasing more equipment, or increase the utilization of equipments that have already been purchased and are either idle or underutilized, often through the implementation of a sample transport network. Ancillary costs, such as those of reagents, typically are tied in complex ways to machine utilization, so the cost of an idle machine may be more than simply its inconvenience or the downstream costs of lost clinical services.After ARV costs, building laboratory capacity is the most capital-intensive component of global ART scale-up. In addition to technical training and patient education, it involves equipment selection, human resource training for operations and maintenance, reagent selection and purchase, and transportation infrastructure for moving samples and results between points of care and laboratory machines [21]. Less well described is the need for laboratory information technology infrastructure, to link samples and results to patients, and return the laboratory data from the testing facilities to the clinicians at the sites of care. It is possible that, with proper development of infrastructure for training, preventive maintenance, and repairs, many countries currently experiencing laboratory service interruptions have sufficient laboratory capacity for ART scale-up. If this is the case, in the short term, rather than adding more machines, Ministries of Health may be better served by development of a flexible and efficient sample referral network to bring samples to the instruments which are currently under-utilized, thereby ensuring the direct and indirect benefits from using that laboratory capacity at its maximum potential. In the long term, investments in laboratory infrastructure and the development of more appropriate, point-of-care devices will be needed to replace potentially unreliable specimen transport networks and shorten the cycle time of laboratory-based information back to the clinical encounter, which can be as long as several weeks in many settings.2.3 Healthcare workforce developmentIn addition to supply chain management and laboratory capacity, the challenge of training and retention of healthcare workers is a third critical logistical aspect of HIV service delivery that has been widely identified as perhaps the single largest constraint on global treatment scale-up. This challenge is greatest in regions worst-hit by the disease due to multiple factors including direct workforce effects of the pandemic (i.e., death and disability), international financing policies restricting investment in the health sector, and the \"brain drain\" – outmigration of qualified practitioners toward urban areas and higher wage countries in Europe and North America [22]. Human resources affect both the scope and speed of treatment scale-up in most resource-limited countries, yet few countries have a \"comprehensive training plan, a clear assessment of ongoing training needs, a plan to operationalize training on a large scale, or adequate funds budgeted for training [23].\"The spectrum of healthcare workers encompasses doctors, nurses, pharmacists, lab technicians, phlebotomists, counselors, program managers, and community health workers, as well as other ancillary staff. Successful scale-up involves relatively complex planning for recruiting and training a mix of healthcare workers providing a wide range of services, which are influenced significantly by the HIV care delivery model chosen by a country or region. In a setting where the demand for healthcare workers enormously exceeds the available resources, OR approaches may be able to provide technical support to program mangers to evaluate the capacity of existing systems and estimate the minimal required amount of healthcare workers for effective scale-up. Furthermore, OR models can be used to evaluate different models of care and to provide insight into the impact on quality and capacity of HIV clinics adopting different HIV care delivery models.3. Applying Operations Research to Scale-upMillions of people living in resource-limited countries need life-long medical care, now including ART, posing a fundamental logistical challenge for effective HIV care and treatment and raising challenges to the technical feasibility of the goal of universal access. Increased funding alone, though necessary, will not solve these issues, since it is not only the procurement of resources but the management of those resources at the point of need that will determine the success or failure of local HIV interventions. This means there is an urgent need for strategies to build and maintain efficient systems of care and pharmaceutical delivery, laboratory capacity, and healthcare worker recruitment and training. In the remainder of this paper, we show how these areas may benefit from the application of operations research methods, as well as anticipated barriers to this application.3.1 What is Operations Research?In the world of HIV treatment and prevention, the term \"operations research\" or, more commonly, \"operational research\" has been applied to a field of study that is somewhat different from what engineers and management scientists mean by OR. To date, most HIV-related operations research studies have focused on the description, analysis, and improvement of day-to-day activities or \"operations\" of HIV program [24]. These operations include training, education, counseling and testing, commodity logistics, hospital and clinic activities, and community- and home-based care, among others. HIV-focused operations research therefore has been used to describe most studies that quantify some aspect of HIV clinic operations and their associated impact on patient outcomes. Because of this inclusive meaning, most observational field studies of ART clinic operations, for example, would be considered HIV-related operations research.By contrast, the engineering/management science discipline of OR refers to the application of a collection of mathematical techniques used to model real-world systems and gain insight into their operations. The Institute for Operations Research and the Management Sciences (INFORMS) – the largest professional society for specialists in the field of operations research – defines operations research as \"the discipline of applying advanced analytical methods to help make better decisions.\" This umbrella concept of operational research covers many analytic approaches and methods, such as mathematical programming, queueing theory, simulation modeling, decision analysis, and forecasting methods. Techniques from other fields such as statistics and computer science are also employed by operations researchers to assist with decision making.OR approaches have been widely implemented for analyzing problems in complex real-life systems. These methods have provided informative insights and yielded substantial economical benefit [25], suggesting that OR models can be used in the following (sometimes overlapping) areas:• The optimal allocation of scarce resources subject to a large number of constraints (e.g., HIV funding allocation to treatment and prevention programs)• The search for efficient solutions among a vast multitude of alternative choices (e.g., clinical and laboratory infrastructure and capacity building)• The analysis of dynamic systems characterized by fluctuating inputs and out puts (e.g., care delivery model evaluation and clinical activities analysis)• The use of inferential processes to derive insights from multivariate statistical analyses (e.g., quality assurance across multiple care delivery and supply chain systems)• Computer simulation of intricate economic and physical systemsThe logistical problems arising in HIV treatment scale-up are precisely the types of problems that OR methodologies were designed to address. The challenge of scaling up HIV treatment in the developing world is unprecedented, and as such will certainly require novel solutions and new ways of thinking. However, certain core aspects of logistical problems in scale-up have been addressed successfully in other industries and settings through the use of OR methodologies.3.2 How can operations research improve global HIV outcomes?Decision makers may employ OR models to address the range of uncertainties in HIV interventions, from assessing treatment capacity requirements and the uncertainty in demand for ARVs, to the optimal staffing of treatment clinics. We categorize these logistics-related uses of OR into two types according to the scope and planning horizon considered: policy-level uses and operational-level uses.3.2.1 Policy level decision supportPolicy level decisions often involve the acquisition or allocation of durable resources intended to be utilized over a long time horizon. For example, decisions related to program funding or resource allocation, healthcare workforce planning, prevention strategy or delivery model selection, typically comprise strategic-level policy decisions, and these present a clear role for the application of operations research methodologies.• Resource AllocationMathematical programming models have been developed to address efficiency and equity in the HIV funding allocation process on the national, state, or municipal levels in the United States [8,10,26,27]. These models seek to determine the best allocation that minimizes the number of potential infections for a given period of time, given an available budget and equity considerations. Recent assessments of ART in developing countries have focused on modeling macro-level distribution of resources for health care delivery. For example, one study examined the impact of healthcare facilities location, catchment area size, and other demographic parameters on an optimally equitable allocation [27]. However, real-world HIV resource allocation typically is driven by a number of criteria aside from cost-effectiveness and equity considerations. OR-based analysis and modeling represent an evaluation methodology with which multiple competing alternatives can be weighed from multiple perspectives, including those of equity or efficiency.• Healthcare Workforce PlanningPerhaps the most glaring deficiency in resource-limited settings is the lack of healthcare workers for the 30+ million people living with HIV. A variety of OR techniques are available to address problems in human resource planning, hiring, and training in knowledge-intensive operations under uncertainty [28-30]. Based on data collected on specific tasks in the delivery of care through process analysis and time-motion studies, OR models can be used to identify the optimal mix of different cadres of healthcare workers for efficient scale-up. Once targets are set for scale-up, models can also be used to estimate the demand for human resources linked to local care delivery models. More important, these estimates will contribute to understanding the capability of existing systems and identify the ideal combination of training methodologies to prepare for scale-up [23].• Laboratory Infrastructure PlanningThe healthcare system infrastructure in most of sub-Saharan Africa has been suffering from decades of underfunding and declining capacity [31]. Adequate financing is a fundamental requirement for building laboratory infrastructure. Yet the development of a national laboratory network into one that improves the utilization of existing laboratory capacity may result in substantial financial savings. This type of problem can be formulated as the traditional capacitated facility location problem (CFLP), which has been studied extensively in the OR literature [25]. CFLP has numerous applications in warehouse location and distribution planning, telecommunication network design, and manufacturing production planning [32]. Given accessible laboratory facilities and their processing capacity, ministries of health can use models to identify the most cost-effective way to re-distribute test demand among laboratory facilities and decide where to locate new laboratory equipment for future scale-up.3.2.2 Operational level decision supportOperational level decisions are local decisions related to situations where operations must be undertaken or executed in a short time frame in response to system status or changes (e.g. order quantity, equipment breakdown, weather or road changes). For instance, material managers have a difficult time determining how much safety stock to hold and when to initiate orders for material from upstream sites, given the various degrees of uncertainty in supply and demand that impact their operations. Model-based support systems can help to anticipate variability and optimize purchasing.• Demand ForecastingForecasting is the process of estimating how much of a selected product will be needed in a given period of time. A number of tools have been developed to assist ministries of health and transnational groups in forecasting resource demand (e.g., Bertozzi, et al. [6]). However, forecasts are generally made using historical data; available data are limited since most countries started to implement their standard guidelines and protocols only in the last several years. For example, simple linear regression models have been used for global ARV demand forecasts, though they have been based on very limited observations [33]. Furthermore, commodity demands for HIV treatment programs can be highly unpredictable, because of the dynamic nature of the problem: evolving treatment protocols and patient populations requiring longitudinal follow-up with changing drug demand patterns. To improve the forecasts, attempts have been made to use a simulation approach to obtain insight into the dynamics of disease progression among existing and expected patient population, and thereby identify trends of resource usage, provide forecasts of drug demands, or even estimate the uncertainty in a forecast [34].• Supply Chain DesignA supply chain is a network of facilities that performs the functions of procurement of material, transformation of material to intermediate and finished products, and distribution of finished products to customers [35]. A major challenge in managing an efficient supply chain is to minimize, to an appropriate extent, inventories and costs along the chain while maximizing customer service performance. OR has a long history of providing supply chain managers the decision support they need to design and improve a supply chain. Many industries have gained tremendous benefit from OR efforts to improve supply chain efficiency. For example, the potential savings from reengineering supply chain design ranges from $14 billion for the food service industry to $30 billion for the grocery industry [36]. However, the design and implementation of an effective HIV drug supply chain poses further challenges for both researchers and practitioners.The strong emphasis of recent treatment scale-up efforts is to place management of pharmaceutical procurement and distribution within the primary healthcare system [5]. Treatment of HIV alone usually requires the simultaneous high-level availability (95%) of at least three drugs, each with relatively short shelf-life. In addition, side-effects of ARVs, especially in the early phase of treatment, occur with calculable but varying frequency, leading to shifting requirements for alternative first-line medications throughout the treatment coverage area. Importantly, HIV is a chronic illness associated with specific conditions that also require treatment, such as tuberculosis or cervical cancer, as well as with routine longitudinal care in a primary health setting. A recent report describes the adaptation of tuberculosis medication procurement systems with large (e.g., three-month) 'buffer stocks\" in order to insure consistent availability of HIV medications, but such systems may not remain financially or logistically viable as treatment volumes increase. The unique characteristics of HIV treatment – requiring an increasing and uninterrupted supply of multiple medications in changing drug combinations – are amenable to models developed specifically for the integration of local, regional, and country-level supply chain management.• Service BenchmarkingWhen applying the same pubic health strategies for HIV treatment programs, outcomes in terms of logistic issues will not necessarily be the same because of diversified settings across the globe. For example, the amount of inventory kept at various locations of the supply chain is an important measure of performance. However, inventories at various points may have different cost structures, varying as a result of service level requirements and supply responsiveness. It is often inappropriate to use the amounts of inventories as a direct performance metric for different supply chains and different points in a supply chain. Furthermore, the uncertainty of supply chains in different countries and regions may differ dramatically in terms of their structures, drug supplies, security, transportation and other infrastructures, etc. Therefore, in the process of scale-up, it is inappropriate to directly adopt successful inventory control rules from other programs. In order to have a better understanding and assessment of existing logistic activities, it is desirable to use OR models to determine the optimal performance needed to support a service target at various locations given the nature of the supply chain.• Service IntegrationThe most cogent critique of many global health programs has been that their implementation involves the delivery of services in vertical silos that by themselves may improve narrowly defined outcomes, but that as a whole enable a fractured system of health care, and perpetuate both inefficiencies and inequities in health care delivery. One oft recognized aspect of the global effort to deliver HIV services is that, because HIV is inherently a longitudinal health condition best treated at a community primary care level, the effective scale-up of HIV care will necessarily target not only the disease but also prevailing vertical systems of care, addressing their inefficiencies and inequities. OR has been used in other industries to recognize the misallocation of resources that results from taking a local perspective to a larger problem, and to redirect resources into an integrated model of service delivery. OR thus provides another tool for policy makers to analyze the benefits of integrating services at a primary care level.3.3 Example: A simulation model for capacity planning at HIV clinicsIn 2005–6, we created a stochastic simulation model to assist capacity planning for HIV treatment clinics, diagramed in Figure 1[37,38]. This simulation model was designed to capture patient characteristics (WHO stage, CD4 count distribution, attrition), disease progression (CD4 decline), treatment protocols, human resource utilization, and the competition for limited resources in a single clinic. We modeled the clinic as a set of interconnected work stations representing different working cadres (e.g., clerk, nurse, doctor). An enrollment plan of the clinic is used as one input to the simulation. Patients scheduled by the enrollment plan, as well as random arrivals, are created by the simulation program and are routed to the clinic, visiting different stations in based on health status (which the model assigns to each patient based on inputted values and evidence-based assumptions) and country-specific treatment protocols (based on health ministry publications). Clinical details ranging from the treatment process, logistics, staffing, to the demand for drugs, are considered in the clinic visit. After each visit, patients may come back or be lost to follow-up visits. During this time, each individual patients disease progression or biological reactions to treatment are modeled. In this way, the simulation program can be used to represent aggregate patient visit dynamics to determine maximal enrollment and visit capacity under steady-state clinic operations in the setting of user-manipulated resource constraints.Figure 1Schematic diagram of simulation model of HIV clinic operations.In early 2008, we created a simulation model to provide a service benchmarking framework, quantifying the physician time saved in a task-shifting program in Rwanda that gave primary HIV treatment responsibility to nurses working under physician supervision [39]. Using data from this pilot program in three health centers, we were able to estimate the person-hour savings of a critical resource, in this case physicians, by modeling the impact of varying provider assignment, the number and duration of visits, and physician time spent supporting nursing staff. We estimated that if task-shifting were scaled up to the national level it would reduce the demand on public-sector physicians for HIV services by 78%. The analysis suggests that implementation of such a program could allow the government of Rwanda to scale-up HIV treatment without overburdening this component of its existing healthcare system.By quantifying the outcomes of task-shifting and other programmatic changes in HIV care and treatment, models like these can assist planners in analyzing and optimizing future medical resource allocation. Similar tools can also provide forecasts for key resources, including staffing, space, drug volumes, laboratory kits and equipment to support operational decisions such as the choice of treatment protocols.3.4 Barriers to Adoption of OR MethodologiesThe relative absence of OR methodologies from global HIV treatment scale-up activities, especially at the operational level, suggests that barriers exist to their implementation. These are likely to include technical complexity of some of the applications (e.g., involving extensive data analysis and computer-based modeling) and the need for outside content experts to assist local governments and their technical support agencies in the appropriate application of various methodologies to specific problems. More fundamentally, OR-based assessments require investment in the collection, storage, and accessibility of valid, current data to yield accurate and applicable conclusions. Funding of this type of operations-related data collection has not always been prioritized in the rush to scale up \"emergency\" treatment; whether this was the intention of major funding organizations (rather than simply a consequence of public health planners and practitioners overwhelmed by immediate treatment needs) is under debate [5]. Clearly, development of an OR workforce that is more cognizant of the specific needs and limitations of working in a health setting – especially in resource-limited settings – can only help this situation. Consequently, one concrete action that may speed the application of OR methodologies to HIV scale up will be \"cross-training\" of engineers in public health and of public health and medical professionals in health systems engineering, either through joint degree programs, special concentrations in existing programs, or post-graduate professional training.4. DiscussionOperations research, the applied field of engineering that is focused on the efficient use of scarce resources, holds great promise to assist in efforts to craft effective and successful scale-up of HIV care and AIDS treatment in resource-limited countries. We have highlighted several areas that parallel HIV scale-up activities and have benefited from OR analyses. However, a number of obstacles to the wide adoption of OR approaches in resource-limited settings, where there are considerable differences in terms of decision making processes when compared to developed countries, remain. A direct transposition of OR techniques and approaches from this setting to another will no doubt encounter many difficulties and may produce misleading results. It is also necessary to take into account the environments in which OR projects are to be carried out. Ethical, cultural, and political considerations, which go beyond maximizing the cost-effectiveness objectives, need to be considered carefully. Furthermore, as noted in a recent review of the PEPFAR program, the general lack of outcomes and operational data – which is being remedied in many programs – is a serious hindrance to carrying out certain OR projects, such as forecasting [5].In addition to the potential impediments in the field, to date only limited funding has been available for operations-related work in scale-up, and what monies are available for this line of investigation and intervention typically have been underutilized. GFATM, for example, allows up to 10% of each grant to be allocated for operations research, but this provision is rarely used by countries and the research community is rarely represented on Country Coordinating Mechanisms (CCMs) [40]. The recent Sydney Declaration, supporting the allocation of 10% of HIV global programming funding on research related to operations, has highlighted the need for and benefits of OR and related study, echoing the results of the evaluation carried out on the first years of the PEPFAR program [5].5. ConclusionWe stand at a critical moment in the global campaign to scale up treatment for HIV-infected people, one in which the magnitude of the remaining task threatens to overwhelm the hard-fought gains that have brought treatments to millions who recently had none. This paper highlights the potential application of classic operations research approaches to a variety of logistical issues that lie at the heart of the scale-up process. HIV-related operational research has provided ministries of health, non-governmental organizations, public health professionals, and clinic managers with a better understanding of impact and cost-effectiveness of various intervention and treatment programs, as well as quantitative data about clinic capacity, performance and other types of outcomes. The engineering field of OR, with its toolbox of systematic quantitative approaches, can provide additional techniques to understand the data unearthed by engaging in and reporting from clinical operations. We believe that using these analytical techniques will allow clinic managers to get even more utility out of their operational research, and will provide insight into management strategies that can minimize the cost of operations while maximizing clinicians' ability to provide high-quality medical care.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsWX, NH, MEO, and WRR conceived of the concept of the paper. WX and NH wrote the first draft and all authors contributed to that and to subsequent drafts of the paper.Pre-publication historyThe pre-publication history for this paper can be accessed here:\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2533329",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533329\nAUTHORS: Marco Sardiello, Stefano Cairo, Bianca Fontanella, Andrea Ballabio, Germana Meroni\n\nABSTRACT:\nBackgroundThe TRIM family is composed of multi-domain proteins that display the Tripartite Motif (RING, B-box and Coiled-coil) that can be associated with a C-terminal domain. TRIM genes are involved in ubiquitylation and are implicated in a variety of human pathologies, from Mendelian inherited disorders to cancer, and are also involved in cellular response to viral infection.ResultsHere we defined the entire human TRIM family and also identified the TRIM sets of other vertebrate (mouse, rat, dog, cow, chicken, tetraodon, and zebrafish) and invertebrate species (fruitfly, worm, and ciona). By means of comparative analyses we found that, after assembly of the tripartite motif in an early metazoan ancestor, few types of C-terminal domains have been associated with this module during evolution and that an important increase in TRIM number occurred in vertebrate species concomitantly with the addition of the SPRY domain. We showed that the human TRIM family is split into two groups that differ in domain structure, genomic organization and evolutionary properties. Group 1 members present a variety of C-terminal domains, are highly conserved among vertebrate species, and are represented in invertebrates. Conversely, group 2 is absent in invertebrates, is characterized by the presence of a C-terminal SPRY domain and presents unique sets of genes in each mammal examined. The generation of independent sets of group 2 genes is also evident in the other vertebrate species. Comparing the murine and human TRIM sets, we found that group 1 and 2 genes evolve at different speeds and are subject to different selective pressures.ConclusionWe found that the TRIM family is composed of two groups of genes with distinct evolutionary properties. Group 2 is younger, highly dynamic, and might act as a reservoir to develop novel TRIM functions. Since some group 2 genes are implicated in innate immune response, their evolutionary features may account for species-specific battles against viral infection.\n\nBODY:\nBackgroundThe TRIM gene family encodes proteins involved in a broad range of biological processes and characterized by the presence of the tripartite motif (hence the name TRIM), which consists of a RING domain, one or two B-box motifs and a Coiled-coil region (RBCC) [1,2]. The tripartite motif is always present at the N-terminus of the TRIM proteins. The order of the domains that compose the motif is also conserved: a RING finger domain precedes the B-box motif(s), and a Coiled-coil (CC) region invariably follows. Even if one of the domains is missing, the order of the remaining ones is maintained. Different C-terminal domains are associated with the tripartite motif in the TRIM family [1-4].Both RING and B-boxes are cysteine-rich zinc-binding domains. The RING finger domain is present, in combination with other domains, in hundreds of proteins and is defined by a linear series of conserved cysteine and histidine residues that represent zinc coordination sites [5]. The B-boxes are the critical determinants of the TRIM family and can be present as B-box1 and B-box2, which share a similar but distinct pattern of cysteine and histidine residues [1]. When both B-box domains are present, type 1 always precedes type 2; when only one B-box domain is present, it is always type 2 [1]. While the tripartite motif is restricted to this protein family, the C-terminal domains are also found in unrelated proteins. A limited choice of C-terminal domains is found in association with the tripartite motif and determined the recent classification of the TRIM proteins in subfamilies [4]. This conserved multi-domain structure appears to behave as an integrated module, rather than a collection of separate motifs, suggesting a possible common function [1,6].We previously classified 35 human RBCC-containing proteins as a gene-protein family and named it TRIM. We observed that these proteins have strong self-association ability, mainly mediated by their CC region, which results in the formation of large protein complexes. In most cases the TRIM proteins identify different discrete nuclear and/or cytoplasmic sub-cellular structures [1].The presence of the RING domain and recent experimental evidence indicate that these proteins can act as E3 ubiquitin ligases, the proteins responsible for mediating the transfer of the ubiquitin moiety to the specific targets [7-10]. Alteration of their activity within ubiquitylation processes might be responsible for the clinical manifestation observed in human diseases caused by mutations in TRIM genes [3,6]. PML, RFP, TIF, and EFP are implicated in tumor insurgence and progression [11-14]. Other TRIM genes are involved in Mendelian inherited disorders: MID1 and MUL are altered in two developmental genetic diseases, Opitz Syndrome and Mulibrey nanism, respectively [15,16]. TRIM32 is involved in both a form of muscular dystrophy and a form of Bardet-Biedl Syndrome (BBS11), and MURF-1 is implicated in muscular atrophy [17-19]. Ro52 is the target antigen of auto-antibodies in both Sjogren syndrome and Systemic Lupus Erythematosus [20]. Finally, TRIM5α has been identified as the major factor restricting HIV-1 during the early phase of infection in Old World monkey cells [3,21].The TRIM family represents one of the largest classes of putative single protein RING-finger E3 ubiquitin ligases, strongly suggesting that the tripartite motif was selectively maintained to carry out a specialized basic common function within the ubiquitylation process. We used a genomic approach to complete the identification of all human TRIM genes and to study their evolutionary relationships in vertebrate and invertebrate organisms. We observed a general paradigm for the evolution of this family and propose a possible relationship between the evolution of TRIM genes and that of their function.ResultsDefining the complete set of TRIM genes in humans and other mammalsTo search for all TRIM genes in humans, mouse, rat, cow, and dog, we screened their genomic sequences, using all known mammalian TRIM sequences as queries, with the BLAST and BLAT algorithms at the NCBI and UCSC genome browsers. We also performed a Pattern-Hit Initiated-Blast (PHI-Blast) search against both redundant and non-redundant databases using the sequence patterns that we previously defined for the two B-box domains as query [1]. In addition, we used representative B-box1 and B-box2 sequences to perform TBLASTN genome screening aimed at identifying all the potential loci encoding for B-box-containing proteins. Each retrieved genomic sequence was compared to available EST/cDNA sequences to infer gene architecture. For those genes that lacked a transcript counterpart, we performed a careful manual examination of the genomic sequences by aligning them to the most closely related TRIM of the same or other species to define exon boundaries.By combining these methods in several iterations, we retrieved the entire set of human TRIM genes. While most of these have been recently reported in the context of other studies, we also report some novel TRIM genes [3,4,22] (http://TRIMbase.tigem.it). Some of the genes we found are present as perfect or almost perfect multiple duplications in the pericentromeric region of chromosome 11 and it is difficult in these cases to establish whether they represent expressed genes (see also below). We also annotated the TRIM complement in mouse, rat, cow, and dog. The inventory of these sets and their comparisons are available at (http://TRIMbase.tigem.it).Domain composition of human TRIM proteinsThe majority of the human proteins reported in http://TRIMbase.tigem.it fulfill the TRIM rule of domain order and composition (RING, B-box(es), CC, C-terminal domain(s)). During our searches, we also found genes encoding 'incomplete' TRIM proteins, i.e. lacking one of the domains present within the tripartite motif (RING, B-box, or CC). Differently from the RING and CC domains, our analysis clearly indicated that B-box domains are virtually always present within the tripartite motif in metazoans. However, there are a few exceptions in which the B-box(es) domain is associated with only one of the domains belonging to the tripartite motif: in humans 6 proteins that possess B-box(es) lack the RING domain (B-box and CC) and 2 have a very short sequence after the B-box and almost entirely lack the CC region (RING and B-box).In the evolutionary analyses reported in this study we included the 68 genes listed in Table 1: the 'orthodox' TRIM genes and the 8 'incomplete' TRIM genes or TRIM-like genes (possessing the B-box domain associated with either the CC or the RING domains). The 'incomplete' TRIM-like genes are included in Table 1 and mentioned within the text with their non-TRIM names to remark their non full adherence to the strict definition of TRIM member; within the databases they are also annotated with a TRIM name, which is reported (Table 1). Moreover, of the chromosome 11 pericentromeric clusters we included in the analyses only representative members.Table 1Human TRIM genes included in our studyaDOMAINSbACC. No.MAPPINGAlternative namesTRIM1R-B1-B2-CC-COS-FN3-SPRYNM_052817Xq22.3MID2TRIM2R-B2-CC-IGFLMN-NHL(6)NM_0152714q31.3RNF86, KIAA0517, NarfTRIM3R-B2-CC-IGFLMN-NHL(6)NM_00645811p15.4BERP, HAC1, RNF22, RNF97TRIM4R-B2-CC-PRY-SpryNM_0330917q22.1RNF87TRIM5R-B2-CC-PRY-SpryNM_03303411p15.4RNF88TRIM6R-B2-CC-PRY-SPRYNM_00100381811p15.4RNF89TRIM7R-B2-CC-PRY-SPRYNM_2032935q35.3GNIP, RNF90TRIM8R-B1-B2-CC-ndNM_03091210q24.32GERP, RNF27TRIM9R-B1-B2-CC-COS-FN3-SPRYNM_01516314q22.1RNF91, SPRING, KIAA0282TRIM10R-B2-CC-PRY-SPRYNM_0067786p21.33RNF9, HERF1, RFB30TRIM11R-B2-CC-PRY-SPRYNM_1452141q42.13BIA1, RNF92TRIM13R-B2-CC-TMNM_00579813q14.2CAR, LEU5, RNF77, RFP2TRIM15R-B2-CC-PRY-SPRYNM_0332296p21.33RNF93, ZNFB7TRIM17R-B2-CC-PRY-SPRYNM_0161021q42.13RBCC, terf, RNF16TRIM18R-B1-B2-CC-COS-FN3-PRY-SPRYNM_000381Xp22.22MID1, FXY, OSX, XPRF, GBBB1, RNF59, ZNFXY,TRIM19R-B1-B2-CC-EXOIIINM_03323815q24.1PML, MYL, RNF71TRIM21R-B2-CC-PRY-SPRYNM_00314111p15.4RO/SSA, SSA, RO52, RNF81TRIM22R-B2-CC-SpryNM_00607411p15.4STAF50, RNF94TRIM23R-B1-B2-CC-ARFNM_0016565q12.3ARD1, RNF46TRIM24R-B1-B2-CC-PHD-BROMONM_0159057q33-q34PTC6, TF1A, RNF82, TIF1A, hTIF1, TIF1ALPHATRIM25R-B1-B2-CC-PRY-SPRYNM_00508217q23.2EFP, Z147, RNF147TRIM26R-B2-CC-PRY-SPRYNM_0034496p21.33AFP, RNF95, ZNF173TRIM27R-B2-CC-PRY-SPRYNM_0065106p22.1RFP, RNF76TRIM28R-B1-B2-CC-PHD-BROMONM_00576219q13.43KAP1, TF1B, RNF96, TIF1BTRIM31R-B2-CC-ndNM_0070286p22.1RING, RNF, HCG1, HCGI, C6orf13TRIM32R-B2-CC-NHL(5)NM_0122109q33.1HT2A, TATIP, LGMD2HTRIM33R-B1-B2-CC-PHD-BROMONM_0159061p13.2TIF1g, PTC7, RFG7, TIF1G, FLJ11429, KIAA1113TRIM34R-B2-CC-SPRYNM_02161611p15.4IFP1, RNF21TRIM35R-B2-CC-PRY-SpryNM_0150668p21.2HLS5, MAIR, KIAA1098, MGC17233TRIM36R-B1-B2-CC-COS-FN3-SpryNM_0187005q22.3RNF98, RBCC728, haprinTRIM37R-B2-CC-MATHNM_01529417q23.2MUL, TEF3, KIAA0898TRIM38R-B2-CC-PRY-SPRYNM_0063556p22.2RNF15TRIM39R-B2-CC-PRY-SPRYNM_0212536p21.33RNF23TRIM40R-B2-CCNM_1387006p21.33RNF35TRIM41R-B2-CC-PRY-SpryNM_0335495q35.3MGC1127, MGC31991TRIM42R-B1-B2-CC-COS-FN3NM_1526163q23FLJ40097TRIM43R-B2-CC-SpryNM_1388002q11.1TRIM45R-B1-B2-CC-IGFLMNNM_0251881p13.1RNF99, FLJ13181TRIM46R-B1-B2-CC-COS-FN3-SpryNM_0250581q22FLJ23229, TRIFICTRIM47R-B1-B2-CC-PRY-SpryNM_03345217q25.1GOA, RNF100TRIM49R-B2-CC-SpryNM_02035811q14.3RNF18TRIM50R-B2-CC-PRY-SpryNM_1781257q11.23TRIM54R-B2-CC-COSNM_0325462p23.3RNF30, MURF1TRIM55R-B2-CC-COSNM_1840858q13.1RNF29TRIM56R-B1-B2-CC-ndNM_0309617q22.1RNF109, DKFZP667O116TRIM58R-B2-CC-PRY-SPRYNM_0154311q44BIA2, FLJ38869, DKFZp434c091TRIM59R-B2-CC-TMNM_1730843q25.33RNF104, TSBF1TRIM60R-B2-CC-PRY-SpryNM_1526204q32.3RNF129, FLJ35882TRIM61R-B2-CC-PRY-SpryNM_0010124144q32.3TRIM62R-B2-CC-PRY-SpryNM_0182071p35.1FLJ16558TRIM63R-B2-CC-COSNM_0325881p36.11RNF28, MURF2, IRISTRIM64R-B2-CC-SpryXM_06189011q14.3TRIM65R-B2-CC-SpryNM_17354717q25.1TRIM67R-B1-B2-CC-COS-FN3-SpryNM_0010043421q42.2TNLTRIM68R-B2-CC-PRY-SPRYNM_01807311p15.4SS-56, DKFZp686D0513, RNF137, FLJ10369TRIM71R-B1-B2-CC-IGFLMN-NHL(6)DQ2328813p23hLIN41TRIM72R-B2-CC-PRY-SpryNM_00100827416p11.2TRIM73R-B2-CCXM_3536287q11.23TRIM50BTRIM74R-B2-CCNM_1988537q11.23TRIM50CTRIM75R-B2-CC-PRY-SPRYXM_9393324q32.3KIAA0129B2-CC-PRY-SPRYNM_0332209q22.33TRIM14, PubEBBPB1-B2-CC-PRY-SPRYNM_00647017p12TRIM16PYRINPAAD-B2-CC-PRY-SPRYNM_00024316p13.3TRIM20, MARENOSTRIN, FMF, MEFVATDCB1-B2-CC-ndNM_01210111q23.3TRIM29DIPBB1-B2-CCNM_01758311p13TRIM44KIAA0298B1-B2-CC-PHD-BROMOXM_08452911p15.4TRIM66RNF101R-B2NM_02411411q11TRIM48, MGC4827RNF102R-B2NM_0327655q35.3TRIM52, MGC16175a The 68 genes listed include 60 TRIM genes and the 8 genes with an incomplete tripartite motif (see text).b R, RING finger; B1, B-box1 domain; B2, B-box2 domain; CC, Coiled-coil; FNIII, Fibronectin type III repeat; PRY, PRY domain; SPRY domain; Spry, SPRY domain detected only by PFAM; NHL, NHL repeats; IGFLMN, Filamin type immunoglobulin domain; MATH, MATH domain; PHD, Plant Homeodomain; BROMO, BROMO domain; ARF, ARF domain; PAAD, PAAD domain; COS, COS-box; ExoIII, Exonuclease III motif; nd, no known domain detected.With the identification of the entire complement of the human TRIM and TRIM-like family, we confirmed and extended the domain composition features of these proteins. Within the TRIM modular structure we found that the spacing between adjacent domains is conserved. In fact, the distance between the RING domain and the first B-box, either type 1 or type 2, ranges from 35 to 55 residues; the distance between the two B-box domains ranges from 13 to 20 amino acids; and the spacing, partly occupied by the CC region, between the B-box2 and the C-terminal domain is usually 170–220 residue-long. The maintenance of the domain scaffold, order and spacing clearly indicates that the TRIM structure is a functional module.The Tripartite MotifWe aligned the sequences of the RING finger domain of all human TRIM proteins to define a general TRIM-specific RING pattern (Additional file 1). Besides the cysteine and histidine residues, which coordinate the two zinc atoms, we found clear preferences for specific residues in positions that are probably required to maintain the cross-brace structure of the RING domain [23]. The loop delimited by the second and third Cys residues has a tighter length range within the TRIM family (on average 11 residues) than within other RING-containing proteins [5]. The second loop, bounded by Cys6 and Cys7, is frequently longer than the 48 residues of the general RING consensus [5]. The RING domain has been associated with the ubiquitylation process and is mainly responsible for the interaction with the ubiquitin conjugating enzymes (E2) in the ubiquitylation cascade process [7]. The different length and composition of the intervening sequences of the RING loops may underlie the binding specificity towards the different E2 enzymes.The comparison of the B-box domains from all TRIM and TRIM-like sequences confirmed that the pattern of Cys and His is similar, although clearly distinct, in the two types of B-boxes (Additional file 1) [1,2]. The B-box1 has a short and tight consensus in which, besides the Cys and His that coordinate two atoms of zinc [24], only two positions show a clear preference for a limited choice of residues. B-box2 sequences are longer than type 1 and their consensus is looser. The cysteine and histidine residues at all 8 possible coordination positions are highly conserved consistent with the recently reported B-box2 structure definition that revealed the coordination of 2 zinc atoms [25] in contrast with previous data [26]. Moreover, additional non-polar or hydrophobic residues are also maintained in defined positions. Twenty-two out of 60 TRIM and 8 TRIM-like proteins possess both B-boxes, with B-box1 always preceding B-box2, whilst the remaining proteins have a single type 2 B-box domain (Table 1).We observed that the third component of the tripartite motif, the Coiled-coil (CC) region, follows the B-box2 in all bona fide human TRIM proteins as well as in six of the eight TRIM-like proteins. Only RNF101/TRIM48* and RNF102/TRIM52* do not possess this Coiled-coil region as they are truncated immediately after the B-box2. In all other cases, the CC region is always confined within 120 amino acids from the end of the B-box2 domain and in approximately 50% of the human TRIM proteins is bipartite (Additional file 2).TRIM C-terminal domainsThe C-terminal domains found in the TRIM and TRIM-like family members are not an exclusive property of this family but are also present in otherwise unrelated proteins [1,4]. The definition of the full complement of human TRIM and TRIM-like proteins allowed us to update the occurrence of C-terminal domains displayed by these proteins (Table 1).The majority of the human TRIM and TRIM-like proteins (40 members) possess either the SPRY domain or the association of PRY and SPRY domain, also known as B30.2 or RFP-like domain. Table 1 reports the presence of the PRY and SPRY domains that we found using the domain detection tools described in Methods and detailed in the legend but, due to the complicated and still debated relationship between PRY-SPRY and B30.2 domains, we will herein simply refer to them as SPRY [27-29]. The SPRY domain in turn can be associated with Fibronectin type III repeat (FN3) [30] and COS microtubule binding domain in different combinations [4]. Five TRIM proteins display NHL and IGFLMN domains, either in association or alone [31,32]. TRIM56 C-terminal region shares sequence similarity with this domain although no clear NHL repeats are detected. Three TRIM and one TRIM-like proteins contain a PHD associated with a BROMO domain, a combination that was demonstrated to cooperate in nucleosome binding [33]. Other domains are present in only one member of the TRIM family: the MATH domain in TRIM37; the ARF domain in TRIM23; and the EXOIII domain in TRIM19/PML [16,34,35]. Fifteen TRIM and TRIM-like proteins do not possess a defined C-terminal domain. In these cases, either their coding region is limited to the tripartite motif or the C-terminal portion is not similar to any other known domains (Table 1).This comprehensive review of TRIM and TRIM-like associated C-terminal domains confirmed that a discrete number of motifs have been selected downstream of the tripartite motif in humans.The TRIM modular structure is metazoan-specificTo trace back in evolution the origin of the TRIM family, we used human B-box1 and B-box2 sequences as queries to investigate the occurrence of these domains in the genomes of prokaryotic and eukaryotic representative species. We did not find any sequences similar to the B-box domains in prokaryotes. B-box sequences are present in plants with a consensus that is more similar to B-box1 than B-box2 (Fig. 1A). We examined 50 B-box containing proteins from 4 plant species (A. thaliana, O. sativa, P. sativum, B. nigra): the B-box is found alone or associated with a second B-box, with the CCT (CONSTANS, CO-like, and TOC1) domain [36], or with both. Differently from mammals, proximal and distal plant B-boxes (we analyzed a total of 60 B-box sequences) are very similar to each other and, consistently, do not separate in distinct branches in phylogenetic analysis (data not shown). No association with RING or Coiled-coil domains was detected in all the plant proteins analyzed.Figure 1TRIM domains in evolution. A) Logo representation of the sequences of Plant B-box (60 B-box sequences; representative species: A. thaliana, O. sativa, P. sativum, B. nigra); Metazoan B-box1 (all the B-box1 sequences in representative species: H. sapiens, D. melanogaster, and C. elegans); Mammalian B-box2 (all B-box2 sequences in representative species: H. sapiens) and Invertebrate B-box2 (all B-box2 sequences in representative species: D. melanogaster, and C. elegans). The overall height of each position is proportional to its information content and, within a given position, the conservation of each residue is represented as the relative height of amino acid symbols. Shaded columns indicate the residues involved in the coordination of zinc atoms. Blue bars represent amino acid segments of variable length; the mean value for each segment is reported. Red bars represent segments of fixed amino acid length that are present only in a proportion (indicated in red above the bar) of proteins. B) TRIM complements of humans, fruitfly (D. melanogaster) and worm (C. elegans). The total number of TRIM and TRIM-like genes in each species is indicated (top). The presence of the TRIM associated C-terminal domains is indicated with the same color code (bottom). The length of each bar in the bottom part is proportional to the number (also indicated) of the relative TRIM C-terminal domains found in each species.Besides plants and metazoans, we found B-box domains in some unicellular eukaryotes (unpublished observation). These protist species possess B-box domains that resemble either the plant or metazoan consensi, but the difficulty in attributing these lineages to specific clades compounds the tracing of the evolution of their B-box domain. In addition, since many of these protists are parasites of metazoans, we cannot rule out the possibility that horizontal gene transfer might have occurred [37].Among the metazoans we also searched the genomes of two invertebrate species, Drosophila melanogaster and Caenorhabditis elegans, for the presence of B-box domains. Distinct proximal (B-box1) and distal (B-box2) domains are found in these species, sharing with mammals the same B-box1 pattern and a similar B-box2 consensus (Fig. 1A). The B-box domains in these species associate with a RING domain and a Coiled-coil region in a tripartite motif as in mammals. The tripartite motif is therefore exclusive to metazoans, despite the fact that its constitutive elements are not.However, these invertebrate organisms have TRIM complements that differ significantly from mammals: the fruitfly has 7 TRIM genes and the worm 18, 12 of which code only for a Tripartite motif (Fig. 1B). Fruitfly and worm TRIM proteins share many of the C-terminal domains found in humans, however, their proportion varies among these species, highlighting lineage-specific expansions, e.g. SPRY in humans (Fig. 1B).The human TRIM family can be subdivided in two distinct groups: group 1 and group 2Given the numerical and structural complexity of TRIM genes in humans, we sought to characterize the relatedness among members of the family. The presence of different combinations of domains characterized by spaced cysteine and histidine residues rendered a global and reliable alignment of all TRIM and TRIM-like proteins along their entire length difficult. We therefore performed an initial alignment using the B-box2 and Coiled-coil portion. The unrooted phylogenetic tree generated from this alignment supports a recent expansion of the genes that contain the SPRY domain and suggests a preliminary separation of the human TRIM proteins in two main groups based on domain composition and branch topology (Fig. 2). Group 1, composed of 34 proteins (29 TRIM and 5 TRIM-like proteins), includes a high proportion of members with a RING-B1-B2-CC module in combination with all the C-terminal domains found in TRIM proteins. Group 2 is composed of the remaining 34 proteins (31 TRIM and 3 TRIM-like proteins), which possess only the B-box2 domain and are mostly organized as RING-B2-CC-SPRY; the 5 proteins of this group that lack the SPRY domain consist of the tripartite motif alone (Fig. 2).Figure 2Relatedness of the human TRIM and TRIM-like proteins. A) Unrooted phylogenetic tree generated upon alignment of the B-box2 and Coiled-coil region of human TRIM and TRIM-like proteins. The numbers indicate the TRIM family members; numbers with an asterisk indicate the 'incomplete' TRIM proteins named with their alternative TRIM name (see Table 1); light blue circles indicate the presence of the B-box1 domain; the colored open circles represent the different C-terminal domains as indicated in the figure. Partition in groups 1 and group 2 is indicated. B) Representative protein structures of the two groups obtained in A); dashed parentheses indicate that B-box 1 may not be present.The alignment of either single or combination of domains that compose the tripartite motif produces similar tree topologies (Additional file 3). This suggests that co-evolution of the domains present within the tripartite motif has occurred, i. e. this module mainly evolved as a single block, with no evidence of large rearrangements leading to domain acquisition or swapping among different TRIM family members; the only exceptions are the incomplete TRIM proteins that have lost one of the tripartite motif domain.We investigated whether the two groups show differences at the level of their genomic organization. Considering solely the coding region, group 2 genes span on average 10.3 kbp split in 5.7 coding exons compared to the 45.4 kbp and 8.3 exons of the genes that belong to group 1, differences that are statistically significant (P < 0.01 for gene length and exon number). Besides the average values, the homogeneity of group 2 with respect to these two parameters is striking. In fact, group 2 gene lengths range from 1.4 to 27 kbp, with only 2 genes larger than 20 kbp, whereas group 1 genes are distributed within a larger range, 1.2 to 143 kbp. Homogeneity of group 2 is also observed for the distribution of the number of coding exons: about two thirds of group 2 TRIM genes are composed of 6 or 7 exons and only in one case they span over 10 exons; again the distribution for group 1 is broader, ranging from 1 to 20 exons.Taken together, group 2 genes are smaller and less complex than group 1. Interestingly, several group 2 genes are clustered in small chromosomal regions, especially within the Major Histocompatibilty Complex region in 6p21.33 (Table 1 and Additional file 4) [38,39]. The high homogeneity of group 2 genes and their organization in clusters suggest a more recent origin of this group.Group 2 TRIM genes evolve more rapidly than group 1To investigate whether group 1 and 2 have different evolutionary dynamics, we compared the members of the human TRIM and TRIM-like set to their mouse counterparts (see http://TRIMbase.tigem.it and below for the definition of orthologous pairs). Ten out of 60 human TRIM and 8 TRIM-like genes (TRIM4, 5, 22, 43, 48*, 49, 52*, 64, 73, 74) do not have a murine ortholog, whereas TRIM31, 15, 20, and 61 are divergent at the level of entire domains compared to the mouse. Interestingly, these 14 non-conserved or divergent genes fall within group 2 and represent an important proportion of this group (41%). Conversely, all human group 1 genes have a mouse ortholog.The degree of conservation of the remaining 54 human/mouse pairs is highly variable, ranging from 49% to more than 99% amino acid identity (peptide comparisons are available at http://TRIMbase.tigem.it). Group 2 pairs present on average 78% amino acid identity versus 89.2% of group 1 (P < 0.01). Furthermore, the majority of group 1 proteins show 90–100% amino acid identity against 50–90% of most group 2 proteins (Fig. 3A).Figure 3Group 1 and group 2 TRIM gene conservation in human and mouse. A) Distribution of the percentage of amino acid identity between human TRIM and TRIM-like proteins and their murine counterparts. Group 2 (yellow) and group 1 (grey). The bars represent the number of human TRIM genes (Y axis) for each percentage of identity interval (X axis); NO indicates absence of a murine counterpart. B) Distribution of the Ka/Ks ratios observed in human-mouse orthologous TRIM pairs considering the two groups separately. The bars represent the number of TRIM pairs (Y axis) for each Ka/Ks value interval (X axis).Fast-evolving genes tend to have a higher ratio of nonsynonymous substitutions per nonsynonymous site (Ka) to synonymous substitutions per synonymous site (Ks) than the slow-evolving ones. The average Ka/Ks ratio between human and rodent coding sequences is 0.18 [40]. We analyzed the Ka/Ks ratio in human-mouse TRIM and TRIM-like orthologous pairs and found that group 1 genes present on average a ratio of 0.13 versus 0.29 of group 2 (P < 0.01). The Ka/Ks distribution is also significantly different (P = 0.01091) between the two groups: the majority of group 1 genes display values below 0.1 while most of group 2 show values above 0.1 (Fig. 3B). Furthermore, only members of group 2 (TRIM38, 40, 60, 75) exceed a Ka/Ks ratio value of 0.5 (Fig. 3B).Taken together, our analyses indicate that group 2 genes evolve more rapidly compared to group 1. This suggests that the two groups may be subject to different evolutionary constraints, likely underlying species-specific adaptations.Group 2 is absent in invertebrates and includes species-specific genes in mammalsPreliminary rounds of sequence alignment and evolutionary analyses allowed us to divide the TRIM and TRIM-like family members into major classes and subsequently generate their phylogenetic trees separately using the full-length protein sequences from man, mouse, fruitfly and worm (see Methods for details). The results show that these proteins are evolutionarily organized in two groups that coincide with group 1 and group 2 (Fig. 4 and 5). The only discrepancy with the previous subdivision is TRIM62, which segregates with group 2 proteins in the evolutionary analysis. Within group 1, TRIM37 did not segregate with any subgroups in preliminary studies and therefore it was used as an outgroup in all phylogenetic analyses.Figure 4Phylogenetic analysis of human (h, blue), mouse (m, blue), fruitfly (CGs, red), and worm (green) group 1 TRIM and TRIM-like proteins. Human and mouse TRIM proteins are indicated with their TRIM numbers; 'incomplete' TRIM proteins are indicated with their alternative TRIM number with an asterisk (see Table 1); fruitfly and worm sequences are indicated with GenBank accession numbers. Bootstrap support values above 50% based on 1000 replicates are shown. The main domain distal to the tripartite motif is indicated on the right; ND indicates no known domain detected. Panels A-E show the evolutionary relationships among the members of TRIM subgroups that belong to group 1; the worm TRIM genes composed of the R-B2-CC motif only (Figure 2B) represent a separate group related to group 1 and are not shown in the figure. TRIM37 (C-terminal domain: MATH) did not segregated within any subgroups in preliminary analyses and was therefore used as an outgroup in all phylogenetic analyses. The trees were drawn to the scale of amino acid sequence divergence indicated at the bottom right corner. A) Subgroup A includes FN3 and FN3-related TRIM sequences. Fruitfly CG31721 and its worm ortholog C39F7 are the only invertebrate proteins present in the FN3 subgroup and segregate with mammalian TRIM9 and 67. B) Subgroup B includes ARF-related TRIM sequences. Genes encoding a protein homologous to TRIM23 are found in worm and in the honeybee Apis mellifera (ENS10667 = ENSAPMT00000010667) but not in D. melanogaster, suggesting that the ARF domain has been acquired by a tripartite-gene precursor before vertebrate-invertebrate lineage separation, and has occasionally been lost in some species. C) Subgroup C includes PHD-BROMO and PHD-BROMO-related TRIM sequences. Fruitfly CG5206 behaves as an outgroup for all human and mouse PHD-BROMO proteins, suggesting that it may be regarded as an ortholog of their protein ancestor. D) Subgroup D includes IGFLMN-related TRIM sequences. This subgroup is the only example of TRIM expansion in invertebrates, because it includes worm and fly genes that do not have any direct correspondent in mammals. E) Subgroup E includes TRIM proteins with B1, B2, and SPRY in various combinations (see Table 1). No invertebrate sequences are found within this subgroup.Figure 5Phylogenetic analysis of human (h) and mouse (m) TRIM and TRIM-like proteins from Group 2. TRIM proteins are indicated with their TRIM number ('incomplete' TRIM proteins are indicated with their alternative TRIM number with an asterisk, see Table 1). No invertebrate TRIM proteins are represented in this group. Bootstrap support values above 50% based on 1000 replicates are shown. Group 1 TRIM37 sequences are used as outgroup. The scale of amino acid sequence divergence is indicated at the bottom right corner. Twenty-four pairs of orthologs can be identified (gray-shadowed branches). The remaining proteins can be subdivided in three different types, based on the phylogenetic relationship with their neighbors: (i) Proteins that are present only in one species and apparently started to diverge from their paralogs before human-mouse split (green clades); (ii) Clades of paralogous proteins that are present only in human and share a single homologous counterpart in mouse (red-shadowed branches); (iii) Clades of paralogous proteins that are present only in mouse and do not have any obvious homologous counterpart in humans (blue-shadowed branches).Group 1 is further divided into subgroups that grossly match with the domains downstream of the tripartite motif (Fig. 4). This analysis confirms that members of group 1 are present in both human and mouse with a strict 1:1 orthologous correspondence and are represented in invertebrates (Fig. 4). By means of this phylogenetic analysis we could appreciate better the mammals-invertebrates TRIM relationship and, since the worm and the fruitfly possess many of the C-terminal domains present in mammals, we could follow the late evolution of the TRIM modular structure as discussed below. Interestingly, no invertebrate sequences are found to be homologous to members of group 1 subgroup E, which includes TRIM and TRIM-like proteins with B1, B2, and SPRY in various combinations (Fig. 4E and Table 1). Group 2 proteins are not represented in invertebrate species as well, and have either a complete or a truncated RING-B2-CC-SPRY domain composition (Fig. 5 and Table 1). The major structural difference between subgroup E and group 2 is the presence of the B-box1, which indicates that group 2 proteins could have derived from a subgroup E member upon loss of B-box1.Interestingly, the evolutionary analysis of group 2 proteins showed, in addition to 24 pairs of orthologs, the presence of species-specific TRIM and TRIM-like proteins, not only in humans (see above) but also in mouse (Fig. 5), indicating high dynamicity in the evolution of this group compared to group 1.The analysis of the TRIM and TRIM-like complements of rat, cow, and dog showed that these species have the same set of group 1 genes as humans and mouse. Conversely, the sets of group 2 genes are different and specific to each species, including closely related ones such as mouse and rat, with only 17 genes (50–70% of group 2 genes) shared among all species (http://TRIMbase.tigem.it). These differences are due to events of gene duplication, deletion, or degeneration that likely occurred during the evolution of each single lineage. Remarkably, in three cases the same gene has undergone degeneration or deletion independently in different lineages: human TRIM38 orthologs, found in mouse and cow, are pseudogenes in rat and dog; human TRIM60 has orthologous loci in all the examined mammals but has become pseudogenes in rat, cow, and dog; human TRIM61 orthologs are found to be pseudogenes or absent in all organisms but mouse. Furthermore, two TRIM genes are present in the five species in three different states: TRIM15 is intact in human and cow, presents premature stop codons in mouse and rat, and is a pseudogene in dog; similarly, TRIM31 is intact in mouse and rat, has distal frameshifts in humans and cow, and is absent in dog. Both premature stop codons and frameshifts result in truncated proteins that have lost the SPRY domain. These losses may represent a degenerative step towards the complete inactivation of these genes.Of note, massive gene duplications and rearrangements had occurred at the level of several genes of group 2 that cluster in the same chromosomal location (Table 1). A thorough characterization of the TRIM-rich 6p21.33 locus is reported in human and chicken [39,41,42]. Indeed, in addition to the human genes here presented, extra-copies of group 2 genes TRIM43, 48*, 49, and 64 are clustered at 2q11.1, 11q11.1, and 11q14.3 for a total of 11 predicted genes and 14 pseudogenes. These clustered loci have paralogs, but not orthologs, in some of the other examined mammalian species (http://TRIMbase.tigem.it).A further example of recent gene evolution is the genomic cluster at 11p15.4 containing TRIM5, a gene involved in HIV-1 viral restriction in some primates [21]. This cluster includes TRIM5 and 22, which are currently considered primate-specific, in addition to TRIM6 and 34, which are not [43,44]. By means of comparative and evolutionary analyses (Additional file 5) we found that the entire cluster, including TRIM5 and 22, was present in the last common ancestor of humans, cow, and dog, supporting a common origin for TRIM5 and its cow functional ortholog LOC516599 rather than evolutionary convergence as previously proposed [43].TRIM genes in other vertebrate speciesGiven the situation in mammals, we asked whether the TRIM complements evolved likewise in other vertebrate species. We searched the databases for TRIM and TRIM-like sequences in representative aves and fish species, chick (Gallus gallus) and a pufferfish (Tetraodon nigroviridis), respectively. In addition, we included in our analysis the urochordata Ciona intestinalis, a representative of the early chordate lineage from which the vertebrates originated. The searches were performed combining different iterations of PHI-BLAST (using the B-box2 pattern) and TBLASTN and BLASTP against nr protein and nucleotide databases at NCBI, starting from both human TRIM proteins and TRIM sequences found in the above species. We found 10 TRIM sequences in ciona, 37 in chick and 58 in pufferfish; all the sequences we retrieved were present in the databases as assessed or predicted genes and only some of them (especially in chicken) were already annotated as TRIM genes (the sequences retrieved for these species are reported at http://TRIMbase.tigem.it). These novel TRIM complements should not necessarily be regarded as complete since in these species there are still regions not yet sequenced or unequivocally assembled. We compared these additional vertebrate sequences to the human sequences using multiple protein alignments and phylogenetic tree constructions using as a paradigm the subdivision into groups and subgroups of Figure 4 and 5. Detailed analyses of these novel genes, at the level of transcript, protein and genomic locus, are required and will be addressed elsewhere, therefore the phylogenetic analyses we present show relationships and are not intended to represent precise evolutionary distances.In all species analyzed, we found clear orthologs for TRIM23 and TRIM37 (Fig. 6A). With respect to subgroup C, there is one representative of this subgroup in ciona, closely related to the fruitfly member. All the known mammalian members of this subgroup are found in chick with recognizable orthologous relationships (Fig. 6B). This is not completely true for tetraodon in which 3 members belonging to this group have been found: one orthologous to TRIM33 and the others representing fish-specific duplications related to the TRIM33-24 clade. Genes strictly related to KIAA0298/TRIM66* and TRIM19 have not been found in tetraodon (Fig. 6B). In ciona there are 5 representative members of subgroup A, 3 representing the ancestors of different subclades (TRIM9-67; TRIM1-18; TRIM54-55-63) and 2 apparently more ancestral genes. DIBP/TRIM44* appears to be uniquely represented in mammals. All the other members are present in chick and fish although orthology is clear only for TRIM9, 67, 1, 18, and 36. TRIM46, if truly absent, may have been lost in chick while of the MURF group (TRIM54, 55, 63) 2 orthologues are present in chick while 4 members with no direct orthologous relationship are present in fish (Fig. 6C). An analogous situation is observed for subgroup D. In fact, ciona has two representative members; orthologous relationship with human is observed for both chick and fish for four members (TRIM2, 3, 45, 71). TRIM32 might have been lost in chick and TRIM56 in both chick and fish (Fig. 6D). For the above group 1 subgroups, we therefore found that most of the mammalian components are present in chick often with clearly recognizable orthologous relationship. Although the number of members within these subgroups is similar also in tetraodon, the TRIM and TRIM-like genes in this species, consistent with a larger evolutionary distance, have duplicated and diverged more extensively, sometimes obscuring orthologies.Figure 6Phylogenetic analysis of TRIM and TRIM-like proteins of representative species of mammals, aves, and fish. Human (Hs, dark blue), chicken (Gg, dark green), tetraodon (Tn, light blue); ciona (Ci, orange), fruitfly (red) and worm (light green) are included. Bootstrap support values based on 1000 replicates are shown. Group 1 TRIM37 sequences are used as outgroups. The scale of amino acid sequence divergence is indicated at the bottom right corner.A different situation is observed with the group 1 subgroup E and, as expected, with group 2. As for the other invertebrates, ciona genes are not represented in this subgroup and in group 2. Orthologous relationship among mammals, chick and fish is observed only for two genes of subgroup E (ATDC/TRIM29* and TRIM65). The other genes within the subgroup are more conserved in chicken while in tetraodon many independent duplication events occurred (Fig. 6E). Even more extensive duplication events and independent divergences are observed for group 2 genes. In this case, close homology among the three species is only recognizable for TRIM35 and 62. A couple of other mammalian TRIM genes are conserved in chick (TRIM7 and 41) but the remaining genes in the three species have been subjected to independent duplications and evolution (Fig. 6F).To confirm in fish the presence of so many TRIM sequences poorly related to the human TRIM genes, we search TRIM and TRIM-like genes also in zebrafish (Danio rerio) using the same criteria and methods described for the chick and tetraodon. Differently from tetraodon, zebrafish presents an elevated number of TRIM and TRIM-like genes; we found 240 entries corresponding to independent genes (the list of zebrafish genes is reported at http://TRIMbase.tigem.it). Also in this case, the number of genes encoding for group 1 TRIM (excluding subgroup E) is comparable to the number in mammals, chick and pufferfish (1 subgroup B; 5 subgroup C; 16 subgroup A; 12 subgroup D) although in many cases clear duplication events occurred. However, the great expansion of the TRIM genes in the zebrafish is associated with members belonging to the group 1 subgroup E and group 2 genes (data not shown).Analyses of TRIM complements in aves and fish corroborate the high conservation of group 1 genes during evolution and highlight the generation of unique sets of group 2 genes in each vertebrate species analyzed. Moreover, the data in non-mammalian vertebrates, especially in tetraodon, confirm that members of the group 1 subgroup E very likely gave rise to group 2 genes.In conclusion, our study indicates the presence of two distinct TRIM gene groups. Group 1 is evolutionary more ancient than group 2 and is likely to contain basic functions that are essential to both vertebrate and invertebrate species. On the other hand, group 2 is younger and more dynamic, possibly acting as a sort of TRIM genes \"reservoir\" to develop novel species-specific functions.DiscussionHere, we report the identification and genomic characterization of the full complement of the human TRIM family examined from an evolutionary perspective by comparison with several vertebrate and invertebrate species.We definitively assessed that the B-box domain is only present within the tripartite module in metazoans, with the few exceptions mentioned above and discussed below, and is therefore the defining domain of the TRIM family. We redefined the B-box1 and B-box2 consensi as well as the TRIM specific RING finger pattern using all human sequences. Within these domains we found conservation not only of the residues putatively involved in metal coordination, but also of other amino acids that compose the novel consensi. It will be interesting to model the RING, B-box1 and B-box2 sequences of the TRIM proteins on these structures to study the possible role of the conserved residues in relation to the ubiquitylation cascade [7-9,24,25].Based on our analyses, we propose a general model of TRIM structure evolution (Fig. 7). Our studies suggest that the origin of the B-box domain is quite ancient and probably dates back to a common ancestor of plants and metazoans. The plants maintained either one or two B-boxes without apparent sequence differentiation into proximal and distal. Conversely, metazoans differentiated a pair of B-boxes into a proximal and a distal type. In an early step of metazoan evolution, a RING domain and a Coiled-coil region associated with the B-box(es) to generate a solid tripartite module that has been maintained from invertebrates to mammals. From then onward, the tripartite motif has evolved as a unique block and it is frequently encoded by a single exon. Interestingly, we found different mammalian and invertebrate B-box2 patterns. This may underlie functional coupling with specific interactors in each lineage, which would have forced convergence at specific sites. A similar species-specific evolutionary convergence was recently described for sulfatase enzymes and their common post-translational modification factor [45].Figure 7Proposed model for TRIM structure evolution (see text). The C-terminal domains probably derived from a single ancestor domain are indicated with the same color.Before invertebrate-vertebrate lineage split, the tripartite module has been associated with a discrete number of C-terminal domains. Addition and loss of C-terminal domains and structure remodeling have then occurred in the various evolutionary lineages (Fig. 7). In at least one case the same domain acquisition has occurred independently in primates and arthropods. In fact, in some species of New World monkeys (Aoutus genus), Cyclophilin A (CypA) is fused with the tripartite motif of TRIM5 [46]. TRIM5 confers a potent block to HIV-1 infection in Old World primates, while Cyclophilin A (CypA) enhances infection by direct interaction with the HIV capsid [21,47]. HIV-1 blockage in Aoutus cells was explained by the exclusive presence of the TRIM5-CypA chimeric gene [46,48,49]. Interestingly, we found that a tripartite motif is associated with a Cyclophilin domain also in fruitfly CG5071 indicating evolutionary convergence. Independent events of gene fusion are considered a hallmark of functional coupling that can be also present in those species in which a similar gene fusion is not observed [50]. Similar processes of fusion between functionally associated domains may have been one of the mechanisms underlying the selection of C-terminal domains during TRIM evolution.The early association of the B-box modules with the RING finger, a domain linked to the ubiquitylation process, has eventually brought the proteins possessing a tripartite motif to exert a common basic biochemical function, i.e. ubiquitin ligase [6]. The large number of proteins belonging to this family in mammals highlights the success of this module to undertake its task. Since the TRIM family represents one of the largest RING finger classes, it is tempting to speculate that, among the myriads of cellular E3 substrates, a large proportion is demanding the unique tripartite structure for reasons yet to be discovered. Whereas the tripartite motif may provide the catalytic E3 activity and the ability to form the scaffold of the TRIM-defined sub-cellular compartments [1], the C-terminal region may contribute to select the specific substrate and/or direct the tagged substrate towards downstream pathways. The PHD-BROMO domain, for example, determines the association with chromatin, and the TRIM and TRIM-like proteins containing the PHD-BROMO domain are consistently involved in chromatin remodeling [51,52]. Along the same way, MID1/TRIM18 and the related TRIM proteins that possess a COS microtubule-binding domain exert their role on the cytoskeleton [4].It should be reaffirmed that not all the proteins we included in our study retain an entire tripartite motif. As mentioned in the 'Results section', we decided to include all the genes encoding for proteins with B-box motifs and that in human correspond to the 'complete' TRIM proteins (with RING, B-box and CC) and few 'incomplete' TRIM proteins (or TRIM-like) presenting only two of the three tripartite motif composing domains (with either 'RING and B-box' or 'B-box and CC'). This might pose a formal problem on what should be classified as a TRIM protein. In the classic definition, a protein family is composed of proteins that have a common phylogenetic origin and share a degree of amino acid identity/similarity above an established threshold. In the case of the TRIM family, the initial definition of family was based on the observation that most members of this protein family share the tripartite arrangement at their N-terminal portion [1]. What was not clear was whether the TRIM proteins had a common origin or rather they were the result of domain swapping from evolutionarily unrelated proteins. Our analyses demonstrate that all the proteins with a RING-B-box-CC module actually have a common evolutionary origin. Therefore, what was raised as an 'operative' definition is now demonstrated to be perfectly adherent to the classic definition of a gene/protein family. Our data allow the same conclusion to be drawn for the 'incomplete' TRIM proteins which possess the B-box motif and which we found evolutionarily belonging to the TRIM family. In support of that, some of the 'incomplete' TRIM proteins also present C-terminal domains characteristic of the 'complete' TRIM proteins. This parallel cannot be used for other domains present within the tripartite motif, e.g. the RING domain has been 'used' to build many different protein families in association with several TRIM unrelated domains [22]. On the other hand, we think that a strict definition of TRIM family based only on function is not feasible at present. As discussed above, the presence of the RING domain suggests a role as E3 ubiquitin ligases for the TRIM proteins. Experimentally, this has been proven for some TRIM proteins and we cannot exclude that some of them, although containing the RING domain in the proper tripartite motif, might have a different biochemical role. What is the role of the 6 RING-less proteins we included in our study? They may be involved in ubiquitylation as well by, for example, acting as regulators of orthodox TRIM proteins through hetero-interaction. Given that the recent solution of the B-box1 and B-box2 domains revealed a strong structural similarity with the RING domain [24,25], it is tempting to speculate that these domains may interact with components of the ubiquitylation machinery and attribute these RING-less proteins the role of E3 ubiquitin ligases. Coherently with these observations, we propose to include within the TRIM family all the proteins that are phylogenetically related to established TRIM members and that have a tripartite motif at their N-terminus, including the few examples in which part of this motif has been lost.The relatively small number of TRIM genes in lower eukaryotes compared to mammals suggests rapid and recent changes of the TRIM family. Our study revealed the presence of two main groups of mammalian TRIM genes that show distinct evolutionary features and that we named group 1 and group 2. Group 1, that is in turn subdivided in several subgroups, is composed of genes that are present in human, mouse, rat, dog, and cow with a one to one relationship. Although orthology with mammals is not always recognizable, this group of genes is highly conserved also in other vertebrates (chick and fish) in number and structure. Our data on the Ka/Ks ratio of human and mouse group 1 genes suggest that they are subject to purifying selection aimed at conserving their function. It is conceivable that group 1 consists of diversified and essential TRIM and TRIM-like functions for which little or no redundancy is present. Consistently, many group 1 genes are involved in basic cellular processes, such as cell cycle progression and transcriptional regulation, and result, when mutated, in developmental disorders, muscular phenotypes, cancer insurgence, etc. [2,6]. Some group 1 TRIM genes have also been found to be involved in viral response, namely TRIM1 [53], TRIM19/PML [3,54,55] and TRIM32 [56]. TRIM19/PML, besides its involvement in acute promyelocytic leukemia, has been shown to interfere with the replicative cycle of many DNA and RNA viruses and evidence indicate that it may represent a broad-spectrum cellular defence factor [3].The important increase of TRIM number in vertebrates is primarily due to the buildup of the genes that constitute group 2. Group 2 is in fact evolutionarily more recent than group 1, is not represented in invertebrates, and evolves at a faster rate. Interestingly, many TRIM proteins that belong to group 2 have been recently associated with cellular innate immunity towards viral infection. In addition to TRIM5α, other members are being investigated as potential retrovirus restriction factors. Among them, TRIM21, 22 and 34 are regulated by interferons, a family of secreted proteins that exert antiviral and immunomodulatory activities [57]. Moreover, other group 2 genes, PYRIN/TRIM20* and TRIM21 are involved in immuno-related diseases [20,58,59]. Interestingly, group 2 proteins share the same C-terminal motif, the SPRY domain. In the case of TRIM5α, this domain is responsible for the species-specific HIV-1 restriction and is subject to positive selection in primates, underlying its possible role in directing and specifying capsid recognition [44,60]. Of note, the SPRY domain is also present in SOCS proteins, involved in cytokine signaling and innate immunity, and in the BTN family of lymphoid expressed proteins, possibly involved in immune regulation [28,61,62]. It has been proposed that the sharing of the SPRY domain between the TRIM and BTN family members located within the MHC locus is somewhat linked to their immunological function [41]. The SPRY domain might therefore confer to group 2 proteins the ability to specifically recognize viral capsids and interfere with early steps of viral infection. Differently from anti-viral group 2 proteins, group 1 TRIM19/PML interferes with general mechanisms of viral replication common to various viruses and consistently is not subject to positive selection [35].Our comparative analysis in five mammalian species shows that subsets of group 2 TRIM and TRIM-like genes are different and specific in each examined lineage. This is more evident in the three non-mammalian vertebrates analyzed, where large numbers of newly identified group 2 genes mainly lie on species-specific evolutionary clades. This observation might underlie dispensability/redundancy of some group 2 genes, which could have provided the basis for novel species-specific roles during evolution. The presence of clusters composed of massively duplicated group 2 genes, in mammals but also in chick, suggests that they may be hot-spots for TRIM gene production and remodeling. In the case of the teleost fish species some of the duplications may be the remnants of the whole genome duplication event early in the teleost lineage. Global duplication is not however enough to explain the large and independent expansion of subgroup E and group 2 genes in the teleosts and a different cause must underlie these expansions. It is interesting to note that similar to the Group 2 TRIM and TRIM-like genes, other families of genes involved in innate immune response and in particular the components that interact with pathogens have been subject to similar large lineage specific expansions in the teleost fish [63]. Moreover, since we observed that group 2 genes tend to have a human/mouse Ka/Ks ratio higher than group 1 genes, it is tempting to speculate that some group 2 genes other than TRIM5α may be subject in some species to positive selection at specific sites to counteract species-specific battles against viral infections, as it has been shown for other family of genes involved in innate cellular immunity [64-66].ConclusionWe found that the TRIM domain structure is an innovation of metazoans. The growing evidence for a common biochemical function of the TRIM proteins as ubiquitin ligases justifies the maintenance of their basic modular structure throughout evolution. Our studies indicate the presence of two distinct TRIM gene groups. Group 1 is evolutionary more ancient than group 2 and is likely to contain basic functions that are essential to both vertebrate and invertebrate species. On the other hand, group 2 is younger and more dynamic, possibly acting as a sort of TRIM genes \"reservoir\" to develop novel functions. Since some of the TRIM genes that belong to this group are implicated in innate immune response, we propose that the different selection we observed for this group of genes underlies pressure towards rapid changes necessary to counteract species-specific battles against viral infection.MethodsGene searchingKnown mammalian TRIM and TRIM-like gene/protein sequences were retrieved from the National Center for Biotechnology Information (NCBI) and re-defined by searching against their respective genome assemblies using BLAT at the UCSC genome browser (http://genome.ucsc.edu). All the 'corrected' sequences were then used as queries to search potential novel TRIM genes within the human, mouse, rat, cow, and dog genomes, using BLAT at UCSC and TBLASTN at NCBI [67] genome browsers, respectively. All searches have been performed in several iterations using default parameters. Human nr and EST databases were screened using the PHI-BLAST in several iterations using the patterns previously defined for B-box1 and B-box2 [1]. We also searched the human, mouse, rat, cow, and dog proteomes using the B-box2 as a bait in five iterations of the PHI-BLAST program [67], which provides a highly sensitive analysis, taking advantage of the fact that the B-box2 is a peculiar constituent of TRIM proteins and does not produce a large amount of background noise in this analysis. The retrieved amino acid sequences were subsequently used to search the respective genomes for identifying their encoding loci. Representative B-box2 sequences were also used as queries for TBLASTN search of the five mammalian genomes to identify all the potential loci encoding TRIM and TRIM-like proteins. All the retrieved genomic sequences were aligned to the available cDNA/EST sequences to infer the gene architectures. For genes that lacked a transcript counterpart in public databases, we performed a careful manual examination of the genomic sequences BLAST-comparing them to the putative more closely related ortholog or paralog, looking for splicing donor and acceptor signals to define the exon-intron boundaries. Constructed open reading frames (ORFs) were conceptually translated into amino acid sequences and checked against their closest homologs. The original genome sequencing traces (Traces-WGS), which are available at the NCBI web site, were checked when the constructed coding sequences presented either stop codons or ORF frame-shifts. When a difference between WGS traces and the genomic assembly was evident, the constructed sequence was properly corrected. Comparison of human, mouse, rat, cow, and dog orthologous TRIM and TRIM-like genes showed that >99% of splicing acceptor and donor sites were conserved at the same relative position in the coding sequence in all species, i.e. the gene structure of TRIM genes is identical among different mammals. To retrieve TRIM genes form ciona (Ciona intestinalis), chick (Gallus gallus), pufferfish (Tetraodon nigroviridis) and zebrafish (Danio rerio) we used a combination of PHI-BLAST and TBLASTN against nr protein and nucleotide databases at NCBI, respectively. The following genome releases have been used for this work: Homo sapiens, May 2004 assembly (NCBI Build 35); Mus musculus, February 2006 assembly (NCBI Build 36); Rattus norvegicus, June 2003 assembly (Baylor College of Medicine HGSC v.3.1); Bos taurus, March 2005 assembly (Baylor College of Medicine Btau_2.0); Canis familiaris, May 2005 assembly (Broad Intitute CanFam2.0). Drosophila melanogaster TRIM genes: CG1624, CG5206, CG12218, CG8419, CG5071, CG10719, CG31721. Caenorabditis elegans TRIM genes: arc1, B0281, ZK1240.1, F43C11.8, ZK1240.2, F43C11.7, ZK1240.9, ZK1240.3, ZK1240.8, ZK1240.6, C28G1.6, K09F6.7, lin41, C39F7, nhl-2, nhl-3, ncl-1, F47G9.Protein domain analysesTo identify and analyze the domain composition of the TRIM and TRIM-like protein products we used the major alternative splicing isoforms, if more than one was available, and utilized different domain prediction programs. First, we submitted the TRIM amino acid sequences to the SMART tool [68] where we analyzed the sequence against contemporary the SMART and Pfam [69] domains databases. The denotation of the C-terminal domains found within the TRIM sequences are the following: MATH, SM00061; PHD, SM00249; BROMO, SM00297; IGFLMN, SM00557; EXOIII, SM00479; FN3, SM00060; PRY, SM00589; SPRY, SM00449; ARF, SM00177; NHL, PF01436 (Pfam). The tripartite motif domains were additionally analyzed as described below. Besides the SMART results, the RING and B-boxes domains were also defined in each TRIM and TRIM-like protein by hand using the previously published patterns [1]. In order to obtain a new profiling, the sequences corresponding to each domain were then aligned using the PRATT 2.1 program [70] and the best scoring consensi were selected and integrated by hand. The order of the sequences in the alignment shown reflects their degree of sequence conservation. The region of each TRIM and TRIM-like protein immediately after the last Cys or His of the B-box2 domain was analyzed for Coiled-coil prediction with the Coil 2.2 program [71]. Analysis was performed with MTIDK and MTK matrices and both the weighted option, which takes into account the polarity of the residue within the predicted Coiled-coil heptad repeat, as well as the unweighted option. When differences of around 20–30% in Coiled-coil prediction were observed between the different methods utilized, the prediction was considered bad and not indicated in the list of Additional file 2. Moreover, only percentages of prediction higher than 50% were considered using two residue windows, 21 and 28 amino acids.Plant B-box containing proteins (from A. thaliana, O. sativa, P. sativum, B. nigra) were retrieved from the SMART B-box database [68].Evolutionary analysesTo perform phylogenetic analysis, TRIM and TRIM-like protein sequences were aligned using MultAlin [72] in a multi-step process. Only proteins containing the complete module R-B1-B2-CC were aligned in a first step, eliminating from each sequence the portion downstream of the coiled-coil domain and all segments that caused a gap to interrupt the alignment. A first phylogenetic tree was produced starting from this multi-alignment. In a successive step, each of the remaining protein sequences was singularly added to the multi-alignment, edited for exceeding amino acids, and assigned to a TRIM subgroup after inspection of the topology of the resulting phylogenetic tree. Once all TRIM and TRIM-like protein sequences were assigned to a subgroup, phylogenetic analyses were performed independently for each subgroup. TRIM37 was not included in any subgroup and therefore was used as an outgroup in all final analyses. Nucleotide sequences of TRIM5/6/22/34 and related non-human sequences were also aligned using MultAlin [72], but in this case a gap-removal step was not necessary due to the high similarity among all considered sequences. Neighbor-Joining and bootstrap analyses were performed with Phylo_win [73], computing the distances among sequences with all the methods available in the package (protein analysis: observed divergence with and without Poisson correction; PAM distance. DNA analysis: observed divergence; Jukes and Cantor distance; Kimura distance; Tajima and Nei distance; HKY distance; Galtier and Gouy distance; and LogDet distance) [73] (and references therein). Gap-removal was set as pairwise rather than global to minimize information loss. Bootstrap values were computed over 1000 repetitions. All tree topologies resulted to coincide in the different methods for branches with a bootstrap value >50. Evaluation of Ka/Ks values for pairs of human-mouse TRIM- and TRIM-like-coding sequences was performed at the Norwegian bioinformatics platform [74].Comparison of the two groups quantitative parameters (gene lengths, exon number, amino acid identity and Ka/Ks ratios) were analyzed using the two samples t-test (two-tail test assuming unequal variances) by the Microsoft Excel statistical package. The comparison of the two groups Ka/Ks distribution has been analyzed using a two-sample Kolmogorov-Smirnov test.Authors' contributionsMS carried out the evolutionary studies and contributed to the identification of TRIM genes in different species; he contributed to the design of the experiments and to the writing of the manuscript. SC identified the entire set of TRIM genes in human and performed the single domains alignments. BF collected all the data in the database and performed statistical analyses. AB contributed to the interpretation of the data and to the drafting the manuscript. GM conceived, designed and coordinated the study and wrote the manuscript.Additional filesThe complete sets of human, mouse, rat, dog and cow TRIM and TRIM-like genes and pseudogenes as well as their sequence comparisons are available at http://TRIMbase.tigem.it. At the same site are the TRIM related sequences from ciona, chick, tetraodon, and a list with the accession numbers of the zebrafish TRIM-like genes. See Additional files 1 to 5.Supplementary MaterialAdditional file 1Includes the alignments of the RING, B-box1 and B-box2 domains of all the human TRIM and TRIM-like proteins, alignments from which the consensi for these domains have been generated.Click here for fileAdditional file 2Reports the values of Coiled-coil predictions for all the human TRIM and TRIM-like proteins.Click here for fileAdditional file 3Shows the unrooted phylogenetic trees generated from the alignments of single domains of the tripartite motif.Click here for fileAdditional file 4Shows a schematic representation of the human TRIM genomic clusters.Click here for fileAdditional file 5Shows comparative and evolutionary analyses of the cluster of TRIM5, 6, 22, and 34 in mammals.Click here for file\n\nREFERENCES:\n1. 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MakalowskiWBoguskiMSEvolutionary parameters of the transcribed mammalian genome: an analysis of 2,820 orthologous rodent and human sequencesProc Natl Acad Sci U S A199895169407941210.1073/pnas.95.16.94079689093\n41. RubyTBed'HomBWittzellHMorinVOudinAZoorobRCharacterisation of a cluster of TRIM-B30.2 genes in the chicken MHC B locusImmunogenetics2005571-211612810.1007/s00251-005-0770-x15744538\n42. HenryJMatherIHMcDermottMFPontarottiPB30.2-like domain proteins: update and new insights into a rapidly expanding family of proteinsMol Biol Evol19981512169617059866204\n43. SiZVandegraaffNO'HuiginCSongBYuanWXuCPerronMLiXMarascoWAEngelmanADeanMSodroskiJEvolution of a cytoplasmic tripartite motif (TRIM) protein in cows that restricts retroviral infectionProc Natl Acad Sci U S A2006103197454745910.1073/pnas.060077110316648259\n44. 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DupontSZacchignaLCordenonsiMSoligoSAdornoMRuggeMPiccoloSGerm-layer specification and control of cell growth by Ectodermin, a Smad4 ubiquitin ligaseCell20051211879910.1016/j.cell.2005.01.03315820681\n53. YapMWNisoleSLynchCStoyeJPTrim5alpha protein restricts both HIV-1 and murine leukemia virusProc Natl Acad Sci U S A200410129107861079110.1073/pnas.040287610115249690\n54. RegadTChelbi-AlixMKRole and fate of PML nuclear bodies in response to interferon and viral infectionsOncogene200120497274728610.1038/sj.onc.120485411704856\n55. MaulGGGuldnerHHSpivackJGModification of discrete nuclear domains induced by herpes simplex virus type 1 immediate early gene 1 product (ICP0)J Gen Virol199374 ( Pt 12)2679269010.1099/0022-1317-74-12-26798277273\n56. FridellRAHardingLSBogerdHPCullenBRIdentification of a novel human zinc finger protein that specifically interacts with the activation domain of lentiviral Tat proteinsVirology1995209234735710.1006/viro.1995.12667778269\n57. 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CompteEPontarottiPColletteYLopezMOliveDFrontline: Characterization of BT3 molecules belonging to the B7 family expressed on immune cellsEur J Immunol20043482089209910.1002/eji.20042522715259006\n63. SteinCCaccamoMLairdGLeptinMConservation and divergence of gene families encoding components of innate immune response systems in the zebrafishGenome Biol2007811R25110.1186/gb-2007-8-11-r25118039395\n64. HughesALOtaTNeiMPositive Darwinian selection promotes charge profile diversity in the antigen-binding cleft of class I major-histocompatibility-complex moleculesMol Biol Evol1990765155242283951\n65. YilmazAShenSAdelsonDLXavierSZhuJJIdentification and sequence analysis of chicken Toll-like receptorsImmunogenetics2005561074375310.1007/s00251-004-0740-815578175\n66. YangZThe power of phylogenetic comparison in revealing protein functionProc Natl Acad Sci U S A200510293179318010.1073/pnas.050037110215728394\n67. NCBI BLASThttp://www.ncbi.nlm.nih.gov/blast/\n68. 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+}

batch_9/PMC2533339.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2533339",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533339\nAUTHORS: Gerald Haidinger, Stephan Madersbacher, Georg Schatzl, Christian Vutuc\n\nABSTRACT:\nBackgroundThe introduction of PSA testing in Austria led to a steep increase of the incidence of prostate cancer. We want to present the course of the number of newly diagnosed cases of prostate cancer in Austria since 1997, and set these numbers in relation to the total of radical prostatectomies (with resection of lymph nodes) in the same time period. All numbers were retrieved from health statistics of Statistics Austria. The report period of cancer cases and of RPE comprises the years 1997–2004. All calculations were performed for totals as well as for 5-year age groups (40–89 years of age).FindingsThe number of prostate cancer cases rose from 1997 to 2004 by 35%, while the number of RPE rose by 94% in the same time period. The proportion of RPE in relation to new cases rose from 41% in 1997 to 59% in 2004.ConclusionA slight decrease of prostate cancer mortality can already be observed in Austria, but the question of over-treatment still awaits analysis.\n\nBODY:\nFindingsOpportunistic Prostate-Specific-Antigen testing (PSA) of healthy men started at the beginning of the 1990s and led to a steep increase of prostate cancer incidence [1-3]. In Austria data on surgical treatment of prostate cancer are available since 1997. The purpose of this paper is to present the correlation between the number of newly detected cases and the number of radical prostatectomies with resection of lymph nodes (RPE) by 5 year age group.Data on the number of newly detected cases and number of prostatectomies where obtained from Statistics Austria. The data on radical prostatectomies by age group are available since 1997 only. The data set covers the age group 40 to 89 years comprising a population of 1.738,655 men in the year 2000. Since cases are anonymized it is indeterminable whether newly detected cases are operated in the year of diagnosis or in the following year, if ever.Cases treated by perineal cryosurgery were excluded, due to the small total number (0 to 2 cases per year).The number of newly detected cases and of RPE (1997 through 2004) as well as the calculated percentage per year by 5 year age group are given in table 1. The absolute number of prostate cancer cases rose from 3999 in 1997 to 5416 in 2004 (+35.4%, all age groups). The absolute number of RPE rose from 1648 in 1997 to 3200 in 2004 (+94.2%, all age groups). The percentage of RPE in relation to the number of newly detected cases rises from 41% in 1997 to 59% in 2004 (all age groups). The increase of this percentage is observed in all age groups younger than 70 years but not in older age groups, the linear trend over time is significant for all age groups and for totals, except for age group 40–44 years.Table 1Number of newly detected cases of prostate cancer in Austria, and number of radical prostatectomies 1997–2004, as well as proportion of RPE/incidence, and P value for linear time trend, totals and 5-year age groups of men aged 40–89.Age19971998199920002001200220032004NndcRPENndcRPENndcRPENndcRPENndcRPENndcRPENndcRPENndcRPEnn%nn%nn%nn%nn%nn%nn%nn%P-value40–4434>100548054801614871815831718>10022177725251000.33845–4926218140379244358074719685819595879212512398109118>100< 0.00150–5412799781491187916615795199171862562409426625997267279>100254258>100< 0.00155–5937130061440382874964338752248292541518965594928859755292585612>100< 0.00160–644903697552835968589454777846888890779888995884891158106792110598089< 0.00165–69841515618805686592456061938666719626857191065172106584279101178978< 0.00170–7483729936873288339762883010423553410353903899837037107450047100137738< 0.00175–7955434665531573738569840667442664426473211916718365< 0.00180–84383413002<1316003331<13282<1339004033893672<1< 0.00185–893670034800340003171<13221<1258002841553251<1< 0.001Total39991648414218182643459319694349232489515128277454508127915557273167555416320059< 0.001Nndc ... Number of newly detected cases.RPE ... Radical Prostatectomy (with resection of lymph nodes).P-value ... P value for linear time trend.The increase of the incidence of prostate cancer is well documented for Austria [1,2], however the absolute numbers are for the first time set in relation to the number of RPE nationwide. Within 8 years, the total number of RPE increased by 94.2%, thus nearly doubled, with the increase mainly occurring in the age group 55–69 years. Starting at age 70 years the percentage of RPE remains more or less stable on a substantially lower level.The number of RPE we used in our calculations represents the official hospital discharge statistics of Austria comprising of the collected data of all hospitals (including private hospitals), but excludes a very small number of self-pay patients. Thus our results negligibly may underestimate the relation of RPE to the number of newly detected cases. A further limitation of our analyses is caused by legal constrains: due to data protection laws we are limited to the use of depersonalized data.The increase of RPE in Austria can be explained by at least three factors: a) operation technique meliorated significantly, now is a standard procedure, perioperative mortality is low and the risk of postoperative morbidity such as incontinence and erectile dysfunction decreased in Austria [4,5], consequently acceptance of RPE in patients increased. b) By the introduction of PSA testing the target group of prostate cancer screening shifted towards younger age groups, meaning that carcinoma more frequently is sought in younger men [3]. c) Because of the slow progression of most cases, RPE only makes sense when health status permits RPE and patients have a life expectancy of at least 10 years [6].The high percentage of RPE in men younger than 70 years surely also is influenced by the fact that counselling (towards RPE or radiation therapy) in Austria is performed by urologists. In this context, it is worth mentioning, that the primary intention of this manuscript is to provide the data which can serve as a basis for a constructive discussion about counselling and the provision of the most adequate therapeutic regimes.The reason for the more or less stable percentage in men aged 70 years or older may lie in the fact that therapy of prostate cancer in these patients more often relies on watchful waiting, active surveillance, external beam radiation, brachytherapy or hormonal treatment. Detailed data for these therapies are not available.In conclusion, currently 59% of prostate cancer cases detected are treated by RPE, a percentage steadily rising from 1997 on. On one hand, the dramatic increase of RPE already leads to a slight reduction of prostate cancer mortality in Austria [3,7], on the other hand the question of over-testing and over-treatment, and added morbidity remains unanswered and analyses are still pending for Austria.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsGH and CV planned the study and drafted the manuscript. CV obtained the data, GH and CV performed the statistical analysis. SM and GS contributed in preparing the manuscript. All authors read and approved the final manuscript.\n\nREFERENCES:\nNo References"
+}

batch_9/PMC2533343.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2533343",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533343\nAUTHORS: Mayur Tamhane, Ramesh Akkina\n\nABSTRACT:\nBackgroundThus far gene therapy strategies for HIV/AIDS have used either conventional retroviral vectors or lentiviral vectors for gene transfer. Although highly efficient, their use poses a certain degree of risk in terms of viral mediated oncogenesis. Sleeping Beauty (SB) transposon system offers a non-viral method of gene transfer to avoid this possible risk. With respect to conferring HIV resistance, stable knock down of HIV-1 coreceptors CCR5 and CXCR4 by the use of lentiviral vector delivered siRNAs has proved to be a promising strategy to protect cells from HIV-1 infection. In the current studies our aim is to evaluate the utility of SB system for stable gene transfer of CCR5 and CXCR4 siRNA genes to derive HIV resistant cells as a first step towards using this system for gene therapy.ResultsTwo well characterized siRNAs against the HIV-1 coreceptors CCR5 and CXCR4 were chosen based on their previous efficacy for the SB transposon gene delivery. The siRNA transgenes were incorporated individually into a modified SB transfer plasmid containing a FACS sortable red fluorescence protein (RFP) reporter and a drug selectable neomycin resistance gene. Gene transfer was achieved by co-delivery with a construct expressing a hyperactive transposase (HSB5) into the GHOST-R3/X4/R5 cell line, which expresses the major HIV receptor CD4 and and the co-receptors CCR5 and CXCR4. SB constructs expressing CCR5 or CXCR4 siRNAs were also transfected into MAGI-CCR5 or MAGI-CXCR4 cell lines, respectively. Near complete downregulation of CCR5 and CXCR4 surface expression was observed in transfected cells. During viral challenge with X4-tropic (NL4.3) or R5-tropic (BaL) HIV-1 strains, the respective transposed cells showed marked viral resistance.ConclusionSB transposon system can be used to deliver siRNA genes for stable gene transfer. The siRNA genes against HIV-1 coreceptors CCR5 and CXCR4 are able to downregulate the respective cell surface proteins and thus confer resistance against viral infection by restricting viral entry. These studies have demonstrated for the first time the utility of the non-viral SB system in conferring stable resistance against HIV infection and paved the way for the use of this system for HIV gene therapy studies.\n\nBODY:\nBackgroundHIV/AIDS continues to be major public health threat with new infections on the rise. Current therapies do not completely cure the disease and there is no effective vaccine available [1,2]. A potentially rewarding approach is intracellular immunization using gene therapy strategies that protect viral susceptible cells from the infecting virus [3]. Thus far, a number of promising intracellular immunization strategies have been employed using different anti-HIV molecules that act by a variety of mechanisms. Among these, nucleic acid-based approaches using ribozymes, antisense constructs, and siRNAs have received considerable attention due to their ease of expression and their non-immunological nature [3,4]. Some of these have entered clinical trials and safety testing with encouraging results [3,4]. In these studies either conventional retroviral vectors or lentiviral vectors were used for gene transfer. Although highly efficient for stable gene transfer, use of retroviral derived vectors poses a degree of risk in terms of viral mediated oncogenesis [5]. Because of this potential risk, non-retroviral mediated gene delivery systems are being currently investigated. In this regard, Sleeping Beauty (SB) transposon system shows considerable promise [6]. This system consists of a synthetic transposon and an associated transposase which functions by a cut and paste mechanism. Gene transposition is mediated by the transposase in a two step process in which the enzyme first recognizes the short inverted/direct (IR/DR) sequences in the transposon followed by the excision of the transposon and later integration of the transposon sequences into a target DNA region with a TA-dinucleotide sequence. The SB system can be deployed either as trans-delivery system in which the transposon and transposase are delivered by independent plasmids or a cis-delivery system in which both the components are incorporated into the same plasmid [7]. Continued progress in this area has resulted in the derivation of more efficient transposases and more efficient gene delivery [8]. Many mammalian cell types have been shown to be substrates for efficient SB mediated gene transfer including mouse embryonic stem cells [9]. Thus, SB system offers a novel way of gene delivery for HIV gene therapy purposes.With regard to effective anti-HIV genes for gene therapy, siRNAs constitute highly effective gene silencing molecules due to their target specificity and improved potency [10]. The siRNAs trigger an innate endogenous RNAi pathway for target recognition and gene silencing. Thus far, siRNAs targeted to a number of HIV genes have shown impressive gene down regulation and consequent viral inhibition both in vitro and in vivo [11-14]. Due to their high target specificity however, a high possibility exists for siRNA viral escape mutants to arise during prolonged treatment. Indeed, such generation of viral escape mutants against specific siRNAs has already been documented [15]. This possibility can be much reduced by targeting essential cellular molecules that aid in viral replication. Among the many cellular molecules shown to be involved in HIV infection and replication, the cell surface coreceptors CCR5 and CXCR4 are essential for viral entry by macrophage tropic R5 and T-cell tropic-X4 HIV respectively [16,17]. The primary HIV infection is established by R5 virus and during the later stages of disease, T-cell tropic X4 virus predominates [17,18]. In nature, a segment of the human population containing a 32-base pair deletion in the CCR5 gene, but apparently physiologically normal, was found to be resistant to infection by R5 tropic HIV-1 [17,19]. Therefore, CCR5 coreceptor is an ideal cellular target to suppress HIV infection. A number of previous studies including ours have successfully targeted both the HIV coreceptors by siRNA mediated gene silencing [12,20-22]. Down regulation of either of these coreceptors resulted in effective viral inhibition. However, retroviral derived vectors were used in these studies.With a long range goal of developing a non-viral gene delivery of anti-HIV genes for gene therapy, here we evaluated the utility of SB transposon system to deliver siRNA genes for stable gene transfer. Two previously well characterized siRNAs against CCR5 and CXCR4 coreceptors were introduced into SB transposon. Our results show that stable cell lines can be derived that harbor and express siRNA genes with concommittent HIV resistance.ResultsStable gene transfer of CXCR4 and CCR5 shRNAs by SB transposon systemTo investigate the utility of SB mediated gene transfer of anti-HIV-1 coreceptor siRNAs against CCR5 and CXCR4 we used the cell lines MAGI-CCR5 and MAGI-CXCR4 that constitutively express the respective individual coreceptors in addition to a GHOST-R3/X4/R5 cell line that constitutively expresses both [23-25]. As described in the methods, the cells were transfected with the respective plasmid SB constructs. Expression of the transposed constructs was monitored by the presence of RFP fluorescence. The gene transposed cells were enriched by FACS sorting and were maintained in culture for six months to confirm stable expression of the transgenes. Expression of RFP was observed throughout the time of culture. We also evaluated cells transfected with SB constructs alone in the absence of the transposase. The RFP expression in these cells was lost within a week post transfection. In a separate set of drug selection experiments to determine the levels of gene transfer using SB system in HeLa cells, it was found that the levels of transposition were 19.5% for the RFP control (above the background 0.6% gene transfer without the transposase). The gene transfer levels for the CXCR4 siRNA and the CCR5 siRNA constructs were 10.5% and 12% respectively. To further confirm transposition mediated transgene integration in stably gene transposed cells, we analysed the genomic DNA for the presence of the respective constructs. This was achieved by PCR amplifying and sequencing the junctional region of transposon and chromosomal DNA [26]. The typical hallmark of transposition is indicated by the presence of the dinucleotide 'TA' which was found at every insertion site analysed. To determine the transposed gene location, both left and right invert/direct repeats were sequenced at the chromosomal junctions. Sequences obtained were analysed using BLASTn software. Multiple integration events were recorded which spanned a range of chromosomal regions. The integration of representative individual SB transposons into the chromosomal DNA is summarized in Table 1. GHOST-R3/X4/R5 cells transposed with the control RFP transposon showed integration in Ch 5 and 17. Cells containing CCR5 siRNA showed Ch 5 and 20 regions at the transposon integration junction, while those transgenic for CXCR4 siRNA were found in Ch 17. In case of MAGI-CCR5 cells, control RFP transposon integrated into Ch 10 and 15. The CCR5 siRNA transposed cells showed integration in Ch 12 and 20. The integration sites for MAGI-CXCR4 cells were in Ch 6 and 12 for control RFP while those for CXCR4 siRNA transposon were in Ch 5 and 7. We also analyzed the copy numbers of integrated genes in GHOST-R3/X4/R5 cells using real time PCR. Our results showed 14.3, 6.5 and 10.8 copies per cell of the RFP control, CXCR4 siRNA and CCR5 siRNA constructs (data not shown).Table 1Chromosomal integration of different SB constructs.Cell LineSB ConstructChromosomal LocationGHOST-R3/X4/R5RFP controlCh 5q34-q35, Ch 17q25.1CXCR4 siRNACh 17q23.3CCR5 siRNACh 5q34-q35.1, Ch 20q13.2MAGI-CXCR4RFP controlCh 6p22.3, Ch 12q14.2CXCR4 siRNACh 5q33.1, Ch 7q31.1MAGI-CCR5RFP controlCh 10p12.31, Ch 15q11CCR5 siRNACh 12p11.2, Ch 20q13.3Down regulation of HIV-1 coreceptors CXCR4 and CCR5 in SB transposed siRNA transgenic cellsThe above data showed that SB transposed siRNAs are stably integrated into respective cells. We next evaluated if the stably gene modified cells show the effect of siRNA mediated gene silencing. Accordingly, the transposed cells were analysed for CXCR4 or CCR5 surface expression by FACS (Figure 2). Our results showed about 94% down-regulation of CXCR4 expression and a 97% down-regulation of CCR5 in GHOST-R3/X4/R5 cells transposed with CXCR4 or CCR5 siRNAs respectively. In the MAGI-CXCR4 cell line, the CXCR4 expression was reduced by 98% by the respective siRNA, while MAGI-CCR5 cells showed a 99% reduction in CCR5 levels as a result of respective transposon mediated siRNA expression (data not shown). Cells transposed with control SB construct without siRNA insert showed no decrease in coreceptor expression with levels similar to that shown by control unmanipulated cells. The levels of coreceptor down regulation obtained with these siRNAs in SB system are similar to that seen with that delivered via lentiviral vectors (data not shown). These results confirmed the efficacy of the respective siRNAs in mediating gene silencing of the HIV-1 coreceptors.Figure 1Schematic representation of siRNA SB constructs. A) Control SB transposon plasmid construct with Neo resistance and RFP reporter genes. RFP is driven by a CMV promoter whereas the Neo resistance is expressed via IRES. B) SB transposon construct incorporating anti-CXCR4 siRNA driven by Pol III U6 promoter. C) SB transposon construct incorporating anti-CCR5 siRNA driven by Pol III H1 promoter. D) Plasmid construct encoding the hyperactive transposase under CMV promoter.Figure 2Cell surface down regulation of CCR5 or CXCR4 coreceptors in siRNA transfected GHOST-R3/X4/R5 cells. GHOST-R3/X4/R5 cells that constitutively express CCR5 and CXCR4 coreceptors were transfected with control RFP, CCR5 or CXCR4 siRNA constructs. RFP expressing transgenic cells were FACS sorted and cultured. To determine the down regulation of respective coreceptors, the cells were stained with respective FITC tagged antibodies and FACS analyzed. The down regulation of CCR5 coreceptor (Panel A) was determined by comparing CCR5 levels in untransfected (A1), control RFP transfected (A2) and CCR5 siRNA transfected (A3) cells. The CXCR4 coreceptor down regulation is shown by comparing CXCR4 levels in untransfected (B1), control RFP transfected (B2) and CXCR4 siRNA transfected (B3) cells. The percent down regulation of CCR5 (A4) or CXCR4 (B4) coreceptors is also indicated.SB transposed anti-CCR5 and CXCR4 siRNAs confer HIV-1 resistanceTo determine if down regulation of CCR5 and CXCR4 coreceptors conferred viral resistance, siRNA transgenic GHOST-R3/X4/R5 cells were challenged with X4-tropic (NL4-3), R5-tropic (BaL-1) and dual coreceptor tropic HIV-1 89.6 strain. Antigen ELISAs to detect viral p24 in culture supernatants were performed on various days post-infection up to three weeks (Figure 3). When challenged with X4-tropic HIV-1 NL4.3, GHOST-R3/X4/R5 cells expressing CXCR4 siRNA showed a 10 fold decrease in virus production as compared to control non-transgenic cells on day 10 post-infection. The level of viral inhibition reached upto 14 fold through day 21 post-infection. In contrast CCR5 siRNA expressing GHOST-R3/X4/R5 cells failed to show any inhibition of virus production against X4 tropic HIV-1 NL4.3. Viral challenge of GHOST-R3/X4/R5 cells expressing CCR5 siRNA with the R5-tropic HIV-1 BaL resulted in an 8 fold reduction in virus production on day 10 post-infection, which doubled to 16 fold on days 14 and 21 post-infection. GHOST-R3/X4/R5 cells expressing CXCR4 siRNA served as a negative control as they showed similar levels of infection seen in control non-transgenic cells with the R5-tropic virus challenge. In dual-tropic HIV-1 89.6 viral challenges, neither of the individual CXCR4 siRNA or CCR5 siRNA expressing GHOST-R3/X4/R5 cells showed significant protection as expected since the challenge virus could use either of the coreceptors. However there was a moderate decrease in the virus production on day 21 as compared to unmanipulated cells. Cells transposed with SB control construct without anti-HIV transgenes showed similar levels of infection as the unmanipulated cells for all three HIV-1 strains. We also challenged SB transposed MAGI-CCR5 and MAGI-CXCR4 cells with R5 or X4 tropic viral strains respectively and found similar levels of resistance (data not shown). These data collectively showed that the respective SB system delivered siRNAs are functional and mediate viral resistance.Figure 3HIV-1 challenge of siRNA transposed GHOST-R3/X4/R5 cells. To determine viral resistance, siRNA transposed transgenic cells were challenged with HIV-1 NL4.3 (CXCR4 tropic virus), HIV-1 BaL (CCR5 tropic virus) or HIV-1 89.6 (dual tropic virus) viruses at an MOI of 0.01. On various days post-infection, cell culture supernatants were collected and analyzed for p24 antigen levels by ELISA to determine the levels of viral inhibition. Untransposed (◆), control RFP transposed (■), CXCR4 siRNA transposed (×) or CCR5 siRNA transposed (○). Panel A – NL4.3, Panel B – BaL, Panel C – 89.6.MethodsConstruction of CCR5 and CXCR4 shRNA expressing SB constructsThe Sleeping Beauty transposon vector pT/BH plasmid was obtained from Dr. Perry Hackett (University of Minnesota). The vector plasmid contains a multiple cloning site (MCS) flanked by a left and right inverted/direct repeat (IR/DR) elements [27,28]. Based on our previous data two well characterized and effective CCR5 and CXCR4 shRNAs were chosen for incorporating into the SB system plasmid [29]. The CCR5 siRNA target sequence is 5'-GUGUCAAGUCCAAUCUAUG-3' whereas the CXCR4 siRNA target sequence is 5'-GAGUCUGAGUCUUCAAGUU-3'. The CXCR4 or CCR5 shRNA DNA cassette was generated by PCR using published protocol [30]. In brief, PCR was done using U6 or H1 forward primer and a reverse primer containing 3'end homologous region of U6 or H1 promoter fused with CXCR4 or CCR5 shRNA sequence. The resulting PCR product was cloned into a Topo vector pCR8GW (Invitrogen, CA). A BglII site was engineered at the 5'end of forward and reverse primers. pT/BH was the transposon vector plasmid used into which a CMV driven RFP, IRES driven neomycin resistance gene and a SV40 polyadenylation signal containing cassette was cloned at the EcoRV site to derive the control RFP SB plasmid. To generate this pIRESneoRFP cassette, RFP gene was cloned as a BamHI – NotI fragment from pDsRed-N2 (Clontech, CA) into pIRESneo3 (Clontech, CA). A U6 promoter driven CXCR4 shRNA or H1 promoter driven CCR5 shRNA DNA cassette was cloned in parallel as a BglII-BglII fragment in the pT/BH plasmid at BamHI site to get pT/BH-U6CXCR4 or pT/BH-H1CCR5. The CMV-RFP-IRES-neo-SV40pA cassette was released as NruI-BstZ17I fragment and cloned at EcoRV site of pT/BH-U6CXCR4 or pT/BH-H1CCR5 plasmid to get pT/BH-U6CXCR4-CMV-RFP-IRES-neo or pT/BH-H1CCR5-CMV-RFP-IRES-neo. A hyperactive transposase expressing plasmid pHSB5, obtained from Dr. Mark Kay (Stanford University) was used to transpose the SB constructs [8]. A schematic representation of SB constructs and transposase plasmid are shown in Figure 1.Cell culture and transfectionRespective coreceptor expressing MAGI-CCR5 and MAGI-CXCR4 cell lines were obtained from the NIH AIDS Reagent Program and maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS, 500 μg/ml G418, 100 μg/ml hygromycin and 1 μg/ml puromycin. Similar culturing conditions were used for GHOST-R3/X4/R5 cells with G418 concentration being 200 μg/ml [23-25]. Cells were transfected with respective SB plasmids using Lipofectamine 2000 (Invitrogen, CA) as we previously described [31].FACS analysis and sortingTo enrich for transgenic cells, the SB transfected cells were subjected to FACS sorting based on RFP expression. The sorted cells were cultured for 4 weeks and analyzed by FACS to determine the cell surface down regulation by the respective siRNAs as described [32]. Briefly, the transfected or untransfected control cells were washed in PBS and resuspended in FACS buffer. FITC conjugated anti-CXCR4 or anti-CCR5 antibody was added to the cells and incubated for 30 minutes at 4°C. Cells were then washed and resuspended in PBS for FACS which was done using a Coulter EPICS-XL MCL (Coulter Corporation, FL) machine and analysed with EXPO32 ADC software.HIV-1 challenge of siRNA transposed cellsTo determine viral resistance conferred by the down regulation of CCR5 and CXCR4 coreceptors, siRNA transposed or non-transposed cells were subjected to viral challenge with HIV-1 BaL (CCR5-tropic), HIV-1 NL4.3 (CXCR4-tropic) or HIV-1 89.6 (Dual-tropic) viral strains. The HIV-1 viral strains were obtained from the AIDS Research and Reference Reagent program, Division of AIDS, National Institute of Allergy and Infectious Diseases. Briefly, 0.5 × 106 transgenic GHOST-X4/R3/R5, MAGI-CXCR4 or MAGI-CCR5 cells in 6 well plates were washed and exposed to virus at an MOI of 0.01 in the presence of polybrene (4 μg/ml). Virus was allowed to adsorb for 2 hours at 37°C. Cells were then washed twice with PBS and 2 ml of complete DMEM was added [21,33]. Culture supernatants collected at different days post-challenge were assayed for p24 antigen by ELISA (Beckman-Coulter, CA).Transposed gene integration analysisTo verify the stable transposition of the siRNA containing genes in the RFP expressing cell lines, the genomic DNA was isolated and subjected to Splinkerette PCR using a published protocol [26]. Transposed cell genomic DNA was digested with Sau3AI (for left IR/DR junctional analysis) or NlaIII (for right IR/DR junction analysis). Splinkerretes were generated by heating equimolar amounts of long primerette (5'-CCTCCACTACGACTCACTGAAGGGCAAGCAGTCCTAACAACCATG-3') with the respective splink to 80°C and cooling it to room temperature. Splink BglII (5'-GATCCATGGTTGTTAGGACCTGGAGGGGAAATCAATCCCCT-3', 5'-phosphate) was used for left IR/DR and splink SphI (5'-GTTGTTAGGACTGCTTGGAGGGGAAAATCAATCAATCCCCT-3', 5'-phosphate) was used for right IR/DR. The splinkerretes were then ligated to the respective digested genomic DNA ends. Ligation was performed with 7.5 μM of splinkerette and 25 ng/μl of genomic DNA with T4 DNA ligase (Fermentas Inc, MD). Primary PCR was done using the ligation reaction as template with primerette short (5'-CCTCCACTACGACTCACTGAAGGGC-3') in conjunction with either long IR/DR (L2) (5'-CTGGAATTTTCCCAAGCTGTTTAAAGGCACAGTCAAC-3') for IR/DR (L) or long IR/DR (R) (5'-GCTTGTGGAGGCTACTCGAAATGTTTGACC-3') for IR/DR (R). Primary PCR was done with 10 cycles of 95°C for 5 sec and 70°C (-0.5°C per cycle) for 2 min followed by 20 cycles of 95°C for 5 sec and 65°C for 2 min. Nested PCR was done by using 1/250 dilution of primary PCR product within the secondary PCR reaction. The second PCR was done using primerette-nested (5'-GGGCAAGCAGTCCTAACAACCATG-3') in conjunction with new L1 (5'-GACTTGTGTCATGCACAAAGTAGATGTCC-3') for IR/DR (L) or IR/DR (R) KJC1 (5'-CCACTGGGAATGTGATGAAAGAAATAAAAGC-3') for IR/DR (R). Nested PCR was done with 30 cycles of 95°C for 5 sec, 61°C for 30 sec and 70°C for 90 sec. Both primary and nested PCR included a hot-start at 95°C for 1 min and a final extension of 70°C for 10 min. Oligonucleotides used for this assay were obtained from IDT (San Jose, CA). The PCR products were cloned using a Topo cloning kit (Invitrogen, CA) and sequenced for the junctional region. The sequencing was done by Laragen (Los Angeles).DiscussionAs a first step towards exploiting a non-viral gene transfer system for HIV gene therapy, here we have shown that SB transposon system can be utilized for deriving stably gene modified cells that display HIV resistance. To achieve this goal, we employed siRNAs with proven efficacy to down regulate expression of the essential HIV-1 coreceptors CCR5 and CXCR4 with a consequent viral resistance phenotype. To our knowledge this is the first report describing gene transfer for viral resistance using a transposon system.GHOST-R3/X4/R5 cells constitutively expressing both CCR5 and CXCR4 coreceptors were used for SB mediated siRNA gene transfer in these proofs of concept studies. Since the general gene transfer efficiency is low relative to that typically obtained with lentiviral vectors [7,33,34], transfected cells were enriched by FACS sorting to evaluate the effectiveness of the stably integrated siRNA transgenes. Our results have shown that transgenic cells could be cultured indefinitely with stable expression of the transposed genes. FACS analysis of the siRNA modified cells showed consistent down regulation of the respective receptors CCR5 and CXCR4 amounting up to a 94% down regulation whereas cells transposed with control SB construct lacking siRNA transgenes showed normal levels of coreceptor expression similar to unmanipulated cells. Thus, down regulation of the respective targeted coreceptors established that siRNA transgenes are functional in a SB transposon system. As determined in the viral challenge experiments, siRNA transgenic cells also showed HIV resistance. With regard to individual siRNAs, GHOST-R3/X4/R5 cells transposed with CCR5 siRNA were found to be resistant to R5 HIV-1 viral challenge, whereas the cells transposed with CXCR4 siRNA were resistant to X4 HIV-1 viral challenge thus confirming the specificity of the respective siRNAs in mediating viral resistance. As expected, no significant protection could be seen from a dual tropic viral challenge of either of the individual siRNA gene modified cells since this viral strain could use either of the coreceptors for cellular entry.To further confirm stable gene transposition of the siRNA genes, we also mapped the integration sites of the SB transposon in respective transfected cells and found that these representative cell clones harbored the transgenes in different chromosomes namely 5, 6, 7, 10, 12, 15, 17 and 20. Previous studies mapped numerous SB-mediated integration sites in cultured and primary cells and found no chromosomal preference for insertion [35,36]. Consistent with this observation, the above clones transposed with siRNAs also represent random transposition events.The non-viral nature of the SB system offers some advantages over the more common retro and lentiviral mediated gene transfer [37]. Among these are that no viral sequences are involved thus minimizing insertion transcriptional activation of cellular genes and risk of generation of replication competent viruses during vector production. However, the gene transfer efficiency with the SB system remains sub-optimal compared to the viral vector systems [9]. Future improvements in the SB system are necessary to achieve higher gene transfer efficiency to be clinically practical [6,9].Although shown to be effective in conferring HIV resistance to cultured cells, the present SB system needs to be further evaluated in a hematopoietic stem cell setting using CD34 progenitor cells with a high efficiency of gene transfer to be clinically useful as shown with lentivirus vectors [3,4]. Even if high enough efficiency gene transfer is not achievable with this system in the near future, other innovative approaches are possible that may show clinical utility. For example, currently human embryonic stem (hESC) cells show great promise in developing novel therapies [38,39]. The hESC have already been shown to be amenable to gene transfer with SB transposon system, and it is now routine to derive hematopoietic CD34 cells from hESC as shown by us and others [40-44]. One can envisage that hESC can be transposed with anti-HIV siRNAs using SB system and high expressing cell clones could be derived. From these transgenic hESC clones, unlimited numbers of siRNA expressing CD34 cells could be derived for HIV gene and cell therapies. Such experiments are currently underway in our laboratory.ConclusionSB gene transposon system can be used to deliver siRNA genes against HIV-1 coreceptors CCR5 and CXCR4 for stable gene expression. The siRNA genes are able to downregulate the respective coreceptor expression on the cell surface and thus confer resistance against HIV-1 infection by restricting viral entry. These studies have demonstrated for the first time the utility of the non-viral SB system to derive viral resistant cells and paved the way for the use of this system for HIV gene therapy studies.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsMT derived the experimental data and RA was responsible for the conception and overall implementation of the project. All authors read and approved the final manuscript.\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2533351",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533351\nAUTHORS: Jérémie JP Lebrec, Hein Putter, Jeanine J Houwing-Duistermaat, Hans C van Houwelingen\n\nABSTRACT:\nBackgroundDespite the current trend towards large epidemiological studies of unrelated individuals, linkage studies in families are still thoroughly being utilized as tools for disease gene mapping. The use of the single-nucleotide-polymorphisms (SNP) array technology in genotyping of family data has the potential to provide more informative linkage data. Nevertheless, SNP array data are not immune to genotyping error which, as has been suggested in the past, could dramatically affect the evidence for linkage especially in selective designs such as affected sib pair (ASP) designs. The influence of genotyping error on selective designs for continuous traits has not been assessed yet.ResultsWe use the identity-by-descent (IBD) regression-based paradigm for linkage testing to analytically quantify the effect of simple genotyping error models under specific selection schemes for sibling pairs. We show, for example, that in extremely concordant (EC) designs, genotyping error leads to decreased power whereas it leads to increased type I error in extremely discordant (ED) designs. Perhaps surprisingly, the effect of genotyping error on inference is most severe in designs where selection is least extreme. We suggest a genomic control for genotyping errors via a simple modification of the intercept in the regression for linkage.ConclusionThis study extends earlier findings: genotyping error can substantially affect type I error and power in selective designs for continuous traits. Designs involving both EC and ED sib pairs are fairly immune to genotyping error. When those designs are not feasible the simple genomic control strategy that we suggest offers the potential to deliver more robust inference, especially if genotyping is carried out by SNP array technology.\n\nBODY:\nBackgroundLinkage analysis of family data have been extensively used in the past in the search for genetic determinants. Nowadays, investigators favor large epidemiological studies of unrelated individuals, however several family datasets are currently being re-analyzed and/or pooled (e.g. [1]). The persistance of interest for linkage is partly triggered by the advent of single-nucleotide-polymorphisms (SNP) array genotyping technology in the field, indeed SNP arrays hold the promise of more reliable linkage maps [2,3]. Although less prone to genotyping error than microsatellites when viewed as singlepoint markers, SNP arrays heavily rely on multipoint algorithms for accurate determination of the identical by descent (IBD) status of alleles. The gain in singlepoint reliability might therefore be annihilated by the propagation of errors across the many SNPs required to infer IBD status.In the search for genetic determinants of complex traits by linkage, the use of selective designs appears to be an efficient way to gain adequate power for detection of typically small gene effects. A few authors have shown by simulation that the impact of genotyping error on evidence for linkage could be particularly severe in affected sib-pair (ASP) designs [4-6], virtually masking most of the evidence for linkage. The impact of error on quantitative traits appears to be less dramatic in random samples, however it is unclear whether the same dramatic power losses hold in selected samples.A method of choice is now emerging for the analysis of quantitative traits arising from selected sib pairs. This method is essentially a regression through the origin of excess identical by descent (IBD) sharing on a function of the trait value, whose slope is an estimate of the linkage parameter. It was first proposed by Sham et al. [7] and turns out to be equivalent to a score test [8]. In a numerical comparison of methods for selected samples, Skatkiewicz et al. [9] and Cuenco et al. [10] showed that this method had good properties in finite samples for extreme proband ascertained sib-pair and discordant sib-pair designs. By use of simple genotyping error models (population frequency error model and false homozygosity model), we show analytically what effects such error generating processes (occurring at rate ϵ per sib pair) induce for an idealized fully informative marker. It is shown that it results in a reduction of the slope estimate (i.e. of the estimated linkage parameter) by a factor 1 - ϵ2 whether sib pairs are selected or not. Since the genotyping error rate ϵ is typically small, the previous effect on the linkage test is minimal. In addition to this slope effect, the regression's intercept is modified and this may have a much more sizable effect on the test for linkage depending on the sampling scheme used to select sib pairs. Surprisingly, this simple result allows us to predict that in extremely concordant (EC) sib pairs designs and in ASP designs, the effect of genotyping error will be milder as the selection becomes more extreme. In extreme discordant (ED) designs, the effect can in theory be either increased type I error or decreased power depending on the definition of discordance, the genotyping error rate and the true linkage effect; in practice however, for small quantitative trait locus (QTL) effects, the result will be an increased type I error. We argue that the basic error generating mechanisms assumed provide reasonable approximations of real-life situations. In the next section, we first describe some common error-generating processes and quantify their effect on IBD sharing in an idealized situation where marker information is complete. We then briefly sketch the inverse regression approach to linkage, we show analytically what the effect of genotyping error is on this regression and quantify the subsequent bias, power and type I error in common selective designs. We argue that under certain assumptions regarding the error model, one can easily implement a linkage test that incorporates a genomic control for genotyping error. Finally, we discuss some assumptions made in our study and the practical relevance of our findings. In particular, we argue that our results generalize to situations where marker information is incomplete and that the smaller error rates observed in SNP chip array compared to microsatellites offer no protection against bias in analysis.ResultsGenotyping error modelsWe consider two mechanisms for the generation of errors in marker data, namely the population frequency error model and the false homozygosity model. In those two models, we consider a single marker with m alleles and further assume that a maximum of one allelic error per sib pair can be made and that this happens with probability ϵ. This restriction to 'one error per sib pair' is just a first order approximation, for small ϵ, of a process where all four alleles would be allowed to be independently erroneous and does not restrict the generalizability of our results.The population frequency error model re-assigns the erroneous allele (chosen at random among the four forming the sib-pair genotype) to one of the possible m alleles with probability equal to population allele frequency. One mathematical advantage of this model is that the marginal distribution of alleles and genotypes is unaltered. The false homozygosity model keeps homozygotes unchanged but re-assigns heterozygotes to homozygotes with alleles equal to one of the two original alleles chosen according to probabilities proportional to population allele frequencies.To our knowledge, false homozygosity is a common type of error: fairly rare alleles go un-reported in samples. The population frequency error model provides an approximation to a process whereby alleles are misread. Errors at the two alleles of a marker's genotype might be correlated, we do not consider this type of process in details here although the effect on linkage will be qualitatively the same as in the two other models. We refer the reader to Sobel et al. [11] for a detailed exposé on genotyping error mechanisms. Note that the two models that we have chosen have been used in the past in order to identify potential genotyping errors [4,11].Impact on IBD sharingLet's denote by π the proportion of alleles shared identical by descent (IBD) at a certain locus by two siblings. Tests for linkage are based on the IBD sharing distribution and although errors as described earlier are made at the genotype level (G is read as Gϵ), the effect of errors on linkage will be entirely mediated via the distortion of the IBD distribution (the true IBD status π of two siblings may be incorrectly inferred as πϵ). We are therefore interested in deriving the probability distribution P(πϵ|π), this is done by conditioning on both the true and observed genotypes as follows:P(πϵ|π)=∑GϵP(πϵ|Gϵ)∑GP(Gϵ|G) P(G|π).Let us consider the case of complete information. This can be conceptualized by means of an idealized marker whose number of alleles is infinite, in particular identity by state (IBS) status is equivalent to IBD status. The unordered genotypes of a sib pair can be partitioned into seven exclusive classes denoted ii/ii, ii/ij, ii/jj, ii/jk, ij/ij, ij/ik and ij/kl depending on the number of homozygous sibs in the pair and the number of distinct alleles in the sib-pair genotype. Sharing 0 alleles IBD corresponds to a sib-pair genotype of the ij/kl class, should an error occur according to the population frequency error model then one of the four alleles would be transformed into yet another type (since the number of alleles is infinite, the probability that the new allele is read as one of i, j, k or l tends to 0), therefore the sib pair genotype will remain in the ij/kl class and the observed IBD status πϵ will still be 0. For the same starting genotype, an error according to the false homozygosity model produces an ii/jk class and πϵ also equals 0 therefore P(πϵ = 0|π = 0) = 1 whatever the genotyping error mechanism considered previously. The same line of reasoning leads to P(πϵ = 0.5|π = 0.5) = 1 - ϵ2, P(πϵ = 0|π = 0.5) = ��2, P(πϵ = 1.0|π = 1.0) = 1 - ϵ, P(πϵ = 0.5|π = 1.0) = ϵ. Those results can be summarized by the transition matrix below, where the (i, j) element is equal to P(πϵ = (j - 1)/2|π = (i - 1)/2)P(πϵ|π)=(100ϵ21−ϵ200ϵ1−ϵ).The overall effect of genotyping error is thus to reduce the observed IBD sharing, indeed E(πϵ|π) = (1 - ϵ/2)π and E(πϵ) = 12 - ϵ/4 while the variance is practically unchanged since var(πϵ)=18−116ϵ2. In selected samples of extremely concordant sib pairs (EC) where linkage is evidenced by an excess in IBD sharing, it therefore seems logical to expect a decrease in power. Conversely, in selected samples of extremely discordant sib pairs (ED) where linkage is evidenced by a reduction in IBD sharing, the test might lead to increased type I error. In the next subsection, we formally quantify this bias in selective samples schemes for quantitative traits under the usual assumption of a normal variance components model.Impact on linkage testingRegression-based linkage testingWe assume that the sib pair phenotypic data x = (x1, x2)' have been adjusted for any relevant covariates (e.g. sex, age, country, ...) and have been standardized so that the (known) population mean, variance and sib-sib correlation are 0, 1 and ρ respectively. Under the additive variance components model, x given IBD information p follows a bivariate normal distribution with zero mean and variance-covariance matrix given by(1γ(π−12)+ργ(π−12)+ρ1),where γ ≥ 0 denotes the proportion of total variance explained by the putative locus. Under this model, an optimal testing strategy first advocated in [7] (and sometimes referred to as the optimal Haseman-Elston regression) is to regress (through the origin) excess IBD sharing π - 12 on the following C function of the trait values:(1)C(x1,x2,ρ)=(1+ρ2)x1x2−ρ(x12+x22)+ρ(1−ρ2)(1−ρ2)2.This test turns out to be a score test for the linkage parameter γ [8] and is based upon the following approximate relation which is valid for small locus effects [12]:(2)E(π−12|x,γ)=γ8C(x,ρ),where 18 = var0(π). In a set of sibships indexed by i, an efficient estimate of the linkage parameter γ is γˆ=8∑i(πi−12)Ci∑iCi2. It is approximately unbiased E(γˆ) = γ and has variance var0(γˆ) = 1/I where I=18∑iCi2 is the corresponding Fisher's information. The test statistic is given by γˆI, it is one-sided, only positive values being regarded as evidence for linkage. For small QTL effects, power of this test can be computed as Φ (Φ-1(α) + γI1/2). Fisher's information I, which depends on sample size and study design, therefore controls power. In the design phase of a study, I should be used as a criterion to differentiate between alternative designs rather than sample size only [12,13].Impact of genotyping error on regressionBy conditioning on the true IBD sharing values, we can compute P(πϵ|x, γ, ϵ) = ∑πP(πϵ|π) P(π|x, γ), using the transition probabilities P(πϵ|π) derived earlier, while the P(π|x, γ)'s are given in [12]. This permits computation of the new regression line in presence of genotyping error as(3)E(πϵ−12|x,γ,ϵ)=−ϵ4+(1−ϵ2)γ8C(x,ρ).As mentioned earlier, the corresponding variance under the null hypothesis is only slightly altered. The effect of genotyping error is thus to shrink the regression line by a factor 1 - ϵ2 and to shift the intercept by -ϵ4. If we ignore genotyping error i.e. we estimate γ using γˆϵ=8∑i(πi−12)Ci∑iCi2, this results in a biased estimator bias(γˆϵ)=E(γˆϵ)−γ=−ϵ(γ2+2A) with A=∑iCi∑Ci2=C¯C2¯. The resulting testing statistic γˆϵI1/2 would then have power equal to(4)Φ(Φ−1(α)+γI1/2+bias(γˆϵ)I1/2).Note that taking γ = 0 in this formula gives the type I error rate. Since I increases with sample size, the impact of genotyping error on both power and type I error will be larger as the sample size increases. In terms of Y versus X regression, the intuition is that the regression through the origin is not affected by a general shift in the Y-variable (IBD sharing) if the X-variable (C variable) has average 0, or takes values far away from 0. The further away the X-variable C is from 0, the smaller A, hence the smaller the bias.Bias and impact on power and type I errorSince bias(γˆϵ)=−ϵ(γ2+2A) and γ is typically small, the distortion of the usual linkage test in presence of genotyping error heavily depends on the design-specific quantity A=C¯/C2¯. Unfortunately, there is little intuition about the distribution of C (hence about the distribution of A) in the whole population or in a selected sample. Nevertheless, Monte Carlo simulations can be used to determine the characteristics of the C and A distributions in the whole population or for a specific ascertainment scheme. In random samples and under the variance components model, C is a score function hence E(C) = 0 therefore its sample estimate C¯ will be close to 0; one can also check that its distribution is negatively skewed (unless ρ = 0). The result is that the bias will be small for random samples. The same finding would hold for any ascertainment scheme where C¯ = 0. An optimal selection scheme [12] that would select sib pairs based on Fisher's information I (i.e. such that |C| ≥ C0) does not warrant that C¯ = 0 because of the skewness of C. In EC designs (both siblings have trait values either larger than a positive threshold or smaller than a negative threshold), C¯ tends to be positive while it tends to be negative in ED designs (one sibling's trait value is larger than a positive threshold while the other sibling's trait value is smaller than a negative threshold), the linkage test will therefore have reduced power in EC designs and increased type I error in ED designs.In the left-hand side of Table 1, we have computed the values of A and C¯ for the three selective schemes considered. The designs are indexed by the sib-sib correlation ρ and the degree of selection. One obvious way to correct for the shift in the intercept induced by genotyping error would be to leave the regression unconstrained, this would correct for most of the bias. Unfortunately, in selected designs where the variance of C is reduced, this results in a very inefficient estimator of the linkage parameter γ. The right-hand side of Table 1 displays the variance of the linkage parameter estimates in constrained (varcon(γˆ)=1/∑iCi2) and unconstrained (varuncon(γˆ)=1/∑i(Ci−C¯)2) regressions. Efficiency losses of unconstrained versus constrained regressions in EC and ED designs are unacceptably large even for moderately extreme selection schemes.Table 1Bias in selective designsAC¯varconvarunconSelectionρECEDIECEDIECEDIECEDI1%0.10.27-0.23-0.073.45-3.93-1.610.080.060.041.130.680.050.20.29-0.21-0.133.28-4.25-3.190.090.050.041.460.520.070.30.30-0.19-0.153.15-4.72-4.630.100.040.031.820.380.100.40.31-0.17-0.143.06-5.29-6.000.100.030.022.270.270.170.50.32-0.14-0.123.01-6.10-7.440.110.020.022.380.180.230.60.31-0.12-0.103.02-7.33-9.330.100.020.011.920.120.1910%0.10.47-0.40-0.061.71-1.87-0.400.280.220.141.480.880.140.20.50-0.36-0.111.66-1.99-0.810.300.180.131.840.660.140.30.52-0.32-0.141.64-2.14-1.300.320.150.112.200.480.130.40.53-0.28-0.161.63-2.35-1.860.320.120.092.370.350.120.50.52-0.24-0.171.64-2.61-2.610.310.090.062.050.230.110.60.47-0.19-0.151.68-3.01-3.640.280.060.041.330.150.1030%0.10.65-0.53-0.040.96-1.01-0.110.680.520.311.801.120.320.20.69-0.46-0.070.95-1.03-0.230.730.440.292.150.850.290.30.71-0.39-0.090.96-1.06-0.360.740.370.252.330.630.260.40.69-0.32-0.110.97-1.13-0.520.710.280.202.170.450.210.50.62-0.25-0.111.00-1.22-0.730.620.210.151.640.300.160.60.50-0.19-0.101.05-1.35-1.010.470.140.100.980.190.11Left-hand side: Values of A, C¯ quantities influencing the effect of genotyping error for a variety of selective designs indexed by degree of Selection and sib-sib trait correlation ρ) – Right-hand side: Comparison of efficiency in constrained and unconstrained regressions – See text for definitions of A, C¯, varcon and varunconIn Table 2, we report the power and type I error for realistic genotyping error rates [14] equal to 0.005 and 0.01 for the same designs as in Table 2. The equivalent sample size used corresponds to samples with Fisher's information equal to 2500 which provides 90% power to detect a QTL explaining 10% of the total variance in absence of genotyping error (pointwise nominal error rate = 10-4). The most visible impact is on type I error rates in ED design which is up to 7 times its nominal value. The I design that combines EC and ED sib pairs appears to be fairly immune to genotyping error while EC designs do not incur power losses greater than 20%. Finally, those computations confirm the intuition expressed earlier that the effect of genotyping error is less severe in more extreme selection schemes.Table 2Impact of genotyping error (rate = ϵ) on type I error and powerError rate ϵSelectionρECEDIPowerType I Error × 10-4PowerType I Error × 10-4PowerType I Error × 10-40.0051%0.10.870.60.921.60.901.10.20.870.60.921.50.911.30.30.870.50.911.50.911.30.40.870.50.911.40.911.30.50.870.50.911.40.911.30.60.870.50.911.30.911.210%0.10.850.40.932.20.901.10.20.850.40.932.00.911.20.30.840.30.921.90.911.30.40.840.30.921.70.911.40.50.840.30.921.60.911.40.60.850.40.911.50.911.330%0.10.830.30.942.80.901.10.20.820.20.932.40.901.10.30.820.20.932.10.911.20.40.820.20.921.90.911.20.50.830.30.921.60.911.20.60.850.40.911.50.911.20.011%0.10.840.30.932.40.911.30.20.830.30.932.20.921.70.30.830.30.932.10.921.80.40.830.30.921.90.921.70.50.830.30.921.70.921.60.60.830.30.921.60.911.510%0.10.780.10.954.50.911.30.20.780.10.953.90.911.50.30.770.10.943.40.921.70.40.770.10.942.90.921.90.50.770.10.932.50.921.90.60.780.10.932.10.921.830%0.10.730.10.967.10.901.20.20.710.10.965.60.911.30.30.710.00.954.40.911.40.40.710.10.943.40.911.50.50.740.10.932.60.911.50.60.780.10.932.10.911.5Impact of genotyping error (rate = ϵ) on power and type I error of linkage test in selective designs (indexed by degree of Selection and sib-sib trait correlation ρ) – Nominal error rate = 10-4, QTL effect γ = 0.1 and sample size equivalent to a Fisher's information = 2500 in each design (provides 90% power in absence of genotyping error)Genomic control for genotyping errorAs we have seen in previous sections, the main effect of genotyping error is to modify the intercept in the regression used to test for linkage. Although an unconstrained regression would correct most of the bias due to genotyping error, the inefficiency of this strategy makes it impractical. In order to obtain an efficient and robust inference, it therefore seems natural to try and constrain the regression through its correct origin a. In this section, we propose a completely data-driven strategy for doing this.At any position, the sample mean IBD sharing has variance 1/8n where n is the number of sib pairs available. If we knew that the position is unlinked or if the sample of sib pairs was random then the deviation of this mean from 12 would provide an estimate of the intercept a in the linkage regression.Unfortunately, detection of a position-specific intercept corresponding to typical error rates would require a sample size of order 104, a number that is almost never reached in linkage studies. In order to obtain an intercept estimate aˆ with sufficient precision, it is therefore essential to combine information across positions. The value of IBD sharing at positions outside of the neighborhood of influencing loci (those positions are subsequently referred to as unlinked) across the genome may serve as control in the test for linkage, this concept of genomic control has been used to make the analysis of association studies more robust [15].Let's assume that the proportions of alleles shared IBD π is computed at a series of approximately regular positions indexed by t across the whole genome. Let yt be the sample mean (among families) excess IBD at position t i.e. yt≡πtϵ¯−0.5. Under the variance components model and for small QTL effect γ, equation (3) implies thatE(yt)≃{a,if position t is unlinked,a+b8γC¯,if position t is linked.In random samples or in any sample where C¯ ≃ 0, taking the average of yt across positions provides an estimate of a. In selected samples, we can use a trimmed version of the mean of y, for example a 20%-trimmed mean of the (yt)t series (i.e. the mean of the yt values after removing the 20% lowest and and 20% highest values) will provide a robust genomic estimate aˆ of a. Because a ≤ 0 and C¯ is positive and negative in EC designs and ED designs respectively, aˆ could be refined by trimming off only the 20% highest and lowest yt values respectively before taking the mean. Of course, how much we trim is arbitrary but 20% can safely be taken as a conservative value for oligogenic traits (Indeed, a 3500 cM genome contains approximately 70 quasi-independent loci, so a 20% trimming of yt values discards 14 positions (including all active gene positions if less than 14 genes) from the sample used to estimate intercept a.). An ad-hoc implementation of the concept of genomic control is then to plug in the estimate of the intercept aˆ into the linkage regression (3). Since most of the bias in the inference is due to the intercept mis-specification, the precise estimate obtained by pooling across the genome will eliminate it. The implicit assumption that we make in this genomic control approach is that the regression intercept is the same at all positions, this will be challenged in the next section.DiscussionUnder two basic error models, we were able to predict quantitatively the consequences of genotyping error on inference in linkage analysis. In the idealized situation of complete IBD information, both error models have the same impact on linkage analysis. As we have seen, the effect is due to a decrease in IBD sharing. A contrario, an error process which would increase IBD sharing would produce opposite results. The true error processes involved in practice are complicated mixtures of the models alluded to here. In our experience however, it seems that processes which lower IBD sharing are predominant. Because genotyping error tends to decrease the estimated number of alleles shared IBD, the effect on evidence for linkage is opposite in EC (reduced power) and ED (increased type I error) designs, it can be dramatic in typical designs and paradoxically less severe for more extreme ascertainment schemes. By analogy, for a dichotomous trait, this means that the effect of genotyping error is less severe in ASP designs for rare diseases than for common diseases. Remarkably, in designs combining both ED and EC pairs like the I (or EDAC designs), the competing effects of genotyping error tend to cancel each other out. We have considered here only three types of basic selection schemes however the approach can be straightforwardly applied to any arbitrary selection scheme. Under the widely accepted variance components model, the important quantity which determines bias, type I error and power is A=C¯/C2¯ and it can be easily estimated by Monte Carlo simulations. Note that the bias is proportional to the error rate so that Equation (4) can easily be adapted to different error rates than those considered in Table 2.Our study used an idealized model where IBD information is assumed to be complete. In practice, IBD is uncertain and it is inferred using marker data and multipoint algorithms as implemented in publicly available software [16,17], the general effect is to shrink the IBD estimate πˆ towards 0.5. The linkage regression (2) is changed into E(πˆ−12|x,γ,ϵ)≃var⁡0(πˆ)γ C(x,ρ) where var0(πˆ)<18 can be either estimated from the data or by simulations. The effect of genotyping error is again mediated via the shift of the intercept in this regression but no general formula can be obtained because it depends in a very complex manner on the whole marker map configuration. Nevertheless, we can quantify this shift under realistic scenarios and compare it to its theoretical value when IBD information is complete. We simulated two different marker maps in 1 million sib pairs without parents and quantified by how much IBD sharing was reduced on average under the population frequency error model (error rate = 0.01). The microsatellites map (MS) had 13 equi-frequent ten-allele markers (heterozygozity = 90%) located 10 cM apart (spanning the 0–120 cM chromosomal region) and the SNP map had 41 equi-frequent SNPs (heterozygozity = 50%) spanning the 50–70 cM chromosomal region (this smaller region was chosen to keep simulation time acceptable). The resulting average reduction in IBD sharing for an error rate of 0.01 was measured every 2 cM in the 50–70 cM region, it ranged from 0.4974 to 0.4976 in the MS map and from 0.4945 to 0.4955 in the SNP map. For these two maps which mimic the two most widespread genotyping paradigms nowadays, those simulations confirm results derived under the complete marker information assumption with a reduction in IBD sharing from 0.5 to 0.5 – 0.01/4 = 0.4975. Our results therefore appear to be applicable to real-life situations where IBD information is incomplete.The genomic-control strategy that we have proposed, although triggered by the specific issue of genotyping error, potentially offers a general robust method for carrying out linkage analysis. It is nonetheless important to recognize its limitations. Firstly, if the trait is highly polygenic with contributing genes scattered across the genome, the high correlation between linkage positions will make it impossible to estimate the IBD sharing at null positions. The genomic control strategy should therefore only be considered with oligogenic traits. Secondly, the concept of genomic control relies on the assumption that the genotyping error rates are similar across markers. For markers with a similar degree of polymorphism (number of alleles and frequencies), this assumption might be acceptable. In a multipoint setting, an additional assumption required to ensure the validity of a genomic control strategy is that inter-marker distances be approximately equal. With microsatellite markers, both these assumptions might fail resulting in differences in the IBD sharing reduction across markers. The 'regression-based linkage testing' view allows one to qualitatively assess how deviation from these assumptions will impact linkage testing. For example, in ASP or EC designs, wrongly assuming that IBD is uniformly reduced across markers will result in inflated type I error at marker positions with low genotyping error rate compared to other markers. The advent of SNP chips in linkage mapping holds the promise of regular marker maps with less variable information content than in classical microsatellites maps [2,3]. The many SNPs used are likely to be subject to similar genotyping error processes, this makes the critical assumption of the genomic control strategy all the more plausible. Alternatives to this genomic-control strategy are possible and they also consist in constraining the linkage regression through a new origin as in the ad-hoc method, the estimation procedure can be adapted to suit particular circumstances. Firstly, in random samples, the assumption regarding exchangeability of positions might be relaxed. Indeed, the reduction in IBD sharing at each position may be used as estimates of the position-specific intercepts (a study sufficiently powered to detect linkage in random samples should have a huge sample size which would ensure sufficient precision of the position-specific intercepts). However, it must be stressed that the advantage of using a genomic control in random samples is limited because the impact of genotyping error is small in such designs. Secondly, one could use previous lab data to estimate by how much IBD sharing deviates from its expected value, this could also be done at each position separately provided sufficient data are available. In practice, such data might not be available or they might not trustfully reflect current error mechanisms.Elston et al. [18] have pointed out that the implicit assumption made in ASP designs, that randomly sampled sib pairs share half of their alleles IBD, might not hold in practice and have argued for including discordant pairs in such studies. The genomic control approach suggested here may be an alternative solution to this issue. Finally we note that, although we have only considered designs involving sib pairs, the approach naturally extends to other types of relative pairs.ConclusionUnder realistic genotyping error scenarios, power losses observed in extremely concordant designs are modest but the effect on type I error in extremely discordant designs can be dramatic. Our analytic approach provides some understanding of the differences in influence of genotyping errors across study designs. The advent of SNP arrays does not eliminate the impact of genotyping errors but it makes genomic control a feasible option with the potential to deliver more robust inference in linkage analysis data subject to genotyping errors or other mechanisms distorting the IBD signal.AbbreviationsASP: affected sib pair; EC: extremely concordant; ED: extremely discordant; EDAC: extremely concordant and extremely discordant; IBD: identical-by-descent; QTL: quantitative trait locus; SNP: single-nucleotide-polymorphism.Authors' contributionsJJPL participated in the method development, carried out the simulations summarized in Table 1, drafted and finalized the manuscript. HP participated in method development and in drafting the manuscript. JJH-D and HCvH both participated in method development. All authors read and approved the final manuscript.\n\nREFERENCES:\nNo References"
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